Impacto de la deficiencia de C3 plasmático en la diferenciación y función leucocitaria

El papel del complemento en la inmunobiología ha evolucionado notablemente desde que fue descrito inicialmente como un elemento clave de la inmunidad innata. Lejos de su visión tradicional como un simple elemento termolábil que complementaba la eliminación de microbios mediada por anticuerpos, hoy día el sistema de complemento es considerado como un puente entre la inmunidad innata y adaptativa1. De las más de 50 proteínas que lo componen, la proteína C3 es el eje central en el que convergen sus tres vías de activación y por ello, tiene un fuerte impacto en la regulación de la biología leucocitaria, especialmente en linfocitos T, B y células dendríticas (DC). Los antígenos solubles opsonizados por fragmentos plasmáticos de C3d(g) se unen a través de su receptor CD21 e inducen el reclutamiento de CD19 y CD81 para formar el co-receptor de linfocitos B, reduciendo significativamente el umbral de activación del receptor de linfocitos B2. El receptor CD21, también conocido como receptor de complemento 2, es expresado por células dendríticas foliculares que capturan antígenos opsonizados con C3d(g) en los centros germinales de los órganos linfoides secundarios, favoreciendo la maduración de la afinidad, el cambio de isotipo y la adquisición de memoria de linfocitos B naíf o primados3. A pesar de que hace años ya se apuntó a una síntesis extra-hepática del complemento, con el tiempo han ido creciendo las evidencias que respaldan su síntesis local por diferentes tipos leucocitarios4. De hecho, los recientes hallazgos que exponen la existencia de un sistema de activación de C3 intracelular diferente al conocido plasmático con importantes consecuencias para la homeostasis y función de los linfocitos T ponen de manifiesto el alcance de la influencia de C3 en mamíferos5. En este contexto se ha visto que tanto los linfocitos T como las DC sintetizan fragmentos de C3 que actúan de forma autocrina y paracrina, favoreciendo la función de presentación antigénica de las DC e induciendo funciones efectoras en los linfocitos T6..

Intraepithelial paracrine Hedgehog signaling induces the expansion of ciliated cells that express diverse progenitor cell markers in the basal epithelium of the mouse mammary gland

The Hedgehog signaling pathway regulates embryo patterning and progenitor cell homeostasis in adult tissues, including epidermal appendages. A role for the Hh pathway in mammary biology and breast cancer has also been suggested. The aim of this study was to analyze Hh signaling in the mouse mammary gland through the generation of transgenic mice that express Sonic Hedgehog (Shh) under the control of the mammary-specific WAP promoter (WAP-Shh mice). To identify mammary cells capable of activating the Hh pathway we bred WAP-Shh mice to Ptch1-lacZ knock-in mice, in which the expression of a nuclear-targeted β-galactosidase reporter protein (β-gal) is driven by the endogenous Patched 1 gene regulatory region. After two cycles of induction of transgenic Shh expression, we detected areas of X-gal reactivity. Immunohistochemical analysis showed nuclear β-gal staining in clusters of mammary cells in WAP-Shh/Ptch1-lacZ bitransgenic mice. These were epithelial cells present in a basal location of displastic ducts and alveoli, adjacent to Shh-expressing luminal cells, and overexpressed epithelial basal markers keratin 5, 14 and 17 and transcription factor p63. Absence of smooth muscle actin expression and a cuboidal morphology differentiated Hh-responding cells from flat-shaped mature myoepithelial cells. Groups of cells expressing stem cell markers integrin β3 and keratins 6 and 15 were also detected within Hh-responding areas. In addition, we found that Hh-responding cells in the mammary glands of WAP-Shh/Ptch1-lacZ mice were ciliated and exhibited a low proliferation rate. Our data show the paracrine nature of hedgehog signaling in the epithelial compartment of the mouse mammary gland, where a subset of basal cells that express mammary progenitor cell markers and exhibit primary cilia is expanded in response to secretory epithelium-derived Shh.This work was supported by MCINN Grant no. SAF2006 03244, Fundación Marcelino Botín and Federación Española Cáncer de Mama (FECMA)

γδ T lymphocytes in the diagnosis of human T cell receptor immunodeficiencies

Supported by grants from MINECO (SAF 2011-24235, BES-2012-055054, SAF2014- 54708-R, and SAF2014-53563-REDT) CAM (S2010/BMD-2316) ISCIII (RD08- 0075-0002 and PI12/02761)Peer Reviewe

Efficient preclinical treatment of cortical T cell acute lymphoblastic leukemia with T lymphocytes secreting anti-CD1a T cell engagers

BACKGROUND: The dismal clinical outcome of relapsed/refractory (R/R) T cell acute lymphoblastic leukemia (T-ALL) highlights the need for innovative targeted therapies. Although chimeric antigen receptor (CAR)-engineered T cells have revolutionized the treatment of B cell malignancies, their clinical implementation in T-ALL is in its infancy. CD1a represents a safe target for cortical T-ALL (coT-ALL) patients, and fratricide-resistant CD1a-directed CAR T cells have been preclinically validated as an immunotherapeutic strategy for R/R coT-ALL. Nonetheless, T-ALL relapses are commonly very aggressive and hyperleukocytic, posing a challenge to recover sufficient non-leukemic effector T cells from leukapheresis in R/R T-ALL patients. METHODS: We carried out a comprehensive study using robust in vitro and in vivo assays comparing the efficacy of engineered T cells either expressing a second-generation CD1a-CAR or secreting CD1a x CD3 T cell-engaging Antibodies (CD1a-STAb). RESULTS: We show that CD1a-T cell engagers bind to cell surface expressed CD1a and CD3 and induce specific T cell activation. Recruitment of bystander T cells endows CD1a-STAbs with an enhanced in vitro cytotoxicity than CD1a-CAR T cells at lower effector:target ratios. CD1a-STAb T cells are as effective as CD1a-CAR T cells in cutting-edge in vivo T-ALL patient-derived xenograft models. CONCLUSIONS: Our data suggest that CD1a-STAb T cells could be an alternative to CD1a-CAR T cells in coT-ALL patients with aggressive and hyperleukocytic relapses with limited numbers of non-leukemic effector T cellsResearch in LA-V laboratory is funded by the Spanish Ministry of Science and Innovation (PID2020-117323RB-100 and PDC2021-121711-100), and the Carlos III Health Institute (DTS20/00089), with European Regional Development Fund (FEDER) cofinancing; the Spanish Association Against Cancer (AECC PROYE19084ALVA) and the CRIS Cancer Foundation (FCRIS-2018-0042 and FCRIS2021-0090). Research in PM laboratory is supported by CERCA/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for core support; 'la Caixa' Foundation under the agreement LCF/PR/HR19/52160011; the European Research Council grant (ERC-PoC-957466); the Spanish Ministry of Science and Innovation (PID2019-108160RB-I00); and the ISCIII-RICORS within the Next Generation EU program (plan de Recuperación, Transformación y Resilencia). MLT is supported by Spanish Ministry of Science and Innovation (PID2019-105623RB-I00) and the Spanish Association Against Cancer (CICPF18030TORI). PP is supported by Carlos III Health Institute (PI21-01834), with FEDER cofinancing and Fundación Ramón Areces. NT was supported by an FPU PhD fellowship from Spain's Ministerio de Universidades (FPU19/00039). OH was supported by an industrial PhD fellowship from the Comunidad de Madrid (IND2020/BMD-17668). LD-A was supported by a Rio Hortega fellowship from the Carlos III Health Institute (CM20/00004). VMD is supported by the Torres Quevedo subprogram of the State Research Agency of the Ministry of Science, Innovation and Universities (Ref. PTQ2020-011056). DSM is partially founded by a Sara Borrell fellowship from Carlos III Health Institute (CD19/00013

Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity

17 p.-4 fig.-1 tab.-1 grph. abst.Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcγR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition, with no indication of 4-1BB mAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo-Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy.Financial support for this work was obtained from the MCIN/AEI/10.13039/501100011033 (SAF2017-89437-P and PDC2021-121711-100 to LA-V, PID2019-104544GB-I00 to CA, and PID2020-113225GB-I00 to FJB), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (PI19/00132 to LS; PI20/01030 to BB), partially supported by the ERDF; the ISCIII-RICORS within the Next Generation EU program (plan de Recuperación, Transformación y Resilencia); the Spanish Association Against Cancer (AECC 19084 to LA-V); the CRIS Cancer Foundation (FCRIS-2018-0042 and FCRIS-2021-0090 to LA-V), the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 years COVID-19 to LA-V); and the Fundació “La Caixa” (HR21-00761 project IL7R_LungCan to LA-V). AD, OAM, and KAH were funded by the Novo Nordisk Foundation, Grant; CEMBID (Center for Multifunctional Biomolecular Drug Design, Grant Number: NNF17OC0028070). OH was supported by an industrial PhD fellowship from the Comunidad de Madrid (IND2020/BMD-17668). AE-L was supported industrial PhD fellowship from the Carlos III Health Institute (IFI18/00045). CD-A was supported by a predoctoral fellowship from the Spanish Ministry of Science Innovation and Universities (PRE2018-083445). LR-P was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck. LD-A was supported by a Rio Hortega fellowship from the Carlos III Health Institute (CM20/00004).Peer reviewe

Complement in basic processes of the cell

The complement system is reemerging in the last few years not only as key element of innate immunityagainst pathogens, but also as a main regulator of local adaptive responses, affecting dendritic cells as wellas T and B lymphocytes. We review data showing that leucocytes are capable of significant autocrine syn-thesis of complement proteins, and express a large range of complement receptors, which in turn regulatetheir differentiation and effector functions while cross talking with other innate receptors such as Toll-like receptors. Other unconventional roles of complement proteins are reviewed, including their impactin non-leukocytes and their intracellular cleavage by vesicular proteases, which generate critical cuesrequired for T cell function. Thus, leucocytes are very much aware of complement-derived information,both extracellular and intracellular, to elaborate their responses, offering rich avenues for therapeuticintervention and new hypothesis for conserved major histocompatibility complex complotypes

Lymphocyte integration of complement cues

We address current data, views and puzzles on the emerging topic of regulation of lymphocytes by complement proteins or fragments. Such regulation is believed to take place through complement receptors (CR) and membrane complement regulators (CReg) involved in cell function or protection, respectively, including intracellular signalling. Original observations in B cells clearly support that complement cues through CR improve their performance. Other lymphocytes likely integrate complement-derived signals, as most lymphoid cells constitutively express or regulate CR and CReg upon activation. CR-induced signals, particularly by anaphylatoxins, clearly regulate lymphoid cell function. In contrast, data obtained by CReg crosslinking using antibodies are not always confirmed in human congenital deficiencies or knock-out mice, casting doubts on their physiological relevance. Unsurprisingly, human and mouse complement systems are not completely homologous, adding further complexity to our still fragmentary understanding of complement-lymphocyte interactions

The Behçet's disease-associated variant of the aminopeptidase ERAP1 shapes a low-affinity HLA-B*51 peptidome by differential subpeptidome processing.

A low-activity variant of endoplasmic reticulum aminopeptidase 1 (ERAP1), Hap10, is associated with the autoinflammatory disorder Behçet's disease (BD) in epistasis with HLA-B*51, which is the main risk factor for this disorder. The role of Hap10 in BD pathogenesis is unknown. We sought to define the effects of Hap10 on the HLA-B*51 peptidome and to distinguish these effects from those due to HLA-B*51 polymorphisms unrelated to disease. The peptidome of the BD-associated HLA-B*51:08 subtype expressed in a Hap10-positive cell line was isolated, characterized by mass spectrometry, and compared with the HLA-B*51:01 peptidome from cells expressing more active ERAP1 allotypes. We additionally performed synthetic peptide digestions with recombinant ERAP1 variants and estimated peptide-binding affinity with standard algorithms. In the BD-associated ERAP1 context of B*51:08, longer peptides were generated; of the two major HLA-B*51 subpeptidomes with Pro-2 and Ala-2, the former one was significantly reduced, and the latter was increased and showed more ERAP1-susceptible N-terminal residues. These effects were readily explained by the low activity of Hap10 and the differential susceptibility of X-Pro and X-Ala bonds to ERAP1 trimming and together resulted in a significantly altered peptidome with lower affinity. The differences due to ERAP1 were clearly distinguished from those due to HLA-B*51 subtype polymorphism, which affected residue frequencies at internal positions of the peptide ligands. The alterations in the nature and affinity of HLA-B*51·peptide complexes probably affect T-cell and natural killer cell recognition, providing a sound basis for the joint association of ERAP1 and HLA-B*51 with BD

Human congenital T-cell receptor disorders

Immunodeficiencies of most T-cell receptor (TCR) components (TCRID) have been reported in almost 40 patients worldwide who have also, at times, shown signs of autoimmunity. We updated their clinical, immunological, and molecular features with an emphasis on practical diagnosis, as the range of the disorder grows in complexity with new partial defects. Cellular and animal models are also reviewed and in some cases reveal their limitations for predicting TCRID immunopathology

Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF-β and PD-L1 enhances antitumor responses

International audienceT cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-β play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-β inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-β, termed AxF (scFv)$_2$, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)$_2$ antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3×EGFR TriTE with the AxF (scFv)$_2$ delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-β by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression