A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity.

Cell-mediated cytotoxicity is a crucial mechanism involved in several fundamental immunological processes such as protection against intracellular pathogens or termination of an immune response. This phenomenon is classically evaluated by the 51Cr release assay, which requires a radioactive isotope and does not permit the characterization of cells involved in the cytotoxic reaction. We describe a new flow cytometry method, developed in the context of CD95-mediated cell death, which allows the precise quantitation of cell-mediated cytotoxicity and the detection of intracellular events involved in the cytotoxic process. This assay uses a combination of two dyes, i.e. 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE) to label effector cells and 7-amino actinomycin D (7-AAD) to stain apoptotic target cells. We show that this assay is more sensitive than the 51Cr release assay and makes it possible to quantitate the percentage of cell lysis and, concomitantly, to immunophenotype target cells. It also facilitates the analysis of some events of the apoptotic pathway such as caspase activation or the expression of mitochondrial molecules. This new assay should contribute to a better understanding of the mechanisms involved in cell-mediated cytotoxicity in normal and pathological situations.

The flexibility of the TCR allows recognition of a large set of naturally occurring epitope variants by HIV-specific cytotoxic T lymphocytes.

Pathogens attempt to evade immune recognition by expressing mutated antigens. The present study shows that two mechanisms happen in vivo during the course of HIV infection to limit the escape of antigenic variants from cytotoxic T lymphocyte (CTL) recognition: recognition of several epitope variants by the same TCR and generation of several CTL populations specific for a single epitope but recognizing different variant sequences. We have studied two CTL populations directed towards the HIV-p24gag amino acids 176--184 QASQEVKNW epitope, presented by HLA-B5301. Both CTL populations were derived from a long-term asymptomatic HIV-infected child and they express different TCR. Each of the two CTL recognizes five of the 10 naturally occurring variants. These variants are distinct for both CTL and thus a total of eight variants are recognized. Thus, polyclonality of CTL specific for the same epitope but differing in variant sequences recognized may improve the control of variant viruses' replication in vivo. In addition to cross-recognition of several variant epitopes, promiscuous recognition of exogenous peptides complexed to allogeneic HLA-B molecules occurs, showing that the TCR can tolerate amino acid changes on both the peptide and the MHC molecule. This flexibility of the TCR is probably of great importance for control of viruses with high genetic variability, such as HIV.

Frequency and phenotyping of human immunodeficiency virus (HIV)-specific CD8+ T cells in HIV-infected children, using major histocompatibility complex class I peptide tetramers.

HLA-A*02 tetramers complexed to human immunodeficiency virus (HIV) Gag SLYNTVATL and HIV Pol ILKEPVHGV peptides were used to characterize HLA class I-restricted CD8(+) T cells in 41 HIV-infected children. The frequencies and the phenotype of specific circulating CD8(+) T cells were determined in whole-blood samples by means of cytometric analysis. Background staining of 13 HLA-A*02-negative patients showed that the frequency of CD8(+) T cells was <0.01%. Of the 28 HLA-A*02-positive patients, blood samples from 26 stained positive at least once the Gag tetramer (mean CD8(+) T cells, 0.87%; range, 0.1%-3.9%), and blood samples from 21 stained positive for the Pol tetramer (mean CD8(+) T cells, 0.59%; range, 0.1%-5.5%). The tetramer-binding cells were CD28(-), CD45RA(-), CD45RO(+), HLA-DR(+), and CD69(-) T lymphocytes. HIV-specific CD8(+) T cells can be detected easily in peripheral blood of HIV-infected children, using HLA tetramers combined with HIV peptides. These cells are memory activated CD28(-)CD8(+) T lymphocytes.

Expansion of HBV-specific memory CTL primed by dual HIV/HBV genetic immunization during SHIV primary infection in rhesus macaques.

We have previously shown the induction of humoral and cytotoxic responses specific for human immunodeficiency virus (HIV) and hepatitis B virus (HBV) antigens, following genetic immunization of rhesus macaques with a plasmid encoding both the third variable domain of the HIV-1 external envelope glycoprotein and the pseudo-viral particle of hepatitis B surface antigen (HBsAg) as presenting molecules. The DNA-immunized primates and two control animals were then challenged with a chimeric simian/human immunodeficiency virus (SHIV). They were all infected. Significant frequencies of SHIV specific cytotoxic T lymphocyte precursors (CTLp) were detected early in peripheral blood. But, in all DNA-immunized macaques, HBV envelope specific CTLp were detected during the primary infection, and they were correlated with the peak of SHIV viremia. Furthermore, HBV or SHIV specific cytotoxicity corresponded in part to CD8(+) T cells presenting a memory phenotype. Several mechanisms could account for this cellular response. But our results suggest that an expansion of memory cytotoxic CD8(+) cells, not restricted to SHIV specific effectors, could occur in peripheral blood during SHIV primary infection.

CD8(+)-Cell antiviral factor activity is not restricted to human immunodeficiency virus (HIV)-specific T cells and can block HIV replication after initiation of reverse transcription.

CD8(+) lymphocytes from human immunodeficiency virus (HIV)-infected patients can suppress in vitro HIV replication in CD4(+) T cells by a noncytolytic mechanism involving secreted CD8(+)-cell antiviral factor(s) (CAF). Using an HIV Nef-specific cytotoxic-T-lymphocyte (CTL) line and autologous CD4(+) T cells infected with a nef-deleted HIV-1 virus, we demonstrated that, after a priming antigenic stimulation, this suppression does not require the presence of the specific antigen during the effector phase. Furthermore, using an Epstein-Barr virus (EBV)-specific CTL line from an HIV-seronegative donor, we demonstrated that the ability to inhibit HIV replication in a noncytolytic manner is not restricted to HIV-specific effector cells; indeed, EBV-specific CTL were as efficient as HIV-specific effectors in suppressing R5 or X4 HIV-1 strain replication in vitro. This HIV-suppressive activity mediated by a soluble factor(s) present in the culture supernatant was detectable for up to 14 days following stimulation of EBV-specific CD8(+) cells with the cognate epitope peptide. Following acute infection of CEM cells with an X4 strain of HIV-1, EBV-specific CTL line supernatant containing HIV-suppressive activity did not block virus entry but was shown to interfere with virus replication after the first template switching of reverse transcription. Our results suggest that the noncytolytic control of HIV replication by EBV-specific CD8(+) T lymphocytes corresponded to a CAF-like activity and thus demonstrate that CAF production may not be restricted to CTL induced during HIV disease. Moreover, CAF acts after reverse transcription at least for X4 isolate replication inhibition.

Zinc and cadmium hyperaccumulation by Thlaspi caerulescens from metalliferous and nonmetalliferous sites in the Mediterranean area: implications for phytoremediation.

Growth, tolerance and zinc and cadmium hyperaccumulation of Thlaspi caerulescens populations from three metal contaminated soils and three normal soils were compared under controlled conditions. Individuals of six populations were cultivated on five soils with increasing concentrations of zinc (50-25000 µg g-1 ) and cadmium (1-170 µg g-1 ). There was no mortality of normal soil populations in the four metal-contaminated soils, but plant growth was reduced to half that of populations from metal-contaminated soils. However, in noncontaminated soil, the growth of individuals from normal soils was greater than that of individuals from metal-contaminated soils. Individuals from normal soils concentrated three times more zinc in the aboveground biomass than those from metal-contaminated soils, but the latter accumulated twice as much cadmium. We conclude that populations of T. caerulescens from both normal and metal-contaminated soils are interesting material for phytoextraction of zinc and cadmium, but to optimize the process of phytoextraction it is necessary to combine the extraction potentials of both type of populations.

In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen.

DNA immunization offers a novel means to induce humoral and cellular immunity in inbred or in outbred animals. Here we have tested the efficiency of genetic immunization with hepatitis B virus (HBV) envelope-based vectors. In naive primates, injection of a plasmid DNA encoding HBV envelope proteins induced an HBV-specific cytotoxic response and appearance of potentially protective anti-HBs antibodies. Moreover, intramuscular and intradermal injections of a DNA expression vector encoding an epitope of the human immunodeficiency virus envelope fused to the surface protein of the hepatitis B virus (HBsAg) induced strong humoral and cytotoxic responses to antigenic determinants of both viruses in mice and nonhuman primates alike. In addition, in protein-primed Rhesus monkeys B-cell memory was successfully boosted by DNA injection of hybrid vectors and animals subsequently developed a multispecific cellular response. This suggests that DNA-based immunization could be used to boost efficiently and broaden the immune response in individuals immunized with conventional vaccines, regardless of their genetic variability. These results also indicate that it might be possible to rationally design HBsAg-based expression vectors to induce multispecific immune responses for vaccination against hepatitis B and other pathogens.

Potential deleterious effect of anti-viral cytotoxic lymphocyte through the CD95 (FAS/APO-1)-mediated pathway during chronic HIV infection.

The potential deleterious effect through a CD95-based pathway of anti-viral cytotoxic lymphocyte (CTL) during HIV-infection was studied. The present paper reports that a Nef specific CTL line derived from an HIV-infected person is able to kill not only Nef-expressing target cells but also CD95+ compliant Jurkat cells. The two mechanisms of cytotoxicity, i.e. perforin-vs-CD95-dependent were differentiated according to their respective Ca(2+)-dependence. The existence of the dual killing machinery in the anti-HIV CTL line was correlated with the coexpression in these cells of perforin and CD95-L molecules. A model of AIDS pathogenesis involving the deleterious effect through the CD95 pathway of the viral specific CTL response is discussed.

CD26 as a positive regulator of HIV envelope-glycoprotein induced apoptosis in CD4+ T cells.

The membrane-expressed HIV-1 envelope glycoprotein complex, gp120/gp41, has been shown to be responsible for the initiation of cell killing by apoptosis in CD4+ T cells. By using two experimental approaches we demonstrate that CD26, independent of its DPP IV activity, appears to be implicated in this function of the gp120/gp41 complex to initiate apoptosis.

Dual function of a human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte clone: inhibition of HIV replication by noncytolytic mechanisms and lysis of HIV-infected CD4+ cells.

CD8+ T cells may play a beneficial role in human immunodeficiency virus (HIV)-infected patients by two mechanisms. HIV-specific cytotoxic activity and secretion of a soluble mediator(s) that inhibits HIV replication in vitro. Here we characterized both activities mediated by an HIV p24gag-specific cytotoxic T lymphocyte (CTL) CD8+ clone derived from an HIV-infected patient. When the CTL clone was mixed with HIV-infected autologous CD4+ T cells, viral replication was suppressed. This viral inhibition was observed in heterologous CD4+ T cells and when CD8+ and CD4+ populations were separated by a semipermeable membrane, demonstrating the involvement of a diffusible factor(s). The lysis of autologous HIV-infected T cells was also detected. However, HIV suppression was more efficient when CD4+ and CD8+ T cells shared major histocompatibility complex alleles and were in direct contact. Thus, one and the same CD8+ T cell population can mediate both lysis of HIV-infected targets and nonlytic suppression of HIV replication. These results underline the multiple roles of CD8+ T lymphocytes in the suppression of HIV-infected cells.

HIV envelope glycoprotein-induced cell killing by apoptosis is enhanced with increased expression of CD26 in CD4+ T cells.

The membrane-expressed HIV-1 envelope glycoprotein complex, gp120 and gp41, has been shown to be responsible for the initiation of cell killing by apoptosis in CD4+ T cells. By using two experimental approaches we demonstrate here that CD26, also known as dipeptidyl peptidase IV (DPP IV), appears to be implicated in this function of the gp120/gp41 complex to initiate apoptosis. In the first experimental model, we used persistently HIV-1-infected H9/IIIB cells expressing the membrane-associated gp120/gp41 complex as effector cells to induce apoptosis in Jurkat CD4+ T cells: parental or transfected in order to express high levels of recombinant CD26, either wild-type or mutated at its Ser-630 which inactivates the DPP IV activity of CD26. Parental Jurkat cells and transfected control cell clones express low but reproducibly detectable levels of endogenous CD26. In coculturing experiments using H9/IIIB and Jurkat cells, the occurrence of apoptosis was found to be retarded by at least 24 hr in Jurkat cells expressing low levels of endogenous CD26, compared to cell clones expressing high levels of either wild-type or mutated catalytically inactive transfected CD26. In the second experimental model, the different Jurkat cell lines were infected with vaccinia recombinant viruses expressing HIV-1 env gene, either wild-type to generate a functional gp120/gp41 complex or mutated to generate an uncleavable membrane-expressed precursor of the envelope glycoprotein gp160. At 18 hr postinfection with such vaccinia recombinant viruses, apoptosis was observed only in Jurkat cells with enhanced levels of CD26 and expressing the gp120/gp41 complex. Apoptosis was not detected in the different Jurkat cell lines expressing the uncleavable precursor gp160. In both of the experimental models used, no significant differences were observed between the transfected cells expressing either the wild-type or the mutated form of CD26, thus suggesting that the DPP IV activity of CD26 is not essential for its function as a cofactor of CD4 in the mechanism of initiation of apoptosis by the HIV envelope gp120/gp41 complex. Taken together, these results indicate that CD26 in CD4+ T cells may determine the rate of initiation of apoptosis by the mature HIV-1 envelope glycoproteins, i.e., CD26 is being involved as a cofactor of CD4 in the mechanism of triggering apoptosis by the gp120/gp41 complex. As signaling through CD26 could lead to T cell activation, we propose that this latter might be modified following the binding of the gp120/gp41 complex to CD4 and thus leading to apoptosis.

Gag-specific cytotoxic responses to HIV type 1 are associated with a decreased risk of progression to AIDS-related complex or AIDS.

The duration of human immunodeficiency virus (HIV-1) infection prior to the development of AIDS is variable, and for most patients the exact time of infection is not known. A group of 38 HIV-1-infected subjects was tested while asymptomatic for comparative cytotoxic lymphocyte responses to the Gag and envelope antigens of HIV-1. Twenty of the 38 patients had no detectable primary cytotoxic T lymphocyte (CTL) response to Gag, and this was associated with a relative risk of 1.89 for progression to ARC or AIDS during the subsequent 3 to 40 months of observation when compared with patients who had Gag-specific CTL activity at the beginning of the observation period. In contrast, no significant association was observed between envelope-specific cytotoxic activity and disease progression. Other patient characteristics, including CD4+ T lymphocyte counts and antibody levels to the p24gag protein, measured at the start of observation, did not correlate with disease progression during the observation period. This suggests that the anti-Gag CTL response may be protective during HIV-1 infection.

A prime-boost approach to HIV preventive vaccine using a recombinant canarypox virus expressing glycoprotein 160 (MN) followed by a recombinant glycoprotein 160 (MN/LAI). The AGIS Group, and l'Agence Nationale de Recherche sur le SIDA.

The safety and the immunogenicity of a recombinant canarypox live vector expressing the human immunodeficiency virus type 1 (HIV-1) gp160 gene from the MN isolate, ALVAC-HIV (vCP125), followed by booster injections of a soluble recombinant hybrid envelope glycoprotein MN/LAI (rgp160), were evaluated in vaccinia-immune, healthy adults at low risk for acquiring HIV-1 infection. Volunteers (n = 20) received vCP125 (10(6) TCID50) at 0 and 1 month, followed randomly by rgp160 formulated in alum or in Freund's incomplete adjuvant (FIA) at 3 and 6 months. Local and systemic reactions were mild or moderate and resolved within the first 72 hr after immunization. No significant biological changes in routine tests were observed in any volunteer. Two injections of vCP125 did not elicit antibodies. Neutralizing antibodies (NA) against the HIV-1 MN isolate were detected in 65 and 90% of the subjects after the first and the second rgp 160 booster injections, respectively. Six months after the last boost, only 55% were still positive. Seven of 14 sera with the highest NA titers against MN weakly cross-neutralized the HIV-1 SF2 isolate; none had NA against the HIV-1 LAI or against a North American primary isolate. Specific lymphocyte T cell proliferation to rgp 160 was detected in 25% of the subjects after vCP125 and in all subjects after the first booster injection and 12 months after the first injection. An envelope-specific cytotoxic lymphocyte activity was found in 39% of the volunteers and characterized for some of them as CD3+, CD8+, MHC class I restricted. The adjuvant formulation did not influence significantly the immune responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterologous HIV-2 challenge of rhesus monkeys immunized with recombinant vaccinia viruses and purified recombinant HIV-2 proteins.

In an attempt to analyse the role of anti-envelope immunity in the protection of rhesus monkeys against an HIV-2 intravenous challenge, rhesus macaques were immunized twice with recombinant HIV-2 ROD vaccinia viruses (10(8) p.f.u. each) at days 0 and 30, followed by booster injections of purified HIV-2 proteins at months 8, 9, 15 and 27. One group of five macaques was immunized with the Gag, Pol, Vif and Nef antigens, whereas a second group received the same antigens with the addition of HIV-2 Env protein. Eight months after the last boost, the animals were challenged by intravenous injection of 100 AID50 of a monkey PBMC-grown stock of HIV-2 SBL. None of the animals was protected in spite of high humoral immune responses on day of challenge as determined by ELISA and Western Blot assays.

Vaccine-induced neutralizing antibodies directed in part to the simian immunodeficiency virus (SIV) V2 domain were unable to protect rhesus monkeys from SIV experimental challenge.

The potential of the simian immunodeficiency virus (SIV) variable 2 (V2) domain as an effective region to boost SIV-neutralizing antibodies and to protect against live SIV challenge was tested in rhesus macaques. In this study, two rhesus macaques were primed with vaccinia virus recombinants expressing the surface glycoprotein gp140 of SIVmac and were given booster injections with the SIVmac V2 domain presented by a highly immunogenic carrier, the hepatitis B surface antigen (HBsAg). The two vaccinated macaques exhibited SIV-neutralizing antibodies after primer injections that were enhanced by the V2/HBsAg injections. Part of these SIV-neutralizing antibodies were directed specifically to the V2 region, as shown by neutralization-blocking experiments. However, despite having consistent SIV-neutralizing antibody titers, animals were not protected against homologous challenge with BK28, the molecular clone of SIVmac251. No SIV envelope-specific cellular cytotoxic response was detected throughout the immunization protocol, suggesting that neutralizing antibodies directed to SIV envelope gp140 and especially to the V2 domain were unable on their own to protect against SIV challenge. Furthermore, the vaccinees seemed to have higher viral loads than control animals after challenge, raising the question of whether neutralizing antibodies induced by vaccination and directed to the SIV envelope selected viral escape mutants, as shown previously in SIV-infected macaques. This mechanism is certainly worthy of intensive investigation and raises some concern for SIV envelope-targeted immunization.

Multispecific and heterogeneous recognition of the gag protein by cytotoxic T lymphocytes (CTL) from HIV-infected patients: factors other than the MHC control the epitopic specificities.

The HIV gag polyprotein is a major target for recognition by CTL in infected humans. Using recombinant vaccinia viruses (rVV) expressing truncations of the p24gag, and the p18gag, p15gag and HIV-2 p56gag proteins, the characterization of epitope regions recognized by in vitro-stimulated peripheral blood mononuclear cells (PBMC) from 18 infected patients has been studied. The gag-specific response of most individuals is polyclonal and multispecific, and interindividual variations between target epitope regions were frequently observed, despite shared MHC alleles. As CTL may play an important role in the control of HIV replication in infected hosts, these results have important implications for designing vaccine strategies.

Strain specificity of cell-mediated cytotoxic responses specific for the human immunodeficiency virus type 1 (HIV-1) envelope protein in seropositive donors: HIV-1Lai is more commonly recognized than HIV-1MN.

Cell-mediated cytotoxic (CMC) responses were measured in a group of human immunodeficiency virus type 1 (HIV-1)-seropositive donors against target cells expressing the envelope protein of either the HIV-1 strain Lai or strain MN. In primary CMC assays using freshly isolated peripheral blood mononuclear cells, seropositive individuals more commonly had CMC responses against HIV-1Lai than HIV-1MN. Moreover, each of the responders to HIV-1MN envelope also had primary CMC responses against HIV-1Lai envelope. Cytotoxic T cells generated by nonspecific in vitro stimulation recognized both strains at a similar frequency. These results may indicate the existence of multiple effector populations or that the cellular immune response is directed at regions of the envelope outside the V3 loop. By contrast, serologic studies done on similar populations have shown that most persons have antibodies capable of recognizing the V3 loop of HIV-1MN but rarely that of HIV-1Lai.

Virus-specific cytotoxic T lymphocyte responses in patients infected with the human immunodeficiency virus, HIV-1.

A vigorous virus-specific cytotoxic response has been detected in individuals infected by the human immunodeficiency virus (HIV). ADCC and CTL activities specific to the envelope glycoproteins could be measured at the same time from the same donor. CTL response have been shown directed to most viral structural and non-structural proteins. The p24gag protein is the most frequent target of HIV-specific CTL. The characterization of CTL epitopes at the level of the human population is of importance for vaccine strategy. The mechanism of control of virus replication by CD8+ T lymphocytes is not elucidated but HIV-specific CTL have been shown to mediate this antiviral activity. The biological relevance of CTL in vivo is discussed.

Efficient antigen presentation to cytotoxic T lymphocytes by cells transduced with a retroviral vector expressing the HIV-1 Nef protein.

In the classic model of antigen processing and presentation, viral antigens must be synthesized within the cytoplasm of infected cells to be processed and presented to CD8+, MHC class I-restricted cytotoxic T lymphocytes (CTLs). We have examined the utility of a retroviral vector (pNeoNef) expressing the human immunodeficiency virus type (HIV-1)Lai Nef protein for the development of target cells to study HIV-specific CTLs. Autologous Epstein-Barr-transformed B cell lines (EBV-B cells) transduced with pNeoNef were efficiently lysed by CTL lines from donors capable of lysing EBV-B cells infected with a recombinant vaccinia virus (rVV) expressing Nef. Also, the transduced cells were efficient stimulator cells for the generation of Nef-specific CTL lines. The CTL lines thus generated recognized the same epitopes as CTL lines from the same donor generated by nonspecific stimulation. The use of similar cell lines transduced with retroviral vectors expressing HIV proteins may be useful in the study of CTLs in HIV-infected donors and in the study of the ability of candidate vaccines, including rVV, to induce HIV-specific CTLs. As antigen-presenting cells, the cell lines may be useful in the generation of antigen-specific CTL lines.

HIV-specific CD8+ T-cell immune responses and viral replication.

AIM: To review current knowledge of CD8+ T cells in relation to their effect on the replication of HIV and on disease progression. PRESENT KNOWLEDGE: Both CD8+ cytotoxic T lymphocytes capable of killing cells expressing HIV antigens and CD8+ lymphocytes that suppress HIV replication in vitro are detectable in response to HIV infection. CONCLUSION: These CD8+ T cells may help to maintain a low viral load in vivo, thus allowing a long asymptomatic period of infection.