Memory CD4+ T cells induce innate responses independently of pathogen

Abstract

Inflammation induced by recognition of pathogen-associated molecular patterns markedly affects subsequent adaptive responses. We asked whether the adaptive immune system can also affect the character and magnitude of innate inflammatory responses. We found that the response of memory, but not naive, CD4(+) T cells enhances production of multiple innate inflammatory cytokines and chemokines (IICs) in the lung and that, during influenza infection, this leads to early control of virus. Memory CD4(+) T cell-induced IICs and viral control require cognate antigen recognition and are optimal when memory cells are either T helper type 1 (T(H)1) or T(H)17 polarized but are independent of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) production and do not require activation of conserved pathogen recognition pathways. This represents a previously undescribed mechanism by which memory CD4(+) T cells induce an early innate response that enhances immune protection against pathogens.

Figure 1. Memory CD4⁺ T cells induce an acute increase in IIC upon flu infection

(a) Naïve B6 mice, or mice primed with A/Phil 60 days prior, were treated with isotype, CD4−, or Thy1.2-depleting antibody prior to A/PR8 challenge and levels of IIC in lungs assessed after 40 hrs (n = 5). (b) Bulk CD4⁺ T cells were isolated from naïve or A/PR8 primed mice (polyclonal memory), and equal numbers transferred to naive hosts or, alternatively, naive or in vivo- or in vitro-generated HNT memory cells were adoptively transferred to naive BALB/c hosts. All recipients were challenged with A/PR8 and lung homogenates assessed for IIC after 40 hrs (n = 5). Dotted lines in all figures represent levels of IIC in the absence of infection. Error bars indicate SD; * P < 0.05, ** P < 0.005, *** P < 0.001 (one-way ANOVA followed by Bonferroni’s post hoc test).

Figure 2. Role of IFN-γ, TNF-α, and CCL3 in IIC upregulation by memory CD4⁺ T cells

(a) Naïve or T_H1-polarized memory populations generated from Ifng^−/− CD4⁺ T cells were transferred to unprimed mice then infected with A/PR8. Fold-increase in IFN-γ detected in lung homogenate with transfer of memory versus naïve cells for days 2 and 3 post-infection is shown (n = 5). (b) Memory OT-II cells were transferred to unprimed B6 YETI hosts and lung cells stained for indicated surface markers to identify IFN-γ producing cells (eYFP+) (n = 5). Naïve or T_H1-polarized memory OT-II cells were transferred to WT, Ifngr^−/−, Tnfrsf1ab^−/−, or Ccr5^−/− hosts then infected with A/PR8. Level of IIC at 40 hours post-infection (c) and (d) viral titer on day 4 post-infection (n = 5 mice per day). Error bars indicate SD; * P < 0.05, ** P < 0.005, *** P < 0.001 (Students t-test).

Figure 3. T_H1- or T_H17-polarization is required for enhanced IIC response and viral control

Naïve, T_H1-, T_H17-, T_H2-polarized or T_H0 unpolarized memory HNT cells were transferred to BALB/c hosts then infected with A/PR8. (a) Levels of IIC in lungs 40 hrs post-infection (n = 5). (b) Pulmonary viral titers (n = 5 per day). Error bars indicate SD; * P < 0.05, ** P < 0.005, *** P < 0.001 (one-way ANOVA followed by Bonferroni’s post hoc test).

Figure 4. Recognition of antigen in the lung is sufficient for IIC upregulation

CFSE-labeled Thy-disparate naïve or T_H1-polarized memory HNT cells were transferred to separate hosts then infected with A/PR8 (n = 5 per group). (a) Numbers of donor cells in spleen, draining lymph node (dLN), and lung 40 hrs post-infection. (b) Representative CFSE and CD69 expression of donor cells 2 and 6 days post-infection. Naïve or memory OT-II cells were transferred to sham treated or splenectomized Lta^−/− hosts and infected with A/PR8-OVA_II. (c) Pulmonary IIC levels at 40 hrs post-infection (n = 5). (d) Pulmonary viral titers (n = 5 per day). Error bars indicate SD; * P < 0.05, ** P < 0.005, *** P < 0.001 (one-way ANOVA followed by Bonferroni’s post hoc test or t-test).

Figure 5. Cognate recognition of antigen on MHC Class II expressing CD11c⁺ cells is sufficient to induce IIC upregulation

(a) Naïve or T_H1-polarized OT-II memory cells were transferred to naive B6 hosts then infected with A/PR8 or A/PR8-OVA_II. To facilitate comparison of the different viruses, the ratio of viral titers on day 4 in mice receiving memory and naïve OT-II cells is shown (n = 5 per group per virus). (b) Levels of IIC in lung homogenates 40 hrs post A/PR8-OVA_II infection in WT, H2-Ab1^−/−, or CD11c Tg.H2-Ab1^−/− mice receiving T_H1-polarized memory OT-II cells (n = 5) and (c) pulmonary viral titers. (d) Number of CD11c⁺ cells in the lung 40 hrs post infection in mice receiving naïve or memory cells and (e) expression of MHC-II, CD40 and CD80 on CD11c⁺ cells. (f) Expression of MHC-II and CD40 on DC cultured with memory cells. (g) Cytokines detected in 48 hr supernatants of naïve or memory HNT cells cultured with DC. Error bars indicate SD; * P < 0.05, ** P < 0.005, *** P < 0.001 (one-way ANOVA followed by Bonferroni’s post hoc test or t-test).

Figure 6. Memory CD4⁺ T cells induce IIC responses independently of PAMP recognition

(a) Naïve or T_H1-polarized OT-II memory cells were transferred to naive WT, Ifnar2^−/−, or Myd88^−/− Trif^−/− hosts then infected with A/PR8-OVA_II. Levels of IIC detected in lungs 40 hrs post-infection (n = 5), and (b) viral titers. (c) Naïve or T_H1-polarized memory OT-II cells were transferred to B6 hosts then administered 100 or 10 μg of soluble LPS-free OVA i.n. and levels of IIC assessed after 40 hrs (n = 5). (d) Memory OT-II cells were transferred to B6 hosts administered LPS-free OVA as in (c) and infected with A/PR8 on the same day or 7 days later, or only infected with A/PR8, and viral titers determined (n = 5 per group). Error bars indicate SD; * P < 0.05, ** P < 0.005, *** P < 0.001 (one-way ANOVA followed by Bonferroni’s post hoc test or t-test).