Eric K. Ho

The role of anti-HLA antibodies in heart transplantation

The major threat to long-term survival of heart allograft recipients is the development of graft atherosclerosis, which seems to be a manifestation of chronic rejection. To assess the role of anti-HLA antibodies in heart allograft rejection we studied 107 patients and compared the survival of recipients who formed anti-HLA antibodies with the survival of recipients who developed no antibodies. At 4 years the actuarial survival was 90% in the nonproducer group and 38% in antibody-producers (P = 0.038). We further explored the possibility that HLA antigens from the injured graft are released into the circulation and can be found in the serum either free or complexed with anti-HLA antibodies. This hypothesis was confirmed by the finding that the frequency of sera containing soluble HLA antigens from the graft or immune complexes of HLA alloantigens with anti-HLA antibodies was significantly higher in patients who rejected compared with patients with successful heart allografts (P less than 0.05). Following depletion of soluble HLA antigens, anti-HLA antibodies became detectable in 53% and 74% sera obtained during the first and second year posttransplantation, respectively, from patients undergoing chronic rejection. Long-term survivors showed a significantly lower (P less than 0.001) frequency of anti-HLA antibodies in sera depleted of HLA antigens. Lastly, studies of anti-anti-HLA-A2 and A3 antibodies in recipient sera suggest that quiescence is maintained by antiidiotypic antibodies.

Monitoring of soluble HLA alloantigens and anti-HLA antibodies identifies heart allograft recipients at risk of transplant-associated coronary artery disease.

The development of accelerated transplant-related coronary artery disease (T-CAD) is the major obstacle to long-term survival of cardiac allografts. We have investigated the role of various demographic and immunologic parameters as prognostic indicators of T-CAD in a population of 274 heart allograft recipients. Our data demonstrate that patients who experience more than 1 episode of acute rejection per year and/or develop antidonor HLA antibodies are at increased risk of developing T-CAD. Using HLA-A2 as a marker for the release of soluble HLA antigens from the donor, we established that recipients displaying circulating donor alloantigens for more than 26 weeks following transplantation are at increased risk of developing T-CAD (P=0.008). This association suggests that the release of alloantigens from the allograft is indicative of chronic injury and/or that it stimulates chronic rejection via the indirect allorecognition pathway. Our findings indicate that patients at risk of developing T-CAD can be identified by monitoring the release of donor alloantigens and production of antidonor HLA antibodies following transplantation.

Regulatory CD8+CD28- T cells in heart transplant recipients.

Human regulatory CD8+CD28- T cells (Ts) generated in vitro were demonstrated to suppress the activation and proliferation of T helper cells (Th) induced by allogeneic cells. This effect requires cell-to-cell contact, is antigen-specific, and results in Th anergy. To study the population of CD8+CD28- T cells present in vivo, flow cytometry was performed on whole blood specimens obtained from 25 heart transplant recipients and 12 normal controls. A significant expansion of CD8+CD28- T cells was found in transplant recipients as compared with normal individuals (p = 0.005). Expression of CD38, human leukocyte antigen-DR, and perforin positive cells within the CD8+CD28- subset was significantly higher in transplant patients than in normal controls, yet there was no correlation between the expression of these markers and acute rejection. Expression of the CD27 marker, however, was significantly higher within CD8+CD28- T cells from patients without rejection as compared with patients in rejection (p = 0.005), indicating that the memory-like CD8+CD28-CD27+ T-cell subset comprises regulatory cells, which play a protective role for the graft. CD8+CD28- T cells isolated from transplant patients did not display cytotoxic activity against donor cells and showed high expression of the killing inhibitory receptor CD94. This study identifies the phenotypic changes that occur in patients with heart transplants and opens new avenues for the induction of specific immunosuppression in transplantation.

Detection of T suppressor cells in patients with organ allografts.

Specific immunosuppression of hosts immune response to donor HLA antigens has been a major goal to clinical transplantation. Recent evidence has been accumulating to show that a distinct population of T cells expressing the CD8(+) CD28(-) phenotype display suppressor function and inhibit Th activation and proliferation by modulating the APC function. To assess the presence of Ts in transplant recipients circulation, we have developed a flow cytometry method that measures the expression of costimulatory molecules on donor APC exposed to recipient Th and Ts. Our results demonstrate that quantitation of the capacity of CD8(+) CD28(-) T cells from patient circulation to suppress the activation of costimulatory molecules (CD80, CD86) on donor APC offers a reliable tool for monitoring specific immunosuppression against the graft in solid organ transplantation.

Immunoglobulin-Like Transcript 3-Fc Suppresses T-Cell Responses to Allogeneic Human Islet Transplants in hu-NOD/SCID Mice

OBJECTIVE—The aim of our study was to explore the immunomodulatory activity of soluble immunoglobulin (Ig)-like transcript (ILT) 3-Fc in pancreatic islet transplantation and to determine its mechanism of action. RESEARCH DESIGN AND METHODS—NOD/SCID mice in which diabetes was induced by streptozotocin injection were transplanted with human pancreatic islet cells. Mice in which the transplant restored euglycemia were humanized with allogeneic peripheral blood mononuclear cells and treated with ILT3-Fc or control human IgG or left untreated. The blood glucose level was monitored twice a week, and rejection was diagnosed after two consecutive readings >350 mg/dl. Tolerated and rejected grafts were studied histologically and by immunostaining for human T-cells and insulin production. CD4 and CD8 T-cells from the spleen were studied for suppressor activity, expression of cytokines, and CD40L. RESULTS—Although human T-cell engraftment was similar in all groups, ILT3-Fc–treated mice tolerated the islets for the entire period of observation (91 days), whereas control mice rejected the graft within 7 weeks (P < 0.0001). ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human CD8+ T suppressor cells that inhibited Th alloreactivity against graft HLA antigens. T-cells allostimulated in vitro in the presence of ILT3-Fc inhibited CD40L-induced upregulation of CD40 in human pancreatic islet cells. Histochemical studies showed dramatic differences between human pancreatic islets from tolerant, ILT3-Fc–treated mice and control recipients rejecting the grafts. CONCLUSIONS—The data indicated that ILT3-Fc is a potent immunoregulatory agent that suppressed islet allograft rejection in humanized NOD/SCID mice.

Soluble Ig-Like Transcript 3 Inhibits Tumor Allograft Rejection in Humanized SCID Mice and T Cell Responses in Cancer Patients

Attempts to enhance patients’ immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8+ T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8+ T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68+ tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.

Distinct mRNA microarray profiles of tolerogenic dendritic cells

Dendritic cells are crucial to the activation as well as suppression of the immune response. Previous reports have illustrated that APC interacting with antigen-specific T suppressor cells become tolerogenic, inducing T helper anergy. To characterize the molecular changes occurring in tolerogenic APC, the mRNA profile of KG-1 dendritic cells exposed to allospecific T helper and T suppressor cells were analyzed. This study now provides evidence that immature dendritic cells stimulated by T suppressor cells differentiate into mature dendritic cells with a distinct phenotype. The identification of Ts induced pathways of dendritic cell differentiation is critical to the development of new therapeutic strategies.

MECHANISM OF LIVER ALLOGRAFT REJECTION : THE INDIRECT RECOGNITION PATHWAY

Transplant rejection is mediated by the direct and indirect pathways. To explore the role of the indirect recognition pathway in the rejection of liver allografts, T cells obtained from peripheral blood were expanded in medium containing IL-2 and tested in LDA for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial investigations of 17 recipients showed that T-cell reactivity to donor HLA-DR peptides was strongly associated with acute rejection episodes. In recipients carrying a graft that was mismatched by two HLA-DR alleles, a single donor antigen was targeted during primary rejection, although allopeptide reactivity against the second HLA-DR antigen was observed during subsequent episodes of acute rejection. The finding that allopeptide reactivity occurs early following transplantation and is predictive of rejection is consistent with the notion that processing of donor alloantigens by recipient APCs activates the indirect T-cell recognition pathway that plays a major role in initiating and amplifying allograft rejection.

Tailoring of immunosuppression in renal and liver allograft recipients displaying donor specific T-suppressor cells.

Although transplantation tolerance cannot be yet reliably achieved in humans, there is evidence that active immunosuppression contributes to the maintenance of quiescence. However, the mechanism that underlies quiescence and the precise identity of regulatory cells are not completely understood. We have demonstrated that allograft recipients who remain rejection-free display allospecific T-suppressor cells (Ts). Ts express the CD8(+) CD28(-) phenotype, recognize major histocompatibility complex (MHC) class I antigens, and suppress the up-regulation of costimulatory molecules induced by CD40 ligation of donor antigen presenting cells. The presence of Ts is inversely correlated with T cell alloreactivity to donor MHC peptides, alloantibody production, and rejection. Monitoring of Ts has been successfully used in our studies for tailoring immunosuppression in kidney and liver allograft recipients.

Immunologic monitoring in lung allograft recipients

To identify patients with increased risk of chronic lung allograft rejection, we assessed the utility of an in vitro biopsy-derived lymphocyte growth assay and serum anti-HLA antibody screening as a complement to currently available methods of monitoring lung allograft recipients. Lymphocyte growth assay was performed on bronchoscopic fragments of tissue cultured in medium with rIL-2. Seventy-nine biopsies from 31 lung transplant recipients were tested by lymphocyte growth assay, and results were correlated with histopathology findings. Positive lymphocyte growth was found in 12/26 (46%) episodes of acute rejection, 5/44 biopsies without rejection (11%), and 0/9 episodes of bronchitis. Positive lymphocyte growth was seen in 7/16 (44%) grade A1 rejections and in 5/10 (50%) grade A2 rejections, as opposed to only 5/44 (11%) grade A0 (no rejection) biopsies (P < 0.01 for both A1 and A2 with respect to A0). Actuarial probability of remaining free from obliterative bronchiolitis (OB)* tended to be higher in patients who did not exhibit lymphocyte growth in biopsies. Sequential samples of sera obtained at the time of the biopsy were screened for lymphocytotoxic anti-HLA antibodies. Twenty-two of 44 recipients (50%) developed anti-HLA antibodies during the first postoperative year, exhibiting greater than 10% reactivity to an HLA reference panel of lymphocytes in four or more consecutive serum samples. Actuarial survival of lung allograft recipients with anti-HLA antibodies (n = 22) was lower than in those without anti-HLA antibodies (n = 22; P = 0.03). Of the 22 antibody producers, 7/12 died as a consequence of OB. Of the 22 non-antibody-producers, 1/2 deaths occurred as a consequence of OB. Anti-HLA antibodies were present in 9/11 instances of OB (82% sensitivity) and in 13/33 patients without OB (61% specificity; P = 0.03). These data indicate that lung transplant recipients with positive lymphocyte growth and anti-HLA antibodies are at an increased risk of chronic allograft rejection.