1. Volume 38, Issue 1, Pages 53-65 (January 2013)
The CD27 and CD70 Costimulatory Pathway Inhibits Effector Function of T Helper 17 Cells and Attenuates Associated Autoimmunity 
Jonathan M. Coquet, Sabine Middendorp, Gerda van der Horst, Jop Kind, Elise A.M. Veraar, Yanling Xiao, Heinz Jacobs, Jannie Borst 
Immunity 
Volume 38, Issue 1, Pages (January 2013)
DOI: /j.immuni
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2. Figure 1
EAE Is Exacerbated in CD27- and CD70-Deficient Mice and Ameliorated in Cd70tg Mice
(A–C) EAE was induced in WT (n = 17), Cd27−/− (n = 16), and Cd70Cre/Cre (n = 7) mice and mice were monitored daily for clinical signs of EAE over a period of 20 days.
(A) Disease development as diagnosed by defined scores.
(B) Overall survival. Mice with a disease score of 4 or more were sacrificed and a survival curve was generated.
(A and B) p < 0.05 for Cd27−/− (indicated by #) and Cd70Cre/Cre (indicated by §) mice as compared to WT.
(C) Cytokine production by CD4+ T cells in the spleen. Splenocytes were isolated at day 6, restimulated with MOG35-55 peptide, and analyzed for IL-17 and IFN-γ production by intracellular staining. Contour plots depict IL-17 and CD44 expression in gated CD4+ T cells. Numbers represent percentage of cells in the indicated gates. Graphs indicate the percentage of cytokine-expressing cells among gated CD4+ T cells. Data are from two experiments with WT (n = 9) and Cd27−/− (n = 10) mice, ∗p < 0.05.
(D and E) EAE was induced in control Cd27+/− (n = 13) and Cd70tg;Cd27+/− (n = 10) mice and mice were monitored daily for clinical signs of EAE over a period of 20 days.
(D) Disease development in both groups, ∗p < 0.05.
(E) Frequency of disease severity. The percentage of mice with a disease score greater than 1 was plotted, ∗∗∗p <
See also Figure S2 and Table S1. Statistical significance was determined by Mann-Whitney U rank test (A, C, and D) and Log rank test (B, E).
Immunity  , 53-65DOI: ( /j.immuni )
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3. Figure 2 Reduced Th17 Cell Generation in Cd70tg Mice with EAE
(A) Cytokine production by CD4+ T cells in the CNS at day 20 after EAE induction. CNS-infiltrating cells were isolated, stimulated with PMA and ionomycin, and analyzed for IL-17 and IFN-γ production by intracellular staining. Contour plots depict IL-17- and IFN-γ-producing cells among gated CD4+ T cells of control Cd27+/− mice and Cd70tg;Cd27+/− mice with progressive (score > 1) and nonprogressive (score < 1) disease. Shown are mice with disease scores of 2 (left plot), 2 (middle), and 0.5 (right). Numbers depict the percentage of cells in each quadrant. Graphs indicate the percentage and absolute number (#) of cytokine-expressing cells among gated CD4+ T cells. Data are from two experiments with control (n = 7), Cd70tg score > 1 (n = 4), and Cd70tg score < 1 (n = 4) mice.
(B) Cytokine production by CD4+ T cells in the spleen at day 20 was analyzed as described in Figure 1C. Graphs depict data from WT (n = 5) and Cd70tg (n = 3) mice.
(C and D) Cytokine production by CD4+ T cells in the CNS (C) and spleen (D) at day 10. Analysis was done as outlined in (A) and (B). Data are from two experiments with control (n = 9) and Cd70tg (n = 6) mice.
Statistical significance was determined by Mann-Whitney U test, ∗p < 0.05.
Immunity  , 53-65DOI: ( /j.immuni )
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4. Figure 3
CD27 Signaling Reduces CCR6 but Not RORγt Expression in Th17 Cells of Mice with EAE
EAE was induced in control Cd27+/− and Cd70tg;Cd27+/− mice and the spleen and CNS were harvested at day 10. CNS-infiltrating lymphocytes and splenocytes were restimulated in vitro as described in Figure 2 and analyzed by intracellular staining.
(A and B) RORγt expression. Histograms depict RORγt expression on IL-17+IFN-γ− (bold black line, unfilled), IL-17+IFN-γ+ (light gray, filled), IL-17−IFN-γ+ (gray line, unfilled), and IL-17−IFN-γ− (dark gray, filled) cells in control and Cd70tg mice from the CNS (A) and the spleen (B). Bar graphs depict the percentage of RORγt+ cells among the gated population indicated.
(C) CCR6 expression. Histograms depict the expression of CCR6 on control (gray line) or Cd70tg (black line) cytokine-producing cells as indicated. Bar graphs depict the percentage of CCR6+ cells among the gated population indicated. Results are comprised from one experiment with littermate control (n = 7) and Cd70tg (n = 3) mice.
Statistical significance was determined by the Student’s t test, ∗p < 0.05.
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5. Figure 4 CD27 Signaling Impairs Th17 Cell Differentiation In Vitro
Naive CD4+ T cells (CD4+CD25−CD44lo) were purified from spleens of WT or Cd27−/− mice and cultured for 4 days with irradiated splenocytes under Th17 or Th1 cell differentiation conditions, in the presence or absence of 1 μg/ml agonistic recombinant CD70 (FcCD70). After culture, cells were stimulated with PMA and ionomycin and analyzed for IL-17 and IFN-γ by intracellular staining.
(A) Contour plots depict IL-17- and IFN-γ-producing cells. Numbers indicate the percentage of cells in the quadrants.
(B) Graphs of the percentage and number of IL-17+ cells per well. Data are pooled from three experiments with duplicate cultures (n = 6).
(C and D) In separate experiments, cells were costimulated with FcCD70 and IL-2Rα mAb (PC-61 mAb, 10 μg/ml) was added to the cultures as indicated. Graphs depict the percentage (C) and number (D) of cells per well that produce IL-17. Data are pooled from two experiments with duplicate cultures (n = 4).
Statistical significance was determined by Mann-Whitney U test, ∗p < 0.05, ∗∗p < See also Figure S3.
Immunity  , 53-65DOI: ( /j.immuni )
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6. Figure 5
CD27 Signaling Inhibits Transcription of Il17a, Il17f, and CCR6 Genes but Does Not Affect Lineage Commitment of Th17 Cells
(A and D) CD4+ T cells were cultured under Th17 cell differentiation conditions in presence or absence of FcCD70. After 4 days, triplicate cell samples were analyzed by quantitative PCR for transcription of the indicated genes. Messenger RNA expression was normalized to 18S ribosomal RNA. Data are representative of two experiments. Statistical significance was determined with the Student’s t test, ∗p < 0.05, ∗∗p < 0.01.
(B and C) Cells were cultured under Th17 cell differentiation conditions in the presence (dotted line) or absence (solid line) of FcCD70, or with CD3 mAb as a control (shaded histogram) and analyzed for STAT-3 phosphorylation (B) after 20 hr or for Foxp3 (C) expression after 4 days of culture. Histograms are representative of two (B) or four (C) experiments.
See also Tables S2–S4.
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7. Figure 6 CD27 Inhibits IL-17 and CCR6 Expression via JNK Signaling
Th17 cell differentiation cultures were performed in the presence or absence of FcCD70.
(A) At 24, 48, or 96 hr, phosho(p)-JNK expression was analyzed by flow cytometry. The histogram shows control (secondary antibody) staining of cells cultured without (dark gray, filled) or with (light gray, filled) FcCD70 and staining with a p-JNK-specific primary antibody and secondary antibody on cells cultured without (solid black line) or with (dotted line) FcCD70 at the 48 hr time point. Graph depicts the percentage of p-JNK+ cells. Data are pooled from three experiments (n = 5–7).
(B) Cells were cultured in the presence of the JNK inhibitor D-JNKI1 (3 μM) or vehicle control (−). At day 4, IL-17 and CCR6 expression was analyzed by flow cytometry as shown in the dot plots, with percentage of cells indicated in the quadrants. Graphs depict the fold increase in IL-17- or CCR6-expressing cells because of D-JNKI1 addition. Data are pooled from four experiments (n = 6).
(C) Cells cultured as in (B) were analyzed for IL-17 mRNA expression by quantitative PCR, with normalization to 18S ribosomal RNA. Results are pooled from two experiments with a total of five separate cultures.
(D) Cultures were performed in the presence of p38 MAP kinase inhibitor SB (1 μM) or vehicle control (−) and IL-17 and CCR6 expression was analyzed as indicated in (B). Data are pooled from two experiments (n = 3–4).
Statistical significance was determined by Mann-Whitney U test, ∗p < 0.05, ∗∗p < 0.01.
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8. Figure 7 CD27 Signaling Epigenetically Silences the Il17a Gene
CD4+ T cells were cultured under Th17 cell differentiation conditions in the presence or absence of FcCD70 for 4 days.
(A–C) Chromatin immunoprecipitations (ChIP) were performed from 5 × 106 cells per sample with antibodies specific for total H3, H3K4me3, H3K14Ac, H3K27me3, and H3K9me2.
(A and B) Isolated DNA was used as a template for quantitative PCR with primers specific for the Il17a gene promoter (A) or the Ifng promoter (B). Signals from ChIP of modified histone H3 were standardized to signals obtained from ChIP of total H3 (recovery was between 3% and 10%). Values from cells cultured in the presence of FcCD70 were normalized to values in its absence to allow for interexperimental comparison. Results are from three experiments.
(C) H3K27me3 ChIP fractions were additionally used for next-generation sequencing. The graph shows the distribution of the H3K27me3 signal (normalized read count) over the Il17a locus, as detected in cells cultured without (black) or with (gray) FcCD70. The gene region presented includes a 1 kb promoter region (black line), the transcriptional start site (TSS), the coding region (gray line), and 3′ untranslated region (UTR).
(D) Genomic demethylation restores IL-17 production by CD27-stimulated cells. The demethylating agent 5-azacytidine was added for the last 16 hr of Th17 cell differentiation culture. Contour plots depict IL-17- and IFN-γ-producing cells. The graph depicts the fold increase in percentage of IL-17+ cells in the presence of 5-aza in cultures with or without FcCD70. Data are pooled from four experiments (n = 9–10).
Statistical significance was determined by Mann-Whitney U test, ∗∗∗p <
Immunity  , 53-65DOI: ( /j.immuni )
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