The Novel Immune Checkpoint GPR56 Is Expressed on Tumor-Infiltrating Lymphocytes and Selectively Upregulated upon TCR Signaling

High levels of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) are associated with a survival benefit in various cancer types and the targeted (re)activation of TILs is an attractive therapeutic anti-cancer approach that yields curative responses. However, current T cell targeting strategies directed at known immune checkpoints have not increased objective response rates for all cancer types, including for epithelial ovarian cancer (EOC). For this reason, the identification of new immune checkpoints that regulate T cell immunity remains of great interest. One yet largely uninvestigated checkpoint of potential interest is the G protein-coupled receptor 56 (GPR56), which belongs to the adhesion GPCR family. GPR56 was originally reported to function in cerebral cortical development and in anti-depressant response, but also in cancer. Recently, GPR56 was identified as an inhibitory receptor expressed on human NK cells that by cis-interaction with the tetraspanin CD81 attenuated the cytotoxic activity of NK cells. This NK cell checkpoint could be blocked by an GPR56 antibody, leading to increased cytotoxicity. Interestingly, GPR56 expression has also been reported on cytokine producing memory CD8 T lymphocytes and may thus represent a T cell checkpoint as well. Here, GPR56 mRNA expression was characterized in the context of TILs, with GPR56 expression being detected predominantly in tumor infiltrating CD8 T cells with a cytotoxic and (pre-)exhausted phenotype. In accordance with this mRNA profile, TILs from ovarian cancer patients expressed GPR56 primarily within the effector memory and central memory T cell subsets. On T cells from healthy donors the expression was limited to effector memory and terminally differentiated T cells. Notably, GPR56 expression further increased on TILs upon T cell receptor (TCR)-mediated stimulation in co-cultures with cancer cells, whereas GPR56 expression on healthy primary human T cells did not. Further, the ectopic expression of GPR56 significantly reduced the migration of GPR56-positive T cells. Taken together, GPR56 is a potential immune-checkpoint in EOC found on (pre-)exhausted CD8 TILs that may regulate migratory behavior

A luminescence-based method to assess antigen presentation and antigen-specific T cell responses for in vitro screening of immunomodulatory checkpoints and therapeutics

Investigations into the strength of antigen-specific responses in vitro is becoming increasingly relevant for decision making in early-phase research of novel immunotherapeutic approaches, including adoptive cell but also immune checkpoint inhibitor (ICI)-based therapies. In the latter, antigen-specific rapid and high throughput tools to investigate MHC/antigen-specific T cell receptor (TCR) activation haven't been implemented yet. Here, we present a simple and rapid luminescence-based approach using the human papillomavirus 16 (HPV16) E7 11-20 peptide as model antigen and E7-TCR transgenic Jurkat.NFAT- luciferase reporter cells. Upon E7 peptide pulsing of HLA-A2 + cell lines and macrophages, an effector to target ratio dependent increase in luminescence compared to non-pulsed cells was observed after co-incubation with E7-TCR expressing Jurkat, but not with parental cells. Analogous experiments with cells expressing full-length HPV16 identified that E7-specific activation of Jurkat cells enabled detection of endogenous antigen processing and MHC-I presentation. As proof of concept, overexpression of established checkpoints/inhibitory molecules (e.g., PD-L1 or HLA-G) significantly reduced the E7-specific TCR-induced luminescence, an effect that could be restored after treatment with corresponding targeting antagonistic antibodies. Altogether, the luminescence-based method described here represents an alternative approach for the rapid evaluation of MHC-dependent antigen-specific T cell responses in vitro. It can be used as a rapid tool to evaluate the impact of the immunosuppressive tumor microenvironment or novel ICI in triggering effective T cell responses, as well as speeding up the development of novel therapeutics within the immune-oncology field. </p

CD24 Is a Potential Immunotherapeutic Target for Mantle Cell Lymphoma

CD24 and its ligand Siglec-10 were described as an innate immune checkpoint in carcinoma. Here, we investigated this axis in B-cell lymphoma by assessing CD24 expression and evaluating pro-phagocytic effects of CD24 antibody treatment in comparison to hallmark immune checkpoint CD47. In mantle cell lymphoma (MCL) and follicular lymphoma patients, high mRNA expression of CD24 correlated with poor overall survival, whereas CD47 expression did not. Conversely, CD24 expression did not correlate with survival in diffuse large B-cell lymphoma (DLBCL), whereas CD47 did. CD24 was also highly expressed on MCL cell lines, where treatment with CD24 antibody clones SN3 or ML5 potently induced phagocytosis, with SN3 yielding >90% removal of MCL cells and triggering phagocytosis of primary patient-derived MCL cells by autologous macrophages. Treatment with CD24 mAb was superior to CD47 mAb in MCL and was comparable in magnitude to the effect observed in carcinoma lines. Reversely, CD24 mAb treatment was less effective than CD47 mAb treatment in DLBCL. Finally, phagocytic activity of clone SN3 appeared at least partly independent of antibody-dependent cellular phagocytosis (ADCP), suggesting CD24/Siglec-10 checkpoint activity, whereas clone ML5 solely induced ADCP. In conclusion, CD24 is an immunotherapeutic target of potential clinical relevance for MCL, but not DLBCL

CD20 positive CD8 T cells are a unique and transcriptionally-distinct subset of T cells with distinct transmigration properties

Abstract The presence of T cells that are dimly positive for the B cell marker CD20 is well-established in autoimmunity and correlates with disease severity in various diseases. Further, we previously identified that the level of CD20-positive T cells was three–fourfold elevated in ascites fluid of ovarian carcinoma patients, together suggesting a role in both autoimmunity and cancer. In this respect, treatment of autoimmune patients with the CD20-targeting antibody Rituximab has also been shown to target and deplete CD20-positive T cells, previously identified as IFN-gamma producing, low proliferative, CD8 cytotoxic T cells with an effector memory (EM) differentiation state. However, the exact phenotype and relevance of CD20-positive T cells remains unclear. Here, we set out to identify the transcriptomic profile of CD20-positive T cells using RNA sequencing. Further, to gain insight into potential functional properties of CD20 expression in T cells, CD20 was ectopically expressed on healthy human T cells and phenotypic, functional, migratory and adhesive properties were determined in vitro and in vivo. Together, these assays revealed a reduced transmigration and an enhanced adhesive profile combined with an enhanced activation status for CD20-positive T cells

CD24 Is a Potential Immunotherapeutic Target for Mantle Cell Lymphoma

CD24 and its ligand Siglec-10 were described as an innate immune checkpoint in carcinoma. Here, we investigated this axis in B-cell lymphoma by assessing CD24 expression and evaluating pro-phagocytic effects of CD24 antibody treatment in comparison to hallmark immune checkpoint CD47. In mantle cell lymphoma (MCL) and follicular lymphoma patients, high mRNA expression of CD24 correlated with poor overall survival, whereas CD47 expression did not. Conversely, CD24 expression did not correlate with survival in diffuse large B-cell lymphoma (DLBCL), whereas CD47 did. CD24 was also highly expressed on MCL cell lines, where treatment with CD24 antibody clones SN3 or ML5 potently induced phagocytosis, with SN3 yielding &gt;90% removal of MCL cells and triggering phagocytosis of primary patient-derived MCL cells by autologous macrophages. Treatment with CD24 mAb was superior to CD47 mAb in MCL and was comparable in magnitude to the effect observed in carcinoma lines. Reversely, CD24 mAb treatment was less effective than CD47 mAb treatment in DLBCL. Finally, phagocytic activity of clone SN3 appeared at least partly independent of antibody-dependent cellular phagocytosis (ADCP), suggesting CD24/Siglec-10 checkpoint activity, whereas clone ML5 solely induced ADCP. In conclusion, CD24 is an immunotherapeutic target of potential clinical relevance for MCL, but not DLBCL

Identification of an antitumoral fraction obtained from the lactic acid bacterium Lactobacillus acidophilus

[ES] El estudio de los probióticos ha cobrado una gran importancia en los últimos años debido a su papel en el mantenimiento de una microbiota intestinal equilibrada, así como en el tratamiento de ciertas enfermedades, incluidos ciertos tipos de cáncer. En este trabajo, se ha analizado el efecto anti-proliferativo de un extracto bacteriano obtenido a partir de la cepa probiótica Lactobacillus acidophilus sobre la línea celular HT29. Este efecto se ha detectado con una técnica de impedancia no invasiva que permite cuantificar la proliferación celular. Además, se ha realizado un análisis del ciclo celular mediante citometría de flujo, ya que una posible hipótesis era que este extracto afectase a la división celular. Finalmente, se observó un efecto sobre la línea celular dependiente de la concentración. Aunque dicho efecto se atribuyó inicialmente a un descenso en la proliferación, los resultados de citometría no parecen reflejar esta idea y dan pie a nuevas hipótesis, incluidas una alteración en la morfología/adhesión celular o un efecto anti-proliferativo que no se detectaría debido a la aparición de mecanismos de resistencia. Además, un segundo análisis con citometría de flujo descarta su efecto sobre la viabilidad celular. Por otro lado, la elevada complejidad del extracto requiere de un fraccionamiento previo, por ejemplo mediante cromatografía líquida, para poder identificar qué moléculas son responsables del efecto observado. Antes de llevar a cabo esta operación, es necesaria una optimización de las condiciones de separación, lo cual se presenta en este trabajo utilizando la técnica de cromatografía en capa fina. De esta forma, se consiguió establecer condiciones óptimas tanto para cromatografía en fase reversa con mezclas de acetonitrilo y agua, como para fase normal con mezclas de hexano y acetato de etilo, acetato de etilo y metanol, y cloroformo y metanol.[EN] The global interest in probiotics has gained a great relevance over the last few years due to its importance in both the maintenance of a balanced gut microbiota, and the treatment of several diseases, including certain type of cancers. In the present study, the anti-proliferative effect from a bacterial extract which has been obtained from probiotic strain, Lactobacillus acidophilus, has been tested on the HT29 cell line, derived from a colon adenocarcinome. This effect could be observed thanks to a non-invasive impedance assay, which allows for cell proliferation quantification. Furthermore, a cell cycle analysis through flow cytometry has been also carried out in order to get a better idea about the action mechanism of the extract, as one of the main hypothesis would be its possible impact on the cell division. Finally it could be observed an effect over the cell line dependent on concentration. Although this effect had been previously associated with a decrease in cell proliferation, it seems that flow cytometry results do not reflect this idea and thus, several hypothesis have been purposed, including changes in cell morphology/adhesion or an anti-proliferative effect which was not detected because of resistance mechanisms. Additionally, a second flow cytometry analysis showed no effect on cell viability. On the other hand, the huge complexity of the extract requires a previous separation through liquid chromatography, in order to be able to identify which molecule or molecules are responsible for the observed effect. Before performing this technique, it is necessary to carry out an optimization through thin layer chromatography, which is included in this study. At the end, several optimal conditions have been reached for both reverse phase with mixtures of acetonitrile and water, and normal phase with mixtures of hexane and ethyl acetate, ethyl acetatePeer reviewe

Tumor infiltrating CD8/CD103/TIM-3-expressing lymphocytes in epithelial ovarian cancer co-express CXCL13 and associate with improved survival

Reactivation of tumor infiltrating T lymphocytes (TILs) with immune checkpoint inhibitors or co-stimulators has proven to be an effective anti-cancer strategy for a broad range of malignancies. However, epithelial ovarian cancer (EOC) remains largely refractory to current T cell-targeting immunotherapeutics. Therefore, identification of novel immune checkpoint targets and biomarkers with prognostic value for EOC is warranted. Combining multicolor immunofluorescent staining’s with single cell RNA-sequencing analysis, we here identified a TIM-3/CXCL13-positive tissue-resident memory (CD8/CD103-positive) T cell (Trm) population in EOC. Analysis of a cohort of ~175 patients with high-grade serous EOC revealed TIM-3-positive Trm were significantly associated with improved patient survival. As CXCL13-positive CD8-positive T cells have been strongly linked to patient response to anti-PD1 immune checkpoint blockade, combinatorial TIM-3 and PD-1 blockade therapy may be of interest for the (re)activation of anti-cancer immunity in EOC