A reactive monocyte subset characterized by low expression of CD91 is expanded during sterile and septic inflammation.

OBJECTIVES: This study was undertaken to assess CD91 expression on monocytes and changes in monocyte subset distribution during acute tissue damage and bloodstream infection (BSI). METHODS: We investigated blood specimens from healthy individuals, trauma and cardiac surgery patients as a model of tissue damage, and patients with BSI, by flow cytometry using a panel of antibodies comprising CD45, HLA-DR, CD14, CD16 and CD91 for the identification of monocyte subsets. RESULTS: While infrequent in healthy subjects, CD91low/neg monocyte levels were markedly high in BSI, trauma and after cardiac surgery. This monocyte subset expanded up to 15-fold in both patient cohorts, whereas CD14+CD16+ inflammatory monocytes were multiplied by a factor of 5 only. CD14+CD91low monocytes displayed a significantly lower density of HLA-DR and markedly reduced expression of CD300e, compared to the other subsets. They also expressed high levels of myeloperoxidase and showed robust phagocytic and oxidative burst activity. CONCLUSIONS: Expansion of CD91low monocytes is a sensitive marker of acute inflammatory states of infectious and non-infectious etiology.

Integrative Analysis of Proteomics and Transcriptomics Reveals Endothelin Receptor B as Novel Single Target and Identifies New Combinatorial Targets for Multiple Myeloma.

Despite the recent introduction of next-generation immunotherapeutic agents, multiple myeloma (MM) remains incurable. New strategies targeting MM-specific antigens may result in a more effective therapy by preventing antigen escape, clonal evolution, and tumor resistance. In this work, we adapted an algorithm that integrates proteomic and transcriptomic results of myeloma cells to identify new antigens and possible antigen combinations. We performed cell surface proteomics on 6 myeloma cell lines based and combined these results with gene expression studies. Our algorithm identified 209 overexpressed surface proteins from which 23 proteins could be selected for combinatorial pairing. Flow cytometry analysis of 20 primary samples confirmed the expression of FCRL5, BCMA, and ICAM2 in all samples and IL6R, endothelin receptor B (ETB), and SLCO5A1 in >60% of myeloma cases. Analyzing possible combinations, we found 6 combinatorial pairs that can target myeloma cells and avoid toxicity on other organs. In addition, our studies identified ETB as a tumor-associated antigen that is overexpressed on myeloma cells. This antigen can be targeted with a new monoclonal antibody RB49 that recognizes an epitope located in a region that becomes highly accessible after activation of ETB by its ligand. In conclusion, our algorithm identified several candidate antigens that can be used for either single-antigen targeting approaches or for combinatorial targeting in new immunotherapeutic approaches in MM.

Curcuma longa and Boswellia serrata Extracts Modulate Different and Complementary Pathways on Human Chondrocytes In Vitro: Deciphering of a Transcriptomic Study.

Objectives: Curcuma longa (CL) and Boswellia serrata (BS) extracts are used to relieve osteoarthritis symptoms. The aim of this in vitro study was to investigate their mechanisms of action at therapeutic plasmatic concentrations on primary human osteoarthritic (OA) chondrocytes. Methods: BS (10-50 µg/ml) and CL (0.4-2 µg/ml corresponding to 1-5 µM of curcumin) were evaluated separately or in combination on primary chondrocytes isolated from 17 OA patients and cultured in alginate beads. Ten patients were used for RNA-sequencing analysis. Proteomic confirmation was performed either by immunoassays in the culture supernatant or by flow cytometry for cell surface markers after 72 h of treatment. Results: Significant gene expression modifications were already observed after 6 h of treatment at the highest dose of CL (2 µg/ml) while BS was significantly effective only after 24 h of treatment irrespective of the concentration tested. The most over-expressed genes by CL were anti-oxidative, detoxifying, and cytoprotective genes involved in the Nrf2 pathway. Down-regulated genes were principally pro-inflammatory cytokines and chemokines. Inversely, BS anti-oxidant/detoxifying activities were related to the activation of Nrf1 and PPARα pathways. BS anti-inflammatory effects were associated with the increase in GDF15, decrease in cholesterol cell intake and fatty acid metabolism-involved genes, and down-regulation of Toll-like receptors (TLRs) activation. Similar to CL, BS down-regulated ADAMTS1, 5, and MMP3, 13 genes expression. The combination of both CL and BS was significantly more effective than CL or BS alone on many genes such as IL-6, CCL2, ADAMTS1, and 5. Conclusion: BS and CL have anti-oxidative, anti-inflammatory, and anti-catabolic activities, suggesting a protective effect of these extracts on cartilage. Even if they share some mechanism of action, the two extracts act mainly on distinct pathways, and with different time courses, justifying their association to treat osteoarthritis.

Machine learning identification of specific changes in myeloid cell phenotype during bloodstream infections.

The early identification of bacteremia is critical for ensuring appropriate treatment of nosocomial infections in intensive care unit (ICU) patients. The aim of this study was to use flow cytometric data of myeloid cells as a biomarker of bloodstream infection (BSI). An eight-color antibody panel was used to identify seven monocyte and two dendritic cell subsets. In the learning cohort, immunophenotyping was applied to (1) control subjects, (2) postoperative heart surgery patients, as a model of noninfectious inflammatory responses, and (3) blood culture-positive patients. Of the complex changes in the myeloid cell phenotype, a decrease in myeloid and plasmacytoid dendritic cell numbers, increase in CD14+CD16+ inflammatory monocyte numbers, and upregulation of neutrophils CD64 and CD123 expression were prominent in BSI patients. An extreme gradient boosting (XGBoost) algorithm called the "infection detection and ranging score" (iDAR), ranging from 0 to 100, was developed to identify infection-specific changes in 101 phenotypic variables related to neutrophils, monocytes and dendritic cells. The tenfold cross-validation achieved an area under the receiver operating characteristic (AUROC) of 0.988 (95% CI 0.985-1) for the detection of bacteremic patients. In an out-of-sample, in-house validation, iDAR achieved an AUROC of 0.85 (95% CI 0.71-0.98) in differentiating localized from bloodstream infection and 0.95 (95% CI 0.89-1) in discriminating infected from noninfected ICU patients. In conclusion, a machine learning approach was used to translate the changes in myeloid cell phenotype in response to infection into a score that could identify bacteremia with high specificity in ICU patients.

Correction: Human bone marrow harbors cells with neural crest-associated characteristics like human adipose and dermis tissues.

[This corrects the article DOI: 10.1371/journal.pone.0177962.].

[Immunomonitoring of patients treated with CAR-T cells for hematological malignancy: Guidelines from the CARTi group and the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)]. / Suivi immunologique des patients traités par cellules CAR-T pour hémopathie maligne: recommandations du groupe CARTi et de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

CAR-T cells represent a new anti-tumor immunotherapy which has shown its clinical efficacy in B-cell malignancies. The results of clinical trials carried out in this context have shown that certain immunological characteristics of patients before (at the time of apheresis) and after the administration of the treatment, or of the CAR-T cells themselves, are correlated with the response to the treatment or to its toxicity. However, to date, there are no recommendations on the immunological monitoring of patients treated in real life. The objectives of this workshop were to determine, based on data from the literature and the experience of the centers, the immunological analyses to be carried out in patients treated with CAR-T cells. The recommendations relate to the characterization of the patient's immune cells at the time of apheresis, the characterization of the injected CAR-T cells, as well as the monitoring of the CAR-T cells and other parameters of immune reconstitution in the patient after administration of the treatment. Harmonization of practices will allow clinical-biological correlation studies to be carried out in patients treated in real life with the aim of identifying factors predictive of response and toxicity. Such data could have a major medico-economic impact by making it possible to identify the patients who will optimally benefit from these expensive treatments.

Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of alpha4 integrin- and CXCR4-mediated engraftment in NOD/SCID beta2-microglobulin-null mice.

BACKGROUND: Several studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of alpha4 integrin, alpha5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34(+) cells. DESIGN AND METHODS: Uncultured or expanded CD34(+) cells were infused in NOD/SCID-beta(2)microglobulin-null mice. The function of alpha4, and alpha5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of alpha4 integrin was further tested by adhesion and migration assays. RESULTS: Neutralization of either alpha4 integrin or CXCR4 abolished engraftment of uncultured CD34(+) cells at 6 week spost-transplant, while alpha5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking alpha4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of alpha5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that alpha4 integrin affinity in fresh CD34(+) cells was low and susceptible to stimulation while in cultured CD34(+) cells, it was high and insensitive to further activation. In addition, stromal cell-derived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34(+) cells but not in cultured CD34(+) cells. CONCLUSIONS: Our data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both alpha4 integrin- and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of alpha4 integrin affinity, which impairs directional migration across vascular cell adhesion molecule-1 in response to stromal cell-derived factor-1. Such changes may underlie the engraftment defect of cytokine-stimulated CD34(+) cells.

Human bone marrow harbors cells with neural crest-associated characteristics like human adipose and dermis tissues.

Adult neural crest stem-derived cells (NCSC) are of extraordinary high plasticity and promising candidates for use in regenerative medicine. Several locations such as skin, adipose tissue, dental pulp or bone marrow have been described in rodent, as sources of NCSC. However, very little information is available concerning their correspondence in human tissues, and more precisely for human bone marrow. The main objective of this study was therefore to characterize NCSC from adult human bone marrow. In this purpose, we compared human bone marrow stromal cells to human adipose tissue and dermis, already described for containing NCSC. We performed comparative analyses in terms of gene and protein expression as well as functional characterizations. It appeared that human bone marrow, similarly to adipose tissue and dermis, contains NESTIN+ / SOX9+ / TWIST+ / SLUG+ / P75NTR+ / BRN3A+/ MSI1+/ SNAIL1+ cells and were able to differentiate into melanocytes, Schwann cells and neurons. Moreover, when injected into chicken embryos, all those cells were able to migrate and follow endogenous neural crest migration pathways. Altogether, the phenotypic characterization and migration abilities strongly suggest the presence of neural crest-derived cells in human adult bone marrow.

[Monoclonal B-cell lymphocytosis: from literature to laboratory practice]. / Lymphocytoses B monoclonales: de la revue de la littérature à la pratique de laboratoire.

Monoclonal B-cell lymphocytosis (MBL) is defined as an asymptomatic condition characterized by the presence of less than 5,000 monoclonal B-cells per microliter and the absence of clinical signs or symptoms of a B-cell lymphoproliferative disorder. Most MBL cases involve B cells presenting an identical phenotype to CLL (CLL-like MBL) with a Catovsky-Matutes score of 3 to 5 and share the same chromosomal abnormalities than CLL. Depending on the absolute B cell count, one may distinguish low-count CLL-like MBL (<500 B cells/µL) which have no evidence of progression, no reduction in overall survival, no increase in infection risk and do not require any specific follow-up. Patients with clinical CLL-like MBL (>500 B cells/µL) have a 1% to 2% per year risk of progression to CLL requiring therapy, a higher risk of infectious complications and mortality implicating an annual follow-up by hematologist. MBL may also express other less common phenotypes and are named atypical MBL in case of CD5 antigen expression (Catovsky-Matutes score: 1-2) and non-CLL-like MBL for CD5 negative cases (Catovsky-Matutes score: 0-2). Their poorer prognosis implicates imaging studies, bone marrow biopsy and cytogenetic analysis in addition to physical examination in order to rule out non-hodgkinien lymphoma, and require a more frequent follow-up. This review focuses on key concepts in the classification, diagnosis, monitoring and biology of MBL in laboratory practice.

Modulation of homing properties of primitive progenitor cells generated by ex vivo expansion.

BACKGROUND AND OBJECTIVES: The maintenance of adequate interactions with the bone marrow (BM) microenvironment is critical to ensure efficient homing of ex vivo-expanded hematopoietic cells. This study was intended to assess adhesion and migration properties of long-term culture-initiating cells (LTC-IC) harvested after self-renewal division in ex vivo culture and to determine their susceptibility to growth-inhibitory signals mediated by adhesion to BM stromal ligands. DESIGN AND METHODS: We used cell tracking to isolate primitive LTC-IC that had accomplished 1 or 2 divisions ex vivo. Adhesion, migration and growth inhibition of divided LTC-IC were determined in the presence of purified BM ligands, and compared to the properties of uncultured LTC-IC. RESULTS: As compared to undivided LTC-IC, adhesion and migration mediated by very late antigen (VLA)-4 integrin across both vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (Fn) were downregulated in post-mitotic LTC-IC. Conversely, binding and motility via VLA-5 across Fn were stimulated. No changes occurred in LTC-IC interactions with intercellular adhesion molecule-1 (ICAM-1) or with E- or P-selectin. Proliferation of uncultured LTC-IC was inhibited by VLA-4-mediated binding to VCAM-1 and the CS-1 domain of Fn, as well as binding to P-selectin. Growth of ex vivo-generated LTC-IC became unresponsive to these 3 ligands but was suppressed through VLA-5 engagement by the cell binding domain of Fn. INTERPRETATION AND CONCLUSIONS: The generation of LTC-IC in expansion culture is associated with functional alterations of adhesion receptors, modulating not only binding and migration in the BM but also responsiveness to adhesion-mediated growth inhibitory signals. Such changes may limit homing and engraftment of expanded primitive stem/progenitor cells.

Imatinib and nilotinib inhibit hematopoietic progenitor cell growth, but do not prevent adhesion, migration and engraftment of human cord blood CD34+ cells.

BACKGROUND: The availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34(+) cells. DESIGN AND METHODS: After a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null) mice. RESULTS: TKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34(+) cell cycle entry. Adhesion of CD34(+) cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT. CONCLUSIONS: These data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe.