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Erratum to “A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens” : [J. Immunol. Methods, 230 (1999) 99–112]
... Several authors have recommended the use of CMFDA for labeling different cell types for long-term studies, which is easy to handle. It integrates into the cytoplasm of viable cells, independent from the cell cycle, and has been used in bone marrow derived stem cells [8] keratinocytes, myocytes and osteocytes [3], lymphocytes and U937 cells [4]. Despite these advantages, the duration of the labeling is not known and only few data are available on its use with chondrocytes. ...
... The longest lasting period for PKH 26 ® labeled cells in vivo was reported to be 4 months for neural cells post transplantation into the caudate putamen [9] . Other groups tracked labeled lymphocytes or peripheral blood mononuclear cells for shorter periods of time [3, 8, 10, 19], as well as neuronal precursors and neuronal cells [9, 23], endothelial cells [18] , lympho- cytes [20], L9292 cells [21] and hematopoetic stem cells In contrast to PKH 26 ® CMFDA labeled chondrocytes revealed fluorescence only until day 14, consistent with findings for myocytes, keratinocytes and osteocytes [3]. After that, no further fluorescence could be detected. ...
... For this purpose, we used CD40-activated donor-derived B cells instead of donor leukocytes from spleen or blood, as stimulator cells, and calculated frequencies of responding recipient T-cells from division patterns measured by flow cytometric analysis of carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye dilution. CFSE-dilution has a sensitivity similar to MHC-tetramer staining [27] [28,29,30], and CD40-B cells are a uniform source of professional APC [31,32,33,34] . The second aim was to determine the kinetics of the direct pathway allo-response after liver transplantation by applying this assay. ...
... Proliferation of T cells started at day 4, and new T cells were recruited to proliferate until day 5, thereafter PF reached a plateau value (Figure 3A). After day 6 PF increased again, which is probably due to non-specific bystander activation [27]. Therefore, we decided to determine PF at day 6 of culture. ...
Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donor-specific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigen-presenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3rd party CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and naïve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4+ and CD8+ T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4+ and CD8+ T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3rd party allo-antigens was observed. One year after LTx numbers of CD4+ and CD8+ T cells reacting to donor antigens, as well as those reacting to 3rd party allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4+ and CD8+ T cells responding to donor-derived, as well as those reacting to 3rd party CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays.
... Proliferative responses of lymphocytes were measured using a flow cytometry-based assay that utilises a fluorescent membrane dye (PKH26) that is partitioned between daughter cells at division. Analysis software (Modfit LT, Verity Software House, Topsham, [14] ME) was employed for mathematical deconvolution of daughter generations to provide a precursor frequency of responding cells in the parent population. The following antigens were tested in triplicates: SIVgp120, SIVgag p27 and HSP70, each at a final concentration of 5, 10 and 20 g/ml. ...
... It should be noted that both A3G mRNA and protein examined were significantly knocked down with the specific as compared with non-specific siRNA. A3G protein was also significantly increased by immunization as demonstrated in the CD4 + CD95 + memory T cells [14] by immunofluorescence. However, unlike the persistence of A3G expression in CD4 + CD95 + T cells up to 47 weeks, the proportion of and iliac lymph node CD4 T cells and in mononuclear cells eluted from the rectal and small intestinal mucosa at the termination of the experiment (week 47); mean (±sem) and p values are given for the difference in A3G mRNA between immunized (n = 15) and unimmunized macaques (n = 5). ...
APOBEC3G is an innate intracellular anti-viral factor which deaminates retroviral cytidine to uridine. In vivo studies of APOBEC3G (A3G) were carried out in rhesus macaques, following mucosal immunization with SIV antigens and CCR5 peptides, linked to the 70kDa heat shock protein. A progressive increase in A3G mRNA was elicited in PBMC after each immunization (p<0.0002 to p< or =0.02), which was maintained for at least 17 weeks. Analysis of memory T cells showed a significant increase in A3G mRNA and protein in CD4(+)CCR5(+) memory T cells in circulating (p=0.0001), splenic (p=0.0001), iliac lymph nodes (p=0.002) and rectal (p=0.01) cells of the immunized compared with unimmunized macaques. Mucosal challenge with SIVmac 251 showed a significant increase in A3G mRNA in the CD4(+)CCR5(+) circulating cells (p<0.01) and the draining iliac lymph node cells (p<0.05) in the immunized uninfected macaques, consistent with a protective effect exerted by A3G. The results suggest that mucosal immunization in a non-human primate can induce features of a memory response to an innate anti-viral factor in CCR5(+)CD4(+) memory and CD4(+)CD95(+)CCR7(-) effector memory T cells.
... Fluorescent dyes such as PKH26 and carboxy- Ž . fluorescein diacetate succinimidyl ester CFSE stain cellular membranes and cytoplasm, respectively, and eventually partition between dividing cells allowing the quantification of precursor frequencies of proliferating cells in response to antigenic stimulation ŽAllsopp et al., 1998; Givan et al., 1999; Noorchashm et al., 1999; Song et al., 1999; Wells et al., . 1997 . ...
... The potential loss of PKH26 dye from parental cells over time in culture may also compromise the separation of Ž . daughter generations Givan et al., 1999 . In this study, we describe one example of how the new method could be used to identify lymphocyte subsets responding to antigen stimulation. ...
One method for examining cell cycle kinetics by flow cytometry uses continuous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA fluorochrome Hoechst 33258. After counterstaining with a secondary DNA fluorochrome (e.g., ethidium bromide), the analyst can distinguish cells in different phases of the cell cycle over a number of mitotic cycles with flow cytometry. In this report, we describe a modification of the flow cytometric BrdU-Hoechst assay that allows combined analysis of cell proliferation and immunophenotyping at the single cell level. To demonstrate an application of this method, human peripheral blood mononuclear cells were stimulated with tetanus toxoid or interleukin-2 for up to 6 days in the presence of BrdU, harvested, and immunostained for the cell surface markers CD3, CD4, CD8, CD14, CD19, and the cytokine receptor, CCR5. We used four-color flow cytometry analyses to simultaneously measure cell proliferation and surface marker expression, for the purpose of immunophenotyping and identifying specific cell subsets responding to antigen stimulation. Our successful application of this method suggests that it may be used to study immune responses at the molecular and cellular level and to identify mechanisms of immune system modulation.
... While dual labelling has been widely used to track cell migration in animal models [52], its application in in vitro human T cell proliferation experiments has, to our knowledge , not previously been reported. Single-colour labelling of distinct human PBMC populations has been used to characterise, isolate, and clone peanut-allergen-specific T cells [53] and to determine precursor frequencies of recallantigen-specific T cells [54]. Measurement of proliferation by means of CFSE has the additional advantages of requiring relatively low numbers of cells and allowing additional phenotypic (cell surface markers) or functional parameters (intracellular cytokine secretion) to be studied in parallel, in distinct subpopulations [54]. ...
... Single-colour labelling of distinct human PBMC populations has been used to characterise, isolate, and clone peanut-allergen-specific T cells [53] and to determine precursor frequencies of recallantigen-specific T cells [54]. Measurement of proliferation by means of CFSE has the additional advantages of requiring relatively low numbers of cells and allowing additional phenotypic (cell surface markers) or functional parameters (intracellular cytokine secretion) to be studied in parallel, in distinct subpopulations [54]. In conclusion, our data indicate that low-dose PIT targets both CD4 þ and CD8 þ memory T cells and induces a population of active suppressor/regulatory T cells within the CD4 þ compartment. ...
Synthetic peptides, representing CD4(+) T cell epitopes, derived from the primary sequence of allergen molecules have been used to down-regulate allergic inflammation in sensitised individuals. Treatment of allergic diseases with peptides may offer substantial advantages over treatment with native allergen molecules because of the reduced potential for cross-linking IgE bound to the surface of mast cells and basophils.
In this study we address the mechanism of action of peptide immunotherapy (PIT) in cat-allergic, asthmatic patients. Cell-division-tracking dyes, cell-mixing experiments, surface phenotyping, and cytokine measurements were used to investigate immunomodulation in peripheral blood mononuclear cells (PBMCs) after therapy. Proliferative responses of PBMCs to allergen extract were significantly reduced after PIT. This was associated with modified cytokine profiles generally characterised by an increase in interleukin-10 and a decrease in interleukin-5 production. CD4(+) cells isolated after PIT were able to actively suppress allergen-specific proliferative responses of pretreatment CD4(neg) PBMCs in co-culture experiments. PIT was associated with a significant increase in surface expression of CD5 on both CD4(+) and CD8(+) PBMCs.
This study provides evidence for the induction of a population of CD4(+) T cells with suppressor/regulatory activity following PIT. Furthermore, up-regulation of cell surface levels of CD5 may contribute to reduced reactivity to allergen.
... The amount of CFSE fluorescence per cell, and surface labelling of CD4 and CD19 was measured using a FACScalibur flow cytometer (Becton Dickinson) and CELLQuesti software. Precursor frequency and proliferation index were calculated using Modfit LT 3.0 (Verity Software) (Givan et al., 1999). ...
... Proliferation was measured by CFSE dilution. The precursor frequency (defined as the proportion of lymphocytes that leave the original parent population to undergo at least two cell divisions) (Givan et al., 1999; Lyons et al., 2001) of the lymphocyte population was measured in patients and controls. There was a nonsignificant trend for lymphocytes taken within 72 h of TBI to have a lower precursor frequency compared to the later time point and that measured for controls (Fig. 2). ...
Murine models of CNS injury show auto-reactive T cell responses directed at myelin antigens, associated with improved neuronal survival and functional recovery. This pilot study shows, for the first time, that similar immune responses against myelin occur in human traumatic brain injury (TBI), with an expansion of lymphocytes recognising myelin basic protein observed in 40% of patients studied. "Reactive" patients did not have greater contusion volume on imaging, but were younger than the "unreactive" subgroup and tended towards a more favorable outcome. These findings are consistent with the concept of "beneficial autoimmunity".
... The state of the population at time t is described by the distribution (density) function n(t, x)(cell/UI), so that the number of cells with the CFSE intensity between x 1 and x 2 is given by At the beginning of the follow-up experiment, the lymphocyte population is stained with CFSE giving rise to the initial (starting) distribution of cells with respect to the CFSE fluorescence. The following phenomenological features of the label-structured lymphocyte proliferation have to be taken into account by the model for the dynamics of the distribution of labelled cells (56723]): ...
... The corresponding computer-based procedures require setting of the spacing between generations , i.e., marking the CFSE fluorescence intensities that separate consecutive generations of dividing cells. Note that when the starting population of cells exhibits a broad range of CFSE fluorescence, the division peaks can be not easily identifiable, making conventional division tracking analysis problematic [3,23,25]. The number of divisions which can be followed is limited by the autofluorescence of unlabelled cells. ...
The flow cytometry analysis of CFSE-labelled cells is currently one of the most informative experimental techniques for studying cell proliferation in immunology. The quantitative interpretation and understanding of such heterogenous cell population data requires the development of distributed parameter mathematical models and computational techniques for data assimilation.
The mathematical modelling of label-structured cell population dynamics leads to a hyperbolic partial differential equation in one space variable. The model contains fundamental parameters of cell turnover and label dilution that need to be estimated from the flow cytometry data on the kinetics of the CFSE label distribution. To this end a maximum likelihood approach is used. The Lax-Wendroff method is used to solve the corresponding initial-boundary value problem for the model equation. By fitting two original experimental data sets with the model we show its biological consistency and potential for quantitative characterization of the cell division and death rates, treated as continuous functions of the CFSE expression level.
Once the initial distribution of the proliferating cell population with respect to the CFSE intensity is given, the distributed parameter modelling allows one to work directly with the histograms of the CFSE fluorescence without the need to specify the marker ranges. The label-structured model and the elaborated computational approach establish a quantitative basis for more informative interpretation of the flow cytometry CFSE systems.
... On the 3 rd day of culture, the cells were harvested and stained with anti-CD4-Pacific blue and whole cells were acquired for analysis by using WinList software (Verity, Topsham ME, USA). Precursor frequency (Pf) was estimated for cells exclusively gated for CD4 + live cells according to the scattering characteristics and using the " Proliferation Wizard " setting of ModFit software (Verity), as described elsewhere [34]. ...
The suppressive function of regulatory T cells (Tregs) is critical to the maintenance of immune homeostasis in vivo and yet, the specific identification of Tregs by phenotypic markers is not perfect. Tregs were originally identified in the CD4+CD25+ fraction of T cells, but FoxP3 expression was later included as an additional marker of Tregs as FoxP3 expression was identified as being critical to the development and function of these cells. Intracellular expression of FoxP3 makes it difficult in using to isolate live and not permeabilized cells for functional assays. As such CD4+CD25+ fraction is still frequently used for functional assays of Tregs. Although, the CD4+CD25+ fraction substantially overlaps with the FoxP3+ fraction, the minor mismatch between CD4+CD25+ and FoxP3+ fractions may confound the functional characteristics of Tregs. In this study, we isolated CD4+FoxP3+ as well as CD4+CD25+ fractions from Foxp3 knock-in mice, and compared their proliferative and suppressive activity in the presence or absence of various concentrations of IL-2. Our results showed comparable patterns of proliferative and suppressive responses for both fractions, except that contrary to the CD4+CD25+ fraction the FoxP3+ fraction did not proliferate in an autocrine fashion even in response to a strong stimulation. In presence of exogenous IL-2, both CD4+CD25+ and CD4+FoxP3+ fractions were more sensitive than the CD4+CD25- responder cells in proliferative responsiveness. In addition, a low dose IL-2 enhanced whereas a high dose abrogated the suppressive activities of the CD4+CD25+ and CD4+FoxP3+ fractions. These results may provide an additional understanding of the characteristics of the various fractions of isolated Tregs based on phenotype and function and the role of varying levels of exogenous IL-2 on the suppressive activity of these cells.
... Comparison of proliferative responses of lymphocytes to TGN1412 air-dried or wet-coated onto polypropylene and polystyrene microtitre plates Proliferative responses of human lymphocytes were measured using a flow cytometry-based assay that uses a fluorescent membrane dye (PKH26) that was partitioned between daughter cells at division. The assay has been described previously (Givan et al., 1999; Stebbings et al., 2007). TGN1412 and an isotype-matched negative control were immobilised by coating onto the walls of wells of polypropylene and polystyrene plates as described above. ...
... Comparison of proliferative responses of lymphocytes to TGN1412 air-dried or wet-coated onto polypropylene and polystyrene microtitre plates Proliferative responses of human lymphocytes were measured using a flow cytometry-based assay that uses a fluorescent membrane dye (PKH26) that was partitioned between daughter cells at division. The assay has been described previously (Givan et al., 1999; Stebbings et al., 2007). TGN1412 and an isotype-matched negative control were immobilised by coating onto the walls of wells of polypropylene and polystyrene plates as described above. ...
In 2006 a near-fatal “cytokine storm” occurred in six healthy volunteers during the phase I clinical trial of TGN1412, a therapeutic superagonistic CD28-specific monoclonal antibody, signalling a failure of pre-clinical safety testing. Subsequently it was established that TGN1412 could stimulate a “cytokine storm” in vitro from human white blood cells but only if presented to the cells by immobilisation onto plastic or if the white blood cells were cultured over a monolayer of human endothelial cells. Data from the novel in vitro procedures suggests that the dose of TGN1412 given to the volunteers was close to the maximum immunostimulatory dose. In contrast to human white blood cells, TGN1412 was found not to be a superagonist in vivo or in vitro for white blood cells from the non-human primates used in the pre-clinical testing. The novel procedures are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system.
... If the characteristics of the logarithmic amplifier on the flow cytometer are known, it is possible to derive, from a histogram of fluorescence intensity, the proportion of cells that have undergone any particular number of divisions (Givan et al., 1999). Generally, a flow cytometer with 1024 channels (channels range) represents a nominal range of 4 log-decades. ...
Herein we introduce flowFit, a Bioconductor package designed to perform quantitative analysis of cell proliferation in tracking dye-based experiments. The software, distributed as an R Bioconductor library, is based on a mathematical model that takes into account the height of each peak, the size and position of the parental population (labeled but not proliferating) and the estimated distance between the brightness of a cell and the brightness of its daughter (in which the dye is assumed to undergo a twofold dilution). While the algorithm does not make any inference on cell types, rates of cell divisions, or rates of cell death, it deconvolutes the actually collected data into a set of peaks, whereby each peak corresponds to a subpopulation of cells that have divided N times. We validated flowFit by retrospective analysis of published proliferation tracking experiments (Quah and Parish, 2012) and demonstrated that the algorithm predicts the same percentage of cells/generation either in samples with discernible peaks (in which the peaks are visible in the collected raw data) or in samples with non discernible peaks (in which the peaks are fused together). To the best of our knowledge, flowFit represents the first open source algorithm in its category and might be applied to numerous areas of cell biology in which quantitative deconvolution of tracking dye-based experiments is desired, including stem cell research.
http://www.bioconductor.org/packages/devel/bioc/html/flowFit.html(Bioconductor software page).http://www.bioconductor.org/packages/2.13/bioc/vignettes/flowFit/inst/doc/HowTo-flowFit.pdf (package vignette)http://rpubs.com/tucano/flowFit (online tutorial).
... Using PBMC, 10d cultures were0 1 0 2 10 3 10 4 10 5 0 200 400 600 800 1000 0 1 0 2 10 3 10 4 10 5 0 500 1000 1500 2000 0 1 0 2 10 3 10 4 10 5 0 500 1000 1500 2000 0 1 0 2 10 3 10 4 10 5 0 200 400 600 800 0 1 0 2 10 3 10 4 10 5 0 500 1000 1500 0 1 0 2 10 3 10 4 10 5 0 100 200 300 400 500 0 1 0 2 10 3 10 4 10 5 0 500 1000 1500 0 1 0 2 10 3 10 4 10 5 0 500 1000 1500 2000 0 1 0 2 10 3 10 4 10 5 0 500 1000 1500 0 1 0 2 10 3 10 4 10 5 0 300 600 900 1200 0 1 0 2 10 3 10 4 10 5 0 500 1000 1500 0 1 0 2 10 3 10 4 10 5 0 300 600 900 used to observe optimal proliferation; in such prolonged proliferation experiments, both the media control (data not shown) as well as the allergen activated cultures yielded 2 CTV bright " negative " peaks (Figure 1A, lower right hand quadrant). Using 12d antigen activated cultures, similar doublet " negative " peaks were described by Givan and Wallace [21,22] and are thought to be an artifact due to homeostatic proliferation during the prolonged culture. PBMC proliferation assays were performed using complete media containing 5% heat inactivated human AB serum (Invitrogen). ...
Th2 cytokine responses are enhanced by all trans retinoic acid (ATRA), the bioavailable form of vitamin A. Retinoic acid receptor alpha (RARalpha) is the high affinity receptor for ATRA that mediates these pro-Th2 effects. We have previously characterized two major human Th2 subpopulations: IL-5- Th2 (IL-5-, IL-4+, IL-13+) and IL-5+ Th2 cells (IL-5+, IL-4+, IL-13+), which represent less and more highly differentiated Th2 cells, respectively. We hypothesized that the pro-Th2 effects of ATRA may differentially affect these Th2 subpopulations.
Specific cytokine producing Th2 subpopulations were identified using intracellular cytokine staining. Proliferation was measured using the Cell Trace Violet proliferation tracking dye. Apoptotic cells were identified using either annexin-V or active caspase 3 staining. Th2 gene expression was measured using quantitative polymerase chain reaction.
ATRA increased the output of Th2 cells from house dust mite allergen (HDM) specific short-term cell lines, and this enhancement was limited to the IL-5+ Th2 subpopulation. Conversely, the RARalpha antagonist Ro415253 decreased Th2 cell output from these cultures, and this effect was again limited to the IL-5+ Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited Th2 cell proliferation, and this affect was more pronounced for the IL-5+ vs. IL-5- Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited the expression of IL5 in a significant manner, which was not found for IL4 or IL13.
We report that the reciprocal regulation of Th2 cytokine expression and proliferation by RARalpha modulators are largely limited to modulation of IL-5 gene expression and to proliferation of the highly differentiated IL-5+ Th2 subpopulation. These results suggest that RARalpha antagonism is a potential means to therapeutically target allergic inflammation.Trial registration: Clinicaltrials.gov identifier: NCT01212016.
... After 8 d, CFSE staining was measured in combination with CD19-APC (BD) and CD5-PE (BD) by flow cytometry. Precursor frequency, defined as the proportion of cells that have responded in the original population, was calculated by FlowJo software as described previously (Givan et al., 1999). The precursor frequency of cells cultured with CD40L expressing fibroblasts was subtracted as background. ...
B cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is a clonal expansion of CD5+CD19+ B lymphocytes. Two types of CLLs are being distinguished as carrying either unmutated or somatically mutated immunoglobulins
(Igs), which are associated with unfavorable and favorable prognoses, respectively. More than 30% of CLLs can be grouped based
on their expression of stereotypic B cell receptors (BCRs), strongly suggesting that distinctive antigens are involved in
the development of CLL. Unmutated CLLs, carrying Ig heavy chain variable (IGHV) genes in germline configuration, express low-affinity, poly-, and self-reactive BCRs. However, the antigenic specificity
of CLLs with mutated IGHV-genes (M-CLL) remained elusive. In this study, we describe a new subset of M-CLL, expressing stereotypic BCRs highly specific
for β-(1,6)-glucan, a major antigenic determinant of yeasts and filamentous fungi. β-(1,6)-glucan binding depended on both
the stereotypic Ig heavy and light chains, as well as on a distinct amino acid in the IGHV-CDR3. Reversion of IGHV mutations to germline configuration reduced the affinity for β-(1,6)-glucan, indicating that these BCRs are indeed affinity-selected
for their cognate antigen. Moreover, CLL cells expressing these stereotypic receptors proliferate in response to β-(1,6)-glucan.
This study establishes a class of common pathogens as functional ligands for a subset of somatically mutated human B cell
lymphomas.
... potential for clonal expansion) of antigenstimulated T-cells is directly measured. Alternative methods, other than tritiated thymidine incorporation, have been proposed in order to avoid the use of a radioactive reagent (Givan et al., 1999; Lyons, 2000; Lyons and Doherty, 2004; Wallace et al., 2008). The number of peripheral blood mononuclear cells (PBMC) needed to run a conventional LPA in 96-well plates can be a limiting factor as frequently experienced in the clinical immunology laboratory. ...
Lymphoproliferation assay (LPA) is used to test specific T-cell responses. LPA is performed in 96-well plates with 2-5×10⁵ PBMC/well. In order to test numerous antigens, as in the case of epitope mapping or screening of antigenic panels from relevant pathogens, PBMC numbers may not be sufficient. We developed a miniaturized and automated procedure to perform LPA in 384- and 1536-well plates with one fourth to one twentieth of PBMC numbers used for standard assays. Here, we demonstrate that the procedure is reliable and robust using recall antigens and protein and peptide antigens from CMV and HIV. By using HIV specific T-cell lines, we also demonstrate that sensitivity ranges overlap with those of standard LPA and that as few as 3 specific cells/well provide a positive signal. This procedure is consistent with our policy to miniaturize assays for specific T-cell immunity, as we have already established for cytokine secretion assays.
... If the TCR specifically recognizes the antigen as foreign, these interactions will activate the tyrosine kinase Lck (associated with co-receptors CD4 and CD8), which in turn will activate downstream cell signaling resulting in activation of transcription factors to induce T cell proliferation and differentiation (Garvin et al., 2009). As TCRs are extraordinarily diverse molecules, only ∼0.01% of naïve T cells will recognize a given antigen (Givan et al., 1999). SAg-mediated T cell activation is both quantitatively and qualitatively distinct from conventional T cell activation (Bueno et al., 2007). ...
Superantigens (SAgs) are a family of potent immunostimulatory exotoxins known to be produced by only a few bacterial pathogens, including Staphylococcus aureus. More than 20 distinct SAgs have been characterized from different S. aureus strains and at least 80% of clinical strains harbor at least one SAg gene, although most strains encode many. SAgs have been classically associated with food poisoning and toxic shock syndrome (TSS), for which these toxins are the causative agent. TSS is a potentially fatal disease whereby SAg-mediated activation of T cells results in overproduction of cytokines and results in systemic inflammation and shock. Numerous studies have also shown a possible role for SAgs in other diseases such as Kawasaki disease (KD), atopic dermatitis (AD), and chronic rhinosinusitis (CRS). There is also now a rich understanding of the mechanisms of action of SAgs, as well as their structures and function. However, we have yet to discover what purpose SAgs play in the life cycle of S. aureus, and why such a wide array of these toxins exists. This review will focus on recent developments within the SAg field in terms of the molecular biology of these toxins and their role in both colonization and disease.
... Comparison of proliferative responses of lymphocytes to TGN1412 air-dried or wet-coated onto polypropylene and polystyrene microtitre plates Proliferative responses of human lymphocytes were measured using a flow cytometry-based assay that uses a fluorescent membrane dye (PKH26) that was partitioned between daughter cells at division. The assay has been described previously (Givan et al., 1999; Stebbings et al., 2007). TGN1412 and an isotype-matched negative control were immobilised by coating onto the walls of wells of polypropylene and polystyrene plates as described above. ...
TGN1412 is a "superagonistic" CD28 monoclonal antibody (IgG4) that caused serious adverse events at its first time in human clinical trial. In the present study, different in vitro methods for detecting and quantifying unwanted pro-inflammatory activity of therapeutic monoclonal antibodies (mAbs) such as TGN1412 are described. The antibody of interest is immobilised by wet-coating or air-drying onto polypropylene or polystyrene 96-well plates prior to the addition of human peripheral blood mononuclear cells (PBMCs). The cells are incubated for 16-24h with the immobilised antibody which allows the accumulation of pro-inflammatory cytokines, quantified by enzyme-linked immunoabsorbent assay (ELISA), in response to the antibody. Cytokine responses stimulated by TGN1412 immobilised by air-drying onto polypropylene and polystyrene plates were much larger than responses to TGN1412 wet-coated onto polypropylene and polystyrene plates, respectively. In additional experiments with other mAbs associated with clinical reactions, air-dried mAbs stimulated larger tumour necrosis factor-alpha (TNF) responses than antibodies added in aqueous phase. Also, TGN1412 air-dried onto plastic plates stimulated large proliferative responses of 3-day cultures of lymphocytes. It was concluded that immobilising mAbs by air-drying offers a useful in vitro method for detecting and quantifying pro-inflammatory activities of therapeutic mAbs.
... Modern techniques allow for simultaneous analyses of peptide-specific proliferative and synthetic activities by cytotoxic and regulatory lymphocytes, enabling to estimate not only the extent but also the direction of the immune response. Furthermore, multimer techniques , in HLA compatible individuals, provide demonstration of response at the very molecular level, allowing for the purest of quantitative data, the absolute count of lymphocytes mounting the peptide-specific TCR [21]. With these additional data, the present study enlightens both mechanisms and extent of response and immune safety issues. ...
Safety and efficacy of adjuvanted vaccines in autoimmune individuals raises growing clinical and scientific interest. Protection from influenza would bring particular benefits in these patients with common cardiac and respiratory impairment. This study evaluates efficacy, clinical safety and immune effects of the administration of a single dose of a virosomal flu vaccine in 46 scleroderma patients. The following parameters were evaluated before and after administration of Inflexal: clinical conditions, inflammation and autoimmunity parameters, humoral response, lymphocyte proliferation and cytokine production upon flu antigen stimulation by specific and non-specific cells. Inflexal was found effective in scleroderma patients. In no subject was worsening of clinical conditions, inflammation and immunological parameters observed.
... Doublecolor analysis was performed with CFSE and anti-CD8-PEcy5 using a FACSCalibur (Becton Dickinson). Flow cytometric data files were analyzed with the " Proliferation Wizard " module in ModFit LT Macintosh software [29, 30]. ...
The graft-versus-leukemia effect of allogeneic marrow transplantation suggests the dramatic effect of the allogeneic T cell to eradicate malignant disease. Preparation and adoptive transfusion of tumor-specific T cells from HLA-mismatched donors might be expected to circumvent CTL tolerance to the tumor. In this study, a soluble, divalent HLA-A2 molecule was constructed with the Fc part of human IgG1 and was pulsed with a peptide related to melanoma tyrosinase 368-376 [Tyr(368-376) (Tyr)] to form the Tyr/HLA-A2 dimer, which allowed loading onto monocytes via interaction of the Fc and FcR. The HLA-A2-negative (HLA-A2-ve) monocytes loaded with the Tyr/HLA-A2 dimer acted as allo-APC with copies of a single allogeneic epitope. After coculture of the HLA-A2-ve PBLs and autologous monocytes loaded with the dimer, CD8+ cells in the coculture show an obvious proliferation and increased frequency of Tyr/HLA-A2 tetramer-stained cells. The sorted Tyr/HLA-A2 tetramer-positive CD8+ cells display an elevated cytotoxic activity against HLA-A2-positive melanoma cells expressing tyrosinase endogenously (i.e., SK-Mel-5) but little against tyrosinase-negative melanoma cells (i.e., A375). The coculture of PBLs and autologous monocytes loaded with allogeneic peptide/HLA complexes offers a novel approach to expand allo-restricted, peptide-specific CTLs, which might be a potential arsenal for treatment of patients with malignant disease, if the tumor-related epitope were defined.
... The spacing value was automatically computed by the program, based on the number of decades and the assumption that each generation has one-half the intensity of the previous generation. With a four-decade log amplifier (and resolution in the software of 0–255 channels), every halving of intensity resulted in a decrease by 19AE19 channels (Givan et al, 1999). The ModFit software deconvolutes the fluorescence intensity histogram with Gaussian distributions centred on peaks at 19AE19 channel intervals. ...
In acute myeloid leukaemia (AML), cell kinetic quiescence has been postulated to contribute to drug resistance. As the anti-apoptotic genes Bcl-2 and Bcl-X(L) have been implicated in cell cycle regulation, we investigated the expression of these genes in non-proliferating (Q) and proliferating (P) AML and normal CD34+ progenitor cells. Using reverse transcription polymerase chain reaction, Bcl-X(L) and Bcl-2 were overexpressed in Q versus P AML cells, whereas no difference in Bcl-XS and Bax expression was found. Furthermore, the Bcl-X(L)/X(S) but not the Bcl-2/Bax ratio was higher in Q AML compared with normal CD34+ Q cells (P = 0.001). An inverse correlation between Bcl-2 expression of leukaemic Q cells and their ability to enter the cell cycle was found. Treatment with all-trans retinoic acid (ATRA) reduced Bcl-2 and Bcl-X(L) expression in the leukaemic Q cells, and enhanced their chemosensitivity to cytosine arabinoside (ara-C). These findings demonstrate overexpression of the anti-apoptotic proteins Bcl-X(L) and Bcl-2 in quiescent CD34+ AML cells and suggest their involvement in the chemoresistance. The observed inverse correlation between Bcl-2 and proliferation suggests a role for Bcl-2 in the cell cycle regulation of AML. These findings could be used in the development of therapies that selectively induce apoptosis in quiescent leukaemic progenitor cells.
... To evaluate the induction and function of PSAspecific CD4 þ and CD8 þ T cell subsets over the course of treatment, a dye dilution proliferation assay (DDPA) was performed [12]. We have previously published data that demonstrates the utilitiy and comparability of DDPA to ELISPOT and tetramer methods of determining T cell precursors. ...
Granulocyte monocyte-colony stimulating factor (GM-CSF) supports the survival, expansion, and differentiation of lymphoid and myeloid derived dendritic cells (DCs). We hypothesized that systemic therapy with GM-CSF in prostate cancer patients could augment prostate cancer-related immunity and induce clinical response.
Eligible patients were randomly assigned to receive either 125 or 250 microg/m(2) GM-CSF subcutaneously three times a week until clinical progression. Prostate-specific antigen (PSA) T cell precursor frequencies were determined by a flow cytometric method.
We were able to show, for the first time, a statistically significant correlation between pre-treatment PSA level and PSA-specific CD4(+) T cell precursors and a trend between pre-treatment PSA level and PSA-specific CD8(+) T cell precursors (P<0.0001 and P=0.059, respectively).
These results suggest that existent immunity to PSA in prostate cancer patients may be a promising target for future immunotherapeutic approaches to prostate cancer.
Background
Fluorescence-labeled MHC class II/peptide tetramer complexes are considered as optimal tools to characterize allergen-specific CD4+ T cells, but this technique is restricted to frequently expressed HLA-class II molecules and the knowledge of immunodominant epitopes. In contrast, allergen-stimulated proliferation assessed by CFSE-dilution is less sophisticated and widely applicable. The major mugwort allergen, Art v 1, contains only one single, immunodominant, HLA-DR1-restricted epitope (Art v 125-36). Thus, essentially all Art v 1-reactive cells should be identified by a HLA-DRB1*01:01/Art v 119-36 tetramer.Methods
We compared specificity and sensitivity of tetramer+ and allergen-induced proliferating (CFSElo) CD4+T cells by flow cytometry.ResultsThe frequency of tetramer+CD4+ T cells determined ex vivo in PBMC of mugwort-allergic individuals ranged from 0 to 0.029%. After 2-3 weeks of in vitro expansion, sufficient tetramer+ T cells for phenotyping were detected in 83% of Art v 125-36-reactive T cell lines (TCL) from mugwort-allergic individuals, but not in TCL from healthy individuals. The tetramers defined bona fide Th2 cells. Notably, Art v 125-36-reactive TCL depleted of tetramer+ T cells still reacted to the peptide and only 44% Art v 125-36-specific T cell clones were detected by the tetramer. CFSElo CD4+ T cells contained only 0.3-10.7% of tetramer+ T cells and very low proportions of Th2 cells.Conclusion
Allergen-specific T cells can be identified by HLA-class II tetramers with high specificity, but unexpected low sensitivity. In contrast, allergen-stimulated CFSElo CD4+ T cells contain extremely high fractions of bystander cells. Therefore, for T cell monitoring either method should be interpreted with caution.This article is protected by copyright. All rights reserved.
Background: Cellular responses to HIV-1 play a critical role in determining the course of infection. We used a flow cytometric assay based on PKH26 dilution to quantitatively evaluate the proliferation of CD4 and CD8 T-cell subsets in a cohort of HIV-1-infected patients.
Carboxyfluorescein diacetate succinimidyl ester (CFSE) is currently used to investigate migration and proliferation of hemopoietic cells. In principle, CFSE is retained by the cells and is shared by the daughter cells at each division, resulting in multimodal flow cytometric CFSE histograms, with each cell generation clustering around half the fluorescence intensity of the previous one. However, intercell variability of CFSE loading results in overlapping peaks, thereby limiting its use with cancer cell lines.
We used IGROV1 ovarian cancer cells loaded with CFSE at the time of seeding; 24 h later cells were treated with an anticancer drug (topotecan). Potential pitfalls of the analysis were examined, and a procedure of evaluation of CFSE efflux was applied to fix the peak positions with good approximation in advance. Histograms were fitted by a series of gaussians, with each representing cells in a given generation.
Effects of topotecan on IGROV1 cells were analyzed in terms of the time course of the percentage of cells that remained undivided or entered the second, third, and subsequent division cycles. A simple algorithm, which combined flow cytometric data with the absolute cell number independently measured by Coulter counter, provided an estimate of the 96-h outcome of the starting cell population by quantifying cells that remained undivided, those able to divide at least once, or those that had died.
We assessed experimental and data analytic procedures for a CFSE-based measurement of antiproliferative activity of drugs in cancer cell lines. A quantitative level was achievable but required a strict procedure for control of the experimental data, which was not straightforward.
In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8+ T cells. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in the T-cell pool that are able to bind tetramer, synthesize cytokines, undergo antigen-driven proliferation, and/or carry out various combinations of these functional responses.
Analysis of antigen-specific proliferative responses represents just one of many functions that can be monitored using cell tracking dyes and flow cytometry. In this third edition, we address issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T-cell functions. We summarize key characteristics of and differences between general protein- and membrane-labeling dyes, discuss determination of optimal staining concentrations, and provide detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking are provided in the form of protocols for (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay and (b) simultaneous monitoring of proliferative responses in effector and regulatory T cells.
To determine whether an autologous dendritic cell (DC) vaccine could induce antitumor immune responses in patients after resection of colorectal cancer metastases and whether these responses could be enhanced by activating DCs with CD40L.
Twenty-six patients who had undergone resection of colorectal metastases were treated with intranodal injections of an autologous tumor lysate- and control protein [keyhole limpet hemocyanin (KLH)]-pulsed DC vaccine. Patients were randomized to receive DCs that had been either activated or not activated with CD40L. All patients were followed for a minimum of 5.5 years.
Immunization induced an autologous tumor-specific T-cell proliferative or IFNγ enzyme-linked immunospot response in 15 of 24 assessable patients (63%) and a tumor-specific DTH response in 61%. Patients with evidence of a vaccine-induced, tumor-specific T-cell proliferative or IFNγ response 1 week after vaccination had a markedly better recurrence-free survival (RFS) at 5 years (63% versus 18%, P = 0.037) than nonresponders. In contrast, no association was observed between induction of KLH-specific immune responses and RFS. CD40L maturation induced CD86 and CD83 expression on DCs but had no effect on immune responses or RFS.
Adjuvant treatment of patients after resection of colorectal metastases with an autologous tumor lysate-pulsed, DC vaccine-induced, tumor-specific immune responses in a high proportion of patients. There was an association between induction of tumor-specific immune responses and RFS. Activation of this DC vaccine with CD40L did not lead to increased immune responses.
Peanut allergy poses significant healthcare problems, because its prevalence is increasing in many countries, and it is rarely outgrown. To explore the immunological mechanisms that underlie peanut allergy and tolerance, we compared the peanut-specific responses of peanut-allergic (PA) and nonallergic (NA) individuals.
We measured peanut-specific peripheral blood mononuclear cells (PBMC) proliferation using tritiated thymidine. The frequency of peanut-specific T cells amongst PBMC was determined by carboxyfluorescein succinimidyl ester labelling. The role of IgE-dependent facilitated antigen presentation (FAP) in modulating proliferation was investigated by depleting IgE from plasma with anti-IgE-coated beads and then assessing PBMC proliferation in the presence of IgE-depleted or nondepleted plasma.
We found that peanut-specific PBMC proliferation is higher and peaks earlier in PA than in NA donors. We investigated the immunological mechanisms that could underlie these differences. We found that both PA and NA have memory responses to peanut, but the frequency of peanut-specific T cells is higher in PA than in NA. Facilitated antigen presentation could cause both the higher proliferation and precursor frequency in PA. Facilitated antigen presentation activity in vitro was confirmed by showing that IgE depletion decreases proliferation, while adding IgE back restores it.
Our results identify FAP as a mechanism that underlies higher responses to peanut in PA. In these individuals, high levels of peanut-specific IgE could furthermore maintain long-term allergic T-cell responses. We raise the question whether, in the future, therapies targeting IgE such as anti-IgE antibodies may be used to suppress these T-cell responses.
Limiting dilution analysis (LDA) has been extensively employed as a quantitative method to estimate the precursor frequency of various T lymphocyte subsets according to their functional properties in vitro. We describe here an example of LDA experiment assessing antigen-specific T cell proliferation of microcultures in the presence or absence of adjuvant and illustrate how to estimate the frequencies of precursor T cells using an online tool that we made publicly available.
Recently, new methods have been introduced describing assessment of antigen-specific CD4+ T-cell immunity according to the induction of CD154 (CD40L) on CD4+ T cells during short-term activation. In our study, we have evaluated the influence of different stimulation conditions on the flow cytometric analysis of CD154 expression after antigenic in vitro activation. We used different cell preparation methods, antigen sources, and time periods of in vitro stimulation and analyzed their impact on intra and extracellular detection of antigen-induced CD154 expression on CD4+ T cells. We could demonstrate that analysis of CD4+ T-cell immunity according to CD154 expression displayed low intra-assay variability and was robust with respect to its induction in the course of a variety of stimulation conditions. For a basic quantitative evaluation of antigen-specific CD4+ T cells, surface CD154 analysis could be employed, enabling the fast analysis of live antigen-specific CD4+ T cells. Intracellular analysis of CD154 in combination with cytokines such as IL-2 and IFNgamma allowed quantitative and qualitative assessment of antigen-specific CD4+ T cells. The cytometric analysis of antigen-specific CD4+ T-cell immunity according to CD154 expression is characterized by robustness, high sensitivity, and low intra-assay variability.
Cell-tracking reagents such as the green-fluorescent protein labeling dye CFSE and the red-fluorescent lipophilic membrane dye PKH26 are commonly used to monitor cell proliferation by flow cytometry in heterogeneous cell populations responding to immune stimuli. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division. Because daughter cell fluorescence intensities are approximately halved after each division, the intensity of a cell relative to its intensity at the time of staining provides information about how many divisions it has undergone. Knowing how many rounds of division have occurred and the relative number of cells in each daughter generation, one can back-calculate the number of cells in the original population (i.e., cells present at the time of stimulus) that went on to respond by proliferating. Using this information, the precursor cell frequencies and extent of expansion to a specific antigen or mitogen of interest can be calculated. Concurrently, the phenotype of the cells can be determined, as well as their ability to bind antigen or synthesize cytokines, providing more detailed characterization of all cells responding to the antigen, not just effector cells. In multiparameter flow cytometric experiments to simultaneously analyze antigen-specific tetramer binding, cytokine production and T-cell proliferation, we found that only approximately half of the cells that exhibited specific binding to influenza tetramer also proliferated, as measured by dye dilution, and synthesized IFNgamma in response to antigen. We expect the advent of new cell tracking dyes emitting from the violet to the near infrared combined with the increasing number of lasers and detectors on contemporary flow cytometers to further expand the usefulness of this approach to characterization of complex antigen-driven immunological responses.
sensitive or provide more information than previously used assays. Importantly, some of these new techniques allow direct ex vivo analysis of T cells without in vitro amplification, thus providing a more accurate picture of the in vivo immune re- sponse. In this review, we describe some of the most widely used techniques for immune monitoring of specific T-cell responses. These various assays can be schematically divided into func- tional assays, which measure the secretion of a particular cy- tokine (ELISPOT and intracellular cytokines); assays which assess the specificity of the T cells irrespective of their func- tionality and which are based on structural features of the TCR (tetramers and immunoscope); and assays aimed at detecting T-cell precursors by amplifying cells that proliferate in re- sponse to antigenic stimulation. The sensitivity and immuno- logical relevance of these various methods are discussed. Ma- jor findings and future applications in basic and clinical immunology are also presented.
The integrin alpha4beta7 binds to MAdCAM-1 and contributes to homing of lymphocytes to gut and other mucosal tissues. In humans, the alpha4beta7(hi) subset of circulating memory cells appears to have been primed in mucosal tissues. The factors that determine whether alpha4beta7(lo) naive cells become alpha4beta(hi) or alpha4beta7(-) cells upon differentiation are poorly understood but could include an influence of the activating antigen-presenting cell. To address this point, the induction of alpha4beta7 following activation of mouse cells with the APC-dependent stimulus soluble anti-CD3 has been examined. Almost all mouse T cells freshly isolated from mesenteric lymph nodes (MLN) and peripheral (PLN; axillary, brachial and inguinal) lymph nodes stained only weakly for alpha4beta7 but a subpopulation became alpha4beta7(hi) upon activation with anti-CD3 in a cell cycle- and accessory cell-dependent manner. A small proportion (approximately 1.5 %) of the starting cells gave rise to alpha4beta7(hi) cells after culture. A higher proportion of alpha4beta7(hi) cells were generated in MLN than PLN cultures. Peyer's patch cultures gave intermediate values. In crossover experiments, MLN dendritic cells (DC) induced higher proportions and numbers of alpha4beta7(hi) cells than PLN DC irrespective of the source of T cells. Therefore, in addition to their other immunoregulatory roles, DC have the potential to shape immune responses by influencing the homing of the lymphocytes they activate.
The objective of this study was to develop a method to simultaneously examine phenotype, proliferation, apoptosis, and death of antigen-stimulated porcine lymphocytes.
Peripheral blood mononuclear cells (PBMCs) were isolated from pigs vaccinated with a Brachyspira hyodysenteriae bacterin.
Once isolated, PBMCs were stained with the fluorescent membrane intercalating dye, PKH67, and cultured with or without B. hyodysenteriae whole-cell sonicate antigen. Serial samples of nonstimulated and B. hyodysenteriae-stimulated PBMCs were harvested for flow cytometric analysis. Fluorochrome excitation was performed with spatially separated air-cooled argon and red helium neon laser beams. Five-color analysis included signal detection of PKH67 (proliferation), phycoerythrin (cell surface antigen), Texas Red phycoerythrin tandem (cell surface antigen), allophycocyanin (annexin V), and 7-amino-actinomysin D (7AAD; viability). For analysis, gates were set on live (annexin V(-), 7AAD(-)), intact apoptotic (annexin V(+), 7AAD(dim)), and live plus intact apoptotic (annexin V(+/-), 7AAD(dim/-)) cells, and the phenotypes of PBMCs within these populations were determined during the course of the in vitro response. Dead cells (i.e., 7AAD(bright)) were excluded from the analysis.
Application of this method for the determination of porcine lymphocyte subset proliferation is presented.
Graft-vs-host disease (GVHD) remains the most life-threatening complication following the transfer of allogeneic bone marrow into immunocompromised hosts. Transferred alloreactive T cells respond in a complex manner. While massive T cell expansion is observed upon entry into an allogeneic environment, anergy, apoptosis, and repertoire selection are also observed. The study presented here shows that alloreactive T cell expansion and differentiation vs anergy and suppression are dramatically influenced by host conditioning. Using alloreactive CD4(+) and CD8(+) TCR transgenic (Tg) T cells, a novel GVHD model is presented that allows for the visualization of how alloreactive T cells behave when host conditioning is manipulated. Following the transfer of alloreactive CD4(+) and CD8(+) TCR Tg T cells into sublethally irradiated hosts, both Tg T cells populations expand, develop effector function, and cause GVHD. In contrast, when Tg T cells are transferred in non-irradiated hosts, expansion is observed, but there is no development of effector function or disease. Assessment of CD4(+) Tg T cell function following transfer into non-irradiated hosts reveals that these CD4(+) Tg cells are profoundly anergic and have acquired a regulatory function, as manifested in their ability to suppress the expansion of naive TCR Tg T cells in vitro and in vivo as well as the development of GVHD. These findings underscore the decisive effect of the inflammatory environment created by irradiation in determining the ultimate fate and function of alloreactive T cells in vivo
Dendritic cells (DC) play a crucial role in controlling the initiation and orientation of antigen (Ag)-specific immune responses. It is widely accepted that optimal T cell priming requires mature DC. Although the molecular events associated with DC activation have been extensively studied, little is known about the consequences of DC maturation on recruitment and expansion of naive T cells. In the present study, we used a model tumor Ag to show that the kinetics of human DC maturation drastically affect the induction of Ag-specific effector CD8(+) T cells. In absence of exogenous cytokines and CD4 help, only DC at early stages of maturation were able to generate high frequencies of CTL. This expansion resulted from both enhanced recruitment and intense proliferation ofT cell precursors and could lead to an increase of up to 1,000-fold in the final number of effector T cells compared to non-matured DC. In our model, larger recruitment of naïve CD8(+) cells did not modify the overall avidity of the Ag-specific T cell population.
Our objective was to characterize T-cell responses to Phleum pratense in grass pollen allergic individuals and healthy controls using the fluorescent dye PKH26. Peripheral blood mononuclear cells were stimulated with P. pratense, or with recall antigens, and CD3+/CD4+ and CD3+/CD8+ T-cells that had proliferated were analysed by flow cytometry. In the presence of P. pratense CD4+/CD3+ T-cells proliferated more in grass pollen sensitive atopic patients than in nonallergic controls or in nongrass pollen sensitive atopic subjects. PPD and TT recall antigens elicited uniformly high proliferation in all T-cell subsets. Only half of pollen sensitive patients also had an increased proliferation of CD3+/CD8+ T-cells in response to P. pratense. We determined precursor frequency of CD4+ T cells in the original population that responded to P. pratense and found values ranging from 1 x 10-3 to 0.6 x 10-1, in the same range as those measured for PPD and TT. In conclusion, grass pollen sensitive atopic patients show enhanced CD4+ T-cell reactivity to P. pratense, and this could be related to the presence of elevated numbers of circulating allergen-specific CD4+ T cells. This flow cytometric method should allow the identification of other phenotypic markers such as intracellular cytokines in allergen specific responding CD4+ T cells.
The murine local lymph node assay (LLNA) is a method for the prospective identification of chemical contact allergens. The current validated protocol assesses lymphocyte proliferation induced in the draining lymph node as a function of in situ incorporation of radiolabeled thymidine. We have explored the potential utility of an alternative nonradioisotopic marker of cell division, the cytoplasmic dye carboxyfluoresein succinimidyl ester (CFSE). Using this method, the cell phenotype and the number of divisions each cell has undergone can be tracked using flow cytometry. BALB/c strain mice were exposed topically to various concentrations of the contact allergens 2,4-dinitrochlorobenzene (DNCB), oxazolone (ox) or hexyl cinnamic aldehyde (HCA), or to the nonsensitizing skin irritant methyl salicylate (MS). Five days later, lymph node cells (LNC) were labeled with CFSE, cultured for 96 h, then incubated with fluorescent labeled anti-CD4 (T helper) and -CD8 (T cytotoxic) cell antibodies, and proliferating CD4+ and CD8+ cells analyzed by flow cytometry. In LNC populations derived from vehicle-treated animals, less than 1% of either cell population had undergone one cell division or more. Topical exposure to MS (2.5 to 20%) did not increase the frequencies of proliferating cells. Exposure to all three allergens, however, resulted in a marked increase in the percentages of both CD4+ and CD8+ cells undergoing division, with up to 5% and 3% of these cells, respectively, proliferating in response to DNCB and oxazolone, and with lower levels of proliferation stimulated by HCA. These preliminary data suggest that this method may be applied to provide the basis of a nonradioisotopic end point for the LLNA, particularly for the identification of potent contact allergens.
There is a breakdown of tolerance to neutrophil components during systemic vasculitis, which is marked by autoantibodies and T cells with specificity for proteinase 3 or myeloperoxidase, expressed on the surface of apoptotic neutrophils. This study was undertaken to investigate the effects of human apoptotic and necrotic neutrophils on human dendritic cell (DC) phenotype and ability to stimulate allogeneic T cell proliferation.
DCs were generated from human peripheral blood mononuclear cells and allowed to interact with human apoptotic and necrotic neutrophils in the presence or absence of tumor necrosis factor alpha (TNFalpha). Effects on DC phenotype and ability to stimulate T cell proliferation were observed.
Immature DCs engulfed apoptotic and necrotic neutrophils, resulting in up-regulation of CD83 and class II major histocompatibility complex molecules, but down-regulation of CD40, CD80, and CD86, and a decreased ability to stimulate T cell proliferation. When TNFalpha was added in combination with apoptotic neutrophils, the inhibitory effects were overcome to some extent.
Our results suggest that DC uptake of apoptotic or necrotic neutrophils alone does not shift the immune response from tolerance to autoimmunity in systemic vasculitis. However, cytokines found at sites of inflammation in vasculitis patients may act as maturation factors for DCs, and in combination with apoptotic neutrophils, may lead to an autoimmune phenotype.
Biological therapy for renal cell carcinoma (RCC) uses agents that mobilize immune effector cells which are able to recognize and destroy cancer. We evaluated the effects of weekly then monthly autologous tumor vaccine combined with daily granulocyte macrophage-colony stimulating factor (GM-CSF) in patients with RCC as a method of stimulating antigen presenting cells.
Eligible patients with pathological stage II to IV RCC were entered into this pilot study. Autologous tumor vaccine (0.5 to 1 x 107 irradiated tumor cells) admixed with 250 microg GM-CSF per vaccine was given subcutaneously weekly for 4 weeks and then monthly for 4 months. GM-CSF (125 microg/m2) was given subcutaneously for 13 days after vaccine injection 1 and injections 4 to 8. Treatment related tumor specific CD4 and CD8 positive T cell precursors were assessed.
A total of 22 patients were entered into this study. Patients were stratified by bulk of disease (group 1, 9 patients with micrometastatic disease, and group 2, 13 patients with macrometastatic disease). In general treatment was well tolerated. Of 9 patients in group 1 7 remained disease-free after nephrectomy. In group 2, 6 patients had stable (46.2%) and 7 patients had progressive disease (53.8%). Statistically significant treatment related increases in CD4 (p = 0.028) and CD8 (p = 0.018) positive tumor specific T cell precursors were observed for the entire group of patients. Changes in CD4 and CD8 positive precursors correlated significantly with each other (p = 0.0001). This correlation was seen in the 2 patient subpopulations as well (group 1 p = 0.003, group 2 p = 0.013). Patients with minimal disease, and with changes in CD4 and CD8 positive tumor specific T cell precursors greater than the median appeared to have an improved time to progression as well as a survival benefit.
GM-CSF and autologous vaccine can be given safely in combination to patients with renal cell cancer. We observed treatment related changes in tumor specific circulating lymphocyte populations.
Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-gamma, whereas effector memory CD8 cells (CD45RAdullCCR7-) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-gamma. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.
The application of molecules that fluoresce in the infrared (IR) region to measure cell products would be enhanced by a flow cytometer capable of measuring them. To our knowledge, none exist at this time. Accordingly, we have developed such an instrument.
A Becton Dickinson LSR flow cytometer was modified to include a small 785-nm IR diode laser the size of a C cell battery with 44-mW output power. The instrument was modified further to accommodate this laser in addition to a 405-nm solid-state laser, a 488-nm air-cooled argon laser, and a 658-nm solid-state laser. Because the IR laser is dangerous to the eye, the laser beams were viewed for optical alignment using a CCD camera and video monitor. An avalanche photodiode was used in place of a photomultiplier tube because its detection sensitivity in the IR region is superior.
To assess performance, scatter and fluorescence measurements were made using microspheres that fluoresce in the IR region, and human leukocytes were stained with CD45 biotin followed by a streptavidin conjugated with an IR dye. An avalanche photodiode was 2.3 to 2.8 times more sensitive than a photomultiplier tube for detecting IR fluorescence. Cells stained with CD45 biotin and avidin conjugated with an IR dye could easily be resolved and their fluorescence quantified; there was virtually no autofluorescence. In addition, a lipophilic membrane dye that emits in the IR region was studied. HL60 cells were stained with this dye and they exhibited bright fluorescence intensity.
A commercial instrument could be modified to accommodate an IR laser for exciting dyes that fluoresce in the IR region. This new capability will extend the range of fluorescence that can be measured by flow cytometry.
Following lymphocyte depletion, homeostatic mechanisms drive the reconstitution of lymphocytes. We prospectively studied this process in 16 patients for 1 year after a single pulse of treatment with Campath-1H, a humanised anti-CD52 monoclonal antibody. We observed two phases of lymphocyte reconstitution. In the first 6 months after treatment the precursor frequency and proliferation index of the patients’ autologous mixed lymphocyte reaction increased; the depleted T cell pool was dominated by memory T cells, especially CD4+CD25high T cells, a putative regulatory phenotype; and there was a non-significant rise in peripheral mononuclear cell FoxP3 mRNA expression and fall in constitutive cytokine mRNA expression. In the later phase, from 6-to-12 months after Campath-1H, these changes reversed and there was a rise in ROG mRNA expression. However, total CD4+ numbers remained below 50% of pre-treatment levels at 12 months, perhaps reflecting a failure in homeostasis. This was not due to an impaired IL-7 response, as in rheumatoid arthritis, nor to a lack of IL-7 receptors, which are found on fewer human CD4+CD25high than naive cells. We speculate that CCL21 and IL-15 responses to lymphopaenia may be suboptimal in multiple sclerosis.
See accompanying commentary: http://dx.doi.org/10.1002/eji.200535385
The objective of this study was to evaluate the relationship between the level and function of circulating immune cells with average daily gain, live and carcass measurements, feed intake, and feed conversion. Production performance was monitored throughout the pig's lifetime. Pigs were moved in weekly batches through the nursery and growing/finishing rooms at specific target weights. Animals were individually weighed at birth and at weaning, and then every two weeks while they were "on test" until they were "off test" and sent to the slaughterhouse. At six to seven weeks of age, the pigs were bled in the nursery. The percentage of immune cell subsets and lymphocyte proliferation was estimated using swine monoclonal antibodies and flow cytometric analysis. The predictive effect of the immune cell subset markers and lymphocyte proliferation on production traits was statistically analyzed. The results indicated that the proportion of several peripheral cell subsets, including CD16+, CD2+/CD16+, and CD8+ lymphocytes, appear to predict growth during the entire productive life of the pig. Larger percentages of lymphocytes expressing CD16+ CD2+/CD16+, and CD8+receptors in blood resulted in a reduction in average daily gain. In addition, high percentages of SLA-DQ+ cells were associated with better carcass weight and feed conversion. The CD16+, CD2+/CD16+, CD8+, and SLA-DQ+/- cell subsets appear to be important biomarkers involved with the inherent ability of the pig to efficiently grow and produce better carcass weight in representative commercial environments.
Proliferation of vascular smooth muscle cells (VSMCs) contributes to restenosis after coronary intervention. We have shown previously that increased expression of plasminogen activator inhibitor type 1 (PAI-1) limits VSMC apoptosis. Because apoptosis and proliferation appear to be linked, we sought to determine whether increased PAI-1 would affect VSMC proliferation.
VSMCs were explanted from control and transgenic mice (SM22-PAI+) in which VSMC expression of PAI-1 was increased. Increased growth of SM22-PAI+-VSMCs (2.3+/-0.4-fold) reflected, at least partially, increased proliferation. Greater expression of FLICE-like inhibitory protein (FLIP; 2.7-fold) and its cleaved active form were seen in SM22-PAI+-VSMCs. The balance between caspase-8 and FLIP favored proliferation in SM22-PAI+-VSMCs. Increased expression of NF-kappaB and activation of extracellular signal-regulated kinase (ERK) were demonstrated in SM22-PAI+-VSMCs (fold=NF-kappaB=2.2+/-0.1, fold=phosphorylated-ERK=1.6+/-0.1). Results were confirmed when expression of PAI-1 was increased by transfection. Inhibition of NF-kappaB and ERK attenuated proliferation in SM22-PAI+-VSMCs. Increased expression of PAI-1 promoted proliferation when VSMCs were exposed to tumor necrosis factor (TNF).
Increased expression of PAI-1 is associated with greater activity of FLIP that promotes VSMC proliferation through NF-kappaB and ERK. Thus, when vascular wall expression of PAI-1 is increased, restenosis after coronary intervention is likely to be potentiated by greater proliferation of VSMC and resistance to apoptosis.
It is well understood how a variety of Ig H and L chains, components of BCR, are generated in the DNA level during B cell development. However, it has remained largely unknown whether and how each component is monitored for its quality and selected before the assembly into the BCR. Here we show that muH chains produced by pre-B cells display a wide spectrum of ability to form the pre-BCR, which is composed of muH and surrogate light (SL) chains and is crucial for B cell development. The level of surface pre-BCR expression varies among pre-B cells, depending on the ability of their muH chains to pair with SL chains. The higher the level of pre-BCR expression by pre-B cells, the stronger their pre-BCR signaling, and the better they proliferate and differentiate. Thus, the extent of survival, proliferation, and differentiation of individual pre-B cells is primarily determined by the SL-pairing ability of their muH chains. Furthermore, IgH chains with higher potential to assemble with IgL chains appear to be positively selected and amplified through the assessment of their ability to pair with SL chains at the pre-BCR checkpoint before the assembly into the BCR. These results indicate that the pre-BCR assesses the quality of muH chains and tunes the pre-B cell repertoire by driving the preferential expansion and differentiation of cells with the higher quality of muH chains.
A nonradioactive multi-parameter flow cytometry assay was developed to identify antigen-specific lymphocytes in human subjects previously vaccinated against rabies virus and was subsequently compared to the standard tritiated thymidine method. A cell tracking dye, carboxyfluorescein succinimidyl ester, was used in combination with surface label for CD4 and CD8 cells in order to determine the response of lymphocytes to killed rabies virus in an antigen recall assay. The rabies virus-specific lymphocyte response was compared to the humoral immune response in each of ten vaccinated and five non-vaccinated subjects. Lymphocyte responses to rabies virus were observed in all ten vaccinated subjects; some noted as early as 3 days after stimulation while others were not until 7 days after stimulation. There was good agreement between the proliferation index of the CFSE assay and the simulation index of the [3H]thymidine assay (kappa statistic=0.73). An inverse relationship was detected between the level of rabies virus neutralizing antibody (RVNA) and the lymphocyte response to inactivated rabies virus in the vaccinated subjects. The association between cytokines production and level of humoral and cellular response was investigated in four representative subjects. Two vaccinated subjects with high proliferation indices and low RVNA titers produced Th1 type cytokines to rabies virus stimulation, whereas two vaccinated individuals with low proliferation indices and high RVNA titers responses did not produce these cytokines.
In patients with progressive malignancy, the natural balance between proinflammatory (Yang) and inhibitory (regulatory or Yin) immune pathways is disrupted and favors cancer-specific immune suppression. Therapy with interleukin 2 (IL-2) can mobilize immune effector cells that recognize and destroy cancer. High-dose IL-2 is the only therapy that has consistently induced complete durable remissions in patients with metastatic renal cell carcinoma (RCC) but only in a few of them. The lack of benefit in most metastatic RCC patients is likely due to the ineffective manipulation of other immune circuits critical in regulating tumor cytotoxic pathways. The limited clinical activity of IL-2, RCC vaccines, and other immune therapies to date leads us to postulate that effective clinical treatment strategies will need to simultaneously enhance proinflammatory pathways and disrupt regulatory pathways. We present preliminary studies in RCC patients to highlight the complexity of the regulatory pathways and our approach to shifting the balance of proinflammatory and regulatory immune pathways using dendritic cell-tumor lysate vaccine followed by cytokine therapy.
In this study, we evaluated alternative possibility for CFSE beryllium flow cytometric test against beryllium blood lymphocyte proliferation test (BeLPT) as a standard radioactive clinical screening method to identify sensitization to beryllium.
Delta PD (the ratio of divided cell population to the total number of cells with subtracted counts of unstimulated cells) of specific beryllium-induced pathogenic CD3+ CD4+ T-lymphocytes and stimulation index (SI) in CFSE proliferation test was compared with delta counts per minute (mean test CPM minus mean control CPM) and SI in radioactive blood BeLPT.
Comparison analysis of CFSE and BeLPT demonstrated excellent agreement between delta PD and delta CPM (kappa = 0.845, P < 0.0001). We determined 6.8% positive subjects in the beryllium-exposed, Be-LPT-negative group. The decreased mean difference of these indexes to percentage of average and the long tail in the plot reflects increased sensitivity. CFSE/CD4+ T-cell proliferation assay has 100% specificity, significantly higher sensitivity and efficiency than BeLPT.
Both delta PD, measured by the precursor frequencies method in CFSE assay and delta CPM, defined by tritiated thymidine in BeLPT, can be used for the enumeration of beryllium specific CD4+ T-cell proliferation and may substantially improve the quality of the early diagnosis of beryllium hypersensitivity.
Several mechanisms that impair the immune response to promote tumour progression are reported. These mechanisms aim to reduce the ability of antigen-presenting cells to present antigen and activate naïve T cells to support an active immune response or to create a suppressive environment that induce non-functional tumour-associated antigen-specific T cells. Prostate cancer (PC) alone accounts for 33% of incident cancer cases and about 9% of all cancer-related deaths among men in the USA during 2006. Whereas androgen deprivation has remained the first line of therapy for advanced PC, other therapies are still required due to progression to an androgen-resistant state and eventually loss of control in patients receiving hormonal therapy. Immunotherapy seems to be a promising approach to enhance tumour-specific T-cell responses in different cancers including prostate. More importantly, clinical trials in advanced PC patients have shown that immunotherapy may generate significant clinical responses. Immunology and immunotherapy aspects of PC with focus on prostate-specific antigen will be presented.
The CD28-specific mAb TGN1412 rapidly caused a life-threatening "cytokine storm" in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.
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