A single-institution pre-post comparison of subcutaneous immunoglobulin replacement therapy in allogeneic haematopoietic cell transplantation recipients.

Immunoglobulin replacement therapy (IgRT) reduces the risk of infection in hypogammaglobulinaemia secondary to chronic lymphocytic leukaemia and multiple myeloma. However, the benefit of IgRT, especially subcutaneous IgRT (ScIgRT), has not been assessed in hypogammaglobulinaemia after allogeneic haematopoietic cell transplantation (allo-HCT). We performed a pre-post comparison of the clinical impact of ScIgRT after allo-HCT in a retrospective analysis of 209 patients who underwent allogeneic HCT at our institution from 2011 to 2019. Since ScIgRT became available at our institution in April 2017, we categorized patients treated from January 2011 to March 2017 as the Pre-ScIgRT group (n = 118) and those treated from April 2017 to December 2019 as the Post-ScIgRT group (n = 91). The 2-year overall survival rate was 65% in the Pre-ScIgRT group and 81% in the Post-ScIgRT group (p = 0.02). The cumulative incidence (CI) of non-relapse mortality at 2 years was 18% and 7% (p = 0.02). There were 78 infectious events in 44 patients in the Pre-ScIgRT group and 28 such events in 19 patients in the Post-ScIgRT group. The CI of the documented infection during the observation period was between 38% and 21% (p = 0.01). Our study suggests that ScIgRT may reduce infection rates and improve prognosis after allo-HCT.

Special AT-Rich Sequence-Binding Protein 1 Supports Survival and Maturation of Naive B Cells Stimulated by B Cell Receptors.

Epigenetic mechanisms underpin the elaborate activities of essential transcription factors in lymphocyte development. Special AT-rich sequence-binding protein 1 (SATB1) is a chromatin remodeler that orchestrates the spatial and temporal actions of transcription factors. Previous studies have revealed the significance of SATB1 in T cell lineage. However, whether and how SATB1 controls B cell lineage development is yet to be clarified. In this study, we show that SATB1 is an important factor during splenic B cell maturation. By analyzing SATB1/Tomato reporter mice, we determined the dynamic fluctuation of SATB1 expression in the B cell lineage. Although SATB1 expression decreased to minimal levels during B cell differentiation in the bone marrow, it resurged markedly in naive B cells in the spleen. The expression was dramatically downregulated upon Ag-induced activation. Splenic naive B cells were subdivided into two categories, namely SATB1high and SATB1-/low, according to their SATB1 expression levels. SATB1high naive B cells were less susceptible to death and greater proliferative than were SATB1-/low cells during incubation with an anti-IgM Ab. Additionally, SATB1high cells tended to induce the expression of MHC class II, CD86, and CD83. Accordingly, naive B cells from B lineage-specific SATB1 conditional knockout mice were more susceptible to apoptosis than that in the control group upon anti-IgM Ab stimulation in vitro. Furthermore, conditional knockout mice were less capable of producing Ag-specific B cells after immunization. Collectively, our findings suggest that SATB1 expression increases in naive B cells and plays an important role in their survival and maturation.

Prolonged gut microbial alterations in post-transplant survivors of allogeneic haematopoietic stem cell transplantation.

Dysbiosis of the gut microbiota has been reported to increase early complications after allogeneic haematopoietic stem cell transplantation (allo-HSCT). However, it remains unclear whether gut microbial alterations persist during late complications, such as chronic graft-versus-host disease (cGVHD) or secondary cancers. Here, we analysed the gut microbiota of 59 patients who survived for 1-21.7 years (median, 6.4 years) after allo-HSCT. Long-term survivors showed lower gut microbial diversity than the age- and sex-matched healthy controls. This decreased diversity was reflected in the reduced abundance of the butyrate-producing bacteria. Patients with a history of grade 3 acute graft-versus-host disease (aGVHD) exhibited higher Veillonella abundance than patients with a history of grade 1-2 or non-aGVHD cases. The abundance of Faecalibacterium showed no decrease only in limited cGVHD cases. Additionally, the microbial structure in the secondary cancer group was significantly different (p < 0.05) from that in the non-secondary cancer group. This study is the first to show that microbial dysbiosis is present over a 10-year lifetime after discharge following allo-HSCT. Our results suggest that these prolonged gut microbial alterations may be associated with the development and exacerbation of late complications in post-transplant survivors.

Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation.

B cells can suppress autoimmunity by secreting interleukin-10 (IL-10). Although subpopulations of splenic B lineage cells are reported to express IL-10 in vitro, the identity of IL-10-producing B cells with regulatory function in vivo remains unknown. By using IL-10 reporter mice, we found that plasmablasts in the draining lymph nodes (dLNs), but not splenic B lineage cells, predominantly expressed IL-10 during experimental autoimmune encephalomyelitis (EAE). These plasmablasts were generated only during EAE inflammation. Mice lacking plasmablasts by genetic ablation of the transcription factors Blimp1 or IRF4 in B lineage cells developed an exacerbated EAE. Furthermore, IRF4 positively regulated IL-10 production that can inhibit dendritic cell functions to generate pathogenic T cells. Our data demonstrate that plasmablasts in the dLNs serve as IL-10 producers to limit autoimmune inflammation and emphasize the importance of plasmablasts as IL-10-producing regulatory B cells.

[Acquired hemophilia A following BNT162b2 mRNA COVID-19 vaccination].

Acquired hemophilia A (AHA) is a rare disease characteized by bleeding symptoms caused by decreased factor VIII activity due to the appearance of inhibitors to factor VIII triggered by malignancy or collagen disease. An 86-year-old woman developed purpura on her extremities after the first dose of the BNT162b2 mRNA COVID-19 vaccine. This symptom subsided after a few days. After the second dose of the BNT162b2 mRNA COVID-19 vaccine, purpura appeared again, and the patient was referred to our hospital Her APTT was remarkably prolonged to 110 seconds, and a cross-mixing test revealed an inhibitor pattern. Since FVIII activity was <1% and FVIII inhibitor was 51.6 BU, she was diagnosed with AHA. Prednisolone therapy was started, and coagulative complete remission was achieved. Because acquired hemophilia can develop after mRNA COVID-19 vaccination, as in this case, it is critical to monitor the appearance of bleeding symptom.

Impaired B cell terminal differentiation in B cell-specific knockout mice of cell death-defying factor anamorsin.

Anamorsin (AM) is an anti-apoptotic molecule cloned by us as a molecule that confers resistance against apoptosis induced by growth factor deprivation. AM-deficient mice are embryonic lethal, which impedes detailed analyses of the roles of AM in various types of adult cells. To overcome the embryonic lethality, we generated AM conditional knockout (AMflox/flox) mice and cell type-specific genetic modification became possible using the Cre-loxP system. CD19-Cre/AMflox/flox mice with AM deleted specifically in CD19+ B cells exhibited less B220+ B cells in their spleen, peripheral blood, and lymph node compared with control CD19-Cre mice. Using flow cytometry to categorize bone marrow and spleen cells into B cell subsets, we observed significantly less follicular type I cells, which are the most mature follicular B cells, compared with control CD19-Cre mice. These data suggest that AM has an important role in the generation of mature B cells.

Autonomous TGFß signaling induces phenotypic variation in human acute myeloid leukemia.

Heterogeneity of leukemia stem cells (LSCs) is involved in their collective chemoresistance. To eradicate LSCs, it is necessary to understand the mechanisms underlying their heterogeneity. Here, we aimed to identify signals responsible for heterogeneity and variation of LSCs in human acute myeloid leukemia (AML). Monitoring expression levels of endothelial cell-selective adhesion molecule (ESAM), a hematopoietic stem cell-related marker, was useful to detect the plasticity of AML cells. While healthy human hematopoietic stem/progenitor cells robustly expressed ESAM, AML cells exhibited heterogeneous ESAM expression. Interestingly, ESAM- and ESAM+ leukemia cells obtained from AML patients were mutually interconvertible in culture. KG1a and CMK, human AML clones, also represented the heterogeneity in terms of ESAM expression. Single cell culture with ESAM- or ESAM+ AML clones recapitulated the phenotypic interconversion. The phenotypic alteration was regulated at the gene expression level, and RNA sequencing revealed activation of TGFß signaling in these cells. AML cells secreted TGFß1, which autonomously activated TGFß pathway and induced their phenotypic variation. Surprisingly, TGFß signaling blockade inhibited not only the variation but also the proliferation of AML cells. Therefore, autonomous activation of TGFß signaling underlies the LSC heterogeneity, which may be a promising therapeutic target for AML.

The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages.

How hematopoietic stem cells (HSCs) produce particular lineages is insufficiently understood. We searched for key factors that direct HSC to lymphopoiesis. Comparing gene expression profiles for HSCs and early lymphoid progenitors revealed that Satb1, a global chromatin regulator, was markedly induced with lymphoid lineage specification. HSCs from Satb1-deficient mice were defective in lymphopoietic activity in culture and failed to reconstitute T lymphopoiesis in wild-type recipients. Furthermore, Satb1 transduction of HSCs and embryonic stem cells robustly promoted their differentiation toward lymphocytes. Whereas genes that encode Ikaros, E2A, and Notch1 were unaffected, many genes involved in lineage decisions were regulated by Satb1. Satb1 expression was reduced in aged HSCs with compromised lymphopoietic potential, but forced Satb1 expression partly restored that potential. Thus, Satb1 governs the initiating process central to the replenishing of lymphoid lineages. Such activity in lymphoid cell generation may be of clinical importance and useful to overcome immunosenescence.

[Exploring new molecules that regulate hematopoietic stem cells and early stages of lymphoid hematopoiesis: the functional significance of ESAM and SATB1].

Hematopoietic stem cells (HSCs) possess multilineage differentiation capability, which sustains the production of blood and immune cells throughout life. However, the precise mechanisms by which HSCs initiate differentiation toward a particular lineage and the factors that attenuate their lymphopoietic potential with aging are yet to be elucidated. Our group has investigated this issue for over two decades. We initially developed a method for segregating early lymphoid progenitors from HSCs and identified two molecules: endothelial cell-selective adhesion molecule (ESAM), highly expressed in HSCs, and special AT-rich sequence binding protein 1 (SATB1), expressed in early lymphoid progenitors. ESAM marks HSCs across species, including humans. In addition to its significance in stress-induced hematopoiesis, ESAM is also useful in identifying features of human acute myeloid leukemia stem cells. Further, we determined the role of SATB1 in the early HSC differentiation processes toward the lymphoid lineage. Remarkably, SATB1 expression in HSCs significantly decreased with aging, whereas its exogenous induction in aged HSCs rejuvenated their lymphopoietic potential. Furthermore, SATB1-expressing HSCs demonstrated robust lymphopoietic and long-term reconstituting capability, whereas HSCs without SATB1 skewed toward the myeloid lineage. Thus, our continuing research has revealed the significance of ESAM and SATB1 in the fundamental biology of HSCs.

Graft-versus-host disease develops in mice transplanted with lymphocyte-depleted bone marrow cells from signal-transducing adaptor protein-2 transgenic mice.

Graft-versus-host disease (GVHD) is the most frequent complication after allogeneic hematopoietic stem cell transplantation (HSCT), and is one of the major causes of non-relapse mortality. Transferred mature lymphocytes are thought to be responsible for GVHD based on the findings that mice transplanted with lymphocyte-depleted bone marrow (BM) cells from MHC-mismatched donors do not develop GVHD. However, we found that overexpression of signal-transducing adaptor protein (STAP)-2 in lymphoid cells could induce GVHD after lymphocyte-depleted BM transplantation. To examine the function of STAP-2, which has been shown to play an important role in development and function of lymphocytes, in GVHD, we transplanted BM cells from STAP-2 deficient, or Lck promoter/IgH enhancer-driven STAP-2 transgenic (Tg) mice into MHC-mismatched recipients. Unexpectedly, mice transplanted with lymphocyte-depleted BM cells from STAP-2 Tg mice developed severe acute GVHD with extensive colitis and atrophy of thymus, while no obvious GVHD developed in mice transplanted with the wild type or STAP-2 deficient graft. Furthermore, mice transplanted with lymphocyte-depleted BM cells from the syngeneic STAP-2 Tg mice developed modest GVHD with colitis and atrophy of thymus. These results suggest that STAP-2 overexpression may enhance survival of allo-, and even auto-, reactive lymphocytes derived from engrafted hematopoietic progenitor cells in lethally irradiated mice, and that clarification of the mechanism may help understanding induction of immune tolerance after HSCT.

Enterococcus: A Predictor of Ravaged Microbiota and Poor Prognosis after Allogeneic Hematopoietic Stem Cell Transplantation.

Intestinal flora plays an essential role in regulating immune responses. Changes in the gut flora are associated with poor prognosis after allogeneic hematopoietic stem cell transplantation (HSCT). We aimed to investigate the impact of diverse intestinal flora on survival after allogeneic HSCT. Using next-generation sequencing of the bacterial 16S ribosomal RNA (rRNA) gene, we found that the intestinal microbiota of patients undergoing allogeneic HSCT differed significantly from that of healthy controls. Furthermore, dysbiosis persisted for at least 1 year after transplantation. Interestingly, increased abundance of the genus Enterococcus detected by 16S rRNA sequencing as early as 1 month after transplantation was correlated with poor survival (overall survival at 2 years post-HSCT, 83.9% for patients with <1% relative abundance of Enterococcus and 47.6% for those with ≥1% relative abundance of Enterococcus), which was undetectable by conventional standard stool culture. These findings suggest that detection of Enterococcus by 16S rRNA analysis reflects compromised intestinal flora and may be a promising prognostic indicator.

Pre- and post-serial metagenomic analysis of gut microbiota as a prognostic factor in patients undergoing haematopoietic stem cell transplantation.

The human gut harbours diverse microorganisms, and gut dysbiosis has recently attracted attention because of its possible involvement in various diseases. In particular, the lack of diversity in the gut microbiota has been associated with complications of haematopoietic stem cell transplantation (HSCT), such as infections, acute graft-versus-host disease and relapse of primary disease, which lead to a poor prognosis. However, few studies have serially examined the composition of the intestinal microbiota after HSCT. In this study, we demonstrated, using next-generation sequencing of the bacterial 16S ribosomal RNA gene, combined with uniFrac distance analysis, that the intestinal microbiota of patients undergoing allogeneic HSCT substantially differed from that of healthy controls and recipients of autologous transplants. Faecal samples were obtained daily throughout the clinical course, before and after transplantation. Notably, the proportions of Bifidobacterium and genera categorized as butyrate-producing bacteria were significantly lower in patients with allogeneic HSCT than in healthy controls. Furthermore, among allogeneic transplant recipients, a subgroup with a preserved microbiota composition showed a benign course, whereas patients with a skewed microbiota showed a high frequency of complications and mortality after transplantation. Thus, we conclude that the stability of intestinal microbiota is critically involved in outcomes of HSCT.

[Processes regulating early lymphocyte differentiation].

Lymphocytes play pivotal roles in innate and adaptive immunity. The differentiation process by which hematopoietic stem cells (HSCs) acquire specific functions has been extensively investigated and is considered the paradigm of cell differentiation. It has been widely accepted that highly enriched HSCs are heterogeneous with respect to their lymphopoietic potential, and aged or stressed HSCs are skewed to the myeloid lineage. Several transcription factors and cytokine signaling pathways have been reported as essential to lymphocyte differentiation. However, the molecular mechanism that modulates the earliest stage remains unclear. Furthermore, the origin and characteristics of early T-lymphoid progenitors that migrate from the bone marrow to the thymus are still unknown in this field. Epigenetic mechanisms likely influence early lineage specification through the regulation of mitochondrial function and modification of nuclear chromatin structure. This review summarizes previous and recent findings on the processes involved in early lymphocyte differentiation. Thus, it provides a foundation for the understanding of the physiology of HSC aging and the pathology of intractable acute lymphocytic leukemia.

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis.

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34+CD38+CD33+ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3+ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.

Genetic abnormalities associated with acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) occurs with high frequency in childhood and is associated with high mortality in adults. Recent technical advances in next-generation sequencing have shed light on genetic abnormalities in hematopoietic stem/progenitor cells as the precursor to ALL pathogenesis. Based on these genetic abnormalities, ALL is now being reclassified into newly identified subtypes. Philadelphia chromosome-like B-lineage ALL is one of the new high-risk subtypes characterized by genetic alterations that activate various signaling pathways, including those involving cytokine receptors, tyrosine kinases, and epigenetic modifiers. Philadelphia chromosome-like ALL is essentially heterogeneous; however, deletion mutations in the IKZF1 gene encoding the transcription factor IKAROS underlie many cases as a key factor inducing aggressive phenotypes and poor treatment responses. Whole-genome sequencing studies of ALL patients and ethnically matched controls also identified inherited genetic variations in lymphoid neoplasm-related genes, which are likely to increase ALL susceptibility. These findings are directly relevant to clinical hematology, and further studies on this aspect could contribute to accurate diagnosis, effective monitoring of residual disease, and patient-oriented therapies.

Estrogen-inducible sFRP5 inhibits early B-lymphopoiesis in vivo, but not during pregnancy.

Mammals have evolved to protect their offspring during early fetal development. Elaborated mechanisms induce tolerance in the maternal immune system for the fetus. Female hormones, mainly estrogen, play a role in suppressing maternal lymphopoiesis. However, the molecular mechanisms involved in the maternal immune tolerance are largely unknown. Here, we show that estrogen-induced soluble Frizzled-related proteins (sFRPs), and particularly sFRP5, suppress B-lymphopoiesis in vivo in transgenic mice. Mice overexpressing sFRP5 had fewer B-lymphocytes in the peripheral blood and spleen. High levels of sFRP5 inhibited early B-cell differentiation in the bone marrow (BM), resulting in the accumulation of cells with a common lymphoid progenitor (CLP) phenotype. Conversely, sFRP5 deficiency reduced the number of hematopoietic stem cells (HSCs) and primitive lymphoid progenitors in the BM, particularly when estrogen was administered. Furthermore, a significant reduction in CLPs and B-lineage-committed progenitors was observed in the BM of sfrp5-null pregnant females. We concluded that, although high sFRP5 expression inhibits B-lymphopoiesis in vivo, physiologically, it contributes to the preservation of very primitive lymphopoietic progenitors, including HSCs, under high estrogen levels. Thus, sFRP5 regulates early lympho-hematopoiesis in the maternal BM, but the maternal-fetal immune tolerance still involves other molecular mechanisms that remain to be uncovered.

Signal-transducing adaptor protein-2 regulates macrophage migration into inflammatory sites during dextran sodium sulfate induced colitis.

Signal-transducing adaptor protein-2 (STAP-2) was cloned as a c-fms/M-CSF receptor interacting protein. STAP-2 is an adaptor protein carrying pleckstrin homology and Src homology 2 like domains, as well as a YXXQ motif. STAP-2 has been indicated to have an ability to bind and modulate a variety of signaling and transcriptional molecules. Especially, our previous in vitro studies showed that STAP-2 is crucial for immune and/or inflammatory responses. Here, we have investigated the role of STAP-2 in intestinal inflammation in vivo. The disruption of STAP-2 attenuates dextran sodium sulfate induced colitis via inhibition of macrophage recruitment. To study whether hematopoietic or epithelial cell derived STAP-2 is required for this phenomenon, we generated BM chimeric mice. STAP-2-deficient macrophages impair the ability of CXCL12-induced migration. Intriguingly, STAP-2 also regulates production of proinflammatory chemokines and cytokines such as CXCL1 and TNF-α from intestinal epithelial cells. Therefore, STAP-2 has a potential to regulate plural molecular events during pathological inflammatory responses. Furthermore, our findings not only indicate that STAP-2 is important in regulating intestinal inflammation, but also provide new insights toward the development of novel therapeutic approaches.

Role of endothelial antigen ESAM in activated hematopoietic stem cells.

Through their differentiation and self-renewal capabilities, hematopoietic stem cells (HSCs) supply the vast amounts of blood cells needed throughout life. The use of surface markers to purify HSCs has been promoted. After bone marrow injury, however, HSCs are activated, and the expression pattern of HSC-related antigens changes dramatically. Endothelial cell-selective adhesion molecule (ESAM), an immunoglobulin superfamily protein, was originally identified as an endothelium-specific molecule. We recently reported ESAM as a new HSC marker. In the present study, we demonstrate the monitoring of ESAM levels to provide a useful indicator of HSC activation. Expression levels of ESAM clearly mirrored shifts in HSC status between quiescence and activation, and the shifts were more prominent than those of other HSC-related antigens. ESAMHi HSCs were active in the cell cycle while maintaining a high capacity for long-term reconstitution. More than 80% of ESAMHi HSCs were located near endothelium in the bone marrow after 5-FU treatment. Furthermore, ESAM is functionally important for HSCs to re-establish homeostatic hematopoiesis. Our data demonstrate ESAM expression levels to be useful for monitoring HSC status, tracing the proliferation of HSCs in vivo and understanding the molecular events underlying HSC activation.

The endothelial antigen ESAM monitors hematopoietic stem cell status between quiescence and self-renewal.

Whereas most hematopoietic stem cells (HSC) are quiescent in homeostasis, they actively proliferate in response to bone marrow (BM) injury. Signals from the BM microenvironment are thought to promote entry of HSC into the cell cycle. However, it has been cumbersome to assess cycle status of viable HSC and thus explore unique features associated with division. In this study, we show that expression of endothelial cell-selective adhesion molecule (ESAM) can be a powerful indicator of HSC activation. ESAM levels clearly mirrored the shift of HSC between quiescence and activation, and it was prominent in comparison with other HSC-related Ags. ESAM(hi) HSC were actively dividing, but had surprisingly high long-term reconstituting capacity. Immunohistochemical analyses showed that most ESAM(hi) HSC were located near vascular endothelium in the BM after 5-fluorouracil treatment. To determine the importance of ESAM in the process of BM recovery, ESAM knockout mice were treated with 5-fluorouracil and their hematopoietic reconstruction was examined. The ESAM deficiency caused severe and prolonged BM suppression, suggesting that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis. With respect to intracellular regulators, NF-κB and topoisomerase II levels correlated with the ESAM upregulation. Thus, our data demonstrate that the intensity of ESAM expression is useful to trace activated HSC and to understand molecular events involved in stem cell states.