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Physiological sphingosine 1-phosphate requirement for optimal activity of mouse CD4⁺ regulatory T Cells ¹

Departments of Medicine and Microbiology-Immunology, University of California, San Francisco, California, USA

² Correspondence: University of California, UB8B, Box 0711, 533 Parnassus at 4th, San Francisco, CA 94143-0711, USA. E-mail: egoetzl{at}itsa.ucsf.edu

SPECIFIC AIMS

The constitutive subset of regulatory CD4⁺25⁺ T cells is vital to maintaining immune homeostasis and suppressing autoimmunity, but little is known of their controls. As normal blood concentrations of sphingosine 1-phosphate (S1P) affect many T cell functions through their type 1 G-protein-coupled receptors (S1P₁ Rs), this study was designed to profile CD4⁺25⁺ T cell S1P Rs and functional responses to S1P.

PRINCIPAL FINDINGS

1. CD4⁺25⁺ regulatory T cells express principally S1P₁ Rs, which are down-regulated by T cell activation, as for S1P₁ Rs of other T cells
mRNA for real-time PCR analyses was isolated from highly purified CD4 T cells of each subset immediately after isolation and 24 h after incubation with anti-CD3 plus anti-CD28 monoclonal antibodies (MoAbs) or IL-2 (Table 1 ). The freshly isolated unstimulated CD4 subsets showed identical patterns of S1P Rs with S1P₁>S1P₄, but no detectable level of any other type (lines 1 and 4 of Table 1 ). T cell receptor-dependent activation of both CD4 subsets by anti-CD3 MoAbs suppressed the levels of S1P₁ and S1P₄ GPCRs, whereas incubation for the same period with IL-2 had no effect on their expression (Table 1) . Activation-induced CD4⁺25⁺ T cells were generated by incubation of the purified CD4⁺25^– T cell subset with adherent anti-CD3 plus anti-CD28 MoAbs and were isolated before extraction of mRNA for comparison with native CD4⁺25⁺ T cells. In sharp contrast to the high levels of expression of S1P₁ and S1P₄ GPCRs by the native subset, T cell receptor activation-induced CD4⁺25⁺ T cells showed only low levels of S1P₁ and S1P₄ GPCRs (Table 1) . T cell mRNA levels of S1P GPCRs always accurately reflect protein levels, so it was not surprising that Western blots of proteins extracted from the same subsets of CD4 T cells confirmed the results of analyses of mRNA (not shown).

2. S1P is required for optimal suppressive activities of CD4⁺25⁺ T cells
Unconventional conditions were identified for T cell stimulation using concentrations of fluid-phase anti-CD28 MoAb much lower than usual and only one-fifth that of anti-CD3 to improve T cell survival after 24 and 48 h without increasing resistance to the suppressive effects of CD4⁺25⁺ T cells. The ratio of CD4⁺25⁺ T cells to CD4⁺25^– T cells was 1:10 in all studies, which reflects that in native T cell populations more closely than in most protocols, so that the level of suppression without S1P was low enough to allow a consistent enhancing effect of S1P. Under these conditions, a mixture of the two subsets showed ³H-thymidine uptake of only 69% of the sum of the separate values or 31% inhibition, reflecting the suppressive effect of the CD4⁺25⁺ T cells (Fig. 1 ). The addition to this mixture of S1P, which had no suppressive effect alone, significantly increased inhibition at 10^–7 M to 10^–6 M, up to a maximum of 50%, which was similar to the value observed with 10% normal FCS supplemented with 100 nM S1P.

View larger version (31K): [in this window] [in a new window]   Figure 1. Mouse CD4+25+ Treg cell requirement for a physiological concentration of S1P. Each column and bar represents the mean ± SD of the results of 3 experiments. Control values (0% inhibition) for the sum of 3H-thymidine uptake by CD4+25– T cells plus that by CD4+25+ T cells stimulated separately with soluble anti-CD3 plus anti-CD28 monoclonal antibodies were 136,256 to 261,708 dpm. Similar control values (0% inhibition) for IL-2 generation were 2.6 to 5.0 ng/mL. S1P preincubation was with CD4+25+ T cells (left) or CD4+25– T cells (center). Statistical significance of differences between each mean value and that for CD4+25– T cells plus CD4+25+ T cells without any lipid or FCS (left-hand bars) was calculated by a paired t test; +P < 0.05 and *P < 0.01.

The dependence of IL-2 secretion on S1P was greater than that of proliferation (Fig. 1) . Stimulated CD4⁺25^– T cells alone secreted 3.8 ± 0.7 ng/mL of IL-2 (mean±SD, n=10) whereas CD4⁺25⁺ T cells produced no detectable IL-2. In a mixture of the two subsets, inhibition of IL-2 secretion by CD4⁺25⁺ T cells in the absence of S1P was a mean of 20%. S1P enhanced this inhibitory effect significantly at 10^–9 M to 10^–6 M, up to a maximum of 51% and 60% at 3 x 10^–7 M and 10^–6 M, respectively, which are indistinguishable from the effect of 10% FCS with a normal level of 100 nM S1P. S1P alone had no effect on IL-2 secretion by CD4⁺25^– T cells. That S1P enhancement of inhibition is dependent on the CD4⁺25⁺ T cell subset was supported by results of preincubating each subset separately with S1P and washing prior to coculture.

3. Optimal generation of IL-10 by CD4⁺25⁺ T cells requires S1P and may contribute to the suppressive activities of these Treg cells
The proposed involvement of IL-10 in the regulatory T cell (Treg) activities of CD4⁺25⁺ T cells is a controversial subject with discrepant, but mostly negative, findings in vitro. We suggest that such a role for IL-10 depends on normal levels of S1P and a physiological ratio of CD4⁺25⁺ T cells to CD4⁺25^– T cells of 1:10. To determine whether S1P is required for generation and secretion of suppressive concentrations of IL-10, CD4⁺25⁺ T cells were stimulated without and with S1P. The CD4⁺25⁺ T cells produced low mean levels of 46 pg/mL, 70 pg/mL, and 120 pg/mL of IL-10 per 10⁶ cells at 24, 48, and 72 h, respectively, in S1P-free medium. At 3 x 10^–9 M to 3 x 10^–8 M S1P, IL-10 generation by CD4⁺25⁺ T cells was increased 2.5-fold after 48 h to attain a level similar to that in 10% normal FCS. After 72 h, the greatest increases in generation of IL-10 of twofold were observed at one of the same concentrations of S1P, and the maximal effect again was similar in magnitude to that of 10% normal FCS. In contrast, IL-10 generation by stimulated CD4⁺25^– T cells had a mean of only 27% that by CD4⁺25⁺ T cells without S1P after 48 h and was decreased by up to 25% by the same concentrations of S1P that optimally augmented IL-10 generation by CD4⁺25⁺ T cells.

To examine possible functional roles of IL-10 generated by S1P-exposed and stimulated CD4⁺25⁺ T cells, replicate suspensions of CD4⁺25⁺ T cells were preincubated without and with 3 x 10^–8 M S1P and neutralizing antibody to IL-10 before addition to CD4⁺25^– T cells, which had been preincubated without and with neutralizing antibody to IL-10 receptor. Inhibition of ³H-thymidine uptake and IL-2 generation was enhanced significantly by S1P; this effect was diminished significantly in the presence of antibody to IL-10 or IL-10 receptor. Neither neutralizing antibody alone affected ³H-thymidine uptake or IL-2 generation by CD4⁺25^– T cells significantly. In two studies, concentrations of purified synthetic IL-10 similar to those generated by S1P-stimulated CD4⁺25⁺ T cells were added to cultures of CD4⁺25^– T cells after 2 h of incubation with anti-CD3 plus anti-CD28 MoAbs, and IL-2 in the supernatant was quantified after 48 h. IL-2 levels were suppressed by a mean of 36% and 58%, respectively, by 100 pg/mL and 200 pg/mL of IL-10. Likely participation of S1P₁ Rs was established by incubating replicate suspensions of CD4⁺25⁺ T cells with 100 nM FTY720 for 16 h at 37°C under conditions where S1P₁ Rs but not S1P₄ Rs are down-regulated. S1P did not enhance Treg activity of FTY720-treated CD4⁺25⁺ T cells.

CONCLUSIONS AND SIGNIFICANCE

The major effects of S1P on T cell functions are highly S1P concentration dependent. At levels of 3–100 nM found in many tissues, S1P exerts principally supportive, permissive, and stimulatory effects on T cells. In this range, S1P evokes direct chemotaxis, enhances chemotaxis to chemokines optimally, suppresses apoptosis, and also supports full activity of cytotoxic T cells. In contrast, blood and lymph concentrations of 0.3 to 3.0 µM S1P are uniformly inhibitory of T cell functions. In this range, S1P inhibits chemokine-elicited chemotaxis by up to 90%. The capacity of S1P but not LPA to inhibit chemotactic responses of naïve and possibly memory T cells to chemokines is a unique and quantitatively striking effect. The chemotactic inhibitory activity of blood and lymph concentrations of S1P is one basis for a new conceptual model of the T cell regulatory activities of S1P and explains one mechanism of action of an immunosuppressive drug designated FTY720, which also acts on many S1P GPCRs

Enhancement by S1P of the Treg activities of CD4⁺25⁺ T cells was significant over the broad concentration range of 10^–9–10^–6 M (Fig. 1) , suggesting contributions of both low- and high-concentration mechanisms. Stimulation of production of IL-10 by CD4⁺25⁺ T cells was maximal at 3 to 100 nM S1P, and thus IL-10 is a candidate mediator for enhancement of Treg activities by lower concentrations of S1P (Fig. 2 ). However, the mechanisms responsible for enhancement of Treg activities by higher concentrations of S1P have not been identified and may involve one or more of the requirements for cell–cell contact. CTLA-4 has been suggested as one of these T cell surface determinants. Preliminary data confirm a role for CTLA-4 in the enhancement of Treg activities of CD4⁺25⁺ T cells by higher concentrations of S1P. At 0.25 µg/mL, mouse CTLA-4-IgG fusion protein (BD-PharMingen, Inc., San Diego, CA, USA), capable of blocking interactions of CTLA-4 with its immune counter ligands, prevented enhancement of CD4⁺25⁺ T cell inhibition of IL-2 generation by 3 x 10^–7 M S1P. Although the mechanisms are not fully defined, trophic enhancement of Treg activities of CD4⁺25⁺ T cells by S1P concentrations of up to normal in vivo physiological levels should be considered one of the immunological functions of the S1P-S1P₁ R axis (Fig. 2) .

View larger version (12K): [in this window] [in a new window]   Figure 2. Schematic diagram depicting the trophic roles of the S1P-S1P1 R axis in regulatory functions of CD4+25+ T cells. TCR = T cell antigen receptor. Solid arrows in cells depict stimulation of expression, secretion, and proliferation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1555fje;

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