GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells

Committed T helper type 1 (Th1) and Th2 effector cells, resulting from chronic antigenic stimulation in interleukin (IL)-12 and IL-4, are implicated in the pathology of autoimmune and allergic diseases. Committed Th1 cells cannot be induced to change their cytokine profiles in response to antigenic stimulation and Th2 cytokine-inducing conditions. Here, we report that ectopic expression of GATA-3 induced Th2-specific cytokine expression not only in developing Th1 cells but also in otherwise irreversibly committed Th1 cells and a Th1 clone, HDK1. Moreover, cAMP, an inhibitor of cytokine production by Th1 cells, markedly augmented Th2 cytokine production in GATA-3-expressing Th1 cells. Ectopic expression of GATA-3 in developing Th1 cells, but not in Th1 clone HDK1, induced endogenous GATA-3, suggesting an autoregulatory mechanism for maintenance of GATA-3 expression in Th2 cells. Structure-function analyses of GATA-3 revealed that the NH(2)-terminal transactivation domain and the COOH-terminal zinc finger domain of GATA-3 were critical, whereas the NH(2)-terminal zinc finger domain was dispensable for the induction of IL-4. Both zinc fingers, however, were required for IL-5 induction. A Th2-specific DNaseI-hypersensitive site of the IL-4 locus was detected in GATA-3-expressing Th1 cells. Thus, GATA-3 can change the phenotype of committed Th1 cells, previously considered to be irreversible.     

Basophils produce IL-4 and accumulate in tissues after infection with a Th2-inducing parasite

Using mice in which the eGfp gene replaced the first exon of the Il4 gene (G4 mice), we examined production of interleukin (IL)-4 during infection by the intestinal nematode Nippostrongylus brasiliensis (Nb). Nb infection induced green fluorescent protein (GFP)pos cells that were FcepsilonRIpos, CD49bbright, c-kitneg, and Gr1neg. These cells had lobulated nuclei and granules characteristic of basophils. They were found mainly in the liver and lung, to a lesser degree in the spleen, but not in the lymph nodes. Although some liver basophils from naive mice express GFP, Nb infection enhanced GFP expression and increased the number of tissue basophils. Similar basophil GFP expression was found in infected Stat6-/- mice. Basophils did not increase in number in infected Rag2-/- mice; Rag2-/- mice reconstituted with CD4 T cells allowed significant basophil accumulation, indicating that CD4 T cells can direct both tissue migration of basophils and enhanced IL-4 production. IL-4 production was immunoglobulin independent and only partially dependent on IL-3. Thus, infection with a parasite that induces a "Th2-type response" resulted in accumulation of tissue basophils, and these cells, stimulated by a non-FcR cross-linking mechanism, are a principal source of in vivo IL-4 production.     

Role of interleukin 6 for differential responsiveness of naive and memory CD4+ T cells in CD2-mediated activation

The present study was undertaken to elucidate different requirements for CD2-mediated activation of naive (CD45RO-) and memory (CD45RO+) CD4+ T cells. A mitogenic combination of anti-CD2 (anti-T11(2) and anti-T11(3] mAbs could effectively induce the proliferation of memory CD4+ T cells even in the absence of monocytes. In marked contrast, naive CD4+ T cells did not disclose any proliferative responses to anti-CD2 mAbs, when monocytes were absent in culture. This differential responsiveness of naive and memory CD4+ T cells appeared to be related largely to a difference in IL-6-producing ability between both populations. IL-6 among monocyte-derived cytokines could correct unresponsiveness of naive CD4+ T cells to anti-CD2 stimulation. Unlike naive CD4+ T cells, memory CD4+ T cells produced IL-6 by themselves, with its mRNA being expressed on anti-CD2 stimulation. Anti-IL-6R mAb significantly inhibited proliferation of memory CD4+ T cells seen in the anti-CD2-stimulated cultures without monocytes, indicating the involvement of their own production of IL-6 in CD2-mediated activation. The results suggest an essential role of IL-6 for triggering of CD4+ T cells via the CD2 molecule.     

A peripheral CD4+ T cell precursor for naive, memory, and regulatory T cells

Mechanisms that control the size of the T cell pool, the ratio between naive cells and memory cells, the number and frequency of regulatory T cells, and T cell receptor (TCR) diversity are necessary to maintain immune integrity and avoid disease. We have previously shown that a subset of naive CD4(+) T cells, defined by the expression on their surface of a very low density of CD44 (CD44(v.low) cells), can inhibit wasting and wasting-associated lymphopenia in mice with cancer. In this study, we further investigate the properties of CD44(v.low) cells and show that they are significantly more efficient than the remaining naive (CD44(low) or CD44(int)) and memory CD4(+) cell subsets in reconstituting the overall size of the CD4(+) T cell pool, creating a T cell pool with a diverse TCR repertoire, generating regulatory T cells that express forkhead box P3 (FoxP3), and promoting homeostatic equilibrium between naive, memory, and Foxp3(+) regulatory T cell numbers. T cell population reconstitution by CD44(v.low) cells is thymus independent. Compared with CD44(int) cells, a higher percentage of CD44(v.low) cells express B cell leukemia/lymphoma 2, interleukin-7 receptor, and CD5. The data support a key role for CD4(+) CD44(v.low) cells as peripheral precursors that maintain the integrity of the CD4(+) T cell pool.     

Resting and anergic B cells are defective in CD28-dependent costimulation of naive CD4+ T cells

Successful antibody production in vivo depends on a number of cellular events, one of the most important of these being cognate B cell-T cell interaction. To examine this phenomenon in vitro, homogeneous populations of hen egg lysozyme (HEL)-specific small resting B cells and naive CD4+ HEL-specific T cells (derived from immunoglobulin [Ig] and T cell receptor transgenic mice, respectively) were cultured together. On addition of intact HEL protein. HEL-specific B cells increase their expression of activation molecules, including a B7- related protein and CD44, and enlarge into blast cells. Within the same cultures, HEL-specific CD4+ T cells also increase expression of the activation markers CD69 and CD44, enlarge, secrete lymphokines, and proliferate. This response is radiation sensitive, supporting the conclusion that HEL-specific B cells present antigen to and activate the naive T cells. By contrast, when a synthetic peptide fragment of HEL is used to bypass B cell antigen-receptor engagement, the naive T cells enlarge and display activation antigens, but fail to produce lymphokines, proliferate, or promote B cell blastogenesis. Presentation of HEL by tolerant B cells, which are no longer able to signal effectively through their antigen receptors, results in an identical pattern of incomplete T cell activation. Addition of a stimulating anti- CD28 antibody and blocking of CD28 signals with CTLA4/Ig fusion protein both show that complete activation of naive CD4+ T cells depends on the initial induction of B7 and related costimulatory molecules after HEL binding to nontolerant HEL-specific B cells. Thus, in the absence of adequate constimulation from the B cell, naive CD4+ T cells undergo a form of "partial activation" in which they upregulate surface expression of certain T cell activation antigens, but fail to efficiently produce lymphokine and proliferate. This may explain the different conclusions that have been reached regarding the consequences of B cell antigen presentation to T cells, in that the ability of B cells to activate naive CD4+ T cells depends both on their specificity and their activation state.     

Activation of CD4+ T cells in the presence of a nondepleting monoclonal antibody to CD4 induces a Th2-type response in vitro

In vitro experiments using purified rat CD4+ T cells in primary and secondary mixed leukocyte cultures (MLC) have been carried out to explore the mechanism of inhibition of cell-mediated autoimmune disease in the rat by a nondepleting monoclonal antibody (mAb) to CD4. Previous work has shown that W3/25, a mouse anti-rat CD4 mAb of immunoglobulin G1 isotype, completely prevents the development of the paralysis associated with experimental allergic encephalomyelitis (EAE) in Lewis rats, but does so without eliminating the encephalitogenic T cells. The in vitro experiments described in this study have shown that when CD4+ T cells were activated in the presence of the anti-CD4 mAb in a primary MLC, the synthesis of interferon (IFN) gamma, but not interleukin (IL) 2, was completely inhibited. After secondary stimulation, now in the absence of the mAb, the synthesis of IL-4 and IL-13 mRNA was greatly enhanced compared with that observed from CD4+ T cells derived from primary cultures in which the mAb was omitted. As IL-4 and IL-13 are known to antagonize cell-mediated immune reactions, and as EAE is cell- mediated disease, the data suggest that the W3/25 mAb controls EAE by modifying the cytokine repertoire of T cells that respond to the encephalitogen. The capacity for the mAb to suppress IFN-gamma synthesis provides, in part, an explanation for this change in cytokine production. These findings are discussed in terms of what is known of the factors that determine which cytokine genes are expressed on T cell activation. Possible implications for the evolution of T cell responses in human immunodeficiency virus infection are also discussed.     

NFATc2 and NFATc3 transcription factors play a crucial role in suppression of CD4+ T lymphocytes by CD4+ CD25+ regulatory T cells

The phenotype of NFATc2(-/-) c3(-/-) (double knockout [DKO]) mice implies a disturbed regulation of T cell responses, evidenced by massive lymphadenopathy, splenomegaly, and autoaggressive phenomena. The population of CD4(+) CD25(+) T cells from DKO mice lacks regulatory capacity, except a small subpopulation that highly expresses glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) and CD25. However, neither wild-type nor DKO CD4(+) CD25(+) regulatory T cells (T reg cells) are able to suppress proliferation of DKO CD4(+) CD25(-) T helper cells. Therefore, combined NFATc2/c3 deficiency is compatible with the development of CD4(+) CD25(+) T reg cells but renders conventional CD4(+) T cells unresponsive to suppression, underlining the importance of NFAT proteins for sustaining T cell homeostasis.     

Antiviral CD4+ memory T cells are IL-15 dependent

Survival and intermittent proliferation of memory CD4(+) and CD8(+) T cells appear to be controlled by different homeostatic mechanisms. In particular, contact with interleukin (IL)-15 has a decisive influence on memory CD8(+) cells, but not memory CD4(+) cells. Past studies of memory CD4(+) cells have relied heavily on the use of naturally occurring memory phenotype (MP) cells as a surrogate for antigen (Ag)-specific memory cells. However, we show here that MP CD4(+) cells contain a prominent subset of rapidly proliferating major histocompatibility complex (MHC) II-dependent cells. In contrast, Ag-specific memory CD4 cells have a slow turnover rate and are MHC II independent. In irradiated hosts, these latter cells ignore IL-15 and expand in response to the elevated levels of IL-7 in the lymphopenic hosts. In contrast, in normal nonlymphopenic hosts where IL-7 levels are low, memory CD4 cells are heavily dependent on IL-15. Significantly, memory CD4(+) responsiveness to endogenous IL-15 reflects marked competition from other cells, especially CD8(+) and natural killer cells, and increases considerably after removal of these cells. Therefore, under normal physiological conditions, homeostasis of CD8(+) and CD4(+) memory cells is quite similar and involves IL-15 and IL-7.     

CD4＋CD25＋调节性T细胞和Th17细胞在特发性血小板减少性紫癜中的表达和意义

目的验证并探讨CD4＋CD25＋调节性T细胞及Th17细胞与特发性血小板减少性紫癜发病的关系及可能机制。方法入选自2009年12月至2010年11月在山东省医学科学院附属济宁市第一人民医院血液内科门诊就诊或住院治疗的41例ITP患者（其中初治19例,复发22例,符合难治标准17例）及同期41例健康查体者为研究对象。所有标本均抽取清晨空腹外周血,以肝素钠抗凝。应用流式细胞术（FCM）测定ITP患者和健康对照外周血的CD4＋CD25＋调节性细胞及Th17细胞百分率;应用酶联免疫吸附试验（ELISA技术）测定ITP患者和健康对照血浆的IL-17水平。结果 ITP病例组外周血CD4＋CD25＋调节T细胞的百分率明显低于对照组（P〈0.05）,而Th17细胞的比例显著高于正常对照组（P〈0.05）,血浆IL-17水平在两组间差异无统计学意义（P〉0.05）;病例组中初治患者与复发患者外周血CD4＋CD25＋调节T细胞的百分率比较差异无统计学意义（P〉0.05）;难治组患者外周血CD4＋CD25＋调节T细胞的百分率低于非难治组（P〈0.05）;初治患者治疗后外周血CD4＋CD25＋调节T细胞百分率显著升高,但仍低于正常对照组（P〈0.05）。ITP病例组的CD4＋CD25＋Treg/Th17较健康对照组明显下降,差异具有统计学意义（P〈0.05）。结论特发性血小板减少性紫癜（ITP）患者外周血CD4＋CD25＋调节性T细胞显著减少,Th17细胞明显增加,出现Treg/Th17细胞失衡,这可能是影响特发性血小板减少性紫癜发生发展的一个重要因素。ITP患者外周血CD4＋CD25＋调节性T细胞减少程度在初治与复发ITP患者中差异无统计学意义;难治性ITP可能与CD4＋CD25＋调节性T细胞数量减少和Treg/Th17失衡尚未纠正有关。初治ITP患者经临床常规治疗后外周血CD4＋CD25＋调节性T细胞比例回升。     

Antigen-independent changes in naive CD4 T cells with aging

In the elderly, a dramatic shift within the CD4+ T cell population occurs, with an increased proportion having a memory phenotype with markedly decreased responsiveness. To determine what aspects of the aged phenotype are dependent upon repeated contact with antigen in the environment, we examined CD4+ cells isolated from TCR Tg mice. There is good evidence that no cross-reacting antigens for the Tg TCR recognizing pigeon cytochrome c are found in the environment of the animal, so that alterations in the Tg CD4+ cells with aging are likely to be due to antigen-independent processes. We found that in aged animals, TCR transgene(pos) CD4+ cells, although decreased in number and antigen responsiveness, maintain a naive phenotype rather than acquiring a prototypical aged memory phenotype. In contrast, the population of transgene(1o-neg) CD4+ cells increase in proportion and express the aged phenotype. Consistent with their naive status, transgene(pos) cells of aged individuals remain CD44lo CD45RBhi, secrete IL-2 and not IL-4 or IFN-gamma upon antigenic stimulation, and require co-stimulation to proliferate to anti-CD3 stimulation. These findings suggest that the aging-associated shift to CD4 cells expressing the memory phenotype is dependent on antigenic stimulation. However, the decrease in antigen responsiveness of naive transgenepos cells, as revealed by a lower secretion of IL-2 and IL-3 and a lower proliferative capacity, suggests that additional intrinsic changes occur with aging that do not depend on encounter with antigen.     

Development and characterization of IL-21-producing CD4+ T cells

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells, functions as an autocrine growth factor for Th17 cells, and plays critical roles in autoimmune diseases. In this study, we investigated the differentiation and characteristics of IL-21-producing CD4(+) T cells by intracellular staining. Unexpectedly, we found that under Th17-polarizing conditions, the majority of IL-21-producing CD4(+) T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21-producing CD4(+) T cells without the induction of IL-4, IFN-gamma, IL-17A, or IL-17F production. On the other hand, TGF-beta inhibited IL-6- and IL-21-induced development of IL-21-producing CD4(+) T cells. IL-2 enhanced the development of IL-21-producing CD4(+) T cells under Th17-polarizing conditions. Finally, IL-21-producing CD4(+) T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6, but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21-producing CD4(+) T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6-rich environment devoid of TGF-beta, and that IL-21 functions as an autocrine growth factor for IL-21-producing CD4(+) T cells.     

Sensory adaptation in naive peripheral CD4 T cells.

T cell receptor interactions with peptide/major histocompatibility complex (pMHC) ligands control the selection of T cells in the thymus as well as their homeostasis in peripheral lymphoid organs. Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56(lck). Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca(2)+ responses to subsequent TCR engagement by anti-CD3 or nominal antigen. Adaptation to peripheral pMHC contact may be important for regulating naive CD4 T cell responsiveness.     

T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis

This report shows that highly self-reactive T cells produced in mice as a result of genetically altered thymic T cell selection spontaneously differentiate into interleukin (IL)-17-secreting CD4+ helper T (Th) cells (Th17 cells), which mediate an autoimmune arthritis that clinically and immunologically resembles rheumatoid arthritis (RA). The thymus-produced self-reactive T cells, which become activated in the periphery via recognition of major histocompatibility complex/self-peptide complexes, stimulate antigen-presenting cells (APCs) to secrete IL-6. APC-derived IL-6, together with T cell-derived IL-6, drives naive self-reactive T cells to differentiate into arthritogenic Th17 cells. Deficiency of either IL-17 or IL-6 completely inhibits arthritis development, whereas interferon (IFN)-gamma deficiency exacerbates it. The generation, differentiation, and persistence of arthritogenic Th17 cells per se are, however, insufficient for producing overt autoimmune arthritis. Yet overt disease is precipitated by further expansion and activation of autoimmune Th17 cells, for example, via IFN-gamma deficiency, homeostatic proliferation, or stimulation of innate immunity by microbial products. Thus, a genetically determined T cell self-reactivity forms a cytokine milieu that facilitates preferential differentiation of self-reactive T cells into Th17 cells. Extrinsic or intrinsic stimuli further expand these cells, thereby triggering autoimmune disease. Intervention in these events at cellular and molecular levels is useful to treat and prevent autoimmune disease, in particular RA.     

Proteome-wide analysis of HIV-specific naive and memory CD4+ T cells in unexposed blood donors

The preexisting HIV-1–specific T cell repertoire must influence both the immunodominance of T cells after infection and immunogenicity of vaccines. We directly compared two methods for measuring the preexisting CD4⁺ T cell repertoire in healthy HIV-1–negative volunteers, the HLA-peptide tetramer enrichment and T cell library technique, and show high concordance (r = 0.989). Using the library technique, we examined whether naive, central memory, and/or effector memory CD4⁺ T cells specific for overlapping peptides spanning the entire HIV-1 proteome were detectable in 10 HLA diverse, HIV-1–unexposed, seronegative donors. HIV-1–specific cells were detected in all donors at a mean of 55 cells/million naive cells and 38.9 and 34.1 cells/million in central and effector memory subsets. Remarkably, peptide mapping showed most epitopes recognized by naive (88%) and memory (56%) CD4⁺ T cells had been previously reported in natural HIV-1 infection. Furthermore, 83% of epitopes identified in preexisting memory subsets shared epitope length matches (8–12 amino acids) with human microbiome proteins, suggestive of a possible cross-reactive mechanism. These results underline the power of a proteome-wide analysis of peptide recognition by human T cells for the identification of dominant antigens and provide a baseline for optimizing HIV-1–specific helper cell responses by vaccination.Only one candidate HIV vaccine, a canarypox vectored gp120 with a protein boost, has shown any efficacy (Rerks-Ngarm et al., 2009). The limited protection correlated with induction of nonneutralizing antibodies to the VI/V2 region of the virus Envelope protein (Env; Rerks-Ngarm et al., 2009; Haynes et al., 2012). This modest success has stimulated efforts to design vaccines that generate more efficient neutralizing antibodies, together with potent CD4⁺ T cell responses capable of providing help to B cells and cytotoxic T cells (Burton et al., 2012). Understanding how the magnitude and specificity of these helper T cells can be optimized will be critical to the design of an effective vaccine.Primary immune responses are probably influenced strongly by the preexisting repertoire of B and T cells. However, characterization and quantification of these repertoires is difficult due to the extremely low number of circulating naive precursor cells (Jenkins et al., 2001; Su et al., 2013). Previous studies of naive CD4⁺ T cell repertoires in humans and mice have relied on magnetic beads to enrich MHC tetramer binding cells (Moon et al., 2007; Kwok et al., 2012; Su et al., 2013). However, although this approach gives precise information on responses to particular MHC-peptide epitopes, it does not measure the total repertoire and misses previously unknown epitopes. An alternative T cell library technique requires no prior knowledge of donor HLA type or epitope specificity (Geiger et al., 2009). The method presorts circulating T cells into naive and memory subsets which are seeded at limiting dilution before polyclonal expansion in the presence of PHA, allogeneic feeder cells, and IL-2. Individual cultures are then screened for proliferative responses to a protein or series of peptides representing the pathogen of interest (Geiger et al., 2009). Combined with epitope mapping and the Poisson distribution, the T cell library technique can provide quantitative data on the specificity of the entire preexisting naive and memory repertoire.The existence of HIV-1–specific memory cells in seronegative donors was originally suggested by studies of highly exposed HIV-1 seronegative (HESN) donors. It has been shown that 25–61% of HESNs have demonstrable HIV-1–specific memory cells, probably primed by exposure to the virus. Surprisingly, HIV-1–specific CD4⁺ T cells were also detected in 24–44% of unexposed donors (Ritchie et al., 2011), although it was not clear whether the latter came from cross-reactive memory T cells or naive T cells primed in vitro. More recently, the existence of low frequency (1–10/million) memory CD4⁺ T cells, specific for a known HIV-1 Gag epitope, was demonstrated by HLA DR4 tetramers in 50% of HIV-1 unexposed HLA DR4⁺ adults (Su et al., 2013), but it was not clear how generalizable the HIV-1 result was beyond the single epitope–HLA DR4 combination.The present study first validates the library technique by direct comparison with the tetramer enrichment method for measuring precursor T cell frequencies. We then use the T cell library technique to provide the first proteome-wide analysis of the frequencies and specificities of preexposure HIV-1–specific naive and memory CD4⁺ T cells in a HLA diverse population of HIV-1 unexposed donors.