SHP-1 Regulates Antigen Cross-Presentation and Is Exploited by Leishmania to Evade Immunity.

Intracellular pathogens have evolved strategies to evade detection by cytotoxic CD8+ T lymphocytes (CTLs). Here, we ask whether Leishmania parasites trigger the SHP-1-FcRγ chain inhibitory axis to dampen antigen cross-presentation in dendritic cells expressing the C-type lectin receptor Mincle. We find increased cross-priming of CTLs in Leishmania-infected mice deficient for Mincle or with a selective loss of SHP-1 in CD11c+ cells. The latter also shows improved cross-presentation of cell-associated viral antigens. CTL activation in vitro reveals increased MHC class I-peptide complex expression in Mincle- or SHP-1-deficient CD11c+ cells. Neuraminidase treatment also boosts cross-presentation, suggesting that Leishmania triggers SHP-1-associated sialic-acid-binding receptors. Mechanistically, enhanced antigen processing correlates with reduced endosomal acidification in the absence of SHP-1. Finally, we demonstrate that SHP-1 inhibition improves CD11c+ cell-based vaccination against the parasite. Thus, SHP-1-mediated impairment of cross-presentation can be exploited by pathogens to evade CTLs, and SHP-1 inhibition improves CTL responses during vaccination.Work in the S.I. laboratory is funded by the Spanish Ministerio de Ciencia e Innovación (MICINN), Agencia Estatal de Investigación, and Fondo Europeo de Desarrollo Regional (RTI2018-094484-B-I00 and RYC-2016-19463). S.C.K. is a recipient of a FPU fellowship (FPU16/03142) from the Spanish Ministry of Education, Culture and Sports. M.M.-L. is a recipient of an EMBO Long-Term Fellowship (EMBO LTF 463-2019).S

Effective cancer immunotherapy by natural mouse conventional type-1 dendritic cells bearing dead tumor antigen

BACKGROUND: The manipulation of dendritic cells (DCs) for cancer vaccination has not reached its full potential, despite the revolution in cancer immunotherapy. DCs are fundamental for CD8+ T cell activation, which relies on cross-presentation of exogenous antigen on MHC-I and can be fostered by immunogenic cancer cell death. Translational and clinical research has focused on in vitro-generated monocyte-derived DCs, while the vaccination efficacy of natural conventional type 1 DCs (cDC1s), which are associated with improved anti-tumor immunity and specialize on antigen cross-presentation, remains unknown. METHODS: We isolated primary spleen mouse cDC1s and established a protocol for fast ex vivo activation and antigen-loading with lysates of tumor cells that underwent immunogenic cell death by UV irradiation. Natural tumor antigen-loaded cDC1s were transferred and their potential for induction of endogenous CD8+ and CD4+ T cell responses in vivo, cancer prevention and therapy were assessed in three grafted cancer models. Further, we tested the efficacy of natural cDC1 vaccination in combination and comparison with anti-PD-1 treatment in two "wildtype" tumor models not expressing exogenous antigens. RESULTS: Herein, we reveal that primary mouse cDC1s ex vivo loaded with dead tumor cell-derived antigen are activated and induce strong CD8+ T cell responses from the endogenous repertoire upon adoptive transfer in vivo through tumor antigen cross-presentation. Notably, cDC1-based vaccines enhance tumor infiltration by cancer-reactive CD8+ and CD4+ T cells and halt progression of engrafted cancer models, including tumors that are refractory to anti-PD-1 treatment. Moreover, combined tumor antigen-loaded primary cDC1 and anti-PD-1 therapy had strong synergistic effects in a PD-1 checkpoint inhibition susceptible cancer model. CONCLUSIONS: This preclinical proof-of-principle study is first to support the therapeutic efficacy of cancer immunotherapy with syngeneic dead tumor cell antigen-loaded mouse cDC1s, the equivalents of the human dendritic cell subset that correlates with beneficial prognosis of cancer patients. Our data pave the way for translation of cDC1-based cancer treatments into the clinic when isolation of natural human cDC1s becomes feasible.Work in the DS laboratory is funded by the CNIC and grant SAF2016–79040-R from Ministerio de Ciencia, Innovación e Universidades (MCIU), Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (FEDER); B2017/BMD-3733 Immunothercan-CM from Comunidad de Madrid; RD16/0015/0018-REEM from FIS-Instituto de Salud Carlos III, MICINN and FEDER; Acteria Foundation; Constantes y Vitales prize (Atresmedia); La Marató de TV3 Foundation (201723); and the European Research Council (ERC-2016-Consolidator Grant 725091). Work at the IM laboratory is funded by grants from MCIU (SAF2014–52361-R and SAF2017–83267-C2–1-R) and by European Commission VII Framework and Horizon 2020 programs (AICR), Fundación de la Asociación Española Contra el Cáncer (AECC), and Fundación BBVA. SKW is supported by a European Molecular Biology Organization Long-term Fellowship (grant ALTF 438–2016) and a CNIC-International Postdoctoral Program Fellowship (grant 17230–2016). SCK is a recipient of a FPU fellowship (FPU16/03142) from the Spanish Ministry of Education, Culture and Sports. IM and DS labs are funded by the European Commission (635122-PROCROP H2020). The CNIC is supported by the MCIU and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). AGRADECIENTOS: ProCNIC; Severo Ochoa (SEV-2015-0505)S

Lung CD103+ dendritic cells restrain allergic airway inflammation through IL-12 production

DCs are necessary and sufficient for induction of allergic airway inflammation. CD11b+ DCs direct the underlying Th2 immunity, but debate surrounds the function of CD103+ DCs in lung immunity and asthma after an allergic challenge. We challenged Batf3-/- mice, which lacked lung CD103+ DCs, with the relevant allergen house dust mite (HDM) as a model to ascertain their role in asthma. We show that acute and chronic HDM exposure leads to defective Th1 immunity in Batf3-deficient mice. In addition, chronic HDM challenge in Batf3-/- mice results in increased Th2 and Th17 immune responses and exacerbated airway inflammation. Mechanistically, Batf3 absence does not affect induction of Treg or IL-10 production by lung CD4+ T cells following acute HDM challenge. Batf3-dependent CD103+ migratory DCs are the main source of IL-12p40 in the mediastinal lymph node DC compartment in the steady state. Moreover, CD103+ DCs selectively increase their IL-12p40 production upon HDM administration. In vivo IL-12 treatment reverts exacerbated allergic airway inflammation upon chronic HDM challenge in Batf3-/- mice, restraining Th2 and Th17 responses without triggering Th1 immunity. These results suggest a protective role for lung CD103+ DCs to HDM allergic airway inflammation through the production of IL-12.We are grateful to members of the DS lab for discussions and critical reading of the manuscript. We thank the staff at the CNIC facilities for technical support, Jesus Sanchez-Ruiz for the graphical illustration, Ruth Conde-Garrosa for technical assistance, Antonio Molina-Iracheta for histological quantification and analysis, and Simon Bartlett for editorial assistance. LC is the recipient of a European Respiratory Society Fellowship (RESPIRE2-2013-3708). Work in the DS laboratory is funded by the CNIC and grants from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) and European Fund for Regional Development (FEDER) (SAF-2013-42920-R and SAF-2016-79040-R); the European Commission (635122-PROCROP H2020); and the Fondation ACTERIA. The CNIC is supported by the MEIC and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Cell death induced by cytotoxic CD8+ T cells is immunogenic and primes caspase-3–dependent spread immunity against endogenous tumor antigens

BackgroundElimination of cancer cells by some stimuli like chemotherapy and radiotherapy activates anticancer immunity after the generation of damage‐associated molecular patterns, a process recently named immunogenic cell death (ICD). Despite the recent advances in cancer immunotherapy, very little is known about the immunological consequences of cell death activated by cytotoxic CD8+ T (Tc) cells on cancer cells, that is, if Tc cells induce ICD on cancer cells and the molecular mechanisms involved.MethodsICD induced by Tc cells on EL4 cells was analyzed in tumor by vaccinating mice with EL4 cells killed in vitro or in vivo by Ag-specific Tc cells. EL4 cells and mutants thereof overexpressing Bcl-XL or a dominant negative mutant of caspase-3 and wild-type mice, as well as mice depleted of Tc cells and mice deficient in perforin, TLR4 and BATF3 were used. Ex vivo cytotoxicity of spleen cells from immunized mice was analyzed by flow cytometry. Expression of ICD signals (calreticulin, HMGB1 and interleukin (IL)-1β) was analyzed by flow cytometry and ELISA.ResultsMice immunized with EL4.gp33 cells killed in vitro or in vivo by gp33-specific Tc cells were protected from parental EL4 tumor development. This result was confirmed in vivo by using ovalbumin (OVA) as another surrogate antigen. Perforin and TLR4 and BATF3-dependent type 1 conventional dendritic cells (cDC1s) were required for protection against tumor development, indicating cross-priming of Tc cells against endogenous EL4 tumor antigens. Tc cells induced ICD signals in EL4 cells. Notably, ICD of EL4 cells was dependent on caspase-3 activity, with reduced antitumor immunity generated by caspase-3–deficient EL4 cells. In contrast, overexpression of Bcl-XL in EL4 cells had no effect on induction of Tc cell antitumor response and protection.ConclusionsElimination of tumor cells by Ag-specific Tc cells is immunogenic and protects against tumor development by generating new Tc cells against EL4 endogenous antigens. This finding helps to explain the enhanced efficacy of T cell-dependent immunotherapy and provide a molecular basis to explain the epitope spread phenomenon observed during vaccination and chimeric antigen receptor (CAR)-T cell therapy. In addition, they suggest that caspase-3 activity in the tumor may be used as a biomarker to predict cancer recurrence during T cell-dependent immunotherapies