Ignacio J. Molina

IL-17-producing CD8+ T lymphocytes from psoriasis skin plaques are cytotoxic effector cells that secrete Th17-related cytokines

IL‐17‐producing CD4+ T lymphocytes (Th17) are currently considered relevant participants in the pathogenesis of psoriasis skin lesions. However, little is known about the potential role of IL‐17‐producing CD8+ T cells, which are also present at the psoriatic plaque. We have addressed the functional characterization of this CD8+ subtype of T lymphocytes from psoriasis patients. Our results show that CD8+IL‐17+ cells from psoriasis‐inflamed skin tissue produce TNF‐α and IFN‐γ (Th1‐related cytokines) as well as IL‐17, IL‐21, and IL‐22 (Th17‐related cytokines) efficiently. A significant up‐regulation of the RORC transcription factor is also observed. These cells are refractory to Tregs but show a proliferative response to anti‐CD3/CD28 stimulation that is enhanced by IL‐12 and IL‐15. Blocking of TNF‐α activity inhibits TCR‐mediated activation and IL‐17 production. CD8+IL‐17+ T cells are cytotoxic cells that display TCR/CD3‐mediated cytotoxic abilities to kill target cells. Thus, CD8+IL‐17+ T cells share some key features with Th17 cells and exhibit remarkable differential abilities attributable to the CD8+ lineage of T lymphocytes, adding new insights into the functional resources of IL‐17‐producing cells from human epidermis that could be of potential interest to our understanding of the pathogenesis of psoriasis.

Expression of killer inhibitory receptors on cytotoxic cells from HIV-1-infected individuals.

Dysfunction of cytotoxic activity of T and natural killer (NK) lymphocytes is a main immunological feature in patients with AIDS, but its basis are not well understood. It has been recently described that T and NK cell‐mediated cytotoxicity can be regulated by HLA killer inhibitory receptors (KIR). In this work, we have determined on cytotoxic T cells and NK cells from HIV‐1‐infected individuals the expression of the following KIR molecules: p58, p70, and ILT2 (immunoglobulin‐like family KIR) as well as CD94 and NKG2A (C‐lectin‐type family KIR). With some exceptions, no significant changes were found on the expression of immunoglobulin‐like KIR in either CD8+ or CD56+ cells. Interestingly, the percentages of CD8+ and CD56+ cells expressing CD94 were significantly increased in these individuals. We also show that, in vitro, IL‐10 up‐regulates CD94 expression on CD8+ and CD56+ cells obtained from normal individuals, suggesting that the augmented expression observed in HIV‐infected individuals could be related to the high levels of IL‐10 previously described in HIV‐1‐infected individuals.

Lentiviral vectors transcriptionally targeted to hematopoietic cells by WASP gene proximal promoter sequences.

The development of vectors that express a therapeutic transgene efficiently and specifically in hematopoietic cells (HCs) is an important goal for gene therapy of hematological disorders. In order to achieve this, we used a 500 bp fragment from the proximal WASP gene promoter to drive the expression of the WASP cDNA in the context of a self-inactivating lentiviral vector. Single-round transduction of WASp-deficient herpesvirus saimiri (HVS)-immortalized cells as well as primary allospecific T cells from Wiskott–Aldrich syndrome (WAS) patients with this vector (WW) resulted in expression levels similar to those of control cells. Non-HCs were transduced with similar efficiency, but the levels of WASp were 135–350 times lower than those achieved in HCs. Additionally, transduction of WASp-deficient cells with WW conferred a selective growth advantage in vitro. Therefore, lentiviral vectors incorporating proximal promoter sequences from the WASP gene confer hematopoietic-specific, and physiological protein expression.

Characterization of gliadin-specific Th17 cells from the mucosa of celiac disease patients.

OBJECTIVES:Celiac disease (CD) is a disorder characterized by a deregulated immune response to ingested wheat gluten and related cereal proteins in susceptible individuals. It has been considered that the onset of CD is mediated by a skewed Th1 response. However, the participation of Th17 cells in the pathogenesis of the disease, a key cell population in other autoimmune disorders, has not been studied in detail. We have investigated the presence of Th17 cells in the mucosa of active CD patients and their functional implications in the pathogenesis of the disease.METHODS:T cells obtained from duodenum biopsies from 15 untreated patients and 11 control individuals were characterized by flow cytometry, immunoassays, and real-time PCR.RESULTS:We found gliadin-specific CD4+ interleukin (IL)-17A-producing T cells in the mucosa of CD patients with a phenotype consisting of TCR (T-cell receptor)αβ+ CD45RO+ CD161+ CCR6+ (C–C chemokine receptor type 6) and IL-23R+. Functional analysis showed that Th17 cells from CD patients are different from those of control individuals in terms of cytokines production. Th17 cells from CD patients, but not from controls, simultaneously express transforming growth factor-β (TGFβ). Th17 CD cells also produce interferon-γ (IFNγ), IL-21, and IL-22. The analysis of the transcription factors revealed a high expression of interferon regulatory factor-4 as a feature of gliadin-specific cells from CD patients with respect to controls.CONCLUSIONS:Gliadin-specific Th17 cells are present in the mucosa of CD patients having a dual role in the pathogenesis of the disease as they produce proinflammatory cytokines (such as IL-17, IFNγ, IL-21), mucosa-protective IL-22, and regulatory TGFβ, which actively modulates IL-17A production by T cells in the celiac mucosa.

Defective B7 expression on antigen-presenting cells underlying T cell activation abnormalities in systemic lupus erythematosus (SLE) patients.

Defective T cell functions, including IL‐2 production and proliferation, have been shown in SLE patients. After T cell stimulation (first signal), a costimulatory signal (second signal) is required to achieve complete T cell activation. Main costimulatory signals are provided to T cells by B7 antigens (CD80 and CD86, expressed on antigen‐presenting cells (APC)) upon interaction with its receptor, the CD28 molecule expressed on T cells. The aim of this study was to investigate the role of CD28/B7 interactions in the impaired T cell responses of SLE patients. We show that stimulation of T cells with phytohaemagglutinin (PHA) in the presence, but not in the absence, of anti‐CD28 MoAb or B7+ cells results in tyrosine phosphorylation of specific substrates, transcription of mRNA and production of IL‐2 that is indistinguishable in SLE patients and healthy controls. Moreover, proliferation of costimulated T cells from SLE and controls was specifically abrogated by blocking the CD28/B7 interactions by means of addition to the culture of the CTLA4‐Ig fusion protein. However, in most patients activated APC failed to up‐regulate B7 molecules, giving rise to ineffective costimulatory signalling to T cells. These results indicate that the CD28/B7 costimulatory pathway is defective in SLE patients.

Expression of CD94 and NKG2 molecules on human CD4(+) T cells in response to CD3-mediated stimulation.

We investigated the ability of human peripheral CD4+ cellsto express CD94 and NKG2 molecules as a consequence ofCD3‐mediated activation. Using highly purified peripheralCD4+ T cells, we found expression of both CD94 and NKG2A 15days after CD3‐mediated stimulation of cells. We also determined byreverse transcriptase‐PCR that all gene members of NKG2 family—namely,NKG2A, ‐C, ‐D, and ‐E—are sequentially expressed on CD4+cells. We found that this expression is tightly regulated by cytokines,and we identified transforming growth factor‐β1 and interleukin‐10 asthe main factors that, on CD3‐dependent stimulation, positivelycontribute to the expression of CD94 and NKG2A on CD4+cells. We also investigated the functional role of NKG2A and found thatcoligation of CD3 and NKG2A by specific monoclonal antibodies resultsin significant inhibition of interferon γ and tumor necrosis factorα production by stimulated CD4+ cells. The presence andfunction of these receptors on CD4+ lymphocytes support amore general role for NKG2 molecules, whose functions were originallythought to be confined to cytotoxic cells, in the immune system.

Hematopoietic-specific lentiviral vectors circumvent cellular toxicity due to ectopic expression of Wiskott-Aldrich syndrome protein.

Efficient and safe gene modification of hematopoietic stem cells is a requirement for gene therapy of primary immunodeficiencies such as Wiskott-Aldrich syndrome. However, deregulated expression or ectopic expression in the progeny of transduced nonhematopoietic progenitor cells may lead to unwanted toxicity. We therefore analyzed the effect of ectopic expression of Wiskott-Aldrich syndrome protein (WASp) and the potential benefits of hematopoietic-specific lentiviral vectors (driven by the WAS proximal promoter). Overexpression of WASp by constitutive lentiviral vectors is highly toxic in nonhematopoietic cells because it causes dramatic changes in actin localization and polymerization that result in decreased cell viability, as evidenced by a significant growth disadvantage of WASp-overexpressing nonhematopoietic cells and increased cell death. These toxic effects do not affect cells of hematopoietic origin because, remarkably, we found that WASp cannot be readily overexpressed in T cells, even after multiple vector integrations per cell. The adverse cellular effects found after transduction of nonhematopoietic cells with constitutive lentiviral vectors are overcome by the use of transcriptionally targeted lentiviral vectors expressing WASp, which, at the same time, are efficient tools for gene therapy of WAS as demonstrated by their ability to reconstitute cellular defects from WASp-deficient mouse and human cells. We therefore postulate that transcriptionally regulated lentiviral vectors represent a safer and efficient alternative for the development of clinical protocols of WAS gene therapy.

Efficient lentiviral transduction of Herpesvirus saimiri immortalized T cells as a model for gene therapy in primary immunodeficiencies

Infection of human T lymphocytes with the Herpesvirus saimiri (HVS) yields immortalized T-cell lines (HVS-T) which retain all the phenotypical and functional characteristics of their parental cells. This represents a new experimental model for studying genetic disorders of T lymphocytes. In spite of the efforts of many laboratories, no satisfactory way has been found so far to modify HVS-T cells genetically. We have analyzed the capacity of oncoretroviral (MLV)- and lentiviral (HIV-1)-based vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSVg) to transduce HVS-T cells. HIV-1-derived vectors efficiently transduced HVS-T cell lines, reaching up to 85% of cells expressing the transgene in a single round of infection. MLV-based vectors, on the other hand, were unable to transduce more than 1% of any of the HVS-T cell lines analyzed. Lentiviral-driven gene expression was maintained constant and stable in HVS-T cells for a minimum of 48 days. We also observed that although the lentiviral transduction efficiency achieved on HVS-T cells is lower than that obtained with tumor or primary endothelial cells, it is nevertheless similar to that found with activated primary T cells.

An exopolysaccharide produced by the novel halophilic bacterium Halomonas stenophila strain B100 selectively induces apoptosis in human T leukaemia cells

Microbial exopolysaccharides (EPSs) are highly heterogeneous polymers produced by fungi and bacteria and have recently been attracting considerable attention from biotechnologists because of their potential applications in many fields, including biomedicine. We have screened the antitumoural activity of a panel of sulphated EPSs produced by a newly discovered species of halophilic bacteria. We found that the novel halophilic bacterium Halomonas stenophila strain B100 produced a heteropolysaccharide that, when oversulphated, exerted antitumoural activity on T cell lines deriving from acute lymphoblastic leukaemia (ALL). Only tumour cells were susceptible to apoptosis induced by the sulphated EPS (B100S), whilst primary T cells were resistant. Moreover, freshly isolated primary cells from the blood of patients with ALL were also susceptible to B100S-induced apoptosis. The newly discovered B100S is therefore the first bacterial EPS that has been demonstrated to exert a potent and selective pro-apoptotic effect on T leukaemia cells, and thus, we propose that the search for new antineoplastic drugs should include the screening of other bacterial EPSs, particularly those isolated from halophiles.

Defective actin reorganization and polymerization of Wiskott-Aldrich T cells in response to CD3-mediated stimulation

The Wiskott-Aldrich syndrome (WAS) is a severe immunodeficiency and platelet deficiency disease arising from mutation(s) in the WASP gene, which in normal cells encodes an intracellular protein able to interact with other proteins relevant to the control of cytoskeleton organization. Immunodeficiency is mainly due to T-cell progressive malfunction. Salient defects of WAS T cells are a CD3-restricted impairment in proliferative responses and cytoskeletal abnormalities, including the frequent appearance of T cells with atypical morphology. We have investigated the possibility that the CD3-restricted defect and some of the cytoskeletal defects of WAS T cells are linked. For this purpose, we immortalized by means of infection with Herpesvirus Saimiri a number of previously described allospecific WAS T-cell lines. The resulting cells preserve the surface, molecular, and functional phenotypes of their parental lines, including a negligible WASP mRNA expression as well as the CD3-restricted defect and cytoskeleton abnormalities. Results show that, in CD3-stimulated WAS T cells, the pattern of temporal changes in cell shape and F-actin distribution is substantially different from that of control cells. Furthermore, polymerization of actin, a critical step in the CD3-mediated cytoskeleton reorganization, does not occur in WAS T-cell lines in response to OKT3 stimulation. In conclusion, our data link both CD3 and cytoskeletal defects in WAS T cells, strongly suggesting that cytoskeleton abnormalities are an underlying cause for WAS immunodeficiency.