RBCeq: A robust and scalable algorithm for accurate genetic blood typing

Background: While blood transfusion is an essential cornerstone of hematological care, patients requiring repetitive transfusion remain at persistent risk of alloimmunization due to the diversity of human blood group polymorphisms. Despite the promise, user friendly methods to accurately identify blood types from next-generation sequencing data are currently lacking. To address this unmet need, we have developed RBCeq, a novel genetic blood typing algorithm to accurately identify 36 blood group systems. Methods: RBCeq can predict complex blood groups such as RH, and ABO that require identification of small indels and copy number variants. RBCeq also reports clinically significant, rare, and novel variants with potential clinical relevance that may lead to the identification of novel blood group alleles. Findings: The RBCeq algorithm demonstrated 99·07% concordance when validated on 402 samples which included 29 antigens with serology and 9 antigens with SNP-array validation in 14 blood group systems and 59 antigens validation on manual predicted phenotype from variant call files. We have also developed a user-friendly web server that generates detailed blood typing reports with advanced visualization (https://www.rbceq.org/). Interpretation: RBCeq will assist blood banks and immunohematology laboratories by overcoming existing methodological limitations like scalability, reproducibility, and accuracy when genotyping and phenotyping in multi-ethnic populations. This Amazon Web Services (AWS) cloud based platform has the potential to reduce pre-transfusion testing time and to increase sample processing throughput, ultimately improving quality of patient care. Funding: This work was supported in part by Advance Queensland Research Fellowship, MRFF Genomics Health Futures Mission (76,757), and the Australian Red Cross LifeBlood. The Australian governments fund the Australian Red Cross Lifeblood for the provision of blood, blood products and services to the Australian community.</p

MicroRNA-146a regulates immune-related adverse events caused by immune checkpoint inhibitors

Immune checkpoint inhibitor (ICI) therapy has shown a significant benefit in the treatment of a variety of cancer entities. However, immune-related adverse events (irAEs) occur frequently and can lead to ICI treatment termination. MicroRNA-146a (miR-146a) has regulatory functions in immune cells. We observed that mice lacking miR-146a developed markedly more severe irAEs compared with WT mice in several irAE target organs in 2 different murine models. miR-146a-/- mice exhibited increased T cell activation and effector function upon ICI treatment. Moreover, neutrophil numbers in the spleen and the inflamed intestine were highly increased in ICI-treated miR-146a-/- mice. Therapeutic administration of a miR-146a mimic reduced irAE severity. To validate our preclinical findings in patients, we analyzed the effect of a SNP in the MIR146A gene on irAE severity in 167 patients treated with ICIs. We found that the SNP rs2910164 leading to reduced miR-146a expression was associated with an increased risk of developing severe irAEs, reduced progression-free survival, and increased neutrophil counts both at baseline and during ICI therapy. In conclusion, we characterized miR-146a as a molecular target for preventing ICI-mediated autoimmune dysregulation. Furthermore, we identified the MIR146A SNP rs2910164 as a biomarker to predict severe irAE development in ICI-treated patients

Immunization against poly--acetylglucosamine reduces neutrophil activation and GVHD while sparing microbial diversity

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly--acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity

Metabolic reprogramming of donor T cells enhances graft-versus-leukemia effects in mice and humans

Acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT) has a dismal prognosis. We found that T cells of patients relapsing with AML after allo-HCT exhibited reduced glycolysis and interferon-gamma production. Functional studies in multiple mouse models of leukemia showed that leukemia-derived lactic acid (LA) interfered with T cell glycolysis and proliferation. Mechanistically, LA reduced intracellular pH in T cells, led to lower transcription of glycolysis-related enzymes, and decreased activity of essential metabolic pathways. Metabolic reprogramming by sodium bicarbonate (NaBi) reversed the LA-induced low intracellular pH, restored metabolite concentrations, led to incorporation of LA into the tricarboxylic acid cycle as an additional energy source, and enhanced graft-versus-leukemia activity of murine and human T cells. NaBi treatment of post-allo-HCT patients with relapsed AML improved metabolic fitness and interferon-y production in T cells. Overall, we show that metabolic reprogramming of donor T cells is a pharmacological strategy for patients with relapsed AML after allo-HCT

Phase II trial of hypomethylating agent combined with nivolumab for acute myeloid leukaemia relapse after allogeneic haematopoietic cell transplantation—Immune signature correlates with response

SummaryAcute myeloid leukaemia (AML) relapse after allogeneic haematopoietic cell transplantation (allo‐HCT) is often driven by immune‐related mechanisms and associated with poor prognosis. Immune checkpoint inhibitors combined with hypomethylating agents (HMA) may restore or enhance the graft‐versus‐leukaemia effect. Still, data about using this combination regimen after allo‐HCT are limited. We conducted a prospective, phase II, open‐label, single‐arm study in which we treated patients with haematological AML relapse after allo‐HCT with HMA plus the anti‐PD‐1 antibody nivolumab. The response was correlated with DNA‐, RNA‐ and protein‐based single‐cell technology assessments to identify biomarkers associated with therapeutic efficacy. Sixteen patients received a median number of 2 (range 1–7) nivolumab applications. The overall response rate (CR/PR) at day 42 was 25%, and another 25% of the patients achieved stable disease. The median overall survival was 15.6 months. High‐parametric cytometry documented a higher frequency of activated (ICOS$^{+}$, HLA‐DR$^{+}$), low senescence (KLRG1$^{−}$, CD57$^{−}$) CD8$^{+}$ effector T cells in responders. We confirmed these findings in a preclinical model. Single‐cell transcriptomics revealed a pro‐inflammatory rewiring of the expression profile of T and myeloid cells in responders. In summary, the study indicates that the post‐allo‐HCT HMA/nivolumab combination induces anti‐AML immune responses in selected patients and could be considered as a bridging approach to a second allo‐HCT. Trial‐registration: EudraCT‐No. 2017‐002194‐18