Eva Reali

Polymorphonuclear neutrophils pulsed with synthetic peptides efficiently activate memory cytotoxic T lymphocytes

Polymorphonuclear neutrophils (PMNs), traditionally considered effector cells in the inflammatory response, have recently been regarded as potential regulators of the immune response. In the present study we investigate whether PMNs are efficient antigen‐presenting cells for reactivation of memory cytotoxic T lymphocytes (CTLs). PMNs were pulsed with synthetic peptides derived from Epstein‐Barr virus (EBV) antigens. We have used the IVTDFSVIK (IVT) peptide derived from the Epstein‐Barr virus—encoded nuclear antigen 4 protein, corresponding to the immunodominant epitope of HLA‐A11–restricted CTL responses, and the CLGGLLTMV (CLG) peptide derived from the latent membrane protein 2 antigen, representing a subdominant epitope of HLA‐A2–restricted CTL responses. The data indicate that peptide‐pulsed PMNs selectively activate specific CTL responses to both immunodominant and subdominant epitopes. The efficiency of CTL induction by PMNs was comparable to that observed with the conventional method of EBV‐specific CTL reactivation with the autologous lymphoblastoid cell line, as well as with peptide‐pulsed monocyte‐enriched adherent cells. On the contrary, unactivated peptide‐pulsed lymphocytes failed to induce an epitope‐specific CTL response. These results demonstrate that PMNs efficiently present antigens to memory virus‐specific CTLs and suggest that they may have a role as antigen‐presenting cells. J. Leukoc. Biol. 60: 207–213; 1996.

Two new formylated peptides able to activate chemotaxis and respiratory burst selectively as tools for studying human neutrophil responses

Two new For-Met-Leu-Phe-OH (FMLP) methyl ester analogues, For-Thp-Leu-Ain-OMe [Thp1, Ain3] and For-Met-delta zLeu-Phe-OMe [delta zLeu2], able to activate selectively chemotaxis and superoxide anion (O2-) release, respectively modulate intracellular cyclic AMP (cAMP) levels in different ways. FMLP and [delta zLeu2] enhance human neutrophil cAMP levels per se, and this effect is potentiated by Ro 201724, a non-xanthinic phosphodiesterase (PDE) inhibitor, whereas it is counteracted by 3-isobutyl-1-methyl-xanthine (IBMX), a blocker of both phosphodiesterase and adenosine receptors. In contrast, [Thp1, Ain3] is ineffective. However, no formylated peptides influence cAMP phosphodiesterase activity. Neutrophil preincubation with Ro 201724 or IBMX drastically reduces chemotaxis and superoxide anion (O2-) production triggered by peptides. Our results suggest that: (1) peptide-induced cAMP increase is probably indirect, and due mainly to the action on adenosine-sensitive adenylate cyclase; (2) formylated peptide, endowed solely with chemotactic activity is unable to increase neutrophil cAMP concentration; (3) cAMP elevation may represent a feed-back mechanism to inhibit the physiological responses induced by formylated peptides.

HIV-1 Tat affects the programming and functionality of human CD8⁺ T cells by modulating the expression of T-box transcription factors

Objective:HIV infection is characterized by several immune dysfunctions of both CD8+ and CD4+ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8+ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4+ T cells; however, its effects on CD8+ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8+ T-cell functionality and programming. Methods:CD8+ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Results:Tat favors the secretion of interleukin-2, interferon-&ggr; and granzyme B in CD8+ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8+ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Conclusion:Tat deeply alters the programming and functionality of CD8+ T lymphocytes.

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein–Barr virus (EBV)‐specific memory CTL responses, reactivated from blood lymphocytes of HLA‐A11‐positive individuals by stimulation with the autologous EBV‐transformed lymphoblastoid cell line (LCL), are often dominated by reactivities directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416–424 of EBV nuclear antigen‐4 (EBNA4). We now report the selective activation of IVT‐specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10‐fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA‐A11‐transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC : peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT‐specific CTL precursors in a donor that failed to mount an IVT‐specific response upon stimulation with the autologous B95.8 virus‐transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.

IL-8 enhances antibody-dependent cellular cytotoxicity in human neutrophils.

Human neutrophils are able to kill in vitro colorectal carcinoma cell line SW11‐16 coated with mAb 17‐1A, but they are not cytotoxic towards a non‐immunized tumour target. Neutrophil exposure to the inflammatory cytokine, IL‐8, produces a significant increase in antibody‐dependent cellular cytotoxicity, which is related to IL‐8 concentration. Oxyradical production is one of the lytic mechanisms used by phagocytes, and IL‐8 is shown to activate this function, which does not occur if neutrophils are pretreated with the protein kinase C inhibitor, staurosporine, but is increased by R59022, a dyacylglycerol kinase inhibitor. The IL‐8 effect is mediated by protein kinase C, which is potentiated by the calcium flux induced by the interaction between antibody coating tumour target and FcγRIII on effector cells, as previously demonstrated. Data suggest a possible new role for IL‐8 in tumour surveillance.

Priming with a very low dose of DNA complexed with cationic block copolymers followed by protein boost elicits broad and long-lasting antigen-specific humoral and cellular responses in mice.

Cationic block copolymers spontaneously assemble via electrostatic interactions with DNA molecules in aqueous solution giving rise to micellar structures that protect the DNA from enzymatic degradation both in vitro and in vivo. In addition, we have previously shown that they are safe, not immunogenic and greatly increased antigen-specific CTL responses following six intramuscular inoculations of a very low dose (1microg) of the vaccine DNA as compared to naked DNA. Nevertheless, they failed to elicit detectable humoral responses against the antigen. To gain further insight in the potential application of this technology, here we show that a shorter immunization protocol based on two DNA intramuscular inoculations of 1microg of DNA delivered by these copolymers and a protein boost elicits in mice broad (both humoral and cellular) and long-lasting responses and increases the antigen-specific Th1-type T cell responses and CTLs as compared to priming with naked DNA. These results indicate that cationic block copolymers represent a promising adjuvant and delivery technology for DNA vaccination strategies aimed at combating intracellular pathogens.

A cyclic peptide T analogue with high chemotactic activity

Abstract Peptide T is an octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH) based on a consensus sequence that has been identified in HIV proteins. Two conformationally-restricted analogues of peptide T fragment (H-Thr-Thr-Asn-Tyr-Thr-OH) were synthesized by cyclisation of the N-terminal amino group of Thr to the α- or β-carboxyl moiety of Asp introduced at the C-terminus of the peptide. The cyclo Thr-Thr-Asn-Tyr-Thr-Asp(OH) (Cα) showed high in vitro chemotactic activity, whereas the conformational restriction introduced into Thr-Thr-Asn-Tyr-Thr-A sp-OH was incompatible with the CD 4 receptor. The cyclic analogue Cα was more active than its ester and the open-chain reference compound H-Thr-Thr-Asn-Tyr-Thr-Asp-OH. It also proved to be highly resistant to degradation by plasma or brain enzymes.

T Cell Hierarchy in the Pathogenesis of Psoriasis and Associated Cardiovascular Comorbidities

The key role of T cells in the pathogenesis of cutaneous psoriasis has been well described in the last decade and the knowledge of the relative role of the different subsets of T cells in psoriasis pathogenesis has considerably evolved. Now, it is clear that IL-17A-producing T cells, including Th17/Tc17, have a central role in the pathogenesis of cutaneous psoriasis and therapies blocking the IL-17A pathway show high clinical efficacy. By contrast, the contribution of IFNγ-producing T cells has progressively become less clear because of the lack of efficacy of anti-IFNγ antibodies in clinical studies. In parallel, the role of CD8+ T cells specific for self-antigens has been revived and increasing evidence now indicates that in psoriatic skin the majority CD8+ T cells are present in the form of epidermal tissue-resident memory T cells. In the last years it also emerged the possibility of a contribution of T cell recirculation in the pathogenesis of psoriasis and its systemic manifestations. The aim of this review is to define a hierarchy for the different subsets of T cells in the T cell-mediated inflammatory cascade in psoriatic skin. This analysis will possibly help to distinguish the subsets that initiate the disease, those involved in the establishment of the self-sustaining amplification loop that leads to the cutaneous clinical manifestations and finally the subsets that act as downstream players in established lesions. Specific T cell subpopulations finally will be considered for their possible role in propagating inflammation at distant sites and for representing a link with systemic inflammation and cardiovascular comorbidities.

GM-CSF Augments the IL-4 Induced Cytotoxic Activity of Human Peripheral Blood Mononuclear Cells in the Presence of the Mouse Monoclonal Antibody 17–1A

The therapeutic effect of modulators of the immune system might be increased if administered in combination. Informations on their activity performed in different schedules may be obtained from in vitro analyses. In this study, human peripheral blood mononuclear cells (lymphocytes and monocytes) (PBMC) were treated simultaneously or sequentially with GM-CSF and IL-4, or vice versa, for different time periods. Cytokine activated PBMC were tested for cytolytic activity towards the human colorectal cell line SW11–16 in an 18 h antibody dependent cellular cytotoxicity (ADCC) assay using the specific MAM7–1A (mouse IgG2a). The simultaneous incubation of effector cells with both cytokines slightly increased the cytotoxicity induced by IL-4 or GM-CSF separately. Priming of PBMC with GM-CSF at the optimal concentration (10-3 µg/ml) for 1,2 and 4 h significantly enhanced the subsequent IL-4 induced activation of cytotoxicity. GM-CSF increased IL-4 induced cytotoxicity also at the suboptimal concentration (10-4 µg/ml), but to a lower extent. Priming of PBMC with IL-4 at optimal or suboptimal concentrations followed by stimulation with GM-CSF had no synergistic effect in ADCC. These results might have relevance as preliminary informations to exploit the concept of combining different biologic therapeutics for useful clinical protocols in cancer patients.