Single-cell NAD(H) levels predict clonal lymphocyte expansion dynamics.

Adaptive immunity requires the expansion of high-affinity lymphocytes from a heterogeneous pool. Whereas current models explain this through signal transduction, we hypothesized that antigen affinity tunes discrete metabolic pathways to license clonal lymphocyte dynamics. Here, we identify nicotinamide adenine dinucleotide (NAD) biosynthesis as a biochemical hub for the T cell receptor affinity-dependent metabolome. Through this central anabolic role, we found that NAD biosynthesis governs a quiescence exit checkpoint, thereby pacing proliferation. Normalizing cellular NAD(H) likewise normalizes proliferation across affinities, and enhancing NAD biosynthesis permits the expansion of lower affinity clones. Furthermore, single-cell differences in NAD(H) could predict division potential for both T and B cells, before the first division, unmixing proliferative heterogeneity. We believe that this supports a broader paradigm in which complex signaling networks converge on metabolic pathways to control single-cell behavior.

Cbl-b mitigates the responsiveness of naive CD8+ T cells that experience extensive tonic T cell receptor signaling.

Naive T cells experience tonic T cell receptor (TCR) signaling in response to self-antigens presented by major histocompatibility complex (MHC) in secondary lymphoid organs. We investigated how relatively weak or strong tonic TCR signals influence naive CD8+ T cell responses to stimulation with foreign antigens. The heterogeneous expression of Nur77-GFP, a transgenic reporter of tonic TCR signaling, in naive CD8+ T cells suggests variable intensities or durations of tonic TCR signaling. Although the expression of genes associated with acutely stimulated T cells was increased in Nur77-GFPHI cells, these cells were hyporesponsive to agonist TCR stimulation compared with Nur77-GFPLO cells. This hyporesponsiveness manifested as diminished activation marker expression and decreased secretion of IFN-γ and IL-2. The protein abundance of the ubiquitin ligase Cbl-b, a negative regulator of TCR signaling, was greater in Nur77-GFPHI cells than in Nur77-GFPLO cells, and Cbl-b deficiency partially restored the responsiveness of Nur77-GFPHI cells. Our data suggest that the cumulative effects of previously experienced tonic TCR signaling recalibrate naive CD8+ T cell responsiveness. These changes include gene expression changes and negative regulation partially dependent on Cbl-b. This cell-intrinsic negative feedback loop may enable the immune system to restrain naive CD8+ T cells with higher self-reactivity.

The T-cell niche tunes immune function through modulation of the cytoskeleton and TCR-antigen forces.

Obesity is a major public health crisis given its rampant growth and association with an increased risk for cancer. Interestingly, patients with obesity tend to have an increased tumor burden and decreased T-cell function. It remains unclear how obesity compromises T-cell mediated immunity. To address this question, we modeled the adipocyte niche using the secretome released from adipocytes as well as the niche of stromal cells and investigated how these factors modulated T-cell function. We found that the secretomes altered antigen-specific T-cell receptor (TCR) triggering and activation. RNA-sequencing analysis identified thousands of gene targets modulated by the secretome including those associated with cytoskeletal regulation and actin polymerization. We next used molecular force probes to show that T-cells exposed to the adipocyte niche display dampened force transmission to the TCR-antigen complex and conversely, stromal cell secreted factors lead to significantly enhanced TCR forces. These results were then validated in diet-induced obese mice. Importantly, secretome-mediated TCR force modulation mirrored the changes in T-cell functional responses in human T-cells using the FDA-approved immunotherapy, blinatumomab. Thus, this work shows that the adipocyte niche contributes to T-cell dysfunction through cytoskeletal modulation and reduces TCR triggering by dampening TCR forces consistent with the mechanosensor model of T-cell activation.

Accumulation of TCR signaling from self-antigens in naive CD8 T cells mitigates early responsiveness.

The cumulative effects of T cell receptor (TCR) signal transduction over extended periods of time influences T cell biology, such as the positive selection of immature thymocytes or the proliferative responses of naive T cells. Naive T cells experience recurrent TCR signaling in response to self-antigens in the steady state. However, how these signals influence the responsiveness of naive CD8+ T cells to subsequent agonist TCR stimulation remains incompletely understood. We investigated how naive CD8+ T cells that experienced relatively low or high levels of TCR signaling in response to self-antigens respond to stimulation with foreign antigens. A transcriptional reporter of Nr4a1 (Nur77-GFP) revealed substantial heterogeneity of the amount of TCR signaling naive CD8+ T cells accumulate in the steady state. Nur77-GFPHI cells exhibited diminished T cell activation and secretion of IFNγ and IL-2 relative to Nur77-GFPLO cells in response to agonist TCR stimulation. Differential gene expression analyses revealed upregulation of genes associated with acutely stimulated T cells in Nur77-GFPHI cells but also increased expression of negative regulators such as the phosphatase Sts1. Responsiveness of Nur77-GFPHI cells to TCR stimulation was partially restored at the level of IFNγ secretion by deficiency of Sts1 or the ubiquitin ligase Cbl-b. Our data suggest that extensive accumulation of TCR signaling during steady state conditions induces a recalibration of the responsiveness of naive CD8+ T cells through gene expression changes and negative regulation, at least in part, dependent on Sts1 and Cbl-b. This cell-intrinsic negative feedback loop may allow the immune system to limit the autoreactive potential of highly self-reactive naive CD8+ T cells.

Strong Basal/Tonic TCR Signals Are Associated with Negative Regulation of Naive CD4+ T Cells.

T cells experience varying intensities of tonic or basal TCR signaling in response to self-peptides presented by MHC (self-pMHC) in vivo. We analyzed four subpopulations of mouse naive CD4+ cells that express different levels of Nur77-GFP and Ly6C, surrogate markers that positively and inversely correlate with the strength of tonic TCR signaling, respectively. Adoptive transfer studies suggest that relatively weak or strong Nur77-GFP intensity in thymocytes tends to be maintained in mature T cells. Two-dimensional affinity measurements were lowest for Nur77-GFPloLy6C+ cells and highest for Nur77-GFPhiLy6C- cells, highlighting a positive correlation between apparent TCR affinity and tonic TCR signal strength. Despite experiencing the strongest tonic TCR signaling, Nur77-GFPhiLy6C- cells were least responsive to multiple concentrations of a cognate or suboptimal pMHC. Gene expression analyses suggest that Nur77-GFPhiLy6C- cells induce a gene expression program that has similarities with that of acutely stimulated T cells. However, strong tonic TCR signaling also correlates with increased expression of genes with inhibitory functions, including coinhibitory receptors. Similarly, assay for transposase-accessible chromatin with sequencing analyses suggested that increased tonic TCR signal strength correlated with increased chromatin accessibility associated with genes that have positive and inhibitory roles in T cell activation. Strikingly, Nur77-GFPhiLy6C- cells exhibited differential accessibility within regions of Cd200r1 and Tox that were similar in location to differentially accessible regions previously identified in exhausted CD8+ T cells. We propose that constitutive strong tonic TCR signaling triggers adaptations detectable at both the transcriptional and epigenetic levels, ultimately contributing to the tuning of T cell responsiveness.

Functional heterogeneity and adaptation of naive T cells in response to tonic TCR signals.

Mature CD4+ and CD8+ T cells constitutively experience weak T cell receptor (TCR) stimulation in response to self-antigens, termed tonic (or basal) signaling. How tonic TCR signal strength impacts T cell responses to foreign antigens is an active area of investigation. Such studies rely on surrogate markers of tonic signal strength, including CD5, Ly6C, and transgenic reporters of Nr4a genes. Recent research indicates that strong tonic TCR signal strength influences basal T cell metabolism, effector differentiation, and TCR signal transduction. T cells that experience the strongest tonic TCR signaling exhibit features of T cell activation and negative regulation. These data suggest a model whereby adaptation to tonic signaling has lasting effects that alter T cell activation and differentiation.

Reverse TCR repertoire evolution toward dominant low-affinity clones during chronic CMV infection.

Adaptive evolution is a key feature of T cell immunity. During acute immune responses, T cells harboring high-affinity T cell antigen receptors (TCRs) are preferentially expanded, but whether affinity maturation by clonal selection continues through the course of chronic infections remains unresolved. Here we investigated the evolution of the TCR repertoire and its affinity during the course of infection with cytomegalovirus, which elicits large T cell populations in humans and mice. Using single-cell and bulk TCR sequencing and structural affinity analyses of cytomegalovirus-specific T cells, and through the generation and in vivo monitoring of defined TCR repertoires, we found that the immunodominance of high-affinity T cell clones declined during the chronic infection phase, likely due to cellular senescence. These data showed that under conditions of chronic antigen exposure, low-affinity TCRs preferentially expanded within the TCR repertoire, with implications for immunotherapeutic strategies.

Circadian regulation of phosphodiesterase 6 genes in zebrafish differs between cones and rods: Implications for photopic and scotopic vision.

A correlation is known to exist between visual sensitivity and oscillations in red opsinand rhodopsin gene expression in zebrafish, both regulated by the clock gene. This indicates that an endogenous circadian clock regulates behavioural visual sensitivity, apart from the regulation exerted by the pineal organ. However, the specific mechanisms for cones (photopic vision) and rods (scotopic vision) are poorly understood. In this work, we performed gene expression, cosinor and immunohistochemical analyses to investigate other key genes involved in light perception, encoding the different subunits of phosphodiesterase pde6 and transducin GαT, in constant lighting conditions and compared to normal light-dark conditions. We found that cones display prominent circadian oscillations in mRNA levels for the inhibitory subunit gene pde6ha that could contribute to the regulation of photopic sensitivity by preventing overstimulation in photopic conditions. In rods, the mRNA levels of the inhibitory subunit gene pde6ga oscillate under normal conditions and dampen down in constant light but continue oscillating in constant darkness. There is an increase in total relative expression for pde6gb in constant conditions. These observations, together with previous data, suggest a complex regulation of the scotopic sensitivity involving endogenous and non-endogenous components, possibly present also in other teleost species. The GαT genes do not display mRNA oscillations and therefore may not be essential for the circadian regulation of photosensitivity. In summary, our results support different regulation for the zebrafish photopic and scotopic sensitivities and suggest circadian regulation of pde6ha as a key factor regulating photopic sensitivity, while the regulatory mechanisms in rods appear to be more complex.

Evolution and expression of the phosphodiesterase 6 genes unveils vertebrate novelty to control photosensitivity.

BACKGROUND: Phosphodiesterase 6 (PDE6) is a protein complex that hydrolyses cGMP and acts as the effector of the vertebrate phototransduction cascade. The PDE6 holoenzyme consists of catalytic and inhibitory subunits belonging to two unrelated gene families. Rods and cones express distinct genes from both families: PDE6A and PDE6B code for the catalytic and PDE6G the inhibitory subunits in rods while PDE6C codes for the catalytic and PDE6H the inhibitory subunits in cones. We performed phylogenetic and comparative synteny analyses for both gene families in genomes from a broad range of animals. Furthermore, gene expression was investigated in zebrafish. RESULTS: We found that both gene families expanded from one to three members in the two rounds of genome doubling (2R) that occurred at the base of vertebrate evolution. The PDE6 inhibitory subunit gene family appears to be unique to vertebrates and expanded further after the teleost-specific genome doubling (3R). We also describe a new family member that originated in 2R and has been lost in amniotes, which we have named pde6i. Zebrafish has retained two additional copies of the PDE6 inhibitory subunit genes after 3R that are highly conserved, have high amino acid sequence identity, are coexpressed in the same photoreceptor type as their amniote orthologs and, interestingly, show strikingly different daily oscillation in gene expression levels. CONCLUSIONS: Together, these data suggest specialisation related to the adaptation to different light intensities during the day-night cycle, most likely maintaining the regulatory function of the PDE inhibitory subunits in the phototransduction cascade.