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Supplementary Methods, Supplementary Figures and Figure Legends. Figure S1. EGR1 is a downstream target of BCR and JAK1 signaling (related to Figure 1). Figure S2. Sensitivity of MCL cell lines to EGR1 shRNAs and statistical analysis (related to Figure 2). Figure S3. EGR1 motif analysis and gene set enrichment analysis of RNA-seq data (related to Figure 3). Figure S4. Enrichment of E2F pathway genes in ABC DLBCL after EGR1 knockdown (related to Figure 4). Figure S5. EGR1 regulates expression of MYC and MYC targets and OCI-Ly10 xenografts (related to Figure 4). Figure S6. Enrichment of EGR1 on EGR1 own promoter and EP300 regulatory (5'UTR and enhancer) regions (related to Figure 4). Figure S7. Inhibition of EGR1 transcription and tumor growth by mithramycin (related to Figure 4). Figure S8. Synergism of mithramycin and lenalidomide in ABC DLBCL (related to Figure 6). Figure S9. Expression of MYC and MYC target genes is higher in ABC DLBCL than GCB DLBCL patient samples (related to the Discussion). Figure S10. No correlation between EGR1 and MYC expression in DLBCL cell lines and patient samples (related to the Discussion).

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell–like subtype (ABC) than germinal center B cell–like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL.Implications:The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Supplementary Table S1 including analyzed RNA-seq and ChIP-seq data

An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4 + /CD8 + double-positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develops from antigen-stimulated CD8 T cells, which become transcriptionally, metabolically, and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when retransplanted into naïve mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.

New non-destructive tools are needed to reliably assess lymphocyte function for immune profiling and adoptive cell therapy. Optical metabolic imaging (OMI) is a label-free method that measures the autofluorescence intensity and lifetime of metabolic cofactors NAD(P)H and FAD to quantify metabolism at a single-cell level. Here, we investigate whether OMI can resolve metabolic changes between human quiescent versus IL4/CD40 activated B cells and IL12/IL15/IL18 activated memory-like NK cells. We found that quiescent B and NK cells were more oxidized compared to activated cells. Additionally, the NAD(P)H mean fluorescence lifetime decreased and the fraction of unbound NAD(P)H increased in the activated B and NK cells compared to quiescent cells. Machine learning classified B cells and NK cells according to activation state (CD69+) based on OMI parameters with up to 93.4% and 92.6% accuracy, respectively. Leveraging our previously published OMI data from activated and quiescent T cells, we found that the NAD(P)H mean fluorescence lifetime increased in NK cells compared to T cells, and further increased in B cells compared to NK cells. Random forest models based on OMI classified lymphocytes according to subtype (B, NK, T cell) with 97.8% accuracy, and according to activation state (quiescent or activated) and subtype (B, NK, T cell) with 90.0% accuracy. Our results show that autofluorescence lifetime imaging can accurately assess lymphocyte activation and subtype in a label-free, non-destructive manner.TeaserLabel-free optical imaging can assess the metabolic state of lymphocytes on a single-cell level in a touch-free system.

A 6-year-old female presenting with an abdominal mass was found to have an unresectable undifferentiated sarcoma. The tumor did not respond to multi-agent chemotherapy. However, molecular testing identified an NTRK3 fusion, and treatment was changed to larotrectinib monotherapy. Following 6 months of therapy, the patient achieved a very good partial response with 96% reduction in tumor size. She underwent proton beam radiation therapy with continued larotrectinib therapy and achieved a complete response. This case report shows that an NTRK fusion positive undifferentiated sarcoma can be safely treated with larotrectinib and radiation therapy and highlights the importance of early molecular testing.

Background Acute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS. Methods Human monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation. Results LPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically. Conclusion LPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.

BackgroundChimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain.MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG™ xenograft neuroblastoma model.ResultsIn comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model.ConclusionsThis study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.

Relapsed/refractory T cell acute lymphoblastic leukemia (T-ALL) is difficult to salvage especially in heavily pretreated patients, thus novel targeted agents are sorely needed. Hyperactivated JAK/STAT and BCL2 overexpression promote increased T-ALL proliferation and survival, and targeting these pathways with ruxolitinib and venetoclax may provide an alternative approach to achieve clinical remissions. Ruxolitinib and venetoclax show a dose-dependent effect individually, but combination treatment synergistically reduces survival and proliferation of Jurkat and Loucy cells in vitro. Using a xenograft CXCR4+ Jurkat model, the combination treatment fails to improve survival, with death from hind limb paralysis. Despite on-target inhibition by the drugs, histopathology demonstrates increased leukemic infiltration into the central nervous system (CNS), which expresses CXCL12, as compared to liver or bone marrow. Liquid chromatography-tandem mass spectroscopy shows that neither ruxolitinib nor venetoclax can effectively cross the blood-brain barrier, limiting efficacy against CNS T-ALL. Deletion of CXCR4 on Jurkat cells by CRISPR/Cas9 results in prolonged survival and a reduction in overall and neurologic clinical scores. While combination therapy with ruxolitinib and venetoclax shows promise for treating T-ALL, additional inhibition of the CXCR4-CXCL12 axis will be needed to eliminate both systemic and CNS T-ALL burden and maximize the possibility of complete remission.

Background aims Several methods to expand and activate (EA) NK cells ex vivo have been developed for the treatment of relapsed or refractory cancers. Infusion of fresh NK cells is generally preferred to the infusion of cryopreserved/thawed (C/T) NK cells because of concern that cryopreservation diminishes NK cell activity. However, there has been little head-to-head comparison of the functionality of fresh versus C/T NK cell products. Methods We evaluated activity of fresh and C/T EA NK cells generated by interleukin (IL)-15, IL-2 and CD137L expansion. Results Analysis of C/T NK cell products demonstrated decreased recovery of viable CD56+ cells, but the proportion of NK cells in the C/T EA NK cell product did not decrease compared with the fresh EA NK cell product. Fresh and C/T EA NK cells demonstrated increased granzyme B compared with NK cells pre-expansion, but only fresh EA NK cells showed increased NKG2D. Compared with fresh EA NK cells, cytotoxic ability of C/T EA NK cells was reduced, but C/T EA NK cells remained potently cytotoxic against tumor cells via both antibody-independent and antibody-dependent mechanisms within 4 h post-thaw. Fresh EA NK cells generated high levels of gamma interferon (IFN-γ), which was abrogated by JAK1/JAK2 inhibition with ruxolitinib, but C/T EA NK cells showed lower IFN-γ unaffected by JAK1/JAK2 inhibition. Discussion Usage of C/T EA NK cells may be an option to provide serial "boost" NK cell infusions from a single apheresis to maximize NK cell persistence and potentially improve NK-induced responses to refractory cancer.

The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.

An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-vs-host disease (GVHD) while preserving the graft-vs-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4+/CD8+ double positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develop from antigen stimulated CD8 T cells which become transcriptionally, metabolically and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when re-transplanted into naive mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.One Sentence SummaryHuman CD4+/CD8+ double positive T cells (DPTs) mediate xenogeneic GVHD but possess limited GVL activity.

Natural Killer (NK) cell therapies are being increasingly used as an adoptive cell therapy for cancer because they can recognize tumor cells in an antigen-independent manner. While promising, the understanding of NK cell persistence, particularly within a harsh tumor microenvironment, is limited. Fluorine-19 ((19)F) MRI is a non-invasive imaging modality that has shown promise in longitudinally tracking cell populations in vivo; however, it has not been studied on murine NK cells. In this study, the impact of (19)F labeling on murine NK cell viability and function was assessed in vitro and then used to quantify NK cell persistence in vivo. While there was no noticeable impact on viability, labeling NK cells with (19)F did attenuate cytotoxicity against lymphoma cells in vitro. Fluorescent microscopy verified (19)F labeling in both the cytoplasm and nucleus of NK cells. Lymphoma bearing mice were given intratumoral injections of (19)F-labeled NK cells in which signal was detectable across the 6-day observation period via (19)F MRI. Quantification from the composite images detected 78–94% of the initially injected NK cells across 6 days, with a significant decrease between days 3 to 6. Postmortem flow cytometry demonstrated retention of (19)F intracellularly within adoptively transferred NK cells with less than 1% of (19)F-containing cells identified as tumor-associated macrophages that presumably ingested nonviable NK cells. This work demonstrates that (19)F MRI offers a specific imaging platform to track and quantify murine NK cells within tumors noninvasively.

BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors.

In the setting of radiation-induced trauma, exposure to high levels of radiation can cause an acute radiation syndrome (ARS) causing bone marrow (BM) failure, leading to life-threatening infections, anemia, and thrombocytopenia. We have previously shown that human macrophages educated with human mesenchymal stem cells (MSCs) by coculture can significantly enhance survival of mice exposed to lethal irradiation. In this study, we investigated whether exosomes isolated from MSCs could replace direct coculture with MSCs to generate exosome educated macrophages (EEMs). Functionally unique phenotypes were observed by educating macrophages with exosomes from MSCs (EEMs) primed with bacterial lipopolysaccharide (LPS) at different concentrations (LPS-low EEMs or LPS-high EEMs). LPS-high EEMs were significantly more effective than uneducated macrophages, MSCs, EEMs, or LPS-low EEMs in extending survival after lethal ARS in vivo. Moreover, LPS-high EEMs significantly reduced clinical signs of radiation injury and restored hematopoietic tissue in the BM and spleen as determined by complete blood counts and histology. LPS-high EEMs showed significant increases in gene expression of STAT3, secretion of cytokines like IL-10 and IL-15, and production of growth factors like FLT-3L. LPS-EEMs also showed increased phagocytic activity, which may aid with tissue remodeling. LPS-high EEMs have the potential to be an effective cellular therapy for the management of ARS.

Stimulatory type 1 conventional dendritic cells (cDC1s) engage in productive interactions with CD8

Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor β (TGF-β) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4 hours at 37°C with or without the addition of 20 ng/mL of each IL10 and TGF-β (n = 7). Naive and cold storage (4 hours at 4°C) livers served as controls (n = 4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGF-β treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGF-β treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGF-β.

BackgroundAllogeneic hematopoietic stem cell transplants (allo-HSCTs) dramatically reduce HIV reservoirs in antiretroviral therapy (ART) suppressed individuals. However, the mechanism(s) responsible for these post-transplant viral reservoir declines are not fully understood but may include pre-transplant conditioning regimens, ART-mediated protection of donor cells, and graft-versus-host (GvH) responses. Therefore, we modeled allo-HSCT in ART-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay.ResultsWe infected four MCMs with CCR5-tropic SHIV162P3 and started ART 6-16 weeks post-infection (p.i.) to establish robust viral reservoirs. We maintained the MCMs on continuous ART during myeloablative conditioning with total body irradiation (TBI) and while transplanting allogeneic MHC-matched α/β T cell-depleted bone marrow cells. Post-transplant, we prophylactically treated the MCMs with cyclophosphamide and tacrolimus to prevent GvH disease (GvHD). The transplants produced ~85% whole blood donor chimerism without causing high-grade GvHD. Consequently, three MCMs had undetectable SHIV DNA in their peripheral blood mononuclear cells post-transplant. However, SHIV-harboring cells persisted in various tissues. We detected viral DNA in lymph node biopsies and terminal analyses of tissues between 38 and 62 days post-transplant. Further, we removed ART from one MCM at 63 days post-transplant, resulting in viral rebound within seven days and viral loads nearing 1×108SHIV RNA copies/ml of plasma after treatment interruption.ConclusionsOur results indicate that myeloablative conditioning and maintaining ART through the peri-transplant period alone are insufficient for eradicating latent viral reservoirs early after allo-HSCTs. Furthermore, our findings suggest that extended ART and GvH responses may be necessary to substantially deplete viral reservoirs after allo-HSCTs.

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell–like subtype (ABC) than germinal center B cell–like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL. Implications: The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Patients with relapsed/refractory osteosarcoma (OSA) have a poor prognosis with few treatment options. Immunotherapy with allogeneic natural killer (NK) cells after allogeneic bone marrow transplant (BMT) is an attractive approach to employ a graft-versus-tumor (GVT) effect against OSA. However, NK cells have had limited success against solid tumors in vivo. CD155 is overexpressed on OSA and interacts with DNAM-1, an activating ligand, and TIGIT, an inhibitory ligand, on NK cells. The impact of blocking CD155 after allogeneic BMT is unknown. IL-15-expanded C57BL/6 (B6, H-2b) and BALB/c (H-2d) murine NK cells were co-cultured with K7M2 murine OSA (H-2d) after CD155 and CD155 ligand blockade, and analyzed by flow cytometry and cytotoxicity assays. BALB/c mice were also transplanted with T cell depleted allogeneic B6 or syngeneic BALB/c bone marrow, challenged with K7M2 and treated with IL-15-expanded NK cells and CD155 blockade. Allogeneic NK cells showed increased degranulation, interferon-gamma production and cytotoxicity against K7M2 OSA in vitro compared to syngeneic NK cells. NK cell activation and cytotoxicity were further enhanced by CD155 and TIGIT blockade, but decreased with DNAM-1 blockade. In vivo, allogeneic NK cell infusion and anti-CD155 treatment decreased tumor growth and increased survival compared to syngeneic NK cell treatment, without induction of graft-versus-host-disease. CD155 blockade augments NK cell activation, cytotoxicity and GVT effects against OSA without toxicity, suggesting TIGIT is the dominant CD155 checkpoint during allogeneic BMT. CD155 blockade with allogeneic NK cell therapy after BMT may be an effective combination immunotherapy platform for treating relapsed/refractory OSA. Supported by NIH grants (T32 CA009135, R01 CA215461)

Background Pediatric patients with relapsed and refractory osteosarcoma have poor prognoses with few treatment options. Allogeneic bone marrow transplant (BMT) has not yet shown a graft-versus-tumor (GVT) effect for osteosarcoma. Natural killer (NK) cells demonstrate antitumor activity against osteosarcoma, but adoptively transferred NK cells have limited proliferation, cytotoxicity, and persistence in vivo. To enhance an NK-specific GVT effect, we propose blocking the poliovirus receptor CD155 checkpoint molecule, which is overexpressed on osteosarcoma and can engage both activating and inhibitory receptors on NK cells. The impact of CD155 blockade on GVT and graft-versus-host-disease (GVHD) is unknown. Methods NK cells from C57BL/6 (B6) mice were expanded with recombinant IL-15/IL-15R and analyzed by flow cytometry. Cytotoxicity assays were performed with IL-15 expanded B6 NK cells and mKate2-expressing K7M2 murine osteosarcoma at a 1:1 ratio with blockade of CD155 and CD155 ligands. To test efficacy of NK cell infusion and CD155 blockade after allogeneic BMT, BALB/c mice were lethally irradiated, transplanted with allogeneic B6 bone marrow, and challenged with luciferase-expressing K7M2 on day 0. At day 7, mice received IL-15 expanded B6 NK cells intravenously with either anti-IgG control or anti-CD155 antibody intraperitoneally and IL-2 subcutaneously on days 7 and 11. Mice were monitored for tumor growth by bioluminescence, and toxicity by GVHD using weight loss and clinical scores. Results Compared to unexpanded murine NK cells, IL-15 expanded NK cells (n = 6) show increased expression of NKG2D (65.33 ± 10.77% NKG2D+, p = 0.0077; 1030 ± 177.0 MFI, p = 0.0101) and an increased ratio of the CD155 activating (CD226) to inhibitory (TIGIT) ligand expression (11.71 ± 4.121, p = 0.0362). In cytotoxicity assays with IL-15 expanded allogeneic murine NK cells (n = 3 replicates), CD155 blockade enhances K7M2 osteosarcoma lysis (60.62 ± 3.19%, p = 0.0189) compared to IgG control (29.01 ± 7.66%). CD226 blockade decreased tumor killing (10.62 ± 8.51%, p = 0.0053) compared to CD155 blockade. In vivo allogeneic murine NK cell infusion and anti-CD155 antibody treatment after allogeneic BMT decreased tumor area under the curve by 44.3% compared to IgG control, without exacerbating GVHD. Conclusions These findings demonstrate that blockade of CD155 enhances an allogeneic NK cell-specific GVT effect for osteosarcoma treatment without exacerbating GVHD. CD155 blockade has the potential to improve usage of allogeneic BMT and NK cell adoptive immunotherapy as a combination treatment for osteosarcoma, and perhaps other pediatric sarcomas. Acknowledgements This work was supported by grants from the National Institute of General Medical Sciences/NIH T32 GM008692 and Training in Cancer Biology Training Grant NIH T32 CA009135 (to MMC), St. Baldrick’s Stand up to Cancer (SU2C) Pediatric Dream Team Translational Research Grant SU2C-AACR-DT-27-17, NCI/NIH R01 CA215461, American Cancer Society Research Scholar Grant RSG- 18-104-01-LIB, and the Midwest Athletes Against Childhood Cancer (MACC) Fund (to CMC). SU2C is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. The contents of this article do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

Background Relapsed pediatric sarcomas have a poor prognosis with no available curative options. Alpha-Tocopheryloxyacetic acid (a-TEA) is a redox-silent analog of alpha-tocopherol that induces apoptotic and immunogenic cell death of tumor cells at doses that are not harmful to healthy normal cells. In a first-in-human clinical trial, a-TEA was well tolerated in adults with advanced solid tumors (NCT02192346), but has not yet been studied in pediatric sarcoma. We used a murine model of rhabdomyosarcoma (M3-9-M RMS) to assess the in vitro and in vivo anti-tumor effects of a-TEA. Methods In vitro studies were performed on the M3-9-M RMS cell line to measure a-TEA-mediated apoptosis using flow cytometry (Annexin V+/7AAD+ cells) and live cell imaging (Annexin V+ cells). In vivo studies involved orthotopic implantation of luciferase+ M3-9-M tumor cells into syngeneic C57BL/6 recipients. Once tumors were palpable, mice were randomized to a control diet or a-TEA-supplemented chow for 21 days and evaluated for bioluminescence, tumor growth and overall survival. Gene expression of tumor-infiltrating and splenic T cells were analyzed by bulk RNA-Seq and flow cytometry respectively. Results M3-9-M RMS treatment with 2.5–100 uM a-TEA induced apoptosis in a dose-dependent manner within 24 hours (p 4 weeks) compared to recipients of matched control chow (p Conclusions These data indicate that a-TEA mediates apoptosis of RMS in vitro and suppresses in vivo tumor growth, leading to prolonged survival likely via enhanced activation of adaptive immunity through CD4+ T cells and suppression of innate immunity through regulation of myeloid cell subsets. Furthermore, a-TEA may have direct effects on tumor cell proliferation through EP300 and c-Jun as well as indirect effects on tumor growth by regulation of immune cell recruitment through CD24 and CXCR4 gene expression. Administration of a-TEA as a potential salvage treatment for RMS is warranted. Acknowledgements The study was supported by NIH TL1 TR002375 (FS), St. Baldrick’s Stand up to Cancer (SU2C) Pediatric Dream Team Translational Research Grant SU2C-AACR-DT-27-17, NIH/NCI R01 CA215461, American Cancer Society Research Scholar Grant RSG- 18-104-01-LIB, and the Midwest Athletes Against Childhood Cancer (MACC) Fund (CMC). SU2C is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. The contents of this article do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government. Ethics Approval The University of Wisconsin-Madison Animal Care and Use Committee approved all protocols (M005915).

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for hematologic cancers and chronic infections such as human immunodeficiency virus (HIV). Its success in these settings is attributed to the ability of engrafting immune cells to eliminate cancer cells or deplete the HIV reservoir (graft-versus-host effect [GvHE]). However, alloHSCT is commonly associated with graft-versus-host diseases (GvHDs) causing significant morbidity and mortality, thereby requiring development of novel allogeneic HSCT protocols and therapies promoting GvHE without GvHD using physiologically relevant preclinical models. Here we evaluated the outcomes of major histocompatibility complex-matched T-cell receptor α/β-depleted alloHSCT in Mauritian cynomolgus macaques (MCMs). Following T-cell receptor α/β depletion, bone marrow cells were transplanted into major histocompatibility complex-identical MCMs conditioned with total body irradiation. GvHD prophylaxis included sirolimus alone in two animals or tacrolimus with cyclophosphamide in another two animals. Posttransplant chimerism was determined by sequencing diagnostic single-nucleotide polymorphisms to quantify the amounts of donor and recipient cells present in blood. Animals treated posttransplant with sirolimus developed nearly complete chimerism with acute GvHD. In the cyclophosphamide and tacrolimus treatment group, animals developed mixed chimerism without GvHD, with long-term engraftment observed in one animal. None of the animals developed cytomegalovirus infection. These studies indicate the feasibility of alloHSCT engraftment without GvHD in an MHC-identical MCM model following complete myeloablative conditioning and anti-GvHD prophylaxis with posttransplant cyclophosphamide and tacrolimus. Further exploration of this model will provide a platform for elucidating the mechanisms of GvHD and GvHE and for testing novel alloHSCT modalities for HIV infection.

PURPOSE OF REVIEW: Innovative and minimally toxic treatment approaches are sorely needed for the prevention and treatment of hematopoietic acute radiation syndrome (H-ARS). Cell therapies have been increasingly studied for their potential use as countermeasures for accidental and intentional ionizing radiation exposures which can lead to fatal ARS. Mesenchymal stem/stromal cells (MSCs) are a cell therapy that have shown promising results in preclinical studies of ARS, and are being developed in clinical trials specifically for H-ARS. MSCs, MSC-educated macrophages (MEMs) and MSC-exosome educated macrophages (EEMs) all have the potential to be used as adoptive cell therapies for H-ARS. Here we review how MSCs have been reported to mitigate inflammation from radiation injury while also stimulating hematopoiesis during ARS. RECENT FINDINGS: We discuss emerging work with immune cell subsets educated by MSCs, including MEMs and EEMs, in promoting hematopoiesis in xenogeneic models of ARS. We also discuss the first placental-derived MSC product to enter phase I trials, PLX-R18, and the challenges faced by bringing MSC and other cell therapies into the clinic for treating ARS. SUMMARY: Although MSCs, MEMs and EEMs are potential cell therapy candidates in promoting hematopoietic HRS, challenges persist in translational clinical development of these products to the clinic. Whether any of these cellular therapies will be sufficient as stand-alone therapies to mitigate H-ARS or if they will be a bridging therapy that insures survival until a curative allogeneic hematopoietic stem cell transplant can be performed are the key questions that will have to be answered.

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Over the past two decades, immune cell therapy has emerged as a potent treatment for multiple cancers, first through groundbreaking leukemia therapy, and more recently, by tackling solid tumors. Developing successful therapeutic strategies using live cells could benefit from the ability to rapidly determine their in vivo biodistribution and persistence. Assaying cell biodistribution is unconventional compared to traditional small molecule drug pharmacokinetic readouts used in the pharmaceutical pipeline, yet this information is critical towards understanding putative therapeutic outcomes and modes of action. Towards this goal, efforts are underway to visualize and quantify immune cell therapy in vivo using advanced magnetic resonance imaging (MRI) techniques. Cell labeling probes based on perfluorocarbon nanoemulsions, paired with fluorine-19 MRI detection, enables background-free quantification of cell localization and survival. Here, we highlight recent preclinical and clinical uses of perfluorocarbon probes and 19F MRI for adoptive cell transfer (ACT) studies employing experimental T lymphocytes, NK, PBMC, and dendritic cell therapies. We assess the forward looking potential of this emerging imaging technology to aid discovery and preclinical phases, as well as clinical trials. The limitations and barriers towards widespread adoption of this technology, as well as alternative imaging strategies, are discussed.

Acute graft-vs-host disease (aGVHD) and cancer relapse remain the primary complications following an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for malignant blood disorders. While post-transplant cyclophosphamide combined with standard GVHD prophylaxis has greatly reduced the overall prevalence and severity of aGVHD, relapse rates remain a concern. There is thus a need to identify the specific human T cell populations mediating GVHD vs GVL activity as a means to develop targeted therapeutics capable of controlling aGVHD without inhibiting GVL activity. In this study, we identify a novel human T cell population that develops after transplant that is predictive and sufficient for GVHD pathology. To determine the role of human T cell populations in aGVHD, we performed xenogeneic transplantation studies using primary human graft tissue from a variety of sources (peripheral blood, G-CSF mobilized peripheral blood, bone marrow and umbilical cord blood) in addition to collecting primary human aGVHD blood samples from our clinic. Using the LD50 dose of human graft tissue, we identified a novel mature CD4 +/CD8αβ + double positive (DP) T cell population that only developed after transplantation. The development of this population was further confirmed in aGVHD patients from our clinic. The presence of DP T cells, irrespective of graft source, was also predictive of lethal GVHD in as early as one week after xenogeneic transplantation. To identify the origin of DP T cells, we transplanted isolated human CD4 or CD8 T cells into mice which showed that DP T cells only arise from the CD8 pool. Furthermore, re-transplantation of flow-sorted CD8 T cells from GVHD mice did not reveal a 2nd wave of DP T cell differentiation. This data, in addition to their highly proliferative state, suggests that DP T cells represent highly activated CD8 T cell clones. The ability of these CD8-derived DP T cells to gain CD4 expression coincides with their co-expression of both RUNX3 and THPOK, the master transcription factors of the CD8 and CD4 lineages respectively, that classically repress each other. Intracellular cytokine staining also revealed that DP T cells are the primary activated T cell population in xenogeneic GVHD, secreting both modulatory and cytotoxic cytokines (e.g. IFNγ, IL-17A, IL-22, perforin and granzyme). Ex vivo re-stimulation or re-transplantation of flow-sorted DP T cells showed that this T cell population is capable of dividing and expanding independent of CD4 and CD8 single positive T cells with the majority of the isolated DP T cells retaining their co-expression of CD4 and CD8. Finally, transplantation of either isolated human peripheral blood CD4 or CD8 T cell populations were capable of causing lethal GVHD. Conversely, re-transplantation of flow-sorted DP, CD8 or CD4 T cells from GVHD mice revealed that DP and CD4 T cells are sufficient to mediate GVHD pathology but re-transplanted CD8 T cell are not. This correlates with the absence of DP T cell differentiation in that re-transplanted CD8 population. The differentiation of DP T cells from chronically activated CD8 T cells represents a novel mechanism of GVHD pathology not previously described. The presence of DP T cells in other chronic inflammatory human diseases also suggests a broader pathology mediated by DP T cells. Further understanding of DP T cell differentiation and pathology may lead to targeted prophylaxis and/or treatment regimens for aGVHD and other human chronic inflammatory diseases. Figure 1 Figure 1. Disclosures Capitini: Nektar Therapeutics: Honoraria; Novartis: Honoraria.

Treatment of relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) remains a challenge particularly in patients who do not respond to chemotherapy or immunotherapy. While a subset of these patients can be cured with allogeneic bone marrow transplant, success of the transplant is contingent upon achieving a minimal residual disease negative complete remission. The objective of this study was to assess the efficacy of fedratinib, a semi selective JAK2 inhibitor and venetoclax, a selective BCL-2 inhibitor, on the B-ALL cell lines RS4;11, NALM-6, and SUP-B15. While select studies have examined the efficacy of these drugs on B-ALL as single agents, data is lacking on whether these drugs work optimally as single agents or in combination. Cell lines were treated with either vehicle (DMSO), single agent fedratinib or venetoclax, or the combination of both inhibitors for 48 and 72 hours. Fedratinib doses were tested from 150 nM to 1 uM and venetoclax doses were tested from 2.5 nM to 500 nM. Live/dead discrimination was performed using flow cytometry and a 72-hour MTS assay to assess proliferation. Drug synergy was calculated using CompuSyn software to generate combination index (CI) scores, with a CI score less than 1 reflecting synergy. RNA was isolated from treated leukemia cells and processed on a Nanostring nCounter platform to assess potential changes in gene expression. Results were then analyzed using Rosalind data processing software. In RS4;11, the combination of 5 nM venetoclax and 525 nM fedratinib significantly increased cell death compared to single agent venetoclax (p=0.0451) and fedratinib (p=0.0077). Furthermore, the combination significantly decreased leukemia cell proliferation compared to single agent fedratinib (p Disclosures Capitini: Novartis: Honoraria; Nektar Therapeutics: Honoraria.

PRMT5, which regulates gene expression by symmetric dimethylation of histones and non-histone target proteins, is overexpressed and plays a pathogenic role in many cancers. In diffuse large B cell lymphoma (DLBCL), the mechanisms of PRMT5 dysregulation and its role in lymphomagenesis remain largely unknown. Here we demonstrate that B cell receptor (BCR) signaling regulates PRMT5 expression in DLBCL cells. Immunohistochemical analysis reveals elevated levels of PRMT5 expression in DLBCL cases and in germinal center (GC) B cells when compared to naive B cells. PRMT5 can be induced in naive B cells by BCR stimulation. We discovered that BTK-NF-κB signaling induces PRMT5 transcription in activated B cell-like (ABC) DLBCL cells while BCR downstream PI3K-AKT-MYC signaling upregulates PRMT5 expression in both ABC and GCB DLBCL cells. PRMT5 inhibition inhibits the growth of DLBCL cells in vitro and patient derived xenografts. Genomic and biochemical analysis demonstrate that PRMT5 promotes cell cycle progression and activates PI3K-AKT signaling, suggesting a feedback regulatory mechanism to enhance cell survival and proliferation. Co-targeting PRMT5 and AKT by their specific inhibitors is lethal to DLBCL cell lines and primary cancer cells. Therefore, this study provides a mechanistic rationale for clinical trials to evaluate PRMT5 and AKT inhibitors for DLBCL.

Finding improved therapeutic strategies against T-cell Non-Hodgkin’s Lymphoma (NHL) remains an unmet clinical need. We implemented a theranostic approach employing a tumor-targeting alkylphosphocholine (NM600) radiolabeled with 86Y for positron emission tomography (PET) imaging and 90Y for targeted radionuclide therapy (TRT) of T-cell NHL. PET imaging and biodistribution performed in mouse models of T-cell NHL showed in vivo selective tumor uptake and retention of 86Y-NM600. An initial toxicity assessment examining complete blood counts, blood chemistry, and histopathology of major organs established 90Y-NM600 safety. Mice bearing T-cell NHL tumors treated with 90Y-NM600 experienced tumor growth inhibition, extended survival, and a high degree of cure with immune memory toward tumor reestablishment. 90Y-NM600 treatment was also effective against disseminated tumors, improving survival and cure rates. Finally, we observed a key role for the adaptive immune system in potentiating a durable anti-tumor response to TRT, especially in the presence of microscopic disease., Hernandez et al. show the effectiveness of 90Y for targeted radionucleotide therapy of T-cell Non-Hodgkin’s Lymphoma (NHL). This study suggests that delivering radiation to all NHL disease sites elicits minimal toxicity and induces a memory T-cell response, inviting combination therapies with immune activating agents.