The phosphatases of regenerating liver control T cell actin rearrangements and immunological synapse architecture through the WD domain repeat 1 regulator

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Medicina, leída el 02-11-2023La regulación del citoesqueleto de actina es muy importante en la respuesta inmunitaria. En los linfocitos T CD4+, esta red de actina es esencial para el establecimiento de la sinapsis inmunitaria (SI), una estructura especializada que se forma entre una célula presentadora de antígeno y un linfocito T. En el contexto de la SI, el reconocimiento por parte del receptor de la célula T (TCR) de un péptido antigénico específico presentado en el complejo mayor de histocompatibilidad conlleva el inicio de la señalización mediada por el TCR y reordenamientos del citoesqueleto que estabilizan el contacto. La dinámica del citoesqueleto de actina en los linfocitos T está controlada por un amplio número de proteínas, como cofilina, un factor que promueve el corte y despolimerización de la actina. La interacción y función de cofilina sobre los filamentos de actina están reguladas por varios factores entre los que se encuentran las proteínas WDR1 y coronina, cuyas mutaciones en humanos resultan en cuadros de inmunodeficiencia. Un correcto establecimiento de la SI es esencial para conseguir una completa activación y función efectora del linfocito T llevada a cabo por la señalización del TCR, el cual inicia una cascada de fosforilaciones y desfosforilaciones de proteínas regulada por quinasas y fosfatasas. Las fosfatasas de regeneración hepática (PRLs: PRL-1, PRL-2 y PRL-3) se expresan en las células T, pero su papel en la activación del linfocito T está aún por explorar. Nuestro grupo ha demostrado que la activación T causa una bajada de expresión de PRL-2, que resulta en un incremento del ratio PRL-1/PRL-2 en la célula, y que la PRL-1 sobreexpresada se polariza a la SI en la línea celular T CD4+ Jurkat (JK). Además, el uso de fármacos inhibidores de la actividad catalítica de las PRLs reduce la polarización de la actina filamentosa (F-actina) a la SI y la secreción de interleuquina-2 (IL-2) tras la estimulación del linfocito T. Estos resultados sugieren que las PRLs poseen un rol regulador durante la activación del linfocito T, aunque el mecanismo molecular que media ese papel es desconocido...Regulation of the actin cytoskeleton is essential for the immune response. In CD4+ T cells, the actin networks are important during the assembly of the immune synapse (IS), a specialized structure formed between an antigen presenting cell and a T lymphocyte. In the IS context, the T cell receptor (TCR) engagement with a specific antigenic peptide presented on the major histocompatibility complex results in TCR downstream signalling and cytoskeletal rearrangements that stabilize the contact. Actin cytoskeleton dynamics in T cells is controlled by a wide range of proteins, including the severing and depolymerization factor cofilin. Cofilin interaction and function on actin filaments is controlled by several factors such as WDR1 and coronin proteins, whose mutations result in human immunodeficiencies.Proper IS formation is essential to achieve full T cell activation and effector function driven by TCR signalling, which starts a cascade of phosphorylation and dephosphorylation of proteins regulated by kinases and phosphatases. The phosphatases of regenerating liver (PRLs: PRL-1, PRL-2 and PRL-3) are expressed in T lymphocytes and their regulatory role during T cell activation is still to explore. Our group have showed that T cell activation downregulates the expression of PRL-2, resulting in an increased PRL-1/PRL-2 ratio in the cell, and that overexpressed PRL-1 is delivered to the IS formed by the Jurkat CD4+ T cell line (JK). Moreover, pharmacological inhibition of the catalytic activity of the PRLs reduces actin polarization at the IS and secretion of interleukin-2 (IL-2). These results suggest a regulatory role of PRLs during T cell activation, but the exact molecular mechanism is unknown...Fac. de MedicinaTRUEunpu

ACLY inhibition and diabetes

Trabajo fin de máster presentado en la Universidad Pablo de Olavide.--04-04-201

Effect of Pharmacological Inhibition of the Catalytic Activity of Phosphatases of Regenerating Liver in Early T Cell Receptor Signaling Dynamics and IL-2 Production

We have previously shown the delivery of phosphatase of regenerating liver-1 (PRL-1) to the immunological synapse (IS) and proposed a regulatory role of the catalytic activity of PRLs (PRL-1, PRL-2 and PRL-3) in antigen-induced IL-2 production. Nonetheless, the expression in T cells and delivery to the IS of the highly homologous PRL-3, as well as the role of the catalytic activity of PRLs in antigen-induced early signaling, has not been investigated. Here, the expression of PRL-3 protein was detected in primary CD4 T cells and in the CD4 T cell line Jurkat (JK), in which an overexpressed GFP-PRL-3 fluorescent fusion protein trafficked through the endosomal recycling compartment and co-localized with PLCγ1 signaling sites at the IS. Pharmacological inhibition was used to compare the role of the catalytic activity of PRLs in antigen-induced early signaling and late IL-2 production. Although the phosphatase activity of PRLs was not critical for early signaling triggered by antigen, it seemed to regulate signaling dynamics and was necessary for proper IL-2 production. We propose that enzymatic activity of PRLs has a higher significance for cytokine production than for early signaling at the IS. However, further research will be necessary to deeply understand the regulatory role of PRLs during lymphocyte activation and effector function

Towards an common strategy for internalizing teaching in the Scholl of Medicine in particular and al UCM in general

Este proyecto se basa en diseñar una estrategia para la internalización de las asignaturas de grado y posgrado de la Facultad de Medicina, especialmente los impartidos por el Departamento de Inmunología, Oftalmología y ORL.This project is based on the design of a strategy to internalize the main courses for the BSc and MSc at the School of Medicine, especially by the subjects taught at the Department of Immunology, Ophthalmology and ORL.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaFALSEsubmitte

Presentación y discusión de trabajos científicos de respuesta inmune a la infección en congresos

El proyecto ha tenido como objetivo proporcionar a los alumnos del máster de Investigación en Inmunología de la UCM una formación teórica y práctica para la presentación de trabajos científicos en congresos. Se ha desarrollado una reunión científica en la que los alumnos han presentado y discutido trabajos científicos relevantes relacionados con la asignatura “Interacción Patógeno Sistema Inmunitario (607653)” en los formatos de presentación oral y presentación en póster que se utilizan en las reuniones científicasFac. de MedicinaFALSEsubmitte

Human T-cell receptor triggering requires inactivation of Lim kinase-1 by Slingshot-1 phosphatase

Abstract Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation

Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers

Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CART19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti- CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies

Engineered T cells secreting anti-BCMA T cell engagers control multiple myeloma and promote immune memory in vivo.

Multiple myeloma is the second most common hematological malignancy in adults and remains an incurable disease. B cell maturation antigen (BCMA)-directed immunotherapy, including T cells bearing chimeric antigen receptors (CARs) and systemically injected bispecific T cell engagers (TCEs), has shown remarkable clinical activity, and several products have received market approval. However, despite promising results, most patients eventually become refractory and relapse, highlighting the need for alternative strategies. Engineered T cells secreting TCE antibodies (STAb) represent a promising strategy that combines the advantages of adoptive cell therapies and bispecific antibodies. Here, we undertook a comprehensive preclinical study comparing the therapeutic potential of T cells either expressing second-generation anti-BCMA CARs (CAR-T) or secreting BCMAxCD3 TCEs (STAb-T) in a T cell-limiting experimental setting mimicking the conditions found in patients with relapsed/refractory multiple myeloma. STAb-T cells recruited T cell activity at extremely low effector-to-target ratios and were resistant to inhibition mediated by soluble BCMA released from the cell surface, resulting in enhanced cytotoxic responses and prevention of immune escape of multiple myeloma cells in vitro. These advantages led to robust expansion and persistence of STAb-T cells in vivo, generating long-lived memory BCMA-specific responses that could control multiple myeloma progression in xenograft models, outperforming traditional CAR-T cells. These promising preclinical results encourage clinical testing of the BCMA-STAb-T cell approach in relapsed/refractory multiple myeloma.Acknowledgments: We would like to thank the cell Sorting Service of the nUcleUS platform (University of Salamanca, Salamanca, Spain) for technical assistance. Funding: Financial support for this work was obtained from the Spanish Ministry of Science and innovation Mcin/Aei/10.13039/501100011033 (PiD2020- 115444GB- i00 to P.r.- n., PiD2019- 108160rB- i00 to P.M., Ple2021- 0075 to c.B., and PiD2020- 117323rB- 100 and PDc2021- 121711- 100 to l.Á.-V.), partially supported by the european regional Development Fund (erDF); the carlos iii health institute (iSciii) (Pi20/01030 to B.B., Pi19/00132 to l.S., Pi21- 01834 to P.P., Pi20/00822 to c.B., and DTS20/00089 to l.Á.-V.), partially supported by the erDF; the iSciii- ricorS within the next Generation eU program (plan de recuperación, Transformación y resilencia) (rD21/0017/0030 to B.B. and J.M.- l. and rD21/0017/0029 to P.M.); the iSciii- ciBeronc program (cB16/12/00400 to A.o.), the criS cancer Foundation (FcriS- 2021- 001 to J.M.- l. and FcriS- 2021- 0090 to l.Á.-V.), the Spanish Association Against cancer (Aecc) (PrYGn234975Mene to P.M., PrYGn211192BUen to c.B., and ProYe19084AlVA and PrYGn234844AlVA to l.Á.-V.); the Accelerator Award- cancer research UK/Airc/Aecc- incAr (GeAcc18001orF to A.o.), the Fundación “la caixa” (lcF/Pr/hr19/52160011 to P.M. and hr21- 00761 project il7r_lungcan to l.Á.-V.), the european research council (erc) (erc- Poc- 957466 to P.M.) and erc under the eU’s horizon Program (grant agreement 101100665 to P.M.), the Fundación de investigación Biomédica 12 de octubre (programa investiga 2022- 0082) to l.Á.-V.; the Fundación ramón Areces to P.P. l.D.-A. was supported by a rio hortega fellowship from the carlos iii health institute (cM20/00004). A.F. was supported by a postdoctoral fellowship from the Spanish Ministry of Science and innovation (FJc2021- 046789- i). A. Mayado was supported by the ciBeronc (PrF- 2869). A.P.- P. was supported by a grant from the Government of castilla y león (orden eDU/556/2019; Valladolid, Spain). M.G.- r. was supported by an industrial PhD ellowship from the comunidad de Madrid (inD2022/BMD- 23732). o.A.- S. was supported by a PhD fellowship from the complutense University of Madrid. c.D.-A. was supported by a PhD fellowship from the Spanish Ministry of Science and innovation (Pre2018- 083445). l.r.- P. was supported by a PhD fellowship from the immunology chair, Universidad Francisco de Vitoria/Merck. o.h. was supported by an industrial PhD fellowship from the comunidad de Madrid (inD2020/BMD- 17668). A.V. is supported by research institute hospital 12 de octubre (imas12). A.G.- o. is supported by hiGeA 2019/0123 Aie project to J.M.- l.S