Introduction We have established an international collaboration between the DKMS (Germany) and the NMDP (USA) to characterize more than 2000 donor peripheral blood stem cell (PBSC) grafts. Our goal is to correlate graft immunophenotype with patient outcomes. Here, we present an exploratory analysis with data from the first 727 recipients. Microbiome-dependent mucosal-associated invariant T (MAIT) and Vδ2 populations have each been associated with favorable HCT outcomes in prior studies. We hypothesized that receiving a graft bearing higher ‘doses’ of these cell populations would be associated with favorable clinical outcomes. Methods Donor PBSC graft samples were collected, processed, freshly stained (34-color panel for immunophenotyping of lymphocytes and hematopoietic stem cells), and analyzed using high dimensional full spectrum flow cytometry at the NMDP contract laboratory (n = 457; Roswell Park, Buffalo, NY) or the DKMS laboratory in Dresden, Germany (n = 270). Clinical data were collected via CIBMTR reporting from each individual transplant center. We focused on patients who were transplanted for AML or MDS, and for whom at least 12 months of follow-up data was available. The Kaplan-Meier estimates and cumulative incidence rates were determined for survival and competing risks outcomes, respectively, as a univariate analysis. The Cox proportional hazard model was fitted to assess the relationship of MAIT and Vδ2 populations (absolute dose/kg recipient weight in the graft) with transplant outcome and identify significant risk factors, including prophylaxis (PT-Cy vs other), graft cryopreservation status, patient age, donor age, refined disease risk index, HCT-CI, conditioning intensity, and HLA matching. Results 727 patients who received unrelated donor allografts for the treatment of AML or MDS were included in the analysis. Patients were treated at 113 different transplant centers within the US between 2021 and 2024. The median recipient age was 64.2 years. 404 (55.57% of the cohort received post-transplant cyclophosphamide (PT-Cy) as GVHD prophylaxis. MAIT and Vδ2 counts in the graft ranged from 0.1-33.9 (interquartile range [IQR]: 2.28-5.69) and 0-21.2 (IQR: 1.31-4.5) cells/µl, respectively. Univariate analyses revealed that higher (above-median) absolute numbers of Vδ2 cells in the graft were associated with increased OS (p = 0.033), and MAIT cells with lower rates of chronic GVHD (p = 0.043). Interestingly, higher (above-median) MAIT numbers were only associated with improved OS in the absence of PT-Cy as part of GVHD prophylaxis (n = 323; p = 0.031). There were no associations between MAIT/Vδ2 numbers and acute GVHD or relapse in the univariate analyses. In multivariable analyses, recipients of grafts bearing above-median absolute number of Vδ2 cells significantly associated with improved overall survival compared with equal to or below-median (HR=1.337, 95% CI 1.032-1.733, p = 0.0281). Additional clinical factors associated with worse overall survival are high/very high disease risk index (HR=1.637, 95% CI 1.234-2.172, p = 0.0006) compared to low/intermediate group and HCT-CI 3+ (HR=1.597, 95% CI 1.252-2.037, p = 0.0002) compared with the HCT-CI 0-2 group. There were no statistically significant differences in acute or chronic GVHD in patients who received above-median Vδ2 doses in our multivariate models. Multivariable analyses did not reveal any associations between MAIT cell graft content and outcome. Given the OS finding, we next characterized the causes of death in the cohort. Notably, infection was reported as the cause of death in 3.9% in the above-median Vδ2 setting and 7.9% in the recipients of grafts containing below-median numbers of Vδ2 cells (p = 0.034; Fisher's exact test). Conclusions In this exploratory analysis, we observed an association between the Vδ2 content of the donor graft and 12-month overall survival in the recipient. In prior studies, this population has been associated with lower rates of GVHD and improved OS when measured after HCT, however Vδ2 cells have not been previously studied in PBSC grafts. Intriguingly, little is known regarding the post-transplant capacity of these T cells, and further studies will focus on uncovering the mechanism by which they may be protective after HCT.

Cell-derived extracellular vesicles (EV) are mediators of intercellular communication with increased circulating levels of endothelial cell-derived EV (EC-EV) reported in cardiovascular diseases (CVD). The EC-EV ability to elicit either detrimental or restorative effects on target EC is thought to be, in part, due to horizontal transfer of their mitochondrial cargo. To understand the role of mitochondrial cargo in EC-EV paracrine effects, large EV were collected from media of cultured human EC, and the number of mitochondria-carrying EV (mitoEV), EV mitochondrial cargo mass, and mitoEV quality/polarization were quantified. EC activation with tumor necrosis factor (TNF)-α caused an increased release rate of EV (TNF-EV), including mitoEV that carried a larger and more depolarized mitochondrial cargo, compared to EV released from control EC (C-EV). EC co-treatment with TNF-α and the mitochondria-targeted antioxidant MitoTEMPO restored both the mitochondrial cargo quality and the number of mitoEV carrying polarized mitochondria to levels similar to C-EV. TNF-EV, but not C-EV, dose-dependently upregulated inflammatory gene expression in target naïve EC. Fluorescence microscopy showed the EV mitochondrial cargo to transfer and colocalize with the target EC mitochondrial network. Mitochondrial cargo depolarization of C-EV using carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone was sufficient for those EV to trigger inflammation in target naïve EC. In conclusion, the mitochondrial redox state of donor EC regulates mitoEV mitochondrial cargo quality that, at least in part, determines their capacity to cause target EC dysfunction and promote CVD. The mitochondrial membrane potential (ΔΨm) in EC-mitoEV may be a new biomarker and therapeutic target in vascular biology and medicine.

The World Cancer Research Fund and the American Institute for Cancer Research recommend a plant-based diet to cancer survivors, which may reduce chronic inflammation and excess adiposity associated with worse survival. We investigated associations of plant-based dietary patterns with inflammation biomarkers and body composition in the Pathways Study, in which 3659 women with breast cancer provided validated food frequency questionnaires approximately 2 months after diagnosis. We derived three plant-based diet indices: overall plant-based diet index (PDI), healthful plant-based diet index (hPDI) and unhealthful plant-based diet index (uPDI). We assayed circulating inflammation biomarkers related to systemic inflammation (high-sensitivity C-reactive protein [hsCRP]), pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, IL-13). We estimated areas (cm 2 ) of muscle and visceral and subcutaneous adipose tissue (VAT and SAT) from computed tomography scans. Using multivariable linear regression, we calculated the differences in inflammation biomarkers and body composition for each index. Per 10-point increase for each index: hsCRP was significantly lower by 6·9 % (95 % CI 1·6%, 11·8%) for PDI and 9·0 % (95 % CI 4·9%, 12·8%) for hPDI but significantly higher by 5·4 % (95 % CI 0·5%, 10·5%) for uPDI, and VAT was significantly lower by 7·8 cm 2 (95 % CI 2·0 cm 2 , 13·6 cm 2 ) for PDI and 8·6 cm 2 (95 % CI 4·1 cm 2 , 13·2 cm 2 ) for hPDI but significantly higher by 6·2 cm 2 (95 % CI 1·3 cm 2 , 11·1 cm 2 ) for uPDI. No significant associations were observed for other inflammation biomarkers, muscle, or SAT. A plant-based diet, especially a healthful plant-based diet, may be associated with reduced inflammation and visceral adiposity among breast cancer survivors.

TPS1140 Background: In PD-L1+ mTNBC patients (pts), the standard of care treatment is chemotherapy and pembrolizumab (P) in the first-line setting. Our group and others have demonstrated that chronic β2-AR signaling suppresses CD8 + cytotoxic T lymphocytes (CTL) function, drives their exhaustion, and increases the number of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the tumor microenvironment (TME), thus supporting tumor proliferation. Consequently, abrogation of β-AR signaling using the pan β-blocker propranolol or β-AR - / - knockout mice increased the intratumoral frequency of CTLs and elevated the CTL:Treg ratio (Bucsek et al. Cancer Res. 2017; PMID: 28819022). Similarly, mouse tumor models also demonstrated decreased exhaustion markers (PD1, TIM3, LAG3) on CTLs when β-AR was blocked, via propranolol (Qiao G et al. Cancer Immunol Res. 2021, PMID: 33762351). Confirming this phenomenon, we have shown, in a prospective clinical trial in metastatic melanoma, that β-AR blockade with propranolol significantly increased response to P with an objective response rate (ORR) of 78%, as opposed to 30-40% with P alone (Gandhi et al. Clin Cancer Res 2021, PMID: 33127652). Moreover, clinical β-AR blockade was associated with higher immune infiltration in the TME (Hiller JG, Clin Cancer Res 2020, PMID: 31754048). Therefore, we hypothesize that using propranolol with chemotherapy and P should improve response for pts with newly metastatic PD-L1+ TNBC. Methods: This is a phase II single-arm, non-randomized multi-center study. Pts are women ≥18 yrs with PD-L1+ mTNBC, who will receive propranolol, chemotherapy (paclitaxel, nab-paclitaxel, gemcitabine-carboplatin) and P in the upfront setting: chemotherapy on days 1, 8 and P on day 1 every 3 weeks in addition to propranolol 30 mg BID, with intra-pt propranolol dose-escalation by 10 mg BID weekly to a total of 80 mg BID as tolerated by blood pressure and heart rate as natural biomarkers for dose. Treatment will continue until disease progression per RECIST. The primary endpoint is ORR, defined as complete or partial response. The secondary endpoint is safety, 6-month progression-free and overall survival. As an exploratory endpoint, changes in TME and blood immune markers will be assessed. In stage 1, n1=23 evaluable pts will be enrolled. If ≥ 13/23 responses are observed, then the study will continue to enroll another n2=14 pts for a total of n=37, otherwise will be suspended for futility. If ≥ 24/37 responses are observed, then the proposed therapy will be considered promising. Pre- and 6-week post-treatment tumor biopsies and blood samples will be analyzed for changes in stress-induced biomarkers (epinephrine, norepinephrine, and frequency of CTL, MDSC, Treg) and exhaustion markers (PD1, TIM3, LAG3). The study is currently open and has accrued one patient. Clinical trial information: NCT05741164 .

Introduction We have established an international collaboration between the DKMS (Germany) and the National Marrow Donor Program (NMDP; USA) to characterize more than 2000 donor peripheral blood stem cell (PBSC) grafts. We aim to correlate graft immunophenotype with patient outcomes and perform additional analyses on biobanked aliquots of selected samples to deepen our understanding of the relationships between features of the donor graft and key patient outcomes, such as graft-versus-host disease, relapse, and overall survival. Here, we present an interim analysis, focused on the CD8 T cell compartment, describing relationships between donor characteristics and graft composition. We have developed a novel computational clustering method to integrate cytometry data from both the US and German analytical laboratories. Methods 300 donor PBSC graft samples were collected, processed, freshly stained (34-color panel for immunophenotyping of lymphocytes and hematopoietic stem cells), and analyzed using an Aurora Cytek instrument at the NMDP contract laboratory (Roswell Park, Buffalo, NY). 53 additional samples were processed in the DKMS laboratory in Dresden, Germany (equivalent antibody panel and instrument). We have developed a novel analytical pipeline for integrating these data. First, we group samples by facility, and by month run for computational convenience, to form “batches” for initial clustering. We perform Leiden clustering, and subclustering, to generate a large number of clusters per batch. To integrate data from all batches, we then cluster all subcluster centroids from all batch-specific clusterings. This gives a global clustering for each stream ( e.g. the CD8+ T cells). Cluster frequencies per sample provide features for quantifying links between immunophenotype and donor metadata ( e.g., CMV serostatus, age, and sex). Our approach offers an important complement to traditional gating and allows full exploration of the data in an unbiased fashion. Results 353 donor products were included in the analysis. The median donor age was 28 years (range 18 - 60), 59% were female and 65% were CMV positive. Initial metaclustering demonstrated that the 53 DKMS samples and the 300 NMDP samples are each well represented in all clusters, and that site-driven batch effects, while present, are mild. We initially focused on the CD8 T cell compartment (UMAP in Fig 1A) and discovered several phenotypic differences when we compared CMV seronegative and seropositive donors. CD8 T cell clusters 10 and 11 (both TCRαβ+ CD45RA+ CCR7+ HLA-DR+ CD57+; cluster 10 CCR9+; cluster 11 CCR9-) were present at higher frequency in CMV+ donors (n = 116) compared with CMV- donors (n = 220; p <0.0001; Fig 1B). Conversely, clusters 9 and 12, enriched for mucosal-associated invariant T (MAIT) cells, were significantly reduced in CMV+ donors (p <0.001). Of note, CMV serostatus was not significantly associated with age (median age in the CMV+ group was 29.4 years; vs 28.4 years in CMV- donors). We also observed an association between cluster 8 (TCRαβ+ CD45RA+ CCR7+ HLA-DR+ CD38+; consistent with an activated phenotype) and age (p = 0.0009; Fig1B). There was a trend toward decreased MAIT frequencies with age (Cluster 9; p = 0.058). When we analyzed the MAIT cell frequency as defined by traditional flow cytometry gating (CD3+ CD8+ CD161+ Vα7.2+), we observed a statistically significant relationship between younger donor age and increased MAIT cell frequency (p = 0.0002), consistent with the trend seen in cluster 9. Sex at birth was not linked with immunophenotype in our analyses thus far. Conclusions In this initial analysis of our multi-center observational study of PBSC graft products, we demonstrate the development and application of new computational tools for large-cohort flow cytometric data analysis. CMV-positive serostatus and younger donor age are both associated with an increased frequency of MAIT cells in the graft. We also found differences in CD8 T cell activation status in association with age and CMV. In the future, we will assess the relationships between these populations and patient outcomes, as well as explore cluster frequencies in association with key patient outcomes including GVHD, relapse, and overall survival. Sample collection and analysis are ongoing at both US and German sites as an ongoing collaborative effort between the NMDP and the DKMS.