Interleukin‐4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γ_C) cytokines decrease CD127 expression on CD4⁺ and CD8⁺ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8⁺ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA⁺) CD8⁺ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8⁺ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8⁺ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8⁺ T cells, IL‐4 is a potential contributor to impaired CD8⁺ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.     

Notch ligands potentiate IL‐7‐driven proliferation and survival of human thymocyte precursors

Notch and IL‐7 are both well‐characterized factors involved in T‐cell development. In contrast to the mouse model, their precise requirements in the differentiation and/or proliferation of various stages of human thymic development have not been fully explored. Here, we demonstrate that IL‐7 alone is sufficient to induce the differentiation of ex vivo purified CD34⁺ triple negative (TN) surface (s) CD3^? CD4^?CD8^? (CD3^?CD4^?CD8^?), CD4 immature single positive (ISP) (sCD3^?CD4⁺CD8^?) and double positive (DP) (sCD3^?CD4⁺CD8⁺) human thymic precursors to mature DP expressing sCD3 (sCD3⁺CD4⁺CD8⁺). We show that activation of Notch signaling by its ligands Delta‐1 or Delta‐4 potentiates IL‐7‐driven proliferation and survival of CD34⁺ TN and to a lesser extent of CD4⁺ ISP precursors. This effect of Notch is related to a sustained induction of IL‐7 receptor α chain expression on thymocytes through a decreased methylation of its gene promoter. Thus, we show here that proliferation and differentiation of T‐cell precursors are differentially modulated by IL‐7 depending on the presence or absence of external signals. These results may have important implications for the clinical use of this cytokine as a strategy aimed at improving immune restoration.     

Enhanced suppressor function of TIM‐3+FoxP3+ regulatory T cells

T‐cell immunoglobulin and mucin domain 3 (TIM‐3) is an Ig‐superfamily member expressed on IFN‐γ‐secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM‐3 is expressed by a subset of activated CD4⁺ T cells, and anti‐CD3/anti‐CD28 stimulation increases both the level of expression and the number of TIM‐3⁺ T cells. In mice, TIM‐3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4⁺ T‐cell differentiation. Here, we examined TIM‐3 expression on human Treg cells to determine its role in T‐cell suppression. In contrast to mice, TIM‐3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM‐3⁺ Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM‐3^? Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT‐3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM‐3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1‐ and Th17‐cell responses.     

Gads‐deficient thymocytes are blocked at the transitional single positive CD4+ stage

Positive selection of T‐cell precursors is the process by which a diverse T‐cell repertoire is established. Positive selection begins at the CD4⁺CD8⁺ double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down‐regulate CD8 to become transitional single positive (TSP) CD4⁺ thymocytes. Then, cells are selected to become either mature single positive CD4⁺ or mature single positive CD8⁺ thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads‐deficient mouse line. In Gads^+/+ mice, most TSP CD4⁺ thymocytes are TCR^hiBcl‐2^hiCD69⁺, suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads^?/? mice could readily generate TSP CD4⁺ thymocytes, many Gads^?/? TSP CD4⁺ cells were TCR^loBcl‐2^loCD69^?, suggesting that Gads^?/? cells proceeded to the TSP CD4⁺ stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4⁺ thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4⁺ stage are separable events and Gads is only required for positive selection.     

1α,25‐dihydroxyvitamin D3 acts via transforming growth factor‐β to up‐regulate expression of immunosuppressive CD73 on human CD4+ Foxp3– T cells

Vitamin D deficiency is associated with increased incidence and severity of various immune‐mediated diseases. Active vitamin D (1α,25‐dihydroxyvitamin D3; 1,25(OH)₂D3) up‐regulates CD4⁺ T‐cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti‐inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)₂D3 on expression of the downstream ecto‐5′‐nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor‐β (TGF‐β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10^?8 to 10^?7 m , 1,25(OH)₂D3 significantly increased expression of CD73 on peripheral human CD4⁺ T cells. Although 1,25(OH)₂D3 did not affect the mRNA expression of latent TGF‐β₁, 1,25(OH)₂D3 did up‐regulate expression of TGF‐β‐associated molecules [latency‐associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin‐1, thrombospondin‐1 and α_v integrin] which is likely to have contributed to the observed enhancement in TGF‐β bioactivity. CD73 was highly co‐expressed with LAP and GARP following 1,25(OH)₂D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4⁺ Foxp3^– T cells, rather than CD4⁺ Foxp3⁺ T cells. Notably, neutralization of TGF‐β significantly impaired 1,25(OH)₂D3‐mediated induction of CD73. Collectively, we show that 1,25(OH)₂D3 enhances expression of CD73 on CD4⁺ Foxp3^– T cells in a process that is at least partially TGF‐β‐dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune‐mediated disease.