Dose-dependent and schedule-dependent effects of interleukin-12 on antigen-specific CD8 responses.

Interleukin-12 (IL-12) has been shown to play a central role in the innate and acquired immune responses. Its activities include enhancement of natural killer (NK) and cytotoxic T lymphocyte (CTL) activity and promotion of CD4 Th1 cell development. It has also been shown to provide potent activity as a vaccine adjuvant in generating antibody and T cell responses. We have investigated the efficacy of IL-12 protein in promoting CD8 T cell responses when it is used as an adjuvant for immunization. Studies using, as antigen, cDNA from an autologous antigen (P1A) as well as studies of responses to vaccinia virus-delivered self (gp100) and non-self (beta-galactosidase) antigens show that the dose and schedule of IL-12 administration can significantly affect adjuvant activity, leading to enhancement or suppression of antigen-specific responses.

IL-12-Dependent enhancement of CTL response to weak class I-restricted peptide immunogens requires coimmunization with T helper cell immunogens.

The effect of in vivo administration of rmIL-12 on the CTL response to immunization with a weakly immunogenic class I-restricted peptide emulsified in incomplete Freund's adjuvant was investigated. In the absence of IL-12, peptide-specific CTL responses were significantly greater following coimmunization with class I-restricted peptide and T helper cell antigens than following immunization with the class I-restricted peptide alone. IL-12-dependent enhancement of the CTL response to peptide immunization was demonstrated in the presence of, but not in the absence of, coimmunization with T helper cell antigen. These findings indicate that IL-12 enhancement of the CTL response to weak class I-restricted immunogens is T helper cell dependent. Treatment with rmIL-12 also enhanced the CTL response to immunization with cDNA encoding both CTL and T helper cell epitopes. These findings are relevant to the design of vaccines containing tumor-associated class I-restricted peptides currently being tested as an immunotherapy for cancer patients.

A CTL assay requiring only 150 microliter of mouse blood.

In this paper, we present a method for measuring antigen specific cytotoxic T lymphocyte (CTL) activity from individual mouse peripheral blood samples without animal sacrifice. Peripheral blood cells are stimulated in vitro with a cocktail of antigen, cytokines, costimulatory molecules and irradiated feeder cells resulting, 7 days later, in a readily detectable antigen specific signal from a well plated under limiting dilution conditions. This highly sensitive and antigen specific assay is more efficient than conventional CTL assays and thus increases the number of mice that can be tested in a single assay. Since blood samples can be assayed from an individual mouse at multiple times during the course of an in vivo study, the assay can facilitate and strengthen correlative studies on CTL responses and in vivo results.

Potent activity of soluble B7-IgG fusion proteins in therapy of established tumors and as vaccine adjuvant.

Fusion proteins consisting of the extracellular region of murine B7.1 or B7.2 and the Fc portion of murine IgG2a (B7-IgG) were evaluated for their ability to promote antitumor responses. Therapeutic administration of soluble B7-IgG in mice with established tumors induced complete regression of the tumor and increased the survival of mice. In three models, MethA, P815, and MB49, mice with 7-day-old established tumors were cured with two to three treatment cycles of B7-IgG, given twice a week. Even in mice with an established B16/F10 tumor (a poorly immunogenic melanoma), therapeutic treatment with B7-IgG alone slowed tumor growth and increased survival significantly. Still stronger antitumor activity was achieved when B7-IgG was used as a vaccine adjuvant mixed with irradiated tumor cells. In 80% of mice with 7-day-old B16 tumors, tumors regressed completely, and mice survived for at least 80 days. In all tumor models, B7.1-IgG and B7.2-IgG had similar antitumor activity. B7-IgG-mediated tumor rejection was dependent on T cells, specifically CD8 cells, as demonstrated by the failure of B7-IgG to induce tumor regression in severe combined immunodeficient or CD8-depleted mice. In addition, mice that were cured of an established tumor were protected against a rechallenge with the same tumor for at least 4 months, suggesting the generation of memory responses. Surprisingly, the antitumor activity of B7-IgG was independent of IFN-gamma, as demonstrated by tumor rejection in IFN-gamma knockout mice. Our findings demonstrate the potent capacity of B7-IgG to generate or enhance antitumor immune responses and suggest the clinical value of B7-IgG.

Immune response enhancement by in vivo administration of B7.2Ig, a soluble costimulatory protein.

The identification of both class I- and class II-restricted tumor-associated peptides recognized by T cells has led to the test of these peptides as immunogens in experimental immunotherapy for cancer patients. However, optimal T cell activation requires signaling both through the T cell receptor for antigen and through costimulatory pathways. B7.1 and B7.2 are powerful costimulatory molecules expressed on the surface of antigen-presenting cells. Using a mouse model, we have sought to optimize costimulatory signals during antipeptide responses by administering a soluble form of B7.2 at the time of peptide immunization. Administration of B7. 2Ig fusion protein significantly enhanced T helper cell and CTL responses. These findings suggest that soluble forms of human B7.2 protein may provide a straightforward and practical method of supplying optimal costimulation during clinical immunotherapy.

B7.1 is a quantitatively stronger costimulus than B7.2 in the activation of naive CD8+ TCR-transgenic T cells.

Using a TCR transgenic mouse bred onto a recombinase-activating gene-2-deficient background, we have examined the influence of B7.1 and B7.2 on activation of naive, CD8+ T cells in vitro. We found that B7.1 was a more potent costimulus than B7.2 for induction of proliferation and IL-2 production by naive CD8+ T cells. This difference appeared to be quantitative in nature, as determined using transfectants expressing various defined levels of B7.1 or B7.2, or using purified B7.1 or B7.2 fusion proteins. In contrast to the quantitative differences seen in stimulation of naive T cells, B7.1 and B7.2 were comparable in their ability to costimulate responses in T cells previously primed in vitro. In addition, primed, but not naive, T cells were capable of proliferating and producing IL-2 in response to a TCR stimulus alone, apparently in the absence of B7 costimulation. Lastly, we found that B7.1 and B7.2 were equivalently capable of driving differentiation of naive CD8+ T cells into an IL-4-producing phenotype when exogenous IL-4 was added to the primary culture or to an IFN-gamma-producing phenotype in the presence of IL-12. These results indicate that signals generated by B7.1 and B7.2 are qualitatively similar, but that B7.1 is quantitatively stronger than B7.2. Further, our results indicate that the activation state of the responding T cell may influence the efficiency with which the T cell can respond to a costimulatory signal provided by either B7.1 or B7.2.