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In their paper the authors describe distinct transcriptomic changes associated to treatment response in core bone marrow biopsies from patients with acute myeloid leukaemia. This finding raises the possibility that stratifying patients for treatment according to their transcriptomic profiles could improve patients' response and prognosis. Commentary on: Treaba et al. Transcriptomics of AML core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles. Br J Haematol 2022 (Online ahead of print). doi: 10.1111/bjh.18513.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive census of the bone marrow immune microenvironment in adult and pediatric patients with AML. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B-cell subtype enriched in patients with high-inflammation AML, as well as an increase in CD8(+)GZMK(+) and regulatory T cells, accompanied by a reduction in T-cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in patients with AML. Addition of the iScore refines current risk stratifications for patients with AML and may enable identification of patients in need of more aggressive treatment. This work provides a framework for classifying patients with AML based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings.

Multiple myeloma (MM) is a plasma cell malignancy characterized by the presence of multiple foci in the skeleton. These distinct tumor foci represent cycles of tumor growth and dissemination that seed new clusters and drive disease progression. By using an intratibial Vk*MYC murine myeloma model, we found that CD169+ radiation-resistant tissue-resident macrophages (MPs) were critical for early dissemination of myeloma and disease progression. Depletion of these MPs had no effect on tumor proliferation, but it did reduce egress of myeloma from bone marrow (BM) and its spread to other bones. Depletion of MPs as a single therapy and in combination with BM transplantation improved overall survival. Dissemination of myeloma was correlated with an increased inflammatory signature in BM MPs. It was also correlated with the production of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) by tumor-associated MPs. Exogenous intravenous IL-6 and TNFα can trigger myeloma intravasation in the BM by increasing vascular permeability in the BM and by enhancing the motility of myeloma cells by reducing the adhesion of CD138. Moreover, mice that lacked IL-6 had defects in disseminating myeloma similar to those in MP-depleted recipients. Mice that were deficient in TNFα or TNFα receptor (TNFR) had defects in disseminating MM, and engraftment was also impaired. These effects on dissemination of myeloma required production of cytokines in the radiation-resistant compartment that contained these radiation-resistant BM MPs. Taken together, we propose that egress of myeloma cells from BM is regulated by localized inflammation in foci, driven in part by CD169+ MPs.

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INTRODUCTION: Injury compromises our epithelial barriers, leaving them vulnerable to external threats. Organismal survival thus impinges on rapid barrier re-establishment after damage. Immunocompromised individuals and immunodeficient animals have profound defects in epithelial repair. However, the precise mechanisms and consequences of immune-epithelial cross-talk after injury remain incompletely defined. RATIONALE: Epithelial repair requires cellular adaptation to the hypoxic wound microenvironment through evolutionarily conserved hypoxia-inducible factors (HIFs). This response has long been thought to depend upon epithelial cell-autonomous sensing of oxygen deprivation and responsiveness through HIFs. However, whether and how supportive microenvironmental signals from immune cells intersect with ancient hypoxia responses during epithelial repair have yet to be explored. To address these questions, we were drawn to the skin, a primary epithelial barrier that is surveilled by immune cells and has evolved sophisticated repair mechanisms. RESULTS: Using unbiased profiling of repair-associated lymphocytes, we uncovered the enrichment of heterogeneous populations of type 17 lymphocytes. Homozygous Rorgt-EGFP (GFI-KI) mice deficient in RORγt(+) cells exhibited a significant defect in epithelial migration and wound re-epithelialization. After damage, we observed a rapid local expansion of preexisting skin-resident RORγt(+) cells that was sufficient to drive repair. Of the myriad wound RORγt(+) populations, spatial transcriptomics and functional studies revealed that innate-like γδ T cells directed tissue repair by localizing to the wound front and boosting epithelial migration. These cells dominantly produced IL-17A/F in the wound microenvironment, which signaled directly to epithelia through IL-17RC to induce a migratory program. Comparative spatial transcriptomics of RORγt(+) cell-deficient and wild-type (WT) wound edges revealed differences in HIF1α signaling. WT migrating epithelia had robust nuclear HIF1α activation that was notably missing from RORγt(+) cell– and epithelial IL-17RC–deficient (Il17rc(EKO)) animals. Accordingly, epithelial-specific loss of HIF1α (Hif1a(EKO)) phenocopied the wound-healing defect of GFP-KI and Il17rc(EKO) mice. However, in contrast to GFP-KI wounds, recombinant IL-17A was unable to augment repair in Hif1a(EKO) mice, underscoring the necessity of this transcriptional effector mediating re-epithelialization downstream of IL-17A signaling. RORγt(+) cell–deficient and control animals had comparable levels of wound hypoxia, indicating that the failure of RORγt(+) cell–deficient animals to induce HIF1α was independent of oxygen sensing. Instead, IL-17A was sufficient to activate epithelial HIF1α both in vivo and in epithelial organoids. IL-17RC signaling rapidly induced activation of both ERK and AKT, which augmented HIF1α protein and transcript levels, but not stability, through mammalian target of rapamycin (mTOR). Acute hypoxia potently activated epithelial HIF1α in vitro, and this response was further boosted by IL-17A. By contrast, we uncovered a striking loss of mTOR and HIF1α in chronic hypoxia. Exogenous IL-17A rescued mTOR, and consequently HIF1α, under conditions of chronic hypoxia, indicating that IL-17A acts as a second signal to enable cell adaptation to low-oxygen environments. IL-17A induced a transcriptional and functional program of glycolytic metabolism in epithelia, which was dependent upon mTOR and HIF1α. The IL-17A-HIF1α-dependent program of glycolysis fueled epithelial migration, and pharmacological inhibition of glycolysis impaired wound re-epithelialization. CONCLUSION: Our findings upend a long-held view that hypoxia is sufficient to cell-autonomously induce HIF1α-mediated metabolic remodeling. We illustrate that IL-17A supplied by RORγt(+) γδ T cells is necessary for optimal HIF1α activation in the wound-edge epithelium. The IL-17A-HIF1α axis directed the metabolic rewiring of damaged epithelium toward a program of glycolysis to fuel migration. HIF1α and glycolysis are drivers of tumor progression and metastasis, raising the possibility that IL-17A or other immune-derived signals could drive these pathways in cancer. IL-17A is also central to the pathology of many autoimmune conditions, including psoriasis and inflammatory bowel disease. Thus, the IL-17A–HIF1α axis unveiled by our study may provide therapeutic opportunities for a range of epithelial inflammatory and metastatic diseases.

Single-cell sequencing approaches offer exploration of tissue architecture at unprecedented resolution. These tools are especially powerful when deconvoluting highly specialized microenvironments, such as stem cell (SC) niches. Here, we review single-cell studies that map the cellular and transcriptional makeup of stem and progenitor niches and discuss how these high-resolution analyses fundamentally advance our understanding of how niche factors shape SC biology and activity. In-depth characterization of the blueprint of SC-niche crosstalk, as well as understanding how it becomes dysregulated, will undoubtedly inform the development of more efficient therapies for malignancies and other pathologies.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive single cell RNA-Sequencing census of the bone marrow immune microenvironment in adult and pediatric AML patients. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B cell subtype enriched in high-inflammation AML patients, as well as an increase in CD8+ GZMK+ and regulatory T cells, accompanied by a reduction in T cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in AML patients. Addition of the iScore refines current risk stratifications for AML patients and may enable identification of patients in need of more aggressive treatment. This work provides a first framework for classifying AML patients based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings. Citation Format: Audrey Lasry, Bettina Nadorp, Maarten Fornerod, Deedra Nicolet, Huiyun Wu, Christopher J Walker, Zhengxi Sun, Matthew T Witkowski, Anastasia N Tikhonova, Maria Guillamot, Geraldine Cayanan, Anna Yeaton, Tanja A Gruber, Ann-Kathrin Eisfeld, Iannis Aifantis. Inflammation remodels the immune microenvironment in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A20.

The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB–POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.

The molecular complexity of the bone marrow (BM) microenvironment and its response to stress are incompletely understood, despite its key role in the regulation of hematopoiesis. Here we map the transcriptional landscape of BM vascular, perivascular, and osteoblast niche populations at single-cell resolution at both homeostasis and under stress hematopoiesis. This analysis revealed a previously unappreciated level of cellular heterogeneity within the BM niche, identified novel cellular subsets, and resolved cellular sources of pro-hematopoietic growth factors, chemokines, and membrane-bound ligands. Under conditions of stress, our studies revealed a significant transcriptional remodeling of these niche elements, including an adipocytic skewing of the perivascular cells. Among the stress-induced changes, we observed that vascular Notch ligand delta-like ligands (Dll1,4) were downregulated. In the absence of vascular Dll4, hematopoietic stem cells (HSC) prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the BM niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress, and illustrate the utility of single cell transcriptomic data in systematically evaluating the regulation of hematopoiesis by discrete niche populations.

Single-cell sequencing approaches have transformed our understanding of stem cell systems, including hematopoiesis and its niche within the bone marrow. Recent reports examined the bone marrow microenvironment at single-cell resolution at steady state, following chemotherapy treatment, leukemic onset, and aging. These rapid advancements significantly informed our understanding of bone marrow niche heterogeneity. However, inconsistent representation and nomenclature among the studies hinder a comprehensive interpretation of this body of work. Here, we review recent reports interrogating bone marrow niche architecture and present an integrated overview of the published datasets.

SUMMARY T cell acute lymphoblastic leukemia (T-ALL) is commonly associated with activating mutations in the NOTCH1 pathway. Recent reports have shown a link between NOTCH1 signaling and intracellular Ca2+ homeostasis in T-ALL. Here, we investigate the role of store-operated Ca2+ entry (SOCE) mediated by the Ca2+ channel ORAI1 and its activators STIM1 and STIM2 in T-ALL. Deletion of STIM1 and STIM2 in leukemic cells abolishes SOCE and significantly prolongs the survival of mice in a NOTCH1-dependent model of T-ALL. The survival advantage is unrelated to the leukemic cell burden but is associated with the SOCE-dependent ability of malignant T lymphoblasts to cause inflammation in leukemia-infiltrated organs. Mice with STIM1/STIM2-deficient T-ALL show a markedly reduced necroinflammatory response in leukemia-infiltrated organs and downregulation of signaling pathways previously linked to cancer-induced inflammation. Our study shows that leukemic T lymphoblasts cause inflammation of leukemia-infiltrated organs that is dependent on SOCE., In Brief T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of T cell progenitors affecting children and adults. Saint Fleur-Lominy et al. show that calcium influx mediated by STIM1 and STIM2 promotes the proinflammatory function of leukemic cells and premature death from leukemia., Graphical Abstract

Riok2 haploinsufficiency leads to aberrant TH22 skewing and interleukin-22-mediated erythroid dysfunction.

Pain-sensing nerve cells can mobilize blood stem cells in mice, with a component of chilli peppers being one stimulus. The finding holds the promise of improving procedures for stem-cell transplantation. Stimulation of nociceptive neurons mobilizes haematopoietic stem cells.

During normal T cell development in the thymus, αβ TCRs signal immature thymocytes to differentiate into mature T cells by binding to peptide–MHC ligands together with CD4/CD8 coreceptors. Conversely, in MHC and CD4/CD8 coreceptor-deficient mice, the thymus generates mature T cells expressing MHC-independent TCRs that recognize native conformational epitopes rather than linear antigenic-peptides presented by MHC. To date, no structural information of MHC-independent TCRs is available, and their structural recognition of non-MHC ligand remains unknown. To our knowledge in this study, we determined the first structures of two murine MHC-independent TCRs (A11 and B12A) that bind with high nanomolar affinities to mouse adhesion receptor CD155. Solution binding demonstrated the Vαβ-domain is responsible for MHC-independent B12A recognition of its ligand. Analysis of A11 and B12A sequences against various MHC-restricted and -independent TCR sequence repertoires showed that individual V-genes of A11 and B12A did not exhibit preference against MHC-restriction. Likewise, CDR3 alone did not discriminate against MHC binding, suggesting VDJ recombination together with Vα/Vβ pairing determine their MHC-independent specificity for CD155. The structures of A11 and B12A TCR are nearly identical to those of MHC-restricted TCR, including the conformations of CDR1 and 2. Mutational analysis, together with negative-staining electron microscopy images, showed that the CDR regions of A11 and B12A recognized epitopes on D1 domain of CD155, a region also involved in CD155 binding to poliovirus and Tactile in human. Taken together, MHC-independent TCRs adopt canonical TCR structures to recognize native Ags, highlighting the importance of thymic selection in determining TCR ligand specificity.

The αβ T cell receptor (TCR) repertoire on mature T cells is selected in the thymus, but the basis for thymic selection of MHC-restricted TCRs from a randomly generated pre-selection repertoire is not known. Here we perform comparative repertoire sequence analyses of pre-selection and post-selection TCR from multiple MHC-sufficient and MHC-deficient mouse strains, and find that MHC-restricted and MHC-independent TCRs are primarily distinguished by features in their non-germline CDR3 regions, with many pre-selection CDR3 sequences not compatible with MHC-binding. Thymic selection of MHC-independent TCR is largely unconstrained, but the selection of MHC-specific TCR is restricted by both CDR3 length and specific amino acid usage. MHC-restriction disfavors TCR with CDR3 longer than 13 amino acids, limits positively charged and hydrophobic amino acids in CDR3β, and clonally deletes TCRs with cysteines in their CDR3 peptide-binding regions. Together, these MHC-imposed structural constraints form the basis to shape VDJ recombination sequences into MHC-restricted repertoires., For T cells, functional antigen receptors are selected in the thymus from a pre-selection repertoire by interaction with self MHCs. Here the authors show that specific, non-germline coded features located in the complementarity determining region 3 of the pre-selection antigen receptors are essential for the selection of MHC-restricted repertoire.

Summary A subset of B cell acute lymphoblastic leukemia (B-ALL) patients will relapse and succumb to therapy-resistant disease. The bone marrow microenvironment may support B-ALL progression and treatment evasion. Utilizing single-cell approaches, we demonstrate B-ALL bone marrow immune microenvironment remodeling upon disease initiation and subsequent re-emergence during conventional chemotherapy. We uncover a role for non-classical monocytes in B-ALL survival, and demonstrate monocyte abundance at B-ALL diagnosis is predictive of pediatric and adult B-ALL patient survival. We show that human B-ALL blasts alter a vascularized microenvironment promoting monocytic differentiation, while depleting leukemia-associated monocytes in B-ALL animal models prolongs disease remission in vivo. Our profiling of the B-ALL immune microenvironment identifies extrinsic regulators of B-ALL survival supporting new immune-based therapeutic approaches for high-risk B-ALL treatment.

Notch signaling drives graft-versus-host-disease (GVHD) pathogenesis in preclinical models of allogeneic hematopoietic cell transplantation (allo-HCT). Short-term systemic blockade of the Notch ligands Delta-like1 (Dll1) and Delta-like4 (Dll4) within two days of allo-HCT conferred long-term protection from acute GVHD lethality in mice, with a dominant impact of Dll4. Surprisingly, critical Notch ligands in GVHD were provided by radioresistant non-hematopoietic fibroblastic stromal cells lineage traced by a Ccl19-Cre transgene in secondary lymphoid organs (SLO) (Chung, JCI 2017). Our discovery revises prevailing GVHD models and identifies a pathogenic role for a stromal cell niche expressing Notch ligands in SLOs. However, little is known about the distribution and regulation of Dll1/Dll4-expressing cells among SLO stromal cells. To address this question, we deployed genetic and biochemical tools to map expression of Notch ligands in SLO stromal cells, characterize the transcriptional landscape of these cells, and identify putative regulators of Notch ligand expression. To map Dll1 and Dll4 expression, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele and Dll1-mCherry or Dll4-mCherry BAC reporters. Using this model, we fluorescently traced all cells derived from Ccl19-Cre+ cells which contain the essential Dll1/Dll4 source in GVHD, while detecting Dll1 vs. Dll4 expression with high sensitivity. Within the lymph node (LN) Ccl19-Cre+ROSA26-YFP+ compartment, Dll4-mCherry was present predominantly among CD157hi CD45-Ter119-CD31-gp38+ fibroblastic stromal cells, including fibroblastic reticular cells (FRCs), marginal reticular cells (MRCs) and follicular dendritic cells (FDCs). In the spleen, Dll4-mCherry was detected among both CD157+ cells (predominant in the T-zone) and CD157- cells. In contrast, Dll1-mCherry was abundant among spleen CD157+ and CD157- cells, but not detected in the corresponding LN compartments. Thus, Dll1 and Dll4 are expressed with a non-overlapping distribution pattern in different SLOs and stromal cell subsets. Next, we studied expression of the dominant Dll4 ligand after MHC-mismatched allo-HCT (BALB/c into C57BL/6). Although Dll4-mCherry fluorescence did not increase, we detected a rise in surface Dll4 protein, peaking 12 hrs after allo-HCT in LN Ccl19-Cre+ CD157hi FRCs, MRCs and FDCs. Increased Dll4 abundance coincided with the critical timing of pathogenic Notch signals within days after allo-HCT and required irradiation conditioning as well as infusion of allogeneic T cells, suggesting a crosstalk between alloreactive T cells and Dll4+ stromal cells. The mechanisms underlying increased surface Dll4 abundance are under investigation, but could involve post-transcriptional effects as the Dll4-mCherry transcriptional reporter did not change. We next characterized transcriptomic features of Dll4+ fibroblastic stromal cells. We performed RNA-Seq on rare SLO stromal cells sort-purified from LNs at baseline and 12 hrs after allo-HCT. We compared CD157hi FRCs that do or do not express Dll4-mCherry, while including CD157lo FRCs that do not express Dll4-mCherry and lymphatic endothelial cells that express Dll4 at high levels as controls. These populations were sort-purified for RNA-Seq analysis, using the data to produce a comprehensive picture of gene expression and regulatory machinery in the SLO niche in alloimmunity, as well as identify new putative regulators of Notch ligand expression. Interestingly, we found only 13 differentially expressed genes between Ccl19-Cre-expressing CD157hi fibroblastic stromal cells that do and do not express the Dll4-mCherry reporter 12 hrs after transplant (log-fold change ≥ 1, corrected p ≤ 0.01). The list of up-regulated transcripts included Cxcl13, encoding a chemokine produced by FDCs, and Rankl, encoding a TNF-family member important in LN development and homeostasis. In summary, our data uncover key new features of the specialized fibroblastic stromal cells expressing Delta-like Notch ligands in SLOs. Ongoing work is testing how qualitative and quantitative differences in Dll1 and Dll4's effects during GVHD may relate to their distribution, regulated expression, or biochemical properties as they interact with Notch receptors in T cells. Our ultimate goal is to design strategies that target Notch ligand expression in SLOs as new prophylactic approaches in GVHD. Disclosures Blazar: Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees. Maillard:Genentech: Consultancy; Regeneron: Consultancy.

As with most cancer types, there remains a subset of B-cell acute lymphoblastic leukaemia (B-ALL) patients who will relapse and succumb to therapy-resistant disease. It is believed that tumour heterogeneity underpins therapy failure leading to a Darwinian model of clonal evolution, however, such studies do not account for the role of the bone marrow microenvironment in supporting leukaemia survival, progression and escape from treatment. Here, we perform single-cell RNA-Sequencing (scRNA-Seq) to generate a comprehensive map of the primary human B-ALL bone marrow immune microenvironment throughout three distinct stages of the human leukemic disease process: diagnosis, remission and relapse. These studies show extensive re-modelling of the immune microenvironment composition and cell-to-cell interactions throughout the course conventional chemotherapy, and uncover a role for inflammatory leukaemia-associated monocytes in promoting B-ALL pathogenesis in vivo. These monocytic subsets are predictive of Ph+ B-ALL patient event-free survival and when targeted in B-ALL animal models, lead to prolonged disease remission. Our profiling of the human B-ALL bone marrow immune microenvironment provides a greater understanding of the potential extrinsic regulators of B-ALL survival and may highlight previously unknown environmental factors influencing immune-based treatment approaches to high-risk B-ALL.

Summary Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αβ T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αβTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αβTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αβTCR repertoire.

Summary Major histocompatibility complex (MHC) restriction is the cardinal feature of T cell antigen recognition and is thought to be intrinsic to αβ T cell receptor (TCR) structure because of germline-encoded residues that impose MHC specificity. Here, we analyzed αβTCRs from T cells that had not undergone MHC-specific thymic selection. Instead of recognizing peptide-MHC complexes, the two αβTCRs studied here resembled antibodies in recognizing glycosylation-dependent conformational epitopes on a native self-protein, CD155, and they did so with high affinity independently of MHC molecules. Ligand recognition was via the αβTCR combining site and involved the identical germline-encoded residues that have been thought to uniquely impose MHC specificity, demonstrating that these residues do not only promote MHC binding. This study demonstrates that, without MHC-specific thymic selection, αβTCRs can resemble antibodies in recognizing conformational epitopes on MHC-independent ligands.

Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-associated signaling pathways of single-positive thymocytes are attenuated to respond appropriately to the peptide-MHC molecules that will be encountered in the periphery. In this study, we explore the mechanisms that regulate the tuning of CD4+ single-positive T cells to MHC class II encountered in the thymic medulla. Experiments with murine BM chimeras demonstrate that tuning can be mediated by MHC class II expressed by either thymic medullary epithelial cells or thymic dendritic cells. Tuning does not require the engagement of CD4 by MHC class II on stromal cells. Rather, it is mediated by interactions between MHC class II and the TCR. To understand the molecular changes that distinguish immature hyperactive T cells from tuned mature CD4+ T cells, we compared their responses to TCR stimulation. The altered response of mature CD4 single-positive thymocytes is characterized by the inhibition of ERK activation by low-affinity self-ligands and increased expression of the inhibitory tyrosine phosphatase SHP-1. Thus, persistent TCR engagement by peptide-MHC class II on thymic medullary stroma inhibits reactivity to self-Ags and prevents autoreactivity in the mature repertoire.

SummaryThe role of the microenvironment in T cell acute lymphoblastic leukemia (T-ALL), or any acute leukemia, is poorly understood. Here we demonstrate that T-ALL cells are in direct, stable contact with CXCL12-producing bone marrow stroma. Cxcl12 deletion from vascular endothelial, but not perivascular, cells impeded tumor growth, suggesting a vascular niche for T-ALL. Moreover, genetic targeting of Cxcr4 in murine T-ALL after disease onset led to rapid, sustained disease remission, and CXCR4 antagonism suppressed human T-ALL in primary xenografts. Loss of CXCR4 targeted key T-ALL regulators, including the MYC pathway, and decreased leukemia initiating cell activity in vivo. Our data identify a T-ALL niche and suggest targeting CXCL12/CXCR4 signaling as a powerful therapeutic approach for T-ALL.

SummaryAccumulating evidence suggests that Notch signaling is active at multiple points during hematopoiesis. Until recently, the majority of such studies focused on Notch signaling in lymphocyte differentiation and knowledge of individual Notch receptor roles has been limited due to a paucity of genetic tools available. In this manuscript we generate and describe animal models to identify and fate-map stem and progenitor cells expressing each Notch receptor, delineate Notch pathway activation, and perform in vivo gain- and loss-of-function studies dissecting Notch signaling in early hematopoiesis. These models provide comprehensive genetic maps of lineage-specific Notch receptor expression and activation in hematopoietic stem and progenitor cells. Moreover, they establish a previously unknown role for Notch signaling in the commitment of blood progenitors toward the erythrocytic lineage and link Notch signaling to optimal organismal response to stress erythropoiesis.

Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.

Mature αβ T cells recognize foreign antigenic peptides presented by MHC molecules but do not recognize native antigenic proteins; features known as MHC restriction. How MHC restriction is imposed on αβ T cells has intrigued immunologists for several decades. One model proposes that germline-encoded elements in the T cell receptor (TCR) variable regions are evolutionarily conserved to only recognize MHC. However, we propose an alternative model that posits that MHC restriction is imposed by CD4 and CD8 co-receptors during thymic selection. Thus, we think that TCRs are structurally able to recognize a huge diversity of ligands but only TCRs with MHC specificity survive thymic selection.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy arising in immature T cell progenitors. Despite improved survival rates, relapse remains a major therapeutic obstacle and intensive treatment regimens expose T-ALL patients to serious long-term side-effects. Hence more effective, less toxic treatment options are required. While the localization of leukemia cells determines the course of disease, little is known about T-ALL migration. Upon profiling chemokine receptor expression by T-ALL cells, we observed high surface levels of the CXCL12 receptor, CXCR4. Intravital imaging revealed that T-ALL cells are in direct contact with CXCL12-producing bone marrow stromal cells in vivo. Genetic targeting of CXCR4 in T-ALL cells resulted in a marked reduction of leukemia burden and significantly prolonged survival. Moreover, disruption of CXCR4 signaling with a small molecule inhibitor severely impaired T-ALL progression in mouse and human xenograft models of the disease. We found that CXCR4 promotes c-Myc expression, which is required in leukemia initiating cells. Given the relatively subtle role for CXCR4 in normal developing T cells, CXCR4 inhibition to our knowledge has not been proposed as a therapy for T cell malignancies. Our data strongly suggest that targeting CXCR4 could be a promising strategy for combating T-ALL. Citation Format: Lauren A. Pitt, Anastasia N. Tikhonova, Thomas Trimarchi, Bryan King, Hai Hu, Yixiao Gong, Aris Tsirigos, Marta Sanchez-Martin, Dan R. Littman, Adolfo Ferrando, Sean J. Morrison, David R. Fooksman, Iannis Aifantis, Susan Schwab. The chemokine receptor CXCR4 is essential for the maintenance of T cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A06.

Notch signals drive pathogenic T cell alloreactivity in mouse models of allogeneic bone marrow transplantation (allo-BMT) leading to lethal graft-versus-host disease (GVHD). In a mouse model of MHC-mismatched allo-BMT with high intensity whole body conditioning (11 Gy), residual recipient or donor hematopoietic cells were dispensable sources of Notch signals to drive T cell alloreactivity. Instead, abrogating the expression of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands in Ccl19-Cre+ secondary lymphoid organ (SLO) fibroblastic stromal cells protected mice from GVHD (Chung et al., JCI 2017). In this study we sought to 1) understand the expression and regulation of Notch ligands in fibroblastic stromal cells during allo-BMT; 2) test whether fibroblastic stromal cells are still critical sources of Notch signals in models of allotransplantation with reduced or no conditioning (when other radiation-sensitive sources of Notch ligands are better preserved); and 3) assess whether SLO fibroblastic stromal also present alloantigens to T cells during GVHD. To characterize stromal sources of Notch ligands early after allo-BMT, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele in Dll4-mCherry BAC reporter mice. We observed combined Ccl19-Cre activity and Dll4-mCherry reporter expression in CD157hi fibroblastic reticular cells, MAdCAM1+ marginal reticular cells, CD21+ follicular dendritic cells, and a subset of MAdCAM1+ lymphatic endothelial cells. 12 hours after high intensity conditioning and allo-BMT, Dll4 mRNA and cell surface expression increased in fibroblastic but not in hematopoietic or endothelial cellular compartments. Dll4 upregulation required transfer of alloreactive T cells and was potentiated by the intensity of myeloablative conditioning, suggesting that fibroblastic stromal cells have increased availability of Notch ligands to drive GVHD after high-intensity conditioning. To assess the functional impact of conditioning on the cellular sources of Notch ligands, we studied a parent-to-F1 allo-BMT model in which recipients are tolerant to donor-derived T cells, and the conditioning intensity can be titrated (C57BL/6 [H-2b] or BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b]). Cell-specific genetic loss of function systems combining floxed alleles for Dll1 and Dll4 with Ccl19-Cre+ and Mx-Cre+ transgenes allowed conditional deletion in SLO fibroblastic stromal cells or hematopoietic cells, respectively. Across all conditioning intensities tested, upregulation of core-2 O-glycosylation machinery in alloreactive T cells was critically regulated by Notch signals. Thus, induction of the core-2 glycoform of CD43 could be used as a sensitive surrogate readout of Notch signals received by alloreactive T cells. In models of allotransplantation with high-intensity (11 Gy), low-intensity (3 Gy), and no conditioning, fibroblastic stromal cells remained essential and hematopoietic cells were dispensable as sources of Notch signals driving both T cell core-2 O-glycosylation and lethal alloimmunity. Because previous studies indicated that non-hematopoietic cells can serve as antigen-presenting cells in CD4+ T cell mediated GVHD, we tested if fibroblastic stromal cells could present MHC class II alloantigens in addition to Notch ligands. We used Ccl19-Cre+ crossed to mice with a floxed I-A allele to delete the cell-intrinsic ability of fibroblastic stromal cells to present MHC class II alloantigens. In multiple models of high-intensity conditioned CD4+ T cell mediated GVHD (BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b] and TEa TCR Tg into BALB/c x C57BL/6 F1 [H-2d/b]), fibroblastic stromal cells were dispensable as sources of alloantigen to drive T cell alloreactivity and lethal GVHD. Together, these results indicate that across varying conditioning intensities, specialized subsets of SLO fibroblastic stromal cells critically regulate T cell alloreactivity through presentation of Notch ligands independently of antigen presentation. Selective targeting of Delta-like Notch ligand expression on SLO fibroblastic cells may prevent T cell immunopathology while preserving other beneficial antigen-dependent T cell responses after both high-intensity conditioned allo-BMT and allotransplantation with reduced intensity conditioning. Disclosures Blazar: Kadmon Corporation, LLC: Consultancy, Research Funding. Siebel:Genentech: Employment.