Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway - PubMed (original) (raw)

. 2013 Apr 8;210(4):715-28.

doi: 10.1084/jem.20112061. Epub 2013 Apr 1.

Julie C Ribot, Nikolina Bąbała, Sabine Middendorp, Gerda van der Horst, Yanling Xiao, Joana F Neves, Diogo Fonseca-Pereira, Heinz Jacobs, Daniel J Pennington, Bruno Silva-Santos, Jannie Borst

Affiliations

Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway

Jonathan M Coquet et al. J Exp Med. 2013.

Abstract

CD4(+)Foxp3(+) regulatory T cells (Treg cells) are largely autoreactive yet escape clonal deletion in the thymus. We demonstrate here that CD27-CD70 co-stimulation in the thymus rescues developing Treg cells from apoptosis and thereby promotes Treg cell generation. Genetic ablation of CD27 or its ligand CD70 reduced Treg cell numbers in the thymus and peripheral lymphoid organs, whereas it did not alter conventional CD4(+)Foxp3(-) T cell numbers. The CD27-CD70 pathway was not required for pre-Treg cell generation, Foxp3 induction, or mature Treg cell function. Rather, CD27 signaling enhanced positive selection of Treg cells within the thymus in a cell-intrinsic manner. CD27 signals promoted the survival of thymic Treg cells by inhibiting the mitochondrial apoptosis pathway. CD70 was expressed on Aire(-) and Aire(+) medullary thymic epithelial cells (mTECs) and on dendritic cells (DCs) in the thymic medulla. CD70 on both mTECs and DCs contributed to Treg cell development as shown in BM chimera experiments with CD70-deficient mice. In vitro experiments indicated that CD70 on the CD8α(+) subset of thymic DCs promoted Treg cell development. Our data suggest that mTECs and DCs form dedicated niches in the thymic medulla, in which CD27-CD70 co-stimulation rescues developing Treg cells from apoptosis, subsequent to Foxp3 induction by TCR and CD28 signals.

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Figures

Figure 1.

Figure 1.

Treg cell development is compromised in Cd27−/− and Cd70Cre/Cre mice. Thymocytes and splenocytes were isolated from WT and CD27- and CD70-deficient mice, enumerated, stained with antibodies to CD4, CD8, CD25, CD44, and Foxp3 and analyzed by flow cytometry. (A) Representative analysis of CD25 versus Foxp3 staining on gated CD4+CD8− cells in thymus and spleen. Numbers indicate the percentage of Foxp3+ cells present in the indicated gate. (B) Absolute number and percentage of CD4+Foxp3+ Treg cells among total CD4+ T cells and absolute numbers of conventional CD4+Foxp3− T cells and CD8+CD4− T cells in the thymus and spleen of WT, Cd27−/−, and Cd70Cre/Cre mice. (C) Absolute number of cells in the double negative (DN; CD4−CD8−) subsets, based on CD44 and CD25 expression, and in the double positive (DP; CD4+CD8+) subset in the thymus of WT, Cd27−/−, and Cd70Cre/Cre mice. Data in B and C are derived from 10–16 mice per genotype, pooled from at least three independent analyses. Numbers (mean + SEM) from WT versus Cd27−/− and Cd70Cre/Cre mice were significantly different according to one-way analysis of variance, followed by a post-Dunn’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Thymic CD4+CD8− cells were analyzed for the percentage of CD25+Foxp3− pre-Treg cells and CD25+ or CD25−Foxp3+ Treg cells. Data are from nine mice pooled from three experiments. Values from WT and Cd27−/− mice were compared by Mann–Whitney U rank test. **, P < 0.01. See also

Fig. S1 A

. (E) Cd27−/− mice were administered 100 µg anti-CD80 and anti-CD86 or the appropriate isotype control antibody i.p. every other day for 2 wk. 2 d after the last administration, Treg cell frequency in the thymus was analyzed by flow cytometry. Percentage and absolute number of Treg and pre-Treg cells in treated Cd27−/− mice are shown. Data shown are from one representative of two independent experiments with three mice per group. Results were analyzed by Student’s t test. *, P < 0.05. See also Fig. S1 B. Error bars are SEM.

Figure 2.

Figure 2.

CD27–CD70 co-stimulation promotes thymic Treg cell development in vitro and in vivo. (A) Independent FTOCs were performed with either 1 or 5 µg/ml CD70-blocking antibody FR70 and an internal control of 1 or 5 µg/ml of control IgG2a antibody (left). Alternatively, cultures were performed in the presence of either 1 or 5 µg/ml CD27-agonist FcCD70 or control IgG1 antibody (right). After 14–16 d, Treg cell development was assessed by disrupting the lobes and performing flow cytometric analyses for Foxp3, TCRβ, and CD4. Bar diagrams depict the percentage of Foxp3+ cells (Treg cells) among TCRβ+CD4+cells. Data are from two to three separate experiments comprising four to six independent wells. Data were analyzed using the Mann–Whitney U rank test (*, P < 0.05). (B) Thymocytes were isolated from WT and Cd27−/− neonates, stained with antibodies to CD4, CD8, CD25, and Foxp3, and analyzed by flow cytometry. Representative plots of CD25 and Foxp3 expression on gated CD4+CD8− thymocytes are shown, and graphs depict the absolute number of Foxp3+ Treg cells and Foxp3−CD4+ thymocytes. Data in histograms are mean + SEM from five WT and six Cd27−/− mice and were analyzed by Mann–Whitney U rank test (*, P < 0.05). (C and D) Mixed BM chimeras were established by lethally irradiating B6 mice of the CD45.2 allotype and reconstituting them the next day with a 1:1 mixture of CD45.1+ WT and CD45.2+ Cd27−/− BM cells. 8 wk later, reconstitution of recipient thymus and spleen was assessed by flow cytometry. (C) The dot plot (top left) depicts a representative analysis of the thymus and the histogram (top right) depicts the expression of CD45.2 on gated CD4+ cells. The histograms in the bottom panels depict the expression of Foxp3 within the gated (arrows) CD45.2− WT (left) or CD45.2+ Cd27−/− (right) CD4+ cell populations. Numbers in each plot represent percentages of the gated population. (D) The percentage of Treg cells among total CD4+ cells and the absolute number of conventional CD4+Foxp3− T cells and CD4+Foxp3+ Treg cells in the thymus and spleen of WT and Cd27−/− mice. Data are mean + SEM from four mice per group and are representative of three separate experiments. The Mann–Whitney U rank sum test was used to calculate significance between groups (*, P < 0.05).

Figure 3.

Figure 3.

CD27–CD70 co-stimulation promotes Treg cell survival but not function. (A) CD4+CD25+ (Treg) cells were sorted from naive spleens of WT or Cd27−/− mice and co-cultured with sorted CFSE-labeled CD4+CD25− responder T (Tresp) cells from WT mice of the CD45.1 allotype on plates coated with anti-CD3 mAb, in the presence of irradiated splenocytes. After 3 d, flow cytometry was performed and gated CD45.1+ cells were analyzed for dilution of CFSE. Plotted is the percentage of divided responder T cells after culture in the presence of various numbers of Treg cells. One representative experiment of four is shown. Genotype labeling indicated in A also applies to B–E. (B and C) CD4+CD8−CD25+CD69+HSA+ (pre-Treg) cells were sorted by flow cytometry and cultured with or without 10 ng/ml IL-2 for 3 d and diagnosed by flow cytometry for Foxp3 expression (B) and Annexin-V binding (C). See also Fig. S1 C. Data in the bar graph in B were normalized to the percentage of Foxp3+ cells derived from WT cultures in the presence of IL-2. Bar diagrams depict mean + SEM of three to four individual experiments. Data were analyzed using the Mann–Whitney U rank test (*, P < 0.05). (D) CD4+CD8−CD25+, CD4+CD8−CD25−, and CD8+CD4− cells from thymus or spleen of WT or Cd27−/− mice were analyzed for Annexin-V binding and propidium iodide (PI) exclusion and the proportion of apoptotic cells (Annexin-V+PI−) in each population is depicted. See also Fig. S1 D. Bar diagrams depict mean + SEM of four individual mice. Data were analyzed by Student’s t test (**, P < 0.01). (E) mRNA was isolated from purified CD4+CD8−CD25+ and CD4+CD8−CD25− cells from WT or Cd27−/− mice in four independent experiments each with five pooled thymi and analyzed by MLPA with primers specific for the indicated genes. Levels of apoptosis-related genes were calculated relative to the housekeeping gene Gusb, and in each experiment the level of gene expression in the Cd27−/− sample was normalized to that of the WT sample. Bar graphs depict the level of mRNA expression in relative units in WT (closed bars) and Cd27−/− (open bars) cells. Data were analyzed according to one-way analysis of variance, followed by a post-Dunn’s test (*, P < 0.05). (F–H) CD45.1+ WT and CD45.2+ Cd27−/− BM cells were harvested and T cell depleted. Cd27−/− cells were transduced with retroviral vectors expressing GFP alone (gray-filled histogram) or Bcl-2IRESGFP (black line), and WT cells were transduced with a vector expressing GFP alone. Empty GFP vector–expressing WT and Cd27−/− cells or GFP vector–expressing WT cells and Bcl-2IRESGFP–expressing Cd27−/− cells were injected at a 1:1 ratio into irradiated recipients, and Treg cell development was analyzed 8 wk later. (F) Expression of the Cd27−/− cell marker CD45.2 on gated GFP+CD4+CD25+ Treg cells. (G) Percentage of Treg cells arising from the Cd27−/− donor cells when cells were transduced to express GFP alone or Bcl-2 and GFP. (H) Absolute number of CD4+CD25+ Treg cells or CD4+CD25− conventional T cells arising from the Cd27−/− donor cells when cells were transduced with GFP alone (empty) or Bcl-2IRESGFP. Results in F–H are from one representative of two independent experiments with five mice per group and were analyzed by Mann–Whitney U rank test (*, P < 0.05; **, P < 0.01). Error bars are SEM.

Figure 4.

Figure 4.

CD70 is expressed by Aire+ and Aire− epithelial cells in the thymic medulla. (I–IV, VII, and VIII) Thymic sections of WT and Cd70Cre/Cre mice were stained to detect CD70 on mTECs using sequential staining with ER-TR5 mAb, Alexa Fluor 488–conjugated anti-IgG, anti-CD70 mAb, and Alexa Fluor 568–conjugated anti-IgG (I, II, VII, and VIII) or sequential staining with anti–Keratin-5 (K5) mAb, Alexa Fluor 488–conjugated anti-IgG, anti-CD70 mAb, and Alexa Fluor 568–conjugated anti-IgG (III and IV). (V and VI) Thymic sections of WT and Cd70Cre/Cre mice were stained to detect CD70 and Aire using sequential staining with anti-CD70 mAb, Alexa Fluor 568–conjugated anti-IgG, and Alexa Fluor 488–conjugated anti-Aire mAb. Sections I–VI were subsequently stained with DAPI to detect the nuclei of all cells present. The same magnification is shown for all panels. High magnification (I–VI) and lower magnification (VII–VIII) images of the thymus are shown. Data for each staining are representative of at least three to four different sections that were stained and analyzed independently.

Figure 5.

Figure 5.

CD27–CD70 co-stimulation promotes positive selection of sAg-specific Treg cells. (A–C) Mixed BM chimeras were established by lethally irradiating recipient DBA/2 or B6 mice and reconstituting them the next day with a 1:1 mixture of T cell–depleted CD45.1+ WT and CD45.2+ Cd27−/− BM cells. 8 wk later, thymocytes were analyzed by flow cytometry for CD4 and Foxp3 expression within the CD45.1+ and CD45.1− populations. (A) Bar diagrams depict the numbers of WT (CD45.1+) or Cd27−/− (CD45.1−) CD4+Foxp3+ or CD4+Foxp3− thymocytes in B6 or DBA/2 recipients. (B) Representative histograms of Vβ5 expression in gated WT and Cd27−/− CD4+Foxp3+ thymocytes from B6 or DBA/2 recipient mice. (C) Percentages of Vβ5+ and Vβ4+ cells among WT and Cd27−/− CD4+Foxp3+ thymocytes in B6 and DBA/2 mice. Data are derived from one experiment with three to four mice per group and are representative of two independent experiments. (D and E) Mixed BM chimeras were established by lethally irradiating recipient DBA/2 mice and reconstituting them the next day with a 1:1 mixture of WT (Cd27+/+) and Cd27−/− BM cells or Cd27+/+;Cd70Cre/Cre and Cd27−/−;Cd70Cre/Cre BM cells, resulting in expression of CD70 on hematopoietic cells or not, as indicated. After 8 wk, the development of Treg cells was analyzed as outlined for A–C. Bar diagrams indicate the percentage of Vβ5+ Treg cells (D) and the absolute number of Treg cells (E) within WT and Cd27−/− cells or Cd70Cre/Cre and Cd27−/−;Cd70Cre/Cre cells. Data are from two independent experiments with six to eight mice per group. All data were analyzed by Student’s t test (*, P < 0.05; **, P < 0.01). Error bars are SEM.

Figure 6.

Figure 6.

CD70 on CD8α+ thymic cDCs contributes to Treg cell development. (A) Thymic sections were stained sequentially using anti-CD70 mAb, Alexa Fluor 568–conjugated anti-IgG, FITC-conjugated anti-CD11c mAb, and APC-conjugated anti-CD8α mAb. Arrowheads indicate cells with double staining for CD70 and CD11c. (B and C) DCs were enriched from WT or Cd70Cre/Cre thymi and analyzed by flow cytometry. (B) Shown are representative plots of CD8α versus SIRPα expression on gated CD11c+B220lo DCs. (C) CD8α+ and SIRPα+ DC subsets were purified from WT and Cd70Cre/Cre thymi, RNA was isolated, and RT-PCR was performed on cDNA. Shown are PCR products from cDNA samples amplified with primers specific for CD70 and HPRT. (D) CD8α+ and SIRPα+ CD11c+B220low/− cDCs and CD11c+B220+ pDCs were purified from the pooled thymi of two WT or Cd70Cre/Cre mice. The purified DC subsets were co-cultured for 5 d with purified CD4+CD25− WT thymocytes. After culture, gated CD4+ cells were analyzed for expression of Foxp3 and CD25. Shown is one representative plot of Foxp3 and CD25 expression on gated CD4+ cells in the presence of the indicated DC population. (E) Data from D, expressed as the percentage of Foxp3+CD25+ cells among gated CD4+ cells in the presence of the indicated DC populations. Results are from four to five separate wells over two independent experiments. The Mann–Whitney U rank sum test was used to analyze results (*, P < 0.05). Error bars are SEM.

Figure 7.

Figure 7.

CD70 expression by both epithelial and hematopoietic cells is required for Treg cell development. (A) CD45.2+ Cd70Cre/Cre mice were lethally irradiated and reconstituted with a 1:1 mixture of BM cells from CD45.1+ WT and CD45.2+ Cd27−/− mice. 8 wk later, mice were analyzed for the presence of CD4+Foxp3+ Treg cells and CD4+Foxp3− conventional T cells in thymus and spleen by flow cytometry. Bar diagrams represent the percentage of Treg cells within the total CD4+ population and the absolute numbers of Treg cells and conventional CD4+ T cells for WT and Cd27−/− cells. Results are from one experiment with four mice per group and are representative of two independent experiments. The Mann–Whitney U rank sum test was used to calculate significance between groups (*, P < 0.05). (B) WT and Cd70Cre/Cre mice were lethally irradiated and reconstituted with BM cells from WT or Cd70Cre/Cre mice. 8 wk later, mice were analyzed for the presence of Foxp3+ Treg cells among total CD4+CD8− cells in thymus and spleen by flow cytometry. Bar diagrams represent the percentage of Treg cells within the total CD4+ population for the indicated combinations. Results are pooled from two independent experiments with three to eight mice per group, except for the setting with Cd70Cre/Cre BM into Cd70Cre/Cre recipients which was included in only one experiment. For the comparison of multiple groups, a one-way analysis of variance followed by a post-Dunn’s test was used to assess significance of the difference with the WT control setting (*, P < 0.05; **, P < 0.01). Error bars are SEM.

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References

    1. Acuto O., Michel F. 2003. CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat. Rev. Immunol. 3:939–951 10.1038/nri1248 - DOI - PubMed
    1. Anderson M.S., Venanzi E.S., Klein L., Chen Z., Berzins S.P., Turley S.J., von Boehmer H., Bronson R., Dierich A., Benoist C., Mathis D. 2002. Projection of an immunological self shadow within the thymus by the aire protein. Science. 298:1395–1401 10.1126/science.1075958 - DOI - PubMed
    1. Apostolou I., Sarukhan A., Klein L., von Boehmer H. 2002. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3:756–763 - PubMed
    1. Aschenbrenner K., D’Cruz L.M., Vollmann E.H., Hinterberger M., Emmerich J., Swee L.K., Rolink A., Klein L. 2007. Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ medullary thymic epithelial cells. Nat. Immunol. 8:351–358 10.1038/ni1444 - DOI - PubMed
    1. Atarashi K., Tanoue T., Shima T., Imaoka A., Kuwahara T., Momose Y., Cheng G., Yamasaki S., Saito T., Ohba Y., et al. 2011. Induction of colonic regulatory T cells by indigenous Clostridium species. Science. 331:337–341 10.1126/science.1198469 - DOI - PMC - PubMed

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