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.

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.

A supergene-linked estrogen receptor drives alternative phenotypes in a polymorphic songbird.

Behavioral evolution relies on genetic changes, yet few behaviors can be traced to specific genetic sequences in vertebrates. Here we provide experimental evidence showing that differentiation of a single gene has contributed to the evolution of divergent behavioral phenotypes in the white-throated sparrow, a common backyard songbird. In this species, a series of chromosomal inversions has formed a supergene that segregates with an aggressive phenotype. The supergene has captured ESR1, the gene that encodes estrogen receptor α (ERα); as a result, this gene is accumulating changes that now distinguish the supergene allele from the standard allele. Our results show that in birds of the more aggressive phenotype, ERα knockdown caused a phenotypic change to that of the less aggressive phenotype. We next showed that in a free-living population, aggression is predicted by allelic imbalance favoring the supergene allele. Finally, we identified cis-regulatory features, both genetic and epigenetic, that explain the allelic imbalance. This work provides a rare illustration of how genotypic divergence has led to behavioral phenotypic divergence in a vertebrate.

Adaptation by naïve CD4+ T cells to self-antigen-dependent TCR signaling induces functional heterogeneity and tolerance.

Naïve CD4+ T cells experience weak T cell receptor (TCR) signals induced by self-peptides presented by MHC II. To investigate how these "basal" TCR signals influence responses to agonist TCR ligand stimulation, we analyzed naïve CD4+ cells expressing varying amounts of CD5, Ly6C, and Nur77-GFP, markers that reflect the strength of basal TCR signaling. Phenotypic analyses indicate that the broadest range of basal TCR signal strength can be visualized by a combination of Nur77-GFP and Ly6C. A range of basal TCR signaling is detectable even in populations that express identical TCRs. Whereas moderate basal TCR signal strength correlates with higher IL-2 secretion at early time points following TCR stimulation, weak basal TCR signaling correlated with higher IL-2 secretion at later time points. We identify a population of Nur77-GFPHI Ly6C- cells that could not be reliably marked by either of CD5, Ly6C, or Nur77-GFP alone. These cells experience the strongest basal TCR signaling, consistently produce less IL-2, and express PD-1 and markers associated with anergy, such as Grail and Cbl-b. We propose that adaptation to the strength of basal TCR signaling drives the phenotypic and functional heterogeneity of naïve CD4+ cells.

Rapid regulatory evolution of a nonrecombining autosome linked to divergent behavioral phenotypes.

In the white-throated sparrow (Zonotrichia albicollis), the second chromosome bears a striking resemblance to sex chromosomes. First, within each breeding pair of birds, one bird is homozygous for the standard arrangement of the chromosome (ZAL2/ZAL2) and its mate is heterozygous for a different version (ZAL2/ZAL2m). Second, recombination is profoundly suppressed between the two versions, leading to genetic differentiation between them. Third, the ZAL2m version is linked with phenotypic traits, such as bright plumage, high aggression, and low parental behavior, which are usually associated with males. These similarities to sex chromosomes suggest that the evolutionary mechanisms that shape sex chromosomes, in particular genetic degeneration of the heterogametic version due to the suppression of recombination, are likely important in this system as well. Here, we investigated patterns of protein sequence evolution and gene expression evolution between the ZAL2 and ZAL2m chromosomes by whole-genome sequencing and transcriptome analyses. Patterns of protein evolution exhibited only weak signals of genetic degeneration, and few genes harbored signatures of positive selection. We found substantial evidence of transcriptome evolution, such as significant expression divergence between ZAL2 and ZAL2m alleles and signatures of dosage compensation for highly expressed genes. These results suggest that, early in the evolution of heteromorphic chromosomes, gene expression divergence and dosage compensation can prevail before large-scale genetic degeneration. Our results show further that suppression of recombination between heteromorphic chromosomes can lead to the evolution of alternative (sex-like) behavioral phenotypes before substantial genetic degeneration.

Estrogen Receptor Alpha as a Mediator of Life-History Trade-offs.

Trade-offs between competitive and parental strategies often are mediated by sex steroids. The mechanisms underlying steroid signaling and metabolism may therefore serve as targets of disruptive selection that leads to alternative behavioral phenotypes. White-throated sparrows exhibit two color morphs that differ in both competitive and parental behavior; white-striped (WS) birds engage in more territorial singing, whereas tan-striped (TS) birds provision nestlings more often. Although WS birds have higher levels of plasma testosterone (T) and estradiol than do TS birds, experimental equalization of these hormones does not abolish morph differences in singing. Neural sensitivity to sex steroids may differ between the morphs because the gene for estrogen receptor alpha (ERα) has been captured by a chromosomal rearrangement found only in the WS birds. We recently showed that expression of this gene differs between the morphs and may drive the behavioral polymorphism. First, the ERα promoter region contains fixed polymorphisms that affect transcription efficiency in vitro. Second, in a free-living population, local expression of ERα depends strongly on morph and predicts both territorial singing and parental provisioning. Differential ERα expression is particularly striking in the medial amygdala; WS birds have three times more ERα mRNA than do TS birds. This difference persists during the non-breeding season and is unaffected by exogenous T treatment. Finally, preliminary data generated by RNA-seq confirm that ERα expression in MeA is both differentially expressed and correlated with territorial singing. Together, these results suggest that ERα may be a target of disruptive selection that leads to alternative behavioral strategies. Our future directions include a more detailed analysis of the ERα promoter regions to determine the molecular basis of differential expression as well as gene network analyses to identify genes connected to ERα.

Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds.

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.

Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes.

The evolution of behavior relies on changes at the level of the genome; yet the ability to attribute a behavioral change to a specific, naturally occurring genetic change is rare in vertebrates. In the white-throated sparrow (Zonotrichia albicollis), a chromosomal polymorphism (ZAL2/2(m)) is known to segregate with a behavioral phenotype. Individuals with the ZAL2(m) haplotype engage in more territorial aggression and less parental behavior than individuals without it. These behaviors are thought to be mediated by sensitivity to sex steroids, and the chromosomal rearrangement underlying the polymorphism has captured a prime candidate gene: estrogen receptor 1 (ESR1), which encodes estrogen receptor α (ERα). We therefore hypothesized that the behavioral effects of the ZAL2(m) rearrangement are mediated by polymorphism in ESR1. We report here that (i) the ESR1 promoter region contains fixed polymorphisms distinguishing the ZAL2(m) and ZAL2 alleles; (ii); those polymorphisms regulate transcription efficiency in vitro and therefore potentially do the same in vivo (iii); the local expression of ERα in the brain depends strongly on genotype in a free-living population; and (iv) ERα expression in the medial amygdala and medial preoptic area may fully mediate the effects of genotype on territorial aggression and parenting, respectively. Thus, our study provides a rare glimpse of how a chromosomal polymorphism has affected the brain and social behavior in a vertebrate. Our results suggest that in this species, differentiation of ESR1 has played a causal role in the evolution of phenotypes with alternative life-history strategies.