Since vascular endothelium is now recognized as an immunologically active tissue, a better understanding of the relationship between endothelial cells and T lymphocytes is critical to the field of solid organ transplantation. Investigations of endothelial cell-T cell interactions have been limited by methodology. We developed a flow cytometric method allowing for concurrent investigation of multiple cell populations within the same culture that can be applied to these complex interactions. Allogeneic CD8+ or CD4+ T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycle-blocking agent. At specific time points, the coculture was analyzed by flow cytometry. T-cell proliferation could be detected by gating on the T-cell subset and evaluating the CFSE fluorescence peaks. By directly analyzing cellular division, we minimized erroneous interpretation of the data encountered by previous studies, which utilized (3)H-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilitated the study of CD8+ lymphocyte activation, differentiation, and acquisition of effector function. By gating on the endothelial cell population, phenotypic changes such as upregulation of surface MHC molecules or immune-mediated apoptosis could be detected. In conclusion, we present a flow cytometric approach that could have important applications for clinical immunological monitoring in allogeneic or xenogeneic transplantation, and might provide the requisite information to better tailor immunotherapy to prevent chronic rejection.

The interaction between CD86, expressed on antigen presenting cells (APCs), and CD28 on T cells is critical for T cell activation. It has been shown that stimulation of peripheral blood lymphocytes with anti-CD3 and IL-2 induces CD86 expression on T cells, which are capable of providing costimulation to naı̈ve T cells in a human allogeneic system. The goal of our experiments was to determine whether human or porcine APCs can induce CD86 expression on human T cells and whether these T cells can function as APCs. Method: CFSE-labeled human T cells were co-cultured with human or porcine APCs. At day 5, the cultures were harvested and stained with labeled CD86, CD80, CD25 and anti-HLA-DR, CD4, CD8 and TOPRO-3 (viability probe). The samples were analyzed by four-color flow cytometry. Furthermore, T cells were isolated from cultures stimulated with anti-CD3 and Il-2, human APC or Pig APC and these cells were fixed with 1% paraformaldehyde. These cells were used to stimulate fresh syngeneic or allogeneic CFSE-labeled human T cells and these cultures were analyzed at day 7 by flow cytometry. Results: At day 5, cultures stimulated with anti-CD3 and IL-2, allogeneic APCs or porcine APCs show a high percentage (>50%) of proliferating cells expressing CD86 whereas few (2%) non-proliferating cells express CD86. The proliferating cells also expressed CD80, CD25, CD54, Class II and CD45RO. CFSE profiles indicated that fixed T cells expressing CD86 induced by these routes trigger the proliferation of both allogeneic and syngeneic T cells. However, 3H-Thymidine incorporation at day 7 was negative. Conclusion: Human T cells, in response to human or porcine APC stimulation, express CD86 and Class II molecules and function as APCs as assessed by CFSE proliferation assays. Thymidine incorporation data indicates that proliferation had ceased by day 7. This is most likely explained by activation-induced cell death at this time point, since, the proliferating T cells show high levels activation markers CD25 (IL-2R) and CD54 (ICAM-1) and are of memory phenotype (CD45RO positive).

The use of porcine organs for clinical transplantation is a promising potential solution to the shortage of human organs. Preformed anti-pig antibody is the primary cause of hyperacute rejection, while elicited antibody can contribute to subsequent “delayed” xenograft rejection. This article will review recent progress to overcome antibody mediated xenograft rejection, through modification of the host immunity and use of genetically engineered pig organs.

Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ((3)H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with (3)H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4-7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical (3)H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient.

BACKGROUNDGraft-resident antigen presenting cells (APCs) are potent stimulators of the alloresponse. To test whether replacement of graft-resident donor-type APCs with those of recipient-type alters allorecognition and the pathogenesis of both acute and chronic rejection, we created chimeric hearts for transplantation into naive recipients.METHODSTo replace donor-type APCs with those of recipient-type, chimeric animals were created by bone marrow transplantation (BMT) in fully allogeneic mouse and rat strain combinations. The degree of APC replacement in chimeric organs was assessed phenotypically and functionally. Chimeric hearts were transplanted heterotopically into untreated recipients.RESULTSFlow cytometric and immunohistochemical analysis did not detect residual bone marrow recipient-type APCs in mouse BMT chimeras. Although semi-quantitative reverse transcription polymerase chain reaction detected 0.001-0.01% residual cells, APCs isolated from chimeric organs were functionally unable to stimulate donor-type cells. When transplanted into naive recipients, chimeric mouse hearts had significantly prolonged survival but were nevertheless rejected acutely. Similar results were obtained in the ACI --> LEW rat strain combination. However, in the PVG --> DA rat model, the majority of chimeric hearts survived >100 days and all long-surviving hearts developed cardiac allograft vasculopathy.CONCLUSIONSBMT leads to near complete replacement of organ-resident APCs. The virtual absence of donor-type APCs in chimeric hearts delays or prevents acute rejection in a strain-dependent manner. In contrast, this type of graft modification does not prevent cardiac allograft vasculopathy. This suggests that, although the CD4+ direct pathway may play a role in acute rejection, it is not essential for the development of chronic rejection in rodent cardiac allografts.

51 T cell activation consequent to direct and indirect allo- and xenorecognition has usually been determined by measuring proliferation of bulk mixed lymphocyte cultures (MLC). Recently, single cell analysis of T cell activation has been reported using flow cytometric analysis of responder cells labeled with the fluorescent carboxyfluorescein diacetate succinimidyl ester (CFSE). Our study is the first to analyze single T cell proliferation in human allo and xeno responses due to direct and indirect recognition and to compare these results to the classical bulk thymidine incorporation assay. Methods: Human peripheral blood lymphocytes (PBL) were obtained by density gradient centrifugation of heparinized whole blood. One-way MLCs were set-up with 4×106 responder cells and 1×106 irradiated (25Gy) stimulator cells in 2 ml of medium. MLCs were set up to test direct recognition (APC-depleted responder PBL + stimulator adherent cells), indirect recognition (APC- depleted responder PBL + responder adherent cells + APC-depleted stimulator PBL) and both pathways (responder PBL + stimulator PBL). The stimulators were from allogeneic or xenogeneic (porcine) origin. Proliferation was assessed by 3H-thymidine incorporation (conventional MLC) and by flow cytometric analysis of CFSE labeled responder cells, counter stained with phycoerythrin labeled monoclonal antibodies for CD4 and CD8, dead cells were eliminated by gating with ViaProbe™. Results: One representative experiment of three is shown below. The conventional MLC results are given in counts per minute (cpm) and the CFSE data is expressed in number of divisions (div.) in the CD4/CD8 positive cell population at day 6. (Table)(Figure)TableFigureConclusions: We have demonstrated that single cell analysis of T-cell proliferation using CFSE can be used to study human allo and xenorecognition pathways (including preliminary studies analyzing costimulatory blockade; data not shown) and that the results mirror conventional MLC, in which indirect responses are less vigorous.

145 Background: Previous studies have shown that depletion of donor-type bone marrow-derived antigen presenting cells (APCs) from rat renal allografts leads to prolonged survival. However, these studies have not examined grafts histologically for the presence of graft vasculopathy. We have depleted donor-type APCs from rat hearts using both "organ parking" and bone marrow transplantation (BMT) and we have confirmed APC replacement using flow cytometry, mixed lymphocyte response (MLR), immunohistochemistry, and reverse transcriptase polymerase chain reaction (RT-PCR). The goal of this study was to use chimeric hearts to test whether donor APC depletion prevents cardiac allograft vasculopathy (CAV). Methods: PVG hearts were depleted of PVG APCs by two methods: DA→PVG BMT and "parking" of PVG hearts in cyclosporine-treated DA recipients. APC replacement was ≥ 99.9% as assessed by two-color flow cytometry, MLR, immunohistochemistry and RT-PCR. Chimeric PVG hearts or PVG hearts bearing PVG APCs were transplanted heterotopically into DA rats. Recipients received no immunosuppression. Rejection was assessed by palpation. Rejected hearts and those surviving > 100 days were examined histologically in a blinded fashion. Results: All PVG hearts bearing PVG APCs (n=21) were acutely rejected, whereas PVG hearts lacking PVG APCs (n=23) had significantly(p<.00001, Breslow) prolonged survival. However all long surviving PVG grafts had histologic evidence of moderate to severe CAV.TableConclusions: Depletion of donor APCs in rat heart grafts leads to prolonged survival, but does not prevent cardiac allograft vasculopathy(CAV). This finding lends credence to the hypothesis that CAV is a consequence of indirect allorecognition, since the lack of donor-type APCs prevents the direct pathway.

110 Background: Although graft resident, bone marrow-derived antigen presenting cells (APCs) are highly immunogenic, they are not required for rejection, as chimeric BALB/c hearts bearing CBA APCs are rejected by CBA recipients, despite a modest prolongation in survival. The goal of this study was to characterize the cellular infiltrate in acutely rejecting chimeric BALB/c hearts to determine the allorecognition pathway(s) responsible. Methods: Chimeric BALB/c hearts were created via CBA→BALB/c bone marrow transplantation. Control or chimeric hearts were transplanted heterotopically into naïve CBA recipients. Immunohistochemistry (IHC) was performed using a standard avidin biotin complex immunoperoxidase technique on frozen sections obtained from rejected hearts. Antibodies against CD4, CD8, CD11b (macrophages[Mφ]), CD11c (dendritic cells[DC]) and CD45R/B220 (B cells) were used. One frozen section from each case was stained with hematoxylin and eosin to grade the rejection. Results: As in previous studies, APC replacement prolonged graft survival, but did not prevent rejection. All specimens showed acute cellular rejection with no difference in ISHLT grade between groups. Cellular infiltrates in both groups were predominantly effector CD8+ T cells and CD11b+ Mφ. Chimeric hearts showed an increase in CD11c+ DC and CD45R/B220+ B cells and CD4+ T cells (p=.01,.03, and.05 respectively, Mann-Whitney U Test). Table,TableConclusion: Chimeric hearts showed a different pattern of cellular rejection with increased numbers of recipient-type APCs (both B cells and DC) and CD4+ T cells. This supports the hypothesis that hearts depleted of donor-type APCs are rejected by the indirect allorecognition pathway.

498 Background: It has been postulated that donor-type bone marrow-derived antigen presenting cells (APCs) may be important for tolerance induction via a mechanism of posttransplant microchimerism. Our goal was to use chimeric hearts created via BMT to test whether donor-type APCs are required for the induction and maintenance of cardiac allograft tolerance in the rat. Methods: Chimeric PVG hearts were created via DA→PVG BMTs. Elimination of PVG APCs was confirmed by flow cytometry, MLR, immunohistochemistry and RT-PCR. Chimeric and naïve hearts were then used for heterotopic transplants, in which APCs were matched or mismatched to the recipient, while organ parenchyma was always mismatched to the recipient. To induce tolerance, oral cyclosporine (CyA, 20 mg/kg, ×10 days) was given following transplantation. Rejection was monitored by palpation of the graft and histological analysis was performed in a blinded fashion. Results: CyA led to permanent survival of PVG→DA heart grafts with minimal vascular changes. 5/7 chimeric PVG hearts survived long-term in untreated DA recipients, but uniformly demonstrated mild cellular rejection and cardiac allograft vasculopathy (CAV). 4/6 chimeric PVG hearts survived long-term in CyA treated recipients, and two of these long-term surviving hearts that have been examined showed focal rejection and moderate CAV. TableAnimals from all experimental groups accepted second naïve PVG hearts(normal histology) without immunosuppression (Group II: 3/3, Group III: 2/2, Group IV: 3/3) and rejected third party F344 hearts (Groups II, IV: 2/2), confirming induction of antigen-specific tolerance in CyA-treated recipients. Conclusions: Donor-type bone marrow-derived APCs are not required for tolerance induction in this rat model. Furthermore, these data support recently published work by Tullius et al showing that donor-specific tolerance can develop despite the presence of vasculopathy in the first graft.

New Zealand Black (NZB) mice spontaneously develop immune dysfunction manifested as autoimmune hemolytic anemia and systemic lupus erythematosus. In later life, a subset of these mice develop clonal CD5+B cell tumors analogous to human chronic lymphocytic leukemia (CLL). NZB disease is marked by B cell hyperactivity characterized by spontaneous immunoglobulin secretion and proliferation. Elimination of autoreactive lymphocytes by apoptosis is a vital mechanism to prevent expansion of self-reactive lymphocyte population. TGF-β appears to be an important factor in normal and abnormal immune regulation and this cytokine may play a role in the development of chronic human B cell tumors. We asked whether the response to or production of TGF-β by NZB B cells was aberrant and could contribute to disease development. In this study, we demonstrated that the apoptotic response to TGF-β was increased in B cells from NZB mice compared to B cells from normal BALB/c mice. The increased apoptosis was related to endogenous activation and was possibly mediated through increased expression of the TGF-β Type II receptor. Despite functional differences between CD5-negative B cells and CD5-positive B cells, TGF-β induced apoptosis in both populations to a similar extent. NZB B cells also secrete increased active TGF-β compared to BALB/c B cells. We suggest that the aberrant secretion of active TGF-β and the increased response to the apoptotic effects of TGF-β by NZB B cells may play a role in the disease process of these mice, perhaps attempting to limit the autoimmune phenomena, but possibly also contributing to generalized immunosuppression. We also suggest that the CD5+tumors in the NZB mouse may not be a fully appropriate model of human CLL, since CLL B cells are abnormally resistant to the apoptotic effects of TGF-β.

Chronic lymphocytic leukemia (CLL) is the most common leukemia of the western world and is characterized by a slowly progressing accumulation of clonal CD5+ B cells. Our laboratory has investigated the role of transforming growth factor-beta (TGF-beta) and interleukin-4 (IL-4) in the pathogenesis of B-cell expansion in CLL. In vitro addition of TGF-beta did not increase spontaneous apoptosis of B cells from most CLL patients, as determined using the TUNEL method, compared with a twofold increase observed in cultures of normal B cells. There was similar expression of TGF-beta type II receptors on both CLL B cells and normal B cells. In contrast to apoptosis, CLL B-cell proliferation was variably inhibited with addition of TGF-beta. In vitro addition of IL-4, previously reported to promote CLL B-cell survival, dramatically reduced spontaneous apoptosis of CLL B cells compared with normal B cells. CLL B-cell expression of IL-4 receptors was increased compared to normal B cells. Thus, our results show aberrant apoptotic responses of CLL B cells to TGF-beta and IL-4, perhaps contributing to the relative expansion of the neoplastic clone.