Eric Ledru

Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods

The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the FITC annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed with FSC/SSC criteria. Comparative analysis of apoptosis in cultured PBMC from HIV-infected patients considering the FSC/SSC parameters, 7-AAD stainability and annexin V fixation revealed that the latter identifies early apoptotic cells, also characterized as 7-AAD(low) with a reduced FSC. Moreover these three methods proved to be reliable and gave statistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of debris/apoptotic bodies, which were still stained by anti-cell surface mAbs and might therefore significantly distort the apoptosis percentage in a given lymphocyte subset. In the present report we also point out that the ISNT assay is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can particularly underestimate the rate of apoptosis in the B-cell subset. This was found to be related to the apoptosis-associated decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethanol used for cell permeabilization. Finally, we propose a three step analytical strategy to accurately phenotype apoptotic peripheral human lymphocytes. It includes two gating steps performed on FSC/SSC criteria and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and debris-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these observations emphasize that it is essential to assess critically the ability of a cytofluorometric method to phenotype apoptotic cells in complex lymphoid populations and that inaccurate identification of cell subsets undergoing apoptosis can be readily overcome by gating properly the lymphoid population, and using assays which preserve cell surface structure.

A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes.

The aim of this study was to define a simple and reliable method to detect simultaneously surface and intracellular antigens in apoptotic peripheral human lymphocytes. This approach requires a permeabilizing procedure for intracellular access of mAbs, which raises the important question of the influence of this procedure on parameters which identify apoptotic cells and on the surface expression of antigens. We compared the effects of three currently used permeabilizing methods (saponin quillaia bark 0.05%, Triton X-100 0.1, ethanol 70%) on the quantification of apoptotic lymphocytes, defined according to FSC/SSC criteria or following 7-AAD staining, and on the detection of surface CD3, CD4, CD8, Fas, CD45R0 molecules. The combined detection of these surface antigens with intracellular molecules, including Bcl-2 and cytokines (IFNgamma, TNFalpha, IL-2) was also analysed in the context of these three permeabilizing procedures. All the experiments were performed on PBMC from HIV-infected donors, known to undergo excessive apoptosis following short-term culture. We report that permeabilization with saponin is the only procedure which allows: (1) the preservation of lymphocyte morphology determined by the FSC/SSC parameters; (2) the quantification of apoptotic lymphocytes following 7-AAD staining; (3) a reliable surface immunophenotyping, maintaining a good antibody binding capacity (ABC); (4) the proper detection of intracellular membrane bound antigens (Bcl-2) and intracellular cytokines (IFNgamma, TNFalpha, IL-2); (5) the combined detection of apoptotic nuclei, surface antigens and intracellular molecules. Altogether these observations demonstrate that the simultaneous analysis of extracellular and intracellular antigens in apoptotic cells belonging to a complex lymphoid populations such as PBMC can be readily overcome provided the detergent used for cell permeabilization is appropriate and the successive staining procedures performed in a defined order.

Lack of control of T cell apoptosis under HAART. Influence of therapy regimen in vivo and in vitro.

Background Increased and premature T cell apoptosis is recognized as a feature of HIV infection, and its normalization during highly active antiretroviral therapy (HAART) is thought to contribute to quantitative CD4 T cell restoration. Design Cross-sectional study of spontaneous, CD3- and CD95-mediated apoptosis in lymphocytes from 53 HIV-infected individuals taking HAART. Methods Overnight stimulation of peripheral blood mononuclear cells (PBMC) with coated anti-CD3 or anti-CD95 monoclonal antibodies or incubation overnight in medium. Apoptosis in CD4 and CD8 T cells was measured by flow cytometry. For in vitro assay of antiretroviral drugs, normal PBMC were prestimulated with anti-CD3 monoclonal antibodies and apoptosis was induced by ligation of CD95. The expression of active caspase-8 and caspase-3 was examined by flow cytometry. Results We report for the first time that important levels of T cell apoptosis may persist under HAART, in spite of a rise in CD4 T cells from baseline and a sustained suppression of plasmatic viral load. Spontaneous CD3- or CD95-induced apoptosis levels were inversely correlated with the in vivo number of CD4 T cells and the CD4/CD8 ratio, but not with the viral load or duration of antiretroviral therapy. Regimens including lamivudine are associated with persistent T cell apoptosis, particularly following CD95 ligation. Lamivudine was also found to stimulate in vitro CD95-induced apoptosis and caspase activation in pre-activated T lymphocytes from healthy donors. Conclusion The immunomodulatory effect of lamivudine may be one of the contributing factor to increased levels of T cell apoptosis under HAART. The data suggest that there is a requirement for physiological apoptosis during HAART.

HIV, cytokines and programmed cell death. A subtle interplay.

Abstract: HIV infection is marked by the progressive destruction of the CD4 T lymphocyte subset, an essential component of the immune system and a vital source of cytokines required for differentiation of natural killer (NK) and γδ T cells, for maturation of B lymphocytes into plasmocytes, and for differentiation of CD8+ T cells into virus‐specific cytotoxic T lymphocytes. CD4 T lymphocytes are also a source of chemokines which control migration of lymphocytes to the site of infection and which also inhibit HIV entry into CD4‐expressing targets. Continuous production of viral proteins leads to an unbalanced immune activation and to the triggering of apoptotic programs, turning mononuclear cells, including CD4 T cells, CD8 T cells and APC, into effectors of apoptosis, leading to fratricidal destruction of healthy uninfected cells expressing the death receptors. Inappropriate PCD is also responsible for the disappearance of T helper cells primed for type‐1 cytokine synthesis, thus contributing to the lack of survival factors which could prevent spontaneous lymphocyte apoptosis. Under potent anti‐retroviral therapies, a significant decrease in spontaneous, TCR‐ and CD95‐induced lymphocyte apoptosis is observed, concomitant with a partial quantitative and qualitative restoration of the immune system in treated patients. However, owing to the suppressive effect of anti‐retroviral drugs on physiological apoptosis, these therapies are associated with alteration of TNF‐α‐regulated T cell homeostasis, leading to an accumulation in the blood of T cells primed for TNF‐α synthesis, and contributing to the development of a new syndrome associated with these treatments, the lipodystrophy syndrome.

T Cell Apoptosis in HIV Infection: Mechanisms and Relevance for AIDS Pathogenesis

Human immunodeficiency virus (HIV) infection results in the progressive destruction of CD4 T lymphocytes, generally associated with disease progression. Despite years of investigation, the mechanisms responsible for the deletion of this lymphocyte subset are still not elucidated (Levy 1993; Weiss 1993; Gougeon 1995). CD4 cell destruction can be mediated directly by virus replication as a consequence of viral gene expression or indirectly by priming of uninfected cells to apoptosis when triggered by different agents. For example, Tat, a viral transcription factor, was shown to affect transciption of genes involved in cell survival. Tat was found to upregulate Bcl-2 expression, protecting cells from apoptosis (Zauli et al. 1995). In contrast, establishment of stable Tat-expressing cell lines or addition of exogenous Tat have been reported to sensitize cells to CD95-, anti-T-cell receptor (TCR)- and anti-CD4-induced apoptosis (Li et al. 1995; Westendorp et al. 1995). Vpr gene, required for productive infection of non-dividing cells (Hattori et al. 1990), was also recently found to induce apoptosis by blocking the cell cycle in the G2 phase (Bartz et al. 1996; Steward et al. 1997). The cytopathic effect of HIV in CD4 T cell cultures, manifested by ballooning of cells and formation of syncytia, was shown to be associated with apoptosis (Laurent-Crawford et al. 1991; Terai et al. 1991).

Increased priming for interleukin-12 and tumour necrosis factor alpha in CD64 monocytes in HIV infection: modulation by cytokines and therapy.

BackgroundA key factor leading to impaired immunity in HIV infection is an alteration of the pattern of cytokine response, although its precise nature remains controversial, particularly the in vivo influence of HIV on interleukin (IL)-12 synthesis. DesignA cross-sectional study in 73 HIV-infected persons (28 of them receiving highly active antiretroviral therapy) and 18 HIV-seronegative healthy donors. MethodsThe frequency of monocytes/macrophages (M/M) synthesizing IL-12, IL-10 and tumour necrosis factor α (TNF-α) was determined in peripheral blood mononuclear cells. The cells were cultured in medium or were stimulated with lipopolysaccharide; proportions of CD64 M/M producing IL-12, TNF-α or IL-10 was determined by cytofluorometric analysis. The influence of exogenous interferon γ (IFN-γ), IL-10 or IL-15 on IL-12 synthesis was tested. ResultsChronic HIV disease is associated with increased priming of M/M for IL-12 (involving both p40 and p70 molecules) and TNF-α synthesis; this was associated with cosynthesis of both cytokines by a fraction of M/M. Priming for IL-12 was physiologically enhanced by IFN-γ and decreased by IL-10; IL-15 had no effect. The proportion of IL-10-producing CD64 M/M was not altered in patients compared with controls but there was an inverse correlation between IL-10-producing M/M and viral load. IL-12 production was not correlated with viral load but was increased following antiretroviral therapy. Following LPS stimulation, IL-12 and TNF-α responses were not altered in HIV-positive patients; however, the IL-10 response was decreased but restored by antiretroviral therapy. ConclusionThese observations argue for a preserved intrinsic CD64 M/M of IL-12 production in HIV pathogenesis.

Vitamin A supplementation and HIV-1 mother-to-child transmission in Africa

Fawzi et al.s Tanzanian study suggested that multivitamin supplementation during pregnancy can reduce adverse pregnancy outcomes in women infected with HIV-1. Before this intervention is implemented however the effect of vitamins on T cell counts should be addressed further. For example the question of whether the mechanism by which vitamins increase CD4 cell counts can slow HIV disease progression requires attention. The global rise noted by Fawzi and colleagues in all peripheral mature T subsets rather than in CD4 cells alone suggests an impact on proliferation of leukocyte progenitors. A trophic effect on both fetal cells and hematologic precursors rather than a real immunologic T cell improvement could result from the use of folic acid and vitamin B12 both of which have key roles in the metabolism of proliferating cells. Also in need of further study is whether the increase in CD4 cells resulting from this whole T lymphocyte population enlargement is protective against opportunistic diseases.