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Special AT-binding protein 1 (SATB1) is a chromatin-binding protein that has been shown to be a key regulator of T-cell development and CD4 + T-cell fate decisions and function. The underlying function for SATB1 in peripheral CD8 + T-cell differentiation processes is largely unknown. To address this, we examined SATB1-binding patterns in naïve and effector CD8 + T cells demonstrating that SATB1 binds to noncoding regulatory elements linked to T-cell lineage-specific gene programs, particularly in naïve CD8 + T cells. We then assessed SATB1 function using N-ethyl-N-nitrosourea-mutant mice that exhibit a point mutation in the SATB1 DNA-binding domain (termed Satb1 m1Anu/m1Anu ). Satb1 m1Anu/m1Anu mice exhibit diminished SATB1-binding, naïve, Satb1 m1Anu/m1Anu CD8 + T cells exhibiting transcriptional and phenotypic characteristics reminiscent of effector T cells. Upon activation, the transcriptional signatures of Satb1 m1Anu/m1Anu and wild-type effector CD8 + T cells converged. While there were no overt differences, primary respiratory infection of Satb1 m1Anu/m1Anu mice with influenza A virus (IAV) resulted in a decreased proportion and number of IAV-specific CD8 + effector T cells recruited to the infected lung when compared with wild-type mice. Together, these data suggest that SATB1 has a major role in an appropriate transcriptional state within naïve CD8 + T cells and ensures appropriate CD8 + T-cell effector gene expression upon activation., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Suppressor of cytokine signaling (SOCS) proteins are key regulators of innate and adaptive immunity. There is no described biological role for SOCS4, despite broad expression in the hematopoietic system. We demonstrate that mice lacking functional SOCS4 protein rapidly succumb to infection with a pathogenic H1N1 influenza virus (PR8) and are hypersusceptible to infection with the less virulent H3N2 (X31) strain. In SOCS4-deficient animals, this led to substantially greater weight loss, dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This was associated with impaired trafficking of influenza-specific CD8 T cells to the site of infection and linked to defects in T cell receptor activation. These results demonstrate that SOCS4 is a critical regulator of anti-viral immunity.

Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen. Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8(+) effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus. These epigenetic modifications are maintained long-term in vivo following adoptive transfer. Even after extended type 2 polarization, however, some CD8(low) effector cells respond to interferon-γ by re-expressing CD8 and a type 1 cytokine profile in association with partial Cd8a demethylation. Cd8a methylation signatures in naive, polarized and repolarized cells are distinct from those observed during the initiation, maintenance and silencing of CD8 expression by developing T cells in the thymus. This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8(+) T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.

Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(α)14-J(α)18) T cell antigen receptor (TCR) α-chain and recognition of the glycolipid α-galactosylceramide (α-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of α-GalCer-reactive NKT cells that expressed a canonical V(α)10-J(α)50 TCR α-chain, which showed a preference for α-glucosylceramide (α-GlcCer) and bacterial α-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCRα sequence identity, the V(α)10 TCR-CD1d-α-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-α-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.

Pathogen-specific responses are characterized by preferred profiles of peptide+class I MHC (pMHCI) glycoprotein-specific T-cell receptor (TCR) Variable (V)-region use. How TCRV-region bias impacts TCRαβ heterodimer selection and resultant diversity is unclear. The D(b)PA(224)-specific TCR repertoire in influenza A virus-infected C57BL/6J (B6) mice exhibits a preferred TCRV-region bias toward the TRBV29 gene segment and an optimal complementarity determining region (CDR3) β-length of 6 aa. Despite these restrictions, D(b)PA(224)-specific BV29(+) T cells use a wide array of unique CDR3β sequences. Structural characterization of a single, TRBV29(+)D(b)P(A224)-specific TCRαβ-pMHCI complex demonstrated that CDR3α amino acid side chains made specific peptide interactions, but the CDR3β main chain exclusively contacted peptides. Thus, length but not amino acid sequence was key for recognition and flexibility in Vβ-region use. In support of this hypothesis, retrovirus expression of the D(b)PA(224)-specific TCRVα-chain was used to constrain pairing within a naive/immune epitope-specific repertoire. The retrogenic TCRVα paired with a diversity of CDR3βs in the context of a preferred TCRVβ spectrum. Overall, these data provide an explanation for the combination of TCRV region bias and diversity within selected repertoires, even as they maintain exquisite pMHCI specificity.

Earlier studies of influenza-specific CD8(+) T cell immunodominance hierarchies indicated that expression of the H2K(k) MHC class I allele greatly diminishes responses to the H2D(b)-restriced D(b)PA(224) epitope (acid polymerase, residues 224-233 complexed with H2D(b)). The results suggested that the presence of H2K(k) during thymic differentiation led to the deletion of a prominent Vβ7(+) subset of D(b)PA(224)-specific TCRs. The more recent definition of D(b)PA(224)-specific TCR CDR3β repertoires in H2(b) mice provides a new baseline for looking again at this possible H2K(k) effect on D(b)PA(224)-specific TCR selection. We found that immune responses to several H2D(b)- and H2K(b)-restricted influenza epitopes were indeed diminished in H2(bxk) F(1) versus homozygous mice. In the case of D(b)PA(224), lower numbers of naive precursors were part of the explanation, though a similar decrease in those specific for the D(b)NP(366) epitope did not affect response magnitude. Changes in precursor frequency were not associated with any major loss of TCR diversity and could not fully account for the diminished D(b)PA(224)-specific response. Further functional and phenotypic characterization of influenza-specific CD8(+) T cells suggested that the expansion and differentiation of the D(b)PA(224)-specific set is impaired in the H2(bxk) F(1) environment. Thus, the D(b)PA(224) response in H2(bxk) F(1) mice is modulated by factors that affect the generation of naive epitope-specific precursors and the expansion and differentiation of these T cells during infection, rather than clonal deletion of a prominent Vβ7(+) subset. Such findings illustrate the difficulties of predicting and defining the effects of MHC class I diversification on epitope-specific responses.

TCR repertoire diversity can influence the efficacy of CD8(+) T-cell populations, with greater breadth eliciting better protection. We analyzed TCR beta diversity and functional capacity for influenza-specific CD8(+) T cells expressing a single TCR alpha chain. Mice (A7) transgenic for the H2K(b)OVA(257-264)-specific V alpha 2.7 TCR were challenged with influenza to determine how fixing this "irrelevant" TCR alpha affects the "public" and restricted D(b)NP(366) (+)CD8(+) versus the "private" and diverse D(b)PA(224) (+)CD8(+) responses. Though both D(b)NP(366) (+)CD8(+) and D(b)PA(224) (+)CD8(+) sets are generated in virus-primed A7 mice, the constrained D(b)NP(366) (+)CD8(+) population lacked the characteristic, public TCRV beta 8.3, and consequently was reduced in magnitude and pMHC-I avidity. For the more diverse D(b)PA(224) (+)CD8(+) T cells, this particular forcing led to a narrowing and higher TCR beta conservation of the dominant V beta 7, though the responses were of comparable magnitude to C57BL/6J controls. Interestingly, although both the TCR beta diversity and the cytokine profiles were reduced for the D(b)NP(366) (+)CD8(+) and D(b)PA(224) (+)CD8(+) sets in spleen, the latter measure of polyfunctionality was comparable for T cells recovered from the infected lungs of A7 and control mice. Even "sub-optimal" TCR alpha beta pairs can operate effectively when exposed in a milieu of high virus load. Thus, TCR beta diversity is important for optimal TCR alpha beta pairing and function when TCR alpha is limiting.

Lipopeptide constructs offer a novel strategy for eliciting effective cellular and humoral immunity by directly targeting the vaccine Ag to dendritic cells. Importantly, it is not known how closely immunity generated after lipopeptide vaccination mimics that generated after natural infection. We have used a novel lipopeptide vaccine strategy to analyze both the quantity and quality of CD8(+) T cell immunity to an influenza A virus epitope derived from the acidic polymerase protein (PA(224)) in B6 mice. Vaccination with the PA(224) lipopeptide resulted in accelerated viral clearance after subsequent influenza virus infection. The lipopeptide was also effective at recalling secondary D(b)PA(224) responses in the lung. Lipopeptide recalled D(b)PA(224)-specific CTL produced lower levels of IFN-gamma and TNF-alpha, but produced similar levels of IL-2 when compared with D(b)PA(224)-specific CTL recalled after virus infection. Furthermore, lipopeptide- and virus-recalled CTL demonstrated similar TCR avidity. Interestingly, lipopeptide administration resulted in expansion of D(b)PA(224)-specific CTL using a normally subdominant TCRBV gene segment. Overall, these results demonstrate that protective CTL responses elicited by lipopeptide vaccines can be correlated with TCR avidity, IL-2 production, and broad TCR repertoire diversity. Furthermore, factors that impact the quality of immunity are discussed. These factors are important considerations when evaluating the efficacy of novel vaccine strategies that target dendritic cells for eliciting cellular immunity.

The H-2Db-restricted CD8 T cell immune response to influenza A is directed at two well-described epitopes, nucleoprotein 366 (NP366) and acid polymerase 224 (PA224). The responses to the two epitopes are very different. The epitope NP366-specific response is dominated by TCR clonotypes that are public (shared by most mice), whereas the epitope PA224-specific response is private (unique within each infected animal). In addition to being public, the NP366-specific response is dominated by a few clonotypes, when T cell clonotypes expressing the Vbeta8.3 element are analyzed. Herein, we show that this response is similarly public when the NP366+Vbeta4+ CD8 T cell response is analyzed. Furthermore, to determine whether these features resulted in differences in total TCR diversity in the NP366+ and PA224+ responses, we quantified the number of different CD8 T clonotypes responding to each epitope. We calculated that 50-550 clonotypes recognized each epitope in individual mice. Thus, although the character of the response to the two epitopes appeared to be different (private and diverse vs public and dominated by a few clonotypes), similar numbers of precursor cells responded to both epitopes and this number was of similar magnitude to that previously reported for other viral CD8 T cell epitopes. Therefore, even in CD8 T cell responses that appear to be oligoclonotypic, the total response is highly diverse.

To study the relation in time between replication and endoreplication and the relation between appearance of platelet-specific proteins and endoreplication in maturing megakaryocytes, peripheral blood mononuclear cells highly enriched in hematopoietic progenitors were cultured in liquid cultures and plasma clots in the presence of either interleukin-3 (IL-3) and stem cell factor (SCF) or medium conditioned by blood mononuclear cells stimulated by phytohemagglutinin (PHA). In plasma clots, megakaryocytic (MK) colonies appeared first on day 5 and reached a maximum by day 8, whereas the number of cells per colony increased until day 10, indicating that there was a single wave of MK colony formation. In liquid cultures, the first immunologically recognizable megakaryocytes appeared on day 5 and expressed GPIIb/IIIa and thrombospondin only, but all other platelet-specific protein markers appeared within 24 hours. Replating cells from liquid medium into plasma clots showed that 92 +/- 8% of day 6 GPIIb/IIIa-positive cells are capable of replicating. Their replicative potential decreased with age, however, so that between days 6 and 11, a linear correlation was noted between the logarithm of the percentage of megakaryocytes with replicative capacity and their age in culture. Replication ceased completely after day 10. In the presence of IL-3, polyploid megakaryocytes appeared at the same time that GPIIb/IIIa was expressed, and the megakaryocyte distribution into ploidy classes remained unchanged until day 20. In the presence of PHA-leukocyte conditioned medium (PHA-LCM), ploidy of megakaryocytes was shifted toward higher classes after day 6, and the process of endoreplication was completed by day 10. No changes in ploidy distribution were noted between days 10 and 20. These findings indicate that in the cohort of megakaryocytes derived from colony-forming units-megakaryocyte (CFU-MK), endoreplication can occur at an early stage of development, proceeds synchronously with replication, and is completed before the megakaryocytes exhaust their replicative potential.

Cultured Nb2 node rat lymphoma cells require lactogenic hormone for their proliferation. We reported previously that dexamethasone (Dex) inhibits prolactin (PRL)-induced mitogenesis and, in the absence of mitogen, induces apoptosis of Nb2 cells. Both antiproliferative and cytolytic effects of Dex on Nb2 cells appear to involve glucocorticoid (Type II) receptor mediation. In this study, we compared Dex effects in PRL-dependent Nb2 cells (Nb2) with SFJCD1 (SF), a clone of Nb2 cells that proliferates independently of exogenous PRL. Proliferative assays involved a 72-hr incubation in a chemically defined, serum-free medium where ovine PRL (1 ng/ml) was added to Nb2 cells but not to SF cells. Both cell lines were responsive to the antiproliferative effects of Dex in a dose (6.25-200 nM)-dependent fashion of comparable sensitivity and magnitude. Co-incubation with the glucocorticoid receptor antagonist, RU 486, prevented the antiproliferative effect of Dex in both cell lines. In the same medium devoid of PRL, Dex was cytolytic to Nb2 cells and fragmented DNA in a fashion reflective of apoptosis, but was ineffective in SF cells. A dual chamber incubation system revealed no evidence that SF cells produced cytokines that were mitogenic or anticytolytic to Nb2 cells. Both Nb2 and SF cells fragmented DNA in a fashion indicative of apoptosis in the presence of the Ca2+ ionophore, A23187 (1 microM). These studies reveal a basic difference in glucocorticoid responsiveness between the PRL-dependent Nb2 cell line and its PRL-independent subclone, SF. While both cell lines exhibit functional glucocorticoid receptors and the necessary intranuclear machinery for apoptosis, the pathway mediating the latter is inhibited or dysfunctional in SF cells.

The interaction between the immunosuppressive effects of glucocorticoids and the mitogenic effects of prolactin (PRL) were examined in Nb2 lymphoma cells, a pre-T cell line. The synthetic glucocorticoid, dexamethasone (Dex), caused a concentration-dependent (6.25-200 nM) inhibition of basal and ovine PRL (oPRL)-stimulated Nb2 cell proliferation. Although Dex was antiproliferative, the steroid had no effect on cell viability in the presence of PRL. However, when PRL was omitted from the medium, Dex increased the proportion of dead Nb2 cells by 24 hr in a concentration (25-200 nM)-dependent fashion without affecting total cell number. The antiproliferative and cytolytic effects of Dex were mimicked by other corticosteroids (cortisol, corticosterone, aldosterone, and deoxycorticosterone) in the expected order of glucocorticoid potency, but not by other steroids (17-beta-estradiol, progesterone, testosterone, 5-alpha-dihydrotestosterone, and dehydroepiandrosterone) or triiodothyronine. In addition, the antiproliferative and cytolytic effects of glucocorticoids were antagonized by the glucocorticoid receptor antagonist RU 486. Since corticosteroid-induced cytolysis was apparent only in the absence of mitogen, the anticytolytic effects of oPRL were tested. In the presence of Dex (100 nM), oPRL (25-1600 pg/ml) caused a concentration-dependent inhibition of cytolysis without changing cell number. Other lactogenic hormones (human growth hormone, human placental lactogen, rat PRL), but not trophic nonlactogenic hormones (rat growth hormone, human chorionic gonadotropin, ACTH), also inhibited Dex (100 nM)-induced cytolysis. Agarose gel electrophoresis of DNA extracted from Nb2 cells revealed that within 12 hr, 100 nM Dex induced DNA fragmentation, indicative of programmed cell death or apoptosis. Coincubation of cells with Dex and oPRL (1 ng/ml) inhibited Dex-induced fragmentation of Nb2 cell genomic DNA. These studies reveal a complex interaction between glucocorticoids and PRL in Nb2 cells. Although a glucocorticoid receptor-mediated antiproliferative effect is evident, PRL (at concentrations that usually stimulate cell proliferation) has the capacity to protect the cell against glucocorticoid-receptor-mediated induction of apoptosis.