Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus - PubMed (original) (raw)

Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus

Norifumi Iijima et al. J Exp Med. 2008.

Abstract

Although the importance of cytotoxic T lymphocytes and neutralizing antibodies for antiviral defense is well known, the antiviral mechanism of Th1 remains unclear. We show that Th1 cells mediate noncytolytic antiviral protection independent of direct lysis through local secretion of IFN-gamma after herpes simplex virus (HSV) 2 infection. IFN-gamma acted on stromal cells, but not on hematopoietic cells, to prevent further viral replication and spread throughout the vaginal mucosa. Importantly, unlike other known Th1 defense mechanisms, this effector function did not require recognition of virally infected cells via MHC class II. Instead, recall Th1 response was elicited by MHC class II(+) antigen-presenting cells at the site of infection. Dendritic cells (DCs) were not required and only partially sufficient to induce a recall response from memory Th1 cells. Importantly, DCs and B cells together contributed to restimulating memory CD4 T cells to secrete IFN-gamma. In the absence of both DCs and B cells, immunized mice rapidly succumbed to HSV-2 infection and death. Thus, these results revealed a distinct mechanism by which memory Th1 cells mediate noncytolytic IFN-gamma-dependent antiviral protection after recognition of processed viral antigens by local DCs and B cells.

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Figures

Figure 1.

Memory CD4 T cells localize in the vagina of immune mice and provide protection against secondary HSV-2 challenge. (A and B) The localization of CD4 and CD8 T cells in the vagina of nonimmunized (A) or TK−HSV-2–immunized mice at 3 wk after immunization (B). (C and D) Congenic (CD45.1+) effector CD4, CD8, or naive CD4 T cells were adoptively transferred into naive recipient (CD45.2+) mice. At 3 d after ivag infection with HSV-2, viral antigen (anti–HSV-2, green; C and D), transferred T cells (anti-CD45.1, red; C and D), and MHC class II (blue; D) were visualized. Images were captured using a 10 (A–C) or 40× (D) objective lens. Arrowheads indicate the basement membrane. Bars, 100 μm. (E and F) CD4 or CD8 T cells were depleted from TK−HSV-2–primed mice and challenged ivag with WT HSV-2. (E) The dot plot represents CD4+ and CD8+ cells in vagina 3 d after challenge. (F) Virus titers in the vaginal fluids (nonimmune, n = 4; immune/control Ab, n = 6; immune/anti-CD4, n = 4; and immune/anti-CD8, n = 4) were measured at the indicated days after HSV-2 secondary challenge. Error bars represent the mean ± SD of the number of mice per group. These data are representative of three similar experiments.

Figure 2.

Direct cytolysis via perforin or Fas–FasL is not required for Th1-mediated protection. C57BL/6 (WT) →WT (n = 3), Perforin−/−→WT (n = 4), and Perforin−/−→lpr chimera (n = 5) were immunized with TK−HSV-2 ivag and, 3 wk later, challenged with lethal WT HSV-2 virus. As a control, nonimmunized C57BL/6 mice were challenged with the virus. (A) FACS analyses of CD4 and CD8 expression in live CD3+ cells in the peripheral blood of WT →WT chimera, Perforin−/−→lpr chimera, and lpr mice. The percentage of the CD3+CD4−CD8−B220+ cells (DN cells) found in these mice is shown in the histogram. (B) Survival, genital mean pathology scores, and viral titers in vaginal wash after secondary challenge are depicted. Error bars represent the mean ± SE of the number of mice per group. These results are representative of three similar experiments.

Figure 3.

Virus clearance mediated by memory CD4 T cells requires IFN-γ responsiveness by the stromal, but not the hematopoietic, compartment. 129 (WT; n = 9), IFN-γR−/− (n = 3), WT→IFN-γR−/− (n = 6), and IFN-γR−/−→WT mice (n = 6) were immunized with TK−HSV-2 ivag and, 4 wk later, challenged with lethal WT HSV-2. As a control, nonimmunized 129svj mice (n = 6) were challenged with WT HSV-2 (A and C). CD4 T cells were depleted from the respective groups of mice before secondary challenge with WT HSV-2 (B and D). Each bar represents the mean ± SE of the number of mice per group. Viral titers in vaginal washes after secondary challenge were measured. These results are representative of two similar experiments.

Figure 4.

Direct recognition of the infected vaginal epithelial cells by Th1 cells is not required for protection. C57BL/6 (WT; n = 7), MHC class II−/− (n = 4), WT thymus-transplanted MHC class II−/− BM chimera, and WT→{MHC II−/− + WT thymus} (n = 9) were immunized with TK−HSV-2 ivag and, 4 wk later, challenged with lethal WT HSV-2. As a control, nonimmunized C57/BL6 mice (n = 6) were challenged with the virus. (A) FACS analyses of CD4 and CD8 T cells (live CD3+ cells) in the peripheral blood of C57BL/6 (WT)→WT, MHC class II−/−, and WT→{MHC II−/−+ WT thymus} mice. (B–D) Viral titers in vaginal washes (B), survival (C), and genital mean pathology scores (D) after secondary challenge were examined. Error bars represent the mean ± SE of the number of mice per group. These data are representative of three similar experiments.

Figure 5.

Conventional DCs are not required but are partially sufficient to elicit recall Th1 responses in the vagina. (A and B) TK−HSV-2-immunized CD11c-DTR→WT chimeras (n = 5) were inoculated with DT or PBS i.p., as shown in Fig. S9 (available at

http://www.jem.org/cgi/content/full/jem.20082039/DC1

). Depletion of vaginal DCs (CD11c+GFP+ and MHC II+GFP+) in the epithelium and lamina propria after DT injection was examined by FACS analysis (A). Viral titers in vaginal secretion were measured at the indicated days after challenge (B). (C–F) To examine the sufficiency of MHC class II expression on CD11c+ cells for protection against WT HSV-2 secondary challenge, Abb mice and MHC class II−/− mice were transplanted with naive polyclonal CD4 T cells (107 cells) 1 d before TK−HSV-2 immunization (C). MHC class II expression on vaginal DCs in TK−HSV-2–immunized mice (4 wk) was analyzed (D). Frozen sections were stained with MHC class II (green) and counterstained with DAPI (blue). Images were captured using a 10 (left) or 40× (right) objective. Bars, 100 μm. (E) Adoptively transferred naive CD4 T cells normally differentiate into Th1 cells in Abb mice immunized with TK−HSV-2. CD4 T cells isolated from the draining LNs of Abb, MHC class II−/− (MHCII−/−), and C57BL6 mice (WT) mice immunized ivag with TK−HSV-2 (7 d after infection) were cocultured with syngeneic splenocytes in the presence of HSV-2 antigens (left) or mock antigen (right) and analyzed for IFN-γ secretion (in nanograms/milliliter). Each column represents the mean ± SD of triplicate samples. (F) 3 wk after TK− immunization, Abb and MHC class II−/− mice were challenged with 104 PFU WT HSV-2 virus. Viral titers in vaginal secretion were measured at the indicated days after challenge. Error bars represent the mean ± SD. These data are representative of two similar experiments.

Figure 6.

DCs and B cells mediate memory Th1 recall response in the vagina. In addition to depletion of DCs, TK−HSV-2–immunized CD11c-DTR→WT chimeras (n = 5) were inoculated with either a control Ab or anti–mouse CD20 i.v., as shown in Fig. S9 (available at

http://www.jem.org/cgi/content/full/jem.20082039/DC1

). B cell depletion in the vaginal tissue of TK−HSV-2–immunized mice (3 wk after infection) after Ab injection was determined by FACS analysis (A). The mortality and mean clinical score are shown in B. Error bars represent the mean ± SD of five mice per group. These data are representative of three similar experiments.

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Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus - PubMed (original) (raw)