Lack of CD28 expression on HIV-specific cytotoxic T lymphocytes is associated with disease progression

During HIV infection, CD8+ T cells lacking the costimulatory molecule CD28 increase in number and proportion. This accumulation is associated with disease activity and possibly with CD8+ T-cell dysfunction. In this study, CD8+CD28+ and CD8+CD28- T cells from 41 HIV-infected individuals at various stages of disease were compared in terms of HIV-specific cytotoxicity, TCR beta V repertoire diversity, and cytokine production. We found that the CD28 phenotype of anti-HIV CTL evolves in parallel with disease progression and disease activity. Absolute numbers of CD4+ T cells and CD4+/CD8+ T-cell ratios progressively decreased in 3 groups with an increasing prevalence of CD28- HIV-specific CTL. Conversely, HIV replication levels progressively increased in parallel with the prevalence of CD28- HIV-specific CTL. Repertoire diversity at the level of TCR beta V gene family expression was maintained at normal levels for both CD28+ and CD28- T cells at all stages of infection. Diversity at the level of junctional length polymorphism was more restricted in the CD8+CD28- T-cell population, but this difference remained relatively constant through different stages of infection. Both CD28+ and CD28- T cells produced IL-2 and IFN-gamma, regardless of disease stage and/or the predominant CD28 phenotype of anti-HIV CTL.     

HIV-1-specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in Nairobi

Understanding how individuals with a high degree of HIV exposure avoid persistent infection is paramount to HIV vaccine design. Evidence suggests that mucosal immunity, particularly virus-specific CTL, could be critically important in protection against sexually acquired HIV infection. Therefore, we have looked for the presence of HIV-specific CD8+ T cells in cervical mononuclear cells from a subgroup of highly HIV-exposed but persistently seronegative female sex workers in Nairobi. An enzyme-linked immunospot assay was used to measure IFN-gamma release in response to known class I HLA-restricted CTL epitope peptides using effector cells from the blood and cervix of HIV-1-resistant and -infected sex workers and from lower-risk uninfected controls. Eleven of 16 resistant sex workers had HIV-specific CD8+ T cells in the cervix, and a similar number had detectable responses in blood. Where both blood and cervical responses were detected in the same individual, the specificity of the responses was similar. Neither cervical nor blood responses were detected in lower-risk control donors. HIV-specific CD8+ T cell frequencies in the cervix of HIV-resistant sex workers were slightly higher than in blood, while in HIV-infected donor cervical response frequencies were markedly lower than blood, so that there was relative enrichment of cervical responses in HIV-resistant compared with HIV-infected donors. HIV-specific CD8+ T cell responses in the absence of detectable HIV infection in the genital mucosa of HIV-1-resistant sex workers may be playing an important part in protective immunity against heterosexual HIV-1 transmission.     

Peripheral blood cytotoxic gammadelta T lymphocytes from patients with human immunodeficiency virus type 1 infection and AIDS lyse uninfected CD4+ T cells,and their cytocidal potential correlates with viral load

Progression of human immunodeficiency virus type 1 (HIV-1) infection in humans is marked by declining CD4+-T-cell counts and increasing virus load (VL). Cytotoxic T lymphocytes (CTL) play an important role in the lysis of HIV-infected cells, especially during the early phase of asymptomatic infection. CTL responses in the later phase of disease progression may not be as effective since progressors with lower CD4+-T-cell counts have consistently higher VL despite having elevated CTL counts. We hypothesized that, apart from antiviral effects, some CTL might also contribute to AIDS pathogenesis by depleting CD4+ T cells and that this CTL activity may correlate with the VL in AIDS patients. Therefore, a cross-sectional study of 31 HIV-1-infected patients at various clinical stages was carried out. Purified CTL from these donors as well as HIV-seronegative controls were used as effectors against different human cell targets by using standard 51Cr release cytolytic assays. A direct correlation between VL and CTL-mediated, major histocompatibility complex (MHC)-unrestricted lysis of primary CD4+-T-cell, CEM.NKR, and K562 targets was observed. CD4+-T-cell counts and duration of infection also correlated with MHC-unrestricted cytolytic activity. Our data clearly show that gammadelta CTL are abnormally expanded in the peripheral blood of HIV-infected patients and that the Vdelta1 subset of gammadelta T cells is the main effector population responsible for this type of cytolysis. The present data suggest that gammadelta CTL can contribute to the depletion of bystander CD4+ T cells in HIV-infected patients as a parallel mechanism to HIV-associated immunopathogenesis and hence expedite AIDS progression.     

Contact of human immunodeficiency virus type 1-infected and uninfected CD4+ T lymphocytes is highly cytolytic for both cells.

Individuals infected with the human immunodeficiency virus (HIV) experience a marked loss of CD4+ T lymphocytes, leading to fatal immunodeficiency. The mechanisms causing the depletion of these cells are not yet understood. In this study, we observed that CD4+ T lymphocytes from HIV type 1 (HIV-1)-infected and uninfected individuals rapidly lysed B lymphoblasts expressing the HIV-1 envelope glycoprotein on the cell surface and Jurkat cells expressing the complete virus. Contact of uninfected CD4+ T cells with envelope glycoprotein-expressing cells also resulted in the lysis of the uninfected CD4+ T cells. Cytolysis did not require priming or in vitro stimulation of the CD4+ T cells and was not restricted by major histocompatibility complex molecules. Cytotoxicity was inhibited by soluble CD4 and anti-CD4 monoclonal antibodies that block binding of CD4 to gp120. In addition, neutralizing anti-CD4 and anti-gp120 monoclonal antibodies which block postbinding membrane fusion events and syncytium formation also inhibited cell lysis, suggesting that identical mechanisms in HIV-infected cultures underlie cell-cell fusion and the cytolysis observed. However, cytotoxicity was not always accompanied by the formation of visible syncytia. Rapid cell lysis after contact of uninfected and HIV-1-infected CD4+ T cells may explain CD4+ T-cell depletion in the absence of detectable syncytia in infected individuals. Moreover, because of its vigor, lysis of envelope-expressing targets by contact with unprimed CD4+ T lymphocytes may at first glance resemble antigen-specific immune responses and should be excluded when cytotoxic T-lymphocyte responses in infected individuals and vaccinees are evaluated.     

Macrophages and CD4+ T lymphocytes from two multiply exposed, uninfected individuals resist infection with primary non-syncytium-inducing isolates of human immunodeficiency virus type 1.

Despite multiple, high-risk sexual exposures, some individuals remain uninfected with human immunodeficiency virus type 1 (HIV-1). CD4+ lymphocytes from these individuals are less susceptible to infection in vitro with some strains of HIV-1, suggesting that the phenotype of the virus may influence its ability to interact with certain CD4+ cells. In the present study, we examined the susceptibility of CD4+ T lymphocytes and macrophages from two exposed uninfected individuals (EU2 and EU3) to infection with a panel of biologically cloned isolates of HIV-1 having either a non-syncytium-inducing (NSI) or a syncytium-inducing (SI) phenotype. Our results indicate that CD4+ T lymphocytes from EU2 and EU3 are resistant to infection with NSI isolates of HIV-1 but are susceptible to infection with primary SI isolates. In addition, we found that macrophages from EU2 and EU3 are resistant to infection with both NSI and SI isolates. The latter finding was confirmed by using several uncloned NSI and SI isolates obtained from patients during acute HIV-1 infection. In further experiments, env clones encoding glycoproteins characteristic of NSI or SI viruses were used in single-cycle infectivity assays to evaluate infection of CD4+ lymphocytes and macrophages from EU2 and EU3. Consistent with our previous results, we found that macrophages from these individuals are resistant to infection with NSI and SI env-pseudotyped viruses, while CD4+ T lymphocytes are resistant to NSI, but not SI, pseudotyped viruses. Overall, our results demonstrate that CD4+ cells from two exposed uninfected individuals resist infection in vitro with primary, macrophage-tropic, NSI isolates of HIV-1, which is the predominant viral phenotype found following HIV-1 transmission. Furthermore, infection with NSI isolates was blocked in both CD4+ T lymphocytes and macrophages from these individuals, suggesting that there may be a common mechanism for resistance in both cell types.     

In vitro infection of natural killer cells with different human immunodeficiency virus type 1 isolates.

Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-Fc gamma RIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considered.     

Evaluation of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses utilizing B-lymphoblastoid cell lines transduced with the CD4 gene and infected with HIV-1.

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.     

T cytotoxic-1 CD8+ T cells are effector cells against pneumocystis in mice

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. A hallmark of HIV infection is Pneumocystis carinii (PC) pneumonia. Recently, CD8+ T cells, which are recruited to the lung in large numbers in response to PC infection, have been associated with some level of host defense as well as contributing to lung injury in BALB/c mice. In this study, we show that CD8+ T cells that have a T cytotoxic-1 response to PC in BALB/c mice, as determined by secretion of IFN-gamma, have in vitro killing activity against PC and effect clearance of the organism in adoptive transfer studies. Moreover, non-T cytotoxic-1 CD8+ T cells lacked in vitro effector activity and contributed to lung injury upon adoptive transfer. This dichotomous response in CD8+ T cell response may in part explain the clinical heterogeneity in the severity of PC pneumonia.     

The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection.

The murine immune response to lymphocytic choriomeningitis virus (LCMV) infection involves the activation of CD8+, class I MHC-restricted and virus-specific CTL. At times coinciding with CTL activation, high levels of IL-2 gene expression and production occur, the IL-2R is expressed, and T cell blastogenesis and proliferation are induced. We have previously found that, although both CD4+ and CD8+ T cell subsets transcribe IL-2, the CD4+ subset appears to be the major producer of IL-2 whereas the CD8+ subset appears to be the major proliferating population when the subsets are separated after activation in vivo. The studies presented here were undertaken to examine the contribution made by the CD4+ subset to lymphocyte proliferation in vivo. Responses to LCMV infection were examined in intact mice and in mice depleted of CD4+ or CD8+ subsets by antibody treatments in vivo. Protocols were such that in vivo treatments with anti-CD4 or anti-CD8 depleted the respective subset by greater than 90%. In situ hybridizations demonstrated that the IL-2 gene was expressed in non-B lymphocytes isolated from either CD4+ cell-depleted or CD8+ cell-depleted mice on day 7 post-infection with LCMV. When placed in culture, however, cells from CD8+ cell-depleted mice produced significantly higher levels of detectable IL-2 than did cells isolated from CD4+ cell-depleted mice on day 7 post-infection. IL-2 was apparently produced in vivo in mice depleted of either CD4+ or CD8+ cells, as expression of the gene for the p55 chain of the IL-2R, IL-2 responsiveness, and lymphocyte proliferation were observed with cells isolated from both sets of mice. Lymphocyte proliferation was shown to be sustained in mice depleted of CD4+ cells in vivo by three criteria: 1) non-B lymphocytes isolated from infected mice depleted of CD4+ cells underwent more DNA synthesis than did those isolated from uninfected mice or from infected mice depleted of CD8+ cells; 2) leukocyte yields were expanded during infection of CD4+ cell-depleted mice; and 3) CD8+ cell numbers were increased during infection of CD4+ cell-depleted mice. The majority of non-B lymphocytes having the characteristics of blast lymphocytes was recovered in the CD8+ populations isolated from infected CD4+ cell-depleted mice. These findings suggest that the requirement for the CD4+ subset to sustain CD8+ lymphocyte proliferation in vivo is limited, and that CD4+ and CD8+ cell types can function independently in many aspects of their responses to viral infections.     

Enhanced dendritic cell-driven proliferation and anti-HIV activity of CD8(+) T cells by a new phenothiazine derivative, aminoperazine

T cell anergy, apoptosis, and chronic activation of T lymphocytes are prevailing features of HIV infection. The inability to develop an efficient natural antiviral activity in infected patients might be the consequence of a failure of the Ag presentation by dendritic cells (DCs) in chronically activated lymphoid tissues. We have identified a new phenothiazine derivative aminoperazine (APR; 2-amino-10-[3'-(1-methyl-4-piperazinyl)propyl]phenothiazine, C(20)H(26)N(4)S; m.w. 354.51) able to increase (effective dose from 0.1 to 100 nM) the Ag-specific DC-driven proliferation and differentiation of in vitro HIV-infected and uninfected normal donor T cells and of T cells from HIV-1-infected patients. The immunomodulatory effect of APR-sensitized DCs were ascribed to soluble factors derived from DCs. APR was also capable of increasing HIV gag-p24-specific proliferation and anti-HIV cytotoxic activity of patients' CD8(+) T cells against autologous B-lymphoblastoid cell lines expressing a HIV gag gene, resulting in the suppression of both proviral DNA and supernatant viral RNA in the HIV-1-infected patients' T cell culture. This new phenothiazine derivative (APR) might be used for boosting the immune response of vaccinated individuals and for restoring the immunity of immunocompromised patients.     

Naturally acquired CD8+ cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

In rodent malaria model systems, protective immunity induced by immunization with irradiated sporozoites is eliminated by in vivo depletion of CD8+ T cells, and adoptive transfer of CTL clones against the circumsporozoite protein protects against malaria. We recently demonstrated that volunteers immunized with irradiated Plasmodium falciparum sporozoites produce CTL against peptide 368-390 of the P. falciparum circumsporozoite protein. To determine whether natural exposure to malaria induced similar CTL, we studied 11 adult, male, life-long residents of a highly malarious area of Kenya, who were selected because their lymphocytes had been shown to proliferate after stimulation with peptides 361-380, 371-390, or 368-390 and because nine had been resistant to malaria in previous studies. In four of the 11 individuals there was peptide-specific, genetically restricted, CTL activity. In all four individuals, this activity was unaffected by depletion of CD4+ T cells. In three volunteers the activity was eliminated or reduced by depletion of CD8+ T cells; in the fourth volunteer the CD8+ T cell depletion was uninterpretable. This first demonstration of CD8+ T cell, genetically restricted, Ag-specific CTL against a malaria protein among individuals exposed to endemic malaria provides a foundation for studying the relationship between circulating CTL and resistance to malaria infection.     

Distinct Mechanisms Trigger Apoptosis in Human Immunodeficiency Virus Type 1-Infected and in Uninfected Bystander T Lymphocytes

Apoptosis is a main feature of AIDS pathogenesis and is thought to play a role in the progressive decrease of CD4⁺ T lymphocytes in infected individuals. To determine whether apoptosis occurs in infected and/or in uninfected peripheral blood T lymphocytes, we have used a recombinant human immunodeficiency virus type 1 (HIV-1) infectious clone expressing the green fluorescent protein (GFP). Using flow cytometry, we have determined the incidence of apoptosis by either terminal transferase dUTP nick end labeling or annexin-V assays in different cell subpopulations, i.e., in CD4⁺ or CD8⁺ T cells that were GFP positive or negative. After HIV-1 infection of purified peripheral blood lymphocytes, we observed that apoptosis occurred mostly in infected CD4⁺ peripheral blood lymphocytes. Remarkably, the presence of monocyte-derived macrophages in the culture increased dramatically the apoptosis of uninfected bystander T lymphocytes, while apoptosis in HIV-infected T lymphocytes was not changed. We therefore demonstrate that HIV-induced apoptosis results from at least two distinct mechanisms: (i) direct apoptosis in HIV-infected CD4⁺ T lymphocytes and (ii) indirect apoptosis in uninfected T cells mediated by antigen-presenting cells.     

Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.     

HIV-infected humans, but not chimpanzees, have circulating cytotoxic T lymphocytes that lyse uninfected CD4+ cells

It has been suggested that autoimmune phenomena contribute to the depletion of CD4+ T cells and the development of AIDS in HIV-1 infected humans based, in part, on observations that some HIV-1-infected humans have autoantibodies reactive with Ag expressed on uninfected CD4+ cells. In this study, 11 of 14 asymptomatic HIV-1-infected homosexuals and hemophiliacs, but none of 17 uninfected homosexuals or heterosexuals, were found to have cytotoxic lymphocytes in blood that can lyse uninfected CD4+ T cells from humans and chimpanzees but not human B lymphoblastoid cells or mouse T cells. The cytotoxic PBL were concluded to be CTL rather than NK cells, with the phenotype being CD3+, TCR-1 alpha beta+, CD8+, CD4-, CD16- based on findings that PBL-mediated lysis of uninfected CD4+ cells was 1) blocked by a mAb to CD3, which inhibits CTL but not NK activity; 2) diminished by treatment of PBL with a mAb to CD8 and C, but not by treatment with mAb to CD4 or CD16 and C; and 3) blocked by mAb WT31 directed against the TCR-1 alpha beta. In contrast, PBL from HIV-1-infected chimpanzees, which to date have not developed AIDS, lacked detectable CTL lytic for uninfected CD4+ cells.     

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.     

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+) T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.     

Activated peripheral CD8 lymphocytes express CD4 in vivo and are targets for infection by human immunodeficiency virus type 1.

There is increasing evidence that CD8 lymphocytes may represent targets for infection by human immunodeficiency virus type 1 (HIV-1) in vivo whose destruction may contribute to the loss of immune function underlying AIDS. HIV-1 may infect thymic precursor cells destined to become CD4 and CD8 lymphocytes and contribute to the numerical decline in both subsets on disease progression. There is also evidence for the induction of CD4 expression and susceptibility to infection by HIV-1 of CD8 lymphocytes activated in vitro. To investigate the relationship between CD8 activation and infection by HIV-1 in vivo, activated subsets of CD8 lymphocytes in peripheral blood mononuclear cells (PBMCs) of HIV-seropositive individuals were investigated for CD4 expression and HIV infection. Activated CD8 lymphocytes were identified by expression of CD69, CD71, and the human leukocyte antigen (HLA) class II, the beta-chain of CD8, and the RO isoform of CD45. CD4(+) and CD4(-) CD8 lymphocytes, CD4 lymphocytes, other T cells, and non-T cells were purified using paramagnetic beads, and proviral sequences were quantified by PCR using primers from the long terminal repeat region. Frequencies of activated CD8 lymphocytes were higher in HIV-infected study subjects than in seronegative controls, and they frequently coexpressed CD4 (mean frequencies on CD69(+), CD71(+), and HLA class II(+) cells of 23, 37, and 8%, respectively, compared with 1 to 2% for nonactivated CD8 lymphocytes). The level of CD4 expression of the double-positive population approached that of mature CD4 lymphocytes. That CD4 expression renders CD8 cell susceptible to infection was indicated by their high frequency of infection in vivo; infected CD4(+) CD8 lymphocytes accounted for between 3 and 72% of the total proviral load in PBMCs from five of the eight study subjects investigated, despite these cells representing a small component of the PBMC population (<3%). Combined, these findings provide evidence that antigenic stimulation of CD8 lymphocytes in vivo induces CD4 expression that renders them susceptible to HIV infection and destruction. The specific targeting of responding CD8 lymphocytes may provide a functional explanation for the previously observed impairment of cytotoxic T-lymphocyte (CTL) function disproportionate to their numerical decline in AIDS and for the deletion of specific clones of CTLs responding to HIV antigens.     

High levels of human immunodeficiency virus infection of CD8 lymphocytes expressing CD4 in vivo

Human immunodeficiency virus (HIV)-infected CD8 lymphocytes have been reported in vivo, but the mechanism of infection remains unclear. Experiments using the thy/hu mouse model support export of intrathymically infected CD8 precursors, while recent in vitro data suggest that mature CD8 lymphocytes upregulate CD4 upon activation (generating a CD8bright CD4dim phenotype) and are susceptible to HIV infection. To determine whether these mechanisms operate in vivo and to assess their relative importance in the generation of circulating HIV-infected CD8 lymphocytes, we quantified HIV long terminal repeat (LTR) DNA in CD8+ CD4- and CD8bright CD4dim lymphocytes isolated from HIV-infected individuals by fluorescence-activated cell sorting. HIV infection of CD8 lymphocytes was demonstrated in 17 of 19 subjects, with a significant inverse relationship between level of infection and CD4 lymphocyte count (R = -0.73; P < 0.001). The level of HIV infection of CD8bright CD4dim lymphocytes was significantly higher (median, 1,730 HIV LTR copies/10(6) cells; n = 9) than that of CD8+ CD4- lymphocytes (undetectable in seven of nine individuals; P < 0.01) and approached that of CD4 lymphocytes from the same individuals (median, 3,660 HIV LTR copies/10(6) cells). CD8bright CD4dim lymphocytes represented 0.8 to 3.3% of total CD8 lymphocytes and were most prevalent in the memory subset. Thus, HIV-infected CD8 lymphocytes commonly circulate in HIV-infected individuals and are generated through infection of activated CD8 lymphocytes rather than through export of intrathymically infected precursors. The high level of infection of CD8bright CD4dim lymphocytes could have a direct role in the decline in CD8 lymphocyte function that accompanies HIV disease progression.