Citations

Abstract Chimeric antigen receptor T (CART) cell therapy has shown unprecedented success in the treatment of hematological malignancies. However, the immunosuppressive tumor microenvironment of both solid and liquid tumors is a major barrier for CART efficacy. Immunosuppressive myeloid cells inhibit CART efficacy. Colony-stimulating factor 1 receptor (CSF1R) promotes macrophage proliferation, survival, and anti-inflammatory response and is upregulated on myeloid cells in many cancers, making CSF1R an appealing therapeutic target. We aimed to test the interactions between CSF1R, myeloid cells, and CART response, and to study CSF1R targeting in the context of CART cell therapy. We utilized CD19-targeted CART cell therapy (CART19) in lymphoma preclinical models and thyroid-stimulating hormone receptor (TSHR)-targeting CART cell therapy (TSHR-CART) in thyroid cancer models. We first measured CSF1R expression on myeloid cells in vitro. Suppressive myeloid cells, polarized from healthy donor monocytes using an anaplastic thyroid cancer (ATC) cell line, 8505c, or mantle cell lymphoma cell line, JeKo-1, significantly upregulate CSF1R (p=0.0002) compared to media control. After polarization, myeloid cells significantly inhibit TSHR-CART and CART19 antigen-specific proliferation compared to freshly isolated monocytes (p=0.012, p<0.01, respectively) indicating that suppressive myeloid cells contribute to CART failure in multiple tumor types. We then performed single cell RNA sequencing on peripheral blood taken at baseline from patients with mantle cell lymphoma treated with CART19. We found a significant increase of CSF1R on monocyte subsets in CART19 non-responders vs responders (padj=0.0037). Next, we determined the impact of CSF1R blockade on CART cell therapy in vivo. Using lymphoma models, B6 mice were engrafted with the luciferase+ CD19+ murine lymphoma cell line TBL-12 (1x106 cells i.v. 1 day after 250 mg/kg cyclophosphamide). Tumor burden was significantly reduced after 7 days of treatment with low dose CART19 (0.5x106 i.v.) + anti-mouse CSF1R antibody (16mg/kg i.p. twice weekly) vs CART19 + IgG control (p=0.0146). We then tested the impact of CSF1R blockade in mice experiencing tumor relapse. After TBL-12 engraftment, mice were treated with anti-mouse CSF1R or IgG control antibody followed two days later with high dose CART19 (1x106 i.v.) to induce remission. Mice were re-challenged with TBL-12 14 days later. Mice treated with anti-mouse CSF1R antibody showed increased tumor control and significantly higher survival (p=0.0416). Using thyroid cancer models, first we identified high macrophage infiltration in biopsy specimens of patients with ATC compared to normal thyroid tissue and benign thyroid tumors through immunohistochemistry (IHC) of patient samples (p<0.01, p<0.0001, respectively). To test if macrophages are associated with CART failure in ATC, NSG mice were subcutaneously engrafted with 1x106 TSHR+ THJ529 tumors, an ATC cell line derived from patient biopsy samples from the Mayo Clinic thyroid cancer biobank, followed by i.v. treatment with 1x107 TSHR-CART or untransduced T cell control (UTD). IHC staining of tumor tissue collected at experimental endpoints revealed a dose-dependent, significant infiltration of human CD3+ T cells into the tumors which inversely correlated with murine F4/80+ myeloid cell and murine YM-1+ M2 macrophage infiltration. This led us to hypothesize that myeloid cell targeting with CSF1R blockade can improve TSHR-CART efficacy. We utilized the same ATC model described above to test CSF1R targeting. When TSHR+ THJ529 tumors reached approximately 100 mm3, mice were randomized by tumor volume and treated with CSF1R inhibitor PLX5622 (600mg/kg) or vehicle control, followed by either UTD or TSHR-CART cells 5x106 i.v. Mice treated with TSHR-CART + PLX5622 showed significantly improved tumor control vs TSHR-CART + vehicle control (p<0.0001) or UTD + PLX5622 (p<0.0001). Together, our results highlight immunosuppressive myeloid cells which express CSF1R as a major cell type limiting CART efficacy in both solid and liquid tumors. We also identified a strategy to overcome myeloid-mediated suppression of CART cells by targeting CSF1R with antibody or small molecule inhibitors.

Abstract Background Chimeric antigen receptor T cell (CART) therapy has revolutionized treatment for hematologic malignancies. However, durable remission following anti-CD19 CART therapy (CART19) remains limited to 40–50% of patients, often due to tumor relapse and CART dysfunction (PMID:31501612). We previously demonstrated that tumor-derived extracellular vesicles (EVs) from patients with chronic lymphocytic leukemia (CLL) can directly impair CART function (PMID:33388419). In this study, we aimed to identify tumor-derived EV microRNA (miRNA) cargo associated with clinical CART failure in B cell malignancies and define how they mechanistically drive CART cell dysfunction. Methods To achieve these aims, we profiled tumor-derived EV miRNA cargo using small RNA sequencing (RNAseq) in two independent patient cohorts. Cohort 1 compared plasma-derived EVs from untreated CLL patients to those from healthy donor EVs to identify miRNAs enriched in CLL (n=23). Cohort 2 included patients with diffuse large B cell lymphoma (DLBCL) treated with axicabtagene ciloleucel (CART19, n=12). Here, plasma EV miRNAs were analyzed at baseline (pre-CART treatment) and 1-month post-CART treatment and compared between CART responders (complete remission at 6 months) and nonresponders (no response at 1 month). Enriched miRNAs from both cohorts were identified following miRNA count normalization and differential expression analysis using adjusted p values of ≤0.05. Functional studies were conducted by transfecting CART19-28ζ cells with synthetic miRNA mimics or scrambled controls. CART cells were also generated with stable overexpression of miRNA via lentiviral shRNA delivery. CART function was assessed via in vitro killing, proliferation, apoptosis assays, RNAseq, and in vivo tumor xenograft models. Results In Cohort 1, we identified several miRNAs including miR-125a to be significantly enriched in CLL-derived EVs relative to healthy controls. Similarly, in Cohort 2, miRNA-125a was also identified among the miRNAs enriched in nonresponders vs. responders to CART treatment; Notably, miR-125a was significantly enriched in nonresponders at baseline (pre-CART treatment) and 1-month post-CART treatment (p=0.0177, p=0.0196). To define the functional effects of miR-125a, we delivered synthetic mimics or stable shRNA constructs encoding miR-125a into CART cells. Delivery of miR-125a was confirmed by RT-qPCR (p<0.0001 for mimic vs. scramble, p=0.0069 for shRNA vs. scramble). Here, miR-125a delivery and overexpression impaired CART cytotoxicity/killing of target tumor cells (~25% reduction, p= 0.0039), decreased antigen-specific proliferation (~47% reduction, p=0.0002), and increased apoptosis following antigen stimulation (1.8-fold increase, p=0.0002) as compared to scramble controls in vitro. We next studied how miR-125a impacts CART functions in vivo. Here, tumor-bearing mice treated with miR-125a–overexpressing CART cells (CART sh125a) exhibited accelerated tumor progression (4.1-fold increase, p=0.0019) and significantly reduced survival as compared to mice treated with scramble control CART treatment (CART shScr) (20% vs 80% survival at day 35, p=0.0064), underscoring the dramatic detrimental impact of miR-125a on therapeutic efficacy. To uncover changes in CART gene expression induced by miR-125a delivery, RNAseq was also performed across stimulated CART sh125a vs. CART shScr cells. CART sh125a cells demonstrated broad transcriptional changes, with gene set enrichment of E2F targets, G2/M checkpoint, and apoptosis pathways (FDR p<0.0001, p<0.0001, and p=0.006) further validating the role of miR-125a in disrupting CART cell cycle control, proliferation, and survival. Conclusion In summary, our study identifies miR-125a as a tumor-derived EV cargo that drives CART dysfunction in two separate B cell malignancies (CLL and DLBCL). miR-125a was enriched in both CLL EVs and nonresponders to CART19 therapy at baseline prior and post CART treatment. Functional studies demonstrate that miR-125a delivery and overexpression directly impair CART cytotoxicity and proliferation, increase CART apoptosis, reduce therapeutic efficacy in tumor xenograft models, and disrupt key pathways related to CART cell cycle regulation and survival. These findings position miR-125a as both a key mechanistic driver of CART failure and a potential predictive biomarker for treatment response, while highlighting it as a possible therapeutic target to improve CART outcomes in the clinic.

Abstract Chimeric antigen receptor T (CART) cell therapy is potentially curative in certain hematological malignancies, resulting in several FDA approvals since 2017. However, widespread adoption is limited by 1) cost and complexity of an autologous platform, 2) severe toxicities, and 3) limited durable remissions. To better predict CART efficacy and toxicity, live CART imaging would allow the realtime assessment of in vivo CART expansion and trafficking. Incorporation of the sodium iodide symporter (NIS)in CART cells (NIS+ CART cells) allows the CART cells to be tracked via PET imaging at high sensitivity. PET signal correlates with CART expansion and toxicity in mouse models (PMID: 34244299). Building on this platform, we aimed to track xenogeneic CART cells to monitor for their rejection, utilizing dogs with spontaneous lymphoma. Canine diffuse large B-cell lymphoma (DLBCL) affects over 250,000 dogs a year and has shared pathologic, biologic, phenotypic, genetic, immunotherapeutic targets, and treatment responses with human DLBCL, providing strong rationale for studying canine lymphoma as a pre-clinical model. We therefore initiated a USDA-CVB regulated pilot study and treated companion dogs with naïve canine CD20 (cCD20)+ DLBCL with human-derived TRAC- and B2M-KO NIS+ CART cells which targeted cCD20. First, we tested and optimized a chemotherapy regimen to achieve optimal lymphodepletion in dogs. Healthy beagles were treated with pentostatin (pen) on days 2-4 (0.125, 0.25, or 0.5 mg/kg i.v.), cyclophosphamide (cy) on day 4 (400 mg/m2 i.v.), or combination cy/pen. The most robust decrease in lymphocytes was seen with combination cy/pen (all cy/pen doses vs all other treatments, p≤0.0001) and in neutrophils with cy or cy/pen (all cy/pen doses vs all pen doses, p≤0.0001; cy vs 0.25 mg/kg pen, p≤0.001; cy vs 0.5 mg/kg pen, p≤0.0001). All lymphodepletion was transient and recovered within two weeks. All regimens were well-tolerated, and we therefore used 0.5 mg/kg pen plus 300 mg/m2 cy for the companion dog pilot study. Next, we assessed the feasibility of generating cCD20-targeted NIS+ human CART cells (CART20-NIS) with double KO (DKO) of TRAC and B2M. Primary T cells from healthy human donors were transduced with bicistronic lentivirus encoding 41BB-costimulated cCD20-targeted CAR and NIS. Two days after transduction, cells were transfected with CRISPR Cas9-gRNA targeted to TRAC and B2M. The resulting CART20-NIS DKO cells were expanded until day 11 and then were further depleted for CD3 and cryopreserved. CART20-NIS DKO cells demonstrated high CAR expression (untransduced T cells (UTD) vs CART20-NIS DKO, p<0.0001) and KO efficiency (TCRαβ and HLA-ABC expression of UTD vs CART20-NIS DKO, p<0.0001). NIS functionality was confirmed via a qualitative in vitro I-125radioactive uptake assay. We then assessed antigen specificity and activity in vitro. CART20-NIS DKO or UTD were co-cultured with either wildtype or cCD20-overexpressing K562 cells for three days. Target cell killing was then quantified via flow cytometry. CART20-NIS DKO cells demonstrated significant cytotoxicity against cCD20+ K562 cells (UTD vs CART20-NIS DKO, p<0.0001). Lastly, we treated two companion dogs in a USDA-CVB regulated pilot study of CART20-NIS DKO cells in naïve cCD20+ DLBCL. Following lymphodepletion on days -5 to -3, on day 0, dogs were i.v. administered 1 x 108 CART20-NIS DKO cells and intratumorally administered 2.5 x 107 CART20-NIS DKO cells in two lymph nodes, with contralateral saline injection as controls. Dogs underwent 18F-TFB-PET imaging on days 1, 3, and 7, which showed evidence of CART20-NIS DKO cells both in the CART-injected lymph nodes and distal lymph nodes, as determined by SUVmax. Both dogs sustained clinical remission for the duration of the 21-day study, as determined by sum of longest diameters and accompanying x-ray radiology and abdominal ultrasound. Histology showed marked reduction of CD20 cells in the lymph nodes. Organ and hematological toxicities were minimal, and dogs remained in remission for 95 and 101 days post-CART. In summary, this study demonstrated that xenogeneic CART cells were well-tolerated, induced remission, and were visualized via PET imaging in companion dogs with lymphoma. These results are promising both for the treatment of companion dogs with novel cell therapies and for the translation of novel CART cell therapies into human clinical trials.

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is marked by impaired T cell responses that limit the efficacy of immunotherapies. CD19-directed chimeric antigen receptor T cell therapy (CART19) received FDA approval to treat CLL in 2024. However, itsefficacy in CLL patients remains low, with durable remission rate of ~20%. Ibrutinib, a Bruton tyrosine kinase inhibitor, has shown synergy with CART19 in preclinical and early clinical studies. We aimed to study the impact of ibrutinib on CART19 utilizing clinical samples from the pivotal clinical trial E1912 (NCT02048813) of ibrutinib/rituximab (IR) vs fludarabine/cyclophosphamide/rituximab (FCR). This trial led to the FDA approval of first line ibrutinib in CLL. Methods: We generated CART19 incorporating either CD28 (28ζ) or 41BB (BBζ) costimulatory domains from T cells isolated from patients with CLL treated with IR or FCR on the E1912 trial. IR samples were collected at baseline (pretreatment), 6- or 12-months during treatment, or at progression. FCR samples were collected at baseline, 15-18 months during treatment, or at progression. For animal experiments, NOD-SCID-γ-/- (NSG) mice were intravenously (i.v.) engrafted with 1×10⁶ luciferase⁺ CD19⁺ JeKo-1 cells. Upon tumor establishment (~10⁸ photons/sec by bioluminescence imaging), mice were randomized and treated i.v. with 1×10⁶ 28ζ or BBζ derived from longitudinal IR- or FCR-treated patient samples. To further assess the impact of prolonged ibrutinib exposure on CART19 function, we chronically stimulated healthy donor-derived 28ζ or BBζ in vitro for 22 days with JeKo-1 cells and 100nM ibrutinib or vehicle control and then tested CART19 functions. Finally, we performed single cell RNA sequencing (scRNAseq) of longitudinal patient T cell samples treated with IR. Results: Ex vivo T cell expansion was significantly enhanced following IR compared to FCR. BBζ generated from IR-treated patients after 6 months of ibrutinib exhibited enhanced in vitro expansion (p=0.016) and antigen-specific proliferation (p=0.038) in the presence of JeKo-1 cells. This corresponded with significantly improved antitumor efficacy and prolonged survival in vivo compared to cells generated at disease progression (p=0.017). In contrast, 28ζ showed no significant differences in efficacy across IR studied treatment timepoints. BBζ from FCR-treated patients did not show differences in tumor control based on treatment timepoints but demonstrated shorter survival compared to BBζ from IR-treated patients. 28ζ from FCR-treated patients generated at disease progression demonstrated significantly reduced tumor control and shorter survival compared to 28ζ produced at baseline (p=0.0002), indicating detrimental effects of FCR treatment on the therapeutic potential of CART products. To further investigate the impact of prolonged ibrutinib exposure, we utilized in vitro models of CART exhaustion. We found that, after repeated antigen stimulation with JeKo-1 cells, ibrutinib-treated BBζ had significantly increased antigen-specific degranulation (p=0.005), and cytokine production (IL-2, p=0.045; TNF-α, p=0.003; IFN-γ, p=0.001), compared to BBζ treated with vehicle control. In contrast, 28ζ did not show improved degranulation or cytokine production in cocultures with ibrutinib. Prolonged ibrutinib exposure led to downregulation of FOXO3, a transcription factor associated with T cell exhaustion which has been shown to cause dysfunction specifically in BBζ. These results further indicate that ibrutinib may be more beneficial to BBζ. Indeed, scRNAseq revealed that after ≥ 6 months of ibrutinib, T cells showed downregulation of inflammatory, oxidative stress, and apoptosis pathways, paralleled by modest activation of MYC and E2F targets. However, at disease progression, T cells under ibrutinib treatment displayed marked upregulation of interferon signaling, inflammation responses, and stress-related programs, indicating a shift toward immune reactivation or dysfunction.Conclusion: Our study shows that BBζ, but not 28ζ, generated 6 months during ibrutinib treatment has superior antitumor activity and persistence compared to similar later timepoints or FCR-treated counterparts. Prolonged ibrutinib exposure enhances BBζ function by modulating stress and apoptotic pathways. These findings highlight the importance of pre-CART treatment timing and support ibrutinib as a potential enhancer of CART cell fitness in CLL.

Abstract Recent studies have highlighted the important but pleiotropic role of IL-4 in response to chimeric antigen receptor T (CART) cell therapy. IL-4 was identified as a regulator of dysfunction in CD19-targeting CART cells with a CD28 costimulatory domain (CART19-28ζ) through a genome-wide CRISPR knockout screen as well as RNA and ATAC sequencing of baseline and exhausted healthy donor CART19-28ζ cells and pre-infusion CART19-28ζ cell products from responders and non-responders in the ZUMA-1 trial (Stewart et al, Nat Commun., 2024). However, interrogation of a single-cell atlas fromactivated pre-infusion CART cell products from patients with acute lymphoblastic leukemia revealed a beneficial role for IL-4 and other type 2 cytokines in the promotion of long-term response (Bai et al, Nature, 2024). In this report, we aim to further understand how IL-4 can be detrimental to CART cell therapy. To study the regulation of CART19-28ζ cells by IL-4, we performed RNA sequencing on CART19-28ζ cells that were stimulated with CD19+target cells in the presence of either 20 ng/mL hrIL-4 or diluent for 24 hours, one week, or two weeks in an in vitro model for exhaustion as previously described (Stewart et al, Nat Commun., 2024). We observed that hrIL-4 treatment drove the development of a distinct transcriptomic profile after chronic stimulation for one week (up genes = 3253 and down genes = 3645) or two weeks (up genes = 6435 and down genes = 6224) but not following 24 hours of activation (up genes = 0 and down genes = 0). Gene set enrichment analysis on the differentially expressed genes following two weeks of chronic stimulation showed that hrIL-4 treatment pushes CART cells to a more exhausted and less naïve phenotype (FDR = 0.015) and leads to a broad downregulation in cytokine signaling (FDR = 0.012). To investigate transcription factors associated with IL-4-induced CART cell dysfunction, we used BETA analysis with publicly available transcription factor binding sites for STAT6, GATA3, and EOMES and our differential expression data. EOMES (p = 0.039), but not STAT6 (p = 0.999) nor GATA3 (p = 0.999) significantly regulated the differentially expressed genes. In support of these findings, the addition of hrIL-4 also significantly upregulated EOMES expression after one (p < 0.0001) and two (p < 0.0001) weeks of chronic stimulation and downregulated the expression of TBX21 (p < 0.0001), a binding competitor of EOMES, following two weeks of chronic stimulation. To evaluate if EOMES is critical for IL-4-induced CART cell dysfunction, we treated either control or EOMES CRISPR knockdown CART cells with 20ng/mL hrIL-4 during chronic stimulation. EOMES knockdown reduced IL-4-induced CART19-28ζ cell dysfunction as seen by an increase in antigen-specific expansion (p = 0.046) and an increase in cytokine production including IL-2 (p < 0.0001), TNF-α (p = 0.006), and IFN-γ (p = 0.020). Having identified that EOMES is at least partially responsible for IL-4-induced CART cell dysfunction, we evaluated EOMES binding sites in human CART19-28ζ cells with CUT&RUN sequencing. Our analysis revealed EOMES binding at hypersensitivity site 2 (HS2) in the IL-4 locus which, in a previous study, was more accessible in both exhausted healthy donor CART19-28ζ cells and pre-infusion CART cell products from non-responders in the ZUMA-1 clinical trial (Stewart et al, Nat Commun., 2024). To determine how HS2 can impact CART cell functions, we generated CART19-28ζ cells from either wildtype (HS2+/+) or HS2-deleted (HS2-/-) murine T-cells and tested them in a syngeneic model of lymphoma generated by engrafting C57BL6 mice with luciferase+ TBL12 cells (1x106, tail vein injection on Day -3) following preconditioning (250mg/kg cyclophosphamide, intraperitoneal injection on Day -4). Following randomization of mice via tumor burden (n = 5 per group), mice were treated with either HS2+/+ or HS2-/- CART19-28ζ cells (1x106 cells, tail vein injection). In this model, HS2-/- CART cells showed enhanced expansion (p = 0.022), increased anti-tumor activity (p = 0.018), and improved overall survival (p = 0.003). Together, our study shows that IL-4 induces a distinct cellular fate in CART19-28ζ cells in the presence of chronic antigen-specific stimulation at least partially through the upregulation of EOMES. Further, the epigenetic regulation of CART cells by the IL-4 enhancer HS2 can significantly influence CART cell activity, and deletion of HS2 can improve CART cell therapy.

Abstract INTRODUCTION Immune checkpoint inhibitors (ICIs) have emerged as a cornerstone of cancer therapy. However, despite their efficacy, ICIs can cause a unique spectrum of autoimmune toxicities known as immune-related adverse events (irAEs). In contrast to irAEs involving other organ systems, hematologic irAEs such as ICI-associated immune thrombocytopenia (ICI-ITP) are less common. As a result, existing data on ICI-ITP are largely confined to isolated case reports, single-center case series, and pharmacovigilance databases lacking granular clinical data. To address this key knowledge gap, we sought to comprehensively characterize the incidence, risk factors, clinical features, treatments, and outcomes associated with ICI-ITP. METHODS We performed a multicenter cohort study of adult patients who initiated ICI therapy at 29 hospitals across 7 major cancer centers in 11 geographically diverse states in the US between 2016-2023. ICI-ITP was defined as a decline in platelet count to <100 x109/L in the absence of an alternative more plausible etiology of thrombocytopenia. Patients with hematologic malignancies were excluded, as were those who received cytotoxic chemotherapy within 30 days prior to thrombocytopenia onset. For each patient identified as having ICI-ITP, we collected data on demographics, comorbidities, medications, laboratory values, treatments, and outcomes. All data were extracted by manual chart review. To identify risk factors for ICI-ITP and to examine its association with death, for each case of ICI-ITP we also collected data on two control patients who initiated ICI therapy at the same site and in the same year but without development of ICI-ITP. We used multivariable logistic regression to identify independent risk factors for ICI-ITP and its recovery (the latter defined according to criteria established by the ITP International Working Group). We used a multivariable Cox model to examine the association between ICI-ITP and its severity with all-cause mortality. RESULTS We identified a total of 214 patients who developed ICI-ITP among 86,648 examined (0.25%). The incidence was highly consistent across sites (range, 0.15–0.30%). Risk factors for ICI-ITP included simultaneous receipt of two different classes of ICIs, additional irAEs, and a lower baseline platelet count. ICI-ITP developed at a median of 8 weeks (IQR, 4–18) following ICI initiation. The median nadir platelet count was 41 x109/L (IQR, 17–64). Six out of 214 patients (2.8%) developed a major bleed, and 16 patients (7.5%) developed a clinically relevant non-major bleed. Cell-bound anti-platelet antibody testing was positive in 6 of 8 patients (75.0%) for whom it was assessed, and median circulating thrombopoietin levels (201 pg/mL [IQR, 178–295]) were similar to those seen in primary ITP. Treatments used included glucocorticoids (49.5%), immune globulin (18.2%), and thrombopoietin receptor agonists (13.6%). Overall, 161 patients (75.2%) recovered from ICI-ITP at a median of 2 weeks (IQR, 1–5) following ICI-ITP onset. Predictors of recovery included additional irAEs and a higher nadir platelet count. Treatment response was observed in 57.0%, 56.4%, and 58.3% of patients within 60 days following initiation of glucocorticoids, immune globulin, and thrombopoietin receptor agonists, respectively. Of 76 patients rechallenged with an ICI, 23 (30.3%) developed recurrent ICI-ITP at a median of 9 weeks (IQR, 2–15); 17 of these 23 patients (73.9%) recovered. Finally, ICI-ITP and its severity were monotonically associated with a higher risk of death (adjusted hazard ratio 2.96 [95% CI, 2.14–4.08] for those with severe ICI-ITP compared to those without ICI-ITP). CONCLUSION In this multicenter study involving patients treated with ICIs at 29 hospitals across the US, we identified 214 patients with ICI-ITP. For the first time, we characterize in detail the incidence, risk factors, clinical features, treatments, and outcomes of ICI-ITP, a relatively novel disease entity. We found that more than half of patients responded to conventional ITP-directed therapies, and that most patients rechallenged did not develop recurrent ICI-ITP. We also found that ICI-ITP was independently associated with an increased risk of death, including a nearly 3-fold increased risk in those with severe ICI-ITP. These findings highlight ICI-ITP as a rare but clinically significant complication of ICI therapy that hematologists must be equipped to recognize and manage.

Abstract Background: Integrin αE (CD103) has been thought to mediate gut-homing Treg retention during GVHD by facilitating E-cadherin interaction on intestinal epithelial cells. However, only a small percentage of natural Tregs express CD103 in peripheral blood (PB). To leverage this mechanism and potentially enhance Treg therapeutic efficacy, we hypothesized that Tregs overexpressing CD103 (CD103high) would show improved gut-homing and GVHD mitigation. Methods: Murine CD4 Tregs were isolated from C57BL/6 (B6) mice via flow sorting and were retrovirally transduced with murine CD103 or truncated epidermal growth factor receptor as a control. Transduced Tregs were then tested in a murine acute GVHD model (B6 → BALB/c). Mouse organs, including small intestine (SI), colon, spleen, PB, axillary (aLN) and mesentric lymph nodes (mLN) were harvested on day 10 post-transplant and analyzed by flow cytometry, multiplex cytokine assay, histopathology, and single-cell RNA sequencing (scRNA-seq). Ex vivo expanded Tregs were subjected to 13C-glucose tracing assay to evaluate glycolytic flux, TCA cycle, and lipid biosynthesis. Results: Despite comparable FoxP3 expression at the end of ex vivo expansion, CD103high Tregs had significantly higher in vitro suppression compared to control Tregs (54% vs. 33% at Treg:Tcon ratio of 1:8, p=0.004). Upon re-stimulation, CD103high Tregs demonstrated increased proliferation compared to control Tregs (3.8 vs 2.8 fold expansion, p <0.0001). Bulk RNA seq on ex vivo expanded CD103high showed upregulation of immuneregulatory and metabolic programs including GTPase signaling and oxidative stress control. These transcriptomic changes suggest that CD103 intrinsically promotes a regulatory phenotype, stress tolerance and metabolic fitness. In vivo, CD103highTregs significantly decreased the severity and mortality of GVHD, compared to control Tregs, as measured by clinical scores, weight loss, and survival (50% vs 0% on day +60, p<0.0001 for three independent experiments combined; n = 24/group). Day 20 histopathology revealed significantly improved colitis scores in mice treated with CD103high Tregs vs controls (9.5 vs. 14.6, p=0.02). While these findings could be partially attributed to CD103 gut-homing function, we observed that CD103high Treg absolute number and frequency were higher not only in the the GI organs and mLN, but also in the spleen, PB, and aLN with similar distribution ratios across different organs (~5-fold higher). In vivo CFSE suppression assay, where CFSE labeled donor T cells were cotransferred with Tregs into B6 Rag1ko mice, demonstrated enhanced suppressive function of CD103high Tregs in the spleen (p=0.02), an effect that could not be accounted for by the conventional gut-homing mechanism attributed to CD103. Plasma cytokine analysis showed significantly elevated IL-10 levels in mice treated with CD103high Tregs (p<0.0001), consistent with increased Treg number and frequency. 13C-glucose tracing assay showed lower palmitate levels in CD103high Tregs, indicating lower de novo lipid synthesis, while glycolysis and TCA cycle tracing were similar. Day 10 scRNA-seq on colon samples revealed seven distinct immune clusters. The neutrophil cluster was absent in CD103high Treg group, suggesting reduced inflammation. Donor-derived dendritic cells (DCs) upregulated genes such as Cstb, and Slamf7, while pro-inflammatory genes including Cd52 and Cd74 were downregulated. STAT5 signaling was upregulated, indicating reprograming of myeloid cells toward an immunoregulatory state. Colon epithelial cells from CD103high Treg-treated mice displayed enhanced stress tolerance, with increased oxidative phosphorylation, fatty acid metabolism, and epithelial repair pathways. CellChat analysis revealed that CD103high Tregs reshaped the intestinal communication landscape, with DCs acting as key signaling hubs through interactions such as Ccl6–Ccr2 and Lgals3–Cd44. Enhanced DC-to-DC and DC-to-T cell communication suggest that CD103high Tregs promote a tolerogenic DC network that modulates downstream immune responses and supports epithelial recovery. Conclusion: Tregs overexpressing CD103 via retroviral transduction significantly improve overall survival and GVHD clinical scores in a murine transplant model. CD103High Tregs orchestrate broad remodeling of cell-cell communication across immune and stromal compartments, promoting an immunoregulatory microenvironment that underlies their protective effects in GVHD.

Abstract B cell maturation antigen (BCMA)-targeted chimeric antigen receptor T (CART) cell therapies have emerged as a potentially curative treatment for multiple myeloma (MM). However, 50-70% of patients relapse within 3 years. The MM tumor microenvironment (TME) is highly immunosuppressive with an abundance of bone marrow (BM) cancer-associated fibroblasts (CAFs), which contribute to BCMA-CART cell failure. Therapeutic targeting of BM-CAFs has been beset by 1) an incomplete understanding of CAF interactions with CART cells and other BM cells and 2) toxicity risks due to prolonged depletion of healthy fibroblasts and inefficient drug delivery to the BM. To elucidate how BM-CAFs drive BCMA-CART resistance, we performed single cell RNA sequencing (scRNAseq) of baseline BM aspirates from responders (R, n = 6) and non-responders (NR, n = 6) to BCMA-CART cell therapy. NR BMs were enriched for inhibitory CAFs (iCAFs) that highly express fibroblast activation protein (FAP). Spatial analyses of BM cells revealed greater iCAF-iCAF and iCAF-inhibitory macrophage interactions in NR. These findings suggest that FAP+ iCAFs in NR are abundant in the BM and exhibit significant interactions with immunosuppressive macrophages. Next, we sought to validate our scRNAseq data in preclinical models. Xenograft mice were intravenously (IV) engrafted with luciferase+ (luc+) BCMA+ OPM2 cells and/or patient-derived (pd) BM-CAFs (1x106 each). Following engraftment, mice received 1x106 BCMA-CART cells (IV). BCMA-CART cells eradicated tumors in mice engrafted with OPM2 cells, but not in mice engrafted with pdBM-CAFs and OPM2 cells (p<0.01). In NSG and humanized mice (NSG mice engrafted with human CD34+ cells in the first 48 hours of life), tumor growth was significantly accelerated in the presence of pdBM-CAFs (NSG: p<0.01; humanized: p<0.001). BCMA-CART, OPM2, and pdBM-CAF co-cultures also showed significant increases in immunosuppressive cytokines including IL-1Rα, IL-4 and IL-10 and inhibited CART cell proliferation compared to co-cultures without pdBM-CAFs (IL-1Rα, IL-10: p<0.01; IL-4: p<0.001; proliferation: p<0.05). To overcome CAF-mediated CART dysfunction, we engineered BCMA-CART cells to secrete molecules to target BM FAP+ iCAFs and remodel the MM-TME (STriKEs). We designed two BCMA-CART-STriKE strategies: 1) a tri-specific composed of IL-15, anti-FAP, and anti-CD16 molecules to activate innate immune cells to kill CAFs and 2) bi-specific anti-FAP, anti-SIRPα molecules linked to an antibody Fc region (Fc fusion) targeting the CD47/SIRPα axis to induce phagocytosis of FAP+ iCAFs. We hypothesized that BCMA-CART-STriKEs would surmount CAF-mediated immunosuppression without toxicity. We generated BCMA-CART-STriKEs from healthy human donor T cells. CAR expression, STriKE secretion, and binding were confirmed via flow cytometry. Co-cultures of BCMA-CART-STriKEs, donor-matched macrophages, OPM2 cells or BCMA+ MM1S cells, and pdBM-CAFs showed significantly higher OPM2 or MM1S killing (p<0.001), immune cell activation (p<0.01), and phagocytosis (p<0.05), compared to control BCMA-CART cells secreting IL-15 or Fc fusions targeting only FAP or only SIRPα. Next, we assessed BCMA-CART-STriKE efficacy in vivo. NOD-SCID-γ-/- (NSG) mice received 1x106 pdBM-CAFs and 1x106 luc+ OPM2 or luc+ MM1S cells via IV or subcutaneous (SQ) injection. Mice were randomized after confirming tumor engraftment (via bioluminescence imaging for IV or tumor caliper for SQ) and received IV treatment with 1x106 BCMA-CART-STriKEs (or controls) and 5x105 donor-matched macrophages or CD16+ immune cells. BCMA-CART-STriKE-treated mice achieved total tumor clearance. Treatment with BCMA-CART cells secreting IL-15 led to lethal weight loss despite tumor clearance while BCMA-CART cells secreting Fc fusions targeting only FAP or SIRPα failed to eradicate tumors. Lastly, we assessed the potential toxicities of BCMA-CART-STriKEs in humanized mice engrafted with OPM2 cells and pdBM-CAFs (1x106 each). CART cells expressing BCMA and FAP CARs (BCMA-FAP CART) served as a control. BCMA-FAP CART treatment resulted in significant toxicity and weight loss while BCMA-CART-STriKE treatment did not (p<0.01). In summary, we have identified iCAF interactions with immunosuppressive immune cells as key contributors to BCMA-CART cell failure and designed novel BCMA-CART-STriKEs that overcome this failure in MM preclinical models. These findings support the translation of BCMA-CART-STriKEs to a phase I clinical trial.

Summary Esophageal high-resolution manometry (HRM) is the gold standard for evaluating esophageal motility disorders but can be limited by patient intolerance. With increasing use of glucagon-like peptide-1 receptor agonists (GLP-1RAs) for obesity and type 2 diabetes, delayed gastric emptying raises concerns for HRM feasibility and safety. This study assessed HRM tolerability in patients on GLP-1RAs. We conducted a retrospective case–control study of adult patients who underwent HRM at our tertiary care center between January 2014 and November 2024. Patients actively taking GLP-1RAs during HRM were identified as cases, while an equal number of consecutive eligible patients not on GLP-1RAs served as controls. Patients with established gastrointestinal dysmotility, prior foregut surgery, esophageal mechanical obstruction, large hiatal hernias, malignancy, or recent opioid use were excluded. From a prospective database of 7194 HRM attempts, 83 cases and 83 matched controls were identified. Among 166 patients, 15 (9.0%) had incomplete HRM due to intolerance, with similar rates between GLP-1RA users and controls (10.84% vs. 7.2%, P = 0.59). Predictors of incomplete HRM included younger age (44.9 ± 17.4 vs. 60.4 ± 13.7 years, P < 0.01), globus (P = 0.02), dyspepsia (P = 0.02), and depression (P = 0.04). No aspiration or adverse events occurred in either group. Duration of HRM before abortion was similar between cases and controls. GLP-1RA use was not associated with increased risk of HRM abortion or adverse events, suggesting these medications do not significantly impact HRM feasibility or safety.

Abstract Description Chronic infection-associated inflammatory arthritis is a poorly understood phenomenon and varies by the associated infection. Infections such as human immunodeficiency virus (HIV), hepatitis B and C, lyme disease, cytomegalovirus, and numerous others can not only cause patients to develop arthralgias, but synovial inflammation with inflammatory infiltrate. Based on our prior studies looking at HIV-infected humanized mouse models, there is significant inflammation that develops in the synovial tissue with elevated levels of GM-CSF, TNF, and IL-17. Our current studies are now focused on studying a diverse group of patients with chronic infections that developed inflammatory arthritis and look for patterns of immune cell distribution and cytokine profiles between the various infections. Patients were compared with healthy controls as well as patients with rheumatoid arthritis who tested negative for these infections. Increased inflammatory cells were seen in patients with chronic infections in the serum which was similar to the rheumatoid arthritis patients. Uniquely, elevated levels of GM-CSF were seen in the patients with HIV and hepatitis B and C that correspond with the prior mouse model data. Our data will help provide additional insight into not only the immunology and pathology associated with various infections, but insights into the development of various forms of inflammatory arthritis. Funding Sources Mayo Clinic Benefactor-funded Early Career Development Award in Rheumatology Topic Categories Immune Mechanisms of Human Disease (HUM)