Killing of Latently HIV-Infected CD4 T Cells by Autologous CD8 T Cells Is Modulated by Nef.

The role of HIV-specific CD8 T cell activity in the course of HIV infection and the way it affects the virus that resides in the latent reservoir resting memory cells is debated. The PBMC of HIV-infected patients contain HIV-specific CD8 T cells and their potential targets, CD4 T cells latently infected by HIV. CD4 T cells and CD8 T cells procured from PBMC of HIV-infected patients were co-incubated and analyzed: Formation of CD8 T cells and HIV-infected CD4 T cell conjugates and apoptosis of these CD4 T cells were observed by fluorescence microscopy with PCR of HIV LTR DNA.

Peripheral blood mononuclear cells of HIV-infected patients contain CD8 T cells that form conjugates with and kill HIV-infected autologous CD4 T cells.

Peripheral blood mononuclear cells (PBMC) of untreated, HIV-infected patients contain HIV-specific CD8 T cells as well as their corresponding targets, HIV-infected CD4 T cells. To determine if CD4 T-cell depletion in HIV-infected patients may result from autologous CD8-CD4 T-cell interaction, CD8 and CD4 T cells procured from PBMC of acute and chronic untreated HIV-infected patients were sorted and co-incubated. Formation of CD8-CD4 T-cell conjugates was observed by fluorescence microscopy.

Cytotoxic T lymphocyte perforin and Fas ligand working in concert even when Fas ligand lytic action is still not detectable.

The reason(s) why individual cytotoxic T lymphocytes (CTL) possess a fast-acting, perforin/granzyme-mediated, as well as a much slower, Fas ligand (FasL) -driven killing mechanism is not clear, nor is the basis for wide variations in killing activity exhibited by individual CTL, ranging from minutes to hours. We show that perforin expression among individual, conjugated CTL varies widely, which can account for the heterogeneity in killing speeds exhibited by individual CTL. Despite a 2-hr lag in FasL-based killing, CTL lytic action is enhanced when the two mechanisms operate in concert.

A cell chip for sequential imaging of individual non-adherent live cells reveals transients and oscillations.

Advances in molecular cell biology, medical research, and drug development are driving a growing need for technologies that enable imaging the dynamics of molecular and physiological processes simultaneously in numerous non-adherent living cells. Here we describe a platform technology and software--the CKChip system--that enables continuous, fluorescence-based imaging of thousands of individual living cells, each held at a given position ("address") on the chip. The system allows for sequential monitoring, manipulation and kinetic analyses of the effects of drugs, biological response modifiers and gene expression in both adherent and non-adherent cells held on the chip.

Switch from perforin-expressing to perforin-deficient CD8(+) T cells accounts for two distinct types of effector cytotoxic T lymphocytes in vivo.

Although CD8(+) cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) -based and perforin-based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing machinery. Yet the origin, rationale for possessing both a slow-acting (FasL) and a fast-acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.

Up-regulation of Fas (CD95) expression in tumour cells in vivo.

Both the function and regulation of Fas expression in tumours is poorly understood. Our laboratory has reported that cultured, low Fas-expressing tumours undergo massive, yet reversible, up-regulation of cell surface Fas expression when injected into mice. The present study was aimed at determining what causes this enhanced Fas expression and whether the newly expressed Fas functions as a death receptor.

Tumoristatic effects of anti-CD40 mAb-activated macrophages involve nitric oxide and tumour necrosis factor-alpha.

Effector cells of the innate immune system have diverse functions that can result in tumour inhibition or tumour progression. Activation of macrophages by CD40 ligation has been shown to induce antitumour effects in vitro and in vivo. Here we investigated the role of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) as mediators in the tumoristatic effects of murine peritoneal macrophages activated with agonistic anti-CD40 monoclonal antibody (alphaCD40) alone and following further stimulation with bacterial lipopolysaccharide (LPS).

In vivo CD40 ligation can induce T-cell-independent antitumor effects that involve macrophages.

We have previously demonstrated T cell-independent antitumor and antimetastatic effects of CD40 ligation that involved natural killer (NK) cells. As CD40 molecules are expressed on the surface of macrophages (Mphi), we hypothesized that Mphi may also serve as antitumor effector cells when activated by CD40 ligation. Progression of subcutaneous NXS2 murine neuroblastomas was delayed significantly by agonistic CD40 monoclonal antibody (anti-CD40 mAb) therapy in immunocompetent A/J mice, as well as in T and B cell-deficient severe combined immunodeficiency (SCID) mice.

Synergistic activation of macrophages via CD40 and TLR9 results in T cell independent antitumor effects.

We have previously shown that macrophages (Mphi) can be activated by CD40 ligation to become cytotoxic against tumor cells in vitro. Here we show that treatment of mice with agonistic anti-CD40 mAb (anti-CD40) induced up-regulation of intracellular TLR9 in Mphi and primed them to respond to CpG-containing oligodeoxynucleotides (CpG), resulting in synergistic activation. The synergy between anti-CD40 and CpG was evidenced by increased production of IFN-gamma, IL-12, TNF-alpha, and NO by Mphi, as well as by augmented apoptogenic effects of Mphi against tumor cells in vitro.

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro.

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines.

Simultaneous monitoring of binding to and activation of tumor-specific T lymphocytes by peptide-MHC.

The recent advent of peptide-MHC tetramers has provided a new and effective tool for studying antigen-specific T cell populations through monitoring tetramer binding to T cells by flow cytometry. Yet information regarding T cell activation induced by the bound tetramers cannot be deduced from binding studies alone; complementary methods are needed to bridge this gap. To this end, we have developed a new approach that now enables monitoring both binding to and activation of T cells by peptide-MHC tetramers at the single-cell level.

Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis.

The RUNX transcription factors are important regulators of lineage-specific gene expression. RUNX are bifunctional, acting both as activators and repressors of tissue-specific target genes. Recently, we have demonstrated that Runx3 is a neurogenic transcription factor, which regulates development and survival of proprioceptive neurons in dorsal root ganglia.

Connexin 37 gene is not mutated in lung carcinomas 3LL and CMT.

Our previous studies have shown that intercellular communication mediated by gap junctions is impaired in most tumors as well as in cancer cell lines. However, connexin genes that encode gap junction proteins are only rarely mutated in cancer cells. On the other hand, it was reported that mutated Connexin 37 (Cx37) is the origin of shared tumor-associated antigenic octa-peptides (MUT 1 and MUT 2) of two independently derived lung carcinomas 3LL and CMT 64 of mouse origin.

Tumor sensitivity to anti-cancer drugs predicted by changes in fluorescence intensity and polarization in vitro.

Background: We used the CellScan, a novel static cytometer, to monitor changes induced by anti-neoplastic drugs in the fluorescence intensity and polarization of fluorescently-labeled tumor cells.

Materials And Methods: T47D and T80 human breast cancer cell lines were exposed to navelbine and to 5-fluorouracil and the fluorescence properties of the treated cells, stained with fluorescein diacetate and rhodamine 123, were measured by the CellScan.

Results: A strong correlation was found between the inhibition of cell growth induced by the two drugs, as estimated from cell counts, and the resulting changes in fluorescence intensity and polarization, as monitored by the CellScan.

Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas (CD95/Apo-1) receptor: Fas activation and apoptosis in hypoxic myocytes.

Objective: Since apoptosis is an important contributor to heart diseases in which ischemia and hypoxia are key elements, we tested the hypothesis that hypoxia predisposes neonatal rat ventricular myocytes (NRVM) to Fas-mediated apoptosis, by shifting the balance between antiapoptotic and proapoptotic proteins towards the latter.

Methods: Normoxic or hypoxic (22 h, 1% O(2)) cultured NRVM were exposed to recombinant Fas L (rFasL) for 7 h, and apoptosis measured thereafter.

Results: Whereas in normoxic NRVM, rFasL did not cause apoptosis measured by the TUNEL assay (4.

Immune privilege and FasL: two ways to inactivate effector cytotoxic T lymphocytes by FasL-expressing cells.

The theory that Fas ligand (FasL)-expressing tumours are immune-privileged and can directly counterattack Fas-expressing effector T lymphocytes has recently been questioned and several alternative mechanisms have been proposed. To address this controversial issue, we analysed the impact of FasL-expressing tumours on in vivo-primed cytotoxic T lymphocytes (CTLs) and the mechanisms involved. CTLs were obtained from the peritoneal cavity (PEL) after in vivo priming with syngeneic or allogeneic murine tumour cells.