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Neutrophil migration to the site of bacterial infection is a critical step in host defense. Exclusively produced in the bone marrow, neutrophil release into the blood is tightly controlled. Although the chemokine CXCL1 induces neutrophil influx during bacterial infections, its role in regulating neutrophil recruitment, granulopoiesis, and neutrophil mobilization in response to lung infection-induced sepsis is unclear. Here, we used a murine model of intrapulmonary Streptococcus pneumoniae infection to investigate the role of CXCL1 in host defense, granulopoiesis, and neutrophil mobilization. Our results demonstrate that CXCL1 augments neutrophil influx to control bacterial growth in the lungs, as well as bacterial dissemination, resulting in improved host survival. This was shown in Cxcl1−/− mice, which exhibited defective amplification of early neutrophil precursors in granulocytic compartments, and CD62L- and CD49d-dependent neutrophil release from the marrow. Administration of recombinant CXCL2 and CXCL5 after infection rescues the impairments in neutrophil-dependent host defense in Cxcl1−/− mice. Taken together, these findings identify CXCL1 as a central player in host defense, granulopoiesis, and mobilization of neutrophils during Gram-positive bacterial pneumonia-induced sepsis.

COVID-19 is a newly diagnosed entity that has been identified for about 6 months. With the US now being the prevalent nation affected, we aimed to identify factors that were relevant to survival and hematological events in COVID patients. Our group had primarily been assessing COVID-19 confirmed patients as per PCR at Ochsner Medical Center in Louisiana; we primarily studied patients who were admitted between January 2020 to May 2020. Our main events that are currently being studied include factors such as mortality, length of stay, and bleeding or thrombotic events. Secondary endpoints included factors such as influence on blood type and also amount of transfusions required by these patients. Our overall analysis of 1773 patients yielded a number of 60 patients who were able to identify as having an active cancer diagnosis. This disproportionate number of patients may be related to factors such as reluctance to visit the hospital among cancer patients, a phenomenon noted similarly in Cardiology where the number of admissions for cardiac causes had declined suddenly during this pandemic timeline. Our half of our population was African American and included an even proportion of males and females. The age range of patients was between 35-93 years old. The most common malignancies that were noted to co-occur with COVID-19 in our population seemed to be lung, prostate, and hematological malignancies. Although final analyses regarding mortality are to be completed, the proportion of patients who had died with a diagnosis of cancer was around 50% per collection of our current data. Twenty three out of 60 (38%) patients required blood transfusions. In comparison to other reports which had reported bleeding events and thrombotic events, our report had revealed a much less rate of bleeding events (5/60) and thrombotic events (5/60). We plan to repeat our analysis to assess for any confounders in identifying these events, as noted, since some earlier literature had reported between ¼ to ⅔ of patients having a thrombotic event. Our analysis also looks at other descriptive variables such as use of anticoagulant and antiplatelet agents, absolute counts of neutrophils and lymphocytes, platelets, and coagulation markers. Also, our assessment includes a study in assessment of any delays noted in chemotherapy dates for these patients. Analysis of assessment of bleeding, thrombosis, and mortality for the entire patient group is currently in process. Disclosures No relevant conflicts of interest to declare.

TO THE EDITOR: Mixed phenotype acute leukemia (MPAL) represents a heterogeneous group of leukemias accounting for 2% to 5% of all newly diagnosed acute leukemias.[1][1][⇓][2][⇓][3][⇓][4]-[5][5] Although most cases of acute leukemia display the differentiation pattern of a single lineage

Epstein-Barr Virus (EBV) is an oncogenic herpes virus associated with the development of malignancies associated with poor outcomes. Current treatments lead to further immune suppression, increasing the risk of EBV reactivation and other opportunistic infections. Strategies aimed at reversing the immune-suppressive microenvironment are likely to result in more durable responses while avoiding deleterious effects of chemotherapy. It is well-established that macrophages play an important role in inhibiting the anti-tumor response as evidenced by poor prognosis associated with increasing tumor-associated macrophage (TAM) density or lower ratio of cytotoxic T lymphocytes. Here we describe a pre-clinical model showing the expansion of cytotoxic TAMs and potential mechanisms to prevent this immunosuppressive environment. To assess soluble factors produced by the interaction of tumor on immune effector cells, we used a co-culture (CoCx) system of EBV-transformed human B cell lymphoblastoid cell lines (LCL) cultured with autologous peripheral blood mononuclear cells (PBMC). Serum-free medium collected after 48 hrs showed increased myeloid-associated factors which were absent from either the LCL or PBMC cultures, suggesting the interaction of tumor cells with immune effectors was leading to the production of chemokines known to attract myeloid cells. When purified autologous CD14+ monocytes were incubated in the presence of CoCx conditioned medium (CM) they generated proliferative foci that were absent in the LCL or PBMC CM alone. To better understand which cellular subsets were necessary for the release of these soluble factors, immune subsets were removed from PBMC prior to incubation with LCL or purified and incubated with LCL. We found that T cells, (CD8+ and CD4+), when incubated in the presence of LCL were necessary and sufficient to stimulate the proliferation of myeloid cells. CD56+, CD14+, or CD19+ subsets within the CM did not appear to contribute to the proliferation of purified myeloid cells. We next used flow cytometry to characterize the monocyte-derived population that expanded when introduced to CoCx CM. We again isolated autologous CD14+ monocytes from PMBC and incubated them for 6 days with CoCx CM followed by immunophenotyping. The monocyte-derived population retained CD14 and CD11b in addition to showing pronounced increases in both canonical M1 and M2 macrophage markers (HLA-DR, and CD163 respectively), and PD-L1, indicating activation consistent with a TAM phenotype. To characterize the functional consequence of this cell population, CoCx CM-derived macrophages were incubated with autologous T cells that were either activated non-specifically with anti-CD3/C28 or left inactivated. Surprisingly, we noted a marked decrease in viable activated T cells (both CD4+ and CD8+) when exposed to conditioned macrophages. However, the inactivated T cell populations were only mildly effected. Together, with previously described experiments, this suggests a mechanism by which exposure of T cells to EBV+ lymphoblastic tumor lines results in cytokine release, polarizing monocytes to TAMs that, in turn, exhibit cytotoxic activity to tumor-specific T cells. The precise mechanism of cytotoxicity is under investigation. We previously reported the eIF4A translational inhibitor silvestrol potentiated a potent immune-mediated anti-tumor response against EBV-driven lymphoma mainly by promoting expansion and activity of EBV-specific cytotoxic T cells. We next questioned if macrophages were important to this mechanism. We found that with silvestrol, when added to CoCx, the resultant CM did not drive the proliferation of TAMs, and Immunophenotype of CoCx showed silvestrol treatment to result in the ablation of CD14+ cells from culture. Interrogation by transcriptome analysis indicated a potential role of aryl hydrocarbon receptor (AhR). Protein analysis by western blotting indicated an increase of AhR in isolated monocytes when exposed to CoCx CM, which was abrogated by addition of silvestrol. Together these results suggest AhR may play a role in the polarization of macrophages within the tumor microenvironment leading to elimination of tumor-specific T cells. While full mechanistic characterization is ongoing, these preliminary results provide a potentially novel approach to modulate tumor specific immunity in this challenging group of EBV-related malignancies. Disclosures Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Background: Inotuzumab ozogamicin (INO) is a humanized, anti-CD22 antibody conjugated to calicheamicin with FDA approval for treatment of adults with relapsed or refractory (r/r) B-acute lymphoblastic leukemia (B-ALL). While it is associated with favorable complete remission rates in r/r B-ALL patients (pts), hepatotoxicity both during treatment and with subsequent hematopoietic cell transplantation (HCT) remains a challenge. In general, venoocclusive disease (VOD) during HCT in pediatric pts is estimated at 10-20%. Following treatment with INO, however, up to 52% of pts undergoing HCT have been reported to develop VOD, of which 18% of cases were fatal (Bhojwani et al. 2019). Here we report our institutional HCT experience in pediatric pts with r/r B-ALL who underwent HCT following INO reinduction and our strategy of using defibrotide prophylaxis to reduce the risk of VOD. Methods: A retrospective chart review was conducted for pts with r/r B-ALL who underwent HCT at our institution from December 2015 to January 2021. Clinical characteristics, laboratory parameters, and radiographic evaluations were reviewed. Patients who received ≥1 course of INO during reinduction were included in the INO group. Development of VOD was defined as a clinical diagnosis of VOD post-HCT based on either the Baltimore, Seattle, or European Society for Blood and Marrow Transplantation (EBMT) diagnostic criteria.Clinical characteristics were summarized. Time to VOD, graft versus host disease (GVHD), non-relapse mortality (NRM), and relapse were estimated under the competing risk setting, in which death without the event of interest was considered to be an absorbing competing risk. Results: Fifty-seven patients with relapsed or refractory B-ALL were transplanted between December 2015 and January 2021. Seventeen (30%) received at least 1 course (3 doses) of INO as part of their pre-HCT reinduction regimen. Fifty-five of 57 pts (97%), including all pts in the INO-group received a myeloablative regimen (51 total body irradiation/cyclophosphamide-based, 4 busulfan-based). Two patients in the non-INO group received a reduced-intensity regimen. Median time from last dose of INO to HCT (Day 0) was 34 days (range 24-71 days). Median number of INO doses received was 6 (range 3-9). Four patients had been transplanted previously, 1 in the INO group and 3 in the non-INO group. Two of these patients developed VOD during their current HCT, both from the non-INO group. Pts receiving INO were more likely to be transplanted in CR3 or higher, and to have required 3 regimens in reinduction to achieve that CR. Median follow-up was 20 months. Rates of VOD were similar between the INO (3 of 17, 18%) and non-INO (4 of 40, 10%) groups (p=0.4, Figure 1). All cases were severe or very severe by EBMT criteria. There was no VOD-related mortality in either group. Defibrotide prophylaxis was used in 82% (14 of 17) of pts in the INO group. Of the 3 patients in the INO group who developed VOD, 2 received defibrotide prophylaxis and 1 did not. Reasons to defer defibrotide prophylaxis included increased risk of CNS bleeding and provider preference. Defibrotide prophylaxis was used in 15% of pts in the non-INO group (6 of 40). In the non-INO group, 1 of 4 patients who developed VOD had received defibrotide prophylaxis. All pts received vitamin E and ursodiol per institutional practice. There were no treatment-related deaths in the INO group and no statistically significant differences were detected in (NRM) (0% vs 10%, p=0.25), median time to engraftment (24 vs 26.5 days, p=0.1), or GVHD (24% vs 55%, p=0.1) in the INO vs non-INO groups. Conclusions: HCT was well tolerated with low rates of VOD and NRM in pediatric pts following reinduction with INO. The results of ongoing prospective studies will provide further insight into the role of INO for reinduction of r/r B-ALL. In our cohort, rates of VOD were similar between pts who had and had not previously received INO, with the majority of pts in the INO group receiving defibrotide prophylaxis during HCT, suggesting that inotuzumab ozogamicin reinduction may safely be used with this approach. Figure 1 Figure 1. Disclosures Margossian: Cue Biopharma: Current Employment. Silverman: Takeda, Servier, Syndax, Jazz Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Other: Stock ownership. OffLabel Disclosure: This study evaluates the safety of transplant following inotuzumab ozogamicin re-induction for children with B lymphoblastic leukemia.

Background: Chronic graft-versus-host disease (CGVHD) is the leading cause of long-term morbidity and mortality following hematopoietic stem cell transplant (HCT) and occurs in over 50% of patients undergoing unrelated donor HCT. Despite its frequency, the mechanisms driving this disease remain incompletely understood, making its prevention and successful treatment challenging. To address this issue, we have undertaken a transcriptomic analysis of T cell reconstitution after unrelated donor HCT, to dissect mechanisms driving CGVHD. Methods: The patients studied were enrolled on a Phase 2, randomized, placebo-controlled trial of abatacept for GVHD prevention in patients receiving 8/8 unrelated-donor HCT for hematologic malignancies (NCT01743131). All immune analyses in the current study were performed on patients randomized to standard GVHD prophylaxis with calcineurin inhibition + methotrexate alone (placebo cohort, n =69), and thus provide insights into the drivers of CGVHD during standard unrelated donor HCT. On Day +100, CD4+ T cells were purified from the peripheral blood of these patients, and then analyzed by RNASeq. To determine the transcriptomic drivers of CGVHD without the confounder of significant prior acute GVHD (AGVHD) or exposure to steroids, we focused on profiling the CD4+ transcriptome of de novo CGVHD (CGVHD which develops in the absence of prior grade II-IV AGVHD, n = 7) and compared these patients to those who were 'operationally tolerant' and never developed either grade II-IV AGVHD or any CGVHD (n= 4). Gene expression from the resulting transcriptomes was quantified using kallisto. Differentially expressed (DE) genes were identified using DESeq2 (threshold for DE, adjusted (for multiple testing) p Results: DE analysis identified 101 genes that were significantly upregulated in CD4+ T cells from de novo CGVHD group and 54 genes that were significantly upregulated in the 'operationally tolerant' group (Figure 1A). GSEA identified that the mostly highly enriched signatures in patients with de novo CGVHD encompassed naïve CD4+ transcriptional programing (Figure 1B-C), in agreement with flow cytometric analysis, which also demonstrated expansion of CD4+ naïve T cells at Day +100 in patients developing de novo CGVHD compared to those demonstrating operational tolerance (Figure 1D). Importantly, the naïve CD4+ T cell signatures that were identified were distinct from those defining CD4+ stem cell memory T cells (which did not enrich in the de novo CGVHD cohort). In contrast, the gene signature of the operationally tolerant patients were enriched for regulatory gene sets (Figure 1C), consistent with a large body of evidence demonstrating that Treg expansion can be protective against CGVHD. Discussion: This study represents, to our knowledge, the first interrogation of the transcriptomic features of patients developing de novo CGVHD versus those operationally tolerant patients who develop neither significant AGVHD nor CGVHD after HCT. These patients may represent a particularly effective cohort in which to study immunologic drivers of CGVHD, given their freedom from prior treatment with corticosteroids, which can confound downstream transcriptomic analyses. Our data provide compelling evidence for a prominent naïve CD4+ T cell signature in patients who develop moderate-to-severe CGVHD despite their lack of antecedent AGVHD. These results are provocative, as they implicate a cell subset that is often considered more quiescent (naïve T cells) as associated with patients who develop immune pathology associated with CGVHD. These results suggest that naïve CD4+ T cells may represent a potent reservoir for alloreactivity, that, once activated, can cause significant disease. This would be in agreement with the implications of previously reported trials of naïve T cell depletion, which resulted in significant control of CGVHD. These results suggest that strategies to restrain naïve T cell pathogenic activation after Day +100 may improve CGVHD outcomes, and that the CD4+ T cell transcriptomic signature at this timepoint could be developed into a robust immunologic biomarker for the risk of developing CGVHD versus operational tolerance after HCT. Figure 1 Disclosures Watkins: Bristol Myers Squib: Honoraria. Qayed:Novartis: Consultancy; Mesoblast: Consultancy. Blazar:Tmunity: Other: Co-founder; KidsFirst Fund: Research Funding; BlueRock Therapeutics: Research Funding; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeuetic: Consultancy; Magenta Therapeutics: Consultancy; Fate Therapeutics Inc.: Research Funding. Horan:Bristol Myers Squib: Honoraria, Research Funding. Langston:Kadmon Corporation: Research Funding; Astellas Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Bristol Myers Squib: Research Funding; Chimerix: Research Funding; Takeda: Research Funding. Kean:fortyseven: Consultancy; regeneron: Research Funding; hifibio: Consultancy; kymab: Consultancy; Bristol Meyers Squibb: Research Funding; gilead: Research Funding; novartis: Consultancy; bluebird bio: Research Funding; magenta: Research Funding.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that affects the function and development of immune cells. Here, we show that recipient mice receiving AhR-/- T cells have improved survival and decreased acute graft-versus-host disease (aGVHD) in 2 different murine allogeneic bone marrow transplant (BMT) models. We also show that CD4+ T cells lacking AhR demonstrate reduced accumulation in secondary lymphoid tissue because of low levels of proliferation 4 days after BMT. Additionally, we found a significant increase in the quantity of peripherally induced regulatory donor T (pTreg) cells in the colon of recipients transplanted with AhR-/- T cells 14 days after transplant. Blockade of AhR using a clinically available AhR antagonist greatly enhanced the in vitro generation of inducible Treg (iTreg) cells from naive CD4+ human T cells. We have identified AhR as a novel target on donor T cells that is critical to the pathogenesis of aGVHD.