Gregg A. Hadley

TGF-β–dependent CD103 expression by CD8+ T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease

Destruction of the host intestinal epithelium by donor effector T cell populations is a hallmark of graft-versus-host disease (GVHD), but the underlying mechanisms remain obscure. We demonstrate that CD8+ T cells expressing CD103, an integrin conferring specificity for the epithelial ligand E-cadherin, play a critical role in this process. A TCR transgenic GVHD model was used to demonstrate that CD103 is selectively expressed by host-specific CD8+ T cell effector populations (CD8 effectors) that accumulate in the host intestinal epithelium during GVHD. Although host-specific CD8 effectors infiltrated a wide range of host compartments, only those infiltrating the intestinal epithelium expressed CD103. Host-specific CD8 effectors expressing a TGF-β dominant negative type II receptor were defective in CD103 expression on entry into the intestinal epithelium, which indicates local TGF-β activity as a critical regulating factor. Host-specific CD8 effectors deficient in CD103 expression successfully migrated into the host intestinal epithelium but were retained at this site much less efficiently than wild-type host-specific CD8 effectors. The relevance of these events to GVHD pathogenesis is supported by the finding that CD103-deficient CD8+ T cells were strikingly defective in transferring intestinal GVHD pathology and mortality. Collectively, these data document a pivotal role for TGF-β–dependent CD103 expression in dictating the gut tropism, and hence the destructive potential, of CD8+ T cells during GVHD pathogenesis.

CD103 Expression Is Required for Destruction of Pancreatic Islet Allografts by CD8+ T Cells

The mechanisms by which CD8 effector populations interact with epithelial layers is a poorly defined aspect of adaptive immunity. Recognition that CD8 effectors have the capacity to express CD103, an integrin directed to the epithelial cell-specific ligand E-cadherin, potentially provides insight into such interactions. To assess the role of CD103 in promoting CD8-mediated destruction of epithelial layers, we herein examined the capacity of mice with targeted disruption of CD103 to reject pancreatic islet allografts. Wild-type hosts uniformly rejected islet allografts, concomitant with the appearance of CD8+CD103+ effectors at the graft site. In contrast, the majority of islet allografts transplanted into CD103−/− hosts survived indefinitely. Transfer of wild-type CD8 cells into CD103−/− hosts elicited prompt rejection of long-surviving islet allografts, whereas CD103−/− CD8 cells were completely ineffectual, demonstrating that the defect resides at the level of the CD8 cell. CD8 cells in CD103−/− hosts exhibited normal effector responses to donor alloantigens in vitro and trafficked normally to the graft site, but strikingly failed to infiltrate the islet allograft itself. These data establish a causal relationship between CD8+CD103+ effectors and destruction of graft epithelial elements and suggest that CD103 critically functions to promote intragraft migration of CD8 effectors into epithelial compartments.

Environmental and Antigen Receptor-Derived Signals Support Sustained Surveillance of the Lungs by Pathogen-Specific Cytotoxic T Lymphocytes

ABSTRACT Viral infections often gain access to the body of their host by exploiting areas of natural vulnerability, such as the semipermeable surfaces of mucosal tissues which are adapted for adsorption of nutrients and other diffusible molecules. Once the microbes have crossed the epithelial barrier, they can disperse to other tissues where eradication may not be possible. The best opportunity for successful immune intervention is immediately after infection while the pathogen is confined to a localized area of the body. Cytotoxic T lymphocytes (CTL) which reside at the site where the infection begins can make an important contribution to immunity by reducing early dissemination of the infection. Because the lungs provide easy access points for many pathogens to enter the body, they require protection from many complementary mechanisms, including pathogen-specific cytotoxic T cells. In this study we show that an enduring response to pathogen-derived peptide antigens facilitates sustained surveillance of the lungs by pathogen-specific CTL during the recovery from influenza virus infection. Our studies show that these processed peptide antigens reinforce expression of two homing receptors (CD69 and CD103) which help recently activated virus-specific CTL colonize the lungs during a mild inflammatory response. We suggest that this requirement for prolonged antigen presentation to reinforce local CTL responses in the lungs explains why protective cellular immunity quickly declines following influenza virus infection and other viral infections that enter the body via mucosal tissues.

Regulation of acute graft-versus-host disease by microRNA-155

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD. Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155-deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic anti-miR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.

Recurrent Autoimmunity Accelerates Destruction of Minor and Major Histoincompatible Islet Grafts in Nonobese Diabetic (NOD) Mice

Recurrent autoimmunity destroys nonobese diabetic (NOD) islet isografts, but whether recurrent autoimmunity contributes to islet graft destruction in immunocompetent allogeneic recipients is unknown. In the NOD, a single dose of streptozocin prevents or delays primary autoimmunity, allowing the detection of alloimmunity alone in chemically diabetic hosts (streptozocin‐NOD) to be compared to the combined effects of autoimmunity and alloimmunity in spontaneously diabetic NODs (autoimmune‐NOD). Islets were isolated from prediabetic NOD (H‐2KdDb), nonobese resistant (NOR) (H‐2KdDb), Balb/cByJ (H‐2d) and B10.BR (H‐2k) donors and transplanted to either the renal subcapsule or the intraportal site in autoimmune‐NODs or streptozocin‐NODs. MHC‐matched NOR islets had indefinite graft survival in streptozocin‐NODs. However, NOR islets showed graft loss at 12.6 ± 3.2 days in renal subcapsule and at 6.8 ± 0.1 days in intraportal site of autoimmune‐NODs. Partially MHC‐matched Balb/cByJ islet grafts failed significantly sooner in autoimmune‐NODs than in streptozocin‐NODs (p < 0.005). Fully MHC‐mismatched B10.BR islet grafts also failed sooner in autoimmune‐NODs, but the difference did not reach significance (p < 0.06). Although the streptozocin‐NOD was functionally tolerant of MHC‐matched NOR islets, NOR islets transplanted into autoimmune‐NODs failed sooner than NOD islets in both renal subcapsule (12.6 ± 3.2 days vs. 26.4 ± 10.5 days, p = 0.009) and intraportal sites (6.8 ± 0.1 days vs. 11.5 ± 1.7 days, p = 0.014). In the autoimmune‐NODs, the intraportal site consistently showed shorter graft survival than the renal subcapsule site (NOD: p = 0.009, NOR: p = 0.014, Balb/cByJ: p = 0.008, B10.BR: p = 0.032). In conclusion, autoimmune processes facilitate the alloimmune response to minor and major histocompatibility antigens and accelerate graft destruction. The same autoimmune processes are more pronounced in the intraportal site.

Acute Humoral Rejection of Renal Allografts in CCR5 -/- Recipients

Increasing detection of acute humoral rejection (AHR) of renal allografts has generated the need for appropriate animal models to investigate underlying mechanisms. Murine recipients lacking the chemokine receptor CCR5 reject cardiac allografts with marked C3d deposition in the parenchymal capillaries and high serum donor‐reactive antibody titers, features consistent with AHR. The rejection of MHC‐mismatched renal allografts from A/J (H‐2a) donors by B6.CCR5–/– (H‐2b) recipients was investigated. A/J renal allografts survived longer than 100 days in wild‐type C57BL/6 recipients with normal blood creatinine levels (28 ± 7 μmol/L). All CCR5–/– recipients rejected renal allografts within 21 days posttransplant (mean 13.3 ± 4 days) with elevated creatinine (90 ± 31 μmol/L). The rejected allografts had neutrophil and macrophage margination and diffuse C3d deposition in peritubular capillaries, interstitial hemorrhage and edema, and glomerular fibrin deposition. Circulating donor‐reactive antibody titers were 40‐fold higher in B6.CCR5–/– versus wild‐type recipients. Depletion of recipient CD8 T cells did not circumvent rejection of the renal allografts by CCR5‐deficient recipients. In contrast, μMT–/–/CCR5–/– recipients, incapable of producing antibody, did not reject most renal allografts. Collectively, these results indicate the rapid rejection of renal allografts in CCR5–/– recipients with many histopathologic features observed during AHR of human renal allografts.

Critical Role for CD103+CD8+ Effectors in Promoting Tubular Injury following Allogeneic Renal Transplantation

Immune destruction of the graft renal tubules is an important barrier to the long-term function of clinical renal allografts, but the underlying mechanisms remain obscure. CD103—an integrin conferring specificity for the epithelial cell-restricted ligand, E-cadherin—defines a subset of CD8 effectors that infiltrate the graft tubular epithelium during clinical rejection episodes, predicting a causal role for CD103+CD8+ effectors in tubular injury. In the present study, we used rodent transplant models to directly test this hypothesis. Surprisingly, CD8 cells infiltrating renal allografts undergoing unmodified acute rejection did not express significant levels of CD103. However, we demonstrate that a brief course of cyclosporine A to rat renal allograft recipients promotes progressive accumulation of CD103+CD8+ cells within the graft, concomitant with the development of tubular atrophy and interstitial fibrosis. As in the known clinical scenario, graft-associated CD103+CD8+ cells exhibited a T effector phenotype and were intimately associated with the renal tubular epithelium. Treatment with anti-CD103 mAb dramatically attenuated CD8 infiltration into the renal tubules and tubular injury. Mouse studies documented that CD103 expression is required for efficient destruction of the graft renal tubules by CD8 effectors directed to donor MHC I alloantigens. Taken together, these data document a causal role for CD103+CD8+ effectors in promoting tubular injury following allogeneic renal transplantation and identify novel targets for therapeutic intervention in this important clinical problem.

Immunogenicity of MHC class I alloantigens expressed on parenchymal cells in the human kidney

The goal of the present study was to examine the capacity of human kidney cell lines (KCL) to elicit T cell responses to MHC class I alloantigens. KCL exhibited the phenotypic characteristics of tubular epithelial cells and were devoid of detectable contamination with leukocytes. Coculture of normal peripheral blood leukocytes (PBL) with allogeneic KCL elicited cytolytic T lymphocytes (CTL) that lysed the stimulating KCL but failed to lyse third-party KCL. Cell panel and antibody blocking studies demonstrated that the CTL were directed to the HLA class I alloantigens expressed on the KCL stimulators. Purified T cells completely failed to mount a CTL response to KCL, but the response could be reconstituted by supplementing the cultures with either autologous non-T cells or supernatant from a mixed lymphocyte culture (MLC). IL-2, but not IL-1, IL-4, IL-6, or gamma-interferon, restored the anti-KCL response, suggesting that IL-2 is the active factor in the MLC supernatant. Induction of class II antigens on the KCL stimulators with gamma-IFN failed to restore a CTL response, suggesting that KCL are deficient in a costimulatory factor important for class II restricted T helper responses. Nonetheless, our data demonstrate that parenchymal cells in the kidney are capable of presenting class I antigens to alloreactive T cells and, therefore, may contribute to the immunogenicity of renal allografts.

Disruption of murine cardiac allograft acceptance by latent cytomegalovirus.

Cytomegalovirus (CMV) reactivation is a well‐described complication of solid organ transplantation. These studies were performed to (1) determine if cardiac allograft transplantation of latently infected recipients results in reactivation of CMV and (2) determine what impact CMV might have on development of graft acceptance/tolerance. BALB/c cardiac allografts were transplanted into C57BL/6 mice with/without latent murine CMV (MCMV). Recipients were treated with gallium nitrate induction and monitored for graft survival, viral immunity and donor reactive DTH responses. Latently infected allograft recipients had ∼80% graft loss by 100 days after transplant, compared with ∼8% graft loss in naïve recipients. PCR evaluation demonstrated that MCMV was transmitted to cardiac grafts in all latently infected recipients, and 4/8 allografts had active viral transcription compared to 0/6 isografts. Latently infected allograft recipients showed intragraft IFN‐α expression consistent with MCMV reactivation, but MCMV did not appear to negatively influence regulatory gene expression. Infected allograft recipients had disruption of splenocyte DTH regulation, but recipient splenocytes remained unresponsive to donor antigen even after allograft losses. These data suggest that transplantation in an environment of latent CMV infection may reactivate virus, and that intragraft responses disrupt development of allograft acceptance.

Kidney cell-restricted recognition of MHC class I alloantigens by human cytolytic T cell clones.

The kidney expresses antigens not generally expressed by other cells in the body. To test the hypothesis that kidney-restricted antigens alter T cell recognition of MHC class I alloantigens, a panel of human T cell clones was established using an allogeneic kidney cell line (KCL) as the source of stimulator cells. Unexpectedly, a majority of these clones lysed the stimulating KCL in an allospecific manner but completely failed to lyse lymphoid cell targets derived from the KCL donor. Three of the KCL-reactive clones have been characterized in detail. All three are CD8+/CD4−, alpha/beta TCR,+ and their lytic activity is blocked by monoclonals to HLA class I framework determinants. Mapping studies using a panel of KCL targets, and blocking studies with allele-specific monoclonals indicated that clone 1–5 is directed to HLA-A3, clone 3–10 to HLA-B62 (putatively), and clone 5–2 to HLA-B51. Direct lysis and cold target inhibition assays demonstrated that clones 1–5 and 3–10 recognize their target class I alloantigens on KCL but not on EBV-transformed B cells or PHA-stimulated T cells. FACS analysis and HLA phenotyping excluded quantitative or qualitative deficiencies in HLA class I expression on the lymphoid cell targets as likely mechanisms of tissue specificity. These data suggest that kidney-restricted antigens may play a role in T cell recognition of MHC class I alloantigens, and they raise the possibility that parenchymal cell-restricted effector populations may contribute to rejection of renal allo-grafts.