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Post-thymic maturation status of naïve polyclonal T cells determines their TNF producing capability. A, The percentages of non-transgenic CD8+ and CD4+ cells (both CD44lo and CD44hi) from thymi and spleens of B6 mice staining positive for TNF cytokine are shown. B, Thymocytes and splenocytes from NG-BAC transgenic mice were stimulated with αCD3+αCD28 for 4 hours and then stained for maturation markers and intracellular TNF. The GFP profile of SP thymocytes, RTEs and MN T cells in the CD8+ and CD4+ compartments is shown. B and C, The percentages of CD44lo TNF producing cells in the 3 different T cell subsets and their respective average MFI for TNF expression are shown. The average MFI of TNF expression were analysed by one-way ANOVA with a Tukey post-test. The data are representative of 4 individual mice. The error bars indicate SD.
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The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after ac...
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Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment....
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... Interestingly, murine naïve T cells also contain preformed Tnf mRNA and produce substantial amounts of TNF-α as early as 2 hours after stimulation [83,84]. The ability of naïve T cells to produce TNF-α protein is however acquired in the periphery [85]. Furthermore, Tnf mRNA in T cells is notoriously unstable [40], and its continuous expression must be linked to continuous de novo transcription of this mRNA, possibly driven by tonic signals T cells receive in peripheral secondary lymphoid organs. ...
T cells are central players of the adaptive immune system by protecting us from recurring infections and by killing malignant cells. Protective T cell responses rely on the concerted production of effector molecules such as cytolytic mediators, granzymes, and perforins, as well as pro-inflammatory cytokines and chemokines. Once activated, T cells drastically change their gene expression and rapidly respond to insults by producing ample amounts of effector molecules. In the absence of antigen, T cells remain in a quiescent state and survey our body for possible pathogenic insults. Resting T cells are, however, not inert, but continuously regulate their protein production to survive and to be prepared for possible reinfections. Here, we review our current knowledge on the regulation of gene expression in activated and quiescent T cells. We specifically focus on post-transcriptional mechanisms that define the protein output and that allow dormant cells to undergo active signaling and selective translation, keeping them poised for activation. Finally, we discuss which signals drive T cell survival and their preparedness to respond to insults and which mechanisms are involved in these processes.
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... There are a variety of markers associated with post-thymic maturation, one of which is the expression of Qa2. As cells mature in the periphery, the expression of surface Qa2 moves from low to high (31). There were no main effects of sex or sex chromosomal complement, nor an interaction of these two in the percentage of cells expressing low levels of Qa2 between males and females for either CD8+ or CD4+ cells (Table 1A). ...
We have used the four core genotypes (FCG) mouse model, which allows a distinction between effects of gonadal secretions and chromosomal complement, to determine when sex differences in the immune system first appear and what influences their development. Using splenic T cell number as a measure that could be applied to neonates with as yet immature immune responses, we found no differences among the four genotypes at postnatal day 1, but by day 7, clear sex differences were observed. These sex differences were unexpectedly independent of chromosomal complement and similar in degree to gonadectomized FCG adults: both neonatal and gonadectomized adult females (XX and XY) showed 2-fold the number of CD4+ and 7-fold the number of CD8+ T cells versus their male (XX and XY) counterparts. Appearance of this long-lived sex difference between days 1 and 7 suggested a role for the male-specific perinatal surge of testicular testosterone. Interference with the testosterone surge significantly de-masculinized the male CD4+, but not CD8+ splenic profile. Treatment of neonates demonstrated elevated testosterone limited mature cell egress from the thymus, whereas estradiol reduced splenic T cell seeding in females. Neonatal male splenic epithelium/stroma expressed aromatase mRNA, suggesting capacity for splenic conversion of perinatal testosterone into estradiol in males, which, similar to administration of estradiol in females, would result in reduced splenic T cell seeding. These sex steroid effects affected both CD4+ and CD8+ cells and yet interference with the testosterone surge only significantly de-masculinized the splenic content of CD4+ cells. For CD8+ cells, male cells in the thymus were also found to express one third the density of sphingosine-1-phosphate thymic egress receptors per cell compared to female, a male characteristic most likely an indirect result of Sry expression. Interestingly, the data also support a previously unrecognized role for non-gonadal estradiol in the promotion of intra-thymic cell proliferation in neonates of both sexes. Microarray analysis suggested the thymic epithelium/stroma as the source of this hormone. We conclude that some immune sex differences appear long before puberty and more than one mechanism contributes to differential numbers and distribution of T cells.
... Mice were bred to contain the following alleles: a K15 promoter-driven Cre recombinase (K15.CrePR1) [33], a R26Stop FL P110* conditional allele that carries a loxP-flanked Neo-STOP cassette preceding a constitutively active PIK3CA allele (encoding p110α protein) targeted to the Gt(ROSA)26Sor locus [34], and a conditional TP53 gene knockout [35]. Induction of the recombinase activity was achieved by applying 100 µL of RU486 (0.2 µg/µL in 70% ethanol) orally or to the shaved dorsal flank skin of 5 to 12 week-old mice for 5 consecutive days. ...
Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.
... CD31 expression identifies CD4 + T cell subsets with unique functional capacities in neonates. Homeostatic expansion of naive CD4 + T cells is accompanied by changes in effector function, including the gain of TNF-α production (23). Differences in effector potential based on fetal homeostatic proliferation may negatively effect ELGAN health by enhancing or dysregulating their inflammatory responses. ...
The inverse relationship between gestational age at birth and postviral respiratory morbidity suggests that infants born preterm (PT) may miss a critical developmental window of T cell maturation. Despite a continued increase in younger PT survivors with respiratory complications, we have limited understanding of normal human fetal T cell maturation, how ex utero development in premature infants may interrupt normal T cell development, and whether T cell development has an effect on infant outcomes. In our longitudinal cohort of 157 infants born between 23 and 42 weeks of gestation, we identified differences in T cells present at birth that were dependent on gestational age and differences in postnatal T cell development that predicted respiratory outcome at 1 year of age. We show that naive CD4+ T cells shift from a CD31-TNF-α+ bias in mid gestation to a CD31+IL-8+ predominance by term gestation. Former PT infants discharged with CD31+IL8+CD4+ T cells below a range similar to that of full-term born infants were at an over 3.5-fold higher risk for respiratory complications after NICU discharge. This study is the first to our knowledge to identify a pattern of normal functional T cell development in later gestation and to associate abnormal T cell development with health outcomes in infants.
... Recent thymic emigrants (RTEs) comprise a unique population of T cells that have recently completed a tightly regulated developmental program in the thymus and entered the naive T cell pool in the periphery (2,(24)(25)(26)(27)(28). The phenotype and function of RTEs are different from SP thymocytes as well as peripheral naive T cells (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). Upon activation, RTEs exhibit lower levels of proliferation, cytokine production, and aerobic glycolysis when compared with mature naive T cells (44). ...
Mature naive T cells circulate through the secondary lymphoid organs in an actively enforced quiescent state. Impaired cell survival and cell functions could be found when T cells have defects in quiescence. One of the key features of T cell quiescence is low basal metabolic activity. It remains unclear at which developmental stage T cells acquire this metabolic quiescence. We compared mitochondria among CD4 single-positive (SP) T cells in the thymus, CD4+ recent thymic emigrants (RTEs), and mature naive T cells in the periphery. The results demonstrate that RTEs and naive T cells had reduced mitochondrial content and mitochondrial reactive oxygen species when compared with SP thymocytes. This downregulation of mitochondria requires T cell egress from the thymus and occurs early after young T cells enter the circulation. Autophagic clearance of mitochondria, but not mitochondria biogenesis or fission/fusion, contributes to mitochondrial downregulation in RTEs. The enhanced apoptosis signal-regulating kinase 1/MAPKs and reduced mechanistic target of rapamycin activities in RTEs relative to SP thymocytes may be involved in this mitochondrial reduction. These results indicate that the gain of metabolic quiescence is one of the important maturation processes during SP-RTE transition. Together with functional maturation, it promotes the survival and full responsiveness to activating stimuli in young T cells.
... CD4 + RTEs are characterized by reduced activationinduced upregulation of the high-affinity IL-2 receptor (CD25) and diminished proliferative capacity [4]. Compared to mature CD4 + T cells, RTEs produce less IL-2, IFN-g, TNF-a, and IL-4 when stimulated under nonpolarizing T H 0 conditions [22,23]. Figure 2 summarizes the different fates that RTEs and mature T cells adopt. ...
... Under non-inflammatory conditions, chicken ovalbumin (OVA)-specific CD8 + T cells produce less IFN-g and IL-2 compared to mature T cells during a primary challenge with Act-mOVA splenocytes [25] and in response to pancreatic OVA [26 ]. Even after exposure to a more inflammatory stimulus (Listeria monocytogenes), CD8 + RTEs produce less IFN-g, TNF-a, and IL-2 [22,25,27] on a per cell basis, despite being skewed toward short lived effectors [27]. This diminished cytokine profile is maintained into the memory time point [28]. ...
... RTEs can be identified as GFP + peripheral T cells in Rag2p-GFP-transgenic (Tg) mice in which the Rag2 promoter drives GFP expression (4,5), allowing for the unambiguous identification and retrieval of live, untouched RTEs from unmanipulated reporter mice. Using this model, it has been shown that, compared with their mature counterparts, RTEs encountering Ag in the absence of inflammation exhibit diminished proliferation, reduced IFN-g, IL-2, and TNF-a production, di-minished expression of early activation markers (including CD25), and increased expression of genes associated with dampened function or anergy (5)(6)(7)(8)(9)(10)(11). These disparities are apparent even in memory T cells derived from RTEs, well after the 3-wk transition period has elapsed, indicating that the stage of maturation at which T cells first meet Ag has a lasting impact on their immune properties (7,8). ...
Recent thymic emigrants (RTEs) are the youngest peripheral T cells that have completed thymic selection and egress to the lymphoid periphery. RTEs are functionally distinct from their more mature but still naive T cell counterparts, because they exhibit dampened proliferation and reduced cytokine production upon activation. In this article, we show that, compared with more mature but still naive T cells, RTEs are impaired in their ability to perform aerobic glycolysis following activation. Impaired metabolism underlies the reduced IFN-γ production observed in activated RTEs. This failure to undergo Ag-induced aerobic glycolysis is caused by reduced mTORC1 activity and diminished Myc induction in RTEs. Critically, exogenous IL-2 restores Myc expression in RTEs, driving aerobic glycolysis and IFN-γ production to the level of mature T cells. These results reveal a previously unknown metabolic component to postthymic T cell maturation.
... Although ~30% of RTEs had the capacity to express TNFα, arguing against a deficiency in CD4 + RTEs to produce any cytokines, the frequency of TNFα + CD4 + RTEs did not alter in response to infection and cytokine production was generally of lower level that that seen in recipient CD4 + T cells from the same mice. This suggests that TNFα expression by CD4 + RTEs is not a response to L. donovani infection per se but rather reflects the intermediate state of " licencing " for TNFα production shown by RTEs relative to their single positive thymic precursors and mature naïve CD4 + T cells[48]. Mechanistically, this is likely due to differences between these cells in chromatin remodelling at cytokine loci[49]. RTEs have been shown to require a period of post-thymic maturation of ~14 days before they become fully functional T cells[28]. ...
Recent thymic emigrants (RTEs) represent a source of antigen-naïve T cells that enter the periphery throughout life. However, whether RTEs contribute to the control of chronic parasitic infection and how their potential might be harnessed by therapeutic intervention is currently unclear. Here, we show that CD4⁺ recent thymic emigrants emerging into the periphery of mice with ongoing Leishmania donovani infection undergo partial activation and are recruited to sites of granulomatous inflammation. However, CD4⁺ RTEs displayed severely restricted differentiation either into IFNγ⁺ or IFNγ⁺TNFα⁺ effectors, or into IL-10-producing regulatory T cells. Effector cell differentiation in the chronically infected host was not promoted by adoptive transfer of activated dendritic cells or by allowing extended periods of post-thymic differentiation in the periphery. Nevertheless, CD4⁺ RTEs from infected mice retained the capacity to transfer protection into lymphopenic RAG2-/- mice. Taken together, our data indicate that RTEs emerging into a chronically inflamed environment are not recruited into the effector pool, but retain the capacity for subsequent differentiation into host protective T cells when placed in a disease-free environment.
... GFP signal strength correlates inversely with the time since the loss of Rag2 expression (McCaughtry et al., 2007), allowing RTEs of varied ages to be distinguished from their GFP − mature naive (MN) counterparts. Using such reporter mice, we and others have shown that RTEs are phenotypically and functionally distinct from MN T cells (Priyadharshini et al., 2010;Bhaumik et al., 2013;Fink, 2013;Paiva et al., 2013;Berkley and Fink, 2014;Hog quist et al., 2015), differences also ascribed to neonatal T cells (Opiela et al., 2009;Zaghouani et al., 2009;PrabhuDas et al., 2011;Smith et al., 2014) and to human RTEs (McFarland et al., 2000;Haines et al., 2009). ...
T cell development requires a period of postthymic maturation. Why this is the case has remained a mystery, particularly given the rigors of intrathymic developmental checkpoints, successfully traversed by only ∼5% of thymocytes. We now show that the first few weeks of T cell residence in the lymphoid periphery define a period of heightened susceptibility to tolerance induction to tissue-restricted antigens (TRAs), the outcome of which depends on the context in which recent thymic emigrants (RTEs) encounter antigen. After encounter with TRAs in the absence of inflammation, RTEs exhibited defects in proliferation, diminished cytokine production, elevated expression of anergy-associated genes, and diminished diabetogenicity. These properties were mirrored in vitro by enhanced RTE susceptibility to regulatory T cell–mediated suppression. In the presence of inflammation, RTEs and mature T cells were, in contrast, equally capable of inducing diabetes, proliferating, and producing cytokines. Thus, recirculating RTEs encounter TRAs during a transitional developmental stage that facilitates tolerance induction, but inflammation converts antigen-exposed, tolerance-prone RTEs into competent effector cells.
... Thus, amongst proliferation competent M1 and M2 thymocytes, only the most mature M2 cells are competent to emigrate. Finally, we assessed at which stage SP thymocytes became licensed to produce the cytokine TNF 18 . Only the M2 subset had a substantial population of TNF-producing cells following stimulation via CD3 and CD28 (Fig. 1d), and this fraction continued to increase in recent thymic emigrants (data not shown), consistent with previous reports 18 . ...
... Finally, we assessed at which stage SP thymocytes became licensed to produce the cytokine TNF 18 . Only the M2 subset had a substantial population of TNF-producing cells following stimulation via CD3 and CD28 (Fig. 1d), and this fraction continued to increase in recent thymic emigrants (data not shown), consistent with previous reports 18 . ...
Positive selection occurs in the thymic cortex, but critical maturation events occur later in the medulla. Here we defined the precise stage at which T cells acquired competence to proliferate and emigrate. Transcriptome analysis of late gene changes suggested roles for the transcription factor NF-κB and interferon signaling. Mice lacking the inhibitor of NF-κB (IκB) kinase (IKK) kinase TAK1 underwent normal positive selection but exhibited a specific block in functional maturation. NF-κB signaling provided protection from death mediated by the cytokine TNF and was required for proliferation and emigration. The interferon signature was independent of NF-κB; however, thymocytes deficient in the interferon-α (IFN-α) receptor IFN-αR showed reduced expression of the transcription factor STAT1 and phenotypic abnormality but were able to proliferate. Thus, both NF-κB and tonic interferon signals are involved in the final maturation of thymocytes into naive T cells.
