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Differential gene expression among naïve CD5lo and CD5hi CD4⁺ human T cells. (A) Representative histograms of CD5 levels on pre‐ and postsort naïve CD45RA⁺CD27⁺CD25⁻ CD4⁺ T cells gated on the top and bottom 15% of CD5 expression and analyzed by flow cytometry. (B) MFI of CD5 on sorted cells submitted for RNAseq analysis. Each dot represents an individual donor (n = 2). (C) Analysis of differential gene expression in CD5lo and CD5hi cells by qPCR. cDNA was prepared from naïve CD4⁺ T cells sorted on CD5 expression (top and bottom 20%) and DNTT (left panel) and ADAM23 (right panel) gene expression was analyzed. Average Ct values from technical replicates were used for analysis. Target gene expression levels are normalized to internal control gene HPRT, and the fold change, calculated as log2‐transformed 2^delta‐delta Ct values, between the CD5lo and CD5hi subsets is shown. Dots represent individual donors (n = 4). (D) Difference in cell surface CD6 levels on CD5lo and CD5hi cells analyzed using flow cytometry. Representative histogram of CD6 (left panel) and MFI of CD6 (right panel) gated on top and bottom 15% of CD5 expression on naïve CD45RA⁺CD27⁺CD4⁺ T cells. Dots indicate individual donors (n = 3) from two independent experiments. (E) Heat map of select DEG between naïve human CD5lo and CD5hi CD4⁺ T cells enriched within the indicated Gene Ontology pathways at a padj value cut‐off of ≤0.1. Color coding is based on log2‐transformed FPKM values. Horizontal lines on dot plots indicate average. **p < 0.01, ***p < 0.001 as determined by paired Student's t‐test (two‐tailed).
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Studies in murine models show that subthreshold T cell receptor (TCR) interactions with self‐peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self‐peptide, as read‐out by cell surface levels of CD5, were associated with distinct effector potent...
Citations
... Our work revealed the effects of varying features of the pMHC-reactivity distribution of responding T cells on pathogen clearance and suggested a differential role between T cells with low-and high-reactivity to pMHC during different phases of the immune response. This is particularly intriguing, given recent observations that in both mice and humans, both CD4 + and CD8 + T cells with lower self-pMHC reactivity (low CD5 surface levels) express higher levels of Dntt, the gene encoding TdT [36,60,61]. Thus, differences in TdT expression level during development in individual thymocytes may ultimately contribute to the numbers of N-nucleotides inserted into the recombining TCR and be a critical variable impacting strength of pMHC reactivity. ...
The breadth of pathogens to which T cells can respond is determined by the T cell receptors (TCRs) present in an individual’s repertoire. Although more than 90% of the sequence diversity among TCRs is generated by terminal deoxynucleotidyl transferase (TdT)-mediated N-nucleotide addition during V(D)J recombination, the benefit of TdT-altered TCRs remains unclear. Here, we computationally and experimentally investigated whether TCRs with higher N-nucleotide diversity via TdT make distinct contributions to acute or chronic pathogen control specifically through the inclusion of TCRs with lower antigen binding strengths (i.e., lower reactivity to peptide-major histocompatibility complex (pMHC)). When T cells with high pMHC reactivity have a greater propensity to become functionally exhausted than those of low pMHC reactivity, our computational model predicts a shift toward T cells with low pMHC reactivity over time during chronic, but not acute, infections. This TCR-affinity shift is critical, as the elimination of T cells with lower pMHC reactivity in silico substantially increased the time to clear a chronic infection, while acute infection control remained largely unchanged. Corroborating an affinity-centric benefit for TCR diversification via TdT, we found evidence that TdT-deficient TCR repertoires possess fewer T cells with weaker pMHC binding strengths in vivo and showed that TdT-deficient mice infected with a chronic, but not an acute, viral pathogen led to protracted viral clearance. In contrast, in the case of a chronic fungal pathogen where T cells fail to clear the infection, both our computational model and experimental data showed that TdT-diversified TCR repertoires conferred no additional protection to the hosts. Taken together, our in silico and in vivo data suggest that TdT-mediated TCR diversity is of particular benefit for the eventual resolution of prolonged pathogen replication through the inclusion of TCRs with lower foreign antigen binding strengths.
... CD5 has been extensively studied as a marker to define the heterogeneity within Tn (5,(8)(9)(10)(11). Its unique feature lies in its T cell receptor (TCR)-dependent upregulation mechanism, which allows the dissection of Ag-inexperienced Tn based on their affinity to self-Ags (5,8,(11)(12)(13)(14). ...
... While most studies on CD5 have been conducted in murine models, certain characteristics have also been demonstrated in humans. Both human thymocytes and peripheral T cells have been shown to upregulate CD5 expression in a TCR strength-dependent manner (10). Furthermore, human CD4 + Tn were shown to have different functionalities depending on their relative CD5 expression (10). ...
... Both human thymocytes and peripheral T cells have been shown to upregulate CD5 expression in a TCR strength-dependent manner (10). Furthermore, human CD4 + Tn were shown to have different functionalities depending on their relative CD5 expression (10). ...
Defining the molecular dynamics associated with T cell differentiation enhances our understanding of T cell biology and opens up new possibilities for clinical implications. In this study, we investigated the dynamics of CD5 expression in CD8+ T cell differentiation and explored its potential clinical uses. Using PBMCs from 29 healthy donors, we observed a stepwise decrease in CD5 expression as CD8+ T cells progressed through the differentiation stages. Interestingly, we found that CD5 expression was initially upregulated in response to T cell receptor stimulation, but diminished as the cells underwent proliferation, potentially explaining the differentiation-associated CD5 downregulation. Based on the proliferation-dependent downregulation of CD5, we hypothesized that relative CD5 expression could serve as a marker to distinguish the heterogeneous CD8+ T cell population based on their proliferation history. In support of this, we demonstrated that effector memory CD8+ T cells with higher CD5 expression exhibited phenotypic and functional characteristics resembling less differentiated cells compared to those with lower CD5 expression. Furthermore, in the retrospective analysis of PBMCs from 30 non-small cell lung cancer patients, we found that patients with higher CD5 expression in effector memory T cells displayed CD8+ T cells with a phenotype closer to the less differentiated cells, leading to favorable clinical outcomes in response to immune checkpoint inhibitor (ICI) therapy. These findings highlight the dynamics of CD5 expression as an indicator of CD8+ T cell differentiation status, and have implications for the development of predictive biomarker for ICI therapy.
... In line with these differential gene expression profiles, these subsets of naive CD8 + T cells showed significant functional differences, with the highest response of CD5 hi Ly6C + cells, intermediate response of CD5 hi Ly6C − cells, and the lowest response of CD5 lo Ly6C − cells observed in the ability to induce IFN-γ production in response to IL-12 and IL-18 exposure as well as IFN-γ and TNF production upon PMA and ionomycine stimulation (34). Similar functional distinction has also been observed in CD5 lo and CD5 hi subsets of naive CD4 + T cell population in mice and humans (53)(54)(55)(56). ...
Immune diversification helps protect the host against a myriad of pathogens. CD8+ T cells are essential adaptive immune cells that inhibit the spread of pathogens by inducing apoptosis in infected host cells, ultimately ensuring complete elimination of infectious pathogens and suppressing disease development. Accordingly, numerous studies have been conducted to elucidate the mechanisms underlying CD8+ T cell activation, proliferation, and differentiation into effector and memory cells, and to identify various intrinsic and extrinsic factors regulating these processes. The current knowledge accumulated through these studies has led to a huge breakthrough in understanding the existence of heterogeneity in CD8+ T cell populations during immune response and the principles underlying this heterogeneity. As the heterogeneity in effector/memory phases has been extensively reviewed elsewhere, in the current review, we focus on CD8+ T cells in a "naïve" state, introducing recent studies dealing with the heterogeneity of naive CD8+ T cells and discussing the factors that contribute to such heterogeneity. We also discuss how this heterogeneity contributes to establishing the immense complexity of antigen-specific CD8+ T cell response.
... 12 Indeed, the self-reactivity of T cells is an important driver of T cell fate even beyond the thymus, not only impacting how well they compete for survival signals in secondary lymphoid organs, but also by establishing prewired heterogeneity in gene expression that modulates T cell lineage choice, expansion and memory potential following activation. [13][14][15][16][17][18][19][20][21][22] In addition, both for CD4 + and CD8 + T cells there is evidence that there is a direct relation between self-reactivity and foreign pMHC binding strength. 14,23 Despite the pivotal role of the TCR sequence in T cell fate outcomes, few tools exist to systematically define sequence patterns among T cells with similar fates. ...
... 10 We and others have shown that the expression of TdT at the gene-level is substantially reduced (>10 fold) in higher-affinity T cells in both naïve CD4 + and CD8 + T cells, in humans and mice, although this had not been previously observed to be reflected by n-nucleotide additions at the TCR sequence level. 17,21,22,37 Together with the relationship between Dntt expression and self-reactivity, our findings raise the intriguing possibility that there could be a trade-off between TCRβ repertoire diversity -which is increased by Dntt activity -and recognition strength -which is decreased by Dntt activity. It will be interesting to test this idea directly in a system where Dntt expression levels can be raised or lowered experimentally, TCRs sequenced, and subsequent cell fates and repertoire diversity studied. ...
The T cell receptor (TCR) determines the specificity and affinity for both foreign and self-peptides presented by MHC. It is established that self-pMHC reactivity impacts T cell function, but it has been challenging to identify TCR sequence features that predict T cell fate. To discern patterns distinguishing TCRs from naive CD4+ T cells with low versus high self-pMHC reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that predicts self-reactivity directly from TCRβ sequences. This approach revealed that n-nucleotide additions and acidic amino acids weaken self- reactivity. We tested our ML predictions of TCRβ sequence self-reactivity using retrogenic mice. Extrapolating our analyses to independent datasets, we found high predicted self-reactivity for regulatory CD4+ T cells and low predicted self-reactivity for T cells responding to chronic infection. Our analyses suggest a potential trade-off between repertoire diversity and self-reactivity intrinsic to the architecture of a TCR repertoire.
... A novel finding from this work is that CD5 levels are modified in association with pre-TCR expression and that treatment with ACY1215 prevents the up-regulation of CD5 (but has a lesser effect on Lef1 up-regulation) during β-selection. At other stages of T cell development and activation, CD5 reports on the strength of TCR signalling, with increasing expression reflecting a stronger TCR signal (Tarakhovsky et al, 1995;Azzam et al, 1998;Sood et al, 2021). Our findings that the correlation of CD5 with TCRβ expression is lost with ACY1215 treatment, but the correlation of CD5 expression with proliferation is retained with ACY1215 treatment, suggest the possibility that CD5 plays a similar role as a reporter of pre-TCR signalling. ...
... to tune the TCR signal and to shift the window and modify the timing of positive selection (Voisinne et al, 2018;Matson et al, 2020;Lutes et al, 2021;Sood et al, 2021). Whether or not such a tuning role might occur for CD5 during β-selection needs further exploration, but a possible role for tuning of the TCRβ repertoire during β selection is made relevant by findings that the pre-TCR can bind with low affinity to MHC-peptide (Mallis et al, 2015;Li et al, 2021). ...
During T cell development, the first step in creating a unique T cell receptor (TCR) is genetic recombination of the TCRβ chain. The quality of the new TCRβ is assessed at the β-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of fate at the β-selection checkpoint is not yet understood. We shed new light on fate determination during β-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the β-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in β-selection, bookended by up-regulation of TCR co-receptors, CD28 and CD2, respectively. Within this "DN3bPre" stage, CD5 and Lef1 are up-regulated to reflect pre-TCR signalling, and their expression correlates with proliferation. These findings suggest a refined model of β-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 allows for modulating TCR signalling strength and culminates in the expression of CD2 to enable exit from the β-selection checkpoint.
... Murine studies identify a correlation between surface expression of CD5, a marker of 187 TCR signal strength on naïve CD4 + and CD8 + T cells, and the intensity of lymphopenia-induced 188 proliferation (25,26). We demonstrate here that naive PLZF + CD4 + T cells expressed higher 189 levels of CD5 as compared to their PLZFcounterparts ( Figure 3F and G), suggesting that the 190 enhanced response to IL-7 was set during thymic selection (27). To confirm that the effects of 191 IL-7 were due to the expansion of a thymus-derived PLZF + CD4 + T population, we sorted on 192 proxy surface markers to enrich and deplete the native population of naïve PLZF + cells 193 (Supplemental Figure 4). ...
The development of human adaptive immunity progresses faster than previously appreciated, with the emergence of memory CD4+ T cells alongside regulatory T (Treg) cells by the second trimester of pregnancy. We previously identified a prenatal-specific subset of PLZF+ CD4+ T cells with heightened effector potential that accounted for most memory T cells in the developing intestine and accumulated in the cord blood of infants exposed to prenatal inflammatory pathologies. However, the signals that drive their tissue distribution and effector maturation are unknown. In this report, we define the transcriptional and functional heterogeneity of prenatal PLZF+ CD4+ T cells and identify compartmentalization of Th-like effector function across the small intestine (SI) and mesenteric lymph nodes (MLN). We find that IL-7, which is more abundant in the SI relative to the MLN, drives the preferential expansion of naive PLZF+ CD4+ T cells via JAK/STAT and MEK/ERK signaling. Exposure to IL-7 induces a subset of PLZF+ CD4+ T cells to acquire a memory-phenotype and rapid effector function, identifying the human analogue of memory-phenotype CD4+ T cells. Further, IL-7 modulates the differentiation of Th1- and Th17-like PLZF+ CD4+ T cells, and thus likely contributes to the anatomic compartmentalization of prenatal CD4+ T cell effector function.
... Thus, neonatal TCRs may integrate all the "Goldilocks" conditions for intrathymic selection and survival in the periphery. Notably, polyreactivity to self-antigens could also favor the commitment of thymocytes bearing neonatal TCRs toward either the regulatory or alternative T cell lineages (Sood et al., 2021;Vrisekoop et al., 2014). This possibility should be explored in future studies. ...
Based on analyses of TCR sequences from over 1,000 individuals, we report that the TCR repertoire is composed of two ontogenically and functionally distinct types of TCRs. Their production is regulated by variations in thymic output and terminal deoxynucleotidyl transferase (TDT) activity. Neonatal TCRs derived from TDT-negative progenitors persist throughout life, are highly shared among subjects, and are polyreactive to self and microbial antigens. Thus, >50% of cord blood TCRs are responsive to SARS-CoV2 and other common pathogens. TDT- dependent TCRs present distinct structural features and are less shared among subjects. TDT- dependent TCRs are produced in maximal numbers during infancy when thymic output and TDT activity reach a summit, are more abundant in subjects with AIRE mutations, and seem to play a dominant role in graft-versus-host disease. Factors decreasing thymic output (age, male sex) negatively impact TCR diversity. Males compensate for their lower repertoire diversity via hyperexpansion of selected TCR clonotypes.
... For instance, Cd5, Folr4, Cd6, Nr4a1, Tox, Ptpn6, and Tcf25 were more highly expressed in CD5 hi cells, while Ly6c1 and Dntt were higher among CD5 lo cells ( Figure 3B). Indeed, as was described in CD5-sorted naive CD8 + T cells (Fulton et al., 2015) and in human naive CD4 + T cells (Sood et al., 2021), one of the top DEGs was Dntt (encoding the DNA polymerase terminal deoxynucleotidyl transferase [TdT], which inserts non-templated nucleotides during V(D)J A B C Figure 1. Cellular variability among naive CD4 + T cells at the singlecell level is driven by TCR signaling gene expression (A) Plot of top 5% most variable genes (blue) across individual sorted naive CD4 + T cells. ...
... Thus, removing competitor T cells, by modulating both IL-2 and TCR signal strength, led to the opposite outcome with regard to T FH differentiation predicted by TCR signal strength alone, in line with recent observations that Nur77 lo CD4 + T cells adoptively transferred into TCRa À/À gave rise to a greater T FH frequency than transferred Nur77 hi cells (Bartleson et al., 2020). A recent study has corroborated the use of CD5 as a marker for the self-ligand reactivity in human T cells (Sood et al., 2021), but it will be important to investigate whether the relationships described between self-reactivity and effector potential hold. ...
... Whether differences among naive CD4 + T cells can ultimately be related back to features of their specific TCRs remains an open question. It is intriguing that in CD5 lo naive CD4 + T cells, one of the top DEGs was Dntt (encoding TdT), as was also observed in mouse naive CD8 + T cells and human naive CD4 + T cells (Fulton et al., 2015;Sood et al., 2021). TdT is responsible for adding n-nucleotides during V(D)J recombination and thus diversifying the TCR repertoire (Benedict et al., 2000;Cabaniols et al., 2001). ...
CD4⁺ T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probe the heterogeneity present among naive CD4⁺ T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and chromatin landscape differences. Single-cell RNA sequencing shows that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a readout of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4⁺ T cells impact follicular helper T (TFH) cell versus non-TFH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4⁺ T cell chromatin landscapes that ultimately shape their effector potential.
... Lin28 regulation of CD4 + ab T-cell functional skewing Mature CD4 + T cells differentiate into T helper cells (Th) 1, Th2, Th17, T follicular helper cells, in addition to Treg, among other subsets, to perform a diverse range of effector functions [125,126]. Differences in the basal TCR signals that accompany thymic selection discussed above could influence these effector T cell fates as the effector biases of T cells with higher basal self-reactivity correlate with neonatal T-cell function and those with lower basal self-reactivity with adultlike T-cell function [127][128][129][130]. Neonatal CD4 + T cells have cell-intrinsic tendencies to elicit Th2 responses by producing more IL-4 and IL-13 in vitro and in vivo, whereas adult CD4 + T cells predominately generate Th1 responses ( Fig. 2A) [9,131,132,133]. ...
T cells comprise a functionally heterogeneous cell population that has important roles in the immune system. While T cells are broadly considered to be a component of the antigen‐specific adaptive immune response, certain T‐cell subsets display innate‐like effector characteristics whereas others perform immunosuppressive functions. These functionally diverse T‐cell populations preferentially arise at different stages of ontogeny and are tailored to the immunological priorities of the organism over time. Many differences in early life versus adult T‐cell phenotypes can be attributed to the cell‐intrinsic properties of the distinct progenitors that seed the thymus throughout development. It is becoming clear that Lin28, an evolutionarily conserved, heterochronic RNA‐binding protein that is differentially expressed among early life and adult hematopoietic progenitor cells, plays a substantial role in influencing early T‐cell development and function. Here, we discuss the mechanisms by which Lin28 shapes the T‐cell landscape to protect the developing fetus and newborn. Manipulation of the Lin28 gene regulatory network is being considered as one means of improving hematopoietic stem cell transplant outcomes; as such, understanding the impact of Lin28 on T‐cell function is of clinical relevance.
... This leads to significant variation in the level of basal 'selfreactivity' between individual cells within the mature T cell population, which influences their response during antigen challenge [18]. CD4 + T cells with relatively higher basal self-reactivity predominate during acute antigen challenge and are preferentially recruited into the memory T cell pool [14,19,20]. In addition, there is evidence of T helper biases among CD4 + T cells with relatively high versus low affinity for self-peptide [19][20][21][22]. ...
... CD4 + T cells with relatively higher basal self-reactivity predominate during acute antigen challenge and are preferentially recruited into the memory T cell pool [14,19,20]. In addition, there is evidence of T helper biases among CD4 + T cells with relatively high versus low affinity for self-peptide [19][20][21][22]. While some of these characteristics might be continually influenced by tonic signals (interactions between naïve T cells and self-pMHC that are necessary for their survival) in secondary lymphoid organs, there is evidence that some functional biases might be imprinted during their differentiation into mature T cells in the thymus based on the strength of positive selection signals [23]. ...
The ability of T cells to identify foreign antigens and mount an efficient immune response while limiting activation upon recognition of self and self-associated peptides is critical. Multiple tolerance mechanisms work in concert to prevent the generation and activation of self-reactive T cells. T cell tolerance is tightly regulated, as defects in these processes can lead to devastating disease; a wide variety of autoimmune diseases and, more recently, adverse immune-related events associated with checkpoint blockade immunotherapy have been linked to a breakdown in T cell tolerance. The quantity and quality of antigen receptor signaling depend on a variety of parameters that include T cell receptor affinity and avidity for peptide. Autoreactive T cell fate choices (e.g., deletion, anergy, regulatory T cell development) are highly dependent on the strength of T cell receptor interactions with self-peptide. However, less is known about how differences in the strength of T cell receptor signaling during differentiation influences the ‘function’ and persistence of anergic and regulatory T cell populations. Here, we review the literature on this subject and discuss the clinical implications of how T cell receptor signal strength influences the ‘quality’ of anergic and regulatory T cell populations.
