Generation of CD4+ or CD8+ regulatory T cells upon mesenchymal stem cell-lymphocyte interaction

Background and Objectives Mesenchymal stem cells (MSC) have been proposed as a way to treat graft-versus-host disease based on their immunosuppressive effect. We analyzed whether regulatory T cells can be generated in co-cultures of peripheral blood mononuclear cells (PBMC) and MSC.Design and Methods MSC were obtained from the bone marrow of four healthy donors and nine patients with acute leukemia in complete remission following chemotherapy. Short-term (4 days) co-cultures of MSC and autologous or allogeneic PBMC were set up, the lymphocytes harvested and their regulatory activity assessed.Results Lymphocytes harvested from MSC-PBMC co-cultures strongly inhibit (up to 95%) mixed lymphocyte reaction (MLR), recall to alloantigen, and CD3- or phytohemagglutinin-induced lymphocyte proliferation. These lymphocytes, termed regulatory cells (Regc), were all CD45+CD2+ with variable proportions of CD25+ cells (range 40–75% n=10) and a minor fraction expressed CTLA4 (2–4%, n=10) or glucocorticoid-induced tumor necrosis factcor receptor-related gene (0.5–4% n=10). Both CD4+ and CD8+ Regc purified from MSC-PBMC co-cultures strongly inhibited lymphocyte proliferation at a 1:100 Regc:responder cell ratio. CD4+ Regc expressed high levels of forkhead box P3 (Foxp3) mRNA while CD8+ Regc did not. The effectiveness of Regc, whether CD4+ or CD8+, was 100-fold higher than that of CD4+CD25+high regulatory T cells. Regc were also generated from highly purified CD25− PBMC or CD4+ or CD8+ T cell subsets. Soluble factors, such as interleukin-10, transforming growth factor-β and prostaglandin E2 did not appear to be involved in the generation of Regc or in the Regc-mediated immuno-suppressive effect. Furthermore, cyclosporine A did not affect Regc generation or the immunosuppression induced by Regc.Interpretation and Conclusions These findings indicate that powerful regulatory CD4+ or CD8+ lymphocytes are generated in co-cultures of PBMC with MSC. This strongly suggests that these regulatory cells may amplify the reported MSC-mediated immunosuppressive effect

Defective Expression and Function of the Leukocyte Associated Ig-like Receptor 1 in B Lymphocytes from Systemic Lupus Erythematosus Patients

Systemic lupus erythematosus (SLE) is characterized by the production of a wide array of autoantibodies and dysregulation of B cell function. The leukocyte associated Immunoglobulin (Ig)-like receptor (LAIR)1 is a transmembrane molecule belonging to Ig superfamily which binds to different types of collagen. Herein, we have determined the expression and function of LAIR1 on B lymphocyte from SLE patients. LAIR1 expression in peripheral blood B lymphocytes from 54 SLE, 24 mixed connective tissue disease (MCTD), 20 systemic sclerosis (SSc) patients, 14 rheumatoid arthritis (RA) and 40 sex and age matched healthy donors (HD) have been analyzed by immunofluorescence. The effect of LAIR1 ligation by specific monoclonal antibodies, collagen or collagen producing mesenchymal stromal cells from reactive lymph nodes or bone marrow on Ig production by pokeweed mitogen and B cell receptor (BCR)-mediated NF-kB activation was assessed by ELISA and TransAM assay. The percentage of CD20+ B lymphocytes lacking or showing reduced expression of LAIR1 was markedly increased in SLE and MCTD but not in SSc or RA patients compared to HD. The downregulation of LAIR1 expression was not dependent on corticosteroid therapy. Interestingly, LAIR1 engagement by collagen or collagen-producing mesenchymal stromal cells in SLE patients with low LAIR1 expression on B cells delivered a lower inhibiting signal on Ig production. In addition, NF-kB p65 subunit activation upon BCR and LAIR1 co-engagement was less inhibited in SLE patients than in HD. Our findings indicate defective LAIR1 expression and function in SLE B lymphocytes, possible contributing to an altered control of B lymphocytes behavior

Sol-gel derived mesoporous Pt and Cr-doped WO(3) thin films: the role played by mesoporosity and metal doping in enhancing the gas sensing properties

Mesoporous Cr or Pt-doped WO(3) thin films to be employed as ammonia gas sensors were prepared by a fast one-step sol-gel procedure, based on the use of triblock copolymer as templating agent. The obtained films were constituted by aggregates of interconnected WO(3) nanocrystals (20-50 nm) separated by mesopores with dimensions ranging between 2 and 15 nm. The doping metals, Pt and Cr, resulted differently hosted in the WO(3) mesoporous matrix. Chromium is homogeneously dispersed in the oxide matrix, mainly as Cr(III) and Cr(V) centers, as revealed by EPR spectroscopy; instead platinum segregated as Pt (0) nanoparticles (4 nm) mainly included inside the WO(3) nanocrystals. The semiconductor layers containing Pt nanoclusters revealed, upon exposure to NH(3), remarkable electrical responses, much higher than Cr-doped and undoped layers, particularly at low ammonia concentration (6.2 ppm). This behavior was attributed to the presence of Pt nanoparticles segregated inside the semiconductor matrix, which act as catalysts of the N-H bond cleavage, decreasing the activation barrier in the ammonia dissociation. The role of the mesoporous structure in influencing the chemisorption and the gas diffusion in the WO(3) matrix appeared less decisive than the electronic differences between the two examined doping metals. The overall results suggest that a careful combination between mesoporous architecture and metal doping can really promote the electrical response of WO(3) toward ammonia

Epitope characterization of a monoclonal antibody that selectively recognizes KIR2DL1 allotypes

Killer immunoglobulin-like receptor (KIR) genes code for a family of inhibitory and activating receptors, finely tuning NK cell function. Numerous studies reported the relevance of KIR allelic polymorphism on KIR expression, ligand affinity, and strength in signal transduction. Although KIR variability, including gene copy number and allelic polymorphism, in combination with HLA class I polymorphism, impacts both KIR expression and NK cell education, only a precise phenotypic analysis can define the size of the different KIRpos NK cell subsets. In this context, reagents recognizing a limited number of KIRs is essential. In this study, we have characterized the specificity of an anti-KIR mAb termed HP-DM1. Testing its binding to HEK-293T cells transfected with plasmids coding for different KIRs, we demonstrated that HP-DM1 mAb exclusively reacts with KIR2DL1. Using site-directed mutagenesis, we identified the four amino acids relevant for HP-DM1 recognition: M44, S67, R68, and T70. HP-DM1 mAb binds to a conformational epitope including M44, the residue crucial for HLA-C K80 recognition by KIR2DL1. Based on the HP-DM1 epitope characterization, we could extend its reactivity to all KIR2DL1 allotypes identified except for KIR2DL1*022 and, most likely, KIR2DL1*020, predicting that it does not recognize any other KIR with the only exception of KIR2DS1*013. Moreover, by identifying the residues relevant for HP-DM1 binding, continuously updating of its reactivity will be facilitated.This work was supported by Italian Ministry of Health (Ricerca Corrente G. Gaslini, Ricerca Corrente Ospedale Policlinico San Martino) (Cristina Bottino and Daniela Pende), Instituto de Salud Carlos III, FIS00/0181 (to Miguel Lopez-Botet), H2020-MSCA-ITN-2017-765104-MATURE-NK (to Miguel Lopez-Botet and Daniela Pende), AIRC 5×1000 2018 id. 21147 (Lorenzo Moretta)

Collagen-producing LMSC regulate Ig production upon engagement of LAIR1: this effect is defective in SLE patients.

<p>A, LMSC from a representative reactive lymph node were surface stained with the indicated mAbs (first row) followed by PE-conjugated anti-isotype specific goat anti-mouse antiserum. Control: cells stained with an unrelated mAb followed by GAM as negative control. Second row: LMSC were cytoplasmic stained after fixation and permeabilization with mAbs to the indicated molecules (ALP: alkaline phosphatase, BSP: bone sialoprotein, collagen or vimentin) followed by PE-conjugated GAM. Results are expressed as log red fluorescence intensity vs number of cells. In each panel are indicated the percentages of positive cells above the horizontal bar set on negative control (first subpanel on the left of each row). B. left: bright field (BF) of LMSC from a representative reactive lymph node (upper left), staining with anti-collagen mAb (upper right, red) without cell permeabilization and the respective negative controls (BF neg control, neg control). B right: staining of LMSC with anti-HLA-I (surface, green), anti-prolyl-4-hydroxylase (P4H, cytoplasmic, red) and anti-HMGB1 mAb (nucleus, blue) analyzed by confocal microscopy. Merge analysis is also shown. 400× (left), 600× (right) magnification. White Bars: 10 µ m; reactivity for collagen is disposed in large and concentrated regions (upper left); the white arrows indicate the intracytoplasmic reactivity for P4H (upper right). C. PBMC of healthy donors (HD, n = 7, left) or SLE patients (n = 9 from SLE2 group, right) were incubated for 5 d on collagen-producing mesenchymal stromal cells (MSC) from reactive lymph node coated plates. Then SN were analyzed for the presence of the human IgM, IgG and IgA by ELISA. In some experiments, F(ab′)₂ of anti-LAIR1 mAb (5 µ g/ml) to compete with the interaction of surface LAIR1 and collagen-producing MSC or an unrelated mAb matched for the isotype as control mAb (5 µ g/ml) was added at the onset of cell culture. Results are expressed as ng/ml/10⁵ CD20⁺ B cells as mean±SD. * p<0.001 vs basal production of Ig. ** p<0.001 vs Ig production on LMSC coated plates. In the right panel is indicated the statistical significance of Ig production in the culture condition PBMC+LMSC in SLE2 patients vs HD.</p

Expression of LAIR1 on PBMC from SLE or RA or SSc patients and HD.

<p>A. Peripheral blood mononuclear cells (PBMC) were isolated from SLE (n = 50) or RA (n = 14) or SSc (n = 20) patients and stained with anti-LAIR1 mAb and anti-CD20 mAb followed by goat anti-isotype specific GAM conjugated with either PE or Alexafluor647 and goat anti-human sIgM-FITC polyclonal antibody. Results are expressed as CD20⁺LAIR1⁻ B cells gated on sIgM⁺ cells. Surface IgM⁺ B cells were 95–100% of all B cells in PBMC of any population tested. The percentages of CD20⁺LAIR1⁻ cells present in PBMC of healthy donors (HD) is shown for comparison in each panel (n = 40). A, right panel: Patients suffering from SLE were subdivided in two groups (with or without steroid therapy). The statistical significance of results is shown in each panel. B. Some representative examples of expression of CD20 and LAIR1 on PBMC of SLE in remission phase (SLE1 and SLE2) or in acute phase (SLE flare) or RA or SSc patients and HD. Results are expressed as Log far red fluorescence intensity vs Log red fluorescence intensity. Each dot plot is subdivided into four quadrants representing CD20⁻LAIR1⁺ (upper left) or CD20⁺LAIR1⁺ (upper right) or CD20⁻LAIR1⁻ (lower left) or CD20⁺LAIR1⁻ (lower right) cells respectively.</p

Epitope characterization of a monoclonal antibody that selectively recognizes allotypes

Killer immunoglobulin-like receptor (KIR) genes code for a family of inhibitory and activating receptors, finely tuning NK cell function. Numerous studies reported the relevance of KIR allelic polymorphism on KIR expression, ligand affinity, and strength in signal transduction. Although KIR variability, including gene copy number and allelic polymorphism, in combination with HLA class I polymorphism, impacts both KIR expression and NK cell education, only a precise phenotypic analysis can define the size of the different KIR pos NK cell subsets. In this context, reagents recognizing a limited number of KIRs is essential. In this study, we have characterized the specificity of an anti-KIR mAb termed HP-DM1. Testing its binding to HEK-293T cells transfected with plasmids coding for different KIRs, we demonstrated that HP-DM1 mAb exclusively reacts with KIR2DL1. Using site-directed mutagenesis, we identified the four amino acids relevant for HP-DM1 recognition: M44, S67, R68, and T70. HP-DM1 mAb binds to a conformational epitope including M44, the residue crucial for HLA-C K80 recognition by KIR2DL1. Based on the HP-DM1 epitope characterization, we could extend its reactivity to all KIR2DL1 allotypes identified except for KIR2DL1*022 and, most likely, KIR2DL1*020, predicting that it does not recognize any other KIR with the only exception of KIR2DS1*013. Moreover, by identifying the residues relevant for HP-DM1 binding, continuously updating of its reactivity will be facilitated