Nancy J. Poindexter

Indirect recognition of donor HLA class I peptides in lung transplant recipients with bronchiolitis obliterans syndrome.

BACKGROUND The presentation of donor MHC class II-derived peptides by host antigen-presenting cells in the context of self-MHC class II molecules has been suggested as a mechanism for the chronic rejection of kidney and heart allografts. The aim of this study was to determine whether indirect allorecognition of HLA class I-derived peptides occurred in lung transplant (LTx) recipients and whether it correlated with the development of bronchiolitis obliterans syndrome (BOS). METHODS Peripheral blood mononuclear cells from LTx recipients were cultured with synthetic peptides corresponding to the hypervariable regions of the mismatched HLA class I antigens of the donor. Proliferation and precursor frequency (PF) of allopeptide reactive T cells were determined by the incorporation of [3H]thymidine into DNA and limiting dilution analysis. RESULTS Peripheral blood leukocytes of LTx recipients with BOS mismatched for HLA class I molecules showed a proliferative response three- to fourfold higher than those observed in mismatched recipients without BOS and in normal control individuals (P<0.001). Similarly, the PF of allopeptide-reactive T cell was 3- to 24-fold higher in recipients with BOS compared with recipients without BOS (P<0.05) as well as normal control individuals (P<0.03). The T cell PF to donor-specific allopeptides, as well as irrelevant allopeptides, was not significantly different in LTx recipients without BOS and normal control individuals. CONCLUSIONS These data suggest that T cells from LTx recipients are sensitized to mismatched HLA class I antigens. The sensitization was significantly higher in LTx recipients with BOS compared with LTx recipients without BOS. Strategies to block T-cell responses generated by indirect allorecognition after lung transplantation may provide a means for the prevention or treatment of BOS in LTx recipients.

Cytolytic T lymphocytes from human renal allograft biopsies are tissue specific.

The cytolytic activity of T lymphocytes infiltrating renal allografts from recipients undergoing episodes of acute cellular rejection was studied. These T-cell populations, composed of both CD4+ and CD8+ cells, demonstrated significant cytolytic activity against both donor-derived KCLs and B-LCLs. In five of 21 biopsy-derived lines greater cytolytic activity was measured against donor KCLs compared to donor B-LCLs, suggesting the presence of kidney antigen-specific, MHC-restricted clones. Clones developed by stimulation with donor B-LCLs lysed both donor B-LCLs and KCLs while clones developed on donor KCLs as stimulator cells showed tissue specificity. Three of 26 clones recognized tissue-specific antigens in the context of donor MHC class I antigens lysing donor KCLs but not B-LCLs. These data demonstrate that a subpopulation of T cells recognizing kidney-specific antigens are present in biopsies of renal allograft recipients undergoing acute cellular rejection. This subpopulation of tissue-specific cytotoxic T lymphocytes may prove to contribute significantly to the pathology of allograft rejection.

Cytotoxic T lyhocytes directed against donor HLA class I antigens on airway epithelial cells are present in bronchoalveolar lavage fluid from lung transplant recipients during acute rejection

BACKGROUND The lung epithelium is among the first donor tissues encountered by the lung allograft recipients immune system. The purpose of this study was to determine whether lung epithelium was recognized by T lymphocytes that are isolated from bronchoalveolar lavage fluid of lung allograft recipients during periods of acute rejection. METHODS Lymphocytes isolated from 45 bronchoalveolar lavage samples (from 41 lung transplant recipients) served as effector cells in standard cell-mediated cytolytic assays with several cell lines as targets: BEAS-2B (an immortalized airway epithelial cell line); B-lymphoblastoid cell lines; and K562 (a natural killer-sensitive cell line). Cytotoxic T-lymphocyte activity of bronchoalveolar lavage lymphocytes was correlated with pathologic status. RESULTS During acute rejection alone (ie, without concomitant cytomegalovirus infection), mean lysis of the airway epithelial target was significantly greater, compared with during no rejection, when these targets expressed donor-specific HLA class I antigens (P =.007). Lysis of donor class I-matched B-lymphoblastoid cell line targets during rejection was not significantly different from lysis during no-rejection periods (P =.18). Mean lysis of K562, a natural killer cell target, did not differ between acute rejection (without concomitant cytomegalovirus infection) and no rejection (P =.30). During cytomegalovirus infection (without concomitant acute rejection), there was no difference in mean lysis of airway epithelial cells, B-lymphoblastoid cell lines, or K562 targets compared with during no cytomegalovirus infection, whereas during acute rejection, compared with cytomegalovirus infection without rejection, there was a significant increase in mean lysis of the airway epithelial target when it expressed donor-specific HLA antigens (P =.01). CONCLUSIONS Donor HLA class I-specific cytotoxic T-lymphocyte activity directed at airway epithelial cells was demonstrated in bronchoalveolar lavage lymphocytes from lung transplant recipients. Lysis of these targets was significantly higher during episodes of acute rejection.

LIVER TRANSPLANT RECIPIENT SERA DERIVED SOLUBLE HLA MEDIATES ALLELE SPECIFIC CTL APOPTOSIS

BACKGROUND Significant levels of donor soluble human leukocyte antigen (HLA) class I (sHLA) are present in patients after transplants. We investigated the possibility that sHLA may inhibit cytolytic T lymphocyte (CTL) activity by inducing apoptosis of the CTL, thereby serving as a mechanism for specific tolerance. METHODS sHLA-A2 and A3 were isolated from the sera of liver transplant recipients by affinity chromatography. T cell bulk lines directed against HLA-A2 and HLA-A3 were generated by stimulation with HLA-A2, A3+ peripheral blood leukocytes and B-lymphoblastoid cells. Induction of T cell apoptosis by sHLA was analyzed by adding sHLA to allospecific CTL 4 or for 24 hr before flow cytometric analysis of propidium iodide and fluorescein isothiocyanate-conjugated annexin V stained cells. T cell receptor (TCR) engagement by sHLA was demonstrated using a monoclonal antibody specific for the TCR. RESULTS sHLA-A3 inhibited CTL activity of a HLA-A3 T cell line by 53%, whereas sHLA-A2 had no effect. sHLA-A3 also increased T cell death by 77% over the control, whereas sHLA-A2 had no significant effect. However, sHLA-A2 induced 21% apoptosis of an anti-HLA-A2 T cell line, whereas sHLA-A3 caused only 3% apoptosis. The antibody complexed form of sHLA was ineffective in the induction of apoptosis. Preincubation of the T cells with anti-T cell receptor monoclonal antibody protected the T cells from sHLA-induced apoptosis, indicating that sHLA-TCR engagement is necessary for this process to occur. CONCLUSION TCR-mediated apoptosis of alloreactive CTL may serve as a mechanism by which sHLA can modulate the immune response.

Renal allograft infiltrating lymphocytes: frequency of tissue specific lymphocytes.

Acute rejection, mediated by T lymphocytes recognizing donor MHC class I and II, is a major factor influencing renal transplant survival. To define the specificity of these effector cells we examined cytolytic activity of graft infiltrating T lymphocytes (GIL) from renal biopsies of individuals undergoing acute cellular rejection. The majority of these cells recognized MHC class I on both donor kidney epithelial cells (KCL) and B-lymphoblastoid cells (LCL) suggesting these T cells recognized peptides from various tissues. However, cold target inhibition experiments demonstrated a significant proportion of GIL T cells were tissue specific. We reported previously that kidney specific CTL can be isolated from biopsies of kidney allografts undergoing acute cellular rejection. Here we extend that observation showing we were able to isolate tissue specific CTL from two additional biopsies. Greater than 10% of the clones isolated (4 of 36 and 5 of 37) from these biopsies were CTL recognizing donor KCL but not LCL targets suggesting that peptides, recognized in the context of donor MHC, were tissue specific. Repeated isolation of significant numbers of tissue specific CTL suggests these T cells play a role in allograft rejection and may be important effector cells mediating rejection in HLA matched transplants.

In vitro T cell proliferation from kidney allograft biopsies with unremarkable pathology: New strategies for an old problem

Acute rejection of renal allografts is mediated by infiltrating alloreactive T cells. The goals of this study were to correlate T cell proliferation with rejection and to determine whether T cell proliferation in the absence of rejection would predict future rejection episodes. Toward this, kidney biopsies (n=100) were cultured in the presence of interleukin-2. Cultures were examined at 4, 24, and 48 hr for T cell proliferation. A strong correlation was observed between T cell proliferation at any time point and rejection. There was not a significant correlation between T cell proliferation in biopsies with no rejection and the occurrence of a rejection episode within 2 months. However, T cell proliferation after 4 hr was a better predictor of the occurrence of rejection within 2 months compared with observations after 24 and 48 hr. Therefore, a subgroup of patients with unremarkable biopsies but T cell proliferation may be at risk for rejection and warrant closer observation and possible tailoring of immunosuppression.

Regulatory function of human CD4+ cytolytic T lymphocytes

Allograft rejection is mediated by both CD4+ and CD8+ T cells. The lytic function of the classic CD8+ cytolytic T lymphocytes (CTL) occurs through recognition of allogeneic major histocompatibility complex (MHC) class I on the surface of the graft. CD4+ CTL recognize MHC class II through a direct recognition pathway or an indirect pathway where MHC peptides are presented in the context of self MHC class II. Lytic CD4+ cells may destroy graft tissue or, we hypothesize, the indirect CD4+ T cell may down regulate CD8+ CTL by recognition of donor MHC peptides presented by self MHC class II expressed on CD8+ T cells. To define the role of CD4+ CTL in allograft outcome we used a CD4+ CTL that is MHC class II restricted, recognizing human leucocyte antigen (HLA)-A1 and HLA-B8 peptides in the context of HLA-DR4. This line (MDSxA1/B8) will lyse DR4+ B lymphoblastoid cells (LCL) pulsed with HLA-A1/B8 peptides (amino acids 60-84 of the alpha1 domain of the MHC class I molecule). These T cells will also lyse peptide-pulsed antigen-specific T cell clones, both CD4+ and CD8+, that express HLA-DR4. These clones must process and present the MHC class I peptides for recognition and lysis to occur. These results suggest a possible mechanism to explain allograft tolerance. Lytic CD4+ T cells, that recognize donor HLA peptides through an indirect antigen presentation pathway, down-regulate donor-specific CTL through peptide-specific lysis resulting in graft tolerance.

Characterization of an HLA-A3 restricted human kidney specific T cell clone.

BACKGROUND The tissue specificity of a cytolytic T lymphocyte is determined by the MHC class I bound peptide it recognizes. We have developed an allorestricted human CTL clone, DBS 1.5, that recognizes an epitope found on HLA-A3+ kidney epithelial cells but not on HLA identical B-lymphoblastoid cells. The peptide recognized by this clone has been isolated from HPLC separated, acid eluted peptides from purified HLA class I molecules from HLA-A3+ kidney tissue. This peptide shares no sequence homology with any known protein. METHODS To confirm the tissue specificity of the HLA-A3 restricted clone and the peptide it recognizes we have transfected the gene for HLA-A3 into a number of tumor cell lines both human and murine not expressing this antigen. The resulting transfected lines, confirmed by immunofluorescent staining, were used as targets to determine if expression of HLA-A3 alone was sufficient to allow recognition and lysis by the HLA-A3 restricted T cell clone. RESULTS The HLA-A3 restricted T cell clone recognized HLA-A3 when expressed on human kidney epithelial cells and to a lesser extent on human lung epithelium and human epidermal cells. Of the tumor lines transfected with HLA-A3 only the human kidney tumor cell line was lysed at a level equal to the original kidney epithelial cell used to develop the clone. CONCLUSION These results confirm that this allorestricted human CTL clone is tissue specific recognizing a peptide found in human epithelial tissue that must be presented in the context of HLA-A3 for recognition.