CD301b+ dendritic cells stimulate tissue-resident memory CD8+ T cells to protect against genital HSV-2

Abstract

Tissue-resident memory CD8+ T (CD8 T_RM) cells are an essential component of protective immune responses at barrier tissues, including the female genital tract. However, the mechanisms that lead to the initiation of CD8 T_RM-mediated protective immunity after viral infection are unclear. Here we report that CD8 T_RM cells established by 'prime and pull' method confer protection against genital HSV-2 infection, and that IFN-γ produced by CD8 T_RM cells is required for this protection. Furthermore, we find that CD8 T_RM-cell restimulation depends on a population of CD301b⁺ antigen-presenting cells (APC) in the lamina propria. Elimination of MHC class I on CD301b⁺ dendritic cells abrogates protective immunity, suggesting the requirement for cognate antigen presentation to CD8 T_RM cells by CD301b⁺ dendritic cells. These results define the requirements for CD8 T_RM cells in protection against genital HSV-2 infection and identify the population of APC that are responsible for activating these cells.

Figure 1. Protection against HSV-2 after prime and pull requires CD8+ T cells.

(a) Experimental schematic. (b) Flow cytometry plots and graph showing total CD8 T cells in the spleen and vagina 1 day post pull. Plots are gated on total lymphocytes from the spleen (top) and vagina (bottom). Numbers in plots indicate percentage of lymphocytes that are CD3+CD8b+. Graph shows total number of donor gBT-I CD8 T cells in the vagina 1 day post pull. After lethal intravaginal challenge with WT HSV-2, all groups shown in a were monitored over 2 weeks for disease score (c), weight loss (d) and survival (e). Viral titres in the vaginal mucosa were measured for the first 5 days post challenge by plaque assay (f). *P<0.05 by unpaired t-test for (b). ****P<0.0001 by repeated-measures analysis of variance for c,d. Data are representative of three independent experiments; n=11 for all experimental groups, and 9 for unimmunized controls. Error bars show s.e.m.

Figure 2. Circulating memory CD8+ T cells are dispensible for protection against HSV-2.

(a) Experimental schematic. (b) Graphs showing total number of donor gBT-I CD8 T cells in the spleen and vagina 1 day after final antibody injection in the indicated groups. After a lethal intravaginal challenge with WT HSV-2, mice in the indicated groups were monitored over 2 weeks for disease score (c), weight loss (d) and survival (e). Viral titres in the vaginal mucosa were measured for the first 5 days post challenge by plaque assay (f). *P<0.05 by two-tailed t-test in b. No statistical difference was measured by two-way repeated-measures analysis of variance between the TK+pull isotype and TK+pull anti-CD8 antibody-treated groups for c,d,f, or by log-rank test for e. Data are representative of three independent experiments; n=7–19 for experimental groups, and 7 for unimmunized controls. Error bars show s.e.m.

Figure 3. IFN-γ produced by CD8 T_RM cells is essential for protection against HSV-2.

(a) Experimental schematic. (b) Flow cytometry plots and graphs showing donor WT or IFN-γ KO T cells in the spleen and vagina at 3 weeks post pull. Plots are gated on total lymphocytes in the spleen (top) and vagina (bottom). Numbers in plots indicate the percentage of total lymphocytes that are WT (left) or IFN-γ KO donor CD8 T cells (right). Graphs show absolute number of the indicated donor CD8 T-cell population in the spleen (left) or vagina (right). After lethal intravaginal challenge with WT HSV-2, all groups shown in a were monitored over 2 weeks for disease score (c), weight loss (d) and survival (e). Viral titres were measured in the vaginal mucosa for the first days post challenge by plaque assay (f). Data are representative of two independent experiments; n=5–7 for TK+PBS and TK+pull groups, n=3 for unimmunized controls. *P<0.05, **P<0.01 by two-way repeated-measures analysis of variance for c,d, with no significant difference for f. Error bars show s.e.m.

Figure 4. CD8 T_RM cells are proximal to CD301b+ APC in the vagina after HSV-2 infection.

(a) Depletion of CD301b+ APC in the vaginae of Mgl2DTR mice treated with DT. Prime and pull immunized Mgl2DTR or WT mice were injected with DT and assessed 1 day later with the indicated markers. White arrowheads point to the basement membrane. (b) Distribution of MHCII⁺ populations in the vagina after DT treatment in WT or Mgl2DTR mice. MHCII⁺ cells were counted in total vaginal sections (left), epithelium only (left mid) or lamina propria only (right mid). Graphs show a ratio of MHC II⁺ cells per 100 total nuclei. Right graph shows the number of CD301b+MHC II⁺ cells per 100 nuclei in the vaginal lanmina propria of the indicated mice. (c) Images show localization of CD8 T cells and CD301b+ MHC II⁺ cells in the vagina of prime and pull immunized Mgl2DTR/GFP mice at 0 and 24 h post challenge, with lethal WT HSV-2. Images on the right show area within the yellow square in middle images at a higher magnification. White arrowheads indicate the basement membrane. Yellow arrowheads show areas of co-localization between CD8+ and CD301b+MHC II⁺ cells. ***P<0.001 by two-tailed t-test. Thirty individual slides were counted from two independent experiments for a,b. Images in c are representative of two independent experiments. Error bars show s.e.m. Scale bars, 75 μm.

Figure 5. CD301b+ DCs drive CD8 T_RM-cell-mediated protection against HSV-2 infection.

(a) Experimental schematic. (b) Graphs showing absolute number of donor gBT-I CD8 T cells in the spleen (left) or vagina (right) in WT or Mgl2DTR prime and pull immunized mice 1 day post-DT treatment. After lethal intravaginal challenge with WT HSV-2, all groups in a were monitored over 2 weeks for disease score (c), weight loss (d) and survival (e). Viral titres were measured in the vaginal mucosa by plaque assay for the first 5 days post challenge (f). *P<0.05, **P<0.01, ***P<0.001 by two-way repeated-measures analysis of variance for c,d. Data are representative of three independent experiments; n=5–11 for experimental groups, and 6 for unimmunized controls. Error bars show s.e.m.

Figure 6. MHC I on CD301b+ DCs is required for CD8+ T_RM-cell-mediated antiviral protection.

(a) Reconstitution of bone marrow chimeras. Histograms show ratio of Mgl2DTR to WT donor cells (top) and Mgl2DTR to MHCI KO donor cells (bottom) 8 weeks after reconstitution. (b) Experimental schematic. Both bone marrow chimera groups were immunized with TK− HSV-2 and treated intravaginally with CXCL9 and CXCL10 5 days post immunization. Four weeks post pull, all mice were injected with DT. One day post-DT treatment, mice were challenged intravaginally with a lethal dose of WT HSV-2. Mice were monitored over 2 weeks for disease score (c), weight loss (d) and survival (e). Data are representative of two independent experiments; n=5–8 per group. Error bars show s.e.m. **P<0.01 by two-way repeated-measures analysis of variance for c,d.