Direct IL-6 Signals Maximize Protective Secondary CD4 T Cell Responses against Influenza

Abstract

Memory T cells can often respond against pathogens that have evaded neutralizing Abs and are thus key to vaccine-induced protection, yet the signals needed to optimize their responses are unclear. In this study, we identify a dramatic and selective requirement for IL-6 to achieve optimal memory CD4 T cell recall following heterosubtypic influenza A virus (IAV) challenge of mice primed previously with wild-type or attenuated IAV strains. Through analysis of endogenous T cell responses and adoptive transfer of IAV-specific memory T cell populations, we find that without IL-6, CD4⁺, but not CD8⁺, secondary effector populations expand less and have blunted function and antiviral impact. Early and direct IL-6 signals to memory CD4 T cells are required to program maximal secondary effector responses at the site of infection during heterosubtypic challenge, indicating a novel role for a costimulatory cytokine in recall responses.

Figure 1. High priming doses of IAV are lethal in the absence of IL-6

WT (black symbols) or Il6^−/− (grey symbols) mice on a BALB/c background were infected with a low dose (500 EID₅₀ = 0.1 LD₅₀ for WT mice) or high dose (2500 EID₅₀ = 0.5 LD₅₀) of A/PR8 (H1N1). Weight loss following (a) low and (b) high dose challenge (n = 5 mice/group; one of 5 experiments). On the indicated days following 2500 EID₅₀ challenge, (c) survival, and (d) viral titers were assessed (n =3–5 mice/group/day). H&E stained lung sections were scored blindly for levels of histopathology on 7 dpi. Summary of analysis (n = 3–5 mice/group/day) (e), and representative H&E images at 10x (f) for categories of inflammation scored in (e) (PV = peri-vascular; PB = peri-bronchial; AI = alveolar inflammation; IE = Intra-epithelial).

Figure 2. IL-6 is required for optimal heterosubtypic protection

WT or Il6^−/− mice on a BALB/c background were primed with a low dose (500 EID₅₀ = 0.1 LD₅₀ for WT mice) of A/PR8 (H1N1). After 45 days mice were challenged with a 150 LD₅₀ dose of A/Philippines (H3N2) virus and (a) morbidity and (b) survival monitored (n=5 mice/group; one of 2 experiments). Viral titers (c) from lungs of A/PR8-primed WT or Il6^−/− mice (n=4 mice/group), or unprimed WT control mice (n=3) were assessed on the stated dpi after 150 A/Phil challenge. Data is representative of one of two experiments. The dashed line in (c) represents sensitivity cutoff of the assay.

Figure 3. CD4 but not CD8 T cell cytokine responses are reduced during heterosubtypic challenge of mice primed with attenuated IAV

WT or Il6^−/− C57BL/6 mice were primed with two doses of 2500 TCID₅₀ A/Alaska ten days apart. At day 45 post-priming mice were challenged with 20 LD₅₀ A/PR8. Survival and weight loss (a) of primed or unprimed control WT and Il6^−/− mice (n=4 mice/group; one of two experiments). IFN-γ responses elicited by immunodominant MHC I-restricted peptides were enumerated by ELISPOT from cells isolated from stated organs 5 days after A/PR8 challenge primed WT or Il6^−/− mice (b) and numbers of CD44^hi CD8 T cells in the lung determined by FACS (c) (n=3 mice/group; one of two experiments). Similarly, IFN-γ (d) and IL-2 (e) responses were enumerated in stated organs by ELISPOT using IAV lysate to stimulate CD4 T cells (n=3 mice/group; one of two experiments) and the number of CD44^hi CD4 T cells in the lung determined by FACS (f). Dual IFN-γ and IL-2 production by CD4 T cells in the stated organs in response to IAV lysate was determined by ICCS and the frequency expressed as a ratio to the WT response (g), representative staining of spleen cells (h), and the enumeration of dual cytokine producing cells (i) is shown.

Figure 4. Memory CD4 T cell formation following IAV priming is not impaired in the absence of IL-6

WT or Il6^−/− C57Bl/6 mice were primed with A/Alaska as described in Figure 3. The (a) percentage and (b) absolute number of NP_311–325 tetramer⁺ CD44^high CD4 T cells was enumerated at 45 dpi (n=4 mice/group; one of 2 similar experiments). In separate experiments, WT or Il6^−/− mice receiving 1×10⁶ Thy-disparate naïve OT-II donor cells were primed with 0.1 LD₅₀ A/PR8-OVA_II. At 45 dpi, the (c) percentage and (d) absolute number of donor cells detected in stated organs was determined (n=4 mice/group; one of 2 experiments). The mean fluorescence intensity of CD127 on donor cells was determined by FACS (e). The percentage of donor cells co-producing IFN-γ and IL-2 was determined by intracellular cytokine staining following ex vivo stimulation (f).

Figure 5. Protection mediated by memory CD4⁺, but not CD8⁺, T cells is compromised in the absence of IL-6

Memory CD8 T cells, 5×10⁶, generated from HA TcR transgenic cells were transferred to naïve WT or Il6^−/− BALB/c mice. Mice were subsequently infected with 10,000 EID₅₀ A/PR8 and their (a) survival and (b) weight loss monitored (n=10 mice/group) and compared to mice not receiving donor memory cells (n=3 mice). WT or Il6^−/− BALB/c mice that received 5×10⁶ memory CD4 T cells generated from HNT TcR transgenic cells were challenged with 10,000 EID₅₀ A/PR8, and (c) survival and (d) weight loss monitored. Analysis was also done on mice challenged with 2500 EID₅₀ A/PR8 (e and f) (n=10 mice/group). In separate experiments, WT mice receiving 5×10⁶ memory HNT CD4 T cells were treated with 0.5 mg of IL-6 neutralizing Ab on the stated days post 10,000 EID₅₀ A/PR8 challenge. Control mice were treated with isotype control Ab on 0, 2, 4, 6, and 8 dpi. Survival (g) and weight loss (h) is depicted for 5 mice/group (one of three experiments).

Figure 6. Initial activation of memory CD4 T cells and early viral control are not impacted by IL-6

Unprimed thy-disparate WT or Il6^−/− mice received 1×10⁶ CFSE-labeled HNT memory CD4 and were challenge with 10,000 EID₅₀ A/PR8. Representative (a) donor CD69 expression, (b) proliferation profile determined by loss of CFSE label, and (c) cell number at 4 dpi were assessed in stated organs (n=3 mice/group). Results from 1 of 3 similar experiments. In separate experiments, (d) pulmonary viral titers were determined on 4 dpi from lungs of WT BALB/c or Il6^−/− mice receiving 5×10⁶ memory HNT cells or not and challenged with 10,000 EID₅₀ A/PR8 (n = 4/group). Results representative of 2 separate experiments.

Figure 7. 2° CD4 T cell effector number and function are reduced in the absence of IL-6

HNT memory cells, 1×10⁶, were transferred to naïve WT BALB/c subsequently infected with 2,500 EID₅₀ A/PR8 and treated with 0.5mg of isotype or IL-6 neutralizing Ab on days 0, 2, 4, and 6 dpi. Donor cells were enumerated at 7 dpi (a). Dual IFN-γ⁺/IL-2⁺ cytokine producing cells (b) and mean fluorescence intensity of IFN-γ signal (c) was assessed at 7 dpi by ICCS (n=4 mice/group; representative of 3 similar experiments). Viral titers (d) were determined on 7 dpi in mice receiving donor HNT memory cells followed by 2,500 EID₅₀ A/PR8 challenge (n=4 mice/group; one of two experiments). WT C57Bl/6 mice were primed with A/Alaska, challenged with A/PR8 as described in Figure 3, and mice treated with isotype or IL-6 neutralizing Ab as described above. On 5dpi, NP_311–325 tetramer⁺ CD4 T cells detected in the lung (e) were assessed for dual IFN-γ⁺/IL-2⁺ cytokine producing cells. The frequency (f and g) and number (h) is shown (n=4 mice/group; one of 2 similar experiments).

Figure 8. Direct IL-6 signals to memory CD4 T cells maximize2° effector responses

Memory populations were generated in vitro from IL6ra^+/+ and Il6ra^−/− OT-II cells. Cytokine production (a) from an equal number of IL6ra^+/+ and Il6ra^−/− memory cells was determined by luminex analysis of supernatants from triplicate wells 8 hours post-stimulation. Results representative of 3 separate experiments. IL6ra^+/+ or Il6ra^−/− OT-II memory cells, 1×10⁶, were transferred to different Thy-disparate hosts followed by infection with 0.1 LD₅₀ A/PR8-OVA_II. At 7 dpi, the total number of donor cells (b) and IFN-γ+ and IL-2+ cytokine production in the lung were determined from 4 mice/group (one of 2 separate experiments). Representative ICCS staining of lung cells in the absence and presence of stimulation is shown in (c) and the percentage of dual IFN-γ+ IL-2+ donor cells in (d). In separate experiments, WT.Thy1.2/Thy1.1 or Il6ra^−/−.Thy1.2 OT-II memory cells, 1×10⁶, were co-transferred to the same WT C57BL/6.Thy1.1 host followed by infection with 0.1 LD₅₀ A/PR8-OVA_II. At 7 dpi, the total frequency of donor cells (e), the ratio of Il6ra^−/− to WT OT-II memory cells (f), and the frequency of CD25⁺ Foxp3⁺ CD4⁺ T cells lymphocytes from the host or donor populations (g and h) was determined from 4 mice/group (one of 2 separate experiments).