Francesco Indiveri

Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway.

Bone marrow mesenchymal progenitor cells (BMSC) are used for regenerating tissues of mesodermal origin, as well as tissues of different embryological derivation. Experimental evidence shows that BMSC are able to suppress the activation of the immune response by mechanisms that are still not completely understood. Thus far, in vitro studies carried using human or mouse cells indicate that autologous or allogeneic BMSC strongly suppress proliferation of T lymphocytes, triggered by cellular stimuli, nonspecific mitogenic stimuli, or antigenic peptides. Using cell proliferation and blocking assays, we demonstrated that BMSC inhibited the activation of murine splenocytes, T, and B lymphocytes. Direct contact of BMSC and target cells in a cognate fashion determined the inhibition of cell proliferation via engagement of the inhibitory molecule programmed death 1 (PD‐1) to its ligands PD‐L1 and PD‐L2, leading the target cells to modulate the expression of different cytokine receptors and transduction molecules for cytokine signaling. Soluble factors present on supernatants of BMSC cultures were effective in suppressing proliferation of B cells to a mitogenic stimulus. Taken together, these results highlight the complexity of the role of BMSC in regulating the immune response, asserting the possibility of their therapeutic application in transplantation and autoimmune diseases.

Soluble HLA-A,-B,-C and -G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation.

There is convincing evidence that soluble HLA‐A,‐B,‐C (sHLA‐A,‐B,‐C) and soluble HLA‐G (sHLA‐G) antigens can induce apoptosis in CD8+ activated T cells although there is scanty and conflicting information about the mechanism(s) by which sHLA‐A,‐B,‐C antigens and sHLA‐G antigens induce apoptosis. In this study we have compared the apoptosis‐inducing ability of sHLA‐A,‐B,‐C antigens with that of sHLA‐G1 antigens in CD8+ T lymphocytes and CD8+ NK cells. Furthermore we have compared the inhibitory effect of sHLA‐A,‐B,‐C antigens and of sHLA‐G1 antigens on theactivity of EBV‐specific CD8+ cytotoxic T lymphocytes (CTL). sHLA molecules were purified from serum and from the supernatant of HLA class I‐negative cells transfected with one gene encoding either classical or non‐classical HLA class I antigens. Both classical and non‐classical sHLA class I molecules trigger apoptosis in CD8+ T lymphocytes and in CD8+ NK cells, which lack the T cell receptor, and their apoptotic potency is comparable. The binding of sHLA‐A,‐B,‐C and sHLA‐G1 molecules to CD8 leads to Fas ligand (FasL) up‐regulation, soluble FasL (sFasL) secretion and CD8+ cell apoptosis by Fas/sFasL interaction. Moreover, classical and non‐classical sHLA class I molecules inhibit the cytotoxic activity of EBV‐specific CD8+ CTL. As the amount ofsHLA‐G molecules detectable in normal serum is significantly lower than that of sHLA‐A,‐B,‐C molecules, the immunomodulatory effects of sHLA class I molecules purified from serum are likely to be mainly attributable to classical HLA class I antigens. As far as the potential in vivo relevance of these findings is concerned, we suggest that classical sHLA class I molecules may play a major immunoregulatory role in clinical situations characterized by activation of the immune system and elevated sHLA‐A,‐B,‐C serum levels. In contrast, non‐classical HLA class I molecules may exert immunomodulatory effects in particular conditions characterized by elevated sHLA‐G levels such as pregnancy and some neoplastic diseases.

Gastroesophageal reflux and pulmonary fibrosis in scleroderma: a study using pH-impedance monitoring.

RATIONALE Interstitial lung disease (ILD) in patients with systemic sclerosis (SSc) is associated with increased morbidity and mortality. Gastroesophageal reflux (GER) is considered a contributing factor in the pathogenesis of ILD. OBJECTIVES To characterize GER (acid and nonacid) in patients with SSc with and without ILD. METHODS Patients with SSc underwent pulmonary high-resolution computer tomography (HRCT) scan and 24-hour impedance-pH monitoring off-proton pump inhibitor therapy. The presence of pulmonary fibrosis was assessed using validated HRCT-scores. Reflux monitoring parameters included number of acid and nonacid reflux episodes, proximal migration of the refluxate, and distal esophageal acid exposure. Unless otherwise specified, data are presented as median (25th-75th percentile). MEASUREMENTS AND MAIN RESULTS Forty consecutive patients with SSc (35 female; mean age, 53 yr; range, 24-71; 15 patients with diffuse and 25 with limited SSc) were investigated; 18 (45%) patients with SSc had pulmonary fibrosis (HRCT score >or= 7). Patients with SSc with ILD had higher (P < 0.01) esophageal acid exposure (10.3 [7.5-15] vs. 5.2 [1.5-11]), higher (P < 0.01) number of acid (41 [31-58] vs. 19 [10-23]) and nonacid (25 [20-35] vs. 17 [11-19]) reflux episodes, and higher (P < 0.01) number of reflux episodes reaching the proximal esophagus (42.5 [31-54] vs. 15 [8-22]) compared with patients with SSc with normal HRCT scores. Pulmonary fibrosis scores (HRCT score) correlated well with the number of reflux episodes in the distal (r(2) = 0.637) and proximal (r(2) = 0.644) esophagus. CONCLUSIONS Patients with SSc with ILD have more severe reflux (i.e., more reflux episodes and more reflux reaching the proximal esophagus). Whether or not the development of ILD in patients with SSc can be prevented by reflux-reducing treatments needs to be investigated.

Impairment of CD8+ T Suppressor Cell Function in Patients with Active Systemic Lupus Erythematosus

Alteration of T cell suppression function has been recognized in patients with systemic lupus erythematosus (SLE). Recently, CD8+ T suppressor lymphocytes (CD8+ Ts) have been generated in vitro by incubating purified CD8+ T cells with IL-2 and GM-CSF. Using this method, we generated CD8+ Ts from patients affected by SLE. No major differences were found in the CD8+ Ts phenotype between SLE patients and healthy subjects. CD8+ Ts from SLE patients with active disease did not inhibit the anti-CD3 mAb-induced proliferation of autologous PBMC, whereas CD8+ Ts from SLE patients in remission exerted an inhibitory activity comparable to normal subjects. The inhibitory effect of CD8+ Ts cells was neither mediated by cytotoxic activity nor by apoptosis induction. Two cytokines, IFN-γ and IL-6, were found to be responsible for the function of CD8+ Ts. In fact, counteraction of CD8+ Ts suppression activity was obtained by blocking IFN-γ with a specific Ab or by inhibiting CD8+ Ts-mediated IL-6 secretion by an antisense oligonucleotide. Interestingly, CD8+ Ts from SLE patients showed a peculiar cytokine pattern characterized by an impaired secretion of IL-6 and an increased secretion of IL-12. Thus, it appears that an altered balance between inhibitory (IL-6) and stimulatory (IL-12) cytokines might be responsible for the functional impairment of CD8+ Ts in SLE patients.

CD8+CD28− T Regulatory Lymphocytes Inhibiting T Cell Proliferative and Cytotoxic Functions Infiltrate Human Cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+CD28− T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+CD28− T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+CD25+ T regulatory lymphocytes associate with CD8+CD28− T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.

Serum HLA class I antigens: markers and modulators of an immune response?

Abstract HLA class I antigens circulate in the serum in soluble form. This article discusses the clinical significance of levels of serum HLA class I antigens, both in patients with viral diseases and following organ transplantation, as well as the potential involvement of such antigens in the immune response.

Small intestinal bacterial overgrowth in patients suffering from scleroderma: clinical effectiveness of its eradication.

OBJECTIVES:After the skin, the gastrointestinal tract is the second most common target of systemic sclerosis (SSc).AIM:Our aims were to investigate orocecal transit time (OCTT) and the presence of small intestinal bacterial overgrowth (SIBO) in SSc as a cause of intestinal symptoms.METHODS:Fifty-five SSc patients and 60 healthy controls, sex and age matched, entered the study. Enrolled subjects completed a questionnaire for intestinal symptoms and a global symptomatic score (GSS) was calculated. OCTT and the presence of SIBO were assessed by a lactulose breath test (LBT). Patients with SIBO were treated with rifaximin 1,200 mg/day for 10 days. Finally, a second questionnaire and LBT were performed 1 month after the end of therapy.RESULTS:The prevalence of SIBO was higher in SSc patients compared with controls (30/54 vs 4/60, respectively, P < 0.001). OCTT was significantly slower in SSc patients compared with controls (150 min, 25–75th percentile 142.5–165 vs 105 min, 25–75th percentile 90–135, respectively, P < 0.001). In patients with SIBO, the median GSS score was 8 (25–75th percentile 3.25–10.75). Eradication of SIBO was achieved in 73.3% of patients, with a significant reduction of symptoms in 72.7% of them (GSS score 2, 25–75th percentile 1–3, P < 0.05).CONCLUSIONS: These data suggest that SIBO occurs more frequently in SSc patients than in controls. Intestinal symptoms in these patients may be related to this syndrome and its eradication seems useful to improve clinical features. OCTT is significantly delayed in SSc patients, suggesting an impairment of intestinal motility, a further risk factor for the development of SIBO.

Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes.

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4+ and CD8+ T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10−3‐10−12M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G1 phase of cell cycle and inhibits both IL‐2 receptor (IL‐2R) expression and IL‐2 secretion. Apoptosis is clearly increased by PDN in PHA‐activated human PBL, and the apoptotic effect of PDN is stronger on CD8+ than on CD4+ T lymphocytes. All these effects are dose‐ and time‐dependent. The addition of exogenous IL‐2 did not rescue lymphocytes from PDN‐increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post‐thymic level.

Isolation of human T lymphocytes: Comparison between nylon wool filtration and rosetting with neuraminidase (VCN) and 2-aminoethylisothiouronium bromide (AET)-treated sheep red blood cells (SRBC)

Isolation of human T lymphocytes by rosetting with SRBC, centrifugation on Ficoll-Hypaque gradients and lysis of SRBC, which reportedly affects the functional activities of lymphocytes, was compared to isolation of T lymphocytes by filtration through a nylon wool column. The two methods were similar in yields and purity of T lymphocytes. No selective loss of T cell subpopulations was found in T lymphocytes isolated by filtration through a nylon wool column as judged by the percentage of T gamma and T micron and the suppressor and helper activity in functional assays. Therefore, filtration through a nylon wool column is a useful method to purify T lymphocytes and has the advantage of being comparatively simple and quick to perform and of avoiding manipulations which may affect the functional activity of T lymphocytes.

Alteration of Th17 and Treg cell subpopulations co-exist in patients affected with systemic sclerosis.

Aim of the study has been to understand the relationship between TH17 and Treg cell subsets in patients affected with systemic sclerosis (SSc). Phenotypes and functions of Th17 and Treg cell subsets were analyzed in a series of 36 SSc patients. Th17 cell concentration in the peripheral blood was found to be increased in SSc patients with respect to healthy controls independently from type or stage of disease. After PBMC stimulation with a polyclonal stimulus or Candida albicans antigens the frequency of Th17 T cell clones was significantly higher in SSc patients with respect to controls suggesting the skewing of immune response in SSc patients toward Th17 cell generation/expansion. Concerning the Treg compartment, both CD4+CD25+ and CD8+CD28- Treg subsets showed quantitative and qualitative alteration in the peripheral blood of SSc patients. Collectively, these data highlight the existence of an imbalanced ratio between Th17 and Treg cell subsets in SSc patients.