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Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. We integrated over 8,000 transcriptomes and 2,000 samples with multilevel genomics of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival, and validated key findings experimentally. Infiltration of cytotoxic lymphocytes was associated with TP53 and myelodysplasia-related changes in acute myeloid leukemia, and activated B cell-like phenotype and interferon-gamma response in lymphoma. CIITA methylation regulating antigen presentation, cancer type-specific immune checkpoints, such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity and predicted survival. Our study provides a resource linking immunology with cancer subtypes and genomics in hematological malignancies.

Gemtuzumab-ozogamicin (GO) is a humanized anti-CD33 antibody, which is conjugated to a cytotoxic calicheamicin. It is used to treat acute myeloid leukemia (AML) in combination with chemotherapy. We describe here two GO-treated acute myeloid leukemia (AML) cases: both patients suffered from a toxic syndrome, which manifested as impaired hemoglobin-haptoglobin scavenging and accumulation of hemolysis-related products. Our observations and earlier reports indicated that the reaction was caused by GO-targeted destruction of CD33 + CD163+ monocytes/macrophages, which are responsible for the clearance of hemoglobin-haptoglobin complexes. The rise of plasma lactate dehydrogenase was an early sign of the reaction, and both patients had high levels of free plasma hemoglobin, but plasma haptoglobin and bilirubin levels were paradoxically normal. Symptoms included septic fever and abnormalities in cardiac tests and in the case of the first patient, severe neurological symptoms which required intensive care unit admittance. Therapeutic plasma exchanges supported the patients until the recovery of normal hematopoiesis. The symptoms may be easily confounded with infectious complications-related organ damage. Regarding the increasing use of gemtuzumab-ozogamicin and other emerging CD33-targeted cell therapies, we want to highlight this mostly unknown and probably underdiagnosed toxicity.

Common variable immunodeficiency and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is however implicated. To study whether somatic mutations in CD4+ and CD8+ T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset common variable immunodeficiency and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2533 immune-associated genes from CD4+ and CD8+ cells. Deep T-cell receptor beta sequencing was used to characterize CD4+ and CD8+ T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in common variable immunodeficiency and 48% in controls. Clonal hematopoiesis-associated variants in both CD4+ and CD8+ cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immune- and proliferative functions, such as STAT5B (two patients), C5AR1 (two patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, common variable immunodeficiency patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4+ and CD8+ cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.

Funding Information: Acknowledgements The authors would like to thank Hanna Läh-teenmäki, Tiina Kasanen, and Jay Klievink from the Hematology Research Unit Helsinki for valuable technical assistance. The authors would also like to thank Noora Aarnio from the Biomedicum Flow Cytometry for her expertise and assistance with cell sorting. Jenni Lahtela and Emma Saarinen from Technology Centre of Institute of Molecular Medicine Finland (FIMM) are acknowledged for their excellent work on single-cell sequencing. Single-cell sequencing, immunogene panel sequencing, and skin sample WES were performed at FIMM Single-cell Analytics and Sequencing units, which are supported by HiLIFE and Biocenter Finland. The authors acknowledge Finnish Red Cross Blood Service for providing healthy control samples. The authors acknowledge Helsinki Biobank for providing bone marrow biopsies and the Finnish Hematology Registry and Clinical Biobank (FHRB) for providing MNC samples of AA patients. The authors thank all the patients for their generous participation. The FHRB Biobank is supported by the Finnish Association of Funding Information: Hematology, Finnish Red Cross Blood Service, Institute for Molecular Medicine Finland, and participating hospitals in Finland. The authors acknowledge IT Center for Science Ltd for data storage and computational resources and the computational resources provided by the Aalto Science-IT project. The research was funded with research grants from the European Research Council (M-IMM and STRATIFY projects), Academy of Finland, Sigrid Juselius Foundation, Cancer Foundation Finland, Blood Disease Research Foundation, and Instrumentarium Science Foundation. Publisher Copyright: © 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved. The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients’ CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4 T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4 T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies. + +, This work was supported by the European Research Council (M-IMM project), Academy of Finland (Decisions 287224, 314442), Finnish special governmental subsidy for health sciences, research and training, Sigrid Juselius Foundation, Instrumentarium Science foundation, Helsinki Institute of Life Sciences Fellow funding, Cancer Foundation Finland, and Finnish Cancer Institute. T.L. was supported by the Academy of Finland (Decisions 311081 and 314557). This study was supported by Finnish Functional Genomics Center, University of Turku, Åbo Akademi University and Biocenter Finland. IT Center for Science LTD (CSC) is acknowledged for their help and computing resources.

Hypoxia-inducible factors (HIFs) play a critical role in inflammatory properties of myeloid-derived cells. The effect of HIFs on myeloid-derived cell functions in organ transplantation remains unknown, however. We transplanted hearts into transgenic mice with myeloid cell-targeted deletions of HIF-1α or its negative regulator von Hippel-Lindau (VHL) to investigate the effects of HIF-1α inactivation or HIF pathway activation, respectively, on ischemia-reperfusion injury (IRI) and acute rejection. Deletion of VHL in myeloid cells enhanced mRNA expression of anti-inflammatory genes IDO, Arg-1, and HO-1 in vitro. In vivo, VHL-/- myeloid-derived cells of allograft recipients alleviated IRI and acute rejection, evidenced by reduced cardiomyocyte damage, decreased proinflammatory cytokine mRNA levels, and absence of inflammatory infiltrate at 5 days after transplantation. Ultimately, allograft survival was significantly prolonged. In vitro, VHL-/- myeloid-derived cells dose-dependently inhibited T-cell proliferation. Myeloid cells with HIF-1α-deletion retained proinflammatory qualities in vitro and in vivo. Deletion of VHL in myeloid cells of nonimmunosuppressed cardiac allograft recipients reduced myocardial injury and acute rejection. We suggest that HIF transcription factors induce a regulatory phenotype in myeloid-derived cells, which may be harnessed as a novel therapeutic strategy to regulate immune responses after heart transplantation.

Graft-versus-host-disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation. GvHD patients have aberrant T cell expansions, which are thought to drive pathological immune activation. Here we report mechanistic insights that somatic mutations may account for persistent clonal T cell expansions in chronic GvHD (cGvHD). In an index patient suffering from cGVHD, we discovered persisting somatic MTOR, NFKB2, and TLR2 mutations in an expanded CD4+ T clone. In the screening cohort (n=135), the MTOR P2229R kinase domain mutation was detected in two additional cGvHD patients, but not in controls. Functional analysis of the discovered MTOR mutation indicated a gain-of-function alteration in translational regulation yielding in up-regulation of phosphorylated S6K1, S6, and AKT. Paired single-cell RNA and T cell receptor alpha and beta sequencing strongly supported cytotoxicity and abnormal proliferation of the clonally expanded CD4+ T cells. Real-time impedance measurements indicated increased cytotoxicity of mutated CD4 + T cells against the patient’s fibroblasts. High throughput drug-sensitivity testing suggested that mutations induce resistance to mTOR inhibitors but increase sensitivity for HSP90 inhibitors. Our findings suggest a novel explanation for the aberrant, persistent T cell activation in cGvHD, and pave the way for novel targeted therapies.

SUMMARYUnderstanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. Here, we integrated over 8,000 transcriptomes and over 1,000 samples with multilevel genomic data of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival. Infiltration of cytotoxic immune cells was associated with distinct microenvironmental responses and driver alterations in different cancers, such asTP53in acute myeloid leukemia andDTX1in diffuse large B cell lymphoma. Epigenetic modification ofCIITAregulating antigen presentation, cancer type-specific immune checkpoints such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity. Prognostic models highlighted the significance of immunological properties in predicting survival. Our study represents the most comprehensive effort to date to link immunology with cancer subtypes and genomics in hematological malignancies, providing a resource to guide future studies and immunotherapy development.

Organ damage and innate immunity during heart transplantation may evoke adaptive immunity with serious consequences. Because lymphatic vessels bridge innate and adaptive immunity, they are critical in immune surveillance; however, their role in ischemia-reperfusion injury (IRI) in allotransplantation remains unknown. We investigated whether the lymphangiogenic VEGF-C/VEGFR3 pathway during cardiac allograft IRI regulates organ damage and subsequent interplay between innate and adaptive immunity. We found that cardiac allograft IRI, within hours, increased graft VEGF-C expression and lymphatic vessel activation in the form of increased lymphatic VEGFR3 and adhesion protein expression. Pharmacological VEGF-C/VEGFR3 stimulation resulted in early lymphatic activation and later increase in allograft inflammation. In contrast, pharmacological VEGF-C/VEGFR3 inhibition during cardiac allograft IRI decreased early lymphatic vessel activation with subsequent dampening of acute and chronic rejection. Genetic deletion of VEGFR3 specifically in the lymphatics of the transplanted heart recapitulated the survival effect achieved by pharmacological VEGF-C/VEGFR3 inhibition. Our results suggest that tissue damage rapidly changes lymphatic vessel phenotype, which, in turn, may shape the interplay of innate and adaptive immunity. Importantly, VEGF-C/VEGFR3 inhibition during solid organ transplant IRI could be used as lymphatic-targeted immunomodulatory therapy to prevent acute and chronic rejection.

The major cause of death during the first year after heart transplantation is primary graft dysfunction due to preservation and ischemia-reperfusion injury (IRI). Angiopoietin-1 is a Tie2 receptor-binding paracrine growth factor with anti-inflammatory properties and indispensable roles in vascular development and stability. We used a stable variant of angiopoietin-1 (COMP-Ang1) to test whether ex vivo intracoronary treatment with a single dose of COMP-Ang1 in donor Dark Agouti rat heart subjected to 4-h cold ischemia would prevent microvascular dysfunction and inflammatory responses in the fully allogeneic recipient Wistar Furth rat. COMP-Ang1 reduced endothelial cell-cell junction disruption of the donor heart in transmission electron microscopy during 4-h cold ischemia, improved myocardial reflow, and reduced microvascular leakage and cardiomyocyte injury of transplanted allografts during IRI. Concurrently, the treatment reduced expression of danger signals, dendritic cell maturation markers, endothelial cell adhesion molecule VCAM-1 and RhoA/Rho-associated protein kinase activation and the influx of macrophages and neutrophils. Furthermore, COMP-Ang1 treatment provided sustained anti-inflammatory effects during acute rejection and prevented the development of cardiac fibrosis and allograft vasculopathy. These results suggest donor heart treatment with COMP-Ang1 having important clinical implications in the prevention of primary and subsequent long-term injury and dysfunction in cardiac allografts.

Immune aplastic anemia (AA) is a life-threatening bone marrow failure syndrome in which the hematopoietic stem cells are destroyed, leading to pancytopenia. Although the exact biological process leading to AA remains largely unknown, bone marrow destruction is thought to be mediated by an autologous T cell response. We hypothesized that the autoimmune process in AA would create a T cell fingerprint unique to aplastic anemia. To decipher this signature, we collected an international, multi-centre cohort of 245 AA-samples from bone marrow and peripheral blood profiled with T-cell receptor beta (TCRβ) -sequencing. CD8+ T cell- and MNC-sorted samples from 153 clinically annotated AA patients were obtained from diagnosis, during remission and at relapse. To compare AA to similar diseases, we gathered 116 samples from other bone marrow failure syndromes, including MDS, PNH and hypoplastic LGL, and 45 samples from other autoimmune disorders. As healthy controls, we profiled 60 CD4+ and CD8+ T cells and utilized 786 MNC samples from public data repositories. To gain insight into T cell phenotypes, we also profiled 6 longitudinal samples with scRNA+TCRαβ-sequencing. As there are 1x1012-16 different TCRs and most of them are exclusive to individuals (private), we reasoned that by studying the most biologically interesting clonotypes from each individual, we could explain differences in disease severities, variation in treatment responses and pathogenesis. From all subjects, we selected private response clonotypes: highly expanded clonotypes (at least 1% of the total repertoire), convergent clonotypes (in which multiple nucleotide sequences converge to encode the same amino acid sequence) and from patients with AA, treatment-responding clonotypes (clonotypes that were suppressed/expanded after immune therapy). To analyse epitope-specificities of these clonotypes we leveraged TCRGP, our recently described Gaussian process method that can predict if TCRs recognize previously known epitopes. Clonotypes recognising viral epitopes (CMV, EBV and Influenza A) were enriched among private response clonotypes in comparison to the total repertoire (Fisher's exact test, p=2e-16), indicating that our filtering strategy indeed enriched for epitope-specific clonotypes. Of interest, the healthy donors' private response clonotypes showed more anti-viral clonotypes than did AA-patients (p=0.003), suggesting that in AA the epitope-specifities of private response clonotypes are not driven by common viral antigens. To identify specifities against unknown epitopes of the private response AA clones, we used an unsupervised learning strategy, GLIPH,that groups TCRs recognising the same epitope based on amino acid level similarities. Clonotypes in AA showed high convergence in their epitope-targets, as 1709 of 5744 (29.75%) clonotypes formed a single, potentially epitope-specific cluster that was not viral-specific. Similar analysis of control samples resulted in fewer clones clustering to the most prominent cluster (23.20%, p=1.63e-10), suggesting for a more homogenous target population within AA patients' clones. After showing sequence-level similarity of the private response clonotypes in AA, we aimed to link these pathological clonotypes to transcriptomes at the single-cell level using scRNA+TCRαβ-sequenced samples. The cells of the private response clonotypes showed multiple T cell phenotypes, but most cells (47.13%) in the bone marrow environment were recently activated CD8+ effector phenotype, marked by expression of GZMH, GNLY and PRF1. In comparison, the anti-viral clonotypes were mostly (37.3%) central memory phenotype (CCR7, TCF7). In serially sampled patients, anti-thymocyte globulin treatment suppressed private response clonotypes in a responding patient (55.03% of T cells to 12.79%), while the amount of these clonotypes increased in a non-responding patient (18.65% to 37.86%), where treatment mostly affected the viral-specific clonotypes. In summary, our data suggest that the private response clonotypes in immune AA patients may recognise a common antigen, which was not predicted to be viral. Further, at the single-cell level AA signature clonotypes are of effector phenotype and fluctuate following immunosuppressive treatment. Monitoring of these clonotypes throughout treatment may provide insight into disease biology and variation in treatment responses. Figure Disclosures Blombery: Janssen: Honoraria; Novartis: Consultancy; Invivoscribe: Honoraria. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding.

Personalized medicine involves a comprehensive analysis of factors affecting a disease. Family history is an important but not a definitive indicator of inherited predisposition to malignancy and thus studying the germline gene aberrations alongside somatic variants is warranted. The significance of germline predisposition has been increasingly recognized in acute myeloid leukemia and is noted in the latest WHO classification.1,2,3Despite the recent progress in acute lymphoblastic leukemia (ALL) therapies, many adult patients with ALL still do poorly and there is a need for new biomarkers and therapy targets. The aim of our study was to identify and determine the frequency of germline mutations in known ALL genes, to discover new genes associated with ALL predisposition, and to compare the germline genetic background and respective consequences of pediatric and adult high-risk ALL. We examined exome sequencing data from biobanked samples of adult (50) and pediatric (68) patients with high-risk ALL (Finnish Hematological Registry and Biobank - FHRB, and clinical repositories). First, a candidate-gene analysis consisted of 92 genes previously associated with germline predisposition to ALL or syndromes predisposing to ALL. Variants with minor allele frequency of Second, an unbiased approach was applied to find novel genes predisposing to ALL by checking pathogenic variants in the same gene in at least two (adult/pediatric) patients and filtering by gene ontologies DNA repair, cell cycle, and lymphocyte differentiation; and by COSMIC cancer census genes. In both analyses, only statistically significantly more common variants in our series compared to normal population were included. We also conducted a mutational signature analysis on the samples. Our analysis (Table 1) demonstrates that 8% of adult and 10% pediatric study patients carried a pathogenic or likely pathogenic mutation in their germline in known ALL predisposing genes. All these mutations were at least 30-fold more frequent in our study series compared with allele frequencies in the normal population (p In conclusion, our results emphasize that germline predisposition is not rare among high-risk ALL patients. In addition to pediatric ALL patients, we show contributing germline variants also in adult patients. At least 20% of the adult ALL patients are transplanted and a potential germline basis of the disease should be considered when choosing the donor. Our analysis also reveals new information on the biology of high-risk ALL and may contribute to the future studies seeking for therapy options in this challenging patient category. Despite the anxiety that acknowledging inheritable factors may cause in patients, families, and caretakers, we encourage clinicians to integrate carefully interpreted germline data into patient care. References 1. Wartiovaara-Kautto U et al. Germline alterations in a consecutive series of acute myeloid leukemia. Leukemia. 2018. 2. Arber DA et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016. 3. Tawana K et al. Universal genetic testing for inherited susceptibility in children and adults with myelodysplastic syndrome and acute myeloid leukemia : are we there yet? Leukemia. 2018. Disclosures Porkka: Daiichi Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.

BACKGROUND: Obliterative bronchiolitis after lung transplantation is characterized by chronic airway inflammation leading to the obliteration of small airways. Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular responses to hypoxia and inflammation. The Von Hippel-Lindau protein (pVHL) drives the degradation of oxygen-sensitive subunit HIF-1 alpha that controls the activity of HIF-1. We investigated the effect of myeloid cell targeted gene deletion of HIF-1 alpha or its negative regulator pVHL on the development of obliterative airway disease (OAD) in the recipients of tracheal allografts, a mouse model for obliterative bronchiolitis after lung transplantation. METHODS: Tracheal allografts were heterotopically transplanted from BALB/c donor mice to fully major histocompatibility complex mismatched recipient mice with HIF-1 alpha or VHL gene deletion in myeloid cells. The recipients were left non-immunosuppressed or received tacrolimus daily. Histologic, immunohistochemical, and real-time reverse transcription polymerase chain reaction analyses were performed at 3, 10, and 30 days. RESULTS: In the absence of immunosuppression, myeloid cell-specific VHL deficiency of the recipient mice improved epithelial recovery, decreased inflammatory cell infiltration and expression of pro-inflammatory cytokines, increased regulatory forkhead box P3 messenger RNA expression, and reduced OAD development in tracheal allografts. In the presence of tacrolimus immunosuppression, loss of HIF-1 alpha activity in myeloid cells of the recipient by HIF-1 alpha gene deletion accelerated OAD development in mouse tracheal allografts. CONCLUSIONS: Activity of the HIF-pathway affects the development of allograft rejection, and our results suggest that myeloid cell-specific VHL-deficiency that potentially increases HIF-activity decreases allograft inflammation and the subsequent development of OAD in mouse tracheal allografts. (C) 2016 International Society for Heart and Lung Transplantation. All rights reserved.

Background. Acquired aplastic anemia (AA) is a bone marrow failure syndrome, in which patients' hematopoietic stem cells are destroyed, resulting in pancytopenia. The exact mechanism and biological process leading to AA remain largely unknown. Bone marrow destruction is perceived as an immune-mediated process, which is supported by elevated cytotoxic T lymphocyte (CTL) counts, responsiveness to immunosuppressive therapy and skewed CTL T cell receptor (TCR) repertoire. Although there is a well-established role of T cells in the pathology of AA, the putative antigen behind the autoimmune response, and thus, the TCRs recognising the antigen are still unrevealed. Methods. Our cohort comprised of 130 samples, consisting of bone marrow and/or peripheral blood samples from AA patients (n=52) from diagnosis and/or follow-up phases and from healthy controls (n=27). We performed TCRβ sequencing (immunoSEQ, Adaptive Biotechnologies) on sorted AA and healthy CTLs (n=25 and n=27) or AA mononuclear cells (MNCs, n=27). To gain in-depth understanding of the TCR-repertoire, we built two novel analysis methods: 1) an unsupervised clustering method to characterize epitope-specific T cells based on the amino acid -level similarities of TCRs and 2) a probability based classifier to identify AA based on TCR data in the CTL (discovery) and MNC (validation) cohorts. Results. To fully appreciate the complex nature of AA pathology, we divided the cytotoxic T cell response in two distinct categories, private and public response. Private response comprises T cell clones found only in individual patients, and this compartment could explain the variation in treatment responses and disease severity across patients. In the CTL TCR repertoire, we identified patient exclusive expanded T cell clones (>1% frequency in TCR repertoire, n = 317) and treatment responding clones (n = 364). Clustering analysis revealed that there is significant amino acid -level similarity between the TCRs of the private clones. We found several epitope-specific TCR clusters that were associated with different treatment responses, disease severities and HLA-DR15 risk-allele positivity. Interestingly, we discovered that the public response (CTL clones that are statistically enriched in AA patients compared to healthy controls) could discriminate AA from healthy TCR repertoire. Based on these publicly enriched TCRs, we built a classifier which could identify AA CTL TCR repertoire from healthy controls with 97% accuracy (F-score, revieved from leave-one-out cross-validation). We tested our classifier with the validation cohort. The accuracy to diagnose AA based on the TCR repertoire data in the MNC cohort was 0.72. Furthermore, the public clones that differentiated best the AA cases from healthy controls showed statistically significant peptide similarity. These public TCRs occurred also in healthy controls, but with smaller frequencies. When combining the private and public response TCRs, we discovered that some of AA patients' most expanded and treatment responding clones clustered in the same putative epitope-specific clusters with the public TCRs, showing an interesting intersection between public and private signatures. In addition, the comparison of the interesting private and public TCRs against databases of TCR sequences of known antigen specificities hinted that some of these clones may originally target known viral species (CMV, EBV and Influenza A), suggesting a role of these common pathogens in the development of AA. Discussion. CTLTCR repertoire analysis of AA patients revealed a TCR signature that was typical of AA patients, but varied between different patients, and it was validated with an independent dataset. Thus, we could design a TCR-based framework which could identify AA patients based on their TCR repertoire, independently of sample type. A future application for our classifier could be distinguishing AA from other AA-like diseases, like hypoplastic myelodysplastic syndrome. Furthermore, we found groups of TCRs that look similar on amino acid level, and hence these clones may target the same epitope. These TCR clusters were associated with clinical features. Amino acid similarity -based TCR signatures or TCR classifiers have not previously been published for AA or any other autoimmune diseases, and thus our pioneering tools could be utilized to study pathogenesis of other T cell mediated diseases as well. Figure 1. Figure 1. Disclosures Ebeling: Boehringer Ingelheim: Consultancy; Celgene: Speakers Bureau; Otsuka Pharma Scandinavia AB: Consultancy. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy. Mustjoki:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria; Ariad: Research Funding; Pfizer: Honoraria, Research Funding.

Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. How cancer-cell intrinsic genomic and epigenetic alterations influence immune signatures in hematological malignancies is not known. Here, we integrated over 8,000 transcriptomes of hematologic cancers and multilevel genomic datasets to investigate associations of immune states to cancer molecular subtypes, genetic and epigenetic alterations, and clinical outcomes. We utilized a resource of over 8,000 transcriptomes collected across 36 hematologic malignancies and normal hematopoietic cells (Hemap), together with multi-omics datasets of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) from The Cancer Genome Atlas and other sources (Figure). In addition to gene expression data, we integrated somatic DNA alterations, methylation data, multiplex immunohistochemistry (mIHC), and flow cytometry to comprehensively map immune-associated features and validate the robustness of the findings. To characterize the composition of the cytolytic immune infiltrate from bulk transcriptomes, we defined a signature of genes most specifically expressed in cytotoxic CD8+ T lymphocytes and natural killer (NK) cells termed cytolytic score. We found significant heterogeneity in the cytotoxic lymphocyte infiltration signature across hematologic malignancies. Highest cytolytic infiltrate was detected in lymphomas and correlated with IFN-γ and myeloid cell infiltration signatures including CXCL9-11 and IDO1, distinguishing the lymphoma microenvironment from leukemias. In addition to transcriptomic microenvironmental properties, specific genetic alterations were associated with cytotoxic lymphocyte infiltration. In DLBCL, driver alterations enriched in the germinal center B-cell like (GCB) molecular subtype including BCL2 translocations and KMT2D were linked to an immune-cold transcriptomic phenotype. In contrast, DTX1 alterations defined immune-infiltrated lymphomas within the GCB molecular subtype. In AML, TP53 mutations and complex karyotype were enriched in a distinct tSNE-based transcriptomic cluster characterized by increased immune infiltration in the bone marrow (BM). Given the importance of effective antigen presentation for adaptive anti-tumor immune responses, we aimed to understand the transcriptional regulation of HLA genes and co-stimulatory and co-inhibitory signaling in subtypes of hematological malignancies. Downregulation of the antigen-presenting HLA II genes was associated with CpG methylation of the promoter region of the HLA class II master regulator CIITA in distinct transcriptomic clusters of AML harboring PML-RARA or NPM1 alterations. Expression of genes encoding immune checkpoint molecules was strongly influenced by the cell-of-origin and microenvironment of each cancer type. We identified novel associations of inhibitory immune checkpoint molecules to disease subtypes, such as VISTA/PD1-H enriched in myeloid malignancies including AML, CML, and MDS, validated by mIHC performed on BM biopsies. Furthermore, variation in the expression of several genes encoding immune checkpoints was associated with somatic mutations (e.g. CD70 in DLBCL), copy-number alterations (e.g. MICB in DLBCL), and DNA methylation (e.g. PDL1 and PDCD1LG2 in AML). Finally, we integrated GTEx gene expression data across tissues to define cancer-germline antigens (CGAs) with an immune privileged tissue expression pattern. CGAs were frequently expressed in multiple myeloma and DLBCL compared to other hematologic malignancies. CGA expression was associated with cytogenetic alterations and increased MYC activity signature in myeloma and CD58 and KLHL6 mutations in DLBCL. In addition, CGA expression in myeloma and DLBCL was linked to reduced antigen gene promoter methylation and decreased survival. In summary, our findings demonstrate that molecular subtypes of hematological malignancies harbor distinct immunological signatures influenced by genetic and epigenetic alterations. Integrating genetic, epigenetic, and transcriptomic data may facilitate the development of precision immune intervention strategies in hematological malignancies. Figure. Figure. Disclosures Leppa: Bayer: Research Funding; Celgene: Consultancy; Roche: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding.

Background—Lymphatic network and chemokine-mediated signals are essential for leukocyte traffic during the proximal steps of alloimmune response. We aimed to determine the role of lymphatic vessels and their principal growth signaling pathway, vascular endothelial growth factor (VEGF)-C/D/VEGFR-3, during acute and chronic rejection in cardiac allografts.Methods and Results—Analysis of heterotopically transplanted rat cardiac allografts showed that chronic rejection increased VEGF-C+inflammatory cell and hyaluronan receptor-1 (LYVE-1)+lymphatic vessel density. Allograft lymphatic vessels were VEGFR-3+, contained antigen-presenting cells, and produced dendritic cell chemokine CCL21. Experiments with VEGFR-3/LacZ mice or mice with green fluorescent protein–positive bone marrow cells as cardiac allograft recipients showed that allograft lymphatic vessels originated almost exclusively from donor cells. Intraportal adenoviral VEGFR-3-Ig (Ad.VEGFR-3-Ig/VEGF-C/D-Trap) perfusion was used to inhibit VEGF-C/D/VEGFR-3 signaling. Recipient treatment with Ad.VEGFR-3-Ig prolonged rat cardiac allograft survival. Ad.VEGFR-3-Ig did not affect allograft lymphangiogenesis but was linked to reduced CCL21 production and CD8+effector cell entry in the allograft. Concomitantly, Ad.VEGFR-3-Ig reduced OX62+dendritic cell recruitment and increased transcription factor Foxp3 expression in the spleen. In separate experiments, treatment with a neutralizing monoclonal VEGFR-3 antibody reduced arteriosclerosis, the number of activated lymphatic vessels expressing VEGFR-3 and CCL21, and graft-infiltrating CD4+T cells in chronically rejecting mouse cardiac allografts.Conclusions—These results show that VEGFR-3 participates in immune cell traffic from peripheral tissues to secondary lymphoid organs by regulating allograft lymphatic vessel CCL21 production and suggest VEGFR-3 inhibition as a novel lymphatic vessel–targeted immunomodulatory therapy for cardiac allograft rejection and arteriosclerosis.

Background. Microvascular dysfunction and cardiomyocyte injury are hallmarks of ischemia-reperfusion injury (IRI) after heart transplantation. Platelet-derived growth factors (PDGF) have an ambiguous role in this deleterious cascade. On one hand, PDGF may exert vascular stabilizing and antiapoptotic actions through endothelial-pericyte and endothelial-cardiomyocyte crosstalk in the heart;and on the other hand, PDGF signaling mediates neointimal formation and exacerbates chronic rejection in cardiac allografts. The balance between these potentially harmful and beneficial actions determines the final outcome of cardiac allografts. Methods and Results. We transplanted cardiac allografts from Dark Agouti rat and Balb mouse donors to fully major histocompatibility complex-mismatched Wistar Furth rat or C57 mouse recipients with a clinically relevant 2-hour cold ischemia and 1-hour warm ischemia. Ex vivo intracoronary delivery of adenovirus-mediated gene transfer of recombinant human PDGF-BB upregulated messenger RNA expression of anti-mesenchymal transition and survival factors BMP-7 and Bcl-2 and preserved capillary density in rat cardiac allografts at day 10. In mouse cardiac allografts PDGF receptor-beta, but not -alpha intragraft messenger RNA levels were reduced and capillary protein localization was lost during IRI. The PDGF receptor tyrosine kinase inhibitor imatinib mesylate and a monoclonal antibody against PDGF receptor-a enhanced myocardial damage evidenced by serum cardiac troponin T release in the rat and mouse cardiac allografts 6 hours after reperfusion, respectively. Moreover, imatinib mesylate enhanced rat cardiac allograft vasculopathy, cardiac fibrosis, and late allograft loss at day 56. Conclusions. Our results suggest that PDGF-B signaling may play a role in endothelial and cardiomyocyte recovery from IRI after heart transplantation.

s S267 model to the classical non-working HHTX models as it offers the capability for functional assessment.

INTRODUCTION The recent success of checkpoint blockade immunotherapies in diverse solid tumors has prompted the evaluation of these treatments in hematologic malignancies such as acute myeloid leukemia (AML). It is critical to identify the patient and disease subsets that could respond to such therapies. Infiltration of tumors by cytotoxic T lymphocytes (CTLs) has been associated with better prognosis and responses to checkpoint inhibition. We hypothesized that the presence of a substantial fraction of activated CTLs and natural killer (NK) cells in the blood and bone marrow samples of hematologic tumors could indicate a preexisting active immune response potentially targeting the tumor cells. Moreover, the density of the immune infiltrate could shape and be shaped by the expression of cancer-germline and leukemia-associated antigens (LAAs), antigen-presenting machinery (APM) and immunosuppressive genes by the tumor cells. Here, we examined these immunological properties of hematological tumors in large-scale gene expression datasets to identify immunologically active patient subsets. METHODS Curated set of 9,544 transcriptomes collected across 36 hematological malignancies (HEMAP), including 1,858 AML cases was utilized to identify subsets of patients with existing, potentially tumor-directed immune responses. Additional multi-omics datasets of 173 AML patients from The Cancer Genome Atlas (TCGA) were integrated to gain insight into the genetic landscape of immunologically active patients. Cytolytic activity (geometric mean of GZMA (granzyme A) and PRF1 (perforin) transcript levels, Rooney et al., Cell 2015) was used as a marker of immunologic activity. Cytolytic activity was correlated to the expression levels of all transcripts, gene sets from collections such as MSigDB and manually curated gene sets representing the APM (HLA-A, -B, -C, B2M), 145 known cancer-germline antigens as well as established LAAs such as WT1 and PRTN3. Furthermore, we used an in silico flow cytometry approach, CIBERSORT (Newman et al., Nat Methods 2015), to infer the relative fractions of 22 immune cell subpopulations from the gene expression data to dissect the immune cell composition of the samples. RESULTS Cytolytic activity showed high correlation with other transcripts expressed in activated CTLs and NK cells (e.g. GZMB, GNLY, KLRB1, CD8A, CD2; Spearman's R ≥ 0.7) as well as lymphocyte activation-related gene sets across both the HEMAP and the TCGA AML datasets, validating it as a robust and specific metric of active cellular immunity. When correlated to the CIBERSORT immune cell populations, cytolytic activity was positively associated with CD8 T cells and showed a negative correlation to the proportion of M2 macrophages. High levels of cancer-germline antigens were associated with decreased expression of components of the APM and low cytolytic activity, suggesting HLA downregulation as a mechanism of immune evasion by cancer-germline antigen-expressing tumor cells. We observed extensive heterogeneity in the cytolytic activity between different diseases and subtypes within the same disease, most prominently in AML. In AML patients, complex karyotype and unfavorable prognosis were correlated with high cytolytic activity, indicating biological similarity of the immune-infiltrated tumors. Furthermore, TP53 mutations, genome fragmentation and immune checkpoint transcripts such as CD274 (PD-L1), PDCD1LG2 (PD-L2), CTLA4 and LAG3 were enriched within the complex karyotype cluster in the TCGA AML dataset. In contrast, mutations in NPM1 and FLT3 showed a modest but significant negative correlation to cytolytic activity. CIBERSORT analysis revealed that AML cases with low cytolytic activity preferentially had enrichment of an eosinophilic phenotype in addition to increased M2 polarization of macrophages. CONCLUSIONS Using large-scale transcriptomics approaches, we were able to identify patient subsets with variable levels of immune cytolytic activity within hematologic malignancies. Furthermore, we identified connections between the cytotoxic immune response and genetic properties of AML tumors. These observations have potential clinical implications, as the choice of patients to clinical trials receiving immune checkpoint blockade immunotherapies would require careful consideration in light of the observed immunological heterogeneity. Disclosures Mustjoki: Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding.

Normal adult vasculature is in a quiescent state. In transplanted hearts, peri- and postoperative ischemic and alloimmune stimuli may be interpreted as inadequate tissue perfusion leading to activation of angiogenic signaling. Although this may have protective functions, improper activation of cardiac allograft endothelial cells and smooth muscle cells may actually result in impaired survival of cardiac allografts. In this paper, we review the current knowledge on angiogenic growth factors, vascular endothelial growth factor, angiopoietins, and platelet-derived growth factor in cardiac allografts. We also discuss the potential for therapies aimed at angiogenic growth factors in preventing and treating cardiac allograft rejection and transplant coronary artery disease.

Background— Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. Methods and Results— Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell–endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1α, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury–induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N -nitro- l -arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-β1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-β1–induced microvascular endothelial-to-mesenchymal transition. Conclusions— Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.

Objective— Chronic rejection is the main reason for the poor long-term survival of heart transplant recipients and is characterized by cardiac allograft inflammation, fibrosis, and arteriosclerosis. We examined the specific roles of different platelet-derived growth factor (PDGF) ligands (A–D)—potent mesenchymal cell mitogens—in rat cardiac allografts. Methods and Results— PDGFR-α mRNA was upregulated in acutely-rejecting, and PDGF-A and PDGF-C mRNA in chronically-rejecting cardiac¢hatn allografts. In acute rejection, PDGFR-α immunoreactivity increased in the media of arteries. In chronically-rejecting allografts, immunoreactivity of all PDGF ligands and receptors—except that of PDGF-B ligand—was found in the intima of arteries, and the expression of PDGF-A and PDGF-C was seen in cardiomyocytes. Intracoronary adeno-associated virus-2 (AAV2)-mediated PDGF-A and -D gene transfer enhanced cardiac allograft inflammation. AAV2-PDGF-A, AAV2-PDGF-C, and AAV2-PDGF-D significantly upregulated profibrotic TGF-β1 mRNA and accelerated cardiac fibrosis and arteriosclerosis. In contrast, AAV2-PDGF-B did not aggravate chronic rejection. Conclusions— We found that alloimmune response induces PDGF-A, PDGF-C, and PDGF-D expression in the graft vasculature. PDGF-A, PDGF-C, and PDGF-D mediated profibrotic and proarteriosclerotic effects in transplanted hearts involving the TGF-β1 pathway. Inhibition of signaling of all PDGF-ligands except that of PDGF-B may thus be needed to inhibit chronic rejection in cardiac allografts.

Angiopoietin-1 (Ang1) and Ang2 regulate the maintenance of normal vasculature by direct endothelial and indirect smooth muscle cell (SMC) effects. Dysfunction of vascular wall cells is considered central in cardiac allograft vasculopathy (CAV), where inflammation and arterial injury initiate subsequent intimal SMC proliferation. In this study, we investigated the effect of exogenous Ang1 and Ang2 in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus (AAV)-mediated gene transfer. Bioluminescent imaging of AAV-transfected syngeneic grafts revealed gradual and stable transgene expression in graft cardiomyocytes. In cardiac allografts, both AAV-Ang1 and AAV-Ang2 decreased inflammation and increased antiapoptotic Bcl-2 mRNA and Bcl-2/Bax ratio at 8 weeks. Only AAV-Ang2 decreased the development of CAV, whereas AAV-Ang1 activated arterial SMC and increased PDGF-A mRNA in the allograft. Collectively, our results show that exogenous Ang1 and Ang2 have similar antiinflammatory and antiapoptotic effects in cardiac allografts. Prolonged AAV-mediated Ang1 transgene expression also induced SMC activation, whereas AAV-Ang2 lacked the SMC activating effects and decreased CAV. Our results thus highlight the common protective and diverse SMC effects of Ang1 and Ang2 in cardiac allograft microenvironment and the importance of timing of angiopoietins to achieve therapeutic effects.

Purpose VEGF-C/D/VEGFR-3 signaling may regulate lymphatic vessel activation and the initiation of alloimmunity after transplantation. We investigated the effect of perioperative intracoronary injection of synthetic VEGF-C/D inhibitor on lymphatic activation, antigen-presenting cell trafficking and subsequent development of alloimmune responses in rat cardiac allografts. Methods and Materials We perfused the coronaries of DA donor rat hearts ex vivo with VEGF-C/D inhibitor or PBS and preserved the grafts in +4°C PBS for 4h and then transplanted the grafts to fully MHC-mismatched WF recipient rats. We analyzed ischemia-reperfusion injury, and acute and chronic rejection responses. In acute and chronic rejection models, the recipients received CyA as immunosuppression. Results At 6h after the reperfusion, VEGF-C/D inhibitor did not affect the myocardial injury but reduced the number of LYVE-1+ lymphatic vessels expressing VEGFR-3, ICAM-1 and VCAM-1 in the allograft and reduced the number of dendritic cells migrating out of the allograft. VEGF-C/D inhibition enhanced allograft survival in acute rejection model. In chronic rejection model, VEGF-C/D inhibitor reduced allograft inflammation and prevented the development of cardiac fibrosis and vasculopathy. Conclusions Intracoronary treatment with VEGF-C/D inhibitor prevented early activation of lymphatic endothelial cells and trapped dendritic cells into the allograft. This interfered with allorecognition resulting in reduced acute and chronic rejection responses. Our results suggest donor single-dose intracoronary treatment with VEGF-C/D inhibitor as a novel clinically feasible lymphatic vessel targeted immunomodulatory approach. [ figure 1 ]