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.
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.
Summary
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.
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► RWJ68198 and RWJ67657 inhibit Plasmodium falciparum replication. ► These p38 MAPK inhibitors interfere with parasite stage differentiation. ► Structural modification may increase ...potency and selectivity of these antimalarials.
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.
•TgMAPK1 is a Toxoplasma gondii mitogen-activated protein kinase (MAPK).•It affects parasite proliferation in an IFN-γ, iNOS and MKK3-dependent manner.•Parasite tissue burden is regulated by TgMAPK1 ...expression in iNOS-replete tissues.•Thus TgMAPK1 ultimately affects virulence by manipulating host IFN-γ-mediated iNOS.
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 (TgMAPK1lo) 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. TgMAPK1lo infection in wild type mice produced ⩾ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1con). 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.
Abstract
Interleukin (IL)-12 is a proinflammatory cytokine generally regarded as a master regulator of type 1 T cell mediated immunity against intracellular pathogens, including T. gondii and ...cancers. A critical measure of IL-12 activity is its ability to induce IFN-γ production by NK and T cells. Using a transgenic T. gondii expressing ovalbumin (OVA), we show here that by flow cytometry analysis T. gondii-OVA infected BL6 IL-12-/- mice generate OVA-pentamer+CD8+ T cells similar to WT and surprisingly, T. gondii-infected IL-12-/- mice produce more IFN-γ +CD8+ T cells than T. gondii-infected WT mice (avg 9.6% in IL-12-/- vs 3.7% in WT). We confirmed that function of OVA-specific CD8+ T cells in IL-12-/- was comparable to BL6 WT using an in vivo OVA+ target lysis assay. As expected, IFN-γ +CD4+ T cells decreased from an avg 29% to 9% in WT mice compared to IL-12-/- mice respectively when infected with T. gondii. These results demonstrate a novel IL-12-independent pathway to CD8+ CTL that may be less dependent of CD4+ T cell help than previously thought.
Abstract
Toxoplasma gondii is a medically important, obligate intracellular protozoan parasite that causes significant morbidity and mortality in hosts with defective cellular immunity. Using ...antisense RNA knockdown, we generated T. gondii tachyzoites expressing 5 - 10-fold less TgMAPK1, a stress-response mitogen-activated protein kinase. In infection, T. gondii converts from the rapidly-dividing tachyzoite to the latent bradyzoite form. We show here that T. gondii tachyzoites with reduced TgMAPK1 display distinctive bradyzoite characteristics, including expressing Bag1 (a bradyzoite-specific antigen) and significantly reducing Sag1 (a tachyzoite-specific antigen). Although knockdown (KD) tachyzoites were dramatically less sensitive to the anti-proliferative effects of IFN-γ than wildtype (WT), these parasites were nonetheless significantly less virulent than WT in BL6 mice, based on: (i) KD parasites achieved only a fraction of the parasite burden in tissues of infected mice relative to control parasites, (ii) mice infected with KD produced substantially less IFN-γ, and (iii) the majority of mice challenged with KD were able to survive lethal challenge. These results show that TgMAPK1 is an important virulence determinant affecting stage conversion, proliferation, and replication. This work was supported by funds from NIH AI060424 and Johnson & Johnson Focused Giving.
Abstract
Treg depletion improves anti-tumor immunity but is unlikely to be curative alone. Denileukin diftitox (DT) depletes Tregs in BL6 mice and prolongs survival 11% at 5 µg/mouse weekly starting ...2 weeks after challenge with ID8 ovarian cancer. Weekly human interferon (hIFN)-α 20,000 U/day on 4 consecutive days of 7 plus weekly DT survival improved 26% vs DT alone. Neither DT nor DT+hIFN-α improved survival in RAG1-/- mice with ID8, showing a requirement for adaptive immunity. hIFN-α alone reduced in vitro Treg suppression ~15% vs PBS, but did not clearly enhance DT-mediated Treg depletion, or increase T cell activation by CD69 expression. A patient in our clinical trial of DT for ovarian cancer failed, with CA-125 increasing 4.5-fold after 4 monthly cycles. Addition of weekly pegylated IFN-α2a to failed monthly DT improved blood Treg depletion 50% over DT alone and reduced CA-125 by 9-fold, demonstrating immunologic and clinical efficacy. To provide additional mechanistic insight we studied dendritic cell (DC) activation in hIFN-α treatment. In mice with ID8 tumor, hIFN-α significantly increased CD86 expression on myeloid and plasmacytoid DC. IFN-α may thus have an expanded role in treatment of epithelial carcinomas when used with Treg depletion, through enhanced Treg depletion or DC activation among other mechanisms.
Supported by FD003118, the Fanny Rippel Foundation, the Voelcker Trust and the Greehey Fund
Abstract
We tested Treg function in B7-H1-/- (KO) mice. 6-10 wk male (M) or female (F) KO and M or F wildtype (WT) had equal numbers of CD4+CD25hiFoxP3+ T cells (Tregs) with similar CD25 and Foxp3 ...MFI. CTLA-4 trended lower in F vs M KO. M and F WT and M KO Tregs suppressed T cell proliferation equally in vitro but F KO Tregs were ~30% less suppressive vs the other 3 groups. Transfer of M KO Tregs fully protected RAG1-/- mice from CD4+RBhi T cell colitis but F KO Tregs did not, with up to 30% weight loss demonstrating poor in vivo Treg function. In M or F WT or KO with subcutaneous B16 melanoma, tumor was different at day 15 in F (159 mm3) vs M (458 mm3, p = 0.04) and F KO vs F WT (593 mm3, p = 0.01). We detected OVA-specific immunity in 0/3 M KO bearing OVA+ B16 vs 3/3 in F KO based on in vivo OT-I cell proliferation. Tregs in pre-pubertal 3-wk F KO had similar phenotype and function vs post-pubertal WT and M KO Tregs. Culture with TGF-β +IL-2 converted 6-wk CD4+CD25- F KO T cells into CD4+CD25hiFoxP3+ T cells with suppression similar to WT, but lower CD25. Thus, F KO T cells can become functional Tregs ex vivo. This previously unknown sex/T cell co-signaling interaction could explain sex-related differences in cancer, autoimmune disease, infection susceptibility and response to hormone antagonists. We hypothesize Treg dysfunction to improve immunity and tumor resistance in KO and estrogen as a B7-H1 co-factor in Treg development. CTLA-4 defects remain undefined.
Supported by CA105207, FD003118, and Voelcker and Greehey Trusts
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