West Nile virus-specific CD4 T cells exhibit direct antiviral cytokine secretion and cytotoxicity and are sufficient for antiviral protection

Abstract

CD4 T cells have been shown to be necessary for the prevention of encephalitis during West Nile virus (WNV) infection. However, the mechanisms used by Ag-specific CD4 T cells to protect mice from WNV encephalitis remain incompletely understood. Contrary to the belief that CD4 T cells are protective because they merely maintain the CD8 T cell response and improve Ab production, in this study we provide evidence for the direct antiviral activity of CD4 T cells that functions to protect the host from WNV encephalitis. In adoptive transfers, naive CD4 T cells protected a significant number of lethally infected RAG(-/-) mice, demonstrating the protective effect of CD4 T cells independent of B cells and CD8 T cells. To shed light on the mechanism of this protection, we defined the peptide specificities of the CD4 T cells responding to WNV infection in C57BL/6 (H-2(b)) mice, and used these peptides to characterize the in vivo function of antiviral CD4 T cells. WNV-specific CD4 T cells produced IFN-gamma and IL-2, but also showed potential for in vivo and ex vivo cytotoxicity. Furthermore, peptide vaccination using CD4 epitopes conferred protection against lethal WNV infection in immunocompetent mice. These results demonstrate the role of direct effector function of Ag-specific CD4 T cells in preventing severe WNV disease.

Figure 1. Protective effect of Naïve CD4 T cells

A. Antibody depletion of CD4 T cells (triangles) renders C57BL/6 mice significantly (**p<0.005, *p<0.04) more susceptible to WNV induced mortality compared to controls (squares). CD4 T cells were depleted using two doses of GK1.5 antibody on day −3 and day 0, then infected with either 400pfu/mouse (left) or 800pfu/mouse (right). Results of one experiment with n=10 mice per group are shown, representative of two independent experiments. B. Adoptive transfer of naïve CD4 T cells provides protection to RAG-1−/− mice against lethal WNV infection. Splenic CD4⁺ T cells (5–10×10⁶) from naive C57BL/6 mice were isolated by negative selection (80–95% purity) and transferred to C57BL/6 RAG-1−/− mice. 24 hours after transfer, mice were challenged with 100 pfu or 200 pfu WNV sc. Survival in these groups was significantly different according to the log-rank test (*** p<0.0005, left) (**p<0.002, right). Two individual experiments are shown n=25 mice per group are shown, representative of a total of 4 experiments completed.

Figure 2. CD4 T cell response during West Nile virus infection

A. Representative example of GzB expression by CD4 T cells after WNV infection as measured by direct ex-vivo intracellular FCM, without in-vitro stimulation. A naïve animal is shown as a control. Results are from one representative mouse of four mice from each time point. One experiment of two is shown. B. Representative example of IFNγ expression in CD4 T cells after WNV infection, measured by ICCS upon stimulation with 0.5μg/ml anti-CD3e (clone 2c11); a naïve animal is shown as a control. Results are from one mouse out of 4. Data is representative one experiment out of two. C. Left panel- Aggregate analysis of GzB expression in CD4 T cells, determined as in panel A. Percentage of CD4 GzB⁺ T cells for the time points given above. There is a significant induction of GzB in CD4 T cells on day 7 (p<0.02), but not on day 10 (p>0.05). Panels show average of four mice per time point (x± S.E.M.), representative of two experiments. Right panel- Aggregate analysis of IFNγ expression in CD4 T cells, determined as in panel B. Percentage of CD4 IFNγ⁺ T cells for the time points given above. Average of four mice per time point (x± S.E.M.), representative of two experiments.

Figure 3. Antigen-specific CD4 T cell IFNγ response to class II epitopes

A. Representative example of CD4 T cell IFNγ ICCS response to the three immunodominant and three sub-dominant CD4 T cell epitopes as measured by 6 hour ICCS. CD4 T cells were from mice 7 days post infection and were stimulated with the peptide (10⁻⁶M) indicated above each plot. One mouse of 5 is shown for one experiment, representative of 3 experiments. B. Aggregate analysis of IFNγ expression in primary and memory responses. Left panel- Quantification of the CD4 T cell IFNγ ICCS response 7 days post infection, shown in panel A. Average of 5 mice per time point (x± S.E.M.), representative of three experiments. Right panel- Quantification of day 50 CD4 T cell IFNγ response. Average of 5 mice per time point (x± S.E.M.), representative of three experiments. C. Quantification of day 7 CD4 T cell IFNγ response (measured by ICCS) to either WNV-infected IC-21s (MOI:40) or peptide pulsed IC-21s 10⁻⁶M (Env₆₄₁ and NS3_1616+2066) peptide. CD4 T cells were stimulated for 6 hours in the presence of BFA. Results depict average values of four mice per time point (x± S.E.M.), representative of two experiments.

Figure 4. Functional potential of antigen specific CD4 T cells

A. Representative example of a CD4 T cell cytokine response 7 days post infection. Following gating on CD4, IFNγ and IL-4 were measured after 6 hr stimulation in the presence of monensin. Cells were stimulated with media, 2c11 (0.5μg/ml) or NS3_1616+2066(10⁻⁶M), and a naïve mouse was used as a control. One mouse of four is shown, from one representative experiment of two. B. Representative example of CD4 T cell ICCS during the course of infection, as indicated above each plot. Following gating on CD4, IFNγ and IL-2 were measured after 6 hr stimulation with NS3_1616+2066(10⁻⁶M) in BFA. Controls and repetitions were as in A. C. In vivo CD4 T cell CFSE cytotoxicity assay. Left panel- Representative histogram of transferred (donor, Ly-5.1+) splenocytes 12 hours after adoptive transfer of target cells into naïve (left histogram) and infected (right histogram) mice. Middle panel- Aggregate quantification of in vivo CD4 T cell CFSE based cytotoxicity assay. CD4 T cells were cytolytic in vivo on day 7 post WNV infection (**p<0.008). Results represent the average of 5 mice per group, and are representative of three independent experiments. Right panel- Quantification of in vivo CD4 T cell CFSE-based cytotoxicity assay completed within WNV infected Perforin−/−, CD4−/− and B6 control mice. B6 or LPR−/− splenocytes were coated with WNV or control peptides, labeled with CFSE and used as targets with different hosts – the x-axis legend denotes which molecule(s) were missing during the interaction of CTL with their targets. B6 (targets) transferred into Perf−/− mice as well as the transfer of LPR−/− deficient targets leads to a reduction in cytotoxicity (*p<0.03). Transfer of LPR−/− targets into perf−/− mice leads to a complete ablation of cytotoxicity (p<0.03). Results represent the compilation 2 independent experiments. D. In vitro CTL activity of purified CD4 T-cells against targets coated with indicated WNV peptides or the control class II-restricted ovalbumin peptide (OT-II, Ova_323-339). IC21 cells were coated with the indicated peptides and labeled with ⁵¹Cr. The cells were incubated with CD4 T-cells purified from spleens of B6 mice infected with WNV 7 days earlier, as described, with minimal purity of 89% and contaminating CD8 or B-cells at <0.5%. Chromium release assay was performed as described in Methods. Results are representative of 3 experiments.

Figure 5. Antigen-specific CD4 T cell responses are essential for protection against WNV

CD4 T cell peptide vaccination leads to a significant (p<0.03) increase in protection of WNV infected mice. Mice were vaccinated using 20μg of NS3_1616+2066 or 20μg of OTII control peptide in Titermax Gold^® emulsion. Ten mice per group were challenged with 1200 pfu of WNV. One representative experiment of three is shown.