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Measurable residual disease (MRD) is a powerful prognostic factor of relapse in acute myeloid leukemia (AML). We applied the single-cell RNA sequencing to bone marrow (BM) samples from patients with (n=20) and without (n=12) MRD after allogeneic hematopoietic stem cell transplantation. A comprehensive immune landscape with 184,231 cells was created. Compared with CD8 + T cells enriched in the MRD-negative group (MRD-_CD8), those enriched in the MRD-positive group (MRD+_CD8) showed lower expression levels of cytotoxicity-related genes. Three monocyte clusters (i.e., MRD+_M) and three B-cell clusters (i.e., MRD+_B) were enriched in the MRD-positive group. Conversion from an MRD-positive state to an MRD-negative state was accompanied by an increase in MRD-_CD8 clusters and vice versa. MRD-enriched cell clusters employed the macrophage migration inhibitory factor pathway to regulate MRD-_CD8 clusters. These findings revealed the characteristics of the immune cell landscape in MRD positivity, which will allow for a better understanding of the immune mechanisms for MRD conversion., (© 2024. Science China Press.)

Purpose: Type I conventional dendritic cells (cDC1s) play a key role in priming anti-tumor cytotoxic T cells and inducing immune tolerance for self-antigens and tumor antigens. However, it remains unclear whether cDC1 has a protective or pathogenic role in multiple myeloma. We investigated a role of cDC1 in myeloma progression., Methods: A myeloma mouse model was performed by intravenous transplantation of Vk*MYC myeloma cells into XCR1-Diphtheria toxin receptor (DTR) knock-in or wild-type mice. Following injection with Diphtheria toxin (DT), monoclonal (M)-proteins and myeloma cells were analyzed by ELISA and flow cytometry., Results: Here we show that inducible depletion of cDC1 after myeloma transplantation markedly suppressed the progression of myeloma in the bone marrow and extramedullary sites, such as the spleen. cDC1 appeared in the bone marrow and spleen of myeloma-transplanted mice, which highly expressed CD103 and lowly produced interleukin (IL)-12. Consequently, the frequencies of exhausted CD8 T cells and regulatory T cells significantly decreased in the bone marrow of cDC1-depleted mice., Conclusions: cDC1 supports the progression of myeloma inducing exhausted CD8 T cells and regulatory T cells., 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 © 2024 Suzuki, Komiya, Tsuda, Yoshino, Kaisho, Bergsagel, Kawamura, Fukuda and Tokoyoda.)

Neutrophil subsets endowed with regulatory/suppressive properties are widely regarded as deleterious immune cells that can jeopardize antitumoral response and/or antimicrobial resistance. Here, we describe a sizeable fraction of neutrophils characterized by the expression of programmed death-ligand 1 (PD-L1) in biological fluids of humans and mice with severe viral respiratory infections (VRI). Biological and transcriptomic approaches indicated that VRI-driven PD-L1 + neutrophils are endowed with potent regulatory functions and reduced classical antimicrobial properties, as compared to their PD-L1 - counterpart. VRI-induced regulatory PD-L1 + neutrophils were generated remotely in the bone marrow in an IFN-γ-dependent manner and were quickly mobilized into the inflamed lungs where they fulfilled their maturation. Neutrophil depletion and PD-L1 blockade during experimental VRI resulted in higher mortality, increased local inflammation, and reduced expression of resolving factors. These findings suggest that PD-L1 + neutrophils are important players in disease tolerance by mitigating local inflammation during severe VRI and that they may constitute relevant targets for future immune interventions.

Certain immune cells, including neutrophils, macrophages, dendritic cells, B cells, Breg cells, CD4 + T cells, CD8 + T cells, and Treg cells, establish enduring residency within the bone marrow. Their distinctive interactions with hematopoiesis and the bone marrow microenvironment are becoming increasingly recognized alongside their multifaceted immune functions. These cells play a dual role in shaping hematopoiesis. They directly influence the quiescence, self-renewal, and multi-lineage differentiation of hematopoietic stem and progenitor cells through either direct cell-to-cell interactions or the secretion of various factors known for their immunological functions. Additionally, they actively engage with the cellular constituents of the bone marrow niche, particularly mesenchymal stem cells, endothelial cells, osteoblasts, and osteoclasts, to promote their survival and contribute to tissue repair, thereby fostering a supportive environment for hematopoietic stem and progenitor cells. Importantly, these bone marrow immune cells function synergistically, both locally and functionally, rather than in isolation. In summary, immune cells residing in the bone marrow are pivotal components of a sophisticated network of regulating hematopoiesis., (© 2024. The Author(s).)

Mucosal-associated invariant T cells (MAIT cells) are a subset of T cells with innate, effector-like properties that play an essential role in the immune response to microbial infections. In humans, MAIT cells are detectable in the blood, liver, and lungs, but little is known about the frequency of these cells in the bone marrow. Also, the pathogenic role, if any, of MAIT cells in the development of aplastic anemia, a disease with an exquisite origin in the bone marrow, is currently unknown. We investigated the frequency and clinical relevance of bone marrow MAIT cells in a cohort of 14 patients (60.6 ± 23 and 57% women) with aplastic anemia. MAIT cells in the bone marrow samples obtained at diagnosis were evaluated by flow cytometry, and their association with various blood cell parameters and the patients' clinical features was analyzed. MAIT cells were detectable in the bone marrow of all patients, with considerable variations among them. Bone marrow MAIT cells expressing the activator receptor natural killer group 2D - NKG2D (NKG2D+ MAIT cells) were significantly more abundant in the specimens of the aplastic anemia patients than in patients with bone marrow failure distinct from aplastic anemia. In addition, the NKG2D+ MAIT cells positively correlated with whole blood cell counts (WBC), platelet counts, and neutrophil counts, as well as with various inflammatory markers, including neutrophil-to-lymphocyte rate (NLR), platelet-to-lymphocyte rate (PLR), and systemic inflammatory index (SII). In functional studies, bone marrow CD34+ hematopoietic cells exposed to phytohemagglutinin or bacterial-derived lipopolysaccharide and acetyl-6-formylpterin upregulated MR1 (major histocompatibility complex, class I-related, known to interact with MAIT cells) and MICA/B (MHC class I chain-related gene A, a ligand of NKG2D) proteins on their cell surface, suggesting that under stress conditions, CD34+ hematopoietic cells are more likely to interact with NKG2D+ MAIT cells. In addition, NKG2D+ MAIT cells upregulated perforin and granzyme B in response to their interaction with recombinant MICA protein in vitro . This study reports for the first time the frequency of MAIT cells in the bone marrow of patients with aplastic anemia and assesses the potential implications of these cells in the pathogenesis or progression of aplastic anemia.

High-throughput immune repertoire (IR) sequencing provides direct insight into the diversity of B cell receptor (BCR) and T cell receptor (TCR), with great potential to revolutionize the diagnosis, monitoring, and prevention of immune system-related disorders. In this study, multiplex PCR was applied to amplify the complementarity-determining regions of BCR and TCR, followed by comprehensive analysis by high-throughput sequencing. We compare the TCR (BCR) of bone marrow fluid (BMF) and peripheral blood (PB) samples from 17 patients in the Epstein-Barr and immunodeficiency groups, respectively. Our study shows that the diversity of the IR of blood samples is very similar to that of bone marrow samples statistically. However, the distributions of the monoclonal genes are significantly different in these 2 samples of most patients. This suggests that the BMFs can be replaced by the PB samples in diversity detection of IR to monitor the immune status of the body, while the detection of the BMFs is unreplaceable when the monoclonal change occurs. We used high-throughput sequencing to assess the TCR and BCR of the patients and provide a basis for the clinical analysis of PB and bone marrow samples and selection of disease diagnosis and monitoring methods., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

This mini review summarizes the immunobiology of myelodysplastic syndromes, specifically focusing on the interactions between immune cells, cytokines, and dysplastic cells within the tumor microenvironment in the bone marrow. We elucidate in detail how immune dysregulation and evasion influence the initiation and progression of myelodysplastic syndromes, as well as resistance to therapy and progression to AML. In addition, we highlight a range of therapeutic strategies, including the most recent breakthroughs and experimental therapies for treating MDS. Finally, we address the existing knowledge gaps in the understanding of the immunobiology of MDS and propose future research directions, promising advancements toward enhancing clinical outcomes and survival for patients with MDS., 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 © 2024 Kannan, Vedia and Molldrem.)

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFβ1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFβ antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFβ. TGFβ1 reduced perforin and TNFα production by T cells in vitro. TGFβ1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFβ1 expression. In four independent patient cohorts, TGFβ1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFβ1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFβ1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFβ antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms., (©2024 American Association for Cancer Research.)

Background: T helper (Th) cell subsets primarily assist B cells in differentiating into plasma cells in the germinal center. The mechanism of malignant transformation of plasma cells is an important target for the clinical treatment of MM; however, the mechanism remains unclear., Methods: We collected the peripheral blood (PB) and bone marrow (BM) samples of 33 patients with MM. In addition, the PB was also collected from 25 normal healthy controls (HCs). We analyzed the percentages of Th cell subsets in the PB and BM samples of patients with MM., Results: Tfh/CD4 + were positively correlated with the proportion of myeloma cells in the BM and PB samples (r = 0.592, P = 0.002 and r = 0.510, P = 0.010 respectively), and showed a strong correlation between the BM and PB samples (r = 0.6559, P = 0.0095). In the PB samples, the percentages of Th2/CD4 + and Tfh2/Tfh cells were significantly lower in patients with MM than in HCs (P = 0.00013 and P = 0.0004, respectively), whereas the percentage of Th17/CD4 + and Tfh17/Tfh was significantly higher in newly diagnosed patients with MM than in HCs (P = 0.0037 and P = 0.03, respectively), and all these cells showed a good predictive value for MM (area under the curve [AUC] 0.781, = 0.792, = 0.837, and 0.723 respectively). In the PB samples, all subsets of PD-1 + ICOS - Tfh showed a noticeable downward trend in MM from newly diagnosed to non-remission and remission groups. In contrast, all subsets of PD-1 - ICOS + Tfh increased gradually., Conclusion: Th cell subsets play an important role in the occurrence and development of MM and may provide a fundamental basis for identifying new immunotherapy targets and prognosis., 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 © 2024 Zhang, Zhong, Fan, Mao and Li.)

Mutational profiles of myelodysplastic syndromes (MDS) have established that a relatively small number of genetic aberrations, including SF3B1 and SRSF2 spliceosome mutations, lead to specific phenotypes and prognostic subgrouping. We performed a multi-omics factor analysis (MOFA) on two published MDS cohorts of bone marrow mononuclear cells (BMMNCs) and CD34 + cells with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, retrotransposon (RTE) expression, and cell-type composition, were derived from these modalities to identify the latent factors with significant impact on MDS prognosis. SF3B1 was the only mutation among 13 mutations in the BMMNC cohort, indicating a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34 + cohort. Interestingly, the MOFA factor representing the inflammation shows a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases show a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Furthermore, MOFA identified RTE expression as a risk factor for MDS. This work elucidates the efficacy of our integrative approach to assess the MDS risk that goes beyond all the scoring systems described thus far for MDS., Competing Interests: SG, NS, SH, WM, IT, SJ, DK, AI, AP, JB, GN, GM No competing interests declared, MK Reviewing editor, eLife, (© 2024, Gerlevik, Seymen, Hama et al.)

Myeloid malignancies arise in bone marrow microenvironments and shape these microenvironments in favor of malignant development. Immune suppression is one of the most important stages in myeloid leukemia progression. Leukemic clone expansion and immune dysregulation occur simultaneously in bone marrow microenvironments. Complex interactions emerge between normal immune system elements and leukemic clones in the bone marrow. In recent years, researchers have identified several of these pathological interactions. For instance, recent works shows that the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), from bone marrow stromal cells contributes to immune dysregulation and the selective proliferation of JAK2V617F+ clones in myeloproliferative neoplasms. Moreover, inflammasome activation and sterile inflammation result in inflamed microenvironments and the development of myelodysplastic syndromes. Additional immune dysregulations, such as exhaustion of T and NK cells, an increase in regulatory T cells, and impairments in antigen presentation are common findings in myeloid malignancies. In this review, we discuss the role of altered bone marrow microenvironments in the induction of immune dysregulations that accompany myeloid malignancies. We also consider both current and novel therapeutic strategies to restore normal immune system function in the context of myeloid malignancies., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Modest response rates to immunotherapy observed in advanced lung cancer patients underscore the need to identify reliable biomarkers and targets, enhancing both treatment decision-making and efficacy. Factors such as PD-L1 expression, tumor mutation burden, and a 'hot' tumor microenvironment with heightened effector T cell infiltration have consistently been associated with positive responses. In contrast, the predictive role of the abundantly present tumor-infiltrating myeloid cell (TIMs) fraction remains somewhat uncertain, partly explained by their towering variety in terms of ontogeny, phenotype, location, and function. Nevertheless, numerous preclinical and clinical studies established a clear link between lung cancer progression and alterations in intra- and extramedullary hematopoiesis, leading to emergency myelopoiesis at the expense of megakaryocyte/erythroid and lymphoid differentiation. These observations affirm that a continuous crosstalk between solid cancers such as lung cancer and the bone marrow niche (BMN) must take place. However, the BMN, encompassing hematopoietic stem and progenitor cells, differentiated immune and stromal cells, remains inadequately explored in solid cancer patients. Subsequently, no clear consensus has been reached on the exact breadth of tumor installed hematopoiesis perturbing cues nor their predictive power for immunotherapy. As the current era of single-cell omics is reshaping our understanding of the hematopoietic process and the subcluster landscape of lung TIMs, we aim to present an updated overview of the hierarchical differentiation process of TIMs within the BMN of solid cancer bearing subjects. Our comprehensive overview underscores that lung cancer should be regarded as a systemic disease in which the cues governing the lung tumor-BMN crosstalk might bolster the definition of new biomarkers and druggable targets, potentially mitigating the high attrition rate of leading immunotherapies for NSCLC., 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 © 2024 Calderon-Espinosa, De Ridder, Benoot, Jansen, Vanhonacker, Heestermans, De Becker, Van Riet, Decoster and Goyvaerts.)

To investigate the role of co-stimulatory and co-inhibitory molecules on immune tolerance in immune thrombocytopenia (ITP), this study mapped the immune cell heterogeneity in the bone marrow of ITP at the single-cell level using Cytometry by Time of Flight (CyTOF). Thirty-six patients with ITP and nine healthy volunteers were enrolled in the study. As soluble immunomodulatory molecules, more sCD25 and sGalectin-9 were detected in ITP patients. On the cell surface, co-stimulatory molecules like ICOS and HVEM were observed to be upregulated in mainly central memory and effector T cells. In contrast, co-inhibitory molecules such as CTLA-4 were significantly reduced in Th1 and Th17 cell subsets. Taking a platelet count of 30×10 9 L -1 as the cutoff value, ITP patients with high and low platelet counts showed different T cell immune profiles. Antigen-presenting cells such as monocytes and B cells may regulate the activation of T cells through CTLA-4/CD86 and HVEM/BTLA interactions, respectively, and participate in the pathogenesis of ITP. In conclusion, the proteomic and soluble molecular profiles brought insight into the interaction and modulation of immune cells in the bone marrow of ITP. They may offer novel targets to develop personalized immunotherapies., (© 2024. Science China Press.)

The prognosis of patients with peripheral T-cell lymphoma (PTCL) depends on bone marrow involvement (BMI). The bone marrow (BM) tumor microenvironment in PTCL remains unclear. We performed single-cell RNA sequencing (scRNA-seq) on 11 fresh BM samples from patients with BMI to reveal the associations of immune landscape and genetic variations with the prognosis of PTCL patients. Compared with PTCL not otherwise specified (NOS), angioimmunoblastic T-cell lymphoma (AITL) had a higher number of T cells, lower number of lymphocytes, and greater inflammation. Immune heterogeneity in AITL is associated with prognosis. In particular, specific T-cell receptor (TCR) T cells are enriched in patients with good response to anti-CD30 therapy. We observed RhoA mutation-associated neoantigens. Chidamide-treated patients had a higher number of CD4+ regulatory cells and a better treatment response compared with other patients. In the nonresponder group, T-cell enrichment progressed to secondary B-cell enrichment and subsequently diffuse large B-cell lymphoma. Moreover, AITL patients with lymphoma-associated hemophagocytic syndrome had more T follicular helper (Tfh) cells with copy number variations in CHR5. To our knowledge, this study is the first to reveal the single-cell landscape of BM microenvironment heterogeneity in PTCL patients with BMI. scRNA-seq can be used to investigate the immune heterogeneity and genetic variations in AITL associated with prognosis., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

In this study, we try to find the pathogenic role of immune-related genes in the bone marrow microenvironment of AML. Through WGCNA, seven modules were obtained, among which the turquoise module containing 1793 genes was highly correlated with the immune infiltration score. By unsupervised clustering, the turquoise module was divided into two clusters: the intersection of clinically significant genes in the TCGA and DEGs to obtain 178 genes for mutation analysis, followed by obtaining 17 genes with high mutation frequency. Subsequently, these 17 genes were subjected to LASSO regression analysis to construct a riskscore model of 8 hub genes. The TIMER database, ImmuCellAI portal website, and ssGSEA elucidate that the hub genes and risk scores are closely related to immune cell infiltration into the bone marrow microenvironment. In addition, we also validated the relative expression levels of hub genes using the TCGA database and GSE114868, and additional expression levels of hub genes in AML cell lines in vitro . Therefore, we constructed an immune infiltration-related gene model that identify 8 hub genes with good risk stratification and predictive prognosis for AML., 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 © 2024 Yu and Jiang.)

Innate lymphoid cells (ILCs) are largely tissue-resident, mostly described within the mucosal tissues. However, their presence and functions in the human draining lymph nodes (LNs) are unknown. Our study unravels the tissue-specific transcriptional profiles of 47,287 CD127 + ILCs within the human abdominal and thoracic LNs. LNs contain a higher frequency of CD127 + ILCs than in BM or spleen. We define independent stages of ILC development, including EILP and pILC in the BM. These progenitors exist in LNs in addition to naïve ILCs (nILCs) that can differentiate into mature ILCs. We define three ILC1 and four ILC3 sub-clusters in the LNs. ILC1 and ILC3 subsets have clusters with high heat shock protein-encoding genes. We identify previously unrecognized regulons, including the BACH2 family for ILC1 and the ATF family for ILC3. Our study is the comprehensive characterization of ILCs in LNs, providing an in-depth understanding of ILC-mediated immunity in humans., (© 2024. The Author(s).)

Iberdomide is a potent cereblon E3 ligase modulator (CELMoD agent) with promising efficacy and safety as a monotherapy or in combination with other therapies in patients with relapsed/refractory multiple myeloma (RRMM). Using a custom mass cytometry panel designed for large-scale immunophenotyping of the bone marrow tumor microenvironment (TME), we demonstrate significant increases of effector T and natural killer (NK) cells in a cohort of 93 patients with multiple myeloma (MM) treated with iberdomide, correlating findings to disease characteristics, prior therapy, and a peripheral blood immune phenotype. Notably, changes are dose dependent, associated with objective response, and independent of prior refractoriness to MM therapies. This suggests that iberdomide broadly induces innate and adaptive immune activation in the TME, contributing to its antitumor efficacy. Our approach establishes a strategy to study treatment-induced changes in the TME of patients with MM and, more broadly, patients with cancer and establishes rational combination strategies for iberdomide with immune-enhancing therapies to treat MM., Competing Interests: Declaration of interests This research was sponsored and funded by Bristol Myers Squibb. M.A., E.F., W.E.P., and A.T. were employees of Bristol Myers Squibb at the time of the analysis and conception of the study. S.J. reports consulting fees for Bristol Myers Squibb (Celgene), Janssen, Karyopharm Therapeutics, Merck, Sanofi, and Takeda Pharmaceuticals. S.P. reports research funding from Bristol Myers Squibb (Celgene), Karyopharm, and Amgen, as well as consulting fees from Foundation Medicine., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid autoimmunity. The specific contribution of apoptosis to normal B cell development and self-tolerance is incompletely understood. Here, we quantify self-reactivity, polyreactivity and apoptosis during physiologic B lymphocyte development. Self-reactivity and polyreactivity are most abundant in early immature B cells and diminish significantly during maturation within the bone marrow. Minimal apoptosis still occurs at this site, however B cell receptors cloned from apoptotic B cells show comparable self-reactivity to that of viable cells. Apoptosis increases dramatically only following immature B cells leaving the bone marrow sinusoids, but above 90% of cloned apoptotic transitional B cells are not self-reactive/polyreactive. Our data suggests that an apoptosis-independent mechanism, such as receptor editing, removes most self-reactive B cells in the bone marrow. Mechanistically, lack of survival signaling rather than clonal deletion appears to be the underpinning cause of apoptosis in most transitional B cells in the periphery., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19 low -BMPC are derived from follicular, while CD19 high -BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19 low - and CD19 high -BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19 high -BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response., (© 2024. The Author(s).)

The recent identification of skull bone marrow as a reactive hematopoietic niche that can contribute to and direct leukocyte trafficking into the meninges and brain has transformed our view of this bone structure from a solid, protective casing to a living, dynamic tissue poised to modulate brain homeostasis and neuroinflammation. This emerging concept may be highly relevant to injuries that directly impact the skull such as in traumatic brain injury (TBI). From mild concussion to severe contusion with skull fracturing, the bone marrow response of this local myeloid cell reservoir has the potential to impact not just the acute inflammatory response in the brain, but also the remodeling of the calvarium itself, influencing its response to future head impacts. If we borrow understanding from recent discoveries in other CNS immunological niches and extend them to this nascent, but growing, subfield of neuroimmunology, it is not unreasonable to consider the hematopoietic compartment in the skull may similarly play an important role in health, aging, and neurodegenerative disease following TBI. This literature review briefly summarizes the traditional role of the skull in TBI and offers some additional insights into skull-brain interactions and their potential role in affecting secondary neuroinflammation and injury outcomes., 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 © 2024 Goodman, Devlin, West and Ritzel.)

There is increasing interest in how immune cells in the meninges-the membranes that surround the brain and spinal cord-contribute to homeostasis and disease in the central nervous system 1,2 . The outer layer of the meninges, the dura mater, has recently been described to contain both innate and adaptive immune cells, and functions as a site for B cell development 3-6 . Here we identify organized lymphoid structures that protect fenestrated vasculature in the dura mater. The most elaborate of these dural-associated lymphoid tissues (DALT) surrounded the rostral-rhinal confluence of the sinuses and included lymphatic vessels. We termed this structure, which interfaces with the skull bone marrow and a comparable venous plexus at the skull base, the rostral-rhinal venolymphatic hub. Immune aggregates were present in DALT during homeostasis and expanded with age or after challenge with systemic or nasal antigens. DALT contain germinal centre B cells and support the generation of somatically mutated, antibody-producing cells in response to a nasal pathogen challenge. Inhibition of lymphocyte entry into the rostral-rhinal hub at the time of nasal viral challenge abrogated the generation of germinal centre B cells and class-switched plasma cells, as did perturbation of B-T cell interactions. These data demonstrate a lymphoid structure around vasculature in the dura mater that can sample antigens and rapidly support humoral immune responses after local pathogen challenge., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir., (© 2022. The Author(s).)

Disability and distress caused by chronic low back pain (LBP) lacking clear pathoanatomical explanations cause huge problems both for patients and society. A subgroup of patients has Modic changes (MC), identifiable by MRI as vertebral bone marrow lesions. The cause of such changes and their relationship to pain are not yet understood. We explored the pathobiology of these lesions using profiling of gene expression in blood, coupled with an edema-sensitive MRI technique known as short tau inversion recovery (STIR) imaging. STIR images and total RNA from blood were collected from 96 patients with chronic LBP and MC type I, the most inflammatory MC state. We found the expression of 37 genes significantly associated with STIR signal volume, ten genes with edema abundancy (a constructed combination of STIR signal volume, height, and intensity), and one gene with expression levels significantly associated with maximum STIR signal intensity. Gene sets related to interferon signaling, mitochondrial metabolism and defense response to virus were identified as significantly enriched among the upregulated genes in all three analyses. Our results point to inflammation and immunological defense as important players in MC biology in patients with chronic LBP., (© 2022. The Author(s).)

B-cell development undergoes a series of steps from the bone marrow to the secondary lymphoid organs. A defect in B-cell development can lead to immunodeficiency or malignant disorders, such as leukaemia or lymphoma. Long non-coding RNAs have been reported to act as important regulators of many pathological processes. However, very little is known regarding the role of lncRNAs during B-cell development and the regulation of their expression. In this study, we explored the expression and role of lncRNA Gme00492 in B-cell development. We observed that lnc00492 was highly expressed in B-cell development and primarily expressed in the nucleus. Lnc00492-deficient mice had fewer marginal zone B cells in the spleen, likely due to a developmental block. Importantly, lnc00492 interacts with CTBP1 and targets it for ubiquitination and degradation during B-cell development, whereas the transcriptional corepressor factor CTBP1 plays a critical role in Notch2 signalling. Thus, we identified a novel regulatory axis between lnc00492 and CTBP1 in B cells, suggesting that lnc00492 is essential for marginal zone B-cell development., (© 2021 John Wiley & Sons Ltd.)

Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High-dimensional single-cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1-polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune-related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1-polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a key role in cancer immunosurveillance thanks to their ability to recognize and kill cancer cells. NKG2D is an activating receptor that binds to MIC and ULBP molecules typically induced on damaged, transformed or infected cells. The release of NKG2D ligands (NKG2DLs) in the extracellular milieu through protease-mediated cleavage or by extracellular vesicle (EV) secretion allows cancer cells to evade NKG2D-mediated immunosurveillance. In this work, we investigated the immunomodulatory properties of the NKG2D ligand MICA*008 associated to distinct populations of EVs (i.e., small extracellular vesicles [sEVs] and medium size extracellular vesicles [mEVs]). By using as model a human MICA*008-transfected multiple myeloma (MM) cell line, we found that this ligand is present on both vesicle populations. Interestingly, our findings reveal that NKG2D is specifically involved in the uptake of vesicles expressing its cognate ligand. We provide evidence that MICA*008-expressing sEVs and mEVs are able on one hand to activate NK cells but, following prolonged stimulation induce a sustained NKG2D downmodulation leading to impaired NKG2D-mediated functions. Moreover, our findings show that MICA*008 can be transferred by vesicles to NK cells causing fratricide. Focusing on MM as a clinically and biologically relevant model of tumour-NK cell interactions, we found enrichment of EVs expressing MICA in the bone marrow of a cohort of patients. All together our results suggest that the accumulation of NKG2D ligands associated to vesicles in the tumour microenvironment could favour the suppression of NK cell activity either by NKG2D down-modulation or by fratricide of NK cell dressed with EV-derived NKG2D ligands., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell genetic mutation disease that causes defective erythrocyte membrane hemolysis. Its pathologic basis is the mutation of the PIG-A gene, whose product is necessary for the synthesis of glycosylphosphatidylinositol (GPI) anchors; the mutation of PIG-A gene results in the reduction or deletion of the GPI anchor, which leads to the deficiency of GPI-anchored proteins (GPI-APs), such as CD55 and CD59, which are complement inhibitors. The deficiency of complement inhibitors causes chronic complement-mediated intravascular hemolysis of GPI-anchor-deficient erythrocyte. PIG-A gene mutation could also be found in bone marrow hematopoietic stem cells (HSCs) of healthy people, but they have no growth advantage; only the HSCs with PIG-A gene mutation in PNH patients have this advantage and expand. Besides, HSCs from PIG-A -knockout mice do not show clonal expansion in bone marrow, so PIG-A mutation cannot explain the clonal advantage of the PNH clone and some additional factors are needed; thus, in recent years, many scholars have put forward the theories of the second hit, and immune escape theory is one of them. In this paper, we focus on how T lymphocytes are involved in immune escape hypothesis in the pathogenesis of PNH., 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 Li, Dong, Wang and Shao.)

Neutrophils (polymorphonuclear cells; PMNs) form a first line of defense against pathogens and are therefore an important component of the innate immune response. As a result of poorly controlled activation, however, PMNs can also mediate tissue damage in numerous diseases, often by increasing tissue inflammation and injury. According to current knowledge, PMNs are not only part of the pathogenesis of infectious and autoimmune diseases but also of conditions with disturbed tissue homeostasis such as trauma and shock. Scientific advances in the past two decades have changed the role of neutrophils from that of solely immune defense cells to cells that are responsible for the general integrity of the body, even in the absence of pathogens. To better understand PMN function in the human organism, our review outlines the role of PMNs within the innate immune system. This review provides an overview of the migration of PMNs from the vascular compartment to the target tissue as well as their chemotactic processes and illuminates crucial neutrophil immune properties at the site of the lesion. The review is focused on the formation of chemotactic gradients in interaction with the extracellular matrix (ECM) and the influence of the ECM on PMN function. In addition, our review summarizes current knowledge about the phenomenon of bidirectional and reverse PMN migration, neutrophil microtubules, and the microtubule organizing center in PMN migration. As a conclusive feature, we review and discuss new findings about neutrophil behavior in cancer environment and tumor tissue., 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 Kraus and Gruber.)

Neutrophils are key pathogen exterminators of the innate immune system endowed with oxidative and non-oxidative defense mechanisms. More recently, a more complex role for neutrophils as decision shaping cells that instruct other leukocytes to fine-tune innate and adaptive immune responses has come into view. Under homeostatic conditions, neutrophils are short-lived cells that are continuously released from the bone marrow. Their development starts with undifferentiated hematopoietic stem cells that pass through different immature subtypes to eventually become fully equipped, mature neutrophils capable of launching fast and robust immune responses. During severe (systemic) inflammation, there is an increased need for neutrophils. The hematopoietic system rapidly adapts to this increased demand by switching from steady-state blood cell production to emergency granulopoiesis. During emergency granulopoiesis, the de novo production of neutrophils by the bone marrow and at extramedullary sites is augmented, while additional mature neutrophils are rapidly released from the marginated pools. Although neutrophils are indispensable for host protection against microorganisms, excessive activation causes tissue damage in neutrophil-rich diseases. Therefore, tight regulation of neutrophil homeostasis is imperative. In this review, we discuss the kinetics of neutrophil ontogenesis in homeostatic conditions and during emergency myelopoiesis and provide an overview of the different molecular players involved in this regulation. We substantiate this review with the example of an autoinflammatory disease, i.e. systemic juvenile idiopathic arthritis., Competing Interests: CW obtained unrestricted grants to KU Leuven from Novartis, Roche, GSK immuno-inflammation and Pfizer. 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 Malengier-Devlies, Metzemaekers, Wouters, Proost and Matthys.)

Objectives: A new regulatory subpopulation of innate lymphoid cells (ILCs), regulatory innate lymphoid cells (ILCregs), has been identified with both innate lymphoid cells and regulatory cells characteristics. The purpose of this study is to explore ILCregs and its associated miRNAs in patients with aplastic anemia (AA) by evaluating ILCregs frequency, associated miRNA quantification, and their significance., Methods: Using 4 color combinations of surface and intracellular antibody staining, the CD45+Lin-CD127+IL-10+ ILCregs from 30 healthy donors and 30 patients newly diagnosed with AA were measured by flow cytometry. Bone marrow cells were studied by next-generation sequence miRNAs quantification., Results: Our results showed that the frequency of ILCregs in bone marrow cells from healthy donors (HD) and AA patients were 0.703 ± 0.941 and 0.171 ± 0.233%, respectively. The frequencies of ILCregs in AA patients were significantly lower than that in HD ( p <0.05). miRNA detection results showed different expression patterns in the AA patient group comparing with HD. Comparing with HD, there were 52 miRNAs up-regulated and 130 miRNAs down-regulated from AA patients. Analysis of miRNAs from ILCregs associated genes demonstrated different miRNAs expression patterns between HD and AA patient., Conclusion: The present study demonstrated the deficiency of ILCregs and differential expression pattern of ILCregs gene-related miRNA in patients with AA. Further studies need to be done to explore the clinical significance of ILCregs and related miRNAs in patients with AA with large samples size and clinical follow-up.

Virtually all patients with multiple myeloma become unresponsive to treatment over time. Relapsed/refractory multiple myeloma (RRMM) is accompanied by the clonal evolution of myeloma cells with heterogeneous genomic aberrations and profound changes of the bone marrow microenvironment (BME). However, the molecular mechanisms that drive drug resistance remain elusive. Here, we analyze the heterogeneous tumor cell population and its complex interaction network with the BME of 20 RRMM patients by single cell RNA-sequencing before/after treatment. Subclones with chromosome 1q-gain express a specific transcriptomic signature and frequently expand during treatment. Furthermore, RRMM cells shape an immune suppressive BME by upregulation of inflammatory cytokines and close interaction with the myeloid compartment. It is characterized by the accumulation of PD1 + γδ T-cells and tumor-associated macrophages as well as the depletion of hematopoietic progenitors. Thus, our study resolves transcriptional features of subclones in RRMM and mechanisms of microenvironmental reprogramming with implications for clinical decision-making., (© 2021. The Author(s).)

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.

Most lineages of blood cells, including immune cells, are generated from hematopoietic stem cells (HSCs) in bone marrow throughout adult life. Since HSCs cannot expand on their own, they require and contact the special microenvironments, termed niches for their maintenance. HSC niches comprise supportive cells that provide adjacent HSCs with critical signals, including cytokines. Although bone marrow microenvironments have been thought to be complex, recent studies have demonstrated that the bone marrow-specific population of fibroblastic reticular cells with long processes, termed CXC chemokine ligand 12 (CXCL12)-abundant reticular (CAR) cells, which overlap strongly with leptin receptor (LepR)-expressing (LepR+) cells, is the major cellular component of niches for HSCs and lymphoid progenitors. CAR cells have salient features, expressing much higher levels of critical HSC niche factors than any other cell populations and function as self-renewing mesenchymal stem cells. Human counterpart of CAR cells is present and affected in diseases, including leukemia. Foxl1+ telocytes recently identified as the niche for intestinal stem cells share some features with CAR cells, suggesting that CAR cells might serve as a prototype for fibroblastic reticular cells creating niche for long-lived cells, including tissue stem cells and memory lymphocytes. These findings provided the basis for future mechanistic studies on the cross-talk between hematopoietic cells and microenvironments in both health and disease., (© The Japanese Society for Immunology. 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acute lymphoblastic leukemia (ALL) is a malignancy with high heterogeneity in its biological features and treatments. Although the overall survival (OS) of patients with ALL has recently improved considerably, owing to the application of conventional chemo-therapeutic agents, approximately 20% of the pediatric cases and 40-50% of the adult patients relapse during and after the treatment period. The potential mechanisms that cause relapse involve clonal evolution, innate and acquired chemoresistance, and the ability of ALL cells to escape the immune-suppressive tumor response. Currently, immunotherapy in combination with conventional treatment is used to enhance the immune response against tumor cells, thereby significantly improving the OS in patients with ALL. Therefore, understanding the mechanisms of immune evasion by leukemia cells could be useful for developing novel therapeutic strategies., 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 Jiménez-Morales, Aranda-Uribe, Pérez-Amado, Ramírez-Bello and Hidalgo-Miranda.)

The expression level of BCMA in bone marrow of 54 MM patients was detected in this study to explore the relationship between the BCMA expression and the classification, stage, and prognostic factors of MM. The BCMA expression level of the stable group and remission group was lower than that of the newly diagnosed group and relapse group (P=0.001). There was no significant difference in BCMA expression of MM patients in different types and stages (P>0.05), but it was found that for the newly diagnosed MM patients, the BCMA expression level of IgG patients was higher than that of IgA or light-chain patients (rank average 11.20 vs 5.44, P=0.014). There was no significant correlation between the BCMA expression and the age and serum creatinine of MM patients (P>0.05). And there was no significant difference in BCMA expression between patients with different levels of age and serum creatinine (P>0.05). But it was found that the BCMA expression level of the newly diagnosed MM patients was moderately positively correlated with their age (P=0.025, r=0.595). There was no significant correlation between the BCMA expression and serum β2-microglobulin, serum lactate dehydrogenase, free kap/lam ratio, and urine β2-microglobulin (P>0.05). But we found that the BCMA expression of patients with high serum β2-microglobulin was higher than that of patients with low serum β2-microglobulin (rank average 28.89 vs 17.54, P=0.017). And the BCMA expression of patients with abnormal serum free kap/lam ratio was higher than that of patients with normal ratio (rank average 28.49 vs 13.55, P=0.004). The BCMA expression was strongly positively correlated with 24-h urine protein, was moderately positively correlated with serum M protein and the percentage of plasma cells in bone marrow, was moderately negatively correlated with albumin and hemoglobin count, and was weakly positively correlated with serum corrected calcium (P<0.05). And it was found that the BCMA expression of positive serum immunofixation electrophoresis patients was higher than that of negative patients (rank average 29.94 vs 16.75, P=0.017). And we try to clarify the relationship between the bone marrow BCMA expression and the peripheral blood sBCMA expression. However, we have not found a clear correlation between them so far (P>0.05)., 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 Ma, Shi, Xiao, Bian, Wang, Hui, Wang and Liu.)

The development of new laser-driven electron linear accelerators, providing unique ultrashort pulsed electron beams (UPEBs) with low repetition rates, opens new opportunities for radiotherapy and new fronts for radiobiological research in general. Considering the growing interest in the application of UPEBs in radiation biology and medicine, the aim of this study was to reveal the changes in immune system in response to low-energy laser-driven UPEB whole-body irradiation in rodents. Forty male albino Wistar rats were exposed to laser-driven UPEB irradiation, after which different immunological parameters were studied on the 1st, 3rd, 7th, 14th, and 28th day after irradiation. According to the results, this type of irradiation induces alterations in the rat immune system, particularly by increasing the production of pro- and anti-inflammatory cytokines and elevating the DNA damage rate. Moreover, such an immune response reaches its maximal levels on the third day after laser-driven UPEB whole-body irradiation, showing partial recovery on subsequent days with a total recovery on the 28th day. The results of this study provide valuable insight into the effect of laser-driven UPEB whole-body irradiation on the immune system of the animals and support further animal experiments on the role of this novel type of irradiation.

In contrast to the curative effect of allogenic stem cell transplantation in acute myeloid leukemia via T cell activity, only modest responses are achieved with checkpoint-blockade therapy, which might be explained by T cell phenotypes and T cell receptor (TCR) repertoires. Here, we show by paired single-cell RNA analysis and TCR repertoire profiling of bone marrow cells in relapsed/refractory acute myeloid leukemia patients pre/post azacytidine+nivolumab treatment that the disease-related T cell subsets are highly heterogeneous, and their abundance changes following PD-1 blockade-based treatment. TCR repertoires expand and primarily emerge from CD8 + cells in patients responding to treatment or having a stable disease, while TCR repertoires contract in therapy - resistant patients. Trajectory analysis reveals a continuum of CD8 + T cell phenotypes, characterized by differential expression of granzyme B and a bone marrow-residing memory CD8 + T cell subset, in which a population with stem-like properties expressing granzyme K is enriched in responders. Chromosome 7/7q loss, on the other hand, is a cancer-intrinsic genomic marker of PD-1 blockade resistance in AML. In summary, our study reveals that adaptive T cell plasticity and genomic alterations determine responses to PD-1 blockade in acute myeloid leukemia., (© 2021. The Author(s).)

Chronic myeloid leukemia (CML) is a form of myeloproliferative neoplasm caused by the oncogenic tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitors have dramatically improved the prognosis of patients with CML, several problems such as resistance and recurrence still exist. Immunological control may contribute to solving these problems, and it is important to understand why CML patients fail to spontaneously develop anti-tumor immunity. Here, we show that differentiation of conventional dendritic cells (cDCs), which are vital for anti-tumor immunity, is restricted from an early stage of hematopoiesis in CML. In addition, we found that monocytes and basophils, which are increased in CML patients, express high levels of PD-L1, an immune checkpoint molecule that inhibits T cell responses. Moreover, RNA-sequencing analysis revealed that basophils express genes related to poor prognosis in CML. Our data suggest that BCR-ABL not only disrupts the "accelerator" (i.e., cDCs) but also applies the "brake" (i.e., monocytes and basophils) of anti-tumor immunity, compromising the defense against CML cells., (© 2021. The Author(s).)

A growing body of evidence suggests that innate immune cells can respond in a memory-like (adaptive) fashion, which is referred to as trained immunity. Only few in vivo studies have shown training effects in neutrophils; however, no in vitro setup has been established to study the induction of trained immunity or tolerance in neutrophils by microbial agents. In light of their short lifespan (up to 48 h), we suggest to use the term trained sensitivity for neutrophils in an in vitro setting. Here, we firstly describe a feasible two-hit model, using different doses of lipopolysaccharide (LPS) in bone marrow neutrophils. We found that low doses (10 pg/mL) induce pro-inflammatory activation (trained sensitivity), whereas priming with high doses (100 ng/mL) leads to suppression of pro-inflammatory mediators such as TNF-α or IL-6 (tolerance) ( p < 0.05). On a functional level, trained neutrophils displayed increased phagocytic activity and LFA-1 expression as well as migrational capacity and CD11a expression, whereas tolerant neutrophils show contrasting effects in vitro. Mechanistically, TLR4/MyD88/PI3Ks regulate the activation of p65, which controls memory-like responses in mouse bone marrow neutrophils ( p < 0.05). Our results open a new window for further in vitro studies on memory-like inflammatory responses of short-lived innate immune cells such as neutrophils.

Ab repertoire diversity plays a critical role in the host's ability to fight pathogens. CDR3 is partially responsible for Ab-Ag binding and is a significant source of diversity in the repertoire. CDR3 diversity is generated during VDJ rearrangement because of gene segment selection, gene segment trimming and splicing, and the addition of nucleotides. We analyzed the Ab repertoire diversity across multiple experiments examining the effects of spaceflight on the Ab repertoire after vaccination. Five datasets from four experiments were analyzed using rank-abundance curves and Shannon indices as measures of diversity. We discovered a trend toward lower diversity as a result of spaceflight but did not find the same decrease in our physiological model of microgravity in either the spleen or bone marrow. However, the bone marrow repertoire showed a reduction in diversity after vaccination. We also detected differences in Shannon indices between experiments and tissues. We did not detect a pattern of CDR3 usage across the experiments. Overall, we were able to find differences in the Ab repertoire diversity across experimental groups and tissues., (Copyright © 2021 The Authors.)

The intracellular fragment of Notch1, a mediator of Notch signaling that is frequently detected in thymic immigrants, is critical for specifying T-cell fate in the thymus, where Delta-like 4 (Dll4) functions as a Notch ligand on the epithelium. However, as such Notch signaling has not been detected in mature T cells, how Notch signaling contributes to their response in secondary lymphoid organs has not yet been fully defined. Here, we detected the marked expression of Dll4 on the stromal cells and the active fragment of Notch1 (Notch1 intracellular domain, N1ICD) in CD4+ T cells in the follicles of Peyer's patches (PPs). In addition, N1ICD-bearing T cells were found in the T-cell zone of PPs, especially in the transcription factor Foxp3+ regulatory T (Treg) cells, with slight expression of Dll4 on the stromal cells. These fragments disappeared in Dll4-deficient conditions. It was also found that Notch1- and Notch2-deficient T cells preferentially differentiated into Treg cells in PPs, but not CXCR5+PD-1+ follicular helper T (Tfh) cells. Moreover, these phenotypes were also observed in chimeric mice reconstituted with the control and T-cell-specific Notch1/2-deficient bone marrow or Treg cells. These results demonstrated that Dll4-mediated Notch signaling in PPs is required for the efficient appearance of Tfh cells in a Treg cell-prone environment, which is common among the gut-associated lymphoid tissues, and is critical for the generation of Tfh-mediated germinal center B cells., (© The Japanese Society for Immunology. 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Adoptive cell therapy (ACT) using tumor-reactive T cells is a promising form of immunotherapy to specifically target cancer. However, the survival and functional maintenance of adoptively transferred T cells remains a challenge, ultimately limiting their efficacy. Here, we evaluated the use of recombinant IL7-Fc in ACT. In a lymphopenic murine melanoma model, IL7-Fc treatment led to the enhanced inhibition of tumor growth with an increased number of adoptively transferred CD8 + T cells in tumor tissue and tumor-draining lymph nodes. Additionally, IL7-Fc further enhanced anti-tumor responses that were induced by recombinant human IL2 in the same mouse model. In contrast, in an immunocompetent murine melanoma model, IL7-Fc dampened the anti-tumor immunity. Further, IL7-Fc decreased the proliferation of adoptively transferred and immune-activated tumor-reactive CD8 + T cells in immunocompetent mice by inducing the massive expansion of endogenous T cells, thereby limiting the space for adoptively transferred T cells. Our data suggest that IL7-Fc is principally beneficial for enhancing the efficacy of tumor-reactive T-cells in lymphopenic conditions for the ACT.

Megakaryocytes (MKs) and their progeny platelets function in a variety of biological processes including coagulation, hemostasis, inflammation, angiogenesis, and innate immunity. However, the divergent developmental and cellular landscape of adult MKs remains mysterious. Here, by deriving the single-cell transcriptomic profiling of MKs from human adult bone marrow (BM), cellular heterogeneity within MKs is unveiled and an MK subpopulation with high enrichment of immune-associated genes is identified. By performing the dynamic single-cell transcriptomic landscape of human megakaryopoiesis in vitro, it is found that the immune signatures of MKs can be traced back to the progenitor stage. Furthermore, two surface markers, CD148 and CD48, are identified for mature MKs with immune characteristics. At the functional level, these CD148 + CD48 + MKs can respond rapidly to immune stimuli both in vitro and in vivo, exhibit high-level expression of immune receptors and mediators, and may function as immune-surveillance cells. The findings uncover the cellular heterogeneity and a novel immune subset of human adult MKs and should greatly facilitate the understanding of the divergent functions of MKs under physiological and pathological conditions., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy. The two-step BCP-ALL pathogenesis requires in utero-induced chromosomal aberrations and additional mutagenic events for overt leukemia. In mouse models, activation-induced cytidine deaminase (AID/AICDA) was suggested to contribute to BCP-ALL pathogenesis by off-target mutagenic activity. The role of AID in patients, however, remains unclear. Moreover, AID is usually not expressed in precursor B-cells but in germinal center B-cells, where it is induced upon T-helper (Th) cell stimulation. We have previously demonstrated that autologous Th-cells supportively interacted with BCP-ALL-cells. Here, we hypothesize that this interaction additionally induces AID expression in BCP-ALL-cells, leading to off-target mutagenic activity. We show that co-culture with autologous bone marrow Th-cells induced high AICDA expression in primary BCP-ALL-cells. This induction was mediated by a mechanism similar to the induction in mature B-cells involving IL-13/Stat6, CD40L/NF-κB and TGFβ/Smad2/3 signaling. Even though Th-cell-induced AID seemed to be active in vitro in a BCP-ALL reporter cell line, extensive mutational signature analysis revealed no major contribution of AID activity to the mutational landscape in BCP-ALL patients. AID activity was neither detected in mutation clusters nor in known AID targets. Moreover, no recurrently mutated gene showed a relevant enrichment of mutations in the AID motif. Together, the lack of AID-induced mutational consequences argues towards a Th-cell-promoted yet AID-independent BCP-ALL pathogenesis and favors therapeutic research focusing on Th-cell-derived support of BCP-ALL-cells rather than AID-induced effects., (© 2021. The Author(s).)

Background/aim: Liver metastases are among the principal mortality causes in cancer patients. Dendritic cell immunotherapies have shown promising results in some tumors by mediating immunological mechanisms that could be involved in liver metastases during primary tumor growth. The present study aimed to evaluate the impact of prophylactic dendritic cell vaccination on the liver of mice with 4T1 mouse breast carcinoma., Materials and Methods: Adult female Balb/c mice were submitted or not to vaccination with dendritic cells before the induction of 4T1 tumor lineage. Liver tissues from mice were analyzed by flow cytometry (markers CD3, CD4, CD8, CD25, IL-10, IL-12, IL-17, TNF-α, IFN-γ, T-bet, GATA3, RORγt, and FoxP3) and hematoxylin-eosin. The dendritic cell vaccine was differentiated and matured ex vivo from the bone marrow., Results: Prophylactic vaccination reduced areas of liver metastases (p=0.0049), induced an increase in the percentage of total T and cytotoxic T lymphocytes (p<0.0001), as well as FoxP3 + (p<0.0001). It also increased the levels of cytokines IL-10 and IL-17 in helper T lymphocytes (p<0.0001)., Conclusion: The prophylactic dendritic cell vaccine changed the cell phenotype in the immune response of liver, and it was able to reduce metastases. Cytotoxic T cells and regulatory T lymphocytes were more present, likewise, the production of IL-10 and IL-7 simultaneously, demonstrating that the vaccine can induce a state of control of pro-inflammatory responses, which can provide a less favorable environment for metastatic tumor growth., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

High expression of the inhibitory receptor programmed cell death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found to play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the programmed cell death 1 (PD-1)/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in multiple myeloma (MM) remains poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSC-mediated regulation of CD8 + T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased compared to that in normal controls (NC) (18·81 ± 1·61 versus 2·78± 0·70%; P < 0·001). Furthermore, the PD-1 expression on CD8 + T cells with NDMM patients was significantly higher than that in normal controls (43·22 ± 2·98 versus 20·71 ± 1·08%; P < 0·001). However, there was no significant difference in PD-1 expression of CD4 + T cells and natural killer (NK) cells between the NDMM and NC groups. Additionally, the co-culture assays revealed that BMSCs significantly suppressed CD8 + T cell function. However, the PD-L1 inhibitor effectively reversed BMSC-mediated suppression in CD8 + T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8 + T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8 + T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8 + T cells to promote the immune escape of MM., (© 2021 British Society for Immunology.)

Natural killer (NK) cells are prominent cytotoxic and cytokine-producing components of the innate immune system representing crucial effector cells in cancer immunotherapy. Presently, various NK cell-based immunotherapies have contributed to the substantial improvement in the reconstitution of NK cells against advanced-staged and high-risk AML. Various NK cell sources, including haploidentical NK cells, adaptive NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, chimeric antigen receptor NK cells, cytokine-induced memory-like NK cells, and NK cell lines have been identified. Devising innovative approaches to improve the generation of therapeutic NK cells from the aforementioned sources is likely to enhance NK cell expansion and activation, stimulate ex vivo and in vivo persistence of NK cells and improve conventional treatment response of myeloid leukemia. The tumor-promoting properties of the tumor microenvironment and downmodulation of NK cellular metabolic activity in solid tumors and hematological malignancies constitute a significant impediment in enhancing the anti-tumor effects of NK cells. In this review, we discuss the current NK cell sources, highlight ongoing interventions in enhancing NK cell function, and outline novel strategies to circumvent immunosuppressive factors in the tumor microenvironment to improve the efficacy of NK cell-based immunotherapy and expand their future success in treating myeloid leukemia., 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 Kaweme and Zhou.)

Chimeric Antigen Receptor (CAR) T-cell therapy has demonstrated high rates of response in recurrent B-cell Acute Lymphoblastic Leukemia in children and young adults. Despite this success, a fraction of patients' experience relapse after treatment. Relapse is often preceded by recovery of healthy B cells, which suggests loss or dysfunction of CAR T-cells in bone marrow. This site is harder to access, and thus is not monitored as frequently as peripheral blood. Understanding the interplay between B cells, leukemic cells, and CAR T-cells in bone marrow is paramount in ascertaining the causes of lack of response. In this paper, we put forward a mathematical model representing the interaction between constantly renewing B cells, CAR T-cells, and leukemic cells in the bone marrow. Our model accounts for the maturation dynamics of B cells and incorporates effector and memory CAR T-cells. The model provides a plausible description of the dynamics of the various cellular compartments in bone marrow after CAR T infusion. After exploration of the parameter space, we found that the dynamics of CAR T product and disease were independent of the dose injected, initial B-cell load, and leukemia burden. We also show theoretically the importance of CAR T product attributes in determining therapy outcome, and have studied a variety of possible response scenarios, including second dosage schemes. We conclude by setting out ideas for the refinement of the model.

Group 2 innate lymphoid cells (ILC2s) are emerging as important cellular regulators of homeostatic and disease-associated immune processes. The cytokine interleukin-33 (IL-33) promotes ILC2-dependent inflammation and immunity, with IL-33 having been shown to activate NF-κB in a wide variety of cell types. However, it is currently unclear which NF-κB members play an important role in IL-33-dependent ILC2 biology. Here, we identify the NF-κB family member c-Rel as a critical component of the IL-33-dependent activation of ILC2s. Although c-Rel is dispensable for ILC2 development, it is critical for ILC2 function in the lung, with c-Rel-deficient ( c-Rel -/- ) mice present a significantly reduced response to papain- and IL-33-induced lung inflammation. We also show that the absence of c-Rel reduces the IL-33-dependent expansion of ILC2 precursors and lower levels of IL-5 and IL-13 cytokine production by mature ILC2s in the lung. Together, these results identify the IL-33-c-Rel axis as a central control point of ILC2 activation and function., 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. The handling editor declared a shared affiliation with the authors at the time of review., (Copyright © 2021 Zaini, Fulford, Grumont, Runting, Rodrigues, Ng, Gerondakis, Zaph and Scheer.)