Food Allergy in Pediatric Recipients of Liver Transplant by Mori et al (1), delineates allergic conditions in children post-liver transplant. While the transfer of allergic diseases and rare eosinophilic gastrointestinal disorders (EGID) after adult hematopoietic stem cell transplant (HSCT) has been reported, at the time of this review, there are no reports of EGID post-pediatric allogeneic HSCT. We share a case of eosinophilic esophagitis (EoE) following pediatric HSCT.A 2-year-old with beta-thalassemia underwent curative matched sibling HSCT. Four months later, he developed abdominal pain, postprandial nausea, and vomiting. Based on symptomatology and 25% eosinophilia in peripheral blood leukocytes, he was empirically diagnosed with upper gastrointestinal graft-versus-host disease (GVHD) and treated with oral prednisone 2 mg/kg/ daily. Symptoms improved but rapidly recrudesced after prednisone discontinuation. Upper endoscopy with biopsies was performed (Fig. 1), with 31, 43, and 35 eosinophils per high power field in the proximal, middle, and distal esophagus, respectively, with no evidence of GVHD.He was diagnosed with EoE and treated with budesonide viscous slurries and pantoprazole, 1 mg/kg/dose twice daily. Based on allergen testing, milk, wheat, eggs, and red food dyes were eliminated from his diet. Symptoms resolved. Repeat endoscopy revealed mucosal healing. Budesonide was discontinued ten months later, with no symptom recurrence, and mucosal healing was maintained.Eosinophilic disorders should be considered in HSCT recipients with gastrointestinal symptoms (2,3). Apparently, new allergies may develop in the HSCT recipient following donor immune reconstitution, often reflecting undiagnosed allergies of the donor (4). Endoscopic biopsies are crucial to diagnosis and definitive management.

Purpose Diffuse Intrinsic Pontine Glioma is a fatal tumor traditionally treated with radiotherapy (RT) and previously characterized as having a non-inflammatory tumor immune microenvironment (TIME). FLASH is a novel RT technique using an ultra-fast dose-rate which is associated with decreased toxicity, effective tumor control and potential immune-sparing properties. However, the effect of FLASH on the DIPG tumor immune microenvironment (TIME) has not yet been explored. Methods Here, we perform single-cell RNA sequencing on immune cells isolated from an orthotopic syngeneic murine model of DIPG following the use of FLASH (90Gy/sec) or conventional (2Gy/min) dose-rate RT (CONV-RT), and compare to unirradiated tumor and normal brainstem. Results Sequencing of immune cells reveals 17 unique populations, most abundant of which is microglia. In the most activated microglia subtypes, both CONV-RT and FLASH show upregulation of type 1 interferon (IFN1) genes and pathway scores compared to unirradiated tumor. In macrophages (MACs) and dendritic cells (DCs), CONV-RT is significantly enriched for IFN1 while this response is less seen with FLASH. Further, FLASH shows an increase in CNS border-associated MACs and upregulation of a myeloid-derived suppressor cell (MDSC) signature in MONOs, less seen with CONV-RT. In the lymphocytes, FLASH yields a higher mature B cell proportion and upregulation of T-cell activation and trafficking markers compared to CONV-RT. Finally, we correlate our data with myeloid cells from cerebrospinal fluid of human DIPG patients and find overlap with our murine tumor- and treatment-associated markers. Conclusion Our work is the first to map CONV-RT and FLASH immune alterations with single-cell resolution in the DIPG TIME. We find that CONV-RT and FLASH sculpt the microglial compartment similarly while recruiting distinct non-resident myeloid subsets and mature B-cell fractions, highlighting the potential to combine each modality with unique immunotherapy regimens in this fatal disease.

BackgroundDiffuse intrinsic pontine gliomas (DIPG’s) are immunologically inert tumors with a median survival of 9–15 months. Radiation therapy (RT) is the mainstay treatment for DIPG but is associated with immunodepletion of the tumor microenvironment (TME) at high dose ranges. FLASH, or ultra-fast dose rate RT, represents a novel ablative technique that may spare TME immune responses while decreasing tumor burden. Here, we present single-cell immune profiling of DIPG tumors treated with FLASH, conventional dose rate RT (CONV) or no RT (SHAM).MethodsMurine H3.3K27M mutant DIPG cells were stereotactically injected and tumor induction confirmed by magnetic resonance imaging (MRI) 15 days later. DIPG-bearing mice were randomly assigned to one of three treatment groups (n=4/group), FLASH, CONV or SHAM. A fourth group with no tumor (NML) was included as a negative biological control. A modified linear accelerator was used to deliver 15 Gy of electron RT to the brainstem at dose rates of 90 Gy/second and 2 Gy/minute, for the FLASH and CONV groups, respectively. Four days post-RT, mice brainstems were harvested, homogenized, stained for CD45 and tagged with a hashtag antibody specific to each group. CD45+ immune cells were isolated and sequenced using the 10X Genomics chromium single-cell 3’ platform. After processing and alignment of the reads using CellRanger with default parameters, the data was quality checked and filtered before hashtag demultiplexing, unsupervised clustering and downstream analysis was implemented following the Seurat R package. Differential expression evaluated based on the non-parametric Wilcoxon rank sum test. Key genes determine by an adjusted p value of < 0.05 based on bonferroni correction and |avg log2FC| > 0.8.ResultsPreliminary analysis identifies 15 clusters with distinct CD45 immune phenotypes (figure 1). Differential gene expression analysis by hashtag antibody (treatment group) reveals 14 clusters differentially expressing key genes, including 3 clusters upregulated in DIPG compared to NML, and 2 clusters upregulated in irradiated tumors compared to SHAM and NML (figure 2). Notably, analysis demonstrates an individual cluster upregulated in FLASH versus all other groups (p = 3.07E-171). Further deconvolution of specific immune phenotypes represented by each cluster is ongoing.Abstract 91 Figure 1tSNE plot based on clustering of RNA signatures, grouped by RNAAbstract 91 Figure 2tSNE plot based on clustering of RNA signatures, grouped by hashtag antibodyConclusionsOur preliminary analysis shows differential immune responses among DIPG tumors compared to NML. We also find several immune cell subsets that are unique to DIPG treated with CONV or FLASH compared to unirradiated samples. Most notably, we identify a single immune cell subset that is exclusive to FLASH alone, indicating that FLASH elicits a unique immune response in murine DIPG.