Systemic and mucosal infection program protective memory CD8 T cells in the vaginal mucosa

Abstract

Whether mucosal immunization is required for optimal protective CD8 T cell memory at mucosal surfaces is controversial. In this study, using an adoptive transfer system, we compare the efficacy of two routes of acute lymphocytic choriomeningitis viral infection on the generation, maintenance, and localization of Ag-specific CD8 T cells in tissues, including the vaginal mucosa. Surprisingly, at day 8, i.p. infection results in higher numbers of Ag-specific CD8 T cells in the vaginal mucosa and iliac lymph node, as well as 2-3x more Ag-specific CD8 T cells that coexpress both IFN-gamma and TNF-alpha in comparison to the intranasal route of infection. Expression of the integrin/activation marker CD103 (alphaEbeta7) is low on vaginal mucosal Ag-specific CD8 T cells in comparison to gut mucosal intraepithelial lymphocytes. At memory, no differences are evident in the number, cytokine production, or protective function of Ag-specific CD8 T cells in the vaginal mucosa comparing the two routes of infection. However, differences persist in the cytokine profile of genital tract vs peripheral Ag-specific CD8 T cells. So although the initial route of infection, as well as tissue microenvironment, appear to influence both the magnitude and quality of the effector CD8 T cell response, both systemic and mucosal infection are equally effective in the differentiation of protective memory CD8 T cell responses against vaginal pathogenic challenge.

Figure 1

Magnitude of the Ag-specific CD8 T cell response differs in the genital tract on day 8. Lymphocytes from spleen, peripheral blood, lung, bone marrow, CLN, MedLN, ILN, vaginal tract, and o-NALT were isolated and surface stained with D^bGP33–41, CD8. A, Representative FACS staining of tissues from a day 8 postinfection LCMV Armstrong i.p. or i.n. infected mouse. The numbers represent the percentage of Ag-specific CD8 T cells in the indicated tissue compartment. B, Total number of Ag-specific CD8 T cells in tissues. C, Percentage of Ag-specific CD8 T cells in PBMC. D, Representative staining and E, Total numbers of Ag-specific T cells in the ILN. F, Representative staining and G, Total number of Ag-specific CD8 T cells in the vaginal tract and o-NALT. Black/filled symbols are i.p. and white/open symbols are i.n. The data represent three experiments; for o-NALT and vaginal tract tissues are pooled from four animals, each experiment. Not shown are uninfected control mice that showed no detectable Ag-specific responses.**, p < 0.01.

Figure 2

Confocal microscopy confirms differences in the magnitude of the Ag-specific CD8 T cell response in the vagina on day 8. Frozen sections of the vaginal tract were fixed and stained with CD8 and Thy 1.1 Abs. Thy1.1 staining shows Ag-specific CD8 T cell staining in the stroma of the vaginal mucosa on day 8 postinfection with LCMV. A, D, and G, CD8 T cells in green and B, E, H, Thy1.1 staining in red. C, F, I, Merged image shows Thy1.1 staining only on CD8 T cells of infected mice. Naive stained only for CD8 T cells. Images were captured using a 20× objective lens. The epithelial layer is indicated by the white arrowheads (luminal edge) and blue arrowheads (basement membrane). J, Percentage of Ag-specific CD8 T cells in vaginal tract of infected mice at day 8 postinfection. Thy1.1⁺ CD8⁺ cells were counted on blinded samples.***, p < 0.001.

Figure 3

Cytokine production is influenced by the route of immunization and tissue localization on day 8. Lymphocytes from spleen and ILN were isolated on day 8 of i.p. or i.n. LCMV infected mice and after 5 h ± peptide stimulation, stained with Thy1.1, CD8, IFN-γ, and TNF-α. A, Representative staining gating on Thy1.1⁺ CD8⁺ Ag-specific cells incubated with GP33–41 peptide. No staining was seen with no peptide (data not shown). B, Percentage of IFN-γ⁺ by Ag-specific T cells and C, Percentage of IFN-γ⁺TNF-α⁺by Ag-specific cells. Data are from three separate experiments.**, p < 0.01;***, p < 0.001.

Figure 4

Cytokine production of memory T cells is influenced by tissue localization. Lymphocytes from spleen and ILN were isolated at day 80 and after 5 h ± peptide stimulation stained with Thy 1.1, CD8, IFN-γ, and TNF-α. Gating on Thy 1.1⁺ CD8⁺ cells, stimulation with GP33–41 specific peptide results in IFN-γ and TNF-α. No staining of IFN-γ or TNF-α was seen with no peptide controls (data not shown). A, Representative staining showing reduced TNF-α coexpressing cells in the ILN compared with spleen. B, Percentage of IFN-γ⁺ and C, Percentage of IFN-γ⁺ TNF-α⁺ Ag-specific cells.**, p < 0.01;***, p < 0.001.

Figure 5

Expression of CD103 on Ag-specific CD8 T cells in genital mucosa. Lymphocytes were isolated from the spleen, PBMC, ILN, IEL, and vaginal tract from day 8 and day 400 LCMV Armstrong i.p. Cells were stained with CD8, CD103, and D^bGP33–41. Representative staining is shown, gating on CD8 T cells. Numbers represent percentage of CD103^high Ag-specific CD8 T cells in indicated tissue compartments. For vaginal tract, tissues are pooled from four to six mice.

Figure 6

Intravaginal rechallenge generate protective secondary responses in the genital tract of systemic and mucosal immune mice. Lymphocytes were isolated from indicated tissues of immune mice and day 5 intravaginal rechallenge with vaccinia expressing the GP33–41 peptide of the LCMV glycoprotein. A, Representative FACS staining of vaginal tract lymphocytes from i.p. and i.n. day 45 memory and day 5 intravaginal rechallenge mice. B, Total number of Ag-specific CD8 T cells in indicated tissue compartments of day 80 i.p. or i.n. immune mice and day 5 intravaginal rechallenge mice. Black bars are i.p. and white bars are i.n. For o-NALT and vaginal tracts, tissues pooled from four animals in each experiment.

Figure 7

Cytokine production of secondary effectors following intra-vaginal rechallenge. A, Representative staining showing IFN-γ⁺ TNF-α⁺ coexpressing cells in the ILN vs spleen. B and C, Production of IFN-γ and TNF-α by Ag-specific CD8 T cells. D, Viral titers in the ovaries on day 5 mice challenged intravaginally with 5 × 10⁵ PFU vaccinia expressing the GP33–41 peptide of the LCMV glycoprotein. Gray bar represents naive mice; black bars are i.p. immune; and white bars are i.n. immune; n = 10–12 mice in each group;*, p < 0.5;***, p < 0.001.