Giuseppe Terrazzano

Recognition of autologous dendritic cells by human NK cells.

NK cells can recognize and kill tumor as well as certain normal cells. The outcome of the NK‐target interaction is determined by a balance of positive and negative signals initiated by different target cell ligands. We have previously shown that human NK cells kill CD40‐transfected tumor targets efficiently, but the physiological significance of this is unclear. We now demonstrate that human NK cells can kill dendritic cells (DC), known to express CD40 and other co‐stimulatory molecules. The killing was observed with polyclonal NK cells cultured short term in IL‐2 as well as with NK cell clones as effectors, and with allogeneic as well as autologous DC as targets. NK cell recognition could be inhibited, but only partially, by preincubation of target cells with monoclonal antibodies against CD40, suggesting that this molecule may be one of several ligands involved. Addition of TNF‐α of the cultures stimulated the development of a more mature DC phenotype, while addition of IL‐10 resulted in a less mature phenotype, with lower expression of CD40 and other co‐stimulatory molecules. Nevertheless, such DC were more NK susceptible than the differentiated DC. This may be partly explained by a reduced MHC class I expression observed on such cells, since blocking of MHC class I molecules on differentiated DC or CD94 receptors of NK cells led to increased NK susceptibility. The results show that NK cells may interact with DC, and suggest that the outcome of such interactions depend on the cytokine milieu.

Celiac Disease Association with CD8+ T Cell Responses: Identification of a Novel Gliadin-Derived HLA-A2-Restricted Epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8+ T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123–132) by CD8+ T lymphocytes from celiac patients. A-gliadin 123–132-specific IFN-γ production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123–132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123–132 epitope recognized human APC pulsed with either whole recombinant α-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.

Gliadin Regulates the NK-Dendritic Cell Cross-Talk by HLA-E Surface Stabilization

We analyzed the autologous NK cell interaction with gliadin-presenting dendritic cells. Gliadin is the known Ag priming the celiac disease (CD) pathogenesis. We demonstrate that gliadin prevents immature dendritic cells (iDCs) elimination by NK cells. Furthermore, cooperation between human NK cells-iDCs and T cells increases IFN-γ production of anti-gliadin immune response. Gliadin fractions were analyzed for their capability to stabilize HLA-E molecules. The α and ω fractions conferred the protection from NK cell lysis to iDCs and increased their HLA-E expression. Gliadin pancreatic enzyme digest and a peptide derived from gliadin α increased HLA-E levels on murine RMA-S/HLA-E-transfected cells. Analysis of HLA-E expression in the small intestinal mucosa of gluten-containing diet celiac patients and organ culture experiments confirmed the in vitro data.

Inhibition of Human NK Cell-Mediated Killing by CD1 Molecules

It is now well established that NK cells recognize classical and nonclassical MHC class I molecules and that such recognition typically results in the inhibition of target cell lysis. Given the known structural similarities between MHC class I and non-MHC-encoded CD1 molecules, we investigated the possibility that human CD1a, -b, and -c proteins might also function as specific target structures for NK cell receptors. Here we report that expression of CD1a, -b, or -c can partially inhibits target cell lysis by freshly isolated human NK cells and cultured NK lines. The inhibitory effects of CD1 molecules on NK cell could be shown upon expression of individual CD1 proteins in transfected NK-sensitive target cells, and these effects could be reversed by incubation of the target cells with mAbs specific for the expressed form of CD1. Inhibitory effects of CD1 expression on NK-mediated lysis could also be shown for cultured human dendritic cells, which represent a cell type that prominently expresses the various CD1 proteins in vivo. In addition, the bacterial glycolipid Ags known to be bound and presented by CD1 proteins could significantly augment the observed inhibitory effects on target cell lysis by NK cells.

Immune dysregulation and dyserythropoiesis in the myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are clonal disorders characterised by ineffective haematopoiesis with high risk of leukaemia progression. The relevance of immune‐dysregulation for emergence, dominance and progression of dysplastic clones has been suggested, but valuable criteria to obtain insight into these connections are lacking. This study showed significant increase of CD8 lymphocytes and mature B cells in the bone marrow (BM) compared to peripheral blood (PB) of low risk MDS patients. Different BM levels of Regulatory T cells (Treg) identified two sub‐groups in these patients; only the sub‐group with lower Treg percentage showed BM recruitment of CD8 lymphocytes. Different levels of CD54 on BM CD8 cells revealed two sub‐groups of Intermediate‐1 (Int‐1) patients. The sub‐group with higher CD54 expression on BM CD8 showed high levels of this molecule also on CD4 cells. BM recruitment of CD8 lymphocytes in the low risk group and/or the presence of high CD54 expression on BM CD8 in Int‐1 patients were associated with more pronounced dyserythropoiesis and erythropoietin treatment. Our data shed light on the involvement of immune‐mediated mechanisms in Low and Int‐1 risk MDS patients and suggest that BM versus PB levels of immune effectors could represent useful criteria for a more homogeneous grouping of MDS patients.

Allelic distribution of human leucocyte antigen in historical and recently diagnosed tuberculosis patients in Southern Italy

This study addresses the analysis of the human leucocyte antigen (HLA) allele distribution in 54 historical and in 68 recently diagnosed tuberculosis (TB) patients. The historical cohort was characterized by the presence of large fibrocavernous lesions effectively treated with therapeutic pneumothorax during the period 1950–55. Patients and healthy controls enrolled in the study were from the Campania region of southern Italy. No significant association between HLA alleles and TB in the population of recently diagnosed TB patients was observed. On the contrary, among the historical TB patients there was a strong association with an increased frequency of the HLA‐DR4 allele alone and/or in the presence of the HLA‐B14 allele (P = 0·000004; Pc = 0·0008), as well as with a decreased frequency of the HLA‐A2+,‐B14−,DR4− allele association (P = 0·00005; Pc = 0·01). In order to exclude any interference from age‐related factors, these results were confirmed by comparing the historical cohort of TB patients with an age‐matched healthy control population of the same ethnic origin (P = 0·00004; Pc = 0·008; and P = 0·0001; and Pc = 0·02, respectively).

T cells from paroxysmal nocturnal haemoglobinuria (PNH) patients show an altered CD40-dependent pathway

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoiesis disorder characterized by the expansion of a stem cell bearing a somatic mutation in the phosphatidylinositol glycan‐A (PIG‐A) gene, which is involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor. A number of data suggest the inability of the PIG‐A mutation to account alone for the clonal dominance of the GPI‐defective clone and for the development of PNH. In this context, additional immune‐mediated mechanisms have been hypothesized. We focused on the analysis of T lymphocytes in three PNH patients bearing a mixed GPI+ and GPI– T cell population and showing a marked cytopenia. To analyze the biological mechanisms underlying the control of T cell homeostasis in PNH, we addressed the study of CD40‐dependent pathways, suggested to be of crucial relevance for the control of autoreactive T cell clones. Our data revealed significant, functional alterations in GPI+ and GPI– T cell compartments. In the GPI– T cells, severe defects in T cell receptor‐dependent proliferation, interferon‐γ production, CD25, CD54, and human leukocyte antigen‐DR surface expression were observed. By contrast, GPI+ T lymphocytes showed a significant increase of all these parameters, and the analysis of CD40‐dependent pathways revealed a functional persistence of CD154 expression on the CD48+CD4+ lymphocytes. The alterations of the GPI+ T cell subset could be involved in the biological mechanisms underlying PNH pathogenesis.

Effects of recombinant trout leptin in superoxide production and NF-κB/MAPK phosphorylation in blood leukocytes.

Studies in mammals indicate that leptin is a multifunctional cytokine involved in regulation of energy metabolism and the modulation of the immune function. However, evidence for an immunomodulatory effect of leptin in fish is still missing. At least in part, this lack of knowledge is due to the absence of materials and models. In this study, we produced trout recombinant leptin (rt-lep) and tested its capacity to trigger cellular pathways, usually active in mammal immune system cells. STAT3, NF-κB, and the three major MAPK cascades (JNK, p38 and ERK), were activated by rt-lep in in vitro incubations with blood leucocytes of the rainbow trout Oncorhynchus mykiss. We also showed that rt-lep causes a decrease in superoxide anion production in trout blood leucocytes. Thus our data indicate that as in mammals also in teleosts leptin plays pleiotropic activities. Importantly, its actions in fishes do not always conform to the picture emerging for mammals.

Eculizumab treatment modifies the immune profile of PNH patients

Paroxysmal Nocturnal Haemoglobinuria (PNH) is due to pathological expansion of a stem progenitor bearing a somatic mutation of PIG-A gene involved in the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor. Numerous data suggest a role for immune-mediated mechanisms in the selection/expansion of GPI-defective clone. Haemolytic anaemia in PNH is dependent on the effect of complement against GPI-defective red cells. Eculizumab, an anti-C5 monoclonal antibody, is dramatically effective in controlling haemolysis and thrombosis, in reducing fatigue and in improving quality of life of patients. However, this therapy presents new challenges that need to be properly faced. Here, we report the decrease in B, Natural Killer (NK) and regulatory T cells (Treg), an altered cytokine profile of invariant-NKT cells (NKTi) and the increasing of C-X-C chemokine receptor type 4 (CXCR4) receptor in PNH patients before the Eculizumab therapy. Treatment significantly affects some of these alterations: after Eculizumab, the number of B lymphocytes, the cytokine secretion of NKTi and CXCR4 expression on CD8 T cells became similar to healthy donors. No effects were observed on NK and Treg. The amplitude of the GPI-defective compartment remained unchanged.

Differential involvement of CD40, CD80, and major histocompatibility complex class I molecules in cytotoxicity induction and interferon-γ production by human natural killer effectors

Natural killer (NK) cells are physiologically involved in the immune response against viruses, intracellular bacteria, and parasites as well as against malignant diseases. In addition to the cytotoxic activity, NK lymphocytes mediate a variety of homeostatic effects by producing cytokines. This study focused on the differential role of CD40 and CD80 costimulatory molecules and major histocompatibility complex class I (MHC‐I) antigens in the regulation of cytotoxicity and of interferon (IFN)‐γ secretion of resting and interleukin (IL)‐2‐activated human NK cells. CD40 and CD80 molecules were observed to play a specific role in the induction of cytotoxic function but not in IFN‐γ production of IL‐2‐activated NK effectors. In addition, a critical role of CD94‐dependent MHC‐I recognition for the regulation of IFN‐γ production and target lysis was demonstrated. These data provide a possible mechanism underlying functional interactions between NK lymphocytes and CD40/CD80‐expressing cell targets, as represented by dendritic cells.