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Background CD19. CAR T-cells for the treatment of relapsed and refractory (r/r) Diffuse Large B-Cell Lymphoma (DLBCL) demonstrated complete responses in 40%-58% of the patients. Recently, others could associate high tumor volume and low CAR T-cell expansion in vivo with poor outcome. We hypothesize, that the expansion and immunphenotype of (CAR) T cells in vivo determine treatment response and depend on patient- and disease associated factors. Materials and Methods Patients with r/r DLBCL (n=34) were treated with either Axi-cel or Tisa-cel at the University Hospitals of Erlangen and Munich (LMU). The CAR T-cell product and peripheral blood were collected on day 0, 4, 7, 14, 30, 60 and 90 post transfusion. CAR T-cells were detected through flow cytometry utilizing a two step-staining with a biotinylated CD19 protein. Effector:Target (E:T) Ratios were estimated as absolute peak expansion of CAR T-cells (/ul) per tumorvolume (cm3). Responder (R, complete or partial remission) were compared to Non-Responder (NR, stable or progressive disease) according to response assessment with PET-CT three months after transfusion. Results CAR T-cell expansion peaked between day 7 and day 14 after transfusion with a greater expansion of CD8+compared to CD8- CAR T-cells on day 14 (59.27% vs 37.42%, p=0.021). The ratio of CD8+ and CD8- CAR T-cells did not differ between R and NR, however R exhibited higher E:T ratios of CD3+ CAR T-cells compared to NR (20.94 vs 12.81, p=0.015) and an increased E:T ratio of CD8+ CAR T-cells correlated with better progression-free survival (p=0.033). Interestingly, high CRP and ferritin levels at baseline were inversely associated with the E:T ratio (p=0.048 and p=0.017). CD3+ CAR T-cells of R showed earlier peak expression of PD-1 than NR (day 7 vs day 21). Further, peak expansion of CD3+ CAR T-cells correlated with higher PD-1 expression in R but not in NR (p=0.003 vs p=0.12). In addition, R revealed an increased relative frequency of effector memory differentiated CD3+ CAR T-cells (CCR7-CD45RA-, p=0.02), whereas CAR T-cells in NR showed an increased relative frequency of a naive phenotype (CCR7+CD45RA+, p=0.001) on day 7 post infusion. Conclusions Flow-based immunomonitoring with longitudinal characterization of CAR T-cells demonstrated a correlation of the E:T ratio with treatment response and survival. Increased inflammatory conditions at baseline correlated with diminished E:T ratios. Notably, in R CAR peak expansion was positively associated with higher PD-1 expression suggestive for superior CAR T-cell activation. In addition, greater memory differentiation was associated with efficacy during the time of peak expansion. Multiparameter analysis with other clinical covariates will show, whether CAR T-cell expansion and immunphenotypes can predict patient outcome. Disclosure Information V. Blumenberg: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Novartis, Gilead Sciences, Janssen, BMS/Celgene. S. Volkl: None. G. Busch: None. S. Baumann: None. M. Winkelmann: None. B. Tast: None. D. Nixdorf: None. G. Hanel: None. L. Frolich: None. C. Schmidt: None. R. Jitschin: None. F. Vettermann: None. W. Kunz: None. D. Mougiakakos: None. M. von Bergwelt: None. V. Bucklein: None. A. Mackensen: None. M. Subklewe: None.

The clinical and immunological impact of B-cell depletion in the context of coronavirus disease 2019 (COVID-19) is unclear. We conducted a prospectively planned analysis of COVID-19 in patients who received B-cell depleting anti-CD20 antibodies and chemotherapy for B-cell lymphomas. The control cohort consisted of age- and sex-matched patients without lymphoma who were hospitalized because of COVID-19. We performed detailed clinical analyses, in-depth cellular and molecular immune profiling, and comprehensive virological studies in 12 patients with available biospecimens. B-cell depleted lymphoma patients had more severe and protracted clinical course (median hospitalization 88 versus 17 d). All patients actively receiving immunochemotherapy (n = 5) required ICU support including long-term mechanical ventilation. Neutrophil recovery following granulocyte colony stimulating factor stimulation coincided with hyperinflammation and clinical deterioration in 4 of the 5 patients. Immune cell profiling and gene expression analysis of peripheral blood mononuclear cells revealed early activation of monocytes/macrophages, neutrophils, and the complement system in B-cell depleted lymphoma patients, with subsequent exacerbation of the inflammatory response and dysfunctional interferon signaling at the time of clinical deterioration of COVID-19. Longitudinal immune cell profiling and functional in vitro assays showed SARS-CoV-2-specific CD8+ and CD4+ T-effector cell responses. Finally, we observed long-term detection of SARS-CoV-2 in respiratory specimens (median 84 versus 12 d) and an inability to mount lasting SARS-CoV-2 antibody responses in B-cell depleted lymphoma patients. In summary, we identified clinically relevant particularities of COVID-19 in lymphoma patients receiving B-cell depleting immunochemotherapies.

Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p < 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p < 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p < 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p < 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p < 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p < 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p < 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p < 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p < 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC > 1 or < -1; p < 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj < 0.0001) and protein secretion (NES=-1.49; p adj < 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.

Endocannabinoids are lipid mediators that signal via several seven-transmembrane domain G protein-coupled receptors. The endocannabinoid receptor CB2 is expressed on blood cells, including stem cells, and mediates the effects of cannabinoids on the immune system. The role of the endocannabinoid system in immature hematopoiesis is largely elusive. Both direct effects of endocannabinoids on stem cells and indirect effects through endocannabinoid-responsive niche cells like macrophages have been reported. Using two different CB2-deficient mouse models, we studied the role of the endocannabinoid system in immature hematopoiesis. Moreover, we utilized both models to assess the specificity of putative CB2 agonists. As heterodimerization of CB2 and CXCR4, which is highly expressed on hematopoietic stem cells, has already been described, we also assessed potential consequences of CB2 loss for CXCR4/CXCL12 signaling. Overall, no differential effects were observed with any of the compounds tested; the compounds barely induced signaling by themselves, whereas they attenuated CXCL12-induced signals in both CB2-competent and CB2-deficient cells. In vivo experiments were therefore by necessity restricted to loss-of-function studies in knockout (CB2–/–) mice: Except for mild lymphocytosis and slightly elevated circulating progenitor cells, homeostatic hematopoiesis in CB2–/– mice appears to be entirely normal. Mobilization in response to pharmacological stimuli, Plerixafor or G-CSF, was equally potent in wild-type and CB2–/– mice. CB2–/– bone marrow cells reconstituted hematopoiesis in lethally irradiated recipients with engraftment kinetics indistinguishable from those of wild-type grafts. In summary, we found the endocannabinoid system to be largely dispensable for normal murine hematopoiesis.

Patients with Acute Myeloid Leukemia (AML) frequently relapse due to chemorefractory AML cells persisting after intensive chemotherapy at levels below the 5% morphological detection threshold (measurable residual disease, MRD). MRD has been established as an important prognostic factor for relapse-free and overall survival, making it highly relevant for post-remission treatment stratification. In contrast to MRD assessment by molecular techniques, multiparameter flow cytometry (MFC)-based MRD measurements are applicable in more than 95% of AML patients, while still offering a sensitivity of 10-4 to 10-5. Current MFC MRD assessment strategies measure 8-10 fluorochromes in parallel, resulting in a high-dimensional data set. However, evaluation of this data is usually performed by scatterplot-based manual, two-dimensional analysis. This leads to loss of information and significant inter-observer variability in MRD diagnostics. We therefore established a computational data analysis strategy for MFC MRD diagnostics, based on the unsupervised FlowSOM algorithm. By comparison with healthy bone marrow (HBM) data, FlowSOM analysis can identify aberrant (sub-)populations of cells, clustered in nodes (according to similarity of their antigen profile). These nodes can be denoted as "nodes of interest" (NOI) to simplify MRD analysis after clustering. Aim of the project was to establish FlowSOM analysis protocols and retrospectively evaluate their prognostic significance in a cohort of 46 patients with known outcomes. Bone marrow samples of these patients were analyzed at aplasia (day 16 after initiation of induction chemotherapy). Only patients with morphological blast clearance at aplasia were included. Healthy reference FlowSOM trees were established by merging flow data of 17 HBM. Analysis protocols were developed to report individual ("any node" approach) and cumulative ("sum node" approach) differences in NOI percentages when comparing HBM and MRD samples. We then performed FlowSOM MRD analyses in a patient subcohort of 19 AML patients. Importantly, for these analyses, we excluded patients who underwent allogeneic stem cell transplantation in first remission (non-HSCT subcohort). Median follow-up time was 8.3 (range 2-40) months for this subcohort. Receiver operating characteristic (ROC) analyses were used to determine optimal threshold values to differentiate relapse (n=5) and non-relapse (n=14) patients within the cohort. For "sum node" analysis strategies (defining MRD levels as cumulative difference of NOI percentages) a threshold of -2.44% was identified, optimized for Youden (Y) index and diagnostic odds ratio (DOR). For the "any node" strategy (defining MRD levels by the maximum difference of any NOI), a threshold of 0.04%, also optimized for the Y-index and DOR, discriminated best between relapse and non-relapse patients. Relapse-free survival (RFS) was significantly shorter for MRD-positive (MRDpos) patients identified by "sum node" analysis (median 8 months vs. not reached, p=0.016) and tended to be shorter for MRDpos patients by "any node" analysis (median 8 months vs. not reached, p=0.1). When applying the thresholds identified in the non-HSCT cohort to the full set of 46 patients (median follow-up interval 10.6 months, range 2-40), median RFS was not reached for the MRD-negative group (both for "sum node" and "any node" analysis), and was 14 ("sum node", p=0.098) and 14 months ("any node", p=0.360) for the MRDpos patients. Median overall survival for MRDpos patients by "sum node" analysis was 27 months, whereas it was not reached for MRD-negative patients. However, this difference did not reach statistical significance (p=0.335), probably due to the small sample size. Taken together, FlowSOM-based analysis strategies seem well suited to identify patients with MRD positivity after intensive induction chemotherapy. MFC MRD positivity at aplasia, defined by FlowSOM-based analysis, is associated with inferior RFS in retrospective analyses of small patient cohorts. Due to the underlying computational, unsupervised data analysis, FlowSOM-based assessment can be a means to harmonize MFC MRD evaluation. These promising results need to be verified in larger cohorts, with inclusion of post-induction assessments, and should be followed by prospective analyses to delineate the diagnostic validity of FlowSOM for AML MRD diagnostics in clinical trials. Disclosures Subklewe: Janssen: Consultancy; Morphosys: Research Funding; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Pfizer: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Research Funding.