The Protein Phosphatase Shp1 Regulates Invariant NKT Cell Effector Differentiation Independently of TCR and Slam Signaling

Abstract

Invariant NKT (iNKT) cells are innate lipid-reactive T cells that develop and differentiate in the thymus into iNKT1/2/17 subsets, akin to T_H1/2/17 conventional CD4 T cell subsets. The factors driving the central priming of iNKT cells remain obscure, although strong/prolonged TCR signals appear to favor iNKT2 cell development. The Src homology 2 domain-containing phosphatase 1 (Shp1) is a protein tyrosine phosphatase that has been identified as a negative regulator of TCR signaling. In this study, we found that mice with a T cell-specific deletion of Shp1 had normal iNKT cell numbers and peripheral distribution. However, iNKT cell differentiation was biased toward the iNKT2/17 subsets in the thymus but not in peripheral tissues. Shp1-deficient iNKT cells were also functionally biased toward the production of T_H2 cytokines, such as IL-4 and IL-13. Surprisingly, we found no evidence that Shp1 regulates the TCR and Slamf6 signaling cascades, which have been suggested to promote iNKT2 differentiation. Rather, Shp1 dampened iNKT cell proliferation in response to IL-2, IL-7, and IL-15 but not following TCR engagement. Our findings suggest that Shp1 controls iNKT cell effector differentiation independently of positive selection through the modulation of cytokine responsiveness.

Figure 1.

Shp1-deficient iNKT cells are biased towards iNKT2 and iNKT17 subsets. (A) Frequency (top row) and absolute numbers (bottom row) of iNKT cells in the indicated tissues of Shp1^fl/fl and Shp1^fl/fl CD4-cre mice. (B, C) iNKT cell subsets in the thymus (B) and spleen (C) of Shp1^fl/fl (fl/fl) and Shp1^fl/fl CD4-cre (cre) mice were assessed by PLZF and RORγt staining. (D) Frequency of iNKT cells in the thymus of single or mixed (mix) bone marrow chimeras. Rag1^−/− mice were reconstituted with wild type CD45.1 and/or Shp1^fl/fl CD4-cre CD45.2 bone marrow cells. (E, F) Frequency of iNKT2 and iNKT17 subsets in the thymus (E) and spleen (F) of 16-week-old Shp1^fl/fl and Shp1^fl/fl CD4-cre mice. Data shows representative dot plots, individual mice and the mean values +/− s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student t test.

Figure 2.

Shp1-deficient iNKT cells produce more T_H2 cytokines. (A) Shp1^fl/fl and Shp1^fl/fl CD4-cre mice were injected i.v. with αGC (0.5 μg). Intracellular FACS analysis of IFN-γ and IL-4 in spleen iNKT cells 90 min post-injection. Data shows representative flow cytometry plots (left), as well as the corresponding frequency (individual mice and mean values +/− s.e.m.) of total, single and double producers of IFN-γ and/or IL-4 (right). (B, C) Thymocytes (B) and splenocytes (C) from Shp1^fl/fl and Shp1^fl/fl CD4-cre mice were stimulated with plate-bound CD1d-αGC, or PMA/ionomycin (P/I) for 6 hours. Intracellular FACS analysis of IFN-γ and IL-4 in iNKT cells. Data shows representative flow cytometry plots (left), as well as individual and mean values +/− s.e.m. of IL-4/IFN-γ ratios. (D) Shp1^fl/fl and Shp1^fl/fl CD4-creER^T2 mice were treated with tamoxifen and subsequently injected i.v. with αGC (0.2 μg). Data shows individual and mean values +/− s.e.m. of IFN-γ⁺ or IL-4⁺ iNKT cells, as well as IL-4/IFN-γ ratios. (E) iNKT cells from Shp1^fl/fl or Shp1^fl/fl CD4-cre mice were sorted and cultured with BMDCs alone, BMDCs pre-loaded with αGC, or BMDCs in the presence of the indicated cytokine for 48 h. in the presence of indicated cytokines. IFN-γ, IL-4, IL-13, IL-17A and IL-22 production was assessed using a multiplex assay. (F) iNKT cells from Shp1^fl/fl or Shp1^fl/fl CD4-cre mice were sorted and cultured with BMDCs with isotype control, anti-CD25 or anti-CD127 antibodies (40 μg/ml) for 48 h. Data shows the mean +/− s.e.m. of triplicate values and is representative of 2 individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student t test (A-D) or two-way ANOVA (E).

Figure 3.

Shp1-deficient iNKT cells drive the expansion of innate memory CD8 T cells in the thymus. (A) Frequency of Eomes⁺ CD8 T cells in the thymus (left) and spleen (right) of Shp1^fl/fl and Shp1^fl/fl CD4-cre mice. (B) BrdU incorporation by CD44^high CD8 T cells from the thymus (left) and spleen (right) of Shp1^fl/fl and Shp1^fl/fl CD4-cre mice. (C) Frequency of Eomes⁺ CD8 T cells in the thymus (top) and spleen (bottom) of Shp1^fl/fl CD1d^+/−, Shp1^fl/fl CD1d^−/−, Shp1^fl/fl CD4-cre CD1d^+/− and Shp1^fl/fl CD4-cre CD1d^−/− mice. Data represents individual experiments and mean values +/− s.e.m of 2 or 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student t test.

Figure 4.

Shp1 deficiency does not affect iNKT TCR signaling. (A) Expression of Egr2 in thymic iNKT cells from Shp1^fl/fl (fl/fl) and Shp1^fl/fl CD4-cre (cre) mice was determined by FACS. (B) Expression of Egr2 and PLZF in thymic stage 0 (CD24^hi CD44^™ NK1.1^™), stage 1 (CD24^™ CD44^™ NK1.1^™), stage 2 (CD24^™ CD44⁺ NK1.1^™) and stage 3 (CD24^™ CD44⁺ NK1.1⁺) iNKT cells from Shp1^fl/fl (fl/fl) and Shp1^fl/fl CD4-cre (cre) mice. (C) Frequency of Nur77⁺ and Egr2⁺ thymic (top) or splenic (bottom) iNKT cells upon stimulation with different concentrations of anti-CD3 antibodies. (D) Splenocytes from Shp1^fl/fl (fl/fl) and Shp1^fl/fl CD4-cre (cre) mice were labelled with CellTrace™ CFSE and stimulated for 72 h with the indicated concentrations of anti-CD3 (top) or anti-CD3/CD28 (bottom) antibodies. Proliferation of iNKT cells (TCRβ⁺ PBS57-CD1d Tetramer⁺) was assessed by FACS. (E) Frequency of Vβ8⁺ and Vβ7⁺ iNKT cells in the thymus (left) and spleen (right) of Shp1^fl/fl and Shp1^fl/fl CD4-cre mice. Data represents individual mice and mean +/− s.e.m of 2-3 independent experiments (A, E) or the mean +/− s.e.m. of 2 to 3 independent experiments (n ≥ 4). *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student t test.

Figure 5.

Shp1 deletion does not affect Slamf6 mediated upregulation of Egr2 and PLZF. (A) Frequency of live PLZF⁺ PSDPs from Shp1^fl/fl and Shp1^fl/fl CD4-cre mice after 48 hours of stimulation with the indicated immobilized antibodies. (B) PSDPs were stimulated stimulation with anti-CD3 and anti-Slamf6 antibodies for 30 min., washed and analyzed for Egr2 expression at the indicated times following stimulus withdrawal. (C) Frequency of iNKT cells in the thymus of SAP^−/− Shp1^fl/fl, SAP^−/− Shp1^fl/fl CD4-cre and SAP^+/− Shp1^fl/fl CD4-cre mice Representative dot plots gated on live cells are shown. Data represents individual mice and mean +/− s.e.m. of 2-3 independent experiments.

Figure 6.

Shp1 regulates cytokine-mediated iNKT cell proliferation. (A) Representative histogram plots (3 mice per group) of CFSE dilution profile of iNKT cells sorted from the thymus (left) or the spleen (right) of Shp1^fl/fl and Shp1^fl/fl CD4-cre mice cultured for 4 days in the presence of 10ng/ml of IL-2, IL-7 or IL-15. One experiment out of two is shown. (B) BrdU incorporation by iNKT cells from the thymus (left) and spleen (right) of Shp1^fl/fl and Shp1^fl/fl CD4-cre mice. (C) Expression of CD25, CD127 and CD122 on thymic (left) and splenic (right) iNKT cells from Shp1^fl/fl and Shp1^fl/fl CD4-cre mice. Data shows representative histogram plots as well as individual mice and the mean values +/− s.e.m of 2-3 independent experiments.