Study of the immunogenicity of the cell death induced by the activation of CD47 in tumor cells

CD47 activation by peptides derived from the C-terminal domain of thrombospondin-1, such as PKHB1, induces regulated cell death (RCD) in different types of cancer cells. The RCD induced by the activation of CD47 is characterized by being independent of caspases, calcium dependent, generating morphological changes in the endoplasmic reticulum (ER) and the exposure of calreticulin (CRT) in chronic lymphocytic leukemia (CLL) cells. On the other hand, ER stress and CRT exposure are characteristics of immunogenic cell death (ICD). Based on the above, the objective of this thesis was to determine the immunogenicity of cell death induced by the activation of CD47 in lymphoma and breast cancer cells. To this end, ICD’s characteristics were evaluated, such as damage associated molecular patterns (DAMPs) exposure or release and their effect on the activation of an antitumor immune response. In addition, the extent of the immunogenicity of death was evaluated by prophylactic and therapeutic vaccinations, with lysates of cancer cells treated with PKHB1 (PKHB1-TCL) on the tumor establishment of lymphoma and breast cancer (in vivo), as well as their capacity to generate a long-term anti-tumor immunological memory. The results obtained allow us to demonstrate that PKHB1 induces CRT exposure and release of CRT and HSP90 in lymphoma and breast cancer cells. It was also demonstrated that the PKHB1-TCL promotes the maturation of dendritic cells (DCs) increasing the expression of maturation markers CD80, CD86 and the release of TNFa. Furthermore, the PKHB1-TCL activated the antitumor immune response (ex vivo), promoting the specific cytotoxicity of tumor cells by T lymphocytes and induced the inflammatory cytokines IL-2 and IFNg release. Finally, it was demonstrated that the PKHB1-TCL is able to prevent the tumor establishment in a lymphoma and murine breast cancer model, and to induce a long-term immunological anti-tumor memory. Finally, when observing that the immunogenic characteristics of death are shared among the different tumor models, we can propose that the CD47 activation by PKHB1 induces a conserved ICD mechanism, enhancing its application and making it a promising antitumor therapy. RESUMEN La activación de CD47 por péptidos derivados del dominio C-terminal de la trombospondina1, como el PKHB1, induce muerte celular regulada (MCR) en diferentes tipos de células cancerosas. La MCR inducida por la activación de CD47 se caracteriza por ser independiente de caspasas, dependiente de calcio, generar cambios morfológicos en el retículo endoplásmico (RE) y exposición de Calreticulina (CRT) en células de leucemia linfocítica crónica (LLC). El estrés del RE y la exposición de CRT son característias de la muerte celular inmunogénica (MCI). Con base en lo anterior, el objetivo de esta tesis fue determinar la inmunogenicidad de la muerte celular inducida por la activación de CD47 en células de linfoma y cáncer de mama. Para ello se evaluaron características de MCI, como la liberación de patrones moleculares asociados a daño (DAMPs) y su efecto en la activación de la respuesta inmune antitumoral. Además, se evaluó el alcance de la inmunogenicidad de la muerte mediante vacunaciones profilácticas y terapéuticas, con lisados de células cancerosas tratadas con PKHB1 (PKHB1-TCL) sobre el establecimiento tumoral de linfoma y cáncer de mama (in vivo), así como su capacidad de generar una memoria inmunológica antitumoral a largo plazo. Los resultados obtenidos nos permiten demostrar que el PKHB1 induce la exposición de CRT y liberación de CRT, HSP90, ATP y HMGB1 en células de linfoma y cáncer de mama. También se demostró que el PKHB1-TCL promueve la maduración de células dendríticas (DCs) aumentando la expresión de marcadores de maduración CD80, CD86 y la liberación de TNFa. Asimismo, el PKHB1-TCL activó la respuesta inmune antitumoral (ex vivo), promoviendo la citotoxicidad específica de células tumorales por linfocitos T e indujo la liberación de citocinas inflamatorias IL-2 e IFNg. Además, se demostró que el PKHB1-TCL es capaz de prevenir y disminuir el establecimiento tumoral hasta lograr la remisión en un modelo de linfoma y cáncer de mama murino, además de inducir una memoria inmunológica antitumoral a largo plazo. Finalmente, al observar que las características inmunogénicas de la muerte se comparten entre los diferentes modelos tumorales, podemos proponer que la activación de CD47 por el PKHB1 induce un mecanismo conservado de MCI, potenciando su aplicación y haciéndola una prometedora terapia antitumoral

Immunotherapies inducing immunogenic cell death in cancer: insight of the innate immune system

Cancer immunotherapies include monoclonal antibodies, cytokines, oncolytic viruses, cellular therapies, and other biological and synthetic immunomodulators. These are traditionally studied for their effect on the immune system’s role in eliminating cancer cells. However, some of these therapies have the unique ability to directly induce cytotoxicity in cancer cells by inducing immunogenic cell death (ICD). Unlike general immune stimulation, ICD triggers specific therapy-induced cell death pathways, based on the release of damage-associated molecular patterns (DAMPs) from dying tumour cells. These activate innate pattern recognition receptors (PRRs) and subsequent adaptive immune responses, offering the promise of sustained anticancer drug efficacy and durable antitumour immune memory. Exploring how onco-immunotherapies can trigger ICD, enhances our understanding of their mechanisms and potential for combination strategies. This review explores the complexities of these immunotherapeutic approaches that induce ICD, highlighting their implications for the innate immune system, addressing challenges in cancer treatment, and emphasising the pivotal role of ICD in contemporary cancer research

Evaluation of the Immune Response of a Candidate Phage-Based Vaccine against <i>Rhipicephalus microplus</i> (Cattle Tick)

Cattle tick (Rhipicephalus microplus) represents a severe problem causing substantial economic losses, estimated in billions of dollars annually. Currently, chemical acaricides represent the most widely used control method. However, several problems such as resistance have been described. Phage-based vaccines represent a fast and low-cost tool for antigen delivery. In this regard, the objective of the present work was to develop a candidate phage-based vaccine displaying a cattle tick antigen (Bm86-derived Sbm7462 antigen) on the surface of bacteriophage M13. Phage ELISA and dot blotting analysis confirmed the display of the antigen. Vaccine immunogenicity was evaluated using a bovine monocyte-derived dendritic cell-based ex vivo assay and a murine in vivo assay. The ex vivo model showed the maturation of dendritic cells after being pulsed with the phage-based vaccine. The humoral response was confirmed in the in vivo assay. These results demonstrated the capacity of the phage-based vaccine to induce both humoral and cellular immune-specific responses. Importantly, this is the first report describing a control method for cattle ticks using a candidate phage-based vaccine. Further studies to evaluate the immunogenicity in a bovine model are needed. The current approach represents a promising alternative to control cattle tick infestations

CD 47 agonist peptide PKHB 1 induces immunogenic cell death in T‐cell acute lymphoblastic leukemia cells

International audienceT-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin-1-derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first-described serum-stable CD47-agonist peptide) on CEM and MOLT-4 human cell lines (T-ALL) and on one T-murine tumor lymphoblast cell-line (L5178Y-R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non-tumor cells after CD47 activation. In vivo, we determined that PKHB1-treatment in mice bearing the L5178Y-R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage-associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high-mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase-independent and calcium-dependent cell death in leukemic cells while sparing non-tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia