Evidence that drug-resistant alloreactive T cells may contribute to human graft rejection

The objective of our study was to determine whether resistance to immunosuppressive drugs by transplant recipient's T cells could contribute to continued graft rejection, in spite of immunosuppressive therapy. The T cell lines used in this series of experiments were originally established from T cells that had infiltrated kidney or liver grafts and initiated rejections in patients receiving immunosuppressive drugs, including the purine analogue azathioprine (AZ). We have used a proliferation assay and the Strauss-Albertini test to analyze the T cell lines. Both assays use 6-thioguanine (6-TG), an amino derivative of AZ, as the selective agent to measure the resistance to AZ. Although the frequency of 6-TG-resistant variants in the majority of T cell lines closely resembled the frequency found in peripheral blood lymphocytes of controls, we identified four cell lines that demonstrated virtually complete resistance to the purine analogue 6-TG in both assays. These observations indicate that a more effective immunosuppressive treatment may be achieved by using a combination of drugs.     

Maturing thymocytes in accelerated rejection of cardiac allografts in presensitized rats.

LBNF1 cardiac allografts are rejected within 36 hr in LEW rats sensitized with BN skin grafts 7 days earlier (acute rejection in unmodified hosts = 8 days). We have studied and compared the function and migration patterns of thymocytes one day after engraftment in sensitized recipients, unmodified hosts, and normal naive rats. Thymocytes from animals experiencing accelerated rejection were more mature and functionally active, as shown by a significant elevation in percentage of OX-44+ (CD37+) cells, increased alloreactivity to BN and WF antigens, and proliferative responses to Con A and exogenous IL-2. However, the cells could neither lyse BN targets in vitro nor trigger rejection of otherwise indefinitely functioning test cardiac allografts in immunologically unresponsive T cell-deficient (B) rats after adoptive transfer. The traffic of 111In-labeled thymocytes was then evaluated. The migration index increased significantly during accelerated graft rejection, with thymocytes preferentially circulating in the blood, penetrating peripheral lymph nodes--and, interestingly, migrating back to the thymus. Thus, immunoresponsive and functionally active thymocytes, which lack the ability to recognize primed specific antigen, appear during accelerated rejection of cardiac allografts in sensitized rats. These cells migrate to the periphery, and then return in large numbers to their site of origin, the thymus. Hence, this study describes a novel behavior of thymocytes in the state of host alloreactivity that is distinct from the physiological one in otherwise normal thymus.     

灵长类动物预致敏后肾移植加速排斥反应模型的建立

目的 建立灵长类动物预致敏后肾移植加速排斥反应模型.方法 取血型相容的正常猕猴配对,预先将供者腹部全层皮肤移植到受者背部,使受者预致敏.2周后再将同一供者的左侧肾脏移植到受者腹腔内,间时切除受者自体双肾,术后予以环孢素A、霉酚酸酯和泼尼松治疗(致敏用药组),不用免疫抑制剂者为对照(致敏对照组),以未致敏的肾移植作为对照组.术后观察受者血肌酐变化、移植物存活时间及病理特点.结果 对照组的4只移植肾存活时间分别为9、18、8、7 d;致敏对照组的3只移植肾存活时间分别为3、3、4 d;致敏用药组的3只移植肾存活时间分别为2、3、4 d.移植皮肤于术后10 d出现排斥反应,至术后14 d被完全排斥.对照组于肾移植1周以后才发牛排斥反应,而致敏者均在肾移植后3 d左右发生较严重的排斥反应.结论 受者被供者皮肤预致敏后再行肾移植,可以加速移植物的排斥,且不能被环孢素A、霉酚酸酯及泼尼松所组成的三联免疫抑制方案逆转.     

The role of TDTH and Tc populations in organ graft rejection. I. Functional analysis of graft-infiltrating T cells

To analyze the role of T cell subpopulations in the rejection of organ allografts, we developed a new model for obtaining large numbers of graft infiltrating cells (GICs). We isolated W3/25+ Th/DTH and OX8+ Ts/c from vascularized, irradiated rat spleen allografts. W3/25+ GICs obtained from spleen allografts transplanted to normal recipients were highly effective in eliciting cardiac allograft rejection when transferred to sublethally irradiated recipients, however, the OX8+ subset was incapable of eliciting rejection. On the other hand, when OX8+ GICs were obtained from spleen allografts transplanted to previously immunized recipients, they were as efficient as the W3/25+ Th/DTH subset in eliciting cardiac allograft destruction. These results indicate that the W3/25+, OX8- T cell is required for the rejection of primary organ allografts, but that the rejection of a secondary allograft by an immune recipient may be mediated, independently, by both W3/25+ and OX8+ cells.     

Exhaustive differentiation of alloreactive CD8+ T cells: critical for determination of graft acceptance or rejection

BACKGROUND: The precise role that CD8+ T cells play in the rejection and acceptance of different types of allograft is unclear and has been shown to vary between donor-recipient combinations. METHODS: The response of adoptively transferred CD8+ T cells reactive to the donor alloantigen H2Kb was examined after transplantation of H2Kb liver, kidney, and heart grafts in mice. RESULTS: After transfer of 6 x 10(6) alloreactive CD8+ T cells to T-cell depleted syngeneic mice spontaneous long-term acceptance of liver grafts was observed, whereas kidney and heart grafts were acutely rejected. Within 5 days of liver transplantation, we found that the entire H2Kb-reactive T-cell pool was stimulated to proliferate and differentiate into memory or effector cells that were detectable within lymphoid tissues as well as the liver graft itself. However, despite the generation of effector or memory T cells, liver allografts were accepted, which correlated with the exhaustion or deletion of such cells. In contrast, although activation and proliferation of H2Kb-reactive CD8+ T cells was observed after transplantation of heart or kidney grafts, unactivated, H2Kb-reactive CD8+ T cells were still present in the spleen even long term. Interestingly, differences in the effector function of liver and kidney graft infiltrating donor-reactive CD8+ T cells were not detected after adoptive transfer into immunodeficient mice, despite a reduction in Th1-type cytokines within liver grafts. CONCLUSIONS: The rapid and extensive initial activation and differentiation of donor-reactive CD8+ T cells that occurs after liver transplantation leads to clonal exhaustion or deletion of the alloreactive CD8+ T-cell repertoire resulting in spontaneous tolerance induction.     

Urinary detected renal antigens in the early diagnosis of kidney graft rejection.

The determination of renal antigens in the urine with an immunoassay, based on monoclonal antibodies (moabs), is a noninvasive test system for the analysis and monitoring of renal injury. New moabs allowing an immunohistologic dissection of the human nephron were generated by a direct intrasplenic immunization of mice with pathologic urine samples. A sandwich enzyme immunoassay was developed to quantitate renal cell membrane antigens in the urine samples. A sandwich enzyme immunoassay was developed to quantitate renal cell membrane antigens in the urine. While antigen excretion in healthy individuals is low, preliminary data of a clinical investigation suggest the usefulness of these assay systems in diagnosis of tubular injury in human kidney transplant recipients. The immunoassay can provide very early hints of renal graft rejection prior to the appearance of clinical symptoms or the detection by routine clinical laboratory investigations.     

Regulation of cytotoxic T lymphocyte-mediated graft rejection following bone marrow transplantation

Cytotoxic T lymphocytes have been implicated as the effector cell mediating graft rejection following human allogeneic bone marrow transplantation. We have studied a BMT patient who rejected haploidentical T cell-depleted marrow. In vitro studies demonstrated that the circulating lymphocytes were CD3+ and CD8+, of recipient origin, and exhibited selective cytotoxicity against donor-specific class I major histocompatibility complex antigens. Cytotoxicity was inhibited by monoclonal antibodies directed against CD3, CD8, CD2, and lymphocyte function-associated antigen-1 on the T cell, and against MHC class I proteins on the target cell. Furthermore, these circulating cells inhibited the in vitro growth and differentiation of enriched donor bone marrow progenitor cells, an inhibition that was partially reversed by anti-CD3 MAb. Donor-specific recipient-derived CTL may mediate resistance to engraftment, and CTL activity may be inhibited by a number of MAb. The implications of these findings for host preparation and treatment are discussed.     

The replacement of graft endothelium by recipient-type cells conditions allograft rejection mediated by indirect pathway CD4(+) T cells

BACKGROUND: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4(+) T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4(+) T cells. METHODS: Transgenic RAG2(-/-) mice expressing I-A(b)-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2(k) male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2(k) male allograft and presented by the recipient's own I-A(b) APC to transgenic T cells. RESULTS: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-A MHC class II molecules. This observation suggested that graft endothelium replacement by I-A(b)-positive cells of recipient origin could stimulate the rejection of male H-2(k) graft by I-A(b)--restricted H-Y--specific T cells. To investigate further this possibility, hearts from H-2(b)--into--H-2(k) irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-A(b)-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2(k) male cardiac allografts transplanted in transgenic recipients. CONCLUSIONS: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4(+) T cells.     

The replacement of graft endothelium by recipient-type cells conditions allograft rejection mediated by indirect pathway CD4+ T cells

BACKGROUND: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4+ T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4+ T cells. METHODS: Transgenic RAG2-/- mice expressing I-Ab-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2k male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2k male allograft and presented by the recipient's own I-Ab APC to transgenic T cells. RESULTS: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-Ab MHC class II molecules. This observation suggested that graft endothelium replacement by I-Ab-positive cells of recipient origin could stimulate the rejection of male H-2k graft by I-Ab-restricted H-Y-specific T cells. To investigate further this possibility, hearts from H-2b-into-H-2k irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-Ab-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2k male cardiac allografts transplanted in transgenic recipients. CONCLUSIONS: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4+ T cells.     

Hyperacute and acute kidney graft rejection due to antibodies against B cells.

Because of the perception of its uncertain clinical significance, the B cell crossmatch is not universally performed before renal transplantation. Even though sporadic cases of hyperacute rejection associated with B cell antibodies have been reported, doubts remain in light of other studies suggesting no effect on graft survival. This report describes 4 cases of graft rejection (3 hyperacute and 1 acute) that occurred in patients with anti-B-cell antibodies specific against donor HLA-DR or DQ antigens. Absence of anti-donor class I antibodies was confirmed in all cases by 2-color flow cytometry. Strong evidence for an antibody-mediated mechanism was found in one patient with anti-class I and anti-class II antibodies in serum transplanted with a class II mismatched kidney. In this case, only anti-class II antibodies were recovered in the eluate of the nephrectomy specimen. These four cases were compiled from three different institutions over a four-year period, which confirms the infrequent occurrence of these events. While anti-class II antibodies may not always be detrimental for graft survival, these results also confirm that they have the potential to cause hyperacute or acute graft loss. We conclude that the information provided by the B cell crossmatch should be available at the time that a decision to proceed with a renal transplant is made.     

Copolymer 1 inhibits manifestations of graft rejection.

BACKGROUND: Copolymer 1 (Cop 1) was previously shown to prevent graft-versus-host disease and interfere in various manifestations of immune rejection. In this study, we tested whether Cop 1 can also hinder the reactivity of host against graft and inhibit graft rejection. METHODS: Cop 1 was tested in two transplantation systems: skin and thyroid grafting assays. The effect of Cop 1 on T cell reactivity was investigated by proliferation and cytokine secretion of spleen and lymph node cells from transplanted mice, as well as the T cell lines generated therefrom. RESULTS: Cop 1 treatment prolonged skin graft survival and inhibited the functional deterioration of thyroid grafts, in various strain combinations, across minor and major histocompatibility barriers, similarly to the potent immunosuppressive drug FK506. Cop 1 inhibited the proliferation of graft-specific T cell lines, as well as their interleukin (IL)-2 and interferon-gamma (IFN-gamma) secretion, when incubated in vitro with the stimulating allogeneic cells. Spleen and lymph node cells from Cop 1-treated mice, as well as the T cell lines generated from them, demonstrated a pronounced inhibition of proliferation and secretion of T helper (Th)1 cytokines in response to graft cells. In addition, cells from Cop 1-treated mice secreted high amounts of Th2 cytokines in response to Cop 1 and small but significant amounts of Th2 cytokines, mainly IL-10, in response to allograft cells. CONCLUSIONS: Cop 1 treatment inhibited the Th1 response to graft and induced a Th2 cytokines secretion in response to both Cop 1 and graft cells, leading to improved survival and function of the transplanted grafts.