Search

CD27 is a member of tumour necrosis factor receptor family. Its expression is predominantly confined to mature lymphocytes and is strongly enhanced after cell activation. Shedding of the CD27 from the surface of activated cells is related to their effector phase. The aim of the present study was to evaluate the levels of soluble CD27 in sera and synovial fluids, together with its expression on circulating and synovial fluid (SF) memory T cells, in children with JIA.Sera from 40 patients with active JIA were studied for soluble CD27. Paired SF samples were available in 20 patients. Sera from 12 age-matched patients affected with various acute infectious diseases and 12 age-matched healthy subjects were used as controls. In 8 JIA patients freshly isolated circulating and SF lymphocytes were stained for CD27 in CD4+CD45 RO+ T cell subpopulation and analyzed by cytometry.Soluble CD27 serum levels were significantly higher in patients with polyarticular JIA and acute systemic infectious diseases than in patients with active oligoarticular or healthy controls. Both polyarticular and oligoarticular JIA patients showed increased levels of soluble CD27 in SF when compared with paired serum samples (p = 0.01). In all the patients tested a significant enrichment of CD27- T cells was seen in the SF (median 39.5%, range 18-56%) when compared to paired CD4+CD45RO+ peripheral lymphocytes (median 19.5%, range 5-43%; p = 0.01).A clear enrichment of CD4+ memory SF T cells with a CD27-phenotype is observed when compared to correspondent circulating T lymphocytes. This issue is conceivably related to re-activation and recruitment of memory T cells to the site of inflammation, and to the subsequent expansion of a subpopulation of "effector" memory T cells.

The expression of genes encoding different polypeptide chains of the TCR-CD3 complex was analyzed in a panel of cloned MHC-unrestricted cytotoxic cells. The clones were derived from CD3+ and CD3- human PBL. After expansion in rIL-2, all clones were able to lyse the NK-sensitive target cell line K562. In contrast, lysis of fresh tumor cells was achieved almost exclusively by CD3- clones. To test whether a known TCR-CD3 complex may be involved in MHC-unrestricted cytotoxicity, total RNA from nine CD3+ and 11 CD3- clones was isolated and hybridized with DNA probes for the TCR alpha-, beta-, and gamma-chains and for the CD3 gamma-, delta-, and epsilon-chains. TCR gamma transcripts were present at high levels in CD3+CD4- CD8- clones but were undetectable in all CD3- clones. Lysis of fresh tumor cells is an activity which can be independent of the TCR alpha beta and TCR gamma complexes because the CD3- clones did not express these TCR genes. Interestingly, all CD3- clones expressed CD3 epsilon transcripts, but not CD3 gamma- or delta-transcripts. CD3- lymphokine-activated cytotoxic cells may therefore be derived from immature T cells which do not yet express a complete CD3 complex. The CD3 epsilon chain, if expressed in CD3- cells in association with other molecules, could be involved in the activation and lytic function of these MHC-unrestricted cytotoxic cells.

The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-gamma by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-gamma+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.

Objective. Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is a multisystemic autoinflammatory condition associated with heterozygous TNFRSF1A mutations, presenting with a variety of clinical symptoms, many of which yet unexplained. In this work, we aimed at deepening into TRAPS pathogenic mechanisms sustained by monocytes.

Monoclonal antibodies (MoAbs) specific for surface molecules involved in lymphocyte activation, represent an useful tool for the analysis of the activation pathways of different effector lymphocytes. We have analyzed the ability of MoAbs directed against "triggering" surface molecules, such as CD3/TCR, CD2 and CD16 to induce activation of the lytic machinery in T and NK lymphocytes, using a redirected killing assay. In addition, we have constructed bispecific monoclonal antibodies (BiMoAbs) directed against both the CD3/TCR complex (or the CD16 molecule) and a tumor associated antigen expressed on the surface of ovarian cancer cells. BiMoAbs were constructed by two different approaches: a) conjugation of two distinct MoAbs by a chemical heterobifunctional agent; b) selection of hybrid-hybridomas secreting BiMoAbs. BiMoAbs were able to focus appropriate lymphoid effector cells on tumor cells expressing the relevant antigen, and to induce tumor cell lysis. Therefore, these reagents were able to confer a new specificity for a given antigen to effector lymphocytes, independently from the original one. For these properties, BiMoAbs may represent a suitable tool for new strategies of adoptive immunotherapy, based on the use of effector cells that have been specifically armed by BiMoAbs against the tumor.

GD2-redirected chimeric antigen receptor (CAR) T lymphocytes represent a promising therapeutic option for immunotherapy of neuroblastoma (NB). However, despite the encouraging therapeutic effects observed in some hematological malignancies, clinical results of CAR T cell immunotherapy in solid tumors are still modest. Tumor driven neo-angiogenesis supports an immunosuppressive microenvironment that influences treatment responses and is amenable to targeting with antiangiogenic drugs. The latter agents promote lymphocyte tumor infiltration by transiently reprogramming tumor vasculature, and may represent a valid combinatorial approach with CAR T cell immunotherapy. In light of these considerations, we investigated the anti-NB activity of GD2-CAR T cells combined with bevacizumab (BEV) in an orthotopic xenograft model of human NB. Two weeks after tumor implantation, mice received BEV or GD2-CAR T cells or both by single intravenous administration. GD2-CAR T cells exerted a significant anti-NB activity only in combination with BEV, even at the lowest concentration tested, which per se did not inhibit tumor growth. When combined with BEV, GD2-CAR T cells massively infiltrated tumor mass where they produced interferon-γ (IFN-γ), which, in turn, induced expression of CXCL10 by NB cells. IFN-γ, and possibly other cytokines, upregulated NB cell expression of PD-L1, while tumor infiltrating GD2-CAR T cells expressed PD-1. Thus, the PD-1/PD-L1 axis can limit the anti-tumor efficacy of the GD2-CAR T cell/BEV association. This study provides a strong rationale for testing the combination of GD2-CAR T cells with BEV in a clinical trial enrolling NB patients. PD-L1 silencing or blocking strategies may further enhance the efficacy of such combination.

We prospectively assessed functional and phenotypic characteristics of γδ T lymphocytes up to 7 months after HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) depleted of αβ+ T cells and CD19+ B cells in 27 children with either malignant or nonmalignant disorders. We demonstrate that (1) γδ T cells are the predominant T-cell population in patients during the first weeks after transplantation, being mainly, albeit not only, derived from cells infused with the graft and expanding in vivo; (2) central-memory cells predominated very early posttransplantation for both Vδ1 and Vδ2 subsets; (3) Vδ1 cells are specifically expanded in patients experiencing cytomegalovirus reactivation and are more cytotoxic compared with those of children who did not experience reactivation; (4) these subsets display a cytotoxic phenotype and degranulate when challenged with primary acute myeloid and lymphoid leukemia blasts; and (5) Vδ2 cells are expanded in vitro after exposure to zoledronic acid (ZOL) and efficiently lyse primary lymphoid and myeloid blasts. This is the first detailed characterization of γδ T cells emerging in peripheral blood of children after CD19+ B-cell and αβ+ T-cell–depleted haplo-HSCT. Our results can be instrumental to the development of clinical trials using ZOL for improving γδ T-cell killing capacity against leukemia cells. This trial was registered at www.clinicaltrials.gov as #{"type":"clinical-trial","attrs":{"text":"NCT01810120","term_id":"NCT01810120"}}NCT01810120.

IL-12 promotes generation of LAK activity in short-term-cultured NK cells, but information on the structure and function of IL-12-induced LAK cells is not yet available. The latter issues have been here investigated with emphasis on interactions between IL-12 and IL-2. Peripheral blood mononuclear cells (MNC) exposed to IL-12 for 5-7 days displayed a decrease in the amount and density of the matrix of large granular lymphocyte (LGL)-associated granules. In cells cultured with IL-12 and IL-2 for 5-7 days, empty vacuoles were predominant and the electron-dense matrix was scanty. In MNC incubated with IL-2 for 5-7 days, most granules were loaded with electron-dense matrix. IL-12 and IL-2 displayed an additive effect on LAK cell cytotoxicity until approximately 48 h in culture which was followed by a sharp decline. Immunocytochemical and biochemical studies demonstrated that MNC cultured for 5-7 days with IL-12 and IL-2 displayed downregulated perforin expression and upregulated granzyme B expression. Fas ligand expression was virtually undetectable in MNC cultured for 5-7 days with or without cytokines. It appears that perforin downregulation plays a major role in the reduced cytotoxicity of MNC cultured with IL-12 and IL-2 for 5-7 days.