[Show abstract][Hide abstract] ABSTRACT: Type 1 diabetes is an autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Among the genetic variants associated with type 1 diabetes, the C1858T (Lyp) polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene alters the function of T cells but also of B cells in innate and adaptive immunity. The Lyp variant was shown to diminish interferon production and responses upon Toll-like receptor stimulation in macrophages and dendritic cells, possibly leading to uncontrolled infections as triggers of the diabetogenic process. The aim of this study was to unravel the yet uncharacterized effects that the variant could exert on the immune and autoimmune responses, particularly regarding the B cell phenotype, in the peripheral blood lymphocytes of diabetic patients and healthy controls in basal conditions and after unmethylated bacterial DNA CpG stimulation. The presence of the Lyp variant resulted in a significant increase in the percentage of transitional B cells in C/T carriers patients and controls compared to C/C patients and controls, in C/T carrier patients compared to C/C controls and in C/T carrier patients compared to C/C patients. A significant reduction in the memory B cells was also observed in the presence of the risk variant. After four days of CpG stimulation, there was a significant increase in the abundance of IgM+ memory B cells in C/T carrier diabetics than in C/C subjects and in the groups of C/T carrier individuals than in C/C individuals. IgM- memory B cells tended to differentiate more precociously into plasma cells than IgM+ memory B cells in heterozygous C/T subjects compared to the C/C subjects. The increased Toll-like receptor response that led to expanded T cell-independent IgM+ memory B cells should be further investigated to determine the putative contribution of innate immune responses in the disease pathogenesis.
PLoS ONE 10/2014; 9(10):e110755. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of biological agents combined with methotrexate (MTX) in rheumatoid arthritis (RA) patients has strongly improved disease outcome. In this study, the effects of abatacept on the size and function of circulating B and T cells in RA patients not responding to anti‐tumour necrosis factor (TNF)‐α have been analysed, with the aim of identifying immunological parameters helpful to choosing suitable tailored therapies. We analysed the frequency of peripheral B and T cell subsets, B cell function and T regulatory cell (Treg) inhibitory function in 20 moderate/severe RA patients, according to the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) criteria, primary non‐responders to one TNF‐α blocking agent, who received abatacept + MTX. Patients were studied before and 6 months after therapy. We found that abatacept therapy significantly reduced disease activity score on 44 joints (DAS)/erythrocyte sedimentation rate (ESR) values without causing severe side effects. The size of the circulating B and T cell compartments in RA patients was not significantly different from healthy donors, but B cell proliferation and plasma cell differentiation was impaired before therapy and restored by abatacept. While Treg cell frequency was normal, its inhibitory function was absent before therapy and was partially recovered 6 months after abatacept. B and Treg cell function is impaired in RA patients not responding to the first anti‐TNF‐α agent. Abatacept therapy was able to rescue immune function and led to an effective and safe clinical outcome, suggesting that RA patients, in whom anti‐TNF‐α failed, are immunologically prone to benefit from an agent targeting a different pathway.
[Show abstract][Hide abstract] ABSTRACT: Immunosuppressive and anti-inflammatory properties of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role such as Systemic Lupus Erythematosus (SLE) and rejection of kidney transplantation. Here, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC co-cultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4+ and CD8+ playing a role. Moreover, cell-cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. In conclusion, the presence of functional T cells, as well as cell-cell contact between MSCs and T cells are crucial for B cell inhibition. This information can be relevant for the use of MSC-based therapeutic immune-modulation in patients in whom T-cell function is impaired.
Stem Cells and Development 07/2014; · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stromal cells (MSCs) are multipotent cells that possess broad immunomodulatory properties; the mechanisms underlying these properties have not been completely clarified. Aim of this study was to compare in vitro immunomodulatory effects of MSCs with those of microvesicles (MVs) released in supernatants from the same MSCs. MSCs were generated from bone marrow of 12 healthy donors (HDs) and MVs were isolated from their supernatant by serial ultracentrifugation according to 2 different procedures. Both MSCs and MVs were characterized by flow-cytometry and incubated in vitro with peripheral blood mononuclear cells (PBMCs) of 12 HDs after stimulation with PHA and CpG. Growth factors and cytokines were quantified by ELISA. MVs were identified as 0.1-1 μm particles positive for CMFDA, CD107 and CD13. MSCs were significantly more capable to inhibit in vitro PHA-induced T-cell proliferation as compared with the corresponding MVs (P<0.01 and <0.05 for MSC:PBMC ratio 1:2 and 1:10, respectively). While MVs displayed similar inhibitory activity on B-cell proliferation (P=0.43 as compared with PBMCs/CpG/MSCs; MSC:PBMC ratio 1:10); they induced lower inhibitory effect on plasmacell differentiation and antibody secretion (P<0.05 as compared with PBMCs/CpG/MSCs). For both T and B cells, MSC co-colture induced a statistically significant increase in IL-10 and TGFβ and decrease of GM-CSF and IFNγ, as compared with MV incubation. Our data indicate a lower in vitro immunomodulatory effect of MVs on T-cell proliferation and antibody formation, as compared with their cellular counterpart. The relative clinical benefit of either MSCs or MVs needs to be compared in proper prospective studies.
Stem Cells and Development 06/2014; · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulated transgene expression may improve safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked Chronic Granulomatous Disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34+ HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.Molecular Therapy (2014); doi:10.1038/mt.2014.87.
[Show abstract][Hide abstract] ABSTRACT: B cells are generated every day in the bone marrow, but only a small fraction integrates the peripheral B-cell pool. In the murine spleen, we can find several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized in two main structures with different functions: the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone and contains the marginal zone B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and cell sorting (FACS).Here, we will focus on marginal zone B cells and their precursors giving some experimental hints to identify, generate, and isolate these cells. We will combine the use of FACS analysis and confocal microscopy to visualize marginal zone B cells in cell suspension and tissue sections, respectively.
[Show abstract][Hide abstract] ABSTRACT: Lymphocyte characterization is primarily based on the differential expression of surface markers. In this context, flow-cytometry analysis (FACS) is an exceptional technique that not only allows the identification of B-cell subsets, but can also be used to evaluate cell function, activation, and division. Here, we will combine the use of FACS analysis and ELISA techniques to identify murine bone marrow and peripheral B-cell subsets. The main function of B cells, derived through a multistage differentiation process from precursor cells, is to produce antibodies. This task is performed by terminally differentiated B cells called antibody-secreting cells (ASC) present at mucosal sites, in the bone marrow and in the spleen. The number and specificity of ASC can be measured by Enzyme-linked immunosorbent spot (ELISPOT) assay, a variation of the enzyme-linked immunosorbent assay (ELISA) used to quantify serum immunoglobulins.
[Show abstract][Hide abstract] ABSTRACT: Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133(+) cells isolated from the CaCo-2 CC cell line were analysed by microarray molecular profiling compared to CD133(-) counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over-expressed in CD133(+) cells were evaluated by quantitative RT-PCR in the CD133(+) fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133(+) cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin-1 (END-1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133(+) and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END-1 and NR4A2 expression levels in CaCo-2 cells, while expression of all three molecules decreased during sodium butyrate-induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression in colon cancer cells.
The Journal of Pathology 06/2011; 225(2):305-14. · 7.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We were interested in the question of whether the congenital lack of B cells actually had any influence on the development of the T cell compartment in patients with agammaglobulinaemia. Sixteen patients with X-linked agammaglobulinaemia (XLA) due to mutations in Btk, nine patients affected by common variable immune deficiency (CVID) with <2% of peripheral B cells and 20 healthy volunteers were enrolled. The T cell phenotype was determined with FACSCalibur and CellQuest Pro software. Mann-Whitney two-tailed analysis was used for statistical analysis. The CD4 T cell memory compartment was reduced in patients with XLA of all ages. This T cell subset encompasses both CD4(+)CD45RO(+) and CD4(+)CD45RO(+)CXCR5(+) cells and both subsets were decreased significantly when compared to healthy controls: P = 0·001 and P < 0·0001, respectively. This observation was confirmed in patients with CVID who had <2% B cells, suggesting that not the lack of Bruton's tyrosine kinase but the lack of B cells is most probably the cause of the impaired CD4 T cell maturation. We postulate that this defect is a correlate of the observed paucity of germinal centres in XLA. Our results support the importance of the interplay between B and T cells in the germinal centre for the activation of CD4 T cells in humans.
[Show abstract][Hide abstract] ABSTRACT: The endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-I peptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4 and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response and depends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed, high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NK inhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in its two-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express high levels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target for cancer immunotherapy.
Cancer Research 03/2011; 71(5):1597-606. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The immunogenicity of a vaccine is conventionally measured through the level of serum Abs early after immunization, but to ensure protection specific Abs should be maintained long after primary vaccination. For hepatitis B, protective levels often decline over time, but breakthrough infections do not seem to occur. The aim of this study was to demonstrate whether, after hepatitis B vaccination, B-cell memory persists even when serum Abs decline. We compared the frequency of anti-hepatitis-specific memory B cells that remain in the blood of 99 children five years after priming with Infanrix -hexa (GlaxoSmithKline) (n=34) or with Hexavac (Sanofi Pasteur MSD) (n=65). These two vaccines differ in their ability to generate protective levels of IgG. Children with serum Abs under the protective level, <10 mIU/mL, received a booster dose of hepatitis B vaccine, and memory B cells and serum Abs were measured 2 wk later. We found that specific memory B cells had a similar frequency in all children independently of primary vaccine. Booster injection resulted in the increase of memory B cell frequencies (from 11.3 in 10(6) cells to 28.2 in 10(6) cells, p<0.01) and serum Abs (geometric mean concentration, GMC from 2.9 to 284 mIU/mL), demonstrating that circulating memory B cells effectively respond to Ag challenge even when specific Abs fall under the protective threshold.
European Journal of Immunology 03/2011; 41(6):1800-8. · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A subset of patients with common variable immunodeficiency (CVID), group 1a of the Freiburg classification, is characterized by increased B cells expressing low levels of CD21 (CD21(low) ), lymphoproliferation and autoimmunity. The CD21(low) B cells have been shown to be profoundly anergic, and defects of BCR-mediated calcium signaling and of T cells have been described in CVID 1a. We found that also the classical naïve B cells from CVID 1a patients, but not from CVID non-1a patients, proliferated poorly. The B cells of CVID 1a patients had a reduced capacity to divide reminiscent of the proliferative arrest associated with replicative senescence. Thus, we investigated whether lymphocyte dysfunction in CVID 1a was related to telomere-dependent replicative senescence, and found that both the B and the T cells from CVID 1a patients had significantly shorter telomeres compared with B and T cells from CVID non-1a patients. Telomere lengths in B and T cells were significantly correlated, indicating that the rate of telomere attrition in lymphocytes is an individual characteristic of CVID patients. Our findings suggest that telomere-dependent replicative senescence contributes to the immune dysfunction of CVID 1a patients, and may provide an important clue for a better understanding of the pathogenesis of CVID.
European Journal of Immunology 03/2011; 41(3):854-62. · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Secondary resistance may be a major problem in the management of autoimmune diseases. P-glycoprotein (P-gp) over-function has been described as a mechanism of drug resistance in autoimmune patients. P-gp function can in vitro be inhibited by cyclosporine A (CSA) and verapamil; moreover, P-gp reduction by CSA in systemic lupus erythematosus and rheumatoid arthritis has been demonstrated. Here, P-gp function before and after CSA administration in three psoriatic arthritis (PsA) patients, who developed a resistance to MTX/SSA, has been evaluated. P-gp function on patient cells was analyzed by measuring the changes in rhodamine-123 (Rh-123) fluorescence after verapamil incubation. CSA treatment resulted in good clinical outcome that was related with a significant P-gp function reduction at CD3+ and CD8+ levels. In addition to its immunosuppressive activity, CSA results may also be related to MTX/SSA effect restoration through P-gp inhibition. This is the first time that CSA has been demonstrated as being able to revert MTX/SSA resistance in PsA.
[Show abstract][Hide abstract] ABSTRACT: TLR9 activation by unmethylated CpG provides a homeostatic mechanism to maintain B cell memory in the absence of Ag. In this study, we demonstrate that CpG also triggers the generation of somatically mutated memory B cells from immature transitional B cells. In response to CpG, a fraction of transitional B cells proliferates and introduces somatic hypermutations in the H chain V regions. The nonproliferating pool of transitional B cells mostly maintains germline configurations. Mutations are VH specific: VH5 is the least mutated family, whereas VH1 and VH4/6 are the most mutated families. CpG stimulation also results in upregulation of VH5 transcripts in proliferating cells. Therefore, early recognition of bacterial DNA preferentially expands VH5-expressing B cells while inducing somatic hypermutations in other families. The mutation frequency, range, and type of substitutions observed in vitro are comparable to those found in memory B cells from the peripheral blood of Hyper IgM type 1 patients and the spleen of normal infants. The process triggered by TLRs may represent a first step leading to additional diversification of the germline repertoire and to the generation of memory B cells that will further refine their repertoire and specificity in the germinal centers.
The Journal of Immunology 11/2010; 185(12):7293-301. · 5.36 Impact Factor