T cell immunity requires the long-term survival of T cells that are capable of recognizing self antigens but are not overtly autoreactive. How this balance is achieved remains incompletely ...understood. Here we identify a homeostatic mechanism that transcriptionally tailors CD8 coreceptor expression in individual CD8+ T cells to the self-specificity of their clonotypic T cell receptor (TCR). 'Coreceptor tuning' results from interplay between cytokine and TCR signals, such that signals from interleukin 7 and other common gamma-chain cytokines transcriptionally increase CD8 expression and thereby promote TCR engagement of self ligands, whereas TCR signals impair common gamma-chain cytokine signaling and thereby decrease CD8 expression. This dynamic interplay induces individual CD8+ T cells to express CD8 in quantities appropriate for the self-specificity of their TCR, promoting the engagement of self ligands, yet avoiding autoreactivity.
For developing T cells, coreceptor choice is matched to T cell antigen receptor (TCR) MHC specificity during positive selection in the thymus, but the mechanism remains uncertain. Here, we document ...that TCR-mediated positive selection signals inactivate the immature CD8
III enhancer in double positive (DP) thymocytes, explaining in part the cessation of CD8 coreceptor transcription that occurs during positive selection. More importantly, by placing CD4 protein expression under the control of CD8 transcriptional regulatory elements, we demonstrate that cessation of CD4 coreceptor transcription during positive selection results in precisely the same lineage fate as cessation of CD8 coreceptor transcription. That is, MHC-II-signaled DP thymocytes differentiated into CD8-lineage cytotoxic T cells, despite the MHC-II specificity and CD4 dependence of their TCRs. This study demonstrates that termination of coreceptor transcription during positive selection promotes CD8-lineage fate, regardless of TCR specificity or coreceptor protein identity.
Abstract
The glycoprotein cell surface marker CD4 is a key mediator in the adaptive immune response and T-cell development. Signaled CD4+ CD8+ double positive cells (DP) upregulate CD4 on the cell ...surface, allowing for a persistent TCR signal in the thymus and differentiating into the CD4+ single positive helper lineage. Previously, we have demonstrated that NCE (novel cis element), located in the first intron of Cd4, acts as an enhancer in RLM11 cells (CD4+, CD8−, Th-Pok− thymoma, arrested at the intermediate stage of development), but not in AKR-1G1 or VL3-3M2 cells (DP thymomas). We have identified a highly conserved region within NCE, labeled core-NCE, which may be vital to the enhancer’s function. To investigate the contribution of core-NCE to the level of CD4 expression, we utilized CRISPR/Cas9 technology to target the region for deletion. Here we describe the construction of plasmids containing guide sequences for targeted deletion of core-NCE by Cas9 cutting and NHEJ repair. We have successfully generated and sequence verified plasmids with three upstream and three downstream guides and introduced them in RLM11 cells. After single cell sorting, we obtained one clone that has the 300bp core-NCE deleted on one Cd4 allele, which resulted in 66% reduction in CD4 expression from that allele. As we removed the core-NCE in cells that have already established CD4 expression, we conclude that the core NCE has an enhancer function at the intermediate/transitional stage of thymocyte development that is separate from the previously described epigenetic function of the same intronic region, making NCE a good candidate for the regulatory element responsible for CD4 upregulation during differentiation into the CD4 lineage.
Abstract
The gut microbiome is known to play an integral role in the regulation of the immune system. Previous studies indicate that species diversity and overall abundance of commensal bacteria in ...the gut influences immune system function by altering total T cell counts and the ratio of CD4+ to CD8+ T cells in peripheral lymphoid organs and blood. In normal individuals, the CD4:CD8 ratio is greater than one. However, the congenic B10.A mouse strain in our conventional facility consistently demonstrates an inverted CD4:CD8 ratio in a sex- and age-dependent manner, in contrast to both the parental C57Bl/10 strain and B10.A kept under SPF conditions. To determine whether this phenomenon is due to the composition and diversity of the gut microbiome affecting the homeostasis of the CD4:CD8 ratio, we altered the microbiome by supplementing the diet with the prebiotic inulin and looked for changes in the number of Peyer’s patches and changes in the CD4:CD8 ratio in gut lymphoid organs and blood in both B10.A and C57Bl/10 strains. In addition, we followed the changes in the microbiome upon treatment. We find that the mice in our facility have lower microbiome diversity and very different composition from mice kept at SPF conditions. The inulin treatment significantly increased the CD4:CD8 ratio in the blood of the C57Bl/10 mice, but not the B10.A mice. In addition, males of both strains had a significant increase in Peyer’s patch number, while the females had a significant increase in the CD4:CD8 ratio in the Peyer’s patches.
The thymus generates major histocompatibility complex (MHC)-restricted αβT cells that only recognize antigenic ligands in association with MHC or MHC-like molecules. We hypothesized that MHC ...specificity might be imposed on a broader αβTCR repertoire during thymic selection by CD4 and CD8 coreceptors that bind and effectively sequester the tyrosine kinase Lck, thereby preventing T cell receptor (TCR) signaling by non-MHC ligands that do not engage either coreceptor. This hypothesis predicts that, in coreceptor-deficient mice, αβ thymocytes would be signaled by non-MHC ligands to differentiate into αβT cells lacking MHC specificity. We now report that MHC-independent αβT cells were indeed generated in mice deficient in both coreceptors as well as MHC (“quad-deficient” mice) and that such mice contained a diverse αβT cell repertoire whose MHC independence was confirmed at the clonal level. We conclude that CD4 and CD8 coreceptors impose MHC specificity on a broader αβTCR repertoire during thymic selection by preventing thymocytes from being signaled by non-MHC ligands.
The mechanism by which CD4/CD8 lineage choice is coordinated with TCR specificity during positive selection remains an unresolved problem in immunology. The stochastic/selection model proposes that ...CD4/CD8 lineage choice in TCR-signaled CD4(+)CD8(+) thymocytes occurs randomly and therefore is highly error-prone. This perspective is strongly supported by "coreceptor rescue" experiments in which transgenic CD4 coreceptors were ectopically expressed on thymocytes throughout their development and caused significant numbers of cells bearing MHC-II-specific TCR to differentiate into mature, CD8 lineage T cells. However, it is not known if forced coreceptor expression actually rescued positively selected thymocytes making an incorrect lineage choice or if it influenced developing thymocytes into making an incorrect lineage choice. We have now reassessed coreceptor rescue and the concept that lineage choice is highly error-prone with a novel CD4 transgene (referred to as E8(I)-CD4) that targets expression of transgenic CD4 coreceptors specifically to thymocytes that have already undergone positive selection and adopted a CD8 lineage fate. Unlike previous CD4 transgenes, the E8(I)-CD4 transgene has no effect on early thymocyte development and cannot itself influence CD4/CD8 lineage choice. We report that the E8(I)-CD4 transgene did in fact induce expression of functional CD4 coreceptor proteins on newly arising CD8 lineage thymocytes precisely at the point in thymic development that transgenic CD4 coreceptors would putatively rescue MHC-II-specific thymocytes that incorrectly adopted the CD8 lineage. However, the E8(I)-CD4 transgene did not reveal any MHC-II-selected thymocytes that adopted the CD8 lineage fate. These results demonstrate that CD4/CD8 lineage choice is neither error-prone nor stochastic.
TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the ...impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.
The lineage fate of developing thymocytes is determined by the persistence or cessation of TCR signaling during positive selection, with persistent TCR signaling required for CD4 lineage choice. We ...now report that transcriptional upregulation of CD4 expression is essential for error-free lineage choice during MHC-II specific positive selection and is especially critical for error-free lineage choice in TCR transgenic mice whose thymocytes compete for the identical selecting ligand. CD4 upregulation occurs only for endogenously encoded CD4 coreceptors as CD4 transgenes are downregulated during positive selection, disrupting MHC-II specific TCR signaling and causing lineage errors regardless of the absolute number or signaling strength of transgenic CD4 proteins. Thus, the kinetics of CD4 coreceptor expression during MHC-II specific positive selection determines the integrity of CD4 lineage choice, revealing an elegant symmetry between coreceptor kinetics and lineage choice.
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