IL-17RA-signaling modulates CD8+ T Cell survival and exhaustion during trypanosoma cruzi infection

The IL-17 family contributes to host defense against many intracellular pathogens by mechanisms that are not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes, and their survival and ability to mount cytotoxic responses are orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. The absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells, while in vitro recombinant IL-17 down-regulated the pro-Apoptotic protein BAD and promoted the survival of activated CD8+ T cells. Phenotypic, functional, and transcriptomic profiling showed that T. cruzi-specific CD8+ T cells derived from IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi.Fil: Tosello Boari, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Araujo Furlan, Cintia Liliana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Fiocca Vernengo, Facundo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Rodriguez, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Ramello, María Cecilia. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Amezcua Vesely, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Gorosito Serran, Melisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Nuñez, Nicolás G.. Institute Curie; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Richer, Wilfrid. Institut National de la Santé et de la Recherche Médicale; Francia. Institute Curie; FranciaFil: Piaggio, Eliane. Institut National de la Santé et de la Recherche Médicale; Francia. Institute Curie; FranciaFil: Montes, Carolina Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Gruppi, Adriana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Acosta Rodriguez, Eva Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

IL-17RA Signaling Reduces Inflammation and Mortality during Trypanosoma cruzi Infection by Recruiting Suppressive IL-10-Producing Neutrophils

Members of the IL-17 cytokine family play an important role in protection against pathogens through the induction of different effector mechanisms. We determined that IL-17A, IL-17E and IL-17F are produced during the acute phase of T. cruzi infection. Using IL-17RA knockout (KO) mice, we demonstrate that IL-17RA, the common receptor subunit for many IL-17 family members, is required for host resistance during T. cruzi infection. Furthermore, infected IL-17RA KO mice that lack of response to several IL-17 cytokines showed amplified inflammatory responses with exuberant IFN-γ and TNF production that promoted hepatic damage and mortality. Absence of IL-17RA during T. cruzi infection resulted in reduced CXCL1 and CXCL2 expression in spleen and liver and limited neutrophil recruitment. T. cruzi-stimulated neutrophils secreted IL-10 and showed an IL-10-dependent suppressive phenotype in vitro inhibiting T-cell proliferation and IFN-γ production. Specific depletion of Ly-6G+ neutrophils in vivo during T. cruzi infection raised parasitemia and serum IFN-γ concentration and resulted in increased liver pathology in WT mice and overwhelming wasting disease in IL-17RA KO mice. Adoptively transferred neutrophils were unable to migrate to tissues and to restore resistant phenotype in infected IL-17RA KO mice but migrated to spleen and liver of infected WT mice and downregulated IFN-γ production and increased survival in an IL-10 dependent manner. Our results underscore the role of IL-17RA in the modulation of IFN-γ-mediated inflammatory responses during infections and uncover a previously unrecognized regulatory mechanism that involves the IL-17RA-mediated recruitment of suppressive IL-10-producing neutrophils

Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors

Background Neuroendocrine tumors (NETs) overexpress somatostatin receptors (SSTRs). Methods We developed a second-generation, ligand-based, anti-SSTR chimeric antigen receptor (CAR) incorporating the somatostatin analog octreotide in its extracellular moiety. Results Anti-SSTR CAR T cells exerted antitumor activity against SSTR+NET cell linesin vitro. The killing activity was highly specific, as demonstrated by the lack of CAR T cell reactivity against NET cells engineered to express mutated variants of SSTR2/5 by CRISPR/Cas9. When adoptively transferred in NSG mice, anti-SSTR CAR T cells induced significant antitumor activity against human NET xenografts. Although anti-SSTR CAR T cells could recognize the murine SSTRs as shown by their killing ability against murine NET cells, no obvious deleterious effects on SSTR-expressing organs such as the brain or the pancreas were observed in mice. Conclusions Taken together, our results establish anti-SSTR CAR T cells as a potential candidate for early phase clinical investigations in patients with NETs. More broadly, the demonstration that a known peptide drug can direct CAR T cell targeting may streamline the potential utility of multiple peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers

Reduced neutrophil chemoattractant production and neutrophil recruitment in the spleen and liver of <i>T. cruzi</i> infected IL-17RA KO mice.

<p>A) Cell numbers in spleen, lymph nodes and liver of WT and IL-17RA KO mice determined at different times after <i>T. cruzi</i> infection. Data are shown as mean ± SD, n = 5 mice per group. P values calculated using two-way ANOVA followed by Bonferroni's posttest. B) Percentage of CD11b+ Gr-1+ neutrophils in spleen and liver of 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice. Plots are representative one out of five mice. C) Absolute numbers of CD11b+ Gr-1+ neutrophils in spleen (left) and liver (right) of 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice. Each symbol represents a different mouse and horizontal line indicates the mean. P values calculated with two-tailed T test. D) Concentration of CXCL1, CXCL2 and CXCL10 in spleen and liver homogenates obtained from WT and IL-17RA KO mice at different times post <i>T. cruzi</i> infection. Data are shown as mean ± SD of biological triplicates, normalized to total protein concentration, n = 5 mice per group. P values calculated with two-way ANOVA followed by Bonferroni's posttest. (* p: spleen WT vs spleen KO; # p: liver WT vs liver KO). E) Absolute number or frequency of transferred WT and IL-17RA KO neutrophils detected in bone marrow, blood, spleen and liver of non-infected (NI) and infected (I) WT and IL-17RA KO mice 3 h after i.v. injection. Data are shown as mean ± SD, n = 5 per group. P values calculated with two-tailed T test. Data in A–C and D–E are representative of four and two independent experiments, respectively.</p

Hepatic lesions in anti-IFN-γ treated <i>T. cruzi</i> infected WT and IL-17RA KO mice at day 20 post-infection.

a<p>Scale: +: mild; ++:moderate; +++: severe; ++++: extremely severe.</p>b<p>PMN: Polymorphonuclear cells; Mo/Ly: monocyte/lymphocytes.</p

Increased IFN-γ production and hepatic transaminases activity in <i>T. cruzi</i> infected WT and IL-17RA KO mice after neutrophil depletion.

<p>A) CD11b+Gr-1+ frequency and absolute numbers in blood, spleen and liver of 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-Ly6G mAbs. Data are shown as mean ± SD, n = 6–8 mice per group. P values calculated by two-tailed T test. B) Concentration of IL-10 determined in 48 h unstimulated culture supernatants of spleen and liver cell suspensions obtained from 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-Ly-6G mAbs. Each symbol represents a different mouse and horizontal line indicates the mean. P values calculated with two-tailed T test. C) Plasma IFN-γ and TNF concentration in 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-Ly-6G mAbs. Each symbol represents a different mouse and horizontal line indicates the mean. P values calculated with two-tailed T test. D) Activity of ALT and AST determined in the plasma of 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-Ly-6G. Data are shown as mean ± SD, n = 6–8 mice per group. P values calculated using two-tailed T test. E) Parasitemia determined in 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-Ly-6G Abs. Data are shown as mean ± SD, n = 6–8 mice per group. P values calculated using two-tailed T test. F) Survival of <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-Ly-6G. P value calculated with a Gehan-Breslow-Wilcoxon test, n = 12 per group. Data in A–D and in E are representative of three and two independent experiments, respectively.</p

IL-10-dependent modulation of IFN-γ production by adoptively transferred neutrophils during <i>T. cruzi</i> infection.

<p>A) Parasitemia determined at day 20 post-infection in infected WT and IL-17RA KO mice adoptively transferred with WT neutrophils. Data are shown as mean ± SD, n = 6 per group. P values calculated with two-tailed T test. B) Concentration of IFN-γ in plasma of 20-day <i>T. cruzi</i> infected WT and IL-17R KO mice adoptively transferred with WT neutrophils. Data are shown as mean ± SD, n = 6 per group. P values calculated with two-tailed T test. C) Survival of <i>T. cruzi</i> infected WT and IL-17R KO mice adoptively transferred with WT neutrophils. P value calculated with a Gehan-Breslow-Wilcoxon test, n = 12 per group. D) Parasitemia determined at day 20 postinfection in infected WT mice adoptively transferred with WT and IL-10 KO neutrophils. Data are shown as mean ± SD, n = 12 per group. P values calculated with two-tailed T test. E) Concentration of IFN-γ in plasma of 20-day <i>T. cruzi</i> infected WT mice adoptively transferred with WT and IL-10 KO neutrophils. Data are shown as mean ± SD, n = 12 per group. P values calculated with two-tailed T test. F) Survival of <i>T. cruzi</i> infected WT mice adoptively transferred with WT and IL-10 KO neutrophils. P value calculated with a Gehan-Breslow-Wilcoxon test, n = 12 per group. Data in A–F are representative of two independent experiments.</p

Augmented production of IFN-γ caused increased hepatic damage and mortality in <i>T. cruzi</i> infected IL-17RA KO mice.

<p>A) Plasma IFN-γ and TNF concentration in 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice. Data are shown as mean ± SD, n = 6 mice per group. P values calculated using two-tailed T test. B) Plasma IFN-γ concentration in 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-IFN-γ. P values calculated using two-tailed T test. C) Parasitemia determined in 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-IFN-γ. Data are shown as mean ± SD, n = 6 mice per group. P values calculated using two-tailed T test. D) Activity of ALT and AST determined in the plasma of 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-IFN-γ. Data are shown as mean ± SD, n = 6 mice per group. P values calculated using two-tailed T test. E) Survival of <i>T. cruzi</i> infected WT and IL-17RA KO mice treated with anti-IFN-γ. P value calculated with a Gehan-Breslow-Wilcoxon test, n = 12 per group. Data in A and in B–E are representative of four and two independent experiments, respectively.</p

Suppression of T cell proliferation and IFN-γ production by IL-10 secreting neutrophils obtained from <i>T. cruzi</i> infected mice.

<p>A–B) Concentration of IL-10 and TNF determined in 48 h culture supernatants of Ly-6G+ neutrophils purified from bone marrow of WT and IL-17RA KO mice (A) and of CD11b+Ly-6G+ neutrophils sorted from spleen 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice (B) and stimulated as indicated. Data are shown as mean ± SD of cuatriplicates. C) Proliferation and percentage of IFN-γ-producing CD3 positive splenocytes from normal mice after 5 day stimulation in anti-CD3 and anti-CD28 coated plates in the presence of CD11b+Ly-6G+ neutrophils obtained from 20-day <i>T. cruzi</i> infected WT and IL-17RA KO mice and of a blocking anti-IL10R Ab. Data are shown as mean ± SD of triplicates. P values calculated using two-tailed T test. Data in A–C are representative of two independent experiments.</p