TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production.

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

B7-H1-dependent sex-related differences in tumor immunity and immunotherapy responses.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1-dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1(-/-) females versus males as a result of reduced regulatory T cell function in the B7-H1(-/-) females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1(-/-) Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.

Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication.

We recently demonstrated that human p38 mitogen-activated protein kinase (MAPK) inhibitors reduced in vitro and in vivo replication of the protozoan parasites Toxoplasma gondii and Encephalitozoon cuniculi. In this study, we assessed the efficacy of five p38 MAPK inhibitors to block the replication of Plasmodium falciparum in human erythrocytes cultured ex vivo and demonstrate that the pyridinylimidazole RWJ67657 and the pyrrolobenzimidazole RWJ68198 reduced P. falciparum replication, yielded trophozoites that were greatly diminished in size at 24h, and that these two agents interfered with stage differentiation. Interestingly, the chloroquine-resistant strain W2 was significantly more sensitive to these drugs than was the chloroquine-sensitive strain HB3. These results suggest that pyridinylimidazoles and pyrrolobenzimidazoles designed to inhibit human p38 MAPK activation can be developed to treat malaria.

Aged regulatory T cells protect from autoimmune inflammation despite reduced STAT3 activation and decreased constraint of IL-17 producing T cells.

Regulatory T cells (Tregs) are specialized CD4(+) T lymphocytes helping defend against autoimmunity and inflammation. Although age is associated with increased inflammation and autoimmunity, few reports address age effects of immune regulation or auto-aggressive T cells. We show here that young and aged naïve CD4(+) T cells are equivalently auto-aggressive in vivo in T cell-driven autoimmune colitis. Young and aged CD4(+) Tregs equally suppressed age-matched T cell proliferation in vitro and controlled clinical and pathologic T cell-driven autoimmune colitis, suggesting equivalent regulatory function. However, whereas young and aged CD4(+) Tregs suppressed interferon (IFN)-γ(+) T cells equivalently in this model, aged CD4(+) Tregs unexpectedly failed to restrain interleukin (IL)-17(+) T cells. Nonetheless, young and aged CD4(+) Tregs equally restrained IL-17(+) T cells in vivo during acute inflammation, suggesting a chronic inflammation-related defect in aged CD4(+) Tregs. In support, aged Tregs expressed reduced STAT3 activation, a defect associated with poor IL-17-producing T cell restraint. Aged naïve mice had markedly increased programmed death (PD)-1(+) T cells, but these exhibited no significant auto-aggressive or regulatory functions in T cell-driven colitis. Young CD8(+) CD122(-) T cells induce autoimmune bone marrow failure, but we show that aged CD8(+) CD122(-) T cells do not. These data demonstrate no apparent age-related increase in auto-aggressive T cell behavior, but disclose previously unrecognized functional defects in aged CD4(+) Tregs during chronic inflammation. IL-17 can be inflammatory and contributes to certain autoimmune disorders. Reduced aged Treg function during chronic inflammation and reduced IL-17 restraint could contribute to age-related inflammation or autoimmunity.

Mitigating age-related immune dysfunction heightens the efficacy of tumor immunotherapy in aged mice.

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.