Pedro Aparicio

Role of ICAM-3 in the initial interaction of T lymphocytes and APCs

Antigen-independent adhesive interactions between T lymphocytes and antigen-presenting cells (APCs) are essential for scanning for specific antigens on the APC surface and for initiating the immune response. Here we show, through time-lapse imaging of live cells, that the intercellular adhesion molecule 3 (ICAM-3, also known as CD50) is clustered specifically at the region of the T lymphocyte surface that initiates contact with APCs. We describe the role of ICAM-3 in T cell–APC conjugate formation before antigen recognition, in early intracellular signaling and in cytoskeletal rearrangement. Our data indicate that ICAM-3 is important in the initial scanning of the APC surface by T cells and, therefore, in generating the immune response.

FoxP3 in peripheral blood is associated with operational tolerance in liver transplant patients during immunosuppression withdrawal.

Background. Human liver allografts do sometimes survive in a recipient after withdrawal of immunosuppression (IS), commonly referred to as “operational tolerance.” Preliminary clinical data have suggested an increase in the frequency of regulatory T cells (Treg) CD4+CD25high and FoxP3 expression in operationally tolerant liver transplant recipients (Gr-T). In the context of human liver transplantation, the dynamics of Treg have not been studied. We designed a prospective study to ascertain the profile of the Treg population and FoxP3 expression during IS withdrawal. Methods. To identify such parameters, we analyzed peripheral blood mononuclear cell populations and FoxP3 mRNA expression in 12 liver allograft recipients under cyclosporine A-based IS, who showed stable function of the allograft for more than 2 years. Results. An increase was observed in the frequency of CD4+CD25high cells when the IS was withdrawn in Gr-T patients (n=5). These patients exhibited a 3.5-fold increase for relative mRNA FoxP3 expression before the complete IS withdrawal and this continued when IS therapy was stopped. In patients who suffered rejection (n=7) there was no increase in the CD4+CD25high cells or FoxP3 expression. Conclusions. With the present study, the first evidence is provided that the increase of CD4+CD25high T cells and FoxP3 transcripts is associated with operational tolerance in liver transplanted patients during IS withdrawal.

Human T cell receptor-mediated recognition of HLA-E

The HLA‐E class Ib molecule presents hydrophobic peptides derived from the leader sequences of other class I molecules, constituting the ligands for CD94/NKG2 lectin‐like receptors. Along the course of our studies on human CD94+ T cells, we characterized an α β CD8+CD94/NKG2C+ CTL clone (K14). In cytolytic assays against the murine TAP‐deficient RMA‐S cells transfected with human β2 microglobulin and HLA‐E (RMA‐S/HLA‐E), loaded with different synthetic peptides, K14 displayed a pattern of specific recognition distinct to that observed in CD94/NKG2C+ NK clones tested in parallel. RMA‐S/HLA‐E cells loaded with some but not all HLA class I leader sequence peptides were efficiently recognized by K14 but not by CD94/NKG2C clones, andvice versa. Remarkably, K14 also reacted with HLA‐E loaded with a peptide derived from the BZLF‐1 Epstein‐Barr virus protein. Anti‐CD94 mAb did not prevent K14 cytotoxicity against RMA‐S/HLA‐E cells, whereas incubation with anti‐clonotypic mAb specific for the K14 TCR markedly inhibited lysis. Soluble HLA‐E tetramers refolded with different peptides (i.e. VMAPRTVLL, VMAPRTLIL, VMAPRTLFL) specifically stained K14 cells. HLA‐E tetramer binding was minimally reduced by pretreatment with anti‐CD94 mAb alone, but was completely prevented in combination with anti‐clonotypic mAb. Altogether, the data unequivocally imply the generation of human T cells potentially recognizing through the α β TCR HLA‐E molecules that bind to class I‐ and virus‐derived peptides.

Haematopoietic progenitor cells from adult bone marrow differentiate into cells that express oligodendroglial antigens in the neonatal mouse brain

Stem cells are self‐renewable, pluripotent cells that, in adult life, proliferate by a characteristic asymmetric division in which one daughter cell is committed to differentiation whereas the other remains a stem cell. These cells are also characterized by their ability to differentiate into various cell types under heterotopic environmental influences. In the present study, we have explored the potential of adult haematopoietic bone marrow cells to differentiate into cells of oligodendroglial lineage under physiological, active myelinating conditions. We present evidence of generation of cells expressing oligodendroglial specific markers from a bone marrow subpopulation enriched on adult haematopoietic progenitor cells (CD117+) in vivo after intracerebral transplantation into the neonatal mouse brain. Our results suggest that adult bone marrow cells have the capacity to undergo differentiation from haematopoietic to oligodendroglial cells and add support the validity of bone marrow transplants as an alternative treatment for demyelinating diseases of the CNS including Multiple Sclerosis.

PARP‐2 deficiency affects the survival of CD4 + CD8 + double‐positive thymocytes

Poly‐(ADP‐ribose) polymerase‐2 (PARP‐2) belongs to a large family of enzymes that synthesize and transfer ADP‐ribose polymers to acceptor proteins, modifying their functional properties. PARP‐2‐deficient (Parp‐2−/−) cells, similar to Parp‐1−/− cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp‐2, but not Parp‐1, produced a two‐fold reduction in CD4+CD8+ double‐positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl‐2 family member Noxa in Parp‐2−/− DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp‐2−/− have a reduced expression of T‐cell receptor (TCR)α and a skewed repertoire of TCRα toward the 5′ Jα segments. Our results show that in the absence of PARP‐2, the survival of DP thymocytes undergoing TCRα recombination is compromised despite normal amounts of Bcl‐xL. These data suggest a novel role for PARP‐2 as an important mediator of T‐cell survival during thymopoiesis by preventing the activation of DNA damage‐dependent apoptotic response during the multiple rounds of TCRα rearrangements preceding a positively selected TCR.

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-β1 (TGF-β1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-β1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-β1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-β1 on the melanogenic activity of B16/F10 cells.

Interleukin-7 rescues human activated T lymphocytes from apoptosis induced by glucocorticoesteroids and regulates bcl-2 and CD25 expression

We studied the ability of several interleukins to inhibit the cellular death of IL-2-dependent human T cells deprived of IL-2 testing viability, DNA integrity, and expression of bcl-2 gene product. Our in vitro results showed that the addition of IL-7, and in a far less efficient manner IL-4, augmented the viability of IL-2-dependent T-cell clones of different origin, specificity, and phenotype. Furthermore, IL-7 reduced the percentage of apoptotic T cells inhibiting DNA fragmentation. In addition, IL-7 but not IL-4 was consistently able to suppress the cell death of IL-2-dependent T cells triggered by DEX, a synthetic GC. The suppression of T-cell death triggered by IL-7 was not affected by the addition of anti-IL-2 antibody. Interestingly, IL-7 inhibited the downregulation of bcl-2 gene product expression that appeared on TCCs after IL-2 withdrawal and also shared with IL-2 the ability to induce the upregulation of CD25 antigen on activated T lymphocytes in the presence of DEX. These experiments establish a novel role for IL-7 in regulating viability and GC-induced apoptosis on activated human T cells and suggest that the maintenance of bcl-2 levels is a general mechanism by which interleukins preserve activated T cells from undergoing apoptosis.

Loss of poly(ADP-ribose) polymerase-2 leads to rapid development of spontaneous T-cell lymphomas in p53-deficient mice

Poly(ADP-ribose) polymerase-2 (Parp-2) belongs to a family of enzymes that catalyse poly(ADP-ribosyl)ation of proteins. Parp-2 deficiency in mice (Parp-2−/−) results in reduced thymic cellularity associated with increased apoptosis in thymocytes, defining Parp-2 as an important mediator of T-cell survival during thymopoiesis. To determine whether there is a link between Parp-2 and the p53 DNA-damage-dependent apoptotic response, we have generated Parp-2/p53-double-null mutant mice. We found that p53−/− backgrounds completely restored the survival and development of Parp-2−/− thymocytes. However, Parp-2-deficient thymocytes accumulated high levels of DNA double-strand breaks (DSB), independently of the p53 status, in line with a function of Parp-2 as a caretaker promoting genomic stability during thymocytes development. Although Parp-2−/− mice do not have spontaneous tumours, Parp-2 deficiency accelerated spontaneous tumour development in p53-null mice, mainly T-cell lymphomas. These data suggest a synergistic interaction between Parp-2 and p53 in tumour suppression through the role of Parp-2 in DNA-damage response and genome integrity surveillance, and point to the potential importance of examining human tumours for the status of both genes.

Signalling via CD70, a member of the TNF family, regulates T cell functions.

In the present work, we provide data supporting that CD70, a tumor necrosis factor (TNF)‐related molecule, defined as the CD27 ligand (CD27L), may actively regulate T cell functions similarly to other members of the TNF family (i.e., CD40L and CD30L). Cross‐linking CD70 with specific monoclonal antibodies (mAb) stimulated cytotoxicity and cytokine production in human T cell clones. Detection of intracellular‐free calcium mobilization and mitogen‐activated protein kinase phosphorylation upon mAb engagement of CD70 further supported an active signaling role for the TNF‐related molecule. Similar results were obtained in the Jurkat leukaemia T cell line stably transfected with CD70; in that system, induction of Akt phosphorylation was detected, indirectly revealing the involvement of the phosphatidylinositol‐3 kinase pathway. Stimulation of CD70+ Jurkat cells, with a CD70‐specific mAb or with COS‐7 cells transiently transfected with CD27, induced transcriptional activity detectable by different reporter gene expression systems. Altogether, our data point out that a reciprocal communication may be established between CD27+ and CD70+ cells during the immune response.

Transcriptional regulation by Poly(ADP-ribose) polymerase-1 during T cell activation

BackgroundAccumulating evidence suggests an important role for the enzyme poly(ADP-ribose) polymerase-1 (PARP-1) as an integral part of the gene expression regulatory machinery during development and in response to specific cellular signals. PARP-1 might modulate gene expression through its catalytic activity leading to poly(ADP-ribosyl)ation of nuclear proteins or by its physical association with relevant proteins. Recently, we have shown that PARP-1 is activated during T cell activation. However, the proposed role of PARP-1 in reprogramming T cell gene expression upon activation remains largely unexplored.ResultsIn the present study we use oligonucleotide microarray analysis to gain more insight into the role played by PARP-1 during the gene expression reprogramming that takes place in T cells upon activation with anti-CD3 stimulation alone, or in combination with anti-CD28 co-stimulation. We have identified several groups of genes with expression modulated by PARP-1. The expression of 129 early-response genes to anti-CD3 seems to be regulated by PARP-1 either in a positive (45 genes) or in a negative manner (84 genes). Likewise, in the presence of co-stimulation (anti-CD3 + anti-CD28 stimulation), the expression of 203 genes is also regulated by PARP-1 either up (173 genes) or down (30 genes). Interestingly, PARP-1 deficiency significantly alters expression of genes associated with the immune response such as chemokines and genes involved in the Th1/Th2 balance.ConclusionThis study provides new insights into changes in gene expression mediated by PARP-1 upon T cell activation. Pathway analysis of PARP-1 as a nuclear signalling molecule in T cells would be of relevance for the future development of new therapeutic approaches targeting PARP-1 in the acquired immune response.