CD8α/α homodimers fail to function as co‐receptor for a CD8‐dependent TCR
Anne‐Marie McNicolGavin BendleAngelika HollerTheres MatjekaEmma DaltonLorna RettigRose ZamoyskaWolfgang UckertShao‐An XueHans J. Stauss
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In this study, we have started to dissect the molecular basis of CD8 dependence of a high and low avidity CTL clone specific for the same peptide epitope. Using anti-CD8alpha and anti-CD8beta antibodies, we found that cytotoxicity and IFN-gamma production by high but not by low avidity CTL was strongly CD8 dependent. We isolated the TCR genes of both types of CTL clones and used retroviral gene transfer to analyse the function of these TCR in primary T cells of wild-type and CD8beta-deficient mice. Both TCR triggered antigen-specific killing in wild-type T cells, and blocking experiments showed that CD8 dependence/independence co-transferred with the TCR into primary T cells, indicating that it was dictated by the TCR itself. Gene transfer experiments into CD8beta-deficient T cells revealed that only the TCR derived from the CD8-independent CTL clone elicited antigen-specific cytotoxicity, while the CD8-dependent TCR was non-functional in the absence of the CD8beta-chain. These data indicate a striking difference between CD8alpha/beta heterodimers and CD8alpha/alpha homodimers as only the former were able to provide co-receptor function for the CD8-dependent TCR.Keywords:
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Abstract Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral infection by recognizing lower Ag densities than those distinguished by low-avidity CTL and 2) they initiate lysis of target cells more rapidly at any given Ag density. Alternative mechanisms were excluded, including: 1) the possibility that low-avidity CTL might control virus given more time (virus levels remained as high at 6 days following transfer as at 3 days) and 2) that differences in efficacy might be correlated with homing ability. Furthermore, adoptive transfer of high- and low-avidity CTL into SCID mice demonstrated that transfer of a 10-fold greater amount of low-avidity CTL could only partially compensate for their decreased ability to eliminate infected cells. Thus, we conclude that high-avidity CTL exploit two complementary mechanisms that combine to prevent the spread of virus within the animal: earlier recognition of infected cells when little viral protein has been made and more rapid lysis of infected cells.
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Abstract Adoptive T cell therapy (ACT) with antitumor CTL is a promising and tailored treatment against cancer. We investigated the role played by the affinity and avidity of the interaction between the tumor and the CTL on the outcome of ACT against a mouse non-Hodgkin B cell lymphoma that expresses OVA as a model neoantigen. ACT was assessed under conditions where antitumor CTL expressed TCR of varying affinity for OVA. We also assessed conditions where the avidity of Ag recognition varied because the lymphoma cells expressed high or low levels of OVA. Efficient eradication of small tumor burdens was achieved by high- or low-affinity CTL. Tumors expressing low levels of OVA could also be eliminated. However, ACT against large tumor burdens was unsuccessful, accompanied by CTL deletion and functional impairment. This negative outcome was not prevented by lowering the affinity of the CTL or the expression of OVA in the lymphoma. Thus, tumor burden, rather than CTL affinity or avidity, appears to be the main determinant of ACT outcomes in our lymphoma model. Insofar as our results can be extrapolated to the clinical setting, they imply that the range of CTL and tumor-associated Ag combinations that may be effectively harnessed in ACT against lymphoma may be wider than generally assumed. CTL expressing low-affinity TCR may be effective against lymphoma, and lowly expressed tumor-associated Ag should be considered as potential targets, but tumor reduction should always be implemented before infusion of the CTL.
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Abstract TCRα- and β-chains cooperatively recognize peptide–MHC complexes. It has been shown that a “chain-centric” TCR hemichain can, by itself, dictate MHC-restricted Ag specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non–chain-centric, hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α-chain–centric HLA-A*02:01(A2)/MART127–35 TCRα, clone SIG35α, into A2-matched and unmatched postthymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127–35. Although the generated A2/MART127–35–specific T cells used various TRBV genes, TRBV27 predominated with >102 highly diverse and unique clonotypic CDR3β sequences. T cells individually reconstituted with various A2/MART127–35 TRBV27 TCRβ genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127–35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-Ags. Our results indicate that, although a chain-centric TCR hemichain determines Ag specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising Ag specificity. TCR chain centricity can be exploited to generate a thymically unselected Ag-specific T cell repertoire, which can be used to isolate high-avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery because of tolerance.
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Abstract CTL that possess a high functional avidity are known to be optimal for the clearance of pathogens in vivo. We have shown that the amount of peptide encountered by a CD8+ CTL determines its functional avidity. Notably, in these studies nonprofessional APC were used. However, it is mature dendritic cells (DC) that are predominantly responsible for the activation of naive T cells in vivo. Whether DC also direct dose dependent-differences in avidity is unknown. In this work we examined the ability of mature DC presenting a high vs low level of peptide to generate CTL of distinct avidities. In contrast to what was observed with nonprofessional APC, CTL generated by stimulation with mature DC were of high avidity regardless of the amount of peptide presented. This DC property may promote generation of highly effective CTL that retain plasticity, which would allow the tuning of avidity in the periphery to promote optimal pathogen recognition and clearance.
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Abstract High avidity CTL are most effective at clearing viruses and cancer cells. Therefore, understanding the mechanisms involved in induction of high avidity CTL is critical for effective vaccines. However, no vaccine approach to selectively induce high avidity CTL in vivo has been discovered. In a new approach, signals from MHC class I (signal 1) and costimulatory molecules (signal 2) were adjusted by varying Ag dose and by use of recombinant poxvirus expressing a triad of costimulatory molecules (B7-1, ICAM-1, and LFA-3), respectively. Independent of CTL avidity, a strong signal 1 resulted in an increased frequency of CD8+ CTL. However, a strong signal 2 was necessary for the induction of high avidity CD8+ CTL that killed target cells more efficiently, and signal 2 played a more crucial role in the absence of a strong signal 1. Only CTL induced with strong signal 2 killed tumor cells endogenously expressing low levels of Ag. Signal 2 contributed to the induction of high avidity CD8+ CTL in both primary and secondary responses. Thus, although signal 2 has been known to increase the quantity of CTL response, in this study we show that it also improves the quality of CTL response. Our data also suggested that dendritic cells play an important role in induction of high avidity CD8+ CTL in vivo. This strategy to selectively induce higher avidity CTL may lead to more effective vaccines for viruses and cancer.
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Abstract All HIV‐1 ‘systemic vaccine trials’ in humans have yielded poor outcomes. Thus, it is important to understand whether the route of delivery influences the quality of protective CTL immunity. Using heterologous poxvirus immunisation we have shown that systemically (i.m./i.m.) immunised CD8 + T cells generated higher levels of IL‐4/IL‐13 compared to mucosal delivery and expression also correlated with i.m./i.m. immunised mice eliciting CTL of lower avidity. Studies using IL‐4 −/− and IL‐13 −/− KO mice have shown that the capacity to express IFN‐γ, IL‐4 and/or IL‐13 by K d Gag 197–205 ‐specific CTL differed between these groups and was inversely correlated with CTL avidity (IL‐13 −/− >IL‐4 −/− >BALB/c), although no significant differences in the magnitude of CTL responses were observed between IL‐13 −/− and wild type mice. When IL‐13 was reconstituted in IL‐13 −/− splenocytes in vitro , their ability to bind tetramers also decreased significantly. Our data reveal that total absence of IL‐13 can greatly enhance CTL avidity. In contrast, extracellular IL‐4 appears to be important in maintaining long‐term Th1/Th2 balance in CTL, even though expression of IL‐4 by CTL markedly reduced avidity. STAT6 −/− mice also showed memory CTL of higher avidity. Furthermore, CCL5 expression in K d Gag 197–205 ‐specific CTL was also regulated by IL‐4/IL‐13.
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Abstract Although CD8+ CTLs are presumed to be an important mediator of protective immunity in HIV-1 infection, the factors that determine CTL antiviral efficiency are poorly understood. Two factors that have been proposed to influence CTL antiviral function are antigenic avidity and epitope specificity. In this study we evaluate these by examining the activity of HIV-1-specific CTL against acutely infected cells. The ability of CTL to kill infected cells is variable and depends more on epitope specificity than functional avidity within the range for the tested clones (50% of maximal killing, 50 pg/ml to 100 ng/ml); killing efficiency is similar for different clones recognizing the same epitope, despite their variation in avidity. When CTL clones are tested for their ability to suppress viral replication, similar results are observed. Inhibition is more dependent on epitope specificity than functional avidity among the tested clones (50% of maximal killing, 20 pg/ml to 20 ng/ml). Thus, CTL specificity can be an overriding factor in the ability of CTL to interact with HIV-1-infected cells, indicating that factors determining the process of epitope presentation on infected cells have a key influence on CTL efficiency. These results suggest that CTL specificity may have a pivotal role in the immunopathogenesis of infection, and that simple quantitative measures of CTL may be insufficient indicators of the CTL response to HIV-1.
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Donor-specific CTL present within the cardiac allograft during a rejection episode are distinct from those that populate the cardiac allograft in the absence of rejection. Whereas the former generally have a high avidity for donor cells, the latter mainly have a low avidity for donor cells. This observation made us reason that high-avidity CTL are implicated in transplant rejection, whereas low-avidity CTL are not. In the present study, we analyse whether both CTL subsets were distinct with respect to their IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) secretion pattern. CTL clones with either a high or a low avidity for donor antigens were stimulated with donor cells, third party cells, or immobilized anti-CD3 MoAb and the amount of cytokine released was measured. High- and low-avidity CTL clones were found to differ with respect to their IFN-gamma production profile. Stimulation with donor cells resulted in IFN-gamma secretion by high-avidity CTL clones, but not by low-avidity CTL clones. CD3 stimulation, in contrast, led to secretion of equivalent amounts of IFN-gamma by both CTL subsets. These observations indicate that low-avidity CTL are fully capable of producing IFN-gamma, but, in contrast to high avidity CTL, fail to do so when they encounter donor cells. As IFN-gamma favours the occurrence of transplant rejection, this observation emphasizes the relevance of high-avidity CTL in the rejection process. Additionally, the data show that the cytokine production profile of CTL depends on the nature of the stimulus.
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