T-cell dysfunction in CLL is mediated through expression of SIGLEC-10 ligands CD24 and CD52 on CLL cells.

Autologous T-cell-based therapies, such as CAR-T-cell therapy, exhibit low success rates in chronic lymphocytic leukemia (CLL) which correlates with a dysfunctional T-cell phenotype observed in patients. Despite various proposed mechanisms of T-cell dysfunction in CLL, the specific CLL-derived factors responsible remain unidentified. This study aims to investigate mechanisms by which CLL cells suppress (CAR) T-cell activation and function. We found that CLL-derived T cells get activated, albeit in a delayed fashion and that specifically re-stimulation of (CAR-) T cells in presence of CLL cells causes impaired cytokine production and reduced proliferation. Notably, co-culture of T cells with CD40-activated CLL cells did not result in T-cell dysfunction and this required direct cell contact between the CD40-stimulated CLL cells and T cells. Inhibition of kinases involved in the CD40-signaling cascade revealed that the SRC-kinase inhibitor dasatinib prevented rescue of T-cell function independent of CD40-mediated increased levels of costimulatory and adhesion ligands on CLL cells. Transcriptome profiling of CD40-stimulated CLL cells with or without dasatinib identified widespread differential gene expression. Selecting for surface receptor genes revealed CD40-mediated downregulation of SIGLEC-10-ligands CD24 and CD52, which was prevented by dasatinib; suggesting a role for these ligands in functional T-cell suppression in CLL. Indeed, blocking CD24 and/or CD52 markedly reduced (CAR) T-cell dysfunction upon co-culture with resting CLL cells. These results demonstrate that T cells derived from CLL patients can be reinvigorated by manipulating CLL-T cell interactions. Targeting CD24- and CD52-mediated CLL-T cell interaction could be a promising therapeutic strategy to enhance T-cell function in CLL.

Ectopic expression of cGAS in Salmonella typhimurium enhances STING-mediated IFN-ß response in human macrophages and dendritic cells.

BACKGROUND: Interferon (IFN)-ß induction via activation of the stimulator of interferon genes (STING) pathway has shown promising results in tumor models. STING is activated by cyclic dinucleotides such as cyclic GMP-AMP dinucleotides with phosphodiester linkages 2'-5' and 3'-5' (cGAMPs), that are produced by cyclic GMP-AMP synthetase (cGAS). However, delivery of STING pathway agonists to the tumor site is a challenge. Bacterial vaccine strains have the ability to specifically colonize hypoxic tumor tissues and could therefore be modified to overcome this challenge. Combining high STING-mediated IFN-ß levels with the immunostimulatory properties of Salmonella typhimurium could have potential to overcome the immune suppressive tumor microenvironment. METHODS: We have engineered S. typhimurium to produce cGAMP by expression of cGAS. The ability of cGAMP to induce IFN-ß and its IFN-stimulating genes was addressed in infection assays of THP-I macrophages and human primary dendritic cells (DCs). Expression of catalytically inactive cGAS is used as a control. DC maturation and cytotoxic T-cell cytokine and cytotoxicity assays were conducted to assess the potential antitumor response in vitro. Finally, by making use of different S. typhimurium type III secretion (T3S) mutants, the mode of cGAMP transport was elucidated. RESULTS: Expression of cGAS in S. typhimurium results in a 87-fold stronger IFN-ß response in THP-I macrophages. This effect was mediated by cGAMP production and is STING dependent. Interestingly, the needle-like structure of the T3S system was necessary for IFN-ß induction in epithelial cells. DC activation included upregulation of maturation markers and induction of type I IFN response. Coculture of challenged DCs with cytotoxic T cells revealed an improved cGAMP-mediated IFN-γ response. In addition, coculture of cytotoxic T cells with challenged DCs led to improved immune-mediated tumor B-cell killing. CONCLUSION: S. typhimurium can be engineered to produce cGAMPs that activate the STING pathway in vitro. Furthermore, they enhanced the cytotoxic T-cell response by improving IFN-γ release and tumor cell killing. Thus, the immune response triggered by S. typhimurium can be enhanced by ectopic cGAS expression. These data show the potential of S. typhimurium-cGAS in vitro and provides rationale for further research in vivo.

Redirecting T-cell Activity with Anti-BCMA/Anti-CD3 Bispecific Antibodies in Chronic Lymphocytic Leukemia and Other B-cell Lymphomas.

T-cell redirecting bispecific antibodies hold high promise for treatment of B-cell malignancies. B-cell maturation antigen (BCMA) exhibits high expression on normal and malignant mature B cells including plasma cells, which can be enhanced by inhibition of γ-secretase. BCMA is considered a validated target in multiple myeloma but whether mature B-cell lymphomas can be targeted by the BCMAxCD3 T-cell redirector teclistamab is currently unknown. BCMA expression on B-cell non-Hodgkin lymphoma and primary chronic lymphocytic leukemia (CLL) cells was assessed by flow cytometry and/or IHC. To assess teclistamab efficacy, cells were treated with teclistamab in presence of effector cells with/without γ-secretase inhibition. BCMA could be detected on all tested mature B-cell malignancy cell lines, while expression levels varied per tumor type. γ-secretase inhibition universally increased BCMA surface expression. These data were corroborated in primary samples from patients with Waldenstrom's macroglobulinemia, CLL, and diffuse large B-cell lymphoma. Functional studies with the B-cell lymphoma cell lines revealed teclistamab-mediated T-cell activation, proliferation, and cytotoxicity. This was independent of the level of BCMA expression, but generally lower in mature B-cell malignancies compared with multiple myeloma. Despite low BCMA levels, healthy donor T cells and CLL-derived T cells induced lysis of (autologous) CLL cells upon addition of teclistamab. These data show that BCMA is expressed on various B-cell malignancies and that lymphoma cell lines and primary CLL can be targeted using teclistamab. Further studies to understand the determinants of response to teclistamab are required to identify which other diseases might be suitable for teclistamab targeting. Significance: Besides reported BCMA expression on multiple myeloma, we demonstrate BCMA can be detected and enhanced using γ-secretase inhibition on cell lines and primary material of various B-cell malignancies. Furthermore, using CLL we demonstrate that low BCMA-expressing tumors can be targeted efficiently using the BCMAxCD3 DuoBody teclistamab.