The Journal of Immunology

Published by American Association of Immunologists
Online ISSN: 1550-6606
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Article
A comparison was made of the abilities of carrier (BGG)-primed T cell populations from young (4-month old), middle-aged (14- and 19-month old) and old (31- and 34-month old) mice to collaborate with hapten (DNP)-primed B cells from young mice in a cell-transfer system. The plaque-forming cell responses to 2,4-dinitrophenol (DNP) were measured by a modification of the Jerne plaque assay. The DNP-specific antibody-forming cell responses of old T cell/young B cell combinations were significantly lower than those of young T cell/young B cell combinations, both in the number of T cells needed for peak response and in the size of that response. These data indicate that the primed T cell populations of old mice are deficient by a factor of 6 in their ability to initiate B cell proliferation and differentiation into antibody-forming cells.
 
Article
Although it has been demonstrated that malignant human B cell lines are capable of producing B cell growth factor (BCGF), production of BCGF by normal B cells has not been shown. In this study, we demonstrate BCGF production by normal B cells, achieved by using human peripheral blood B cells prepared by a positive selection technique and stimulated with Staphylococcus aureus Cowan I (SAC) for 12 hr. SAC was removed from the supernatants by anti-SAC-coupled Sepharose. Supernatants absorbed with this antibody were functionally free of SAC, as demonstrated by their inability to activate resting B cells. B cells stimulated with SAC for 12 hr produced BCGF activity that was generally unmeasurable in supernatants by 36 hr. Characterization of BCGF produced by SAC-stimulated B cells revealed a m.w. of 32,000 by high-performance liquid chromatography sieving and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; this BCGF was found to have an isoelectric point of 6.7. Furthermore, this BCGF lacked interleukin 1, interleukin 2, interferon, and B cell differentiation factor activity. This observation that BCGF can be produced by normal human B cells is significant because it demonstrates for the first time that normal B cells have the ability to provide their own growth factors or the growth factors for other B cells.
 
Article
CD5+ B cells have attracted considerable interest because of their association with self-reactivity, autoimmunity, and leukemia. In mice, CD5+ B cells are readily generated from fetal/neonatal precursors, but inefficiently from precursors in adult. One model proposed to explain this difference is that their production occurs through a distinctive developmental process, termed B-1, that enriches pre-B cells with novel germline VDJs and that requires positive selection of newly formed B cells by self-Ag. In contrast, follicular B cells are generated throughout adult life in a developmental process termed B-2, selecting VDJs that pair well with surrogate L chain, and whose maturation appears relatively independent of antigenic selection. In the present study, I focus on processes that shape the repertoire of mouse CD5+ B cells, describing the differences between B-1 and B-2 development, and propose a model encompassing both in the generation of functional B cell subpopulations.
 
Article
It was recently reported that human and mouse melanoma cells express Fas ligand (FasL) but almost no Fas, which may contribute to their immune privilege. AS101 (ammonium trichloro(dioxoethylene-0,0')tellurate), a synthetic immunomodulator with minimal toxicity, was found to have antitumor effects in various tumor models. Our present study shows that AS101 has direct and indirect effects on tumor cells; AS101 inhibits the clonogenicity of B16 melanoma cells in vitro. Moreover, wild-type P53 expression, which is required for induction of Apo-1 expression, increased significantly in AS101-treated cells. We therefore investigated Fas expression in AS101-treated B16 cells and found that Fas, but not FasL, expression was significantly increased; moreover, Fas receptors were functional. Longer incubation with AS101 resulted in spontaneous apoptosis triggered by the Fas-FasL system. To explore the relationship of these results to the antitumor effects of AS101, we injected B16-F10 mouse melanoma cells into syngeneic C57BL/6 mice carrying the lpr mutation in the Fas gene and to gld mutant mice that lack functional FasL. Tumor development in control groups was lowest in the lpr mice, while no difference was observed between gld and wild-type mice. Among the AS101-treated groups, the most pronounced effect appeared in the lpr mice, while the lowest was seen in the gld mutant mice. Our study suggests that AS101 may render melanoma tumor cells more sensitive to Fas/FasL-induced apoptosis and may therefore have clinical potential.
 
Differences between HLA-B*57:01(Bw4) and HLA-B*08:01(Bw6). (A) Conservation of residues between HLA-B*57:01 and HLAB*08:01. Differences are colored orange. Positions that are bound by KIR3DL1*001 are colored green. Positions 80 and 83, which differ between HLAB*57:01 and HLA-B*08:01 and are bound by KIR3DL1*001, are colored blue. (B) Partial sequence alignments of HLA-B*57:01 and HLA-B*08:01. The Bw4/Bw6 motif is highlighted in gray, and HLA-B*57:01 regions involved in KIR3DL1*001 binding are noted.
KIR3DL1 + NK cell recognition of HLA class I molecules is influenced by residues 80 and 83. (A) Surface expression of mutant HLAB*57:01 molecules transfected into 721.221 (221) cells was assessed by flow cytometry, staining with either the pan class I Ab W6/32 (black histograms) or a specific anti-Bw4 Ab (dashed histograms). Filled, gray histograms represent background staining with a secondary anti-mouse IgG-FITC alone. (B) PBMCs were isolated from healthy donors and incubated overnight with equal numbers of 221 or 221.B5701 mutant transfectants in the presence of brefeldin A. Cells were assayed for the production of IFN-g, gating on CD56 + , CD3 2 NK cells expressing KIR3DL1. IFN-g production was normalized against the maximum observed upon incubation with parental, untransfected 221 cells. Data were averaged from five KIR3DL1*001 + donors with the SEM represented and analyzed via a one-way ANOVA, applying a Tukey's multiple comparison test (only values significant compared with inhibition obtained with wild type HLA-B*57:01 are depicted). (C) Mutations were introduced into the Bw6 molecule HLA-B*08:01 and transfected into 721.221 cells. Their surface expression was assessed by flow cytometry staining with the pan class I Ab W6/32 and anti-Bw4. (D) PBMC were incubated overnight with equal numbers of 221.B0801 transfectants in the presence of brefeldin A, and cells were then assayed for the production of IFN-g, gating on CD56 + , CD3 2 NK cells expressing KIR3DL1. IFN-g production was normalized against the maximum observed upon incubation with 221.B0801 cells. The SEM is depicted from the five KIR3DL1*001 + donors and was analyzed using a one-way ANOVA with Tukey test (depicting only differences compared with wild-type HLA-B*08:01). **p , 0.01, ***p , 0.001. 
R83G and I80N mutations to HLA-B*57:01 confer susceptibility to lysis by NK cell lines expressing KIR3DL1*001. (A) YTS cells were transfected with FLAG-tagged KIR3DL1*001, and the surface expression was examined via staining with anti-NKB1 FITC. (B) The 221 transfectants labeled with 150 mCi 51 Cr/10 6 cells were incubated with YTS cells or YTS cells transfected with KIR3DL1*001. Experiments were performed twice in triplicate with one data set shown. Open circles represent percentage specific lysis by YTS cells, and filled circles lysis by YTS.KIR3DL1*001 cells at the indicated E:T ratio with the SEM depicted. 
Analysis of the conformation of the Bw4 motif. (A) Conformation of the Bw4 and Bw6 motifs. HLA-B*08:01 (green) superposed onto HLA-B*57:01 (gray cartoon representation with yellow side chains). The Bw4 motif is characterized by a central isoleucine at position 80, a salt bridge between Glu 76 and Arg 83 , and a wider interhelical spacing than Bw6. (B) Overlay of HLA-B*57:01 (yellow) with HLA-B*57:01.I80N (cyan) and their relation to L166 of KIR3DL1 (orange). The mutation replicates the conformation observed in HLAB*08:01. Upon mutation, the salt bridge between Glu 76 and Arg 83 is lost. Furthermore, the position of Tyr 84 is altered, with a coincident narrowing of the spacing between the a1 and a2 helices. (C) KIR3DL1*001 in relation to the Bw4 motif. Residues of the Bw4 motif are colored yellow. The salt bridge between Glu 76 and Arg 83 is indicated. Residues Leu 166 and His 278 of KIR3DL1*001 that interact with the Bw4 motif are colored orange. (D) Conformations of Glu 76 in HLA-B*08:01 structures (blue, dark blue, and magenta) and in HLA-B*57:01.I80N.L82R. R83G (green). The lack of constraints by an interaction with Arg 83 is coincident with conformations that sterically clash with Leu 166 of KIR3DL1*001 (orange). These conformations are overlaid with the conformation observed in the Bw4 motif (yellow). 
Interhelical spacing for HLA-Bw4 and HLA-Bw6. Distances between the a1 and a2 helices measured between positions 80 and 146. The dichotomy observed between Bw4 and Bw6 is mirrored in the mutational swaps in HLAB*57:01 and HLA-B*08:01. 
Article
The killer cell Ig-like receptor 3DL1 (KIR3DL1) inhibits activation of NK cells upon interaction with HLA class I molecules such as HLA-B*57:01, which contains the Bw4 epitope spanning residues 77-83 (e.g., NLRIALR), and not with HLA allomorphs that possess the Bw6 motif (e.g., HLA-B*08:01), which differ at residues 77, 80, 81, 82, and 83. Although Bw4 residues Ile(80) and Arg(83) directly interact with KIR3DL1*001, their precise role in determining KIR3DL1-HLA-Bw4 specificity remains unclear. Recognition of HLA-B*57:01 by either KIR3DL1(+) NK cells or the NK cell line YTS transfected with KIR3DL1*001 was impaired by mutation of residues 80 and 83 of HLA-B*57:01 to the corresponding amino acids within the Bw6 motif. Conversely, the simultaneous introduction of three Bw4 residues at positions 80, 82, and 83 into HLA-B*08:01 conferred an interaction with KIR3DL1*001. Structural analysis of HLA-B*57:01, HLA-B*08:01, and mutants of each bearing substitutions at positions 80 and 83 revealed that Ile(80) and Arg(83) within the Bw4 motif constrain the conformation of Glu(76), primarily through a salt bridge between Arg(83) and Glu(76). This salt bridge was absent in HLA-Bw6 molecules as well as position 83 mutants of HLA-B*57:01. Mutation of the Bw4 residue Ile(80) also disrupted this salt bridge, providing further insight into the role that position 80 plays in mediating KIR3DL1 recognition. Thus, the strict conformation of HLA-Bw4 allotypes, held in place by the Glu(76)-Arg(83) interaction, facilitates KIR3DL1 binding, whereas Bw6 allotypes present a platform on the α1 helix that is less permissive for KIR3DL1 binding. Copyright © 2015 by The American Association of Immunologists, Inc.
 
Culture at lower than physiological temperatures increases cell-surface expression of KIR3DL1*004. A, NKL cells transfected to express KIR3DL1*002-GFP (line histograms) or KIR3DL1*004GFP (filled histograms) were incubated for 16 h at the temperatures indicated (top right of histogram). Cellsurface expression of KIR3DL1 was detected by flow cytometry after staining with anti-KIR3DL1 mAb 177407 (black) or an isotype-matched control (gray). B, MFI ratios for histograms shown in A are plotted as surface (mAb)/ total (GFP) KIR3DL1. Mean and SD across three independent experiments are shown. Temperature-induced changes in KIR3DL1*004 but not KIR3DL1*002 expression are statistically significant as assessed by one-way ANOVA (p , 0.0001 and p = 0.2054, respectively). For KIR3DL1*004 expression, post-test analysis for linear trend at temperatures permitting exocytosis (37-22˚C) is also statistically significant. p , 0.0001.
KIR3DL1*004 is largely misfolded. A-C, NKL cells expressing KIR3DL1*002-GFP (*002) or KIR3DL1*004-GFP (*004) were incubated for 16 h at the temperatures indicated (˚C). A, Equivalent amounts of whole-cell lysates were resolved by reducing SDS-PAGE and probed for the proteins indicated. B, Quantification of the Western blots shown in A. Band intensities are plotted as upper band/lower band. Mean and SD across three independent experiments are shown. Temperature-induced changes in KIR3DL1*004 but not KIR3DL1*002 expression are statistically significant as assessed by oneway ANOVA (p = 0.0025 and p = 0.7252, respectively). For KIR3DL1*004 expression, post-test analysis for linear trend is also statistically significant (p = 0.0004). C, KIR3DL1 was immunoprecipitated with anti-KIR3DL1 (upper panel) or-GFP (lower panel) Abs and detected by Western blotting for GFP. Ctrl, untransfected NKL (upper panel) or NKL transfected to express KIR3DL1*002 lacking GFP (lower panel). The migration position of molecular mass markers (kDa) is shown at the left of A and C. D, NKL expressing KIR3DL1*004-GFP were incubated at 37˚C (upper panels) or 25˚C (middle and lower panels) for 12 h in the presence of DMSO (GP 2, upper and middle panels) or GolgiPlug (GP +, lower panels) to inhibit protein transport through the ER and Golgi complex. Surface epitopes were blocked by staining with unlabeled anti-KIR3DL1 mAb (intracellular, right panels) or an isotype-matched control (total, left panels) prefixation and permeabilization. Cells were then stained for KIR3DL1 with PE-conjugated mAb (black line histogram) or an isotype-matched control (gray filled histogram).
Transient surface expression of KIR3DL1*004 is functional for inhibition of NK cell activation. Expression of KIR3DL1 (A) and IFNg production (B) was assessed in NKL cells transfected to express GFP
Article
Variable interaction between the Bw4 epitope of HLA-B and the polymorphic KIR3DL1/S1 system of inhibitory and activating NK cell receptors diversifies the development, repertoire formation, and response of human NK cells. KIR3DL1*004, a common KIR3DL1 allotype, in combination with Bw4(+) HLA-B, slows progression of HIV infection to AIDS. Analysis in this study of KIR3DL1*004 membrane traffic in NK cells shows this allotype is largely misfolded but stably retained in the endoplasmic reticulum, where it binds to the chaperone calreticulin and does not induce the unfolded protein response. A small fraction of KIR3DL1*004 folds correctly and leaves the endoplasmic reticulum to be expressed on the surface of primary NK and transfected NKL cells, in a form that can be triggered to inhibit NK cell activation and secretion of IFN-γ. Consistent with this small proportion of correctly folded molecules, trace amounts of MHC class I coimmunoprecipitated with KIR3DL1*004. There was no indication of any extensive intracellular interaction between unfolded KIR3DL1*004 and cognate Bw4(+) HLA-B. A similarly limited interaction of Bw4 with KIR3DL1*002, when both were expressed by the same cell, was observed despite the efficient folding of KIR3DL1*002 and its abundance on the NK cell surface. Several positions of polymorphism modulate KIR3DL1 abundance at the cell surface, differences that do not necessarily correlate with the potency of allotype function. In this context, our results suggest the possibility that the effect of Bw4(+) HLA-B and KIR3DL1*004 in slowing progression to AIDS is mediated by interaction of Bw4(+) HLA-B with the small fraction of cell surface KIR3DL1*004.
 
Article
KIR3DL1 is an inhibitory HLA-B receptor of human NK and T cells that exhibits genetic and phenotypic polymorphism. KIR3DL1*004, a common allotype, cannot be detected on the surface of PBLs using the KIR3DL1-specific Ab DX9. The nature of this phenotype was investigated through comparison of 3DL1*004 with 3DL1*002, an allele giving high DX9 binding to cell surfaces. Analysis of Jurkat T cell transfectants with 3DL1*004 cDNA showed that 3DL1*004 is poorly expressed at the cell surface, but detectable intracellularly. Analysis of recombinant mutants made between 3DL1*004 and 3DL1*002 showed that polymorphism in Ig domains 0 and 1 (D0 and D1) causes the intracellular retention of 3DL1*004. Reciprocal point mutations were introduced into 3DL1*004 and 3DL1*002 at positions 44 and 86 of the D0 domain, where 3DL1*004 has unique residues, and at position 182 of the D1 domain, where 3DL1*004 resembles 3DL1*005, an allotype giving low DX9-binding phenotype. Leucine 86 in 3DL1*004 is the principal cause of its intracellular retention, with a secondary and additive contribution from serine 182. By contrast, glycine 44, which is naturally present in 3DL1*004, slightly increased cell surface expression when introduced into 3DL1*002. In 3DL1*004, the presence of leucine at position 86 corrupts the WSXPS motif implicated in proper folding of the KIR D0 Ig-like domain. This study demonstrates how a difference between KIR3DL1 allotypes in the D0 domain profoundly affects cell surface expression and function.
 
Article
TRAIL promotes apoptotic tumor cell death; however, TRAIL-resistant tumors need to be sensitized to reverse resistance. Proteasome inhibitors potentiate TRAIL apoptosis in vitro and in vivo and correlate with up-regulation of death receptor 5 (DR5) via an unknown mechanism. We hypothesized that the proteasome inhibitor NPI-0052 inhibits the transcription repressor Yin Yang 1 (YY1) which regulates TRAIL resistance and negatively regulates DR5 transcription. Treatment of PC-3 and Ramos cells with NPI-0052 (</=2.5 nM) and TRAIL sensitizes the tumor cells to TRAIL-induced apoptosis. By comparison to bortezomib, a 400-fold less concentration of NPI-0052 was used. NPI-0052 up-regulated DR5 reporter activity and both surface and total DR5 protein expression. NPI-0052-induced inhibition of NF-kappaB activity was involved in TRAIL sensitization as corroborated by the use of the NF-kappaB inhibitor dehydroxymethylepoxyquinomicin. NPI-0052 inhibited YY1 promoter activity as well as both YY1 mRNA and protein expression. The direct role of NPI-0052-induced inhibition of YY1 and up-regulation of DR5 in the regulation of TRAIL sensitivity was demonstrated by the use of YY1 small interfering RNA. The NPI-0052-induced sensitization to TRAIL involved activation of the intrinsic apoptotic pathway and dysregulation of genes that regulate apoptosis. The NPI-0052 concentrations used for TRAIL sensitization were not toxic to human hematopoetic stem cells. The present findings demonstrate, for the first time, the potential mechanism by which a proteasome inhibitor, like NPI-0052, inhibits the transcription repressor YY1 involved in TRAIL resistance and DR5 regulation. The findings also suggest the therapeutic application of subtoxic NPI-0052 concentrations in combination with TRAIL/agonist DR4/DR5 mAbs in the treatment of TRAIL-resistant tumors.
 
Viral load kinetics in SIVmac239-infected Mamu-B*00801 + rhesus macaques. (A) Viral load profiles for the study animals during the first 28 wk of acute infection. Dashed lines indicate 10,000 V/ ml (C animals) and 1,000 V/ml (EC animals). (B) Geometric mean viral loads for the 15 animals plotted by infection outcome status for each weekly time point. Some animals did not have viral load data for weeks 5, 6, and 7 postinfection. Viral loads in EC animals are statistically lower than P animals beginning at 6 wk postinfection. Geometric mean viral loads were compared using the Kruskal-Wallis one-way ANOVA followed by the Dunn's multiple comparison posttest. *p , 0.05, **p , 0.01.
Consensus viral sequence during acute infection for 12 Mamu-B*00801–restricted SIVmac239 T cell epitopes. This representation of the viral quasispecies lists all variants from wild-type sequence detected in .1% of sequenced reads. (A) Subdominant Mamu-B*00801–restricted T cell epitopes display very little viral quasispecies diversity within the first 8 wk of acute infection, suggesting minimal CD8 + T lymphocyte escape in these regions of the virus. (B) Immunodominant Mamu-B*00801–restricted T cell epitopes demonstrate CD8 + T lymphocyte escape during the first 8 wk of acute SIV infection. Of note, CD8 + T lymphocyte escape in the Vif RL8 epitope appears more diverse in P animals when compared with EC and C animals. Nef RL9b/c naming convention is based upon the order in which each epitope was discovered and is consistent with previously published studies. For most data points with available sample, sequencing was performed twice independently on separate samples of plasma from the same animal and time point.  
Analysis of nonsynonymous Mamu-B*00801-restricted Vif epitope variation over time. Nonsynonymous variation (d N ) within the Vif epitopes increases over time in the P animals, but not in the EC or C. In conjunction with unchanged measurements of synonymous variation (d S ), this suggests selective viral evolution in response to Vif-specific CD8 + T cell pressure in the P animals.
Article
A small number of HIV-infected individuals known as elite controllers experience low levels of chronic phase viral replication and delayed progression to AIDS. Specific HLA class I alleles are associated with elite control, implicating CD8(+) T lymphocytes in the establishment of these low levels of viral replication. Most HIV-infected individuals that express protective HLA class I alleles, however, do not control viral replication. Approximately 50% of Mamu-B*00801(+) Indian rhesus macaques control SIVmac239 replication in the chronic phase in a manner that resembles elite control in humans. We followed both the immune response and viral evolution in SIV-infected Mamu-B*00801(+) animals to better understand the role of CD8(+) T lymphocytes during the acute phase of viral infection, when viral control status is determined. The virus escaped from immunodominant Vif and Nef Mamu-B*00801-restricted CD8(+) T lymphocyte responses during the critical early weeks of acute infection only in progressor animals that did not control viral replication. Thus, early CD8(+) T lymphocyte escape is a hallmark of Mamu-B*00801(+) macaques who do not control viral replication. By contrast, virus in elite controller macaques showed little evidence of variation in epitopes recognized by immunodominant CD8(+) T lymphocytes, implying that these cells play a role in viral control.
 
A Mamu-A*01-restricted gag-specific bulk CTL line preferentially recognizes the SIV gag 9 mer CTPYDINQM. MamuA*01-transfected 221 cells were pulsed with varying amounts of different SIV gag peptides. As a negative control, target cells were pulsed with an irrelevant influenza NP CTL epitope (SNEGSYFF) identified in the cotton-top tamarin (79). As a positive control, autologous B LCL were pulsed with the CTPYDINQML peptide. CTLs were tested at an E:T cell ratio of 20:1 (A) or 2:1 (B). C, Five different gag peptides were tested using a freshly stimulated bulk CTL line at an E:T cell ratio of 20:1 to compare the abilities of these peptides to sensitize target cells for lysis. 
Two of three CTL clones preferentially recognize the SIV gag 9 mer CTPYDINQM. Mamu-A*01-restricted gag-specific CTL clones were tested at an E:T cell ratio of 5:1 against four different SIV gag peptides using various peptide concentrations. A, Target cells were peptide-pulsed B LCLs. B, Target cells were peptide-pulsed Mamu-A*01-transfected C1R cells. 
Article
The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.
 
Cell-surface expression of the seven HLA monochains in a H-2 class I null context. Ficoll-purified splenocytes from individual HLA class I monochain transgenic, H-2 class I null (closed gray histograms), and H-2 K b , D b , mouse b 2-m triple-KO (negative control, open histograms) mice (n = 1) were labeled with anti-b 2-m mAb Tü 99 and flow cytometrically analyzed. This experiment was performed more than five times.
Cell-surface expression of the seven monochains in a H-2 class I + context. Ficoll-purified splenocytes from individual HLA class I monochain transgenic H-2 class I null (dark gray histograms), HLA class I monochain transgenic 3 OF1 F1 hybrid (light gray histograms), and H-2 K b , D b , mouse b 2 -m triple-KO (negative control, open histograms) mice (n = 1) were labeled with anti–b 2 -m mAb Tü 99 and flow cytometrically analyzed. This experiment was performed thrice.  
CD8 + and CD4 + T cells TCR AV peripheral repertoire. Relative peripheral representation in percentage of the AV segment families (International Immunogenetics Information System nomenclature, http://www.imgt.org) as evaluated by quantitative RT-PCR in purified CD8 + and CD4 + T cells from HLA-A*01.03, -A*24:02, -B*08:01, -B*27:05, -B*33:01, -B*44:02, and -C*07:01 monochain transgenic and from C57BL/6 (B6) mice. Open and closed bars correspond to C57BL/6 and monochain strains, respectively. CD4 + T cell AV profiles of A*01:03 versus C57BL/6 mice are illustrated in the upper left panel, similar CD4 + AV profiles (data not shown) being obtained with the six additional monochains. The results are means of the values from at least four individually tested mice. Statistical analyses of the data were carried out with the unpaired t test with Welch's correction, *p , 0.05, **p , 0.005, ***p , 0.0005.  
CD8 + and CD4 + T cells TCR BV peripheral repertoire. Relative peripheral representation in percentage of the BV segment families (International Immunogenetics Information System nomenclature, http://www.imgt.org) as evaluated by quantitative RT-PCR in purified CD8 + and CD4 + T cells from HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, and -C*07:01 monochain transgenic and from C57BL/6 (B6) mice. Open and closed bars correspond to C57BL/6 and monochain strains, respectively. CD4 + T cell BV profiles of A*01:03 versus C57BL/6 mice are illustrated in the upper left panel, similar CD4 + BV profiles (not shown) being obtained with the six additional monochains. The results are means of the values from at least four individually tested mice. Statistical analyses of the data were carried out with the unpaired t test with Welch's correction, *p , 0.05, **p , 0.005, ***p , 0.0005.  
Peptide–HLA-C*07:01 dissociation. Dissociation of predicted HLA-C*07:01 binding peptides RRRPVTRPL (T1/2 16.1 h) and RRARYWLTY (T1/2 29.4 h) from HLA-C*07:01 measured by scintillation proximity assay.  
Article
We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α1α2 H chain domains fused with a mouse α3 domain and covalently linked to human β2-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8(+) T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ-producing CD8(+) T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8(+) T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell-based vaccines.
 
Detection of B51-TI8-specific CTLs expressing public TCRs with the TRAV17/TRBV7-3 genes. (A) TCR ab-chain usage of B51-TI8specific CTL clones established from seven HLA-B*51:01 + individuals infected with HIV-1. (B) Bulk TCR sequence analysis of ex vivo HLA-B*51:01TI8 tetramer binding CD8 + T cell populations from four of the individuals shown in (A). (C) TCR analysis of sorted single cells of ex vivo tetramer binding CD8 + T cells from two patients. TCRs with the TRAV17/TRBV7-3 genes are highlighted in blue. The conserved TCR CDR3a Arg 97 residue is shown in red.
Article
CD8(+) CTL responses directed toward the HLA-B*51:01-restricted HIV-RT128-135 epitope TAFTIPSI (TI8) are associated with long-term nonprogression to AIDS. Clonotypic analysis of responses to B51-TI8 revealed a public clonotype using TRAV17/TRBV7-3 TCR genes in six out of seven HLA-B*51:01(+) patients. Structural analysis of a TRAV17/TRBV7-3 TCR in complex with HLA-B51-TI8, to our knowledge the first human TCR complexed with an 8-mer peptide, explained this bias, as the unique combination of residues encoded by these genes was central to the interaction. The relatively featureless peptide-MHC (pMHC) was mainly recognized by the TCR CDR1 and CDR2 loops in an MHC-centric manner. A highly conserved residue Arg(97) in the CDR3α loop played a major role in recognition of peptide and MHC to form a stabilizing ball-and-socket interaction with the MHC and peptide, contributing to the selection of the public TCR clonotype. Surface plasmon resonance equilibrium binding analysis showed the low affinity of this public TCR is in accordance with the only other 8-mer interaction studied to date (murine 2C TCR-H-2K(b)-dEV8). Like pMHC class II complexes, 8-mer peptides do not protrude out the MHC class I binding groove like those of longer peptides. The accumulated evidence suggests that weak affinity might be a common characteristic of TCR binding to featureless pMHC landscapes.
 
Tetramer-guided epitope mapping of influenza matrix protein epitopes. (A) Pool mapping: T cells from a DR0801 donor were stimulated with overlapping peptide mixtures spanning H1MP and stained using pooled peptide–loaded tetramers after 2 wk. Peptide pool 3 gave positive staining. (B) Individual peptide mapping: T cells from the positive panel shown in (A) were stained again using tetramers loaded with each individual peptide from peptide pool 3. Peptide H1MP 89–108 was identified as a peptide that contains a DR0801restricted epitope. 
Binding register of the test peptide. Observed relative binding affinities for derivatives of the H1MP 98-110 peptide with nonconservative substitutions are shown. RBA values were determined experimentally as the IC 50 value of the unsubstituted peptide divided by the IC 50 value of the substituted peptide. Values are shown on a logarithmic scale centered on 0.1
Amino acid preferences for pocket 1 (A), pocket 4 (B), pocket 6 (C), and pocket 9 (D) of the DR0801 motif. Black bars represent values within 2.5-fold of the reference peptide (RBA $ 0.4, " preferred " ), hatched bars represent values within 10-fold of the reference peptide (RBA $ 0.1, " tolerated " ), and open bars represent values within ,10-fold of the reference peptide (RBA # 0.1, " excluded " ). 
(A) TCR view of pocket 4 of the DR0801/KLYRKLKREITFH complex (anchor residues in bold type). Like residues pair (b26Phe with b74Leu and b70Asp with b71Arg) with the first pair into the pocket and the second hydrophilic pair into the solvent and toward the reader. Anchor p4Leu interacts well with b26Phe-b74Leu and has ample space because of b13Gly. The figure is tilted by 5˚with5˚with respect to the y-axis (left-hand side out of the plane of the paper, right-hand side into the plane of the paper). Atomic color conventions: oxygen: red; nitrogen: blue; carbon: orange in DR8 and green in the antigenic peptide; hydrogen: white; sulfur: yellow. Residues of DR8 are depicted in stick with transparent van der Waals surface representation, with color-coded surface atomic charges (blue, positive; gray, neutral; red, negative; partial charges, colors in-between), and the antigenic peptide is shown in space-filling form. (B) TCR view of pocket 6 with anchor Arg residue in space-filling mode and the pocket residues in stick form. Secondary structural elements depicted in red for the a-helix, turquoise for the b-sheet, and gray for random coil. The figure is tilted by 20˚with20˚with respect to the y-axis (left-hand side out of the plane of the paper, right-hand side into the plane of the paper) for better visibility. The inset at bottom right reveals the full set of putative interactions around p6Arg, depicting the anchor in ball and stick. Other color conventions are the same as in (A). (C) TCR view of pocket 9 with Thr in space-filling mode. The figure is tilted by 5˚with5˚with respect to the y-axis for better visibility (left-hand side into the plane of the paper, right hand side out of the plane of the paper). Depiction and color conventions are identical to (A). 
T cell responses to PDC-E2 peptides. (A) Tetramer staining of cultured CD4 + T cells from a representative DR0801 + healthy subject. (B) Tetramer staining of cultured CD4 + T cells from a representative DR1101 + healthy subject. (C) Cumulative tetramer staining results for PDC-E2 responses in multiple healthy DR0801 + subjects. Tetramerpositive populations .0.4% above background were considered positive (indicated by dotted line). (D) Direct ex vivo tetramer analysis of CD4 + T cells from a representative DR0801 + healthy subject. Staining of nonenriched PBMCs, for which staining is undetectable (upper left panel). Remaining panels depict staining of anti-PE–enriched PBMCs for H1MP 89–108-specific, PDC-E2 145–159-specific, and PDC-E2 249–263-specific CD4 + T cells versus CD45RA. 
Article
DRB1*08:01 (DR0801) and DRB1*11:01 (DR1101) are highly homologous alleles that have opposing effects on susceptibility to primary biliary cirrhosis (PBC). DR0801 confers risk and shares a key feature with other HLA class II alleles that predispose to autoimmunity: a nonaspartic acid at beta57. DR1101 is associated with protection from PBC, and its sequence includes an aspartic acid at beta57. To elucidate a mechanism for the opposing effects of these HLA alleles on PBC susceptibility, we compared the features of epitopes presented by DR0801 and DR1101. First, we identified DR0801- and DR1101-restricted epitopes within multiple viral Ags, observing both shared and distinct epitopes. Because DR0801 is not well characterized, we deduced its motif by measuring binding affinities for a library of peptides, confirming its key features through structural modeling. DR0801 was distinct from DR1101 in its ability to accommodate charged residues within all but one of its binding pockets. In particular, DR0801 strongly preferred acidic residues in pocket 9. These findings were used to identify potentially antigenic sequences within PDC-E2 (an important hepatic autoantigen) that contain a DR0801 motif. Four peptides bound to DR0801 with reasonable affinity, but only one of these bound to DR1101. Three peptides, PDC-E2145-159, PDC-E2249-263, and PDC-E2629-643, elicited high-affinity T cell responses in DR0801 subjects, implicating these as likely autoreactive specificities. Therefore, the unique molecular features of DR0801 may lead to the selection of a distinct T cell repertoire that contributes to breakdown of self-tolerance in primary biliary cirrhosis, whereas those of DR1101 promote tolerance.
 
Summary motif of Mamu-B*01. Summary map of primary and secondary anchor preferences derived from single amino acid substitution analysis. Main and secondary anchor positions are indicated. Preferred and tolerated anchor residues were defined in the text. M at position 2, D at position 5, and M at C terminus have been included on the basis of chemical similarity. A correct binding motif must include at least one preferred and one tolerated residue at the main anchor positions (position 2 and C terminus). Preferred residues at secondary positions were associated with increased binding affinity, but were not required. 
Article
Although the SIV-infected Indian rhesus macaque (Macaca mulatta) is the animal model most widely used for studying HIV infection, our current understanding of the functional macaque MHC class I molecules is limited. To date, SIV-derived CD8+ T lymphocyte epitopes from only three high frequency macaque MHC class I molecules have been extensively characterized. In this study, we defined the peptide-binding properties of the high frequency Indian rhesus macaque class I molecule, Mamu-B*01 ( approximately 26%). We first identified a preliminary binding motif by eluting and sequencing endogenously bound Mamu-B*01 ligands. We further characterized the peptide-binding characteristics using panels of single amino acid substitution analogs. Using this detailed motif, 507 peptides derived from SIV(mac)239 were identified and tested for their Mamu-B*01 binding capacity. Surprisingly, only 11 (2.2%) of these motif-containing peptides bound with IC50 values < or =500 nM. We assessed the immunogenicity of these peptides using freshly isolated PBMC from ten Mamu-B*01+ SIV-infected rhesus macaques in IFN-gamma ELISPOT and IFN-gamma/TNF-alpha intracellular cytokine staining assays. Lymphocytes from these SIV-infected macaques responded to none of these peptides. Furthermore, there was no sequence variation indicative of escape in the regions of the virus that encoded these peptides. Additionally, we could not confirm previous reports of SIV-derived Mamu-B*01-restricted epitopes in the Env and Gag proteins. Our results suggest that the high frequency MHC class I molecule, Mamu-B*01, is not involved in SIV-specific CD8+ T lymphocyte responses.
 
Schematic representation of the residues at the HLA heavy ␣ -chain involved in the contact with ␤ 2 m (modified from reference 17). A , The contact residues in the ␣ 1 chain are represented by black spheres ( F ). The replacement at position 48 (arginine by proline) is shown as a white sphere ( E ). B , The substitution R48P causes significant physicochemical changes (positively charged and hydrophilic Arg by the nonpolar, hydrophobic Pro) in the interaction of residue complex (Gln 32 , Arg 35 , and Arg 48 ) at the ␣ 1 chain with Arg 53 of ␤ 2 m. 
Article
We identified and characterized an HLA-A1 aberrant allele (A*0118N) resulting from a novel molecular mechanism; this allele was present in an unusually informative family with a near identical parental HLA haplotype (c d) differing only by one nucleotide substitution in one HLA-A allele, A*0118N, of the maternal HLA haplotype (c) and not of the paternal HLA haplotype (a). Although serologic HLA typing showed a "blank," DNA molecular HLA typing detected a HLA-A*0118N allele. Sequence based typing identified the substitution of guanine by cytosine at the nucleotide position 215, which resulted in the replacement of arginine by proline at position 48 of the HLA-A1 H chain. The loss of surface protein expression was also found by FACS analysis. Isoelectric-focusing analysis detected a HLA-A H chain with a unique isoelectric-focusing pattern, which does not associate with the L chain (beta(2)-microglobulin). These results suggest that the residue 48-containing interaction site on the alpha(1) domain plays a critical role in the association between HLA class I H chain and beta(2)-microglobulin. Functional studies showed that the T cells of the propositus (HLA haplotypes c d) carrying this null allele recognized its wild-type counterpart, HLA-A*010101, in her HLA-identical son that carries the HLA-A*0101 heterodimer. This is the first example of the generation of cytotoxic T cells in the absence of proliferation of CD4(+) T cells (mixed lymphocyte culture) and the description of an aberrant allele, A*0118N, that may behave as a minor histocompatibility Ag, with implications in allorecognition by cytolytic T cells in solid organ and stem cell transplantation.
 
Article
Evidence from C57BL/6 mice suggests that CD8(+) T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2(b)-restricted epitope (gB498-505), protect against ocular herpes infection and disease. However, the possible role of CD8(+) T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1-seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01-restricted CD8(+) T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive, HSV-1-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8(+) T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342-350 and gB561-569. In contrast, in 10 HLA-A*02:01-positive, HSV-1-seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8(+) T cell responses were directed mainly against nonoverlapping epitopes (gB183-191 and gB441-449). ASYMP individuals had a significantly higher proportion of HSV-gB-specific CD8(+) T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell-based herpes vaccine.
 
Article
Genetic susceptibility to multiple sclerosis (MS) has been linked to the HLA-DR15 haplotype consisting of DRB1*15:01(DR2b) and DRB5*01:01(DR2a) alleles. Given almost complete linkage disequilibrium of the two alleles, recent studies suggested differential roles in susceptibility (DR2b) or protection from MS (DR2a). Our objective was to assess the potential contribution of DR2a to disease etiology in MS using a humanized model of autoimmunity. To assess the potential contribution of DR2a to disease etiology, we created DR2a humanized transgenic (Tg) mice and subsequently crossed them to Tg mice expressing TL3A6, an MS patient-derived myelin basic protein 83-99-specific TCR. In TL3A6/DR2a Tg mice, CD4 Tg T cells escape thymic and peripheral deletion and initiate spontaneous experimental autoimmune encephalomyelitis (EAE) at low rates, depending on the level of DR2a expression. The ability to induce active EAE was also increased in animals expressing higher levels of DR2a. Inflammatory infiltrates and neuronal damage were present throughout the spinal cord, consistent with a classical ascending EAE phenotype with minor involvement of the cerebellum, brainstem, and peripheral nerve roots in spontaneous, as well as actively induced, disease. These studies emphasize the pathologic contribution of the DR2a allele to the development of autoimmunity when expressed as the sole MHC class II molecule, as well as strongly argue for DR2a as a contributor to the CNS autoimmunity in MS.
 
Article
Allopurinol (ALP) hypersensitivity is a major cause of severe cutaneous adverse reactions and is strongly associated with the HLA-B*58:01 allele. However, it can occur in the absence of this allele with identical clinical manifestations. The immune mechanism of ALP-induced severe cutaneous adverse reactions is poorly understood, and the T cell-reactivity pattern in patients with or without the HLA-B*58:01 allele is not known. To understand the interactions among the drug, HLA, and TCR, we generated T cell lines that react to ALP or its metabolite oxypurinol (OXP) from HLA-B*58:01(+) and HLA-B*58:01(-) donors and assessed their reactivity. ALP/OXP-specific T cells reacted immediately to the addition of the drugs and bypassed intracellular Ag processing, which is consistent with the "pharmacological interaction with immune receptors" (p-i) concept. This direct activation occurred regardless of HLA-B*58:01 status. Although most OXP-specific T cells from HLA-B*58:01(+) donors were restricted by the HLA-B*58:01 molecule for drug recognition, ALP-specific T cells also were restricted to other MHC class I molecules. This can be explained by in silico docking data that suggest that OXP binds to the peptide-binding groove of HLA-B*58:01 with higher affinity. The ensuing T cell responses elicited by ALP or OXP were not limited to particular TCR Vβ repertoires. We conclude that the drug-specific T cells are activated by OXP bound to HLA-B*58:01 through the p-i mechanism.
 
p15m tetramer staining of peripheral blood CD8 T lymphocytes of immunized and control macaques. Data are presented as in the legend to Fig. 2.
p54m tetramer staining of peripheral blood CD8 T lymphocytes of immunized and control macaques. Data are presented as in the legend to Fig. 2.
T cell proliferative responses of PBMC to SIV gp120 and aldrithiol-2-inactivated SIV. Positive proliferative responses are depicted as follows for immunized macaques: u , post-Ad-recombinant immunizations; f , postsubunit boosts; and for control macaques: s , post-Ad-vector administrations; XI , postadjuvant administrations. 
Article
CTL are important in controlling HIV and SIV infection. To quantify cellular immune responses induced by immunization, CD8(+) T cells specific for the subdominant Env p15m and p54m epitopes and/or the dominant Gag p11C epitope were evaluated by tetramer staining in nine macaques immunized with an adenovirus (Ad) 5 host range mutant (Ad5hr)-SIVenv/rev recombinant and in four of nine which also received an Ad5hr-SIVgag recombinant. Two Ad5hr-SIV recombinant priming immunizations were followed by two boosts with gp120 protein or an envelope polypeptide representing the CD4 binding domain. Two mock-immunized macaques served as controls. IFN-gamma-secreting cells were also assessed by ELISPOT assay using p11C, p15m, and p54m peptide stimuli and overlapping pooled Gag and Env peptides. As shown by tetramer staining, Ad-recombinant priming elicited a high frequency of persistent CD8(+) T cells able to recognize p11C, p15m, and p54m epitopes. The presence of memory cells 38 wk postinitial immunization was confirmed by expansion of tetramer-positive CD8(+) T cells following in vitro stimulation. The SIV-specific CD8(+) T cells elicited were functional and secreted IFN-gamma in response to SIV peptide stimuli. Although the level and frequency of response of peripheral blood CD8(+) T cells to the subdominant Env epitopes were not as great as those to the dominant p11C epitope, elevated responses were observed when lymph node CD8(+) T cells were evaluated. Our data confirm the potency and persistence of functional cellular immune responses elicited by replication competent Ad-recombinant priming. The cellular immunity elicited is broad and extends to subdominant epitopes.
 
Article
The infection of rhesus macaques (Macaca mulatta) by the SIV is the best animal model for studying HIV infection and for AIDS vaccine development. A prevalent MHC class I allele, Mamu-A*01, is known to correlate with containment of SIV, which has been extensively explored in studies of CTL-based vaccination concepts. We determined the crystal structures of Mamu-A*01 complexed with two immunodominant SIV epitopes: the nonamer CM9 of group-specific Ag (Gag, 181-189; CTPYDINQM) and the octamer TL8 of transcription activator (Tat, 28-35; TTPESANL). The overall structures of the two Mamu-A*01 complexes are similar to other MHC class I molecules. Both structures confirm the presence of an absolutely conserved proline anchor residue in the P3 position of the Ag, bound to a D pocket of the Mamu-A*01 H chain with optimal surface complementarity. Like other MHC/peptide complex structures, the P2 and C-terminal residues of the epitopes are also important for anchoring to the MHC molecule, whereas the middle residues form an arch and their side chains are directed into solvent. These two structures reveal details of how Mamu-A*01 interacts with two well-studied epitopes at the atomic level. We discuss the structural basis of CTL escape, based on molecular models made possible by these two structures. The results we present in this study are most relevant for the rational design of Mamu-A*01-restricted CTL epitopes with improved binding, as a step toward development of AIDS vaccines.
 
C terminus fine specificity of Mamu A*01 ligands. A, Relative Mamu A*01 binding capacity of peptides bearing specific residues at the C terminus. ARB values were calculated as described in Materials and Methods, and indexed relative to the residue with the highest ARB. The average (geometric) binding capacity of the 520 peptides considered was 5237 nM. B, Graphic summary of the data shown in A.
Position 3 fine specificity of Mamu A*01 ligands. A, Relative Mamu A*01 binding capacity of peptides bearing specific residues in position 2. ARB values were calculated as described in Materials and Methods, and indexed relative to the residue with the highest ARB. The average (geometric) binding capacity of the 191 peptides considered was 2286 nM. Except for C, when the number of peptides bearing specific residues was low, residues were grouped on the basis of chemical similarity, as previously described (7). B, Graphic summary of the data shown in A.
Summary maps of secondary effects influencing the Mamu A*01 binding capacity of 8-mer ( A ), 9-mer ( B ), 10-mer ( C ), and 11-mer ( D ) peptides, as described in Tables V-VIII. Shown are the preferred and deleterious residues associated with an ARB capacity of at least 5-fold greater, or 5-fold worse, compared with peptides of the same size carrying other residues. 
Article
Single amino acid substitution analogs of the known Mamu A*01 binding peptide gag 181-190 and libraries of naturally occurring sequences of viral or bacterial origin were used to rigorously define the peptide binding motif associated with Mamu A*01 molecules. The presence of S or T in position 2, P in position 3, and hydrophobic or aromatic residues at the C terminus is associated with optimal binding capacity. At each of these positions, additional residues are also tolerated but associated with significant decreases in binding capacity. The presence of at least two preferred and one tolerated residues at the three anchor positions is necessary for good Mamu A*01 binding; optimal ligand size is 8-9 residues. This detailed motif has been used to map potential epitopes from SIVmac239 regulatory proteins and to engineer peptides with increased binding capacity. A total of 13 wild type and 17 analog candidate epitopes were identified. Furthermore, our analysis reveals a significantly lower than expected frequency of epitopes in early regulatory proteins, suggesting a possible evolutionary- and/or immunoselection directed against variants of viral products that contain CTL epitopes.
 
Article
The HSV type 1 tegument virion phosphoprotein (VP) 11/12 (VP11/12) is a major Ag targeted by CD8(+) T cells from HSV-seropositive individuals. However, whether and which VP11/12 epitope-specific CD8(+) T cells play a role in the "natural" protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8(+) T cell epitopes from the 718-aa sequence of VP11/12. Three of 10 epitopes exhibited high-to-moderate binding affinity to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive and HSV-1-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional effector CD8(+) T cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107(a/b) cytotoxic degranulation, IFN-γ, and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266-74, VP11/12220-228, and VP11/12702-710. Interestingly, ASYMP individuals had a significantly higher proportion of CD45RA(low)CCR7(low)CD44(high)CD62L(low)CD27(low)CD28(low)CD8(+) effector memory CD8(+) T cells (TEMs) specific to the three epitopes, compared with symptomatic individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8(+) TEM cell epitopes induced robust and polyfunctional epitope-specific CD8(+) TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8(+) T cells that should guide the development of an effective T cell-based herpes vaccine. Copyright © 2015 by The American Association of Immunologists, Inc.
 
Article
The expression of HLA-DR1 (DRB1*0101) is associated with an enhanced risk for developing rheumatoid arthritis (RA). To study its function, we have solved the three-dimensional structure of HLA-DR1 complexed with a candidate RA autoantigen, the human type II collagen peptide CII (259-273). Based on these structural data, the CII peptide is anchored by Phe263 at the P1 position and Glu266 at P4. Surprisingly, the Lys at the P2 position appears to play a dual role by participating in peptide binding via interactions with DRB1-His81 and Asn82, and TCR interaction, based on functional assays. The CII peptide is also anchored by the P4 Glu266 residue through an ionic interaction with DRB1-Arg71 and Glu28. Participation of DRB1-Arg71 is significant because it is part of the shared epitope expressed by DR alleles associated with RA susceptibility. Potential anchor residues at P6 and P9 of the CII peptide are both Gly, and the lack of side chains at these positions appears to result in both a narrower binding groove with the peptide protruding out of the groove at this end of the DR1 molecule. From the TCR perspective, the P2-Lys264, P5-Arg267, and P8-Lys270 residues are all oriented away from the binding groove and collectively represent a positive charged interface for CII-specific TCR binding. Comparison of the DR1-CII structure to a DR1-hemagglutinin peptide structure revealed that the binding of these two peptides generates significantly different interfaces for the interaction with their respective Ag-specific TCRs.
 
Article
Antigenic peptides are presented to CD4+ T cells by MHC class II molecules via a highly polymorphic peptide-binding groove. The two HLA-DR alleles isotypically expressed on HLA-DR15Dw2-positive cells, DRB1*1501 (DR2b) and DRB5*0101 (DR2a) molecules, show a number of differences in polymorphic residues of the beta-chain, including the Gly-Val-dimorphism at position beta 86. Therefore, different requirements for interaction of peptides with these alleles must be expected. In this study, naturally processed self-peptides were eluted from purified HLA-DR15Dw2 molecules and related to DRB1*1501 or DRB5*0101 molecules by binding assays. An alignment of self-peptides and foreign peptides allowed the delineation of putative anchor motifs. N- and C-terminally truncated and alanine-substituted derivatives of the DR15Dw2 restricted myelin basic protein epitope MBP(85-105) confirmed their validity. Thus, DRB5*0101 requires a bulky hydrophobic residue (F or Y) at position i as a primary anchor, and Q or an aliphatic residue, such as V, I, or M, at position i + 3; positively charged residues at positions i + 7 and i + 8 are secondary anchors. For DRB1*1501, a nonaromatic, hydrophobic anchor (L, V, or I) at position i is supplemented by a bulky hydrophobic residue (F or Y) at position i + 3 as primary anchor; an additional hydrophobic side chain represented by M, I, V, or F occurs at position i + 6. Therefore, MBP(85-105) seems to contain two MHC interaction sites for DRB1*1501 and DRB5*0101, respectively, that may contribute to its immunodominance. Because HLA-DR15 Dw2 is associated with susceptibility to develop multiple sclerosis, the delineation of ligand motifs of the two DR2 alleles may help to study the interaction between potential autoantigenic peptides and these molecules in the future.
 
Determination of A1-HY candidate peptides. Aliquots of each splitter fraction (1.2 10 9 cell equivalents) were tested for their ability to reconstitute epitope activity as described in Materials and Methods . Ion abundances of candidate masses were determined using the FTMS.  
CAD mass spectra of A1-HY candidate peptide ions. Mass spectra were recorded as described in Materials and Methods. Ions observed in the spectrum are underlined. X represents isoleucine or leucine. A, Candidate peptide (M 2H) 2 ion with monoisotopic m/z of 611.23. B, Candidate peptide (M 2H) 2 ion with monoisotopic m/z of 603.25.  
A1-HY epitope reconstitution with synthetic peptides. The indicated synthetic peptides were assayed for epitope reconstituting activity as described in Materials and Methods. A, Candidate peptide ions 611.23 2 (XVDC*XTEM ox Y) and 669.79 2 (M ox TEXYDYPKY). B, Effects of posttranslational modification of the core sequence XVDCXTEMY.  
Comparative recognition of A1-HY-related peptides derived from DFFRY and DFFRX. Peptides corresponding to the A1-HY epitope (IVDCLTEMY), its cysteinylated and oxidized derivatives, and the DFFRX-encoded homologue (IVDSLTEMY) were assayed for epitope reconstituting activity as described in Materials and Methods.  
Article
In this report, we describe the use of novel mass spectrometry instrumentation to identify a male-specific minor histocompatibility Ag restricted by HLA-A*0101 (A1-HY). This Ag has the sequence IVDC*LTEMY, where C* represents a cysteine disulfide bonded to a second cysteine residue. The core peptide sequence is found in the protein product of DFFRY, a Y chromosome gene not previously identified as the source of an HY Ag. The male-specific form of the peptide differs from its X chromosomal counterpart by the substitution of serine for the C* residue. Both peptides are expressed on the cell surface at 30 or fewer copies per cell. However, A1-HY-specific CTL recognize the DFFRY-derived peptide at a 1500-fold lower dose than the female homologue. Thus, these studies have identified a new source of HY epitopes and provide additional information about the influence of posttranslational modifications of class I-associated peptides on T cell recognition.
 
Article
We have examined the role of 12 polymorphic residues of the beta-chain of the HLA-DR1 class II molecule in T cell recognition of an epitope of pertussis toxin. Murine L cell transfectants expressing wild-type or mutant DR1 molecules (containing single amino acid substitutions in DR(beta 1*0101)) were used as APC in proliferation assays involving nine DR1-restricted T cell clones specific for peptide 30-42 of pertussis toxin. Four different patterns of recognition of the mutants were found among the pertussis-specific clones. Residues in the third hypervariable region (HVR) of DR(beta 1*0101) are critically important for all the T cell clones; amino acid substitutions at positions 70 and 74 abrogated recognition by all of the T cell clones, and substitutions at positions 67 and 71 eliminated recognition by most of the clones. In contrast, most single amino acid substitutions in the first and second HVR, predicted to be located in the floor of the peptide binding groove, had little or no effect on the proliferative responses of these clones. However, the involvement of beta-chain first and second HVR residues was demonstrated by the inability of transfectants expressing wild-type DR(beta 1*0404) (DR4Dw14) or DR(beta 1*1402) (DR6Dw16) to present peptide to these clones. These beta-chains have completely different first and second HVR compared with DR(alpha,beta 1*0101) although the third HVR are identical. These results illustrate the functional importance of third HVR residues of DR(beta 1*0101) and allow definition of the molecular interactions of the DR1 molecule with the 30-42 peptide.
 
Article
A recurring epitope in the human acetylcholine receptor (AChR) alpha subunit (alpha146-160) is presented to specific T cells from myasthenia gravis patients by HLA-DRB3*0101-"DR52a"-or by DR4. Here we first map residues critical for DR52a in this epitope by serial Ala substitution. For two somewhat similar T cells, this confirms the recently deduced importance of hydrophobic "anchor" residues at peptide p1 and p9; also of Asp at p4, which complements this allele's distinctive Arg74 in DRbeta. Surprisingly, despite the 9 sequence differences in DRbeta between DR52a and DR3, merely reducing the bulk of the peptide's p1 anchor residue (Trp149-->Phe) allowed maximal cross-presentation to both T cells by DR3 (which has Val86 instead of Gly). The shared K71G73R74N77 motif in the alpha helices of DR52a and DR3 thus outweighs the five differences in the floor of the peptide-binding groove. A second issue is that T cells selected in vitro with synthetic AChR peptides rarely respond to longer Ag preparations, whereas those raised with recombinant subunits consistently recognize epitopes processed naturally even from whole AChR. Here we compared one T cell of each kind, which both respond to many overlapping alpha140-160 region peptides (in proliferation assays). Even though both use Vbeta2 to recognize peptides bound to the same HLA-DR52a in the same register, the peptide-selected line nevertheless proved to depend on a recurring synthetic artifact-a widely underestimated problem. Unlike these contaminant-responsive T cells, those that are truly specific for natural AChR epitopes appear less heterogeneous and therefore more suitable targets for selective immunotherapy.
 
Article
The individual amino acid contacts responsible for peptide binding to DRB1*0101 and/or DRB1*0401 were defined using a quantitative binding assay. The differential contribution of each amino acid in two well studied T cell determinants, HA307-319 and RMBP 90-102, was delineated by comparing the IC50 values of analogues of varying length. This analysis confirmed the importance of a hydrophobic amino acid located near the amino-terminus for binding to both alleles and revealed that the contribution of the carboxyl-terminal amino acids differed between DRB1*0101 and DRB1*0401. Taking advantage of previous experiments demonstrating that all of the residues could be replaced with alanine, with the exception of the key hydrophobic amino acid, simplified analogues composed of polyalanines were used to prove 1) optimal binding depended on the position of the hydrophobic side chain relative to the amino- and carboxyl-termini; 2) aromatic amino acids were superior to aliphatic side chains at this position; and 3) a significant amount of free energy of binding arises from hydrogen bonding between the class II binding site and the amide bonds of the ligand. The role of each carbonyl and amide nitrogen was measured by assaying analogues containing reduced peptide bonds or N-methyl amino acids. Serine, but not glycine, could be used as a framework amino acid for peptide ligands, indicating that the beneficial aspects of these simplified structures was the combination of retaining the correct orientation of the peptide bonds, the restriction of the conformational freedom by limiting the possible phi/psi angles of the peptide, and avoidance of deleterious side-chain contacts. Collectively, these data were consistent with the peptide binding in a nonrepeating conformation with the vast majority of the free energy of binding arising from hydrogen bonds with the peptide backbone and a single, key hydrophobic side chain interacting in a conserved pocket in both DRB1*0101 and DRB1*0401.
 
Relative binding affinities of HLA-DR1 (DRB1*0101) and HLA-DR4 (DRB1*0401) for Ala-substituted peptides. Ala analog peptides based on CII(259 –273) were used in a competitive binding assay to determine relative affinities based on IC 50 concentrations. Despite polymorphic differences in the 1 domain of DR1 and DR4, identical anchor residues, F at 263 and K at 264 (striped bars), are used for binding CII(259 –273). Dotted lines indicate IC 50 of wild-type CII(259 –273). Open bars indicate Ala substitutions that increased the binding affinity of the CII peptide.  
Identification of TCR contact residues and DR binding residues in CII(259 –273). The ability of the Ala-substituted peptides to stimulate DR-restricted T cell proliferation is compared with the ability of the same peptides to bind to the DR molecules. DR-binding residues were identified by low relative affinity (high IC 50 values, abscissa) and poor T cell stimulation (ordinate). TCR interactions residues were identified by high relative affinity (low IC 50 values) and poor T cell stimulation. Numbers in the plot tokens indicate the position of the amino acid substituted with Ala. Data are derived from Figs. 1 and 6. P1 through P8 indicate the core of the CII peptide determinant, starting with residue 263 through 268.  
DR1 has a higher affinity for the CII immunodominant peptide than DR4. Purified DR1 and DR4 (10 nM) were tested for their ability to bind various concentrations of CII(257–274) in a competitive binding assay using biotinylated HA(307–319) as the indicator peptide (A), or in a direct binding assay using biotinylated (bio) CII(257–274) (B, n 3 for both alleles). The amount of HA peptide bound was determined by the addition of streptavidin-europium and measurement of fluorescence. The concentration of CII peptide inhibiting 50% of the HA peptide binding (IC 50 ) was calculated from the linear portion of the curves. Data are representative of a minimum of four experiments for the competitive studies, and three experiments for the direct binding studies.  
Identification of anchor residues in CII(259 –273) for binding to HLA-DR1 and DR4. Competitive binding assays with soluble DR1 and DR4 and Ala-substituted CII peptides were performed as described in Fig. 4. Substitution of only the Phe 263 (F263A) and the Lys 264 (K264A) decreased the binding of the CII(259 –273) peptide. Ala replacement of the Glu 266 (E266A), an amino acid expected to participate in DR4 binding, actually increased the binding affinity of the CII peptide for both DR1 and DR4. Data are representative of three experiments.  
Article
Rheumatoid arthritis is an autoimmune disease in which susceptibility is strongly associated with the expression of specific HLA-DR haplotypes, including DR1 (DRB1*0101) and DR4 (DRB1*0401). As transgenes, both of these class II molecules mediate susceptibility to an autoimmune arthritis induced by immunization with human type II collagen (hCII). The dominant T cell response of both the DR1 and DR4 transgenic mice to hCII is focused on the same determinant core, CII(263-270). Peptide binding studies revealed that the affinity of DR1 and DR4 for CII(263-270) was at least 10 times less than that of the model Ag HA(307-319), and that the affinity of DR4 for the CII peptide is 3-fold less than that of DR1. As predicted based on the crystal structures, the majority of the CII-peptide binding affinity for DR1 and DR4 is controlled by the Phe(263); however, unexpectedly the adjacent Lys(264) also contributed significantly to the binding affinity of the peptide. Only these two CII amino acids were found to provide binding anchors. Amino acid substitutions at the remaining positions had either no effect or significantly increased the affinity of the hCII peptide. Affinity-enhancing substitutions frequently involved replacement of a negative charge, or Gly or Pro, hallmark amino acids of CII structure. These data indicate that DR1 and DR4 bind this CII peptide in a nearly identical manner and that the primary structure of CII may dictate a different binding motif for DR1 and DR4 than has been described for other peptides that bind to these alleles.
 
Article
Desmogleins (Dsg) are transmembrane glycoproteins of the desmosome that allow a cell-cell adhesion between keratinocytes and comprise four different isoforms (Dsg1 to Dsg4). Two Dsg are targeted by pathogenic autoantibodies produced in the course of autoimmune bullous skin diseases, Dsg1 in pemphigus foliaceus (PF), and Dsg3 and Dsg1 in pemphigus vulgaris. The genetic susceptibility to PF is associated with certain HLA class II alleles, which are thought to participate in disease pathogenesis through their capacity to accommodate autoantigen-derived peptides and present them to autoreactive T cells. So far, a unique isoform of Dsg1 has been described in humans, which includes several immunodominant T cell epitopes. In this study, we describe an alternative transcript of DSG1, which contains a 101-bp insertion corresponding to the 3' end of DSG1-intron 6 and introducing a stop codon in the nucleotide sequence. This alternative transcript leads to the synthesis of a truncated isoform of Dsg1 expressed in normal human epidermis. This isoform bears a specific peptide sequence that binds to the PF-associated HLA class II DRbeta1*0102 molecule as shown in a HLA-DR peptide-binding assay, and induces PF T cell proliferation. These data provide an illustration of an autoantigen encoded by alternative spliced transcript that may participate in the pathogenesis of the disease by bearing PF-associated HLA class II restricted-epitope.
 
Flow cytometric analysis of HLA-DQ allelic proteins expressed on EBV-transformed B-LCLs. EBV-transformed B-LCLs and BLS-1 (HLA-DQ negative) were stained with SPVL3 and IgG2a isotype control mAbs at concentrations ranging from 0.033 g/ml to 3.3 g/ ml. Bound mAbs were detected with fluorescein FITC goat anti-mouse IgG and measured with a Becton Dickinson FACSort using CELLQuest Software. A, Cell number vs log fluorescence intensity plotted for BLS-1 and EBV-transformed B-LCLs (DQ0501, DQ0602, DQ0201, DQ0301, DQ0302) stained with the IgG2a isotype control mAb (dotted line) and the SPVL3 mAb (solid line at 3.3 g/ml). B, Quantitative comparison of the expression of HLA-DQ allelic proteins on EBV-transformed B-LCLs. The fluorescence signal to noise ratio was calculated from the median fluorescence intensity obtained with the SPVL3 mAb divided by the median fluorescence intensity obtained with the IgG2a isotype control mAb, over a range of mAb concentrations. Each point represents the mean SD, n 3.
SDS stability of HLA-DQ allelic dimers in PBLs. Cell lysates from human PBLs with homozygous HLA-DQ alleles (40 g protein) were diluted 1:1 in 2 sample buffer containing 0.4% SDS and incubated at room temperature for 30 min (A and B) or 2 sample buffer containing 4% SDS with 10% 2-ME and boiled for 2 min (C). Samples were electrophoresed on a 4-20% Tris-glycine gel and transferred to Immobilon-P. Western analysis was performed with SPVL3 (anti-HLA-DQ dimer) (A), GS200.1 (anti-HLA-DQ dimer) (B), and 2H3 (anti-HLA-DQ) (C). Bound Ab was detected with goat anti-mouse IgG horseradish peroxidase and enhanced chemiluminescence. A: lane 1, 25 ng purified DQ0602 (positive control); lane 2, m.w. markers; lane 3, DQ0501 cell lysate; lane 4, DQ0602 cell lysate; lane 5, DQ0201 cell lysate; lane 6, DQ0301 cell lysate; lanes 7 and 8, DQ0302 cell lysate. B and C: lane 1, 25 ng purified DQ0602 (positive control); lane 2, m.w. markers; lane 3, DQ0501 cell lysate; lane 4, DQ0602 cell lysate; lanes 5 and 6, DQ0302 cell lysate.
Article
HLA-DQ alleles are closely associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM) but the immunologic mechanisms involved are not understood. Structural studies of the IDDM-susceptible allele, HLA-DQA1*0301/DQB1*0302, have classified it as a relatively unstable dimer, particularly at neutral pH. This is reminiscent of studies in the nonobese diabetic mouse, in which I-A(g7) is relatively unstable, in contrast to other murine I-A alleles, suggesting a correlation between unstable MHC class II molecules and IDDM susceptibility. We have addressed this question by analysis of dimer stability patterns among various HLA-DQ molecules. In EBV-transformed B-lymphoblastoid cell lines and PBL, the protein encoded by the IDDM-protective allele HLA-DQA1*0102/DQB1*0602 was the most SDS stable when compared with other HLA-DQ molecules, including HLA-DQA1*0102/DQB1*0604, a closely related allele that is not associated with protection from IDDM. Expression of six different HLA-DQ allelic proteins and three different HLA-DR allelic proteins in the bare lymphocyte syndrome cell line, BLS-1, revealed that HLA-DQA1*0102/DQB1*0602 is SDS stable even in the absence of HLA-DM, while other HLA class II molecules are not. These results suggest that the molecular property of HLA-DQ measured by resistance to denaturation of the alphabeta dimer in SDS may play a role in IDDM protection.
 
HLA-DQ expression on BLS-1 DQ0604 mutant cell lines. BLS-1 DQ0602, BLS-1 DQ0604, and BLS-1 DQ0604 mutants were examined by flow cytometric analysis for expression of HLA-DQ. The fluorescence signal to noise (FSN) ratio was calculated from the median fluorescence intensity obtained with the anti-HLA-DQ dimer Abs SPVL3 (A), GS200.1 (B), and 1a3 (C) divided by the median fluorescence intensity obtained with an isotype control Ab. Each FSN ratio represents the mean SD of three independent experiments.
Article
Studies of the stability of HLA-DQ have revealed a correlation between SDS stability of MHC class II alphabeta dimers and insulin-dependent diabetes mellitus (IDDM) susceptibility. The MHC class II alphabeta dimer encoded by HLA-DQA1*0102/DQB1*0602 (DQ0602), which is a dominant protective allele in IDDM, exhibits the greatest SDS stability among HLA-DQ molecules in EBV-transformed B-lymphoblastoid cells and PBLs. DQ0602 is also uniquely SDS stable in the HLA-DM-deficient cell line, BLS-1. We addressed the molecular mechanism of the stability of DQ0602 in BLS-1. A panel of mutants based on the polymorphic differences between HLA-DQA1*0102/DQB1*0602 and HLA-DQA1*0102/DQB1*0604 were generated and expressed in BLS-1. An Asp at beta57 was found to be critical for SDS stability, whereas Tyr at beta30, Gly at beta70, and Ala at beta86 played secondary roles. Furthermore, the level of class II-associated invariant chain peptide bound to HLA-DQ did not correlate with SDS stability, suggesting that class II-associated invariant chain peptide does not play a direct role in the unique SDS stability of DQ0602. These results support a role for DQB1 codon 57 in HLA-DQ alphabeta dimer stability and IDDM susceptibility.
 
Article
HLA-DQA1*0102/DQB1*0602 (DQ0602) is observed at a decreased frequency in insulin-dependent diabetes mellitus in different ethnic groups, suggesting a protective role for DQ0602. Analysis of overlapping peptides from human insulin found that insulin B(1-15) bound well to DQ0602 and exhibited a high degree of allelic specificity. Truncation analysis of insulin B(1-15) identified insulin B(5-15) as the minimal peptide for DQ0602 binding. Insulin B(5-15) bound to DQ0602 with an apparent KD of 0.7 to 1.0 microM and peptide binding reached equilibrium at 96 h. Single arginine substitutions at each position of the insulin B(5-15) peptide identified amino acids 6, 8, 9, 11, and 14 (relative positions P1, P3, P4, P6, and P9) as important for binding. Extensive substitutions for each of these amino acids revealed that amino acids 11 and 14 (P6 and P9) exhibited the highest specificity. Amino acid 11 (P6) preferred large aliphatic amino acids, while amino acid 14 (P9) preferred smaller aliphatic and hydroxyl amino acids. Binding of an overlapping series of peptides from a randomly chosen protein, the herpes simplex virus-2 tegument protein UL49, correlated completely with the presence or absence of the DQ0602 peptide binding motif. Peptides 11 amino acids long were selected from GAD65, IA-2, and proinsulin, that contained the DQ0602 peptide binding motif. Of these, 79% (19 of 24) were able to bind DQ0602. This study identifies a peptide binding motif for DQ0602 and peptides from insulin-dependent diabetes mellitus autoantigens that bind DQ0602 in vitro.
 
Article
Endotoxin protein co-purifies with lipopolysaccharide after extraction of Gram-negative bacteria with aqueous n-butyl alcohol or trichloroacetic acid and is known to elicit diverse biologic activies from cells of the mammalian reticuloendothelial system. However, to date the biologically active component(s) in endotoxin protein have not been identified. We have now characterized endotoxin protein prepared from Escherichia coli 0111 by the above 2 extraction procedures. At least 78% of endotoxin protein is derived from the outer membrane of the bacterial cell when extracted with aqueous n-butyl alcohol, and at least 13 outer membrane proteins were identified. Outer membrane porins, protein II* and lipoprotein, known mitogens and polyclonal activators of lymphocytes, are present at the levels of 24%, 4%, and 1% respectively, of the total protein. At least 95% of endotoxin protein is derived from the outer membrane when extracted with trichloroacetic acid, and at least 12 outer membrane proteins were identified. Porins, protein II*, and lipoprotein are present at the levels of 45%, 10% and <1%, respectively, of the total protein. Identification is based on co-migration of endotoxin proteins with outer membrane proteins during gel electrophoresis and reactivity of endotoxin proteins with antibody prepared aginst specific outer membrane proteins. When complexed to lipopolysaccharide, outer membrane porins are partially resistant to pronase and totally resistant to trypsin, whereas aprotein II* and lipoprotein are sensitive to both enzymes. In addition, proteolytic degradation fragments remain associated with lipopolysaccharide after protease treatment. If some of these fragments retain biologic activity, both the known protease resistance of endotoxin protein activities and discrepancies in previously reported m.w. for the active components may be explained.
 
Article
The effect of complement (C) components on the intracellular killing of E. coli 0111:B4 by human PMN was studied. Various intermediate bacteria were prepared by opsonizing IgM-coated 0111:B4 with yeast cell-treated human serum (BAC1-3), a C6-deficient human serum (BAC1-5), a C8-deficient human serum (BAC1-7), a C8-deficient human serum, and with partially purified C8 (BAC1-8). All these bacterial preparations were phagocytosed by human PMN, but only BAC1-8 and, to a lesser extent, BAC1-5 were killed. Similar results were obtained when the 400 x G postnuclear supernatant (PNS) of PMN homogenate was used instead of intact leukocytes. The C9 nature of the killing factor in the PMN homogenate was ruled out by its inability to lyse EAC1-8 and by the finding that the killing of BAC1-8 by the PMN factor was not inhibited by the antiserum against human C9. The anti-C5 and anti-C8 antisera were unable to inhibit the killing by the PNS of BAC1-5 and BAC1-8, respectively, suggesting that bound C5 and C8 do not provide a binding site for the killing factor.
 
Article
The fifth C component (C5) exhibits a different stability when bound to sheep E or Escherichia coli 0111:B4, being fairly stable on the bacterial intermediate sensitized E. coli 0111:B4 coated with C components up to C5 (BAC1-5) and extremely labile on the RBC intermediate sensitized sheep E coated with C components up to C5 (EAC1-5). We examined the possibility that molecular changes of membrane-bound C5 might be responsible for the different functional behavior of the two intermediates using mAb to C5 and sensitive immunoassays to detect bound C5. The decay of EAC1-5 over 30 min of incubation at 37 degrees C was associated with a significant drop in the reactivity of bound C5 with three of four mAb used. These results contrasted with those obtained with BAC1-5, which showed unchanged reactivity with all mAb tested over the same period of incubation. The effect of mAb on the activity of C5 was then investigated in an attempt to relate the change of the reactivity pattern of EAC1-5 with the functional modification of bound C5. MAb 1.5 and 1.6 were the only antibodies that interfered with the functional activity of C5, although through a different mechanism. In particular, mAb 1.5 was active both on fluid-phase and on membrane-bound C5 and is therefore likely to interact with the binding site for the late components on C5. Conversely, mAb 1.6 was only effective on fluid-phase C5 and acted by promoting a decay of BAC1-5 similar to the spontaneous decay of EAC1-5. We suggest that the bacterial outer membrane may protect C5 from functional decay and that mAb 1.6 interferes with the stabilizing effect of the bacteria in an as yet unclear manner.
 
Article
The antigen specificity of two immunoprotective monoclonal antibodies derived from mice immunized with Escherichia coli 0111:B4 bacteria and boosted with purified lipopolysaccharide (LPS) were investigated. One of the antibodies, B7, was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunostaining to bind to the O-antigen containing LPS species, whereas the other antibody, 5B10, reacted with both O-antigen containing homologs and the O-antigen-deficient LPS. 5B10 did not bind to LPS from E. coli J5, an Rc mutant of E. coli 0111:B4 that lacks both the O-antigen and outer core sugars. 5B10 did not cross-react with LPS from several other E. coli strains. Thus 5B10 appeared to recognize a type-specific epitope in the outer core of LPS exclusive of Rc determinants. The monoclonal antibody specific for the polymeric O-antigen is of the IgG3 subclass, and the monoclonal antibody 5B10 specific for the outer core of LPS is an IgG2a. Although B7 and 5B10 were equally able to protect mice from a lethal challenge of E. coli 0111:B4 organisms, the outer core-specific IgG2a antibody was much more efficient at mediating the binding of human complement C3 than the O-antigen-specific IgG3 monoclonal antibody.
 
Killing of bacterial strains by purified C5-C9 by using acid activation to form C5b6"' 
Article
Our previous experiments showed that immune IgG and F(ab')2, but not Fab', mediated serum killing of Escherichia coli 0111B4, strain 12015 (12015), without significantly increasing the extent of terminal complement (C) component attachment to the bacterial surface. We concluded that bactericidal antibody must change either the site or the nature of C5b-9 bacterial attachment. To pursue this possibility, conditions necessary for elution of C5b-9 from the bacterial surface were examined. Forty-two to 44% of 125I-C9 was released from the serum-resistant nonpresensitized 12015 by 1 M NaCl or 0.1% trypsin, compared with the 21 to 24% release from the serum-sensitive presensitized isolate under the same condition. When strain 12015 bearing 125I-C9 was lysed in a French pressure cell, 73.1% of 125I-C9 was released with the capsular fraction if the organisms had not been presensitized. In contrast, on presensitized 12015, 70.2% of 125I-C9 remained associated with the outer membrane after such lysis. These results suggested that C5b-9 was trapped within or underneath the capsule of 12015 in the absence of bactericidal antibody, but that addition of antibody led to C5b-9 insertion into the outer membrane with bacterial killing. The requirement of C components preceding C5 for bacterial killing was next examined. Minimal killing of presensitized 12015 occurred when a terminal C complex was formed by acid activation from purified C5, C6, C7, C8, and C9 in the absence of C3 or earlier components. In contrast, between 1.2 and 3 log killing of nonpresensitized rough Salmonella minnesota and rough E. coli was observed in the same system. Killing of 12015 was examined with bacteria incubated in C5-deficient serum (C5D), followed by washing and the addition of purified C5, C6, C7, C8, and C9 to permit C5b-9 formation. Antibody was added before or after incubation in C5D serum, or after the addition of purified C5-C9. Under conditions of equivalent C3 and C9 binding, significant killing occurred only when antibody was added before incubation in C5D serum. These results show that antibody must be present at or before the time of C5 convertase formation to mediate killing of 12015 by C5b-9. Therefore, antibody is unlikely to be functioning primarily to alter the bacterial surface to expose sites for C5b-9 insertion, nor is the effect of antibody simply to increase C3 and terminal component binding. We postulate that antibody mediates killing of 12015 by localizing C5b-9 around antibody-clustered sites of C3 and C5 convertase formation.
 
Article
The mechanism of antibody-dependent complement-(C) mediated killing of Escherichia coli 0111B4, strain 12015 (12015), was examined. 12015 was resistant to serum killing when incubated in hypogammaglobulinemic serum (H gamma S) or pooled normal human serum (NHS) that had been previously adsorbed to remove specific antibody (Abs NHS). Presensitization with immune rabbit serum or purified immune rabbit IgG resulted in 1 to 3 log killing when 5 X 10(8) colony forming units (CFU)/ml were incubated in 10 to 40% Abs NHS. Binding of 125I-C3 and 131I-C9 to the bacterial surface of the presensitized and the nonpresensitized strain was quantitated when these organisms were incubated in 10, 20, and 40% Abs NHS. Stable binding of up to 3.0 X 10(5) molecules of C3 and 8.0 X 10(4) molecules of C9 to presensitized and nonpresensitized isolates occurred in the highest concentration of serum, but there was no killing without presensitization. Similar results were found when Abs NHS was chelated with ethylene bis glycoltetraacetic acid containing 2 mM MgCl2 (Mg EGTA) to block classical pathway activation, indicating that antibody mediated the bactericidal reaction through the alternative pathway. Deposition of C3 and C9 and killing of 120 15 in 10% Abs NHS or 10% H gamma S was measured after presensitization with increasing amounts of IgG, F(ab')2, or Fab'. There was a dose-dependent increase in C3 deposition and killing, but only minimal change in C9 binding when 1.0 X 10(3) to 3.2 X 10(4) IgG or F(ab')2/CFU were bound to the bacterial surface. In contrast, there was no increase in C3 or C9 binding and no bacterial killing when 1 X 10(3) to 3.4 X 10(4) molecules Fab'/CFU were bound to the bacterial surface. These experiments show that immune IgG and F(ab')2 can mediate killing of E. Coli 0111B4 by the alternative pathway without changing the extent of terminal C component attachment to the bacterial surface.
 
Article
The binding of serum C3 to the O-antigen capsule (OAg Cap), lipopolysaccharide (LPS), and outer membrane proteins (OMP) of Escherichia coli 0111B4 was examined. Bacteria were intrinsically labeled with [3H] or [14C]galactose (*gal) in the OAg Cap and LPS moieties or with [14C]leucine (*leu) to label proteins. Organisms were then incubated in serum containing differentially labeled C3, the above fractions were separated, and the proportion of each binding to a column containing anti-C3 was measured. The OAg Cap fraction bound 72 to 82% of the C3, which bound to E. coli 0111B4 during incubation in absorbed 10% pooled normal human serum (10% PNHS) or absorbed 40% C8-deficient serum (C8D). This distribution did not change when the organism was presensitized with immune IgG before serum incubation. A total of 2.93% +/- 0.48 of OAg Cap and 0.52% +/- 0.16 of LPS *gal bound specifically to Sepharose-containing antibodies to C3 (A:C3-Seph) after incubation in 10% PNHS; these values increased to 10.1% +/- 4.5 and 1.8% +/- 0.3, respectively, when C3 deposition was increased fourfold by incubation in 40% C8D. When encapsulated E. coli 0111B4 was incubated in 10% PNHS containing biotinylated C3, specific attachment of OAg Cap *gal to avidin-Sepharose was demonstrated in 1% sodium dodecyl sulfate (SDS), and complete release of bound *gal but not C3 occurred with 1 M NH2OH. When a mutant of E. coli 0111B4 lacking OAg Cap was incubated in 40% C8D, the outer membrane (OM) bound 85% of C3. Five percent of OM *gal from the unencapsulated organism bound to A:C3-Seph in 0.05% SDS, indicating that the fraction of LPS molecules with bound C3 increased threefold in the absence of OAg Cap. OAg Cap does not contain protein, and no net specific binding of *leu from OAg Cap fractions to A:C3 was detectable; 2.4 to 3.6% of OM *leu bound to A:C3-Seph. Immunoprecipitation of 82.9% of OAg Cap *gal with antisera that were directed to E. coli 0111B4 was associated with co-precipitation of 69.5% of C3 in the capsular fraction. Therefore, the majority of C3 bound to E. coli 0111B4 was covalently attached to OAg Cap and LPS. As corroboration of experiments with whole bacteria, purified OAg Cap and purified LPS consumed C3 when incubated in serum in the fluid phase. These results are the first to evaluate the acceptor site for C3 deposition on a Gram-negative organism incubated in serum, and show that LPS, OAg Cap, and OMP are all major acceptor sites for C3 in nonimmune serum.
 
Conference Paper
Recognition of viral genetic material takes place via several different receptor systems, such as retinoic acid-inducible gene I-like receptors and TLRs 3, 7, 8, and 9. At present, systematic comparison of the ability of different types of RNAs to induce innate immune responses in human immune cells has been limited. In this study, we generated bacteriophage 6 and influenza A virus-specific ssRNA and dsRNA molecules ranging from 58 to 2956 nt. In human monocyte-derived dendritic cells (moDCs), short dsRNAs efficiently upregulated the expression of IFN (IFN-α, IFN-β, and IFN-λ1) and proinflammatory (TNF-α, IL-6, IL-12, and CXCL10) cytokine genes. These genes were also induced by ssRNA molecules, but size-specific differences were not as pronounced as with dsRNA molecules. Dephosphorylation of short ssRNA and dsRNA molecules led to a dramatic reduction in their ability to stimulate innate immune responses. Such a difference was not detected for long ssRNAs. RNA-induced cytokine responses correlated well with IFN regulatory factor 3 phosphorylation, suggesting that IFN regulatory factor 3 plays a major role in both ssRNA- and dsRNA-activated responses in human moDCs. We also found that IFN gene expression was efficiently stimulated following recognition of short dsRNAs by retinoic acid-inducible gene I and TLR3 in human embryonic kidney 293 cells, whereas ssRNA-induced responses were less dependent on the size of the RNA molecule. Our data suggest that human moDCs are extremely sensitive in recognizing foreign RNA, and the responses depend on RNA size, form (ssRNA versus dsRNA), and the level of 5' phosphorylation.
 
Article
Cellular cooperation in the immune response to Escherichia coli 0127 was investigated. Spleen cell cultures prepared from BDF1 and congenitally athymic “nude” mice responded in vitro to E. coli bacteria, and removal of adherent cells from the spleen cell populations had no effect on the response to the bacterial antigen. Nude mice immunized with sheep erythrocytes (SRBC) responded poorly to the SRBC antigen, but the injection of 7.5 × 107 thymocytes with the SRBC antigen increased the response 10-fold. In contrast, nude mice immunized with E. coli responded well to the bacterial antigen, and the injection of thymocytes did not alter this response. These results support the view that the immune response to E. coli 0127 is independent of thymus-derived cells, and demonstrate that the response ia also independent of adherent cells.
 
Article
By means of a hemagglutination procedure, the antibody response of premature infants with diarrhea in an epidemic caused by Escherichia coli 0127:B8 was studied. In preliminary experiments with two adult volunteers who had ingested large numbers of these microorganisms, the specificity of the hemagglutinating-antibody was established by the patterns of antibody response and by absorption tests. Hemagglutinating-antibodies were detected in normal adults and transplacental transfer of such antibody was demonstrated. In infected newborn infants, the antibody response was distinguished from transplacental antibody by its pattern of development.
 
Article
A possible regulatory effect of thymus-derived lymphocytes on the immune response to a thymus independent antigen, Escherichia coli 0127 somatic antigen in mice, was investigated. Antilymphocyte globulin prepared in rabbits caused a substantial suppression of the antibody response to a thymus dependent antigen (sheep erythrocytes) but exerted no effect on the immune response to the bacterial antigen. Administration of E. coli somatic antigen to mice did not induce an immunosuppressive effect on a subsequently injected unrelated antigen indicating that this thymus independent antigen was incapable of inducing antigenic competition. Likewise, the immune response to the bacterial antigen was not influenced by prior immunization of the mice with sheep erythrocytes. Thus, in these immune reactions in which thymus-derived lymphocytes were affected the response to the bacterial antigen was not modified. These results contradict recent observations which indicated that thymus-derived lymphocytes exert a suppressive effect on the immune response to thymus independent antigens.
 
Article
NK cell activity is regulated by the integration of positive and negative signals. One important source of these signals for human NK cells is the killer Ig-like receptor (KIR) family, which includes both members that transduce positive and those that generate negative signals. KIR3DL1 inhibits NK cell activity upon engagement by its ligand HLA-Bw4. The highly homologous KIR3DS1 is an activating receptor, which is implicated in the outcome of a variety of pathological situations. However, unlike KIR3DL1, direct binding of KIR3DS1(+) cells to HLA has not been demonstrated. We analyzed four key amino acid differences between KIR3DL1*01502 and KIR3DS1*013 to determine their role in KIR binding to HLA. Single substitutions of these residues dramatically reduced binding by KIR3DL1. In the reciprocal experiment, we found that the rare KIR3DS1 allotype KIR3DS1*014 binds HLA-Bw4 even though it differs from KIR3DS1*013 at only one of these positions (position 138). This reactivity was unexpectedly dependent on residues at other variable positions, as HLA-Bw4 binding was lost in receptors with KIR3DL1-like residues at both positions 199 and 138. These data provide the first evidence, to our knowledge, for the direct binding of KIR3DS1(+) cells to HLA-Bw4 and highlight the key role for position 138 in determining ligand specificity of KIR3DS1. They also reveal that KIR3DS1 reactivity and specificity is dictated by complex interactions between the residues in this region, suggesting a unique functional evolution of KIR3DS1 within the activating KIR family.
 
LF 15-0195 treatment reduces the MBP-specific cellular immune response. Lymph node cells from MBP-immunized LEW rats treated () or not treated (f) with LF 15-0195 (1 mg/kg) for 12 days starting from the day of MBP sensitization were collected and stimulated in vitro with the indicated concentrations of MBP. A, Proliferation was assessed with an 18-h [ 3 H]thymidine pulse added after 48 h of culture, and results are expressed as the mean [ 3 H]thymidine incorporation adjusted for background proliferation (cpm) SD. Tissue culture supernatants were assayed at 48 h for IFN-(B) and IL-10 (C) proteins using capture ELISA. The results of one representative experiment of three are shown and are expressed as the mean (SD) of values obtained from four individual rats per group. TNF-(D), TGF-(E), and IL-4 (F) mRNA expression were assayed by quantitative RT-PCR in lymph node cells following MBP stimulation (20 g/ml) for 24 h. Results are expressed as the mean cytokine/hypoxanthine phosphoribosyltransferase ratio SD of values obtained from four individual rats in each group and represent two independent experiments. , p 0.05 (by Mann-Whitney U test). 
LF 15-0195 treatment reduces MBP-specific IgG responses. LEW rats were immunized with MBP in CFA and treated (; six rats) or not treated (f; four rats) with LF 15-0195 at 1 mg/kg for 30 days, starting from the day of immunization. The IgG (A), IgG1 (B), IgG2a (C), and IgG2b (D) antiMBP Ab titers were measured by ELISA on days 5, 11, 17, 24, 31, and 39 after immunization. Results are expressed as absorbance at 450 nm of a pool of sera diluted 1/800 for total IgG and 1/1600 for IgG subclasses. 
MBP-specific CD4 T cells from LF 15-0195-treated rats have a diminished capacity to transfer EAE. Lymph node cells from animals in which EAE was completely suppressed by LF 15-0195 treatment at 1 mg/kg, starting from the day of MBP challenge (; n 11 for A and B and n 4 for C and D) or from control PBS-treated diseased rats (f; n 7 for A and B and n 4 for C and D) were adoptively transferred. Lymph nodes were removed on day 10 or 12 following MBP immunization, and cells were either cultured in vitro with MBP (2 g/ml) or Con A (1 g/ml) for 3 days. Viable MBP-stimulated leukocytes (25 10 6 ; A and B) or 10 8 viable Con A-stimulated lymphocytes (C and D) were injected i.v. into each naive syngeneic recipient. The results shown represent in A and C the mean clinical scores for paralysis of each experimental group and in B and D the mean total disease score of each experimental group. Results in A and B are from two independent experiments, and those in C and D are from one experiment. , p 0.05; , p 0.01 (by Mann-Whitney U test). 
Article
Experimental autoimmune encephalomyelitis (EAE) is a T cell-dependent autoimmune disease induced in susceptible animals by a single immunization with myelin basic protein (MBP). LF 15-0195 is a novel immunosuppressor that has been shown to have a potent immunosuppressive effect in several pathological manifestations. The purpose of this study was to investigate the effect of this drug on the induction and progression of established rat EAE and to dissect the mechanisms involved. We show that LF 15-0195 administration at the time of MBP immunization reduces the incidence and severity of EAE in Lewis rats. This drug also inhibits ongoing and passively induced EAE, indicating that LF 15-0195 affects already differentiated pathogenic lymphocytes. Compared with lymph node cells from untreated rats, lymphocytes from MBP-immunized rats treated with LF 15-0195 proliferated equally well in response to MBP in vitro, while their ability to produce effector cytokines and to transfer EAE into syngeneic recipients was significantly reduced. This phenomenon is stable and long-lasting. Indeed, neither IL-12 nor repeated stimulation with naive APC and MBP in vitro rendered MBP-specific CD4 T cells from protected rats encephalitogenic. In conclusion, LF 15-0195 treatment suppresses EAE by interfering with both the differentiation and effector functions of autoantigen-specific CD4 T cells.
 
Proliferation and production of IFN-, IL-10, and IL-2 by thymus CD4 , CD4 CD25 , and CD4 CD25 cells from LF15-0195-treated recipients. Purified total CD4 or CD4 CD25 or CD4 CD25 thymocytes from naive or from tolerant LF15-0195-treated recipients (100 days after transplantation) were stimulated by a LEW.1W donor-irradiated APC-enriched cell population for 72 h. Values represent the cpm SD of all triplicates after 3 days of culture for thymidine incorporation (A). IFN(B), IL-10 (C), and IL-2 (D) were measured in the supernatants of triplicates by ELISA, as described in Materials and Methods, and the results are expressed in picograms per milliliter. Results are representative of three independent experiments.
Article
A 20-day treatment with LF15-0195, a deoxyspergualine analogue, induced allograft tolerance in a fully MHC-mismatched heart allograft model in the rat. Long-term allografts displayed minimal cell infiltration with no signs of chronic rejection. CD4+ spleen T cells from tolerant LF15-0195-treated recipients were able to suppress in vitro proliferation of allogeneic CD4+ T cells and to transfer tolerance to second syngeneic recipients, demonstrating dominant suppression by regulatory cells. A significant increase in the percentage of CD4+CD25+ T cells was observed in the thymus and spleen from tolerant LF15-0195-treated recipient. In vitro direct stimulation with donor APCs demonstrated that CD4+ regulatory T cells proliferated weakly and expressed low levels of IFN-gamma, IL-10, and IL-2. CD4+CD25+ cell depletion increased IL-2 production by CD4+CD25- thymic cells, but not splenic cells. Moreover, tolerance was transferable with splenic and thymic CD4+CD25+ cells, but also in 50% of cases with splenic CD4+CD25- cells, demonstrating that CD25 can be a marker for regulatory cells in the thymus, but not in the periphery. In addition, we presented evidences that donor APCs were required to induce tolerance and to expand regulatory CD4+ T cells. This study demonstrates that LF15-0195 treatment induces donor APCs to expand powerful regulatory CD4+CD25+/- T cells present in both the central and peripheral compartments.
 
Both V 8.2 and V 8.2 MBP-specific CD4 T cells from LF-protected rats have a reduced ability to transfer EAE. MBP-specific T cell lines were generated from MBP-immunized and LF 15-0195-treated rats (LFMBP lines) or from control, MBP-immunized, untreated rats (MBP lines) after two rounds of stimulation with MBP, APCs, and rat IL-2. T cells among these lines were separated according to their expression of V8.2 TCR using FITC-conjugated R78 mAb and anti-FITC magnetic beads. A, The results are depicted as histograms of V8.2 expression by a representative MBP-specific line before (left panel) and after fractionation into V8.2 (middle panel) and V8.2 (right panel) T cells. The percentage of V8.2 T cell subsets is indicated and represents the purity of each cell population. B-D, Naive syngeneic LEW recipients were i.v. injected with 0.5 10 6 viable V8.2 T cells (B, n 7 for MBP lines and n 6 for LFMBP lines) or with 0.5 10 6 (C, n 4) or 3 10 6 (D, n 4) V8.2 T cells purified from the LFMBP lines () or MBP lines (f). The results are expressed as the mean daily clinical score of each experimental group (upper panels) and as the mean cumulative disease score of each experimental group (lower panels). Results represent the pooled data of two independent experiments. , p 0.05; , p 0.01.
MBP-specific CD4 T cells from LF-protected rats overexpress Foxp3 and have a particular pattern of chemokine receptors. MBP-specific T cell lines were generated from MBP-immunized and LF 15-0195-treated rats (LFMBP lines, n 3, ) or from control, MBP-immunized, untreated rats (MBP lines, n 3, f) rats after four rounds of stimulation with MBP, APCs, and rat IL-2 as described in Materials and Methods. Both cell lines were tested for expression of regulatory T cell markers (A) and chemokine receptors (B) by quantitative RT-PCR. The results are relative to HPRT and expressed in AU, where the 1 value represents the mean of the MBP lines. Results are representative of three independent experiments. , p 0.01
Article
Experimental autoimmune encephalomyelitis (EAE) is an instructive model for the human demyelinating disease multiple sclerosis. Lewis (LEW) rats immunized with myelin-basic protein (MBP) develop EAE characterized by a single episode of paralysis, from which they recover spontaneously and become refractory to a second induction of disease. LF 15-0195 is a novel molecule that has potent immunosuppressive effects in several immune-mediated pathological manifestations, including EAE. In the present study, we show that a 30-day course of LF 15-0195 treatment not only prevents MBP-immunized LEW rats from developing EAE but also preserves their refractory phase to reinduction of disease. This effect is Ag driven since it requires priming by the autoantigen during the drug administration. In contrast to other immunosuppressive drugs, short-term treatment with this drug induces a persistent tolerance with no rebound of EAE up to 4 mo after treatment withdrawal. This beneficial effect of LF 15-0195 on EAE does not result from the deletion of MBP-specific Vbeta8.2 encephalitogenic T cells. In contrast, this drug favors the differentiation of MBP-specific CD4 T cells into Foxp3-expressing regulatory T cells that, upon adoptive transfer in syngeneic recipients, prevent the development of actively induced EAE. Finally, we demonstrate that the tolerance induced by LF 15-0195 treatment is not dependent on the presence of TGF-beta. Together, these data demonstrate that short-term treatment with LF 15-0195 prevents MBP-immunized LEW rats from EAE by favoring the development of Foxp-3-expressing regulatory CD4 T cells.
 
Article
MHC class I-restricted CD8+ T cells play an important role in controlling HIV and SIV replication. In SIV-infected Indian rhesus macaques (Macaca mulatta), comprehensive CD8+ T cell epitope identification has only been undertaken for two alleles, Mamu-A*01 and Mamu-B*17. As a result, these two molecules account for virtually all known MHC class I-restricted SIV-derived CD8+ T cell epitopes. SIV pathogenesis research and vaccine testing have intensified the demand for epitopes restricted by additional MHC class I alleles due to the shortage of Mamu-A*01+ animals. Mamu-A*02 is a high frequency allele present in over 20% of macaques. In this study, we characterized the peptide binding of Mamu-A*02 using a panel of single amino acid substitution analogues and a library of 497 unrelated peptides. Of 230 SIVmac239 peptides that fit the Mamu-A*02 peptide-binding motif, 75 peptides bound Mamu-A*02 with IC50 values of < or = 500 nM. We assessed the antigenicity of these 75 peptides using an IFN-gamma ELISPOT assay with freshly isolated PBMC from eight Mamu-A*02+ SIV-infected macaques and identified 17 new epitopes for Mamu-A*02. The synthesis of five Mamu-A*02 tetramers demonstrated the discrepancy between tetramer binding and IFN-gamma secretion by SIV-specific CD8+ T cells during chronic SIV infection. Bulk sequencing determined that 2 of the 17 epitopes accumulated amino acid replacements in SIV-infected macaques by the chronic phase of infection, suggestive of CD8+ T cell escape in vivo. This work enhances the use of the SIV-infected macaque model for HIV and increases our understanding of the breadth of CD8+ T cell responses in SIV infection.
 
Allogeneic HLA-A*0201–WT1 tetramer-positive T cell clones recognize HLA-A*0201–positive targets even in the absence of exogenously loaded WT1 peptide. Multiple HLA-A*0201–WT1 tetramer-positive CD8 + T cell clones were isolated from healthy HLA-A*02–negative donors using three different strategies: 1) stimulation with peptide-loaded allogeneic HLA-A*0201–positive DCs (HD-allo-D); 2) stimulation with peptide-loaded HLA- A*0201–transduced autologous EBV-LCL cells (HD-allo-C), and 3) by direct isolation using HLA-A*0201–WT1 tetramers (HD-allo-A). A, Representative HLA-A*0201–WT1 tetramer stainings of WT1-reactive CD8 + T cell clones. Absence of staining with HLA-A*0201–CMVpp65 tetramer is shown as negative control. B, Cytotoxic activity of WT1-reactive CD8 + T cell clones against TAP-deficient T2 cells and HLA-A*0201–positive EBV-LCL cells in the absence (white bars) or presence (black bars) of exogenously loaded WT1 peptide (1 mM). Cytotoxicity was measured in standard 5-h 51 Cr release assays at an E:T ratio of 3:1 (n = 5, bars show mean percentages of lysis; error bars represent SDs). C, IFN-g (IFNg) production by WT1-reactive CD8 + T cell clones after stimulation with T2 cells or HLA-A*0201–positive EBV-LCL cells loaded with 0 (white bars), 10 nM (light gray bars), 0.1 mM (gray bars), 1 mM (dark gray bars), or 10 mM (black bars) exogenously loaded WT1 peptide is shown at an E:T ratio of 1:3. No IFN-g was produced in the absence of stimulation (NS). n = 5 (the result of one representative experiment is shown). D, IFN-g production by WT1-reactive CD8 + T cell clones after stimulation with HLA-A*0201–positive EBV-LCL cells, PBMCs, PHA blasts, CD34 + precursor cells, or primary human fibroblasts that were either unpulsed (white bars) or pulsed with WT1 126–134 peptide at a concentration of 1 mM (black bars) (E:T ratio 1:3). ND, not done.  
HLA-A*0201–WT1 tetramer-positive T cell clones isolated from HLA-A*0201– positive donors and patients exhibit low functional avidities. Multiple HLA-A*0201–WT1 tetramer-positive CD8 + T cell clones were isolated from HLA-A*0201–positive donors (HDauto ) and an HLA-A*02–positive patient after in vivo WT1 peptide vaccination (WT1-vacc.). A, Representative HLA-A*0201-WT1 tetramer stainings of WT1-reactive CD8 + T cell clones. Absence of staining with HLA-A*0201–CMVpp65 tetramer is shown as negative control. B, Cytotoxic activity of WT1-reactive CD8 + T cell clones against TAP-deficient T2 cells that were either unpulsed (white bars) or pulsed with WT1 126–134 peptide at concentrations of 100 nM (light gray bars), 1 mM (dark gray bars), or 10 mM (black bars). Cytotoxicity was measured in  
Differential reactivity of self-HLA and allo-HLA WT1- reactive CD8 + T cell clones against WT1–HLA-A*0201 complexes presented on artificial Ag-presenting beads. A, IFN-g (IFNg) production by HLA-A*0201–WT1 tetramer-positive CD8 + T cell clones isolated from HLA-A*0201–positive (HD-auto-2 [diamond]) and HLA-A*02–negative (HD-allo-C9 [gray circle], -C10 [gray square], -C12 [black circle], and -C102 [black square]) individuals is shown after stimulation with beads loaded with increasing densities of WT1–HLA-A*0201 complexes (cell/bead ratio 1:20). WT1–HLA-A*0201 complexes were titrated with CMVpp65–HLA-A*0201 complexes, making the total number of stabilized peptide–HLA-A*0201 monomers constant on all beads. Whereas clones HD-auto-2 and HD-allo-C10 and -102 showed profound reactivity against WT1–HLA-A*0201–coated artificial beads, clones HD-allo-C9 and -C12 only showed modest WT1-specific functional reactivity. B, As a control, a CMVpp65–HLA-A*0201–specific clone (black triangle) was tested against the same beads. Dose-dependent IFN-g production was observed in response to stimulation with beads coated with increasing densities of CMVpp65–HLA-A*0201 complexes (cell/bead ratio 1:20).  
Article
T cells recognizing tumor-associated Ags such as Wilms tumor protein (WT1) are thought to exert potent antitumor reactivity. However, no consistent high-avidity T cell responses have been demonstrated in vaccination studies with WT1 as target in cancer immunotherapy. The aim of this study was to investigate the possible role of negative thymic selection on the avidity and specificity of T cells directed against self-antigens. T cell clones directed against the HLA-A*0201-binding WT1(126-134) peptide were generated from both HLA-A*02-positive (self-HLA-restricted) and HLA-A*02-negative [nonself (allogeneic) HLA [allo-HLA]-restricted] individuals by direct ex vivo isolation using tetramers or after in vitro priming and selection. The functional avidity and specificity of these T cell clones was analyzed in-depth. Self-HLA-restricted WT1-specific clones only recognized WT1(126-134) with low avidities. In contrast, allo-HLA-restricted WT1 clones exhibited profound functional reactivity against a multitude of HLA-A*02-positive targets, even in the absence of exogenously loaded WT1 peptide, indicative of Ag-binding promiscuity. To characterize this potential promiscuity, reactivity of the T cell clones against 400 randomly selected HLA-A*0201-binding peptides was investigated. The self-HLA-restricted WT1-specific T cell clones only recognized the WT1 peptide. In contrast, the allo-HLA-restricted WT1-reactive clones recognized besides WT1 various other HLA-A*0201-binding peptides. In conclusion, allogeneic HLA-A*02-restricted WT1-specific T cells isolated from mismatched donors may be more tumor-reactive than their autologous counterparts but can show specific off-target promiscuity of potential clinical importance. As a result of this, administration of WT1-specific T cells generated from HLA-mismatched donors should be performed with appropriate precautions against potential off-target effects.
 
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Mariana Kaplan
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases
Gabriel Nunez
  • University of Michigan
Yoichiro Iwakura
  • The University of Tokyo
Jay Kolls
  • Tulane University
Nico Van Rooijen
  • Amsterdam University Medical Center