Cellular therapy in lymphoma.

CD19-directed chimeric antigen receptor (CAR) T-cell therapy has had a dramatic impact on the natural history and survival of patients with high-risk B-cell non-Hodgkin lymphoma. Accompanying this success has been the development of new fields of medicine and investigation into toxicity risks and mitigation therapies, mechanisms of resistance and the development of novel and next generation products and strategies in order to address relapse, and issues related to global access and health care economics. This article is a survey of each of these areas as it pertains to the rapidly evolving field of CAR T-cell therapy, written by an International community of lymphoma experts, who also happen to be women.

Clinical impact of multiple DNA virus infections in nondepleted haploidentical and unrelated allogeneic hematopoietic stem cell transplantation.

Few studies have compared the clinical impact of multiple DNA-virus infections in haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with posttransplant cyclophosphamide (PTCy) and unrelated donor allogeneic hematopoietic stem cell transplantation (UD-HSCT) with thymoglobulin, so we retrospectively analyzed viral infections in the first 6 mo posttransplant in these scenarios. Fifty-nine patients underwent to haplo-HSCT, and 68 to UD-HSCT. The most frequent infection was cytomegalovirus (CMV) (76.3% in haplo-HSCT and 69.1% in UD-HSCT) (P = .878) and in the group of patients with CMV reactivation, maximal CMV viral load over 2500 UI/ml correlated with worse overall survival-hazard ratio (HR) 1.93 (95% confidence interval [CI] 1.04-3.59) P = .03. The cumulative incidence of multiple DNA virus within 180 d of posttransplant was 78.7% for one virus and 28.4% for two or more viruses with no difference regarding the type of transplant. Viral infections, age, and acute graft versus host disease (GVHD) grades II-IV were risk factors for worse overall survival in multivariate analyses: one virus HR 2.53 (95% CI 1.03-6.17) P = .04, two or more viruses HR 3.51 (95% CI 1.37-9) P < .01, age HR 1.03 (95% CI 1.02-1.05) P < .01 and acute GVHD II-IV HR 1.97 (95% CI 1.13-3.43) P = .01. Also, age over 50 y HR 4.25 (95% CI 2.01-8.97) P < .001, second CMV reactivation or having both CMV and BK polyomavirus (BKV) HR 2.65 (95% CI 1.26-5.56) P = .01 and acute GVHD grades II-IV HR 2.23 (95% CI 1.12-4.43) P = .022 were risk factors for nonrelapse mortality in the multivariate analyses. In conclusion, multiple DNA-virus infections are frequent in both haplo-HSCT and UD-HSCT and a risk factor for worse overall survival.

An Extended Flow Cytometry Evaluation of ex Vivo Expanded NK Cells Using K562.Clone1, a Feeder Cell Line Manufactured in Brazil.

Natural killer (NK) cells play a crucial role in the immune system's response against cancer. However, the challenge of obtaining the required quantity of NK cells for effective therapeutic response necessitates the development of strategies for their ex vivo expansion. This study aimed to develop a novel feeder cell line, K562.Clone1, capable of promoting the ex vivo expansion of NK cells while preserving their cytotoxic potential. he K562 leukemic cell line was transduced with mbIL-21 and 4-1BBL proteins to generate K562.Clone1 cells. NK cells were then co-cultured with these feeder cells, and their expansion rate was monitored over 14 days. The cytotoxic potential of the expanded NK cells was evaluated against acute myeloid leukemia blasts and tumor cell lines of leukemia and glial origin. Statistical analysis was performed to determine the significance of the results. The K562.Clone1 co-cultured with peripheral NK showed a significant increase in cell count, with an approximate 94-fold expansion over 14 days. Expanded NK cells demonstrated cytotoxicity against the tested tumor cell lines, indicating preservation of their cytotoxic characteristics. Additionally, the CD56, CD16, inhibitory KIRs, and activation receptors were conserved and present in a well-balanced manner. The study successfully developed a feeder cell line, K562.Clone1, that effectively promotes the expansion of NK cells ex vivo while maintaining their cytotoxic potential. This development could significantly contribute to the advancement of NK cell therapy, especially in Brazil.

Activated B cells suppress T-cell function through metabolic competition.

BACKGROUND: B cells play a pivotal role in regulating the immune response. The induction of B cell-mediated immunosuppressive function requires B cell activating signals. However, the mechanisms by which activated B cells mediate T-cell suppression are not fully understood. METHODS: We investigated the potential contribution of metabolic activity of activated B cells to T-cell suppression by performing in vitro experiments and by analyzing clinical samples using mass cytometry and single-cell RNA sequencing. RESULTS: Here we show that following activation, B cells acquire an immunoregulatory phenotype and promote T-cell suppression by metabolic competition. Activated B cells induced hypoxia in T cells in a cell-cell contact dependent manner by consuming more oxygen via an increase in their oxidative phosphorylation (OXPHOS). Moreover, activated B cells deprived T cells of glucose and produced lactic acid through their high glycolytic activity. Activated B cells thus inhibited the mammalian target of rapamycin pathway in T cells, resulting in suppression of T-cell cytokine production and proliferation. Finally, we confirmed the presence of tumor-associated B cells with high glycolytic and OXPHOS activities in patients with melanoma, associated with poor response to immune checkpoint blockade therapy. CONCLUSIONS: We have revealed for the first time the immunomodulatory effects of the metabolic activity of activated B cells and their possible role in suppressing antitumor T-cell responses. These findings add novel insights into immunometabolism and have important implications for cancer immunotherapy.

KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape.

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. II: CAR-T cell therapy for patients with CD19+ acute lymphoblastic leukemia.

Chimeric antigen receptor T (CAR-T) cell therapy is a novel therapeutic modality for acute lymphoblastic leukemia (ALL) with robust outcomes in patients with refractory or relapsed disease. At the same time, CAR-T cell therapy is associated with unique and potentially fatal toxicities, such as cytokine release syndrome (CRS) and neurological toxicities (ICANS). This manuscript aims to provide a consensus of specialists in the fields of Hematology Oncology and Cellular Therapy to make recommendations on the current scenario of the use of CAR-T cells in patients with ALL.

Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

Combining AFM13, a Bispecific CD30/CD16 Antibody, with Cytokine-Activated Blood and Cord Blood-Derived NK Cells Facilitates CAR-like Responses Against CD30+ Malignancies.

PURPOSE: Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation. EXPERIMENTAL DESIGN: We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood-derived NK cells was investigated in vitro and in vivo. RESULTS: We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo. CONCLUSIONS: We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

Challenges and opportunities in primary CNS lymphoma: A systematic review.

BACKGROUND: Historically, high-dose methotrexate (HD-MTX) plus consolidation chemotherapy and/or whole brain radiotherapy (WBRT) has been the gold standard on Primary Central Nervous System Lymphoma (PCNSL) management. We sought to examine and summarize the data, on clinical trial (CT) setting, investigating multi-modality treatment to PCNSL. METHODS: We performed a systematic review of electronic databases (Medline, EMBASE, Cochrane Database and clinicaltrials.gov) and a manual search to identify original PCNSL phase 2 and phase 3 CT from the last 10years. After a 4stage Prisma based selection process, 32 published (3 Randomized CT and 29 phases 2 CT) studies ultimately were selected for review. Four ongoing clinical trials found on clinicaltrial.gov were reviewed. Two investigators reviewed titles, abstracts, and articles independently. Two investigators abstracted data sequentially and evaluated each study independently. FINDINGS: Treatment of PCNSL requires a multidisciplinary approach. HD-MTX represents the most accepted standard of care induction therapy for newly diagnosed PCNSL. When HD-MTX is given with WBRT for consolidation delayed neurotoxicity can be an important complication, particularly in elderly patients. Studies have suggested that WBRT may be deferred until relapse without compromising survival and deferring WBRT may be the best approach in elderly patients. Results from dose-reduced WBRT and consolidative HD-Ara-C are encouraging. High-dose chemotherapy in combination with autologous stem cell transplantation (HDC-ASCT) as chemotherapy alone has emerged as an important consolidative treatment for selected population. The optimal salvage therapy is still to be defined. CONCLUSION: WBRT for consolidation is a well-studied modality; however emerging options to selected population such as HDC-ASCT, dose-reduced WBRT or chemotherapy alone are associated with similar survival outcome and less neurotoxicity in selected series. Ongoing and future clinical trials will better define the best approach on this rare disease.

Effect of isolation rearing on the expression of AMPA glutamate receptors in the hippocampal formation.

Reduced glutamatergic signaling may contribute to cognitive dysfunction in schizophrenia. Glutamatergic synapses might be the site of primary abnormalities in this disorder with the dopaminergic changes being secondary to altered glutamatergic transmission. Isolation rearing of rats from weaning has been used as an experimental model for affective disorders like schizophrenia. In this immunohistochemistry study we evaluate the changes in the expression of GluR1 and GluR2 AMPA receptors in the hippocampus, amygdala and entorhinal cortex induced by isolation rearing. Two groups of Wistar rats (grouped and isolated, n = 6/each) were used. Isolation rearing induced a significant decrease in GluR1- and GluR2-immunopositive cells in the hippocampus. For GluR1 the reduction was 31% in the hilus of dentate gyrus (p = 0.02) and 47% in CA3 (p = 0.002). For GluR2 the reduction was 52% in the hilus of dentate gyrus (p < 0.0001) and 29% in CA1 (p = 0.002). Isolation rearing induced a non-significant decrease in GluR1-immunopositive cells in the basolateral amygdala (p = 0.066) while no alteration was found in the lateral nucleus (p = 0.657). For GluR2 no changes were induced by isolation in both nuclei of the amygdala. In the entorhinal cortex no apparent difference was seen in GluR1- or GluR2-immunopositive cells when isolated reared rats were compared to grouped rats. The results suggest that isolation rearing from weaning induces changes on the expression of AMPA glutamate receptors in the hippocampus similar to those reported for postmortem human brains with schizophrenia. These findings also contribute to additional evidence for using isolation rearing of rats from weaning as an animal model for schizophrenia.