Shp1 regulates T cell homeostasis by limiting IL-4 signals

Abstract

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell–intrinsic role of Shp1, we characterized mice with a T cell–specific Shp1 deletion (Shp1fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1(fl/fl) CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4⁺ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1- deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.

Figure 1.

T cell specific deletion of Shp1. (A) The indicated thymocyte populations were FACS sorted and Shp1 expression was determined by immunoblot. (B) Frozen sections of organs from Shp1^fl/fl and Shp1^fl/fl CD4-cre littermates were stained with hematoxylin and eosin. Images are representative of four littermate pairs. Bars, 250 µm.

Figure 2.

Thymocytes develop normally in the absence of Shp1. (A and B) Thymocytes from Shp1^fl/fl and Shp1^fl/fl CD4-cre mice were stained with antibodies specific for various surface markers. CD8 and CD4 profiles for total thymocytes and CD44 and CD25 expression for gated DN thymocytes are shown in A. Total cell numbers for each population are shown in B; n = 4. (C) Mature CD4⁺ and CD8⁺ SP thymocytes were evaluated by flow cytometry for the expression of the surface markers CD24, CD69, and CD5. Thymocytes from P14 Shp1^fl/fl and P14 Shp1^fl/fl CD4-cre mice were evaluated using a similar panel of antibodies. (D and E) Thymocytes were stained with α-CD4 and α-CD8 antibodies (D), and absolute number of the various subsets are shown in E; n = 3. (F) CD8 SP P14 thymocytes were gated and CD24, CD69, CD5, and Vα2 expression was assessed by flow cytometry. (G) CD8 SP P14 thymocytes stained with CFSE were cultured for 2 d with irradiated splenocytes and the indicated concentration of gp33. Statistical analyses were performed by one-way ANOVA (B) or Student’s t test (E); ns, P ≥ 0.05. Values for B and E are displayed as ± standard error.

Figure 3.

Shp1 restricts the development of memory phenotype T cells. T cells from Shp1^fl/fl or Shp1^fl/fl CD4-cre mice were stained with monoclonal antibodies against the indicated cell surface molecules. (A) Mature SP thymocytes and splenic T cells were assayed for CD44 expression by flow cytometry. (B) Percentage of splenic T cells with CD44^hi phenotype, displayed as ± standard error; n = 8. (C) Splenic T cells, gated based on expression of CD4, CD8, and CD44, were stained with antibodies against the indicated markers. (D) Expression of CD44 on splenic T cells from P14⁺ mice. Cells are gated on the CD8⁺ Vα2⁺ population. Statistical analysis of data in B was performed by Student’s t test; **, P < 0.01.

Figure 4.

Shp1-deficient T cells exhibit normal responses to TCR stimulation. (A) T cells were isolated from spleens, labeled with CFSE, and cultured on a 96-well plate coated with cross-linked antibodies against CD3 ± CD28. Cells were harvested 3 d later, stained for CD4 and CD8, and analyzed by flow cytometry. (B) Splenic T cells were sorted into CD44^hi and CD44^lo populations by FACS, labeled with CFSE, and cultured on a 96-well plate coated with cross-linked antibodies against CD3 and CD28 at the indicated concentrations. Cells were analyzed as in A. (C and D) Cells were treated as in A, harvested 6 h after stimulation, and analyzed for IL-2 (C) and TNF (D) expression by intracellular staining; n = 2. Data are representative of three independent experiments. Values for C and D are displayed as ± standard error.

Figure 5.

T cells skew to Th2 in the absence of Shp1. (A) Naive (CD44^lo) CD4⁺ T cells were isolated, stimulated for 3 d with α-CD3/CD28, and then restimulated with PMA/Ionomycin 4 d later. Cells were harvested and stained 6 h after restimulation. (B) Cells were stimulated and analyzed as in A. Percentages of cells staining positive for IFN γ or IL-4. Data are representative of three independent experiments and are displayed as ± standard error; n = 4. (C) Concentration of IgE and IgG2a in sera from the indicated unmanipulated age-matched mice. Horizontal bars represent sample means; n = 8. (D) T cells were stimulated with the indicated concentrations of IL-4 for 30 min and then fixed and stained for p-Stat6. Histograms are gated on CD44^lo T cells. (E) T cells were stimulated with 10 ng/ml IL-4 for 30 min and were then washed three times with media. Cells were harvested at the indicated times and analyzed as in D. Data are representative of three independent experiments. Statistical analyses of data in B and C were performed by Student’s t test; ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01.

Figure 6.

IL-4 is required for the accumulation of CD44^hi T cells in Shp1 conditional knockout mice. (A) Bone marrow from Thy1.1 and/or Shp1^fl/fl CD4-cre mice was transferred into irradiated CD45.1 host animals to generate mixed bone marrow chimeras. CD44 expression on splenic T cells was analyzed by flow cytometry. Flow plots are gated on CD45.2⁺Thy1.1⁺Thy1.2⁻ (Thy1.1) or CD45.2⁺Thy1.1⁻Thy1.2⁺ (Shp1 ^fl/fl CD4-cre) populations, as well as CD4⁺ or CD8⁺. (B) Percentage of splenic T cells with a CD44^hi phenotype in bone marrow chimeras from A.; n = 6–7. (C) CD44 expression on T cells in the blood of mice of the indicated genotypes. (D) Percentage of T cells with a CD44^hi phenotype in the indicated tissues of IL-4 and Shp1-deficient mice; n = 6–7. BLN, brachial LN; CLN, cervical LN; ILN, inguinal LN. (E) CD44 expression of T cells in blood of mice given 200 µg α-IL-4 or an isotype control 5 d before sacrifice. (F) Percentage of T cells with a CD44^hi phenotype in the blood of mice treated as in E. Data are representative of two independent experiments; n = 4. Statistical analyses of data in B, D, and F were performed by two-way ANOVA and Bonferroni’s post-test analysis; ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01. Horizontal bars for B, D, and F represent sample means.