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. Furthermore, conjugation of CD8 T cells with CD4 T cells and apoptosis of CD4 T cells was observed and quantified by imaging flow cytometry using anti-human activated caspase 3 antibody and TUNEL assay. The conjugation activity and apoptosis were found to be much higher in patients with acute HIV infection or AIDS compared to patients in chronic infection on antiretroviral therapy (ART) or not. Patients on ART had low grade conjugation and apoptosis of isolated CD69, CD25, and HLA-DR-negative CD4 T cells (latent reservoir cells) by CD8 T cells. Using
PCR The latent reservoir CD4 T cells were shown to contain most of the HIV DNA. We demonstrate in HIV-infected patients, that CD8 T cells conjugate with and kill HIV-infected CD4 T cells, including HIV-infected resting memory CD4 T cells, throughout the course of HIV infection. We propose that in HIV-infected patients CD4 T cell annihilation is caused in part by ongoing activity of HIV-specific CD8 T cells. HIV Nef protein interacts with ASK 1 and inhibits its pro-apoptotic death signaling by Fas/FasL, thus protecting HIV-infected cells from CD8 T cells killing. A peptide that interrupts Nef-ASK1 interaction that had been delivered into CD4 T cells procured from patients on ART resulted in the increase of their apoptosis inflicted by autologous CD8 T cells. We suggest that elimination of the HIV-infected latent reservoir CD4 T cells can be achieved by Nef inhibition.
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. Here we report that Runx3 and Runx1 are highly expressed in thymic medulla and cortex, respectively, and function in development of CD8 T cells during thymopoiesis. Runx3-deficient (Runx3 KO) mice display abnormalities in CD4 expression during lineage decisions and impairment of CD8 T cell maturation in the thymus. A large proportion of Runx3 KO peripheral CD8 T cells also expressed CD4, and in contrast to wild-type, their proliferation ability was largely reduced. In addition, the in vitro cytotoxic activity of alloimmunized peritoneal exudate lymphocytes was significantly lower in Runx3 KO compared with WT mice. In a compound mutant mouse, null for Runx3 and heterozygous for Runx1 (Runx3-/-;Runx1+/-), all peripheral CD8 T cells also expressed CD4, resulting in a complete lack of single-positive CD8+T cells in the spleen. The results provide information on the role of Runx3 and Runx1 in thymopoiesis and suggest that both act as transcriptional repressors of CD4 expression during T cell lineage decisions.
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 (Mφ), we hypothesized that Mφ 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. Although NK cells can be activated by anti‐CD40 mAb, anti‐CD40 mAb treatment also induced a significant antitumor effect in SCID/beige mice in the absence of T and NK effector cells, even when noncytolytic NK cells and polymorphonuclear cells (PMN) were depleted. Furthermore, in vivo treatment with anti‐CD40 mAb resulted in enhanced expression of cytokines and cell surface activation markers, as well as Mφ‐mediated tumor inhibition in A/J mice, C57BL/6 mice, and SCID/beige mice, as measured in vitro. A role for Mφ was shown by reduction in the antitumor effect of anti‐CD40 mAb when Mφ functions were inhibited in vivo by silica. In addition, activation of peritoneal Mφ by anti‐CD40 mAb resulted in survival benefits in mice bearing intraperitoneal tumors. Taken together, our results show that anti‐CD40 mAb immunotherapy of mice can inhibit tumor growth in the absence of T cells, NK cells, and PMN through the involvement of activated Mφ.
The CTL's kiss of death Berke, Gideon
Cell,
04/1995, Letnik:
81, Številka:
1
Book Review, Journal Article
Recenzirano
Odprti dostop
The potent and specific lytic activity of CTLs can occur by at least two distinct pathways. In the secretion and perforin-mediated pathway, the direct effect(s) on the target cell membrane of the ...pore-forming agent perforin, probably in conjunction with granzymes, also secreted from the CTLs, causes the target's demise. Intercytoplasmic transfer of granzymes is believed to be involved in inducing target apoptosis. In the Fas-mediated pathway, engagement of a CTL membrane ligand with an apoptosis-inducing target cell surface receptor, such as the FasL with Fas, triggers programmed disintegration of the CTL-bound target; secretion of granzymes and pore formation by perforin are not involved in this receptor-mediated mechanism. Despite the fundamental differences in their onset for both pathways, the downstream sequence of events that culminate in target cell apoptosis appears to be similar. Further studies will resolve this enigma.
Killer lymphocytes are primary immune effectors of virus, certain bacteria, and tumor immunity and play a role in autoimmunity and transplant rejection. This article reviews progress in deciphering ...the mechanisms by which they kill target cells through induction of apoptosis by either the secretory, perforin/granzyme-based pathway or the nonsecretory pathway, (i.e., by triggering the cell-surface death receptor Fas (CD95) by the membrane-bound Fas ligand of the killer).
Killer lymphocytes Berke, Gideon; Clark, William R
2005, 2005-05-10
eBook, Book
This extensively documented, comprehensive survey of cell-mediated cytotoxicity (CMC) traces the history of killer lymphocytes from 1960 to the present, providing a definitive resource for ...specialists and non-specialists alike. It offers an advanced analysis of CMC, including a comprehensive examination of key papers underlying its evolution, and provides a thorough discussion of the most recent advances in the field.
Cytolytic T lymphocytes (CTLs) kill by inducing apoptosis within minutes after contact with their target in a process requiring neither new gene expression nor protein synthesis of the target cells. ...Killer lymphocytes possess at least two molecularly distinct, fast-acting lytic mechanisms. In the first, the granule exocytosis lytic mechanism, the secreted lytic protein perforin produces poly-perforin pores (i.d. 10-20 nm) in the target cell's membrane. Granzymes, cosecreted with perforin, are thought to penetrate the target cell through these pores to bring about apoptosis by activating the ICE/ced-3 pathway of apoptosis induction. The second, nonsecretory lytic mechanism involves neither the secretion nor the action of the pore-forming protein perforin nor of cosecreted granzymes. Instead, a surface membrane ligand of the killer cell (Fas-ligand), cross-linking with the apoptosis-inducing target cell surface death receptor Fas (CD95), triggers a cascade of intracellular protein-protein interactions and proteolytic activities involving ICE/ced-3 and culminating in apoptosis of the target cell. Despite fundamental differences in the onset of apoptosis induced by perforin/granzyme and by triggering the Fas pathway, the downstream sequences of events that culminate in apoptosis appear to be quite similar. Although the two CTL lytic pathways appear to work in concert, the specific function of each mechanism and its regulation in a given setting in vivo and even in vitro remain to be elucidated.
CTL and NK cells use two distinct cytocidal pathways: 1) perforin and granzyme based and 2) CD95L/CD95 mediated. The former requires perforin expression by the effectors (CTL or NK), whereas the ...latter requires CD95 (Fas/APO-1) expression by the target. We have investigated how these two factors contribute to tumor immune surveillance by studying the immunity of perforin-deficient mice against the progressor C57BL/6 Lewis lung carcinoma 3LL, which expresses no CD95 when cultured in vitro. Unexpectedly, the results indicated that the perforin-independent CD95L/CD95 pathway of CTL/NK plays a role in acting against D122 and Kb39.5 (39.5) high and low metastatic sublines, respectively, derived from the 3LL tumor. Although no membrane-bound CD95 was detected on cultured D122 and 39. 5 cells, surface CD95 expression on both D122 and 39.5 was considerably up-regulated when the tumors were grown in vivo. A similarly enhanced expression of CD95 was observed with three additional tumors; LF-, BW, and P815, injected into syngeneic and allogeneic mice. The finding of up-regulated CD95 expression on tumor cells placed in vivo suggests that a CD95-based mechanism plays a role in tumor immunity at early stages of tumor growth. Consequently, the progressive down-regulation of CD95 expression during tumor progression may indeed be an escape mechanism as previously reported. Together, these results suggest a role for CD95-dependent, perforin-independent immunity against certain tumors.
Summary
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. This is explained by finding that the two pathways in fact are jump‐started simultaneously with the lag in FasL lytic action reflecting pre‐lytic caspase‐8 activation and BH3‐interacting domain (BID) cleavage. The complementary action of the two lytic pathways, co‐expressed at varying levels among individual CTL, facilitates the lytic action of late‐stage poor perforin‐expressing CTL, ensuring optimal cytocidal action throughout the CTL response.
Summary
Peripheral blood mononuclear cells (
PBMC
) of untreated,
HIV
‐infected patients contain
HIV
‐specific
CD
8 T cells as well as their corresponding targets,
HIV
‐infected
CD
4 T cells. To ...determine if
CD
4 T‐cell depletion in
HIV
‐infected patients may result from autologous
CD
8–
CD
4 T‐cell interaction,
CD
8 and
CD
4 T cells procured from
PBMC
of acute and chronic untreated
HIV
‐infected patients were sorted and co‐incubated. Formation of
CD
8‐
CD
4 T‐cell conjugates was observed by fluorescence microscopy. Apoptosis of
CD
4 T cells in conjugation was recorded by digitized images and was further observed and measured by
FACS
using Annexin staining. Perforin expression in the
CD
8 T cells was measured using intracellular monoclonal perforin antibody staining.
HIV DNA
in the conjugated
CD
4 T cells was detected by
in situ
PCR
. We found that 6·1 ± 0·5% of
CD
4 T cells from acute
HIV
‐infected patients and 3·0 ± 0·5% from chronic
HIV
‐infected patients formed
CD
8–
CD
4 T‐cell conjugates. Annexin binding and cell morphology typical of apoptosis were observed in the conjugated
CD
4 T cells. The majority of
CD
8 T cells that had conjugated to
CD
4 T cells expressed perforin. The conjugated
CD
4 T cells exhibited nuclear
HIV DNA
.
CD
8 T cells and
HIV
‐infected
CD
4 T cells, both procured from the
PBMC
of untreated
HIV
‐infected patients, form conjugates. Apoptotic lytic activity has been observed in the conjugated
CD
4 T cells. We propose that
CD
4 T‐cell annihilation in
HIV
‐infected patients results, at least in part, from the interactions of perforin‐rich
CD
8 T cells with autologous,
HIV
‐infected
CD
4 T cells.
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