Protective cell-mediated immune responses in cancer are critically dependent on T-helper type 1 (T(H)1) cytokines such as interferon-gamma (IFN-gamma). We have previously shown that the combination of CD40 stimulation and interleukin-2 (IL-2) leads to synergistic antitumor responses in several models of advanced metastatic disease. We now report that after this treatment and other immunotherapy regimens, the CD4+ T-cell population, in contrast to CD8+ T cells, did not significantly increase but rather exhibited a substantial level of apoptosis that was dependent on IFN-gamma. Mice immunized with tumor cells and treated with an immunotherapy regimen that was initially protective were later unable to mount effective memory responses compared with immunized mice not receiving immunotherapy. Immunotherapy given to tumor-bearing Ifngr-/- mice resulted in restoration of secondary responses. Thus, although immunotherapeutic regimens inducing strong IFN-gamma responses can lead to successful early antitumor efficacy, they may also impair the development of durable antitumor responses.

Immunostimulatory glucose polymers known as beta-glucans have been studied for many years. Our laboratory has prepared and characterized a novel microparticulate beta-glucan (MG) from the budding yeast Saccharomyces cerevisiae. Because MG particles are rapidly phagocytized by murine peritoneal macrophages and induce the expression of B7 costimulatory molecules, we hypothesized that MG could serve as a vaccine adjuvant to enhance specific immune responses. Here, we describe a procedure for conjugating the test vaccine antigen bovine serum albumin (BSA) to MG via water-soluble carbodiimide linkage. Conjugates with up to 0.4 mg of BSA/mg MG were prepared. MG/BSA conjugates were still actively phagocytized by mouse peritoneal macrophages. When used to immunize mice by the intradermal route, these conjugates enhanced the primary IgG antibody response to BSA in a manner comparable to the prototypic complete Freund's adjuvant. Although primary oral immunization with MG/BSA caused no increase in serum anti-BSA antibody titers, booster immunization elicited a significant anti-BSA antibody response. These results suggest that protein antigens can be conjugated to MG via a carbodiimide linkage and that these conjugates provide an adjuvant effect for stimulating the antibody response to the protein antigens.

Recently, our laboratory reported that secondary CD8+ T cell-mediated antitumor responses were impaired following successful initial antitumor responses using various immunotherapeutic approaches. Although immunotherapy stimulated significant increases in CD8+ T cell numbers, the number of CD4+ T cells remained unchanged. The current investigation revealed a marked differential expansion of CD4+ T cell subsets. Successful immunotherapy surprisingly resulted in an expansion of CD4+Foxp3+ regulatory T (Treg) cells concurrent with a reduction of conventional CD4+ T (Tconv) cells, despite the marked antitumor responses. Following immunotherapy, we observed differential up-regulation of PD-1 on the surface of CD4+Foxp3+ Treg cells and CD4+Foxp3− Tconv cells. Interestingly, it was the ligand for PD-1, B7-H1 (PDL-1), that correlated with Tconv cell loss after treatment. Furthermore, IFN-γ knockout (IFN-γ−/−) and IFN-γ receptor knockout (IFN-γR−/−) animals lost up-regulation of surface B7-H1 even though PD-1 expression of Tconv cells was not changed, and this correlated with CD4+ Tconv cell increases. These results suggest that subset-specific expansion may contribute to marked shifts in the composition of the T cell compartment, potentially influencing the effectiveness of some immunotherapeutic approaches that rely on IFN-γ.

Microparticulate β-glucan (MG) conjugated to vaccine antigen has been shown to serve as an effective adjuvant in vivo. To further study antigen presentation by MG:vaccine conjugates, bone marrow-derived dendritic cells (BMDC) were treated with MG conjugated to ovalbumin (OVA), then interacted with splenocytes from DO11.10 transgenic mice expressing an OVA peptide-specific T cell receptor. BMDC treated with MG:OVA induced significantly higher numbers of activated (CD25+CD69+) OVA-specific CD4+ T cells than BMDC treated with OVA alone. BMDC treated with MG:OVA upregulated CD86 and CD40 expression as well as MG alone, indicating that conjugation of OVA does not alter the immunostimulatory capacity of MG. Activation of CD8+ OVA-specific OT-1 cells showed that MG:OVA is also capable of enhancing cross-presentation by BMDC to CD8+ cytotoxic T cells. These results show that MG acts as an adjuvant to enhance antigen presentation by dendritic cells to naïve, antigen-specific CD4 and CD8 T cells.

Disease relapse remains a principle cause of mortality following autologous hematopoietic transplant. Strategies to enhance immune function in the post-transplant setting are needed to eliminate residual disease. CD40 stimulation through the use of agonistic CD40 antibodies has been shown to promote anti-tumor activity both as a single agent and in combination with IL-2 administration with increases in dendritic cell numbers being observed. We hypothesized that CD40 stimulation in combination with myeloablative syngeneic hematopoietic cell transplantation can act together to promote anti-tumor responses. We developed a model in which anti-CD40 given with syngeneic BMT was assessed in advanced tumor-bearing mice. We have found that delaying administration of anti-CD40 for one week post-BMT could obviate the severe toxicity associated with immediate post-BMT administration. BALB/c were given a syngeneic renal cell carcinoma cell line (Renca) i.v. After 4 days, at which time lung metastases were established, the mice were treated with syngeneic BMT followed one week later with initiation of anti-CD40 treatment (100 μg/dose; daily, 4 days per week for 2 weeks). CD40 stimulation resulted in significant expansion of GR-1+/CD11b+ myeloid and CD11c+/I-A+ dendritic cells in the spleen, similar to the expansion of these populations observed in mice that received anti-CD40 and no BMT. However, while B220+ IgM+ B cells are significantly expanded following anti-CD40 administration in mice that do not receive a transplant, B cell recovery is reduced following transplant in mice that also received anti-CD40. T cell recovery post-transplant did not differ significantly in animals that received anti-CD40 or control antibody. Importantly, significant increases in survival of anti-CD40 treated, tumor-bearing recipients of syngeneic BMT were observed compared to either treatment alone (log-rank test p< 0.001). This promotion of anti-tumor activity by CD40 stimulation was seen in mice that were profoundly T cell deficient due to the transplant. These data suggest that despite severe immune depletion that occurs after lethal TBI, immunomodulation by anti-CD40 can result in significant protection from the residual disease despite the severe lack of T cells at this time period.