Affinity For Self Research Articles

TCR signal strength shapes functional imprinting during CD4 T cell development

Abstract Naïve CD4 and CD8ab T cells are selected in the thymus in response to low-avidity TCR interactions. Such weak TCR signals install a quiescent program that guides naïve T cells to the lymph nodes where they differentiate upon encounter of cognate antigen. Thymocytes expressing self-specific TCRs can be agonist-selected in response to a strong TCR signal and differentiate in the thymus to functionally committed double negative T cells, NKT cells or regulatory T cells. Although the role for strength of TCR activation is clear in instructing these different outcomes, the impact of self-recognition in the thymus on “naïve” T cells is unclear. Previous work demonstrated that negative selection of self-specific T cells is not all encompassing and that affinity for self could even enhance immune protection against pathogens. We have used Stim1fl/fl Stim2fl/fl conditional deletion mice that fail to sustain calcium entry into the cytosol upon TCR activation to assess the role of TCR strength during selection. We confirmed that agonist selection is drastically affected in these mice whereas numbers of conventional T cells are not. Interestingly, viSNE representation of multi-parameter flow cytometry revealed single cell heterogeneity among the CD4SP thymocyte subset that is controlled by Stim1/Stim2. This indicates that differential TCR activation levels give rise to phenotypically different CD4 T cells. Purification of a strongly signaled (Stim1/2-dependent) subset of CD4 thymocytes showed functional differences in terms of their ability to proliferate in response to TCR activation. Our work points to a role for self-recognition during T cell selection that shapes the response of individual CD4 T cells to TCR activation in the periphery.

Affinity war: forging immunoglobulin repertoires.

B cell immunoglobulin (Ig) repertoire composition shapes immune responses. The generation of Ig diversity begins with Ig variable region exon assembly from gene segments, random inter-segment junction sequence diversity, and combinations of Ig heavy and light chain. This generates vast preemptive sequence freedom in early developing B lineage cell Ig genes that can anticipate a great diversity of threats. This freedom is met with large restrictions that ultimately define the naïve (i.e. preimmune) Ig repertoire. Activation-induced somatic hypermutation (SHM), which further diversifies Ig V regions, is also met with strong selection that shapes Ig affinity maturation. While individual repertoire features, such as affinity for self and competition for foreign antigen, are known to drive selection, the selection filters themselves may be subject to regulation. Large sequence freedom coupled with strong selection for each diversification process provides flexibility for demand-driven regulation to dynamically balance antigen recognition capacities and associated autoimmune risks according to host needs.

Islet-infiltrating effector and regulatory T cells specific for a single epitope possess distinct, but overlapping TCR repertoires.

Abstract Regulatory T cells (Tregs) have been shown to play a critical protective role in type 1 diabetes. However, TCR specificity of islet-infiltrating Tregs and its influence on T cell function during autoimmune diabetes is largely unknown. It has been shown that Tregs have a higher affinity for self and possess a unique TCR repertoire. Accordingly, we observed increased TCR signaling in islet-infiltrating insulin-tetramer labeled Tregs compared to effector T cells (Teffs), as measured by the Nur77-GFP reporter of TCR activation. In order to obtain the profile of Treg and Teff TCR repertoires specific for a single epitope, we isolated and sequenced insulin tetramer binding Teffs and Tregs from islets of NOD mice expressing a fixed alpha chain of an insulin specific TCR. Surprisingly, TCR sequence analysis showed relatively high similarity (Morisita-Horn index: 0.27) between Treg and Teff TCR repertoires, where a substantial portion (46.4%) of Tregs expressed shared TCRs. Collectively, our data suggest that Treg and Teff specific for a single epitope can express the same TCRs, which they exploit to exert different effector or suppressive functions.

Searching for natural Treg peptide-MHC ligands by engineering increased affinity/avidity Treg TCRs. (IRC5P.621)

Abstract Natural Tregs (nTregs) are produced in the thymus by selection to self peptide-MHC (spMHC) complexes. There is abundant data in the literature that suggests nTreg TCRs are higher affinity for self than conventional T cells and respond to spMHC in the periphery. To date, no nTreg ligands have been found, in part, possibly because nTregs may have TCR affinities that fall somewhere between the affinties required for positive and negative selection in the thymus for conventional T cells. This implies that although nTreg TCRs may be higher affinity for self, this affinity may still be lower than a conventional T cell TCR has for it's immunological antigen. To overcome this, we have developed a way to increase the affinity for some nTreg TCRs for pMHC while still mainting peptide specificity. Using these higher affinity nTreg TCRs, we can scan peptide-MHC libraries in order to find peptide mimitopes which may lead us to the actual self-peptides which are able stimulate specific nTregs.

Affinity for self drives the preferential accumulation of promiscuous CD4+ T cells over the lifespan. (LYM2P.726)

Abstract T cells discriminate self from foreign peptides presented in the context of self-major histocompatibility complex (pMHC) molecules via clonotypic T cell receptors (TCRs). CD8+ T cell recognition and responsiveness to foreign pMHC is known to diminish over the lifespan, which is consistent with gradual thymic involution over time. How the affinity of the CD4+ T cell compartment for self-pMHC, and its capacity to bind foreign-pMHC change over the lifespan are basic aspects of T cell biology that remain largely unexplored. Experimentally restricting thymic selection is known to allow degenerate CD4+ T cells to develop. This suggests that they might accumulate in the CD4+ T cell compartment over time. We report that, while old mice (18-22 months) contain fewer CD4+ T cells than adults (8-12 weeks), those that remain have a higher intrinsic affinity for self-pMHC. Old mice also have more cells that bind distinct foreign-pMHCs, either alone or in combination. The numerical increase of these subsets with age directly correlates with their affinity for self-pMHC. However, no relationship was observed between affinity for self-pMHC and responsiveness to foreign-pMHC. These data demonstrate that the CD4+ T cell compartment preferentially accumulates promiscuous constituents with age as a consequence of higher affinity T cell receptor interactions with self-pMHC. These results have important implications for the design of immunotherapeutics targeting CD4+ T cells for long-term efficacy.

Dysregulated THEMIS results in the escape of pathogenic auto-reactive T cells. (LYM6P.719)

Abstract THEMIS is required for setting the threshold for conventional selection of thymocytes by dampening TCR signals, resulting in the accumulation of “naïve” T cells in the periphery. In contrast, agonist selection, which is an alternative way to induce self tolerance, requires strong TCR signals and does not involve regulation by THEMIS. Using the OVA-specific monoclonal MHC class II restricted DO11.10 TCR transgenic system, we found that many DO11.10 thymocytes are selected along the agonist selection path and appear as co-receptor double negative (DN) TCRab cells in the intestine. Interesting, some DO11.10 cells also appear as conventional-like CD4 T cells in the spleen, suggesting they escaped agonist selection. Compared to the DN DO11.10 T cells or normal WT conventional selected T cells, these escaped DO11.10 CD4 T cells expressed much higher levels of THEMIS, suggesting that excessive levels of THEMIS might have diverted DO11.10 thymocytes to be selected along the conventional path, resulting in the accumulation of T cells with high affinity for self in the naïve pool. Although these aberrant selected THEMIShigh CD4 T cells remain quiescent at steady state, they caused severe intestinal pathology in RAG-/- recipient mice that were fed an OVA-diet. These data indicate that dysregulated THEMIS can lead to the accumulation of auto-reactive T cells with high potential to induce immune pathology when exposed to their cognate antigen in the periphery.

CD4+ T regulatory and effector cells display a similar wide range of affinities for self during autoimmune disease (BA13P.129)

Abstract The affinity of a T cell receptor (TCR) from a regulatory T cell (Treg) for endogenous self peptide MHC complex is unknown. It is thought that thymic derived Tregs (nTregs) are enriched for high affinity, self-reactive TCRs as an alternative to negative selection. During experimental autoimmune encephalomyelitis (EAE), MOG self-antigen pMHC tetramers identified few (<10%) of the infiltrating CD4+ Tregs and Teffs. The micropipette adhesion frequency assay has previously proven more sensitive for assessing TCR specificity, affinity and frequency of antigen specific cells. Using this assay, it was determined that most of the Tregs and Teffs (>80% each) localized in the central nervous system (CNS) at peak EAE are in fact MOG self antigen specific. Tregs and Teffs possessed similar broad 1000 fold ranges in affinities for MOG with geometric means of 1 and 4 X 10-5 μm4. In the CNS, greater than 90% of the Tregs expressed helios which was consistent with frequencies in the thymus (94%), which suggests an enrichment of nTregs in the CNS during peak EAE. Contrary to the hypothesis that nTregs arise as an alternative to negative selection due to a high affinity for self, our data show that TCR affinity of MOG specific Tregs mimics Teffs with no enrichment of high affinity cells.

Steroids improve T cell fitness

Endogenous glucocorticoids promote the selection of thymocytes that have an appropriate affinity for self.

Thymocyte responsiveness to endogenous glucocorticoids is required for immunological fitness

Generation of a self-tolerant but antigen-responsive T cell repertoire occurs in the thymus. Although glucocorticoids are usually considered immunosuppressive, there is also evidence that they play a positive role in thymocyte selection. To address the question of how endogenous glucocorticoids might influence the adaptive immune response, we generated GRlck-Cre mice, in which the glucocorticoid receptor gene (GR) is deleted in thymocytes prior to selection. These mice were immunocompromised, with reduced polyclonal T cell proliferative responses to alloantigen, defined peptide antigens, and viral infection. This was not due to an intrinsic proliferation defect, because GR-deficient T cells responded normally when the TCR was cross-linked with antibodies or when the T cell repertoire was "fixed" with αβ TCR transgenes. Varying the affinity of self ligands in αβ TCR transgenic mice showed that affinities that would normally lead to thymocyte-positive selection caused negative selection, and alterations in the TCR repertoire of polyclonal T cells were confirmed by analysis of TCR Vβ CDR3 regions. Thus, endogenous glucocorticoids are required for a robust adaptive immune response because of their promotion of the selection of T cells that have sufficient affinity for self, and the absence of thymocyte glucocorticoid signaling results in an immunocompromised state.

Enumeration and characterization of self-reactive T cells in polyclonal populations (111.25)

Abstract The thymic processes of positive and negative selection are crucial for the formation of a functional T cell repertoire. TCR transgenic models have provided crucial insight into these processes, but it has not been possible to quantitate positive and negative selection in normal diverse polyclonal populations. We developed a Nur77GFP transgenic reporter mouse that specifically expresses GFP in T cells in response to TCR stimulation. Using Nur77GFP mice crossed to mice deficient in the pro-apoptotic molecule Bim, we found that two times more DP thymocytes are negatively selected by high affinity interactions than are positively selected by low affinity interactions. We also identified the fraction of SP thymocytes undergoing further deletion in the medulla, after positive selection. Cells that are rescued from negative selection have increased Nur77GFP compared to foxP3+ T regulatory cells, suggesting that T cells undergoing negative selection have TCRs with higher affinity for self compared to developing T regulatory cells. Finally, our results identify a population of self-reactive CD25-CD44+ CD4 T cells in mesenteric LNs, which have a phenotype consistent with clonal anergy. Future studies using this model will allow us to identify and quantitate selection defects in various mutant or autoimmune prone animals.

T‐cell sensitivity: A microRNA regulates the sensitivity of the T‐cell receptor

T‐cell sensitivity: A microRNA regulates the sensitivity of the T‐cell receptor

CD4+CD25+ regulatory T cells are activated in vivo by recognition of self

Naturally occurring CD4(+)CD25(+) regulatory T cells (nT(R)) comprise a separate lineage of T cells that are essential for maintaining immunological tolerance to self. Here we demonstrate that the level of phosphorylation of the TCR zeta-chain is approximately 1.5- to 4-fold higher in nT(R) as compared with CD4(+)CD25(-) T cells. The increased level of TCR zeta-chain phosphorylation is presumably secondary to their higher affinity for self, resulting in a stronger TCR signal as it was completely blocked by treatment with anti-MHC class II. The enhanced level of TCR zeta-chain phosphorylation was correlated with the capacity of nT(R) to develop non-specific suppressor effector function following culture with IL-2 or IL-4 in the absence of TCR stimulus. Thus, a sub-population of nT(R) is activated by recognition of self-peptide-MHC class II ligands in vivo, resulting in their capacity to be induced to mediate suppressor function in vitro in the absence of TCR stimulation.

Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling

A transcription profiling study, together with genetic linkage data, provides a molecular map of the T-cell negative selection response in vivo.

Quantitative and Qualitative Adjustment of Thymic T Cell Production by Clonal Expansion of Premigrant Thymocytes

In normal mice, single-positive thymocytes proliferate before being exported into the peripheral T cell pool. We measured the in vivo proliferation rates of mature thymocytes in several TCR transgenic mice. Different monoclonal TCR transgenic single-positive thymocytes proliferated at different rates in a given MHC context. Conversely, mature thymocytes expressing a given TCR, generated in mice of different MHC haplotypes, also showed different rates of proliferation. In p59(fyn)-deficient mice, the proliferation rate of mature thymocytes was diminished. Thus, premigrant thymocyte expansion is TCR mediated and depends on TCR affinity for self peptide/MHC ligands. In addition, we show that mature thymocyte expansion is clonotypic, increases the daily thymic T cell output, and modifies the TCR repertoire of newly produced T cells.

Selecting T cell receptors with high affinity for self-MHC by decreasing the contribution of CD8.

Selective events during T cell repertoire development in the thymus include both the positive selection of cells whose receptors recognize self-major histocompatibility complex (MHC) molecules and negative selection (tolerance) of cells whose interaction with self-MHC is of high affinity. The affinity of T cell interactions with class I MHC molecules includes contributions by both the T cell receptor and the CD8 coreceptor. Therefore, by decreasing the affinity of the interaction with CD8, T cells whose receptors have relatively high affinities for self-MHC may survive negative selection. Such T cells were generated and those T cells reactive with self-MHC plus antigen also displayed low affinity for self.

T cell activation and thymic tolerance induction require different adhesion intensities of the CD8 co-receptor.

Activation of mature lymphocytes requires in addition to the TCR contact with the corresponding antigen the binding of the CD8 or CD4 co-receptors to MHC class I or class II proteins respectively. To investigate the contribution of the CD8-class I interaction to the elimination of autoreactive T cells during negative selection in the thymus we generated two types of transgenic mice. One set expressed a modified Kb molecule which contained a human HLA-A2 alpha 3 domain, thereby missing the binding residues for the murine CD8 molecules. The second set of mice expressed an anti-Kb specific TCR. Both lines were crossed and in the resulting double transgenic mice the development of Kb-reactive T cells was followed with an anti-clonotypic antibody. Surprisingly, efficient clonal deletion in the thymus was still observed, although the reduced CD8-class I adhesion abrogated effector functions in vivo and in vitro. These results imply that even T cells with intermediate affinity for self are negatively selected in the thymus despite the fact that they are not able to react against self antigens in the periphery. Thus a safety window is created which decreases the risk of autoaggression.

Rescue of thymocytes and T cell hybridomas from glucocorticoid‐induced apoptosis by stimulation via the T cell receptor/CD3 complex: a possible in vitro model for positive selection of the T cell repertoire

Positive and negative selection events are involved in determining useful T cell clones to mature in the thymus. Accumulating evidence suggests that immature self-reactive thymocytes undergo apoptotic death (negative selection) upon stimulation via the T cell receptors (TcR). A similar phenomenon of activation-induced death has been reported in T cell hybridomas. On the other hand, little is known about the mechanism of the positive selection. Apoptosis in rodent thymocytes or T cell hybridomas is also known to be induced by glucocorticoids in vitro at concentrations within the physiologic range. We report here that the TcR/CD3-mediated stimulation and glucocorticoids mutually inhibit the apoptosis in T cell hybridomas. The production of interleukin 2 by the rescued cells indicated that the TcR/CD3-mediated signal was transduced into the cells. Thymocytes were also rescued from glucocorticoid-induced apoptosis by the stimulation with antibodies to TcR/CD3 molecules. The rescue of thymocytes, however, was observed only at a narrow concentration range of each of the antibodies, suggesting that the proper stimulation via the TcR/CD3 is required for the rescue. If thymocytes in situ are differentially stimulated according to the affinity of the TcR towards self, only the thymocytes whose TcR have proper affinity towards self may be rescued from glucocorticoid-induced apoptosis. Therefore, we propose a hypothesis that the positive selection of the T cell repertoire is based on the inhibition of glucocorticoid-induced apoptosis in immature thymocytes bearing TcR with proper affinity for self by the TcR-mediated signals in situ. Furthermore, the selection may be influenced by the peak level of glucocorticoid concentration, since the proper concentration range of the anti-TcR/CD3 antibody for the rescue was variable depending on the glucocorticoid concentration.

Modification of the cytotoxic T cell repertoire in neonatal tolerance. Evidence for preferential survival of cells with low avidity for tolerogen.

Clonal deletion of developing lymphocytes with potential reactivity for self is thought to play a crucial role in the establishment of self tolerance. One prediction of the clonal deletion hypothesis is that cells bearing receptors with high affinity for self are more likely than cells with low affinity receptors to be deleted from the repertoire. Experimental models of B cell tolerance have provided evidence for the preferential survival of low affinity cells with specificity for tolerogen in tolerant animals, but no comparable evidence exists for T cells. To examine this issue in T cells, cytotoxic T cell lines specific for the Kb mutant class I H-2 molecule, bm1, were generated from C57BL/6 mice rendered neonatally tolerant of bm1 and compared with anti-bm1 lines generated from normal mice. Compared with normal lines, those from tolerant mice differed in five ways: 1) they grew more slowly; 2) they were less efficient at lysing bm1 targets; 3) they showed different patterns of lysis against a panel of third party targets; 4) their cytotoxic activity against bm1 could be increased in the presence of leukoagglutinin, whereas the activity of normal lines was not increased by leukoagglutinin; and 5) their cytotoxic activity was more susceptible to inhibition by anti-Lyt-2 antibody. Taken together, these results demonstrate that the repertoire of the remaining tolerogen-specific cytotoxic T cells in neonatally tolerant mice is different from the normal C57BL/6 anti-bm1 repertoire, and the results are consistent with the idea that the remaining tolerogen-specific cells are low avidity cells that have preferentially escaped the clonal deletion process.

A dual recognition model for cytotoxic T cells based on thymic selection of precursors with low affinity for Self H-2 antigens.

Scandinavian Journal of ImmunologyVolume 7, Issue 3 p. 181-190 A Dual Recognition Model for Cytotoxic T Cells Based on Thymic Selection of Precursors with Low Affinity for Self H-2 Antigens R. V. Blanden, R. V. Blanden Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., AustraliaSearch for more papers by this authorG. L. Ada, G. L. Ada Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., AustraliaSearch for more papers by this author R. V. Blanden, R. V. Blanden Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., AustraliaSearch for more papers by this authorG. L. Ada, G. L. Ada Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., AustraliaSearch for more papers by this author First published: March 1978 https://doi.org/10.1111/j.1365-3083.1978.tb00442.xCitations: 55AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume7, Issue3March 1978Pages 181-190 RelatedInformation