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Contribution of direct and indirect recognition to alloreactivity
26
Abstracts
C 3.3.39
CONTRIBUTION OF DIRECT AND INDIRECT RECOGNITION TO ALLOREACTIVITY. Z. Liu, B. Hong, E. Reed and N. Suciu-Foca. Department of Pathology, Columbia University, New York, NY. Two forms of allorecognition are involved in alloreactivity. One form, accounting primarily for cytotoxic T cell responses, is the direct recognition of intact allogeneic MHC molecule with the peptide bound to it. The other form, which may be involved in T helper activation, is the indirect recognition of allopeptides processed and presented on self MHC by host APCs. To determine the relative contribution of direct and indirect recognition to alloreactivity we have evaluated the frequency of allo-peptide specific and allo-MHC specific T cells in a population of T cells primed in 10-days MLC with allogeneic PBMC from a DRB 1"0101 positive stimulator. Primed T cells were tested in limiting dilutions for reactivity to A) DR1 stimulating cells and B) overlapping synthetic 20-met peptides spanning the first domain of DRBI*0101, in the presence of autologous APCs. In the absence of APCs autologous to the responder, T cells primed to allogeneic HLA-DR1 reacted in secondary cultures to "intact" DR1 on allogeneic cells indicating that they recognize this molecule directly. There was no reactivity to HLADR1 negative stimulating cells. When autologous APCs were added to the cultures, these DR1 specific T cells also reacted to a cocktail of DR1 peptides indicating that they recognize processed forms of DR 1. Hence, allo-stimulation resulted in the activation of T cells capable of direct and indirect recognition. Results of limiting dilution assays indicated that T cells capable of indirect recognition are about 100 fold less frequent than T cells exhibiting direct recognition of native DR1 antigen. Given their frequency of 1/44,000 it is evident that they represent an important segment of the population which is likely to contribute to the alloimmune response. The development of HLA-specific antibodies in patients undergoing chronic allograft rejection may require the help of such allopeptide specific T cells. Study of TCR usage in T cells participating in direct and indirect recognition showed distinct but limited repertoires.
C 3.4.40
CHANGES IN THE TCR-VB REPERTOIRE OBSERVED IN THE DEVELOPMENT OF DONOR ANTIGEN-SPECIFIC HYPOREACTIVlTY (DASH). AM Jackson, P Donahue, P Santamaria, K Butters, D Cranger, NL Reinsmoen, Department of Surgery, University of Minnesota, Minneapolis, MN. We have shown that 25% of renal allograft recipients (R) develop in vitro DASH as characterized by a reduced p~oliferative response of R' cells (post vs. pretx) when stimulated with donor cells or HTCs expressing donor class II ag; R with DASH have fewer late rejections episodes (p<0.05), and im;~ro,md graft outcome. We have investigated changes in the T-cell receptor VB (TCR-VB) repertoire pre and posttx in two kidney R displaying DASH. R #1's cells demonstrated a decrease in donor specific (Dw4) MLC of 40 to 8% RR when tested pre vs. posttx. R #2's cells demonstrated hypo-reactivity to HTCs defining the donor's Dw4 ag. Pre and posttx cells from these R were primed in vitro against donor ag and third party ag in the presence of IL2. We then analyzed the TCR-VB repertoire pretx and posttx by r,eans of PCR amplification and VB-family specific primers. When primed against a third party ag there were no major differences in VB usage pre and posttx, supporting the concept that any changes seen are related to the donor ag-specific response and not related to the immunosuppression. Cells primed against the specific donor ag however, did result in quantitative changes in select TCR-VB families. Posttx cells from R #I displayed lower levels of TCR-VBI6.9, VBI0 and V ~ - ] 3 when compared to pretx. R #2 also displayed reduced levels of VBL6.9 posttx and in addition showed a reduction in VBI4 and VBI2.1. We are performing additional quantitative PCR studies and constructing eDNA libraries to determine the clonal frequency of different elonotypes within these VB-families. These studies are of import in addressing the issue of anergy vs. clonal deletion in the generation of DASH.
Abstracts
C 3.3.39
CONTRIBUTION OF DIRECT AND INDIRECT RECOGNITION TO ALLOREACTIVITY. Z. Liu, B. Hong, E. Reed and N. Suciu-Foca. Department of Pathology, Columbia University, New York, NY. Two forms of allorecognition are involved in alloreactivity. One form, accounting primarily for cytotoxic T cell responses, is the direct recognition of intact allogeneic MHC molecule with the peptide bound to it. The other form, which may be involved in T helper activation, is the indirect recognition of allopeptides processed and presented on self MHC by host APCs. To determine the relative contribution of direct and indirect recognition to alloreactivity we have evaluated the frequency of allo-peptide specific and allo-MHC specific T cells in a population of T cells primed in 10-days MLC with allogeneic PBMC from a DRB 1"0101 positive stimulator. Primed T cells were tested in limiting dilutions for reactivity to A) DR1 stimulating cells and B) overlapping synthetic 20-met peptides spanning the first domain of DRBI*0101, in the presence of autologous APCs. In the absence of APCs autologous to the responder, T cells primed to allogeneic HLA-DR1 reacted in secondary cultures to "intact" DR1 on allogeneic cells indicating that they recognize this molecule directly. There was no reactivity to HLADR1 negative stimulating cells. When autologous APCs were added to the cultures, these DR1 specific T cells also reacted to a cocktail of DR1 peptides indicating that they recognize processed forms of DR 1. Hence, allo-stimulation resulted in the activation of T cells capable of direct and indirect recognition. Results of limiting dilution assays indicated that T cells capable of indirect recognition are about 100 fold less frequent than T cells exhibiting direct recognition of native DR1 antigen. Given their frequency of 1/44,000 it is evident that they represent an important segment of the population which is likely to contribute to the alloimmune response. The development of HLA-specific antibodies in patients undergoing chronic allograft rejection may require the help of such allopeptide specific T cells. Study of TCR usage in T cells participating in direct and indirect recognition showed distinct but limited repertoires.
C 3.4.40
CHANGES IN THE TCR-VB REPERTOIRE OBSERVED IN THE DEVELOPMENT OF DONOR ANTIGEN-SPECIFIC HYPOREACTIVlTY (DASH). AM Jackson, P Donahue, P Santamaria, K Butters, D Cranger, NL Reinsmoen, Department of Surgery, University of Minnesota, Minneapolis, MN. We have shown that 25% of renal allograft recipients (R) develop in vitro DASH as characterized by a reduced p~oliferative response of R' cells (post vs. pretx) when stimulated with donor cells or HTCs expressing donor class II ag; R with DASH have fewer late rejections episodes (p<0.05), and im;~ro,md graft outcome. We have investigated changes in the T-cell receptor VB (TCR-VB) repertoire pre and posttx in two kidney R displaying DASH. R #1's cells demonstrated a decrease in donor specific (Dw4) MLC of 40 to 8% RR when tested pre vs. posttx. R #2's cells demonstrated hypo-reactivity to HTCs defining the donor's Dw4 ag. Pre and posttx cells from these R were primed in vitro against donor ag and third party ag in the presence of IL2. We then analyzed the TCR-VB repertoire pretx and posttx by r,eans of PCR amplification and VB-family specific primers. When primed against a third party ag there were no major differences in VB usage pre and posttx, supporting the concept that any changes seen are related to the donor ag-specific response and not related to the immunosuppression. Cells primed against the specific donor ag however, did result in quantitative changes in select TCR-VB families. Posttx cells from R #I displayed lower levels of TCR-VBI6.9, VBI0 and V ~ - ] 3 when compared to pretx. R #2 also displayed reduced levels of VBL6.9 posttx and in addition showed a reduction in VBI4 and VBI2.1. We are performing additional quantitative PCR studies and constructing eDNA libraries to determine the clonal frequency of different elonotypes within these VB-families. These studies are of import in addressing the issue of anergy vs. clonal deletion in the generation of DASH.