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OBJECTIVES: Explore short-term effects of “The Caregiver Pathway,” an intervention for family caregiver follow-up, on Post-Intensive Care Syndrome symptoms among families (PICS-F). DESIGN: A randomized controlled trial. SETTING: A medical ICU at a Norwegian University Hospital. PARTICIPANTS: One hundred ninety-six family caregivers of critically ill patients randomized to an intervention (n = 101) or control group (n = 95). INTERVENTIONS: “The Caregiver Pathway” four-step model offers individual and structured follow-up, including: 1) mapping family caregivers’ needs and concerns with an assessment tool followed by a conversation with an ICU nurse within the first days at the ICU, 2) a supportive card when leaving the ICU, 3) offer for the family caregivers to receive a phone call after ICU patient discharge, and 4) a follow-up conversation within 3 months. MEASUREMENTS AND MAIN RESULTS: Data were collected at baseline and after 3 months and analyzed using linear regression. No significant effects were detected when comparing all participants completing 3-month outcome measurements (n = 144). A subgroups analysis stratified on patient survival, however, showed statistically significant effect for family caregivers of patients surviving the ICU stay receiving the intervention compared with controls. Caregivers of surviving patients reported improved symptoms related to post-traumatic stress disorder, measured by Impact of Event Scale-Revised (B = –8.2 [95% CI, –14.2 to –2.2]; p = 0.008), anxiety (B = –2.2 [95% CI, –4.0 to –0.5]; p = 0.014), and depression (B = –1.5 [95% CI, –2.9 to –0.1]; p = 0.035); measured by the Hospital Anxiety and Depression Scale, subscore physical functioning in health-related quality of life (B = 9.7 [95% CI, 0.3–19.0]; p = 0.043); measured by Short Form 12-Item Health Survey; and hope (B = 2.4 [95% CI, 0.4–4.3]; p = 0.017) and measured by the Herth Hope Index. At 3-month, the model did not appear to improve the outcomes for family caregivers of nonsurviving patients. CONCLUSIONS: “The Caregiver Pathway” intervention was associated with reduced symptoms of PICS-F in family caregivers of surviving ICU patients compared with controls.

Background As the use of flow diverters has expanded in recent years, predicting successful outcomes has become more challenging for certain aneurysms. Objective To provide neurointerventionalists with an understanding of the available machine learning algorithms for predicting the success of flow diverters in occluding aneurysms. Methods This study followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, and the four major medical databases (PubMed, Embase, Scopus, Web of Science) were screened. The study included original research articles that evaluated the predictive abilities of various machine learning algorithms for determining the success of flow diverters in achieving aneurysm occlusion. Results Five studies out of 217 were included based on our criteria. The included studies used various variables (patient demographics, aneurysm and parent artery characteristics, flow diverter and hemodynamic-related features, and angiographic parametric imaging) to predict flow diverter treatment outcomes. The machine learning algorithms used, along with their respective accuracy rates, were as follows: logistic regression (61% and 85%), support vector machine (88%), Gaussian support vector machine (90%), linear support vector machine (85%), decision tree (80%), random forest (87%), k-nearest neighbors (83% and 85%), XGBoost (87%), CatBoost (86%), deep neural networks (77.9%), and recurrent neural networks (74%).Two studies trained the machine learning models with both all features and the most significant features. Both studies observed that the accuracy of machine learning models decreased by removing the insignificant features. Conclusion The current literature indicates that machine learning algorithms can be trained to predict the success of flow diverters with an accuracy of up to 90%.

Despite the high initial responses to chimeric antigen receptor T (CART) cell therapy, most patients relapse within the first 1-2 years. T cell quality influences the success of CART cell therapies greatly, and T cells from cancer patients are prone to transition into dysfunctional states, such as exhaustion or senescence. The role of exhaustion in CART cell failures has been studied extensively; however, the role of T cell senescence has remained mostly unexplored. In this study, we aimed to determine the development of CART cell senescence and its impact on CART therapeutic efficacy. First, we studied the development of CART cell senescence using an in vitro model to induce CART cell dysfunction. Here, we cyclically activated CD19-directed chimeric antigen receptor T (CART19) cells (either costimulated via 4-1BB (BBζ) or CD28 (28ζ)). Activation cycles are designed to mimic recurrent activation of CART cells to study the impact of T cell activation on CART cell fates throughout treatment. We added a rest time in between activation cycles to focus on senesced CART cell populations since, unlike exhaustion, senescence is irreversible even after rest. We examined CART immunophenotype, cell cycle regulators, transcriptomic profile, senescence marker gene expressions and T cell effector functions on days 0 (D0-T cell), 8 (D8-baseline CART), and 15 (D15-CART cell after one activation/rest cycles). Following an activation/rest cycle, T cell exhaustion markers CTLA4, LAG3, and PD1 returned to basal levels after an initial increase due to activation, while only TIM-3 levels remained elevated (BBζ: p=0.0090, 28ζ: p=0.0394). Increased TIM-3 expression is associated with T cell senescence, which prompted us to further investigate the development of a senescence phenotype. We found a prominent T cell senescence phenotype in BBζ compared to 28ζ, as evident by increased expression of p53 (BBζ: p=0.0248), p21 (BBζ: p=0.0483), CD27 (BBζ: p=0.0080), CD28 (BBζ: p=0.0327), uPAR (BBζ vs. 28ζ: p<0.0001), and KLRG1 (BBζ vs. 28ζ: p=0.0029) in recurrently activated-rested CART cells. Next, we evaluated the transcriptomic signature of recurrently activated-rested CART cells. Gene set enrichment analysis (GSEA) indicated enrichment of gene sets related to senescent T cells as well as senMayo, a novel gene set which is reported to be selectively enriched in senescing cells or tissues, in BBζ, but not in 28ζ, compared to T cell D0 or in BBζ D15 compared to 28ζ D15 (p=0.00014 and p=0.089, respectively). MYC-related gene sets were found to be significantly enriched in recurrently activated/rested BBζ and depleted in 28ζ (p<0.1). MYC expression is increased in BBζ (p= 0.0010) while decreased in 28ζ (p=0.0036) upon recurrent activation and is higher in BBζ (D8: p=0.0018, D15: p= 0.0022) compared to 28ζ at indicated time points by qPCR. Given these results, we induced senescence in CART cells using two alternative strategies: MYC-T58A activation (oncogene-induced senescence) and irradiation. Senescence induced by MYC-T58A overexpression ameliorated only BBζ killing (p=0.0057), whereas senescence induced by irradiation improved 28ζ killing (p= 0.0086), indicating versatile impact of senescence on treatment outcome diverse CART cell therapies. Finally, analysis with two independent patient data sets correlated the emergence of senescence with contradicting clinical outcomes to BBζ and 28ζ treatments. The presence of senescent 28ζ cells in the peripheral blood of 28ζ-treated patients was reported to be associated with complete response in the clinic in an independent analysis. In another cohort, 28ζ-treated patients with higher senescence score were correlated with longer disease-free survival (p=0.0029), while senMayo gene set enrichment in BBζ-treated patients was associated with non-response in the clinic (p=0.014). In the same cohort, MYC-related gene sets-the same genes sets enriched in our recurrently activated/rested BBζ-are also enriched in nonresponsive BBζ-treated patients. In summary, our study indicates that (i) CART cells are susceptible to a state of senescence, as demonstrated using three independent approaches (repeated activation/rest, irradiation, and MYC modulation), (ii) CART senescence is more prominent in BBζ than 28ζ, and (iii) senescence is associated with worse outcomes following BBζ but not 28ζ treatment.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies. However, CAR T-cell therapy still has low durable response rates and common associated toxicities such as cytokine release syndrome (CRS). We recently reported that sequential therapy with the CD19 monoclonal antibody tafasitamab and CD19-directed CAR T (CART19, 41BBζ costimulated) cells significantly ameliorated CAR T-cell apoptosis and tumor cell pyroptosis, improving CAR T safety and efficacy (PMID: 37879074). Building on these findings, we hypothesized that controlling massive initial CAR T-cell stimulation by diminishing extensive tumor cell pyroptosis is critical to improve CAR T safety and durability. To test this hypothesis, we evaluated several caspase (Casp) inhibitors, including Casp1, Casp3, Casp7, and Casp11, in combination with CAR T cells. We found that the Casp1 inhibitor VX765 did not inhibit CAR T antigen-specific cytotoxicity and thus focused on VX765 in subsequent assays. We first assessed CAR T proliferation upon stimulation with the CD19+ JeKo-1 mantle cell lymphoma cell line in the presence of VX765. CAR T with vehicle control showed greater proliferation at day 3, but CAR T with VX765 showed higher proliferation at day 5 (p=0.03, absolute CD3+ counts by flow cytometry). We then co-cultured CAR T and JeKo-1 for 24 hours and observed a significant reduction in apoptotic CAR T (Annexin V+ and 7-AAD- via flow cytometry) in VX765-treated conditions (p=0.05). Additionally, there was significantly reduced pyroptosis (Annexin V+ 7-AAD+) (p=0.02) but higher apoptosis (Annexin V+ 7-AAD-) in JeKo-1 in VX765-treated conditions (p=0.04). VX765-treated CAR T showed higher Bcl2 expression via flow cytometry (p=0.03). We also studied tumor cell pyroptosis by measuring the level of HMGB-1. CART19, JeKo-1, and VX765 or vehicle control were co-cultured for 24 hours, and the level of HMGB-1 was found to be significantly reduced in VX765-treated conditions, indicating reduced tumor pyroptosis (p=0.006). We then assessed the impact of Casp1 inhibition in our in vitro model of CAR T exhaustion. We stimulated CAR T with CD19+ JeKo-1 every other day for 2 weeks and then assessed CCR7 and CD45RA expression on CAR T. VX765-treated CAR T showed higher central memory (p=0.08) and naïve (p=0.02) and lower terminally differentiated effector memory phenotypes (p=0.03). We next studied the effect of Casp1 inhibition with VX765 in vivo. To recapitulate CAR T-cell toxicities, including CRS, we utilized an immunocompetent mouse model. First, we generated murine CART19 (mCART19, CD28ζ costimulated) cells. Recipient mice were then engrafted with luciferase+ murine CD19+ TBL12 cells, a murine lymphoma cell line. Mice were treated 12 days later with cyclophosphamide (300 mg/kg, ip) and 24 hours after mice were imaged and randomized to receive 1 x 107, 1) untransduced T cell control (UTD), 2) mCART19 + vehicle control, or 3) mCART19 + VX765 (25 mg/kg). VX765 was given immediately before the CART19 injection and on day 2 post-CAR T injection. Mice were serially monitored and weighed to assess CAR T-cell toxicity. Tumor burden was assessed via weekly bioluminescent imaging. Mice treated with vehicle control showed a significant reduction in weight compared to the UTD control (p=0.02), while mice treated with VX765 maintained their weight and remained active without signs of CRS, including motor weakness and hunched body. UTD mice reached humane endpoints within 3 weeks after T-cell injection. Three satellite mice from each group were euthanized and spleens were harvested. Flow cytometric analysis showed a significant reduction in apoptotic CAR T (p=) and pyroptotic tumor cells in mice treated with VX765 (p=0.03). Western blot showed lower cleaved GSDME and GSDMD in mice treated with VX765. VX765-treated mice demonstrated prolonged CAR T persistence with durable anti-tumor activity. In summary, Casp1 inhibition with VX765 significantly reduced CAR T apoptosis, improved memory phenotype, and increased delayed CAR T proliferation. VX765 also reduced tumor cell pyroptosis and CRS incidence without impacting CAR T anti-tumor activity in vivo. Given the favorable preclinical outcomes and existing clinical data on VX765, this approach holds substantial translational potential for enhancing the safety and efficacy of CAR T-cell therapy in patients with large B-cell lymphoma.

Brexucabtagene autoleucel (brexu-cel), a CD28-costimulated CD19-targeted chimeric antigen receptor T cell (CART19) product, was FDA-approved in 2020 after demonstrating remarkable efficacy in the pivotal ZUMA-2 trial of patients with mantle cell lymphoma (MCL). However, despite the impressive initial activity of CART19 cell therapy in MCL, most patients relapse within the first 3 years following treatment. Myeloid cells, such as monocytes and macrophages, have been demonstrated to be major inhibitors of other immune cells, including T cells. Growing evidence shows that monocytes and macrophages contribute to CART cell failure, although the exact mechanisms are not fully understood. To better understand the mechanisms contributing to CART treatment resistance in MCL, we first utilized brexu-cel products from patients with MCL on the ZUMA-2 trial to uncover their transcriptomic features by single cell RNA sequencing (scRNAseq) analysis. To understand if myeloid cells had negative impacts on clinical outcomes, we then analyzed autologous baseline peripheral blood mononuclear cell (PBMC) samples by scRNAseq. Our analyses of the brexu-cel samples revealed reduced response to IL-1β and its downstream signaling pathway as well as impaired T cell functions in non-responders. Transcriptomic analyses of the autologous baseline monocyte populations showed downregulation of IL-1β production and its associated pathways as well as upregulation of M2 phenotype-associated genes and IL-1 receptor antagonist (IL-1ra) in non-responder monocytes, implying a potential role of myeloid cells in CART resistance through impairment of IL-1 signaling in brexu-cel products. To validate our findings from the ZUMA-2 samples, we further studied the interactions between macrophages, MCL cells, and CART19 cells in preclinical models. First, CD28-costimulated CART19 cells generated from healthy donors were cocultured with JeKo-1, a CD19+ MCL cell line, and freshly isolated monocytes or ex vivo differentiated M2-like macrophages. M2-like macrophages were generated by incubating fresh monocytes with rhGM-CSF followed by coculturing with JeKo-1. CART19 antigen-specific proliferation was significantly inhibited by M2-like macrophages but not fresh monocytes (JeKo-1+CART19 vs JeKo-1+M2+CART19, p=0.00503). Transwell assays indicated that suppression by M2-like macrophages was contact-independent. Cytokine profile analysis of coculture supernatants showed significant elevation of IL-1ra in M2-like macrophage cocultures (JeKo-1+Mono+CART19 vs JeKo-1+M2+CART19, p=0.0292). This led us to hypothesize that M2-like macrophages inhibit CART19 by secreting IL-1ra to block IL-1 signaling in CART19. To test this hypothesis, we interrogated the role of IL-1ra in CART19. CART19 + JeKo-1 cocultures were supplemented with IL-1β + IL-1ra or IL-1β + IL-1ra + IL-1ra neutralizing antibody (neuAb). CART19 proliferation was improved by IL-1β (vehicle control vs IL-1β, p<0.0001). IL-1ra inhibited IL-1β-dependent CART19 proliferation (IL-1β vs IL-1β+IL1ra, p=0.0006), which was restored by IL-1ra neutralization (IL-1β+IL-1ra vs IL-1β+IL-1ra+IL-1ra neuAb, p=0.0215). Next, we assessed the impact of IL-1ra on CART functions in mouse MCL xenografts in the presence of M2-like macrophages. NOD-SCID-γ-/- (NSG) mice were subcutaneously injected with 0.5 million of human macrophages and 1 million of luciferase+ JeKo-1. Mice were then randomized once tumor engraftment was confirmed by bioluminescence imaging (BLI), followed by treatment with 2 million CART19 with 10 mg/kg IL-1ra neuAb or control IgG for 3 weeks. Combination of CART19 cells and IL-1ra neuAb led to improved antitumor activity based on BLI measurements compared to control IgG (IL-1ra neuAb vs control IgG, p=0.0058). Additionally, expression of IL-1RI, receptor of IL-1β and IL-1ra, was measured on T cells after coculturing CART19, JeKo-1, and M2-like macrophages. M2-like macrophages downregulated IL-1RI expression on CART cells, which might potentially desensitize CART response to IL-1β, resulting in further suppression in CART proliferation (JeKo-1+M2+CART19 vs JeKo-1+CART19, p=0.001). Overall, our study elucidates a potential mechanism of IL-1 pathway modulation mediated by myeloid cells in contributing to CART19 failure in MCL.

Introduction: Scant data exist on prognostic factors, survival and treatment (Tx)-related outcomes in SCNSL. We conducted a multicenter international analysis to identify prognostic factors and outcomes in SCNSL at diagnosis (de novo), or after frontline Tx (1L) either with (concomitant) or without (isolated) systemic (syst) relapse. Methods: This study collected data from patients (pts) treated between 2001-2023 at 35 centers. Overall (OS) and progression-free survival (PFS) were calculated from SCNSL diagnosis to first event (death for OS, relapse/death for PFS) or last follow-up. Uni- (UVA) and multivariable (MVA) Cox analyses were performed to assess prognostic factors; Kaplan-Meier method was used to estimate survival. Results: 1173 (US: 863, UK: 273, Canada: 37) pts with SCNSL were included. Baseline characteristics were median age: 62 years (interquartile range 52.5-69), male sex: 58.7%, elevated LDH: 71.1%, stage III/IV: 88.6%, HIV: 3%, transformed LBCL (tLBCL): 13.8%, non-germinal center B-cell like (non-GCB) subtype: 52.3%, double expressor (DE): 40%, MYC rearrangement (MYC-R): 28.3%, MYC/BCL2 double hit: 16.6%, MYC/BCL2/BCL6 triple hit (TH): 6.1%. Among 522 pts with de novo SCNSL, 84.2% had involvement of a single CNS compartment (44.7% brain parenchyma; 40.4% leptomeningeal [lepto]). 1L regimens included 36.2% R-CHOP with high-dose methotrexate (HD-MTX), 12.5% MARIETTA-type, and 9.8% R-CODOX-M/IVAC; 54.6% received intrathecal chemotherapy. Consolidation Thiotepa-based autologous stem cell transplant (TT-ASCT) occurred in 15.3%. The majority of pts who underwent TT-ASCT had previously achieved CR or PR in syst (92.5%) and CNS (72.5%) compartments. At median follow-up 25.7 months (mos), subsequent relapse was observed in 27.5% TT-ASCT recipients: 36.3% had isolated CNS, 27.4% syst, and 36.3% concomitant. 588 pts presented with SCNS after 1L (66.2% isolated, 33.8% concomitant). Brain parenchyma involvement was observed in 56.8%, lepto in 22.6%. Tx included 31.6% HD-MTX combination (eg, R-MPV, MTR); 18.4% HD-MTX +/- rituximab; 16.2% MARIETTA-type; and 9.4% platinum-based regimen. Consolidation TT-ASCT occurred in 22.4% (n=132), and 13.8% (n=81) received CAR-T cell therapy (excluding subsequent relapses in de novo group). Syst (65.1%) & CNS (69.1%) responses (CR/PR) were higher before TT-ASCT compared to CAR-T cell (49.5% & 54.5%, respectively). After TT-ASCT, 52 pts (39.3%) relapsed, mainly in the CNS (n=30, 57.7%), with only 4 (7.7%) concomitant. After CAR-T, 48 pts (59.3%) relapsed: 15 (31.2%) isolated in CNS and 15 (31.2%) concomitant. 12-month cumulative incidence of relapse (with death as competing risk) was 21.1% after TT-ASCT and 55.8% after CAR-T. In 63 pts (5.4%), the first CNS event occurred >2 lines of Tx. With median follow-up of the entire cohort of 24.6 mos, median PFS and OS were 11 and 21 mos, respectively. mPFS and mOS were significantly better in pts with de novo SCNSL (13.4 & 47.4 mos) compared to SCNSL after 1L (10.4 & 14.2 mos) and >2 lines of Tx (6.5 & 9.7 mos); P<0.0001. Factors associated with significantly shorter OS in UVA across all presentations were age >60, male sex, GCB subtype, MYC-R, tLBCL, DE, TH, elevated LDH, ECOG PS ≥2, SCNSL after 1L (vs. de novo), and failure to achieve syst/CNS response to 1L. Of these, age >60, male sex, MYC-R, ECOG PS ≥2, and failure to achieve response to 1L remained significant in MVA. In de novo cases, age >60, DE, ECOG PS≥2, and failure to achieve syst/CNS response to 1L were associated with poorer OS; for pts with SCNSL after 1L, age >60, GCB subtype, and ECOG PS ≥2 were significant. Pts treated with HD-MTX-based regimens after 1L experienced longer OS than those treated with platinum regimens or high-dose cytarabine. In de novo cases, TT-ASCT was significantly associated with longer OS (HR=0.58; P=0.025) in MVA after adjusting for significant factors, 1L type, and response. For SCNSL at relapse, TT-ASCT (HR=0.34; P<.0001) and isolated SCNSL presentation (vs. concomitant HR=0.70, P=0.007) were associated with longer OS, adjusting for significant factors, type of second-line Tx, & site of CNS relapse. Conclusions: This study represents the largest analysis of SCNSL inclusive of all modes of presentation. Concomitant SCNSL at relapse was associated with poor outcomes, while TT-ASCT consolidation was associated with improved OS in de novo & relapsed disease. Despite advances in LBCL SCNSL remains an unmet therapeutic need.

Background: The majority of patients respond to steroid treatment for graft-vs-host disease (GVHD), but the typically high doses and prolonged courses can cause substantial morbidity. GVHD can be classified at onset into two groups with significant differences in treatment response and non-relapse mortality (NRM) by clinical criteria (Minnesota risk) but the more favorable standard risk group still experiences high rates of treatment failure and NRM [MacMillan, BBMT 2015]. We previously validated a serum biomarker-based (ST2+REG3α) risk stratification system, MAGIC algorithm probabilities (MAPs), that can stratify patients both at initiation and during treatment into risk groups for treatment response and NRM [Hartwell, JCI Insight 2017, Srinagesh, Blood Adv 2019]. GVHD that is Minnesota standard risk and has a low MAP (i.e., Ann Arbor 1) at start of treatment represents a low risk group with >80% response rate to steroids and ~10% NRM [Etra, Blood 2023]. We hypothesized that serial monitoring of GVHD symptom severity and MAPs twice (days (d) 7 and 14) in patients with clinical and biomarker defined low risk GVHD could further stratify patients into clinically meaningful groups. Methods: We retrospectively determined MAPs at d7 and 14 in 450 patients with low risk GVHD who participated in a prospective, multi-center, observational trial and were treated with systemic steroids between 2015-2023. MAPs <0.291 at days 7 and 14 were defined as low risk based on a previously validated threshold [Major-Monfried Blood 2018]. Clinical responses were scored as complete (CR), partial (PR), or non-response (NR) by standard criteria. Initiation of second line therapy was considered NR. Patients were categorized into three groups (A-C) based on clinical responses and MAPs at days d7 and 14. A: CR/PR by d14 and low MAP at both d7+14; B: stable or worse GVHD (NR) by d14 with low MAP at both d7 and 14; C: any response with a high MAP at d7 or 14. The endpoints were clinical response at day 28 (overall response rate, ORR=CR or PR), regardless of earlier response status, and 6-month NRM. Results: The median age of the study population was 54 years (range: 0-79); 19% of patients received PT-Cy for GVHD prophylaxis. Target organ involvement was skin ± upper GI (66%), lower GI (23%), isolated UGI (11%), and skin+liver (1%), and GVHD severity was grade I (35%), II (61%), and III (4%) at start of treatment. Patients in group A (n=310, 69%), clinical responders with low MAPs through day 14, had excellent d28 ORR (93%) and very low NRM (4%). GVHD in this group was considered ultra-low risk (ULR) and subset analyses revealed highly consistent d28 ORR and NRM, regardless of target organ severity, age (<18, 18-59, >60), HCT-CI score, conditioning intensity, and GVHD prophylaxis. Group B patients comprised clinical non-responders with low MAPs through day 14 (n=112, 25%); half of them responded by d28 and their NRM was low (8%), albeit double that of group A (p=0.079). Group C was small (n=28, 6%) and included both clinical responders (n=20, 4%) and clinical non-responders (n=8, 2%) who had at least one high MAP through day 14. Their d28 ORR was 54% but NRM increased four-fold (32%) and was significantly worse than patients who maintained low MAPs regardless of clinical response (p<0.001). Conclusion: Serial monitoring by clinical response and biomarkers during the first two weeks of treatment identifies a small number of high risk patients whose GVHD is initially low risk (group C). This group experiences high NRM regardless of clinical response to steroid treatment and may benefit from treatment escalation. Patients with low risk GVHD who do not have an early clinical response to steroids but maintain low MAPs, (group B) have reasonably good long-term outcomes with standard treatment. Finally, serial monitoring identifies a very large subset of patients with ULR GVHD (group A) who respond exceptionally well to standard treatment and experience very low rates of 6-month NRM regardless of pre-transplant characteristics and GVHD prophylaxis. Such patients may benefit from treatment de-escalation strategies such as shorter courses of lower dose steroids, a strategy we are currently testing in a clinical trial (NCT05090384).

CD19-targeted chimeric antigen receptor T (CART19) cell therapy has shown outstanding clinical success. However, patients can develop CART-induced toxicities, including cytokine release syndrome (CRS) and neurotoxicity. Studies have found that activation of myeloid cells by granulocyte monocyte-colony stimulating factor (GM-CSF) can contribute to toxicity development. GM-CSF depletion leads to reduced myeloid activation, prevention of CART-induced toxicities, and improved CART efficacy. CART cells upregulate cytokine receptors upon activation, and our results indicate that CART19 cells significantly upregulate GMCSFR upon antigen-specific stimulation (<10% vs >30%, p<0.01). This led us to hypothesize that GMCSFR signaling contributes to CART cell activity. To understand the role of GM-CSFR upregulation on CART cell functions, we compared the impacts of neutralizing GM-CSF or blocking GM-CSF receptor alpha (GM-CSFRα) on CART19. First, 4-1BB-costimulated CART19 cells generated from healthy donors were stimulated with CD3/CD28 beads at a 1:3 ratio for 6 days. CART cells were maintained at 1 x 106/mL throughout the culture and supplemented with IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade at 10 μg/mL, 20 μg/mL, or 100 μg/mL. On day 6, cells were debeaded and rested for 2 days. Then, CART cells were cocultured with NALM6, a CD19+ acute lymphoblastic leukemia cell line, at a ratio of 1:1 for 3 days. The cocultures were supplemented with the same doses of antibodies listed above. Antigen-specific proliferation of CART cells was measured by flow cytometry. Our data suggested that CART19 proliferated is not impacted by GM-CSF neutralization but is inhibited by GM-CSFRα blockade (IgG isotype vs 10 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 20 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 100 μg/mL GM-CSFRα blockade, p=0.009). CART cells were assessed in a cytotoxicity assay with NALM6 luciferase+ cells as well, which did not show significant impacts from GM-CSF neutralization or GM-CSFRα blockade. To evaluate the impacts of GM-CSF neutralization and GM-CSFRα blockade in a mouse model, we engrafted NSG mice with 1 million NALM6 luciferase+ cells intravenously. Mice were then treated with 1.4 million CART19 plus 10 mg/kg IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade intraperitoneally. GM-CSFRα blockade drastically suppressed antitumor activities of CART (CART19+GM-CSFRα blockade vs CART19+IgG isotype control, p<0.0001; CART19+GM-CSFRα blockade vs CART19+GM-CSF neutralizing antibody, p<0.0001; CART19+ GM-CSFRα blockade vs untransduced T cell, n.s.). Serum cytokines were analyzed 2 days after CART treatment, showing that mice with GM-CSFRα blockade had significantly lower levels of human IFNγ (p=0.0189) and IL-2 (p=0.0121) compared to IgG isotype control group, while GM-CSF neutralization did not show significant impacts. Mice treated with CART plus IgG isotype control or GM-CSF neutralizing antibody showed prolonged survival compared to mice treated with GM-CSFRα blockade or untransduced T cells (p=0.0285). To unravel the mechanisms behind functional differences between GM-CSFRα blockade and GM-CSF neutralization, CART cells (n = 3 biological replicates) were cocultured with NALM6 at a 1:1 ratio supplemented with 10 μg/mL GM-CSF neutralizing antibody, GM-CSFRα blockade, or IgG isotype control overnight, followed by T cell isolation, bulk RNA isolation, and RNA sequencing (RNAseq) analysis. Our RNAseq data indicated significant upregulation (Log2FC>0.5, padj. <0.05) of T cell exhaustion- and dysfunction-associated genes (RGS16, PDCD1, CTLA4, NR4A3, CD101, LAG3) as well as negative regulators of T cell functions (G0S2, PIK3IP1, LGALS3) in the GM-CSFRα blockade group compared to GM-CSF neutralizing antibody or IgG isotype control groups. T cell activation-associated pathways were enriched in the GM-CSFRα blockade group. Overall, our data show that GM-CSFRα blockade impacts CART functions differently from GM-CSF neutralization. Specifically, GM-CSFRα blockade inhibits CART proliferation in vitro and antitumor activity in vivo. Interestingly, GM-CSFRα blockade leads to enrichment of T cell exhaustion- and dysfunction-associated genes in CART19, which is worth further investigation. These findings may be applicable to other cytokine receptors upregulated on activated CART cells.

Background: Chemoimmunotherapy using rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunomycin), vincristine sulfate (Oncovin), and prednisone (R-CHOP) has been the standard of care treatment of DLBCL for over 20 years. Despite its widespread usage, there is no accepted standard for the dose of prednisone in this setting which varies from fixed-dose (100 mg daily for 5 days) to different levels of BSA-adjusted dosage (ranging from 40-100 mg/m2 daily for 5 days). BSA-adjusted dosage results in high doses of prednisone administered to patients with increased body surface area which may lead to significant adverse events. We performed a systematic review and meta-analysis to determine if fixed-dose prednisone during R-CHOP therapy results in equivalent outcomes compared to BSA-adjusted dosing. Methods: A comprehensive search of several databases from each database's inception to April 29th, 2024, English language, was conducted. The databases included Ovid MEDLINE and Epub Ahead of Print, In-Process & Other Non-Indexed Citations, and Daily, Ovid EMBASE, Ovid Cochrane Central Register of Controlled Trials, Ovid Cochrane Database of Systematic Reviews, and Scopus. The search strategy was designed and conducted by an experienced librarian with input from the study's principal investigator. Controlled vocabulary supplemented with keywords was used to search for prospective clinical studies of R-CHOP for adult patients with untreated DLBCL. Search results were reviewed independently by 2 members of the study team for final inclusion. Discrepancies were adjudicated by a third member and reviewed by the senior author. Prednisone dosage and clinical outcomes were abstracted from the available public data. Meta-analysis was performed using the metafor package in R version 4.2.2 by applying a mixed-effects model to combine results from the multiple studies. The analysis was first conducted overall to determine the general effect, and then stratified by prednisone dose (fixed- vs. BSA-adjusted) to explore dose-dependent variations in outcomes. Results: Our search resulted in 2287 entries which were individually reviewed by the study team. A total of 30 studies with a combined sample size of 3573 treated patients fulfilled the inclusion criteria and were included in the final analysis set. Nineteen (63%) studies with a combined sample size of 1930 patients (median 155, range 12-399) used fixed-dose prednisone. Eleven (37%) studies with a combined sample size of 1643 patients (median 70, range 9-439) used BSA-adjusted dosing. Overall survival (OS) at 2 years data were available for 11 (37%) of the studies. Progression free survival (PFS) at 2 years data were available for 8 studies (27%). Overall response rate (ORR) and complete response rate (CRR) data were available for 16 (54%) and 14 (47%) of the included studies, respectively. OS at 2 years for all studies was 84% (SD 78%-91%) and did not differ between fixed-dose (88%, SD 79%-97%) and BSA-adjusted dose (82%, 73%-91%). PFS at 2 years for all studies was 77% (SD 68%-85%) and did not differ between fixed-dose (80%, SD 68%-93%) and BSA-adjusted dose (74%, SD 62%-86%). ORR for all studies was 86% (SD 79%-92%) and did not differ between fixed-dose (86%, SD 76%-96%) and BSA-adjusted dosage (84%, SD 80%-88%). CRR for all studies was 70% (SD 61%-80%) and did not differ between fixed-dose (67%, SD 54%-81%) and BSA-adjusted dose (77%, SD 64%-89%). Heterogeneity tests performed according to available clinical outcomes yielded results between 0% and 76%. Conclusion: The result of our systematic review and meta-analysis combining 30 prospective studies and a total of 3573 patients with newly diagnosed DLBCL treated with frontline R-CHOP found no difference in clinical outcomes between patients treated with fixed-dose and BSA-adjusted dose prednisone. Our findings provide scientific evidence for the widespread practice of capping prednisone at the maximum dose of 100 mg daily for 5 days, potentially avoiding unnecessary adverse effects from BSA-adjusted dosing.