Gisella Puga

• University of Geneva

Natural Killer (NK) cells are pivotal in immune responses to viral infections, malignancies, autoimmune diseases, and transplantation. Assessment of NK cell adhesion, migration, and cytotoxicity is fundamental for in vitro studies. We propose a novel live-cell tracking method that addresses these three major aspects of NK cell function using human...

Natural killer (NK) cells play an important role in the surveillance of viral infections and cancer. NK cell antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity are mediated by the recognition of antibody-coated target cells through the Fc gamma receptor IIIA (FcγRIIIa/CD16) and by ligands of activating/inhibitory NK receptors,...

The production of multiple transcripts per gene is a process regulated by inherited genetic variants and epitranscriptomic modifications, and plays a prominent role in modulating complex traits and diseases. To simultaneously characterize the effect of genetic variants on transcript abundance and N6-methyladenosine (m6A) modifications, we produced...

NK cell‐mediated Ab‐dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc‐gamma receptor IIIA (FcγRIIIA)/CD16a expressed on NK cells remain unknown. The aim of th...

Background: Tolerogenic dendritic cells (DCs) represent a promising approach to promote transplantation tolerance. In this study, the potential of autologous bone marrow (BM)-derived murine DC to protect rat-to-mouse islets xenografts was analyzed. Methods: Tolerogenic DCs were generated by differentiating BM cells in the presence of granulocyte...

Regulatory T‐cells (Treg) induce immunologic tolerance by suppressing effector functions of conventional lymphocytes in the periphery. On the other hand, immune silencing is mediated by recognition of phosphatidylserine (PS) on apoptotic cells by phagocytes. Here we describe expression of the PS‐binding protein Annexin‐V (ANXA5) in CD4+CD25hi Treg...

Small-molecule immunosuppressive drugs (ISD) prevent graft rejection mainly by inhibiting T lymphocytes. Therapeutic immunoglobulins (IVIg) are used for substitution, antibody-mediated rejection (AbMR) and HLA-sensitized recipients by targeting distinct cell types. Since the effect of ISD and IVIg on natural killer (NK) cells remains somewhat contr...

Simultaneous determination of degranulation and intracellular IFNγ in NK cells treated with immunosuppressive drugs. Purified NK cells were analyzed for degranulation by staining for CD107a surface expression and intracellular IFNγ production by flow cytometry, following overnight culture with IL2 (50 U/ml) and IL12 (0.5 ng/ml) in the presence or a...

NK cell viability after culture with ISD. NK cells were incubated overnight in the presence of CsA (0.1 and 1.0 μg/ml), TAC (0.01 and 0.1 μg/ml), MPA (0.5 and 5 μg/ml), EVE (0.01 and 0.1 μg/ml), and MePRD (0.05, 0.5 and 5.0 μg/ml). 7AAD was added to the treated NK cells at a final concentration of 1 μg/ml, followed by flow cytometry. Data are prese...

Effect of immunosuppressive drugs on the expression of NK cell markers and receptors. PBMC were incubated with or without 50 U/ml IL2 (control); with 50 U/l IL2 plus CsA (0.1 μg/ml), TAC (0.01 μg/ml), MPA (5 μg/ml), EVE (0.0 1μg/ml), or MePRD (0.5 μg/ml) for 24 h. NK cell marker and receptor expression was analyzed by FACS. Data are shown as mean ±...

Background: In pig-to-human xenotransplantation, early cellular rejection reactions are mediated by natural killer cells (NK cells). Human NK cells are inhibited by HLA-E via CD94/NKG2A receptors. To protect porcine grafts against human NK cell responses, transgenic GTKO pigs expressing hCD46 and HLA-E have been generated. The aim of this study wa...

Recruitment of human NK cells to porcine tissues has been demonstrated in pig organs perfused ex vivo with human blood in the early 1990s. Subsequently, the molecular mechanisms leading to adhesion and cytotoxicity in human NK cell-porcine endothelial cell (pEC) interactions have been elucidated in vitro to identify targets for therapeutic interven...

Background: In pig-to-human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non-classical human leukocyte antigen-E (HLA-E) on pEC. Thus, the a...

Worldwide, there is a constant rise in the number of patients with end-stage organ failure in critical need for transplants, but the number of organs/cells availa-ble from deceased or living human donors is limited. Xenotransplantation using pig organs/tissues repre-sents a potential solution for this shortage; however, it has been hampered by a nu...

Background: Dendritic cells (DC) play a major role in natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activation leading to cell-mediated xenogeneic responses. In contrast, the use of in vitro differentiated regulatory DC may represent an attractive approach to protect porcine endothelial cells (pEC) from human cell-mediated immune respo...

Significant immunological obstacles are to be negotiated before xenotransplantation becomes a clinical reality. An initial rejection of transplanted vascularized xenograft is attributed to Galα1,3Galβ1,4GlcNAc-R (Galα1,3-Gal)-dependent and -independent mechanisms. Hitherto, no receptor molecule has been identified that could account for Galα1,3-Gal...

Physical as well as psychological stress increases the number of circulating peripheral blood NK cells. Whereas some studies found a positive correlation between exercise and NK cell counts and cytotoxic activity, others showed that, for example, heavy training leads to a decrease in per cell NK cytotoxicity. Thus, the impact of exercise on NK cell...

Gating strategy to quantify the frequency of CD56bright NK cells. Pre-exercise and post-exercise PBMC were isolated, stained with live/dead fixable Aqua stain, with either the isotype controls, or anti-CD3/anti-CD56/anti-CD16 Ab followed by quantification by flow cytometry. Single living lymphocytes were gated and NK cells were defined by the expre...

Flow cytometry histograms of NK marker analysis. Representative multicolor flow cytometry experiment depicting the histograms of several NK cell surface markers as indicated. PBMC were isolated before and after running and stained with fluorescent mAb. Single living lymphocytes were gated and CD3–CD56+ NK cells were analyzed. The expression in pre-...

Functional analysis of CD56bright and CD56dim NK cells. NK cells from five different donors were purified and stimulated overnight with IL-2 and IL-12. Following the addition or not of K652 cells as described in the M&M section degranulation of CD56total, CD56bright, and CD56dim NK cells is shown as percentage of positive cells for CD107a (A), wher...

Natural killer (NK) cells are considered to play a critical role in liver disease. However, the available numbers of intrahepatic lymphocytes (IHL) derived from liver biopsies (LB) for ex vivo analysis of intrahepatic NK cells is very limited; and the isolation method may hamper not only yields and viability, but also phenotype and function of IHL....

Anti-Galα1,3Galβ-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3...

Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood. This study evaluated how STZ-induced diabetes affects adaptive immunity and...

The possibility that allogeneic hematopoietic stem cell transplantation performed across the ABO blood group-barrier is associated with an increase of graft-versus-host disease, in particular endothelial damage, has not been elucidated so far. For this reason, we investigated the level of endothelial cell chimerism after allogeneic hematopoietic st...

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Primer sequences used for real time PCR are listed in Table S1. (0.03 MB DOC)

Humoral response and complement deposition analysis of rejecting and tolerant mice. Immunohistology for humoral immune responses to concordant islet xenografts in untreated C57BL/6 mice and in combination therapy (MR1+RAPA) treated mice at 200 days post transplantation. Sections were stained by anti-mouse IgG (A/D), IgM (B/E) and complement (C3, C/...

Concordant xeno- and allo-responses are diminished in late tolerant mice. White bars, naïve mice; gray bars: Group 6, (late anti-IL2 treatment); black bars: Group 8 (late anti-CD25 mAb treatment). Alternatively Sprague donor strain splenocytes, Lewis splenocytes, BALB/c splenocytes or human PBMC were used as stimulators. In late anti-IL2 mAb and la...

Anti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in the induction and maintenance o...

Pediatric Crohn's disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohn's disease (CD) is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP) have been identified as ubiquitous antigens...

To summarize the current knowledge of the immune response generated against xenografts stemming from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs. In particular, we will address the nature of potentially remaining Gal epitopes, the role of non-Gal xenoantigens, and the cellular response directed against GalT-KO tissues. New findings suppo...

ABO-incompatible kidney transplantation using immunoadsorption to remove anti-A/B antibodies has become a successful clinical practice. Since the data on the specificity of the ABO columns are controversial, the present study assessed the efficiency and specificity of the ABO immunoadsorption, the effect on total immunoglobulins and antibodies prev...

Due to different detection methods, a comparison of anti-A/B antibody (Ab) levels among transplantation centers after living donor ABO-incompatible kidney transplantation is problematic. In the present study, anti-A/B Ab levels were determined prior to, and after, blood group A-to-O kidney transplantation using a recently established semiquantitati...

Objective To identify epitopes on Escherichia coli heat-shock protein (HSP) dnaJ or on homologous human HSP dnaJ involved in the induction/modulation of autoimmune inflammation in patients with oligoarticular juvenile idiopathic arthritis (JIA).Methods We used a proliferation assay and cytokine production to evaluate the immune responses of synovia...

Despite a rapidly accumulating clinical experience with autologous stem cell transplantation (ASCT) as a treatment for severe refractory autoimmune disease, data on the mechanisms by which ASCT induces immune tolerance are still very scarce. In this study it is shown that ASCT restores immunologic self-tolerance in juvenile idiopathic arthritis (JI...

Innate and adaptive immunity contribute to the pathogenesis of autoimmune arthritis by generating and maintaining inflammation, which leads to tissue damage. Current biological therapies target innate immunity, eminently by interfering with single pro-inflammatory cytokine pathways. This approach has shown excellent efficacy in a good proportion of...

Modulation of epitope-specific immune responses would represent a major addition to available therapeutic options for many autoimmune diseases. The objective of this work was to induce immune deviation by mucosal peptide-specific immunotherapy in rheumatoid arthritis (RA) patients, and to dissect the related immunological mechanisms by using a tech...

Rheumatoid arthritis (RA) is an inflammatory disease that primarily involves the joints and has a worldwide prevalence of about one percent, with a female to male ratio of 3:1. This chapter summarizes some of the recent progress in molecular immunology, and discusses the application of this new knowledge for therapeutic purposes. We focus on our re...

MHC class II expression defects have been evidenced in several human tumor cell lines originating from lung cancers or retinoblastoma. Accordingly, the mouse adenocarcinoma and fibrosarcoma cell lines, RAG and L(tk-), do not express I-A and I-E molecules even when treated with IFN-gamma. Here we show that fusion of both cell lines restores the indu...