FTY720 therapy exerts differential effects on T cell subsets in multiple sclerosis.

BACKGROUND: The oral immunomodulator FTY720 has shown efficacy in patients with relapsing multiple sclerosis (MS). FTY720 functionally antagonizes sphingosine 1-phosphate receptor-1 (S1P1) on T cells and consequently inhibits S1P/S1P1-dependent lymphocyte egress from secondary lymphoid organs. Little is known about the phenotype and function of T cells remaining in peripheral blood during long-term FTY720 treatment. METHODS: T cells from FTY720-treated, interferon-beta (IFNbeta)-treated and untreated patients with MS, and healthy donors (HD) were analyzed with respect to T cell subpopulation composition, proliferation, and cytokine production. RESULTS: In FTY720-treated patients (n = 16), peripheral blood CD4+ and CD8+ T cell counts were reduced by approximately 80% and 60% when compared to the other groups (IFN beta: n = 7; untreated: n = 5; HD: n = 10). This related to selective reduction of naive (CCR7+CD45RA+) and central memory (CCR7+CD45RA-) T cells (TCM), and resulted in a relative increase of peripheral effector memory (CCR7-CD45RA- [TEM] and CCR7-CD45RA+ [TEMRA]) T cells. The remaining blood T cell populations displayed a reduced potential to secrete IL-2 and to proliferate in vitro, but rapidly produced interferon-gamma upon reactivation, confirming a functional TEM/TEMRA phenotype. Neither FTY720 nor FTY720-P directly suppressed proliferation or cytokine production by T cells. CONCLUSION: Therapeutic dosing of FTY720 reduces naïve T cells and TCM, but not TEM, in blood, without affecting T cell function. This is presumably because naive T cells and TCM express the homing receptor CCR7, allowing recirculation to secondary lymphoid tissues on a regular basis and, thus, trapping of the cells by FTY720 in lymph nodes.

Activation of STAT proteins and cytokine genes in human Th1 and Th2 cells generated in the absence of IL-12 and IL-4.

We have shown previously that human CD4+ 45RO- T cells could be primed for a Th2 phenotype independent of IL-4 if they were activated by anti-CD28 mAb plus IL-2. If additional TCR signals were provided, the cells differentiated toward Th1 independent of IL-12. Here we show that anti-CD28/IL-2-primed Th2 cells expressed high levels of activated STAT6, but no cytokine mRNA. Moreover, both Th1 and Th2 cells expressed active STAT1 and -3, but not STAT2, -4, and -5. Restimulation of Th1 or Th2 cells via CD3 plus CD28 induced production of IFN-gamma or IL-4, respectively, but did not alter the activation status/DNA binding activity of STATs. Addition of IL-4 (or anti-IL-4 mAb) to restimulated Th2 cells did not modulate STAT6 activation or IL-4 expression, confirming the full commitment. However, Th2 cells remained responsive to IL-12, which repressed STAT6 DNA binding but activated STAT4, and this coincided with a suppression of IL-4/IL-5 and an induction of IFN-gamma. In Th1 cells, IL-12 activated both STAT6 and STAT4, and IL-4 activated STAT6, but in both cases the Th1 phenotype remained. Together the data show that CD28/IL-2-dependent Th2 priming activated STAT6 without inducing IL-4 expression. The primed Th cells resembled memory cells and produced IL-4 upon the first CD3/CD28 costimulus without detectable modulation of STATs. Th2 cells remained responsive to IL-12, which repressed STAT6 DNA binding and activated STAT4, and switched the cells to Th1. The effects of IL-12 may depend on the commitment of the cells, since IL-12 phosphorylated STAT6 in Th1 and dephosphorylated STAT6 in Th2 cells.

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.

Role of IL-13 in CD4 T cell-dependent IgE production in atopy.

IgE isotype switching of human B cells requires physical interaction of T and B cells via surface molecules, and either IL-4 or IL-13 secreted by T cells. In this study we analyzed the role of IL-4 versus IL-13 in IgE production in atopy. We found that peripheral blood mononuclear cells (PBMC) from atopic individuals but not from nonatopic subjects secreted IgE without addition of IL-4 or IL-13, if T and B cells were simultaneously activated by anti-CD3 mAb and soluble CD40L, respectively. IgE production by atopic PBMC was dependent on endogenously secreted IL-4 and IL-13, since it could be blocked by a combination of anti-IL-4 plus anti-IL-13 antibodies. No differences in the B cell compartment of nonatopics and atopics were detectable, since PBMC from both donor populations secreted comparable amounts of IgE, if only the B cells were activated by soluble CD40L plus either exogenous IL-4 or IL-13. Further phenotypic analysis of T cells from atopics revealed that activated CD4+45RO- secreted IL-4 but no IL-13, whereas CD4+45RO+ memory T cells secreted low amounts of IL-4, but large amounts of IL-13. Accordingly, prolonged activation of native CD4+45RO- T cells in vitro induced expression of CD45RO, and strongly favored secretion of IL-13 rather than IL-4. Addition of exogenous IL-4 during activation further increased both IL-4 and IL-13 production to a similar degree. However, the potential of CD4 T cells from atopics to deliver contact-dependent activation signals to B cells and to induce IgE production (in the absence of soluble CD40L) increased with prolonged activation, and coincided with IL-13 rather than IL-4 production. Under similar conditions, CD8 effector cells secreted IL-13 but no IL-4, did not express CD40L, and could not help Ig(E) production by B cells. These results suggest that, in atopy, persistently stimulated CD4+45RO+memory/effector T cells provide contact-dependent activation signals to B cells, and that these cells may induce IgE switching largely via secretion of IL-13.

TCR-independent activation of human CD4+ 45RO- T cells by anti-CD28 plus IL-2: Induction of clonal expansion and priming for a Th2 phenotype.

In this study we show that uncommitted human CD4+ CD45RA+ RO- CD25- CD71- HLA-DR- T cells can be primed for a Th2 phenotype before they encounter TCR signals and before they are exposed to IL-4. We found that >99% of uncommitted T cells proliferated upon costimulation by immobilized anti-CD3 plus anti-CD28 mAbs and differentiated into pure Th1 cells. In contrast, uncommitted T cells did not respond to stimulation by either anti-CD3 or anti-CD28, or by IL-2 alone. Interestingly, 5% of uncommitted T cells proliferated efficiently in response to stimulation by immobilized anti-CD28 plus IL-2 (in the absence of TCR/CD3 signals) and differentiated into pure Th2 "precursor" cells. Like murine CD4+ NK1.1+ T cells, human Th2 precursors promptly expressed mRNA for Th2 cytokines upon stimulation via the TCR/CD3 complex by anti-CD3 mAb or staphylococcal enterotoxin B, and secreted up to 50 ng of IL-4, IL-5, and IL-13 per 10(6) cells. Th2 "precursors" developed only in the complete absence of IL-4, as addition of 0.1 U (5 pg) of exogenous IL-4 suppressed their clonal expansion by >90%, whereas addition of neutralizing anti-IL-4 mAb had no effect. Together these results suggest that, in vivo, a significant fraction of uncommitted T cells may be primed for a Th2 phenotype independent of Ag and IL-4 if they are exposed to Th1 cell-derived IL-2 and simultaneously interact with accessory cells bearing the natural CD28 ligands B7-1 and B7-2. When stimulated by specific Ag, such primed Th2 precursor cells may provide a source of IL-4 to promote Th2 immunity.

Regulation by corticosteroids of Th1 and Th2 cytokine production in human CD4+ effector T cells generated from CD45RO- and CD45RO+ subsets.

Corticosteroids (CS) are widely used as immunosuppressive and anti-inflammatory agents, but their mechanism of action is not well understood. In this study we analyzed the effects of CS on the growth and differentiation of human CD4+45RO- "naive" and CD4+45RO+ "memory" T cells. To generate effector T cells secreting large amounts of Th1 and Th2 cytokines, FACS-sorted naive and memory subsets were primed and restimulated in vitro via the TCR in the presence of IL-2. CS added during priming reduced clonal expansion of both T cell populations, but the memory subset was 100-fold less sensitive. At lower concentrations, CS favored the development of effector T cells (from both subsets), which upon restimulation produced large amounts of the anti-inflammatory cytokine IL-10, but low amounts of IL-4, IL-5, or IFN-gamma. Interestingly, CS displayed different effects if it was added only during the restimulation of effector T cells. CS were unable to suppress clonal expansion of restimulated effector T cells. In effector T cells derived from the naive subset, CS induced production of IL-4 and IL-10, but blocked production of IL-5 and IFN-gamma. In effector T cells generated from the memory subset, CS blocked production of IL-4, IL-5, and IL-10, but inhibited production of IFN-gamma by only 50%, even if 100-fold higher concentrations of CS were applied. These results indicate that persistent TCR stimulation, e.g., in chronic infection, may reduce the sensitivity of T cells to the antiproliferative effects of CS. Furthermore, the potential of CS to increase or suppress IL-4 and IL-10 production depending on the stage of T cell activation may explain in part the beneficial effects of CS in the treatment of acute inflammation and chronic allergic/asthmatic diseases.

TCR-stimulated naive human CD4+ 45RO- T cells develop into effector cells that secrete IL-13, IL-5, and IFN-gamma, but no IL-4, and help efficient IgE production by B cells.

It is currently believed that IgE production by B cells is induced by activated CD4+ Th2-like cells that produce increased amounts of IL-4 but low levels of IFN-gamma. We found that "naive" CD4+ 45RO- T cells primed and restimulated in vitro via the TCR developed into effector cells that produced large amounts of IL-13, IL-5, and IFN-gamma, but no IL-4. Such CD4 T cells induced surface IgD+E- B cells to produce large amounts of IgE. The IgE response could be blocked completely by neutralizing anti-IL-13 Abs, whereas anti-IL-4 had no effect. Addition of exogenous IL-4 during priming of CD4 cells suppressed clonal expansion and the development of Th cells including helpers for IgE, but increased endogenous production of IL-4 and IL-5, and suppressed production of IFN-gamma. Addition of exogenous IFN-alpha, IFN-gamma, or neutralizing anti-IFN-gamma mAbs affected neither priming of CD4 T cells nor B cell help. In contrast to CD4+ 45RO- naive T cells, CD4+ 45RO+ "memory" T cells primed and restimulated in vitro secreted IL-4 in addition to IL-13, IL-5, and IFN-gamma, and the IgE response induced by such cells could be blocked only by a combination of anti-IL-4 plus anti-IL-13 Abs. Unlike CD4 cells, CD8 cells could not be primed to help IgE production. The results indicate that TCR/CD3 cross-linking on naive human CD4 T cells is sufficient to induce the development of potent IgE helper cells, which secrete large amounts of IL-13, IL-5, and IFN-gamma, but no IL-4. Such Th cells may exacerbate atopic inflammation, because all by themselves they could drive IL-13-dependent IgE production, IL-5-dependent eosinophilia, and IFN-gamma-dependent macrophage activation, but could not suppress IFN-gamma production by other T cells via IL-4.