Human CD4+ Memory T Cells Can Become CD4+IL-9+ T Cells

Background

IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-β directs mouse CD4⁺CD25⁻CD62L⁺ T cells to commit to inflammatory IL-9 producing CD4⁺ T cells.

Methodology/Principal Findings

Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-β induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4⁺CD25⁻CD45RO⁺ T cells as compared to naïve CD4⁺CD25⁻CD45RA⁺ T cells. In addition, as compared to pbCD3/sCD28 plus TGF-β stimulation, IL-4+TGF-β stimulated memory CD4⁺CD25⁻CD45RO⁺ T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4⁺IL-9⁺ T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNγ or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-β stimulated resting memory CD4⁺ T cells demonstrated that the addition of IL-1β, IL-12, and IL-21 further enhance IL-9 production.

Conclusions/Significance

Taken together these data show both the differences and similarities between mouse and human CD4⁺IL9⁺ T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4⁺ T cells to antigen.     

IL-33 Induces IL-9 Production in Human CD4+ T Cells and Basophils

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.     

The IL-4 receptor alpha-chain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD25+CD4+ regulatory T cells from CD25-CD4+ precursors

The mechanisms underlying the extrathymic generation of CD25+CD4 regulatory T cells (Tregs) are largely unknown. In this study the IL-4R alpha-chain-binding cytokines, IL-4 and IL-13, were identified as inducers of CD25+ Tregs from peripheral CD25-CD4 naive T cells. IL-4-induced CD25+ Tregs phenotypically and functionally resemble naturally occurring Tregs in that they are anergic to mitogenic stimulation, inhibit the proliferation of autologous responder T cells, express high levels of the Forkhead box P3 and the surface receptors glucocorticoid-induced TNFR family-related protein and CTLA-4, and inhibit effector T cells in a contact-dependent, but cytokine-independent, manner. The IL-4-induced generation of peripheral Tregs was independent of the presence of TGF-beta or IL-10, but was dependent on Ag-specific stimulation and B7 costimulation. The significance of the IL-4Ralpha-binding cytokines in the generation of Ag-specific Tregs was emphasized in a mouse model of oral tolerance, in which neutralization of IL-4 and IL-13 in mice transgenic for the TCR specific for OVA completely inhibited the expansion of OVA-specific Tregs that can be induced in untreated mice by feeding the nominal Ag. Together, our results demonstrate that IL-4 and IL-13 play an important role in generating Forkhead box P3-expressing CD25+ Tregs extrathymically in an Ag-dependent manner and therefore provide an intriguing link between the well-established immunoregulatory capacity of Th2 cells and the powerful CD25+ Treg population. Moreover, our findings might provide the basis for the design of novel therapeutic approaches for targeted immunotherapy with Tregs to known Ags in autoimmune diseases or graft-vs-host reactions.     

Frequency analysis of CD4+CD8+ T cells cloned with IL-4

The coexpression of both CD4 and CD8 molecules on T cells occurs in the peripheral blood at a low frequency and can be generated transiently on CD4+ peripheral blood T cells by treatment with lectin which induces CD8 biosynthesis and cell surface expression. We have cloned T cells in a nonselective fashion from normal subjects in the presence of either IL-2, rIL-4 and IL-2, or rIL-4 and have examined the phenotypic expression of CD4 and CD8. The addition of excess rIL-4 increased the expression of CD8 on the surface of CD4+ T cell clones but did not increase CD4 expression on CD8+ T cell clones. There were three patterns of CD4 and CD8 expression observed: high density CD8 with no CD4 expression; high density CD4 with low CD8 expression; or high density CD4 with higher cell surface CD8 expression which was regulated by the presence of rIL-4. CD4+ T cell clones originally cultured in IL-2 and rIL-4 and subsequently grown in IL-2 alone exhibited decreased expression of the CD8 molecule. The increased expression of CD8 did not correlate with NK activity or lectin-dependent cytotoxicity in an antigen independent system. In addition, rIL-4 alone or in combination with IL-2 appeared to accelerate the growth curve of T cell clones as compared to IL-2 alone. These results show that IL-4 can upregulate CD8 expression on CD4+ T cell clones while not effecting CD4 expression on CD8+ T cell clones. As class I MHC is the ligand for the CD8 molecule, expression of CD8 induced by IL-4 on CD4+ T cells may allow for increased nonspecific cell to cell contact during the course of an inflammatory response.     

Simultaneous production of IL-2, IL-4, and IFN-gamma by activated human CD4+ and CD8+ T cell clones

In the present study, we have investigated the ability of human T cells to secrete IL-2, IL-4, and IFN-gamma. IL-4 and IFN-gamma were quantified with enzymatic immunoassays and IL-2 with a biologic assay by using the murine IL-2-dependent cell line CTLL-2. PBL, stimulated with Con A or with a combination of the phorbol ester 13-O-tetradecanoylphorbol-12-acetate and the Ca2+ ionophore A23187 secreted IL-2, IL-4, and IFN-gamma. The kinetics of the secretion of the three lymphokines was investigated with two CD4+ clones; one (GEO-2) that produced IL-2, IL-4, and IFN-gamma and another (HY640), that produced only IL-2 and IFN-gamma. Significant IL-2, IL-4, and IFN-gamma production was observed after only 8 h of activation. Maximal levels of IL-2 and IL-4 were found 20 h after the onset of the stimulation which subsequently decreased. In contrast, IFN-gamma levels continued to increase in a period up to 40 h and then leveled off. In spite of these differences in secretion, the kinetics of accumulation of mRNA did not differ. The IL-2, IL-4, and IFN-gamma mRNA were detectable 2 h after stimulation and continued to accumulate for a period up to 20 h. In a series of 22 CD4+ clones, 21 were able to secrete all three lymphokines upon stimulation. Almost all CD8+ clones were able to produce IL-2 and IFN-gamma, but only six of the 23 CD8+ T cell clones secreted IL-4. In addition, five CD4+ (allo)antigen-specific T cell clones were tested for IL-2, IL-4, and IFN-gamma secretion upon specific stimulation. Two alloantigen-specific and two tetanus toxoid-specific T cell clones secreted IL-2, IL-4, and IFN-gamma simultaneously, whereas one alloantigen-specific T cell clone secreted IL-2 and IFN-gamma, but not IL-4. A supernatant of the CD4+ T cell clone GEO-2, that contained high levels of IFN-gamma and IL-4, was unable to induce the low affinity receptor for IgE, CD23, on a Burkitt lymphoma cell line. However, after separation of IL-4 from IFN-gamma by using HPLC, the IL-4-containing fraction-induced CD23, which could be blocked by the fraction that contained IFN-gamma and by a polyclonal rabbit anti-IL-4 antiserum. Finally, the partly purified IL-4, that was devoid of IL-2, promoted the growth of the clone GEO-2.     

Cross-talk between activated human NK cells and CD4+ T cells via OX40-OX40 ligand interactions

It is important to understand which molecules are relevant for linking innate and adaptive immune cells. In this study, we show that OX40 ligand is selectively induced on IL-2, IL-12, or IL-15-activated human NK cells following stimulation through NKG2D, the low affinity receptor for IgG (CD16) or killer cell Ig-like receptor 2DS2. CD16-activated NK cells costimulate TCR-induced proliferation, and IFN-gamma produced by autologous CD4+ T cells and this process is dependent upon expression of OX40 ligand and B7 by the activated NK cells. These findings suggest a novel and unexpected link between the natural and specific immune responses, providing direct evidence for cross-talk between human CD4+ T cells and NK receptor-activated NK cells.     

IL-2 and IL-15 regulate CD154 expression on activated CD4 T cells

The cellular and humoral immune system is critically dependent upon CD40-CD154 (CD40 ligand) interactions between CD40 expressed on B cells, macrophages, and dendritic cells, and CD154 expressed primarily on CD4 T cells. Previous studies have shown that CD154 is transiently expressed on CD4 T cells after T cell receptor engagement in vitro. However, we found that stimulation of PBLs with maximal CD28 costimulation, using beads coupled to Abs against CD3 and CD28, led to a very prolonged expression of CD154 on CD4 cells (>4 days) that was dependent upon autocrine IL-2 production. Previously activated CD4 T cells could respond to IL-2, or the related cytokine IL-15, by de novo CD154 production and expression without requiring an additional signal from CD3 and CD28. These results provide evidence that CD28 costimulation of CD4 T cells, through autocrine IL-2 production, maintains high levels of CD154 expression. This has significant impact on our understanding of the acquired immune response and may provide insight concerning the mechanisms underlying several immunological diseases.     

Lectin-mediated induction of IL-4-producing CD4+ T cells.

Cultured murine CD4+ T cells have been shown to differentiate into IL-2 or IL-4-producing subsets. The factors responsible for the development of CD4+ T cells which produce IL-2 but not IL-4 and cells capable of producing IL-4 but not IL-2 are unknown. Here we describe a system that allows the controlled induction of IL-2- or IL-4-producing T cells after one single round of activation. Freshly isolated CD8-depleted T cells were activated with various polyclonal T cell activators for 48 h, washed, and then expanded under different conditions. IL-2 and IL-4 production were induced by restimulation of T cells and were measured with CTLL cells that respond to both cytokines and mAb to IL-2 and IL-4. T cells produced mainly IL-2 and small amounts of IL-4 when restimulated after expansion culture for 12 days with rIL-2 alone. However, after expansion for 12 days in the presence of rIL-2 plus Con A, we observed a 30- to 100-fold up-regulation of IL-4 activity and a 100-fold down-regulation of IL-2 when assessed by responses of CTLL cells incubated with the supernatant of restimulated T cells and by responses of CTLL cells cocultured with restimulated cells. An increase of IL-4 and decrease of IL-2 was also observed when the results were based on the cell numbers at the beginning of the expansion culture. The induction of IL-4 and the down-regulation of IL-2 1) were not reproduced with alpha-methyl-mannoside-treated supernatant of Con A-stimulated spleen cells, 2) were not dependent on the presence of large numbers of APC, 3) did not result from differential consumption of lymphokines after restimulation, 4) were not due to a difference in the time course of IL-2 or IL-4 release in either T cell population, and 5) were obtained regardless of the agents used to activate or to restimulate the T cells. Because Con A remained detectable on the T cell surface and because expansion of activated T cells with IL-2 plus Con A for several days was necessary, our results indicate that mainly IL-4-producing CD4+ T cells can be induced by prolonged engagement of T cell surface molecules.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.     

CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10

The mechanisms by which the immune system achieves constant T cell numbers throughout life, thereby controlling autoaggressive cell expansions, are to date not completely understood. Here, we show that the CD25(+) subpopulation of naturally activated (CD45RB(low)) CD4 T cells, but not CD25(-) CD45RB(low) CD4 T cells, inhibits the accumulation of cotransferred CD45RB(high) CD4 T cells in lymphocyte-deficient mice. However, both CD25(+) and CD25(-) CD45RB(low) CD4 T cell subpopulations contain regulatory cells, since they can prevent naive CD4 T cell-induced wasting disease. In the absence of a correlation between disease and the number of recovered CD4(+) cells, we conclude that expansion control and disease prevention are largely independent processes. CD25(+) CD45RB(low) CD4 T cells from IL-10-deficient mice do not protect from disease. They accumulate to a higher cell number and cannot prevent the expansion of CD45RB(high) CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25(+) CD45RB(low) CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25(-) CD45RB(low) or CD45RB(high) CD4 T cells. We conclude that CD25(+) CD45RB(low) CD4 T cells from nonmanipulated mice control the number of peripheral CD4 T cells through a mechanism involving the production of IL-10 by regulatory T cells.     

Human bone marrow hosts polyfunctional memory CD4+ and CD8+ T cells with close contact to IL-15-producing cells

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4(+) and CD8(+) T cells have not yet been elucidated. In this study, we demonstrate that CD4(+) and CD8(+) effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4(+) and CD8(+) T cells are in the immediate vicinity of IL-15-producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4(+) and CD8(+) T cell subsets. Moreover, the IL-15-inducible molecules Bcl-x(L), MIP-1α, MIP-1β, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15-producing cells, is important for the maintenance of a polyfunctional memory CD4(+) and CD8(+) T cell pool.     

Regulatory roles of IL-2 and IL-4 in H4/inducible costimulator expression on activated CD4+ T cells during Th cell development

We found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4(+) T cells obtained from six different murine strains. BALB/c-activated CD4(+) T cells expressed approximately 10-fold more H4/ICOS on their surfaces and produced approximately 10-fold more IL-4 upon restimulation than C57BL/6-activated CD4(+) T cells. BALB/c naive CD4(+) T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4(+) T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4(+) T cells strongly down-regulated both H4/ICOS expression on activated CD4(+) T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4(+) T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4(+) T cells from C57BL/6 or IL-4(-/-) mice increased H4/ICOS expression on activated CD4(+) T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4(+) T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4(+) T cells.     

Functional and biochemical characterization of mouse P40/IL-9 receptors

The existence of saturable and specific binding sites for mouse P40/IL-9 was demonstrated on a variety of factor-dependent T cell lines derived from Th clones by long term culture in the presence of P40-containing T cell supernatants. Scatchard transformation of the data obtained with one such line was consistent with the existence of a single class of receptors with a Kd of approximately 100 pM and a density of 3000/cell. P40 binding to these cells was followed by rapid internalization of the ligand. P40-receptors (P40-R)3 were also found on certain Th clones maintained in conventional cultures, especially after stimulation with Ag and APC. Only T cell clones that proliferated in response to P40 showed significant levels of binding, suggesting that the regulation of P40-R expression is an important element in the control of P40-responsiveness. In accord with this idea, fresh T cells, cytolytic T cell clones and a wide variety of other cells including B cells and fibroblasts, which do not proliferate in response to P40, showed no significant binding. However, P40-R were not restricted to a few unusual Th clones. They were also detected on several T cell tumors, on macrophages and on mast cell lines. The latter point is of particular interest in view of the mast cell growth factor activity recently ascribed to P40. Cross-linking studies with T-cell lines and mast cells indicated that the P40-R consists of a 64-kDa glycoprotein, the molecular mass of which is reduced to 54 kDa on treatment with N-glycosidase F.     

CD8+ T cell-mediated airway hyperresponsiveness and inflammation is dependent on CD4+IL-4+ T cells

CD4+ T cells, particularly Th2 cells, play a pivotal role in allergic airway inflammation. However, the requirements for interactions between CD4+ and CD8+ T cells in airway allergic inflammation have not been delineated. Sensitized and challenged OT-1 mice in which CD8+ T cells expressing the transgene for the OVA(257-264) peptide (SIINFEKL) failed to develop airway hyperresponsiveness (AHR), airway eosinophilia, Th2 cytokine elevation, or goblet cell metaplasia. OT-1 mice that received naive CD4+IL-4+ T cells but not CD4+IL-4- T cells before sensitization developed all of these responses to the same degree as wild-type mice. Moreover, recipients of CD4+IL-4+ T cells developed significant increases in the number of CD8+IL-13+ T cells in the lung, whereas sensitized OT-1 mice that received primed CD4+ T cells just before challenge failed to develop these responses. Sensitized CD8-deficient mice that received CD8+ T cells from OT-1 mice that received naive CD4+ T cells before sensitization increased AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged with allergen. In contrast, sensitized CD8-deficient mice receiving CD8+ T cells from OT-1 mice without CD4+ T cells developed reduced AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged. These data suggest that interactions between CD4+ and CD8+ T cells, in part through IL-4 during the sensitization phase, are essential to the development of CD8+IL-13+ T cell-dependent AHR and airway allergic inflammation.     

Expression and regulation of IL-22 in the IL-17-producing CD4＋ T lymphocytes

IL-22 is a novel cytokine in the IL-10 family that functions to promote innate immunity of tissues against infection. Although CD4＋ helper T lymphocytes （TH） were found as a source of IL-22, the regulation of this cytokine has been poorly understood. Here, we show that IL-22 is expressed at both mRNA and protein levels by a novel subset of TH cells that also makes IL-17. IL-22 and IL-17 were found to be coordinately regulated by TGFI3 and IL-6 during TH differentiation by real-time PCR as well as ELISA analysis. However, IL-22 does not regulate TH differentiation; exogenous IL-22 or an IL-22 antagonist had no effect on TH differentiation. These data demonstrate a novel cytokine expressed by IL-17-producing T cells, and suggest interaction and synergy of IL-22 and IL-l 7 signaling pathways in tissue inflammation and autoimmune diseases.     

A common precursor for CD4+ T cells producing IL-2 or IL-4.

To investigate whether CD4+ T cells are predetermined to produce a given pattern of lymphokines, we have used a culture system that allows the controlled induction of either IL-2- or IL-4-producing CD4+ T cells. Single, freshly isolated murine CD4+ T cells were activated with Con A, rIL-2, and APC; the developing clones were split and then cultured for an additional 14 days with either rIL-2 alone or with rIL-2 and anti-CD3 stimulation. Subclones expanded in the presence of rIL-2 alone produced predominantly IL-2, although subclones derived from the same precursor and expanded in the presence of rIL-2 and a mitogenic antibody to CD3 released predominantly IL-4. Subclones expanded for 2 wk in the presence of rIL-2 plus a mitogenic mAb to CD3 released up to 60 times more IL-4 but only 1/90 the amount of IL-2 released by subclones derived from the same precursor cell and expanded with rIL-2. Both phenotypes can be derived from IL-2-producing precursor cells. These results demonstrate that IL-2-producing clones can be derived from the same cells as IL-4-producing clones and are most consistent with the view that the IL-2-producing Th1 or the IL-4-producing Th2 phenotype of a T cell clone is acquired during T cell differentiation and is not secondary to the expansion of distinct subpopulations that are predetermined to produce a specific cytokine pattern.     

CD40 engagement enhances antigen-presenting langerhans cell priming of IFN-gamma-producing CD4+ and CD8+ T cells independently of IL-12

The delivery of CD40 signaling to APCs during T cell priming enhances many T cell-mediated immune responses. Although CD40 signaling up-regulates APC production of IL-12, the impact of this increased production on T cell priming is unclear. In this study an IL-12-independent T cell-mediated immune response, contact hypersensitivity (CHS), was used to further investigate the effect of CD40 ligation on the phenotypic development of Ag-specific CD4(+) and CD8(+) T cells. Normally, sensitization for CHS responses induces hapten-specific CD4(+) T cells producing type 2 cytokines and CD8(+) T cells producing IFN-gamma. Treatment of mice with agonist anti-CD40 mAb during sensitization with the hapten 2,4-dinitrofluorobenzene resulted in CHS responses of increased magnitude and duration. These augmented responses in anti-CD40 Ab-treated mice correlated with increased numbers of hapten-specific CD4(+) and CD8(+) T cells producing IFN-gamma in the skin draining lymph nodes. Identical results were observed using IL-12(-/-) mice, indicating that CD40 ligation promotes CHS responses and development of IFN-gamma-producing CD4(+) and CD8(+) T cells in the absence of IL-12. Engagement of CD40 on hapten-presenting Langerhans cells (hpLC) up-regulated the expression of both class I and class II MHC and promoted hpLC migration into the T cell priming site. These results indicate that hpLC stimulated by CD40 ligation use a mechanism distinct from increased IL-12 production to promote Ag-specific T cell development to IFN-gamma-producing cells.