Differential immunologic modulatory effects of tetrahydrocannabinol as a function of age.

Tetrahydrocannabinol (THC) the major psychoactive component of marijuana, differentially modulates the immune response of lymphoid cells from young and old mice. This effect was previously shown when cells from mice of different ages were treated with THC in vitro. In the present study THC was given in vivo to mice of different ages. Lymphoid cells from the young and old mice had different immunologic potential in terms of ability to produce cytokines following stimulation with either Con A or anti-CD3 antibody. IL-4 and IL-10 production was consistently up-regulated in spleen cell cultures from the older animals. In addition, in vivo administration of THC up-regulated the proliferative response of lymphoid cells from young adult mice. Such enhancement was not evident for cells from older animals. Although the proliferative response of spleen cells in the old mice tended to be suppressed, statistical significance was not evident possibly because of marked variation of the responses of cells from the older mice. Since marijuana is used by persons of a wide range of ages, aging should be an important variable that must be considered when assessing the immunomodilatory effect of this drug.

Multiple lineages of tumors express a common tumor antigen, P1A, but they are not cross-protected.

Whether tumors of different lineages share common Ags is a critical issue for understanding anti-tumor immune responses and for designing Ag-specific tumor immunotherapy. Because of lack of cross-protection among individually derived tumors, it has been proposed that tumor Ags are specific for individual tumors. Here we show that lack of cross-protection is not due to lack of a shared tumor Ag. Thus, a plasmocytoma J558 transfected with the costimulatory molecule B7 activates a cross-reactive CTL response in vivo. The major Ag recognized by the cross-reactive CTL is P1A, which is expressed in mastocytoma P815, plasmocytoma J558, and fibrosarcoma Meth A. Surprisingly, no significant cross-protection can be detected among P1A-expressing tumors after immunization with either P1A-expressing or B7-transfected P815 cells. Our results demonstrate that multiple lineages of tumors are not cross-protected even though they share a tumor Ag that can be recognized by CTL. These results have important implications for tumor immunotherapy.

Gene-targeted B-deficient mice reveal a critical role for B cells in the CD4 T cell response.

The role of B cells in promoting T cell responses is still controversial. In this study, we use JHD mice which have a targeted mutation in the JH gene and are thus rendered deficient in B cells to address this issue. We show here that immunization of JHD mice with soluble antigen fails to prime CD4 T cells, for either clonal expansion or delivery of immunological help for antibody responses. This lack of CD4 T cell priming in JHD mice corresponds to a 3- to 9-fold lower co-stimulatory activity of antigen-presenting cells (APC) from the JHD mice, as measured by anti-CD3-induced proliferative responses of CD4 T cells. This in turn is due to a defect of APC from JHD mice in response to T cell-mediated induction of co-stimulatory activity. As the development of macrophages and dendritic cells is unaffected in the JHD mice, our results demonstrate that B cells play a critical role in CD4 T cell priming, possibly by delivering a critical co-stimulatory activity for clonal expansion of CD4 T cells.

T cell costimulation by B7/BB1 induces CD8 T cell-dependent tumor rejection: an important role of B7/BB1 in the induction, recruitment, and effector function of antitumor T cells.

A successful antitumor T cell immune response involves induction, recruitment, and effector function of T cells. While B7/BB1 is known as a major costimulatory molecule in the induction of T cell responses, its role in T cell recruitment and effector function is still unclear. In this study, we show that introducing a major costimulatory molecule B7/BB1 into a major histocompatibility complex class II-negative tumor cell line, J558, results in a drastic reduction of its tumorigenicity. The tumor rejection depends on CD8 T cells but not CD4 T cells. However, unlike the previous reports on melanoma cell lines, B7/BB1-transfected J558 cells fail to induce cross-protection against parental J558 cells. The B7/BB1-transfected (J558-B7), but not untransfected J558 cells (J558-Neo) induce a CD8 T cell-dominant inflammatory response, and the T cells isolated from the tumor infiltrating lymphocytes (TIL) are polyclonal in terms of their T cell receptor V beta usage. Most surprisingly, the freshly prepared TIL have a potent, CD8 T cell-mediated cytotoxicity on tumor cells without any in vitro stimulation. The cytotoxic T lymphocyte (CTL) activity can be blocked by anti-CD8 monoclonal antibody (mAb). Interestingly, the CTL lyse J558-B7 about 10- to 80-fold more efficiently than untransfected J558-Neo cells. This preferential lysis cannot be attributed to recognition of B7/BB1-derived antigen by the T cells. This finding, together with the lack of the cross-protection between the J558-B7 and J558-Neo, suggests that B7/BB1 can also function at the effector phase of CTL responses. This notion is confirmed by our findings that the lysis of J558-B7 can be blocked by anti-B7 mAbs. Taken together, our results indicate that not only can the B7/BB1 molecule function as a costimulatory molecule at the initiation of immune response, it can also play a major role in T cell recruitment and effector function. This conclusion has significant implications for immunotherapy of tumors.

Ity influences the production of IFN-gamma by murine splenocytes stimulated in vitro with Salmonella typhimurium.

The Ity-Lsh-Bcg genetic locus in the mouse has been documented to confer innate resistance to at least three intracellular pathogens: Salmonella typhimurium, Leishmania donovani, and Mycobacterium. Expression of the resistance gene(s) results in a slower net growth of these pathogens in the reticuloendothelial system early postinfection. Although it is clear that the resident macrophages in resistant mice are functionally superior with regard to antimicrobial activity, the exact mechanism(s) underlying the control exerted by this gene is not understood. Using S. typhimurium infection as a model, we have examined the influence of this resistance gene(s) on the production of IFN-gamma, a cytokine known to play an important role in host-defense against several intracellular pathogens. We compared IFN-gamma production by splenocytes from resistant (Ity(r)) and sensitive (Ity(s)) inbred mouse strains after stimulation in vitro with S. typhimurium. Spleen cells from Ity(r) mouse strains produced significantly higher levels of IFN-gamma when compared to spleen cells obtained from Ity(s) mouse strains. Enhanced IFN-gamma production was not a generalized response to bacteria. Listeria monocytogenes induced comparable levels of IFN-gamma production from both Ity(r) (CBA/J) and Ity(s) (C57BL/6) mice. Splenocytes from Ity congenic mouse strains displayed similar differences in the level of IFN-gamma produced after S. typhimurium stimulation, with spleen cells from the Ity(r) strain producing significantly higher levels of IFN-gamma when compared to spleen cells from the Ity(s) strain. A requirement for adherent cells and/or adherent cell-derived factors has been documented for IFN-gamma production by S. typhimurium-stimulated splenocytes. Interestingly, supernatant from adherent cells obtained from Ity(r) mouse strains was found to induce the production of significantly higher levels of IFN-gamma when compared to adherent cell supernatant from Ity(s) strains. Nylon wool nonadherent cells from Ity(s) mouse strains produced high levels of IFN-gamma when exposed to supernatants obtained from adherent cells of Ity(r) mouse strains. In contrast, nylon wool nonadherent cells from Ity(r) mouse strains produced reduced levels of IFN-gamma when exposed to supernatant obtained from adherent cells of Ity(s) mouse strains. Thus, modulation of IFN-gamma production appears to be a function of the Ity(r) gene(s). This study documents for the first time that the Ity locus may play a role in controlling resistance to Salmonella infection by regulating IFN-gamma production by NK cells.

Salmonella typhimurium induces IFN-gamma production in murine splenocytes. Role of natural killer cells and macrophages.

IFN-gamma is a cytokine known to play an important role in host defense against Salmonella typhimurium. The lymphoid cells required for in vitro production of IFN-gamma after S. typhimurium stimulation of mouse spleen cells was investigated. Spleen cells depleted of cells bearing NK1.1, asialo GM1, Thy 1.2, or CD5 resulted in a significant reduction in IFN-gamma production after stimulation with S. typhimurium. In contrast, Con A-induced IFN-gamma production was only slightly reduced after depletion of NK1.1- or asialo GM1-bearing cells. Spleen cells from SCID mice produced elevated levels of IFN-gamma after stimulation with S. typhimurium. IFN-gamma production by SCID spleen cells was dependent upon asialo GM1+ T cells, suggesting that NK cells were the cells producing IFN-gamma in response to S. typhimurium. Splenic adherent cells were required for optimal IFN-gamma production. However, direct contact between the adherent and nylon wool nonadherent (NWNA) cell populations was not essential. IFN-gamma production was observed when the adherent and NWNA cell populations were physically separated or when supernatant from S. typhimurium-stimulated adherent cells was added to NWNA cells. Optimal IFN-gamma production was dependent on the presence of TNF-alpha, inasmuch as addition of antibody to TNF-alpha to spleen cell or NWNA cell cultures significantly reduced IFN-gamma production. However, addition of rTNF-alpha did not induce IFN-gamma production by NWNA cells. These findings document the existence of a T-independent mechanism for early IFN-gamma production in response to S. typhimurium, and show that TNF-alpha is necessary but not sufficient for the production of IFN-gamma.

Interferon gamma (IFN-gamma) production by gut-associated lymphoid tissue and spleen following oral Salmonella typhimurium challenge.

Although IFN-gamma has been shown to play an important role in protection against a systemic S. typhimurium challenge, the in vivo and in vitro production of this cytokine following S. typhimurium infection of the gastrointestinal tract has not been investigated. In this study, IFN-gamma production by gut-associated lymphoid tissue and spleen was investigated in mice following oral challenge with S. typhimurium. Cells obtained from the Peyer's patches (PP), mesenteric lymph nodes (MLN) and spleen (Sp) of mice orally challenged with S. typhimurium were assessed for levels of IFN-gamma mRNA after varying times following in vivo infection. RNA obtained from the above tissues was subjected to reverse transcription followed by PCR amplification using primers specific for murine IFN-gamma. Elevated levels of IFN-gamma mRNA were first detected in the PP at 6 h post-challenge. Elevated levels of IFN-gamma mRNA were then detected in the MLN at 24 h and in the spleen at 4 days post-challenge. These in vivo results were in agreement with the ability of these lymphoid tissues to produce IFN-gamma upon in vitro stimulation with killed S. typhimurium. Neutralization of endogenously produced IFN-gamma by administration of mAb to IFN-gamma completely abrogated resistance to an oral challenge of S. typhimurium. A significant difference in the percent mortality was observed between the antibody-treated and control groups. Evaluation of bacterial spread in the antibody treated group versus the control group at 4 days following oral challenge revealed higher numbers of bacteria in the spleen and liver of antibody treated mice. These results clearly show that IFN-gamma is rapidly produced by gut-associated lymphoid tissue and spleen following oral S. typhimurium infection, and that endogenous production of IFN-gamma is essential in host resistance to S. typhimurium.