Enhanced IL-2 in early life limits the development of TFH and protective antiviral immunity

Abstract

T follicular helper cell (TFH)-dependent antibody responses are critical for long-term immunity. Antibody responses are diminished in early life, limiting long-term protective immunity and allowing prolonged or recurrent infection, which may be important for viral lung infections that are highly prevalent in infancy. In a murine model using respiratory syncytial virus (RSV), we show that TFH and the high-affinity antibody production they promote are vital for preventing disease on RSV reinfection. Following a secondary RSV infection, TFH-deficient mice had significantly exacerbated disease characterized by delayed viral clearance, increased weight loss, and immunopathology. TFH generation in early life was compromised by heightened IL-2 and STAT5 signaling in differentiating naive T cells. Neutralization of IL-2 during early-life RSV infection resulted in a TFH-dependent increase in antibody-mediated immunity and was sufficient to limit disease severity upon reinfection. These data demonstrate the importance of TFH in protection against recurrent RSV infection and highlight a mechanism by which this is suppressed in early life.

Figure S1.

Age-related reduction in adaptive immune responses to RSV infection. 8-wk-old BALB/c mice were infected 8 × 10⁵ ffu RSV A2 i.n. (A) T_FH (CD19⁻CD8⁻Foxp3⁻CXCR5⁺PD1⁺CD49d⁺CD11a⁺CD4⁺) and GC B cell (IgD⁻IgM⁻CD38⁻GL7⁺CD19⁺) numbers were determined in the lungs, mediastinal LNs, and spleen p.i. (B and C) RSV-specific Ig, IgG₁, and IgG_2a in the serum (B) and IgA in the airways (C). (D–G) 1-, 2-, and 8-wk-old BALB/c mice were infected 2.5 × 10⁵ ffu RSV A2 i.n. and sacrificed at day 14 p.i. (D) The total number of CD4 T cells, B cells, and Foxp3⁺ CD4 T cells was enumerated. (E) Cytokine production by LN CD4 T cells after RSV peptide stimulation. (F) Virus-specific CD8 T cells in the lungs and LNs. (G) LN and spleen (SP) from 1- and 8-wk-old mice were imaged by immunofluorescence microscopy for IgD, CD4, and GL7. Data are from n = 5–10 mice per time point and representative of two independent experiments, except in G, where a representative image from n = 6 mice per group across two independent repeats is shown. *, P < 0.05; and **, P < 0.01.

Figure 1.

Early-life RSV infection is associated with reduced GC activity. 1-, 2-, and 8-wk-old BALB/c mice were infected with 2.5 × 10⁵ ffu RSV A2 i.n. (A–C) Endpoint titers of RSV-specific Ig, IgG1, and IgG2a in the serum (A) and RSV-specific IgA in the bronchoalveolar lavage (BAL; B) and nasal lavage (NAL; C) were determined by ELISA. (D) RSV L relative to 18S in lung tissue measured by RT-qPCR at day 4 p.i. (E and F) The number and frequency of antigen-experienced CD11a⁺CD49d⁺Foxp3⁻ CD4 T cells (E) and CXCR5⁺PD1⁺ T_FH and B cells and GC B cells (F) in the lung dLNs were determined by flow cytometry at day 14 p.i. (G) The frequency of T_FH, GC B cells, and IgG1⁺ memory (IgD⁻GL7⁻CD38⁺IgG1⁺) B cells in the LNs was determined by flow at 56 d p.i. (H) Mice were rechallenged with 1 × 10⁶ ffu RSV A2 i.n. 8 wk after primary infection, and weight loss was monitored. Area under the curve at 1 wk (*) and 2 wk (#) compared with 8 wk. Data are shown with mean ± SEM. Data in A–C, E, and H are n = 10–12 mice per time point and are representative of two independent experiments. Data in D and G are from n = 5 mice from one experiment and are representative of two independent experiments presented as *, P < 0.05; ** or ##, P < 0.01; and ***, P < 0.001.

Figure 2.

T cell expression of Bcl6 is essential for humoral immunity but does not affect disease severity after primary RSV infection. 7- to 8-wk-old WT (Bcl6^fl/fl) or T_FH-deficient KO (Cd4cre Bcl6^fl/fl) mice were infected with 1 × 10⁶ ffu RSV A2 i.n. (A) Weight was monitored throughout infection. (B) RSV viral load in the lungs at day 4 p.i. as detected by RT-qPCR. (C) Virus-specific CD8 and CD4 T cells were analyzed in the lungs at day 14 p.i. (D and E) LN T_FH (CXCR5⁺ PD1⁺) CD4 T cells (D) and GC (CD38⁻GL7⁺) B cells (E) were analyzed by flow cytometry. (F) RSV-specific IgG1 and IgG2a were measured in the serum at day 14 p.i. Data in A and F represent n = 12–13 mice per group from two independent experiments. Data in B–E are from n ≥ 5 mice per group and are representative of two independent experiments. Data are shown as mean ± SEM. **, P < 0.01; ****, P < 0.0001.

Figure 3.

Impaired humoral immunity limits protection from RSV reinfection. 7- to 8-wk-old WT (Bcl6^fl/fl) or T_FH-deficient KO (Cd4cre Bcl6^fl/fl) mice were infected with 1 × 10⁶ ffu RSV A2 i.n. and 4 wk later reinfected with 1 × 10⁶ ffu RSV A2 i.n. (A) Weight change was monitored throughout infection. (B) RSV viral load in the lungs at day 4 p.i. detected by RT-qPCR. (C and D) Airway and lung cell numbers. (E and F) Proportion of eosinophils and neutrophils (E) and number of NK cells in the airways at day 4 p.i. as measured by flow cytometry (F). (G) Virus-specific CD8 and CD4 T cells from the lungs were analyzed by flow cytometry at day 14 p.i. (H) T_FH and GC B cell numbers were determined in the lungs and LN by flow cytometry, and total numbers were enumerated. (I) RSV-specific Ig, IgG₁, and IgG_2a in the serum and IgA in the airways were determined by ELISA. Data are representative of n = 12–13 mice per group from two independent experiments. Data are shown with mean ± SEM. *, P < 0.05; and ***, P < 0.001.

Figure S2.

Bcl6 deficiency in T cells during IAV primary infection and secondary infection reduces antibody responses, but does not affect weight loss. 7- to 8-wk-old WT (Bcl6^fl/fl) or T_FH-deficient KO (Cd4cre Bcl6^fl/fl) mice were infected with 10⁶ ffu RSV A2 i.n. (A and B) Virus-specific CD4 and CD8 T cells were stimulated ex vivo with RSV peptides to induce cytokine production and analyzed by flow cytometry. (C–F) 7- to 8-wk-old WT (Bcl6^fl/fl) or T_FH-deficient KO (Cd4cre Bcl6^fl/fl) mice were infected with 40 PFU IAV PR8 and weighed daily to monitor disease severity. (D) T_FH and GC B cell populations were analyzed by flow cytometry. (E) IAV-specific Ig, IgG₁, and IgG_2c in the serum as determined by ELISA. (F) WT or T_FH-deficient KO mice were rechallenged with 40 PFU IAV PR8 8 wk after primary challenge and weighed daily. Data are from n ≥ 5 mice per group and representative of two independent experiments. Data are shown with mean ± SEM. *, P < 0.05; and **, P < 0.01.

Figure S3.

BCL6 deficiency in early life does not affect the acute immune response. WT (Bcl6^fl/fl) or T_FH-deficient KO (Cd4cre Bcl6^fl/fl) mice were infected as neonates (7 d old) with 2.5 × 10⁵ ffu RSV A2 i.n. (A) Mice infected as neonates were weighed daily. (B and C) Virus-specific T cell populations were analyzed in the lungs (B) and LN (C) of infected neonates at day 14 p.i. (D–F) Mice were reinfected 8 wk later with 8 × 10⁵ ffu RSV A2 i.n. (D) Airway, lung, and LN cells before (day 0) and after (days 4 and 7) reinfection. (E) Virus-specific T cell populations were analyzed by flow cytometry at all time points. (F) Cytokine concentrations in the airways was measured by ELISA. Data are from n ≥ 4 mice per group and representative of two independent experiments. Data are shown with mean ± SEM.

Figure 4.

Early-life immune responses to RSV are T_FH independent. WT (Bcl6^fl/fl) or T_FH-deficient KO (Cd4cre Bcl6^fl/fl) mice were infected as neonates (7 d old) with 2.5 × 10⁵ ffu or as adults (7–8 wk old) with 1 × 10⁶ ffu RSV A2 i.n. (A) Virus-specific CD4 T cells and T_FH populations in the LNs of mice infected as neonates or adults were analyzed by flow cytometry at day 14 p.i. Gating for CXCR5⁺PD1⁺ (purple line) and CXCR5^hiPD1⁺ (orange line) are highlighted. (B) LN GC B cells were analyzed by flow cytometry at day 14 p.i. in mice infected as neonates. (C) Virus-specific serum IgG1 and IgG2c was measured by ELISA. (D) Mice infected as neonates were reinfected 8 wk later with 8 × 10⁵ ffu RSV A2 i.n. and weighed daily. (E–G) Virus-specific IFN-γ (E) and IL-4 and IL-13 (F) production by CD4 and CD8 T cells (G) was analyzed by flow cytometry before reinfection and at days 4 and 7 p.i. (H and I) Airway eosinophils (H) and NK cells (I) were analyzed by flow cytometry. (J) Serum IgE was quantified during primary and secondary infections by ELISA. (K) Lung viral load was detected by RT-qPCR at all time points. Data are from n = 4–9 mice per time point and representative of two independent experiments. Data are shown with mean ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Figure 5.

IL-2 and IFN-γ signaling limit antibody-mediated immunity after early-life infection. 7-d-old C57B/L6 mice were infected with 2.5 × 10⁵ PFU RSV A2 i.n. and treated with anti-IFN-γ, anti-IL-2, or isotype-matched antibodies i.p. on days −1, 2, and 5 p.i. (A) At day 14 p.i., lung and LN cells were counted. (B and C) Virus-specific CD4 and CD8 T cell populations in the lung (B) and dLN (C) were analyzed by flow cytometry. (D–G) Total and virus-specific T_FH and ICOS and Bcl6 expression on total T_FH (D and E) and GC (F and G) B cell subsets were analyzed by flow cytometry. (H) RSV-specific Ig, IgG1, and IgM were measured by ELISA. Data are from n = 8–12 mice per group and representative of two independent experiments. Data are shown with mean ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Figure S4.

IL-2 neutralization does not limit T reg cell numbers in early-life RSV infection but limits inflammation on reinfection as adults. Mice were infected with 2.5 × 10⁵ ffu RSV A2 at 7 d of age and treated with anti-IFN-γ, anti-IL-2, or isotype control i.p.; mice were sacrificed at day 14 p.i. (A) Foxp3⁺ T reg cells were enumerated in the lung, LNs, and spleen. (B) The number and frequency of Foxp3⁺CXCR5⁺PD1⁺ CD4 T cells in the lung dLNs. (C) ICOS and Bcl6 expression by LN T_FH was determined by flow cytometry, and representative histograms are shown. (D) Total airway, lung, and LN cell counts at day 7 after reinfection with 8 × 10⁵ PFU RSV A2 i.n. in mice initially infected with 2.5 × 10⁵ PFU RSV A2 i.n. as neonates. Data represent two independent experiments with n = 8–12 mice per group. Data are shown with mean ± SEM. **, P < 0.01; and ***, P < 0.001.

Figure 6.

Neutralizing IL-2 in early life enhances protective immunity to secondary infection in a T_FH-dependent manner. 7-d-old Bcl6^WT or Bcl6^ΔCD4 mice were infected with 2.5 × 10⁵ PFU RSV A2 i.n. and treated with either anti-IL-2 or isotype-matched antibodies i.p. on days −1, 2, and 5 p.i. On day 56 p.i., they were then reinfected with 8 × 10⁵ PFU RSV A2 i.n. (A) Experimental design. (B) RSV-specific serum Ig, IgG1, and IgG2c were measured by ELISA. (C) Weight change on reinfection. (D) At day 7 after challenge, the number of T_FH, GC B cells, and CD138⁺CD3⁻ plasma cells were enumerated in the LNs by flow cytometry. (E and F) RSV L expression in the lungs was determined by RT-qPCR (E), and the number of airway eosinophils, neutrophils, and activated (CD44⁺PD1⁺) CD8 and CD4 T cells was analyzed by flow cytometry (F). (G) Following polyclonal stimulation, the frequency of airway CD8 and CD4 T cells producing IFN-γ or IL-13 was measured. (H) Foxp3⁺ T reg cell frequencies in the lungs and LNs were determined by flow cytometry. Data represent two independent experiments with n = 8–12 mice per group. Data are shown with mean ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Figure 7.

Enhanced pSTAT5 in early-life CD4 T cells is associated reduced T_FH differentiation. (A and B) 7-d-old Bcl6^WT or Bcl6^ΔCD4 mice were infected with 2.5 × 10⁵ PFU RSV A2 i.n. Bcl6^WT mice were treated with either anti-IL-2 or isotype-matched antibodies i.p., while Bcl6^ΔCD4 mice received isotype control. T_FH were analyzed in the dLNs at day 3 p.i. (C) pSTAT3 and pSTAT5 expression measured directly ex vivo in naive (CD44⁻Foxp3⁻GITR⁻) and antigen-experienced (CD44+Foxp3⁻GITR⁻) CD4 T cells in the spleen and dLNs of day 3 p.i. 1- and 8-wk-old C57BL/6 mice. (D) Il2 and Il6 expression in the spleen of 1-, 2-, and 8-wk-old uninfected C57BL/6 mice. (D and E) IL-2 production following polyclonal stimulation in LNs from uninfected or day 3 p.i. RSV-infected 1- and 8-wk-old C57BL/6 mice (D), and (E) the geometric mean fluorescence intensity (MFI) of IL-2 in IL-2⁺ Foxp3⁻ CD4 T cells following polyclonal stimulation (left graph), and the percentage of IL-2⁺ in the presence of brefeldin A alone were measured by flow cytometry. (F) CD4 T cells were isolated from the spleens of 1 and 8-wk-old C57BL/6 mice and stimulated for 15 min with rIL-2 or rIL-6. STAT phosphorylation was quantified by flow cytometry. Data are from n = 4–6 mice per group and are representative of two independent experiments. Data are shown with mean ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Figure S5.

Assessment of IL-2 and IL-6 production and signaling in secondary lymphoid tissue. (A) Neonatal LN T_FH and non-T_FH, as shown in Fig. 7 A, were assessed for expression of PSGL1 and Ly6C. (B) Expression of Il6 and Il2 relative to GAPDH in the spleen of 1-, 2-, and 8-wk-old uninfected C57B/L6 mice was determined by RT-qPCR. (C) IL-2 was measured by flow cytometry in different cell populations from lung dLNs of neonatal C57B/L6 at day 3 after RSV infection. A gating strategy for the cell types and a representative IL-2 histogram following either polyclonal (PMA/I and brefeldin A [BFA]) or unstimulated (brefeldin A alone) are shown. (D) CD25, CD122, and CD132 expression by splenic CD44⁻CD62L⁺Foxp3⁻ and Foxp3⁺ CD4 T cells in 1- and 8-wk-old C57BL/6 mice. FMO, fluorescence minus one. (E) Percentage of CD25⁺ splenic CD44⁻CD62L⁺Foxp3⁻ and Foxp3⁺ CD4 T cells in 1-wk-old C57BL/6 mice. Data in A and B are from n = 4–5 mice per group and are representative of two independent experiments. Data in D and E are from n = 6 mice and are representative of two independent experiments. (C) Representative gating strategy from an RSV-infected mouse; data are enumerated in Fig. 7, D and E. Data are shown with mean ± SEM. *, P < 0.05; and **, P < 0.01.