CD27 is a thymic determinant of the balance between interferon-gamma- and interleukin 17-producing gammadelta T cell subsets

Abstract

The production of cytokines such as interferon-gamma and interleukin 17 by alphabeta and gammadelta T cells influences the outcome of immune responses. Here we show that most gammadelta T lymphocytes expressed the tumor necrosis factor receptor family member CD27 and secreted interferon-gamma, whereas interleukin 17 production was restricted to CD27(-) gammadelta T cells. In contrast to the apparent plasticity of alphabeta T cells, the cytokine profiles of these distinct gammadelta T cell subsets were essentially stable, even during infection. These phenotypes were established during thymic development, when CD27 functions as a regulator of the differentiation of gammadelta T cells at least in part by inducing expression of the lymphotoxin-beta receptor and genes associated with trans-conditioning and interferon-gamma production. Thus, the cytokine profiles of peripheral gammadelta T cells are predetermined mainly by a mechanism involving CD27.

Figure 1

CD27 expression defines distinct subsets of peripheral γδ T cells. (a) Flow cytometry of cells obtained from various tissues (above plots) of adult wild- type C57BL/6 mice, stained for CD3ε, CD27 and TCRγδ and gated on CD3ε⁺TCRγδ⁺ cells. a+i(LN), axillary and inguinal lymph nodes; mLN, mesenteric lymph nodes. Data are representative of three to eight independent experiments with three to five mice each. (b) Flow cytometry of splenocytes stained for CD3ε, CD27 and TCRγδ, plus CD62L or CD44 and gated on CD3ε⁺TCRγδ⁺CD27⁺ cells (γδ²⁷⁺) and CD3ε⁺TCRγδ⁺CD27⁻cells (γδ²⁷⁻). Data are representative of three independent experiments with three to five mice each. (c) Flow cytometry of γδ²⁷⁺ or γδ²⁷⁻ T cells sorted from pooled spleens and lymph nodes and cultured separately for 3 d with antigen-presenting cells and anti-CD3ε (1 mg/ml; left) or injected separately intravenously into RAG-2- deficient mice and analyzed after 2 weeks (right), then recovered, stained for CD3ε, CD27 and TCRδ and gated on CD3ε⁺TCRδ⁺ cells. Data are representative of three independent experiments. (d–f) Flow cytometry analyzing the proliferation of γδ²⁷⁺ or γδ²⁷⁻ T cells sorted from pooled spleens and lymph nodes, labeled with CFSE and cultured separately with antigen- presenting cells in presence of various doses of anti-CD3e (horizontal axis; d) or injected intravenously into RAG-2-deficient mice separately (e) or together at ratio of 1:1 (f), and assessed on the basis of CFSE-dilution kinetics at day 3. Data are representative of four independent experiments.

Figure 2

CD27 expression segregates IFN-γ- versus IL-17-producing γδ cells in naive and malaria-infected mice. (a) Quantitative real-time PCR of the expression of Ifnγ and II17 by γδ²⁷⁺ and γδ²⁷⁻ T cells sorted from pooled spleens and lymph nodes of naive C57BL/6 mice, normalized to Efa1 expression and presented as the percent of maximum expression of the gene of interest. (b) Intracellular staining for IFN-γ or IL-17 in γδ²⁷⁺ and γδ²⁷⁻ T cells sorted from pooled spleens and lymph nodes of naive or Plasmodium berghei ANKA–infected C57BL/6 mice (at day 4 after infection), then stimulated for 4 h with PMA and ionomycin. FSC, forward scatter. Numbers adjacent to outlined areas indicate percent IFN-γ⁺ cells (top row) or IL-17⁺ cells (bottom row). (c) Quantitative real-time PCR of the expression of II2rb (encoding CD122) and the gene encoding SCART2 (National Center for Biotechnology Information identification number 5830411N06Rik; called ‘Scart2’ here) by γδ²⁷⁺ and γδ²⁷⁻ T cells, as described in a. (d) Flow cytometry of splenocytes from naive mice, stained for CD3ε, CD27, TCRγδ and CD122 and gated on CD3ε⁺TCRγδ⁺ cells. Numbers in quadrants indicate percent cells in each. (e) Absolute number of total γδ T cells (left) and percent CD69⁺ cells (middle) and CD44^hiCD62L^lo cells (right) among γδ²⁷⁺ and γδ²⁷⁻ T cells from splenocytes obtained from naive mice (0) and mice at days 3 and 6 after infection with P. berghei ANKA and stained for CD3ε, CD27, TCRγδ and CD69, plus CD62L or CD44. (f) Absolute number of total γδ cells (left), γδ²⁷⁺ T cells (middle) and γδ²⁷⁻ T cells (right) among splenocytes obtained from naive or naive mice and mice at day 4 after infection with ‘titrated’ doses of P. berghei ANKA (5 ×10⁴, 5 ×10⁵ or 5 ×10⁶ parasites per mouse; to achieve variable parasitemia) and stained for CD3ε, CD27 and TCRγδ, presented relative to parasitemia (percent P. berghei ANKA in blood). Each symbol represents an individual mouse (n=4 mice/group). Data are representative of three independent experiments (a,c,e; error bars, s.d.; n=3 samples of four to five mice, pooled) or four to five (b), three (d) or two (f) independent experiments.

Figure 3

Constitutive expression of IL-17 and RORγt by γδ²⁷⁻ cells. (a) Quantitative real-time PCR analysis of II17 expression by CD4⁺CD25⁻ αβ T cells (4⁺25⁻) and γδ²⁷⁺ or γδ²⁷⁻ T cells sorted from pooled spleens and lymph nodes of C57BL/6 mice and assessed without activation (Fresh) or after activation for 4 d with plate-bound anti-CD3ε plus IL-2, with (+) or without (−) TGF-b, presented as described in Figure 2a. Data are representative of three independent experiments (error bars, s.d.; n=3 mice). (b) Intracellular staining of IFN-γ and IL-17 in CD27⁺ or CD27⁻ γδ and CD4⁺CD25⁻αβ⁻ T cells sorted from pooled spleens and lymph nodes from C57BL/6 mice, assessed without preactivation (Fresh) or after preactivation for 4 d on plate-bound anti- CD3ε and IL-2 (PreAct) with or without TGF-β, followed by stimulation with PMA and ionomycin. Data are representative of four independent experiments. (c) Intracellular staining for IL-17 and GFP in CD4⁺CD25⁻, γδ²⁷⁺ and γδ²⁷⁻ T cells isolated from pooled lymph nodes of RORγt-GFP mice. Data are representative of triplicate experiments with five mice each. (d) Intracellular staining for IFN-γ and IL-17 in flow cytometry–sorted γδ²⁷⁻ and γδ²⁷⁺ splenocytes activated in vitro for 3 d with anti-CD3ε and IL-2 in the presence (+) of various cytokines (above plots). Data are representative of three independent experiments. Numbers in quadrants (b–d) indicate percent cells in each. (e) Staining for surface V_γ1 or V_γ4 on and intracellular IL-17 in γδ²⁷⁺ and γδ²⁷⁻ cells sorted from pooled spleens and lymph nodes of C57BL/6 mice, preactivated for 4 d on plate-bound anti-CD3ε plus IL-2 and TGF-β, then stimulated with PMA and ionomycin. Data are representative of three independent experiments.

Figure 4

Both γδ²⁷⁺ and γδ^27– cells originate from common CD27⁺CD25⁺ thymic γδ progenitors. (a) Flow cytometry of thymocytes of C57BL/6 mice (n=3) gated on single-positive stage 4 cells (SP4; CD4⁺CD8^–) or γδ cells (TCRγδ⁺CD3ε). Dashed line, isotype control. (b) Flow cytometry of thymocytes (left) and splenocytes (right) from C57BL/6 mice, stained for CD3ε, CD27, CD25 and TCRγδ, gated on CD3ε⁺TCRγδ⁺ cells. Numbers in plots indicate percent cells in each area. (c) Proportion of the subsets defined n b among total γδ thymocytes from embryonic (E14–E18), newborn (days 1–9 (D0–D9)) or adult (6-week-old (W6)) C57BL/6 mice. (d) Flow cytometry of the cell size of the subsets defined in b. (e) Staining of thymocytes from newborn mice (day 2) with antibodies specific for various markers (below plots), gated on the subsets defined in b. (f) Incorporation of the thymidine analog BrdU (5-bromodeoxyuridine) by gated subsets (defined in b) of thymocytes obtained from C57BL/6 mice given BrdU daily intraperitoneally and then stained for CD27, CD25, TCRγδ and BrdU. (g) Intracellular staining of thymocyte subsets (defined in b) with 7- amino- actinomycin D (7-AAD), an indicator of DNA content. 27,25+, γδ^27,25+; 27+, γδ²⁷⁺; 27–, γδ^27–. (h) Flow cytometry of γδ^25,27+ thymocytes after sorting (Post-sort; left) and after the sorted cells were mixed with thymic stromal cell samples from E15 C57BL/6 mice, depleted of thymocytes and incubated for 7 d (RTOC; right). Numbers adjacent to or in outlined areas indicate percent cells in each. (i) Quantitative RT-PCR analysis of the expression of Ifnγ and II17 in various cell subsets, presented as described in Figure 2a. (j) Semiquantitative RT-PCR analysis of the expression of Rorc, Runx1 and Tbx21 in various cell subsets, normalized to Actb expression (encoding b-actin). For Rorc and Runx1, cells were sorted from pooled organs of over four mice; for Tbx21, γδ thymocyte subsets were purified from pooled FTOCs at day 6 (n>5 cultures). 4⁺8⁺, CD4⁺CD8⁺. Data are representative of three (a,d,e,h,j) or over ten (b) independent experiments or three (c,g,i) or two (f) independent experiments with three mice (f,g,i) or over three mice (c; error bars (c,f,g,i), s.d.).

Figure 5

CD27 controls the functional potential of γδ T cells. (a) Staining of TCRγδ and CD3 surface proteins on wild-type (WT) C57BL/6 and CD27-deficient (Cd27^−/−) thymocytes (left), and absolute number of TCRγδ⁺CD3⁺ thymocytes (right). Data are representative of five independent experiments (error bars, s.d.; n=3 mice). (b) Intracellular staining of various cytokines (left margin) in sorted total γδ T cells obtained from pooled spleens and lymph nodes of wild-type and CD27-deficient mice and stimulated with PMA and ionomycin. Right, percent of total γδ T cells. NS, not significant. *, P<0.05 (Student's t-test). Data are representative of four independent experiments with four to five mice each. (c,d) Absolute number of γδ T cells (c) or IFN-γ⁺ or IFN-γ^hi γδ T cells (d) among spleen cells of naive or P berghei ANKA–infected wild-type or Cd27^−/− mice. D4PI, day 4 after infection. *, P<0.05 (Student's t-test). Data are representative of two independent experiments (error bars, s.d.; n=5 mice).

Figure 6

CD27 signals regulate the differentiation of γδ thymocytes. (a) Quantitative RT-PCR analysis of the expression of Ifng and Tnfa in γδ²⁷⁺ cells sorted from TCRα-deficient mice (a) and TCRβ-deficient mice (b), presented as described in Figure 2a. Data are representative of two independent experiments (error bars, s.d.; n=3 mice). (b) Gene expression and fluorescence intensity of microarray analysis of wild-type and CD27- deficient (KO) γδ T cells. Black, genes with differences in expression of over twofold (WT/KO); gray, all other genes analyzed. P<0.00003 (adjusted) for genes selected as having differences in expression. Data are representative of two independent experiments. (c) Semiquantitative RT-PCR analysis of the expression of various genes (left margin) in total γδ thymocytes from wild-type or CD27-deficient mice. Data are representative of two independent experiments with triplicate samples of cells purified from four to six mice. (d) Quantitative RT-PCR analysis of the expression of Ltbr in total γδ thymocytes sorted from wild-type and CD27-deficient mice, presented as described in Figure 2a. *, P<0.001 (Student's t-test). Data are representative of two independent experiments (n=3 mice). (e) Semiquantitative RT-PCR analysis of the expression of various genes (left margin) in γδ subsets from wild-type thymi. Data are representative of two independent experiments with triplicate samples of cells purified from four to six mice. (f) Quantitative RT-PCR analysis of the expression of Ifng and II17 in γδ T cell subsets sorted at day 7 from FTOCs treated with a soluble recombinant fusion protein of CD70 and immunoglobulin (10 mg/ml; sCD70 +) or control human immunoglobulin G (sCD70 -), presented as described in Figure 2a. Data are representative of two independent experiments (error bars, s.d.; n=3 mice). *, P<0.05 (Student's t-test). (g) Flow cytometry of FTOCs at day 7, treated with medium alone, anti-CD3ε (0.67 mg/ml), anti–keyhole limpet hemocyanin (0.67 mg/ml; isotype control) or cyclosporin A (0.5 mM). Data are representative of two independent experiments with a pool of three E14 thymic lobes each.