Silvia Bruno

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.

Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m‐AMSA) trigger endonucleolytic activity in myelogenous (HL‐60 or KGl), but not lymphocytic (MOLT‐4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G1 were resistant; the sensitivity of G2 or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G2 or M, to these inhibitors. The data show that γ‐irradiation (0.5–15 Gy) of HL‐60 cells does not alter their pattern of sensitivity, i.e. G1 cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT‐4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL‐60 cells arrested in G2 either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL‐60 cells in G1, G2 or M, or MOLT‐4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.

Dual Effect of CD85/Leukocyte Ig-Like Receptor-1/Ig-Like Transcript 2 and CD152 (CTLA-4) on Cytokine Production by Antigen-Stimulated Human T Cells

The functional outcome of a T cell response to Ag is the result of a balance between coactivation and inhibitory signals. In this study we have investigated the effects of the CD85/leukocyte Ig-like receptor (LIR)-1/Ig-like transcript (ILT) 2 and of CD152 (CTLA-4) inhibitory receptors on the modulation of cell-mediated immune responses to specific Ags, both at the effector and at the resting/memory cell level. Proliferation and cytokine production of CD4+ T lymphocytes stimulated by recall Ags have been evaluated. Cross-linking of CD85/LIR-1/ILT2 or CD152 molecules on cultured T cells using specific mAb and goat anti-mouse antiserum inhibits Ag-specific T cell proliferation. This inhibition is always paralleled by increased production of cytokines that down-regulate immune responses, e.g., IL-10 and TGF-β. In contrast, the production of cytokines that support T cell expansion and function (e.g., IL-2, IFN-γ, and IL-13) is significantly decreased. A long-term effect of CD85/LIR-1/ILT2 and of CD152 occurs during Ag-specific T cell activation and expansion. T cells, primed in the presence of anti-CD85/LIR-1/ILT2 and anti-CD152 blocking mAb (but in the absence of cross-linking), proliferate at higher rates and produce higher amounts of IL-2, IFN-γ, and IL-13, in comparison with T cells stimulated with the Ag alone. We also show that the inhibitory receptors exert a similar effect during Ag activation of specific CD4+ effector T cells. Ag-specific polyclonal CD4+ T cell lines exhibit increased proliferation and IL-2, IFN-γ, and IL-13 production when the CD85/LIR-1/ILT2 receptor is blocked by specific mAb. In contrast, cross-linking of this receptor down-regulates Ag-specific CD4+ T cell proliferation and increases IL-10 and TGF-β production.

Effect of tamoxifen on endometrial proliferation

PURPOSE An increase in the incidence of endometrial cancer and a potential increase in related mortality has been associated with the administration of 20 mg tamoxifen, the dose adopted in breast cancer chemoprevention trials, thus urging studies on intermediate markers of risk. PATIENTS AND METHODS Thirty-three women who received 20 mg tamoxifen as adjuvant breast cancer treatment underwent endometrial biopsy. Samples were divided for histologic examination, including a quantitative analysis of stromal:epithelial ratio, and an assessment of DNA ploidy and proliferation by flow cytometry. Results were compared with 37 symptomatic subjects. RESULTS All histograms were DNA diploid. Compared with controls, a significant increase in the risk of proliferation as measured by the hyperdiploid fraction was associated with tamoxifen duration (< or = 36 months: cumulative odds ratio = 16.5, 95% confidence interval, 1.85 to 146.5; > 36 months: cumulative odds ratio = 28.2, 95% confidence interval, 2.56 to 310.6, P for trend < .05). Tamoxifen-induced risk was significantly reduced by the extent of menopausal status. No cases of cancer or epithelial hyperplasia were observed in the tamoxifen group, whereas seven cases of epithelial hyperplasia without atypia were observed in the control group. The effect of tamoxifen on proliferation was associated with an increase in the stromal component. CONCLUSION Tamoxifen at 20 mg/d exerts a time-dependent proliferative effect on the endometrium, particularly in premenopausal and early postmenopausal women. This effect appears to be mediated by the stromal component, which accounts for the discrepancy between flow cytometry and histology. Our study provides preliminary evidence that the DNA flow cytometric hyperdiploid fraction may be a useful tool for monitoring endometrial cell proliferation in women exposed to tamoxifen.

Changes in cell nuclei during S phase : progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34

Using multiparameter flow cytometry we have measured the nuclear DNA content of exponentially growing HL-60 cells in conjunction with protein content, nuclear forward light scatter, DNA in situ sensitivity to denaturation, DNA accessibility to 7-aminoactinomycin D (7-AMD), and content of the proliferation-associated proteins: cyclin (PCNA), p105, p34, and Ki-67. Multivariate analysis of the data made it possible to correlate changes in each parameter with the degree of cell advancement through S phase (amount of replicated DNA). A decrease of the protein/DNA ratio, lowered DNA accessibility to 7-AMD, increased sensitivity of DNA to denaturation, and increased ability of isolated nuclei to scatter light all paralleled cell progression through S phase. These changes indicate that during S phase chromatin progressively condenses and suggest that the condensation is associated with the efflux of nonhistone proteins from the nucleus. The increase in the content of the antigen detected by the Ki-67 antibody was observed to exceed the increase in DNA content during S phase and the rate of the Ki-67/DNA increase was higher during the second half of S phase. Thus, this protein appears to be primarily synthesized during S, especially late in S phase, and is degraded in G1. The ratio of cyclin (PCNA)/DNA remained rather constant whereas the contents of p105 and p34 proteins, when expressed per unit of DNA, both decreased during S phase. The data indicate that significant changes in structure and composition of chromatin take place during S phase and suggest that the composition of chromatin associated with the nonreplicated DNA is different compared to chromatin associated with the newly replicated DNA.

Ventricular loading is coupled with DNA synthesis in adult cardiac myocytes after acute and chronic myocardial infarction in rats

To determine whether the overload associated with myocardial infarction and ventricular failure in rats is coupled with activation of DNA synthesis in the remaining left and right ventricular myocytes, flow cytometric analysis was performed on myocyte nuclei prepared from both ventricles 7 and 30 days after coronary occlusion. In addition, oral captopril was administered in separate groups of control and experimental rats to establish whether a relation existed between attenuation of ventricular loading and magnitude of DNA synthesis in myocytes. Results demonstrated that left ventricular failure and right ventricular dysfunction at 7 days after infarction were biventricularly associated with marked increases in the number of myocyte nuclei in the G2M phase of the cell cycle. In contrast, the fraction of nuclei in the G0+G1 phase decreased. In comparison with the earlier time point, the 30-day interval was characterized by a significant magnitude of cardiac hypertrophy, a moderate amelioration of ventricular pump function, and a decrease in the percentage of myocyte nuclei in the G2M phase in both ventricles. However, 30 days after infarction, the number of right ventricular myocyte nuclei in the S and G2M phases remained elevated with respect to control animals. Captopril therapy reduced the extent of ventricular loading and the population of myocyte nuclei in the cell cycle at 7 days. In conclusion, DNA synthesis in myocyte nuclei may represent an important adaptive component of the myocardial response to infarction.

Specific recognition of the viral protein UL18 by CD85j/LIR-1/ILT2 on CD8+ T cells mediates the non-MHC-restricted lysis of human cytomegalovirus-infected cells

Immune evasion mechanisms of human CMV are known; however, the immune control of infection remains poorly elucidated. We show that interaction between the viral protein UL18 on infected cells and the invariant receptor CD85j/LIR-1/ILT2 expressed on CTL is relevant for the control of infection. Resting and activated CD8+ T cells lysed UL18 expressing cells, whereas cells infected with CMV defective for UL18 were not killed. Lysis was not dependent on CD8+ T cell Ag specificity, MHC-unrestricted and specifically blocked by anti-CD85j and anti-UL18 mAb. Moreover, soluble recombinant UL18Fc immunoprecipitated CD85j from T cells. Activation is mediated by CD85j and its pathway is unrelated to CD3/TCR engagement. UL18 is detected in immunocompromised patients with productive infection and the mechanism used in vivo by human CMV to ensure survival of the immunocompetent host may be mediated by activation signals delivered by infected cells to T lymphocytes via UL18/CD85j interactions.

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems

In 1998 we reported that an L‐peptide derived from H1 of c‐Myc (Int‐H1‐S6A,F8A), linked to an internalization sequence from the third α‐helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro‐inverso (RI‐) peptides can assume a 3D conformation very close to their corresponding L‐forms and can be recognized by the same monoclonal antibody. We synthesized a RI‐peptide form of our original L‐peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI‐form of the original Antennapedia internalization sequence was perfectly capable of carrying a D‐peptide into human cells. We have studied three different potentially active peptides. L‐peptides: Int‐H1wt, Int‐H1‐S6A,F8A. D‐peptides: RI‐Int‐H1‐S6A,F8A. We have also studied three presumed control peptides: Int and RI‐Int (no H1 motif), H1‐S6A,F8A (no internalization sequence). Both ‘active’ and ‘control’ peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 μM) of the control peptide RI‐Int, non‐Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c‐Myc (and molecules of the family), we chose an iso‐amphipathic modified peptide of the H1 motif, with a proximity coefficient >50% and where the major change was at position 7 (F→A). From a family of 73 H1 motifs belonging to (H1‐Loop‐H2) human sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N‐Myc, L‐Myc, c‐Myc, H1‐S6A,F8A of c‐Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int‐H1isoamph and the corresponding RI‐Int‐H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF‐7 cells) a colon cancer line (HCT‐116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso‐amphipathic‐modified H1 sequence were always very clearly (3‐10 times) less active than the corresponding peptides carrying a conserved “H1 of Myc” motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF‐7 cells treated with L‐peptides; MCF‐7 cells treated with RI‐peptides; HCT‐116 cells treated with L‐peptides; PBLs treated with L‐peptides; PBLs treated with RI‐peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF‐7 cells. On a molar basis, RI‐peptides were about 5–10 times more potent and 30–35 times more stable in complete culture medium, than their corresponding L‐forms. RI‐Int can probably internalize longer peptido‐mimetic molecules (for instance molecules mimetic of (H1‐Loop‐H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors—two steps closer to a potential drug.

BH3-only proteins: the death-puppeteer's wires.

Most cell death in vertebrates proceeds through the intrinsic pathway of apoptosis and results from unregulated increase of mitochondrial membrane permeability. Bcl2‐associated X protein (Bax) and Bcl2‐antagonist/killer protein (Bak), the effector proapoptotic members of the Bcl‐2 family, are, in their active state, the principal accomplices for this permeabilization process. How exactly Bax and Bak are activated has been a matter of major investigation in the last decade, and suitable tools offered by quantitative cytometric methodologies have significantly contributed to the understanding of the function of Bcl‐2 family members. Here, we review the most relevant findings in this field and highlight one common trait that has emerged from the diverse new theories: a crucial role in the control of Bax/Bak activation has to be attributed to the BH3‐only subset of the Bcl‐2 family. BH3‐only proteins exert their proapoptotic activity by hierarchical and tightly tuned interactions with other Bcl‐2 family members and operate as sensors of intracellular/extracellular death signals and vectors of information to the core apoptotic machinery. Given their essential role in apoptosis, BH3‐only molecules are proposed as molecular targets for the cure of diseases associated with abnormal cell death, as in the case with neurodegenerative conditions. As well, they are explored as possible tools for cancer therapy, according to the concept that molecules mimicking the BH3 domain of these proteins could selectively and efficiently cooperate in the cell killing by chemotherapeutic drugs. A few BH3 mimetics are currently being tested in clinical trials of hematologic and solid tumors. Nevertheless, the knowledge about the cellular and molecular mechanisms that regulate responsiveness to BH3 therapy has to be further expanded and will benefit from recent advances in cytometric quantitative technologies.

CD85/LIR-1/ILT2 and CD152 (Cytotoxic T Lymphocyte Antigen 4) Inhibitory Molecules Down-Regulate the Cytolytic Activity of Human CD4 T-Cell Clones Specific for Mycobacterium tuberculosis

ABSTRACT Antigen-specific cytolytic CD4+ T lymphocytes controlMycobacterium tuberculosis infection by secreting cytokines and by killing macrophages that have phagocytosed the pathogen. However, lysis of the latter cells promotes microbial dissemination, and other macrophages engulf the released bacteria. Subsequently, CD4+ T-cell-mediated killing of macrophages goes on, and this persistent process may hamper control of infection, unless regulatory mechanisms maintain a subtle balance between lysis of macrophages by cytolytic CD4+ cells and activation of cytolytic CD4+ cells by infected macrophages. We asked whether inhibitory molecules expressed by CD4+ cytolytic T lymphocytes could play a role in such a balance. To this end, human CD4+ T-cell clones specific for M. tuberculosis were produced that displayed an autologous major histocompatibility complex class II-restricted lytic ability against purified protein derivative (PPD)-pulsed antigen-presenting cells. All T-cell clones expressed CD152 (cytotoxic T-lymphocyte antigen 4 [CTLA-4]) and CD85/leukocyte immunoglobulin-like receptor 1 (LIR-1)/immunoglobulin-like transcript 2 (ILT2) inhibitory receptors, but not CD94 and the killer inhibitory receptor (or killer immunoglobulin-like receptor [KIR]) p58.2. CD3-mediated activation of the clones was inhibited in a redirected killing assay in which CD152 and CD85/LIR-1/ILT2 were cross-linked. Specific antigen-mediated proliferation of the clones was also sharply reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked by specific monoclonal antibody (MAb) followed by goat anti-mouse antiserum. In contrast, blockade of the receptors by specific MAb only increased their proliferation. Production of interleukin 2 (IL-2) and gamma interferon (IFN-γ) by the T-cell clones was also strongly reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked. The lytic activity of the T-cell clones against PPD-pulsed autologous monocytes or Epstein-Barr virus-activated B cells was increased by blockade and decreased by cross-linking of the receptors. These results indicate that CD152 and CD85/LIR-1/ILT2 play a role in the regulation of the antigen-specific activity of CD4+ cytolytic T lymphocytes against PPD-presenting cells.