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Thymocyte development and differentiation in sγc-transgenic mice. (a) Total thymocyte numbers in sγ cTg and WT control mice. M, sγ c medium expresser; H, sγ c high expresser. Results are the mean and SEM of 12 independent experiments. (b) CD4 versus CD8 profile of total thymocytes (top) and TCRβ hi gated thymocytes (bottom) in WT and sγ cTg mice. (c) CD4/CD8 ratio of mature SP thymocytes (left) and frequency of DN thymocytes (right) in WT and sγ cTg mice. (d) Cell numbers of thymocyte subsets in WT and sγ cTg mice. Data are summary of 8 independent experiments with each 10 WT and 15 sγ cTg mice.
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The soluble γc protein (sγc) is a naturally occurring splice isoform of the γc cytokine receptor that is produced by activated T cells and inhibits γc cytokine signaling. Here we show that sγc expression is also highly upregulated in immature CD4⁺CD8⁺ thymocytes but then downregulated in mature thymocytes. These results indicate a developmentally c...
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Context 1
... sγ c cDNA was placed under the control of a human CD2 mini-cassette so that sγ c is overexpressed in all T lineage cells. Increased sγ c expression significantly reduced total thymocyte numbers, and we observed an inverse correlation of sγ c expression and total thymocyte numbers in WT, sγ c medium (M) and sγ c high (H) expresser transgenes (Fig. 3a). All further experiments in this study were done with the sγ c high expresser line. Assessing thymocyte profiles of sγ cTg mice did not reveal any significant changes in TCRβ hi mature T cell generation (Fig. 3b) or in CD4/CD8 lineage commitment (Fig. 3c left). However, we did find a significant increase in DN cell frequency (Fig. 3c ...
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... an inverse correlation of sγ c expression and total thymocyte numbers in WT, sγ c medium (M) and sγ c high (H) expresser transgenes (Fig. 3a). All further experiments in this study were done with the sγ c high expresser line. Assessing thymocyte profiles of sγ cTg mice did not reveal any significant changes in TCRβ hi mature T cell generation (Fig. 3b) or in CD4/CD8 lineage commitment (Fig. 3c left). However, we did find a significant increase in DN cell frequency (Fig. 3c right), suggesting a developmental defect in DN to DP cell transition, which would also explain the reduc- tion in thymocyte numbers in sγ cTg mice (Fig. 3d) 26 ...
Context 3
... and total thymocyte numbers in WT, sγ c medium (M) and sγ c high (H) expresser transgenes (Fig. 3a). All further experiments in this study were done with the sγ c high expresser line. Assessing thymocyte profiles of sγ cTg mice did not reveal any significant changes in TCRβ hi mature T cell generation (Fig. 3b) or in CD4/CD8 lineage commitment (Fig. 3c left). However, we did find a significant increase in DN cell frequency (Fig. 3c right), suggesting a developmental defect in DN to DP cell transition, which would also explain the reduc- tion in thymocyte numbers in sγ cTg mice (Fig. 3d) 26 ...
Context 4
... transgenes (Fig. 3a). All further experiments in this study were done with the sγ c high expresser line. Assessing thymocyte profiles of sγ cTg mice did not reveal any significant changes in TCRβ hi mature T cell generation (Fig. 3b) or in CD4/CD8 lineage commitment (Fig. 3c left). However, we did find a significant increase in DN cell frequency (Fig. 3c right), suggesting a developmental defect in DN to DP cell transition, which would also explain the reduc- tion in thymocyte numbers in sγ cTg mice (Fig. 3d) 26 ...
Context 5
... reveal any significant changes in TCRβ hi mature T cell generation (Fig. 3b) or in CD4/CD8 lineage commitment (Fig. 3c left). However, we did find a significant increase in DN cell frequency (Fig. 3c right), suggesting a developmental defect in DN to DP cell transition, which would also explain the reduc- tion in thymocyte numbers in sγ cTg mice (Fig. 3d) 26 ...
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... In particular, cytokines of the common γc-chain (γc) family play critical roles in the lineage choices and maturation of conventional T cells as demonstrated by the severely impaired thymopoiesis of γc-deficient mice [12,13]. Despite its importance in T cell development, however, the expression of the γc cytokine receptor is actively suppressed in preselection CD4 + CD8 + double-positive (DP) thymocytes [14,15]. We recently demonstrated that such a DP thymocyte-specific decrease in γc abundance is mediated by the transcription factor RORγt [16], which is selectively expressed in DP cells among thymocytes [17,18]. ...
... *P < 0.05 and **P < 0.01 (Student's two-tailed t test) then from stage II to III, is mediated by a combination of TCR and cytokine signals, known as β-selection and positive selection, respectively [20]. While the TCR levels progressively increase with maturation, the expression of cytokine receptors is distinct and stage-specific [14]. The IL-7Rα, for example, is highly abundant on stage I and stage III thymocytes but conspicuously absent on immature stage II cells, which correspond to preselection DP thymocytes (Fig. 1A) [14,21]. ...
... While the TCR levels progressively increase with maturation, the expression of cytokine receptors is distinct and stage-specific [14]. The IL-7Rα, for example, is highly abundant on stage I and stage III thymocytes but conspicuously absent on immature stage II cells, which correspond to preselection DP thymocytes (Fig. 1A) [14,21]. The IL-4Rα, on the other hand, is highly expressed on stage II preselection DP thymocytes, as previously reported ( Fig. 1A and Supplemental Fig. 1A) [21]. ...
Innate-like T (iT) cells comprise a population of immunoregulatory T cells whose effector function is imposed during their development in the thymus to provide protective immunity prior to antigen encounter. The molecular mechanism that drives the generation of iT cells remains unclear. Here, we report that the cytokine receptor γc plays a previously unappreciated role for thymic iT cells by controlling their cellular abundance, lineage commitment, and subset differentiation. As such, γc overexpression on thymocytes dramatically altered iT cell generation in the thymus, as it skewed the subset composition of invariant NKT (iNKT) cells and promoted the generation of IFNγ-producing innate CD8 T cells. Mechanistically, we found that the γc-STAT6 axis drives the differentiation of IL-4-producing iNKT cells, which in turn induced the generation of innate CD8 T cells. Collectively, these results reveal a cytokine-driven circuity of thymic iT cell differentiation that is controlled by the abundance of γc proteins.
... First, the increased abundance of IL-2Rb proteins in IL-2Rb Tg mice could increase the binding and consumption of IL-15, thus diminishing the availability of IL-15 for iNKT cell development. IL-15 is critical for the generation of iNKT cells in general and specifically for NKT1 cells (11,36). Consequently, the detrimental effect of IL-2Rb Tg on iNKT cells could have been due to insufficient IL-15 availability, partly phenocopying the effect of IL-15-deficiency (37). ...
Invariant NKT (iNKT) cells are thymus-generated innate-like T cells, comprised of three distinct subsets with divergent effector functions. The molecular mechanism that drives the lineage trifurcation of immature iNKT cells into the NKT1, NKT2, and NKT17 subsets remains a controversial issue that remains to be resolved. Because cytokine receptor signaling is necessary for iNKT cell generation, cytokines are proposed to contribute to iNKT subset differentiation also. However, the precise roles and requirements of cytokines in these processes are not fully understood. Here, we show that IL-2Rβ, a nonredundant component of the IL-15 receptor complex, plays a critical role in both the development and differentiation of thymic iNKT cells. While the induction of IL-2Rβ expression on postselection thymocytes is necessary to drive the generation of iNKT cells, surprisingly, premature IL-2Rβ expression on immature iNKT cells was detrimental to their development. Moreover, while IL-2Rβ is necessary for NKT1 generation, paradoxically, we found that the increased abundance of IL-2Rβ suppressed NKT1 generation without affecting NKT2 and NKT17 cell differentiation. Thus, the timing and abundance of IL-2Rβ expression control iNKT lineage fate and development, thereby establishing cytokine receptor expression as a critical regulator of thymic iNKT cell differentiation.
... IL-2Rb is a receptor subunit shared by IL-2 and IL-15, and it is essential for the ligand binding and signal transduction of both cytokines (45). During T cell development in the thymus, IL-2Rb is mostly absent on immature thymocytes, but it is present on postselection thymocytes, such as invariant NKT cells, CD8 T cells, and Foxp3 + Treg cells (43,51). The molecular mechanisms that control IL-2Rb expression have been extensively investigated (45), and multiple transcription factors, such as Ets1, T-bet, and eomesodermin, were found to induce its expression on T cells (52,53). ...
Lymphopenia-induced homeostatic proliferation (LIP) is a critical mechanism for restoring T cell immunity upon lymphodepleting insults or infections. LIP is primarily driven by homeostatic cytokines, such as IL-7 and IL-15, but not all T cells respond with the same efficiency to homeostatic proliferative cues. Although CD8 T cells vigorously proliferate under lymphopenic conditions, naive CD4 T cells are substantially impaired in their response to homeostatic cytokines, and they fail to fully expand. In this study, we show that the availability of IL-2Rβ (CD122), which is a receptor subunit shared by IL-2 and IL-15, affects both the cytokine responsiveness and the LIP of naive CD4 T cells in the mouse. The enumeration of surface IL-2Rβ molecules on murine naive CD4 and naive CD8 T cells revealed a 5-fold difference in IL-2Rβ abundance. Notably, it was the limited availability of IL-2Rβ that impaired CD4 T cell responsiveness to IL-15 and suppressed their LIP. As such, forced IL-2Rβ expression on CD4 T cells by transgenesis bestowed IL-15 responsiveness onto naive CD4 T cells, which thus acquired the ability to undergo robust LIP. Collectively, these results identify IL-2Rβ availability as a new regulatory mechanism to control cytokine responsiveness and the homeostatic proliferation of murine CD4 T cells.
... were from BD Biosciences; promyelocytic leukemia zinc finger (PLZF; 9E12) was from BioLegend (San Diego, CA, USA). CD1d tetramers loaded with PBS-57 and unloaded controls were obtained from the NIH tetramer facility (Emory University, Atlanta, GA, USA) and used to identify thymic iNKT cells as previously described (24). ...
Invariant NKT (iNKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of iNKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich iNKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched iNKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24⁺ immature thymocytes as an alternative method to enrich iNKT cells. We found that the overall recovery in iNKT cell numbers did not differ between these 2 methods. However, enrichment by CD24⁺ cell depletion preserved the subset composition of iNKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic iNKT cells in further detail.
... sγc interferes with IL-2 and IL-15 signaling in CD8 + T cells (6,35,36). Thus, we assessed the effect of siAp8 on IL-2 or IL-15 signaling in CD8 + T cells with anti-apoptotic Bcl-2 expression (Figures 4A,B). Bcl-2 expression upregulated by IL-2 ( Figure 4A) or IL-15 ( Figure 4B) was decreased in the presence of sγc and was rescued by siAp8 treatment (Figures 4A,B). ...
IL-17 produced by Th17 cells has been implicated in the pathogenesis of rheumatoid arthritis (RA). It is important to prevent the differentiation of Th17 cells in RA. Homodimeric soluble γc (sγc) impairs IL-2 signaling and enhances Th17 differentiation. Thus, we aimed to block the functions of sγc by inhibiting the formation of homodimeric sγc. The homodimeric form of sγc was strikingly disturbed by sγc-binding DNA aptamer. Moreover, the aptamer effectively inhibited Th17 cell differentiation and restored IL-2 and IL-15 signaling impaired by sγc with evidences of increased survival of T cells. sγc was highly expressed in SF of RA patients and increased in established CIA mice. The therapeutic effect of PEG-aptamer was tested in CIA model and its treatment alleviated arthritis pathogenesis with impaired differentiation of pathogenic Th17, NKT1, and NKT17 cells in inflamed joint. Homodimeric sγc has pathogenic roles to exacerbate RA progression with differentiation of local Th17, NKT1, and NKT17 cells. Therefore, sγc is suggested as target of a therapeutic strategy for RA.
... Recently, we discovered sγc that is generated by alternative splicing of the γc pre-mRNA and that is highly released by activated T cells, negatively regulating γc cytokine signaling [13,14], resulting in the modulation of T-cell homeostasis and differentiation. It is interesting to speculate on whether sγc overexpression would ameliorate the development of IL-7-mediated lymphoproliferative disorders (LPDs) through the dampening of IL-7 signaling [15]. ...
... In previous reports, we demonstrated that sγc regulates γc cytokine signaling with the generation of sγc-overexpressing transgenic (sγcTg) mice [13,14,16]. We found with in vitro and in vivo analysis of γc cytokine responsiveness that an elevated level of sγc dramatically dampens IL-2, IL-7, and IL-15 signaling. ...
... Since homeostatic expansion of nonconventional T cells, such as NKT and γδT cells, is dependent on IL-7 and IL-15 [21][22][23][24][25], we next analyzed the role of sγc in their proliferation. Consistent with our previous studies [14], we found that an elevated level of sγc reduces the frequency and numbers of NKT cells (Figure 4a,b). Interestingly, while significant reduction of NKT cell frequency was observed in IL-7Tg mice (Figure 4a), implying that is due to relative reduction by increased frequency of conventional T cells, NKT cell numbers in IL-7Tg were comparable to WT mice (Figure 4b). ...
IL-7 is an essential, nonredundant growth factor for T and B cell generation and maintenance. While IL-7 deficiency results in lymphopenia, overexpression of IL-7 can cause neoplasia in experimental models. IL-7’s involvement in neoplasia has been appreciated through studies of IL-7 transgenic (Tg) mice models and human lymphoma patients. Since we recently found that a soluble form of the common γ-chain (γc) cytokine receptor (sγc) antagonistically regulates IL-7 signaling, IL-7 and sγc double-Tg mice were generated to investigate the effects of sγc overexpression in IL-7-mediated lymphoproliferative disorders (LPDs). The overexpression of sγc prevents IL-7Tg-induced abnormal increase of LN cell numbers and the development of splenomegaly, resulting in striking amelioration of mortality and disease development. These results suggest that modification of γc cytokine responsiveness by sγc molecules might control various γc cytokine-associated hematologic malignancy, and also provide an alternative view to approach antitumor therapy.
... The abundance of c at the cell surface is markedly reduced on preselection DP thymocytes, and it is reinduced at the cell surface only upon positive selection (7,16). This reduction in c abundance on DP cells is specific to c because other cytokine receptors, such as IL-4R and IL-21R, are found in large amounts on DP thymocytes before positive selection (19)(20)(21)(22). Thus, the reduction in c abundance is a developmentally controlled event, but the molecular basis for such transient loss of c from developing thymocytes is unclear. ...
... Consequently, several mechanisms act in a redundant manner to insulate preselection thymocytes from exposure to indiscriminate prosurvival cytokine signaling. In the mouse thymus, these mechanisms include reductions in the amounts of c and IL-7R proteins (7,20,45), an increase in the abundance of suppressor of cytokine signaling 1 (46), and a paucity of IL-7 in the thymic cortex (47,48). Whereas apoptosis and the removal of nonselected DP thymocytes are essential for thymic selection, it is also important that preselection thymocytes can survive long enough to be tested for their reactivity to self-MHC. ...
... However, few studies about Il2rg mRNA expression during T cell differentiation have been reported. We previously found a posttranscriptional mechanism that suppresses Il2rg expression upon T cell activation and during T cell development in the thymus (20,25). We found that Il2rg pre-mRNA transcripts can be alternatively spliced into a new splice isoform that lacks the transmembrane domain and results in the production of sc at the expense of membrane c receptors. ...
The cytokine receptor subunit γc provides critical signals for T cell survival and differentiation. We investigated the molecular mechanism that controls the cell surface abundance of γc during T cell development in the thymus. We found that the amount of γc was low on CD4⁺CD8⁺ double-positive (DP) thymocytes before their positive selection to become mature T cells. The transcription factor RORγt was abundant in immature DP thymocytes, and its loss resulted in an increase in the abundance of surface γc, specifically on preselection DP cells. Rather than directly repressing expression of the gene encoding γc, RORγt acted through the antiapoptotic protein Bcl-xL to reduce the abundance of surface γc, which resulted in decreased cytokine signaling and was associated with inhibition of cell metabolism and mitochondrial biogenesis. Accordingly, overexpression of Bcl-xL in RORγt-deficient thymocytes restored the amount of surface γc to that present on normal preselection DP cells. Together, these data highlight a previously unappreciated role for RORγt and Bcl-xL in limiting γc abundance at the cell surface and reveal a signaling circuit in which survival factors control cytokine signaling by limiting the abundance and surface distribution of a receptor subunit shared by several cytokines.
... [6][7][8][9] sgc is generated via alternative splicing of gc mRNA, which can then be translated to a truncated form of membrane gc (mgc), 4 and the secreted sgc protein forms a homodimeric structure that binds directly to IL-2Rb, another subunit of the cytokine receptors, 10 resulting in the inhibition of IL-2 and IL-15 signaling. 4,5,11 The proliferation of na€ ıve CD8 1 T cells that is responsible to IL-2 unlike CD4 1 T cells were significantly inhibited by sgc. 4 In previous report, however, whether sgc produced during TCR-stimulation regulates the functional properties of activated CD8 1 T cells has not been determined. ...
... We have previously shown an antagonistic effect of sgc on IL-2 and IL-15 signaling. 4,5 Consistent with this, we found that sgc dampened IL-2 and IL-15 signaling and induced the suboptimal effector functions and survival of CD8 1 T cells by showing that sgc could inhibit the function of TCR stimulated CD8 1 T cells, not only by significantly reducing the production of Th1 cytokine IFNg but also by inflammatory cytokines TNFa and granzyme B; sgc deficient CD8 1 T cells demonstrated superior anti-tumor responses and persistence in recipient mice. ...
... sgc has previously been shown to skew CD4 1 T cells to Th17 by dampening IL-2 signals 4 and suppress NKT cell development by downregulating of IL-15 signals. 5 Here, we have confirmed these previous study findings, and went a step further by showing that sgc deficiency could augment IFNg, TNFa and ganzyme B expressions of activated CD8 1 T cells. In addition to increased cytokine production, we have also shown that sgc-deficient antigen specific CD8 1 T cells demonstrate in vitro and in vivo superior cytotoxicity compared with WT or sgc-overexpression CD8 1 T cells. ...
Previous studies have shown that soluble common γ‐chain (sγc) modulates CD4⁺ T cell immunity with antagonistic functions in γc cytokine signaling. However, the role of sγc in functional properties of effector CD8⁺ T cells has not been fully defined. In this study, we report a new mechanism by which the anti‐tumor activity of mouse CD8⁺ T cells is suppressed in sγc of their own producing. While sγc significantly inhibits cytotoxicity of CD8⁺ T cells, blocking sγc production by genetic modification leads to potentiated effector function of CD8⁺ T cells, establishing persistent CD8⁺ T cells. This is due to the modulation of IL‐2 and IL‐15 signaling, which is required for expansion and survival of CD8⁺ T cells as well as for optimal cytotoxic activity. More efficient management of tumor growth was achieved by an adoptive transfer of sγc‐deficient CD8⁺ T cells than that of wild‐type or sγc‐overexpressing CD8⁺ T cells. Blocking of IL‐2 and IL‐15 signaling by sγc attenuates the capacity of CD8⁺ T cells to mount an optimal response to the tumor, with both quantitative and qualitative effects on antigen‐specific CD8⁺ T cells. These results could have a critical implication for the generation and survival of optimal effector T cells for adoptive immunotherapy of cancer. This article is protected by copyright. All rights reserved.
... In the thymus, IL-15 expression contributes to the development and cytotoxic features of CD8 T cells (13,14), and it is also critical for iNKT cell development and differentiation (46)(47)(48). Based on studies of thymic iNKT cell development, it was assumed that medullary epithelial cells would be the source of intrathymic IL-15, since the medulla is the site of final maturation of iNKT cells which requires IL-15 (49,50). ...
... Thus, our results do not support a T cell-autocrine effect of IL-15 in effector T cell generation and differentiation (Fig. 1) It is currently not clear to us why experiments using the same IL-15-deficient CD4 T cells would show different efficiencies of Th17 cell differentiation (23,25). As a potential explanation, we consider that Il15 −/− CD4 T cells are generated and maintained in an IL-15-deficient environment that lack pro-inflammatory NK and iNKT cells (26,48,77). Consequently, the activation status and phenotype of IL-15-deficient naïve CD4 T cells might differ from those of wildtype naïve CD4 T cells depending on the housing environment of Il15 −/− mice. ...
IL-15 is a cytokine of the common γ-chain family that is critical for natural killer (NK), invariant natural killer T (iNKT), and CD8 memory T cell development and homeostasis. The role of IL-15 in regulating effector T cell subsets, however, remains incompletely understood. IL-15 is mostly expressed by stromal cells, myeloid cells, and dendritic cells (DCs). Whether T cells themselves can express IL-15, and if so, whether such T cell-derived IL-15 could play an autocrine role in T cells are interesting questions that were previously addressed but answered with mixed results. Recently, three independent studies described the generation of IL-15 reporter mice which facilitated the identification of IL-15-producing cells and helped to clarify the role of IL-15 both in vitro and in vivo. Here, we review the findings of these studies and place them in context of recent reports that examined T cell-intrinsic IL-15 expression during CD4 effector T cell differentiation.
... 21 We recently reported that the soluble form of common gamma chain (sγc), generated by alternative splicing, regulates T cell response and survival with an antagonistic effect in γc cytokine signaling. 22,23 The inhibitory function of soluble common gamma chain (sγc) in γc cytokine signaling exacerbated the inflammation by promoting the differentiation of pathogenic Th17 cells both in vitro and in vivo. 22 Since COPD is developed with T cell-mediated immunopathogenesis by CS, 24 sγc would be involved in the progression of diseases such as COPD. ...
Cigarette smoking (CS) is a major cause of considerable morbidity and mortality by inducing lung cancer and COPD. COPD, a smoking-related disorder, is closely related to the alteration of immune system and inflammatory processes that are specifically mediated by T cells. Soluble common gamma chain (sγc) has recently been identified as a critical regulator of the development and differentiation of T cells. We examined the effects of sγc in a cigarette smoke extract (CSE) mouse model. The sγc level in CSE mice serum is significantly downregulated, and the cellularity of lymph node (LN) is systemically reduced in the CSE group. Overexpression of sγc enhances the cellularity and IFNγ production of CD8 T cells in LN and also enhances Th1 and Th17 differentiation of CD4 T cells in the respiratory tract. Mechanistically, the downregulation of sγc expression mediated by CSE is required to prevent excessive inflammatory T cell responses. Therefore, our data suggest that sγc may be one of the target molecules for the control of immunopathogenic progresses in COPD.
