CD98 Heavy Chain Is a Potent Positive Regulator of CD4+ T Cell Proliferation and Interferon-γ Production In Vivo

Upon their recognition of antigens presented by the MHC, T cell proliferation is vital for clonal expansion and the acquisition of effector functions, which are essential for mounting adaptive immune responses. The CD98 heavy chain (CD98hc, Slc3a2) plays a crucial role in the proliferation of both CD4⁺ and CD8⁺ T cells, although it is unclear if CD98hc directly regulates the T cell effector functions that are not linked with T cell proliferation in vivo. Here, we demonstrate that CD98hc is required for both CD4⁺ T cell proliferation and Th1 functional differentiation. T cell-specific deletion of CD98hc did not affect T cell development in the thymus. CD98hc-deficient CD4⁺ T cells proliferated in vivo more slowly as compared with control T cells. C57BL/6 mice lacking CD98hc in their CD4⁺ T cells could not control Leishmania major infections due to lowered IFN-γ production, even with massive CD4⁺ T cell proliferation. CD98hc-deficient CD4⁺ T cells exhibited lower IFN-γ production compared with wild-type T cells, even when comparing IFN-γ expression in cells that underwent the same number of cell divisions. Therefore, these data indicate that CD98hc is required for CD4⁺ T cell expansion and functional Th1 differentiation in vivo, and suggest that CD98hc might be a good target for treating Th1-mediated immune disorders.     

Helminth Infections Coincident with Active Pulmonary Tuberculosis Inhibit Mono- and Multifunctional CD4+ and CD8+ T Cell Responses in a Process Dependent on IL-10

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4⁺ and CD8⁺ T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections—Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4⁺ and CD8⁺ T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial–specific frequencies of mono- and multi–functional CD4⁺ Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4⁺ and CD8⁺ T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4⁺ T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4⁺ Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4⁺ T cell responses as well as protective systemic cytokine responses in active pulmonary TB.     

Mycobacterium tuberculosis-Specific IL-21+IFN-γ+CD4+ T Cells Are Regulated by IL-12

In the current study of Mycobacterium tuberculosis (MTB)-specific T and B cells, we found that MTB-specific peptides from early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) induced the expression of IL-21 predominantly in CD4⁺ T cells. A fraction of IL-21-expressing CD4⁺ T cells simultaneously expressed Th1 cytokines but did not secrete Th2 or Th17 cytokines, suggesting that MTB-specific IL-21-expressing CD4⁺ T cells were different from Th1, Th2 and Th17 subpopulations. The majority of MTB-specific IL-21-expressing CD4⁺ T cells co-expressed IFN-γ and IL-21⁺IFN-γ⁺CD4⁺ T cells exhibited obviously polyfunctionality. In addition, MTB-specific IL-21-expressing CD4⁺ T cells displayed a CD45RO⁺CD62L^lowCCR7^lowCD40L^highICOS^high phenotype. Bcl-6-expression was significantly higher in IL-21-expressing CD4⁺ T cells than IL-21^-CD4⁺ T cells. Moreover, IL-12 could up-regulate MTB-specific IL-21 expression, especially the frequency of IL-21⁺IFN-γ⁺CD4⁺ T cells. Taken together, our results demonstrated that MTB-specific IL-21⁺IFN-γ⁺CD4⁺ T cells from local sites of tuberculosis (TB) infection could be enhanced by IL-12, which have the features of both Tfh and Th1 cells and may have an important role in local immune responses against TB infection.     

Peripheral CD4CD8 cells are the activated T cells expressed granzyme B (GrB), Foxp3, interleukin 17 (IL-17), at higher levels in Th1/Th2 cytokines

Peripheral CD4⁺CD8⁺ T cells have been identified as a T cell subset existing in animals and humans. However, the characterization of CD4⁺CD8⁺ T cells, their relationship with T memory (T_M), T effector (T_E), Th1/Th2, Treg and Th-17, remain unclear. This study was to characterize the CD4⁺CD8⁺ T cells. The results from human subjects showed that activated T cells were CD4⁺CD8⁺ T cells, comprised CD4^hiCD8^lo, CD4^hiCD8^hi and CD4^loCD8^hi subsets. They expressed CD62L^hi/lo, granzyme B (GrB), CD25, Foxp3, interleukin 17 (IL-17) and the cytokines of both Th1 and Th2, and had cytolytic function. These findings suggested that CD4⁺CD8⁺ T cells had over-lap function while they kept diversity, and that T cells could be divided into two major populations: activated and inactivated. Hence, the hypotheses of Th1/Th2, Treg and Th-17 might reflect the positive/negative feedback regulation of immune system. When compared to GrB⁺CD62L^lo T effector (T_E) cells, GrB⁺CD62L^hi T central memory effector (T_CME) cells had a quicker response to virus without CD62L loss.     

Development of a unique epigenetic signature during in vivo Th17 differentiation

Activated naive CD4⁺ T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4⁺ T cells, Th1 and Th17 cells. We could demonstrate that naive CD4⁺ T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.     

Functional involvement of dual specificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, in the regulation of T helper cell differentiation

Naïve CD4⁺ T helper (Th) cells differentiate into distinct subsets of effector cells (Th1, Th2, Th17, and induced regulatory T cells (iTreg)) expressing different sets of cytokines upon encounter with presented foreign antigens. It has been well established that Th1/Th2 balance is critical for the nature of the following immune responses. Previous reports have demonstrated important roles of c-Jun N-terminal kinase (JNK) in Th1/Th2 balance, whereas the regulatory mechanisms of JNK activity in Th cells have not been elucidated. Here, we show that dual specificity phosphatase 16 (DUSP16, also referred to as MKP-M or MKP-7), which preferentially inactivates JNK, is selectively expressed in Th2 cells. In the in vitro differentiation assay of naïve CD4⁺ cells, DUSP16 expression is up-regulated during Th2 differentiation and down-regulated during Th1 differentiation. Chromatin immunoprecipitation revealed the increased acetylation of histone H3/H4 at the dusp16 gene promoter in CD4⁺ T cells under the Th2 condition. Adenoviral transduction of naïve CD4⁺ T cells with DUSP16 resulted in increased mRNA expression of IL-4 and GATA-3 in Th2 and decreased expression of IFNγ and T-bet in Th1 differentiation. In contrast, transduction of a dominant negative form of DUSP16 had the reverse effects. Furthermore, upon immunization, T cell-specific dusp16 transgenic mice produced antigen-specific IgG2a at lower amounts, whereas DN dusp16 transgenic mice produced higher amounts of antigen-specific IgG2a accompanied by decreased amounts of antigen-specific IgG1 and IgE than those of control mice. Together, these data suggest the functional role of DUSP16 in Th1/Th2 balance.     

The Effect of PGE2 on Murine Helper-T-Cell Lymphokines

Prostaglandin E₂ (PGE₂) has selective effects on the production of murine helper-T-cell lymphokines. PGE₂ inhibits production of the Th1-associated lymphokines IL-2 and IFN-γ, but does not inhibit production of the Th2-associated lymphokines IL-4 and IL-5. This could have been due to differences in the Th1 and Th2 cells themselves or to differences in the cytokines. To discriminate between these models we first examined the effect of PGE₂ on IL-3 production, a lymphokine produced by both Th1 and Th2 cells. IL-3 production was inhibited by PGE₂ in some cells and enhanced in others, indicating that some property of the cell was critical. However, the effect on IL-3 production did not cleanly discriminate between Th1 and Th2 cells. Second, we examined the effect of PGE₂ on lymphokine production from Th0 cells. In some cells, production of IL-2, IL-3, and IL-4 was inhibited by PGE₂. In other cells, IL-2 and IL-3 were inhibited while IL-4 production was enhanced. These data again indicated that it was a property of the T cell, not necessarily the lymphokine itself, that determined the response to PGE₂. In Th1 and Th2 clones, both the mode of primary stimulation (antigen and antigen-presenting cells or calcium ionophore) and the presence of costimulation also were found to affect the response of IL-3 production to PGE₂. Therefore, the effect of PGE₂ on lymphokine production appears to depend upon the intracellular signaling pathways that are activated within a particular T cell.     

Cytokine Gene Expression in CD4 Positive Cells of the Japanese Pufferfish,Takifugu rubripes

CD4⁺ T (Th) cells are a central component of the adaptive immune response and are divided into distinct sets based on their specific cytokine production pattern. Several reports have suggested that fish possess Th subset activity similar to that of mammals. The aim of the present study was to isolate CD4⁺ T cells from the blood of Japanese pufferfish, Fugu rubripes, and to characterize their cytokine expression profile. We produced a specific antibody against Fugu CD4 and performed cell sorting with the magnetic activated cell sorting system. Sorted Fugu CD4⁺ cells were characterized by morphology and expression analysis of cell marker genes. Fugu CD4⁺ cells expressed T-cell marker genes but not macrophage or B-cell marker genes. In addition, peripheral blood lymphocytes were stimulated with lipopolysaccharide (LPS), polycytidylic acid (polyI:C), concanavalin A (ConA) prior to sorting, and then Multiplex RT-PCR was used to examine the expression of Th cytokines by the stimulated Fugu CD4⁺ cells. LPS and polyI:C stimulation upregulated the expression of Th1, Th17 and Treg cytokines and downregulated the expression of Th2 cytokines. ConA stimulation upregulated the expression of all Th cytokines. These results suggest that fish exhibit the same upregulation of Th-specific cytokine expression as in mammals.     

Use of Antiviral T-Lymphocyte Clones to Characterize Antigen Presentation and T-Lymphocyte Subsets

The ability to isolate homogeneous populations of antiviral T lymphocytes from immune mice has led to insight into a variety of areas in cellular immunology. It has permitted the characterization of the distinct pathways of antigen processing and presentation to CD8⁺, Class I MHC-restricted and CD4⁺, Class II MHC-restricted cytolytic T lymphocytes as well as the identification of antigenic epitopes for T lymphocytes. In addition,in vivoeffector function of CD8⁺and CD4⁺cytolytic T-cell clones in protection from lethal viral pneumonia in a murine model of influenza virus infection has been demonstrated. Since the identification of CD4⁺T-lymphocyte helper subsets based on the lymphokine profiles of clonal populations, much interest has been focused on the role of specific cytokines in ultimately determining the effector functions of those cells. The protocol presented in this paper has been used to isolate Th1 and Th2 clones in a viral infectious disease model that has enabled us to further investigate the role of specific cytokines in controlling viral infection.     

High Peripheral Blood Th17 Percent Associated with Poor Lung Function in Cystic Fibrosis

People with cystic fibrosis (CF) have been reported to make lung T cell responses that are biased towards T helper (Th) 2 or Th17. We hypothesized that CF-related T cell regulatory defects could be detected by analyzing CD4⁺ lymphocyte subsets in peripheral blood. Peripheral blood mononuclear cells from 42 CF patients (6 months–53 years old) and 78 healthy controls (2–61 years old) were analyzed for Th1 (IFN-γ⁺), Th2 (IL-4⁺), Th17 (IL-17⁺), Treg (FOXP3⁺), IL-10⁺ and TGF-β⁺ CD4⁺ cells. We observed higher proportions of Treg, IL-10⁺ and TGF-β⁺ CD4⁺ cells in CF adults (≥ 18 years old), but not children/adolescents, compared with controls. Within the CF group, high TGF-β⁺% was associated with chronic Pseudomonas aeruginosa lung infection (p < 0.006). We observed no significant differences between control and CF groups in the proportions of Th1, Th2 or Th17 cells, and no association within the CF group of any subset with sex, CFTR genotype, or clinical exacerbation. However, high Th17% was strongly associated with poor lung function (FEV1 % predicted) (p = 0.0008), and this association was strongest when both lung function testing and blood sampling were performed within one week. Our results are consistent with reports of CF as a Th17 disease and suggest that peripheral blood Th17 levels may be a surrogate marker of lung function in CF.     

Profiling calcium signals of in vitro polarized human effector CD4+ T cells

Differentiation of naïve CD4⁺ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca²⁺ signals, mainly mediated by the store operated Ca²⁺ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4⁺ T cell subsets show differential Ca²⁺ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4⁺ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca²⁺ release activated Ca²⁺ currents (I_CRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca²⁺ profiles of helper CD4⁺ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4⁺ T cells.     

Th Cells Promote CTL Survival and Memory via Acquired pMHC-I and Endogenous IL-2 and CD40L Signaling and by Modulating Apoptosis-Controlling Pathways

Involvement of CD4⁺ helper T (Th) cells is crucial for CD8⁺ cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4⁺ Th’s signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4⁺ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4⁺ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K^b/OVA_257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4⁺ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4⁺ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4⁺ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.     

Modeling anti-tumor Th1 and Th2 immunity in the rejection of melanoma

Recent experiments indicate that CD4⁺ Th2 cells can reject skin tumors in mice, while CD4⁺ Th1 cells cannot ( [Mattes et al., 2003] and [Zhang et al., 2009]). These results are surprising because CD4⁺ Th1 cells are typically considered to be capable of tumor rejection. We used mathematical models to investigate this unexpected outcome. We found that neither CD4⁺ Th1 nor CD4⁺ Th2 cells could eliminate the cancer cells when acting alone, but that tumor elimination could be induced by recruitment of eosinophils by the Th2 cells. These recruited eosinophils had unexpected indirect effects on the decay rate of type 2 cytokines and the rate at which Th2 cells are inactivated through interactions with cancer cells. Strikingly, the presence of eosinophils impacted tumor growth more significantly than the release of tumor-suppressing cytokines such as IFN-γ and TNF-α. Our simulations suggest that novel strategies to enhance eosinophil recruitment into skin tumors may improve cancer immunotherapies.     

Hyperreactive Onchocerciasis is Characterized by a Combination of Th17-Th2 Immune Responses and Reduced Regulatory T Cells

Clinical manifestations in onchocerciasis range from generalized onchocerciasis (GEO) to the rare but severe hyperreactive (HO)/sowda form. Since disease pathogenesis is associated with host inflammatory reactions, we investigated whether Th17 responses could be related to aggravated pathology in HO. Using flow cytometry, filarial-specific cytokine responses and PCR arrays, we compared the immune cell profiles, including Th subsets, in individuals presenting the two polar forms of infection and endemic normals (EN). In addition to elevated frequencies of memory CD4⁺ T cells, individuals with HO showed accentuated Th17 and Th2 profiles but decreased CD4⁺CD25^hiFoxp3⁺ regulatory T cells. These profiles included increased IL-17A⁺, IL-4⁺, RORC2⁺ and GATA3⁺CD4⁺ T cell populations. Flow cytometry data was further confirmed using a PCR array since Th17-related genes (IL-17 family members, IL-6, IL-1β and IL-22) and Th2-related (IL-4, IL-13, STAT6) genes were all significantly up-regulated in HO individuals. In addition, stronger Onchocerca volvulus-specific Th2 responses, especially IL-13, were observed in vitro in hyperreactive individuals when compared to GEO or EN groups. This study provides initial evidence that elevated frequencies of Th17 and Th2 cells form part of the immune network instigating the development of severe onchocerciasis.     

Distinct regulation of lymphokine production is found in fresh versus in vitro primed murine helper T cells

The kinetics of lymphokine RNA induction and secretion of biologically active lymphokine from CD4-enriched splenic T cell populations was investigated. Cells stimulated immediately after isolation from murine spleen ("fresh" T cells) and cells restimulated after 4 days of in vitro culture ("primed" T cells) were compared. Northern blot analysis and bioassays were used to analyze and quantitate production of eight lymphokines and the IL-2R. Fresh T cells produced high levels of IL-2 and low to moderate levels of IL-3, granulocyte/macrophage-CSF, and IFN-gamma. In vitro primed T cells produced IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte/macrophage-CSF, IFN-gamma, and high levels of IL-2R RNA. Comparison of RNA levels and bioassays of supernatants from these populations indicated that primed T cells produced at least 10-fold more of six of the lymphokines than fresh T cells. Only IL-2 was produced in near equal amounts by fresh and primed T cells. There were also marked differences in the kinetics of lymphokine production by fresh and primed CD4+ T cells. After restimulation with Con A and PMA, primed cells produced a short burst of lymphokine RNA that peaked between 7.5 and 13 h and declined after 18 h. Fresh T cells lagged in the initial production of lymphokine RNA, with levels peaking 18 to 44 h after mitogenic stimulation. Depletion of CD4+ cells indicated that cells of helper phenotype were responsible for the majority of lymphokine production from the primed cells. Thus different subpopulations of Th cells defined by their respective ability to respond either directly (fresh T cells) or only after culture and restimulation (primed T cells) show different patterns of lymphokine gene regulation. Other studies suggest that the activity of "fresh" Th cells is due to a population with a "memory" phenotype, while the cells which require culture have a "precursor" phenotype. These distinct patterns of lymphokine gene regulation in the two populations of Th cells may account in part for differences seen in the kinetics and magnitude of the naive and memory immune responses which are regulated by Th cells.