Different expression of adhesion molecules on myeloid and B-lymphoid CD34+ progenitors in normal bone marrow

The expression of adhesion molecules was studied on B lymphoid and myeloid CD34+ precursors in normal bone marrow. Bone marrow aspirates were labelled in a double fluorescence procedure with the CD34 monoclonal antibody 43A1 and with antibodies directed against maturation and differentiation antigens and adhesion molecules. Three clusters of CD34+ cells could be distinguished by their light scatter characteristics in flow cytometry. The population with the lowest forward scatter contained B-lymphoid precursors while the two others showed phenotypic characteristics of, respectively, early and late myeloid precursors. Nearly all CD34+ cells in the 3 subpopulations expressed VLA-4, VLA-5, LFA-3 and H-CAM. B lymphoid progenitors showed a higher density of VLA-4 and VLA-5 than the myeloid progenitors. Myeloid precursors, and particularly the late subset, expressed more HCAM than the B-lymphoid progenitors. The majority of the CD34+ cells also expressed LFA-1 and L-selectin. Higher numbers of positive cells were found in the myeloid subset. The early myeloid subset showed the highest positivity for L-selectin. We conclude that B lymphoid and early and late myeloid CD34+ precursors in normal bone marrow show a different profile of adhesion molecules. These profiles could reflect a higher tendency of the myeloid CD34+ precursors to circulate.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Preclinical evaluation of invariant natural killer T cells in the 5T33 multiple myeloma model.

Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting

Association Between Immunosenescence Phenotypes and Pre-frailty in Older Subjects: Does Cytomegalovirus Play a Role?

Frailty is highly prevalent in old age and confers an important mortality risk. Although the causes of frailty are multiple, immunosenescence (IS)—predominantly driven by cytomegalovirus (CMV)—has been implicated in its pathophysiology. Thus far, research examining the association between IS and frailty states is sparse and equivocal. On the other hand, evidence is mounting in support of the view that frailty can be reversed, especially for those in the pre-frail stage. Therefore, we aimed to clarify the impact of CMV on IS and its relevance to pre-frailty. One hundred seventy-three persons aged 80 to 99 years were enrolled. Pre-frailty was defined according to Fried’s criteria. Anti-CMV IgG and serum IL-6 were measured using Architect iSystem and Luminex, respectively. T-cell phenotypes were determined using flow cytometry. The prevalence of pre-frailty was 52.6%, increased with age (p = .001), and was greater in men than women (p = .044). No relationship was found between pre-frailty and positive CMV serology. Further, CMV-seropositivity was significantly associated with less naïve cells, more memory and senescence-prone phenotypes (all p < .001). After adjusting for potential confounders, only IL-6, age and sex were predictive of pre-frailty. We conclude that the presence of pre-frailty is independent from CMV infection in very old subjects

iNKT activity in vivo.

<p>(<b>A</b>) In vivo cytokine secretion after i.p. administration of 2 µg α-GalCer in 200 µl of PBS to healthy, non-terminal and terminal sick 5T33MM mice. Serum was collected during a time course, responses were followed by determining IFN-γ and IL-4 levels by ELISA (mean ± SD is shown from 5 mice in each group). (<b>B</b>) Kaplan-Meier survival assay. Mice were intravenously injected with a single dose of matured DCs (6×10⁵ cell/mouse) loaded with α-GalCer (100 ng/mL) or unloaded at the same day of inoculation with 5T33MM cells (n = 8 mice/group, p = 0.0366). Vehicle mice had an average survival of 22 days, α-GalCer treated mice 29 days.</p

iNKT numbers in the 5T33MM model.

<p>(<b>A</b>) Representative FACS analysis of murine iNKT cells from blood, BM, spleen and liver in naive and 5T33MM mice. Live cells (7-AAD negative) were stained with α-GalCer/CD1d tetramer which specifically binds to Vα14 of the invariant TCR and with TCR-β. Double positive iNKT cells were gated. The percentages are indicated in each plot. (<b>B</b>) Box plots of the distribution of iNKT number data, obtained from 6 mice from independent experiments. Differences between naive and 5T33MM cells in BM, liver and spleen are significant (* and ** indicate p<0.05 and p<0.005, Mann-Whitney test).</p

iNKT frequency and activity during the development of the disease in the 5T33MM model.

<p><b>(A, upper panel)</b> Frequency of iNKTs in liver and spleen during the course of the disease, analyzed by flow cytometry as was done in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065075#pone-0065075-g001" target="_blank">figure 1A</a>. Mice were isolated at different time points and liver and MACS-sorted splenic iNKTs were stained with α-GalCer/CD1d tetramer. Plasmacytosis (tumor load) was assessed on May-Grünwald Giemsa stained cytosmears of the isolated BM. The mean ± SD of 3 mice in independent experiment is shown. <b>(A, lower panel)</b> The mean % iNKT cells in liver and spleen of naive and end stage 5T33 mice (n = 6, ** indicates p<0.01, Mann-Whitney test). <b>(B, upper panel)</b> In vitro iNKT activity. Mice were isolated at different time points and liver iNKTs were co-cultured with α-GalCer loaded or unloaded DCs for 72 h. IFN-γ secretion in the co-culture was measured by ELISA. The mean ± SD of 3 mice in independent experiment is shown. <b>(B, lower panel)</b> The mean IFN-γ secretion of DC co-cultures with liver iNKT cells of naïve and end stage 5T33 mice (n = 6, ** indicates p<0.01, Mann-Whitney test). (<b>C</b>) Gene expression of the Vα14 receptor of liver iNKTs of healthy and diseased 5T33MM was assessed by RT-PCR (n = 6). <b>(D, upper panel)</b> Expression of CD1d in live spleen and BM cells in the 5T33MM model during the course of the MM disease compared to tumor load. Mice were isolated at different time points of the disease and spleen and BM cells were double stained for CD1d and 5T33MM. The mean ± SD of 3 mice in independent experiment is shown. <b>(D, lower panel)</b> The mean CD1d expression in spleen and BM cells of naive and end stage 5T33 mice (n = 6, ** indicates p<0.01, Mann-Whitney test).</p

iNKT numbers in MM patients.

<p>Human iNKT (number/mL) were analyzed in blood samples of 51 Patients (mean age is 64 years old) and 62 healthy donors (mean age is 66 years old) by flow cytometry. Total number of iNKT cells were calculated by determining the % iNKT cells (CD3, TCRVα24 and TCRVβ11+ cells) on total T lymphocyte number (* and ** indicate p<0.05 and p<0.01, Student t-test, ns =  not significant).</p