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Volume 21, Issue 7, Pages 1839-1852 (November 2017)
Th1-like Plasmodium-Specific Memory CD4+ T Cells Support Humoral Immunity Ryan A. Zander, Rahul Vijay, Angela D. Pack, Jenna J. Guthmiller, Amy C. Graham, Scott E. Lindner, Ashley M. Vaughan, Stefan H.I. Kappe, Noah S. Butler Cell Reports Volume 21, Issue 7, Pages (November 2017) DOI: /j.celrep Copyright © 2017 The Author(s) Terms and Conditions
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Cell Reports 2017 21, 1839-1852DOI: (10.1016/j.celrep.2017.10.077)
Copyright © 2017 The Author(s) Terms and Conditions
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Figure 1 Therapeutic Ligation of OX40 during Acute P. yoelii Infection Increases the Magnitude of the P. yoelii GP61–80-Specific CD4 T Cell Response (A) Experimental design. Mice were infected with 1 × 106 transgenic Py-GP and administered either control rIgG or agonistic α-OX40 antibodies on days 7 and 10 p.i. (B) Parasite growth and clearance kinetics. (C) Proportions of tetramer+ GP61–80-specific splenic CD4+ T cells on day 45 p.i. in Py-GP-infected rIgG- and α-OX40-treated mice. (D and E) Kinetics of the (D) proportion and (E) total number of GP61–80-specific CD4+ T cells. Data (mean ± SEM) in (B), (D), and (E) are pooled from two to three independent experiments per time point with three to five mice per group per experiment and were analyzed using multiple Student’s t tests while correcting for multiple comparisons using the Holm-Sidak method. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S1. Cell Reports , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions
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Figure 2 Therapeutic Ligation of OX40 during Primary P. yoelii Infection Induces T-bet Expression and Expands Polyfunctional Ly6C+ Th1-like Memory CD4 T Cell Populations (A–D) Mice were infected with 1 × 106 transgenic Py-GP and administered either control rIgG or agonistic α-OX40 antibodies on days 7 and 10 p.i. (A) Proportions of Ly6C+ GP61–80-specific CD4 T cells and their expression of T-bet relative to Ly6Clo GP61–80-specific CD4 T cells on day 14 p.i. (B–D) Kinetics of the (B) proportion and (C) total number of Ly6C+ GP61–80-specific CD4 T cells and (D) total number of T-bet+ GP61–80-specific CD4 T cells in rIgG- and α-OX40-treated mice. (E) Experimental design. 10,000 naive CD90.1+ SMARTA CD4 T cells were transferred to naive CD90.2+ mice. Recipients were infected with Py-GP and treated with either agonistic α-OX40 or control IgG antibodies on days 7 and 10 p.i. (F) Proportions of Ly6C+T-bet+ memory SMARTA cells on day 45 p.i. (G) Proportions of memory SMARTA cells expressing IFN-γ, TNF, and IL-2 on day 45 p.i. (H) Summary of cytokine profiles of memory SMARTA cells from rIgG- and α-OX40-treated mice on day 45 p.i. (I–L) Expression of Ly6C (I), IFN-γ (J and L), and IL-2/TNF (K) by total polyclonal (CD44hi) memory CD4 T cells on day 45 p.i. Data (mean ± SEM) in (B)–(D) are pooled from two to three independent experiments per time point with three to five mice per group per experiment and were analyzed using multiple Student’s t tests while correcting for multiple comparisons using the Holm-Sidak method. Data (mean ± SD) in (H)–(L) derive from four to five mice per group and are representative of two independent experiments. ∗p < 0.05 ∗∗p < 0.01, N.S., not significant. See also Figure S2. Cell Reports , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions
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Figure 3 Therapeutic Ligation of OX40 during Acute Plasmodium Infection Expands GP61–80-Specific CXCR5+ Memory CD4 T Cells that Express Bcl-6 during Recall (A–C) Mice were infected with 1 × 106 transgenic Py-GP and administered either control rIgG or agonistic α-OX40 antibodies on days 7 and 10 p.i. (A) Proportions of CXCR5+PD-1intBcl-6+ memory CD4 T cells on day 60 p.i. (B and C) Kinetics of the proportion (B) and total number (C) of CXCR5+PD-1int memory CD4 T cells. (D and E) Py-GP-immune mice were challenged with 5 × 106 Py-GP parasites on day 60 p.i. (D) Representative flow plots showing the proportion of CXCR5+PD-1int GP61–80-specific CD4 Tfh cells and their expression of Bcl-6. (E) Summary data showing the relative amount (gMFI) of Bcl-6 in GP61–80-specific secondary effector CD4 T cells 6 days after challenge of Py-GP-immune mice. (F) CD44hiCXCR5+PD-1int memory CD4 T cells from rIgG- and α-OX40-treated mice were co-cultured with naive B cells in the presence of α-CD3ε and α-IgM. Supernatants were assayed for secreted IgG on day 7. (G) Py-GP-immune mice were challenged as in (D), and Hep17-specific serum IgG was measured before and 6 days later. (H–J) Py-GP-immune mice harboring memory CD90.1+ SMARTA cells were challenged with 2 × 106 plaque-forming units (p.f.u.) of LCMV. Frequency (H), total number (I), and expression of Bcl-6 (J) were evaluated in secondary effector SMARTA cells. Data (mean ± SEM) in (B), (C), and (E) are pooled from two to three independent experiments per time point with three to five mice per group per experiment and were analyzed using Student’s t tests while correcting for multiple comparisons using the Holm-Sidak method. Data (mean ± SD) in (F) derive from four replicates, represent two independent experiments, and were analyzed by Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S3. Cell Reports , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions
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Figure 4 Therapeutic Ligation of OX40 during Acute Plasmodium Infection Enhanced Both Th1- and Tfh-like Recall Responses and per Cell Protective Capacity of Memory CD4 T Cells (A) Experimental design. Mice were infected with 1 × 106 Py and administered either control rIgG or α-OX40 antibodies on days 7 and 10 p.i. On 75 day p.i., memory CD4 T cells were sorted and transferred (2 × 105) into groups of Tcrα−/− mice. Recipients were challenged with P. yoelii, and parasite growth kinetics were measured. Cells were analyzed on day 14 p.i. (B and C) Kinetics of parasite growth (B) and summary data (C) showing parasitemia on day 14 p.i. Statistical analyses (∗p < 0.05) reflect comparisons between recipients of memory cells from rIgG- and α-OX40-treated donor mice. (D–F) Proportions of donor-derived CD4 T cells expressing either Ly6C (D), IFN-γ (D and E), or CXCR5+PD1+ Tfh cells expressing Bcl-6 (F). (G) Total number of CXCR5+PD1+Bcl-6+ Tfh cells recovered from recipients. (H) MSP119-specific serum antibody titers in mice seeded with memory CD4 T cells derived from rIgG- and α-OX40-treated donors. Data (mean ± SEM) in (B), (C), (E), (G), and (H) are pooled from two independent experiments with three to four mice per group per experiment and were analyzed using either one-way ANOVA (B and C) or Student’s t tests (E, G, and H). ∗p < 0.05; ∗∗p < N.S., not significant. See also Figure S4. Cell Reports , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions
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Figure 5 Both Ly6Clo and Ly6C+ Plasmodium-Specific Memory CD4 T Cells Can Give Rise to Tfh-like Secondary Effectors (A) Experimental design. CD45.1+ mice were infected with 1 × 106 Py and administered either control rIgG or α-OX40 on days 7 and 10 p.i. On day 48 p.i., 2 × 105 total (CD11ahiCD44hi) infection-induced Ly6C+ memory CD4+ T cells were sorted and transferred into congenic CD45.2+ mice. Recipient mice were infected with Py 1 day later. CD4 T cell responses were assessed on day 7 p.i. (B and C) Representative (B) flow plots and (C) summary graph showing expansion of CD45.1+ cells on day 7 p.i. (D) Phenotype and CXCR5 and Bcl-6 expression among recovered CD45.1+ Ly6Clo and Ly6C+ secondary effector CD4 T cells. (E) Summary data showing the proportion of Ly6Clo and Ly6C+ CD45.1+ secondary effector cells expressing CXCR5 and Bcl-6. (F) Summary data of Bcl-6 expression (gMFI) in rIgG- and α-OX40-treated donor-derived CD45.1+ secondary effector CD4 T cells. Data (mean ± SD) in (C) were analyzed using one-way ANOVA. Data (mean ± SEM) in (E) and (F) are pooled from two independent experiments with five mice per group and were analyzed using Student’s t tests. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S5. Cell Reports , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions
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Figure 6 Ligation of OX40 during an Acute P. yoelii Infection Bolsters the Anti-parasitic and B Cell Helper Capacity of Plasmodium-Specific, Th1-like Ly6C+ Memory CD4 T Cells Mice were infected with 1 × 106 Py and administered either control rIgG or agonistic α-OX40 antibodies on days 7 and 10 p.i. (A) MSP119-specific serum IgG2b titers in Plasmodium-infected mice seeded with either Ly6Clo or Ly6Chi memory CD4 T cells. (B) On day 50 p.i., Ly6C+ and Ly6Clo memory CD4 T cells were sort-purified from rIgG- and α-OX40-treated mice and co-cultured with naive (CD19+IgDhi) B cells in the presence of α-CD3ε and α-IgM. Secreted IgG was measured on day 5. (C) Ly6C+ and Ly6Clo memory CD4 T cells from rIgG- and α-OX40-treated mice were sort purified and transferred (2 × 105) into Tcrα−/− mice. Recipients were infected the following day. Parasite burdens on day 16 p.i. are shown. (D and E) Proportion (D) and summary (E) of class-switched (IgDloIgMlo) germinal center B cells (GL-7+CD95+) in Tcrα−/− mice seeded with Th1-like Ly6C+ memory CD4 T cells derived from either rIgG- or α-OX40-treated donors. (F) MSP119-specific serum IgG titers in memory CD4 T cell recipient Tcrα−/− mice on day 16 p.i. Data (mean ± SD) are from five mice per group, represent two to three independent experiments, and were analyzed by either Student’s t tests (A, B, and D–F) or one-way ANOVA (C). ∗p < 0.05; ∗∗p < N.S., not significant. Cell Reports , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions
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