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Journal: Blood advances


Patients with juvenile idiopathic arthritis (JIA) can experience a severe disease course, with progressive destructive polyarthritis refractory to conventional therapy with disease-modifying antirheumatic drugs including biologics, as well as life-threatening complications including macrophage activation syndrome (MAS). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative immunomodulatory strategy for patients with such refractory disease. We treated 16 patients in 5 transplant centers between 2007 and 2016: 11 children with systemic JIA and 5 with rheumatoid factor-negative polyarticular JIA; all were either refractory to standard therapy, had developed secondary hemophagocytic lymphohistiocytosis/MAS poorly responsive to treatment, or had failed autologous HSCT. All children received reduced toxicity fludarabine-based conditioning regimens and serotherapy with alemtuzumab. Fourteen of 16 patients are alive with a median follow-up of 29 months (range, 2.8-96 months). All patients had hematological recovery. Three patients had grade II-IV acute graft-versus-host disease. The incidence of viral infections after HSCT was high, likely due to the use of alemtuzumab in already heavily immunosuppressed patients. All patients had significant improvement of arthritis, resolution of MAS, and improved quality of life early following allo-HSCT; most importantly, 11 children achieved complete drug-free remission at the last follow-up. Allo-HSCT using alemtuzumab and reduced toxicity conditioning is a promising therapeutic option for patients with JIA refractory to conventional therapy and/or complicated by MAS. Long-term follow-up is required to ascertain whether disease control following HSCT continues indefinitely.

Concepts: Disease, Infectious disease, Infection, Graft-versus-host disease, Rheumatoid arthritis, Organ transplant, Hematopoietic stem cell transplantation, Juvenile idiopathic arthritis


Murine models showed that CD8+CD44himemory T ™ cells could eradicate malignant cells without inducing graft-versus-host disease (GVHD). We evaluated the feasibility and safety of infusing freshly isolated and purified donor-derived phenotypic CD8+TMcells into adults with disease relapse after allogeneic hematopoietic cell transplantation (HCT). Phenotypic CD8 TMcells were isolated after unmobilized donor apheresis using a tandem immunomagnetic selection strategy of CD45RA depletion followed by CD8+enrichment. Fifteen patients received CD8+TMcells at escalating doses (1 × 106, 5 × 106, or 10 × 106cells per kg). Thirteen received cytoreduction before CD8+TMcell infusion, and 9 had active disease at the time of infusion. Mean yield and purity of the CD8+TMinfusion were 38.1% and 92.8%, respectively; >90% had CD8+T effector memory phenotype, cytokine expression, and secretion profile. No adverse infusional events or dose-limiting toxicities occurred; GVHD developed in 1 patient (grade 2 liver). Ten patients (67%) maintained or achieved response (7 complete response, 1 partial response, 2 stable disease) for at least 3 months after infusion; 4 of the responders had active disease at the time of infusion. With a median follow-up from infusion of 328 days (range, 118-1328 days), median event-free survival and overall survival were 4.9 months (95% confidence interval [CI], 1-19.3 months) and 19.6 months (95% CI, 5.6 months to not reached), respectively. Collection and enrichment of phenotypic CD8+TMcells is feasible, well tolerated, and associated with a low incidence of GVHD when administered as a manipulated infusion of donor lymphocytes in patients who have relapsed after HCT. This trial was registered at as #NCT01523223.

Concepts: Gene, Cancer, Medical terms, Graft-versus-host disease, Hematopoietic stem cell transplantation, Normal distribution, Thymus, Infusion


Relapses in acute myelogenous leukemia (AML) are a result of quiescent leukemic stem cells (LSCs) in marrow stromal niches, where they resist chemotherapy. LSCs employ CXCL12/CXCR4 to home toward protective marrow niches. Heparin disrupts CXCL12-mediated sequestration of cells in the marrow. CX-01 is a low-anticoagulant heparin derivative. In this pilot study, we combined CX-01 with chemotherapy for the treatment of AML. Induction consisted of cytarabine and idarubicin (7 + 3) with CX-01. Twelve patients were enrolled (median age, 56 years; 3 women). Three, 5, and 4 patients had good-, intermediate-, and poor-risk disease, respectively. Day 14 bone marrows were available on 11 patients and were aplastic in all without detectable leukemia. Eleven patients (92%) had morphologic complete remission after 1 induction (CR1). Eight patients were alive at a median follow-up of 24 months (4 patients in CR1). Three patients received an allogeneic stem cell transplant in CR1. Median disease-free survival was 14.8 months. Median overall survival was not attained at the maximum follow-up time of 29.4 months. No CX-01-associated serious adverse events occurred. Median day to an untransfused platelet count of at least 20 × 109/L was 21. CX-01 is well tolerated when combined with intensive therapy for AML and appears associated with enhanced count recovery and treatment efficacy.

Concepts: Bone marrow, Cure, Chemotherapy, Leukemia, Organ transplant, Acute myeloid leukemia, Blood disorders, Myeloid sarcoma


A National Cancer Institute consensus study on prioritization of cancer antigens ranked the Wilms tumor 1 (WT1) protein as the top immunotherapy target in cancer. We previously reported a pilot study of a multivalent WT1 peptide vaccine (galinpepimut-S) in acute myeloid leukemia (AML) patients. We have now conducted a phase 2 study investigating this vaccine in adults with AML in first complete remission (CR1). Patients received 6 vaccinations administered over 10 weeks with the potential to receive 6 additional monthly doses if they remained in CR1. Immune responses (IRs) were evaluated after the 6th and 12th vaccinations by CD4+ T-cell proliferation, CD8+ T-cell interferon-γ secretion (enzyme-linked immunospot), or the CD8-relevant WT1 peptide major histocompatibility complex tetramer assay (HLA-A*02 patients only). Twenty-two patients (7 males; median age, 64 years) were treated. Fourteen patients (64%) completed ≥6 vaccinations, and 9 (41%) received all 12 vaccine doses. Fifteen patients (68%) relapsed, and 10 (46%) died. The vaccine was well tolerated, with the most common toxicities being grade ½ injection site reactions (46%), fatigue (32%), and skin induration (32%). Median disease-free survival from CR1 was 16.9 months, whereas the overall survival from diagnosis has not yet been reached but is estimated to be ≥67.6 months. Nine of 14 tested patients (64%) had an IR in ≥1 assay (CD4 or CD8). These results indicated that the WT1 vaccine was well tolerated, stimulated a specific IR, and was associated with survival in excess of 5 years in this cohort of patients. This trial was registered at as #NCT01266083.

Concepts: Immune system, Cancer, Vaccine, Chemotherapy, Major histocompatibility complex, Leukemia, Acute myeloid leukemia, Wilms' tumor


CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.

Concepts: Blood, Stem cell, Bone marrow, Chemotherapy, Hematology, Leukemia, Glycoprotein, Selectin


We hypothesized that third-party fecal microbiota transplantation (FMT) may restore intestinal microbiome diversity after allogeneic hematopoietic cell transplantation (allo-HCT). In this open-label single-group pilot study, 18 subjects were enrolled before allo-HCT and planned to receive third-party FMT capsules. FMT capsules were administered no later than 4 weeks after neutrophil engraftment, and antibiotics were not allowed within 48 hours before FMT. Five patients did not receive FMT because of the development of early acute gastrointestinal (GI) graft-versus-host disease (GVHD) before FMT (n = 3), persistent HCT-associated GI toxicity (n = 1), or patient decision (n = 1). Thirteen patients received FMT at a median of 27 days (range, 19-45 days) after HCT. Participants were able to swallow and tolerate all FMT capsules, meeting the primary study endpoint of feasibility. FMT was tolerated well, with 1 treatment-related significant adverse event (abdominal pain). Two patients subsequently developed acute GI GVHD, with 1 patient also having concurrent bacteremia. No additional cases of bacteremia occurred. Median follow-up for survivors is 15 months (range, 13-20 months). The Kaplan-Meier estimates for 12-month overall survival and progression-free survival after FMT were 85% (95% confidence interval, 51%-96%) and 85% (95% confidence interval, 51%-96%), respectively. There was 1 nonrelapse death resulting from acute GI GVHD (12-month nonrelapse mortality, 8%; 95% confidence interval, 0%-30%). Analysis of stool composition and urine 3-indoxyl sulfate concentration indicated improvement in intestinal microbiome diversity after FMT that was associated with expansion of stool-donor taxa. These results indicate that empiric third-party FMT after allo-HCT appears to be feasible, safe, and associated with expansion of recipient microbiome diversity. This trial was registered at as #NCT02733744.

Concepts: Bacteria, Estimator, Patient, Graft-versus-host disease, Interval finite element, Hematopoietic stem cell transplantation, Normal distribution, Human feces


Practical methods are needed to increase the applicability and efficacy of chimeric antigen receptor (CAR) T-cell therapies. Using donor-derived CAR-T cells is attractive, but expression of endogenous T-cell receptors (TCRs) carries the risk for graft-versus-host-disease (GVHD). To remove surface TCRαβ, we combined an antibody-derived single-chain variable fragment specific for CD3ε with 21 different amino acid sequences predicted to retain it intracellularly. After transduction in T cells, several of these protein expression blockers (PEBLs) colocalized intracellularly with CD3ε, blocking surface CD3 and TCRαβ expression. In 25 experiments, median TCRαβ expression in T lymphocytes was reduced from 95.7% to 25.0%; CD3/TCRαβ cell depletion yielded virtually pure TCRαβ-negative T cells. Anti-CD3ε PEBLs abrogated TCRαβ-mediated signaling, without affecting immunophenotype or proliferation. In anti-CD3ε PEBL-T cells, expression of an anti-CD19-41BB-CD3ζ CAR induced cytokine secretion, long-term proliferation, and CD19+leukemia cell killing, at rates meeting or exceeding those of CAR-T cells with normal CD3/TCRαβ expression. In immunodeficient mice, anti-CD3ε PEBL-T cells had markedly reduced GVHD potential; when transduced with anti-CD19 CAR, these T cells killed engrafted leukemic cells. PEBL blockade of surface CD3/TCRαβ expression is an effective tool to prepare allogeneic CAR-T cells. Combined PEBL and CAR expression can be achieved in a single-step procedure, is easily adaptable to current cell manufacturing protocols, and can be used to target other T-cell molecules to further enhance CAR-T-cell therapies.

Concepts: Protein, Gene, Amino acid, B cell, T cell, T cells, Major histocompatibility complex, T cell receptor


We studied the association between non-HLA donor characteristics (age, sex, donor-recipient relationship, blood group [ABO] match, and cytomegalovirus [CMV] serostatus) and transplant outcomes after T-cell-replete HLA-haploidentical transplantation using posttransplantation cyclophosphamide (PT-Cy) in 928 adults with hematologic malignancy transplanted between 2008 and 2015. Siblings (n = 358) and offspring (n = 450) were the predominant donors, with only 120 patients having received grafts from parents. Although mortality risks were higher with donors aged 30 years or older (hazard ratio, 1.39;P< .0001), the introduction of patient age to the Cox regression model negated the effect of donor age. Two-year survival adjusted for CMV seropositivity, disease, and disease risk index was lower in patients aged 55 to 78 years after transplantation of grafts from donors younger than 30 years (53%) or aged at least 30 years (46%) compared with younger patients who received grafts from donors younger than 30 years (61%) and at least 30 years (60%;P< .0001). Similarly, 2-year survival in patients aged 55 to 78 years was lower after transplantation of grafts from siblings (45%) or offspring (48%) compared with patients aged 18 to 54 years after transplantation of grafts from siblings (62%), offspring (58%), and parents (61%;P< .0001). Graft failure was higher after transplantation of grafts from parents (14%) compared with siblings (6%) or offspring (7%;P= .02). Other non-HLA donor characteristics were not associated with survival or graft failure. The current analyses suggest patient and disease, rather than non-HLA donor characteristics, predominantly influence survival in adults.

Concepts: Regression analysis, Death, Blood, Senescence, Proportional hazards models, Survival analysis, Parent, ABO blood group system


Randomized clinical trials comparing direct oral anticoagulants (DOACs) to warfarin in cancer patients have not been performed. We evaluated the effectiveness and associated risk of DOACs vs warfarin, as well as comparisons of DOACs, in a large population of cancer patients with nonvalvular atrial fibrillation (AF). Using the MarketScan databases, we identified 16 096 AF patients (mean age, 74 years) initiating oral anticoagulant and being actively treated for cancer between 2010 and 2014. Anticoagulant users were matched by age, sex, enrollment date, and drug initiation date. Study end points were identified with diagnostic codes and included ischemic stroke, severe bleeding, other bleeding, and venous thromboembolism (VTE). Cox regression was used to estimate associations of anticoagulants with study end points. Compared with warfarin, rates of bleeding (hazard ratio [95% confidence interval]) were similar in rivaroxaban (1.09 [0.79, 1.39]) and dabigatran (0.96 [0.72, 1.27]) users, whereas apixaban users experienced lower rates (0.37 [0.17, 0.79]). Rates of ischemic stroke did not differ among anticoagulant users. Compared with warfarin, rate of VTE (hazard ratio [95% confidence interval]) was lower among rivaroxaban (0.51 [0.41, 0.63]), dabigatran (0.28 [0.21, 0.38]), and apixaban (0.14 [0.07, 0.32]) users. In head-to-head comparisons among DOACs, dabigatran users had lower rates of VTE than rivaroxaban users; apixaban users had lower rates of VTE and severe bleeding than rivaroxaban users. In this population of patients with AF and cancer, DOAC users experienced lower or similar rates of bleeding and stroke compared with warfarin users, and a lower rate of incident VTE.

Concepts: Stroke, Atrial fibrillation, Thrombosis, Warfarin, Anticoagulant, Heparin, Dabigatran, Anticoagulants


To evaluate the immunological mechanisms associated with clinical outcomes after autologous hematopoietic stem cell transplantation (AHSCT), focusing on regulatory T- (Treg) and B- (Breg) cell immune reconstitution, 31 systemic sclerosis (SSc) patients underwent simultaneous clinical and immunological evaluations over 36-month posttransplantation follow-up. Patients were retrospectively grouped into responders (n = 25) and nonresponders (n = 6), according to clinical response after AHSCT. Thymic function and B-cell neogenesis were respectively assessed by quantification of DNA excision circles generated during T- and B-cell receptor rearrangements. At the 1-year post-AHSCT evaluation of the total set of transplanted SSc patients, thymic rebound led to renewal of the immune system, with higher T-cell receptor (TCR) diversity, positive correlation between recent thymic emigrant and Treg counts, and higher expression of CTLA-4 and GITR on Tregs, when compared with pretransplant levels. In parallel, increased bone marrow output of newly generated naive B-cells, starting at 6 months after AHSCT, renovated the B-cell populations in peripheral blood. At 6 and 12 months after AHSCT, Bregs increased and produced higher interleukin-10 levels than before transplant. When the nonresponder patients were evaluated separately, Treg and Breg counts did not increase after AHSCT, and high TCR repertoire overlap between pre- and posttransplant periods indicated maintenance of underlying disease mechanisms. These data suggest that clinical improvement of SSc patients is related to increased counts of newly generated Tregs and Bregs after AHSCT as a result of coordinated thymic and bone marrow rebound.

Concepts: Immune system, Bone marrow, Immunology, T helper cell, Humoral immunity, Major histocompatibility complex, Thymus, Autoimmunity