Steven C. Hoffmann

Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T-cell depletion

T‐cell depletion facilitates reduced immunosuppression following organ transplantation and has been suggested to be pro‐tolerant. However, the characteristics of post‐depletional T cells have not been evaluated as they relate to tolerance induction. We therefore studied patients undergoing profound T‐cell depletion with alemtuzumab or rabbit anti‐thymocyte globulin following renal transplantation, evaluating the phenotype and functional characteristics of their residual cells. Naïve T cells and T cells with potential regulatory function (CD4+CD25+) were not prevalent following aggressive depletion. Rather, post‐depletion T cells were of a single phenotype (CD3+CD4+CD45RA‐CD62L‐CCR7‐) consistent with depletion‐resistant effector memory T cells that expanded in the first month and were uniquely prevalent at the time of rejection. These cells were resistant to steroids, deoxyspergualin or sirolimus in vitro, but were calcineurin‐inhibitor sensitive. These data demonstrate that therapeutic depletion begets a limited population of functional memory‐like T cells that are easily suppressed with certain immunosuppressants, but cannot be considered uniquely pro‐tolerant.

results From A Human Renal Allograft Tolerance Trial Evaluating The Humanized Cd52-specific Monoclonal Antibody Alemtuzumab (campath-1h)

Background. Profound T‐cell depletion before allotransplantation with gradual posttransplant T‐cell repopulation induces a state of donor‐specific immune hyporesponsiveness or tolerance in some animal models. Alemtuzumab (Campath‐1H, Millennium Pharmaceuticals, Cambridge, MA) is a humanized CD52‐specific monoclonal antibody that produces profound T‐cell depletion in humans and reduces the need for maintenance immunosuppression after renal transplantation. We therefore performed a study to determine if pretransplant T‐cell depletion with alemtuzumab would induce tolerance in human renal allografts and to evaluate the nature of the alloimmune response in the setting of T‐cell depletion. Methods. Seven nonsensitized recipients of livingdonor kidneys were treated perioperatively with alemtuzumab and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically by peripheral flow cytometry, protocol biopsies evaluated immunohistochemically, and real‐time polymerase chain reaction‐based transcriptional analysis. Results. Lymphocyte depletion was profound in the periphery and secondary lymphoid tissues. All patients developed reversible rejection episodes within the first month that were characterized by predominantly monocytic (not lymphocytic) infiltrates with only rare T cells in the peripheral blood or allograft. These episodes were responsive to treatment with steroids or sirolimus or both. After therapy, patients remained rejection‐free on reduced immunosuppression, generally monotherapy sirolimus, despite the recovery of lymphocytes to normal levels. Conclusions. T‐cell depletion alone does not induce tolerance in humans. These data underscore a prominent role for early responding monocytes in human allograft rejection.

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.

Functionally significant renal allograft rejection is defined by transcriptional criteria

Renal allograft acute cellular rejection (ACR) is a T‐cell mediated disease that is diagnosed histologically. However, many normally functioning allografts have T‐cell infiltrates and histological ACR, and many nonimmune processes cause allograft dysfunction. Thus, neither histological nor functional criteria are sufficient to establish a significant rejection, and the fundamental features of clinical rejection remain undefined. To differentiate allograft lymphocyte infiltration from clinically significant ACR, we compared renal biopsies from patients with ACR to patients with: sub‐clinical rejection (SCR, stable function with histological rejection); no rejection; and nontransplanted kidneys. Biopsies were compared histologically and transcriptionally by RT‐PCR for 72 relevant immune function genes. Neither the degree nor the composition of the infiltrate defined ACR. However, transcripts up‐regulated during effector TH1 T‐cell activation, most significantly the transcription factor T‐bet, the effector receptor Fas ligand and the costimulation molecule CD152 clearly (p = 0.001) distinguished the patient categories. Transcripts from other genes were equivalently elevated in SCR and ACR, indicating their association with infiltration, not dysfunction. Clinically significant ACR is not defined solely by the magnitude nor composition of the infiltrate, but rather by the transcriptional activity of the infiltrating cells. Quantitative analysis of selected gene transcripts may enhance the clinical assessment of allografts.

Results from a human renal allograft tolerance trial evaluating T-cell depletion with alemtuzumab combined with deoxyspergualin.

Background. Perioperative lymphocyte depletion induces allograft tolerance in some animal models, but in humans has only been shown to reduce immunosuppressive requirements. Without maintenance immunosuppression, depleted human renal allograft recipients experience rejection characterized by infiltration of the allograft with monocytes and macrophages. T-cell depletion combined with a brief course of deoxyspergualin (DSG), a drug with inhibitory effects on monocytes and macrophages, induces tolerance in nonhuman primates. We therefore performed a trial to determine if lymphocyte depletion with alemtuzumab combined with DSG would induce tolerance in humans. Methods. Five recipients of live donor kidneys were treated perioperatively with alemtuzumab and DSG and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically, by flow cytometry, and by protocol biopsies analyzed immunohistochemically and with real-time polymerase chain reaction. Results were compared to previously studied patients receiving alemtuzumab alone or standard immunosuppression. Results. Despite profound T-cell depletion and therapeutic DSG dosing, all alemtuzumab/DSG patients developed reversible rejection that was similar in timing, histology, and transcriptional profile to that seen in patients treated with alemtuzumab alone. Chemokine expression was marked prior to and during rejections. Conclusions. We conclude that treatment with alemtuzumab and DSG does not induce tolerance in humans. Chemokine production may not be adequately suppressed using this approach.

Molecular evaluation of BK polyomavirus nephropathy.

Understanding at a molecular level, the immunologic response of polyomavirus nephropathy (PVN), a critical cause of kidney graft loss, could lead to new targets for treatment and diagnosis. We undertook a transcriptional evaluation of kidney allograft biopsies from recipients with PVN or acute rejection (AR), as well as from recipients with stable allograft function (SF). In both the PVN and AR groups, Banff histologic scores and immunohistochemical analysis of inflammatory infiltrates were similar. Despite their different etiologies, the transcriptional profiles of PVN and AR were remarkably similar. However, transcription of genes previously linked to AR including CD8 (65.9 ± 18.8) and related molecules IFN‐γ(55.1 ± 17.0), CXCR3 (49.9 ± 12.8) and perforin (153.8 ± 50.4) were significantly higher in PVN compared to AR (30.9 ± 2.0, 14.0 ± 7.3, 12.1 ± 7.3 and 15.6 ± 3.8‐fold, respectively; p < 0.01). Importantly, transcription of molecules associated with graft fibrosis including matrix collagens, TGFβ, MMP2 and 9, as well as markers of epithelial‐mesenchymal transformation (EMT) were significantly higher in PVN than AR. Thus, renal allografts with PVN transcribe proinflammatory genes equal in character and larger in magnitude to that seen during acute cellular rejection. BK infection creates a transcriptional microenvironment that promotes graft fibrosis. These findings provide new insights into the intrarenal inflammation of BK infection that promotes graft loss.

Disruption of CD40/CD40-Ligand Interactions in a Retinal Autoimmunity Model Results in Protection without Tolerance

We examined the role of CD40/CD40L interactions on the development of experimental autoimmune uveoretinitis (EAU), a cell-mediated, Th1-driven autoimmune disease that serves as a model for autoimmune uveitis in humans. EAU-susceptible B10.RIII mice immunized with the retinal autoantigen interphotoreceptor retinoid binding protein in CFA and treated with anti-CD40L Ab (MR1) had reduced incidence and severity of disease. Real-time PCR analysis revealed that the innate and adaptive responses of protected mice were reduced, without an obvious shift toward a Th2 cytokine profile. In contrast to some other reports, no evidence was found for regulatory cells in adoptive transfer experiments. To determine whether CD40L blockade resulted in long-term tolerance, mice protected by treatment with MR1 Ab were rechallenged for uveitis after circulating MR1 Ab levels dropped below the detection limit of ELISA. MR1-treated mice developed severe EAU and strong cellular responses to interphotoreceptor retinoid binding protein, comparable to those of control mice. These responses were higher than in mice that had not received the primary immunization concurrently with anti-CD40L treatment. We conclude that 1) CD40/CD40L interaction is required for EAU and its disruption prevents disease development; 2) CD40L blockade inhibits the innate response to immunization and reduces priming, but does not result in immune deviation; and 3) protection is dependent on persistence of anti-CD40L Abs, and long-term tolerance is not induced. Furthermore, immunological memory develops under cover of CD40L blockade causing enhanced responses upon rechallenge. Taken together, our data suggest that ongoing CD40/CD40L blockade might be required to maintain a therapeutic effect against uveitis.

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.