[Show abstract][Hide abstract] ABSTRACT: The CD154-specific monoclonal antibody (Mab) hu5c8 greatly prolongs allograft survival in primates. The CD25-specific Mab daclizumab has not, to date, been paired with hu5c8. We evaluated the effects of hu5c8 in vitro, alone and in combination with daclizumab on rhesus-mixed lymphocyte reactions (MLRs). We then evaluated therapy with hu5c8 and daclizumab in four monkey renal allograft recipients compared with monkeys untreated or contemporaneously treated with hu5c8 alone. Lymphocyte proliferation in MLR was reduced by both daclizumab and hu5c8, and their combined effects were additive. Rejection-free allograft survival in monkeys treated with both hu5c8 and daclizumab (74-479 days) was not significantly better than animals treated with hu5c8 alone (257-587 days), and one combined therapy animal rejected while still on hu5c8 therapy, a condition not typically seen with hu5c8 monotherapy. Although daclizumab and hu5c8 are additively effective in MLR, they do not appear to be synergistic in vivo in rhesus monkeys.
American Journal of Transplantation 12/2003; 3(11):1350-4. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular techniques have become a mainstay for most biomedical research. In particular, sensitive methods for gene transcript detection and advanced flow cytometry have been crucial in fostering our understanding of the basic mechanisms promoting allosensitization and adaptive immune regulation. These technologies have been validated in vitro, and in pre-clinical settings, and as such their clinical application is now clearly appropriate. It is becoming increasingly clear that these robust techniques hold much promise to better elucidate human transplant biology, and more importantly, guide clinical decision making with mechanistically-based information. This article will discuss our laboratory's use of several novel technologies, including gene polymorphism analysis, real-time polymerase chain reaction transcript quantification, and multi-color flow cytometry in clinical human renal transplantation. Specific technical methodology will be presented outlining keys for effective clinical application. Clinical correlations will be presented as examples of how these techniques may have clinical relevance. Suggestions for the adaptation of these methods for therapeutic intervention will be given. We propose that clinical transplantation should proceed in close step with modern molecular diagnostics.
Frontiers in Bioscience 10/2003; 8:e444-62. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND Following allotransplantation, renal ischemia-reperfusion (I/R) injury initiates a series of events that provokes counter-adaptive immunity. Though T cells clearly mediate allospecific immunity, the manner in which reperfusion events augment their activation has not been established. In addition, comprehensive analysis of I/R injury in humans has been limited. METHODS To evaluate the earliest events occurring following allograft reperfusion and gain insight into those factors linking reperfusion to alloimmunity, we examined human renal allografts 30 to 60 minutes postreperfusion (n=10) and compared them with allografts with normal function that had resolved their I/R injury insult (>1 month posttransplant, n=6) and to normal kidneys (living donor kidneys before procurement, n=8). Biopsies were processed both for immunohistochemical analysis as well as for transcript analysis by real-time quantitative polymerase chain reaction (RT-PCR). RESULTS Reperfusion injury was characterized by increased levels of gene transcripts known to be involved in cellular adhesion, chemotaxis, apoptosis, and monocyte recruitment and activation. T-cell-associated transcripts were generally absent. However, recovered allografts exhibited increased levels of T-cell and costimulation-related gene transcripts despite normal allograft function. Consistent with these findings, the immediate postreperfusion state was characterized histologically by tubular injury and monocyte infiltration, while the stable posttransplant state was notable for T-cell infiltration. CONCLUSIONS These data suggest that monocytes and transcripts related to their recruitment dominate the immediate postreperfusion state. This gives way to a T-cell dominant milieu even in grafts selected for their stable function and absence of rejection. These data have implications for understanding the fundamental link between I/R injury and alloimmunity.
[Show abstract][Hide abstract] ABSTRACT: Polymorphisms in the regulatory regions of cytokine genes are associated with high and low cytokine production and may modulate the magnitude of alloimmune responses following transplantation. Ethnicity influences allograft half-life and the incidence of acute and chronic rejection. We have questioned whether ethnic-based differences in renal allograft survival could be due in part to inheritance of cytokine polymorphisms. To address that question, we studied the inheritance patterns for polymorphisms in several cytokine genes (IL-2, IL-6, IL-10, TNF-alpha, TGF-beta, and IFN-gamma) within an ethnically diverse study population comprised of 216 Whites, 58 Blacks, 25 Hispanics, and 31 Asians. Polymorphisms were determined by allele-specific polymerase chain reaction and restriction fragment length analysis. We found striking differences in the distribution of cytokine polymorphisms among ethnic populations. Specifically, significant differences existed between Blacks and both Whites and Asians in the distribution of the polymorphic alleles for IL-2. Blacks, Hispanics and Asians demonstrated marked differences in the inheritance of IL-6 alleles and IL-10 genotypes that result in high expression when compared with Whites. Those of Asian descent exhibited an increase in IFN-gamma genotypes that result in low expression as compared to Whites. In contrast, we did not find significant ethnic-based differences in the inheritance of polymorphic alleles for TNF-alpha. Our results show that the inheritance of certain cytokine gene polymorphisms is strongly associated with ethnicity. These differences may contribute to the apparent influence of ethnicity on allograft outcome.
American Journal of Transplantation 08/2002; 2(6):560-7. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As glucose-induced insulin expression is mainly regulated at the translational level, and such regulation often involves the 5'-untranslated region (5'UTR), we examined the human proinsulin gene 5'UTR. RT-PCR and sequencing demonstrated that a proinsulin splice variant (SPV) generated from a cryptic 5'-splice site and retaining the first 26 bp of intron 1 was present in human pancreatic islets from normal donors. The expression of this SPV was metabolically regulated, as shown by quantitative real-time RT-PCR, revealing a more than 10-fold increase in the SPV in isolated human islets incubated at 16.7 mM compared with 1.67 mM glucose. In vitro wheat-germ translation and in vivom transfection studies demonstrated that the altered 5'UTR of the SPV increased translation. The SPV yielded 4-fold more in vitro translated preproinsulin protein than the native proinsulin mRNA, and the SPV 5'UTR inserted upstream from a luciferase reporter gene resulted in a more than 6-fold higher luciferase activity, suggesting enhanced translation in vivo. Retention of the 26 bp changed the proposed secondary RNA structure of the SPV, which may facilitate ribosomal binding and explain the increase in translation efficiency. These results suggest a novel mechanism by which metabolic changes can modulate the expression of 5'UTR SPVs and thereby regulate translation efficiency.
[Show abstract][Hide abstract] ABSTRACT: Genetic variations in cytokine genes are thought to regulate cytokine protein production. However, studies using T cell mitogens have not always demonstrated a significant relationship between cytokine polymorphisms and in vitro protein production. Furthermore, the functional consequence of a polymorphism at position -330 in the IL-2 gene has not been described. We associated in vitro protein production with cytokine gene polymorphic genotypes after costimulation of cultured peripheral blood lymphocytes.
PBL were isolated from forty healthy volunteers. Cytokine protein production was assessed by enzyme-linked immunosorbent assay. Polymorphisms in interleukin- (IL) 2, IL-6, IL-10, tumor necrosis factor (TNF-alpha), tumor growth factor (TGF-beta), and interferon (IFN-gamma) were determined by polymerase chain reaction (PCR).
Statistical difference between protein production and cytokine polymorphic variants in the IL-10, IFN-gamma, and TNF-alpha genes was not evident after 48-hour stimulation with concanavalin-A. In contrast, after anti-CD3/CD28 stimulation significant differences (P<0.05) were found among high and low producers for IL-2, IL-6, and among high, intermediate, and low producers for IFN-gamma, and IL-10. Augmented levels of IL-2 in individuals that were homozygous for the polymorphic IL-2 allele were due to an early and sustained enhancement of IL-2 production. No association was found among TNF-alpha and TGF-beta genotypes and protein production.
Polymorphisms in IL-2, IL-6, IL-10, and IFN-gamma genes are associated with their protein production after anti-CD3/CD28 stimulation. The profound effect of the IL-2 gene polymorphism in homozygous individuals may serve as a marker for those that could mount the most vigorous allo- or autoimmune responses, or perhaps become tolerant more easily.
[Show abstract][Hide abstract] ABSTRACT: Polymorphisms in the regulatory regions of cytokine genes affect protein production and are associated with allograft outcome. Ethnic origin has been identified as a significant prognostic factor for several immune-mediated diseases and for outcome after allotransplantation. A clear relationship between cytokine polymorphisms and ethnicity has not been shown.
One hundred sixty subjects including 102 whites and 43 African-Americans were studied. Using polymerase chain reaction-based assays and, in some cases, restriction enzyme digestion, we determined genetic polymorphisms for the cytokines interleukin (IL) -2, IL-6, IL-10, tumor necrosis factor-alpha, transforming growth factor-beta, and interferon-gamma (IFN-gamma). Genetic polymorphism frequencies were then compared to ethnicity using chi-square analysis and Fisher's exact two-tailed tests.
For both the IL-2 and IL-6 genes, we found that whites and African-Americans differed significantly (P <0.05) in their allelic distribution and genotype frequency. A trend toward ethnic distribution was noted among the alleles and genotypes for the IL-10 and IFN-gamma genes. We found no correlation between ethnicity and either allelic distribution or genotype frequency for the tumor necrosis factor-alpha or transforming growth factor-beta genes. When comparisons were made between patients with or without a history of kidney failure, the allelic or genotypic distributions for the IL-6 and IFN-gamma genes were found to significantly differ.
Our work demonstrates a correlation between ethnicity and polymorphisms in several cytokine genes. In addition, we found that patients requiring renal transplantation differ from the general population with regard to certain cytokine gene polymorphisms. These findings may have relevance in making prognostic determinations or tailoring immunomodulatory regimens after renal transplantation.
[Show abstract][Hide abstract] ABSTRACT: CD154 plays a critical role in determining the outcome of a transplanted organ. This simple statement is amply supported by experimental evidence demonstrating that anti-CD154 antibodies are potent inhibitors of allograft rejection in many rigorous transplant models. Unfortunately, despite intensive investigation over the past ten years, the precise mechanisms by which antibodies against CD154 exert their anti-rejection effects have remained less obvious. Though originally classified with reference to B-cell function, CD154-CD40 interactions have also been shown to be important in T cell-antigen-presenting cell interactions. Accordingly, CD154 has been classified as a T-cell co-stimulatory molecule. However, mounting data suggest that treatment with anti-CD154 antibodies does not simply block costimulatory signals, but rather that the antibodies appear to induce signalling in receptor-bearing T cells. Other data suggest that anti-CD154 effects may be mediated by endothelial cells and possibly even platelets. In fact, the current literature suggests that CD154 can either stimulate or attenuate an immune response, depending upon the model system under study. CD154 has secured a fundamental place in transplant biology and general immunology that will no doubt be the source of considerable investigation and therapeutic manipulation in the coming decade.
Philosophical Transactions of The Royal Society B Biological Sciences 06/2001; 356(1409):691-702. · 6.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the relationship between ICOS, CD28, CTLA-4, and IL-2 to gain a better understanding of this family of costimulatory receptors in the immune response. Using magnetic beads coated with anti-CD3 and varying amounts of anti-ICOS and anti-CTLA-4 Abs, we show that CTLA-4 ligation blocks ICOS costimulation. In addition to inhibiting cellular proliferation, CTLA-4 engagement prevented ICOS-costimulated T cells from producing IL-4, IL-10, and IL-13. Both an indirect and direct mechanism of CTLA-4's actions were examined. First, CTLA-4 engagement on resting cells was found to indirectly block ICOS costimulation by interferring with the signals needed to induce ICOS cell surface expression. Second, on preactivated cells that had high levels of ICOS expression, CTLA-4 ligation blocked the ICOS-mediated induction of IL-4, IL-10, and IL-13, suggesting an interference with downstream signaling pathways. The addition of IL-2 not only overcame both mechanisms, but also greatly augmented the level of cellular activation suggesting synergy between ICOS and IL-2 signaling. This cooperation between ICOS and IL-2 signaling was explored further by showing that the minimum level of IL-2 produced by ICOS costimulation was required for T cell proliferation. Finally, exogenous IL-2 was required for sustained growth of ICOS-costimulated T cells. These results indicate that stringent control of ICOS costimulation is maintained initially by CTLA-4 engagement and later by a requirement for exogenous IL-2.
The Journal of Immunology 05/2001; 166(8):4943-8. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the relationship between ICOS, CD28, CTLA-4, and IL-2 to gain a better understanding of this family of costimulatory receptors in the immune response. Using magnetic beads coated with anti-CD3 and varying amounts of anti-ICOS and anti-CTLA-4 Abs, we show that CTLA-4 ligation blocks ICOS costimulation. In addition to inhibiting cellular proliferation, CTLA-4 engagement prevented ICOS-costimulated T cells from producing IL-4, IL-10, and IL-13. Both an indirect and direct mechanism of CTLA-4's actions were examined. First, CTLA-4 engagement on resting cells was found to indirectly block ICOS costimulation by interferring with the signals needed to induce ICOS cell surface expression. Second, on preactivated cells that had high levels of ICOS expression, CTLA-4 ligation blocked the ICOS-mediated induction of IL-4, IL-10, and IL-13, suggesting an interference with downstream signaling pathways. The addition of IL-2 not only overcame both mechanisms, but also greatly augmented the level of cellular activation suggesting synergy between ICOS and IL-2 signaling. This cooperation between ICOS and IL-2 signaling was explored further by showing that the minimum level of IL-2 produced by ICOS costimulation was required for T cell proliferation. Finally, exogenous IL-2 was required for sustained growth of ICOS-costimulated T cells. These results indicate that stringent control of ICOS costimulation is maintained initially by CTLA-4 engagement and later by a requirement for exogenous IL-2. The Journal of Immunology, 2001, 166: 4943- 4948.
The Journal of Immunology 04/2001; 166(8). · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retroviral gene transfer studies targeting bone marrow CD34(+)CD38(-) stem cells have been disappointing because of the rarity of these cells, their G(0) cell cycle status, and their low or absent expression of surface retroviral receptors. In this study, we examined whether preincubation of bone marrow CD34(+)CD38(-) stem cells with a hematopoietically supportive porcine microvascular endothelial cell line (PMVECs) could impact the cell cycle status and expression of retroviral receptors in pluripotent CD34+CD38- cells and the efficiency of gene transfer into these primitive target cells. PMVEC coculture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand induced >93% of the CD34(+)CD38(-) population to enter the G(1) or G(2)/S/M phase while increasing this population from 1.4% on day 0 to 6.5% of the total population by day 5. Liquid cultures supplemented with the identical cytokines induced 73% of the CD34(+)CD38(-) population into cell cycle but did not maintain cells with the CD34(+)CD38(-) phenotype over time. We found no significant increase in the levels of AmphoR or GaLVR mRNA in PMVEC-expanded CD34(+)CD38(-) cells after coculture. Despite this, the efficiency of gene transfer using either amphotropic vector (PA317) or GaLV vector (PG13) was significantly greater in PMVEC-expanded CD34(+)CD38(-) cells (11.4 +/- 5.6 and 10.9 +/- 5.2%, respectively) than in either steady state bone marrow CD34(+)CD38(-) cells (0.6 +/- 1.7 and 0.2 +/- 0.6%, respectively; p < 0.01 and p < 0.01) or liquid culture-expanded CD34(+)CD38(-) cells (1.4 +/- 3.5 and 0.0%, respectively; p < 0.01 and p < 0.01). Since PMVEC coculture induces a high level of cell cycling in human bone marrow CD34(+)CD38(-) cells and expands hematopoietic cells capable of in vivo repopulation, this system offers potential advantages for application in clinical gene therapy protocols.
Human Gene Therapy 12/2000; 11(18):2515-28. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study has examined the stimuli required for secretion of regulated upon activation, normal T-cell expressed, presumed secreted (RANTES) from T lymphocytes and found that stimuli such as phorbol 12-myristate 13-acetate (PMA), which are unable to support T-cell proliferation and interleukin-2 (IL-2) production, are nevertheless able to elicit strong secretion of RANTES. Conversely, stimuli such as CD2 and CD28 ligation, which are able to support T-cell proliferation, are unable to elicit RANTES secretion. Coligation of CD3 and CD28 drives T-cell proliferation to a similar degree as CD2 and CD28 coligation, yet also supports modest RANTES secretion. Furthermore, CD28 ligation enhances the secretion of RANTES stimulated by PMA and this costimulatory effect is abrogated by the phosphoinositide 3-kinase inhibitor wortmannin. Our data also indicate that the observed effects of PMA on RANTES secretion are probably due to activation of protein kinase C (PKC) isoenzymes, since RANTES secretion was unaffected by the non-PKC activating 4alpha-phorbol ester, whilst the general PKC inhibitor Ro-32-0432 inhibits PMA-stimulated RANTES secretion. Moreover, the effect of PMA appears to be chemokine-specific because PMA was unable to increase secretion of the related CC chemokine MIP-1alpha. Under stimulation conditions where increases in [Ca2+]i occur (e.g. PMA plus ionomycin or CD3 plus CD28 ligation) RANTES secretion can be severely reduced compared with the levels observed in response to the phorbol ester PMA. Hence, whilst PKC-dependent pathways are sufficient for strong RANTES secretion, a calcium-dependent factor is activated which negatively regulates RANTES secretion. This correlates well with the observation that ligation of cytolytic T lymphocyte-associated antigen-4 (CTLA-4) (expression of which has been reported to be dependent on a sustained calcium signal), inhibits RANTES secretion induced by CD3/CD28, but has no effect on PMA-stimulated RANTES secretion.
[Show abstract][Hide abstract] ABSTRACT: CD4 T cells activated in vitro by anti-CD3/28-coated beads are resistant to infection by CC chemokine receptor 5 (CCR5)-dependent HIV-1 isolates. In vivo, antigen-presenting cells (APCs) activate CD4 T cells in part by signaling through the T cell receptor and CD28, yet cells stimulated in this manner are susceptible to HIV-1 infection. We show that cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement counteracts the CD28 antiviral effects, and that the ratio of CTLA-4 to CD28 engagement determines the susceptibility of HIV-1 infection. Furthermore, unopposed CTLA-4 signaling provided by CD28 blockade promotes vigorous HIV-1 replication, despite minimal T cell proliferation. Finally, CTLA-4 antibodies decrease the susceptibility of antigen-activated CD4 T cells to HIV, suggesting a potential approach to prevent or limit viral spread in HIV-1-infected individuals.
Journal of Experimental Medicine 07/2000; 191(11):1987-97. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Signals generated through CD28-B7 and CD40 ligand (CD40L)-CD40 interactions have been shown to be crucial for the induction of long-term allograft survivability. We have recently demonstrated that humanized anti-CD40L (hu5C8) prevents rejection of mismatched renal allografts in primates. To investigate potential mechanisms of CD40L-induced allograft acceptance, we coimmobilized hu5C8 with suboptimal amounts of anti-CD3 to stimulate CD4(+) T cells. We now report that anti-CD3/CD40L costimulation results in CD28-independent activation and subsequent deletion of resting T cells. Coligation of CD3 and CD40L increased expression of CD69, CD25, and CD54 on CD4(+) T cells. We also found that costimulation with anti-CD3/CD40L resulted in enhanced production of interleukin (IL)-10, interferon gamma, and tumor necrosis factor alpha but not IL-2 or IL-6. Interestingly, after several days, anti-CD3/CD40L-mediated activation was followed by apoptosis in a significant population of cells. Consistent with that observation, anti-CD3/CD40L did not enhance the antiapoptotic proteins Bcl-2 and Bcl-xL. Further, the addition of CD28 at 24 h failed to rescue those cells induced to die after costimulation with anti-CD3/CD40L. Together, these data suggest that the graft-sparing effect of hu5C8 in vivo may result in part from early and direct effects on CD4(+) T cells, including a vigorous induction of immunomodulatory cytokines and/or apoptosis of allograft-specific T cells.
Journal of Experimental Medicine 03/2000; 191(4):651-60. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clinical success has not been routinely achieved for composite tissue allotransplantation (CTA). Although most of the technical details of CTA have been overcome, the immunological aspects of these procedures have proved complex. Many traumatic conditions requiring CTA contraindicate acute global immunosuppression. Moreover, the risk of long-term immunosuppression is difficult to reconcile with non-life-threatening defects that can be adequately palliated. Recently, several successful immunomodulating strategies have been introduced for solid organ transplantation. They include therapies that alter costimulatory signals at engraftment. One approach, using treatment with a monoclonal antibody directed against CD154, has shown promise in rodent and nonhuman primate models and is discussed as a potential strategy for CTAs.
[Show abstract][Hide abstract] ABSTRACT: Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.
The Journal of Immunology 04/1999; 162(6):3237-48. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cells within an organism undergo two common forms of cell death. Sudden injury resulting from physical or chemical insult leads to a form of cell death called necrosis. A more subtle programmed form of cell death is termed apoptosis. Apoptosis describes a genetically encoded pathway that plays an important role in regulating the immune response (1,2). Apoptotic cell death is characterized by distinct biochemical and morphologic changes and the fragmentation of DNA into nucleosomal-sized multimers (3). Apoptosis plays a crucial role in viral infections and in the host response to viral insult (4).
[Show abstract][Hide abstract] ABSTRACT: Fusion and entry of the human immunodeficiency virus (HIV) into CD4(+) T lymphocytes requires expression of CD4 and a coreceptor. At least eight chemokine receptors can serve as coreceptors for HIV. Accumulating evidence indicates that multiple factors, including the state of cellular differentia- tion and activation, regulate the expression of alpha- and beta-chemokine receptors on lymphocytes. For example, binding of antibodies to the CD28 coreceptor can downregulate expression of beta-chemokine receptors, and this appears to have important consequences on the susceptibility of CD4(+) T lymphocytes to infection by HIV-1. In contrast, binding of the natural CD28 ligand B7 or antibodies to the CD28 homologue CTLA-4 can upregulate CCR5 expression, sug- gesting a reciprocal interaction between CD28 and CTLA-4 and the regulation of beta-chemokine receptor expression. Thus, the CD28/CTLA-4/B7 co-stimulation pathway is identi- fied as a potential novel target for the control of susceptibility to some strains of HIV-1 infection.
Seminars in Immunology 07/1998; 10(3):195-202. · 5.93 Impact Factor