The role of antigenic peptide in CD4+ T helper phenotype development in a T cell receptor transgenic model

CD4+ Th1 cells play a critical role in the induction of cell-mediated immune responses that are important for the eradication of intracellular pathogens. Peptide-25 is the major Th1 epitope for Ag85B of Mycobacterium tuberculosis and is immunogenic in I-Ab mice. To elucidate the role of the TCR and IFN-gamma/IL-12 signals in Th1 induction, we generated TCR transgenic mice (P25 TCR-Tg) expressing TCR alpha- and beta-chains of Peptide-25-reactive cloned T cells and analyzed Th1 development of CD4+ T cells from P25 TCR-Tg. Naive CD4+ T cells from P25 TCR-Tg differentiate into both Th1 and Th2 cells upon stimulation with anti-CD3. Naive CD4+ T cells from P25 TCR-Tg preferentially develop Th1 cells upon Peptide-25 stimulation in the presence of I-Ab splenic antigen-presenting cells under neutral conditions. In contrast, a mutant of Peptide-25 can induce solely Th2 differentiation. Peptide-25-induced Th1 differentiation is observed even in the presence of anti-IFN-gamma and anti-IL-12. Furthermore, naive CD4+ T cells from STAT1 deficient P25 TCR-Tg also differentiate into Th1 cells upon Peptide-25 stimulation. Moreover, Peptide-25-loaded I-Ab-transfected Chinese hamster ovary cells induce Th1 differentiation of naive CD4+ T cells from P25 TCR-Tg in the absence of IFN-gamma or IL-12. These results imply that interaction between Peptide-25/I-Ab and TCR may primarily influence determination of the fate of naive CD4+ T cells in their differentiation towards the Th1 subset.     

4-1BB triggers IL-13 production from T cells to limit the polarized, Th1-mediated inflammation

4-1BB (CD137) triggering typically induces Th1 response by increasing IFN-gamma from T cells upon TCR ligation. We found recently that 4-1BB costimulation increased the expression of IL-13 from CD4(+) T cells, as well as CD8(+) T cells. The enhanced IL-13 expression by agonistic anti-4-1BB treatment was mediated via MAPK1/2, PI-3K, JNK, mammalian target of rapamycin, NF-AT, and NF-kappaB signaling pathways. The signaling for IL-13 induction was similar to that of IFN-gamma production by anti-4-1BB treatment in T cells. When the anti-4-1BB-mediated IL-13 expression was tested in an in vivo viral infection model such as HSV-1 and vesicular stomatitis virus, 4-1BB stimulation enhanced IL-13 expression of CD4(+) T, rather than CD8(+) T cells. Although IL-13 was enhanced by anti-4-1BB treatment, the increased IL-13 did not significantly alter the anti-4-1BB-induced Th1 polarization of T cells--increase of T-bet and decrease of GATA-3. Nevertheless, anti-4-1BB treatment polarized T cells excessively in the absence of IL-13 and even became detrimental to the mice by causing liver inflammation. Therefore, we concluded that IL-13 was coinduced following 4-1BB triggering to maintain the Th1/2 balance of immune response.     

Dendritic cells generated in the presence of interferon-alpha stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells

Dendritic cells (DCs) generated in the presence of IFN-alpha (IFN-DCs) exhibit high expression of major histocompatibility and co-stimulatory molecules and a potent ability to stimulate CD8(+) T-cell responses. Here, we found that IFN-DCs were more potent stimulators of bulk and purified CD8(+) T-cell proliferation, as compared with IL-4-DCs. In contrast, IFN-DCs were less efficient than IL-4-DCs in stimulating allogeneic CD4(+) T-cell proliferation, due to a weak induction of naive CD4(+)CD45RO(-) T-cell proliferation by these DCs. However, both DC populations induced similar levels of proliferation of memory CD4(+)CD45RO(+) T cells. IFN-DCs and IL-4-DCs exhibited a similar phenotype and production of IL-10 following maturation induced by CD40 ligation. In contrast, IFN-DCs produced higher levels of IL-10 during the first days of differentiation. In addition, neutralization of IL-10 during the differentiation of DCs increased the expression of DC-LAMP and MHC class II by IFN-DCs, and the ability of IFN-DCs to stimulate allogeneic CD4(+) T-cell proliferation at similar levels, than IL-4-DCs. Independently of IL-10 production, IFN-DCs were found to induce higher levels of CD4(+)T-cell apoptosis, this effect being more sticking on naive T cells. Finally, we demonstrated that IFN-DCs induced a differentiation bias of naive CD4(+) T cells towards Th1 and Tr1 cells, compared to IL-4-DCs. Taken together, these results indicate that, despite the induction of Tr1 cells and enhanced apoptosis of naive CD4(+) T cells, IFN-DCs are potent stimulators of CD8(+) and memory CD4(+) T cells, and induce a strong polarization of naive CD4(+) T cells towards Th1 cells, further supporting their use in immune-based therapy.     

CD38 identifies a hypo-proliferative IL-13-secreting CD4+ T-cell subset that does not fit into existing naive and memory phenotype paradigms

CD38 is commonly regarded as an activation marker for human T cells. Herein, we show that CD38 expression identifies a hypo-proliferative CD4(+) T-cell subset that, following TCR stimulation, retains expression of naive cell surface markers including CD45RA, CD62L and CCR7. Hypo-proliferation was mediated by reduced CD25 up-regulation upon TCR stimulation compared to CD4(+) CD38(-) cells and lack of responsiveness to exogenous IL-2. Instead, CD4(+) CD38(+) T cells expressed CD127, and hypo-proliferation was reversed by addition of IL-7, further associated with increased STAT5 phosphorylation. This phenotype was exacerbated by addition of an agonistic CD38-binding antibody, suggesting that signaling through CD38 promotes this cell profile. Activated CD4(+) CD38(+) cells had a bias towards IL-13 secretion, but not other Th2 cytokines such as IL-4 or IL-5. In comparison, the CD4(+) CD38(-) cells had a clear bias towards secretion of Th1-associated cytokines IFN-γ and TNF. The existence of such CD4(+) CD38(+) T cells may play an important role in pathologies such as asthma, which are associated with IL-13, but not IL-4 and IL-5. Coupled with responsiveness to IL-7 but not IL-2, and the involvement of CD38 ligation, our results highlight a unique T-cell subpopulation that does not fit into existing naive and memory cell paradigms.     

T-cell receptor antagonist modifies cytokine secretion profile of naive CD4+ T cells and their differentiation into type-1 and type-2 helper T cells

A T-cell receptor (TCR) antagonist is an analog of a peptide ligand for TCR that inhibits T-cell responses to the original peptide. We investigated the effects of a TCR antagonist on cytokine secretion of naive CD4+ T cells and their differentiation into type-1 and type-2 helper T cells (Th1 and Th2) induced by stimulation with varying doses of an antigenic peptide. In the presence of a TCR antagonist peptide, proliferation of naive CD4+ T cells and antigen dose-dependent secretion of interferon-gamma, a typical Th1-type cytokine, by these cells was down-regulated. With respect to the secretion of interleukin-4 (IL-4), a typical Th2-type cytokine, the TCR antagonist raised the concentration of the antigenic peptide required to elicit maximal IL-4 production and, surprisingly, significantly increased the maximum level of IL-4 secretion. Similar effects induced by the TCR antagonist were observed on the Th1/Th2 differentiation of naive CD4+ T cells. These results clearly indicate that, for naive CD4+ T cells, a TCR antagonist has the potential to change the balance of Th1/Th2 cytokine secretion and even enhance Th2 responses.     

IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells.

Interleukin-27 (IL-27) supports proliferation of naive CD4(+) T cells and enhances interferon-gamma (IFN-gamma) production by activated T cells and natural killer (NK) cells. We report here that IL-27 induces Stat1 and Stat3 phosphorylation and activation in human and murine cell lines and primary human T cells. IL-27 also induces T-Bet, a Stat1-dependent gene crucial to Th1 cell commitment. Similarly, IFN-alpha activates Stat1 and Stat3 and T-Bet expression in naive T cells. Induction of T-Bet results in upregulation of IL-12Rbeta2 on naive T cells, which is essential for responsiveness to IL-12 and differentiation to a Th1 phenotype. Both IL-27 and IFN-alpha induce expression of IL-12Rbeta2 in T cells. In contrast, IFN-gamma, which activates Stat1 but not Stat3, induces expression of T-Bet but not IL-12Rbeta2 in naive T cells. We propose that IL-27 and IFN-alpha are important for early Th1 commitment and act upstream of IL-12 and IFN-gamma in this pathway.     

Impaired IL-4 production by CD8+ T cells in NOD mice is related to a defect of c-Maf binding to the IL-4 promoter

CD8(+) T cells play an important role in the induction of the autoimmune response in non-obese diabetic (NOD) mice. Here we describe abnormalities in the control of cytokine production by NOD CD8(+) T cells. NOD CD8(+) T cells had an increased propensity to produce IFN-gamma upon TCR activation, in both adult and 2-week-old mice. NOD CD8(+) T cells had a reduced capacity to produce IL-4 in type 2 conditions compared to CD8(+) T cells from the diabetes-resistant strains BALB/c and C57BL/6. Both GATA-3 and c-Maf, two positive transactivators for IL-4 gene expression, were expressed in type 2 conditions at comparable levels in NOD CD8(+) T cells. The GATA-3 was functional since normal levels of IL-5 were produced and the IL-4 promoter was hyperacetylated in NOD CD8(+) T cells. In contrast, c-Maf failed to bind to its responsive element as determined by chromatin immunoprecipitation (ChIP) assay. These results suggest that NOD CD8(+) T cells possess an increased propensity to produce IFN-gamma and impaired c-Maf-dependent DNA binding activities in vivo that lead to reduced IL-4 production following TCR activation. These defects may facilitate the development of the autoimmune response by inducing an overall type 1-biased immune response in NOD mice.     

Notch1 expression on T cells is not required for CD4+ T helper differentiation

Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.     

Th2 cells shape the differentiation of developing T cell responses during interactions with dendritic cells in vivo

During priming, naive CD4(+) Th cells differentiate into cells that produce either IFN-gamma or IL-4. Even though the cascade of pathways that induces IL-4-producing Th2 cells has been determined in vitro, the signals promoting Th2 differentiation under physiological conditions remain enigmatic, especially the natural role of the single most important Th2-inducing signal,IL-4. Using Th2 and naive Th cells, each expressing a distinct transgenic TCR, here we show that Th2 cells migrate with the same dynamics as naive Th cells in draining lymph nodes and bind to the same DC, when driven by antigen in complete Freund's adjuvant (CFA). Th2-cell-derived IL-4 deviates CFA-induced Th1 development toward a Th2 phenotype, if both cell populations co-localize in the same T cell area, and are activated simultaneously. Thus, intranodal Th2 cells directly influence Th cell differentiation in vivo, but only under restricted conditions. These findings have implications for the design of cytokine-based therapies and explain the spreading of Th2 responses to multiple aeroallergens in allergic asthma, where naive Th and Th2 cells co-localize in lung-draining lymph nodes.     

Stat5 activation plays a critical role in Th2 differentiation

Upon TCR engagement, naive CD4 T cells differentiate toward the Th1 or Th2 phenotype. IL-4, acting through Stat6, plays a major role in Th2 differentiation; IL-2 has also been reported to be essential. Here, we report that retroviral (RV)-mediated expression of a constitutively active Stat5A mutant (STAT5A1*6) can fully restore IL-4 production when naive CD4 T cells are primed in the absence of IL-2. Furthermore, STAT5A1*6 expression causes Th2 differentiation in the absence of IL-4 or in Stat6- or IL-4Ralpha-deficient cells. Infection with STAT5A1*6-NGFR-RV does not enhance GATA-3 expression. STAT5A1*6-NGFR-RV and GATA-3-GFP-RV each render the Il4 gene accessible, but the sites of restriction enzyme accessibility are different. Stat5A binds to HSII and HSIII sites of the Il4 gene. Coinfection with STAT5A1*6-NGFR-RV and GATA-3-GFP-RV results in optimal Th2 priming.     

Host resistance and immune deviation in pigeon cytochrome c T-cell receptor transgenic mice infected with Toxoplasma gondii

Resistance to Toxoplasma gondii has been shown to be mediated by gamma interferon (IFN-gamma) produced by NK, CD4(+), and CD8(+) T cells. While studies of SCID mice have implicated NK cells as the source of the cytokine in acute infection, several lines of evidence suggest that IFN-gamma production by CD4(+) T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T-cell receptor (TCR)-dependent stimulation by the parasite, we have examined the resistance to T. gondii infection of pigeon cytochrome c transgenic (PCC-Tg) Rag-2(-/-) mice in which all CD4(+) T lymphocytes are unreactive with the protozoan. When inoculated with the ME49 strain, PCC-Tg animals exhibited only temporary control of acute infection and succumbed by day 17. Intracellular cytokine staining by flow cytometry revealed that, in contrast to infected nontransgenic controls, infected PCC-Tg animals failed to develop IFN-gamma-producing CD4(+) T cells. Moreover, the CD4(+) lymphocytes from these mice showed no evidence of activation as judged by lack of upregulated expression of CD44 or CD69. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses measured after in vitro antigen restimulation displayed a strong Th1 bias which was shown to be dependent on endogenous interleukin 12 (IL-12). The above findings argue that, while T. gondii-induced IL-12 cannot trigger IFN-gamma production by CD4(+) T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against nonparasite antigens, an effect that may explain the immunostimulatory properties of T. gondii infection.