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What’s known on the subject? and What does the study add? Treg overexpression has been demonstrated in several neoplasms, including liver, breast, pancreas and melanoma, while it has not been well evaluated in renal cancer. In renal cancer patients versus controls we found an increased expression of these cells, especially in tumour-infiltrating lymphocytes. Moreover, Treg frequency significantly correlated with pathological stage, nuclear grade and prognostic models. OBJECTIVE • To compare the frequency of T regulatory cells (Tregs) in peripheral blood of patients (pPB) affected by renal cell carcinoma (RCC) both with the frequency of Tregs found in PB of healthy donors (hPB) and that of Tregs present in tumour infiltrating lymphocytes (TILs). To verify in vitro the inhibitory activity of tumour isolated Tregs on the effector T cells and, finally, to assess the prognostic role of Treg frequency determination. PATIENTS AND METHODS • Treg frequency in hPB, pPB and TILs was evaluated in 30 patients and 20 healthy controls by measuring both membrane-CD25 and intracytoplasmic-Foxp3 expression by flow cytometry. • Treg inhibitory activity was evaluated by an in vitro proliferation assay performed on total, CD25-depleted mononuclear cells (MNC) and CD25-depleted MNC cultured in the presence of purified CD25+ Tregs. • Finally, Treg frequency in pPB and TIL were correlated with conventional prognostic factors and scores of University of California Los Angeles and Kattan predictive models. RESULTS • Treg frequency was higher in TILs than in pPB (P= 0.002), whereas there were no important differences between hPB and pPB. CD25+ cells isolated either from PB and tumours showed the ability to significantly suppress in vitro both proliferation and interferon-γ production by CD25-depleted MNC, thus demonstrating that they are active Tregs. • Treg frequency was found to significantly correlate both with pathological stage (pPB, P= 0.03; TIL, P= 0.04) and nuclear grade (TIL, P= 0.005), both for UCLA and Kattan models (all: P < 0.05 for both pPB and TIL). CONCLUSION • Treg frequency is significantly higher in TIL than in pPB of patients with RCC. Tregs showed in vitro an inhibitory activity on effector T cells isolated from kidney tumours. The increase in both peripheral and intratumoral Tregs in subjects affected with RCC were associated with worse prognosis.

Objective In patients with hepatitis C virus (HCV)/HIV co-infection, a faster progression of liver fibrosis to cirrhosis has been reported. In this study, an investigation was carried out to determine whether gp120, an HIV envelope protein, modulates the biology of human hepatic stellate cells (HSCs), key cell types in the pathogenesis of fibrosis. Methods Myofibroblastic HSCs were isolated from normal human liver tissue. Gene expression was measured by real-time PCR. Cell migration was assessed in Boyden chambers. Intracellular signalling pathways were evaluated using phosphorylation-specific antibodies or by transfection of a reporter plasmid. Results Transcripts for the chemokine receptors CCR5 and CXCR4, which bind gp120, were detectable in human HSCs. Upon exposure to M-tropic recombinant gp120, which binds CCR5, a significant increase in HSC chemotaxis was observed (1.6±0.3-fold, p=0.03). The effects of gp120 were prevented by protein inactivation. gp120 also resulted in a significant increase in secretion (1.5±0.3-fold, p=0.03) and gene expression (1.47±0.13-fold, p=0.02) of the proinflammatory chemokine monocyte chemoattractant protein-1, and in increased gene expression of tissue inhibitor of metalloprotease-1 and interleukin-6 (2.03±0.57-fold, p=0.02). gp120-induced migration required Akt activation. gp120 also induced activation of nuclear factor-κB (NF-κB) and p38 MAPK . Preincubation of HSCs with TAK779, a CCR5 receptor antagonist, prevented gp120-mediated chemotaxis and monocyte chemoattractant protein-1 secretion. Expression of CCR5 was detectable in areas of inflammation and fibrogenesis in liver biopsies of patients with HCV/HIV co-infection. Conclusions This study shows that HIV gp120 modulates different aspects of HSC biology, including directional cell movement and expression of proinflammatory cytokines. These results identify a direct pathway possibly linking HIV infection with liver fibrogenesis via envelope proteins.

T helper (Th) 17 cells represent a novel subset of CD4+ T cells that are protective against extracellular microbes, but are responsible for autoimmune disorders in mice. However, their properties in humans are only partially known. We demonstrate the presence of Th17 cells, some of which produce both interleukin (IL)-17 and interferon (IFN)-gamma (Th17/Th1), in the gut of patients with Crohn's disease. Both Th17 and Th17/Th1 clones showed selective expression of IL-23R, CCR6, and the transcription factor ROR gamma t, and they exhibited similar functional features, such as the ability to help B cells, low cytotoxicity, and poor susceptibility to regulation by autologous regulatory T cells. Interestingly, these subsets also expressed the Th1-transcription factor T-bet, and stimulation of these cells in the presence of IL-12 down-regulated the expression of ROR gamma t and the production of IL-17, but induced IFN-gamma. These effects were partially inhibited in presence of IL-23. Similar receptor expression and functional capabilities were observed in freshly derived IL-17-producing peripheral blood and tonsillar CD4+ T cells. The demonstration of selective markers for human Th17 cells may help us to understand their pathogenic role. Moreover, the identification of a subset of cells sharing features of both Th1 and Th17, which can arise from the modulation of Th17 cells by IL-12, may raise new issues concerning developmental and/or functional relationships between Th17 and Th1.

PF-4/CXCL4 is a member of the CXC chemokine family, which is mainly produced by platelets and known for its pleiotropic biological functions. Recently, the proteic product of a nonallelic variant gene of CXCL4 was isolated from human platelets and named as CXCL4L1. CXCL4L1 shows only 4.3% amino acid divergence in the mature protein, but exhibits a 38% amino acid divergence in the signal peptide region. We hypothesized that this may imply a difference in the cell type in which CXCL4L1 is expressed or a difference in its mode of secretion. In different types of transfected cells, CXCL4 and CXCL4L1 exhibited a distinct subcellular localization and a differential regulation of secretion, CXCL4 being stored in secretory granules and released in response to protein kinase C activation, whereas CXCL4L1 was continuously synthesized and secreted through a constitutive pathway. A protein kinase C-regulated CXCL4 secretion was observed also in lymphocytes, a cell type expressing mainly CXCL4 mRNA, whereas smooth muscle cells, which preferentially expressed CXCL4L1, exhibited a constitutive pathway of secretion. These results demonstrate that CXCL4 and CXCL4L1 exhibit a distinct subcellular localization and are secreted in a differentially regulated manner, suggesting distinct roles in inflammatory or homeostatic processes.

Regenerative medicine represents a critical clinical goal for patients with ESRD, but the identification of renal adult multipotent progenitor cells has remained elusive. It is demonstrated that in human adult kidneys, a subset of parietal epithelial cells (PEC) in the Bowman’s capsule exhibit coexpression of the stem cell markers CD24 and CD133 and of the stem cell–specific transcription factors Oct-4 and BmI-1, in the absence of lineage-specific markers. This CD24 + CD133 + PEC population, which could be purified from cultured capsulated glomeruli, revealed self-renewal potential and a high cloning efficiency. Under appropriate culture conditions, individual clones of CD24 + CD133 + PEC could be induced to generate mature, functional, tubular cells with phenotypic features of proximal and/or distal tubules, osteogenic cells, adipocytes, and cells that exhibited phenotypic and functional features of neuronal cells. The injection of CD24 + CD133 + PEC but not of CD24 − CD133 − renal cells into SCID mice that had acute renal failure resulted in the regeneration of tubular structures of different portions of the nephron. More important, treatment of acute renal failure with CD24 + CD133 + PEC significantly ameliorated the morphologic and functional kidney damage. This study demonstrates the existence and provides the characterization of a population of resident multipotent progenitor cells in adult human glomeruli, potentially opening new avenues for the development of regenerative medicine in patients who have renal diseases.

Background The clinical efficacy and safety of sublingual immunotherapy (SLIT) for aeroallergens has been demonstrated in several trials, whereas the immunological changes induced by this treatment, which may account for the clinical improvement, are still unclear. Objective To investigate the effects of a successful SLIT on the in vitro allergen-driven T cell response and cytokine secretion as well as on the serum levels of chemokines and of IgE, IgG1 and IgG4 antibodies (Abs). Materials and methods Twenty-five Dermatophagoides pteronyssinus (Dp)-sensitive patients with perennial rhinitic and/or rhinitic and asthmatic symptoms were randomized into two groups (13 untreated (UT) and 12 SLIT-treated) for a 1 year and half study. The proliferative response of peripheral blood mononuclear cell (PBMC) to purified Der p1 allergen, their cytokines (IFN-gamma, IL-4, IL-10 and TGF-beta) production and serum levels of chemokines associated with T helper type 1 (Th1) (CXCL10) or T helper type 2 (Th2) (CCL22) responses and of Dp-specific IgE, IgG1 and IgG4 Abs were evaluated before and after 6 months of treatment. Results SLIT induced a significant reduction of symptom medication scores after 6, 12 and 18 months of treatment in comparison with UT patients. SLIT-treated patients showed a significant decrease in serum levels of DP-specific IgE Abs, whereas total IgE, and specific IgG1 and IgG4 Abs remained unchanged. The proliferative response of allergen-specific T cells to Der p1 in vitro after 6 months of treatment was reduced, while no effect was observed on T cell proliferation to recall antigen (streptokinase). Moreover, Der p1-driven IFN-gamma and IL-10 were significantly increased in culture supernatants of PBMC from 6 month-treated patients in comparison with those detected at the beginning of therapy. Conclusions These data suggest that the allergen-driven enhancement of IL-10- and IFN-gamma-producing T cells precedes and associates with SLIT-induced down-regulation of specific IgE, thus providing a rationale to explain the clinical benefit of SLIT in allergic patients.

• To compare the frequency of T regulatory cells (Tregs) in peripheral blood of patients (pPB) affected by renal cell carcinoma (RCC) both with the frequency of Tregs found in PB of healthy donors (hPB) and that of Tregs present in tumour infiltrating lymphocytes (TILs). To verify in vitro the inhibitory activity of tumour isolated Tregs on the effector T cells and, finally, to assess the prognostic role of Treg frequency determination.• Treg frequency in hPB, pPB and TILs was evaluated in 30 patients and 20 healthy controls by measuring both membrane-CD25 and intracytoplasmic-Foxp3 expression by flow cytometry. • Treg inhibitory activity was evaluated by an in vitro proliferation assay performed on total, CD25-depleted mononuclear cells (MNC) and CD25-depleted MNC cultured in the presence of purified CD25(+) Tregs. • Finally, Treg frequency in pPB and TIL were correlated with conventional prognostic factors and scores of University of California Los Angeles and Kattan predictive models.• Treg frequency was higher in TILs than in pPB (P= 0.002), whereas there were no important differences between hPB and pPB. CD25(+) cells isolated either from PB and tumours showed the ability to significantly suppress in vitro both proliferation and interferon-γ production by CD25-depleted MNC, thus demonstrating that they are active Tregs. • Treg frequency was found to significantly correlate both with pathological stage (pPB, P= 0.03; TIL, P= 0.04) and nuclear grade (TIL, P= 0.005), both for UCLA and Kattan models (all: P0.05 for both pPB and TIL).• Treg frequency is significantly higher in TIL than in pPB of patients with RCC. Tregs showed in vitro an inhibitory activity on effector T cells isolated from kidney tumours. The increase in both peripheral and intratumoral Tregs in subjects affected with RCC were associated with worse prognosis.

We have previously shown that human Th17 lymphocytes are characterized by the selective expression of IL-23 receptor (IL-23R), CCR6, CD161, and the transcription factor retinoic acid-related orphan receptor C (RORC), and originate from a CD161(+)CD4(+) naive T-cell precursor in response to the combined activity of IL-1β and IL-23. We show here that not only CD4(+)TCRαβ(+), but also CD8(+)TCRαβ(+), CD4(-)CD8(-) TCRαβ(+), and CD4(-)CD8(-) TCRγδ(+) circulating lymphocytes that produce IL-17 express the distinctive marker CD161 on their surface. In addition, we demonstrate that CD161 expression identifies CD8(+) and CD4(-)CD8(-) umbilical cord blood T cells that already express RORC and IL-23R mRNA and that can be induced to differentiate into IL-17-producing cells in the presence of IL-1β and IL-23. Finally, we provide evidence that umbilical cord blood naive CD4(+)CD161(-) T cells, upon lentivirus-mediated transduction with RORC2 can acquire the ability to express IL-23R, IL-1RI, and CD161, as well as to produce IL-17. Taken together, these data allow to conclude that T-cell subsets able to produce IL-17, as well as precursors of IL-17-producing T cells, exhibit surface expression of CD161, and that this feature is at least in part RORC2-dependent.

Background IL-17A has been suggested to play a pathogenic role in bronchial asthma and other allergic disorders. Objective Study of the relationship between human IL-17A–producing CD4 + T H cells (T H 17) and IL-4–producing CD4 + T H (T H 2) cells. Methods T-cell clones generated from the CCR6 + CD161 + fraction of human circulating CD4 + T cells, which contains virtually all T H 17 cells, as well as circulating CD4 + T cells from both healthy subjects and patients with asthma, were assessed by flow cytometry for their cytokine production profile. Results A small proportion of CCR6 + CD161 + CD4 + T-cell clones showed the ability to produce both IL-17A and IL-4 (T H 17/T H 2). T H 17/T H 2 clones also produced IL-5, IL-8, IL-9, IL-13, IL-21, and IL-22 and displayed the ability to induce the in vitro secretion of IgE. A very few T H 17/T H 2 cells were found among circulating CD4 + T cells from normal subjects, but their proportions were significantly increased in the circulation of patients with chronic asthma. T H 17/T H 2 cells could not be derived from naive umbilical cord blood CD4 + T cells under any experimental condition. However, when circulating memory CCR6 + CD161 + CD4 + T cells were cloned under appropriate polarizing conditions, T H 17/T H 2 clones originated in the presence of IL-4, suggesting that an IL-4–rich microenvironment may induce the shifting of memory T H 17 cells into T H 17/T H 2 cells. Conclusion Because of its peculiar functional properties and the increased numbers in the circulation of patients with bronchial asthma, this previously unknown population of T H 17/T H 2 cells may play some role in the pathogenesis of this disease.

We demonstrate that CD161 is a highly up-regulated gene in human interleukin (IL) 17 T helper cell (Th17) clones and that all IL-17–producing cells are contained in the CD161+ fraction of CD4+ T cells present in the circulation or in inflamed tissues, although they are not CD1-restricted natural killer T cells. More importantly, we show that all IL-17–producing cells originate from CD161+ naive CD4+ T cells of umbilical cord blood, as well as of the postnatal thymus, in response to the combined activity of IL-1β and IL-23. These findings implicate CD161 as a novel surface marker for human Th17 cells and demonstrate the exclusive origin of these cells from a CD161+CD4+ T cell progenitor.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of the colorectum, with mucosal infiltration by activated leukocytes, which are the result of complex interactions between lymphocytes, antigen, and dendritic cells (DCs). We carried out lymphocyto-plasmapheresis (LCPA) in a UC patient with the aim of removing lymphocytes, DCs and inflammatory cytokines (ICKs). A 42-year-old female with UC in moderate activity phase was submitted to 5 weekly LCPA treatments. Before and after LCPA we monitored: (i) the percentage of T, B, NK lymphocytes, monocytes, and peripheral blood lymphoid and myeloid DCs; (ii) the T lymphocyte subpopulations; (iii) the ICKs; and (iv) the immune complexes (IC). We achieved the interruption of all pharmacological therapies, and so far the clinical and histological remission has lasted for 24 months. The flow cytometric assessment of the leukocyte subpopulations did not show any relevant variation of their numbers after LCPA, while TNFalpha, IL-6, IL-12 and serum IgG-C1q ICs decreased. In the present case, the contemporary depletion of plasma, lymphocytes and DCs, allowed LCPA to emerge as an efficient alternative to UC pharmacological therapy without affecting the number of white blood cells, DCs and leukocyte subpopulations that were assessed. Further studies are needed both to address LCPA mechanism of action and optimal apheresis protocol, and to compare this form of therapy to a placebo control group.

Background CD4 + CD25 + Foxp3 + T-regulatory (Treg) cells play a fundamental role in the control of autoimmunity. Whether human CD4 + CD25 + Foxp3 + Treg cells that recognize foreign antigens also exist is less clear. Objective To investigate the existence in humans of circulating Treg cells able to recognize exogenous antigens, including allergens. Methods CD4 + CD25 high Foxp3 + and CD4 + CD25 - Foxp3 - cells were purified from human peripheral blood and cultured for 15 days with autologous dendritic cells (DCs), unloaded, or loaded with Der p 1 allergen or the bacterial antigen streptokinase (SK). Results CD4 + CD25 high Foxp3 + circulating T cells obtained from healthy nonatopic subjects and cultured with Der p 1–loaded DCs, but not those cultured with either unloaded or SK-loaded DCs, suppressed the proliferative response to Der p 1 of autologous Der p 1–specific T cells generated from the CD4 + CD25 - Foxp3 - subset. The antigen specificity of either Der p 1–CD4 + CD25 high Foxp3 + or SK–CD4 + CD25 high Foxp3 + T cells was confirmed even at clonal level. Finally, under the same experimental conditions, functionally active Der p 1–specific Treg cells were obtained from the pool of circulating CD4 + CD25 high Foxp3 + T cells of Der p 1–sensitive, atopic individuals. Conclusion These data provide undoubted demonstration of the existence of human CD4 + CD25 high Foxp3 + circulating Treg cells specific for exogenous antigens, including the Der p 1 allergen, and indicate that CD4 + CD25 high Foxp3 + Treg cells specific for Der p 1 are present and functionally active in both nonatopic and Der p 1–sensitive, atopic individuals. Clinical implications Caution should be advised in interpreting allergic disorders as simply resulting from defective Treg cell activity.

Background: The identification of mycobacteria represents the gold standard in the diagnosis of tuberculosis (TB), but it is not applicable in all patients, and immunological tests, such as the tuberculin skin test (TST), are not specific and sensitive enough. Methods: By flow cytometry, we measured the CD4 T-cell response to purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6) protein using the intracellular cytokine staining technique on whole blood samples obtained from active TB (n = 16), latent TB (n = 17), Bacille Calmette-Guérin (BCG)-vaccinated (n = 11) and healthy (n = 10) donors. All the patients were also tested with conventional TST. Results: The identification by flow cytometry of PPD-specific T lymphocytes upon antigen stimulation of whole blood enables the discrimination of active TB, latent TB and BCG-vaccinated subjects from healthy individuals, whereas the ESAT-6 response discriminated active TB from healthy and BCG-vaccinated individuals. Moreover, this test enables identification of active TB patients who were negative on TST and to distinguish between TB and non-typical mycobacteria TB infections. Conclusions: The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than TST, and could represent a further tool in the diagnosis of TB infection.

Endothelial progenitor cells (EPCs) seem to be a promising tool for cell therapy of acute myocardial infarction, but their nature is still unclear. We show here that EPCs obtainable from peripheral blood (PB) derive from the adhesion-related selection in culture of a subset of CD14+ cells, which, when assessed by the highly-sensitive antibody-conjugated magnetofluorescent liposomes (ACMFL) technique, were found to express CD34. These CD14+CD34 low cells represented a variable proportion at individual level of CD14+ cells, ranging from 0.6% to 8.5% of all peripheral-blood leukocytes, and constituted the dominant population among circulating KDR+ cells. By using the ACMFL technique, virtually all CD14+ cells present in the bone marrow were found to be CD14+CD34 low double-positive cells. EPCs, as well as purified circulating CD14+CD34 low cells, exhibited high expression of embryonic stem cell (SC) markers Nanog and Oct-4, which were downregulated in a STAT3-independent manner when they differentiated into endothelial cells (ECs). Moreover, circulating CD14+CD34 low cells, but not CD14+CD34− cells, proliferated in response to SC growth factors, and exhibited clonogenicity and multipotency, as shown by their ability to differentiate not only into ECs, but also into osteoblasts, adipocytes, or neural cells. The results of this study may reconcile apparently contradictory data of the literature, showing the generation of PB-derived EPCs from either CD34+ or CD14+ cells. We suggest that the use of this previously unrecognized population of circulating CD14+CD34 low cells, which exhibit both phenotypic and functional features of SCs, may be useful in improving cell-based therapies of vascular and tissue damage.

Background The mechanisms by which human dendritic cells (DCs) activate a T H 1-polarizing or T H 2-polarizing program are still partially unclear. Objective Study of the mechanisms responsible for the T H 1/T H 2-polarizing activity of human circulating myeloid DCs before and after ligation of their Toll-like receptors (TLRs). Methods IL-4 and IFN-γ production by CD4 + T cells was assessed in cocultures with myeloid DCs before or after TLR triggering. Expression of Jagged-1 and Delta-4 Notch ligands and of GATA-3 and T-box expressed in T cells transcription factors was evaluated by real-time quantitative PCR. Signal transducer and activator of transcription 4 and 6 phosphorylation was assessed by flow cytometry. Knockdown of Jagged-1 or Delta-4 was performed by transfection of DCs with appropriate silencing mRNAs. Results Myeloid immature DCs constitutively expressed Jagged-1, which induces in CD4 + T cells a T H 2 polarization, as shown by Jagged-1 gene silencing. The T H 2 polarization associated with high GATA-3/T-box expressed in T cells ratio and was at least partially dependent on the early induction of IL-4. Maturation of DCs by TLR ligation resulted in the reduction of Jagged-1 and upregulation of Delta-4, which was at least in part responsible for the polarization of CD4 + T cells to the T H 1 phenotype. Conclusion CD4 + T-cell responses are usually characterized by a prevalent T H 2 phenotype unless TLRs are triggered on DCs by microbial components.