Dendritic Cell Precursors Research Articles

Discovery of highly immunogenic spleen-resident FCGR3+CD103+ cDC1s differentiated by IL-33-primed ST2+ basophils

Recombinant interleukin-33 (IL-33) inhibits tumor growth, but the detailed immunological mechanism is still unknown. IL-33-mediated tumor suppression did not occur in Batf3−/− mice, indicating that conventional type 1 dendritic cells (cDC1s) play a key role in IL-33-mediated antitumor immunity. A population of CD103+ cDC1s, which were barely detectable in the spleens of normal mice, increased significantly in the spleens of IL-33-treated mice. The newly emerged splenic CD103+ cDC1s were distinct from conventional splenic cDC1s based on their spleen residency, robust effector T-cell priming ability, and surface expression of FCGR3. DCs and DC precursors did not express Suppressor of Tumorigenicity 2 (ST2). However, recombinant IL-33 induced spleen-resident FCGR3+CD103+ cDC1s, which were found to be differentiated from DC precursors by bystander ST2+ immune cells. Through immune cell fractionation and depletion assays, we found that IL-33-primed ST2+ basophils play a crucial role in the development of FCGR3+CD103+ cDC1s by secreting IL-33-driven extrinsic factors. Recombinant GM-CSF also induced the population of CD103+ cDC1s, but the population neither expressed FCGR3 nor induced any discernable antitumor immunity. The population of FCGR3+CD103+ cDC1s was also generated in vitro culture of Flt3L-mediated bone marrow-derived DCs (FL-BMDCs) when IL-33 was added in a pre-DC stage of culture. FL-BMDCs generated in the presence of IL-33 (FL-33-DCs) offered more potent tumor immunotherapy than control Flt3L-BMDCs (FL-DCs). Human monocyte-derived DCs were also more immunogenic when exposed to IL-33-induced factors. Our findings suggest that recombinant IL-33 or an IL-33-mediated DC vaccine could be an attractive protocol for better tumor immunotherapy.

Acute Myeloid Leukemia, Version 3.2023, NCCN Clinical Practice Guidelines in Oncology.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow, and/or other tissues. It is the most common form of acute leukemia among adults and accounts for the largest number of annual deaths from leukemias in the United States. Like AML, blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a myeloid malignancy. It is a rare malignancy characterized by the aggressive proliferation of precursors of plasmacytoid dendritic cells that frequently involves the bone marrow, skin, central nervous system, and other organs and tissues. This discussion section focuses on the diagnosis and management of BPDCN as outlined in the NCCN Guidelines for AML.

Immunomics analysis of rheumatoid arthritis identified precursor dendritic cells as a key cell subset of treatment resistance

ObjectivesLittle is known about the immunology underlying variable treatment response in rheumatoid arthritis (RA). We performed large-scale transcriptome analyses of peripheral blood immune cell subsets to identify immune cells that...

RAF-Independent MEK Mutations Drive Histiocytic Neoplasms In Vivo and Are Sensitive to Single-Agent ERK Inhibition in Patients

Histiocytic neoplasms are clonal disorders of the monocyte/macrophage lineage that include Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD). Histiocytoses are defined by recurrent, mutually exclusive mutations activating MAP kinase signaling, including BRAFV600E mutations in ~50% of LCH and ECD patients, which confer robust lasting clinical responses to BRAF inhibition. Mutations in MEK1/2, ARAF, and a diverse array of other kinases are present in BRAF wild-type histiocytoses patients and are broadly responsive to the allosteric MEK1/2 inhibitor cobimetinib. Following genomic analysis of ~300 pediatric and adult patients with diverse histiocytoses, the single most common genomic alteration following BRAFV600E mutations are MEK1 E102_I103 in-frame deletions. In contrast to BRAFV600E, the biological impact of activating mutations in MEK1/2 kinases are unknown, and no cancer associated MEK1/2 mutation has ever been modeled in vivo. MEK1/2 mutations are recurrent across a wide variety of human cancers and RASopathies and are now often found at resistance to emerging KRAS-mutant selective inhibitors. These findings motivated us to generate Map2k1E102_I103del conditional knock-in mice within the endogenous locus of Map2k1 (encoding MEK1 kinase), which represent the first genetically engineered mouse model of MEK1/2 mutations. We first crossed Map2k1E102_I103del mice to inducible Mx1-cre driver mice. Interestingly, Mx1-cre Map2k1E102_I103del knock-in mice developed a 100% penetrant lethal myelomonocytic disorder by 5-6 weeks of age while their cre-negative Map2k1E102_I103del littermates did not develop disease (Figure A). Notably, recombination of the Map2k1E102_I103del alleleoccurred spontaneously without pIpC administration and resulted in strong activation of phosphorylated ERK1/2 in bone marrow and spleen mononuclear cells. Mx1-cre Map2k1E102_I103del mice developed a myelomonocytic neoplasm with suppression of B-lymphopoiesis and expansion of classical and non-classical monocyte subsets, macrophages, and Cd11b+ conventional dendritic cells. Monocyte, macrophage, and dendritic cells infiltrated hematopoietic organs (bone marrow, spleen, and peripheral blood), as well as liver and skin, each of which are common sites of LCH and ECD disease involvement. The Mx1-cre Map2k1E102_I103del disease was hematopoietic cell autonomous and could be propagated by serial hematopoietic reconstitution in irradiated recipient mice, as well as using Vav1-cre Map2k1E102_I103del knock-in mice (where Cre is expressed only in hematopoietic cells from earliest hematopoietic development). Finally, restricting MEK1 mutant expression to monocyte and dendritic cell precursors using CD11c-cre resulted in similar lethal expansions of monocyte, macrophage, and dendritic cells as seen in the Mx1-cre and Vav1-cre models. Prior in vitro work has demonstrated that MEK1 p.E102_I103del activates MEK and ERK without requiring activated GTP-bound RAS and is associated with resistance to allosteric MEK inhibitors. Consistent with this, we identified nine histiocytosis patients with this specific MEK1 mutation, three of which had de novo resistance to cobimetinib. These three patients and one treatment-naïve patient were treated with an oral ERK1/2 inhibitor ulixertinib under the FDA single-patient compassionate use program. Three of 4 patients had a clinical or radiological partial or complete response to ulixertinib (Figure B). Overall, these data identify the impact of oncogenic MEK kinase mutations in vivo as found in children and adults with histiocytoses and RASopathies. Expression of a RAF-independent MEK1 mutation in vivo gave rise to disease reminiscent of human systemic histiocytic neoplasms. Importantly, this molecular entity is resistant to MEK inhibition in some patients, a phenomenon previously undocumented, but responsive to ERK inhibition, which may be a promising therapeutic approach to histiocytic neoplasms unresponsive to allosteric MEK inhibition. The clinical observation of efficacy of ERK inhibition in MEK inhibitor resistant histiocytoses patients has resulted in a soon-to-be initiated focused phase 2 clinical trial of ulixertinib in adults with histiocytoses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Distinct responses of newly identified monocyte subsets to advanced gastrointestinal cancer and COVID-19.

Monocytes are critical cells of the immune system but their role as effectors is relatively poorly understood, as they have long been considered only as precursors of tissue macrophages or dendritic cells. Moreover, it is known that this cell type is heterogeneous, but our understanding of this aspect is limited to the broad classification in classical/intermediate/non-classical monocytes, commonly based on their expression of only two markers, i.e. CD14 and CD16. We deeply dissected the heterogeneity of human circulating monocytes in healthy donors by transcriptomic analysis at single-cell level and identified 9 distinct monocyte populations characterized each by a profile suggestive of specialized functions. The classical monocyte subset in fact included five distinct populations, each enriched for transcriptomic gene sets related to either inflammatory, neutrophil-like, interferon-related, and platelet-related pathways. Non-classical monocytes included two distinct populations, one of which marked specifically by elevated expression levels of complement components. Intermediate monocytes were not further divided in our analysis and were characterized by high levels of human leukocyte antigen (HLA) genes. Finally, we identified one cluster included in both classical and non-classical monocytes, characterized by a strong cytotoxic signature. These findings provided the rationale to exploit the relevance of newly identified monocyte populations in disease evolution. A machine learning approach was developed and applied to two single-cell transcriptome public datasets, from gastrointestinal cancer and Coronavirus disease 2019 (COVID-19) patients. The dissection of these datasets through our classification revealed that patients with advanced cancers showed a selective increase in monocytes enriched in platelet-related pathways. Of note, the signature associated with this population correlated with worse prognosis in gastric cancer patients. Conversely, after immunotherapy, the most activated population was composed of interferon-related monocytes, consistent with an upregulation in interferon-related genes in responder patients compared to non-responders. In COVID-19 patients we confirmed a global activated phenotype of the entire monocyte compartment, but our classification revealed that only cytotoxic monocytes are expanded during the disease progression. Collectively, this study unravels an unexpected complexity among human circulating monocytes and highlights the existence of specialized populations differently engaged depending on the pathological context.

The therapeutic potential of bone marrow-derived macrophages in neurological diseases.

Circulating monocytes are precursors of both tissue macrophages and dendritic cells, and they can infiltrate the central nervous system (CNS) where they transform into bone marrow-derived macrophages (BMDMs). BMDMs play essential roles in various CNS diseases, thus modulating BMDMs might be a way to treat these disorders because there are currently no efficient therapeutic methods available for most of these neurological diseases. Moreover, BMDMs can serve as promising gene delivery vehicles following bone marrow transplantation for otherwise incurable genetic CNS diseases. Understanding the distinct roles that BMDMs play in CNS diseases and their potential as gene delivery vehicles may provide new insights and opportunities for using BMDMs as therapeutic targets or delivery vehicles. This review attempts to comprehensively summarize the neurological diseases that might be treated by modulating BMDMs or by delivering gene therapies via BMDMs after bone marrow transplantation.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN): A promising future in the era of targeted therapeutics.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy arising from precursor dendritic cells. BPDCN cells characteristically express several markers on their cell surfaces including CD123, CD4, and CD56. Because of its rarity and challenging clinical presentation, there was no standard of care in managing BPDCN for decades and its prognosis overall was poor. However, as understanding of this rare neoplasm has increased, so have treatment options. The conventional cytotoxic chemotherapy regimens once used in the treatment of BPDCN were modest in their impact on disease relapse until paired with hematopoietic stem cell transplant. Although recent data suggest that there still remains a role for chemotherapeutic agents, targeted modalities have expanded the overall BPDCN treatment landscape. The CD123-targeted agent, tagraxofusp, was the first Food and Drug Administration-approved monotherapy in the treatment of BPDCN. Since its inception, several CD123-targeted and other cell-surface agents have been investigated, with many agents still in the preclinical stages. Although relapsed/refractory disease and central nervous system disease both remain formidable areas of research, there are several promising therapeutic approaches that could have a significant impact on the trajectory of treatment. This review will provide detailed insight on the novel drugs currently in use and those being explored in the management of BPDCN.

Ly6D+Siglec-H+ precursors contribute to conventional dendritic cells via a Zbtb46+Ly6D+ intermediary stage

Plasmacytoid and conventional dendritic cells (pDC and cDC) are generated from progenitor cells in the bone marrow and commitment to pDCs or cDC subtypes may occur in earlier and later progenitor stages. Cells within the CD11c+MHCII−/loSiglec-H+CCR9lo DC precursor fraction of the mouse bone marrow generate both pDCs and cDCs. Here we investigate the heterogeneity and commitment of subsets in this compartment by single-cell transcriptomics and high-dimensional flow cytometry combined with cell fate analysis: Within the CD11c+MHCII−/loSiglec-H+CCR9lo DC precursor pool cells expressing high levels of Ly6D and lacking expression of transcription factor Zbtb46 contain CCR9loB220hi immediate pDC precursors and CCR9loB220lo (lo-lo) cells which still generate pDCs and cDCs in vitro and in vivo under steady state conditions. cDC-primed cells within the Ly6DhiZbtb46– lo-lo precursors rapidly upregulate Zbtb46 and pass through a Zbtb46+Ly6D+ intermediate stage before acquiring cDC phenotype after cell division. Type I IFN stimulation limits cDC and promotes pDC output from this precursor fraction by arresting cDC-primed cells in the Zbtb46+Ly6D+ stage preventing their expansion and differentiation into cDCs. Modulation of pDC versus cDC output from precursors by external factors may allow for adaptation of DC subset composition at later differentiation stages.

Lymph node stromal cells support the maturation of pre-DCs into cDC-like cells via colony-stimulating factor 1.

Conventional dendritic cells (cDCs) arise from committed precursor dendritic cells (pre-DCs) in the bone marrow that continuously seed the periphery. Pre-DCs and other upstream progenitors proliferate and mature in response to Fms-related receptor tyrosine kinase 3 ligand, which is considered the key cytokine for cDC development. However, other cytokines such as stem cell factor and colony-stimulating factor 1 (CSF1) were also shown to induce pre-DC maturation into DC-like cells. Yet, it is still only incompletely understood which cells contribute to cDC development once pre-DCs arrive in peripheral tissues. Here, we analysed the impact of lymph node (LN) fibroblastic stromal cells (FSCs) on the maturation of pre-DCs into cDC-like cells. We could demonstrate that ex vivo isolated LN FSCs co-cultured with pre-DCs induce precursor maturation into DC-like cells, which were capable of efficiently promoting the proliferation of naïve CD4+ T cells. Interestingly, FSCs isolated from distinct LNs induced DC-like cells with highly comparable transcriptomes, characterized by the expression of signature genes of both ex vivo isolated DCs and macrophages. Finally, by performing supplementation and receptor blocking studies, we could demonstrate that CSF1 is a driving factor for LN FSC-mediated pre-DC maturation into DC-like cells. In summary, we could identify CSF1 as a stromal cell-derived factor that has the potential to support the maturation of pre-DCs into cDC-like cells within secondary lymphoid organs.

Clinical Insights into the Management of Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is aggressive hematologic malignancy derived from plasmacytoid dendritic cell precursors of myeloid cell lineage. Patients frequently present with bruise-like skin lesions, which typically are followed months later by progressive cytopenias. Historically, BPDCN prognosis has been dismal, with median overall survival ranging from 9 to 13 months. In the past 2 decades, our understanding of BPDCN pathogenesis has led to the successful development of novel therapeutics. In December 2018, the FDA approved tagraxofusp-erzs for adults and pediatric patients older than 2 years who have either treatment-naïve or relapsed/refractory BPDCN. Acute lymphoblastic leukemia (ALL)-based chemotherapy regimens also provide comparable outcomes to tagraxofusp. In our practice, for patients with good performance status, we use tagraxofusp, ALL-based chemotherapy regimens, or clinical trials as frontline induction therapy, followed by consolidation with allogeneic stem cell transplant once the first complete response has been achieved. Our induction regimen also includes intrathecal chemotherapy for central nervous system prophylaxis. Patients with poor performance status who are treatment-naïve or patients with relapsed/refractory disease have limited therapeutic options, and we strongly recommend enrollment in clinical trials; several novel agents and combinations are currently under clinical investigation for both treatment-naïve and relapsed/refractory BPDCN.

Seed or soil: Tracing the immune subsets in metastatic tumors.

Mapping the tumor-infiltrating immune subsets to an origin of malignancy or tissue niche is important for designing effective immunotherapies, yet it remains a challenging task. In this issue of Cancer Cell, Liu etal. pieced together this puzzle through a comprehensive single-cell analysis of colorectal cancer patients with liver metastases.

CD24 + MDSC-DCs Induced by CCL5-Deficiency Showed Improved Antitumor Activity as Tumor Vaccines.

Background Dendritic cell (DC) tumor vaccine has been extensively utilized in preclinical and clinical studies; however, this technique has encountered many difficulties, particularly in late-stage tumor patients. For those, ex vivo-induced DCs are actuallymyeloid-derived suppressive cells-derived DCs (MDSC-DCs). MDSCs with immunosuppressive activity, but not monocytes, became the major DC precursor. Thus, how to enhance antitumor activity of MDSC-DCs is urgent need to address. Methods We utilized 4T1 and MC38 tumor-bearing both wildtype and CC chemokine ligand 5 −/− (CCL5 −/− ) mice as animal models. MDSC-DCs were induced from splenocytes of these mice by granulocyte macrophage–colony stimulating factor/interleukin-4 with or without all-trans-retinoic acid (ATRA) in vitro for 7 days, then incubated with tumor-cell-lysis to treat mouse models for total three doses. For human MDSC-DCs, peripheral bloods from colorectal cancer patients were induced in vitro as murine cells with or without T- lymphocytes depletion to get rid of CCL5. Results Flow cytometry analysis showed that MDSCs from CCL5 −/− mice could be induced into a new type of CD24 + MDSC-DCs in the presence of ATRA, which had more antitumor activity than control. Antibody blocking and adoptive transfer experiments demonstrated that downregulation of regulatory T cells (Tregs) mediated the inhibition of CD24 + MDSC-DCs on tumor growth. Mechanically, CD24 + MDSC-DCs inhibited Tregs' polarization by secreting cytokine or coactivators' expression. What's important, decreasing CCL5 protein levels by T- lymphocytes depletion during both murine and human MDSC-DCs in vitro induction could also acquire CD24 + MDSC-DCs. Conclusion Knockdown of CCL5 protein during MDSC-DCs culture might provide a promising method to acquire DC-based tumor vaccines with high antitumor activity.

Endothelial cell-specific expression of serine/threonine kinase 11 modulates dendritic cell differentiation

In the bone marrow, classical and plasmacytoid dendritic cells (DC) develop from the macrophage-DC precursor (MDP) through a common DC precursor (CDP) step. This developmental process receives essential input from the niche in which it takes place, containing endothelial cells (EC) among other cell types. Here we show that targeted deletion of serine/threonine kinase 11 (Stk11) encoding tumor suppressor liver kinase b1 (Lkb1) in mouse ECs but not DCs, results in disrupted differentiation of MDPs to CDPs, severe reduction in mature DC numbers and spontaneous tumorigenesis. In wild type ECs, Lkb1 phosphorylates polypyrimidine tract binding protein 1 (Ptbp1) at threonine 138, which regulates stem cell factor (Scf) pre-mRNA splicing. In the absence of Lkb1, exon 6 of Scf is spliced out, leading to the loss of Scf secretion. Adeno-associated-virus-mediated delivery of genes encoding either soluble Scf or the phosphomimetic mutant Ptbp1T138E proteins rescued the defects of MDP to CDP differentiation and DC shortage in the endothelium specific Stk11 knockout mice. In summary, endothelial Stk11 expression regulates DC differentiation via modulation of Scf splicing, marking the Stk11-soluble-Scf axis as a potential cause of DC deficiency syndromes.

P103 Identifying CES-1 positive myeloid cells with mass cytometry as a potential therapeutic target for Crohn’s disease

Abstract Background Myeloid cells are key players in the sustained inflammatory process in Crohn’s disease (CD), and are therefore therapeutic targets. Human mononuclear myeloid cells express the cell selective expression of carboxylesterase 1 (CES-1). Esterase-sensitive motif (ESM) technology has been developed to specifically target ESM tagged small molecules on human mononuclear myeloid cells, based on their CES-1 expression, creating a larger therapeutic window with less side effects. This study investigates the presence of CES-1 expressing cells in PBMCs, intestinal biopsies and fistula cells of CD patients, highlighting the potential of ESM-technology in CD. This could benefit CD treatment in non-responsive patients or patients with a fistulizing phenotype. Methods Intestinal biopsies were collected from CD patients who underwent endoscopy. PBMCs were collected from CD patients who were receiving Vedolizumab (VDZ) treatment and categorized as responders or non-responders. Criteria for response was based on clinical (HBI ≤4) and biochemical (fecal calprotectine ≤250 µg/g) and/or endoscopic assessment (≥50% reduction in SES-CD score). Fistula tissue was of CD patients was obtained during surgery. CES1-positive cells were phenotyped by mass cytometry with a panel of 32-markers, including the myeloid markers CES-1, CD14, CD16, HLA-DR, CD11c, CD206. Results We demonstrated the presence of CD14+HLADR+CD11a+CD44+CD11b+ myeloid cells exclusively expressing CES-1 in intestinal biopsies of CD patients (n=7). CES-1 expression did not differ between inflamed and uninflamed tissue. In PBMCs of VDZ responders (n=5) and non-responders (n=6), we were able to identify different subsets of myeloid cells such as classical (CD14++CD16-), intermediate(CD14+CD16+) and non-classical (CD14-CD16++) monocytes, DCs and CD2+ DC precursors all demonstrating CES-1 expression. Percentage of CES-1 positive cells in inflamed PBMCs (or non-responders) was higher than in uninflamed PBMCs (responders) in all of the above-mentioned subsets. Furthermore, we demonstrated different myeloid populations within fistula cells (n=13) such as pDCs (CD123+CD68+HLA-DR+), DCs (CD11c+CD11b+HLA-DR+ CD44+), CD141+DCs (CD141+CD49d+HLA-DR+),Macrophage (CD14+CD11c+CD11b+HLA-DR+CD44+) and M2 Macrophages (CD14+CD163+CD206+CD11c+CD11b+HLA-DR+CD44+), all expressing CES-1. Conclusion We demonstrated specific CES-1 positive myeloid cells within intestinal biopsies, PBMCs of non-responsive patients, and fistulae samples of CD patients. Targeting CES-1+ with ESM-technology could provide new therapeutic targets for CD.

DNMT1 Deficiency Impacts on Plasmacytoid Dendritic Cells in Homeostasis and Autoimmune Disease.

Dendritic cells (DCs) are heterogeneous immune regulators involved in autoimmune diseases. Epigenomic mechanisms orchestrating DC development and DC subset diversification remain insufficiently understood but could be important to modulate DC fate for clinical purposes. By combining whole-genome methylation assessment with the analysis of mice expressing reduced DNA methyltransferase 1 levels, we show that distinct DNA methylation levels and patterns are required for the development of plasmacytoid DC and conventional DC subsets. We provide clonal in vivo evidence for DC lineage establishment at the stem cell level, and we show that a high DNA methylation threshold level is essential for Flt3-dependent survival of DC precursors. Importantly, reducing methylation predominantly depletes plasmacytoid DC and alleviates systemic lupus erythematosus in an autoimmunity mouse model. This study shows how DNA methylation regulates the production of DC subsets and provides a potential rationale for targeting autoimmune disease using hypomethylating agents.

A Sampling of Highlights from the Literature

TAMs expressing TREM2 are a potential therapeutic target (from Fig. 2F of Cao et al., Cancer Immunol Res 2019)Understanding the mechanisms underlying resistance to anti–PD-1 therapy is key to developing new therapeutics. Binnewies et al. find that human and mouse immunosuppressive tumor-associated macrophages (TAM) express triggering receptor expressed on myeloid cells 2 (TREM2) and that the presence of these cells in the tumor microenvironment correlates with an exhausted T-cell state in human tumors and anti–PD-1 resistance in mouse models of cancer. A TREM2-specific antibody promotes antitumor immunity in mice by depleting TAMs, leading to enhanced CD8+ T-cell function. It also reverses anti–PD-1 resistance, highlighting the potential of targeting TREM2 to treat patients with anti–PD-1–resistant tumors.Binnewies M, …, Sriram V. Cell Rep 2021 Oct 19;37:109844.Loss of antigen can have an impact on therapy efficacy (by Silverisdead via Flickr)Tumor loss of target antigen(s) can lead to resistance to treatments such as adoptive cell therapy and chimeric antigen receptor T-cell therapy. Xue et al. demonstrate that engineered, tumor-specific Th9 cells can overcome this hurdle and eliminate tumor cells regardless of antigen expression. This effect is due to both direct killing and induction of bystander responses driven by intratumoral accumulation of extracellular ATP—because Th9 cells lack CD39—which leads to monocyte recruitment and production of type I IFN. This study identifies a novel role of Th9 cells in antitumor responses and highlights their potential use as an “unconventional” cell type for cell-based therapy.Xue G, …, Lu Y. Cancer Cell 2021 Oct 21. DOI: 10.1016/j.ccell.2021.09.011.Engineered E. coli bacteria can improve immunotherapy (by Janice Haney Carr via PIXNIO)An increase in L-arginine concentration in tumors is associated with improved antitumor T-cell function, but modulating intratumoral L-arginine concentrations is challenging. Canale et al. engineered probiotic Escherichia coli to constitutively convert ammonia, often abundant in the tumor microenvironment, into L-arginine. Intratumoral and intravenous delivery of the engineered bacteria into tumor-bearing mice synergizes with anti–PD-L1 to improve tumor clearance and increase survival. The effect is dependent on T cells and induces long-lasting immune memory against tumor rechallenge. The advent of this synthetic biotic therapy opens a new dimension to immunotherapy-augmenting strategies for future therapeutic development.Canale FP, …, Geiger R. Nature 2021 Oct 6;598:662–6.Each ICB therapy has separate effects but can be combined to increase efficacy (by Fauxto_digit via Flickr)Immune checkpoint blockade (ICB) is effective in only some patients with melanoma, and the effects of ICB on systemic T-cell responses are not fully elucidated. Gangaev et al. analyzed ex vivo peripheral blood CD8+ T-cell responses to melanoma-associated epitopes. Patient treatment with PD-1 blockade does not affect the breadth of the responses, but anti–CTLA-4 enhances the breadth of the responses. The data highlight that ICB strategies have distinct effects and suggest that those induced by anti–PD-1 act primarily at the tumor site, supporting combinatorial use of ICB therapies.Gangaev A, …, Kvistborg P. Proc Natl Acad Sci U S A 2021 Oct 26;118:e2102849118.Ly6c+CD103+ DC differentiation from precursors is inhibited by BTK and IDO (from Fig. 6 of Neumüller et al., Intech Open 2016)The Ly6c+CD103+ dendritic cells (DC) that cross-present tumor antigens are largely absent from the tumor microenvironment. Sharma et al. find that either pharmacologic or genetic inhibition of BTK and IDO in mouse models of melanoma promotes differentiation of these DCs and antitumor activity. This occurs because the BTK–IDO axis inhibits IFNγ-induced GATOR2–mTORC1 activation, which drives differentiation of monocyte-lineage DC precursors into Ly6c+CD103+ DCs. Elucidating this pathway highlights a molecular process that may be targeted to improve immunotherapy response in tumors refractory to current treatments.Sharma MD, …, Munn DH. Immunity 2021 Oct 12;54:2354–71.e8.Microbiota promote IFN-I production in the TME (from Fig. 1C of Hakozaki et al., Cancer Immunol Res 2020)Interactions between tumor microenvironment (TME) components influence tumor development and response to therapy. Lam et al. show that microbiota-derived stimulator of interferon genes (STING) agonists induce monocytes in the TME to produce type I IFN (IFN-I), which promotes macrophage polarization toward an antitumorigenic phenotype and natural killer cell recruitment, activation, and cross-talk with dendritic cells. This IFN-I pathway is triggered and promotes antitumor immunity in mice fed a high-fiber diet or transplanted with fecal microbiota from patients with melanoma responding to immune checkpoint blockade. The data provide insight that could be used to harness microbiota to treat cancer.Lam KC, …, Goldszmid RS. Cell 2021 Oct 14;184:5338–56.e21.

Education on Blastic Plasmacytoid Dendritic Cell Neoplasm Significantly Impacts the Interdisciplinary Physician Team

Education on Blastic Plasmacytoid Dendritic Cell Neoplasm Significantly Impacts the Interdisciplinary Physician Team

Immunophenotypic and Single-Cell Transcriptional Profiling of CD34+ Hematopoietic Stem and Progenitor Cells Mobilized with Motixafortide (BL-8040) and G-CSF Versus Plerixafor and G-CSF Versus Placebo and G-CSF: A Correlative Study of the Genesis Trial

▪Background: CD34 expression remains the most common immunophenotypic cell surface marker defining human hematopoietic stem and progenitor cells (HSPCs). Recently, use of multicolor fluorescence-activated cell sorting (mFACS) and single-cell RNA sequencing (scRNA seq) has illustrated the heterogenous nature of CD34+ HSPCs, with immunophenotypically and transcriptionally distinct subsets ranging from primitive hematopoietic stem cells (HSCs) capable of long-term multilineage potential to differentiated, lineage-committed progenitors. Meanwhile, the addition of CXCR4 inhibitors (CXCR4i) to G-CSF (G) has increased mobilization of CD34+ HSPCs for stem cell transplantation (SCT). Yet, the effect of CXCR4i +/- G on mobilization of specific immunophenotypic and transcriptional CD34+ HSPC subsets is not well-characterized. Motixafortide (M) is a novel cyclic peptide CXCR4i with a low receptor off rate and extended in vivo action vs plerixafor (Px). M was recently evaluated in the phase 3, double blind, placebo controlled GENESIS Trial as an HSPC mobilizer prior to autologous SCT (ASCT) in multiple myeloma (MM).Methods: GENESIS Trial patients were prospectively randomized (2:1) to receive either M+G or placebo (P)+G for HSPC mobilization. Demographically similar patients undergoing mobilization with Px+G prior to ASCT for MM were prospectively enrolled on a parallel tissue banking protocol. All patients received G (10 mcg/kg) on days 1-5 (and 6-8 if needed). Patients also received either M (1.25 mg/kg) or P on day 4 (and 6 if needed); or Px (0.24 mg/kg) on day 4 (and 5-7 if needed). Apheresis began day 5 (and 6-8 if needed). HSPCs were purified from apheresis product on day 5 via CD34+ immunomagnetic selection. CD34+ HSPC subset profiling was performed via mFACS and scRNA seq. CXCR4 expression and receptor occupancy was evaluated by antibody binding capacity of 12G5 and 1D9 clones.Results: Demographics were similar between the M+G (n=24), P+G (n=13) and Px+G (n=14) cohorts. By mFACS, M+G mobilized a 5.5 fold higher absolute number (#) of HSCs, multipotent progenitors (MPP) and common myeloid progenitors (CMP) vs P+G (p<0.0001); and a 2.6 fold higher # of these subsets vs Px+G (p=0.0327) (Figure 1A). M+G mobilized a 10.5 fold higher absolute # of primitive HSCs vs P+G (p<0.0001) and a 1.1 fold higher # vs Px+G (p=0.7876) (Figure 1B). M+G and Px+G mobilized a 7-8 fold higher # of plasmacytoid dendritic cell precursors vs P+G (p-values >0.05). 1D9 binding to CXCR4 on CD34+ HSPCs was similar between all 3 arms (p-values 0.45-0.75). 12G5 binding (which competes with CXCR4i's for binding to CXCR4) was significantly lower with M+G (MFI:11) vs P+G (MFI:74; p<0.0001) and Px+G (MFI:271; p<0.0001).By scRNA seq, UMAP clustering identified 3 transcriptionally similar HSC sub-clusters (HSC-I, -II and -III) mobilized by all 3 regimens; and 1 distinct HSC-PL cluster mobilized by P+G expressing heat shock protein genes (HSPA1 A/B) (Figure 1C). Differentially expressed genes (DEGs) of HSCs I-III included CD52, FTH1, HLA-E, KLF2 and LMNA. AVP was unique to HSC-I while EGR1, JUN and ZFP36 were unique to HSC-III. MPPs clustered into 3 sub-clusters (MPP-I, -II and -III). MPP-I clustered closely to HSC-II/III with low expression of genes of differentiation. MPP-II expressed DEGs (PLEK) on a continuum toward megakaryocyte-erythroid progenitors (MEP-I/II), which expressed DEGs of erythroid differentiation (HBD/HBB). MPP-I and -II contained cells from all 3 regimens. However, MPP-III was specific to M+G with DEGs (MT-ATP8, HIST1H1) associated with monocyte, lymphocyte and NK cell differentiation.Conclusions: Extended CXCR4i with M+G mobilized significantly higher #s of combined CD34+ HSCs, MPPs and CMPs vs Px+G and P+G (p-values <0.05). Additionally, M+G mobilized a 10.5 fold higher # of immunophenotypically primitive CD34+ HSCs capable of broad multilineage hematopoietic reconstitution vs P+G (p<0.0001) and similar #s vs Px+G. CXCR4 expression on CD34+ HSPCs by 1D9 binding was similar across all arms. Whereas, 12G5 binding was much lower with M+G vs P+G and Px+G, consistent with extended CXCR4 receptor occupancy by M. All regimens mobilized transcriptionally similar HSC-I-III subsets. However, lack of CXCR4i (M or Px) resulted in mobilization of more-differentiated HSCs (HSC-PL subset). Whereas extended CXCR4i with M+G (but not Px+G) mobilized a unique MPP-III subset with DEGs related to leukocyte differentiation. [Display omitted] DisclosuresCrees: BioLineRx Ltd.: Research Funding. Retting: BioLineRx Ltd.: Research Funding. Jayasinghe: WUGEN: Consultancy; MMRF: Consultancy. Vainstein: BioLineRx LTD: Current Employment. Sorani: BioLineRx LTD: Current Employment. Ickowicz: BioLineRx Ltd.: Current Employment. Shemesh-Darvish: BioLineRx LTD: Current Employment. Kadosh: BioLineRx Ltd.: Current Employment.

Changes in phenotypic patterns of blood monocytes after kidney transplantation and during acute rejection.

Peripheral blood monocytes, which serve as precursors for tissue macrophages and dendritic cells (DC), play a key role in the immune response to kidney allograft, reparation processes and homeostasis regulation. In this prospective study, we used multicolor flow cytometry to monitor the phenotypic patterns of peripheral monocytes in subjects with uncomplicated outcomes and those with acute rejection. We found a reciprocal increase in the proportion of "classical monocytes" (CD14+CD16-) along with a decline in pro-inflammatory "intermediary" (CD14+CD16+) and "non-classical" (CD14lowCD16+) monocytes in subjects with normal outcomes. In subjects with acute rejection, we observed no reduction in "intermediary" monocytes and no increase in "classical" monocytes. Patients with uncomplicated outcomes exhibited downregulated HLA-DR in all three monocyte subpopulations. However, non-classical monocytes were unaffected in subjects with acute rejection. Expression of CD47 was downregulated after transplantation, while patients with antibody-mediated rejection and donor-specific antibodies showed higher pre-transplant values. In monocytes isolated at the time of biopsy, CD47 expression was higher in individuals with acute rejection compared to patients with normal outcomes one year post-transplant. Expression of CD209 (DC-SIGN) and the proportion of CD163+CD206+ subpopulations were upregulated during the first week after kidney transplantation. CD209 was also upregulated in samples taken on the day of biopsy confirming acute rejection. Our data demonstrate that kidney allograft transplantation is associated with phenotypic changes in peripheral blood monocytes during acute rejection.

Immunohistochemistry Aids in the Diagnosis of Blastic Plasmacytoid Dendritic Cell Neoplasm in a Patient with Multiple Cutaneous Plaques and Nodules

Abstract Introduction/Objective Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is a rare hematologic malignancy of plasmacytoid dendritic cell precursors with an estimated incidence of 0.04 cases per 100,000 in the US. Given the cutaneous tropism of BPDCN, it should be differentiated from other CD56+ hematopoietic neoplasms with skin involvement such as CD56+ AML, extranodal NK/T-cell lymphoma, and other T-cell lymphomas. Methods/Case Report A 71-year-old male presented to the emergency department with asymptomatic, pink- violaceous plaques/nodules on the trunk for 3 months. One month prior, a dermatologist diagnosed epidermal inclusion cysts that were left untreated. The nodules progressed and a course of steroids prescribed by a primary care physician provided short term improvement before the lesions flared again. Review of systems and vitals were normal. Biopsies of representative lesions sent for histologic examination showed a diffuse dermal infiltrate of small to medium atypical cells with irregular nuclear contours, fine chromatin, one to several nucleoli and scant cytoplasm. Immunohistochemistry showed these cells were positive for CD2, CD4, CD56 and CD45 with strong expression of BCL2 and focal CD123. The cells were negative for CD3, CD20, CD79a, CD8, CD30, ALK-1, MUM-1, CD10, Cyclin- D1, C-MYC, EBER, BCL6, Langerin, Granzyme, TIA1, CD68, CD163, MPO, and Lysozyme. The histology and immunoprofile were consistent with BPDCN. A bone marrow biopsy showed cells with similar morphology and staining pattern, including expression of CD123.Treatment with chemotherapy and Tagraxofusp, was initiated. Within a week, the patient showed near resolution of cutaneous lesions. Repeat bone marrow aspirate and flow cytometry a month later showed no malignant cells. Results (if a Case Study enter NA) NA Conclusion We present this case as a rare hematologic malignancy with a challenging clinical and histopathologic diagnosis. The histologic findings suggested either a high grade myeloid or lymphoid malignancy. The combination of CD3-/CD56+/CD4+/CD123+ in the lesional cells helped establish the diagnosis of BPDCN, allowing for prompt treatment.