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Cellular immunotherapy with chimeric antigen receptor (CAR)-T cells (CARTs) represents a breakthrough in the treatment of hematologic malignancies. CARTs are genetically engineered hybrid receptors that combine antigen-specificity of monoclonal antibodies with T cell function to direct patient-derived T cells to kill malignant cells expressing the target (tumor) antigen. CARTs have been introduced into clinical medicine as CD19-targeted CARTs for refractory and relapsed B cell malignancies. Despite high initial response rates, current CART therapies are limited by a long-term loss of antitumor efficacy, the occurrence of toxicities, and the lack of biomarkers for predicting therapy and toxicity outcomes. In the past decade, the gut microbiome of mammals has been extensively studied and evidence is accumulating that human health, apart from our own genome, largely depends on microbes that are living in and on the human body. The microbiome encompasses more than 1000 bacterial species who collectively encode a metagenome that guides multifaceted, bidirectional host-microbiome interactions, primarily through the action of microbial metabolites. Increasing knowledge has been accumulated on the role of the gut microbiome in T cell-driven anticancer immunotherapy. It has been shown that antibiotics, dietary components and gut microbes reciprocally affect the efficacy and toxicity of allogeneic hematopoietic cell transplantation (allo HCT) as the prototype of T cell-based immunotherapy for hematologic malignancies, and that microbiome diversity metrics can predict clinical outcomes of allo HCTs. In this review, we will provide a comprehensive overview of the principles of CD19-CART immunotherapy and major aspects of the gut microbiome and its modulators that impact antitumor T cell transfer therapies. We will outline i) the extrinsic and intrinsic variables that can contribute to the complex interaction of the gut microbiome and host in CART immunotherapy, including ii) antibiotic administration affecting loss of colonization resistance, expansion of pathobionts and disturbed mucosal and immunological homeostasis, and ii) the role of specific gut commensals and their microbial virulence factors in host immunity and inflammation. Although the role of the gut microbiome in CART immunotherapy has only been marginally explored so far, this review may open a new chapter and views on putative connections and mechanisms., Competing Interests: M-LS: consultancy for Kite/Gilead, Takeda. Advisory board Kite/Gilead, Janssen. None of these sources were involved in the writing of this review. MS: research grants from Apogenix, Hexal and Novartis. Travel grants from Hexal and Kite. Financial support for educational activities and conferences from bluebird bio, Kite and Novartis. Advisory board member of MSD. (Co-)PI of clinical trials of MSD, GSK, Kite and BMS. Co-Founder and shareholder of TolerogenixX Ltd. None of these sources were involved in the writing of this review. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schubert, Rohrbach, Schmitt and Stein-Thoeringer.)

Chimeric antigen receptor T (CAR-T) cells targeting CD19 came into clinical practice for the treatment of B cell lymphoma in 2018. However, patients being treated for B cell lymphoma often suffer from comorbidities such as chronic pain, cardiovascular diseases and arthritis. Thus, these patients frequently receive concomitant medications that include nonsteroidal anti-inflammatory drugs (NSAIDs) like cyclooxygenase (COX) inhibitors. Celecoxib, a selective COX-2 inhibitor, and aspirin, a non-selective COX-1 and COX-2 inhibitor, are being used as anti-inflammatory, analgesic and anti-pyretic drugs. In addition, several studies have also focused on the anti-neoplastic properties of COX-inhibitors. As the influence of COX-inhibitors on CD19.CAR-T cells is still unknown, we investigated the effect of celecoxib and aspirin on the quantity and quality of CD19.CAR-T cells at different concentrations with special regard to cytotoxicity, activation, cytokine release, proliferation and exhaustion. A significant effect on CAR-T cells could be observed for 0.1 mmol/L of celecoxib and for 4 mmol/L of aspirin. At these concentrations, we found that both COX-inhibitors could induce intrinsic apoptosis of CD19.CAR-T cells showing a significant reduction in the ratio of JC-10 red to JC-10 green CAR-T cells from 6.46 ± 7.03 (mean ± SD) to 1.76 ± 0.67 by celecoxib and to 4.41 ± 0.32 by aspirin, respectively. Additionally, the ratios of JC-10 red to JC-10 green Daudi cells were also decreased from 3.41 ± 0.30 to 0.77 ± 0.06 by celecoxib and to 1.26 ± 0.04 by aspirin, respectively. Although the cytokine release by CD19.CAR-T cells upon activation was not hampered by both COX-inhibitors, activation and proliferation of CAR-T cells were significantly inhibited via diminishing the NF-ĸB signaling pathway by a significant down-regulation of expression of CD27 on CD4 + and CD8 + CAR-T cells, followed by a clear decrease of phosphorylated NF-ĸB p65 in both CD4 + and CD8 + CAR-T cells by a factor of 1.8. Of note, COX-inhibitors hampered expansion and induced exhaustion of CAR-T cells in an antigen stress assay. Collectively, our findings indicate that the use of COX-inhibitors is a double-edged sword that not only induces apoptosis in tumor cells but also impairs the quantity and quality of CAR-T cells. Therefore, COX-inhibitors should be used with caution in patients with B cell lymphoma under CAR-T cell therapy., Competing Interests: MS received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, he received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, he is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite and BMS, as well as co-founder and shareholder of TolerogenixX Ltd. AS received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt and is co-founder of TolerogenixX Ltd. AS and LW are part- or full-time employers of TolerogenixX Ltd. LS was employed by Takeda Pharma Vertrieb GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Wang, Ni, Neuber, Wang, Gong, Sauer, Schubert, Hückelhoven-Krauss, Xia, Ge, Kleist, Eckstein, Sellner, Müller-Tidow, Dreger, Schmitt and Schmitt.)

Chimeric antigen receptor (CAR) T cell therapy has shown promising responses in patients with refractory or relapsed aggressive B-cell malignancies that are resistant to conventional chemotherapy or stem cell transplantation. A potentially combinatorial therapeutic strategy may be the inhibition of anti-apoptotic Bcl-2 family proteins, overexpressed in most cancer cells. In this study we investigated the combination of 3rd-generation CD19.CAR-T cells and the BH3 mimetics venetoclax, a Bcl-2 inhibitor, or S63845, a Mcl-1 inhibitor, under three different treatment conditions: pre-sensitization of cancer cells with BH3 mimetics followed by CAR-T cell treatment, simultaneous combination therapy, and the administration of BH3 mimetics after CAR-T cell treatment. Our results showed that administration of CAR-T cells and BH3 mimetics had a significant effect on the quantity and quality of CD19.CAR-T cells. The administration of BH3 mimetics prior to CAR-T cell therapy exerted an enhanced cytotoxic efficacy by upregulating the CD19 expression and pro-apoptotic proteins in highly sensitive tumor cells, and thereby improving both CD19.CAR-T cell cytotoxicity and persistence. In simultaneous and post-treatment approaches, however, the quantity of CAR-T cells was adversely affected. Our findings indicate pre-sensitization of highly sensitive tumor cells with BH3 mimetics could enhance the cytotoxic efficacy of CAR-T cell treatment., Competing Interests: MS received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite, and BMS, as well as a co-founder and shareholder of TolerogenixX Ltd. AS received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt, and is a co-founder of TolerogenixX LtD. AS and LW are part- or full-time employers of TolerogenixX Ltd. LS was employed by Takeda Pharma Vertrieb GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past collaboration with one of the authors TS., (Copyright © 2020 Yang, Wang, Ni, Neuber, Wang, Gong, Sauer, Sellner, Schubert, Hückelhoven-Krauss, Hong, Zhu, Kleist, Eckstein, Müller-Tidow, Dreger, Schmitt and Schmitt.)

CD56 bri natural killer (NK) cells play an important role in the pathogenesis of graft-vs. -host disease (GVHD) and immune defense in the early period after allogeneic hematopoietic stem cell transplantation. Extracorporeal photopheresis (ECP) as an immunomodulating therapy has been widely used for GVHD treatment. However, the mechanism of action of ECP still remains to be elucidated, particularly the influence of ECP on NK cells. Thirty-four patients with steroid-refractory/resistant acute GVHD (aGVHD) ≥ °II and moderate to severe chronic GVHD (cGVHD) received ECP therapy. Patient samples obtained during intensive and long-term treatment were analyzed. Immunomonitoring with respect to cell phenotype and function was performed on rested peripheral blood mononuclear cells (PBMCs) using multiparametric flow cytometry. NK activity in terms of cytokine release was analyzed by intracellular cytokine staining after co-culture with K562 cells. Moreover, the proliferative capacity of NK cells, CD4 + , and CD8 + T cells was determined by carboxyfluorescein succinimidyl ester (CFSE) staining. Clinically, 75% of aGVHD and 78% of cGVHD patients responded to ECP therapy. Moreover, our data show that aGVHD, cGVHD patients and healthy donors (HDs) present distinct NK patterns: aGVHD patients have a higher frequency of CD56 bri NK subsets with stronger NKG2D and CD62L expression, while CD56 - CD16 + NK cells with higher expression of CD57 and CD11b stand out as a signature population for cGVHD. ECP therapy could significantly decrease CD56 bri CD16 - NK cells with shifting the quality from a cytotoxic to a regulatory pattern and additionally mature CD56 dim NK cells via upregulation of CD57 in complete responding aGVHD patients. Moreover, ECP could keep the anti-viral and anti-leukemic effects intact via maintaining specialized anti-viral/leukemic CD57 + NKG2C + CD56 dim NK cells as well as remaining the quality and quantity of cytokine release by NK cells. The proliferative capacity of effector cells remained constant over ECP therapy. In conclusion, ECP represents an attractive option to treat GVHD without compromising anti-viral/leukemic effects. Shaping of CD56 bri NK cell compartment by downregulating the cytotoxic subset while upregulating the regulatory subset contributes to the mechanisms of ECP therapy in aGVHD.

Introduction: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (T N ) vs. effector (T E ) T cells, T N cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the T N /T E ratio of CART cells., Materials and Methods: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays., Results: IL-7/IL-15 preferentially induced differentiation into T N , stem cell memory (T SCM : naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (T EM ), CD56+ and CD4+ T regulatory (T Reg ) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CART N cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CART N cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CART N cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CART N cells in untreated CLL patients. Final T N /T E ratio stayed <0.3 despite stimulation condition for patients, whereas this ratio was >2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CART N expansion., Conclusion: Untreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of T N among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the T N /T E ratio within the CART product.