Patrick J. Blair

Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes.

Background. 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. Methods. 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-&agr;), tumor growth factor (TGF-&bgr;), and interferon (IFN-&ggr;) were determined by polymerase chain reaction (PCR). Results. Statistical difference between protein production and cytokine polymorphic variants in the IL-10, IFN-&ggr;, and TNF-&agr; 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-&ggr;, 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-&agr; and TGF-&bgr; genotypes and protein production. Conclusion. Polymorphisms in IL-2, IL-6, IL-10, and IFN-&ggr; 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.

Ethnicity greatly influences cytokine gene polymorphism distribution.

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‐α, TGF‐β, and IFN‐γ) 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‐γ 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‐α. 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.

Cytokine polymorphic analyses indicate ethnic differences in the allelic distribution of interleukin-2 and interleukin-6

BACKGROUND 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. METHODS 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 Fishers exact two-tailed tests. RESULTS 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. CONCLUSIONS 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.

Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25+ regulatory T cells.

CD28 and CTLA‐4 are opposing regulators of T cell activation, triggered by the two ligands CD80 and CD86. How these ligands promote either T cell activation via CD28 or inhibition via CTLA‐4 is not understood. Using CD80 and CD86 molecules expressed on transfected cells, we have identified a major difference between these ligands in that CD80 transfectants have the ability to inhibit activation of resting human peripheral blood T cells via interaction with CTLA‐4, whereas CD86 transfectants do not. Rather, CTLA‐4–CD86 interactions appear to contribute towards T cell proliferation. We also observed that CTLA‐4 function is strongly influenced by TCR stimulation, effects being observed only at relatively low levels of TCR stimulation. The kinetics of CD80–CTLA‐4 interactions revealed that CTLA‐4 inhibition took place within the first 8 h of T cell stimulation, despite there being little measurable CTLA‐4 expression on the majority T cells. However, significant amounts of CTLA‐4 were observed in the CD25+ CD4+ subset of T cells which, when removed from the cultures, accounted for the CTLA‐4 inhibition observed. Overall, these data provide evidence that CD80 and CD86 differ in their interactions with CTLA‐4 and that CD80 appears to be the preferential inhibitory ligand for CTLA‐4 working via a population of CD4+ CD25+ CTLA‐4+ regulatory T cells.

Measurement of CD69 induction in the assessment of immune function in asymptomatic HIV-infected individuals

Peripheral blood mononuclear cells from many asymptomatic HIV-infected patients exhibit defects in cytokine production and impaired proliferative responses in vitro but the mechanisms underlying this subclinical immune deficiency are controversial. To determine whether abnormalities in the earliest events following receptor engagement may help to explain the in vitro immune dysfunction, we measured the inducibility of the early activation marker CD69 in T cells from asymptomatic HIV-infected individuals in response to stimulation with anti-CD2 or anti-CD3 mAb. In a whole blood assay, we found that induction of CD69 was markedly impaired in CD4+ T cells from later-stage HIV-infected patients (CD4 counts 200-400/mm3) compared to uninfected controls. Among early stage patients (CD4 > 400/mm3), a subset (29%) had impaired CD69 induction. CD69 responses were equally depressed after stimulation through the CD3 or CD2 receptor pathways. Survey of a panel of immunophenotypic markers and propensity for apoptosis demonstrated a significant association between depressed induction of CD69 and decreased percentages of CD4+CD26+ and CD4+CD95+ cells but no association with the level of apoptosis. These data indicate that defects in T lymphocyte activation through CD3 and CD2 can be measured within hours of receptor stimulation in asymptomatic HIV-infected individuals and might be useful to monitor as an indicator of immune function in these patients.

Immune profiling: molecular monitoring in renal transplantation.

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 laboratorys 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.

Impaired Induction of the Apoptosis-Protective Protein Bcl-xL in Activated PBMC from Asymptomatic HIV-Infected Individuals

Progression to AIDS in asymptomatic HIV-infected individuals is characterized by a gradual but progressive loss of CD4+ T cells. While the mechanisms underlying this decline are currently unknown, recent evidence suggests that these cells are abnormally sensitive to apoptosis in response to activation signals. Recent work has implicated downregulation of Bcl-2 with the increased spontaneous apoptosis in lymphocytes from HIV-infected patients. We have evaluated the roles of the apoptosis-protective proteins Bcl-2 and Bcl-x in stimulated PBMC from asymptomatic HIV-infected and HIV-uninfected individuals. We found that Bcl-2 was constitutively expressed in PBMC from both HIV-infected and uninfected samples. However, Bcl-x induction was delayed and responses were decreased in stimulated HIV-infected samples. Additionally, single-cell intracellular staining of Bcl-x revealed a significant inverse correlation between PWM-induced Bcl-x expression and apoptosis (r = −0.695, P = 0.005). This was confirmed at the single-cell level in direct experiments when stimulated cells were sorted based on Bcl-x induction and then measured for apoptosis. Furthermore, low Bcl-x expression was not due to reduced lymphocyte activation following PWM stimulation. Our data indicate that the induction of Bcl-x is markedly impaired in asymptomatic HIV-infected patients and that stimuli which induce inadequate expression of Bcl-x are associated with increased levels of apoptosis in these cells.

Effects of Combined Treatment with CD25- and CD154-Specific Monoclonal Antibodies in Non-Human Primate Allotransplantation

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.

Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34(+)CD38(-) progenitor cells.

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.

Detection of Apoptosis in HIV-Infected Ceil Populations using TUNEL.

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).