Immunological unresponsiveness induced by ultraviolet-B-irradiated and nonirradiated skin grafts.

We previously reported that ultraviolet-B-irradiated B10.AQR tail skin grafts were permanently accepted by B10.T6R recipients in about half the cases. Such a beneficial effect on graft survival could only be demonstrated in this particular combination. We have now investigated whether these animals had become tolerant to the donor strain antigens. Nonirradiated B10.AQR tail skin grafted 50 days after acceptance of a UVB-irradiated B10.AQR graft was accepted in 9/9 cases, indicating that these animals had become tolerant to the B10.AQR alloantigens. However, secondary grafts on animals that had rejected the first graft also showed a prolonged or definite survival. This tolerance was specific; B10.T6R mice tolerant to B10.AQR grafts rejected B10 skin grafts, while F1(B10A X B10.AQR) and F1(B10 X B10A) grafts, sharing class II antigens with B10.AQR, had a slightly prolonged graft survival. Cells of tolerant animals showed normal proliferative responses against B10.AQR antigens. However, when autologous serum was added, proliferation was specifically suppressed. Likewise, this specific tolerance could be transferred with serum or serum and cells but not with cells only. Analysis of the sera of these animals showed long-lasting and donor-specific high-titered cytotoxic antibody titers, which are likely to play a pivotal role in the observed suppression.

An increase of donor-specific T helper precursors resulting from blood transfusions.

In order to study the effect of blood transfusions on the donor-specific helper T cell repertoire, donor-specific interleukin 2-producing precursors (Th precursors [Thp]) were examined in 10 patients before and after transfusion. These patients were selected to be male, had received no previous transfusions or transplantation, and had no cytotoxic antibodies against MHC antigens in their serum. The limiting dilution curves were linear before as well as after transfusion, showing that only the cell under study was limiting. It was found that the transfusion donor-specific Thp frequencies increased significantly after transplantation in 10 of 10 patients (ratio: 1.5 to 7.1). In addition, the IL-2 production by individual clones tended to be higher after transfusion. In contrast, Thp frequencies against a third-party control showed a small increase in 4 and was unchanged in 6 patients. These data indicate that blood transfusions prime the donor-specific Th compartment.

Increase of donor-specific cytotoxic T lymphocyte precursors after transfusion.

Cytotoxic T lymphocyte precursor (CTLp) frequencies were determined in the blood of 10 patients before and after transfusion. The male patients were selected from those on the waiting list for a heart or kidney transplantation, who had received no previous blood transfusions and had no panel-reactive antibodies. Both the limiting dilution curves obtained before as well as after transfusion were linear and applied to zero-order kinetics, indicating that only cytotoxic T cells were limiting in the assay. In 9 out of 10 patients CTLp frequencies against the blood donor antigens significantly increased (up to 107 times) compared to the CTLp frequencies measured before transplantation. The CTLp frequencies against controls, sharing no HLA antigens with the blood transfusion donor, were only slightly increased (up to 5 times) in 7 patients and decreased in 3.

Evidence that antibody formation against a certain HLA alloantigen is associated not with a quantitative but with a qualitative change in the cytotoxic T cells recognizing the same antigen.

Previous studies in highly sensitized patients waiting for a renal transplant, showed a lack of correlation between the B cell and T cell allorepertoire. The cytotoxic T cell precursor (CTLp) frequencies against HLA-antigens, toward which patients had formed antibodies (not-acceptable mismatches, NAM) were similar to those against HLA antigens, toward which no antibodies were present (acceptable mismatches, AM). In the present study we have tested whether the immunological triggering leading to antibody formation might have resulted in a different population of cytotoxic T cells. Limiting dilution assays performed in the absence or presence of antibodies against CD8 showed that CTL directed against NAM were significantly less inhibited by anti-CD8 compared to AM. A possible clinical relevance of these findings is suggested by experiments showing that the CTL against NAM were also more resistant to cyclosporine than CTL against AM.

Functional and molecular characterization of graft-infiltrating T lymphocytes propagated from different biopsies derived from one heart transplant patient.

Alloreactive T lymphocytes play an important role in graft rejection. In the present study, we have analyzed the cytolytic capacity against donor cells of graft infiltrating T lymphocyte cell lines, which were propagated from various endomyocardial biopsies taken at different time points after transplantation, including during a rejection crisis. Also, T cell clones were generated from the rejection biopsy and evaluated for their cytolytic capacity and nucleotide composition of the TCR alpha and beta chains. The results of these studies revealed a strong cytolytic activity against donor cells by T cells derived from the rejection biopsy, whereas from the other biopsies, no cytolytic T cell clones could be established. The T cells that were responsible for this activity, as detected by T cell cloning and TCR gene analysis, could not been identified in earlier biopsies, indicating that these cytolytic cells were recently recruited toward the endomyocardium.

The amplifier role of T cells in the human in vitro B cell response to type 4 pneumococcal polysaccharide.

The human B cell response to T cell independent type 2 antigens is regulated by thymus-derived lymphocytes. We analyzed the role of T cells in the in vitro antibody response to type 4 pneumococcal polysaccharide (PS4). We here show that the amplifying effect of T cells, which has previously been shown to be radioresistant and confined to T cell preparations enriched for CD4+ cells, is MHC non-restricted as demonstrated in cultures carried out in the presence of allogeneic T cells. Also, T cell clones derived from non-related donors are able to enhance the B cell response to PS4. All TCR alpha beta +, CD 4+ T cell clones, but none of the TCR alpha beta +, CD 8+ T cell clones tested, enhanced the B cell response to PS4. Furthermore, 3 out of 6 TCR gamma delta+ T cell clones were capable of enhancing the anti-PS4 B cell response. Experiments using recombinant lymphokines and glutaraldehyde-fixed T cells indicated that both lymphokines and T-B cell interactions are required for an optimal antibody response to PS4.

Detection and characterization of HLA class I molecules in the supernatant of an hepatocarcinoma cell line and of EBV-transformed B cell lines.

Human leukocyte antigens (HLA) class I molecules can be detected in "soluble" form in the supernatant of cultured cell lines and in serum and plasma of humans. These "soluble" HLA class I molecules are assumed to play a role in liver transplantation. In order to define the nature and composition of HLA class I molecules found in solution, we studied the HLA class I production of an hepatoma carcinoma cell line (HepG2) and of EBV-transformed B-cell lines. Based on molecular weight (MW) analysis, it was demonstrated that different forms of HLA class I molecules were produced by HepG2 cells and EBV B-cells. Monoclonal antibodies (mAbs) specific for HLA class I alleles were able to recognize the mature 45 kDa form, but failed to interact with the 42 kDa and 39 kDa MW forms of HLA class I. Of these different MW forms of HLA class I molecules the mature 45 kDa product was found predominantly to be associated with subcellular vesicles whereas the alternative MW forms of 42 kDa and 39 kDa exist as truly free entities in supernatants.

Transcriptional control of MHC genes in fetal trophoblast cells.

Tight control of MHC expression is essential for the outcome of a successful pregnancy. The lack of MHC class II and class I mediated antigen presentation by fetal trophoblast cells is an important mechanism to evade maternal immune recognition. Interestingly, the deficient expression of MHC class II molecules (HLA-DR, -DQ and -DP) and of the classical MHC class I molecules HLA-A and HLA-B is also noted after IFN-gamma treatment in trophoblast-derived cell lines. Our studies show that in trophoblast cell lines the IFN-gamma induced transactivation of HLA-A and HLA-B promoters is repressed. Furthermore, it was found that trophoblast cells lacked IFN-gamma mediated induction of the class II transactivator (CIITA). This lack of CIITA expression in trophoblast cells is due to CIITA promoter hypermethylation. In addition to lack of CIITA expression, trophoblast cells also displayed a repressed expression of RFX5. Together, these observations reveal a silencing of multiple activation pathways that are critical to the transcriptional control of MHC class II and class I antigen presentation functions by trophoblast cells.

HLA-C expression on platelets: studies with an HLA-Cw1-specific human monoclonal antibody.

BACKGROUND AND OBJECTIVES: The expression of HLA-C on the surface of platelets is rarely studied due to the lack of proper alloantisera. We addressed this question using an IgM human monoclonal antibody directed against HLA-Cw1 (VP6G3). MATERIAL AND METHODS: Both flow cytometry and complement dependent cytotoxicity studies were used in the current analysis. RESULTS: The expression of the HLA-Cw1 antigen on platelets is lower than on peripheral blood lymphocytes as shown by flow cytometry. Variation in expression levels between individuals is also observed. Using this antibody in a complement-dependent cytotoxicity assay, we did not observe lysis using platelets as targets, whereas peripheral blood lymphocytes of the same blood donors were adequately lysed. CONCLUSIONS: These results confirm that platelets indeed express HLA-C. Furthermore, the results support the insignificant role of HLA-C in immunological platelet refractoriness.

Cytotoxic T lymphocytes are the prime mediators of suppression of the mixed lymphocyte reaction by alloactivated cells.

An alloactivated secondary mixed lymphocyte reaction (MLR) culture (SMC), which was suppressive in the MLR with autologous responder cells, was studied in more detail. In particular, we investigated whether the suppressive activity of this SMC was mediated by cytotoxic T cells or whether bona fide suppressor cells were involved. The SMC suppressed an MLR when the stimulator cells shared Bw57, Bw60, or Dw19 with the original stimulator. Separation of the SMC into CD4+ and CD8+ fractions demonstrated that the CD8+ fraction contained suppressive and cytotoxic activity against Bw57 or Bw60 antigens, while the CD4+ fraction contained both activities against the Dw19 specificity. The CD8+ fraction was also suppressive in the reverse MLR, while the CD4+ fraction was not. Limiting dilution analysis demonstrated that all CD8+-suppressive cultures were also cytotoxic, whereas in the CD4+ fraction both cytotoxic and non-cytotoxic suppressor cultures were found.

Definition of a human suppressor T-cell epitope.

The quality of the response produced by regulatory or helper T (Th) cells presently receives much attention because of its possible implications for vaccine development and immunomodulation. Apart from cytokines and so-called costimulatory signals, antigens and the presenting major histocompatibility complex (MHC) molecules may play a role in determining the type of T-cell response generated toward antigens. To examine the role of antigen and/or HLA in control of T-cell subset activation, we have studied a special case, namely CD4+ suppressor T (Ts) cells in leprosy. Mycobacterium leprae-induced Ts cell clones have been previously isolated from peripheral blood and skin lesions of lepromatous leprosy patients and were shown to specifically down-regulate mycobacterium-specific Th cell responses. Despite considerable effort, the antigens recognized by these Ts cells have thus far not been identified. Here we report that all HLA-DR2-restricted CD4+ Ts cell clones derived from a lepromatous leprosy patient recognize an epitope that maps between the amino acid residues 439 and 448 of the mycobacterial hsp65. The peptide was presented to these Ts cells by HLA-DRB1*1503, a recently discovered HLA-DR2 variant. Non-suppressor T-cell clones derived from the same patient recognized antigens other than the hsp65 and were also stimulated by other HLA-DR2 variants. In independent cloning experiments peptide 435-449 and recombinant hsp65 induced exclusively Ts cells in this lepromatous leprosy patient. The Ts clones recognizing this particular epitope were derived from at least seven different progenitors, as they expressed different T-cell receptor alpha and beta chains. Thus, our data indicate that a specific peptide-HLA class II combination may exclusively activate Ts cells.

Analysis of the donor-specific cytotoxic T lymphocyte repertoire in a patient with a long term surviving allograft. Frequency, specificity, and phenotype of donor-reactive T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ clones.

In the present study the transplant specific CTL repertoire of a patient (HLA:A1,3, B8,18, Cw5,7 DR3, DQw2, DPw3) with a long term surviving HLA mismatched kidney graft (HLA: A1,24 B8,27 Cw2,7, DR3, w13 DQw2,6 DPw1,3) has been investigated. This patient was unable to generate specific cytolytic activity against donor-derived PHA-blasts in the MLC in which donor spleen cells or B lymphoblastoid cell line were used as stimulator cells. In addition, the CTL precursor frequencies against donor alloantigens were very low (1/67,000). The patient had otherwise normal immune responses in vivo and in vitro and no signs of transplant rejection. Transplant specific CTL clones were generated in high frequencies (1/195) from T cell bulk cultures activated by PHA in the absence of any sensitization by donor Ag in vitro. The repertoire of 14 donor-reactive CTL clones (12 TCR-alpha beta+ and 2 TCR-gamma delta+) was analyzed. Two TCR-alpha beta+ CD8+ clones were specific for B27. Ten TCR-alpha beta+ CTL clones directed against class II HLA Ag were isolated. Seven of these were CD4+ and recognized DRw13 (3), DQw6 (3), and DPw1 (1), whereas three of these clones were CD4-CD8+ recognizing DRw13 (1) and DQw6 (2). In addition, two donor-specific TCR-gamma delta+ CTL clones were obtained recognizing HLA-A9(23,24) and DQw6. Our data indicate that the precursors of CTL clones specifically directed against donor class I or II HLA Ag are not deleted from the repertoire and that part of this reactivity resides in the TCR-gamma delta+ fraction.