An IL-1ß-driven neutrophil-stromal cell axis fosters a BAFF-rich protumor microenvironment in individuals with multiple myeloma.

Human bone marrow permanently harbors high numbers of neutrophils, and a tumor-supportive bias of these cells could significantly impact bone marrow-confined malignancies. In individuals with multiple myeloma, the bone marrow is characterized by inflammatory stromal cells with the potential to influence neutrophils. We investigated myeloma-associated alterations in human marrow neutrophils and the impact of stromal inflammation on neutrophil function. Mature neutrophils in myeloma marrow are activated and tumor supportive and transcribe increased levels of IL1B and myeloma cell survival factor TNFSF13B (BAFF). Interactions with inflammatory stromal cells induce neutrophil activation, including BAFF secretion, in a STAT3-dependent manner, and once activated, neutrophils gain the ability to reciprocally induce stromal activation. After first-line myeloid-depleting antimyeloma treatment, human bone marrow retains residual stromal inflammation, and newly formed neutrophils are reactivated. Combined, we identify a neutrophil-stromal cell feed-forward loop driving tumor-supportive inflammation that persists after treatment and warrants novel strategies to target both stromal and immune microenvironments in multiple myeloma.

Bone marrow inflammation in haematological malignancies.

Tissue inflammation is a hallmark of tumour microenvironments. In the bone marrow, tumour-associated inflammation impacts normal niches for haematopoietic progenitor cells and mature immune cells and supports the outgrowth and survival of malignant cells residing in these niche compartments. This Review provides an overview of our current understanding of inflammatory changes in the bone marrow microenvironment of myeloid and lymphoid malignancies, using acute myeloid leukaemia and multiple myeloma as examples and highlights unique and shared features of inflammation in niches for progenitor cells and plasma cells. Importantly, inflammation exerts profoundly different effects on normal bone marrow niches in these malignancies, and we provide context for possible drivers of these divergent effects. We explore the role of tumour cells in inflammatory changes, as well as the role of cellular constituents of normal bone marrow niches, including myeloid cells and stromal cells. Integrating knowledge of disease-specific dynamics of malignancy-associated bone marrow inflammation will provide a necessary framework for future targeting of these processes to improve patient outcome.

A Single-Cell Taxonomy Predicts Inflammatory Niche Remodeling to Drive Tissue Failure and Outcome in Human AML.

Cancer initiation is orchestrated by an interplay between tumor-initiating cells and their stromal/immune environment. Here, by adapted single-cell RNA sequencing, we decipher the predicted signaling between tissue-resident hematopoietic stem/progenitor cells (HSPC) and their neoplastic counterparts with their native niches in the human bone marrow. LEPR+ stromal cells are identified as central regulators of hematopoiesis through predicted interactions with all cells in the marrow. Inflammatory niche remodeling and the resulting deprivation of critical HSPC regulatory factors are predicted to repress high-output hematopoietic stem cell subsets in NPM1-mutated acute myeloid leukemia (AML), with relative resistance of clonal cells. Stromal gene signatures reflective of niche remodeling are associated with reduced relapse rates and favorable outcomes after chemotherapy across all genetic risk categories. Elucidation of the intercellular signaling defining human AML, thus, predicts that inflammatory remodeling of stem cell niches drives tissue repression and clonal selection but may pose a vulnerability for relapse-initiating cells in the context of chemotherapeutic treatment. SIGNIFICANCE: Tumor-promoting inflammation is considered an enabling characteristic of tumorigenesis, but mechanisms remain incompletely understood. By deciphering the predicted signaling between tissue-resident stem cells and their neoplastic counterparts with their environment, we identify inflammatory remodeling of stromal niches as a determinant of normal tissue repression and clinical outcomes in human AML. See related commentary by Lisi-Vega and Méndez-Ferrer, p. 349. This article is featured in Selected Articles from This Issue, p. 337.

Proceedings from the Blood and Marrow Transplant Clinical Trials Network Myeloma Intergroup Workshop on Immune and Cellular Therapy in Multiple Myeloma.

The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Myeloma Intergroup conducted a workshop on Immune and Cellular Therapy in Multiple Myeloma on January 7, 2022. This workshop included presentations by basic, translational, and clinical researchers with expertise in plasma cell dyscrasias. Four main topics were discussed: platforms for myeloma disease evaluation, insights into pathophysiology, therapeutic target and resistance mechanisms, and cellular therapy for multiple myeloma. Here we provide a comprehensive summary of these workshop presentations.

Identification of High-Risk Multiple Myeloma With a Plasma Cell Leukemia-Like Transcriptomic Profile.

PURPOSE: Primary plasma cell leukemia (pPCL) is an aggressive subtype of multiple myeloma, which is distinguished from newly diagnosed multiple myeloma (NDMM) on the basis of the presence of ≥ 20% circulating tumor cells (CTCs). A molecular marker for pPCL is currently lacking, which could help identify NDMM patients with high-risk PCL-like disease, despite not having been recognized as such clinically. METHODS: A transcriptomic classifier for PCL-like disease was bioinformatically constructed and validated by leveraging information on baseline CTC levels, tumor burden, and tumor transcriptomics from 154 patients with NDMM included in the Cassiopeia or HO143 trials and 29 patients with pPCL from the EMN12/HO129 trial. Its prognostic value was assessed in an independent cohort of 2,139 patients with NDMM from the HOVON-65/GMMG-HD4, HOVON-87/NMSG-18, EMN02/HO95, MRC-IX, Total Therapy 2, Total Therapy 3, and MMRF CoMMpass studies. RESULTS: High CTC levels were associated with the expression of 1,700 genes, independent of tumor burden (false discovery rate < 0.05). Of these, 54 genes were selected by leave-one-out cross-validation to construct a transcriptomic classifier representing PCL-like disease. This not only demonstrated a sensitivity of 93% to identify pPCL in the validation cohort but also classified 10% of NDMM tumors as PCL-like. PCL-like MM transcriptionally and cytogenetically resembled pPCL, but presented with significantly lower CTC levels and tumor burden. Multivariate analyses in NDMM confirmed the significant prognostic value of PCL-like status in the context of Revised International Staging System stage, age, and treatment, regarding both progression-free (hazard ratio, 1.64; 95% CI, 1.30 to 2.07) and overall survival (hazard ratio, 1.89; 95% CI, 1.42 to 2.50). CONCLUSION: pPCL was identified on the basis of a specific tumor transcriptome, which was also present in patients with high-risk NDMM, despite not being clinically leukemic. Incorporating PCL-like status into current risk models in NDMM may improve prognostic accuracy.

The multiple myeloma microenvironment is defined by an inflammatory stromal cell landscape.

Progression and persistence of malignancies are influenced by the local tumor microenvironment, and future eradication of currently incurable tumors will, in part, hinge on our understanding of malignant cell biology in the context of their nourishing surroundings. Here, we generated paired single-cell transcriptomic datasets of tumor cells and the bone marrow immune and stromal microenvironment in multiple myeloma. These analyses identified myeloma-specific inflammatory mesenchymal stromal cells, which spatially colocalized with tumor cells and immune cells and transcribed genes involved in tumor survival and immune modulation. Inflammatory stromal cell signatures were driven by stimulation with proinflammatory cytokines, and analyses of immune cell subsets suggested interferon-responsive effector T cell and CD8+ stem cell memory T cell populations as potential sources of stromal cell-activating cytokines. Tracking stromal inflammation in individuals over time revealed that successful antitumor induction therapy is unable to revert bone marrow inflammation, predicting a role for mesenchymal stromal cells in disease persistence.

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation.

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR+ cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.

Intestinal-derived ILCs migrating in lymph increase IFNγ production in response to Salmonella Typhimurium infection.

Innate lymphoid cells (ILCs) are enriched in mucosae and have been described as tissue-resident. Interestingly, ILCs are also present within lymph nodes (LNs), in the interfollicular regions, the destination for lymph-migratory cells. We have previously shown that LN ILCs are supplemented by peripheral tissue-derived ILCs. Using thoracic duct cannulations, we here enumerate the intestinal lymph ILCs that traffic from the intestine to the mesenteric LNs (MLNs). We provide, for the first time, a detailed characterisation of these lymph-migratory ILCs. We show that all ILC subsets migrate in lymph, and while global transcriptional analysis reveals a shared signature with tissue-resident ILCs, lymph ILCs express migration-associated genes including S1PRs, SELL (CD62L) and CCR7. Interestingly, we discovered that while Salmonella Typhimurium infections do not increase the numbers of migrating ILCs, infection changes their composition and cytokine profile. Infection increases the proportions of RORyt+ T-bet+ ILCs, levels of IFNγ, and IFNγ/GM-CSF co-expression. Infection-induced changes in migratory ILCs are reflected in colon-draining MLN ILCs, where RORyt+ T-bet+ ILCs accumulate and display corresponding increased cytokine expression. Thus, we reveal that ILCs respond rapidly to intestinal infection and can migrate to the MLN where they produce cytokines.

Fibroblast-derived IL-33 is dispensable for lymph node homeostasis but critical for CD8 T-cell responses to acute and chronic viral infection.

Upon viral infection, stressed or damaged cells can release alarmins like IL-33 that act as endogenous danger signals alerting innate and adaptive immune cells. IL-33 coming from nonhematopoietic cells has been identified as important factor triggering the expansion of antiviral CD8+ T cells. In LN the critical cellular source of IL-33 is unknown, as is its potential cell-intrinsic function as a chromatin-associated factor. Using IL-33-GFP reporter mice, we identify fibroblastic reticular cells (FRC) and lymphatic endothelial cells (LEC) as the main IL-33 source. In homeostasis, IL-33 is dispensable as a transcriptional regulator in FRC, indicating it functions mainly as released cytokine. Early during infection with lymphocytic choriomeningitis virus (LCMV) clone 13, both FRC and LEC lose IL-33 protein expression suggesting cytokine release, correlating timewise with IL-33 receptor expression by reactive CD8+ T cells and their greatly augmented expansion in WT versus ll33-/- mice. Using mice lacking IL-33 selectively in FRC versus LEC, we identify FRC as key IL-33 source driving acute and chronic antiviral T-cell responses. Collectively, these findings show that LN T-zone FRC not only regulate the homeostasis of naïve T cells but also their expansion and differentiation several days into an antiviral response.

B cell zone reticular cell microenvironments shape CXCL13 gradient formation.

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.

Yap1-Driven Intestinal Repair Is Controlled by Group 3 Innate Lymphoid Cells.

Tissue repair requires temporal control of progenitor cell proliferation and differentiation to replenish damaged cells. In response to acute insult, group 3 innate lymphoid cells (ILC3s) regulate intestinal stem cell maintenance and subsequent tissue repair. ILC3-derived IL-22 is important for stem cell protection, but the mechanisms of ILC3-driven tissue regeneration remain incompletely defined. Here we report that ILC3-driven epithelial proliferation and tissue regeneration are independent of IL-22. In contrast, ILC3s amplify the magnitude of Hippo-Yap1 signaling in intestinal crypt cells, ensuring adequate initiation of tissue repair and preventing excessive pathology. Mechanistically, ILC3-driven tissue repair is Stat3 independent, but it involves activation of Src family kinases. Our findings reveal that ILC3-driven intestinal repair entails distinct transcriptional networks to control stem cell maintenance and epithelial regeneration, which implies that tissue repair and crypt proliferation can be influenced by targeting innate immune cells independent of the well-established effects of IL-22.

Frequencies of circulating regulatory TIGIT+CD38+ effector T cells correlate with the course of inflammatory bowel disease.

Disease heterogeneity hampers achieving long-term disease remission in inflammatory bowel disease (IBD). Monitoring ongoing tissue-localized regulatory and inflammatory T-cell responses in peripheral blood would empower disease classification. We determined whether regulatory and inflammatory phenotypes of circulating CD38+ effector (CD62LnegCD4+) T cells, a population enriched for cells with mucosal antigen specificity, classify disease course in pediatric IBD patients. In healthy individuals, circulating CD38+ effector T cells had a predominant regulatory component with lower frequencies of IFNγ-secreting T cells, higher frequencies of IL-10-secreting T cells and higher frequencies of inhibitory molecule T-cell immunoglobulin and ITIM domain+ (TIGIT) cells than CD38neg effector T cells. TIGIT expression was stable upon stimulation and marked CD38+ T cells with inhibitory properties. In IBD patients with active intestinal inflammation this predominant regulatory component was lost: circulating CD38+ effector T cells had increased activated CD25+CD45RAneg and decreased TIGIT+ cell frequencies. TIGIT percentages below 25% before treatment associated with shorter duration of clinical remission. In conclusion, phenotypic changes in circulating CD38+ effector T cells, in particular the frequency of TIGIT+ cells, classify pediatric IBD patients and predict severity of disease course. These findings have relevance for IBD and can be exploited in graft-versus-host-disease and checkpoint inhibitor-induced inflammation in cancer.

Expression of Plet1 controls interstitial migration of murine small intestinal dendritic cells.

Under homeostatic conditions, dendritic cells (DCs) continuously patrol the intestinal lamina propria. Upon antigen encounter, DCs initiate C-C motif chemokine receptor 7 (CCR7) expression and migrate into lymph nodes to direct T cell activation and differentiation. The mechanistic underpinnings of DC migration from the tissues to lymph nodes have been largely elucidated, contributing greatly to our understanding of DC functionality and intestinal immunity. In contrast, the molecular mechanisms allowing DCs to efficiently migrate through the complex extracellular matrix of the intestinal lamina propria prior to antigen encounter are still incompletely understood. Here we show that small intestinal murine CD11b+ CD103+ DCs express Placenta-expressed transcript 1 (Plet1), a glycophoshatidylinositol (GPI)-anchored surface protein involved in migration of keratinocytes during wound healing. In the absence of Plet1, CD11b+ CD103+ DCs display aberrant migratory behavior, and accumulate in the small intestine, independent of CCR7 responsiveness. RNA-sequencing indicated involvement of Plet1 in extracellular matrix-interactiveness, and subsequent in-vitro migration assays revealed that Plet1 augments the ability of DCs to migrate through extracellular matrix containing environments. In conclusion, our findings reveal that expression of Plet1 facilitates homeostatic interstitial migration of small intestinal DCs.

ILC2: at home in the thymus.

The relevance of innate lymphoid cells (ILC) for anti-infectious immunity remains a matter of constant debate. At the same time, evidence for additional, non-immune related functions of ILC is steadily increasing. In the thymus, non-immune functions of ILC were shown for group 3 ILC (ILC3), which regulate differentiation and proliferation of thymic epithelial cells. In this issue of the European Journal of Immunology, Withers and colleagues [Eur. J. Immunol. 2018. 48: 1481-1491] now show that ILC2, a subset of ILCs specialized in tissue protection and regeneration, are the major ILC subset in the adult thymus, heavily outnumbering ILC3. These findings raise novel questions on the function of thymic ILC, and warrant re-evaluation of the importance of ILC2 and their cytokines during thymic function and repair.

IL-7-dependent maintenance of ILC3s is required for normal entry of lymphocytes into lymph nodes.

IL-7 is essential for the development and homeostasis of T and B lymphocytes and is critical for neonatal lymph node organogenesis because Il7-/- mice lack normal lymph nodes. Whether IL-7 is a continued requirement for normal lymph node structure and function is unknown. To address this, we ablated IL-7 function in normal adult hosts. Either inducible Il7 gene deletion or IL-7R blockade in adults resulted in a rapid loss of lymph node cellularity and a corresponding defect in lymphocyte entry into lymph nodes. Although stromal and dendritic cell components of lymph nodes were present in normal numbers and representation, innate lymphoid cell (ILC) subpopulations were substantially decreased after IL-7 ablation. Testing lymphocyte homing in bone marrow chimeras reconstituted with Rorc-/- bone marrow confirmed that ILC3s in lymph nodes are required for normal lymphocyte homing. Collectively, our data suggest that maintenance of intact lymph nodes relies on IL-7-dependent maintenance of ILC3 cells.

Characterization of Endothelial Cells Associated with Hematopoietic Niche Formation in Humans Identifies IL-33 As an Anabolic Factor.

Bone marrow formation requires an orchestrated interplay between osteogenesis, angiogenesis, and hematopoiesis that is thought to be mediated by endothelial cells. The nature of the endothelial cells and the molecular mechanisms underlying these events remain unclear in humans. Here, we identify a subset of endoglin-expressing endothelial cells enriched in human bone marrow during fetal ontogeny and upon regeneration after chemotherapeutic injury. Comprehensive transcriptional characterization by massive parallel RNA sequencing of these cells reveals a phenotypic and molecular similarity to murine type H endothelium and activation of angiocrine factors implicated in hematopoiesis, osteogenesis, and angiogenesis. Interleukin-33 (IL-33) was significantly overexpressed in these endothelial cells and promoted the expansion of distinct subsets of hematopoietic precursor cells, endothelial cells, as well as osteogenic differentiation. The identification and molecular characterization of these human regeneration-associated endothelial cells is thus anticipated to instruct the discovery of angiocrine factors driving bone marrow formation and recovery after injury.

Cross-Tissue Transcriptomic Analysis of Human Secondary Lymphoid Organ-Residing ILC3s Reveals a Quiescent State in the Absence of Inflammation.

A substantial number of human and mouse group 3 innate lymphoid cells (ILC3s) reside in secondary lymphoid organs, yet the phenotype and function of these ILC3s is incompletely understood. Here, we employed an unbiased cross-tissue transcriptomic approach to compare human ILC3s from non-inflamed lymph nodes and spleen to their phenotypic counterparts in inflamed tonsils and from circulation. These analyses revealed that, in the absence of inflammation, lymphoid organ-residing ILC3s lack transcription of cytokines associated with classical ILC3 functions. This was independent of expression of the natural cytotoxicity receptor NKp44. However, and in contrast to ILC3s from peripheral blood, lymphoid organ-residing ILC3s express activating cytokine receptors and have acquired the ability to be recruited into immune responses by inflammatory cytokines. This comprehensive cross-tissue dataset will allow for identification of functional changes in human lymphoid organ ILC3s associated with human disease.