Wen-Gong Yu

IL-12-induced tumor regression correlates with in situ activity of IFN-gamma produced by tumor-infiltrating cells and its secondary induction of anti-tumor pathways.

Administration of recombinant interleukin‐12 (rIL‐12) into CSA1M fibrosarcoma‐bearing mice results in complete regression of growing tumors. This tumor regression is associated with massive lymphoid cell infiltration to tumor sites and is completely blocked by injection of anti‐interferon‐γ (IFN‐γ) monoclonal antibody (mAb). We investigated whether anti‐IFN‐γ mAb exerts its suppressive effect on tumor regression by blocking the IL‐12‐induced lymphoid cell migration to tumor sites or by inhibiting the secondary effects of IFN‐γ produced by infiltrating cells. Injection of anti‐IFN‐γ mAb to CSA1M‐bearing mice before IL‐12 treatment prevented the induction of tumor regression, whereas this treatment affected only marginally the infiltration of lymphoid cells to tumor masses. In accordance with this, IFN‐γ mRNA was expressed inside tumor masses by infiltrating cells after IL‐12 therapy irrespective of whether anti‐IFN‐γ mAb was injected. However, anti‐IFN‐γ mAb treatment almost completely abrogated the in situ expression of inducible nitric oxide synthase (iNOS) as well as IFN‐inducible protein‐10 (IP‐10) genes as examples of IFN‐γ‐inducible genes. Immunohistochemical analyses also revealed that the expression of iNOS protein was completely inhibited by anti‐IFN‐γ injection. These results suggest that the implementation of in situ IFN‐γ activity and its secondary induction of antitumor pathways such as iNOS and IP‐10 expression are important processes in the IL‐12‐induced tumor regression. J. Leukoc. Biol. 62: 450–457; 1997.

ESTABLISHMENT OF AN IL-12-RESPONSIVE T CELL CLONE : ITS CHARACTERIZATION AND UTILIZATION IN THE QUANTITATION OF IL-12 ACTIVITY

We previously demonstrated that proliferation of terminally differentiated Thl clones depends primarily on an interleukin‐12 (IL‐12)‐paracrme mechanism mediated by their interactions with antigen‐presenting cells (APC) rather than on an IL‐2‐autocrine mechanism. Such a Thl clone (4‐86, C57BL/6 origin) was cultured with recombinant IL‐12 (rIL‐12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL‐12, and by limiting dilution, several clones highly reactive to rIL‐12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL‐12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) αβ+, Vβ11+, NK‐1.1‐; CD4‐ CD8‐; LFA‐1+, ICAM‐1+; and CD28+, CD80+, CD86+, CTLA‐4‐. In accordance with high responsiveness to IL‐12, 2D6 cells were also found to express IL‐12 receptor (IL‐12R) as detected by incubation with rIL‐12 and then staining with anti‐IL‐12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL‐12 resulted in the expression of interferon‐γ (IFN‐γ) and IL‐10 mRNAs and production of these cytokines. The 2D6 clone responded to IL‐2 (vigorously), IL‐7 (moderately), and IL‐4 (mildly) in addition to IL‐12. However, the Ab capture assay using anti‐IL‐12 mAb enabled us to quantify IL‐12‐specific activity contained in a given sample. Thus, this study describes the unique features of the IL‐12‐responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL‐12 activity. J. Leukoc. Biol. 61: 346–352; 1997.

Requirement for Distinct Janus Kinases and STAT Proteins in T Cell Proliferation Versus IFN-γ Production Following IL-12 Stimulation

While IL-12 is known to activate JAK2 and TYK2 and induce the phosphorylation of STAT4 and STAT3, little is known regarding how the activation of these signaling molecules is related to the biologic effects of IL-12. Using an IL-12-responsive T cell clone (2D6), we investigated their requirements for proliferation and IFN-gamma production of 2D6 cells. 2D6 cells could be maintained with either IL-12 or IL-2. 2D6 lines maintained with IL-12 (2D6(IL-12)) or IL-2 (2D6(IL-2)) exhibited comparable levels of proliferation, but produced large or only small amounts of IFN-gamma, respectively, when restimulated with IL-12 after starvation of either cytokine. 2D6(IL-12) induced TYK2 and STAT4 phosphorylation. In contrast, their phosphorylation was marginally induced in 2D6(IL-2). The reduced STAT4 phosphorylation was due to a progressive decrease in the amount of STAT4 protein along with the passages in IL-2-containing medium. 2D6(IL-12) and 2D6(IL-2) similarly proliferating in response to IL-12 induced comparable levels of JAK2 activation and STAT5 phosphorylation. JAK2 was associated with STAT5, and IL-12-induced STAT5 phosphorylation was elicited in the absence of JAK3 activation. These results indicate that IL-12 has the capacity to induce/maintain STAT4 and STAT5 proteins, and that TYK2 and JAK2 activation correlate with STAT4 phosphorylation/IFN-gamma induction and STAT5 phosphorylation/cellular proliferation, respectively.