Key role for CD4 T cells during mixed antibody-mediated rejection of renal allografts.

We utilized mouse models to elucidate the immunologic mechanisms of functional graft loss during mixed antibody-mediated rejection of renal allografts (mixed AMR), in which humoral and cellular responses to the graft occur concomitantly. Although the majority of T cells in the graft at the time of rejection were CD8 T cells with only a minor population of CD4 T cells, depletion of CD4 but not CD8 cells prevented acute graft loss during mixed AMR. CD4 depletion eliminated antidonor alloantibodies and conferred protection from destruction of renal allografts. ELISPOT revealed that CD4 T effectors responded to donor alloantigens by both the direct and indirect pathways of allorecognition. In transfer studies, CD4 T effectors primed to donor alloantigens were highly effective at promoting acute graft dysfunction, and exhibited the attributes of effector T cells. Laser capture microdissection and confirmatory immunostaining studies revealed that CD4 T cells infiltrating the graft produced effector molecules with graft destructive potential. Bioluminescent imaging confirmed that CD4 T effectors traffic to the graft site in immune replete hosts. These data document that host CD4 T cells can promote acute dysfunction of renal allografts by directly mediating graft injury in addition to facilitating antidonor alloantibody responses.

The pathogenesis of acute allograft dysfunction in desensitized renal transplant recipients.

BACKGROUND: Acute allograft rejection after HLA desensitization is common early post-transplant but the sequence of histopathologic changes leading to graft dysfunction has not been well defined. METHODS: We evaluated the early pathogenesis and sequence of antibody-mediated graft damage of 35 desensitized living donor kidney recipients by studying the course of biopsies taken in the very early post-transplant period (<1 month). RESULTS: A total of 14 of the 35 patients met criteria for acute antibody-mediated rejection (AMR). In these patients, the chronologic sequence of pathologic changes was C4d peritubular capillary deposition, acute tubular injury, and peritubular capillaritis, followed by glomerulitis and interstitial inflammation. Classic AMR lesions occurred early, followed by mononuclear cellular infiltration, which comprised CD4 and CD8 T cells and monocytes. Development of graft dysfunction in most patients occurred concurrently with the emergence of graft cellular infiltration, rather than at the earlier time of antibody deposition as detected via C4d deposition. CONCLUSION: These data provide novel insight into the sequence of pathologic changes in patients with AMR post-transplant after HLA desensitization.

An anti-CD103 immunotoxin promotes long-term survival of pancreatic islet allografts.

Previous studies using knockout mice document a key role for the integrin CD103 in promoting organ allograft rejection and graft-versus-host disease. However, a determination of whether blockade of the CD103 pathway represents a viable therapeutic strategy for intervention in these processes has proven problematic due to the lack of reagents that efficiently deplete CD103+ cells from wild type hosts. To circumvent this problem, we conjugated the nondepleting anti-CD103 monoclonal antibody, M290, to the toxin, saporin, to produce an immunotoxin (M290-SAP) that efficiently depletes CD103+ cells in vivo. Herein, we show that M290-SAP dramatically reduces the frequency and absolute numbers of CD103-expressing leukocytes in the blood, spleen, mesenteric lymph nodes and intestinal epithelium of treated mice. We further demonstrate that M290-SAP promotes indefinite islet allograft survival in a fully MHC mismatched mouse model. The prolonged islet allograft survival resulting from M290-SAP treatment was associated with multiple effects in the host immune system including not only depletion of CD103-expressing leukocytes, but also an increase in CD4+CD25+FoxP3+ T regulatory cells and a predominance of effector-memory CD8 T cells. Regardless of the underlying mechanisms, these data document that depletion of CD103-expressing cells represents a viable strategy for therapeutic intervention in allograft rejection.

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 approximately 80% graft loss by 100 days after transplant, compared with approximately 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-alpha 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.

Tubulitis and epithelial cell alterations in mouse kidney transplant rejection are independent of CD103, perforin or granzymes A/B.

One of the defining lesions of kidney allograft rejection is epithelial deterioration and invasion by inflammatory cells (tubulitis). We examined epithelial changes and their relationship to effector T cells and to CD103/E-cadherin interactions in mouse kidney allografts. Rejecting allografts showed interstitial mononuclear infiltration from day 5. Loss of epithelial mass, estimated by tubular surface area, and tubulitis were minimal through day 7 and severe by day 21. Tubules in day 21 allografts manifested severe reduction of E-cadherin and Ksp-cadherin by immunostaining with redistribution to the apical membrane, indicating loss of polarity. By flow cytometry T cells isolated from allografts were 25% CD103+. Laser capture microdissection and RT-PCR showed increased CD103 mRNA in the interstitium and tubules. However, allografts in hosts lacking CD103 developed tubulitis, cadherin loss, and epithelial deterioration similar to wild-type hosts. The loss of cadherins and epithelial mass was also independent of perforin and granzymes A and B. Thus rejection is characterized by severe tubular deterioration associated with CD103+ T cells but not mediated by CD103/cadherin interactions or granzyme-perforin cytotoxic mechanisms. We suggest that alloimmune effector T cells mediate epithelial injury by contact-independent mechanisms related to delayed type hypersensitivity, followed by invasion of the altered epithelium to produce tubulitis.

CD103+ CTL accumulate within the graft epithelium during clinical renal allograft rejection.

BACKGROUND: We have previously reported that activated CD8+TCRalphabeta+ cells that express high levels of the beta7 integrin CD103 (formerly alphaE, MLA) are present at the graft site during clinical renal allograft rejection. This observation potentially provides new insight into the mechanisms underlying renal allograft destruction because the ligand of CD103 is the epithelial cell-specific molecule E-cadherin, which is known to be expressed by critical graft functional elements such as the renal tubular epithelium. We herein used combined fluorescence-activated cell sorter (FACS) and immunohistochemical (IHC) analyses of transplant nephrectomy (TN) specimens to demonstrate that CD103+ cytolytic T lymphocytes (CTLs) specifically home to the graft epithelium during rejection episodes. METHODS: Serial sections of TN specimens undergoing histologically confirmed cellular rejection (n=7) were stained with anti-CD8 or anti-CD103 and were scored for the presence of positively stained cells within the tubular basement membrane. Freshly isolated graft-infiltrating lymphocytes were subjected to three-color FACS analyses to define the extended phenotypic characteristics of CD103+ cells detected by IHC. RESULTS: CD103+ cells in all specimens were biased towards an intratubular localization. On average, the percentage of CD103+ cells with an intraepithelial localization was 52.2+/-13.1 compared to 12.0+/-3.5 for pan CD8+ cells (mean+/-SE, n=5). FACS analyses confirmed that CD103+ cells detected by IHC exhibited the salient characteristics of CD8+ CTLs (large CD8+TCRalphabeta+CD62L-CD11a(hi)perforin+). The CD103- subset of graft-infiltrating CD8 cells also exhibited a CTL phenotype, but these were predominantly restricted to the graft interstitium. CONCLUSIONS: These data implicate CD103 as a homing receptor that targets graft-infiltrating CD8+ CTLs to the graft epithelium. Given the strong association of tubulitis with clinical rejection, these data are consistent with a role for the CD103+ CTL subset as an effector mechanism in renal allograft destruction.

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 in definite 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.

Regulation of the epithelial cell-specific integrin, CD103, by human CD8+ cytolytic T lymphocytes.

BACKGROUND: The destruction of the graft epithelium by CD8+ cytolytic T lymphocytes (CTL) is an important aspect of organ allograft rejection. Our recent finding in a mouse model that the epithelial cell-specific integrin, CD103, defines a subset of CD8+ CTL potentially sheds new light onto such interactions. The goal of the present study was to assess the relevance of these data to the human system. METHODS: CD103 expression by human T-cell populations generated in mixed lymphocyte cultures or isolated from transplant nephrectomy specimens was quantitated using multiparameter FACS analyses. RESULTS: CD103 defined a major subset (26-76%) of CD8+ CTL generated in human mixed lymphocyte cultures; cell sorting experiments confirmed that the CD103+ and CD103- subsets both possess allospecific lytic activity. Anti-transforming growth factor (TGF)-beta blocked the appearance of the CD103+ CTL subset, and persistent expression of CD103 by CD8+ CTL was dependent on bioactive TGF-beta. Isolated CD103+ and CD103- CD8 subsets maintained their phenotypic integrity during in vitro expansion, although optimal CD103 expression on the former was TGF-beta dependent. Although CD103+ cells were rare among activated CD8 cells in peripheral lymphoid compartments (< 10%), analyses of transplant nephrectomy specimens revealed that a major subset (21-61%) of CD8 memory/effector cells that infiltrate rejecting renal allografts express high levels of CD103. CONCLUSIONS: We conclude that CD103 defines a discrete and stable subset of human CD8+ CTL and that CD103 expression by such cells is initiated and maintained by bioactive TGF-beta. These data point to the existence of a human effector subset that is uniquely specialized for the destruction of the graft epithelium.

Intracellular delivery of a cytolytic T-lymphocyte epitope peptide by pertussis toxin to major histocompatibility complex class I without involvement of the cytosolic class I antigen processing pathway.

A CD8(+) cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion by Bordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.

Integrin-mediated interactions influence the tissue specificity of CD8+ cytolytic T lymphocytes.

We previously reported that CD8+ cytotoxic T lymphocytes (CTL) elicited in response to allogeneic renal epithelial cells (anti-REC CTL) preferentially lyse REC targets as compared to conventional lymphoid cell (LC) targets. It is often tacitly assumed that such cell type specificity results from CTL recognition of tissue-restricted MHC/peptide complexes. However, we herein report that anti-REC CTL uniquely express CD103, an integrin with known specificity for the epithelial cell-restricted ligand E-cadherin, and are deficient in expression of CD11a (LFA-1), an integrin known to play a critical accessory role in promoting lysis of LC targets. We demonstrate that CD8+ CTL clones with disparate CD103/CD11a phenotypes but identical specificities for allo-MHC/peptide can exhibit marked differences in cell type specificity. Antibody blocking studies provided direct evidence that CD103 serves as an accessory molecule that promotes lysis of REC targets. Taken together, these data indicate that integrin-mediated accessory interactions can influence the capacity of CD8+ CTL to discriminate between different cell types.

The epithelial cell-specific integrin, CD103 (alpha E integrin), defines a novel subset of alloreactive CD8+ CTL.

The interaction of CD8+CTL with epithelial layers is an important but poorly defined aspect of organ allograft rejection. We herein report that CD103 (formerly alpha E integrin), a known receptor for the epithelial cell-specific ligand E-cadherin, is expressed by a major subset of CD8 + CTL elicited in response to allogeneic renal epithelial cells (REC). In contrast, CD103 was expressed poorly on CD8 + CTL generated in the conventional manner by stimulation with allogeneic leukocytes, although expression could be dramatically up-regulated by supplementing cultures with REC or exogenous TGF-beta 1. That TGF-beta controls the expression of CD103 on CD8+ CTL was further supported by the capacity of anti-TGF-beta mAb to block the generation of such cells in anti-REC cultures. Clonal analyses of anti-REC cultures revealed that individual CD8+ CTL clones were discretely CD103+ or CD103-, nd maintained their respective phenotypes independently of the cell type used for clonal restimulation. In a mouse model of graft-vs-host disease, 16.4 +/- 2.7% of CD8 cells that infiltrated host kidneys were CD103+ (n = 4). CD8 kidney-infiltrating lymphocytes were predominantly of donor origin and displayed an activated/memory phenotype (CD62L-, CD44high), consistent with expression of CD103 on a CD8 effector subset elicited in vivo following allogeneic transplantation. Taken together, the present data demonstrate that CD103 identifies a novel CD8 effector subset and, moreover, that such cells may comprise a significant component of the response to allogeneic tissues. The potential for CD103+ CTL as an important effector mechanism in organ allograft rejection, and more generally, as a mechanistic basis for tissue-specific immune phenomena, is discussed.

Interaction between cytolytic T-cells and immobilized renal tubular epithelial cells: evidence of polarity on both effector and target cells.

The mode by which cytolytic T-lymphocytes (CTL) deliver the lethal hit to their targets has been studied extensively. The CTL exhibits a characteristic polarization, with the attacking end formed by the uropod (containing the Golgi complex and associated granules). The effect on the target cell (TC) has been considered as a form of apoptosis or of both necrosis. In this communication we describe the particular morphological steps that relate to the participation of the target renal epithelial cell to its own destruction. Thus, the TC forms tight membrane contacts with the CTL that are invested by a dense network of stress fibers and microtubules within the TC cytoplasm in a manner similar to the areas of attachment between the epithelial cells or of the latter to the underlying surface. The TC mitochondria at the site of the CTL attachment are markedly swollen, in contrast to the more distally located ones that appear initially normal. The TC plasma membrane surrounding the CTL attachment site shows focally intense blebbing. In later stages, associated with overt apoptosis, global TC blebbing is present. In summary, the TC shows a polarization pattern, that corresponds roughly to the polarity exhibited by the attacking cell (uropod-protopod), and to some degree treats the latter cell as a junctional partner.

Prevention of autoimmune islet allograft destruction by engraftment of donor T cells.

The results of clinical islet transplantation have remained poor when compared with the consistent success of pancreas transplantation. Autoimmunity has usually been discounted as a cause of islet transplant failure. Previously, we demonstrated that pancreas transplants from the diabetes resistant BB rat (BB-DR) function indefinitely in autoimmune diabetic hosts, but islets from the same donor are vulnerable to recurrent autoimmunity. Addition of 100 million pancreatic lymph node cells (PLNC) to BB-DR islets restores resistance to autoimmunity and leads to repletion of a T cell subset (RT6.1) in the recipients. Autoimmune (BB-Ac) and streptozocin (BB-Sz) diabetic BB rats were recipients of Wistar Furth (WF) intraportal islet or islets plus PLNC transplants with cyclosporine 5 mg/kg/day recipient treatment. One cohort of Brown Norway (BN) islet transplants to BB-Ac with CsA was performed. At the termination of the experiment, recipient peripheral blood lymphocytes (PBL) were characterized by flow cytometry (FACS) for class I, CD4, CD8, RT6.1, and RT6.2, a T cell maturation marker found in WF but not BB rats. All (14/14) WF and 75% (6/8) BN islet transplants to BB-Ac recipients failed after a mean of 42 and 36 days, respectively, despite CsA immunosuppression. WF islets were successful in 6/8 (75%) transplants to BB-Sz recipients (P<0.001 vs. BB-Ac recipients), confirming that autoimmunity is the major cause of islet failure in BB-Ac rats. Addition of PLNC to WF islets increased the survival in BB-Ac to 82% (9/11) (P<0.0001 vs. WF islets alone). Recipients of islet+PLNC express 19.7% RT6.2 compared with 4.6% and 4.0% for WF islets alone in BB-Ac (P<0.01) and BB-Sz (P<0.01), respectively. Autoimmunity is an important factor leading to islet transplant failure in autoimmune diabetic BB rats. Addition of donor PLNC prevent islet allograft failure and leads to recipient chimerism for a donor T cell subset (RT6.2) associated with resistance to autoimmunity.

Dominance of tissue-restricted cytotoxic T lymphocytes in the response to allogeneic renal epithelial cell lines.

Our current knowledge of cytotoxic T lymphocytes (CTL) is largely derived from studies of effector populations generated in allogeneic mixed leukocyte cultures (MLC) and assayed for lytic activity to lymphoid cell (LC) targets. We herein report that the CTL response to allogeneic renal epithelial cell lines (REC) is dominated by effectors that efficiently lyse REC targets but show little cross-reactivity with LC targets. In contrast, CTL generated against allogeneic spleen cell stimulators (ie., in MLC) lysed REC and LC targets at comparable levels. Lytic activity in both types of cultures was mediated by CD8+TCRalpha/beta+ cells directed to classical H2 class I alloantigens. Anti-REC effectors cross-reacted with fibroblast and macrophage targets but not with targets commonly used to detect alloreactive CTL, such as lipopolysaccharide- or Con A-stimulated lymphoblasts or lymphoid tumor lines, whereas MLC-elicited effectors efficiently lysed all targets. CTL clones propagated from anti-REC cultures exhibited the same allospecificity and tissue specificity as bulk anti-REC effectors. Individual CTL clones were highly heterogeneous in their capacity to recognize the same class I alloantigen expressed on cells derived from different tissues. These data demonstrate that the cellular environment in which CD8 precursors encounter class I alloantigens can have a profound effect on the cell-type specificity of CTL populations. An important implication of these data is that conventional assays of CTL lytic activity may fail to reveal a significant component of the host response to allogeneic tissues.

Prolonged cardiac xenograft survival is induced by intrathymic splenocyte injection.

The shortage of suitable human organs currently limits widespread utilization of clinical transplantation. Successful xenotransplantation could potentially eliminate the shortage of suitable organs for transplantation. The present study determines the effect of intrathymic donor spleen cell injection on the survival of cardiac xenografts performed without post-transplant immunosuppression. Wistar-Furth rats served as recipients of Lewis rat cardiac allografts or Golden Syrian hamster cardiac xenografts either with or without intrathymic donor spleen cell injection. Some xenograft cohorts were pretreated with four doses of cyclophosphamide prior to cardiac transplantation. Graft function was assessed by daily graft palpation and rejection was defined as cessation of graft function. Cardiac allograft median survival time (MST) was 8 days in untreated recipients. Pretreatment with intrathymic donor spleen cell injection led to essentially indefinite allograft survival (MST > 100 days). Control xenograft survival was 3 days with no significant prolongation seen when intrathymic donor spleen cell injection was performed (MST = 2.5 days). The addition of pulse-dose cyclophosphamide resulted in markedly prolonged xenograft survival (MST = 30 days) compared to control (P < 0.01) and to cyclophosphamide alone (MST = 9.5 days, P < 0.01). Cardiac xenografts undergo a vigorous rapid rejection. The time course of rejection was not altered by the intrathymic presence of donor cells under conditions which lead to indefinite survival of cardiac allografts. A brief period of pretransplant cyclophosphamide treatment markedly improved the success of the thymic tolerance protocol in xenografts. This finding suggests that temporary inhibition of humoral immunity may be permissive for the development of thymic tolerance to cell-mediated immunity in xenotransplants.

Composite kidney-islet transplantation prevents recurrent autoimmune beta-cell destruction.

BACKGROUND: Clinically successful islet transplantation has been rare despite adequate isolation techniques. Reenactment of the original autoimmune beta-cell destruction may contribute to the poor results. Distinguishing autoimmune effects from rejection can be accomplished with isogeneic transplants exchanged between diabetes-prone (BB-DP) and diabetes-resistant (BB-DR) rats. These experiments determine the relative sensitivity of islet, whole pancreas, and composite kidney-islet transplants to recurrent autoimmunity. METHODS: Acutely diabetic (BB-Ac) BB rats served as recipients of vascularized pancreas, intraportal (IPo) or renal capsular (KC) islet transplants, or vascularized composite kidney-islet grafts from BB-DR or BB-DP donors. Graft function was assessed by daily blood glucose level, and the outcome was confirmed on histologic examination. Cyclosporine 5 mg/kg/day intramuscularly was administered to assess its effect on recurrent beta-cell injury. RESULTS: BB-DP pancreases developed recurrent autoimmunity in 55% of cases; cyclosporine afforded complete protection if maintained. Diabetes resistance was transplanted with 23 of 23 BB-DR pancreas grafts; however, islet isolation led to a loss of diabetes resistance for islet grafts to the KC and IPo. Cyclosporine protected KC but not IPo islets. Composite BB-DR kidney-islet transplants functioned indefinitely in all cases. CONCLUSIONS: Transplanted islets initially survive by passive diffusion but are ultimately revascularized by capillary ingrowth. The finding that composite kidney-islet transplants function indefinitely suggests that the revascularizing endothelium may play a role in resistance or susceptibility to autoimmune beta-cell destruction.

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 allografts.

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.