Sorina Tugulea

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.

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.

Target cell susceptibility to lysis by human natural killer cells is augmented by α(1,3)-galactosyltransferase and reduced by α(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 α(1,3)GlcNAc, the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the α(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.

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.

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.

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.

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.

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.