Antoine Blancher

Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells.

Like mesenchymal stem cells from bone marrow (BM‐MSCs), adipose tissue‐derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well‐characterized immunosuppressive properties of BM‐MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM‐MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin‐10 and transforming growth factor‐β (TGF‐β), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM‐MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM‐MSCs. Therefore, ADAS cell‐based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM‐MSCs to treat allogenic conflicts.

FOXP3 mRNA levels are decreased in peripheral blood CD4+ lymphocytes from HIV-positive patients.

The impact of HIV infection on regulatory CD4+CD25high (Treg) lymphocyte subpopulations was evaluated by FOXP3 quantitative reverse transcriptase polymerase chain reaction and by flow cytometry. FOXP3 mRNA was quantified in peripheral blood mononuclear cells or purified CD4+ lymphocytes from HIV+ lymphopenic patients. Patients were distributed among clinical stages A, B, and C and received highly active antiretroviral therapy. The frequency of CD4+CD25high lymphocytes, measured by flow cytometry, was decreased in HIV+ patients (n = 38) compared with the group of uninfected subjects (n = 39). FOXP3 mRNA levels were found decreased in HIV+ patients (n = 25) compared with controls (n = 17) when expression of CD3γ or β-actin but not that of TATA box binding protein 1 was used for data normalization. Our results are compatible with a decrease of the Treg lymphocytes during HIV infection. The consequences of a Treg decrease are discussed in the context of immunologic anomalies observed during HIV infection.

Sequence, evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines

The Duffy antigen/receptor for chemokine, DARC, acts as a widely expressed promiscuous chemokine receptor and as the erythrocyte receptor for Plasmodium vivax. To gain insight into the evolution and structure/function relations of DARC, we analyzed the binding of anti-human Fy monoclonal antibodies (mAbs) and human chemokines to red blood cells (RBCs) from 11 nonhuman primates and two nonprimate mammals, and we elucidated the structures of the DARC genes from gorilla, gibbon, baboon, marmoset, tamarin, night monkey and cattle. CXCL-8 and CCL-5 chemokine binding analysis indicated that the promiscuous binding profile characteristic of DARC is conserved across species. Among three mAbs that detected the Fy6 epitope by flow cytometric analysis of human and chimpanzee RBCs, only one reacted with night monkey and squirrel monkey. Only chimpanzee RBCs bound a significant amount of the anti-Fy3 mAb. Fy3 was also poorly detected on RBCs from gorilla, baboon and rhesus monkey, but not from new world monkeys. Alignment of DARC homologous sequences allowed us to construct a phylogenetic tree in which all branchings were in accordance with current knowledge of primate phylogeny. Although DARC was expected to be under strong internal and external selection pressure, in order to maintain chemokine binding and avoid Plasmodium vivax binding, respectively, our present study did not provide arguments in favor of a selection pressure on the extracellular domains involved in ligand specificity. The amino acid variability of DARC-like polypeptides was found to be well correlated with the hydrophylicity indexes, with the highest divergence on the amino-terminal extracellular domain. Analysis of the deduced amino acid sequences highlighted the conservation of some amino acid residues, which should prove to be critical for the structural and functional properties of DARC.

Polymerase chain reaction on cerebrospinal fluid cells in the detection of leptomeningeal involvement by B‐cell lymphoma and leukaemia: a novel strategy and its implications

In the search of B‐cell lymphoma/leukaemia dissemination to cerebrospinal fluid (CSF), we used the highly sensitive semi‐nested PCR (snPCR) for the analysis of IgH gene rearrangements. This method detects a rearranged IgH gene from a single B lymphocyte which may or may not represent the neoplastic B‐cell population. We therefore performed multiple snPCR (three to five) experiments on the same CSF sample, postulating that the detection of a band of the same size and sequence in different PCR runs was highly indicative of a clonal population. 17 consecutive cases with a differential diagnosis of primary (PcnsL) (n=10) or secondary (ScnsL) (n=7) CNS lymphoma or leukaemia were investigated by the new strategy. The clonal nature of the B‐cell population was confirmed in 3/10 of suspected PcnsL, and in six other cases the PCR study was indicative of reactive lymphocytosis. One case revealed a clonal B‐cell population in the clinical context of an autoimmune disorder. Evidence of clonal B‐cell population was found in 4/7 of suspected ScnsL. In one of these cases the detected band and its sequence proved identical to that of the primary nodal lymphoma. We believe that the evaluation of B‐cell clonality in CSF requires multiple snPCR amplification on the same sample to compare the size of the products and, if necessary, the DNA sequences to ascertain the diagnosis of malignancy in equivocal cytologic and clinical findings.

Antibody BNH9 detects red blood cell-related antigens on anaplastic large cell (CD30+) lymphomas.

Two new monoclonal antibodies--BNH9 and BNF13--were generated by using a human lung adenocarcinoma cell line and standard hybridoma techniques. Both were found to react with epithelial and endothelial cells in routinely fixed and embedded tissues. Unexpected membrane labelling of some large cell lymphomas while non-reacting with normal lymphoid cells, prompted further characterisation. The antibodies were found to recognise red blood cell-related oligosaccharide antigens. The specificities were directed towards H and Y determinants. A distinctive pattern of reactivity was found for BNH9 in studying 480 cases of various lymphoid neoplasms. Strong expression of H and/or Y antigens was observed in 65/127 (51%) cases of anaplastic large cell(ALC) (CD30+) lymphomas, which are also known to co-express epithelial membrane antigen (EMA) frequently. Only a minority (less than 6%) of other non-Hodgkins lymphomas (NHL) (CD30-,EMA-; 208 cases) and Hodgkins disease (HD) (CD30+, EMA-; 126 cases) were positive. Expression of H and Y antigens was inducible on normal lymphocytes by mitogenic stimulation and by Epstein-Barr virus infection. The data suggest remarkable biological differences of ALC lymphomas within NHL and from HD.

Heterogeneous molecular background of the weak C, VS+, hrB-, HrB-phenotype in black persons

BACKGROUND: The rare HrB– phenotype is encoded by the (C)ces haplotype when present at the homozygous state. This haplotype contains two altered genes: a hybrid RHD‐CE‐Ds gene segregated with a ces allele of RHCE (733C>G and 1006G>T substitutions in Exon 5 and Exon 7 respectively). The aim of this study was to further investigate the molecular background of the (C)ces haplotype.

Antigen topography is critical for interaction of IgG2 anti‐red‐cell antibodies with Fcγ receptors

IgG antibodies to the Rh D polypeptide on red cells are normally IgG1 or IgG3, whereas antibodies produced in response to carbohydrate antigens such as the A and B blood groups are predominantly IgG2. The consequences of this isotype restriction for the immune destruction of red cells were investigated. Human IgG2 anti‐D and IgG2 anti‐A were isolated by affinity purification from an unusual anti‐D serum (DEL) and anti‐A sera, respectively. These antibodies were compared with IgG1 and IgG3 monoclonal anti‐D in in vitro functional assays of the interaction between IgG‐coated red cells (EA‐IgG) and cells bearing IgG Fc receptors (FcγR). Dimeric IgG2 anti‐D bound efficiently to cell lines transfected with FcγRIIa‐H131, an allotypic form of FcγRIIa which readily interacts with IgG2, IgG1 and IgG3. Unexpectedly, however, ‐D‐phenotype red cells coated with IgG2 anti‐D did not form rosettes with these cells, whereas EA‐IgG2 anti‐A and EA‐IgG1 and EA‐IgG3 anti‐D effectively formed rosettes with these transfectants at the same sensitization level (100 000 molecules IgG/red cell). In antibody‐dependent cell‐mediated cytotoxicity (ADCC) assays, lysis of EA‐IgG2 anti‐A was mediated via FcγRIIa, whereas lysis of EA‐IgG1 and EA‐IgG3 anti‐D was mediated via FcγRI or FcγRIII; EA‐IgG2 anti‐D was inactive in all functional assays. These experiments suggest that both IgG subclass and antigen structure affect functional IgG–FcγR interactions. The topography of the Rh D antigen, an integral membrane protein, ensures that anti‐D is bound near the lipid bilayer surrounded by the glycocalyx. This may sterically hinder access of FcγRIIa‐H131 to the FcγR recognition site on the relatively inflexible IgG2 anti‐D, but not to that of IgG1 or IgG3 anti‐D. In contrast, IgG2 bound to the A antigen on glycoproteins is not so constrained. The topography of the D and A antigens may thus determine whether functional interactions of red‐cell‐bound IgG2 anti‐D and IgG2 anti‐A with cells bearing Fcγ receptors can occur.

Blood Concentrations of Hydroxychloroquine and Its Desethyl Derivative Correlate Negatively with the Percentage of CD45RO+ Cells among CD4+ Lymphocytes in Hydroxychloroquine-Treated Lupus Patients

Abstract:  The objective of the study was to investigate the influence of the blood concentrations of hydroxychloroquine ([HCQ]) and its derivative desethylhydroxychloroquine ([DHCQ]) on lymphocyte activation or differentiation in HCQ‐treated lupus patients. We studied the correlations between [HCQ], [DHCQ], and the frequency of various lymphocyte subsets in 58 HCQ‐treated lupus patients (mean HCQ dose: 4.93 ± 1.58 mg/kg/day; mean duration of the disease: 122 ± 64 months). [HCQ] and [DHCQ] were determined by high‐performance liquid chromatography (HPLC). Lymphocyte markers were studied by flow cytometry using monoclonal anti‐CD3, ‐CD4, ‐CD8, ‐CD25, ‐DR, ‐CD45RA,‐CD45RO, ‐CD19, ‐CD38, and ‐CD86 antibodies. sIL2‐R serum concentrations were measured by enzyme‐linked immunosorbent assay (ELISA). [HCQ] and [DHCQ] were 599.9 ng/mL (median: 529.5; range: 55–1935) and 353.43 (median: 286 ng/mL; range: 118–1090). In a multiple regression analysis, [HCQ] and [DHCQ] were associated with the HCQ prescribed dose in mg/kg/day (P= 0.0002 and P= 0.03) and with compliance to the treatment (P= 0.004 and P= 0.03). We found a negative correlation between [HCQ], [DHCQ], and the CD45RO+ cell frequency among CD3+CD4+ cells (P= 0.03 and P= 0.007, respectively). Other lymphocyte subset markers (LSMs) and sIL2‐R concentrations were not significantly associated with [HCQ] or [DHCQ]. In the multiple regression analysis, CD45RO+ expression was negatively influenced by [HCQ] (P= 0.005), and positively influenced by smoking habits (P= 0.005) and age (P= 0.005). Similar results were found in the multivariate model including [DHCQ]. Disease activity and taking more than 10 mg/day of corticosteroids or an immunosuppressive drug did not influence CD45RO+ expression. Lupus patients had less CD3+CD4+CD45RO+ cells than controls (P= 0.03). In lupus patients, HCQ and DHCQ may alter the generation or the blood circulation of CD4+CD45RO+ lymphocytes in a concentration‐dependent pattern.

Decrease of lewis frequency in HIV-infected patients : possible competition of fucosylated antigens with HIV for binding to DC-SIGN

We explored the impact of human ABO glycosyltransferase and Lewis and secretor fucosyltransferase polymorphisms in HIV infection. We found that, compared with healthy blood donors, HIV-infected patients display a significant decrease in Lea−b+ phenotype frequencies. We showed that HIV binding on DC-SIGN-transduced Jurkat cells was inhibited by fucosyl bovine serum albumin. Our results suggest a slight protective effect of Lewis b antigen on HIV infection, possibly by the competition of Lewis antigens with HIV for binding to DC-SIGN.

Immunoglobulin IgA, IgD, IgG, IgM and IgG subclass reference values in adults

*Corresponding author: Pr. Antoine Blancher, Laboratoire d’Immunologie du CHU de Toulouse, Hôpital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France, Phone: +33 5 61 323432, Fax: +33 5 61 323424, E-mail: blancher.a@chu-toulouse.fr; and Laboratoire d’Immunogénétique Moléculaire, EA 3034, Université Paul Sabatier, Toulouse III, France Bénédicte Puissant-Lubrano, Pol-André Apoil and Nicolas CongyJolivet: Laboratoire d’Immunogénétique Moléculaire, EA 3034, Université Paul Sabatier, Toulouse III, France; and Laboratoire d’Immunologie, CHU de Toulouse, France Michael Peres: Laboratoire d’Immunologie, CHU de Toulouse, France Francis Roubinet: EFS Pyrénées-Méditerranée, Toulouse, France Letter to the Editor