Cell Infusion Research Articles

Abstract 3636: Myeloid cell rebound associated with GD2-CAR-T cell therapy in solid tumor patients

Abstract GD2 is a validated cancer immunotherapy target with highly restricted normal tissue expression. Promising activity of GD2-CAR-T cell therapy in diffuse midline glioma and neuroblastoma has promoted interest in this therapy. We initiated three phase 1 clinical trials of escalating doses of intravenously administered autologous GD2-CAR-T cells in patients with advanced GD2-positive solid tumors. Emerging evidence suggests that CAR-T cells may increase circulating myeloid derived suppressor cells (MDSCs) and that this event may be mediated and/or triggered by CAR-T cell-derived cytokines and chemokines. These findings may help to explain the limited persistence of CAR-T cell therapy in solid tumor patients. Hence, we sought evidence of MDSC generation and secretion of relevant cytokines and chemokines in our clinical trial patients. The CARPETS trial (www.anzctr.org.au: ACTRN12620000622909) recently closed to recruitment after enrolling 12 patients (7 melanoma, 4 colorectal cancer, and 1 sarcoma). No significant adverse events were attributable to CAR-T cell therapy. We opened two other GD2-CAR-T cell therapy trials in 2023: LEVI’S-CATCH (ACTRN12622000675729) in patients with diffuse midline glioma at Sydney Children’s Hospital and KARPOS (ACTRN12622001514796) in adult patients with recurrent glioblastoma at Royal Adelaide Hospital. To date, 3 patients have enrolled to LEVI’S-CATCH with the first patient clearing the dose limiting (DLT) evaluation period. Three patients completed enrolment to the first dose cohort of KARPOS without DLTs. In all CARPETS patients, CAR-T cell expansion, which was measured by by DNA quantitative PCR and flow cytometry, peaked during the 6-week post-CAR-T infusion DLT evaluation period. In 6 CARPETS patients for whom peripheral blood samples were available, we found by flow cytometry, newly formed or increased size of pre-existing subpopulations of non-classical monocytes and/or MDSCs of mononuclear and/or polymorphonuclear class, which peaked at or after the time of peak CAR-T cell expansion. Typically, serum levels of inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL2, GM-CSF, and CXCL10, were detected only after the CAR-T cell infusion with variable patterns of the timing and concentration of analyte observed among the 12 patients. At the time of writing, blood samples from the first KARPOS patient showed CAR-T cell expansion and the subsequent increases in circulating myeloid derived suppressor cells and inflammatory cytokines. We expect that full data sets for these parameters will be presented for current and future enrolled patients to both studies. To improve CAR-T cell persistence in solid tumor patients, these data indicate the need for new therapeutic strategies to mitigate effects of CAR-T cell induced myeloid cells with antitumor activity. Citation Format: Michael P. Brown, Tessa Gargett, Nga T. Truong, Orazio Vittorio, David S. Ziegler. Myeloid cell rebound associated with GD2-CAR-T cell therapy in solid tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3636.

Abstract 3610: Utilizing fructose metabolism to fuel anti-tumor immunity

Abstract Consumption of fructose in the form of high fructose corn syrup is elevated in the western diet, but it is not fully understood how fructose metabolism influences anti-tumor immunity. Fructose is canonically metabolized in the intestine and the liver and can shunt into glycolysis. In certain tumor types, fructose metabolism becomes deregulated, allowing tumor cells to metabolize fructose to power their biosynthetic processes. Similarly, reprogramming immune cells to be able to efficiently metabolize fructose may help us power immunotherapy to work in difficult to treat cancers, especially in individuals who have high fructose in their blood or in the tumor microenvironment (TME). Imparting effector T cells with the ability to use fructose for fuel can overcome one of the major metabolic limitations of the TME: limited nutrient supply. As T cells become activated, they alter their metabolism to support growth and proliferation. Activated CD8+ T cells upregulate their glycolysis which results in increased biosynthetic processes, such as synthesis of macromolecules and effector cytokines. However, tumor cells also consume substantial amounts of glucose, resulting in a glucose-depleted microenvironment, which could be one of the major barriers to immunotherapy working in solid tumors. Therefore, we engineered T cells to overexpress a fructose transporter, GLUT5, which is normally not expressed in the immune compartment at high levels. We hypothesized that increased fructose uptake by CD8+ T cells will ameliorate exhaustion caused by the glucose-low tumor microenvironment. Indeed, we determined that GLUT5 T cells are more efficient in killing in low glucose supplemented with fructose. We also observed that GLUT5 expression restores T cell glycolytic capacity under fructose to that of unexhausted T cells under glucose replete conditions. Similarly, GLUT5-expressing T cells proliferate faster in vitro and exhibit an improved ability to control B16 tumor growth in vivo. Additionally, combining GLUT5 cell infusion with checkpoint inhibition resulted in durable tumor control and prolonged mouse survival. Further, engineered CD19-targeted CAR T cells with expression of GLUT5 were more metabolically active and cytotoxic in low glucose conditions, mimicking the TME. In conclusion, GLUT5 expression in effector CD8+ and CAR T cells may allow immunotherapy to function in solid tumors under low glucose conditions. Citation Format: Tanya Schild, Rachel Chirayil, Lyric Haughton, Patrick Wallisch, Marjan Berishaj, David A. Scheinberg, Kayvan R. Keshari. Utilizing fructose metabolism to fuel anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3610.

Abstract 3997: PARP inhibitors plus anlotinib as bridging therapy for TGFβ-insensitive CAR-T cell therapy targeting MSLN and CD19 in advanced ovarian cancer

Abstract Background: The efficacy of chimeric antigen receptor (CAR) T cell therapy remains modest for the treatment of solid tumors due to restricted T cell infiltration and immunosuppressive tumor microenvironment (TME). Leveraging the window of opportunity between leukapheresis and infusion using bridging therapy might improve TME and potentiate CAR-T cell therapy. Here, we assessed the potential of poly (ADP-ribose) polymerase (PARP) inhibitors plus anlotinib as bridging therapy for CAR-T cells with resistance to TGFβ and capability of rapid expansion by bait-and-switch strategy of dual-targeting mesothelin (MSLN) and CD19 in preclinical models of advanced ovarian cancer. Methods: Immunocompetent mice bearing ID8 tumors were treated with niraparib (21 days) plus anlotinib (14 days) and euthanized at different timepoints (0, 7, or 14 days since discontinuation) to quantify tumor-infiltrating T cells and profile T cell functions. CAR-T cells transfected with dominant-negative TGFβRII dual-targeting MSLN and CD19 were produced by IASO Biotherapeutics. NSG mice bearing MSLN-positive cell- or patient-derived xenografts (CDX or PDX) of ovarian cancer received CAR-T cells alone or after bridging therapy (niraparib plus anlotinib followed by a wash-out period of 7 days). Tumor volume was balanced before CAR-T cell infusion. Replication-deficient NALM6 cells pre-treated with mitomycin C were injected to provide CD19 antigen. A PET scan that visualized granzyme B release was used to track CAR-T cells and assess immune-mediated tumor killing. Results: Niraparib combined with anlotinib increased tumor-infiltrating T cells without impairing T cell functionality. The effects of promoting T cell infiltration remained significant at 14 days after discontinuation, which might associate with lasting activation of cGAS-STING pathway and secretion of downstream chemokines (CXCL10 and CCL5), and normalized tumor vasculature featured by better perfusion and reduced leakage. The engineered CAR-T cells could resist TGFβ and displayed improved proliferation, cytotoxicity, cytokine release, and in vivo antitumor activities upon CD19 stimulation. Importantly, bridging therapy significantly increased CAR-T cell infiltration, curbed tumor growth, and prolonged survival in mice bearing SKOV3 CDX, HRD-negative PDX, and another multidrug-resistant PDX. PET imaging showed that bridging therapy increased tumor-infiltrating CAR-T cells and boosted tumor-killing capability. Conclusions: PARP inhibitors plus anlotinib as bridging therapy facilitated CAR-T cell infiltration and enhanced antitumor activities in multiple preclinical models of advanced ovarian cancer, and an early phase I trial (NCT05141253) is ongoing to evaluate PARP inhibitors plus anlotinib as bridging therapy for CAR-T cell therapy in patients with refractory MSLN-positive ovarian cancer. Citation Format: Huayi Li, Minghua Xiang, Jiahao Liu, Wei Mu, Li Zhu, Xuejiao Zhao, Gordon B. Mills, Qinglei Gao, Yong Fang. PARP inhibitors plus anlotinib as bridging therapy for TGFβ-insensitive CAR-T cell therapy targeting MSLN and CD19 in advanced ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3997.

The role of TIM3+ NK and TIM3- NK cells in the immune pathogenesis of severe aplastic anemia.

Natural killer (NK) cells play important immunoregulatory roles in the immune pathogenesis of severe aplastic anemia (SAA). Our previous research showed that SAA caused a decrease in T cell immunoglobulin mucin-3 (TIM3) expression on NK cells. Here we investigated the expression of surface receptors, and the cytotoxicity of peripheral TIM3+ NK and TIM3- NK cells in patients with SAA. The expressions of surface receptors and cytoplasmic protein of TIM3+ NK and TIM3- NK cells from peripheral blood were detected by FCM. The functions of mDCs, and apoptosis rate of K562 cells after co-culture with TIM3+ NK and TIM3- NK cells were maesured by FCM. Westren-blot was used to detect the changes of TIM3+ NK and TIM3- NK signaling pathway proteins (AKT, P-AKT) and compare the functional activity of the two groups. Activating receptors NKG2D and Granzyme B were higher, while inhibiting receptors NKG2A, CD158a and CD158b were lower on TIM3- NK cells compared with TIM3+ NK cells in patients with SAA. In SAA, the expression of CD80 and CD86 on mDCs (Myeloid dendritic cells) was significantly decreased after incubation with TIM3- NK cells. The apoptosis rate (AR) of K562 cells was significantly increased after being incubated with TIM3- NK cells in SAA. The level of signal pathway protein AKT of TIM3- NK cells in SAA was similar to that of TIM3+ NK cells, and the levels of P-AKT and P-AKT/AKT ratio of TIM3- NK cells were significantly higher than those of TIM3+ NK cells. Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell activity which in turn inhibits the immune activation state of SAA and improves the disease state. Our research may aid the development of new therapeutic strategies based on TIM3-NK cells infusion for the treatment of SAA.

Incidence of Catheter-Associated Bloodstream Infections in Stem Cell Recipients-Should We Be "PICCy"?

The management of patients undergoing HSCT requires a multipurpose central venous catheter. Peripheral catheters (PCs), such as peripherally inserted central catheters (PICCs) and MidLine catheters (MLCs), appear to be adequate vascular catheters to be used for stem cell infusion, although their utilization in this indication is not yet common. We analyzed the infectious complications such as blood stream infection (BSI), febrile neutropenia (FN) and central line-associated bloodstream infection (CLBSI) in patients undergoing stem cell infusion through PC and conventionally inserted central catchers (CICCs), and evaluated their impacts on transplantation outcomes. Our results reveal no statistically significant differences between different types of catheter in terms of FN, BSI and CLABSI. Moreover, transplantation outcomes were comparable between the groups. Interestingly, according to our data, there were no differences in terms of abovementioned infectious complications between individuals who received antibiotic prophylaxis and those who did not. Our study has shown that infection complications are independent of the intravenous device and antibiotic prophylaxis. Considering that PCs are not associated with life-threatening complications, they should be considered more frequently in the stem cell transplantation setting.

Coupling Osmotic Efficacy with Biocompatibility in Peritoneal Dialysis: A Stiff Challenge.

Peritoneal dialysis (PD) is a home-based efficacious modality for the replacement of renal function in end-stage kidney failure patients, but it is still under-prescribed. A major limitation is the durability of the dialytic technique. Continuous exposure of the peritoneum to bioincompatible conventional glucose-based solutions is thought to be the main cause of the long-term morpho-functional peritoneal changes that eventually result in ultrafiltration failure. Poor PD solution biocompatibility is primarily related to the high glucose content, which is not only detrimental to the peritoneal membrane but has many potential metabolic side effects. To improve the clinical outcome and prolong the survival of the treatment, PD-related bioincompatibility urgently needs to be overcome. However, combining dialytic and osmotic efficacy with a satisfactory biocompatible profile is proving to be quite difficult. New approaches targeting the composition of the PD solution include the replacement of glucose with other osmotic agents, and the addition of cytoprotective or osmo-metabolic compounds. Other strategies include the infusion of mesenchymal cells or the administration of orally active agents. In the present article, we review the current evidence on efforts to improve the biocompatible and functional performance of PD, focusing on studies performed in vivo (animal models of PD, human subjects on PD).

Serum cytokine profiles during engraftment syndrome and acute graft-versus-host disease in adult patients after hematopoietic stem cell transplantation

BackgroundThe underlying biology of engraftment syndrome (ES) following allogeneic hematopoietic stem cell transplantation (HSCT) is not fully elucidated, and the extent of its overlap with acute graft-versus-host disease (aGvHD) remains unclear. In order to establish potential indicator to distinguish ES more accurately, we conducted a retrospective analysis of cytokine levels during HSCT. MethodsA total of 121 consecutive adult patients who underwent HSCT were enrolled in this study. Blood samples for interleukin (IL)-2, IL-2R, IL-4, IL-5, IL-6, IL-8, IL-10, IL-1β, IL-12p70, interferon (IFN)-γ, IFN-α, tumor necrosis factor alpha (TNF-α) and C-reactive protein CRP were regularly assessed after transplantation and during transplantation related adverse events. Additionally, the balance of naïve, central memory and effector memory of CD4+ and CD8+ was analyzed around 30 and 60 days after stem cell infusion, respectively. ResultsThirty (24.79 %) and 33 (27.27 %) patients were diagnosed with ES and aGvHD, respectively. ES was characterized by a significant increase in level of IL-5, IL-6, IL-8 and sIL-2R, while aGvHD was associated with a significant upregulation of IL-6, IL-5, IL-10 and sIL-2R in the patients from grade I to grade IV. Notably, patients got much higher levels of IL-6, IL-5 and sIL-2R when developed to ES than to aGvHD. Moreover, a pronounced shift from naïve to memory cells, both in CD4+ and CD8+ subsets, was found in ES patients. ConclusionsThese findings suggest that cytokine profiles could serve as potential indicators for detecting and differentiating ES and aGvHD, enabling timely clinical intervention. Prospective clinical trials involving larger, independent patient cohorts are required to validate these observations.

Bioreactor-based stem cell therapy for liver fibrosis

Stem cell therapy, achieved using mesenchymal stem cells (MSCs), has been highlighted for the treatment of liver fibrosis. Infusion into the circulatory system is a traditional application of MSCs; however, this approach is limited by phenotypic drift, stem cell senescence, and vascular embolism. Maintaining the therapeutic phenotype of MSCs while avoiding adverse infusion-related reactions is the key to developing next-generation stem cell therapy technologies. Here, we propose a bioreactor-based MSCs therapy to avoid cell infusion. In this scheme, 5% liver fibrosis serum was used to induce the therapeutic phenotype of MSCs, and a fluid bioreactor carrying a co-culture system of hepatocytes and MSCs was constructed to produce the therapeutic medium. In a rat model of liver fibrosis, the therapeutic medium derived from the bioreactor significantly alleviated liver fibrosis. Therapeutic mechanisms include immune regulation, inhibition of hepatic stellate cell activation, establishment of hepatocyte homeostasis, and recovery of liver stem cell subsets. Overall, the bioreactor-based stem cell therapy (scheme) described here represents a promising new strategy for the treatment of liver fibrosis and will be beneficial for the development of ‘cell-free’ stem cell therapy.

Early CAR- CD4+ T-lymphocytes recovery following CAR-T cell infusion: A worse outcome in diffuse large B cell lymphoma.

CAR- CD4+ T cell lymphopenia is an emerging issue following CAR-T cell therapy. We analyzed the determinants of CD4+ T cell recovery and a possible association with survival in 31 consecutive patients treated with commercial CAR-T for diffuse large B-cell (DLBCL) or mantle cell lymphoma. Circulating immune subpopulations were characterized through multiparametric-flow cytometry. Six-month cumulative incidence of CAR- CD4+ T cell recovery (≥200 cells/μL) was 0.43 (95% confidence interval [CI]: 0.28-0.65). Among possible determinants of CD4+ T cell recovery, we recognized infusion of a 4-1BB product (tisagenlecleucel, TSA) in comparison with a CD28 (axicabtagene/brexucabtagene, AXI/BRX) (hazard ratio [HR] [95% CI]: 5.79 [1.16-24.12] p=0.016). Higher CD4+ T cell counts resulted with TSA at month-1, -2 and -3. Moderate-to-severe infections were registered with prolonged CD4+ T cell lymphopenia. Early, month-1 CD4+ T cell recovery was associated with a worse outcome in the DLBCL cohort, upheld in a multivariate regression model for overall survival (HR: 4.46 [95% CI: 1.12-17.71], p=0.03). We conclude that a faster CAR- CD4+ T cell recovery is associated with TSA as compared to AXI/BRX. Month-1 CAR- CD4+ T cell subset recovery could represent a "red flag" for CAR-T cell therapy failure in DLBCL patients.

Early Chimeric Antigen Receptor T Cell Expansion Is Associated with Prolonged Progression-Free Survival for Patients with Relapsed/Refractory Multiple Myeloma Treated with Ide-Cel: A Retrospective Monocentric Study

The outcomes of patients with relapsed and refractory multiple myeloma (RRMM) previously treated with the 3 main classes of myeloma therapy-immunomodulatory drugs, proteasome inhibitors, and anti-CD38 antibodies-remain poor. Recently, based on the phase II pivotal KarMMa trial showing prolonged overall survival (OS) and progression-free survival (PFS) in heavily treated patients, idecabtagene vicleucel (ide-cel), a B cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cell therapy (CAR-T) product, was approved in the United States for the treatment of RRMM. In France, since June 2021, an early access program has authorized the use of ide-cel in the setting of RRMM (defined as progressive myeloma after at least 3 previous regimens, including the 3 main antimyeloma therapies). We report the first French experience through this early access program in a retrospective study of 24 consecutive patients treated with ide-cel at our institution. The patients were evaluated according to International Myeloma Working Group criteria and by positron emission tomography computed tomography (PET-CT) at 1, 3, 6, 9, and 12 months after ide-cel infusion. Most patients had adverse cytogenetic abnormalities, and RRMM with triple-refractory drugs were seen in 79%. Bridging therapy was required for 19 of 24 patients. Before CAR-T cell infusion, lymphodepletion with fludarabine and cyclophosphamide was systematically performed. The median follow-up was 15.2 months. At 3 months after ide-cel infusion, 92% of patients achieved at least a partial response, and 50% achieved a complete response or better (≥CR). At 6 months, 70% of patients had a persistent ≥CR. At 3 and 6 months, bone marrow minimal residual disease (10-6 level) was undetectable in 79% and 75% of patients, respectively. At 6 months, CR as assessed by PET-CT was achieved in 15 of 20 patients (75%). The median PFS was 14.8 months, and median OS was not reached. Notably, an expansion of circulating CAR-T cells to >180/mm3 after infusion was strongly associated with prolonged PFS. Additionally, the level of soluble BCMA measured before infusion was identified as a prognostic factor for PFS, likely correlated to the tumor burden. Grade 1-2 cytokine release syndrome (CRS) occurred in 22 of 24 patients (92%). Only 1 patient (4%) experienced grade ≥3 CRS. The occurrence of neurologic toxicity was infrequent (12.5%) and reversible in all cases. Hematologic toxicity was relatively common, and secondary hypogammaglobulinemia occurred in most patients. Infections (mostly viral) were frequent but most often nonsevere. This study echoes the promising results of the KarMMa trial and identifies possible prognostic indicators in RRMM patients treated with ide-cel, potentially refining treatment strategies and improving outcomes in this challenging context.

Indicators describing the tumor lesion aggregation and dissemination and their impact on the prognosis of patients with diffuse large B cell lymphoma receiving chimeric antigen receptor T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has markedly improved the prognosis of patients with diffuse large B-cell lymphoma (DLBCL). The relative positioning of tumor lesions in lymphoma varies among patients, manifesting as either aggregation (clumped together) or dissemination (spread throughout the body). Prognostic significance of factors indicating the relative positioning of tumor lesions in CAR T cell therapy remains underexplored. For aggregation, prior research proposed the tumor volume surface ratio (TVSR), linking it to prognosis in chemotherapy. Regarding dissemination, indicators such as disease stage or extranodal involvement, commonly used in clinical practice, have not demonstrated prognostic significance in CAR T cell therapy. This study aims to analyze current indicators of tumor aggregation or dissemination and introduce a novel indicator to assess the prognostic value of tumor lesions' relative positioning in DLBCL patients undergoing CAR T cell therapy. This retrospective study included 42 patients receiving CAR T cell therapy. Lesion image information was obtained from the last PET/CT scan prior to CAR T cell infusion, including total metabolic tumor volume, total tumor surface, diameter of lymphoma masses, and the sites of tumor lesions. We evaluated TVSR and bulky disease as descriptors of tumor aggregation. We refined existing indicators, stage III&IV and >1 site extranodal involvement, to distill a new indicator, termed 'extra stage', to better represent tumor dissemination. The study examined the prognostic significance of tumor aggregation and dissemination. Our findings indicate that TVSR, while prognostically valuable in chemotherapy, lacks practical prognostic value in CAR T cell therapy. Conversely, bulky disease emerged as an optimal prognostic indicator of tumor aggregation. Both bulky disease and extra stage were associated with poor prognosis and exhibiting synergistic prognostic impact in CAR T cell therapy. Overall, the relative positioning of tumor lesions significantly influences the prognosis of patients with DLBCL receiving CAR T cell therapy. The ideal scenario involves tumors with minimal dissemination and no aggregation.

Role and limitation of cell therapy in treating neurological diseases.

The central role of the brain in governing systemic functions within human physiology underscores its paramount significance as the focal point of physiological regulation. The brain, a highly sophisticated organ, orchestrates a diverse array of physiological processes encompassing motor control, sensory perception, cognition, emotion, and the regulation of vital functions, such as heartbeat, respiration, and hormonal equilibrium. A notable attribute of neurological diseases manifests as the depletion of neurons and the occurrence of tissue necrosis subsequent to injury. The transplantation of neural stem cells (NSCs) into the brain exhibits the potential for the replacement of lost neurons and the reconstruction of neural circuits. Furthermore, the transplantation of other types of cells in alternative locations can secrete nutritional factors that indirectly contribute to the restoration of nervous system equilibrium and the mitigation of neural inflammation. This review summarized a comprehensive investigation into the role of NSCs, hematopoietic stem cells, mesenchymal stem cells, and support cells like astrocytes and microglia in alleviating neurological deficits after cell infusion. Moreover, a thorough assessment was undertaken to discuss extant constraints in cellular transplantation therapies, concurrently delineating indispensable model-based methodologies, specifically on organoids, which were essential for guiding prospective research initiatives in this specialized field.

Retinoic acid receptor activation reprograms senescence response and enhances anti-tumor activity of natural killer cells

Cellular senescence can exert dual effects in tumors, either suppressing or promoting tumor progression. The senescence-associated secretory phenotype (SASP), released by senescent cells, plays a crucial role in this dichotomy. Consequently, the clinical challenge lies in developing therapies that safely enhance senescence in cancer, favoring tumor-suppressive SASP factors over tumor-promoting ones. Here, we identify the retinoic-acid-receptor (RAR) agonist adapalene as an effective pro-senescence compound in prostate cancer (PCa). Reactivation of RARs triggers a robust senescence response and a tumor-suppressive SASP. In preclinical mouse models of PCa, the combination of adapalene and docetaxel promotes a tumor-suppressive SASP that enhances natural killer (NK) cell-mediated tumor clearance more effectively than either agent alone. This approach increases the efficacy of the allogenic infusion of human NK cells in mice injected with human PCa cells, suggesting an alternative therapeutic strategy to stimulate the anti-tumor immune response in "immunologically cold" tumors.

Multi-centers experience using therapeutic plasma exchange for corticosteroid/tocilizumab-refractory cytokine release syndrome following CAR-T therapy

The chimeric antigen receptor T (CAR-T) cell therapy significantly enhances the prognosis of various hematologic malignancies; however, the systemic expansion of CAR-T cells also gives rise to severe cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS). Despite the successful application of corticosteroids and tocilizumab in alleviating severe CRS in most patients, there are still individuals who experience life-threatening CRS without responding to the aforementioned therapies. In our retrospective cohort, we conducted an analysis of clinical and laboratory parameters, including inflammatory cytokines, in 17 patients from three centers who underwent therapeutic plasma exchange (TPE) for refractory CRS with or without ICANS following CAR-T products treatment. Our findings demonstrate a significant improvement in both clinical symptoms and laboratory parameters subsequent to TPE treatment. The rapid decrease in temperature and levels of inflammatory indexes indicates the remarkable scavenging efficacy of TPE against cytokine storm following CAR-T therapy. In conclusion, TPE may serve as a valuable and safe adjunct to corticosteroids and tocilizumab in the management of severe CRS resulting from CAR-T cell infusion. We eagerly await further prospective studies to validate this finding.

Impact of tissue factor expression and administration routes on thrombosis development induced by mesenchymal stem/stromal cell infusions: re-evaluating the dogma.

Hyperactive coagulation might cause dangerous complications such as portal vein thrombosis and pulmonary embolism after mesenchymal stem/stromal cell (MSC) therapy. Tissue factor (TF), an initiator of the extrinsic coagulation pathway, has been suggested as a predictor of this process. The expression of TF and other pro- and anticoagulant genes was analyzed in xeno- and serum-free manufactured MSCs. Furthermore, culture factors affecting its expression in MSCs were investigated. Finally, coagulation tests of fibrinogen, D-dimer, aPPTs, PTs, and TTs were measured in patient serum after umbilical cord (UC)-MSC infusions to challenge a potential connection between TF expression and MSC-induced coagulant activity. RESULTS: Xeno- and serum-free cultured adipose tissue and UC-derived MSCs expressed the highest level of TF, followed by those from dental pulp, and the lowest expression was observed in MSCs of bone marrow origin. Environmental factors such as cell density, hypoxia, and inflammation impact TF expression, so in vitro analysis might fail to reflect their in vivo behaviors. MSCs also expressed heterogeneous levels of the coagulant factor COL1A1 and surface phosphatidylserine and anticoagulant factors TFPI and PTGIR. MSCs of diverse origins induced fibrin clots in healthy plasma that were partially suppressed by an anti-TF inhibitory monoclonal antibody. Furthermore, human umbilical vein endothelial cells exhibited coagulant activity in vitro despite their negative expression of TF and COL1A1. Patients receiving intravenous UC-MSC infusion exhibited a transient increase in D-dimer serum concentration, while this remained stable in the group with intrathecal infusion. There was no correlation between TF expression and D-dimer or other coagulation indicators. The study suggests that TF cannot be used as a solid biomarker to predict MSC-induced hypercoagulation. Local administration, prophylactic intervention with anticoagulation drugs, and monitoring of coagulation indicators are useful to prevent thrombogenic events in patients receiving MSCs. Trial registration NCT05292625. Registered March 23, 2022, retrospectively registered, https://www. gov/ct2/show/NCT05292625?term=NCT05292625&draw=2&rank=1 . NCT04919135. Registered June 9, 2021, https://www. gov/ct2/show/NCT04919135?term=NCT04919135&draw=2&rank=1 .

The Use of a High Flow PICC Catheter for Stem Cell and Lymphocyte Apheresis: The Initial Experience of a Pediatric Oncology Center in Brazil

BackgroundAutologous hematopoietic stem cell transplant (HSCT), characterized by high intensity chemotherapy followed by the infusion of HSC previously collected from the peripheral blood, is a procedure used in the treatment of several malignancies. In pediatrics, the apheresis procedure represents a challenge, due to the need for insertion of a rigid central venous catheter (CVC) in small children. The CVC is usually used for stem cell collection and then removed. Later, the patient will need a new device for cell infusion. AimWe propose the use of one single catheter for both apheresis and infusion. MethodsWe present five children between 1 and 13 years of age who underwent apheresis using a high flow PICC catheter surgically inserted. ResultsAll patients utilized a PICC line double lumen 5Fr (PowerPICC™ 5Fr DL BARD/USA) placed in the brachiocephalic vein tunneled on the chest, inserted under 24 h prior to apheresis to assure the devices were pervious. Three of the patients were diagnosed with solid tumor and one with acute lymphoblastic leukemia (ALL) awaiting Car-T Cell therapy. The four children who underwent autologous HSCT used the same catheter for cell infusion and remained with the catheter following discharge. The child who was submitted for Car-T Cell still awaits infusion and the catheter was removed. ConclusionsHigh flow PICC is a viable alternative for apheresis to maintain an adequate flow of 5 ml/s and can be used as a single catheter throughout the HSCT process, reducing the risks from anesthesia and the catheter insertion procedure. Type of StudyClinical Research.

Differentiation of regulatory myeloid and T-cells from adult human hematopoietic stem cells after allogeneic stimulation.

Donor hematopoietic stem cell (DHSC) infusions are increasingly being studied in transplant patients for tolerance induction. To analyze the fate of infused DHSCs in patients, we developed an in vitro culture system utilizing CD34+DHSCs stimulated with irradiated allogeneic cells in cytokine supplemented medium long-term. Flow cytometric analyses revealed loss of the CD34 marker and an increase in CD33+ myeloid and CD3+ T-cell proportion by 10.4% and 72.7%, respectively, after 21 days in culture. T-cells primarily expressed TcR-αβ and were of both CD4+ and CD8+ subsets. Approximately 80% of CD3+ T cells lacked expression of the co-stimulatory receptor CD28. The CD4+ compartment was predominated by CD4+CD25+CD127-FOXP3+ Tregs (>50% CD4+CD127- compartment) with <1% of all leukocytes exhibiting a CD4+CD127+ phenotype. Molecular analyses for T-cell receptor excision circles showed recent and increased numbers of TcR rearrangements in generated T cells over time suggesting de novo differentiation from DHSCs. CD33+ myeloid cells mostly expressed HLA-DR, but lacked expression of co-stimulatory receptors CD80 and CD83. When studied as modulators in primary mixed lymphocyte reactions where the cells used to stimulate the DHSC were used as responders, the DHSC-lines and their purified CD8+, CD4+, CD33+ and linage negative subsets inhibited the responses in a dose-dependent and non-specific fashion. The CD8+ cell-mediated inhibition was due to direct lysis of responder cells. Extrapolation of these results into the clinical situation would suggest that DHSC infusions into transplant recipients may generate multiple subsets of donor "chimeric" cells and promote recipient Treg development that could regulate the anti-donor immune response in the periphery. These studies have also indicated that T cell maturation can occur in vitro in response to allogeneic stimulation without the pre-requisite of a thymic-like environment or NOTCH signaling stimulatory cell line.

The characteristics and impact on prognosis of cytopenia after anti-BCMA-CAR-T therapy in patients with relapsed and refractory multiple myeloma

Objective: To investigate the characteristics of cytopenia and its impact on prognosis in patients with relapsed and refractory multiple myeloma (RRMM) after B-cell maturation antigen (BCMA) chimeric antigen receptor T-cell (CAR-T) immunotherapy therapy. Methods: Clinical data of 36 RRMM patients received BCMA CAR-T therapy at the First Affiliated Hospital of Nanjing Medical University from April 2017 to March 2023 were retrospectively collected. Among them, there were 17 males and 19 females, with an age [M (Q1, Q3)] of 62 (53, 67) years. The follow-up deadline was August 31, 2023, and the follow-up time [M (Q1, Q3)] was 33 (10, 30) months. The characteristics of cytopenia at different time points before lymphodepleting chemotherapy and after CAR-T cell infusion in all patients were analyzed. Kaplan-Meier method was used to compare the differences in progression-free survival (PFS) and overall survival (OS) in patients with different clinical characteristics. Single-cell sequencing analysis was used to analyze the changes in hematopoietic stem cells in three patients after CAR-T cell therapy. Results: The incidence of cytopenia after BCMA CAR-T cell therapy in 36 RRMM patients reached 100%. The incidence of neutropenia peaked on the 7th and 28th day after cell infusion with a biphasic pattern of change.Patients with all grade neutropenia reached 61.1% (22/36) and grade 3 or higher reached 33.3% (12/36) on the 7th day, while patients with all grade neutropenia reached 67.9% (19/28) and grade 3 or higher reached 28.6% (8/28) on the 28th day (P<0.001),respectively. The occurrence rate of lymphopenia reached a peak on the day of CAR-T cell infusion [97.2% (35/36) patients showed lymphopenia, while 80.6% (29/36) patients showed grade 3 or higher lymphopenia] (P<0.001).The incidence of all grade of thrombocytopenia and severe thrombocytopenia (grade 3 or higher) peaked on the 14th day after cell infusion, with the rates of 69.4% (25/36) and 30.6% (11/36) respectively, which had a prolonged duration(P<0.001). Even after 12 months, 40% (8/20) of patients still experienced thrombocytopenia.The incidence of anemia peaked on the 7th and 14th day after cell infusion, with a rate of 100% (36/36) (P<0.001). 50% (10/20) of patients still had anemia even 12 months after cell infusion. Kaplan-Meier survival analysis showed that patients with thrombocytopenia < grade 3 had undefined OS, while patients with thrombocytopenia ≥grade 3 had shorter OS [17 (95%CI: 2-32) months, χ2=4.154, P=0.042], indicating a poorer prognosis. However, there was no statistically significant difference in the relationship between other cytopenia and survival (all P>0.05). Single-cell sequencing analysis of bone marrow cells revealed decreased proliferation, increased apoptosis, and cell cycle arrest of hematopoietic stem cells after CAR-T cell infusion. Conclusions: All patients experienced varying degrees of cytopenia after receiving BCMA CAR-T cell infusion, and patients with thrombocytopenia ≥grade 3 had shorter OS and poorer prognosis.

Extracorporeal photopheresis as a promising strategy for the treatment of graft-versus-host disease after CAR T-cell therapy

AbstractGraft-versus-host disease (GVHD) occurs in about 10% to 33% of patients receiving “allogeneic” or “autologous” chimeric antigen receptor T (CAR-T) cells after preceding allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to the substantial presence of alloreactive T cells. Extracorporeal photopheresis (ECP) shows promising clinical outcomes in the treatment of GVHD after allo-HSCT without hampering antitumor and antiviral effects. This raises an interesting question: whether ECP might constitute a new way to treat patients with GVHD after CAR T-cell therapy without compromising CAR-T cells significantly. Third-generation CD19-specific CAR-T cells were generated and an in vitro ECP protocol was established. The impact of ECP on CAR-T cells was comprehensively investigated in 2 models: the nondilution model reflects days after CAR T-cell infusion and the dilution model weeks after infusion. The therapeutic effect of ECP on GVHD was examined in an in vitro mixed lymphocyte reaction (MLR) assay. We found, ECP-treated CAR-T cells demonstrated reduced potency in inducing alloreaction compared with that of the group without ECP treatment in MLR assay. ECP could selectively induce apoptosis, thereby enriching the naive and central memory CAR-T cells with a reduced alloreactivity. The cytokine milieu of CAR-T cells could be switched from immune stimulation to immune tolerance in both models. Moreover, ECP could modulate the proliferative capacity of CAR-T cells without hampering their long-term functionality in the dilution model. In conclusion, ECP constitutes a promising treatment strategy for GVHD after allo-HSCT and CAR T-cell transfusion, as ECP reduces the alloreactivity without hampering CAR T-cell functionality.

Negative Vaccination Strategies for Promotion of Transplant Tolerance.

Organ transplantation requires the use of immunosuppressive medications that lack antigen specificity, have many adverse side effects, and fail to induce immunological tolerance to the graft. The safe induction of tolerance to allogeneic tissue without compromising host responses to infection or enhancing the risk of malignant disease is a major goal in transplantation. One promising approach to achieve this goal is based on the concept of "negative vaccination." Vaccination (or actively acquired immunity) involves the presentation of both a foreign antigen and immunostimulatory adjuvant to the immune system to induce antigen-specific immunity. By contrast, negative vaccination, in the context of transplantation, involves the delivery of donor antigen before or after transplantation, together with a "negative adjuvant" to selectively inhibit the alloimmune response. This review will explore established and emerging negative vaccination strategies for promotion of organ or pancreatic islet transplant tolerance. These include donor regulatory myeloid cell infusion, which has progressed to early-phase clinical trials, apoptotic donor cell infusion that has advanced to nonhuman primate models, and novel nanoparticle antigen-delivery systems.