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Immunotherapy has shown efficacy in relapsed multiple myeloma (MM). However, these therapies may depend on a functional tumor immune microenvironment (iTME) for their efficacy. Characterizing the evolution of the iTME over the disease course is necessary to optimize the timing of immunotherapies. We performed mass cytometry, cytokine analysis, and RNA sequencing on bone marrow samples from 39 (13 newly diagnosed [NDMM], 11 relapsed pre-daratumumab exposure [RMM], and 13 triple-refractory [TRMM]) MM patients. Three distinct cellular iTME clusters were identified; cluster 1 comprised mainly of NDMM and RMM patients; and clusters 2 and 3 comprised primarily of TRMM patients. We showed that naive T cells were decreased in clusters 2 and 3, cluster 2 was characterized by increased senescent T cells, and cluster 3 by decreased early memory T cells. Plasma cells in clusters 2 and 3 upregulated E2F transcription factors and MYC proliferation pathways, and downregulated interferon, TGF-beta, interleuking-6, and TNF-αlpha signaling pathways compared to cluster 1. This study suggests that the MM iTME becomes increasingly dysfunctional with therapy whereas the MM clone may be less dependent on inflammation-mediated growth pathways and less sensitive to IFN-mediated immunosurveillance. Our findings may explain the decreased sensitivity of TRMM patients to novel immunotherapies.

MicroRNAs (miRNAs) post-transcriptionally repress almost all genes in mammals and thereby form an additional layer of gene regulation. As such, miRNAs impact on nearly every physiological process and have also been associated with cancer. Prominent examples of such miRNAs can be found in the miR-15 family, composed of the bicistronic clusters miR-15a/16-1, miR-15b/16-2, and miR-497/195. In particular, the miR-15a/16-1 cluster is deleted in almost two thirds of all chronic B lymphocytic leukemia (CLL) cases, a phenotype that is also recapitulated by miR-15a/16-1-deficient as well as miR-15b/16-2-deficient mice. Under physiological conditions, those two clusters have been implicated in T-cell function, and B-cell and natural killer (NK) cell development; however, it is unclear whether miR-497 and miR-195 confer similar roles in health and disease. Here, we have generated a conditional mouse model for tissue-specific deletion of miR-497 and miR-195. While mice lacking miR-15a/16-1 in the hematopoietic compartment developed clear signs of CLL over time, aging mice deficient for miR-497/195 did not show such a phenotype. Likewise, loss of miR-15a/16-1 impaired NK and early B-cell development, whereas miR-497/195 was dispensable for these processes. In fact, a detailed analysis of miR-497/195-deficient mice did not reveal any effect on steady-state hematopoiesis or immune cell function. Unexpectedly, even whole-body deletion of the cluster was well-tolerated and had no obvious impact on embryonic development or healthy life span. Therefore, we postulate that the miR-497/195 cluster is redundant to its paralog clusters or that its functional relevance is restricted to certain physiological and pathological conditions., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Multiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported., Methods: This study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment., Results: Using the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8 + T cell numbers; (ii) activated NK cells and CD8 + T cells and (iii) upregulated effector-memory CD8 + T cells. Moreover, increased effector-memory CD8 + T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes., Conclusion: These data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority., Competing Interests: Competing interests: MC is affiliated with Oncolytics Biotec Inc., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Multiple myeloma (MM), a malignant neoplasm of plasma cells that reside in the bone marrow (BM), is universally preceded by a precursor state termed monoclonal gammopathy of undetermined significance (MGUS). Many individuals with MGUS never progress to MM or progress over many years. Therefore, MGUS provides a unique opportunity to surveil changes in the BM tumor microenvironment throughout disease progression. It is increasingly appreciated that MGUS cells carry many of the genetic changes found in MM. Prior studies have also shown that MGUS cells can be recognized by the immune system, leading to early changes in the BM immune environment compared to that of healthy individuals, including alterations in both innate and adaptive immunity. Progression to clinical MM is associated with attrition of T cells with stem memory-like features and instead accumulation of T cells with more terminally differentiated features. Recent clinical studies have suggested that early application of immune-modulatory drugs, which are known to activate both innate and adaptive immunity, can delay the progression to clinical MM. Understanding the biology of how the immune response and tumors coevolve over time is needed to develop novel immune-based approaches to achieve durable and effective prevention of clinical malignancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 McCachren, Dhodapkar and Dhodapkar.)

CD4 expression identifies a subset of mature T cells primarily assisting the germinal center reaction and contributing to CD8+ T-cell and B-cell activation, functions, and longevity. Herein, we present a family in which a novel variant disrupting the translation-initiation codon of the CD4 gene resulted in complete loss of membrane and plasma soluble CD4 in peripheral blood, lymph node, bone marrow, skin, and ileum of a homozygous proband. This inherited CD4 knockout disease illustrates the clinical and immunological features of a complete deficiency of any functional component of CD4 and its similarities and differences with other clinical models of primary or acquired loss of CD4+ T cells. The first inherited loss of any functional component of CD4, including soluble CD4, is clinically distinct from any other congenital or acquired CD4 T-cell defect and characterized by compensatory changes in T-cell subsets and functional impairment of B cells, monocytes, and natural killer cells., (Published by Oxford University Press for the Infectious Diseases Society of America 2021.)

Hemorrhagic shock induces an aberrant immune response characterized by simultaneous induction of a proinflammatory state and impaired host defenses. The objective of this study was to evaluate the impact of conditionally immortalized neutrophil progenitors (NPs) on this aberrant immune response. We employed a mouse model of hemorrhagic shock, followed by the adoptive transfer of NPs and subsequent inoculation of Staphylococcus aureus to induce pneumonia. We observed that transplant of NPs decreases the proportion of host neutrophils that express programmed death ligand 1 and intercellular adhesion molecule 1 in the context of prior hemorrhage. Following hemorrhage, NP transplant decreased proinflammatory cytokines in the lungs, increased neutrophil migration into the airspaces, and enhanced bacterial clearance. Further, hemorrhagic shock improved NP engraftment in the bone marrow. These results suggest that NPs hold the potential for use as a cellular therapy in the treatment and prevention of secondary infection following hemorrhagic shock., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: In coronavirus disease 2019 (COVID-19), the adaptive immune response is of considerable importance, and detailed cellular immune reactions in the hematological system of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are yet to be clarified., Methods: This study reports the morphological characterization of both bone marrow and spleen in 11 COVID-19 decedents with respect to findings in the peripheral blood and pulmonary SARS-CoV-2 burden., Results: In the bone marrow, activation and left shift were found in at least 55% of patients, which was mirrored by peripheral anaemia, granulocytic immaturity and multiple thromboembolic events. Signs of sepsis-acquired immunodeficiency were found in the setting of an abscess-forming superinfection of viral COVID-19 pneumonia. Furthermore, a severe B cell loss was observed in the bone marrow and/or spleen in 64% of COVID-19 patients. This was reflected by lymphocytopenia in the peripheral blood. As compared to B cell preservation, B cell loss was associated with a higher pulmonary SARS-CoV-2 burden and only a marginal decrease of of T cell counts., Conclusions: The results of this study suggest the presence of sepsis-related immunodeficiency in severe COVID-19 pneumonia with superinfection. Furthermore, our findings indicate that lymphocytopenia in COVID-19 is accompanied by B cell depletion in hematopoietic tissue, which might impede the durability of the humoral immune response to SARS-CoV-2., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CD19-directed chimeric antigen receptors (CAR) T cells induce impressive rates of complete response in advanced B-cell malignancies, specially in B-cell acute lymphoblastic leukemia (B-ALL). However, CAR T-cell-treated patients eventually progress due to poor CAR T-cell persistence and/or disease relapse. The bone marrow (BM) is the primary location for acute leukemia. The rapid/efficient colonization of the BM by systemically infused CD19-CAR T cells might enhance CAR T-cell activity and persistence, thus, offering clinical benefits. Circulating cells traffic to BM upon binding of tetrasaccharide sialyl-Lewis X (sLeX)-decorated E-selectin ligands (sialofucosylated) to the E-selectin receptor expressed in the vascular endothelium. sLeX-installation in E-selectin ligands is achieved through an ex vivo fucosylation reaction. Here, we sought to characterize the basal and cell-autonomous display of sLeX in CAR T-cells activated using different cytokines, and to assess whether exofucosylation of E-selectin ligands improves CD19-CAR T-cell activity and BM homing. We report that cell-autonomous sialofucosylation (sLeX display) steadily increases in culture- and in vivo-expanded CAR T cells, and that, the cytokines used during T-cell activation influence both the degree of such endogenous sialofucosylation and the CD19-CAR T-cell efficacy and persistence in vivo. However, glycoengineered enforced sialofucosylation of E-selectin ligands was dispensable for CD19-CAR T-cell activity and BM homing in multiple xenograft models regardless the cytokines employed for T-cell expansion, thus, representing a dispensable strategy for CD19-CAR T-cell therapy., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Transcription factor (TF) reporter mice have proved integral to the characterization of murine innate lymphoid cell (ILC) development and function. Here, we implemented a CRISPR/Cas9-generated combinatorial reporter approach for the simultaneous resolution of several key TFs throughout ILC development in both the fetal liver and adult bone marrow. We demonstrate that the Tcf7-expressing early innate lymphoid precursor (EILP) and the common helper ILC precursor (CHILP) both contain a heterogeneous mixture of specified ILC and lymphoid tissue inducer (LTi) precursors with restricted lineage potential rather than a shared precursor. Moreover, the earliest specified precursor to the LTi lineage was identified upstream of these populations, before Tcf7 expression. These findings match dynamic changes in chromatin accessibility associated with the expression of key TFs (i.e., GATA3 and RORγ(t)), highlighting the distinct origins of ILC and LTi lineages at the epigenetic and functional levels, and provide a revised map for ILC development., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Kasal and Bendelac.)

Hematopoietic stem/progenitor cells (HSPCs) participate in cardiovascular (CV) homeostasis and generate different types of blood cells including lymphoid and myeloid cells. Diabetes mellitus (DM) is characterized by chronic increase of pro-inflammatory mediators, which play an important role in the development of CV disease, and increased susceptibility to infections. Here, we aimed to evaluate the impact of DM on the transcriptional profile of HSPCs derived from bone marrow (BM). Total RNA of BM-derived CD34 + stem cells purified from sternal biopsies of patients undergoing coronary bypass surgery with or without DM (CAD and CAD-DM patients) was sequenced. The results evidenced 10566 expressed genes whose 79% were protein-coding genes, and 21% non-coding RNA. We identified 139 differentially expressed genes ( p -value < 0.05 and |log2 FC| > 0.5) between the two comparing groups of CAD and CAD-DM patients. Gene Set Enrichment Analysis (GSEA), based on Gene Ontology biological processes (GO-BP) terms, led to the identification of fourteen overrepresented biological categories in CAD-DM samples. Most of the biological processes were related to lymphocyte activation, chemotaxis, peptidase activity, and innate immune response. Specifically, HSPCs from CAD-DM patients displayed reduced expression of genes coding for proteins regulating antibacterial and antivirus host defense as well as macrophage differentiation and lymphocyte emigration, proliferation, and differentiation. However, within the same biological processes, a consistent number of inflammatory genes coding for chemokines and cytokines were up-regulated. Our findings suggest that DM induces transcriptional alterations in HSPCs, which are potentially responsible of progeny dysfunction.

Background: This study investigates the effect of autologous bone marrow transfusion (BMT) on the reconstruction of both bone marrow and the immune system in patients with AIDS-related lymphoma (ARL)., Methods: A total of 32 patients with ARL participated in this study. Among them, 16 participants were treated with conventional surgery and chemotherapy (control group) and the remaining 16 patients were treated with chemotherapy followed by autologous bone marrow transfusion via a mesenteric vein (8 patients, ABM-MVI group) or a peripheral vein (8 patients, ABM-PI group). Subsequently, peripheral blood and lymphocyte data subsets were detected and documented in all patients., Results: Before chemotherapy, no significant difference in indicators was observed between three groups of ARL patients. Unexpectedly, 2 weeks after the end of 6 courses of chemotherapy, the ABM-MVI group, and the ABM-PI group yielded an increased level of CD8 + T lymphocytes, white blood cells (WBC), and platelet (PLT) in peripheral blood in comparison to the control group. Notably, the number of CD4 + T lymphocytes in the ABM-PI group was significantly higher than that in the other two groups. Additionally, no significant difference in haemoglobin levels was observed before and after chemotherapy in both the ABM-MVI and ABM-PI groups, while haemoglobin levels in the control group decreased significantly following chemotherapy., Conclusions: Autologous bone marrow transfusion after chemotherapy can promote the reconstruction of both bone marrow and the immune system. There was no significant difference in bone marrow recovery and reconstruction between the mesenteric vein transfusion group and the peripheral vein transfusion group.

To investigate the utility of noninvasive µPET-CT with 64 Cu-DOTA-anti-CD11b ( 64 Cu-αCD11b) in assessing bone marrow status after anticancer therapies, and the protective role of anti-CSF-1 (αCSF-1) against bone marrow suppression induced by Abraxane. Methods: MDA-MB-435 tumor-bearing mice were treated with Abraxane, αCSF-1, or αCSF-1 plus Abraxane. µPET-CT and biodistribution of 64 Cu-αCD11b were performed after intravenous injection of the radiotracer. Cells from mouse bone marrow and MDA-MB-435 tumor were analyzed by flow cytometry. A humanized αCSF-1 was investigated for its role in protecting bone marrow cells, using a transgenic mouse model that expresses functional human CSF-1. Results: μPET-CT showed that 64 Cu-αCD11b had high uptake in the bone marrow and spleen of both normal and tumor-bearing mice. Abraxane significantly reduced 64 Cu-αCD11b uptake in the bone marrow and spleen of treated mice compared to untreated mice. Interestingly, 64 Cu-αCD11b μPET-CT revealed that αCSF-1 alleviated the depletion of bone marrow cells by Abraxane. These changes in the bone marrow population of CD11b + myeloid cells were confirmed by flow cytometry. Moreover, αCSF-1 potently enhanced tolerance of bone marrow granulocytic myeloid cells to Abraxane, decreased cell migration, and suppressed recruitment of myeloid cells to the tumor microenvironment. The humanized αCSF-1 also alleviated the effects of Abraxane on bone marrow cells in transgenic mice expressing human CSF-1, suggesting clinical relevance of αCSF-1 in prevention of bone marrow suppression in addition to its role in reducing tumor-infiltrating myeloid cells. Conclusions: Abraxane-induced bone marrow CD11b + myeloid cell depletion in tumor-bearing mice could be noninvasively assessed by μPET-CT with 64 Cu-αCD11b and prevented by αCSF-1., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis -derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces., Competing Interests: E.R., E.P., N.A., and C.M. are co-inventors on a patent on a tuberculosis vaccine held by the University of Zaragoza and Biofabri. E.R. and E.P. are employees of Biofabri. J.T. is an employee of the TuBerculosis Vaccine Initiative and an advisor to Biofabri. M.G.N. holds a patent on the inhibition of trained immunity with nanobiologics, and a patent on the stimulation of trained immunity with nanobiologics. M.G.N. is also a scientific founder of Trained Therapeutix and Discovery (TTxD). The remaining authors of the Radboud UMC and the authors from the BPRC declare no competing interests., (© 2020 The Author(s).)

Background: The PD-1/PD-L1 axis has recently emerged as an immune checkpoint that controls antitumor immune responses also in hematological malignancies. However, the use of anti-PD-L1/PD-1 antibodies in multiple myeloma (MM) patients still remains debated, at least in part because of discordant literature data on PD-L1/PD-1 expression by MM cells and bone marrow (BM) microenvironment cells. The unmet need to identify patients which could benefit from this therapeutic approach prompts us to evaluate the BM expression profile of PD-L1/PD-1 axis across the different stages of the monoclonal gammopathies., Methods: The PD-L1/PD-1 axis was evaluated by flow cytometry in the BM samples of a total cohort of 141 patients with monoclonal gammopathies including 24 patients with Monoclonal Gammopathy of Undetermined Significance (MGUS), 38 patients with smoldering MM (SMM), and 79 patients with active MM, including either newly diagnosed or relapsed-refractory patients. Then, data were correlated with the main immunological and clinical features of the patients., Results: First, we did not find any significant difference between MM and SMM patients in terms of PD-L1/PD-1 expression, on both BM myeloid (CD14 + ) and lymphoid subsets. On the other hand, PD-L1 expression by CD138 + MM cells was higher in both SMM and MM as compared to MGUS patients. Second, the analysis on the total cohort of MM and SMM patients revealed that PD-L1 is expressed at higher level in CD14 + CD16 + non-classical monocytes compared with classical CD14 + CD16 - cells, independently from the stage of disease. Moreover, PD-L1 expression on CD14 + cells was inversely correlated with BM serum levels of the anti-tumoral cytokine, IL-27. Interestingly, relapsed MM patients showed an inverted CD4 + /CD8 + ratio along with high levels of pro-tumoral IL-6 and a positive correlation between %CD14 + PD-L1 + and %CD8 + PD-1 + cells as compared to both SMM and newly diagnosed MM patients suggesting a highly compromised immune-compartment with low amount of CD4 + effector cells., Conclusions: Our data indicate that SMM and active MM patients share a similar PD-L1/PD-1 BM immune profile, suggesting that SMM patients could be an interesting target for PD-L1/PD-1 inhibition therapy, in light of their less compromised and more responsive immune-compartment., Competing Interests: NG received research funding and honoraria from Amgen, Bristol Mayers Squibb, Celgene, Millenium Pharmaceutical, and Janssen Pharmaceutical. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Costa, Vescovini, Marchica, Storti, Notarfranchi, Dalla Palma, Toscani, Burroughs-Garcia, Catarozzo, Sammarelli and Giuliani.)

B cells are primarily known for their capacity to differentiate into antibody-secreting cells (ASCs). ASCs are usually viewed as terminally differentiated cells sharing a unique phenotype. However, it lately became evident that ASCs exist in a variety of subsets differing by their lifespan, anatomic location, and immunological function. Thus, ASCs can exist as long-lived plasma cells (LLPC) that can persist for years in a nonproliferating state within particular niches in the bone marrow (BM), or as short-lived plasma cells (SLPC) that are primarily found in secondary lymphoid organs or inflamed tissues and wane upon the termination of the associated immune response. Another layer of ASC diversity was uncovered with the discovery of their capacity to produce various pro- or anti-inflammatory cytokines. Notably, a subset of natural regulatory plasma cells characterized by the distinctive expression of the inhibitory receptor lymphocyte activation gene (LAG)-3 and a unique capacity to produce interleukin (IL)-10 upon stimulation was recently identified. Here, we describe how to immunophenotypically characterize murine plasma cells as well as how to isolate them using cell sorting, with a special focus on these recently described natural regulatory plasma cells.

Immunofluorescence is an indispensable method for the identification, localization and study of the expression of target antigens in formalin-fixed, paraffin-embedded (FFPE) tissue sections of human bone marrow. However, the procedure shows technical limitations because of the chemical and physical treatments required for sample processing before imaging. Here we describe a revisited protocol to obtain high-resolution images of human bone marrow trephine biopsies, improving the antigen-antibody recognition and preserving the morphology and the architecture of the bone marrow microenvironment.

Background: Older cancer patients with locally advanced or metastatic disease may benefit from chemotherapy alone or combined with radiotherapy. However, chemotherapy is often omitted either because of physician bias or because of its underlying comorbidity, thus compromising their survival. The coronavirus disease 19 (COVID-19) pandemic is compounding this issue because of the fear of immunosuppression induced by chemotherapy on the elderly which makes them more vulnerable to the virus., Summary: Immunotherapy has less effect on the patient bone marrow compared to chemotherapy. The potential synergy between radiotherapy and immunotherapy may improve local control and survival for older patients with selected cancer. Preliminary data are encouraging because of better survival and local control in diseases which are traditionally resistant to radiotherapy and chemotherapy such as melanoma and renal cell carcinoma. Key Message: We propose a new paradigm combining immunotherapy at a reduced dose and/or extended dosing intervals and hypofractionated radiotherapy for older patients with selected cancer which needs to be tested in future clinical trials., (© 2021 S. Karger AG, Basel.)

Young donors are reported to be associated with better transplant outcomes than older donors in allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the mechanism is still unclear. The current study compared the different subsets of haematopoietic stem cells (HSCs) and their progenitors as well as immune cells in bone marrow (BM) between young and older donors. The frequencies of HSCs, multipotent progenitors (MPPs) and myeloid progenitors, including common myeloid progenitors (CMPs) and megakaryocyte-erythroid progenitors (MEPs), were decreased, whereas those of lymphoid progenitors, including multi-potent lymphoid progenitors (MLPs) and common lymphoid progenitors (CLPs), were increased in the BM of young donors compared with in that of older donors. Lower reactive oxygen species (ROS) levels were observed in BM HSCs and six progenitor lines in young donors. Furthermore, young donors demonstrated higher frequencies of naive T cells and immune suppressor cells, such as alternative macrophages (M2) and lower frequencies of memory T cells and immune effectors, including T helper-1 and T cytotoxic-1 cells, in BM than older donors. Multivariate analysis demonstrated that donor age was independently correlated with BM HSC frequency. Although further validation is required, our results suggest that the differences in the frequency and immune differentiation potential of HSCs in BM between young donors and older donors may partly explain the different outcomes of allo-HSCT., (© 2020 British Society for Immunology.)

The diagnosis of juvenile myelomonocytic leukaemia (JMML) is based on clinical, laboratory and molecular features but immunophenotyping [multiparametric flow cytometry (MFC)] has not been used routinely. In the present study, we describe the flow cytometric features at diagnosis with special attention to the distribution of monocytic subsets and the relation between MFC and molecular subgroups. MFC was performed with an eight-colour platform based on Euroflow. We studied 33 JMML cases. CD34 + /CD117 + /CD13 + cells >2% was found in 25 cases, and 51·5% presented an aberrant expression of CD7. A decrease of CD34 + /CD19 + /CD10 + cells was seen in eight cases and in four they were absent. The granulocytic population had a decreased side scatter in 29 cases. Bone marrow monocytic precursors were increased in 28 patients, with a decrease in classical monocytes (median 80·7%) and increase in CD16 + (intermediate and non-classical). A more pronounced increase in myeloid CD34 + cells was seen in patients with Neurofibromatosis type 1 (NF1) and tyrosine-protein phosphatase non-receptor type 11 (PTPN11), with aberrant CD7 expression in four of six and 10/12 patients respectively. Thus, JMML shows an immunophenotypic profile similar to myelodysplastic syndromes, and a different monocyte subset distribution when compared with chronic MML. MFC proved to be an important diagnostic tool that can help in differential diagnosis with other clonal diseases with monocytosis., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Until recently, immunologic memory was considered an exclusive characteristic of adaptive immunity. However, recent advances suggest that the innate arm of the immune system can also mount a type of nonspecific memory responses. Innate immune cells can elicit a robust response to subsequent inflammatory challenges after initial activation by certain stimuli, such as fungal-derived agents or vaccines. This type of memory, termed trained innate immunity (also named innate immune memory), is associated with epigenetic and metabolic alterations. Hematopoietic progenitor cells, which are the cells responsible for the generation of mature myeloid cells at steady-state and during inflammation, have a critical contribution to the induction of innate immune memory. Inflammation-triggered alterations in cellular metabolism, the epigenome and transcriptome of hematopoietic progenitor cells in the bone marrow promote long-lasting functional changes, resulting in increased myelopoiesis and consequent generation of trained innate immune cells. In the present brief review, we focus on the involvement of hematopoietic progenitors in the process of trained innate immunity and its possible role in cardiometabolic disease.

B-1 cells are fetal-origin B lymphocytes with unique developmental and functional characteristics that can generate natural, polyreactive antibodies with important functions in tissue homeostasis and immune defense. While B-1 cell frequencies in bone marrow and secondary lymphoid tissues are low, relative high frequencies exist within peritoneal and pleural cavities of mice, including both CD5 + and CD5 - B-1 cells. These cells represent B-1 reservoirs that, when activated, migrate to lymphoid tissues to secrete antibodies and/or cytokines. Here, we outline efficient methods for the extraction and magnetic isolation of CD5 + B-1 cells from the peritoneal and pleural cavities as well as the separation and phenotypic characterization of CD5 + and CD5 - B-1 cells by flow cytometry.

Background & Aims: Histidine decarboxylase (HDC), the histamine-synthesizing enzyme, is expressed in a subset of myeloid cells but also marks quiescent myeloid-biased hematopoietic stem cells (MB-HSCs) that are activated upon myeloid demand injury. However, the role of MB-HSCs in dextran sulfate sodium (DSS)-induced acute colitis has not been addressed., Methods: We investigated HDC+ MB-HSCs and myeloid cells by flow cytometry in acute intestinal inflammation by treating HDC-green fluorescent protein (GFP) male mice with 5% DSS at various time points. HDC+ myeloid cells in the colon also were analyzed by flow cytometry and immunofluorescence staining. Knockout of the HDC gene by using HDC-/-; HDC-GFP and ablation of HDC+ myeloid cells by using HDC-GFP; HDC-tamoxifen-inducible recombinase Cre system; diphtheria toxin receptor (DTR) mice was performed. The role of H2-receptor signaling in acute colitis was addressed by treatment of DSS-treated mice with the H2 agonist dimaprit dihydrochloride. Kaplan-Meier survival analysis was performed to assess the effect on survival., Results: In acute colitis, rapid activation and expansion of MB-HSC from bone marrow was evident early on, followed by a gradual depletion, resulting in profound HSC exhaustion, accompanied by infiltration of the colon by increased HDC+ myeloid cells. Knockout of the HDC gene and ablation of HDC+ myeloid cells enhance the early depletion of HDC+ MB-HSC, and treatment with H2-receptor agonist ameliorates the depletion of MB-HSCs and resulted in significantly increased survival of HDC-GFP mice with acute colitis., Conclusions: Exhaustion of bone marrow MB-HSCs contributes to the progression of DSS-induced acute colitis, and preservation of quiescence of MB-HSCs by the H2-receptor agonist significantly enhances survival, suggesting the potential for therapeutic utility., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the growing success for osteochondral allograft (OCA) transplantation in treating large articular cartilage lesions in multiple joints, associated revision and failure rates are still higher than desired. While immunorejection responses have not been documented, the effects of the host's immune responses on OCA transplantation failures have not been thoroughly characterized. The objective of this study was to systematically review clinically relevant peer-reviewed evidence pertaining to the immunology of OCAs to elucidate theragnostic strategies for improving functional graft survival and outcomes for patients undergoing OCA transplantation. This systematic review of Cochrane Central Register of Controlled Trials, the Cochrane Database of Systematic Reviews, MEDLINE, PubMed, and EMBASE suggests that host immune responses play key roles in incorporation and functional survival of OCA transplants. OCA rejection has not been reported; however, graft integration through creeping substitution is reliant on host immune responses. Prolonged inflammation, diminished osteogenic potential for healing and incorporation, and relative bioburden are mechanisms that may be influenced by the immune system and contribute to undesirable outcomes after OCA transplantation. Based on the safety and efficacy of OCA transplantation and its associated benefits to a large and growing patient population, basic, preclinical, and clinical osteoimmunological studies on OCA transplantation that comprehensively assess and correlate cellular, molecular, histologic, biomechanical, biomarkers, diagnostic imaging, arthroscopic, functional, and patient-reported outcome measures are of high interest and importance., Competing Interests: A. M. S. reports personal fees from Musculoskeletal Transplant Foundation, outside the submitted work. J. P. S. reports grants and personal fees from Arthrex, Inc., grants from DePuy Synthes, other from Journal of Knee Surgery, grants from National Institutes of Health (NIAMS and NICHD), personal fees and other from Thieme, grants from U.S. Department of Defense, other from AO Foundation, other from American Orthopaedic Association, other from AO North America, grants from Coulter Foundation, other from Mid-America Orthopaedic Association, personal fees from Orthopedic Designs North America, personal fees from Smith & Nephew, outside the submitted work. J. L. C. reports grants and personal fees from Arthrex, Inc., personal fees from AthleteIQ, grants from ConforMIS, personal fees from CONMED Linvatec, grants from Coulter Foundation, grants from DePuy Synthes, grants and personal fees from Eli Lilly, other from The Journal of Knee Surgery, grants from Merial, other from Midwest Transplant Network, grants, personal fees, and other from Musculoskeletal Transplant Foundation, grants from National Institutes of Health (NIAMS and NICHD), grants from Purina, grants from Sites Medical, personal fees, and other from Thieme, grants from TissueGen Inc, personal fees from Trupanion, grants from U.S. Department of Defense, grants from Zimmer-Biomet, outside the submitted work. All the other authors report no conflict of interest., (Thieme. All rights reserved.)

Flow cytometry is a critical tool that can be employed to detect unique cells and to isolate cells from tissues based on their antigen profiles. While mouse eosinophils can be readily detected by one or more distinct antigen profiles, many of these strategies do not result in accurate eosinophil counts. We present here our basic protocol, which permits quantitative detection of eosinophils and isolation of eosinophils from bone marrow, spleen, and lung tissue of allergen-challenged wild-type and unchallenged IL5 transgenic mice. With small protocol variations, eosinophils can be isolated from small intestines and muscle tissue, the latter from infiltrates characteristic of muscular dystrophy (mdx) mice.

Acute myeloid leukemia (AML) is a hematological malignancy with a poor prognosis attributed to elevated reactive oxygen species (ROS) levels. Thus, agents that inhibit ROS generation in AML should be exploited. Azelaic acid (AZA), a small molecular compound, can scavenge ROS and other free radicals, exerting antitumor effects on various tumor cells. Herein, this study evaluated the antileukemic activity of AZA against AML via regulation of the ROS signaling pathway. We found that AZA reduced intracellular ROS levels and increased total antioxidant capacity in AML cell lines and AML patient cells. AZA suppressed the proliferation of AML cell lines and AML patient cells, expending minimal cytotoxicity on healthy cells. Laser confocal microscopy showed that AZA-treated AML cells surged and ruptured gradually on microfluidic chips. Additionally, AZA promoted AML cell apoptosis and arrested the cell cycle at the G1 phase. Further analysis demonstrated that peroxiredoxin (Prdx) 2 and Prdx3 were upregulated in AZA-treated AML cells. In vivo , AZA prolonged survival and attenuated AML by decreasing CD33 + immunophenotyping in the bone marrow of a patient-derived xenograft AML model. Furthermore, mice in the AZA-treated group had an increased antioxidant capacity and Prdx2/Prdx3 upregulation. The findings indicate that AZA may be a potential agent against AML by regulating the Prdxs/ROS signaling pathway., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Dongdong Zhang et al.)

Glucocorticoids are considered first-line therapy in a variety of eosinophilic disorders. They lead to a transient, profound decrease in circulating human eosinophils within hours of administration. The phenomenon of glucocorticoid-induced eosinopenia has been the basis for the use of glucocorticoids in eosinophilic disorders, and it has intrigued clinicians for 7 decades, yet its mechanism remains unexplained. To investigate, we first studied the response of circulating eosinophils to in vivo glucocorticoid administration in 3 species and found that the response in rhesus macaques, but not in mice, closely resembled that in humans. We then developed an isolation technique to purify rhesus macaque eosinophils from peripheral blood and performed live tracking of zirconium-89-oxine-labeled eosinophils by serial positron emission tomography/computed tomography imaging, before and after administration of glucocorticoids. Glucocorticoids induced rapid bone marrow homing of eosinophils. The kinetics of glucocorticoid-induced eosinopenia and bone marrow migration were consistent with those of the induction of the glucocorticoid-responsive chemokine receptor CXCR4, and selective blockade of CXCR4 reduced or eliminated the early glucocorticoid-induced reduction in blood eosinophils. Our results indicate that glucocorticoid-induced eosinopenia results from CXCR4-dependent migration of eosinophils to the bone marrow. These findings provide insight into the mechanism of action of glucocorticoids in eosinophilic disorders, with implications for the study of glucocorticoid resistance and the development of more targeted therapies. The human study was registered at ClinicalTrials.gov as #NCT02798523.

Objective: Assessment of role of the bone marrow colony-forming efficiency in plasma cell myeloma patients at different stages of treatment as a prognostic criterion for the disease course., Materials and Methods: The colony forming efficiency (CFE) was assayed in stage I-II plasma cell myeloma (PCM)patients (n = 37) aged 42-73, namely in patients survived after the Chornobyl NPP accident (n = 21) and persons notexposed to ionizing radiation (n = 16). There were 11 males exposed to ionizing radiation and having got stage I PCM,9 males and 3 females exposed and having got stage II PCM, 3 males and 3 females not exposed and having got stageI PCM, 6 males and 2 females not exposed and having got stage II PCM. Healthy persons (n = 20) were included in thecontrol group., Results: Number of the bone marrow (BM) granulocyte-macrophage colony-forming units (CFU-GM) in both exposedand not exposed PCM patients depended on a disease stage. CFU-GM was (16.7 ± 1.2) in the stage I PCM patients vs.(11.1 ± 1.1) in the stage II PCM ones both being lower (p < 0.05) compared to control (64.5 ± 2.2). Changes in cluster formation were similar, i.e. (37.7 ± 1.6) and (19.4 ± 1.3) correspondingly in the stage I and stage II PCM patients.Respective values in control were (89.8 ± 3.6). The CFE in stage I and stage II PCM patients at the time of diagnosiswas lower (5.7 ± 1.5 and 2.4 ± 1.1 respectively) vs. control (39.5 ± 1.51, p < 0.05), but has increased in remission upto (29. 6 ± 1.8) and (13.8 ± 1.2) respectively. There was no difference at that between the irradiated and non-irradiated patients. Number of the fibroblast colony-forming units (CFU-F) in the stage I and stage II PCM patients duringdiagnosis, namely (43.9 ± 5.4) and (22.5 ± 3.7), was lower (p < 0.05) vs. control (110.5 ± 4.9). Upon reaching remission the CFU-F value increased significantly (p < 0.05), reaching (87.4 ± 4.2) and (55.6 ± 2.7) correspondingly in thestage I and stage II PCM patients., Conclusion: Dependence of the BM cell CFE on the stage of PCM and presence or absence of remission was established. Prognostic value of the CFE of BM CFU-GM in terms of life span of patients was shown (Ro Spearm = 0.39,p < 0.02), namely in case of CFE > 20 before the polychemotherapy administration the life span of PCM patients wassignificantly longer vs. cases of CFE < 20., (Zh. M. Minchenko, T. F. Liubarets, V. V. Balan, O. O. Dmytrenko, T. Yu. Shlyakhtichenko, V. O. Moyseyenko, Yu. O. Silayev, V. G. Bebeshko.)

Emerging evidence indicates that metabolic programs regulate B cell activation and Ab responses. However, the metabolic mediators that support the durability of the memory B cell and long-lived plasma cell populations are not fully elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved serine/threonine kinase that integrates cellular energy status and nutrient availability to intracellular signaling and metabolic pathways. In this study, we use genetic mouse models to show that loss of ΑMPKα1 in B cells led to a weakened recall Ab response associated with a decline in the population of memory-phenotype B cells. AMPKα1-deficient memory B lymphocytes exhibited aberrant mitochondrial activity, decreased mitophagy, and increased lipid peroxidation. Moreover, loss of AMPKα1 in B lymphoblasts was associated with decreased mitochondrial spare respiratory capacity. Of note, AMPKα1 in B cells was dispensable for stability of the bone marrow-resident, long-lived plasma cell population, yet absence of this kinase led to increased rates of Ig production and elevated serum Ab concentrations elicited by primary immunization. Collectively, our findings fit a model in which AMPKα1 in B cells supports recall function of the memory B cell compartment by promoting mitochondrial homeostasis and longevity but restrains rates of Ig production., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The aim of the present work was to evaluate counts and functional properties of PD-1 + and TIM-3 + T cells in peripheral blood (PB) and bone marrow (BM) of multiple myeloma (MM) patients following the induction therapy. Sixty patients were enrolled in the study, CD4 + and CD8 + T cells expressing PD-1 and TIM-3, intracellular production of IFNγ and intracellular expression of Granzyme B were assessed. Relative counts of the majority of circulating PD-1 + , TIM-3 + and PD-1 + TIM-3 + T cells were higher in MM patients with disease progression compared with individuals in remission. Frequencies of almost all evaluated PD-1 + and TIM-3 + T cell subsets were higher in BM samples compared with PB; circulating CD4 + PD-1 + , CD8 + PD-1 + , CD8 + TIM-3 + , CD8 + PD-1 + TIM-3 + T cells positively correlated with the same BM subsets. Circulating CD4 + T cells, expressing PD-1 and TIM-3 (including co-expressing subset), as well as CD8 + PD-1 + TIM-3 + T cells, and BM CD8 + PD-1 + T cells correlated with serum B2-M levels. Sufficient frequencies of GrB + and IFNγ + subsets in PD-1-expressing T cells indicated their retained functional properties. TIM-3-expressing T cells and double positive PD-1 + TIM-3 + populations showed diminished cytotoxic and cytokine-producing ability and therefore might be attributed to the exhausted compartment. To identify T cell exhaustion, it is necessary to evaluate T cells co-expressing PD-1, TIM-3 and other inhibitory signal molecules and to study their functional properties. Sustained functionality of PD-1-positive T cells may explain low efficacy and frequent immune-mediated adverse events during anti-PD-1 therapy in MM.

Lifelong blood production is maintained by bone marrow (BM)-residing hematopoietic stem cells (HSCs) that are defined by two special properties: multipotency and self-renewal. Since dysregulation of either may lead to a differentiation block or extensive proliferation causing dysplasia or neoplasia, the genomic integrity and cellular function of HSCs must be tightly controlled and preserved by cell-intrinsic programs and cell-extrinsic environmental factors of the BM. The BM had been long regarded an immune-privileged organ shielded from immune insults and inflammation, and was thereby assumed to provide HSCs and immune cells with a protective environment to ensure blood and immune homeostasis. Recently, accumulating evidence suggests that hemato-immune challenges such as autoimmunity, inflammation or infection elicit a broad spectrum of immunological reactions in the BM, and in turn, influence the function of HSCs and BM environmental cells. Moreover, in analogy with the emerging concept of "trained immunity", certain infection-associated stimuli are able to train HSCs and progenitors to produce mature immune cells with enhanced responsiveness to subsequent challenges, and in some cases, form an inflammatory or infectious memory in HSCs themselves. In this review, we will introduce recent findings on HSC and hematopoietic regulation upon exposure to various hemato-immune stimuli and discuss how these challenges can elicit either beneficial or detrimental outcomes on HSCs and the hemato-immune system, as well as their relevance to aging and hematologic malignancies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Sezaki, Hayashi, Wang, Johansson, Umemoto and Takizawa.)

Immunophenotyping of bone marrow (BM) precursors has been used as an ancillary diagnostic tool in myelodysplastic syndromes (MDS), but there is no general agreement about which variables are the most relevant for prognosis. We developed a parsimonious prognostic model based on BM cell populations well-defined by phenotype. We analyzed 95 consecutive patients with primary MDS diagnosed at our Institution between 2005 and 2012 where BM immunophenotyping had been performed at diagnosis. Median follow-up: 42 months (4-199). Median age: 67 years (33-79). According to IPSS-R, 71 cases were low or intermediate risk. Flow variables significant in the univariate Cox analysis: "%monocytes/TNCs", "% CD16 + monocytes/TNCs", "total alterations in monocytes", "% myeloid CD34 + cells", "number of abnormal expressions in myeloblasts" and "% of B-cell progenitors". In the multivariate model remained independent: "% myeloid CD34 + cells", B-cell progenitors" and "% CD16 + monocytes/TNCs". These variables were categorized by the extreme quartile risk ratio strategy in order to build the score: % myeloid CD34 + cells" (≥ 2.0% = 1 point), B-cell progenitors" (< 0.05% 1 point) and "CD16 + monocytes/TNCs" (≥ 1.0% 1 point). This score could separate patients with a different survival. There was a weak correlation between the score and IPSS-R. Both had independent prognostic values and so, the flow score adds value for the prognostic evaluation in MDS.

Coronavirus disease 2019 (COVID-19) is a global public health emergency with many clinical facets, and new knowledge about its pathogenetic mechanisms is deemed necessary; among these, there are certainly coagulation disorders. In the history of medicine, autopsies and tissue sampling have played a fundamental role in order to understand the pathogenesis of emerging diseases, including infectious ones; compared to the past, histopathology can be now expanded by innovative techniques and modern technologies. For the first time in worldwide literature, we provide a detailed postmortem and biopsy report on the marked increase, up to 1 order of magnitude, of naked megakaryocyte nuclei in the bone marrow and lungs from serious COVID-19 patients. Most likely related to high interleukin-6 serum levels stimulating megakaryocytopoiesis, this phenomenon concurs to explain well the pulmonary abnormal immunothrombosis in these critically ill patients, all without molecular or electron microscopy signs of megakaryocyte infection.

Background: Diffuse large B cell lymphoma (DLBCL) is the commonest lymphoma that is highly aggressive where one-third of the patients relapse despite effective treatment. Interaction between the lymphoma cells and the non-clonal immune cells within the bone marrow microenvironment is thought to play a critical role in the pathogenesis of DLBCL., Methods: We used flow cytometry to characterize the proportion of B cell subpopulations in the bone marrow (N = 47) and peripheral blood (N = 54) of 75 DLBCL patients at diagnosis and study their impact on survival., Results: Anergic B cells in the bone marrow (BM), characterized as having CD21(-/low)/CD38- expression, influenced survival with high numbers (defined as > 13.9%) being associated with significantly shorter overall survival (59.7 months vs 113.6 months, p = 0.0038). Interestingly, low numbers of anergic B cells in the BM (defined as ≤13.9%) was associated with germinal center B cell type of DLBCL (p = 0.0354) that is known to have superior rates of survival when compared to activated B cell type. Finally, Cox regression analysis in our cohort of patients established that the inferior prognosis of having high numbers of anergic B cells in the bone marrow was independent of the established Revised International Prognostic Index (R-IPI) score., Conclusions: High proportion of anergic B cells in the BM characterized by CD21(-/low)/CD38- expression predicts poor survival outcomes in DLBCL.

Plasma cells (PCs) are essential for protection from infection, and at the origin of incurable cancers. Current studies do not circumvent the limitations of removing PCs from their microenvironment and confound formation and maintenance. Also, the investigation of PC population dynamics has mostly relied on nucleotide analog incorporation that does not label quiescent cells, a property of most PCs. The main impediment is the lack of tools to perform specific genetic manipulation in vivo. Here we characterize a genetic tool (Jchain creERT2 ) in the mouse that permits first-ever specific genetic manipulation in PCs in vivo, across immunoglobulin isotypes. Using this tool, we found that splenic and bone marrow PC numbers remained constant over-time with the decay in genetically labeled PCs being compensated by unlabeled PCs, supporting homeostatic population turnover in these tissues. The Jchain creERT2 tool paves the way for an in-depth mechanistic understanding of PC biology and pathology in vivo, in their microenvironment., Competing Interests: AX, RB, DC No competing interests declared, (© 2020, Xu et al.)

Objective To investigate the effect of Brucella abortus mutant strain S2308δrfbE on the maturation and immune response of mouse bone marrow-derived dendritic cells (BMDCs). Methods The expression of major histocompatibility complex I (MHC I), MHC II, CD40, CD86 were detected by flow cytometry; the secretion of cytokines tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-10 was detected by ELISA in S2308δrfbE-infected mouse BMDCs. Results The number of cells expressing MHC I, MHC II, CD40, CD86 and the secretion of cytokines TNF-α, IL-6 and IL-10 significantly increased in BMDCs infected with S2308δrfbE as compared with control groups. Conclusion S2308δrfbE promotes the maturation and enhances the inflammatory cytokines secretion in BMDCs.

Objective: Major ABO incompatible hematopoietic progenitors from bone marrow (HPC(M)) donor collections that are destined for clinical transplantation are typically processed to deplete products of red blood cells (RBCs). The purpose of this study was to compare RBC depletion when using the Spectra Optia® relative to a 2-step method involving a COBE2991 instrument to obtain a buffy coat followed by a hydroxyethyl starch (HES) density gradient (COBE+HES) of the buffy coat., Methods: Post-processing recoveries of products undergoing 4, 8, and 10 bone marrow processing (BMP) cycles (i.e. 1 cycle=1 volume of HPC(M)) with the Spectra Optia® were determined for volume, RBC content, viable total nucleated cells (vTNC), viable CD34+ cells (vCD34), viable CD3+ cells (vCD3) and colony-forming-cells (CFC). Subsequent RBC depletions with Spectra Optia® were then performed with 10 BMP cycles on additional HPC(M) collections and were compared against a retrospective analysis of historical COBE+HES post-processing data., Results: Ten BMP cycles of HPC(M) (n=6) products were identified as optimal with volume reductions of 81.3±1.6 % and RBC reductions of 97.0±0.6 % with the Spectra Optia®. This also resulted in an average of 0.28 ±0.14 mL of RBC/kg (mean±SD; n=6) with vTNC yields of 65.0±10.9%, vCD34+ yields of 98.5±12.7%, and vCD3+ yields of 90.6±10.0%. Recoveries with the COBE+HES methodology resulted in vTNC recoveries of 62.9±20.5% (mean±SD; n=30) and 0.63±0.71 mL of RBC/kg (mean±SD; n=30)., Conclusions: The Spectra Optia® is a viable option for depleting HPC(M) harvests of contaminating RBC in situations of ABO incompatibility. Target cells from a MNC rich fractionation were preserved through processing while eliminating RBC contaminants., (© 2020 by the Association of Clinical Scientists, Inc.)

Acute infection is characterized by eosinopenia. However, the underlying mechanism(s) are poorly understood and it is unclear whether decreased mobilization/production of eosinophils in the bone marrow (BM) and/or increased homing to the tissues play a role. The objective of this study was to investigate the differentiation and activation status of eosinophils in the human BM and blood upon experimental human endotoxemia, a standardized, controlled, and reproducible model of acute systemic inflammation. A BM aspirate and venous blood was obtained from seven healthy volunteers before, 4 h after, and 1 week after intravenous challenge with 2 ng/kg endotoxin. Early progenitors (CD34+/IL-5Rα+), eosinophil promyelocytes, myelocytes, metamyelocytes, and mature eosinophils were identified and quantified in the bone marrow and blood samples using flowcytometry based on specific eosinophil markers (CD193 and IL-5Rα). Activation status was assessed using antibodies against known markers on eosinophils: Alpha-4 (CD49d), CCR3 (CD193), CR1 (CD35), CEACAM-8 (CD66b), CBRM 1/5 (activation epitope of MAC-1), and by plasma cytokine analysis. Four hours after endotoxin administration, numbers of mature eosinophils in the blood and in the BM markedly declined compared with baseline, whereas numbers of all eosinophil progenitors did not change. The remaining eosinophils did not show signs of activation or degranulation despite significantly increased circulating levels of eotaxin-1. Furthermore, the expression of CD49d and CD193 on eosinophils was lower compared to baseline, but normalized after 7 days. Together these data imply that circulatory eosinopenia after an innate immune challenge is mediated by CD49d-mediated homing of eosinophils to the tissues., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4 + T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4 + T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.

Tris(2-butoxyethyl) phosphate (TBEP) is a major organophosphorus flame retardant and has been widely increasing as a substitute for brominated flame retardants. TBEP may have adverse effects on human health; however, its impact on immune and allergic responses remains largely uncharacterized. In this study, the effects of low-dose TBEP comparable with the level of actual human exposure to that of human tolerable daily intake on allergic asthmatic mice were explored. Five-week-old C3H/HeJSlc male mice consumed a diet containing approximately 0.02, 0.2 or 2 μg/kg/day TBEP and were intratracheally administrated ovalbumin (OVA) (1 μg/mouse every 2 weeks from 5 to 11 weeks of age). Exposure to 2 μg/kg/day TBEP with OVA tended to enhance allergic pulmonary inflammation and significantly elevated mRNA levels of interleukin-5, eotaxin-1 and estrogen receptor alpha (ERα) compared with OVA alone. In mediastinal lymph nodes (MLNs), TBEP (0.2 or 2 μg/kg/day) with OVA significantly increased in total cell number and promoted conventional dendritic cell activation than OVA alone; MLN cell proliferation by OVA restimulation was also enhanced in these groups. In the bone marrow (BM), TBEP (0.02 or 0.2 μg/kg/day) with OVA resulted in a net decrease in total cell number and fraction of CCR2 + Gr-1 + cells; the fraction of Gr-1 + cells increased. In conclusion, oral exposure to low-dose TBEP levels equivalent to tolerable daily intake may exacerbate allergic pulmonary inflammation by promoting a skewed T-helper 2 cell response, upregulation of ERα and dysregulation of both MLN and BM microenvironments., (© 2020 John Wiley & Sons, Ltd.)

Myelotoxicity is a serious side effect of anticancer drugs. The search for drugs that can reduce the hematological complications of chemotherapy through modulation of hematopoietic stem cells is an urgent task of oncopharmacology. In the present study we showed that administration of Tussilago farfara L. polysaccharides to C57BL/6 mice treated with cyclophosphamide can increase the number of hematopoietic stem cells (CD117 + 34 + ) in the bone marrow.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF., (© 2020 by The American Society of Hematology.)

The sympathetic nervous system plays critical roles in the differentiation, maturation and recruitment of immune cells under homeostatic conditions, and in responses to environmental stimuli, although its role in the migratory control of immune cells during acute inflammation remains unclear. In this study, using an advanced intravital bone imaging system established in our laboratory, we demonstrated that the sympathetic nervous system locally regulates neutrophil egress from the bone marrow for mobilization to inflammatory foci. We found that sympathetic neurons were located close to blood vessels in the bone marrow cavity; moreover, upon lipopolysaccharide (LPS) administration, local sympathectomy delayed neutrophil egress from the bone marrow and increased the proportion of neutrophils that remained in place. We also showed that vascular endothelial cells produced C-X-C motif chemokine ligand 1 (CXCL1), which is responsible for neutrophil egress out of the bone marrow. Its expression was up-regulated during acute inflammation, and was suppressed by β-adrenergic receptor blockade, which was accompanied with inhibition of neutrophil egress into the systemic circulation. Furthermore, systemic β-adrenergic signaling blockade decreased the recruitment of neutrophils in the lung under conditions of acute systemic inflammation. Taken together, the results of this study first suggested a new regulatory system, wherein local sympathetic nervous activation promoted neutrophil egress by enhancing Cxcl1 expression in bone marrow endothelial cells in a β-adrenergic signaling-dependent manner, contributing to the recruitment of neutrophils at the onset of inflammation in vivo., (© The Japanese Society for Immunology. 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In recent years, porcine dendritic cells (DCs) have been identified from pig tissues. However, studying the interaction of porcine DCs with pathogens is still difficult due to the scarcity of DCs in tissues. In the present work, the Flt3-ligand (Flt3L)-based in vitro derivation system was further characterized and compared with other cytokine derivation models using a combination of factors: stem cell factor (SCF), GM-CSF, and IL-4. The method using Flt3L alone or combined with SCF supported the development of pig bone marrow hematopoietic cells into in vivo equivalent conventional DCs (cDCs). The equivalent cDC1 (the minor population in the cultures) were characterized as CADM1 + CD14 - MHC-II + CD172a -/ lo CD1 - CD163 - DEC205 + CD11R3 lo CD11R1 + CD33 + CD80/86 + . They expressed high levels of FLT3, ZBTB46, XCR1, and IRF8 mRNA, were efficient in endocytosing dextran and in proliferating allogenic CD4 + CD8 + T cells, but were deficient in phagocyting inactivated Staphylococcus aureus ( S. aureus ). Also, after poly I:C stimulation, they predominantly produced IL-12p40a and matured as indicated by the increase of MHC-I, MHC-II, and CD80/86. The equivalent cDC2 (the main population) were CADM1 + CD14 - MHC-II + C D172a + CD1 + CD163 -/ lo DEC205 lo CD11R3 + CD11R1 + CD33 + CD80/86 + ; meanwhile, they overexpressed FcεR1α and IRF4 mRNA. They showed high efficiency in the endocytosis of dextran, but weak in phagocytosing bacteria. They supported allogenic CD4 + CD8 - /CD4 + CD8 + T cell proliferation and were high producers of IL-12p40 (upon TLR7 stimulation) and IL-10 (upon TLR7 stimulation). TLR ligand stimulation also induced their maturation. In addition, a CD14 + population was identified with the phenotype CADM1 + CD14 + MHC-II + CD172a + CD1 + CD163 + DEC205 - CD11R3 + CD11R1 + CD33 -/ lo CD80/86 + . They shared some functional similarities with cDC2 and were distinguishable from macrophages. This CD14 + population was efficient in phagocyting S. aureus but showed less maturation upon TLR ligand stimulation than cDC1 or cDC2. The alternative methods of DC derivation including GM-CSF and/or IL-4 produced mostly CADM1 - cells that did not fulfill the canonical phenotype of bona fide porcine DCs. Our study provides an exhaustive characterization of Flt3L-derived DCs with different methods that can help the in vitro study of the interaction of DCs with porcine-relevant pathogens., (Copyright © 2020 Li, Puebla-Clark, Hernández, Díaz and Mateu.)

Patients with relapsed or refractory acute myeloid leukemia (AML) have a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell leukemias and lymphomas and could prove highly efficacious in AML. However, a significant number of patients with AML may not receive treatment with an autologous product due to manufacturing failures associated with low lymphocyte counts or rapid disease progression while the therapeutic is being produced. We report the preclinical evaluation of an off-the-shelf CAR T cell therapy targeting Fms-related tyrosine kinase 3 (FLT3) for the treatment of AML. Single-chain variable fragments (scFvs) targeting various epitopes in the extracellular region of FLT3 were inserted into CAR constructs and tested for their ability to redirect T cell specificity and effector function to FLT3 + AML cells. A lead CAR, exhibiting minimal tonic signaling and robust activity in vitro and in vivo, was selected and then modified to incorporate a rituximab-responsive off-switch in cis. We found that allogeneic FLT3 CAR T cells, generated from healthy-donor T cells, eliminate primary AML blasts but are also active against mouse and human hematopoietic stem and progenitor cells, indicating risk of myelotoxicity. By employing a surrogate CAR with affinity to murine FLT3, we show that rituximab-mediated depletion of FLT3 CAR T cells after AML eradication enables bone marrow recovery without compromising leukemia remission. These results support clinical investigation of allogeneic FLT3 CAR T cells in AML and other FLT3 + hematologic malignancies., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Anemia of inflammation (AI) is the second most prevalent anemia after iron deficiency anemia and results in persistent low blood erythrocytes and hemoglobin, fatigue, weakness, and early death. Anemia of inflammation is common in people with chronic inflammation, chronic infections, or sepsis. Although several studies have reported the effect of inflammation on stress erythropoiesis and iron homeostasis, the mechanisms by which inflammation suppresses erythropoiesis in the bone marrow (BM), where differentiation and maturation of erythroid cells from hematopoietic stem cells (HSCs) occurs, have not been extensively studied. Here we show that in a mouse model of acute sepsis, bacterial lipopolysaccharides (LPS) suppress medullary erythroblastic islands (EBIs) and erythropoiesis in a TLR-4- and MyD88-dependent manner with concomitant mobilization of HSCs. LPS suppressive effect on erythropoiesis is indirect as erythroid progenitors and erythroblasts do not express TLR-4 whereas EBI macrophages do. Using cytokine receptor gene knock-out mice LPS-induced mobilization of HSCs is G-CSF-dependent whereas LPS-induced suppression of medullary erythropoiesis does not require G- CSF-, IL- 1-, or TNF-mediated signaling. Therefore suppression of medullary erythropoiesis and mobilization of HSCs in response to LPS are mechanistically distinct. Our findings also suggest that EBI macrophages in the BM may sense innate immune stimuli in response to acute inflammation or infections to rapidly convert to a pro-inflammatory function at the expense of their erythropoietic function., (Copyright © 2020 Bisht, Tay, Wellburn, McGirr, Fleming, Nowlan, Barbier, Winkler and Levesque.)

Primary immune deficiency (PID) disorders are clinically and molecularly heterogeneous diseases. T cell receptor excision circles (TRECs) and κ (kappa)-deleting excision circles (KRECs) are markers of T and B cell development, respectively. They are useful tools to assess T and B cell function and immune reconstitution and have been used for newborn screening for severe combined immunodeficiency disease (SCID) and agammaglobulinemia, respectively. Their profiles in several genetically confirmed PIDs are still lacking. The objective of this study was to determine TREC and KREC genomic profiling among various molecularly confirmed PIDs. We used real-time-quantitative polymerase chain reaction (RT-qPCR)-based triplex analysis of TRECs, KRECs and β-actin (ACTB) in whole blood genomic DNA isolated from 108 patients with molecularly confirmed PIDs. All agammaglobulinemia patients had low KREC counts. All SCIDs and Omenn syndrome patients secondary to mutations in RAG1, RAG2, DCLRE1C and NHEJ1 had low TREC and KREC counts. JAK3-deficient patients had normal KREC and the TREC count was influenced by the type of mutation. Early-onset ADA patients had low TREC and KREC counts. Four patients with zeta-chain-associated protein kinase 70 (ZAP70) had low TREC. All purine nucleoside phosphorylase (PNP) patients had low TREC. Combined immunodeficiency (CID) patients secondary to AK2, PTPRC, CD247, DCLREC1 and STAT1 had normal TREC and KREC counts. Most patients with ataxia-telangiectasia (AT) patients had low TREC and KREC, while most DOCK8-deficient patients had low TRECs only. Two of five patients with Wiskott-Aldrich syndrome (WAS) had low TREC counts as well as one patient each with bare lymphocyte syndrome (BLS) and chronic granulomatous disease. All patients with Griscelli disease, Chediak-Higashi syndrome, hyper-immunoglobulin (Ig)M syndrome and IFNGR2 had normal TREC and KREC counts. These data suggest that, in addition to classical SCID and agammaglobulinemia, TREC/KREC assay may identify ZAP70 patients and secondary target PIDs, including dedicator of cytokinesis 8 (DOCK8) deficiency, AT and some individuals with WAS and BLS., (© 2020 British Society for Immunology.)

Next-generation sequencing (NGS) at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 loci was performed on 5,266 southern Chinese unrelated donors of the Hong Kong Bone Marrow Donor Registry. High-resolution HLA genotypes defined by full sequencing of class I loci and extended coverage of class II loci were attained to determine allele frequencies and estimate haplotype frequencies. This study provides allele and haplotype frequencies on 11 loci estimated for the first time in the Hong Kong Chinese population. These results describe extended haplotypes including the less frequently typed HLA-DPA1, -DPB1 and -DQA1 loci and distinctive haplotype associations. The present data are timely in that they allow the permissible matching in HLA-DPB1 for Chinese patients awaiting haematopoietic stem cell transplantation upon applying the latest requirement of NMDP matching guidelines. Overall, these results provide a useful reference source for population genetics studies, HLA-disease association studies and for improving donor recruitment and selection strategies of bone marrow registries. The allele and haplotype data are available in the Allele Frequencies Net Database under the population name ''Hong Kong Chinese HKBMDR, HLA 11 loci'' and the identifier (AFND3724) [1]., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The role of the bone marrow microenvironment in supporting the proliferation and survival of the abnormal plasma cells in multiple myeloma (MM) is well established. Such microenvironment is rich of cytokines like IL-6, TGF-β, IL-1 and IL-23 which are known to promote the differentiation of Th17 lymphocytes, a T helper subpopulation. Th17 cells secrete IL-17, a cytokine involved in the pathophysiology of several auto-immune diseases. Yet, its involvement in cancers remains unclear. Herein, we aimed to try to understand the role of Th17 lymphocytes in multiple myeloma. Bone marrow samples were prospectively collected from 29 MM patients and 23 healthy bone marrow donors for allograft. Mononuclear bone marrow cells were isolated by Ficoll-Hypaque gradient and CD138 + plasma cells were depleted using magnetic beads. The quantification of Th17 cells was performed by flow cytometry in the CD138 negative cells. The mRNA expression of IL17 and RORc was quantified using real time PCR in the same subset. The mRNA expression of IL17R was analyzed in plasma cells (CD138 + cells). Data obtained from patients and healthy donors were compared by both non-parametric Mann-Whitney U test and Spearman test. A significant increase of IL17 and RORC mRNA expression was found in the bone marrow microenvironment of MM patients compared to healthy donors. Th17 cells were also increased in the bone marrow of MM patients compared to healthy donors. Interestingly, the mRNA expression of IL17R was significantly decreased in MM patients. Yet, no correlation was found between the gene expression IL17, RORC and IL17R and the bone marrow infiltration or the stage of the disease. Collectively, our results suggest the involvement of Th17 cells in the pathophysiology of MM. Such data further support the use of anti-IL-17 antibodies as a therapeutic approach in MM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)