Therapeutic CD4(+)Foxp3(+) natural regulatory T cells (Tregs) can control experimental graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) by suppressing conventional T cells (Tconvs). Treg-based therapies are currently tested in clinical trials with promising preliminary results in allo-HCT. Here, we hypothesized that as Tregs are capable of modulating Tconv response, it is likely that the inflammatory environment and particularly donor T cells are also capable of influencing Treg function. Indeed, previous findings in autoimmune diabetes revealed a feedback mechanism that renders Tconvs able to stimulate Tregs by a mechanism that was partially dependent on tumor necrosis factor (TNF). We tested this phenomenon during alloimmune response in our previously described model of GVHD protection using antigen specific Tregs. Using different experimental approaches, we observed that control of GVHD by Tregs was fully abolished by blocking TNF receptor type 2 (TNFR2) or by using TNF-deficient donor T cells or TNFR2-deficient Tregs. Thus, our results show that Tconvs exert a powerful modulatory activity on therapeutic Tregs and clearly demonstrate that the sole defect of TNF production by donor T cells was sufficient to completely abolish the Treg suppressive effect in GVHD. Importantly, our findings expand the understanding of one of the central components of Treg action, the inflammatory context, and support that targeting TNF/TNFR2 interaction represents an opportunity to efficiently modulate alloreactivity in allo-HCT to either exacerbate it for a powerful antileukemic effect or reduce it to control GVHD.

The treatment of multiple myeloma has evolved significantly over the last decades from primarily alkylator-based chemotherapeutic agents with minimal efficacy to the introduction of more effective agents including immune modulators and proteasome inhibitors, which have changed the landscape of therapy for this disease. We are now entering a new era that will increasingly integrate immunotherapy into standard treatment. This review discusses the current immune-based strategies currently approved, as well as various immune approaches being actively investigated including monoclonal antibodies, checkpoint inhibitors, vaccines, and adoptive T-cell therapies.

Megakaryocyte (MK) differentiation occurs within the bone marrow (BM), a complex 3-dimensional (3D) environment of low stiffness exerting local external constraints. To evaluate the influence of the 3D mechanical constraints that MKs may encounter in vivo, we differentiated mouse BM progenitors in methylcellulose (MC) hydrogels tuned to mimic BM stiffness. We found that MKs grown in a medium of 30- to 60-Pa stiffness more closely resembled those in the BM in terms of demarcation membrane system (DMS) morphological aspect and exhibited higher ploidy levels, as compared with MKs in liquid culture. Following resuspension in a liquid medium, MC-grown MKs displayed twice as much proplatelet formation as cells grown in liquid culture. Thus, the MC gel, by mimicking external constraints, appeared to positively influence MK differentiation. To determine whether MKs adapt to extracellular stiffness through mechanotransduction involving actomyosin-based modulation of the intracellular tension, myosin-deficient (Myh9-/-) progenitors were grown in MC gels. Absence of myosin resulted in abnormal cell deformation and strongly decreased proplatelet formation, similarly to features observed for Myh9-/- MKs differentiated in situ but not in vitro. Moreover, megakaryoblastic leukemia 1 (MKL1), a well-known actor in mechanotransduction, was found to be preferentially relocated within the nucleus of MC-differentiated MKs, whereas its inhibition prevented MC-mediated increased proplatelet formation. Altogether, these data show that a 3D medium mimicking BM stiffness contributes, through the myosin IIA and MKL1 pathways, to a more favorable in vitro environment for MK differentiation, which ultimately translates into increased proplatelet production.

Ibrutinib is not known to confer risk for Pneumocystis jirovecii pneumonia (PCP). We observed 5 cases of PCP in 96 patients receiving single-agent ibrutinib, including 4 previously untreated. Clinical presentations included asymptomatic pulmonary infiltrates, chronic cough, and shortness of breath. The diagnosis was often delayed. Median time from starting ibrutinib to occurrence of PCP was 6 months (range, 2-24). The estimated incidence of PCP was 2.05 cases per 100 patient-years (95% confidence interval, 0.67-4.79). At the time of PCP, all patients had CD4 T-cell count >500/μL (median, 966/μL) and immunoglobulin G (IgG) >500 mg/dL (median, 727 mg/dL). All patients underwent bronchoalveolar lavage. P jirovecii was identified by polymerase chain reaction in all 5 cases; direct fluorescence antibody staining was positive in 1. All events were grade ≤2 and resolved with oral therapy. Secondary prophylaxis was not given to 3 patients; after 61 patient-months of follow up, no recurrence occurred. Lack of correlation with CD4 count and IgG level suggests that susceptibility to PCP may be linked to Bruton tyrosine kinase (BTK) inhibition. If confirmed, this association could result in significant changes in surveillance and/or prophylaxis, possibly extending to other BTK inhibitors. This trial was registered at www.clinicaltrials.gov as #NCT01500733 and #NCT02514083.

Reestablishment of competent regulatory pathways has emerged as a strategy to reduce the severity of graft-versus-host disease (GVHD), and recalibrate the effector and regulatory arms of the immune system. However, clinically feasible, cost-effective strategies that do not require extensive ex vivo cellular manipulation have remained elusive. In the current study, we demonstrate that inhibition of the interleukin-27p28 (IL-27p28) signaling pathway through antibody blockade or genetic ablation prevented lethal GVHD in multiple murine transplant models. Moreover, protection from GVHD was attributable to augmented global reconstitution of CD4+ natural regulatory T cells (nTregs), CD4+ induced Tregs (iTregs), and CD8+ iTregs, and was more potent than temporally concordant blockade of IL-6 signaling. Inhibition of IL-27p28 also enhanced the suppressive capacity of adoptively transferred CD4+ nTregs by increasing the stability of Foxp3 expression. Notably, blockade of IL-27p28 signaling reduced T-cell-derived-IL-10 production in conventional T cells; however, there was no corresponding effect in CD4+ or CD8+ Tregs, indicating that IL-27 inhibition had differential effects on IL-10 production and preserved a mechanistic pathway by which Tregs are known to suppress GVHD. Targeting of IL-27 therefore represents a novel strategy for the in vivo expansion of Tregs and subsequent prevention of GVHD without the requirement for ex vivo cellular manipulation, and provides additional support for the critical proinflammatory role that members of the IL-6 and IL-12 cytokine families play in GVHD biology.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited blood disorders characterized by pathological mitochondrial iron deposition in erythroid precursors. Each known cause has been attributed to a mutation in a protein associated with heme biosynthesis, iron-sulfur cluster biogenesis, mitochondrial translation, or a component of the mitochondrial respiratory chain. Here, we describe a recurring mutation, c.276_278del, p.F93del, in NDUFB11, a mitochondrial respiratory complex I-associated protein encoded on the X chromosome, in 5 males with a variably syndromic, normocytic CSA. The p.F93del mutation results in respiratory insufficiency and loss of complex I stability and activity in patient-derived fibroblasts. Targeted introduction of this allele into K562 erythroleukemia cells results in a proliferation defect with minimal effect on erythroid differentiation potential, suggesting the mechanism of anemia in this disorder.

CD4(+)CD25(+)FoxP3(+) thymic-derived regulatory T cells (tTregs) are indispensable for maintaining immune system equilibrium. Adoptive transfer of tTregs is an effective means of suppressing graft-versus-host disease (GVHD) in murine models and in early human clinical trials. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an ubiquitin-conjugating enzyme that mediates nuclear factor κB (NF-κB) activation, plays an essential role in modulating regulatory T cell survival and function. MicroRNAs (miRNAs) are noncoding RNAs, which mediate RNA silencing and posttranscriptional gene repression. By performing comprehensive TaqMan Low Density Array miRNA assays, we identified 10 miRNAs differentially regulated in human tTreg compared with control T cells. One candidate, miR-146b, is preferentially and highly expressed in human naive tTregs compared with naive CD4 T cells. miRNA prediction software revealed that TRAF6 was the one of the top 10 scored mRNAs involved tTreg function with the highest probability as a potential miR-146b target. Antagomir-mediated knockdown of miRNA-146b, but not another miRNA-146 family member (miRNA-146a), enhanced TRAF6 expression. TRAF6, in turn, increases NF-κB activation, which is essential for tTreg function as well as Foxp3 protein and antiapoptotic gene expression, and downregulates proapoptotic gene expression. miR-146b knockdown increased the nuclear localization and expression of genes regulated by NF-κB, which was associated with enhanced tTreg survival, proliferation, and suppressive function measured in vitro and in vivo. TRAF6 inhibition had the opposite effects. We conclude that an miR-146b-TRAF6-NF-κB-FoxP3 signaling pathway restrains regulatory T cell survival, proliferation, and suppressor function. In vitro exposure of human tTregs to miR-146b antagomirs can be exploited to improve the clinical efficacy of human adoptive tTreg transfer in a GVHD setting.

Despite advances in the treatment of acute myeloid leukemia (AML), relapse and drug resistance frequently occur. Therefore, detailed mechanisms of refractoriness, including leukemia-initiating cell (LIC) biology, should be elucidated to treat AML. The self-degradative property of cytosolic macromolecules is central to autophagy and can contribute to homeostasis and stress response. Recent reports suggest the importance of autophagy in hematopoietic stem cells and various tumors. Thus, this study investigated the functional role of autophagy in AML maintenance and drug resistance using tamoxifen-inducible conditional knockout mice of Atg5 or Atg7, which are essential genes for autophagy, combined with an mixed lineage leukemia-eleven nineteen leukemia-induced murine AML model. Inactivation of autophagy by deletion of Atg5 or Atg7 prolonged survival in leukemic mice and reduced functional LICs. Atg7-deficient LICs displayed enhanced mitochondrial activity and reactive oxygen species production together with increased cell death. In addition, Atg7 deletion markedly decreased peripheral blood leukemia cells, concurrent with increased apoptosis, suggesting a higher dependency on autophagy compared with bone marrow leukemia cells. Finally, cytarabine (AraC) treatment activated autophagy in LICs, and Atg7 deletion potentiated the therapeutic effects of AraC, which included decreased LICs and prolonged survival, suggesting that autophagy contributes to AraC resistance. Our results highlight the intratumoral heterogeneity related to autophagy in AML and the unique role of autophagy in leukemia development and drug resistance.

Agents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4(dim)CD5(bright) subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4(bright)CD5(dim) cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α-mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies.

The mechanisms regulating the sequential steps of terminal erythroid differentiation remain largely undefined, yet are relevant to human anemias that are characterized by ineffective red cell production. Erythroid Krüppel-like Factor (EKLF/KLF1) is a master transcriptional regulator of erythropoiesis that is mutated in a subset of these anemias. Although EKLF's function during early erythropoiesis is well studied, its role during terminal differentiation has been difficult to functionally investigate due to the impaired expression of relevant cell surface markers in Eklf(-/-) erythroid cells. We have circumvented this problem by an innovative use of imaging flow cytometry to investigate the role of EKLF in vivo and have performed functional studies using an ex vivo culture system that enriches for terminally differentiating cells. We precisely define a previously undescribed block during late terminal differentiation at the orthochromatic erythroblast stage for Eklf(-/-) cells that proceed beyond the initial stall at the progenitor stage. These cells efficiently decrease cell size, condense their nucleus, and undergo nuclear polarization; however, they display a near absence of enucleation. These late-stage Eklf(-/-) cells continue to cycle due to low-level expression of p18 and p27, a new direct target of EKLF. Surprisingly, both cell cycle and enucleation deficits are rescued by epistatic reintroduction of either of these 2 EKLF target cell cycle inhibitors. We conclude that the cell cycle as regulated by EKLF during late stages of differentiation is inherently critical for enucleation of erythroid precursors, thereby demonstrating a direct functional relationship between cell cycle exit and nuclear expulsion.

Pediatric lower extremity deep vein thrombosis (LE-DVT) can lead to postthrombotic syndrome (PTS) and other adverse events. We investigated the outcomes of LE-DVT in children. Three groups were compared: non-line-related (Non-LR) DVT, LR DVT in neonates (LRneonates), and LR DVT in non-neonates (LRnon-neonates). A total of 339 children were included (Non-LR, n = 56; LRneonates, n = 95; and LRnon-neonates, n = 188). We found a statistically significant difference in the frequency of PTS (P = .04; 62.5%, 40.0%, and 46.3% in Non-LR, LRneonates, and LRnon-neonates, respectively), of recurrent LE-DVT (P = .001; 10.7% and 2.0% in Non-LR and LRnon-neonates, respectively), and pulmonary embolism (PE) (P < .001; 19.6% and 3.2% in Non-LR and LRnon-neonates, respectively) among groups. There was no difference in DVT resolution (P = .41). Multivariable analysis showed that DVT resolution, triggering event, and sex predicted Modified Villalta Scale (MVS; for pediatric PTS) scores >1; there was an interaction between DVT triggering event and sex. The time to reach an MVS >1 was significantly different when comparing groups (log-rank test, P < .001). Moreover, we found a significant difference in baseline MVS scores among groups, but the difference did not appear to change over time. In conclusion, LR LE-DVT had more benign outcomes than Non-LR DVT. Sex, DVT triggering event, and DVT resolution predicted LE-PTS in our cohort.