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(A) Induction of an MDSC phenotype by AML-derived soluble factors, (B) Induction of IDO-activity by AML-EVs, (C) ZetaView-based analysis of AML-EVs, and (D) Impact of chemotherapy on EV release by AML cells.

(A) Impact of anti-TLR4 antibodies on MDSC induction, (B) Impact of anti-TLR2 antibodies on pCREB activation, (C) Impact of PrK pre-treatment of AML-EV uptake, (D) Impact of chemotherapy on protein palmitoylation in AML-cells, and (E) Impact of 2-BP on EV release by AML-cells.

Changes of mitochondrial (A) membrane potential, (B) ROS production, and (C) mass in monocytes upon AML-EV uptake.

(A) CD33 expression on AML-EVs, (B) Cytokine-content of AML-EVs, (C) Localization analysis of AML-EVs in monocytes, (D) Control experiment regarding EV-independent uptake of PKH26.

(A) Induction of MDSCs by AML-EVs released by chemotherapy-pretreated AML celllines, (B) Phagocytotic activity of monocytes upon AML-EV uptake, (C) Induction of MDSCs by AML-EVs isolated by gradient ultracentrifugation, (D) Induction of pCREB by AML-EVs.

Acute myeloid leukemia (AML) represents the most common acute leukemia among adults. Despite recent progress in diagnosis and treatment, long-term outcome remains unsatisfactory. The success of allogeneic stem cell transplantation underscores the immunoresponsive nature of AML, creating the basis for further exploiting immunotherapies. However, emerging evidence suggests that AML, similar to other malignant entities, employs a variety of mechanisms to evade immunosurveillance. In light of this, T-cell inhibitory myeloid-derived suppressor cells (MDSC) are gaining interest as key facilitators of immunoescape. Accumulation of CD14+HLA-DRlow monocytic MDSCs has been described in newly diagnosed AML patients, and deciphering the underlying mechanisms could help to improve anti-AML immunity. Here, we report that conventional monocytes readily take-up AML-derived extracellular vesicles (EV) and subsequently undergo MDSC differentiation. They acquired an CD14+HLA-DRlow phenotype, expressed the immunomodulatory indoleamine-2,3-dioxygenase, and upregulated expression of genes characteristic for MDSCs, such as S100A8/9 and cEBPβ. The Akt/mTOR pathway played a critical role in the AML-EV–induced phenotypical and functional transition of monocytes. Generated MDSCs displayed a glycolytic switch, which rendered them more susceptible toward glycolytic inhibitors. Furthermore, palmitoylated proteins on the AML-EV surface activated Toll-like receptor 2 as the initiating event of Akt/mTOR-dependent induction of MDSC. Therefore, targeting protein palmitoylation in AML blasts could block MDSC accumulation to improve immune responses.Significance:These findings indicate that targeting protein palmitoylation in AML could interfere with the leukemogenic potential and block MDSC accumulation to improve immunity.

Information regarding patient characteristics, antibodies, and primers.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the only curative treatment option for a number of hematologic malignancies. Its therapeutic potential relies on the potency of donor T cells to eliminate residual malignant cells, the so-called graft-versus-leukemia (GVL) effect. Disease relapse remains the most frequent treatment failure and is associated with poor outcome. Therefore, it is inevitable to decipher mechanisms that weaken GVL. In recent years, studies of tumor biology have revealed that metabolic remodeling of the micromilieu can critically regulate immune responses. Accumulation of reactive oxygen species leads to a metabolic condition known as oxidative stress, which can severely hamper T cells. Currently, only a few studies, mainly using preclinical models, have demonstrated the occurrence of oxidative stress after allo-SCTs. Therefore, we sought to investigate oxidative stress in a well-characterized group of patients who underwent allo-SCT and its impact on reconstituting T cells. We identified high concentrations of serum 8-hydroxydeoxyguanosine (8-OHdG) as an established biomarker for oxidative stress. 8-OHdG is one of the major products of DNA oxidation, which is normally rapidly removed. After allo-SCT, T cells accumulated oxidative DNA damage. High cellular 8-OHdG content (8-OHdGhi) was associated not only with signs of enhanced T-cell activation but also premature exhaustion. The inability of 8-OHdGhi T cells to efficiently target malignant cells or produce cytotoxic granzyme B and interferon gamma was associated with a significantly increased relapse risk and a shorter overall survival. Taken together, our novel findings could give reason to focus on bolstering DNA repair in reconstituting T cells as a means to improve GVL efficacy.

Mesenchymal stromal cells (MSCs) are characterized by their multipotency, regenerative potential, and immunoregulatory properties. Nowadays, MSCs represent a promising cell-therapeutic option for hyperinflammatory conditions such as graft-vs-host disease following allogeneic hematopoietic stem cell transplantation. A better understanding of their biology is a prerequisite for improving their treatment efficacy. Emerging evidence suggests that immunosuppressive properties are not constitutively active in MSCs. Instead, microenvironmental inflammatory stimuli such as the cytokines interferon (IFN)-γ or tumor necrosis factor (TNF)-α license MSCs to acquire a tolerance-promoting phenotype. The immunological checkpoint molecule programmed death-ligand 1 (PD-L1) is an important regulator of T-cell responses. Binding of PD-L1 to the programmed cell death protein 1 (PD-1) receptor on T-cells suppresses their activation, proliferation, and induces apoptosis. Previous studies have revealed that cell surface expression and secretion of PD-L1 are part of the MSCs' immunomodulatory armamentarium. Here, we report that inflammatory licensing leads to an enhanced PD-L1 cell surface expression and secretion, which are both accompanied by an increased posttranslational protein N-glycosylation. These post-translational modifications have been shown to be critical for key biological processes such as cell trafficking, receptor signaling, and immunohomeostasis. In fact, promoting N-glycosylation in MSCs yielded increased PD-L1 levels. We report for the first time that PD-L1 N-glycosylation plays a decisive role for its transport to the MSCs' cell surface and its subsequent secretion (in response to proinflammatory trigger). Our data offer insights into a novel regulatory mechanism with the potential to be exploited as a means to foster the immunosuppressive potency of human MSCs.

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.