Roles for CD40, B7 and major histocompatibility complex in induction of enhanced immunity by cryptococcal polysaccharide-pulsed antigen-presenting cells

Abstract

Immunization of mice with activated antigen-presenting cells (APC) pulsed ex vivo with cryptococcal capsular polysaccharide, a glucuronoxylomannan (GXM-APC) results in prolongation of survival and delayed-type hypersensitivity (DTH) responsiveness following infection with Cryptococcus neoformans (NU-2). GXM-APC has both non-specific and GXM-specific effects that influence the immune responses that develop in mice after infection with NU-2. Type 1 cytokine responses are augmented after immunization with APC alone, while GXM must be present for the vaccine to influence survival and DTH reactions. This investigation evaluated the role that major histocompatibility complex (MHC) and co-stimulatory molecules play in the non-specific and GXM-specific responses induced by GXM-APC. APC from CD40 knockout mice were as effective as wild-type APC for the induction of non-specific and GXM-specific responses. Blocking activity of B7-1 and B7-2 by treatment of immunized mice with monoclonal antibodies specific for these molecules just before and for 6 days following GXM-APC immunization decreased the splenic interferon-gamma response of mice subsequently infected with NU-2, but only in mice that were treated with both antibodies. These antibody treatments had no effect on DTH reactivity in similarly treated animals. MHC class I molecules were not involved in the antigen non-specific or GXM-specific activities of the vaccine. MHC class II molecules were not required for augmentation of type 1 cytokine responses but were needed for induction of the GXM-specific response that regulates the expression of DTH reactivity. This investigation has shown that an MHC class II-restricted, GXM-specific response is responsible for altering DTH responsiveness which is the correlate of immunity in this model.

Figure 1

Role of CD40 in augmenting Th1 cytokine responses in GXM-APC-immunized mice. Open bars, spleen cells cultured in medium; solid bars, spleen cells cultured in medium containing cryptococcal CneF antigen. Sham-infected mice were not immunized and were treated with PBS intratracheally at the time that other groups were infected. Naïve mice were not immunized but were infected. Immunized and infected mice were immunized with GXM-APC obtained from wild-type (WT) mice or from CD40 knockout (CD40KO) mice. Peak responses, which occurred on day 15 of infection, are shown. IL-2 data are means ± standard errors of the means for three to five individual C57BL/6J mice per group. IFN-γ data are means ± standard errors of the means for eight or nine individual C57BL/6J mice per group.

Figure 2

Role of CD40 in prolonging DTH responses in GXM-APC-immunized mice. Sham-infected mice were not immunized and were treated with PBS intratracheally at the time that other groups were infected. Naïve mice were not immunized but were infected. Immunized and infected mice were treated with GXM-APC obtained from wild-type (WT) mice or from CD40 knockout (CD40KO) mice. Mice were skin tested with cryptococcal CneF 16 days after infection. Data are means ± standard errors of the means for seven individual C57BL/6J mice per group.

Figure 3

Role of B7-1 and B7-2 in augmenting IFN-γ response and prolonging DTH reactions in GXM-APC-immunized mice. For the IFN-γ analysis open bars represent spleen cells cultured in medium and solid bars represent spleen cells cultured in medium containing cryptococcal CneF antigen. Sham-infected mice were not immunized and were treated with PBS intratracheally at the time that other groups were infected. Naive mice were not immunized but were infected. Immunized and infected mice were treated with 200 μg normal rat immunoglobulin (IgG), 100 μg anti-B7-1, 100 μg anti-B7-2, or 100 μg anti-B7-1 and 100 μg anti-B7-2. Antibody reagents were administered on the day of immunization and days 3 and 6 after immunization. Peak IFN-γ responses, which occurred on day 15 of infection, are shown. DTH responses were measured on the 16th day after infection. Data are means ± standard errors of the means for three to five individual CBA/J mice per group for cytokine analysis and seven or eight individual mice for the DTH analysis.

Figure 4

Role of MHC class I and class II molecules in augmenting Th1 cytokine responses in GXM-APC-immunized mice. Open bars, spleen cells cultured in medium. Solid bars, spleen cells cultured in medium containing cryptococcal CneF antigen. Sham-infected mice were not immunized and were treated with PBS intratracheally at the time that other groups were infected. Naive mice were not immunized but were infected. Immunized and infected mice were immunized with GXM-APC obtained from wild-type (WT) mice, MHC class I deficient mice (Class I) or MHC class II deficient mice (Class II). Peak responses, which occurred on day 14 of infection, are shown. Data are means ± standard errors of the means for three to five individual C57BL/6J mice per group.

Figure 5

Role of MHC class I and MHC class II in prolonging DTH responses in GXM-APC-immunized mice. Sham-infected mice were not immunized and were treated with PBS intratracheally at the time that other groups were infected. Naïve mice were not immunized but were infected. Immunized and infected mice were treated with GXM-APC obtained from wild-type (WT) mice, MHC class I deficient (Class I) or MHC class II deficient (class II) mice. Mice were skin tested with cryptococcal CneF 16 days after infection. Data are means ± standard errors of the means for eight individual C57Bl/6J mice per group.