No abstract

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-gamma (IFN-gamma), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-gamma bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 by murine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-gamma production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.

The mechanisms underlying the profound suppression of cell-mediated immunity (CMI) accompanying measles are unclear. Interleukin-12 (IL-12), derived principally from monocytes and macrophages, is critical for the generation of CMI. Measles virus (MV) infection of primary human monocytes specifically down-regulated IL-12 production. Cross-linking of CD46, a complement regulatory protein that is the cellular receptor for MV, with antibody or with the complement activation product C3b similarly inhibited monocyte IL-12 production, providing a plausible mechanism for MV-induced immunosuppression. CD46 provides a regulatory link between the complement system and cellular immune responses.

Protection induced by vaccination depends on the capacity of the vaccine to elicit an appropriate immune response. In leishmaniasis, protection requires leishmanial-specific CD4+ T helper (TH) cells. Vaccination of BALB/c mice with leishmanial antigens and interleukin-12 (IL-12) promoted the development of leishmanial-specific CD4+ TH1 cells. These mice were resistant to subsequent infection with Leishmania major. Thus, IL-12 is an effective adjuvant for the initiation of protective cell-mediated immunity against leishmaniasis and may be an important component in other vaccines that need to induce cell-mediated immunity.

Several cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 microgram/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-gamma production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-gamma production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1-3 ng/ml at 3-6 h after LPS injection, whereas IFN-gamma production was observed only in BCG-primed mice. The priming effect of BCG on IFN-gamma production appears to be mostly due to its ability to increase TNF-alpha production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-gamma production, as shown by the ability of injection of TNF-alpha and LPS (1 microgram/mouse), but not LPS alone, to induce IFN-gamma production. However, in addition to TNF-alpha, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-gamma production, because co-injection of TNF-alpha and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-gamma production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-gamma production, also completely protect BCG-primed mice injected with up to 10 micrograms of LPS from shock-induced death. Thus, IL-12 is required for IFN-gamma production and lethality in an endotoxic shock model in mice.

The antitumor effect and mechanisms activated by murine IL-12 and IL-18, cytokines that induce IFN-gamma production, were studied using engineered SCK murine mammary carcinoma cells. In syngeneic A/J mice, SCK cells expressing mIL-12 or mIL-18 were less tumorigenic and formed tumors more slowly than control cells. Neither SCK.12 nor SCK.18 cells protected significantly against tumorigenesis by distant SCK cells. However, inoculation of the two cell types together synergistically protected 70% of mice from concurrently injected distant SCK cells and 30% of mice from SCK cells established 3 d earlier. Antibody neutralization studies revealed that the antitumor effects of secreted mIL-12 and mIL-18 required IFN-gamma. Interestingly, half the survivors of SCK.12 and/or SCK.18 cells developed protective immunity suggesting that anti-SCK immunity is unlikely to be responsible for protection. Instead, angiogenesis inhibition, assayed by Matrigel implants, appeared to be a property of both SCK.12 and SCK.18 cells and the two cell types together produced significantly greater systemic inhibition of angiogenesis. This suggests that inhibition of tumor angiogenesis is an important part of the systemic antitumor effect produced by mIL-12 and mIL-18.

No abstract

We have used cDNA arrays to investigate gene expression patterns in peripheral blood mononuclear cells from patients with leukemic forms of cutaneous T cell lymphoma, primarily Sezary syndrome (SS). When expression data for patients with high blood tumor burden (Sezary cells >60% of the lymphocytes) and healthy controls are compared by Student's t test, at P < 0.01, we find 385 genes to be differentially expressed. Highly overexpressed genes include Th2 cells–specific transcription factors Gata-3 and Jun B, as well as integrin β1, proteoglycan 2, the RhoB oncogene, and dual specificity phosphatase 1. Highly underexpressed genes include CD26, Stat-4, and the IL-1 receptors. Message for plastin-T, not normally expressed in lymphoid tissue, is detected only in patient samples and may provide a new marker for diagnosis. Using penalized discriminant analysis, we have identified a panel of eight genes that can distinguish SS in patients with as few as 5% circulating tumor cells. This suggests that, even in early disease, Sezary cells produce chemokines and cytokines that induce an expression profile in the peripheral blood distinctive to SS. Finally, we show that using 10 genes, we can identify a class of patients who will succumb within six months of sampling regardless of their tumor burden.