Figure 1.
Localization and kinetics of HVEM expressing cells in the spleen of VACV infected mice.
(A–D) Groups of C57BL/6 wild-type (WT) mice were infected i.p. with VACV-WR (3×104 PFU/mouse). Uninfected (naïve) mice were used as controls. (A) Frozen sections of naïve (day 0), day 4, day 6 and day 8 VACV infected mice were stained with rat anti-mouse CD3-PE, HVEM-APC and CD169-FITC antibodies. The images were captured by 20× objective using EVOS fl inverted microscope. The micrographs arranged vertically in 1st, 2nd, and 3rd column (left to right), showing localization of CD3 (red), HVEM (purple) and CD3+HVEM expression in the splenic white pulp respectively. CD169 (green) was used to identify splenic marginal zones. (B) B cell follicles (f) identified by B220 (green channel), perilymphatic sheath (PALS) identified by CD3 (red channel) and co-localization of HVEM and CD3 antibodies in PALS region of white pulp. On days 4, 6, 15, 64 (C), and day 120 (D) postinfection splenocytes were harvested and stained for CD8, CD44, VACV-specific tetramers (B8R, A8R, or B2R), and HVEM. Representative plots of HVEM staining on total CD8 T cells and tetramer (B8R, A8R, and B2R) positive cells after gating on CD8 cells. The numbers in each plot indicate the percentage of total CD8 T cells (CD44low and CD44high) or tetramer-positive cells that stained for HVEM.
Figure 2.
HVEM is required for optimal accumulation of virus-specific effector CD8 T cells directed against subdominant but not dominant VACV epitopes.
Groups of C57BL/6 WT or HVEM-deficient (HVEM−/−) mice were infected i.p. with the indicated inoculums of VACV-WR. (A) On day 7-post infection, IFN-γ-secreting CD8 cells were assessed by intracellular cytokine staining after stimulation with the indicated VACV peptides (B8R, A3L, A8R, A23R, or B2R). Total numbers ± SEM of CD8+CD62LlowIFN-γ+ T cells per spleen from four individual mice. *p<0.05 (WT vs HVEM−/−). Similar results were obtained in three separate experiments. Representative plots of IFN-γ staining in gated CD8 T cells after infection with 3×104 PFU VACV-WR (B) or 3×102 PFU VACV-WR (C). Numbers indicate the percentage of CD8+IFN-γ- positive cells. Similar results were obtained in three separate experiments.
Figure 3.
HVEM-deficient CD8 T cells have intact activation and early division in response to VACV infection or lymphopenic environment.
(A) WT or HVEM−/− mice were infected i.p. with VACV-WR (3×104 PFU/mouse). On day 6-post infection, splenocytes were harvested and stained for CD8, CD44, and B8R or A8R tetramer. Top panel, Representative plots of tetramer staining, gating on CD8 cells. Percentages of CD44high expressing B8R-tetramer (left panels) or A8R-tetramer (right panels) positive CD8 T cells are indicated. CD8 T cell activation was assessed by up-regulation of CD25, and down-regulation of CD62L and IL-7R (CD127) on tetramer-positive CD44high cells. Naive (CD44low tetramer-negative) CD8 T cells were used as controls. Percentages that stained positive for each marker are indicated. (B) One×105 WT or HVEM −/− CFSE-labeled OT-I CD8 T cells were adoptively transferred into naïve WT mice. One day later, mice were infected i.p. with VACV-WR-OVA. After 2 or 3 days, OT-I CD8 T cells were analyzed. Top, Representative contour plots of co-staining for Vα2 and Vβ5 after gating on CD8+ T cells. Numbers indicate the percentage of CD8+Vα2+Vβ5+-positive cells. Middle, Representative dot plots of CFSE dilution after gating on CD8+Vα2+Vβ5+ cells. Bottom, Representative histogram of CFSE dilution gating on CD8+Vα2+Vβ5+ cells. (C) One×105 highly purified CFSE-labeled polyclonal WT or HVEM−/− CD8 T cells were adoptively transferred into RAG−/− mice. After 4 days, splenocytes were harvested and stained with anti-CD3, anti-CD8, and anti-CD44. Top, Representative dot plots of CFSE dilution after gating on CD3+CD8+ cells. Bottom, Representative histogram of CFSE dilution gating on CD3+CD8+ cells. Results are the mean number ± SEM (n = 4 mice/group) from one experiment. Similar results were obtained in two separate experiments.
Figure 4.
Impaired generation of CD8 memory cells to both dominant and subdominant VACV epitopes in HVEM-deficient mice.
Groups of C57BL/6 WT or HVEM-deficient (HVEM−/−) mice were infected i.p. with the indicated inoculums of VACV-WR. At day 40 (A, B) day 7 (B), day 14 (B), or day 210 (C and D), splenocytes were harvested and stimulated with VACV peptides as indicated and CD8 T cell number and functionality was assessed by tetramer and intracellular IFN-γ staining. (A and B) Total numbers of CD8+IFN-γ+cells per spleen. Results are mean number ± SEM (n = 4 mice/group) from one experiment. (C) Representative plots of B8R, A3L, and A8R tetramer staining, gating on CD8 cells, are shown. Percentages of activated tetramer+ CD8 T cells (CD8+CD44+B8R+) are indicated. (D) Representative plots of IFN-γ staining in gated CD8 T cells. Percent positive indicated. Bottom, Total numbers of CD8+IFN-γ+ cells per spleen. Results are mean number ± SEM (n = 4 mice/group) from one experiment. *p<0.05 (WT mice vs knockout) as determined by Student’s t test. Similar results were obtained in three separate experiments.
Figure 5.
T cell–expressed HVEM is required for accumulation of large populations of memory CD8 T cells.
(A) Experimental scheme. Naïve OVA-specific CD8 T cells were purified from WT or HVEM−/− OT-I mice. CD8 T cells (>99% CD8+Vα2+Vβ5+; 1 x104/mouse) were injected i.v. into naive WT mice. One day later, recipient mice were infected i.p. with recombinant VACV-WR expressing full-length OVA (VACV-WR-OVA; 2×105 PFU/mouse). (B) On the indicated days, CD8 T cell expansion (Day 8; Top panels) and late (Day 60; Bottom panels) memory formation were analyzed by tracking the transgenic TCR. Contour plots, representative co-staining for IFN-γ after gating on Vα2+Vβ5+ CD8 cells. Percent positive indicated. Right, Total numbers of CD8+Vα2+Vβ5+ and CD8+Vα2+Vβ5+IFN-γ+ cells per spleen after stimulation with OVA SINFEKEL peptide. Results are mean number ± SEM (n = 4 mice/group) from one experiment. *p<0.05 (WT mice vs knockout) as determined by Student’s t test. Similar results were obtained in two additional experiments. (C) Purified splenic dendritic cell (DC) populations from WT or HVEM−/− mice were cultured with recombinant VACV-WR expressing GFP in vitro. Twenty-four hours later, GFP expression in CD8α+, CD4+, and CD8α/CD4-double negative (DN) DC populations was examined. Blue line, HVEM−/− DCs; red line, WT DCs. Percentages of GFP-positive cells are indicated. (D) WT or HVEM−/− mice were infected i.v. with VACV-WR-OVA. Twenty-four hours later, splenocytes were harvested and sorted for CD11c+CD8+ DC and then cultured with naive CFSE-labeled WT OT-I cells. On day 2 and 3, division of OT-I cells was examined. Top, Experimental scheme; Middle, representative dot plots of CFSE dilution on day 2 after gating on CD8+Vα2+Vβ5+ cells; Bottom, representative dot plots of CFSE dilution on day 3 after gating on CD8+Vα2+Vβ5+ cells. Results are the mean number ± SEM (n = 4 mice/group) from one experiment. Similar results were obtained in three separate experiments.
Figure 6.
Impaired generation of CD8 memory cells to both dominant and subdominant VACV epitopes in BTLA-deficient mice.
Groups of C57BL/6 WT, BTLA-deficient (BTLA−/−), or HVEM −/− mice were infected i.p. with the indicated inoculums of VACV-WR (A and B) and VACV-Lister (C and D). At day 64, splenocytes were harvested and stimulated with the indicated VACV peptides and CD8 T cell numbers and functionality was assessed by tetramer and intracellular IFN-γ staining. (A) Representative plots of B8R, A3L, A8R, and A23R tetramer staining, gating on CD8 cells, are shown. Percentages of activated tetramer+ CD8 T cells (CD8+CD44+B8R+) are indicated. (A) Right, Total numbers of CD8+CD44+tetramer+ cells per spleen (B) Representative plots of IFN-γ staining in gated CD8+CD62LLow T cells. Percent positive indicated. Right, Total numbers of CD8+IFN-γ+ cells per spleen. Results are mean number ± SEM (n = 4 mice/group) from one experiment. *p<0.05 (WT mice vs gene-knockout) as determined by Student’s t test. Similar results were obtained in two separate experiments.
Figure 7.
BTLA expressed on a non-T cell is required for accumulation of large populations of virus-specific CD8 T cells.
(A) Groups of C57BL/6 wild-type (WT) mice were infected i.p. with VACV-WR (3×104 PFU/mouse). Uninfected (naïve) mice were used as controls. On days 4, 6, 15, and 64 post-infection splenocytes were harvested and stained for CD8, CD44, VACV-specific tetramers (B8R, A8R, or B2R), and BTLA. Representative plots of BTLA staining on total CD8 T cells and tetramer (B8R, A8R, and B2R) positive cells after gating on CD8 cells. The numbers in each plot indicate the percentage of total CD8 T cells (CD44low and CD44high) or tetramer-positive cells that stained for BTLA. (B and C) Naïve OVA-specific CD8 T cells were purified from WT or BTLA−/− OT-I mice. CD8 T cells (>99% CD8+Vα2+Vβ5+; 1 x104/mouse) were injected i.v. into naive WT (B) or BTLA−/− (C) mice. One day later, recipient mice were infected i.p. with recombinant VACV-WR expressing full-length OVA (VACV-WR-OVA; 2×105 PFU/mouse). (B) On the indicated days, CD8 T cell expansion (Top panel), and memory formation (Bottom panel) were analyzed by tracking the transgenic TCR. Contour plots, representative costaining for IFN-γ after gating on Vα2+Vβ5+ CD8 cells. Percent positive indicated. Right, Total numbers of CD8+Vα2+Vβ5+IFN-γ+ cells per spleen after stimulation with SINFEKEL peptide. Results are mean number ± SEM (n = 4 mice/group) from one experiment. *p<0.05 (WT mice vs knockout) as determined by Student’s t test. Similar results were obtained in two additional experiments.
Figure 8.
BTLA expressed on CD8α+ DCs contributes to accumulation of large populations of virus-specific CD8 T cells.
(A–C) Groups of C57BL/6 wild-type (WT) mice were infected i.p. with VACV-WR (3×104 PFU/mouse). Spleen was harvested on the indicated days after VACV-WR infection. Frozen sections were stained with rat anti-mouse BTLA-PE, CD169-APC for marginal zone macrophages, and CD11c-FITC for dendritic cells. The images were captured by 10×/20×/40× objectives using EVOS fl inverted microscope. WP, white pulp; PALS, perilymphatic sheath; RP, red pulp. Note small clusters of BTLA+ dendritic cells in the PALS. (B) CD11C and BTLA staining from the indicated areas in A (right panels (i) and (ii)). Colocalization signal is indicated as yellow/orange. (C) Splenic tissue 4 days post infection. Magnified view of HVEM+ cells in contact with BTLA+ cells in the PALS region of splenic white pulp. (D) BTLA expression on DCs 24 hr after in vitro (left panel) or in vivo (right panel) infection with VACV-WR. (E) WT or BTLA−/− mice were infected i.v. with VACV-WR-OVA. Twenty-four hours later, splenocytes were harvested and sorted for CD11c+CD8α+ DC and then cultured with naive CFSE-labeled WT OT-I cells. Three days later, division of OT-I cells was examined. Representative histograms of CFSE dilution after gating on CD8+Vα2+Vβ5+ cells. (F) Absolute numbers of CD8+Vα2+Vβ5+ cells that accumulated after the indicated cell division. Results are the mean number ± SEM (n = 4 mice/group) from one experiment. Similar results were obtained in two separate experiments.
Figure 9.
HVEM-deficient CD8 T cells fail to protect against VACV-infection.
Ten million highly purified polyclonal WT, HVEM−/−, or BTLA−/− CD8 T cells were adoptively transferred into RAG−/− mice. One day later, mice were infected i.p. with VACV-WR (2×104 PFU/mouse). Animals were weighed daily and euthanized if weight loss was greater than 25% body weight. (A) Mean % of initial body weight from indicated numbers of mice. Thirty-four days after infection, virus-specific CD8 T cells were assessed in the spleen (B and C) and lungs (B and D) by intracellular cytokine staining after ex vivo stimulation with VACV B8R or A3L peptide. Data are representative plots of IFN-γ staining in gated CD8 T cells, with percent positive indicated, or total numbers ± SEM of CD8+IFN-γ+ T cells per tissue from four individual mice. *p<0.05 (WT vs gene-knockout treated) as determined by Student’s t test. Similar results were obtained in two separate experiments.