Takao Kobayashi, PhD, Koji Iijima, PhD, Alexander L

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Follicular helper T cells mediate IgE antibody response to airborne allergens Takao Kobayashi, PhD, Koji Iijima, PhD, Alexander L. Dent, PhD, Hirohito Kita, MD Journal of Allergy and Clinical Immunology Volume 139, Issue 1, Pages 300-313.e7 (January 2017) DOI: 10.1016/j.jaci.2016.04.021 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 IL-33 promotes development of 2 distinct subsets of IL-4–competent CD4+ T cells. A, Schematic representation of a mouse airway sensitization model. Naive 4get mice (BALB/c background) were exposed intranasally to OVA with or without IL-1β or IL-33. B, On day 14, plasma was analyzed for anti-OVA antibodies (Ab) by means of ELISA. *P < .05 and **P < .01 compared with mice exposed to OVA alone. #P < .05 compared with mice exposed to OVA plus IL-33. Data are presented as means ± SEMs (n = 4-5 in each group). C, Day 11 mLN cells were cultured with or without OVA. Cytokine levels in supernatants were analyzed by mean of ELISA. #P < .05 compared with OVA plus IL-33. Data are presented as means ± SEMs (n = 4-5 in each group). D-G, Day 11 mLN cells were gated on lymphocytes, and expression of CD4, IL-4eGFP, ST2, and CXCR5 was analyzed by means of FACS. Fig 1, D, Representative scattergrams showing expression of CD4 and IL-4eGFP in a total lymphocyte population and expression of ST2 and CXCR5 in CD4+IL-4eGFP+ lymphocytes. Fig 1, E, Numbers of each cell population are presented as means ± SEMs (n = 4 in each group). *P < .05 between the groups indicated by horizontal lines. Fig 1, F, Naive 4get mice were exposed intranasally to OVA with different doses of IL-33, as described in Fig 1, A, and day 11 mLN cells were analyzed. Fig 1, G, Numbers and ratios of each cell population are presented as means ± SEMs (n = 4 in each group). **P < .01 between groups indicated by horizontal lines or compared with 1 ng of IL-33. In Fig 1, C, E, and G, data are a pool of 2 separate experiments. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 ST2−CXCR5+ CD4+ T cells are bona fide TFH cells. A-D, Naive 4get mice (BALB/c background) were exposed intranasally (i.n.) to OVA plus IL-33, as described in Fig 1, A. The cell populations indicated (CD4+IL-4eGFP−, CD4+IL-4eGFP+ST2+CXCR5−, and CD4+IL-4eGFP+ST2−CXCR5+) were sorted from day 11 mLN cells, and mRNA was analyzed by means of microarray and qRT-PCR. Fig 2, A, Heat map shows microarray gene expression data (genes selected for fold difference >10) based on cells isolated from 2 separate experiments. Fig 2, B, Venn diagram shows the number of gene probes in each gene expression group. Fig 2, C, Representative genes in each group are presented by using the criteria described in the Table. Fig 2, D, mRNA expression of select genes was analyzed by means of qRT-PCR, and results were normalized to 18s RNA levels and expressed as a ratio to the CD4+IL-4eGFP− population (mean ± SEM, n = 4). *P < .05 and **P < .01 between the groups indicated by horizontal lines. E and F, Naive BALB/c mice were exposed intranasally to OVA plus IL-33, as described in Fig 1, A. Day 11 mLN cells were sorted into CD3+CD4+ST2−CXCR5−, CD3+CD4+ST2+CXCR5−, and CD3+CD4+ST2−CXCR5+ cell populations and cultured with B cells isolated from mLNs in the presence of OVA. Fig 2, F, Cytokine levels in supernatants were analyzed by means of ELISA. Data are presented as means ± ranges from duplicate cultures. The figure is representative of 2 separate experiments. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 TH2 and TFH cells preferentially mediate allergic airway inflammation and IgE antibody production, respectively. A, Schematic representation of an adoptive transfer model to Tcrb−/− mice (C57BL/6 background). B, Plasma antibody levels were analyzed by means of ELISA. Results are means ± SEMs (n = 3 in each group). **P < .01 compared with mice that received TH2 cells. C, Schematic representation of an adoptive transfer model to Il7−/− mice (C57BL/6 background). D and E, BAL fluids were analyzed for cytokine levels (Fig 3, D) and inflammatory cell numbers (Fig 3, E). Results are means ± SEMs (n = 3 in each group). **P < .01 between groups indicated by horizontal lines. Fig 3, B, D, and E, are representative of 2 separate experiments. Eos, Eosinophils; i.n., intranasal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 TH2 and TFH cells localize differently during allergic immune responses. A, A schematic representation of a mouse airway sensitization and challenge model. B, Representative scattergrams show expression of ST2 and CXCR5 in the CD4+IL-4eGFP+ population. C, Percentages of each cell population are presented as means ± SEMs (n = 8 in each group). **P < .01 between the groups indicated by horizontal lines. Data are a pool of 2 separate experiments. i.n., Intranasal. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Airway exposure to natural allergens induces TFH and TH2 cells. A, Schematic representation of the airway allergen exposure model. Naive 4get mice (BALB/c background) were exposed intranasally (i.n.) to allergen extracts without any adjuvants. Day 11 mLN cells were gated on lymphocytes, and expression of CD4, IL-4eGFP, ST2, and CXCR5 was analyzed by means of FACS. B, Representative scattergrams are shown. C, Numbers of each cell population are presented as means ± SEMs (n = 3 in each group). Data are representative of 2 separate experiments, except for HDM, for which the experiment was performed only once. i.n., Intranasal. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 TFH cell numbers are diminished by depletion of Bcl6 in CD4+ T cells. A, Schematic representation of a mouse airway sensitization model. B, Representative scattergrams show the expression of ST2, CXCR5, and PD-1 in the CD4+ T-cell population. mLN cells were analyzed by gating on CD4+ T cells in the lymphocyte population. C, Cell numbers in each cell population are presented as means ± SEMs (n = 5-7 in each group). *P < .05 and **P < .01 between the groups indicated by horizontal lines. Data are a pool of 3 separate experiments. D and E, Expression of B220, PNA, and FAS in mLN lymphocytes was analyzed by means of FACS. Fig 6, D, Representative scattergrams show expression of B220 in the lymphocyte population and expression of PNA and FAS in the B220+ cell population. Fig 6, E, Percentages and cell numbers in each cell population are presented as means ± SEMs (n = 4 in each group). *P < .05 and **P < .01 between the groups indicated by horizontal lines. F, Day 11 mLN cells were cultured with or without OVA, and cytokine levels in supernatants were analyzed by means of ELISA. Results are shown as means ± SEMs (n = 4 in each group). *P < .05 between the groups indicated by horizontal lines. i.n., Intranasal. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 TFH cells are necessary for production of allergen-specific IgE antibody. A, Schematic representation of a mouse airway sensitization and challenge model. B, Plasma levels of anti-OVA IgE and IgG1 antibodies and total IgE, IgG1, IgM, and IgA were analyzed by means of ELISA. **P < .01 compared with Bcl6 fl/fl mice previously exposed to OVA alone. ##P < .01 compared with Bcl6 fl/fl mice previously exposed to Alternaria alternata extract plus OVA. Data are presented as means ± SEMs (n = 5 in each group). C and D, Cytokine levels in lung homogenates (Fig 7, C) and numbers of inflammatory cells in BAL fluids (Fig 7, D) were analyzed on day 45. In Fig 7, C and D, results are presented as means ± SEMs (n = 5 in each group). *P < .05 and **P < .01 between the groups indicated by horizontal lines. Data are representative of 2 separate experiments. Eos, Eosinophils; i.n., intranasal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 OX40 ligand contributes to type 2 cytokine responses and airway inflammation but does not affect development of TFH cells or IgE antibody production. A-C, Naive C57BL/6 (WT) and OX40 ligand knockout (Tnfsf4−/−) mice were exposed intranasally to OVA and Alternaria alternata extract on days 0 and 7, and mLN cells were analyzed on day 11. A, Representative scattergrams show expression of ST2 and CXCR5 in the CD4+ T-cell population. B, Numbers of each cell population are presented as means ± SEMs (n = 4 in each group). C, mLN cells were cultured with or without OVA, and cytokine levels in supernatants were analyzed by means of ELISA. Results are means ± SEMs (n = 5-7 in each group). *P < .05 between the groups indicated by horizontal lines. Data are representative of 2 separate experiments. D, Schematic representation of a mouse airway sensitization and challenge model. E and F, Cytokine levels in lung homogenates (Fig 7, E) and numbers of inflammatory cells in BAL fluids (Fig 7, F) were analyzed on day 25. Results are means ± SEMs (n = 5-7 in each group). *P < .05 and **P < .01 between the groups indicated by horizontal lines. G, Plasma levels of anti-OVA IgE, IgG1, and IgG2a antibodies were analyzed by means of ELISA. *P < .05 and **P < .01 between groups indicated by horizontal lines. Data are presented as means ± SEMs (n = 5-7 in each group). In Fig 8, C, E, F, and G, data are a pool of 2 separate experiments. Eos, Eosinophils; i.n., intranasal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Antibody production in mice exposed to OVA with different doses of IL-33. Naive BALB/c mice were exposed intranasally to 100 μg of OVA plus 0, 1, 10, or 100 ng of IL-33 on days 0 and 7. On day 14, plasma levels of anti-OVA IgE, IgG1, and IgG2a antibodies were analyzed by means of ELISA. **P < .01 compared with OVA plus no IL-33. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Kinetics of the development of ST2+CXCR5− cells and ST2−CXCR5+ cells. A, Naive 4get mice were exposed intranasally (i.n.) to OVA plus IL-33. Day 4, 7, or 11 mLN cells were analyzed by means of FACS by gating on lymphocytes. B, Representative scattergrams show the expression of CD4 and IL-4eGFP (upper panels) and ST2 and CXCR5 in the CD4+IL-4eGFP+ cell population (lower panels; n = 3 at each time point). Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 T-cell populations sorted for cell culture (Fig 2, E and F) and adoptive transfer (Fig 3) experiments. Naive WT C57BL/6 or BALB/c mice were exposed intranasally to OVA plus IL-33 on days 0 and 7. Day 11 mLN cells were collected and enriched for CD4+ T cells and sorted into CD4+ST2−CXCR5− (blue square), CD4+ST2+CXCR5− (black square), and CD4+ST2−CXCR5+ (red square) subpopulations. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Naive B cells from the spleen did not induce cytokine production by TFH cells. A and B, Naive BALB/c mice were exposed intranasally (i.n.) to OVA plus IL-33 on days 0 and 7. Day 11 mLN cells were sorted into CD3+CD4+ST2−CXCR5− (DN), CD3+CD4+ST2+CXCR5− (ST2+), and CD3+CD4+ST2−CXCR5+ (CXCR5+) T-cell subpopulations. T cells were cultured with B cells isolated from the spleens of naive BALB/c mice at a T-cell/B-cell ratio of 1:1 in the presence of OVA for 7 days. Fig E4, B, Cytokine levels in supernatants were analyzed by means of ELISA. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 ICOS is critical for the development of TFH cells and IgE antibody production. A-E, WT C57BL/6 and Icos−/− mice were exposed intranasally (i.n.) to OVA with or without Alternaria alternata extract on days 0 and 7 and then challenged intranasally with OVA alone, as described. Fig E5, B and C, Day 11 mLN cells were analyzed by means of FACS. Fig E5, B, Representative scattergrams show expression of ST2 and CXCR5 in the Ki-67+CD4+ T-cell population. Fig E5, C, Numbers of each cell population are presented as means ± SEMs (n = 5 in each group). *P < .05 and **P < .01 between the groups indicated by horizontal lines. Fig E5, D, CD4+ T cells were isolated from day 11 mLN cells and cocultured with CD3-depleted splenocytes from naive WT mice with or without 100 μg/mL OVA for 4 days. Cytokine levels in supernatants were measured by means of ELISA. Results are shown as means ± SEMs (n = 5 in each group). *P < .05 and **P < .01 between the groups indicated by horizontal lines. Fig E5, E, Plasma levels of anti-OVA IgE, IgG1, and IgG2a antibodies were analyzed by means of ELISA. *P < .05 and **P < .01 compared with WT mice previously exposed to OVA alone. ##P < .01 compared with WT mice previously exposed to Alternaria alternata extract plus OVA. Data are presented as means ± SEMs (n = 5 in each group). Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Schematic diagram to depict the roles of TFH and TH2 cells in allergic immune responses to airborne allergens. Journal of Allergy and Clinical Immunology 2017 139, 300-313.e7DOI: (10.1016/j.jaci.2016.04.021) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions