A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo.

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.

Type I interferons enhance production of IFN-gamma by NK cells.

In murine models, challenge with different viral and parasitic infection is closely associated with the production of type I interferons (IFN-alpha/beta) and NK cell production of interferon-gamma (IFN-gamma). Therefore, we wished to determine if IFN-alpha/beta had a role in the regulation of NK cell production of IFN-gamma. IFN-alpha/beta alone stimulated low levels of IFN-gamma production by purified populations of IL-2 activated NK cells but in combination with IL-12 resulted in the production of significant levels of IFN-gamma. Interestingly, maximal production of IFN-gamma by NK cells stimulated with IL-2 plus IFN-alpha/beta was dependent on endogenous tumor necrosis factor-alpha (TNF-alpha). Further studies revealed that TNF-alpha enhanced the ability of IFN-alpha/beta to stimulate production of IFN-gamma by NK cells. In contrast to the stimulatory effect of IFN-alpha/beta on NK cell production of IFN-gamma, IFN-alpha/beta inhibited IL-2 induced proliferation of NK cells. This inhibitory effect was not reversed by the addition of neutralizing antibodies specific for IFN-gamma or TNF-alpha. These data demonstrate that the type I interferons enhance NK cell production of IFN-gamma and suggest that they may be important in the regulation of NK cell production of IFN-gamma during infection.

Mesenteric lymph node T cells but not splenic T cells maintain their proliferative response to concanavalin-A following peroral infection with Toxoplasma gondii.

The suppression of T cell responsiveness which occurs after infection with Toxoplasma gondii in mice has been widely studied using spleen cells. Because the natural route of infection with T. gondii is the peroral route, we examined the proliferative responses of mesenteric lymph node (MLN) cells, in addition to spleen cells, to Concanavalin-A (Con-A) in mice perorally infected with T. gondii. Proliferative responses of spleen cells were significantly suppressed seven and ten days after infection when compared with spleen cells from uninfected mice (62% and 91% reduction, respectively). In contrast, proliferative responses of MLN cells from these infected mice did not differ from those of normal MLN cells. Since IFN-gamma-induced reactive nitrogen intermediate (RNI) production has been reported to play a major role in suppression of proliferative responses in spleen cells of infected mice, we compared production of IFN-gamma and RNI by spleen and MLN cells following infection. MLN cells produced as much IFN-gamma as did spleen cells, but produced 70% less nitrite (as a measure of RNI) after Con-A stimulation. Proliferative responses of MLN cells were suppressed when co-cultured with spleen cells from infected mice, and addition of an inhibitor of RNI to these co-culture inhibited this suppression, suggesting that reduced RNI production by MLN cells contributes to their maintenance of higher proliferative responses. These results demonstrated a clear difference in activity of T cells in the MLN and spleen during the acute stage of the infection.

Role of interleukin-10 in regulation of T-cell-dependent and T-cell-independent mechanisms of resistance to Toxoplasma gondii.

Interleukin-10 (IL-10) is a cytokine which can inhibit T-cell and natural killer (NK) cell functions associated with cell-mediated immunity to intracellular infections. The production of IL-10 by mice infected with Toxoplasma gondii has been implicated in the suppression of lymphocyte proliferation observed during acute toxoplasmosis, as well as susceptibility to infection with this parasite. We have used C57BL/6 mice which lack a functional IL-10 gene (IL-10(-/-) mice) to investigate the role of IL-10 in acute toxoplasmosis. Intraperitoneal infection of IL-10(-/-) mice with T. gondii resulted in 100% mortality by day 13, whereas wild-type C57BL/6 (WT) mice survived acute infection. IL-10(-/-) mice infected with T. gondii had significantly higher serum levels of IL-12 and gamma interferon (IFN-gamma) than WT mice. Early mortality of infected IL-10(-/-) mice was prevented by treatment with IL-10 and significantly delayed by neutralizing antibodies to IL-12 and IFN-gamma. Further studies revealed that SCID/IL-10(-/-) mice infected with T. gondii had delayed time to death compared to IL-10(-/-) mice, indicating that lymphocytes contributed to death of IL-10(-/-) mice. In addition, infected SCID/IL-10(-/-) mice survived longer than infected SCID mice. These latter data indicate that in mice lacking lymphocytes, endogenous IL-10 is associated with increased susceptibility to T. gondii. However, the lack of IL-10 does not alter the infection-induced suppression of T cell and NK cell functions. Our experiments reveal that IL-10 is associated with protection or increased susceptibility to infection with T. gondii, depending on whether mice possess lymphocytes, and demonstrate the important roles of IL-12 and IFN-gamma in the early infection-induced mortality observed in the IL-10(-/-) mice.

IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii.

The role for IL-10 in the immunopathogenesis of acute toxoplasmosis following peroral infection was examined in both genetically susceptible C57BL/6 and resistant BALB/c mice. C57BL/6-background IL-10-targeted mutant (IL-10-/-) mice all died in 2 wk after infection with 20 cysts of the ME49 strain, whereas only 20% of control mice succumbed. Histological studies revealed necrosis in the small and large intestines and livers of infected IL-10-/- mice. The necrosis in the small intestine was the most severe pathologic response and was not observed in control mice. Treatment of infected IL-10-/- mice with either anti-CD4 or anti-IFN-gamma mAb prevented intestinal pathology and significantly prolonged time to death. Treatment of these animals with anti-IL-12 mAb also prevented the pathology. Significantly greater amounts of IFN-gamma mRNA were detected in the lamina propria lymphocytes obtained from the small intestine of infected IL-10-/- mice than those from infected control mice. In common with C57BL/6-background IL-10-/- mice, BALB/c-background IL-10-/- mice all died developing intestinal pathology after infection. Control BALB/c mice all survived even after infection with 100 cysts and did not develop the intestinal lesions. Treatment with anti-IFN-gamma mAb prevented the pathology and prolonged time to death of the infected IL-10-/- mice. These results strongly suggest that IL-10 plays a critical role in down-regulating IFN-gamma production in the small intestine following sublethal peroral infection with Toxoplasma gondii and that this down-regulatory effect of IL-10 is required for prevention of development of IFN-gamma-mediated intestinal pathology and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice.

The role of the CD28/B7 interaction in the regulation of NK cell responses during infection with Toxoplasma gondii.

We examined the role of the CD28/B7 interaction in regulation of NK cell activity. Cells transfected with B7 enhanced IL-12-induced production of IFN-gamma by IL-2-activated, CD28+ NK cells, but not by resting CD28- NK cells. The ability of B7 transfectants to enhance NK cell production of IFN-gamma was dependent on the intracellular adhesion molecule-1/LFA-1 interaction and could be inhibited by TGF-beta, but not IL-10. Since IL-12-induced production of IFN-gamma by NK cells is associated with resistance to certain infections, we examined whether the CD28/B7 interaction regulated NK cell responses during infection. Infection of SCID mice with Toxoplasma gondii resulted in the appearance of a population of CD28+ NK cells, NK cell production of IFN-gamma, and increased NK cell cytolytic activity. Administration of CTLA4-Ig to SCID mice infected with T. gondii inhibited these latter two effects and resulted in a significant increase in parasite burden. The stimulus for CD28 expression by NK cells in SCID mice infected with T. gondii appeared to be independent of IL-2. However, mRNA for IL-15, a cytokine with properties similar to those of IL-2, was detected in tissues of SCID mice infected with T. gondii. In vitro experiments demonstrated that IL-15 could stimulate resting NK cells to express functionally active CD28 as well as enhance the production of IFN-gamma by SCID splenocytes stimulated with T. gondii. Together our data demonstrate that the interaction of CD28+ NK cells with B7 regulates NK cell production of IFN-gamma associated with resistance to infection and that IL-15 may be involved in these events.