Role of Cytokines in Thymus- Versus Peripherally Derived-Regulatory T Cell Differentiation and Function

International audienceCD4 + CD25 + Foxp3 + regulatoryT cells (Tregs) are essential players in the control of immune responses. Recently, accordingly to their origin, two main subsets of Tregs have been described: thymus-derived Tregs (tTregs) and peripherally derived Tregs (pTregs). Numerous signaling pathways including the IL-2/STAT5 or theTGF-β/Smad3 pathways play a crucial role in segregating the two lineages. Here, we review some of the information existing on the distinct requirements of IL-2, TGF-β, and TNF-α three major cytokines involved in tTreg and pTreg generation, homeostasis and function. Today it is clear that signaling via the IL-2Rβ chain (CD122) common to IL-2 and IL-15 is required for proper differentiation of tTregs and for tTreg and pTreg survival in the periphery. This notion has led to the development of promising therapeutic strategies based on low-dose IL-2 administration to boost the patients' own Treg compartment and dampen autoimmunity and inflammation. Also, solid evidence points to TGF-β as the master regulator of pTreg differentiation and homeostasis. However, therapeutic administration of TGF-β is difficult to implement due to toxicity and safety issues. Knowledge on the role of TNF-α on the biology of Tregs is fragmentary and inconsistent between mice and humans. Moreover, emerging results from the clinical use of TNF-α inhibitors indicate that part of their anti-inflammatory effect may be dependent on their action on Tregs. Given the profusion of clinical trials testing cytokine administration or blocking to modulate inflammatory diseases, a better knowledge of the effects of cytokines on tTregs and pTregs biology is necessary to improve the efficiency of these immunotherapies

Inhibition of the JAK/STAT Signaling Pathway in Regulatory T Cells Reveals a Very Dynamic Regulation of Foxp3 Expression

The IL-2/JAK3/STAT-5 signaling pathway is involved on the initiation and maintenance of the transcription factor Foxp3 in regulatory T cells (Treg) and has been associated with demethylation of the intronic Conserved Non Coding Sequence-2 (CNS2). However, the role of the JAK/STAT pathway in controlling Foxp3 in the short term has been poorly investigated. Using two different JAK/STAT pharmacological inhibitors, we observed a detectable loss of Foxp3 after 10 min. of treatment that affected 70% of the cells after one hour. Using cycloheximide, a general inhibitor of mRNA translation, we determined that Foxp3, but not CD25, has a high turnover in IL-2 stimulated Treg. This reduction was correlated with a rapid reduction of Foxp3 mRNA. This loss of Foxp3 was associated with a loss in STAT-5 binding to the CNS2, which however remains demethylated. Consequently, Foxp3 expression returns to normal level upon restoration of basal JAK/STAT signaling in vivo. Reduced expression of several genes defining Treg identity was also observed upon treatment. Thus, our results demonstrate that Foxp3 has a rapid turn over in Treg partly controlled at the transcriptional level by the JAK/STAT pathway

Systemic IL-2/anti-IL-2Ab complex combined with sublingual immunotherapy suppresses experimental food allergy in mice through induction of mucosal regulatory T cells

Therapeutic tolerance restoration has been proven to modify food allergy in patients and animal models and although sublingual immunotherapy (SLIT) has showed promise, combined therapy may be necessary to achieve a strong and long‐term tolerance. In this work, we combined SLIT with systemic administration of IL‐2 associated with an anti‐IL‐2 monoclonal antibody (IL‐2/anti‐IL‐2Ab complex or IL‐2C) to reverse the IgE‐mediated experimental allergy. Balb/c mice were sensitized with cholera toxin and milk proteins and orally challenged with allergen to elicit hypersensitivity reactions. Then, allergic mice were treated with a sublingual administration of very low amounts of milk proteins combined with intraperitoneal injection of low doses of IL‐2C. The animals were next re‐exposed to allergens and mucosal as well as systemic immunological parameters were assessed in vivo and in vitro. The treatment reduced serum specific IgE, IL‐5 secretion by spleen cells and increased IL‐10 and TGF‐β in the lamina propria of buccal and duodenal mucosa. We found an augmented frequency of IL‐10‐secreting CD4+CD25+Foxp3+ regulatory T cells (Treg) in the submaxilar lymph nodes and buccal lamina propria. Tregs were sorted, characterized and adoptively transferred to naïve mice, which were subsequently sensitized. No allergy was experienced in these mice and we encouragingly discovered a faster and more efficient tolerance induction with the combined therapy compared with SLIT. The combination of two therapeutic strategies rendered Treg‐mediated tolerance more efficient compared to individual treatments and reversed the established IgE‐mediated food allergy. This approach highlights the ability of IL‐2C to expand Tregs, and it may represent a promising disease‐modifying therapy for managing food allergyInstituto de Estudios Inmunológicos y FisiopatológicosConsejo Nacional de Investigaciones Científicas y Técnica

Systemic IL-2/anti-IL-2Ab complex combined with sublingual immunotherapy suppresses experimental food allergy in mice through induction of mucosal regulatory T cells

Therapeutic tolerance restoration has been proven to modify food allergy in patients and animal models and although sublingual immunotherapy (SLIT) has showed promise, combined therapy may be necessary to achieve a strong and long‐term tolerance. In this work, we combined SLIT with systemic administration of IL‐2 associated with an anti‐IL‐2 monoclonal antibody (IL‐2/anti‐IL‐2Ab complex or IL‐2C) to reverse the IgE‐mediated experimental allergy. Balb/c mice were sensitized with cholera toxin and milk proteins and orally challenged with allergen to elicit hypersensitivity reactions. Then, allergic mice were treated with a sublingual administration of very low amounts of milk proteins combined with intraperitoneal injection of low doses of IL‐2C. The animals were next re‐exposed to allergens and mucosal as well as systemic immunological parameters were assessed in vivo and in vitro. The treatment reduced serum specific IgE, IL‐5 secretion by spleen cells and increased IL‐10 and TGF‐β in the lamina propria of buccal and duodenal mucosa. We found an augmented frequency of IL‐10‐secreting CD4+CD25+Foxp3+ regulatory T cells (Treg) in the submaxilar lymph nodes and buccal lamina propria. Tregs were sorted, characterized and adoptively transferred to naïve mice, which were subsequently sensitized. No allergy was experienced in these mice and we encouragingly discovered a faster and more efficient tolerance induction with the combined therapy compared with SLIT. The combination of two therapeutic strategies rendered Treg‐mediated tolerance more efficient compared to individual treatments and reversed the established IgE‐mediated food allergy. This approach highlights the ability of IL‐2C to expand Tregs, and it may represent a promising disease‐modifying therapy for managing food allergyInstituto de Estudios Inmunológicos y FisiopatológicosConsejo Nacional de Investigaciones Científicas y Técnica

TLR3 Activation of Intratumoral CD103+ Dendritic Cells Modifies the Tumor Infiltrate Conferring Anti-tumor Immunity

An important challenge in cancer immunotherapy is to expand the number of patients that benefit from immune checkpoint inhibitors (CI), a fact that has been related to the pre-existence of an efficient anti-tumor immune response. Different strategies are being proposed to promote tumor immunity and to be used in combined therapies with CI. Recently, we reported that intratumoral administration of naked poly A:U, a dsRNA mimetic empirically used in early clinical trials with some success, delays tumor growth and prolongs mice survival in several murine cancer models. Here, we show that CD103+ cDC1 and, to a much lesser extent CD11b+ cDC2, are the only populations expressing TLR3 at the tumor site, and consequently could be potential targets of poly A:U. Upon poly A:U administration these cells become activated and elicit profound changes in the composition of the tumor immune infiltrate, switching the immune suppressive tumor environment to anti-tumor immunity. The sole administration of naked poly A:U promotes striking changes within the lymphoid compartment, with all the anti-tumoral parameters being enhanced: a higher frequency of CD8+ Granzyme B+ T cells, (lower Treg/CD8+ ratio) and an important expansion of tumor-antigen specific CD8+ T cells. Also, PD1/PDL1 showed an increased expression indicating that neutralization of this axis could be exploited in combination with poly A:U. Our results shed new light to promote further assays in this dsRNA mimetic to the clinical field

Aberrant fucosylation enables breast cancer clusterin to interact with dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN)

Clusterin is a glycoprotein able to mediate different physiological functions such as control of complement activation, promotion of unfolded protein clearance and modulation of cell survival. Clusterin is overexpressed in many types of cancers and a large body of evidence suggests that it promotes carcinogenesis and tumor progression. We have previously described a novel clusterin glycoform present in human semen, but not in serum, highly enriched in terminal fucose motifs. Here we show that human luminal breast cancer (LBC) clusterin also bears terminal fucosylated glycans, conferring clusterin the ability to interact with DC-SIGN, a C-type lectin receptor expressed by myeloid cells. This clusterin glycosylation pattern was absent or diminished in non-involved juxtatumoral tissue, suggesting that fucosylated clusterin might represent a cancer associated glycoform. We also found that DC-SIGN is expressed by luminal breast cancer intratumoral macrophages. Moreover, experiments performed in vitro using semen fucosylated clusterin and monocyte derived macrophages showed that the interaction of semen clusterin with DC-SIGN promoted a proangiogenic profile, characterized by a high production of VEGF, IL-8 and TNF-α. Our results reveal an unexpected complexity on the structure and function of secretory clusterin produced by tumors and suggest that fucosylated clusterin produced by luminal breast cancer cells might play a role in tumor progression by promoting the release of pro-angiogenic factors by intratumoral macrophages.Fil: Merlotti Ippólito, Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina. Institute Curie; FranciaFil: López Malizia, Álvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Michea, Paula. Institute Curie; FranciaFil: Bonte, Pierre Emmanuel. Institute Curie; FranciaFil: Goudot, Christel. Immunite Et Cancer; FranciaFil: Carregal, María Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Nuñez, Nicolás. Immunite Et Cancer; FranciaFil: Sedlik, Christine. Institute Curie; FranciaFil: Ceballos, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Soumelis, Vassili. Institute Curie; FranciaFil: Amigorena, Sebastián. Institute Curie; FranciaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Piaggio, Eliane. Institute Curie; FranciaFil: Sabatte, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin

Blockade of Stat3 oncogene addiction induces cellular senescence and reveals a cell-nonautonomous activity suitable for cancer immunotherapy

Stat3 is constitutively activated in several tumor types and plays an essential role in maintaining their malignant phenotype and immunosupression. To take advantage of the promising antitumor activity of Stat3 targeting, it is vital to understand the mechanism by which Stat3 regulates both cell autonomous and non-autonomous processes. Here, we demonstrated that turning off Stat3 constitutive activation in different cancer cell types induces senescence, thus revealing their Stat3 addiction. Taking advantage of the senescence-associated secretory phenotype (SASP) induced by Stat3 silencing (SASP-siStat3), we designed an immunotherapy. The administration of SASP-siStat3 immunotherapy induced a strong inhibition of triplenegative breast cancer and melanoma growth associated with activation of CD4 + T and NK cells. Combining this immunotherapy with anti-PD-1 antibody resulted in survival improvement in mice bearing melanoma. The characterization of the SASP components revealed that type I IFN-related mediators, triggered by the activation of the cyclic GMP-AMP synthase DNA sensing pathway, are important for its immunosurveillance activity. Overall, our findings provided evidence that administration of SASP-siStat3 or low dose of Stat3- blocking agents would benefit patients with Stat3-addicted tumors to unleash an antitumor immune response and to improve the effectiveness of immune checkpoint inhibitors.Fil: de Martino, Mara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Tkach, Mercedes. Institute Curie; FranciaFil: Bruni, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rocha, Darío Gastón. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; ArgentinaFil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cenciarini, Mauro Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Chervo, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Proietti Anastasi, Cecilia Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Dingli, Florent. Institute Curie; FranciaFil: Loewy, Ruth Miriam. Institute Curie; FranciaFil: Fernández, Elmer A.. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas. Universidad Católica de Córdoba. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas; ArgentinaFil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Piaggio, Eliane. Institute Curie; FranciaFil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Treg-targeted IL-2/anti-IL-2 complex controls graft-<i>versus</i>-host disease and supports anti-tumor effect in allogeneic hematopoietic stem cell transplantation

Modulating an immune response in opposite directions represents the holy grail in allogeneic hematopoietic stem cell transplantation (allo-HSCT) to avoid insufficient reactivity of donor T cells and hematologic malignancy relapse while controlling the potential development of graft-versus-host disease (GVHD), in which donor T cells attack the recipient’s tissues. IL-2/anti-IL-2 complexes (IL-2Cx) represent a therapeutic option to selectively accentuate or dampen the immune response. In dedicated experimental models of allo-HSCT, including also human cells injected in immunodeficient NSG mice, we evaluated side-by-side the therapeutic effect of two IL-2Cx designed either to boost regulatory T cells (Treg) or alternatively to activate effector T cells (Teff), on GVHD occurrence and tumor relapse. We also evaluated the effect of the complexes on the phenotype and function of immune cells in vivo. Unexpectedly, both pro-Treg and pro-Teff IL-2Cx prevented GVHD development. They both induced Treg expansion and reduced CD8+ T-cell numbers, compared to untreated mice. However, only mice treated with the pro-Treg IL-2Cx, showed a dramatic reduction of exhausted CD8+ T cells, consistent with a potent anti-tumor effect. When evaluated on human cells, pro-Treg IL-2Cx also preferentially induced Treg expansion in vitro and in vivo, while allowing the development of a potent anti-tumor effect in NSG mice. Our results demonstrate the clinical relevance of using a pro-Treg, but not a pro-Teff IL2Cx to modulate alloreactivity after HSCT, while promoting a graft-versus-leukemia effect

Imaging and multi-omics datasets converge to define different neural progenitor origins for ATRT-SHH subgroups

Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-Cre$^{ERT2}$::Smarcb1$^{flox/flox}$ model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway