EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

EBV has been reported to impair monocyte in vitro differentiation into dendritic cells (DCs) and reduce cell survival. In this study, we added another layer of knowledge to this topic and showed that these effects correlated with macroautophagy/autophagy, ROS and mitochondrial biogenesis reduction. Of note, autophagy and ROS, although strongly interconnected, have been separately reported to be induced by CSF2/GM-CSF (colony stimulating factor 2) and required for CSF2-IL4-driven monocyte in vitro differentiation into DCs. We show that EBV infects monocytes and initiates a feedback loop in which, by inhibiting autophagy, reduces ROS and through ROS reduction negatively influences autophagy. Mechanistically, autophagy reduction correlated with the downregulation of RAB7 and ATG5 expression and STAT3 activation, leading to the accumulation of SQSTM1/p62. The latter activated the SQSTM1-KEAP1- NFE2L2 axis and upregulated the anti-oxidant response, reducing ROS and further inhibiting autophagy. ROS decrease correlated also with the reduction of mitochondria, the main source of intracellular ROS, achieved by the downregulation of NRF1 and TFAM, mitochondrial biogenesis transcription factors. Interestingly, mitochondria supply membranes and ATP required for autophagy execution, thus their reduction may further reduce autophagy in EBV-infected monocytes. In conclusion, this study shows for the first time that the interconnected reduction of autophagy, intracellular ROS and mitochondria mediated by EBV switches monocyte differentiation into apoptosis, giving new insights into the mechanisms through which this virus reduces immune surveillance. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CSF2: colony stimulating factor 2; CT: control; CYCS (cytochrome C: somatic); DCs: dendritic cells; EBV: Epstein-Barr virus; GSR: glutathione-disulfide reductase; KEAP1: kelch like ECH associated protein 1; IL4: interleukin 4; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MET: metformin; NAC: N-acetylcysteine; NFE2L2/NRF2 nuclear factor: erythroid 2 like 2; NRF1 (nuclear respiratory factor 1); clPARP1: cleaved poly(ADP-ribose) polymerase; Rapa: Rapamycin; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFAM: (transcription factor A: mitochondrial); TUBA1A: tubulin alpha 1a.

KSHV reduces autophagy in THP-1 cells and in differentiating monocytes by decreasing CAST/calpastatin and ATG5 expression.

We have previously shown that Kaposi sarcoma-associated herpesvirus (KSHV) impairs monocyte differentiation into dendritic cells (DCs). Macroautophagy/autophagy has been reported to be essential in such a differentiating process. Here we extended these studies and found that the impairment of DC formation by KSHV occurs through autophagy inhibition. KSHV indeed reduces CAST (calpastatin) and consequently decreases ATG5 expression in both THP-1 monocytoid cells and primary monocytes. We unveiled a new mechanism put in place by KSHV to escape from immune control. The discovery of viral immune suppressive strategies that contribute to the onset and progression of viral-associated malignancies is of fundamental importance for finding new therapeutic approaches against them.

Dexamethasone induces apoptosis in human T cell clones expressing low levels of Bcl-2.

Previous results of ours have demonstrated that the same clonotype can express both a sensitive and a resistant phenotype to Dex-mediated PCD induction depending on its cell cycle phase. In particular, we demonstrated that human T lymphocytes, arrested in the G0/G1 phase of the cell cycle, are susceptible, while proliferating T cells are resistant to Dex-mediated apoptosis. In this paper, we have further characterized the sensitive and resistant phenotypes and investigated whether a different expression of the apoptotic genes Fas, FasL, Bcl-2, Bcl-x and Bax is involved in the regulation of Dex-mediated apoptosis. The results show that the amount of Bcl-2 expression, that changes during cell cycle phases, determines susceptibility or resistance to apoptosis induced by Dex. In fact, undetectable expression of Bcl-2 in sensitive cells favors Dex-mediated apoptosis while high expression of Bcl-2 in proliferating cells counterbalances apoptosis induction. Moreover, the addition of exogenous IL-2, in the presence of Dex, fails to up-regulate Bcl-2 expression and to revert Dex-mediated apoptotic phenomena.

Cutting edge: CD4+ T cells kill CD8+ T cells via Fas/Fas ligand-mediated apoptosis.

To explore the possibility that CD4+ T cells, described to mediate the elimination of themselves or B lymphocytes, could also mediate the elimination of CD8+ T cells, we analyzed apoptotic phenomena in cocultures of CD4+ and CD8+ autologous T cell lines. The data show that CD8+ T cells were lysed by activated CD4+ helper T cells by a Fas/FasL-mediated mechanism. CD4+ T cells were not lysed by activated CD8+ T cells, although Fas and FasL were equally expressed and anti-Fas Abs induced apoptosis in both CD4+ and CD8+ T cell populations. The results allowed us to speculate that CD4+ T cells not only help CD8+ T lymphocytes to mature into effector killer cells and to sustain this function but can also limit their growth.

Analysis of susceptibility of mature human T lymphocytes to dexamethasone-induced apoptosis.

We present evidence that dexamethasone (Dex), a synthetic glucocorticosteroid, causes apoptosis in mature human T cells, similarly to what has been reported for murine T lymphocytes. Human T cell clones and short-term activated T lymphocytes treated with Dex show the characteristic pattern of apoptotic cells, such as hypodiploid nuclei, chromatin condensation and DNA fragmentation into oligonucleosomal fragments. However, Dex susceptibility of T cells to apoptosis is cell cycle-dependent. The progression in the proliferative cell cycle (G1 versus S) rescues Dex-treated T cells from apoptosis. Moreover, occupancy of the T cell receptor reverses Dex-induced apoptotic phenomena. These observations suggest that glucocorticoids contribute to the regulation of the proliferative or the suicidal response of antigen-activated human T cells.

Limiting dilution analysis of T cell unresponsiveness to mycobacteria in advanced disseminated tuberculosis.

Peripheral blood mononuclear cells from patients with advanced disseminated tuberculosis (Dis-TB) do not respond to purified protein derivative (PPD) measured as cell proliferation, lymphokine production and interleukin (IL)-2 receptor (Tac antigen) expression. Limiting dilution analysis revealed "multi-hit" curves and low frequencies of PPD-reactive T cells in cultures of Dis-TB, and "single-hit" curves and high frequencies of PPD-reactive T cells in cultures of patients with localized form of pulmonary tuberculosis. Moreover, a strict relationship between Tac antigen expression and ability of exogenous IL-2 to enhance bulk culture cell proliferation was observed in Dis-TB patients.

Engagement of CD4 before TCR triggering regulates both Bax- and Fas (CD95)-mediated apoptosis.

In the present study, we have aimed at clarifying the CD4-dependent molecular mechanisms that regulate human memory T cell susceptibility to both Fas (CD95)-dependent and Bcl-2-dependent apoptotic pathways following antigenic challenge. To address this issue, we used an experimental system of viral and alloantigen-specific T cell lines and clones and two ligands of CD4 molecules, Leu-3a mAb and HIV gp120. We demonstrate that CD4 engagement before TCR triggering suppresses the TCR-mediated neosynthesis of the Flice-like inhibitory protein and transforms memory T cells from a CD95-resistant to a CD95-susceptible phenotype. Moreover, evidence that the apoptotic programs were executed while Fas ligand mRNA expression was inhibited led us to analyze Bcl-2-dependent pathways. The data show that the engagement of CD4 separately from TCR influences the expression of the proapoptotic protein Bax independently of the anti-apoptotic protein Bcl-2, whereas Ag activation coordinately modulates both Bax and Bcl-2. The increased expression of Bax and the consequent dissipation of the mitochondrial transmembrane potential (DeltaPsim) suggest a novel immunoregulatory function of CD4 and demonstrate that both passive cell death and activation-induced cell death are operative in CD4+ memory T cells. Furthermore, analysis of the mechanisms by which IL-2 and IL-4 cytokines exert their protective function on CD4+ T cells in the presence of soluble CD4 ligands shows that they were able to revert susceptibility to Bax-mediated but not to CD95-dependent apoptotic pathways.

Differential susceptibility to monomeric HIV gp120-mediated apoptosis in antigen-activated CD4+ T cell populations.

To support the hypothesis that indirect mechanisms mediated by viral products like the HIV envelope glycoprotein gp120 could be responsible for T lymphocyte depletion in HIV infection, we developed a system in which the impairment of T cell functions could be investigated in vitro. In particular, we characterized the conditions that allow T lymphocytes repeatedly stimulated with an antigen to be sensitive or resistant to gp120-mediated apoptotic signals. To achieve this goal, a panel of antigen-specific CD4+ T cell clones and primary CD4+ T lymphocytes were treated for 2 and 18 h with saturating amounts of monomeric gp120 (without cross-linking with specific antibodies) and antigen-driven T cell proliferation and apoptosis were analyzed. We show that monomeric gp120 induces apoptosis only in T lymphocytes repeatedly stimulated with the antigen, that primary T lymphocytes are resistant to programmed cell death mediated by monomeric gp120, but are sensitive to anti-CD4 antibodies, and that gp120-mediated apoptosis is dependent on the period of time between the binding of gp120 to CD4 and the encounter with antigen. To investigate the different susceptibility to gp120 induced apoptosis of primary CD4+ and T cell clones further, the number of membrane CD4 molecules and their affinity for gp120, together with Bcl-2 and Fas expression, were studied. Our data suggest that a down-modulation of membrane CD4 together with high expression of the Bcl-2 gene and protein characterizes the susceptibility to apoptosis of gp120-treated cells. In conclusion, our results define the phenotypic features of T cells susceptible to HIV gp120-induced apoptosis and demonstrate that the same clonotype, depending on the activation state, may present a differential sensitivity to apoptosis induction.

A new tripeptide, Pol 509, influences biochemical events associated with antigen presentation efficiency of PPD-specific EBV-B cells.

A synthetic tripeptide (pGLU-LEU-TRP-OCH3) Pol 509, derived from snake venom, was studied directly by analyzing the interactions with synthetic lipid bilayers using NMR spectroscopy. Functional studies were also performed by measuring the effects: i), on early biochemical events (adenyl cyclase and phospholipase C activation products), intermediate (surface Ag expression) and late (DNA synthesis) parameters following B-cell activation elicited by PPD-linkage to specific membrane Ig; and ii), on the presentation of PPD to Ag-specific T-cell lines. Comparative experiments using PMA and IFN-gamma were also performed. We found that all parameters studied were affected by Pol 509 treatment. In fact, while PPD linkage to mlg reversed the balance between cAMP and IP3 existing in unstimulated EBV-B cells, Pol 509 reduced the PPD-induced accumulation of cAMP to control values and induced a further decrease of IP3 level. Pol 509-mediated decrease of these second messenger levels was accompanied by a slight increase of HLA-DR molecule expression and DNA synthesis inhibition. Furthermore, Pol 509 enhanced the efficiency of PPD presentation to T-cell lines. Taken together, these observations suggest that Pol 509, which enhances Ag presentation by modifying second messenger levels, may be considered as a new immunomodulatory drug with immunopotentiating activity.

Regulation of self-major histocompatibility complex reactive human T-cell clones.

The proliferative response of human T-lymphocyte clones, (TLC) specific for self-major histocompatibility complex (MHC) products either alone or associated with PPD epitopes are inhibited in vitro by dexamethasone (DEX) and by a non-specific inhibitory factor(s) (nsINH) produced by PPD-activated T-cells. The inhibiting effect has been investigated by preincubating autoreactive and PPD-specific TLC with nsINH or DEX. Results obtained indicate that T-lymphocytes are the target of these two immunoregulatory molecules. Moreover, the addition of exogenous recombinant interleukin 2 (rIL-2) substantially reverses the inhibition observed in both nsINH- or DEX-treated cultures.

TCR engagement regulates differential responsiveness of human memory T cells to Fas (CD95)-mediated apoptosis.

In this work, we have tried to establish whether human memory T cells may be protected from Fas (CD95)-induced apoptosis when correctly activated by Ag, and not protected when nonspecifically or incorrectly activated. In particular, we wanted to investigate the molecular mechanisms that regulate the fate of memory T cells following an antigenic challenge. To address this issue, we chose an experimental system that closely mimics physiological T cell activation such as human T cell lines and clones specific for viral peptides or alloantigens. We demonstrate that memory T cells acquire an activation-induced cell death (AICD)-resistant phenotype when TCRs are properly engaged by specific Ag bound to MHC molecules. Ag concentration and costimulation are critical parameters in regulating the protective effect. The analysis of the mechanisms involved in the block of CD95 signal transduction pathways revealed that the crucial events are the inhibition of CD95-associated IL-1beta-converting enzyme (ICE)-like protease (FLICE) activation and poly(ADP)-ribose polymerase cleavage, and the mRNA expression of FLICE-like inhibitory protein. Furthermore, we have observed that TCR-mediated neosynthesis of FLICE-like inhibitory protein mRNA is suppressed either by protein tyrosine kinase inhibitors or cyclosporin A. In conclusion, the present analysis of the effects of TCR triggering on the regulation of AICD suggests that AICD could be inhibited in human memory T cells activated in vivo by a foreign Ag, but may become operative when the Ag has been cleared.

Beta-endorphin receptors on cultured and freshly isolated lymphocytes from normal subjects.

[125I]-[D-Ala2]-beta EP*) binding to Epstein-Barr Virus (EBV) transformed B lymphocytes or to freshly isolated lymphocytes was characterized. The binding was time-, temperature- and pH-dependent; furthermore, it was reversible and cell concentration dependent. Maximum binding appeared at 15C in Tris buffer (pH 7.6) containing both BSA 0.5% and Bacitracin 0.1 mg/ml. beta EP inhibited BEP* binding to transformed lymphocytes and to freshly isolated lymphocytes by 50% at approximately 4 x 10(-8) M and 8 x 10(-9) M, respectively. Peptides representing amino-acid sequences 1-5, 1-16 and 1-17 of beta EP did not inhibit beta EP binding, neither did the opiates compounds Naloxone, Morphine, Bremazocine and Ethylketocyclazocine. On the contrary, beta EPd (6-31) inhibited beta EP* binding as effectively as beta EP, thus indicating that beta EP* binding to lymphocytes does not involve the N-terminal region of beta EP. 2 x 10(7)/ml freshly isolated lymphocytes bound 1.53 +/- 75%, whereas 2 x 10(6)/ml transformed lymphocytes bound 1.64 +/- 0.75% (mean +/- SD) beta EP* at tracer concentration. EBV transformed lymphocytes and freshly isolated lymphocytes may be useful to study beta EP receptors in humans.

Dissociation of T cell anergy from apoptosis by blockade of Fas/Apo-1 (CD95) signaling.

Induction of anergy and deletion due to apoptosis are two of the mechanisms involved in peripheral tolerance. To clarify the relationship between these two phenomena we have used an in vitro system of T cell Ag presentation. The recognition of Ag displayed by MHC class II-expressing T cells (T-APC) induces partial signals in Ag-specific T cell clones. This leads to a blunted intracellular calcium flux, and the T cells become unable to proliferate in response to further challenge with professional APC. These T cells are unable to produce IL-2, but retain the ability to release IL-4. In the present study, we report that for some T cell clones, the predominant outcome of Ag recognition on T cells is cell death. For susceptible T cell clones, the number of cells that die is proportional to the peptide concentration. This cell death resulted from Fas/Apo-1 (CD95)/Fas-ligand interactions between the T cells, in that Fas ligand expression was detected following overnight culture of T cells with T-APC and neutralizing anti-CD95 Ab protected from death. Most notably, following anti-CD95-mediated protection from apoptosis, the rescued T cells remained unable to respond to rechallenge with Ag-pulsed, professional APC. These data suggest that anergy and apoptosis can be separated as consequences of partial T cell signaling.

The combined treatment of human peripheral blood mononuclear cells with thymolymphotropin and interleukin 2 increases PPD-driven T-cell proliferation and IL-2 induced cellular cytotoxicity against HIV-infected cells.

Two in vitro systems (the DNA synthetic response to mycobacterial antigens and cytotoxicity against lymphoid cells) were used to analyse the effect of thymolymphotropin (TLT) on peripheral blood mononuclear cells (PBMC). Purified protein derivative of mycobacteria (PPD)-driven T-cell proliferation in low-responder donors was increased by the combined treatment with TLT and suboptimal doses of recombinant interleukin 2 (IL-2). Similarly, the activities of natural killer (NK) cells and lymphokine-activated killer (LAK) cells have been enhanced in PBMC cultures pretreated with TLT. Also, TLT showed an enhancing effect on the development of LAK cells capable of lysing Epstein-Barr virus (EBV)-transformed B-lymphocytes infected or uninfected with the human immunodeficiency virus (HIV).

Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.

Increased autoreactive T cell frequency in tuberculous patients.

The development of putative self-MHC-reactive T cells and their precursor frequency was estimated in peripheral blood lymphocyte cultures stimulated in vitro with PPD. The role of foreign antigen in the generation of self-MHC-reactive T cells in vivo was analyzed by comparing the frequency of autoreactive T cells in the peripheral blood of tuberculous patients with that observed in healthy individuals. It was found that PPD in vitro and Mycobacterium tuberculosis infection in vivo increased substantially the generation of autoreactive T cells. Autoreactive T cell clones were shown (1) to recognize self MHC class II products; (2) to release gamma interferon in the absence of exogenous antigen, and (3) to express autocytotoxic activity. All these findings suggest that self-MHC-reactive T cells may be involved in the inflammatory response to M. tuberculosis.