Lenalidomide IS Able to Restore Immune SYSTEM IN MULTIPLE MYELOMA PATIENTS.

Abstract Abstract 4909 LENALIDOMIDE IS ABLE TO RESTORE IMMUNE SYSTEM IN MULTIPLE MYELOMA PATIENTS Annalisa Chiarenza, Nunziatina Parrinello, Piera La Cava, Eleonora Spina, Daniele Tibullo, Cesarina Giallongo, Maide Cavalli, Alessandra Romano, Paolo Fiumara, Giuseppe A. Palumbo, Francesco Di Raimondo Background Multiple myeloma (MM) is a malignant plasma-cell proliferative disorder associated with dysfunctional T-cell responses. The immunomodulatory Thal derivative (IMiD) CC-5013 (lenalidomide) appears to be a promising agent for the treatment of myeloma. Although the exact antitumor mechanism of action of lenalidomide is unknown, a number of mechanisms are postulated to be responsible for it's activity (inhibition of angiogenesis, direct antiproliferative and proapoptotic effects on MM cells, suppression of pro-inflammatory cytokines, modulation of myeloma-stromal cells adhesive interactions). In addition, it has been demonstrated that lenalidomide in vitro is able to enhance T cell proliferation and to promotes ADCC. In this study we evaluated if MM patients have a deficit of T-reg (CD4+, CD25+, and FOXP3+) and of T lymphocytes bearing CD200 (a tolerogenic molecule) and the effect of lenalidomide treatment on these parameters. In addition, we investigated whether lenalidomide could improve ex vivo the ADCC against myeloma cells. Materials and methods Eight patients with previously untreated MM (median age 56 years) were treated with lenalidomide plus dexamethasone as first line therapy. Lenalidomide was given orally 25 mg daily on days 1 to 21 of a 28-day cycle. Dexamethasone was given orally 40 mg daily on days 1, 8, 15, 22 of each cycle. All patients were evaluable for response and toxicity. Peripheral blood mononuclear cells (PBMNc) were obtained from MM patients using density gradient centrifugation (Fycoll) under sterile condictions, at the beginning of treatment and after 4 cycles of therapy. The percentage of T-reg (CD4+CD25+FOXP3+) and the expression of CD200 on T- lymphocytes were evaluated by cytometry. Twelve healthy subjects were used as control. Moreover, PBMNc (effector cells, E) were incubated with MM cells line ARH-77 (target cells, T), previously labelled with CFDA,SE (carboxyfluorescein diacetate, succinimidyl ester) as a tracing fluorescent marker, in culture medium (RPMI-1640, 10%FCS, 1%penicillin/streptomycin) at different concentration (T/E ratio 1:20, 1:40). After 18-24 h co-colture cells were analyzed by flow cytometry and MM plasma cells cytotoxicity was calculated as the percentage of positive CFDA,SE/propidium cells. Myeloma cell viability was determined by tripan blue esclusion and apoptosis was also evaluated using Annexin V/propidium assay. Two MM patients treated in first line with a combination of Velcade, Thalidomide and Dexamethasone (VTD) were used as control and the experiments were performed in duplicate. Results MM patients have a significantly lower rate of CD4+/CD25+/FOXP3+ and CD200+/CD3+ than normal (28,3±14,9/mmc and 37,8±24,7 /mmc vs 79,3±27,8 and 79,5± 48,9)(p=0,0001 and p=0,01 respectively). In our study, lenalidomide treatment resulted in an increase both of Treg cells and T-lymphocytes espressing CD200. This improvement is not statistically significant probably due to the low number of patients examined (tab I). More important, we observed that PBMC derived from patients treated with lenalidomide showed an increase ability to kill a target MM cell line compared to PBMC collected at diagnosis (CFDA,SE/propidium cells 11% vs 68%). This effect was more prominent in patients treated with lenalidomide than in MM patients treated with VTD (CFDA,SE/propidium cells 12% vs 39%), Fig.1. Conclusions Our data emphasize the role of lenalidomide in modulating the endogenous tumor-specific immune response and underline the anti-myeloma activity of these new class of drugs. Disclosures No relevant conflicts of interest to declare.

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Increased Capacity Of Monocytes From Patients With Multiple Myeloma Treated With Thalidomide Or Lenalidomide To Generate Tissue Factor

Blood ◽

10.1182/blood.v122.21.3628.3628 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3628-3628

Author(s):

Abraham Kornberg ◽

Marina Izak ◽

Yossi Cohen

Keyword(s):

Multiple Myeloma ◽

Tissue Factor ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Line Treatment ◽

First Line ◽

Peripheral Blood Mononuclear ◽

Before And After

Abstract Treatment of multiple myeloma (MM) with thalidomide or lenalidomide is associated with increased incidence of thrombosis in contrast to treatment with bortezomib. The mechanism of thrombosis is unknown. Monocytes generate potent tissue factor (TF), the main activator of coagulation, in response to various stimuli (endotoxin, cytokins and others) and in diseases with increased incidence of thrombosis. We investigated the capacity of monocytes from patients with MM to generate TF in relation to different modalities of treatments and activation of coagulation. Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll-Hypaque centrifugation and monocytes by adhesion. TF activity was assayed by modified PT using the cells as source of TF and TF antigen by ELISA, in endotoxin stimulated (10 ug/ml) and unstimulated PBMC ( all patients) and purified monocytes (50% of patients), before and after 3-4 courses of treatment. Monocytes were identified by anti CD14 and Plasma Fragment1.2 was assayed by ELISA. No TF activity@ (X10-5 U/monocyte) TF antigen@ (X10-5 pg/monocyte) Plasma fragment 1.2@ (nM) Endotoxin - + - + Bortezomib based treat* 12 before 2.2 4.8 22 64 0.28 after 1.8 4.2 18 57 0.36 Thalidomide based treat* 4 before 2.4 5.5 26 60 0.24 after 1.8 34.8# 20 440# 2.48# Lenalidomide based treat^ 6 before 2.1 6.2 28 62 0.30 after 4.1 58.6# 16 312# 3.42# @Mean *First line treatment ^Second line treatment # P<0.05 One patient on thalidomide and 2 on lenalidomide developed thrombosis. The results show that TF was low in unstimulated monocytes from all patients before treatment. It was increased mildely in stimulated monocytes from all patients before treatment and also after treatment with bortezomib (X 2-3). TF in stimulated monocytes and also plasma fragment1.2 were increased significantly after treatment with thalidomide and lenalidomide (x 14-22 and X 10). The phenomenon of enhanced capacity of monocytes to generate potent TF after treatment with thalidomide and lenalidomide but not bortezomib, and the correlation with activation of coagulation suggest a role of monocyte TF in thrombus formation by these drugs. The mechanism of the enhanced capacity is unknown and may be attributed to the immunomodulatory effect of the drugs. Disclosures: No relevant conflicts of interest to declare.

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Ex vivo induction of multiple myeloma–specific cytotoxic T lymphocytes

Blood ◽

10.1182/blood-2002-09-2828 ◽

2003 ◽

Vol 102 (4) ◽

pp. 1435-1442 ◽

Cited By ~ 90

Author(s):

Toshiaki Hayashi ◽

Teru Hideshima ◽

Masaharu Akiyama ◽

Noopur Raje ◽

Paul Richardson ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

T Cell ◽

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Mononuclear Cells ◽

Ex Vivo ◽

Stimulation Index ◽

T Cell Proliferation ◽

Autologous Tumor

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by immunosuppression. In this study, we identified factors in patients' bone marrow (BM) sera inhibiting autologous anti-MM immunity and developed an ex vivo strategy for inducing MM-specific cytotoxic T lymphocytes (CTLs). We found that sera from BM of MM patients inhibited induction of dendritic cells (DCs), evidenced by both phenotype and only weak stimulation of T-cell proliferation. Anti–vascular endothelial growth factor (anti-VEGF) and/or anti–interleukin 6 (anti–IL-6) antibodies neutralized this inhibitory effect, confirming that VEGF and IL-6, at least in part, mediate immunosuppression in MM patients. To induce MM-specific CTLs ex vivo, immature DCs were generated by culture of adherent mononuclear cells in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days and then cocultured with apoptotic MM bodies in the presence of tumor necrosis factor α (TNF-α) for 3 days to induce their maturation. Autologous BM or peripheral blood mononuclear cells were stimulated weekly with these DCs, and cytotoxicity was examined against the MM cells used to pulse DCs. DCs cultured with apoptotic bodies stimulated significantly greater T-cell proliferation (stimulation index [SI] = 23.2 at a T-DC ratio of 360:1) than T cells stimulated by MM cells only (SI = 5.6), DCs only (SI = 9.3), or MM lysate–pulsed DCs (SI = 13.5). These CTLs from MM patients demonstrated specific cytotoxicity (24.7% at the effector-target [E/T] ratio of 40:1) against autologous primary MM cells. These studies therefore show that CTLs from MM patients can recognize and lyse autologous tumor cells and provide the framework for novel immunotherapy to improve patient outcome in MM.

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Myeloid Impairment Contributes to Immunoparesis in Multiple Myeloma but Not MGUS

Blood ◽

10.1182/blood.v120.21.1831.1831 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1831-1831

Author(s):

Alessandra Romano ◽

Nunziatina Parrinello ◽

Calogero Vetro ◽

Piera La Cava ◽

Annalisa Chiarenza ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

T Cell ◽

T Lymphocytes ◽

Peripheral Blood ◽

Healthy Subjects ◽

Conflicts Of Interest ◽

Myeloid Suppressor Cells ◽

Hla Dr ◽

Healthy Donors

Abstract Abstract 1831 Introduction In Multiple Myeloma (MM), but not in the monoclonal gammopathy of unknown significance (MGUS), the immune function is impaired as consequence of an immunologically hostile microenvironment and cellular defects, including reduction of immuno-surveillance and T-cell immunoparesis. We conducted an study focused on the myeloid compartment in MM, and its role in the progression from MGUS to MM. Methods Between January 2009 and April 2011 peripheral blood obtained from 60 consecutive newly diagnosed MM and 70 MGUS plus 30 healthy subjects was studied for evaluation of myeloid subpopulations and lymphoid paresis. Myeloid dysfunction was evaluated as percentage and absolute count of circulating myeloid suppressor cells (MDSC) in peripheral blood assessed by flow cytometry as follows: im-MDSC (CD34+/CD11b+/CD13+/CD14-/ HLA-DR-/CD45+), neutrophilic-like N-MDSC (CD11b+/CD13+/CD15+/CD14-/HLA-DR-/Lin-) and monocytic-like mo-MDSC (CD14+/HLA-DRlow/-). Myeloid function was evaluated by phagocytic activity using a commercially available kit (Phagotest R). Further, we investigated whether MM-neutrophils were able to induce anergy in T-cells. Neutrophils isolated from healthy donor (N=6), MGUS (N=3) or MM (N=6) peripheral blood (PB) were co-cultured with T-lymphocytes obtained from healthy donors. Expression of markers of activation in response to stimulation with PHA-P for 2 hours was assessed by flow cytometry as antigen density expressed as normalized mean of fluorescence intensity (N-MFI) of CD71 at 48 hours. Results The capability of phagocytosis of in neutrophils and monocytes from MM patients at diagnosis was significantly reduced compared to healthy subjects (p<0.001) and MGUS (p<0.0001). While the mature suppressive N-MDSC subset was not increased in MGUS and MM patients, the mo-MDSC subpopulation showed an increasing trend from healthy donors through MM (p=0.06) and the im-MDSC subset was significantly higher in MM vs healthy (p=0.002) and MGUS (p=0.001). After PHA-P stimulation, expression of CD71 (a marker of activation) in normal T-lymphocytes was increased (2954 ± 240.6 arbitrary units, au), and it was reduced (751.3 ± 30.48 au, p=0.0001) when co-coltured with MM-neutrophils, while no differences were evident in co-colture with MGUS- (2783 ± 206.1 au, p=0.61) or healthy donors-neutrophils (2588 ± 135.4, p=0.38). Conclusion Taken together, our findings suggest that in MM but not in MGUS there is a myeloid cell dysfunction that is correlated to impairment of T- cell arm. These alterations may have a role in the development of MM. Disclosures: No relevant conflicts of interest to declare.

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Fiber-modified adenoviruses generate subgroup cross-reactive, adenovirus-specific cytotoxic T lymphocytes for therapeutic applications

Blood ◽

10.1182/blood-2003-07-2449 ◽

2004 ◽

Vol 103 (3) ◽

pp. 1011-1019 ◽

Cited By ~ 72

Author(s):

Ann M. Leen ◽

Uluhan Sili ◽

Barbara Savoldo ◽

Alan M. Jewell ◽

Pedro A. Piedra ◽

...

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Mononuclear Cells ◽

Ex Vivo ◽

Target Cells ◽

Cell Transplant ◽

Peripheral Blood Mononuclear ◽

Hematopoietic Stem ◽

Considerable Morbidity

AbstractAdenovirus (Ad) infections are responsible for considerable morbidity and mortality, particularly in pediatric hematopoietic stem cell transplant (HSCT) recipients. To date there is no therapy. The present study was motivated by the potential for using adoptive immunotherapy as either prophylaxis or treatment for Ad infections and associated diseases. The authors have developed a protocol to reactivate Ad-specific memory T cells from peripheral blood mononuclear cells (PBMCs) using a clinical-grade adenoviral vector. Such lines contain a specific CD4 and CD8 T-cell component and are capable of recognizing and lysing target cells infected with wild-type Ad serotypes from different Ad groups. Furthermore, the frequency of Ad-specific precursors can be determined in PBMCs ex vivo and used as a means to assess changes in Ad-specific T-cell memory responses after infusion. This is the first report of a simple and reproducible method to activate and expand Ad-specific cytotoxic T lymphocytes (CTLs), which should be protective against the range of different Ad subtypes that affect transplant recipients. (Blood. 2004;103:1011-1019)

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Rwd Study for Epidemiology and Characteristics of Patients with Multiple Myeloma in Italy

Blood ◽

10.1182/blood.v128.22.5693.5693 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5693-5693

Author(s):

Giovanni Corrao ◽

Vittorio Montefusco ◽

Francesco De Solda ◽

Paola Finsinger ◽

Elena Lanati ◽

...

Keyword(s):

Multiple Myeloma ◽

Literature Review ◽

Conflicts Of Interest ◽

National Level ◽

Data Availability ◽

Italian Population ◽

First Line ◽

First Line Therapy ◽

Line Therapy ◽

Number Of Patients

Abstract Executive Summary Objective The aim of this study was to estimate the prevalence, incidence and characteristics (comorbidities, treatments) of patients with multiple myeloma (MM) in Italy based on literature review and on analysis from two Italian registries: a representative regional registry (Lombardy), and the National Cancer Institute in Milan (INT). Methods The analysis has been performed using prevalence and incidence information reported in the literature and real-world data from two sources: the Lombardy region registry and the MM database of the INT. Results from this analysis were then compared with national data. The study has been conducted on the Lombardy population because of its relevance in terms of number of inhabitants, patients' characteristics and data availability. Data collected from these two databases were used to calculate the number of patients with MM and their characteristics at a national level. The literature review focused on a retrospective study conducted in 2010 from the Lombardy registry (DENALI) that reported the incidence of MM in this region, and on the INT Milan database. The Lombardy registry compared administrative and clinical data on 436 hospitalized patients diagnosed with MM who received chemotherapy treatment in 2010. The INT database reports clinical and demographic data from 166 patients diagnosed with MM and treated within the period 2010-2015. Results In Italy, the literature reports an age-adjusted incidence for MM of 3.7/100,000 for males and 2.7/100,000 for females; the reported prevalence is 0.040%. The Lombardy registry shows a prevalence of 0.031% and an age-adjusted mortality rate of 1.6/100,000 for males and 1.1/100,000 for females. The percentage of patients with MM beyond first-line therapy is 31%; 28% are in second-line therapy, 2% in third line and 1% in fourth line or more. The type of first-line therapy and baseline comorbidities are summarized below (Table 1). As reported by the Lombardy registry, 26% of patients with MM have cardiovascular or renal comorbidities, compared with 16% reported in the INT database. According to reports from the INT database, 17% of patients with MM do not receive treatment. Applying data from the Lombardy registry, it is possible to estimate that in Italy, in 2016, there are 25,800 patients with MM, 8,000 patients beyond first-line therapy and 2,000 patients with cardiovascular or renal comorbidities. The prevalence used in this study is the one from the literature because it is considered more reliable than the data found in the regional registry. Conclusions This analysis shows that various sources need to be assessed to obtain valuable clinical information that is not always available in the literature, such as comorbidities and patients not treated, even if prevalence data are not precisely consistent with values for Italy in the literature. Based on this analysis we were able to extrapolate the results to the entire Italian population. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

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Productive Infection of Neonatal CD8+ T Lymphocytes by HIV-1

Journal of Experimental Medicine ◽

10.1084/jem.187.7.1139 ◽

1998 ◽

Vol 187 (7) ◽

pp. 1139-1144 ◽

Cited By ~ 68

Author(s):

Liang Peng Yang ◽

James L. Riley ◽

Richard G. Carroll ◽

Carl H. June ◽

James Hoxie ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

T Lymphocytes ◽

Hiv Infection ◽

Cd8 T Cells ◽

Productive Infection ◽

Target Cells ◽

Cd8 T Lymphocytes ◽

Hiv 1

CD8+ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8+ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell–tropic (T-tropic) HIV strains. Physiological activation of CD8-α/β+ CD4− T cell receptor–α/β+ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1–activated cord blood CD8+ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8+ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.

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Absence of allogeneic restriction in human T-cell-mediated cytotoxicity to Epstein-Barr virus-infected target cells. Demonstration of an HLA-linked control at the effector level.

Journal of Experimental Medicine ◽

10.1084/jem.150.6.1310 ◽

1979 ◽

Vol 150 (6) ◽

pp. 1310-1322 ◽

Cited By ~ 45

Author(s):

M Lipinski ◽

W H Fridman ◽

T Tursz ◽

C Vincent ◽

D Pious ◽

...

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Cell Surface ◽

Target Cell ◽

Epstein Barr Virus ◽

Target Cells ◽

Specific Lysis ◽

Barr Virus ◽

Positive Target ◽

Epstein Barr

Peripheral T lymphocytes from patients with infectious mononucleosis (IM) are sensitized in vivo against the Epstein-Barr virus (EBV). The expression of HLA-A, B, or C molecules at the target cell surface is necessary for the cytotoxic reaction because (a) EBV-positive Daudi cells lacking HLA-A, B, and C determinants are resistant to anti-EBV T-cell lysis, (b) cytolysis of EBV-positive target cells can be consistently inhibited by anti-HLA-A, B, and C and anti-beta 2 microglobulin antibodies. However, no evidence for allogeneic restriction in this system was apparent as (a) cytotoxic T lymphocytes (CTL) from one given individual could exert a cytotoxicity of a similar magnitude on different EBV-positive target cells, regardless of the number of HLA-A or B specificities shared by the effectors and targets; (b) CTL from IM patients were able to kill target cells without any HLA-A or B antigen in common; and (c) T5-1 variants lacking one or two HLA antigens at the A, B, or D locus are killed to the same extent as the parental cells. 7 of the 9 IM patients with detectable circulating anti-EBV CTL carried the HLA-A1 antigen, whereas none of the 16 IM patients lacking detectable peripheral CTL were HLA-A1 positive (mean specific lysis of T5-1 target cells by T cells from HLA-A1 positive patients: 29.3 vs. 0.6% in HLA-A1-negative patients) (P less than 10(-9)). These data suggest an HLA-A1-linked gene control of the magnitude of the anti-EBV CTL response. Thus, the HLA region appears to act at two different level sin the T-cell-mediated lysis of EBV-infected cells by controlling first, the development of anti-EBV and second, the expression of HLA-A, B, and C molecules involved as recognition structures at the target cell surface.

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Detection of hyperreactive T cells in multiple myeloma by multivalent cross-linking of the CD3/TCR complex [see comments]

Blood ◽

10.1182/blood.v78.7.1770.1770 ◽

1991 ◽

Vol 78 (7) ◽

pp. 1770-1780 ◽

Cited By ~ 33

Author(s):

M Massaia ◽

A Bianchi ◽

C Attisano ◽

S Peola ◽

V Redoglia ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Mononuclear Cells ◽

Interleukin 2 ◽

Normal Subjects ◽

Cross Linking ◽

Peripheral Blood Mononuclear ◽

Hla Dr ◽

Lower Ratio

Abstract Cellular immunity was investigated in 43 patients with multiple myeloma (MM) by assessing 3HTdR uptake induced by monocyte-dependent [CD3 monoclonal antibodies (MoAbs), phytohemagglutinin (PHA)] and monocyte- independent (CD2 MoAbs, ionomycin + phorbolester) stimulations. The former were evaluated in peripheral blood mononuclear cells (PBMNC) and purified T cells; the latter were evaluated in purified T-cell preparations only. MM showed significantly lower PBMNC responses to PHA (P less than .001), soluble OKT3 (CD3) (P = .01), and immobilized OKT3 MoAbs (P = .01). On purification of T cells, MM responses were still defective to soluble T11(2) + T11(3) (CD2) MoAbs (P = .004), phorbol myristate acetate (PMA) plus ionomycin (P less than .001), but significantly higher to plastic-immobilized OKT3 (P = .004). In some MM, 3HTdR uptake, interleukin-2 (IL-2) receptor (CD25) expression, and IL-2 production were as high on stimulation with plastic-immobilized OKT3 as that observed in normal subjects under optimal conditions (ie, plastic-immobilized OKT3 plus accessory signals). CD3 hyperreactivity correlated with the number of CD8+ HLA-DR+ cells in MM T-cell preparations. MM patients with more than 10% CD8+ HLA-DR+ cells had significantly higher responses to immobilized OKT3 (P less than .001), but lower responses to T11(2) plus T11(3) (P = .01), and PMA plus ionomycin (P = .03) than patients with less than 10% CD8+ HLA-DR+ cells. Phenotyping of CD45RA (naive) and CD45R0 (memory) expressions in resting MM T cells showed a lower ratio of CD45RA to CD45R0 in both CD4 (P less than .05) and CD8 (P less than .001) subpopulations. These data indicate that (a) some MM T cells require significantly fewer accessory signals (if any) to express the IL-2 receptor fully, secrete IL-2, and proliferate on multivalent cross-linking of the CD3/TCR complex; and (b) this peculiar state of activation is associated with high HLA-DR expression in CD8+ lymphocytes.

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Transcriptome Based Projection of Single Cells to Uncover Development and Heterogeneity of Abnormal Hematopoietic Cells

Blood ◽

10.1182/blood-2019-130587 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2520-2520

Author(s):

Parashar Dhapola ◽

Mikael Sommarin ◽

Mohamed Eldeeb ◽

Amol Ugale ◽

David Bryder ◽

...

Keyword(s):

Single Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Single Cells ◽

Hematopoietic Cells ◽

Leukemic Cells ◽

Target Cells ◽

Cell Identity ◽

Large Patient ◽

Low Dimensional

Single-cell transcriptomics (scRNA-Seq) has accelerated the investigation of hematopoietic differentiation. Based on scRNA-Seq data, more refined models of lineage determination in stem- and progenitor cells are now available. Despite such advances, characterizing leukemic cells using single-cell approaches remains challenging. The conventional strategies of scRNA-Seq analysis map all cells on the same low dimensional space using approaches like tSNE and UMAP. However, when used for comparing normal and leukemic cells, such methods are often inadequate as the transcriptome of the leukemic cells has systematically diverged, resulting in irrelevant separation of leukemic subpopulations from their healthy counterpart. Here, we have developed a new computational approach bundled into a tool called Nabo (nabo.readthedocs.io) that has the capacity to directly compare cells that are otherwise unalignable. First, Nabo creates a shared nearest neighbor graph of the reference population, and the heterogeneity of this population is subsequently defined by performing clustering on the graph and calculating a low dimensional representation using t-SNE or UMAP. Nabo then calculates the similarity of incoming cells from a target population to each cell in the reference graph using a modified Canberra metric. The reference cells with higher similarity to the target cells obtain higher mapping scores. The built-in classifier is used to assign each target cell a reference cluster identity. We tested Nabo's accuracy on control datasets and found that Nabo's performance in terms of accuracy and robustness of projection is comparable to state-of-art methods. Moreover, Nabo is a generalized domain adaptation algorithm and hence can perform classification of target cells that are arbitrarily dissimilar to reference cells. Nabo could identify the cell-identity of sorted CD19+ B cells, CD14+ monocytes and CD56+ by projecting these unlabeled cells onto labelled peripheral blood mononuclear cells with an average specificity higher than 0.98. The general applicability of Nabo was demonstrated by successfully integrating pancreatic cells, sequenced in three different studies using different sequencing chemistries with comparable or better accuracy than existing methods. Also, it was conclusively demonstrated that Nabo can predict the identity of human HSPC subpopulations to the same accuracy as can be achieved by established cell-surface markers. Having Nabo at hand, we aimed to uncover the heterogeneity of hematopoietic cells from different stages of AML. Nabo showed that AML cells lacked the heterogeneity of normal CD34+ cells and were devoid of cells with HSC gene signature. A large patient-to-patient variability was found where leukemic cells mapped to distinct stages of myeloid progenitors. To ask whether this variability could reflect differences in leukemia-initiating cell identity, we induced leukemia in murine granulocyte-monocyte-lymphoid progenitors (GMLPs) using an inducible model for MLL-ENL-driven AML. On projection, more than 70% of MLL-ENL-activated cells mapped to a distinct Flt3+ subpopulation present within healthy GMLPs. Statistical validity of this projection was verified using two novel null models for testing cell projections: 1) ablated node model, wherein the mapping strength of target cells are evaluated after removal of high mapping score source nodes, and 2) high entropy features model, which rules out the background noise effect. By separating Flt3+ and Flt3- cells prior to activation of the fusion gene and performing in vitro replating assays, we could demonstrate that Flt3+ GMLPs contained 3-4 fold more leukemia-initiating cells (1/1.34 cells) than Flt3- GMLPs (1/4.89 cells), indicating that leukemia-initiating cells within GMLPs express Flt3. Taken together, Nabo represents a robust cell projection strategy for relevant analysis of scRNA-Seq data that permits an interpretable inference of cross-population relationships. Nabo is designed to compare disparate cellular populations by using the heterogeneity of one population as a point of reference allowing for cell-type specification even following perturbations that have resulted in large molecular changes to the cells of interest. As such, Nabo has critical implementation for delineation of leukemia heterogeneity and identification of leukemia-initiating cell population. Disclosures No relevant conflicts of interest to declare.

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In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6).

Journal of Experimental Medicine ◽

10.1084/jem.167.5.1659 ◽

1988 ◽

Vol 167 (5) ◽

pp. 1659-1670 ◽

Cited By ~ 217

Author(s):

P Lusso ◽

P D Markham ◽

E Tschachler ◽

F di Marzo Veronese ◽

S Z Salahuddin ◽

...

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Cell Population ◽

Mononuclear Cells ◽

Human Herpesvirus ◽

Human Herpesvirus 6 ◽

Infected Cells ◽

Cellular Tropism ◽

Herpesvirus 6

We investigated the cellular tropism of human B-lymphotropic virus (HBLV) (also designated Human Herpesvirus-6) in vitro by infecting fresh MN cells from normal human adult peripheral blood, umbilical cord blood, bone marrow, tonsil, and thymus. Cultures from all the sources examined contained infectable cells, as shown by the appearance of characteristic enlarged, round-shaped, short-lived cells expressing HBLV-specific markers. Detailed immunological analysis demonstrated that the vast majority of these cells expressed T cell-associated antigens (i.e., CD7, CD5, CD2, CD4, and to a lesser extent, CD8). The CD3 antigen and the TCR-alpha/beta heterodimer were not detectable on the surface membrane, but were identified within the cytoplasm of HBLV-infected cells, by both immunofluorescence and radioimmunoprecipitation assay. A proportion of the HBLV-infected cell population also expressed the CD15 and class II MHC DR antigens. By means of immunoselection procedures it was possible to show that a consistent proportion of HBLV-infectable cells were contained within the CD3-depleted immature T cell population, while the depletion of CD2+ cells completely abrogated the infectability of the cultures. Northern blot analysis confirmed the T cell origin of HBLV-infected cells, demonstrating the expression of full size TCR-alpha and -beta chain mRNA. In addition to fresh T cells, HBLV was able to infect normal T lymphocytes expanded in vitro with IL-2 for greater than 30 d. These results indicate that HBLV is selectively T cell tropic in the course of the in vitro infection of normal mononuclear cells and may therefore be directly involved in the pathogenesis of T cell related hematological disorders. In particular, in light of the cytopathic effect exerted in vitro on CD4+ T lymphocytes, a possible role of HBLV in immune deficiency conditions should be considered.

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