Immune reconstitution dynamics after unrelated allogeneic transplantation with post-transplant cyclophosphamide compared to classical immunosuppression with anti-thymocyte globulin: a prospective cohort study.

Post-transplant cyclophosphamide has contributed to the success of haploidentical hematopoietic stem cell transplantation (HSCT) and is also been used in transplantation from matched donors. However, limited data on the immune reconstitution after this type of immunosuppression is available. We aimed to evaluate immune reconstitution after HSCT from unrelated donors, comparing anti-thymocyte globulin (ATG) and post-transplant cyclophosphamide (PTCy). Consecutive patients undergoing HSCT from unrelated donors and receiving either ATG or PTCy were prospectively included. Immune reconstitution analyses were performed by flow cytometry pre transplant and on days 30, 60, 90, and 180 post-transplant. We included 36 patients, 20 in the ATG group and 16 in the PTCy group. In the early post-transplant period (D+30), the ATG group showed a higher number of total lymphocytes, T, B, and NK cells compared to the PTCy group. However, at D+180, the PTCy group exhibited a higher number of B cells. On D+60 and D+90, the ATG group displayed higher number of NK cells CD56dim compared to the PTCy group, while on D+180, the PTCy group showed higher number of CD56neg, CD16pos, and, NKG2Dpos NK cells. Naive CD4+, transition CD4+, and naive CD8+ T cells on D+60 were identified as risk factors for acute graft-versus-host disease (GVHD) grades II-IV, and a higher count of CD4+ memory cells on D+180 was identified as a risk factor for chronic GVHD. In the context of unrelated allogeneic transplantation, immunosuppression with PTCy was associated with later B, T and NK cells reconstitution compared to ATG.

Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors.

BACKGROUND: Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in B-cell cancers. However, CAR T cells can induce substantial toxic effects, and the manufacture of the cells is complex. Natural killer (NK) cells that have been modified to express an anti-CD19 CAR have the potential to overcome these limitations. METHODS: In this phase 1 and 2 trial, we administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkin's lymphoma or chronic lymphocytic leukemia [CLL]). NK cells were transduced with a retroviral vector expressing genes that encode anti-CD19 CAR, interleukin-15, and inducible caspase 9 as a safety switch. The cells were expanded ex vivo and administered in a single infusion at one of three doses (1×105, 1×106, or 1×107 CAR-NK cells per kilogram of body weight) after lymphodepleting chemotherapy. RESULTS: The administration of CAR-NK cells was not associated with the development of cytokine release syndrome, neurotoxicity, or graft-versus-host disease, and there was no increase in the levels of inflammatory cytokines, including interleukin-6, over baseline. The maximum tolerated dose was not reached. Of the 11 patients who were treated, 8 (73%) had a response; of these patients, 7 (4 with lymphoma and 3 with CLL) had a complete remission, and 1 had remission of the Richter's transformation component but had persistent CLL. Responses were rapid and seen within 30 days after infusion at all dose levels. The infused CAR-NK cells expanded and persisted at low levels for at least 12 months. CONCLUSIONS: Among 11 patients with relapsed or refractory CD19-positive cancers, a majority had a response to treatment with CAR-NK cells without the development of major toxic effects. (Funded by the M.D. Anderson Cancer Center CLL and Lymphoma Moonshot and the National Institutes of Health; ClinicalTrials.gov number, NCT03056339.).

Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

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.

Risk factors for adverse outcomes following haploidentical hematopoietic cell transplantation with posttransplant cyclophosphamide: a two-center analysis.

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative therapy for several malignant hematologic diseases and alternative donors, including haploidentical, play a significant role in HCT. Despite the increasing use of haplo-HCT with PTCy, some questions remain open. The objective of the present study was to investigate risk factors for adverse outcomes after haplo-HCT with PTCy. This is a retrospective study conducted at two Brazilian centers. A total of 103 patients with hematologic malignancies who underwent first allogeneic, haploidentical HCT with PTCy were included. Risk factors for death were age at transplant (HR = 1.03 for each year; p = 0.002) and high/very high disease risk index (DRI; HR = 2.77; p = 0.0007) and mother as the donor compared with other donors (HR = 3.53; p = 0.005). In multivariate analysis, PFS was significantly poorer for older patients (HR = 1.02; p = 0.006), high/very high DRI (HR = 2.39; p = 0.003), and mother as the donor compared with other donors (HR = 3.18; p = 0.006). Relapse rate was higher for high/very high DRI (HR = 4.01; p = 0.002) and mother as the donor compared with other donors (HR = 2.52; p = 0.05). NRM was higher for older patients (HR = 1.03 for each year; p = 0.03). Tacrolimus was a protective factor for grades II-IV aGVHD (HR = 0.46; p = 0.04) compared with cyclosporine. Peripheral blood (PBSC) was a risk factor for cGVHD (HR = 3.48; p = 0.006), while tacrolimus was protective (HR = 0.30; p = 0.009). Mother as the donor compared with other donors was also a risk factor for poorer OS, PFS, and relapse, suggesting that this combination should be avoided. Tacrolimus was protective for both grades II-IV aGVHD and cGVHD, suggesting that tacrolimus may be more effective than cyclosporine in preventing GVHD. PBSC was a risk factor for cGVHD without any impact on relapse. Prospective studies comparing tacrolimus with cyclosporine are awaited.

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.

Severe hemorrhagic cystitis caused by the BK polyomavirus is associated with decreased survival post-allogeneic hematopoietic stem cell transplantation.

BACKGROUND: BK polyomavirus reactivation can occur following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may lead to hemorrhagic cystitis (BKPyV-HC). We hypothesized that development of BKPyV-HC is associated with increased mortality post allo-HSCT. METHODS: We retrospectively reviewed data on 133 adult patients (≥18 years old) who underwent allo-HSCT from 2007 until 2014 at Hospital Israelita Albert Einstein in São Paulo, Brazil. RESULTS: Thirty-six patients presented with BKPyV-HC after a median time of 42 days, with a 1-year cumulative incidence probability of 28.9% (95% CI 21.5%-36.7%). In a multivariate Cox model, risk factors for development of BKPyV-HC included younger age, male sex, development of grade 2-4 acute graft-versus-host disease and recipients of umbilical cord blood grafts. Development of grade 3-4 BKPyV-HC (but not grade 1-2) was associated with a decreased overall survival (OS) in a multivariate Cox model (hazard ratio [HR] 7.51, P < 0.0001) and an increased risk of TRM (HR 3.66, P < 0.0001). Grade 3-4 BKPyV-HC was also associated with an increased risk of relapse that did not reach statistical significance (HR 3.01, P = 0.07). Median overall survival (OS) post-BKPyV-HC was 4.7 months, and cidofovir had no impact on survival. CONCLUSION: Development of BKPyV-HC appears to be associated with decreased survival following allo-HSCT.

Clinical outcomes in allogeneic haematopoietic stem cell transplantation: A comparison between young and elderly patients. Observational study.

OBJECTIVES: To analyse clinical outcomes comparing two age groups of patients undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT), and to identify risk factors associated with older patients' mortality. METHODS: In this retrospective study, the medical charts of all consecutive patients admitted in one hospital for allo-HSCT were reviewed. Overall survival (OS) and other outcomes were compared between patients aged up to 55 years (YG) and older than 55 (EG). RESULTS: From January 2007 to August 2014, 111 adult patients were admitted for allo-HSCT and were included 75 in the YG and 36 in the EG group. The OS rate at D+ 100 was 84% for YG individuals in contrast to 75% in the EG (p = 0.01), and 71% vs. 50% at one year after HSCT (p = 0.01) respectively. Therapy-related mortality (TRM) rates for the YG and EG were, respectively, 14% vs. 17% (p = 0.04) at D+ 100 and 17% vs. 32% (p = 0.04) at one year. Haploidentical donor type and active disease status significantly increased mortality risk in the EG (hazard ratio 2.42; p = 0.018; and 2.04; p = 0.033). CONCLUSION: YG and EG have similar TRM rates early after allo-HSCT, but the elderly had higher TRM during the critical period from 100 days to one year.

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.

BATF is a major driver of NK cell epigenetic reprogramming and dysfunction in AML.

Myelodysplastic syndrome and acute myeloid leukemia (AML) belong to a continuous disease spectrum of myeloid malignancies with poor prognosis in the relapsed/refractory setting necessitating novel therapies. Natural killer (NK) cells from patients with myeloid malignancies display global dysfunction with impaired killing capacity, altered metabolism, and an exhausted phenotype at the single-cell transcriptomic and proteomic levels. In this study, we identified that this dysfunction was mediated through a cross-talk between NK cells and myeloid blasts necessitating cell-cell contact. NK cell dysfunction could be prevented by targeting the αvß-integrin/TGF-ß/SMAD pathway but, once established, was persistent because of profound epigenetic reprogramming. We identified BATF as a core transcription factor and the main mediator of this NK cell dysfunction in AML. Mechanistically, we found that BATF was directly regulated and induced by SMAD2/3 and, in turn, bound to key genes related to NK cell exhaustion, such as HAVCR2, LAG3, TIGIT, and CTLA4. BATF deletion enhanced NK cell function against AML in vitro and in vivo. Collectively, our findings reveal a previously unidentified mechanism of NK immune evasion in AML manifested by epigenetic rewiring and inactivation of NK cells by myeloid blasts. This work highlights the importance of using healthy allogeneic NK cells as an adoptive cell therapy to treat patients with myeloid malignancies combined with strategies aimed at preventing the dysfunction by targeting the TGF-ß pathway or BATF.

Need for hemodialysis in patients undergoing hematopoietic stem cell transplantation: risk factors and survival in a retrospective cohort.

INTRODUCTION: Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) patients are exposed to acute and chronic nephrotoxic events (drugs, hypotension, infections, and microangiopathy). The need for hemodialysis (HD) may be associated with high mortality rates. However, the risk factors and clinical impact of HD are poorly understood. AIM: To analyze survival and risk factors associated with HD in allo-HSCT Patients and methods: single-center cohort study 185 (34 HD cases versus 151 controls) consecutive adult allo-HSCT patients from 2007-2019. We performed univariate statistical analysis, then logistic regression and competing risk regression were used to multivariate analysis. Survival was analyzed by Kaplan-Meier and Cox proportional-hazards models. RESULTS: The one-year HD cumulative incidence was 17.6%. Univariate analysis revealed that HD was significantly associated with male gender, age (p 0.056), haploidentical donor, grade II-IV acute GVHD, polymyxin B, amikacin, cidofovir, microangiopathy, septic shock (norepinephrine use) and steroid exposure. The median days of glycopeptides exposure (teicoplanin/vancomycin) was 16 (HD) versus 10 (no HD) (p 0.088). In multivariate analysis, we found: norepinephrine (hazard ratio, HR:3.3; 95% confidence interval, 95%CI:1.2-8.9; p 0.024), cidofovir drug (HR:11.0; 95%CI:4.6- 26.0; p < 0.001), haploidentical HSCT (HR:1.94; 95%CI:0.81-4.65; p 0.14) and Age (HR:1.01; 95%CI: 0.99-1.03; p 0.18) . The HD group had higher mortality rate (HR:6.68; 95% CI: 4.1-10.9; p < 0.001). CONCLUSION: HD was associated with decreased survival in allo-HSCT. Carefully use of nephrotoxic drugs and improving immune reconstitution could reduce severe infections (shock) and patients requiring cidofovir, which taken together may result in lower rates of HD, therefore improving survival.

ATG in HLA-Matched, Peripheral Blood, Hematopoietic Cell Transplantation in Acute Myeloid Leukemia and Myelodysplastic Syndrome: A Secondary Analysis of a CIBMTR Database.

w?>Peripheral blood stem cells (PBSC) are the preferred grafts for hematopoietic cell transplantation (HCT), according to the CIBMTR. Donor recovery is faster with PBSC harvest, but PBSC is associated with higher chronic graft-versus-host disease (GVHD) and poorer quality of life. Anti-T-cell globulin (ATG) is polyclonal IgG from rabbits or horses immunized with human thymocytes or a human T-cell line, which may reduce GVHD in HCT and improve outcomes. The objective of this study was to analyze the impact of ATG in HLA-matched related (MRD) and matched (HLA 8/8) unrelated donor (MUD) HCT. We used a freely available CIBMTR database published online for secondary analyses. The database included patients ≥ 40 years old who have undergone their first PBSC MRD or MUD HCT for acute myeloid leukemia or myelodysplastic syndrome with or without ATG between 2008 and 2017. Patients who received posttransplant cyclophosphamide or alemtuzumab were excluded. Overall survival was not different with ATG (hazard ratio [HR] = 1.09; 95% confidence interval [CI], 1.00-1.19; P = .06) compared with no ATG. Relapse rate was higher with ATG (HR = 1.29; 95% CI, 1.17-1.43; P < .001) and non-relapse mortality was lower with ATG (HR = 0.84; 95% CI, 0.72-0.98; P = .03). Grades II-IV acute GVHD was significantly lower with ATG (HR = 0.77; 95% CI, 0.69-0.87; P < .001) but not grades III-IV acute GVHD (HR = 0.85; 95% CI, 0.69-1.04; P = .11). Both chronic GVHD (HR = 0.54; 95% CI, 0.48-0.60; P < .001) and moderate/severe chronic GVHD (HR = 0.45; 95% CI, 0.38-0.52; P < .001) were lower with ATG. There was an interaction between ATG and conditioning regimen for relapse rate and overall survival. Relapse rate was higher in those who received reduced-intensity (RIC) or non-myeloablative (NMA) conditioning regimens and ATG, compared with MAC ± ATG or RIC without ATG (interaction test, P = .003). Overall survival was also poorer with ATG and RIC or NMA conditioning regimens (interaction test, P = .03). Our results show that ATG can mitigate the more severe forms of chronic GVHD without impairing overall survival in HLA-matched HCT with PBSC grafts and myeloablative conditioning regimen. ATG should be standard in this population. © 2023 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Cardiac iron overload evaluation in thalassaemic patients using T2* magnetic resonance imaging following chelation therapy: a multicentre cross-sectional study.

INTRODUCTION: Magnetic resonance imaging (MRI) T2* technique is used to assess iron overload in the heart, liver and pancreas of thalassaemic patients. Optimal iron chelation and expected tissue iron response rates remain under investigation. The objective of this study was to analyse serum ferritin and the iron concentration in the heart, liver and pancreas measured by MRI T2*/R2* during regular chelation therapy in a real-world cohort of patients with thalassemia. METHODS: We evaluated thalassaemic patients ≥ 7 years old undergoing chelation/transfusion therapy by MRI and assessed serum ferritin at baseline and follow-up from 2004-2011. RESULTS: We evaluated 136 patients, 92% major thalassaemic, with a median age of 18 years, and median baseline ferritin 2.033ng/ml (range: 59-14,123). Iron overload distribution was: liver (99%), pancreas (74%) and heart (36%). After a median of 1.2 years of follow-up, the iron overload in the myocardium reduced from 2,63 Fe mg/g to 2,05 (p 0.003). The optimal R2* pancreas cut-off was 148 Hertz, achieving 78% sensitivity and 73% specificity. However, when combining the R2* pancreas cut off ≤ 50 Hertz and a ferritin ≤ 1222 ng/ml, we could reach a negative predictive value (NPV) of 98% for cardiac siderosis. Only 28% were undergoing combined chelation at baseline assessment, which increased up to 50% on follow up evaluation. CONCLUSIONS: Chelation therapy significantly reduced cardiac siderosis in thalassaemic patients. In patients with moderate/severe liver iron concentration undergoing chelation therapy, ferritin levels and myocardium iron improved earlier than the liver siderosis.

Molecular profile of patients with myelofibrosis: a 10-year experience.

OBJECTIVE: To analyze the karyotype test and myeloid panel with next-generation sequencing findings in patients with myelofibrosis, and to compare transplant characteristics in patients referred for bone marrow transplantation. METHODS: Retrospective, single-center study with patients diagnosed with myelofibrosis treated at Hospital Israelita Albert Einstein between 2010 and 2020. RESULTS: A total of 104 patients with myelofibrosis were examined. Patients who had not been submitted to tests in our service were excluded. The final sample comprised 69 patients. Of these 69, 56 were submitted to karyotyping and 22 to myeloid panel with next-generation sequencing. Karyotype was normal in 60% of the patients and altered in 40%. The prevalence of high-risk molecular mutations was higher in patients referred for bone marrow transplantation (100% versus 50%). The median follow-up of transplant patients was 2.4 years and the overall survival at 2 years was 80% (95%CI: 62-100%). CONCLUSION: The molecular analysis enables estimating the patient's risk and thus instituting more aggressive treatment such as bone marrow transplant for patients at higher risk, being a relevant tool to guide therapy. Given the significance of molecular analysis for therapeutic decision-making in myelofibrosis, collection and disclosure of data on the prevalence of cytogenetic changes and findings of next-generation sequencing in affected patients is important.

Targeted Marrow Irradiation Intensification of Reduced-Intensity Fludarabine/Busulfan Conditioning for Allogeneic Hematopoietic Stem Cell Transplantation.

Reduced-intensity conditioning (RIC) regimens frequently provide insufficient disease control in patients with high-risk hematologic malignancies undergoing allogeneic hematopoietic stem cell transplantation (HSCT). We evaluated intensification of fludarabine/busulfan (Flu/Bu) RIC with targeted marrow irradiation (TMI) in a dose escalation with expansion phase I clinical trial. TMI doses were delivered at 1.5 Gy in twice daily fractions on days -10 through -7 (dose levels: 3 Gy, 4.5 Gy, and 6 Gy), Flu (30 mg/m2 for 5 days) and Bu (area under the curve, 4800 µM*minute for 2 days). Eligible patients were age ≥18 years with high-risk hematologic malignancy and compromised organ function ineligible for myeloablative transplantation (n = 26). The median patient age was 64 years (range, 25 to 76 years). Nineteen patients (73%) had active or measurable residual disease at transplantation. One-year disease-free survival and overall survival were 55% (95% confidence interval [CI], 34% to 76%) and 65% (95% CI, 46% to 85%), respectively. Day +100 and 1 year transplantation-related mortality were 4% (95% CI, 0.6% to 27%) and 8.5% (95% CI, 2% to 32%), respectively. The 1-year cumulative incidence of relapse was 43% (95% CI, 27% to 69%). Rates of grade II-IV and III-IV acute GVHD rates were 57% (95% CI, 39% to 84%) and 22% (95% CI, 9% to 53%), respectively. Whole blood immune profiling demonstrated enrichment of central/transitional memory-like T cells with higher TMI doses, which correlated with improved survival compared with control samples from patients undergoing allogeneic HSCT. Intensification of a Flu/Bu RIC regimen with TMI is feasible with a low incidence of transplantation-related mortality in medically frail patients with advanced malignancies. The recommended phase 2 TMI dose is 6 Gy.

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.

GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A- and HLA-B- K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

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.