c-myc overexpression activates alternative pathways for intracellular proteolysis in lymphoma cells.

Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein-Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

Epitope-dependent selection of highly restricted or diverse T cell receptor repertoires in response to persistent infection by Epstein-Barr virus.

The T cell receptor (TCR) repertoires of cytotoxic responses to the immunodominant and subdominant HLA A11-restricted epitopes in the Epstein-Barr virus (EBV) nuclear antigen-4 were investigated in four healthy virus carriers. The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT). Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis. Blocking of cytotoxic function by antibodies specific for the CD8 coreceptor indicated that, while AVF-specific TCRs are of high affinity, the oligoclonal response to the IVT epitope includes both low- and high-affinity TCRs. Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.

HLA-A11 epitope loss isolates of Epstein-Barr virus from a highly A11+ population.

Cytotoxic T lymphocytes (CTLs) control viral infections by recognizing viral peptides presented by major histocompatibility complex (MHC) class I molecules. Human leukocyte antigen (HLA)-A11-restricted CTLs that recognize peptide residues 416 to 424 of the Epstein-Barr virus (EBV) nuclear antigen-4 frequently dominate EBV-induced responses in A11+ Caucasian donors. This epitope is conserved in type A EBV strains from Caucasians and central African populations, where A11 is relatively infrequent. However, strains from highly A11+ populations in New Guinea carry a lysine-to-threonine mutation at residue 424 that abrogates CTL recognition and binding of the peptide to nascent A11 molecules. The results suggest that evolution of a widespread and genetically stable virus such as EBV is influenced by pressure from MHC-restricted CTL responses.

Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

Synthesis and biological evaluation of new vinyl ester pseudotripeptide proteasome inhibitors.

Here we report the synthesis and biological activities of new tripeptidic-based vinyl ester derivative proteasome inhibitors. Starting from Hmb-Val-Ser-Leu-VE prototype, we investigated P2 position and N-terminal substitution. The more effective inhibitors of the series showed remarkable inhibition and selectivity for the trypsin-like (beta2) subunit and were revealed to be specific for the proteasome. In vitro metabolic stability studies of the new vinyl ester analogues are also reported here.

Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: chemical behaviour and biological activity.

Copper complex formation equilibria of glycyl-L-histidyl-L-lysine (Gly-His-Lys, GHK) and of two synthetic analogues, where the histidine residue was replaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid), have been carefully investigated using different experimental techniques: potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies. All the ligands formed complexes having different stoichiometries and stabilities; evidence for the formation of binuclear species is also shown. The structures of the main complexes are discussed. It is suggested that the lateral lysine amino group participates in complex formation, but only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The two synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell culture media, even in the presence of serum. Relevant information on the GHK action mechanism as cell growth factor has been obtained: the formation of copper complexes, driven by the first (Gly) residue, appears necessary while the second residue (His) does not appear to play a specific role; the presence of the free side chain of the third residue (Lys) appears to be of fundamental importance.

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-AII-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.

Deficiency of neutrophil membrane antigen detected by monoclonal antibody in rheumatoid arthritis.

Monoclonal antibodies (mAbs) against cell surface antigens and receptors are instrumental in defining specific membrane markers. mAbs GF 26.7.3 and MF 25.1 against human neutrophils modulated the activation mechanism of superoxide anion production induced by formyl-peptide and PMA in all subject. However, treatment with mAb MF 25.1 of neutrophils from patients with rheumatoid arthritis did not have any effect. This may suggest that the antigen which MF 25.1 binds is absent in rheumatoid conditions. This confirms our previous data showing that defective expression of membrane components is associated with neutrophil dysfunction.

High structural side chain specificity required at the second position of immunogenic peptides to obtain stable MHC/peptide complexes.

Peptides binding to HLA-A11 contain a hydrophobic or a small polar amino acid at position 2 and a lysine at the carboxy terminus. Synthetic peptides carrying natural and unnatural amino acids in position 2 were used to determine the requirements for formation of stable HLA-A11/peptide complexes. By kinetic analysis we demonstrate that a stereospecific interaction between the side chain residue in position 2 and a subsite of pocket B is required to obtain stable HLA/peptide complexes. This specific interaction is mediated by a methyl group or by an ethyl group bound to the asymmetric Cbeta atom with the correct configuration. Experiments performed with different peptide sequences suggest that the presence of adequate anchor residues may be sufficient to produce stable HLA/peptide complexes.

Peptide analogues of a subdominant epitope expressed in ebv-associated tumors: synthesis and immunological activity.

H-Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val-OH (CLG) peptide is an EBV subdominant epitope that represents the target of HLA-A2 restricted CTL responses. The CLG peptide has low affinity for HLA-A2 and does not produce stable complexes, both factors that determine weak CTL responses. In contrast, the [Tyr(1), Ala(3)]CLG (YLA) analogue showed high affinity for HLA-A2 molecules and efficiently stimulated CLG-specific CTL precursors. Nevertheless, this modified epitope showed low enzymatic stability. To further improve the immunotherapeutical potential of this "improved epitope", we have synthesized and tested YLA analogues containing different modifications next to the scissile peptide bond. Among the analogues we found three peptides, with higher enzymatic resistance, that efficiently stimulate CTL responses. These peptides may be used for EBV-specific immunotherapies.

HLA-A11-mediated protection from NK cell-mediated lysis: role of HLA-A11-presented peptides.

The capacity of MHC class I to protect target cells from NK is well established, but the mechanism by which these molecules influence NK recognition and the physical properties associated with this function remain poorly defined. We have examined this issue using as a model the HLA-A11 allele. HLA-A11 expression correlated with reduced susceptibility to NK and interferon-activated cytotoxicity in transfected sublines of the A11-defective Burkitt's lymphoma WW2-BL and the HLA class I A,B-null C1R cell line. Protection was also achieved by transfection of HLA-A11 in the peptide processing mutant T2 cells line (T2/A11), despite a very low expression of the transfected product at the cell surface. Induction of surface HLA-A11 by culture of T2/A11 cells at 26 degrees C or in the presence of beta 2m did not affect lysis, whereas NK sensitivity was restored by culture in the presence of HLA-All-binding synthetic peptides derived from viral or cellular proteins. Acid treatment rendered T2/A11 and C1R/A11 cells sensitive to lysis, but protection was restored after preincubation with peptide preparations derived from surface stripping of T2/A11 cells. Similar peptide preparations from T2 cells had no effect. The results suggest that NK protection is mediated by HLA-A11 molecules carrying a particular set of peptides that are translocated to the site of MHC class I assembly in the ER in a TAP-independent fashion.

HLA B51 antigen associated with neutrophil hyper-reactivity.

HLA B51 specificity is strongly associated with Behcet's disease (BD) (for references see Baricordi et al., 1986), a multisystem vasculitis of unknown aetiology, for which an immunological pathogenesis has been proposed (O'Duffy et al., 1983; O'Duffy et al., 1990). Neutrophil abnormalities observed in BD patients even during clinical remission suggest prominent involvement of these phagocytic cells in the pathogenesis of the disease (Niwa et al., 1982). In order to clarify how HLA B51 antigen might confer susceptibility to BD, we have investigated neutrophil function in 13 B51-positive and 13 B51-negative healthy subjects. Lymphocyte spontaneous proliferation and circulating immune complexes were also evaluated. Whereas neutrophils from B51-positive subjects showed an increase in the chemotactic response toward casein (P = 0.003) and LPS (P = 0.033) and also in the PMA-induced superoxide production (P = 0.008) no evidence of enhanced lymphocyte activation emerged. These results suggest that the HLA region can exert a regulatory control on PMN functions.

Design of dimeric peptides obtained from a subdominant Epstein-Barr virus LMP2-derived epitope.

The latent membrane protein 2 (LMP2) is expressed in EBV-associated tumours. LMP2 is a target of HLA-A2 restricted EBV-specific CTL responses and consequently it may represent a good target for specific CTL-based immunotherapies. However, the efficacy of such therapy is limited by the poor immunogenicity of the protein that induces weak cytotoxic T lymphocyte (CTL) responses directed against the CLGGLLTMV (CLG) epitope. Indeed, the CLG peptide presents low affinity for HLA-A2 and does not produce stable complexes. Therefore we synthesized and tested CLG-dimeric analogues with the purpose of characterizing new compounds with the capacity to bind HLA-A2 molecules. By these studies we have identified a few peptides which, compared to the natural epitope, showed higher affinity for HLA-A2 molecules and superior capacity to form a complex. These dimeric peptides may have the potential to induce efficient CTL responses directed to the natural epitope.

Response of human neutrophils to formyl-peptide modified at the terminal amino and carboxyl groups.

Two analogs of chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine were examined for their capacity to activate several functions of human neutrophils. The C-terminus methyl ester derivative of the chemotactic peptide was found to possess strong biological activity and was able to induce levels of chemotaxis, enzyme secretion, and superoxide generation comparable to those observed with the same concentrations of N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The analog containing a tert-butyloxycarbonyl group at the N-terminus, as well as the C-terminal methyl ester, was completely devoid of activity towards neutrophils. From these results, it appears that the free carboxyl group is not necessary for biological function. In contrast, the substituent at the N-terminus may play a critical role in the induction of the cellular response.

Modulation of neutrophil functions by activated platelet release factors.

Platelets activated with physiological agonists, such as thrombin, ADP, or collagen, released products able to modulate neutrophil functions. In particular, platelet supernatant contained an inhibitor of superoxide anion generation induced by phorbol ester and a chemotactic factor for human neutrophils. The proteolytic digestion of platelet supernatant completely abrogated chemotactic activity without interfering with the inhibitory effect, indicating the presence of different molecules involved in the modulation of different neutrophil functions. This was further confirmed by the pretreatment of platelets with aromatic benzamidine which abolished chemotactic activity, but did not affect superoxide inhibition of neutrophils. This report provides evidence for interaction of platelets and inflammatory cells, suggesting that platelets are able to induce accumulation of neutrophils and control their respiratory burst, which also has a critical role in tissue damaging in inflammation.

Chemotactic response of human monocytes to pentapeptide analog derived from immunodeficiency virus protein gp 120.

The octapeptide sequence of peptide T is contained within the envelope of HIV and seems to mediate the viral binding to CD4 expressing cells, including monocytes. The biological activity of the alpha-aminobutyric acid pentapeptide derived from the C-terminal sequence of peptide T, in which the polar side chain of threonine in position 4 is substituted by a hydrophilic group, is measured by the monocyte chemotaxis assay. The chemotactic activity of human monocytes is assessed by determining the concentration at which the pentapeptide analog is maximally active and the effectiveness at that concentration, in comparison with peptide T and two shorter homologs, the pentapeptide and tetrapeptide. These experiments suggest that the synthetic analog is a potent chemotactic factor active at picomolar concentrations and that it competes with peptide T for the monocyte binding site.

Phosphinic platinum complexes with 8-thiotheophylline derivatives: synthesis, characterization, and antiproliferative activity.

The platinum mixed-phosphine complexes (SP-4,2)-[PtCl(8-MTT)(PPh3)(PTA)] (2) and cis-[Pt(8-MTT)2(PPh3)(PTA)] (3) (MTTH2 = 8-(methylthio)theophylline, PTA = 1,3,5-triaza-7-phosphaadamantane) have been prepared from the precursor cis-[PtCl2(PPh3)(PTA)] (1), which has been fully characterized by X-ray diffraction determination. Antiproliferative activity tests indicated that the presence of one lipophilic PPh3 and one hydrophilic PTA makes 1-3 more active than the analogues bearing two PPh3 or two PTA. The reactivity of cis-[PtCl2(PPh3)2], cis-[PtCl2(PTA)2], and cis-[PtCl2(PPh3)(PTA)] with the bis(thiopurines) bis(S-8-thiotheophylline)methane (MBTTH2), 1,2-bis(S-8-thiotheophylline)ethane (EBTTH2), and 1,3-bis(S-8-thiotheophylline)propane (PBTTH2) has also been investigated. New binuclear complexes have been prepared and identified by spectroscopic techniques and their antiproliferative activities on T2 and SKOV3 cell lines evaluated.

T-cell engineering by a chimeric T-cell receptor with antibody-type specificity for the HIV-1 gp120.

Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficiency virus type 1 (HIV-1) could be of primary importance for the control of this viral infection. Here, we designed a chimeric cell surface receptor (105TCR) to provide primary human T-lymphocytes with antibody-type specificity for the HIV-1 envelope glycoprotein. This receptor includes the single chain Fv domain of the neutralizing anti-gp120 human monoclonal antibody F105, CD8alpha hinge and the transmembrane and the cytoplasmic domains of TCRzeta. Our results show that 105TCR is expressed at the cellular surface and is capable of recognizing the HIV-1 envelope glycoprotein inducing highly efficient effector T-cell responses, including extracellular signal-regulated kinase phosphorylation and cytokine secretion. Moreover, human primary CD8+ T-lymphocytes transduced by oncoretroviral and lentiviral vectors containing the 105TCR gene are able to mediate in vitro-specific cytolysis of envelope-expressing cells and HIV-1-infected CD4+ T-lymphocytes. These findings suggest that 105TCR is particularly suited for in vivo efficacy studies.