Indirect recognition of donor HLA-DR peptides in organ allograft rejection.

To determine whether indirect allorecognition is involved in heart allograft rejection T cells obtained from peripheral blood and graft biopsy tissues were expanded in the presence of IL-2 and tested in limiting dilution analysis (LDA) for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial studies of 32 patients showed that T cell reactivity to donor allopeptides was strongly associated with episodes of acute rejection. The frequency of allopeptide reactive T cells was 10-50-fold higher in the graft than in the periphery indicating that T cells activated via the indirect allorecognition pathway participate actively in acute allograft rejection. In recipients carrying a graft differing by two HLA-DR alleles the response appeared to target only one of the mismatched antigens of the donor. Indirect allorecognition was restricted by a single HLA-DR antigen of the host and directed against one immunodominant peptide of donor HLA-DR protein. However, intermolecular spreading was demonstrated in patients with multiple rejection episodes by showing that they develop allopeptide reactivity against the second HLA-DR antigen. These data imply that early treatment to suppress T cell responses through the indirect pathway of allorecognition, such as tolerance induction to the dominant donor determinant, may be required to prevent amplification and perpetuation of the rejection process.

High-level porcine endothelial cell expression of alpha(1,2)-fucosyltransferase reduces human monocyte adhesion and activation.

BACKGROUND: Monocyte binding to and activation by human endothelium requires a number of interactions, including those involving sialylated endothelial cell ligands. As porcine endothelial cell transfection with alpha(1,2)-fucosyltransferase has been shown to reduce terminal sialylation, we investigated whether high-level expression of alpha(1,2)-fucosyltransferase by porcine endothelium would reduce human monocyte adhesion and functional activation. METHOD: Purified human monocytes were labeled with 51Cr, and measured for adherence to human or porcine endothelial cell monolayers in the presence of either medium or monoclonal antibodies against monocyte lectins or sialylated endothelial cell ligands. Monocyte production of prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) was measured by enzyme-linked immunosorbent assay, using supernatants collected from cultures performed between human monocytes and human or porcine endothelial cell monolayers. Finally, monocyte adhesion and activation were measured after culture with a porcine endothelial cell line transfected with alpha(1,2)-fucosyltransferase, expressing reduced surface expression of terminal Gal alpha(1,3)-Gal and sialic acid residues. RESULTS: Human monocytes adhered by 50% higher levels to porcine endothelium than to human endothelium. This increased level of adherence was associated with augmented monocyte activation, as defined by 3.3-fold higher levels of PGE2 production and 7.3-fold higher levels of IL-1beta production. Monoclonal antibodies against CD62L (L-selectin) on monocytes or CD15s (sialylated Lewis X) on porcine endothelium reduced monocyte adhesion by 38% and 52%, respectively. Porcine endothelial cell transfection with alpha(1,2)-fucosyltransferase reduced terminal sialic acid expression by 65%, monocyte adherence by 50%, and the production of PGE2 and IL-1beta by 67% and 38%, respectively. CONCLUSIONS: Together, these results demonstrate that human monocytes use surface lectins to bind to sialylated carbohydrate structures on porcine endothelium, and indicate that reduction in porcine endothelial cell surface expression of terminally sialylated structures by high-level alpha(1,2)-fucosyltransferase activity reduces monocyte adherence and activation.

New strategies for early diagnosis of heart allograft rejection.

BACKGROUND: Allograft rejection is mediated by T cells that recognize allogeneic major histocompatibility complex (MHC) molecules via the direct and indirect pathway. The direct pathway involves T cells that react against MHC/peptide complexes expressed on the surface of donor antigen-presenting cells (APCs). In contrast, T cells involved in the indirect pathway recognize peptides derived from processing and presentation of allogeneic MHC molecules by self (recipient) APCs. To explore the relative contribution of these two pathways to rejection, we have evaluated the response of peripheral blood T cells from 50 heart transplant recipients against donor APCs (direct recognition) and against self APCs pulsed with synthetic peptides corresponding to the hypervariable region of the mismatched HLA-DR antigens of the donor (indirect recognition). METHODS: T cell reactivity against donor APCs was quantitated by measuring the expression of CD69 on allostimulated CD3+ LDA1+ cells. Reactivity to synthetic allopeptides was determined in limited dilution assays. RESULTS: Serial studies of the kinetics of direct and indirect recognition showed that both pathways contribute to early acute rejection episodes. Primary rejection was accompanied invariably by indirect recognition of a dominant allopeptide. Intermolecular spreading of T cell epitopes was observed during recurrent rejections. Enhanced recognition of donor alloantigens via the direct pathway was found predominantly during early rejection episodes. A single form of allorecognition was shown to occur in some rejection episodes. CONCLUSIONS: Monitoring of the direct and indirect pathway of allorecognition provides a reliable method for prediction and differential diagnosis of acute rejection of heart allografts.

Influence of HLA class I and class II antigens on the peripheral T-cell receptor repertoire.

Using TCR-specific mAbs, we analyzed the TCR distribution in the CD4 and CD8 compartments of peripheral blood T cells from members of six families, including two which were informative for an HLA-B/DR crossover. TCR distribution in the CD4 compartment is influenced by MHC class II antigens as it was similar in the child carrying the HLA-B/DR recombination and in the HLA-DR-identical sibling. There was also a relatively small degree of difference between TCR distribution in the CD8 compartment of a child with an HLA-B/DR crossover and his HLA class-I-identical sibling. Analysis of TCR V beta gene usage in the CD4 compartment of unrelated individuals sharing an HLA-DR allele showed an increased usage of V beta 8a and V beta 12a in DR4-positive subjects. These data suggest that studies of TCR V beta gene usage in individuals with HLA-associated autoimmune diseases may provide further information about MHC-dependent biases in the T-cell repertoire.

Inhibition of CD40 signaling pathway in antigen presenting cells by T suppressor cells.

Understanding the mechanism which underlies the induction of immunologic tolerance is crucial to the development of strategies for treatment of autoimmune diseases and allograft rejection. Although the concept that T suppressor cells (Ts) downregulate the immune response has long been accepted, the existence of a distinct population of lymphocytes that mediates suppression has not been convincingly demonstrated. In previous studies, we have utilized human T cell lines (TCLs) to analyze the suppressive effects of CD8+CD28 T cells in allogeneic, peptide specific and xeno-specific responses. In each case, CD8+CD28- T cells inhibit proliferation of CD4+ T helper lymphocytes (Th) with cognate antigen specificity. These CD8+CD28- T cells display the critical functional characteristics of T suppressor cells. Similar to the induction of CD8+ cytotoxic T cells (Tc) by Th, this process depends on antigen presenting cells (APC) acting as a "bridge" between MHC-class I specific CD8+ and class II specific CD4+ T cells. A possible explanation of Ts-mediated suppression is their ability to modulate the function of APCs. The present studies show that CD8+CD28- Ts directly inhibit the CD40 signaling pathway of APC by a contact-dependent mechanism that renders bridging APCs incapable of inducing CD4+ Th activation. The effects of Ts on the functional state of APC supports the concept that the order in which Ts and Th cells interact with cognate APCs determines the functional outcome of immune responses.

Induction of MHC-class I restricted human suppressor T cells by peptide priming in vitro.

The induction of regulatory T cells may offer an effective means for specific immunosuppression of autoimmune disease and allograft rejection. The existence of suppressor T cells has been previously documented, yet their mechanism of action remains poorly characterized. Our studies demonstrate that T suppressor (Ts) cell lines can be generated by in vitro immunization of human PBMCs, with synthetic peptides or soluble proteins coupled to beads. Such Ts cells express the CD8+CD28- phenotype and show the following characteristics: (a) antigen specificity and restriction by self MHC Class I molecules; (b) limited TCR V beta gene usage; (c) ability to inhibit antigen-specific, MHC Class II restricted, Th proliferative responses; and (d) capacity to downregulate and/or inhibit the upregulation by Th of CD40, CD80, and CD86 molecules on APCs. The inhibitory activity of Ts on Th proliferation requires the tripartite interaction between Th, Ts, and APCs and results from inefficient costimulation of Th.

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.

The influence of concordant xenografts on the humoral and cell-mediated immune responses to subsequent allografts in primates.

The humoral and cell-mediated immune responses to subsequent allografts were determined in primate recipients after concordant xenotransplantation as a bridge to allotransplantation. Heterotopic heart transplants (n = 4) were performed from cynomolgus monkeys into ABH type-matched olive baboons followed 2 weeks later by allotransplantation from ABH type-matched baboon donors. Allografts were explanted at 8 weeks. All recipients underwent splenectomy at the time of xenotransplantation and received immunosuppression with cyclosporine, azathioprine, and methylprednisolone. Concordant xenotransplantation in these primates did not induce humoral or cell-mediated immune responses that jeopardized subsequent allografts. The degree of xenospecific immune reactivity, as determined by specific cytotoxicity of recipient T-cell lines derived from the xenograft and extent of histologic xenograft rejection, did not predict the severity of subsequent allograft rejection. In two of the four recipients, xenotransplantation induced an alloreactive humoral response against antigens expressed by the B cells of more than 50% of members from a panel of 12 unrelated baboons. In all recipients, priming with xenogeneic splenocytes in vitro induced an accelerated proliferative T-cell response to allogeneic lymphocytes from 16% of this panel. This study affirms the role of concordant xenografts as appropriate biologic bridges to human allotransplantation. However, our results suggest that xenoreactive baboon memory CD4 T cells may recognize major histocompatibility complex class II--like structures shared between the xenogeneic and allogeneic targets. The potential allorecognition induced by a xenograft may affect the process of subsequent allograft donor selection.

Specific suppression of T helper alloreactivity by allo-MHC class I-restricted CD8+CD28- T cells.

Specific suppression of the host's immune response to donor HLA antigens remains the ultimate goal for clinical transplantation. In spite of considerable effort, however, allospecific human suppressor T cells (Ts) have been difficult to generate. Here we show that allospecific and xenospecific Ts can be raised by multiple priming of human T cells in mixed lymphocyte cultures. Ts derive from the CD8+CD28- subset and recognize specifically the MHC class I antigens expressed by antigen-presenting cells (APC) used for in vitro immunization. Allospecific Ts prevent the up-regulation of B7 molecules on target APC, interfering with the CD28-B7 interaction required for T helper (Th) activation. These findings provide a basis for the development of specific immunosuppressive therapy.

Naturally processed tissue- and differentiation stage-specific autologous peptides bound by HLA class I and II molecules of chronic myeloid leukemia blasts.

Structural analysis of naturally processed peptides bound to the HLA class I and class II molecules of chronic myeloid leukemia (CML) blast cells was performed to characterize the antigen processing and autoantigen repertoire in this hematopoietic malignancy. Self-peptides derived from the carboxy-terminal end of the breakpoint cluster region (bcr) protein, as well as several differentiation stage- and tissue-specific self-antigens characteristic of early stages of myeloid differentiation, such as c-fes, c-pim, granulocyte-macrophage colony-stimulating factor receptor alpha chain, proteinase 3, and cathepsin G, were identified. A common characteristic of several of the high copy-number self-peptides identified in this study is the participation of their parent proteins in signal transduction or myeloid effector function. Because bcr-abl junctional peptides bind to a limited number of major histocompatibility complex (MHC) class I alleles, an effective peptide-based immunotherapy strategy for CML requires identification of further tumor-associated or tissue-specific peptide antigens binding to common MHC alleles such as HLA-A2. The differentiation stage- and tissue-specific MHC-bound peptides found in this study, as well as the naturally processed proteins from which they are derived, may represent autoantigens towards which T-cell responses may potentially be developed for immunotherapy of hematopoietic malignancies such as CML.

Predominant HLA-class II bound self-peptides of a hematopoietic progenitor cell line are derived from intracellular proteins.

Human myeloid progenitor cells temporarily express HLA class II molecules during the differentiation pathway to granulocytes and macrophages. The significance of major histocompatibility complex (MHC) class II molecules at this stage of development is unknown. As a first stop of inquiry into their function, we have characterized the profile of major self-peptides bound to the HLA-DR molecules expressed by KG-1 cells, a line that shares many of the phenotypic characteristics of colony-forming unit-granulocyte-macrophage progenitors. Searches of protein data bases showed that all matching peptides bound to the HLA-DR molecules of KG-1 cells corresponded to intracellular, rather than exogenous or transmembrane, precursor proteins. Because the absence of a conventional self-peptide repertoire could be related to altered trafficking of class II molecules, the biosynthesis of HLA-DR and the invariant chain proteins was determined. The MHC class II associated invariant chain protein is synthesized normally in KG-1 cells, but processed fragments of invariant chain, class II-associated invariant chain peptides (CLIPs), occupy the antigen-binding groove of KG-1 class II molecules at a much lower frequency compared with that of mature antigen-presenting cells. Low CLIP occupancy of HLA-DR is a characteristic shared by KG-1 cells, normal CD34+ progenitor cells, and HLA-DR+ breast carcinoma cells. The unusual profile of MHC class II bound peptides and the low level of CLIP bound to HLA-DR suggest that the antigen-processing pathway of KG-1 is different from that characterized in professional antigen-presenting cells and that exogenous antigen-processing may be a developmentally acquired characteristic in the myeloid lineage.

Target cell susceptibility to lysis by human natural killer cells is augmented by alpha(1,3)-galactosyltransferase and reduced by alpha(1, 2)-fucosyltransferase.

Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal alpha(1,3)Gal beta(1,4)GlcNAc [corrected], the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the alpha(1,2)-fucosyltransferase gene reduced susceptibility to human NK lysis. These results indicate that surface carbohydrate remodeling profoundly affects target cell susceptibility to NK lysis, and suggest that successful transgenic strategies to limit xenograft rejection by NK cells and xenoreactive antibodies will need to incorporate carbohydrate remodeling.

Activation-induced T-cell death and immune dysfunction after implantation of left-ventricular assist device.

BACKGROUND: Cardiac transplantation is a limited option for end-stage heart failure because of the shortage of donor organs. Left-ventricular assist devices (LVADs) are currently under investigation as permanent therapy for end-stage heart failure, but long-term successful device implantation is limited because of a high rate of serious infections. To examine the relation between LVAD-related infection and host immunity, we investigated immune responses in LVAD recipients. METHODS: We compared the rate of candidal infection in 78 patients with New York Heart Association class IV heart failure who received either an LVAD (n=40) or medical management (controls, n=38). Fluorochrome-labelled monoclonal antibodies were used in analyses of T-cell phenotype. Analysis of T-cell function included intradermal responses to recall antigens and proliferative responses after stimulation by phytohaemagglutinin, monoclonal antibodies to CD3, and mixed lymphocyte culture. We measured T-cell apoptosis in vivo by annexin V binding, and confirmed the result by assessment of DNA fragmentation. Activation-induced T-cell death was measured after T-cell stimulation with antibodies to CD3. All immunological tests were done at least 1 month after LVAD implantation. Between-group comparisons were by Kaplan-Meier actuarial analysis and Student's t test. FINDINGS: By 3 months after implantation of LVAD, the risk of developing candidal infection was 28% in LVAD recipients, compared with 3% in controls (p=0.003). LVAD recipients had cutaneous anergy to recall antigens and lower (<70%) T-cell proliferative responses than controls after activation via the T-cell receptor complex (p<0.001). T cells from LVAD recipients had higher surface expression of CD95 (Fas) (p<0.001) and a higher rate of spontaneous apoptosis (p<0.001) than controls. Moreover, after stimulation with antibodies to CD3, CD4 T-cell death increased by 3.2-fold in LVAD recipients compared with only 1.2-fold in controls (p<0.05). INTERPRETATION: LVAD implantation results in an aberrant state of T-cell activation, heightened susceptibility of CD4 T cells to activation-induced cell death, progressive defects in cellular immunity, and increased risk of opportunistic infection.

Harvest quality and factors affecting collection and engraftment of CD34+ cells in patients with breast cancer scheduled for high-dose chemotherapy and peripheral blood progenitor cell support.

The use of CFU-GM and CD34+ cell enumeration for assessing harvest quality and factors affecting peripheral blood progenitor cell (PBPC) harvest and engraftment were investigated in 45 women with high-risk and metastatic breast cancer scheduled for dose-intensive cyclophosphamide, thiotepa, and carboplatin (CTCb). PBPC were mobilized with standard breast cancer regimens or cyclophosphamide (1.5 g/m2) and 5 micrograms/kg/day G-CSF and used together with G-CSF for hematopoietic support post-CTCb. There was a significant correlation between peripheral blood CD34+ cells/microliter and harvest CD34+/kg (r = 0.73, p < 0.0001) and between harvest CFU-GM and CD34+ cells/kg (r = 0.5, p < 0.0001). CFU-GM clonogenic assays were of no clinical use beyond that of CD34+ cell enumeration, with the latter allowing for real-time decisions regarding harvesting. Multiple stepwise regression identified the number of prior chemotherapy cycles as the only significant clinical predictor of CD34+ cell yield. For 34 patients proceeding to CTCb with PBPC support, multiple stepwise regression identified as the best predictors for engraftment CFU-GM and CD34+ cells/kg for neutrophils and CFU-GM, CD34+ cells/kg, and the number of prior cycles of chemotherapy for platelets, respectively. A threshold dose of 1 x 10(6) CD34+ cells/kg, obtained in 87% of these heavily pretreated breast cancer patients, was adequate to ensure engraftment within 15 days. There was no significant difference in length of hospital stay or blood product use between patients receiving 1-2.5 x 10(6) CD34+ cells/kg and greater than 2.5 x 10(6) CD34+ cells/kg, although median time to engraftment of neutrophils (9 days versus 8 days, p = 0.007) and platelets (12 days versus 9 days, p = 0.006) was significantly longer. The established threshold of > or = 1 x 10(6) CD34+ cells/kg will allow for more confident consideration of using aliquots of this threshold dose for hematopoietic support in sequential high-dose regimens inclusive of CTCb.