A. D'Agostino

The interaction between NK cells and dendritic cells in bacterial infections results in rapid induction of NK cell activation and in the lysis of uninfected dendritic cells.

NK and DC reciprocal interactions have only recently been investigated. In this study, we focused on the interplay between NK cells and DC in two models of bacterial infection. Immature monocyte‐derived DC were cultured in the presence of live Escherichia coli or bacillus Calmette–Guérin. Upon exposure to either extracellular or intracellular bacteria, DC underwent maturation as assessed by the increased levels of expression of CD80, CD86, and HLA molecules and the de novo expression of CD83 and CCR7. Significant amounts of TNF‐α and IL‐12 were released by DCupon infection, whereas IL‐2 and IL‐15 were barely detectable in culture supernatants. Both infected and uninfected DC were capable of inducing in fresh autologous NK cells the expression of CD69 and HLA‐DR and of inducing cell proliferation. Remarkably, however, infected DC were much stronger inducers of NK cell activation and proliferation than uninfected DC. Thus, after just 24 h of NK/DC coculture, only those NK cells that had been exposed to bacteria‐infected DC had acquired the ability to lyse autologous immature DC. In addition, infected DC were more resistant to NK‐mediated lysis as a consequence of the up‐regulation of HLA class I molecule expression on their surface. This study suggests a regulatory circuit involving NK cells and DC in which DC‐induced NK cell activationis effectively enhanced by the presence of pathogens. Activated NK cells, by limiting the supply of immature DC, may then exert a control on subsequent innate and adaptive immune responses.

Highly stable oligomerization forms of HIV‐1 Tat detected by monoclonal antibodies and requirement of monomeric forms for the transactivating function on the HIV‐1 LTR

The use of newly generated murine monoclonal antibodies directed against distinct epitopes of a functionally active, chemically synthesized HIV‐1 Tat protein has permitted the identification of several molecular forms including monomers, dimers and trimers. Dimers and trimers are particularly stable and resistant to strong reducing conditions. Through epitope mapping it has been possible to demonstrate that the major immunodominant epitope is contained within the basic region of the Tat protein and is lost after oligomerization of the molecule. In contrast, N‐terminal, C‐terminal and conformation‐dependent epitopes are still accessible to mAb specific recognition after Tat oligomerization. Moreover, by using a quantitative HIV‐LTR transactivation assay depending upon exogenous Tat, we could extrapolate the amount of functional Tat produced by cell lines stably transfected with the viral transactivator. More importantly, we could show that only the monomeric form of exogenous Tat is the relevant functional form acting in cells harbouring the HIV‐1 LTR promoter.

Distinct regulation of HLA class II and class I cell surface expression in the THP-1 macrophage cell line after bacterial phagocytosis

Expression of HLA and CD1b molecules was investigated in the THP‐1 macrophage cell line within 2 weeks following phagocytosis of mycobacteria or Escherichia coli. During the first 2 – 3 days, cell surface expression of HLA class II and CD1b was drastically down‐modulated, whereas HLA class I expression was up‐modulated. In the following days both HLA class II and CD1b expression first returned to normal, then increased and finally returned to normal with kinetics similar to that observed for the steadily increased HLA class I. The initial down‐modulation of HLA class II and CD1b cell surface antigens was absolutely dependent on phagocytosis of bacteria. Further studies indicated that initial HLA class II cell surface down‐modulation (1) was not due to reduced transcription or biosynthesis of mature HLA class II heterodimers, (2) was only partially, if at all, rescued by treatment with IFN‐γ, although both mRNA and corresponding intracellular proteins increased up to sixfold with respect to untreated cells, and (3) resulted in failure of THP‐1 cells to process and present mycobacterial antigens to HLA‐DR‐restricted antigen‐specific T cell lines. The existence of a transient block of transport of mature HLA class II heterodimers to the cell surface in the first days after phagocytosis of bacteria may have negative and positive consequences: it decreases APC function early but it may increase it later by favoring optimal loading of bacterial antigens in cellular compartments at high concentration of antigen‐presenting molecules.

HIV-1 Tat mutants in the cysteine-rich region downregulate HLA class II expression in T lymphocytic and macrophage cell lines.

Human macrophage and T cell lines were stably transfected with HIV‐1 wild‐type Tat or Tat mutants in the cysteine‐rich region displaying trans‐dominant negative effects on HIV‐1 life cycle. The expression of HLA class I and class II molecules was not affected by wild‐type Tat. Tat mutants, instead, profoundly down‐regulated in a dose‐dependent fashion the expression of class II, but not of class I, in both cell types by acting at the transcriptional level. Down‐regulation was manifested on constitutive and IFN‐γ‐induced class II gene expression and did not correlate with reduced transcription of the AIR‐1 gene product CIITA, the major transcriptional activator of class II genes, indicating that Tat mutants did not act by inhibiting AIR‐1 gene expression. Class II down‐modulation had important functional implications in macrophages, as both antigen processing and presenting capacity were inhibited. These results represent the first evidence that a modified HIV‐1 Tat product can act as a potent immunosuppressor by inhibiting the HLA class II expression necessary for triggering both cellular and humoral responses against pathogens. The use of these HIV‐1 Tat mutants also discloses new opportunities to investigate the molecular mechanisms underlying the coordinate HLA class II gene transcription.

Block of Stat-1 activation in macrophages phagocytosing bacteria causes reduced transcription of CIITA and consequent impaired antigen presentation

The IFN‐γ‐induced HLA class II expression in human macrophages was drastically reduced after phagocytosis of Escherichia coli. HLA class II down‐modulation depended on phagocytosis of bacteria and could not be reproduced by phagocytosis of inert particles or by treatment with lipopolysaccharide. Study of the kinetics and molecular analysis showed that class II molecules and corresponding mRNA were up‐regulated at 6 h after phagocytosis of bacteria. Subsequently, a progressive reduction of mRNA occurred, and, at 72 h, as little as 25% of the class II mRNA level of IFN‐γ‐treated control cells was found. This was due to reduced transcription of the class II transcriptional activator CIITA, as a consequence of reduced immediate‐early inducible factor (IRF‐1) and particularly of reduced phosphorylated Stat‐1 homodimers, nuclear factors both necessary for optimal triggering of the CIITA promoter. Failure to sustain IFN‐γ‐induced CIITA up‐modulation during phagocytosis of bacteria had functional implications, as human macrophages could not adequately process and present antigenic peptides to HLA‐DR‐restricted antigen‐specific T cells. This is the first evidence that phagocytosis of bacteria can down‐modulate HLA class II expression in normal human macrophages by acting at the level of expression of CIITA.

Neo-adjuvant chemo/immunotherapy in the treatment of stage III (N2) non-small cell lung cancer: a phase I/II pilot study.

In a previous randomized study, we showed that adjuvant immunotherapy with tumor-infiltrating lymphocytes and recombinant interleukin-2 (rIL-2) significantly improved survival in resected N2-Non Small Cell Lung Cancer (NSCLC) patients. The present study assesses feasibility, safety and potential efficacy of combined neo-adjuvant chemotherapy and immunotherapy with peripheral blood mononuclear cells (PBMC) and rIL-2 in resectable N2-NSCLC patients. Eighty-two consecutive N2-NSCLC patients underwent neo-adjuvant chemotherapy with cisplatin and gemcitabine. Out of the 82 patients, 23 were also subjected to leukapheresis prior to neo-adjuvant chemotherapy while the remaining 59 did not. Collected PBMC were analyzed for viability and phenotype and then stored frozen in liquid nitrogen. Thawed PBMC were infused intravenously, 5 days before surgery. After the infusion, rIL-2 was administered subcutaneously until surgery. Only patients with a partial or complete response to neoadjuvant chemotherapy underwent surgery: 13 patients in the experimental immunotherapy group (A) and 32 in the reference group (B). The two groups were homogeneous for all major prognostic factors. Median leukapheresis yield was 10 billion PBMC, (range 3–24 billions). Two to six billion PBMC were infused. The phenotypic analysis showed that similar proportions of CD4 and CD8 cells were present in leukapheresis products, and thawed PBMC, as well as in T lymphocytes isolated from the removed tumours. No severe adverse effects were observed following immunotherapy. No significant differences in overall survival (OS) and event-free survival (EFS) were seen between the two groups. However, the 5-year OS in group A was almost twice as much compared to group B (59% vs 32%). After adjustment for major prognostic factors, a statistically significant 66% reduction in the hazard of death was seen in patients receiving immunotherapy. The OS benefit was more evident in patients with adenocarcinoma than in those with squamous cell carcinoma. This study supports the favorable toxicity profile and potential efficacy of combining neo-adjuvant chemotherapy and immunotherapy with PBMC and rIL-2 in the treatment of N2-NSCLC patients.