Emerging data demonstrate that the activity of immune cells can be modulated by microbial molecules. Here, we show that the short-chain fatty acids (SCFAs) pentanoate and butyrate enhance the anti-tumor activity of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells through metabolic and epigenetic reprograming. We show that in vitro treatment of CTLs and CAR T cells with pentanoate and butyrate increases the function of mTOR as a central cellular metabolic sensor, and inhibits class I histone deacetylase activity. This reprogramming results in elevated production of effector molecules such as CD25, IFN-γ and TNF-α, and significantly enhances the anti-tumor activity of antigen-specific CTLs and ROR1-targeting CAR T cells in syngeneic murine melanoma and pancreatic cancer models. Our data shed light onto microbial molecules that may be used for enhancing cellular anti-tumor immunity. Collectively, we identify pentanoate and butyrate as two SCFAs with therapeutic utility in the context of cellular cancer immunotherapy.

Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.

Despite recent therapeutic advances, the prognosis of multiple myeloma (MM) patients remains poor. Thus, new strategies to improve outcomes are imperative. Chimeric antigen receptor (CAR) T-cell therapy has changed the treatment landscape of B-cell malignancies, providing a potentially curative option for patients who are refractory to standard treatment. Long-term remissions achieved in patients with acute lymphoblastic leukemia and Non-Hodgkin Lymphoma encouraged its further development in MM. B-cell maturation antigen (BCMA)-targeted CAR T-cells have established outstanding results in heavily pre-treated patients. However, several other antigens such as SLAMF7 and CD44v6 are currently under investigation with promising results. Idecabtagene vicleucel is expected to be approved soon for clinical use. Unfortunately, relapses after CAR T-cell infusion have been reported. Hence, understanding the underlying mechanisms of resistance is essential to promote prevention strategies and to enhance CAR T-cell efficacy. In this review we provide an update of the most recent clinical and pre-clinical data and we elucidate both, the potential and the challenges of CAR T-cell therapy in the future.

Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.