Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia.

Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL-selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity.

Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity.

Burden of hospitalizations and outpatient visits associated with moderate and severe acute graft-versus-host disease in Finland and Sweden: a real-world data study.

PURPOSE: The aim of this study was to describe patient characteristics and quantify hospital stays and outpatient visits (H&OV) following diagnosis with moderate-to-severe acute graft-versus-host disease (aGVHD) in Finland and Sweden. METHODS: A retrospective chart audit collected data from patient medical records of 3 specialized centers performing allogeneic hematopoietic stem cell transplantation (HSCT; Finland, n = 2; Sweden, n = 1). Eligible patients received allogeneic HSCT (January 1, 2016-June 30, 2017) from any donor source, were diagnosed with grade II-IV aGVHD (MAGIC or modified Glucksberg criteria) at any time from transplantation to 12 months before data collection, and were ≥ 18 years old at diagnosis. Criteria for comparing patients graded with modified Glucksberg and MAGIC severity scales were defined. RESULTS: Fifty-five patients (Finland, n = 45; Sweden, n = 10) were included. Myeloablative conditioning was the most common conditioning regimen (81.8%); immunosuppression regimens were based on combinations of methotrexate (96.4%), in vivo T-cell depletion (80.0%), cyclosporine (63.6%), mycophenolate (40.0%), and tacrolimus (34.5%). Sixteen patients (29.1%) developed grade III/IV aGVHD; skin was the most common organ involved (80.0%). Most patients required ≥ 1 hospital stay (89.1%; median of 2 hospitalizations per patient); 7 patients (14.3%) required admission to an intensive care unit. Median hospitalization duration from HSCT to discharge was 26 days. Most patients also required outpatient or emergency department visits (90.9%). Subgroup analyses showed longer hospital stays for patients receiving multiple treatment lines; no clear differences in H&OV were observed between prophylactic regimens. CONCLUSION: Based on this retrospective study, moderate-to-severe aGVHD is associated with considerable healthcare resource utilization in Finland and Sweden, particularly in patients who received multiple lines of therapy.

Somatic mutations and T-cell clonality in patients with immunodeficiency.

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.

Hypoxia-inducible factor controls immunoregulatory properties of myeloid cells in mouse cardiac allografts - an experimental study.

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.

T cells and cancer - why do the killers become exhausted?

Nascent tumors are mostly eliminated by the immune system. During carcinogenesis mutated cells find a way to escape from the immune system´s surveillance. As the tumor microenvironment evolves it becomes increasingly difficult for T cells to recognize and kill cancer cells. Recently, novel immunological targets have been recognized and potent immunomodulatory drugs discovered in clinical trials. This has resulted in the emergence of immunotherapy as a novel potent therapy for cancer in addition to chemotherapy, targeted therapy, operative therapy and radiotherapy.

Somatic mutations associate with clonal expansion of CD8+ T cells.

Somatic mutations in T cells can cause cancer but also have implications for immunological diseases and cell therapies. The mutation spectrum in nonmalignant T cells is unclear. Here, we examined somatic mutations in CD4+ and CD8+ T cells from 90 patients with hematological and immunological disorders and used T cell receptor (TCR) and single-cell sequencing to link mutations with T cell expansions and phenotypes. CD8+ cells had a higher mutation burden than CD4+ cells. Notably, the biggest variant allele frequency (VAF) of non-synonymous variants was higher than synonymous variants in CD8+ T cells, indicating non-random occurrence. The non-synonymous VAF in CD8+ T cells strongly correlated with the TCR frequency, but not age. We identified mutations in pathways essential for T cell function and often affected lymphoid neoplasia. Single-cell sequencing revealed cytotoxic TEMRA phenotypes of mutated T cells. Our findings suggest that somatic mutations contribute to CD8+ T cell expansions without malignant transformation.

Treatment Patterns and Clinical Outcomes of Patients with Moderate to Severe Acute Graft-Versus-Host Disease: A Multicenter Chart Review Study.

Acute graft-versus-host disease (aGVHD) remains a barrier to successful allogeneic hematopoietic stem cell transplantation (HSCT) outcomes. Contemporary comprehensive analyses of real-world clinical outcomes among patients who develop aGVHD post-HSCT are needed to better understand the unmet needs of this patient population. This multicenter, retrospective chart review describes treatment patterns and clinical outcomes among patients (≥18 years old) from Finland, Sweden, and France who developed grades II-IV aGVHD after their first HSCT (January 2016-June 2017). From 13 participating centers, 151 patients were included. The median (Q1, Q3) age at HSCT was 56 (45, 62) years old. One line of aGVHD treatment was received by 47.7%, and the most common first-line treatment was methylprednisolone (alone or in a combination regimen, 74.2%; monotherapy, 25.8%). Among patients treated with methylprednisolone, 79.5% achieved a complete or partial response. The median (Q1, Q3) number of treatment lines was 2.0 (1.0, 3.0). The median (Q1, Q3) time to obtain an aGVHD diagnosis from transplant was 29.5 (21.0, 44.0) days, and 14.5 (7.0, 34.0) days to achieve the best response for 110 evaluable patients. At 6 and 12 months, 53.6% and 49.0%, respectively, achieved a complete response. Chronic GVHD occurred in 37.7% of patients, and aGVHD reoccurred in 26.5%. Following aGVHD diagnosis, mortality rates were 30.0% at 6 months and 37.3% at 12 months. Findings from this study demonstrate a continuing unmet need for new therapies that control aGVHD and improve mortality.

Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.

Donor simvastatin treatment abolishes rat cardiac allograft ischemia/reperfusion injury and chronic rejection through microvascular protection.

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-ß1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-ß1-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.

Somatic compensation of inherited bone marrow failure.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by insufficient blood cell production and increased risk of transformation to myeloid malignancies. While genetically diverse, IBMFS are collectively defined by a cell-intrinsic hematopoietic stem cell (HSC) fitness defect that impairs HSC self-renewal and hematopoietic differentiation. In IBMFS, HSCs frequently acquire mutations that improve cell fitness, a phenomenon known as somatic compensation. Somatic compensation can occur via distinct genetic processes such as loss of the germline mutation or somatic alterations in pathways affected by the disease-causing gene. While the clinical implications of somatic compensation in IBMFS remain to be fully discovered, understanding these mutational processes can help understand disease pathophysiology and may inform future diagnostic and therapeutic approaches. In this review, we highlight current understanding about somatic compensation in IBMFS.

Enrichment of cancer-predisposing germline variants in adult and pediatric patients with acute lymphoblastic leukemia.

Despite recent progress in acute lymphoblastic leukemia (ALL) therapies, a significant subset of adult and pediatric ALL patients has a dismal prognosis. Better understanding of leukemogenesis and recognition of germline genetic changes may provide new tools for treating patients. Given that hematopoietic stem cell transplantation, often from a family member, is a major form of treatment in ALL, acknowledging the possibility of hereditary predisposition is of special importance. Reports of comprehensive germline analyses performed in adult ALL patients are scarce. Aiming at fulfilling this gap of knowledge, we investigated variants in 93 genes predisposing to hematologic malignancies and 70 other cancer-predisposing genes from exome data obtained from 61 adult and 87 pediatric ALL patients. Our results show that pathogenic (P) or likely pathogenic (LP) germline variants in genes associated with predisposition to ALL or other cancers are prevalent in ALL patients: 8% of adults and 11% of children. Comparison of P/LP germline variants in patients to population-matched controls (gnomAD Finns) revealed a 2.6-fold enrichment in ALL cases (CI 95% 1.5-4.2, p = 0.00071). Acknowledging inherited factors is crucial, especially when considering hematopoietic stem cell transplantation and planning post-therapy follow-up. Harmful germline variants may also predispose patients to excessive toxicity potentially compromising the outcome. We propose integrating germline genetics into precise ALL patient care and providing families genetic counseling.

Anti-COX-2 autoantibody is a novel biomarker of immune aplastic anemia.

In immune aplastic anemia (IAA), severe pancytopenia results from the immune-mediated destruction of hematopoietic stem cells. Several autoantibodies have been reported, but no clinically applicable autoantibody tests are available for IAA. We screened autoantibodies using a microarray containing >9000 proteins and validated the findings in a large international cohort of IAA patients (n = 405) and controls (n = 815). We identified a novel autoantibody that binds to the C-terminal end of cyclooxygenase 2 (COX-2, aCOX-2 Ab). In total, 37% of all adult IAA patients tested positive for aCOX-2 Ab, while only 1.7% of the controls were aCOX-2 Ab positive. Sporadic non-IAA aCOX-2 Ab positive cases were observed among patients with related bone marrow failure diseases, multiple sclerosis, and type I diabetes, whereas no aCOX-2 Ab seropositivity was detected in the healthy controls, in patients with non-autoinflammatory diseases or rheumatoid arthritis. In IAA, anti-COX-2 Ab positivity correlated with age and the HLA-DRB1*15:01 genotype. 83% of the >40 years old IAA patients with HLA-DRB1*15:01 were anti-COX-2 Ab positive, indicating an excellent sensitivity in this group. aCOX-2 Ab positive IAA patients also presented lower platelet counts. Our results suggest that aCOX-2 Ab defines a distinct subgroup of IAA and may serve as a valuable disease biomarker.

Targeting lymphatic vessel activation and CCL21 production by vascular endothelial growth factor receptor-3 inhibition has novel immunomodulatory and antiarteriosclerotic effects in cardiac allografts.

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.

Gemtuzumab-Ozogamicin-Related Impaired Hemoglobin-Haptoglobin Scavenging as On-Target/Off-Tumor Toxicity of Anti-CD33 AML Therapy: A Report of Two Cases.

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.

Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia.

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.

PDGF-A, -C, and -D but not PDGF-B increase TGF-beta1 and chronic rejection in rat 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-alpha mRNA was upregulated in acutely-rejecting, and PDGF-A and PDGF-C mRNA in chronically-rejecting cardiac centhatn allografts. In acute rejection, PDGFR-alpha 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-beta1 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-beta1 pathway. Inhibition of signaling of all PDGF-ligands except that of PDGF-B may thus be needed to inhibit chronic rejection in cardiac allografts.

Immunogenomic Landscape of Hematological Malignancies.

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-γ 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.

Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease.

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.