Quantitative proteomics reveals GIMAP family proteins 1 and 4 to be differentially regulated during human T helper cell differentiation

T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4(+) cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation.     

Role of L-type calcium channels in the calcium response and interleukin 4 (IL-4) synthesis by Th2 lymphocytes]

CD4+ T lymphocytes are divided in Th1 cells that produce interferon (IFN) gamma and Th2 cells that synthesize IL-4. These subsets may arise from a common precursor: a combination of IL-12 plus anti-IL-4 monoclonal antibody (mAb) drives Th1 cell differentiation while IL-4 plus anti-IFN gamma mAb favor Th2 cell development. TCR stimulation activates protein kinase C that controls a calcium entry through L type calcium channels in Th2 cells. L type calcium channels are induced during Th2 but not Th1 cell differentiation. In addition, L type calcium channel inhibitors may be successfully used in the treatment of an experimental model of Th2 cell-mediated immunopathology. Thus, this signaling pathway that characterizes Th2 cells can be a target for the treatment of Th2 diseases.     

B cell response to T helper cell subsets. II. Both the stage of T cell differentiation and the cytokines secreted determine the extent and nature of helper activity.

Helper activity of several murine CD4+ T cell subsets was examined. Effector Th, derived from naive cells after 4 days of in vitro stimulation with alloantigen, when generated in the presence of IL-4, secreted high levels of IL-4, IL-5, and IL-6, and low levels of IL-2 and IFN-gamma, and induced the secretion of all Ig isotypes particularly IgM, IgG1, IgA, and IgE from resting allogeneic B cells. Effectors generated with IL-6 secreted IL-2, IL-4, IL-5, IL-6, and IFN-gamma, and induced similar levels of total Ig, 25 to 35 micrograms/ml, but with IgM, IgG3, IgG1, and IgG2a isotypes predominating. Helper activity of these Th was significantly greater than that of effectors generated with IL-2 (10-15 micrograms/ml Ig) and of 24-h-activated naive and memory cells (2-4 micrograms/ml), both of which induced mainly IgM. Unlike other isotypes, IgE was induced only by effector Th generated with IL-4. Blocking studies showed that secretion of all isotypes in response to IL-6-primed effectors was dependent on IL-2, IL-5, and IL-6. IL-4 was required for optimal IgM, IgG1, and IgA secretion, but limited secretion of IgG2a, whereas IFN-gamma was required for optimal IgG2a secretion, and limited IgM, IgG1, and IgA. In contrast, secretion of all isotypes in response to IL-4-primed effectors was dependent on IL-5, although IL-4 and IFN-gamma were also essential for IgE and IgG2a, respectively. Addition of exogenous IL-5 to B cell cultures driven by IL-6-primed effectors did not obviate the requirement for IL-2, IL-4, and IL-6, suggesting that interaction of IL-4-primed effectors with B cells was qualitatively different from that of IL-6-primed effectors, driving B cells to a stage requiring only IL-5 for differentiation. Addition of exogenous factors to IL-2-primed effector Th, particularly IL-4 in the presence of anti-IFN-gamma, resulted in levels of Ig, including IgE, comparable to those induced with other effectors. These results show that functionally distinct Th cell subsets can be generated rapidly in vitro, under the influence of distinct cytokines, which vary dramatically in their levels of help for resting B cells. The cytokines involved in responses to distinct Th cells differ depending on the quality of interaction with the B cell, and the extent of help is strongly determined by the quantity and nature of cytokines secreted by the T cells.     

IL-4 plays a dominant role in the differential development of Tho into Th1 and Th2 cells.

We have analyzed the evolution of the pattern of lymphokine secretion by Th cell lines specific for either the synthetic terpolymer Glu60Ala30Tyr10 (GAT) or killed bacillus Calmette Guérin. When cultured in the presence of exogenous rIL-2 as a growth factor, GAT-specific Th cell lines secreted mainly IL-4, whereas bacillus Calmette Guérin-specific lines produced predominantly IL-2. However, culturing in the presence of rIL-4 or of anti-IL-4 mAb and rIL-2 led to the establishment of Th2-like and Th1-like lines, respectively, regardless of their Ag specificity. Inasmuch as we show that the proliferative response of mature Th1 and Th2 cells was identical in the presence of IL-4, these results indicate that IL-4 influences the development of Th cell subsets. To understand the mode of IL-4 action, we isolated immature GAT-specific Tho clones able to secrete IL-2 and IL-4. Two types of Tho cells were isolated: ThoA cells that secreted IL-2 and IL-4, but not IFN-gamma, and ThoB cells that secreted IL-2, IL-4, and IFN-gamma. We show for the first time that such cells are indeed Th precursors able to differentiate into Th1 or Th2 cells. We demonstrate that IL-4 positively and negatively controls the differentiation of Tho cells into Th2 and Th1 cells, respectively. When cultured in rIL-4, Tho cells stop secreting IL-2 and IFN-gamma, but maintain IL-4 secretion. Moreover, endogenous IL-4 produced by Tho cells has similar effects: when cultured in rIL-2 alone, Tho cells either keep their immature phenotype or become Th2 cells, but do not become Th1 cells. In contrast, neutralization of secreted IL-4 completely prevents the differentiation of Tho into Th2 cells, but permits the development of Th1 cells. The presence of exogenous IFN-gamma does not affect the development of Tho into Th1 and Th2 cells, because it does not modify either mode of IL-4 action. However, it influences the ratio between the two types of Tho cells: when IL-4 is neutralized, added IFN-gamma can induce IFN-gamma secretion by ThoA cells and thereby facilitate their passage into ThoB cells. Taken together, our results demonstrate that IL-4, in addition to mediating T cell growth, is a principal factor that controls the differential development of Tho cells into Th1 and Th2 cells.     

Characterization of T helper 1 and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells

We have shown that the requirements for the production of IL-4 and IL-5 by normal L3T4+T cells from murine spleen are very different from those for the production of IL-2. Secretion of detectable quantities of IL-4 and IL-5 and induction of the mRNA for each lymphokine occurs in vitro only after cells are primed and re-stimulated. This priming can be achieved by mitogens (Con A), by antibodies to the TCR (anti-T3) or by stimulation with alloantigen. In contrast, requirements for induction of lymphokine production after priming resemble those for initial production of IL-2. Thus the majority of T cells of helper phenotype that have the potential to become IL-4- and IL-5-secreting T cells, exist in the form of precursors requiring stimulation and several days of culture as well as re-stimulation with mitogen or Ag before they become detectable as lymphokine-secreting cells. In contrast, among fresh CD4+T cells, secretion of IL-2, IL-3, granulocyte/macrophage CSF, and IFN-gamma is easily detected within 24 h of stimulation with mitogen or Ag. These observations establish that distinct phenotypes of Th cells are found at different times after stimulation and support the concept that synthesis and secretion of different lymphokines or groups of lymphokines are regulated independently. Furthermore the patterns of lymphokines secreted by fresh vs primed Th cells, which largely correspond to the patterns that have been used to define the Th1 and Th2 subsets among Th cell lines, provides evidence that different subsets of normal T cells exist that may correspond to these designations. Secretion of different lymphokines by two subsets of Th cells at different times in an immune response, and perhaps in different places, suggests a model in which the ratio of the two T cell subsets (Th1 vs Th2) and state of differentiation of each (precursor vs effector), influence or determine the direction of the response, with variations in these parameters leading to differing responses.     

Antibodies to the IL-12 receptor beta 2 chain mark human Th1 but not Th2 cells in vitro and in vivo

Great attention has been placed on the possibility of distinguishing Th1 from Th2 cells on the basis of differential expression of surface receptors. We have recently shown that the differential expression of the IL-12R beta 2 chain in Th1 and Th2 cells, as measured at the mRNA level, accounts for an important regulatory mechanism in the differentiation of the two cell subsets. In this study, we identify IL-12R expression at the protein level. We have generated an anti-IL-12R beta 2-specific mAb and analyzed IL-12R beta 2 expression on polarized Th cell populations generated in vitro and on T cells derived from patients with Th1- or Th2-mediated inflammatory conditions. Although IL-12R beta 2 was absent in freshly isolated PBMC and in cord blood cells, we were able to detect IL-12R beta 2 expression selectively in differentiated Th1 and T cytotoxic 1, but not Th2 or T cytotoxic 2 cells. In the presence of IL-12, cell surface expression of the IL-12R beta 2 subunit was readily detected on T cells after 24 h, reached the maximum at day 5, and declined thereafter. Most importantly, the anti-IL-12R beta 2 mAb recognizes lung T cells from patients with sarcoidosis, a disease characterized by a typical cell-mediated, Th1-type inflammatory response. In contrast, IL-12R beta 2 was absent in lung T cells from patients with allergic asthma, a disease characterized by a Th2-type inflammatory response. The mAb reported in this study should represent a powerful tool to investigate the role of Th1 and Th2 cells in inflammatory conditions and to monitor therapies aimed at altering the balance of Th cell subsets.     

Peptide dose, affinity, and time of differentiation can contribute to the Th1/Th2 cytokine balance.

Opposing viewpoints exist regarding how Ag dose and affinity modulate Th1/Th2 differentiation, with data suggesting that both high and low level stimulation favors Th2 responses. With transgenic T cells bearing a single TCR, we present novel data, using peptides differing in affinity for the TCR, that show that the time period of differentiation can determine whether Th1 or Th2 responses predominate as the level of initial stimulation is altered. Over the short term, IFN-gamma-producing cells were induced by lower levels of stimulation than IL-4-producing cells, although optimal induction of both was seen with the same high level of stimulation. Over the long term, however, high doses of high affinity peptides led selectively to IFN-gamma-secreting cells, whereas IL-4- and IL-5-secreting cells predominated with lower levels of initial signaling, brought about by moderate doses of high affinity peptides. In contrast, too low a level of stimulation at the naive T cell stage, with low affinity peptides at any concentration, promoted only IL-2-secreting effectors or was not sufficient for long term T cell survival. These results demonstrate that the level of signaling achieved through the TCR is intimately associated with the induction of distinct cytokine-secreting T cells. We show that dose, affinity, time over which differentiation occurs, and initial production of IL-4 and IFN-gamma all can contribute to which T cell subset will predominate. Furthermore, these data reconcile the two opposing views on the effects of dose and affinity and provide a unifying model of Th1/Th2 differentiation based on strength of signaling and length of response.     

Effect of VIP on the balance between cytokines and master regulators of activated helper T cells

CD4T helper cells are decisive in the struggle against pathogens and in maintaining immune homeostasis. Nevertheless, they also drive immune-mediated disease. Recently, emerging evidence suggests that seemingly committed Th cells possess plasticity and may convert into other types of effector cells. Vasoactive Intestinal Peptide (VIP) is an immunomodulator neuropeptide, which is able to promote or inhibit individually the differentiation or function of some T-helper subsets. We conducted ex vivo study with erythrocyte-depleted spleen cells from healthy mice to check the balance between cytokines and master regulators of different T-helper subsets. This neuropeptide adversely affected the differentiation and functionality phases of Th17 cells and had a negative influence on cytokines related to Th1 function, increasing Th17 cells over those of the Th1 cell subset. With respect to Th2 subsets, VIP augmented the interleukin (IL)-4/IL-9 mRNA ratio, and a negative correlation between IL-4 and IL-9 was observed in culture supernatants. VIP augmented Th2 relative to Th1 in cell subsets. VIP decreased the iTreg/Th17 balance. Regarding the induced T-regulatory (iTreg)/Th1 balance, VIP increased the presence of immunoregulatory cytokines in relation to IFNγ. Although additional studies are needed to clarify the role of VIP on the balance between cytokines and master regulators during T-helper differentiation, our data show that VIP reduces Th17/Th1 and Th1/Th2 ratios. However, the iTreg/Th17 ratio was differently counterbalanced, probably because of culture conditions. Finally, this is the first study showing that VIP also modulates Th2/Th9 and iTreg/Th1 ratios.     

The susceptibility to experimental myasthenia gravis of STAT6-/- and STAT4-/- BALB/c mice suggests a pathogenic role of Th1 cells

Autoantibodies to the muscle acetylcholine receptor (AChR) cause the symptoms of human and experimental myasthenia gravis (EMG). AChR-specific CD4+ T cells permit development of these diseases, but the role(s) of the Th1 and Th2 subsets is unclear. The STAT4 and STAT6 proteins, which mediate intracellular cytokine signaling, are important for differentiation of Th1 and Th2 cells, respectively. Wild-type (WT) BALB/c mice, which are prone to develop Th2 rather than Th1 responses to Ag, are resistant to EMG. We have examined the role of Th1 and Th2 cells in EMG using STAT4 (STAT4-/-)- or STAT6 (STAT6-/-)-deficient BALB/c mice. After AChR immunization, STAT6-/- mice were susceptible to EMG: they developed more serum anti-AChR Ab, and had more complement-fixing anti-AChR IgG2a and 2b and less IgG1 than WT or STAT4-/- mice. The susceptibility to EMG of STAT6-/- mice is most likely related to the Th1 cell-induced synthesis of anti-AChR Ab, which trigger complement-mediated destruction of the neuromuscular junction. CD4+ T cells of the STAT6-/- mice had proliferative responses to the AChR comparable to those of WT and STAT4-/- mice, and recognized similar AChR epitopes. STAT6-/- mice had abundant AChR-specific Th1 cells, which were nearly absent in WT and STAT4-/- mice. Spleen and lymph nodes from STAT6-/- mice contained cells that secreted IL-4 when cultured with AChR: these are most likely STAT6-independent cells, stimulated in a non-Ag-specific manner by the cytokines secreted by AChR-specific Th1 cells.     

Heterogeneity of helper/inducer T lymphocytes. III. Responses of IL-2- and IL-4-producing (Th1 and Th2) clones to antigens presented by different accessory cells

Murine CD4+ T cell clones have been classified into at least two subsets, Th1 and Th2, on the basis of their distinct lymphokine secretion profiles and functions. In the present study, we compared the functional responses of Th1 and Th2 clones to Ag presentation by splenic B cells and peritoneal macrophages. Th2 clones secreted IL-4 in response to Ag presented by resting B cells, but their optimal proliferation required the addition of IL-1 or a source of IL-1. The degree of IL-1 dependence varied among the four Th2 clones examined. In contrast, Th1 clones secreted IL-2 and proliferated in response to Ag presented by both B cells and macrophages, without any requirement for exogenous IL-1. Furthermore, the proliferation of Th2 clones in response to Ag presented by splenocytes or macrophages was inhibited by an IL-1R antagonist. These results indicate that IL-1 is an important costimulator for the expansion of the Th2 subset of CD4+ T cells. The different requirements for the proliferation of Th1 and Th2 cells may be responsible for the preferential expansion of one or the other subset under different conditions of immunization.     

The homeostasis but not the differentiation of T cells is regulated by p27(Kip1)

The cyclin-dependent kinase inhibitor p27(Kip1) is a critical regulator of T cell proliferation. To further examine the relationship of T cell proliferation and differentiation, we examined the ability of T cells deficient in p27(Kip1) to differentiate into Th subsets. We observed increased Th2 differentiation in p27(Kip1)-deficient cultures. In addition to increases in CD4(+) and CD8(+) T cells, there is a similar increase in gamma delta T cells in p27(Kip1)-deficient mice compared with wild-type mice. The increase in Th2 differentiation is correlated to an increase of IL-4 secretion by CD4(+)DX5(+)TCR alpha beta(+)CD62L(low) T cells but not to increased expansion of differentiating Th2 cells. While STAT4- and STAT6-deficient T cells have diminished proliferative responses to IL-12 and IL-4, respectively, proliferative responses are increased in T cells doubly deficient in p27(Kip1) and STAT4 or STAT6. In contrast, the increased proliferation and differentiative capacity of p27(Kip1)-deficient T cells has no effect on the ability of STAT4/p27(Kip1)- or STAT6/p27(Kip1)-deficient CD4(+) cells to differentiate into Th1 or Th2 cells, respectively. Thus, while p27(Kip1) regulates the expansion and homeostasis of several T cell subsets, it does not affect the differentiation of Th subsets.