Abstract: Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disorder due to autoantibodies against ADAMTS13. The case of identical twins suffering from acute acquired TTP with severe autoantibody-mediated ADAMTS13 deficiency (Studt JD et al., Blood2004;103:4195) led us to speculate on unknown genetic determinants of acquired TTP and on the immunological basis of ADAMTS13 autoimmunity. We investigated 37 patients (pts) with acute TTP with acquired severe ADAMTS13 deficiency of 〈 10% of the normal, as well as 60 healthy controls. We measured ADAMTS13 activity, anti-ADAMTS13 IgG by ELISA and searched for a functional ADAMTS13 inhibitor. The ADAMTS13 gene was sequenced and 15 short tandem repeats (STR) at loci not linked to the ADAMTS13 gene were analyzed. Genotypic data of 25 polymorphic markers on the ADAMTS13 gene were used for haplotype inference. Inter-allelic diversity was assessed by summing up the number of individual heterozygous base positions for the polymorphic markers on the ADAMTS13 gene and the STR loci separately. Identified ADAMTS13 mutations were expressed in COS-7 cells and their effect on biosynthesis, secretion and activity was determined. We found that 4 of 37 pts with severe acquired ADAMTS13 deficiency were heterozygous carriers of an ADAMTS13 mutation. The mutation P457L, found in two pts, is a known mutation reported in hereditary TTP. R1096H and A1145T are new mutations. Expression studies revealed that P457L and A1145T mutants were secreted normally, but showed reduced activity of 44% and 11%, respectively. Secretion of the R1096H mutant, however, was reduced. A particular ADAMTS13 haplotype (designated H6) inferred from single nucleotide polymorphism (SNP) data was significantly more frequent in pts than in controls (p 〈 0.0001). Moreover, individual ADAMTS13 inter-allelic diversity was significantly higher in pts than in controls (p=0.012). Primarily, non-synonymous SNPs, which encode amino acid exchanges, contributed to this phenomenon (p=0.0014). The increased inter-allelic diversity in pts was found to be restricted to and specific for the ADAMTS13 gene, as individual inter-allelic diversity at 15 neutral STR markers was similar in pts and controls. In conclusion, heterozygous ADAMTS13 missense mutations are found in a high frequency in pts with acquired TTP. The mutations P457L, R1096H and A1145T, reduce baseline ADAMTS13 activity in vitro by at least 50% and may render heterozygous carriers more susceptible for TTP. In addition, ADAMTS13 mutations, such as R1096H, may promote autoantibody production due to increased intracellular degradation and MHC presentation. The ADAMTS13 haplotype H6 and particularly the increased inter-allelic diversity represent risk factors for autoimmunity towards ADAMTS13 and acute acquired TTP. ADAMTS13 autoimmunity as a result of increased inter-allelic diversity represents the first example of genetic out-breeding depression in humans. As autoantigens in general seem to have increased numbers of SNPs, increased inter-allelic diversity may represent an important pathophysiological feature in other autoimmune disorders as well.

Abstract: Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 T cells, are enriched in B-cell non-Hodgkin lymphoma (NHL) and constitute a barrier to potent antitumor immune responses. Despite extensive studies, the significance of tumor-infiltrating Tregs on disease outcome is unclear and while Tregs may express co-inhibitory and co-stimulatory receptors, the role of intratumoral Tregs in the context of immune checkpoint therapy remains elusive. Emerging evidence suggests heterogeneity among Tregs and their suppressive capacities in cancer, emphasizing the need for additional markers to identify highly suppressive Tregs. Therefore, an in-depth characterization of Treg heterogeneity in NHL could provide important insight into the disease pathogenesis and have implications for rational drug design. Methods: Expression of checkpoint receptors in Tregs was characterized by fluorescence flow cytometry and mass cytometry analysis of single-cell suspensions from diffuse large B-cell lymphoma (DLBCL; n = 16), follicular lymphoma (FL; n = 8), mantle cell lymphoma (MCL; n = 10), marginal zone lymphoma (MZL; n = 2), chronic lymphocytic lymphoma (CLL; n = 7), as well as tonsils (n = 8) and peripheral blood (n = 4) from healthy donors. Functional characterization of intratumoral Tregs was performed by a proliferation assay using FACS-sorted Tregs as suppressor cells and autologous CellTrace Violet-labelled T effector cells as responder cells. Single-cell RNA sequencing (scRNA-seq) was performed on FACS-sorted CD4 T cells from 3 DLBCL, 3 FL and 3 healthy donor tonsils using the 10X Genomics single cell 5' based library construction and VDJ libraries for TCR-sequencing. Additionally, for simultaneous profiling of phenotypic features with the mRNA expression in single cells, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) was applied. The Treg compartment was characterized by clustering into distinct transcriptional Treg states and differential expression of marker genes. Results: TIGIT and CTLA-4 were identified as common markers of intratumoral Tregs, in addition to FOXP3 and CD25. Unsupervised computational analysis revealed two distinct Treg subsets, based on contrasting expression of PD-1, OX40, CD226 and ICOS (Figure 1A). One subset displayed a checkpoint receptorlow phenotype that corresponded to peripheral blood Tregs. The second subset had a checkpoint receptorhigh phenotype with elevated levels of PD-1, OX40, ICOS, TIGIT, CTLA-4 and increased levels of activation markers CD28, CD69 and CD95/Fas. The frequency of checkpoint receptorhigh Tregs was significantly increased in NHL tumors, compared to PBMCs and tonsils from healthy donors. FL tumors had the highest frequency of Tregs with receptorhigh phenotype among the NHL entities (median frequency of 86%, range 71-92%) and DLBCL had the highest donor-to-donor variation (median frequency of 77%, range 35-98%) (Figure 1B). This phenotypic heterogeneity of the Treg compartment reflected different suppressive capacities of the two subsets. Checkpoint receptorhigh Tregs were more potent mediators of immunosuppression in terms of suppressing the proliferation of autologous effector CD4 and CD8 T cells (Figure 1C). Furthermore, transcriptomic analysis of CD4 T cells by scRNA-seq and CITEseq revealed distinct transcriptomic signatures of the checkpoint receptorhigh and -receptorlow subsets. In addition, a third subset of Tregs, characterized by increased expression of LAG3 and immunosuppression-associated genes (CTLA-4, IL10, CD38, KLRB1) but lack of FOXP3, was identified (Figure 1D-E). Analysis of scTCR-sequences to compare TCR repertoires and to identify developmental trajectories will further add to our knowledge of intratumoral Tregs. Conclusions: These results reveal heterogeneity within the Treg compartment in NHL based on expression of checkpoint receptors, transcriptional profiles and suppressive capacities. As intratumoral Treg phenotypes differ from peripheral blood Tregs, this presents new therapeutic opportunities. Specific targeting of intratumoral Tregs would lead to stronger antitumor effects while limiting immune-related adverse events. A deeper understanding of Treg heterogeneity within the tumor microenvironment could therefore open new paths for rational design of immune checkpoint therapy. Disclosures Kolstad: Merck: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

Abstract: Myeloid transformation occurs via stepwise acquisition of driver mutations that have distinct positions in the clonal hierarchy. BCOR mutations are common in myelodysplastic syndromes (MDS), but rarely occur as initiating events, instead typically arising in the context of other pre-existing mutations, such as TET2. BCOR encodes a subunit of the Polycomb Repressive Complex 1.1 (PRC1.1) which mediates gene repression by regulating H2AK119 via RING1 ubiquitin ligase activity. We hypothesized that BCOR mutations exert context dependent effects on chromatin regulation, thereby mediating conditional clonal selection during disease progression. We used CRISPR/Cas9 genome editing to establish an isogenic human leukemia cell line model harboring BCOR, TET2, or concurrent BCOR and TET2 mutations. Whereas TET2 deficiency alone caused no effect on cell growth properties, isolated loss of BCOR caused a significant decrease in cell proliferation. Concomitant inactivation of TET2 and BCOR reversed the negative impact of BCOR mutations and resulted in enhanced proliferation suggesting that TET2 and BCOR deficiencies cooperate to drive a distinct cell growth phenotype that is not evident when either gene is knocked out alone. To define the impact of BCOR deficiency on PRC1.1 complex assembly we performed PCGF1 co-immunoprecipitation followed by mass spectrometry (IP/MS) in cells with and without BCOR deficiency. BCOR mutations caused a marked decrease in PRC1.1 complex abundance without disrupting complex integrity. Even in the absence of BCOR, PCGF1 interactions with other PRC1 components (RING1A/B, SKP1, KDM2B, USP7) were maintained. By contrast, reciprocal BCOR IP/MS in cells with and without PCGF1 deletion showed that PCGF1 is required for RING1A/B and RYBP binding, but does not regulate complex abundance. These data suggest that BCOR is critical for maintaining PRC1.1 stability while PCGF1 selectively recruits core enzymatic components to the complex. To evaluate the role of PRC1.1 enzymatic function in regulating target loci, we performed RNA-seq and ChIP-seq analysis in PCGF1 deficient cells that lack PRC1.1-RING1 interactions. 931 genes were significantly upregulated (FC 〉 2, padj≤0.05) in PCGF1 deficient cells compared to control. In wild type cells, these genes were bound by PRC1 (RING1B) and PRC2 (SUZ12) and marked by high levels of repressive histone marks (H3K27me3, H2AK119ub). In PCGF1 deficient cells, RING1B binding and repressive marks were lost at these loci, while BCOR binding was maintained and the active H3K27ac mark significantly increased. These data suggest that selective loss of PRC1.1 enzymatic activity causes derepression of PRC1.1 target loci. To determine whether derepression of PRC1.1 targets underlies the cooperative phenotypic effect of TET2 and BCOR inactivation, we compared the transcriptional and epigenetic state of BCOR/TET2 deficient cells to that of control, TET2, and BCOR deficient cells. Indeed, a subset of genes that were derepressed in PCGF1 deficient cells also showed increased expression, loss of repressive and gain of activating marks in BCOR-TET2 deficient cells, but not in cells with sole BCOR or TET2 deficiency. Derepressed genes were specifically enriched for involvement in developmental processes and cell signaling, including the leukemia-associated HOXA gene cluster. In contrast to PCGF1 deficient cells, BCOR deficient cells also displayed significant transcriptional downregulation of genes involved in metabolism and cell cycle progression, consistent with the negative effect of BCOR mutations on cell growth properties. In wild type cells, these BCOR-dependent loci had high levels of PRC1.1 (BCOR) binding, active histone marks, and high chromatin accessibility. Remarkably, expression of nearly all (590/622) BCOR-dependent genes was restored after introduction of concurrent TET2 deletion. Our data suggest that the BCOR-PRC1.1 complex fulfills a dual epigenetic role via RING1-dependent regulation of gene repression at some target loci and RING1-independent maintenance of active transcription at other loci. Accordingly, inactivation of BCOR alone is sufficient to derepress key leukemia-associated targets, but incurs a metabolic liability that limits transformation potential. Concurrent inactivation of TET2 may therefore enable transformation by attenuating negative impacts of BCOR mutations on active transcription. Disclosures No relevant conflicts of interest to declare.

Abstract: Abstract 1209 Poster Board I-231 Background: The prognosis of patients with cytogenetically normal acute myeloid leukemia (CN-AML) ranges from relatively favorable to extremely poor. Recently, based on the presence or absence of well defined mutations, molecular subgroups have been identified, which allow an estimate of a patient's prognosis at the time of diagnosis. Allogeneic stem cell transplantation (SCT) is the only curative treatment for the majority of these patients. However, only limited data is available to describe the role of alloSCT in different molecular subgroups of CN-AML, particularly in advanced stages of the disease. Methods: We retrospectively analyzed the data on 247 patients with CN-AML, who uniformly had received the FLAMSA-RIC conditioning regimen for alloSCT in 14 European centers between 1996 and 2008. Results: Patients suffered from de novo AML (76%), sAML/MDS (21%), and tAML (4%). Median age was 52.1 (19-71) years. Donors were matched or mismatched family, and matched or mismatched unrelated donors in 30%, 2%, 50% and 18%, respectively. SCT was performed in untreated disease (6%), after primary induction failure (PIF, median time from diagnosis to transplantation 134 days; 23%), in first complete remission (CR1, 14%), and beyond CR1 (57%). Median follow-up of survivors was 19 months. Overall survival (OS) and leukemia-free-survival (LFS) of the entire cohort at 2 years from SCT was 51% and 47%, respectively. The disease stage at transplant was the most important variable for outcome (p=.001 for OS, 〈 .001 for LFS): Encouraging results were achieved in patients transplanted in CR1 (2y OS and LFS: 76%), and in patients with PIF (2y OS and LFS: 69%), whereas results were inferior after transplantation in previously untreated disease (2y OS and LFS: 34%), or beyond CR1 (2y OS: 42%, LFS: 34%). Age, sex, de novo vs. secondary leukemia, donor type and CD34+ cell counts showed no influence on outcome. Information on molecular markers was available in 183 patients (74%). As suggested by Schlenk et al. (NEJM 2008), analysis was based on two subgroups: 22 patients with isolated NPM1 mutation (NPM1mut), and 161 patients with other genotypes (FLT3 internal tandem duplication [FLT3-ITD], n=66; or wildtype FLT3/wildtype NMP1 [FLT3wt/NPM1wt] , n=95). Patients with NPM1mut had a 4y OS/LFS of 75/63%. Results were not significantly different, when these patients were transplanted in PIF, CR1, or beyond CR1. Patients with other genotypes showed an OS/LFS of 51%/48% at 2y and of 40%/39% at 4y, without differences among patients with FLT-ITD and FLT3wt/NPM1wt. However, in this subgroup, outcome was highly dependent on the disease stage at SCT, with excellent results after transplantation in PIF (2y OS/LFS: 75%/74%) or in CR1 (2y OS and LFS: 76%), but inferior outcome after transplantation beyond CR1 (2y OS/LFS 38%/33%; p=.004 for OS and .001 for LFS). Conclusion: Allogeneic SCT following the FLAMSA-RIC conditioning produces excellent survival rates in patients with CN-AML, particularly when performed in CR1. Encouraging results in PIF support an early transplant, regardless of molecular subgroup, when CR is not reached after double induction therapy. In patients with an NPM1 mutation, transplantation in advanced disease achieved identical results as in early stage, which supports the strategy not to transplant these patients in CR1, but to delay alloSCT until relapse has occurred. In contrast, patients with FLT3-ITD or FLT3wt/NPM1wt achieved significantly worse results when transplanted beyond first relapse, arguing in favor of transplantation in CR1 for this molecular subgroup. Disclosures: Mayer: BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy; Fresenius: Consultancy; Roche: Research Funding; Pfizer: Research Funding.

Abstract: Abstract 3907 Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the vast majority of patients initially respond to up-front chemotherapy, relapses occur in approximately 20% of cases and have a poor prognosis. Thus, novel therapeutic strategies are required to treat minimal residual disease and improve long-term survival. B cell precursor (BCP)-ALL cells express low levels of costimulatory and antigen-presenting molecules and therefore are poorly recognized by the immune system. Previous reports show that CpG oligodeoxynucleotides (CpG) can induce immunogenicity of non-Hodgkin's lymphomas including B-CLL, and that CpG may decrease BCP-ALL tumor burden in a murine xenograft model. In the present study we investigated the effect of various combinations of known potent B cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of both primary BCP-ALL cells and a series of established BCP-ALL cell lines. Using combinations of CpG, IL-4 and CD40L we were able to enhance expression of CD40, CD54, CD86 and MHC class II on BCP-ALL cells, resulting in an increased capacity of BCP-ALL cells to induce proliferative T cell responses and to generate cytotoxic T cells (CTLs). Of note, these CTLs exhibited significantly enhanced anti-leukemic cytotoxicity not only towards treated but also towards untreated BCP-ALL cells, with similar effects found in both allogeneic and syngeneic settings. The strongest effects were observed after exposure of BCP-ALL cells to a combination of CpG, IL-4 and CD40L, whereas untreated control BCP-ALL cells induced minimal T-cell proliferation and cytotoxicity only. Importantly, CpG in combination with IL-4 and CD40L was significantly more effective than CpG alone. Our results demonstrate that combined treatment with CpG, IL-2 family cytokines and CD40L is more efficient than CpG alone in inducing an immunogenic phenotype in BCP-ALL cells. Further studies using a humanized leukemia mouse model may open novel therapeutic approaches in the management of BCP-ALL, for example by using adoptive T cell transfer after in-vitro stimulation of autologous T cells with activated BCP-ALL cells. Disclosures: No relevant conflicts of interest to declare.

Abstract: Angiocrine Bmp2 signaling in the liver controls tissue and serum iron concentrations via regulation of hepcidin expression in hepatocytes. Liver-specific angiocrine signaling is essential for the metabolic homeostasis of the whole organism.

Abstract: Homozygous or compound heterozygous mutations in the ADAMTS13 gene cause hereditary thrombotic thrombocytopenic purpura (TTP), an inherited disorder characterized by severe deficiency of plasma ADAMTS13 activity resulting in occlusive microvascular thrombosis. More than eighty ADAMTS13 mutations have been identified, of which 15 have been found in homozygous state. Homozygous mutations are of special interest to unravel a correlation between the ADAMTS13 genotype and the often variable clinical phenotype in hereditary TTP. We studied 5 ADAMTS13 mutations found in homozygous state in 6 patients with hereditary TTP. Detailed patients’ histories were recorded and plasma ADAMTS13 activity and antigen levels were determined. Recombinant ADAMTS13 mutants were transiently expressed in COS-7 cells and stably transfected HEK293 cell lines were generated for all five mutants. The biosynthesis, secretion and specific enzymatic activity under both static and flow conditions were studied. Intracellular localization of the mutants was also determined by immunofluorescent microscopy. Patients homozygous for W688X, R692C or C804R became manifest with acute TTP as neonates and several siblings had died of a TTP-like disorder. Plasma ADAMTS13 activity was & lt; 1% of the normal and ADAMTS13 antigen was undetectable. In patients with homozygous R1060W mutation, milder courses with adult onset of TTP after strong triggering factors were observed. One patient presented with the first TTP attack at the age of 19 years during her first pregnancy while a second patient experienced a first bout at the age of 61 years during a severe pneumonia. Both patients had 3% of residual plasma ADAMTS13 activity and 6% of antigen of normal human plasma. Homozygous mutation G1239V was found in a patient with frequent TTP bouts in childhood starting at 3 years of age and an attenuated course in adulthood with attacks triggered by excessive alcohol consumption. His plasma ADAMTS13 activity was & lt;1% along with undetectable antigen. Recombinant mutants R692C, C804R, R1060W and G1239V migrated at 190 kDa like wild-type ADAMTS13. Secretion of all 4 missense mutants was severely reduced and was highest for R1060W. The truncated mutant W688X was efficiently secreted with a molecular weight of 97 kDa. This mutant could cleave the VWF73 peptide as did wild-type ADAMTS13. Proteolytic activity towards multimeric von Willebrand factor (VWF) under static conditions was moderately impaired and even substantially reduced under fluid shear stress as compared to wild-type. Immunofluorescent staining showed the mutant ADAMTS13 proteases within endoplasmatic reticulum and Golgi apparatus, consistent with the distribution of secretory proteins. We conclude that all 5 investigated ADAMTS13 mutations are causative for hereditary TTP. The homozygous missense mutations in the carboxyl-terminal part of ADAMTS13 severely impair the secretion of the protease while the truncating mutation affects the proteolytic activity towards multimeric VWF under shear stress. Our findings highlight the significance of the middle and distal carboxylterminal domains of ADAMTS13 for secretion and enzymatic activity in vivo. This study of homozygous patients showed an early onset and severe course of TTP for mutations in the 2nd and 3rd TSP-1 repeat while the mutations in the 7th TSP-1 and the first CUB domain led to a later onset and a milder course of TTP with bouts requiring strong triggering factors. The two patients homozygous for R1060W showed low levels of residual plasma ADAMTS13 activity and antigen and the mildest clinical courses which is consistent with the cell culture studies. Together, our findings suggest a phenotype-genotype correlation in hereditary TTP.

Abstract: Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 + T cells, represent a key challenge in the tumor microenvironment by limiting potent antitumor immune responses. While high densities of tumor-infiltrating Tregs are associated with poor prognosis in patients with various types of solid cancers, their prognostic impact in B-cell non-Hodgkin lymphoma (NHL) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotype and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Our in-depth characterization of Tregs in NHL tumors could open new paths for rational drug design, facilitating selective therapeutic manipulation of Tregs to reduce immunosuppression and improve anti-tumor immunity. Methods: Single-cell suspensions from NHL patients (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and healthy donors (tonsils and peripheral blood)) were analyzed by fluorescent flow- and mass cytometry to characterize Tregs, focusing on their expression of co-stimulatory and co-inhibitory checkpoint receptors. The immunosuppressive capacity of Tregs was measured by in vitro co-culture of FACS-sorted subsets of Tregs together with autologous CellTrace Violet-labelled T effector cells as responder cells, using samples from FL and tonsils. Live CD4 + T cells were obtained by FACS sorting from DLBCL (n = 3), FL (n = 3) and healthy donor tonsils (n = 3) and subjected to single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and scTCR-seq by the 10X Genomics platform. The computational framework of CIBERSORTx was used to generate unique signature matrices for the three Treg subsets identified by scRNA-seq, to facilitate validation in separate scRNA-seq cohorts (King, Sci Immunol 2021; Roider, Nat Cell Biol 2020), and to impute frequencies of the Treg subsets in cohorts with bulk RNA-seq data (Chapuy, Nat Med 2018; Schmitz, NEJM 2018; Pastore, Lancet Oncol 2015). Results: Immunophenotyping by mass cytometry revealed a subset of activated Tregs identified by co-expression of TIGIT, CTLA-4, PD-1, ICOS and OX40, and higher expression of FOXP3, CD25 and CD45RO, that was present in DLBCL and tonsils, but lacking in peripheral blood. This was validated by fluorescent flow cytometry, demonstrating significantly higher frequencies of activated Tregs in NHL tumors compared to PBMCs and tonsils from healthy donors. The phenotypic heterogeneity of intratumoral Tregs reflected different suppressive capacities as activated Tregs more potently suppressed the proliferation of autologous effector CD4 + and CD8 + T cells than naïve Tregs. For global transcriptomic profiling of CD4 + T cells from FL, DLBCL and tonsillar samples, we integrating scRNA-seq and CITE-seq data from 17,774 cells, revealing 13 distinct cellular states including three states of Tregs: naïve, activated and non-conventional LAG3 +FOXP3 - Tregs. Activated Tregs had higher expression of checkpoint receptors (TNFRSF4, TNFRSF18, ICOS), phosphatases (DUSP2, DUSP4), NF-κB pathway (NFKBIA, TNFAIP3, NFKBIZ, REL), chemokine receptors (CXCR4) and transcription factors (JUNB, IRF1, STAT3) as compared to naïve Tregs. We next used a computational approach to develop unique signature matrices for each Treg subset. This approach demonstrated strong concordance between CIBERSORTx estimated cell abundances of the three Treg subsets and the ground truth, and was validated in two external scRNA-seq cohorts. The development of unique signature matrices for Treg subsets facilitated imputation of their frequencies in bulk RNA-seq datasets. These analyses revealed that higher frequency of activated Tregs was enriched in the germinal B cell subtype of DLBCL and was associated with adverse outcome in FL. Conclusion: This study demonstrates that Tregs infiltrating NHL tumors are transcriptionally and functionally diverse and include highly immunosuppressive activated Tregs co-expressing several checkpoint receptors, which distinguish them from peripheral blood Tregs. Activated intratumoral Tregs could hamper clinical responses to checkpoint blockade, and identifying and targeting their vulnerabilities has the potential to improve anti-tumor immune responses. Disclosures Holte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Alizadeh: Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding.