Research ArticleINFECTIOUS DISEASE

Sustained T follicular helper cell response is essential for control of chronic viral infection

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Science Immunology  01 Dec 2017:
Vol. 2, Issue 18, eaam8686
DOI: 10.1126/sciimmunol.aam8686
  • Fig. 1 Specific depletion of CD4-DTR–derived CD4 T cells.

    (A) Lethally irradiated CD4−/− mice were reconstituted with CD4-DTR (CD45.2+) and CD45.1+ B6 BM and persistently infected with 2 × 106 FFU LCMV clone 13. From d20 pi onward, continuous DTx treatment every 3 to 4 days was initiated. (B) Longitudinal analysis of CD4 and CD8 T cell ratios in blood. CD4-DTR–derived CD4/CD8 T cells (CD45.2+) and CD45.1+ B6–derived CD4/CD8 T cells are separately plotted. (C) Ratios and total cell numbers of CD4-DTR–derived CD4 T cells to CD45.1+ B6–derived CD4 T cells in the spleen, mLN, and lung at d40 pi. (D) Viral titers in the spleen on d40 pi. Statistical analysis was performed using Mann-Whitney U test; P = 0.8413. (E) Ratios and numbers of CD4-DTR–derived (CD45.2+) and CD45.1+ B6–derived non-TFH (red gate; CXCR5) and TFH (blue gate; CXCR5+ PD-1+) cells in the spleen at d40 pi in untreated or DTx-treated animals. Pregated on CD4+ single lymphocytes. One representative experiment of two is shown, n = 4. Error bars represent means ± SD. ns, not significant.

  • Fig. 2 Specific depletion of TFH and LCMV-specific CD4 T cells.

    (A) Experimental approach. Lethally irradiated CD4−/− mice were reconstituted with CD4-DTR (CD45.2+) BM and CD45.1+ B6 (control chimeras), CXCR5−/− (CXCR5−/− chimeras), or M25 (M25 chimeras) BM and persistently infected with 2 × 106 FFU LCMV clone 13. From d20 pi onward, continuous DTx treatment was initiated. (B) Longitudinal analysis of ratios of CD4-DTR–derived (CD45.2+) and CD45.1+ B6 (left), CXCR5−/− (CD45.1+; middle), or M25 (CD45.1+, right)-derived CD4 T cells in blood in untreated (upper) or DTx-treated (lower) mixed BM chimeras. (C) Ratios of CD4-DTR–derived (CD45.2+, white) and CD45.1+ B6 (black), CXCR5−/−(CD45.1+, red), or M25 (CD45.1+, blue)-derived CD4 T cells in the spleen (left) and mLN (right) on d70 pi in DTx-treated chimeras. (D) Total CD4 T cells in the spleen d70 pi of untreated or DTx-treated control (black), CXCR5−/− (red), or M25 (blue) chimeras. (E) Flow cytometric analysis of TFH cells (CXCR5+ PD-1+) in the spleen on d70 pi in untreated or DTx-treated control (black), CXCR5−/− (red) and M25 (blue) chimeras. (F) Number of gp61-tetramer+ CD4 T cells in untreated or DTx treated control (dark gray) or CXCR5−/− (red) chimeras at d100 pi in the spleen. (B, C, and F) One representative experiment of three is shown, n = 3 to 4 mice per group. Error bars represent means ± SD. (D and E) Pooled data of two independent experiments, n = 3 to 4 mice per group. Error bars represent means ± SEM. Statistical analysis was performed using Mann-Whitney U test, P > 0.05.

  • Fig. 3 CXCR5+/+ TFH cells are dispensable for maintenance of the LCMV-specific antibody response.

    (A) GC B cell (CD95+ CD38−/lo) and memory B cells (CD38+) in the spleen as identified by gating on CD19+ and switched IgD IgM B cells. Representative FACS blots of untreated (upper) and DTx-treated (lower) control (left), CXCR5−/− (middle), and M25 (right) chimeras are shown. Below quantifications of total cell counts are depicted. Data of three independently conducted experiments were pooled, n = 3 to 5 mice per group. (B) Number of LCMV-specific ASC as determined by ELISPOT in the spleen. (C) Number of LCMV-specific ASC as determined by ELISPOT in BM. One representative of two experiments is shown, n = 3 to 5 mice per group. (A to C) Error bars represent means ± SD. Mann-Whitney U test was used for statistical analysis. P > 0.05. (D) Titers of LCMV-specific IgG in the sera of control, CXCR5−/−, and M25 chimeras on d20 pi and (E) d100 pi in untreated (filled squares) and DTx-treated (empty squares) mice as determined by ELISA. Multiple unpaired t test was used for statistical analysis, *P = 0.0086. d20 pi corresponds to the first day of DTx treatment. (F) Titers of LCMV clone 13–GP1–Fc-specific or (G) LCMV clone 13 NP-specific antibodies in untreated (left) and DTx-treated (right) control, CXCR5−/−, and M25 chimeras. Multiple unpaired t test was used for statistical analysis, *P = 0.007. (D to G) Serum was prediluted 1:20 for ELISA analysis and further diluted in a threefold dilution series. Serum from naïve C57BL/6 mice was used as negative control. (D to G) One representative experiment of two is shown, n = 3 to 5 mice per group. Error bars represent means ± SD.

  • Fig. 4 CXCR5+/+ TFH cells are essential for the generation of LCMV-neutralizing antibodies and clearance of a persistent LCMV infection.

    (A) Neutralization of LCMV by serum of untreated or DTx-treated control, CXCR5−/−, and M25 chimeras. Pooled data of three independent experiments, n = 3 to 5 mice per group. Error bars represent means ± SD. (B) Viral titers in the spleen of naïve C57BL/6 mice infected with clone 13 together with either naïve (gray, −DTx) or serum derived from untreated (−DTx), DTx-treated (+DTx) control, or CXCR5−/− chimeras at d4 pi. Representative data of two independent experiments, n = 3. (C) Viral titers in blood at d20 and d70 pi in untreated and DTx-treated control, CXCR5−/−, and M25 chimeras. (D) Viral titers in the spleen at d100 pi in untreated and DTx-treated control, CXCR5−/−, and M25 chimeras. (C and D) Pooled data of three independent experiments, n = 3 to 5 mice per group. Mann-Whitney U test was used for statistical analysis, P > 0.05.

  • Fig. 5 GCs contain CXCR5−/− CD4 T cells in the absence of CXCR5+/+ TFH cells.

    (A) Immunofluorescence staining of splenic thin sections of untreated (upper) or DTx-treated (lower) control (left), CXCR5−/− (middle), and M25 (left) chimeras for GC B cells (PNA+, blue), B cell follicles (IgD+, red), and CD4 T cells (yellow). Scale bars, 100 μm. (B) Ratio of CD4/PNA MFI and (C) quantification of CD4 T cells per square micrometer in the area of the GC. Twenty to 30 GCs in two independent experiments were analyzed in total, n = 3 to 4 mice per group. Error bars represent means ± SD. (D) Immunofluorescence staining of splenic thin sections of untreated (upper) or DTx-treated (lower) control (left) and CXCR5−/− (red) splenocyte chimeras at d50 pi. Staining for GC B cells (PNA, blue), CD4 (yellow), mature B cells (IgD, red), and FDCs (CD35/21+, white). LZ (white circle) identified as FDC-containing PNA+ area, DZ (green marked area) as non–FDC-containing PNA+ area. Scale bars, 100 μm. (E) Quantification of ratio of CD4 T cells per cubic micrometer contained in LZ to CD4 T cells per μm3 contained in the DZ. Twenty to 50 GC were analyzed in total, n = 3 per group. Error bars represent means ± SD. Mann-Whitney U test was used, P > 0.05.

  • Fig. 6 TFH cells drive the adaptation of the neutralizing antibody response toward contemporary virus species.

    (A) Neutralization of sera isolated from single chimeras (indicated by letters) isolated at d20 (upper) or d70 pi (lower) against the inoculum (left) or the contemporary virus isolates (right). Untreated chimeras are marked in green, and DTx-treated chimeras are marked in orange. Lines indicate effective neutralization (>1.5 SD of control). One representative experiment of two is shown. (B) Correlation between neutralization against the inoculum (upper) or the contemporary isolate (lower) and viral clearance in untreated (green) and DTx-treated (orange) control, CXCR5−/−, and M25 chimeras at d70 pi. Pooled data of three (inoculating virus) or two (contemporary isolates) independent experiments are shown. (C) Total number of IFN-γ or (D) TNF-producing CD8 T cells from mice acutely infected with the inoculum and restimulated for 6 hours with macrophages infected with the inoculum (red) or contemporary virus isolates from d70 pi of untreated (green), DTx-treated (orange) control, CXCR5−/−, and M25 chimeras. Uninfected macrophages (white) were used as negative control and PMA/ionomycin restimulation as positive control (black). Lines indicate cytokine expression by CD8 T cells stimulated by thio-macrophages infected with the inoculating virus. One representative experiment of two is shown. PMA, phorbol 12-myristate 13-acetate.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/18/eaam8686/DC1

    Materials and Methods

    Fig. S1. Basic characterization of the CD4-DTR mouse line.

    Fig. S2. Sustained activity of CXCR5 or Bcl6-expressing TFH cells is required for the development of LCMV-neutralizing antibodies and control of protracted infection.

    Fig. S3. Immunofluorescence stainings in splenic thin sections.

    Fig. S4. Viral escape is not driven by pressure of CTLs.

    Fig. S5. CD8 T cell responses are unaffected by the absence of TFH or LCMV-specific CD4 T cells.

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  • Supplementary Materials

    Supplementary Material for:

    Sustained T follicular helper cell response is essential for control of chronic viral infection

    Ute Greczmiel, Nike Julia Kräutler, Alessandro Pedrioli, Ilka Bartsch, Paola Agnellini, Gregor Bedenikovic, James Harker, Kirsten Richter, Annette Oxenius*

    *Corresponding author. Email: oxenius{at}micro.biol.ethz.ch

    Published 1 December 2017, Sci. Immunol. 2, eaam8686 (2017)
    DOI: 10.1126/sciimmunol.aam8686

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Basic characterization of the CD4-DTR mouse line.
    • Fig. S2. Sustained activity of CXCR5 or Bcl6-expressing TFH cells is required for the development of LCMV-neutralizing antibodies and control of protracted infection.
    • Fig. S3. Immunofluorescence stainings in splenic thin sections.
    • Fig. S4. Viral escape is not driven by pressure of CTLs.
    • Fig. S5. CD8 T cell responses are unaffected by the absence of TFH or LCMV-specific CD4 T cells.

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