HIV-specific CD8+ T-cell proliferation is prospectively associated with delayed disease progression

Human immunodeficiency virus (HIV)-specific CD8(+) T-cell proliferation is consistently correlated with enhanced host HIV immune control, but whether proliferative responses are a cause or consequence of immune protection is unclear. We measured Env-specific CD8(+) T-cell proliferation and interferon (IFN)-γ secretion in HIV-infected participants with CD4 counts >200, who then completed 121 person-years of prospective follow-up to monitor HIV disease progression. In all, 13 of 31 participants (42%) reached end point during longitudinal follow-up. Strong Env-specific CD8(+) T-cell proliferation (>10% of CD8(+) T cells) was observed in 14/31 participants at baseline, and this was associated with a longer time to HIV disease progression end point, stratified baseline CD4 count (P=0.016). No associations were observed for IFN-γ ELISPOT responses and progression (P>0.2). Strong proliferation remained significant in multivariate Cox regression analyses (P=0.044) as an independent predictor of delayed HIV disease progression, along with baseline CD4 count (P=0.04). Duration of HIV infection was associated with more rapid progression in univariate, but not multivariate, analysis (P=0.112). Age and baseline viral load were not predictive of progression. HIV-specific CD8(+) T-cell proliferation was a correlate of protective immunity in this prospective study; such responses may be important for HIV vaccine protection.     

Cell-mediated suppression of HIV-specific cytotoxic T lymphocytes

CTL specific for HIV have been described in lungs of infected patients at early stages of HIV disease. In order to characterize the evolution over time of HIV-specific CTL, we have analyzed the cytotoxic function and the cell surface phenotype of the alveolar lymphocytes from 41 patients at various stages of HIV disease. We demonstrated a progressive decline of alveolar anti-HIV CTL activity and detected Ts cells from the lungs of patients with advanced HIV disease. These alveolar T cells strongly suppressed the effector phase of anti-HIV CTL lysis. They lacked a marked specificity of function because they also block anti-HLA CTL response and were not restricted by the HLA-class-I transplantation Ag. They displayed the CD3, CD8, and HNK1 markers, were CD4 and CD16 negative, and lacked NK activity. The presence of Ts cells at late stages of HIV disease could thus partly explain the inefficiency of host defenses against HIV.     

Presence of suppressor HIV-specific CD8+ T cells is associated with increased PD-1 expression on effector CD8+ T cells

Mechanisms leading to the observed immune dysregulation in HIV-1 infection are not well understood. HIV-specific IL-10-positive CD8(+) T cells are increased in advanced HIV disease. We have previously reported that Gag-specific IL-10-positive CD8(+) T cells suppressed cytolysis. In this study we describe the suppressive effect of Nef-specific IL-10-positive CD8(+) T cells. Interestingly, simultaneous removal of both Gag- and Nef-specific IL-10-positive CD8(+) T cells led to higher HIV-specific cytolysis compared with the removal of Nef-specific IL-10-positive CD8(+) T cells alone. We also examined the level of programmed cell death-1 (PD-1) as a measure of immune dysfunction in association with IL-10-positive suppressor CD8(+) T cells. The level of PD-1 expression on CD107-positive effector CD8(+) T cells was significantly increased when IL-10-positive suppressor CD8(+) T cells were present (p < 0.05). Our results suggest that IL-10-positive suppressor CD8(+) T cells contribute to the immune dysfunction observed in advanced HIV infection and that the concomitant presence of multiple IL-10-positive CD8(+) T cell populations may have an additive suppressive effect.     

Unusually high frequencies of HIV-specific cytotoxic T lymphocytes in humans

CTL specific for the HIV belong to the CD8 subset of T lymphocytes, and their activity is restricted by class I HLA transplantation Ag. In this report, HIV-specific CTL and their precursor cells were quantified by limiting dilution analysis. CTL were recovered from the lungs, lymph nodes, and blood of asymptomatic seropositive carriers and of patients with AIDS. HIV was found to be very immunogenic. High frequencies of both HIV-specific CTL and CTL precursor cells were detected in infected individuals. These CTL killed autologous HIV-infected macrophages and T4 lymphoblasts. They also killed doubly transfected P815-A2-env-LAV mouse tumor cells, which express the human HLA-A2 gene and the HIV-1 env gene. In the longitudinal studies of two HIV-infected patients, CTL and CTL precursor cell frequencies decreased as the clinical and immunologic status of the patients deteriorated. Most surprisingly, PBL from seronegative donors also responded to HIV stimulation in vitro and generated large numbers of HLA-restricted, HIV-specific CTL.     

Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction

Functional impairment of HIV-specific CD4(+) T cells during chronic HIV infection is closely linked to viral replication and thought to be due to T cell exhaustion. Programmed death 1 (PD-1) has been linked to T cell dysfunction in chronic viral infections, and blockade of the PD-1 pathway restores HIV-specific CD4(+) and CD8(+) T cell function in HIV infection. This study extends those findings by directly examining PD-1 expression on virus-specific CD4(+) T cells. To investigate the role of PD-1 in HIV-associated CD4(+) T cell dysfunction, we measured PD-1 expression on blood and lymph node T cells from HIV-infected subjects with chronic disease. PD-1 expression was significantly higher on IFN-gamma-producing HIV-specific CD4(+) T cells compared with total or CMV-specific CD4(+) T cells in untreated HIV-infected subjects (p = 0.0001 and p < 0.0001, respectively). PD-1 expression on HIV-specific CD4(+) T cells from subjects receiving antiretroviral therapy was significantly reduced (p = 0.007), and there was a direct correlation between PD-1 expression on HIV-specific CD4(+) T cells and plasma viral load (r = 0.71; p = 0.005). PD-1 expression was significantly higher on HIV-specific T cells in the lymph node, the main site of HIV replication, compared with those in the blood (p = 0.0078). Thus, PD-1 expression on HIV-specific CD4(+) T cells is driven by persistent HIV replication, providing a potential target for enhancing the functional capacity of HIV-specific CD4(+) T cells.     

HIV-specific cytotoxic T lymphocytes directed against alveolar macrophages in HIV-infected patients

A CD8+ lymphocytic infiltration of the lungs is frequently observed in HIV-infected patients, even prior to the onset of opportunistic infections. In such patients, we could demonstrate that most of these CD8+ alveolar T lymphocytes displayed the D44 marker and were functional cytolytic T lymphocytes directed against autologous HIV-infected alveolar macrophages. This primary cytolytic activity was HLA-restricted and, at least partially, specific for the HIV envelope protein, since HLA-A2 alveolar T lymphocytes could specifically lyse cell lines expressing both the HLA-A2 and Env antigens. In contrast to data obtained in peripheral blood, no ADCC activity was observed against the Env antigen.HIV-specific alveolar T-lymphocyte cytolytic activity decreased with progression towards AIDS as shown by studies of a serie of 40 patients.Functional abnormalities of the lung epithelium could be associated with the specific lysis of alveolar macrophages, suggesting that local tissue injury could result from the in vivo immune conflict between alveolar HIV-specific CTL and HIV-infected macrophages.     

HIV-specific cytotoxic T lymphocytes against alveolar macrophages: specificities and downregulation

To analyse the evolution of alveolar-lymphocyte-mediated cytotoxic activity directed against autologous alveolar macrophages (AM), cytotoxic assays against various HIV⁺ target cells were performed in a cohort of 75 patients with HIV-associated lymphoid interstitial pneumonitis (LIP) studied at distinct stages of HIV infection. Our data confirm that alveolar HIV-specific cytotoxic T lymphocytes (CTL) against AM were detectable before AIDS in patients with CD8⁺ LIP. Mild CD8⁺ lymphocytic alveolitis occurs silently in 62% of stage II and III patients with no respiratory symptoms. In these cases, the lack of spontaneous alveolar-lymphocyte-mediated cytotoxic activity against autologous AM may contrast with the detection of primary alveolar CTL specific for HIV proteins such as nef. In AIDS patients, the alveolar CTL lytic efficiency against both AM- and HIV-antigen-expressing cells can be inhibited by a suppressore factor produced by alveolar CD8⁺ CD57⁺ cells. Therefore, spontaneous CTL lysis of AM may be (1) limited to a subgroup of patients with active LIP and (2) controlled by distinct mechanisms, including suppressor phenomenons, and HIV replication levels in AM.     

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.     

HIV-specific IL-10-positive CD8+ T cells are increased in advanced disease and are associated with decreased HIV-specific cytolysis

IL-10-producing T cells have been shown to inhibit Ag-specific CD8+ T cell responses, and may play a role in the immune dysregulation observed in HIV-1 infection. We characterized the Gag-specific IL-10 responses by CD8+ T cells in HIV-1-positive volunteers from Uganda. HIV-specific IL-10 responses were detected in 32 of 61 (52.4%) antiretroviral naive and 2 of 15 (13.3%) volunteers with a complete virologic response on antiretroviral therapy (< 400 copies/ml). The frequency of HIV-specific IL-10-positive cells was significantly higher in volunteers with advanced disease (CD4+ T cell count <200 cells/mm3; p = 0.0004), and correlated positively with plasma HIV RNA (r = 0.43, p = 0.0004). Interestingly, the frequency of Gag-specific CD107a/b-, but not IFN-gamma-, positive cells was significantly lower in individuals with detectable IL-10-positive CD8+ T cells (p = 0.004). Gag-specific IL-10-positive CD8+ T cells demonstrated a pattern of surface memory marker expression that is distinct compared with CD107a/b- and IFN-gamma-positive CD8+ T cell populations (p < 0.0001). Our study describes a distinct population of IL-10-positive CD8+ T cells that may play a role in HIV-associated immune dysfunction.     

HIV-specific CD8+ lymphocytes in semen are not associated with reduced HIV shedding

Sexual contact with HIV-infected semen is a major driving force behind the global HIV pandemic. Little is known regarding the immune correlates of virus shedding in this compartment, although HIV-1-specific CD8+ T cells are present in semen. We collected blood and semen from 27 chronically HIV-infected, therapy-naive men without common sexually transmitted infections or urethral inflammation and measured HIV-1 RNA viral load and cytokine/chemokine levels in both compartments. HIV-1 RNA levels were 10-fold higher in blood than semen, but discordantly high semen shedding was associated with higher semen levels of the proinflammatory cytokines IL-6, IL-8, IL-12, and IFN-gamma. Virus-specific CD8+ T cell epitopes were mapped in blood by IFN-gamma ELISPOT, using an overlapping HIV-1 clade B peptide matrix, and blood and semen CD8+ T cell responses were then assayed ex vivo using intracellular IFN-gamma staining. HIV-specific CD8+ responses were detected in 70% of semen samples, and their frequency was similar to or higher than blood. There was no correlation between the presence of virus-specific CD8+ T cells in semen and levels of HIV-1 RNA shedding. Among participants with detectable CD8+ IFN-gamma semen responses, their relative frequency was not associated with reduced HIV-1 RNA shedding, and their absolute number was correlated with higher levels of HIV-1 RNA semen shedding (r = 0.6; p = 0.03) and of several proinflammatory cytokines. Neither the presence nor the frequency of semen HIV-specific CD8+ T cell IFN-gamma responses in semen correlated with reduced levels of HIV RNA in semen.     

Cutting edge: RANTES regulates Fas ligand expression and killing by HIV-specific CD8 cytotoxic T cells.

Based on the previous observation that RANTES mediates the cytotoxic activity of human HIV-specific CD8+ T cells via the chemokine receptor CCR3, we studied the effect of this chemokine on different effector CD8+ cytolytic cells requiring Fas/Fas ligand (FasL) or perforin-dependent pathway. In CTLs derived from PBMCs of HIV-infected patients, both the spontaneous and the RANTES-induced cytotoxicity were inhibited by anti-FasL neutralizing Abs. In contrast, allogeneic CTLs or NK cells killing through perforin were not affected by RANTES and anti-FasL Ab. Accordingly, RANTES enhanced the expression of FasL in a concentration- and time-dependent manner in HIV-specific CTLs, whereas anti-RANTES Ab decreased markedly FasL expression. Finally, cell surface expression of FasL protein in HIV-specific CTLs was also up-regulated by eotaxin, a selective ligand for CCR3. Our observations show that the action of RANTES via CCR3 is necessary to regulate FasL expression on HIV-specific CD8+ T cells that kill through the Fas/FasL pathway.     

Ligation of CD80 is critical for high-level CD25 expression on CD8+ T lymphocytes

CD80 and CD86 have been shown to play a critical role in the optimal activation of T cells. Although these two molecules bind the same ligand, CD28, the question of whether CD80 and CD86 provide unique signals or serve redundant roles remains controversial. Previous studies have suggested that CD80 binding to CD28 may be superior to CD86 for the activation of naive CD8+ T cells. This study provides a potential mechanism to explain these observations. Our study demonstrates a previously unappreciated role for CD80, its superiority over CD86 in promoting CD25 expression, increasing both the number of cells that express CD25 and the level expressed on a per cell basis. These findings provide new insights into the role of CD80 vs CD86 and have important implications for the design of vaccines and immunotherapeutics aimed at the generation of a robust CD8+ T cell response in vivo.     

Lack of adenosine deaminase activity in cultured murine cytotoxic T lymphocytes

The level of adenosine deaminase (ADA) activity was investigated in various populations of IL 2-dependent, cultured cytotoxic T lymphocytes (CTL), from bulk cultures as well as from CTL lines (CTL-A and CTL-B types). The study of C57BL/6 derived, cytotoxic bulk cultures yielded the following mean values of ADA activity: 12,500 U/mg in the cortical, immature region of the thymus, 1500 U/mg in the immunocompetent, cortisone-resistant medullary thymocytes, and 2000 U/mg in the T cell population from the spleen. These results are in agreement with previous studies on separated T lymphocyte populations of known origin and further indicate that a fall in ADA activity accompanies T cell maturation. ADA activity was measured in C57BL/6-derived CTL-A lines obtained from the thymic and splenic bulk cultures. All lines were characterized by a very low level of ADA activity, compared with the T cell bulk cultures freshly initiated from the thymic medulla or from the spleen, and to a variety of T tumor lines established in long term culture. Some showed undetectable ADA activity (less than or equal to 20 units/mg), whereas others maintained significant activity (50 to 500 U/mg). No correlation was found between the residual ADA activity level and the killing activity, at the time of the enzyme assay. Identical properties were observed for CTL-B cloned lines of various genetic backgrounds. These results suggest that the level of ADA activity of the CTL in the mouse is lower than the average value of mature T cells of the thymic medulla, and might constitute a differentiation marker specific to the CTL population. A possibility remains that low ADA activity levels in these CTL lines may be the consequence of an extinction of the ADA gene during in vitro growth, as it is observed for the cytotoxic activity itself. In either case, a low ADA activity level is a remarkable property of IL 2-dependent CTL clones, when compared to various established T tumor lines, which exhibit high and stable ADA levels during long term in vitro growth (5000 to 15,000 U/mg).     

Functional analysis of HIV-specific cytotoxic T lymphocytes in antiviral-treated- and-naive patients: a preliminary report

HIV-specific CTL functions were analyzed in HIV-infected individuals who did or did not receive antiretroviral therapy (ART). Results showed that gp 160 (env)-stimulated perforin- and granzyme-expressing CTL, as well as perforin and granzyme-specific mRNA, were reduced in treated patients whereas TNFalpha was increased in ART-treated compared to naive individuals. Reduction of perforin and granzyme-expressing cells was not secondary to impaired IFNgamma production. A defect of CTL is observed in ART-treated individuals; this defect is not dependent on impaired Th cell function. These results reinforce the need for immunomodulants to successfully approach therapy of HIV infection.     

4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.     

Lack of correlation between cytotoxic T lymphocytes and lethal murine lymphocytic choriomeningitis

Adoptive transfer of lymph node and spleen cells from mice infected with LCM virus to similarly infected immunocompromised recipients has been the classic way to demonstrate the lethal role of T cells in the CNS disease caused by this virus. Isolation and adoptive transfer techniques are presented here which show that Thy-1+ cells isolated from the meningeal infiltrates (MI) of LCM virus-infected mice possess this property. We compared various T cell functions of MI cells taken from mice infected with two strains of LCM virus differing markedly in their pathogenicities. One of these strains, termed aggressive, caused a typical, invariably fatal, CNS disease within 7 to 10 days after infection. The other virus, termed docile, killed few mice after the standard intracerebral inoculation, and could persist in the mice for 6 mo or more. The yields of MI leukocytes from mice infected with docile virus varied from 50 to 100% of those found in mice infected with aggressive virus (3 X 10(6) cells/brain). On a cell-to-cell basis, the CTL activity in the MI of mice infected with docile virus ranged from 50 to 100% of that found in the MI of mice infected with aggressive virus. MI cells from mice infected with aggressive virus consistently caused lethal disease by adoptive transfer into immunocompromised (irradiated) recipients infected with either strain of virus. All attempts to induce lethal disease by adoptive transfer of MI cells (or splenocytes) from mice infected with docile virus into irradiated recipients failed. The latter experiments with the docile-MI cells were performed with six times the number of aggressive-MI cells needed to kill irradiated recipients by adoptive transfer. The possible reasons for this discordance between CTL and in vivo killer function are discussed.     

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.     

Relative dominance of Gag p24-specific cytotoxic T lymphocytes is associated with human immunodeficiency virus control

Conflicting data on the role of total virus- and protein-specific cytotoxic-T-lymphocyte (CTL) responses in the control of human immunodeficiency virus (HIV) disease progression exist. We present data generated from a Peruvian cohort of untreated, clade B-infected subjects, demonstrating that the proportion of Gag-specific, and in particular p24-reactive, CTL responses among the total virus-specific CTL activity is associated with individuals' CD4 counts and viral loads. Analyses in a second cohort in the United States confirm these findings and point towards a dominant role of Gag-specific immunity in effective control of HIV infection, providing important guidance for HIV vaccine development.     

Circulating cytotoxic CD8+ CD28- T cells in ankylosing spondylitis

Circulating CD8⁺ CD28^- T cells were found to be expanded more in patients with ankylosing spondylitis than in an age-matched healthy population (41.2 ± 17.7% versus 18.6 ± 7.6%). The level of CD8⁺CD28^- T cells was dependent on the disease status, but was independent of age. Most of the CD8⁺ CD28^- T cells produced perforin after stimulation in vitro, in contrast to their CD8⁺CD28⁺ counterparts. From the clinical perspective, the percentage of the cytotoxic CD8⁺ CD28^- T cells reflected a more severe course of disease, as it correlated with distinct movement restrictions, as well as the metrology score summarizing cervical rotation (in sitting position), chin-to-jugulum distance, thoracic Schober, chest expansion, and fingers-to-floor distance (P = 0.032).