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Evidence for amplification of the allogeneic response by heat-shock protein 60 [HSP60]
10
Abstracts
6.6
EVIDENCE FOR AMPLIFICATION OF THE ALLOGENEIC RESPONSE BY HEAT-SHOCK PROTEIN 60 [HSP60]. JD Tyler, Transplant Lab, Department of Surgery, University of Tennessee Medical Center, Knoxville, TN. It seems conceivable that stress proteins [aka heat-shock proteins; HSP] might play a role in the generation of allogeneic immunity since substantial cellular stress often accompanies transplantation. The effect of adding exogenous HSPs to the human allogeneic MLR was examined. Varying doses [1-1000 ng/ml] of recombinant human HSP27, HSP60, HSP70 or HSP90 were added to adherent PBMN from normal healthy donors. After overnight incubation [37C/5%C02], allogeneic responder PBL were added and the co-cultures were incubated for an additional 6-7 days. Cell proliferation, via 3H_ thymidine incorporation, was measured during the last 18-24 hr. HSP60 [at doses ~40 ng/ml] routinely induced a significant increase [2.1 ± 0.34 fold at optimal concentration] in proliferation whereas modest or no increase was observed in the presence HSP70, HSP27 or HSP90. Optimal cell proliferation occurred in the presence of 200-1 000 ng/ml of HSP60. Increased cell proliferation observed in allogeneic MLR containing HSP60 did not appear to be due solely to additive antigenic responses of alloreactive T cells plus HSP60 reactive T cells: 1] similar enhancement of the allo-MLR by HSP60 was observed in both serum-free and FBS containing media indicating that xenoantigens cannot eclipse the effect of HSP60. 2] Cell proliferation in the autologous MLR was not increased suffiCiently by HSP60 to account for the increase seen in allo-MLR. To assess the responding cells in more detail, limiting dilution analysis [LOA] of primed cultures was performed. Results of LOA indicate that both allo- and HSP60- reactive T cells exist in the responder population. However, only the frequency of alloreactive T cells was significantly increased when both stimuli are present. In conclusion, these initial stUdies suggest that HSP60 can amplify the allogeneic MLR and consequently, that HSP60 release in transplanted tissues may accelerate the rejection response.
6.6
IDENTIFICATION OF TWO TYPES OF SELF-APC REACTIVE T CELL CLONES FROM CARDIAC ALLOGRAFT-INFILTRATING CELLS INCUBATED WITH MYCOBACTERIAL HSP71. K Uu, RA Molitemo, Xiaa-Fe Fu, D Attfield and RJ Duquesnoy. Division of Transplantation Pathology, University of Pittsburgh. Recent stUdies in several laboratories have advanced the concept that during cellular rejection, the allograft undergoes a stress response which upregulates the expression of stress proteins (or heat shock proteins, hsp) and triggers the recruitment and activation of hsp-reactive lymphocytes. In a rat model of heterotopic heart transplants we have found that allograft-infiltrating lymphocytes respond to recombinant mycobacterial hsp and irradiated syngeneic spleen cells as a source of self-APC. T cell lines and clones have been generated by culturing ACI into Lewis rat cardiac allograft-derived lymphocytes with mycobacterial hsp71 , autologous APC and 11-2. Two groups of T cell clones have been distinguished, all of them are CD3+,CD4+,CD8-. One group is referred to as hsp71-dependent, self-APC reactive T cells because these clones respond to self-APC but only in the presence of hsp71. No reactivity is seen with mycobacterial hsp65 or when hsp71 is tested with allo-APC from ACI donors or third-party APC from BN rats. Treatment of hsp71 with proteolytic enzymes or polymyxin B abrogates the hsp71 effect thus indicating that structurally intact hsp71 molecules must interact with self-APC which then activate hsp71-dependent, self-APC reactive T-cells. The second group of clones reacts to self-APC and while their reponses does not require the presence of hsp71 , their proliferation is often augmented by hsp71 but not by hsp65. These hsp71-independent, self-APC reactive clones do not respond to allo-APC from ACI donors nor third-party APC from BN rats. Polymyxin or trypsin treatment had no significant effect on the proliferative responses of hsp71-independent, self-APC reactive T cell clones. The data with the anti-TCR-a~ monoclonal antibody R73 offer additional evidence for two functionally different types of self-APC reactive CD4 cells infiltrating the allograft. R73 inhibits the proliferation of self-APC induced responses of hsp71-independent clones as well as the alla-APC induced responses of alloreactive T cell clones. In contrast, this antibody augments the responses of hsp71-dependent T cells. Moreover, these clones can also proliferate in response to self-APC when hsp71 is substituted by R73. The hsp71-dependency of self-APC restricted T-cell reactivity represents a previously unrecognized mechanism of cellular immunity to allografts. This mechanism might be related to the peptide-binding properties of hsp71 and the ability of stress proteins to function as molecular chaperones in antigen processing.
Abstracts
6.6
EVIDENCE FOR AMPLIFICATION OF THE ALLOGENEIC RESPONSE BY HEAT-SHOCK PROTEIN 60 [HSP60]. JD Tyler, Transplant Lab, Department of Surgery, University of Tennessee Medical Center, Knoxville, TN. It seems conceivable that stress proteins [aka heat-shock proteins; HSP] might play a role in the generation of allogeneic immunity since substantial cellular stress often accompanies transplantation. The effect of adding exogenous HSPs to the human allogeneic MLR was examined. Varying doses [1-1000 ng/ml] of recombinant human HSP27, HSP60, HSP70 or HSP90 were added to adherent PBMN from normal healthy donors. After overnight incubation [37C/5%C02], allogeneic responder PBL were added and the co-cultures were incubated for an additional 6-7 days. Cell proliferation, via 3H_ thymidine incorporation, was measured during the last 18-24 hr. HSP60 [at doses ~40 ng/ml] routinely induced a significant increase [2.1 ± 0.34 fold at optimal concentration] in proliferation whereas modest or no increase was observed in the presence HSP70, HSP27 or HSP90. Optimal cell proliferation occurred in the presence of 200-1 000 ng/ml of HSP60. Increased cell proliferation observed in allogeneic MLR containing HSP60 did not appear to be due solely to additive antigenic responses of alloreactive T cells plus HSP60 reactive T cells: 1] similar enhancement of the allo-MLR by HSP60 was observed in both serum-free and FBS containing media indicating that xenoantigens cannot eclipse the effect of HSP60. 2] Cell proliferation in the autologous MLR was not increased suffiCiently by HSP60 to account for the increase seen in allo-MLR. To assess the responding cells in more detail, limiting dilution analysis [LOA] of primed cultures was performed. Results of LOA indicate that both allo- and HSP60- reactive T cells exist in the responder population. However, only the frequency of alloreactive T cells was significantly increased when both stimuli are present. In conclusion, these initial stUdies suggest that HSP60 can amplify the allogeneic MLR and consequently, that HSP60 release in transplanted tissues may accelerate the rejection response.
6.6
IDENTIFICATION OF TWO TYPES OF SELF-APC REACTIVE T CELL CLONES FROM CARDIAC ALLOGRAFT-INFILTRATING CELLS INCUBATED WITH MYCOBACTERIAL HSP71. K Uu, RA Molitemo, Xiaa-Fe Fu, D Attfield and RJ Duquesnoy. Division of Transplantation Pathology, University of Pittsburgh. Recent stUdies in several laboratories have advanced the concept that during cellular rejection, the allograft undergoes a stress response which upregulates the expression of stress proteins (or heat shock proteins, hsp) and triggers the recruitment and activation of hsp-reactive lymphocytes. In a rat model of heterotopic heart transplants we have found that allograft-infiltrating lymphocytes respond to recombinant mycobacterial hsp and irradiated syngeneic spleen cells as a source of self-APC. T cell lines and clones have been generated by culturing ACI into Lewis rat cardiac allograft-derived lymphocytes with mycobacterial hsp71 , autologous APC and 11-2. Two groups of T cell clones have been distinguished, all of them are CD3+,CD4+,CD8-. One group is referred to as hsp71-dependent, self-APC reactive T cells because these clones respond to self-APC but only in the presence of hsp71. No reactivity is seen with mycobacterial hsp65 or when hsp71 is tested with allo-APC from ACI donors or third-party APC from BN rats. Treatment of hsp71 with proteolytic enzymes or polymyxin B abrogates the hsp71 effect thus indicating that structurally intact hsp71 molecules must interact with self-APC which then activate hsp71-dependent, self-APC reactive T-cells. The second group of clones reacts to self-APC and while their reponses does not require the presence of hsp71 , their proliferation is often augmented by hsp71 but not by hsp65. These hsp71-independent, self-APC reactive clones do not respond to allo-APC from ACI donors nor third-party APC from BN rats. Polymyxin or trypsin treatment had no significant effect on the proliferative responses of hsp71-independent, self-APC reactive T cell clones. The data with the anti-TCR-a~ monoclonal antibody R73 offer additional evidence for two functionally different types of self-APC reactive CD4 cells infiltrating the allograft. R73 inhibits the proliferation of self-APC induced responses of hsp71-independent clones as well as the alla-APC induced responses of alloreactive T cell clones. In contrast, this antibody augments the responses of hsp71-dependent T cells. Moreover, these clones can also proliferate in response to self-APC when hsp71 is substituted by R73. The hsp71-dependency of self-APC restricted T-cell reactivity represents a previously unrecognized mechanism of cellular immunity to allografts. This mechanism might be related to the peptide-binding properties of hsp71 and the ability of stress proteins to function as molecular chaperones in antigen processing.