Research ArticleINFECTIOUS DISEASE

Memory-phenotype CD4+ T cells spontaneously generated under steady-state conditions exert innate TH1-like effector function

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Science Immunology  16 Jun 2017:
Vol. 2, Issue 12, eaam9304
DOI: 10.1126/sciimmunol.aam9304
  • Fig. 1 MP cells are generated from naïve cells in the periphery.

    (A) SPF and GF mice have an equal number of MP cells. Splenic CD4+ T lymphocytes from 8-week-old wild-type mice housed in SPF or GF conditions were analyzed for expression of CD44 and CD62L. The representative dot plots indicate the frequency of MP and naïve cells among CD4+ T cells. The bar graphs indicate the number (mean ± SD) of MP and naïve CD4+ T cells from each group (n = 3 to 4). Data are representative of two independent experiments. (B and C) Kinetics of splenic MP and naïve CD4+ T cell development and their proliferative capacity. At the indicated ages, (B) the absolute cell number (mean ± SD) of MP and naïve CD4+ T lymphocytes and (C) the frequency (mean ± SD) of Ki67+ cells among MP and naïve cells were monitored in cohort of wild-type mice [n = 2 to 4 and 2 to 8 per time point in (B) and (C), respectively]. The representative dot plots show the frequency of MP and naïve cells among CD4+ T cells of 1- and 28-day-old mice. Data are pooled from at least two independent experiments. (D) MP CD4+ T cells in Rag2-GFP reporter mice are GFP. The histogram shows the GFP+ fraction among splenic CD4+ T cells in Rag2-GFP reporter mice, whereas the dot plots and the bar graph indicate the frequency of MP and naïve cells among GFP and GFP+ CD4+ T cells (n = 5). Data are representative of two independent experiments. (E and F) A normal-sized MP cell population is generated from peripheral CD4+ T cells in the absence of the thymus. (E) The graph shows the frequency (mean ± SD) of CD4+ T cells in the blood from sham-operated and TXD1 mice (n = 3 to 17 per time point) over a 6-month period. (F) Numbers in the representative plots indicate the frequency of MP and naïve cells among peripheral blood CD4+ T lymphocytes on day 141. The bar graphs show the number (mean ± SD) of MP and naïve CD4+ T cells from each group (n = 5 to 6). Data are pooled from at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 MP cell conversion occurs in both neonates and adults dependently on TCR and CD28 signaling.

    (A and B) Naïve CD4+ T lymphocytes give rise to MP cells in neonatal and adult mice. Sorted naïve CD4+ T cells from CD45.2 wild-type mice were transferred into 1-day-old neonatal or 8-week-old adult CD45.1/2 animals, and donor cells were analyzed in the spleen 1, 2, 4, and 8 weeks later. Representative dot plots show CD44 and CD62L expression by the donor cells, and the graphs show (A) the number (mean ± SD) of MP and naïve donor cells and (B) the frequency (mean ± SD) of Ki67+ cells among MP donor cells (n = 3 to 8). Data are pooled from two independent experiments. (C) The MP conversion rate correlates with age of host animals. Sorted naïve CD4+ T cells from CD45.2 mice were transferred into 1-, 3-, 7-, 14-, and 28-day-old CD45.1/2 mice, and the donor cells were analyzed for CD44 and CD62L expression 6 weeks later. The graphs show the frequency and the number (mean ± SD) of MP cells among the donor cells at each time point (n = 3 to 7). Data are pooled from three independent experiments. (D) The MP conversion rate decreases, but the absolute number of generated MP cells increases with age. The number of MP cells that naïve CD4+ T cells from 1-, 3-, 7-, 14-, and 28-day-old mice can generate 6 weeks later was calculated based on the absolute number of naïve CD4+ T lymphocytes obtained in Fig. 1B and the MP conversion rate obtained in (C). (E) Fast proliferation of naïve CD4+ T lymphocytes requires TCR and CD28 signaling in neonates and adults. Sorted, CFSE-labeled naïve CD4+ T cells from CD45.2 wild-type mice were transferred into 1-day-old neonatal or 8-week-old adult CD45.1/2 recipients that were treated with anti–I-Aβ, CTLA4-Ig, or control IgG and were analyzed 3 weeks later. Representative histograms indicate CFSE dilution of the donor cells in the spleen, whereas the bar graph shows the frequency (mean ± SD) of CFSE cells among donor cells from each group (n = 2 to 3). (F) MP cell generation requires host MHC II expression. CD45.1 naïve CD4+ T cells were transferred into CD45.2 wild-type or MHC II KO mice and were analyzed 3 weeks later. Representative dot plots display CD44 and CD62L expression of donor cells, whereas the graph shows the frequency (mean ± SD) of MP population among donor cells (n = 3). (G) MP cell generation depends on CD28 signaling. The dot plots show expression of CD44 and CD62L by CD4+ T cells from wild-type and CD28 KO mice, whereas the bar graph shows the frequency (mean ± SD) of MP cell population among CD4+ T cells (n = 3). (H) CD5hi naïve cells can generate more MP cells than CD5lo cells. Sorted CD5hi and CD5lo naïve CD4+ T cells from CD45.2 and CD45.1 mice, respectively, were transferred at a 1:1 ratio into 1-day-old and 8-week-old CD45.1/2 recipients and were analyzed 3 weeks later. Representative plots indicate CD44 and CD62L expression by the donor cells in the spleen, whereas the graphs show the number of MP and naïve cells generated from paired CD5hi and CD5lo naïve donor cells in an individual host mouse (n = 4 to 8). Data are pooled from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

  • Fig. 3 MP cells consist of rapidly proliferating CD5hi and relatively quiescent CD5lo cells, both of which require CD28 signaling for Ki67 expression.

    (A and B) MP cells are composed of proliferating CD5hi and relatively quiescent CD5lo cells in both SPF and GF mice. (A) Representative dot plots of CD5 and Ki67 expression on splenic MP cells. (B) Histograms of CD5 expression by MP cells used to define 10 bins. The graph depicts the frequency (mean ± SD) of Ki67+ fraction among MP cells of each CD5 bin (n = 3 to 4). Data are representative of two independent experiments. (C and D) CD5hi MP cells die and proliferate more rapidly than do CD5lo MP cells. (C) Splenic CD5hi and CD5lo MP and naïve cells were analyzed for BrdU incorporation and 7-AAD staining 1 hour after in vivo BrdU pulse of wild-type mice. The numbers in the dot plots show the frequency of BrdU+ (S phase) and BrdU 7-AAD+ (G2-M phase) cells among each CD4+ T cell subpopulation, whereas the bar graph indicates the frequency (mean ± SD) of cells in S and G2-M phase (n = 3). (D) Splenic MP and naïve cells were stained with amine-reactive dye and annexin V. Dot plots indicate the frequency of amine-reactive dye+ annexin V+ (dead) and amine-reactive dye annexin V+ (early apoptotic) cells among each CD4+ T cell subpopulation, whereas the bar graph shows the frequency (mean ± SD) of dead and early apoptotic cells (n = 3). (E) CD5 expression is maintained in MP conversion. CD5lo and CD5hi naïve CD4+ T cells from CD45.1 (blue line) and CD45.2 (red line) mice, respectively, were mixed at a 1:1 ratio and transferred into 1-day-old neonatal and 8-week-old adult CD45.1/2 mice, and 3 weeks later, their CD5 expression was reanalyzed. Representative histograms show CD5 expression by naïve and MP donor cells and were overlaid with respective host populations shown in gray. Data are representative of four to eight animals. (F) Short-term MP cell proliferation requires CD28 but not TCR signaling. Wild-type mice were injected intraperitoneally with anti–I-Aβ, CTLA4-Ig, or control IgG on days 0 and 2, and splenic MP cells were analyzed on day 4. The graph indicates the frequency (mean ± SD) of Ki67+ cells among MP cells in each CD5 bin (n = 3 to 4). Data are representative of two independent experiments. (G) CD28 is required for optimal Ki67 expression on MP cells. The graph shows the frequency (mean ± SD) of Ki67+ population among MP cells from wild-type and CD28 KO mice (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 MP cells contain T-bethi subpopulation, which is induced by IL-12.

    (A) Genes associated with TH1 but not TH2 differentiation are highly expressed in MP cells. Gene expression by CD5hi and CD5lo MP and naïve CD4+ T lymphocytes was analyzed using RNA-seq. Each gray dot shows the average of relative gene expression (log2) by CD5hi (x axis) and CD5lo MP (y axis) cells compared with naïve cell population, whereas black dots indicate genes associated with TH1- or TH2-type immune responses (n = 3). (B and C) MP CD4+ T cells express T-bet. T-bet expression in MP and naïve cells was analyzed by (B) intracellular staining of wild-type CD4+ T lymphocytes and (C) detection of ZsGreen reporter expression in CD4+ T cells. Data are representative of three independent experiments. (D) Generation of T-bethi MP cells requires IL-12p40. FACS-sorted naïve CD4+ T cells from CD45.1 mice were transferred into CD45.2 wild-type or IL-12p40 KO animals, and T-bet expression in MP cells from donor and recipient cells was analyzed 3 weeks later. Representative histograms of T-betlo, T-betint, and T-bethi subpopulations among MP cells from each group and bar graphs showing the corresponding frequency (mean ± SD) are depicted (n = 3 to 5). Data are representative of two independent experiments. (E and F) Efficient transcription of T-bet requires T-bet expression. (E) ZsGreen expression in MP CD4+ T lymphocytes was analyzed in T-bet–ZsGreen reporter mice that were either T-bet+/+ or T-bet−/−. (F) Sorted naïve CD4+ T cells from CD45.2 T-bet+/+ or T-bet−/− T-bet reporter mice were transferred into CD45.1 wild-type mice, and T-bet expression by the MP donor cells was analyzed 3 weeks later. The representative dot plots and the graph show the frequency (mean ± SD) of T-betlo, T-betint, and T-bethi cells among MP cells from each group (n = 5). Data are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 MP cells, but not pathogen-elicited effector CD4+ T lymphocytes, rapidly produce IFN-γ during T. gondii infection independently of pathogen antigens.

    (A to C) MP cells rapidly produce IFN-γ during T. gondii infection in the absence of pathogen recognition. (A) IFN-γ–eYFP reporter mice treated with anti–I-Aβ mAb or control IgG were infected with T. gondii, and CD44hi CD62Llo CD25lo CD4+ T cells from the PC and spleen were analyzed at the indicated days. The numbers in the representative histograms indicate the frequency of IFN-γ–eYFP+ cells among each group. The graphs show the number (mean ± SD) of IFN-γ–YFP+ cells from each group (n = 2 to 3). Data are representative of two independent experiments. (B) T. gondii–infected IFN-γ–eYFP reporter mice were treated with or without CsA, and splenic CD44hi CD62Llo CD25lo CD4+ T cells were analyzed for IFN-γ–eYFP expression on days 2 and 7. The graph shows the number (mean ± SD) of IFN-γ+ cells (n = 2 to 3). (C) IFN-γ mRNA expression in sorted eYFPhi and eYFPlo MP cells obtained from PC and spleen was measured by qPCR. The graph shows its expression (mean ± SD) relative to β-actin (n = 3). (D to F) Existing MP cells are the major source of IFN-γ on day 2 after T. gondii infection. (D) Experimental design. CD45.1/1 Rag KO mice, which received, first, 1 × 106 total CD4+ T cells from CD45.1/2 IFN-γ–eYFP reporter mice (day −28) and, 3 weeks later, 107 naïve CD4+ T cells from CD45.2/2 IFN-γ–eYFP reporter mice (day −7), were infected with T. gondii (day 0), and CD45.2+ donor cells including both CD45.1/2 and CD45.2/2 populations were analyzed on days 0, 2, and 7. iv, intravenous injection. (E) The histogram shows IFN-γ–eYFP expression by naïve and MP CD45.2+ CD4+ T cells, whereas the numbers in the representative dot plots show the frequency of CD45.1/2 and CD45.2/2 cells among naïve and MP CD45.2+ CD4+ T lymphocytes on day 0 (n = 6). (F) IFN-γ–eYFP expression by naïve and MP CD45.2+ CD4+ T cells is shown in the histograms, whereas the dot plots show the frequency of CD45.1/2 and CD45.2/2 donor cell populations among IFN-γ+ CD44hi CD62Llo CD25lo CD45.2+ CD4+ T cells in the spleen on days 2 and 7. The graph shows the frequency (mean ± SD) of CD45.1/2 and CD45.2/2 donor cells among each group (n = 4). Data are pooled from four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 T-bethi MP cells produce IFN-γ in response to IL-12.

    (A) IL-12p70 induces IFN-γ production by MP cells. IFN-γ–eYFP reporter mice were treated intraperitoneally with IFN-γ, IL-12p70, or IL-18 on days 0 and 1, and on day 2, MP cells were analyzed for IFN-γ expression in the PC and spleen. The representative histograms indicate the frequency of IFN-γ+ cells among MP cells, and the bar graph shows the frequency (mean ± SD) of IFN-γ+ cells (n = 4). Data are pooled from two independent experiments. (B) Neutralization of IL-12 abolishes IFN-γ production by MP cells during T. gondii infection. IFN-γ–eYFP reporter mice infected with T. gondii were treated with blocking anti–IL-12 mAb or control IgG, and MP cells in the PC and spleen were analyzed for IFN-γ expression on day 2. The numbers in the histograms show the frequency of IFN-γ+ cells among MP cells, and the graphs indicate the number (mean ± SD) of IFN-γ+ MP cells from each group (n = 3 to 6). Data are pooled from two independent experiments. (C) T-bethi but not T-betlo MP cells produce IFN-γ in response to IL-12p70. IFN-γ–eYFP T-bet–AmCyan double reporter mice were treated with IL-12p70, as described in (A), and MP cells from the PC and spleen were analyzed for IFN-γ–eYFP and T-bet–AmCyan expression. The representative histograms indicate the frequency of IFN-γ+ cells among T-bethi and T-betlo MP cells, and the bar graphs show the frequency (mean ± SD) of IFN-γ+ cells among each MP subset (n = 3 to 5). Data are pooled from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 7 MP cells established in Rag γc KO mice are Toxoplasma antigen–unspecific T-bet+ population.

    (A) MP cells established in Rag γc KO mice do not contain a major Toxoplasma antigen AS15-specific population. Wild-type and Rag γc KO mice that had received CD4+ T cells 4 weeks earlier were infected with T. gondii or left uninfected, and 7 days later, CD4+ T lymphocytes from the PC and spleen of these animals were analyzed for CD44 expression and AS15 tetramer binding. The representative plots depict CD44 expression and AS15 tetramer binding for each group, whereas the bar graphs show the number (mean ± SD) of naïve, CD44hi, and CD44hi AS15 tetramer+ cells (n = 5). Data are representative of two independent experiments. (B) MP cells generated in Rag γc KO mice express T-bet. The histograms show T-bet expression by MP cells generated in Rag γc KO mice, as shown in (A). Filled histograms show negative control staining. Representative data from three animals were analyzed. (C) MP cells established in Rag γc KO mice do not produce IFN-γ in response to Toxoplasma antigen stimulation. Wild-type and Rag γc KO mice reconstituted with MP cells, as shown in (A), were infected with T. gondii, and on day 7 after infection, whole splenocytes and sorted CD44hi CD62Llo CD4+ T cells from the indicated groups were stimulated with medium, anti-CD3 mAb, STAg, or AS15 peptide in vitro. The graph indicates the concentration (mean ± SD) of IFN-γ in the culture supernatant from each group (n = 5). Data are representative of two independent experiments performed. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 8 MP cells can mediate resistance in infectious models that induce TH1-type immunity.

    (A to G) MP cells ameliorate toxoplasmosis via the IL-12–IFN-γ axis in the absence of pathogen antigen recognition. (A) Survival of T. gondii–infected wild-type, Rag γc KO, and Rag γc KO animals that had received CD4+ T cells from wild-type, IFN-γ KO, or IL-12Rβ2 KO mice 4 weeks earlier was monitored daily (n = 4 to 5). (B) T. gondii–infected Rag γc KO and Rag γc KO mice with MP cells were treated with anti–I-Aβ mAb or control IgG, and survival was assessed (n = 9 to 10). (C) Relative pathogen load (mean ± SD) and (D) concentration of IFN-γ in PC of T. gondii–infected wild-type, Rag γc KO, and Rag γc KO animals that received CD4+ T cells 4 weeks earlier (n = 3 to 10). AU, arbitrary units. (E and F) Serum IFN-γ and IL-12p70 concentration (mean ± SD) at the indicated days after infection (n = 3 to 5). (G) T. gondii–infected Rag γc KO mice with or without MP cells from wild-type or IFN-γ KO mice were treated with IL-12, and survival was assessed (n = 7 to 8). Data are representative of two (A and D to F), pooled from two (B and C), and pooled from three (G) independent experiments performed. (H to J) IFN-γ produced by MP cells augments antigen-specific effector CD4+ T cell responses. (H) Experimental design. Rag γc KO mice that had received CD4+ T cells from wild-type or IFN-γ KO mice at day −28 were infected with T. gondii at day 0 and transferred with sorted naïve CD4+ T cells from CD45.1 mice at day 1. (I) The representative dot plots show CD44 expression versus AS15 tetramer staining by CD45.1 donor cells from each group at day 8, whereas the bar graphs show the number (mean ± SD) of CD44hi CD45.1 donor cells (left) and the frequency (mean ± SD) of CD44hi AS15 tetramer+ cell population among CD45.1 donor cells (right; n = 5). (J) CD44hi CD62Llo CD45.1 donor cell population sorted from splenocytes of each group was stimulated with STAg. The bar graph shows the concentration (mean ± SD) of IFN-γ in the culture supernatant from each group (n = 4 to 5). (K and L) MP cells produce IFN-γ and control bacterial growth in M. bovis BCG infection. Wild-type, Rag KO, and Rag KO mice that had received CD4+ T cells 4 weeks earlier were infected with M. bovis BCG, and (K) bacterial burden (CFU; mean ± SD) in the indicated organs and (L) concentration (mean ± SD) of IFN-γ in the spleen were analyzed 17 days after infection (n = 4 to 5). (M) MP cells are protective in M. tuberculosis infection. Wild-type, Rag KO, and Rag KO mice that had received CD4+ T cells were infected with M. tuberculosis, and survival was assessed (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/12/eaam9304/DC1

    Fig. S1. MP cell conversion in LN.

    Fig. S2. Slow and fast homeostatic proliferations generate naïve and MP cells, respectively.

    Fig. S3. Naïve CD4+ T cells generate MP cells independently of commensal antigens.

    Fig. S4. CD5 expression is maintained in MP cell population.

    Fig. S5. MP cell proliferation is less dependent on TCR signaling.

    Fig. S6. T-bet expression on MP cells in PC depends on IL-12.

    Fig. S7. Relative contribution of NK, MP CD4+ T, NKT, γδT, and MP CD8+ T cells to early IFN-γ production during T. gondii infection.

    Fig. S8. MP cells established in Rag γc KO mice exert protective function in the absence of cognate antigen recognition.

    Fig. S9. Gating strategy.

    Table S1. RNA-seq data.

    Table S2. Raw data sets.

  • Supplementary Materials

    Supplementary Material for:

    Memory-phenotype CD4+ T cells spontaneously generated under steady-state conditions exert innate TH1-like effector function

    Takeshi Kawabe,* Dragana Jankovic, Shuko Kawabe, Yuefeng Huang, Ping-Hsien Lee, Hidehiro Yamane, Jinfang Zhu, Alan Sher,* Ronald N. Germain,* William E. Paul

    *Corresponding author. Email: takeshi.kawabe{at}nih.gov (T.K.); asher{at}niaid.nih.gov (A.S.); rgermain{at}niaid.nih.gov (R.N.G.)

    Published 16 June 2017, Sci. Immunol. 2, eaam9304 (2017)
    DOI: 10.1126/sciimmunol.aam9304

    This PDF file includes:

    • Fig. S1. MP cell conversion in LN.
    • Fig. S2. Slow and fast homeostatic proliferations generate na?ve and MP cells, respectively.
    • Fig. S3. Naïve CD4+ T cells generate MP cells independently of commensal antigens.
    • Fig. S4. CD5 expression is maintained in MP cell population.
    • Fig. S5. MP cell proliferation is less dependent on TCR signaling.
    • Fig. S6. T-bet expression on MP cells in PC depends on IL-12.
    • Fig. S7. Relative contribution of NK, MP CD4+ T, NKT, γδT, and MP CD8+ T cells to early IFN-γ production during T. gondii infection.
    • Fig. S8. MP cells established in Rag γc KO mice exert protective function in the absence of cognate antigen recognition.
    • Fig. S9. Gating strategy.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). RNA-seq data.
    • Table S2 (Microsoft Excel format). Raw data sets.

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