Memory CD4+ T cells protect against influenza through multiple synergizing mechanisms

Abstract

Memory CD4+ T cells combat viral infection and contribute to protective immune responses through multiple mechanisms, but how these pathways interact is unclear. We found that several pathways involving memory CD4+ T cells act together to effectively clear influenza A virus (IAV) in otherwise unprimed mice. Memory CD4+ T cell protection was enhanced through synergy with naive B cells or CD8+ T cells and maximized when both were present. However, memory CD4+ T cells protected against lower viral doses independently of other lymphocytes through production of IFN-γ. Moreover, memory CD4+ T cells selected for epitope-specific viral escape mutants via a perforin-dependent pathway. By deconstructing protective immunity mediated by memory CD4+ T cells, we demonstrated that this population simultaneously acts through multiple pathways to provide a high level of protection that ensures eradication of rapidly mutating pathogens such as IAV. This redundancy indicates the need for reductionist approaches for delineating the individual mechanisms of protection mediated by memory CD4+ T cells responding to pathogens.

Figure 1. Memory CD4⁺ T cell protection against lethal IAV infection.

5 × 10⁶ naive or in vitro–generated memory HNT cells, polarized as stated, were transferred to unprimed WT hosts that were then infected with 10,000 EID₅₀ PR8. (A) Survival for n = 10/group and (B) viral titers for n = 4/group (representative of 3 similar experiments). (C) 5 × 10⁶ in vivo, IAV-primed memory HNT cells were transferred to WT host mice, then infected as in A (representative of 2 independent experiments with n = 5/group). (D) 5 × 10⁶ Ifng^–/– T_H1 or T_H17-polarized or in vivo–primed memory HNT cells were transferred to unprimed WT hosts, then infected with 10,000 EID₅₀ PR8. Survival for n = 10/group (representative of 3 separate experiments).

Figure 2. Memory CD4⁺ T cell protection in the absence of host T or B cells.

5 × 10⁶ T_H1-polarized memory HNT cells were transferred to either (A) nude or (B) J_HD hosts, then infected with stated doses of PR8 and survival monitored. Results representative of 3 separate experiments with each host (n = 10/group for nude and n = 5 for J_HD hosts). WT or Ifng^–/– T_H1-polarized memory HNT cells were transferred to (C) nude and (D) J_HD hosts and weight loss monitored after 2500 EID₅₀ PR8 infection (n = 10/group for nude and n = 10 for J_HD hosts) and (E) and (F) viral titers determined at 7 dpi (n = 5/group).

Figure 3. Memory CD4⁺ T cell protection in the absence of both host B cells and T cells.

5 × 10⁶ T_H1-polarized memory HNT cells were transferred to SCID hosts, then infected with 2,500 EID₅₀ PR8. Survival (A) and weight loss (B) for n = 5–10/group. (C) Memory HNT cells were transferred to SCID or J_HD mice and viral titer determined on indicated dpi, n = 5–10/group. (D) Recovery of donor cells in SCID hosts and (E) ability of 3 × 10⁶ d22 reisolated cells from infected SCID hosts to protect WT hosts infected with 10,000 EID₅₀ PR8. (F) Memory HNT cells were transferred to SCID hosts, then infected with 500 EID₅₀ PR8 and morbidity monitored. n = 10/group. Data represent at least 2 independent experiments.

Figure 4. Memory CD4⁺ T cells synergize with unhelped B cell Ab responses.

Memory HNT cells (5 × 10⁶) were transferred to SCID hosts, then infected with 2,500 EID₅₀ PR8 (A) in conjunction with WT naive B cells or mIg Tg Bonnie B cells or (B) followed by administration of naive, PR8-, or X31-immune serum at 7 and 8 dpi and weight loss monitored for n = 5/group. Memory HNT cells were transferred to nude hosts, then infected with 2,500 EID₅₀ PR8 and treated daily with 500 μg of isotype or anti-CD40L ab. (C) Mean PR8-specific total IgG (horizontal bar) endpoint titers at 14 dpi of n = 4/group (circles) and (D) viral titers at 14 dpi, n = 4/group as well as (E) weight loss and (F) survival were determined (n = 5/group). Data representative of at least 2 independent experiments.

Figure 5. Memory CD4⁺ T cells synergize with CD8⁺ T cell effectors in the absence of B cells.

Memory HNT cells (5 × 10⁶) were transferred to J_HD hosts treated with 1 mg of isotype or CD8-depleting ab followed by infection with 2,500 EID₅₀ PR8. (A) Survival and (B) weight loss for n = 5–10/group. (C) Survival and (D) weight loss following memory HNT cell transfer and depletion of CD8 T cells on the indicated days. n = 5–10/group. Memory HNT cells (5 × 10⁶) were transferred to SCID hosts ± naive CD8⁺ T cells followed by infection with 2,500 EID₅₀ PR8. (E) Survival and (F) weight loss for n = 5–10/group. Data representative of 2 independent experiments.

Figure 6. Memory CD4⁺ T cells select for viral escape mutants through a perforin-dependent mechanism.

5 × 10⁶ WT or Prf1^–/– memory HNT cells were transferred to SCID hosts, then infected with 2,500 EID₅₀ PR8. (A) Survival, (B) weight loss, and (C) viral titers at 9 dpi are shown for at least n = 10/group (summary of 3 separate experiments). (D) Schematic of the amplicon used for sequencing PR8 and PR8-OVA_II isolates. Naive splenocytes were infected with concentrated PR8-OVA_II stock or viral isolates from SCID mice receiving memory DO11.10 cells and cocultured with naive CFSE-labeled TCR Tg CD4⁺ T cells for 72 hours. The percentage of maximal division from triplicate cultures for each of the 4 viral isolates or stock PR8-OVA_II cultured with naive (E) DO11.10 and (F) HNT cells.

Figure 7. Memory CD4⁺ T cell protection requires IFN-γ in the absence of host lymphocytes.

5 × 10⁶ WT or Ifng^–/– memory HNT cells were transferred to unprimed SCID hosts, then infected with 2,500 EID₅₀ PR8. Recipients of WT cells were treated with 500 μg of isotype or anti–IFN-γ neutralizing ab throughout the infection. (A) Survival, (B) weight loss, and (C) mean viral titers (horizontal bar) at 9 dpi are shown for at least n = 5/group (circles) (1 of 2 separate experiments, *P < 0.005; **P < 0.005; ****P < 0.0001). (D) SCID recipients of 5 × 10⁶ WT or Ifng^–/– memory HNT cells were challenged with 50 EID₅₀ PR8 and weight loss determined (n = 6/group). SCID recipients of Ifng^–/– memory HNT cells were also reconstituted with no additional cells, unprimed Ifng^–/– B cells, or unprimed Ifng^–/– CD8 T cells and (E) survival and (F) weight loss determined. n = 5/group (1 of 2 separate experiments).