Karen F.T. Copeland

CD8+ T-Cells: Function and Response to HIV Infection

CD8+ T-cells are a critical component of the cellular immune response and they play an important role in the control of viral infection. During HIV infection, CD8+ T-cells are able to recognize infected cells through an MHC-I dependent process and are able to lyse cells harboring viral infection by the secretion of perforin and granzymes. These cytotoxic T-lymphocytes (CTL) can also eliminate virally infected cells through the engagement of death-inducing ligands expressed by CD8+ T-cells with death receptors on the surface of the infected cell. In addition, CD8+ CTL secrete soluble factors such as beta-chemokines and the CD8+ antiviral factor (CAF) that suppress viral binding and transcription, respectively. In order for HIV to survive the pressures placed upon it by the immune system, the virus has adopted numerous strategies to evade the CD8+ T-cell response. The high mutation rate of HIV has allowed the virus to escape CD8+ T-cell recognition in addition to its ability to down-regulate surface MHC-I expression from infected cells. Also, by altering the pattern of cytokine production and engagement of cellular receptors, HIV disrupts proper CD8+ T-cell signaling. The resultant improper T-cell receptor (TcR) stimulation creates an anergic state in these cells. By affecting the function of CD4+ T-cells and antigen presenting cells that are required for proper CD8+ T-cell maturation, HIV is able to decrease the circulating pool of effector and memory CD8+ T-cells that are able to combat viral infection. The end result is the aberration of CD8+ T-cell function.

Cd8+ T-cell-mediated suppression of Hiv-1 long terminal repeat-driven gene expression is not modulated by the Cc chemokines Rantes, macrophage inflammatory protein (mip)-1α and Mip-1β

Objective:To assess the role of RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β in modulation of HIV-1 long terminal repeat (LTR)-mediated gene expression and determine whether these chemokines share identity with CD8+ T-lymphocyte-derived HIV-1 LTR-suppressive factors. Design:HIV-1 LTR-directed reporter gene expression is a model for transcription that is susceptible to inhibition by factors produced by CD8+ lymphocytes of HIV-1-infected individuals. The effect of recombinant chemokines on LTR-directed gene expression was examined. The ability of chemokines found to be present in CD8 supernatants to suppress HIV-1 LTR-mediated gene expression was determined by antibody inhibition assays. Methods:The concentrations of RANTES, MIP-1α and MIP-1β in a panel of CD8+ T-lymphocyte-derived supernatants were determined by enzyme-linked immunosorbent assay. Recombinant chemokines were added to freshly transfected (pLTR-CAT and pSV40-tat) human Jurkat T cells. Excessive polyclonal neutralizing antibodies to these chemokines were added to transfected Jurkat T cells cultured in the presence of strongly inhibitory CD8+ T-cell-derived supernatants with known chemokine concentrations. Results:The concentrations of RANTES, MIP-1α and MIP-1β in a panel of CD8+ lymphocyte-derived supernatants were found to correlate with their relative ability to suppress the LTR-mediated gene expression (r = 0.679, 0.764 and 0.48, respectively). The addition of recombinant CC chemokines had no effect over a broad range of doses on HIV-1 LTR-mediated gene expression. The CD8-suppressive effect on HIV-1 LTR-driven gene expression was not abrogated by a combination of antibodies to RANTES, MIP-1α and MIP-1β. Conclusions:RANTES, MIP-1α and MIP-1β do not alter HIV-1 LTR-directed gene expression at doses up to 100 ng/ml. Although present in varying concentrations in supernatants derived from CD8+ lymphocytes from HIV-positive individuals, these chemokines are not responsible for the powerful CD8-derived suppressive effect on HIV-1 LTR-mediated gene expression observed in our system.

RANTES Production by T Cells and CD8-Mediated Inhibition of Human Immunodeficiency Virus Gene Expression before Initiation of Potent Antiretroviral Therapy Predict Sustained Suppression of Viral Replication

A prospective blinded study was conducted to determine whether immunological differences exist between patients receiving potent antiretroviral therapy who are able to achieve and maintain an undetectable virus load (<50 copies/mL) and those who are not. Eleven patients receiving protease inhibitor-containing antiretroviral therapy were studied for 1 year. After analysis of all baseline samples, patient virus load was disclosed, and patients were classified as suppressors (those who maintained undetectable virus load for 1 year) and nonsuppressors. Baseline virus load and CD4+ T cell count did not differ significantly between the 2 groups. Levels of RANTES production by CD4+ and CD8+ T cells and CD8-mediated inhibition of human immunodeficiency virus type 1 gene expression before initiation of antiretroviral therapy were significantly associated with an undetectable virus load maintained for 1 year (P<.05). Thus, a functionally intact T cell-mediated immune system at the time of initiation of potent antiretroviral therapy may predict long-term virus suppression.

T cell-derived suppressive activity: evidence of autocrine noncytolytic control of HIV type 1 transcription and replication.

The ability of CD8+ T lymphocytes to suppress the transcription and replication of HIV-1 is well documented. We have demonstrated that the factor(s) responsible for the suppression of HIV-1 LTR-mediated gene expression are not the CC chemokines RANTES, MIP-1alpha, and MIP-1beta. Interestingly, these and other chemokines and cytokines are produced by both CD8+ and CD4+ T lymphocytes. On the presumption that CD4+ T lymphocytes may also be able to modulate HIV-1 expression in vitro we assessed the LTR-modulatory effects of a panel of culture supernatants derived from stimulated CD4+ T lymphocytes from HIV-positive patients and uninfected controls. Supernatants of both CD4+ and CD8+ T cells mediated a suppression of LTR-driven gene expression in Jurkat T cells and an enhancement of gene expression in U38 monocytic cells. On the basis of these results, and using a herpesvirus saimiri (HVS)-transformed CD4+ T lymphocyte clone (HVSCD4), we demonstrate that both suppressive and enhancing effects are dose dependent. Furthermore, we have shown that supernatants of both HVSCD4 and HVSCD8 cells suppress LTR-mediated gene expression and HIV-1 replication in transfected/infected T cells. In U1 monocytic cells, supernatants of both CD4+ and CD8+ lymphocytes from an HIV-1-infected individual enhanced LTR-mediated gene expression, HIV-1 replication, and TNF-alpha production. However, only these effects as induced by CD8+ T cells were sensitive to the G protein inhibitor pertussis toxin. These results indicate that factors produced by both CD4+ and CD8+ T cells exert dichotomous effects on HIV-1 gene expression and replication in T cells and monocytes.

Proportion of HIV-1 Infected CD8+CD4- T Lymphocytes In Vivo

The proportion and significance of HIV-1 infection of CD8+ T-cells was examined in a patient cohort of HIV-1 seropositive (n=28) and seronegative individuals (n=4). It was hypothesized that irrespective of the clinical status of the patients, productively HIV-1 infected CD8+ T-cells would be found and these cells would contribute to the plasma viral load in vivo. Flow cytometric analysis using fluorochrome-conjugated antibodies, RT-PCR analysis using HIV-1(pol) specific primers, and quantification of HIV-1 viral transcripts by ex vivo culture of isolated CD8+ T-cells were employed. In 22 of the 28 patient samples analyzed, a significantly higher proportion of cells with expression of CD8+HIV-1(gag)+ than of CD4+HIV-1(gag)+ T-cells was observed (36.9% +/- 10.0% vs 26.4% +/- 13.1% respectively, p< 0.01). No correlation was observed between absolute CD4 counts, CD8 counts, plasma viral load and CD8+ T cell infection. RT-PCR analysis indicated the presence of HIV-1 transcripts in the ex vivo isolated CD8+ T-cell population. Ex vivo isolated CD8+ T-cells demonstrated productive infection over time. We conclude, with three lines of evidence detecting and measuring HIV-1 infection of CD8+ T-lymphocytes, that this cellular target and reservoir may be central to HIV-1 pathogenesis.

Infection of CD8+CD45RO+ Memory T-Cells by HIV-1 and Their Proliferative Response

CD8+ T-cells are involved in controlling HIV-1 infection by eliminating infected cells and secreting soluble factors that inhibit viral replication. To investigate the mechanism and significance of infection of CD8+ T-cells by HIV-1 in vitro, we examined the susceptibility of these cells and their subsets to infection. CD8+ T-cells supported greater levels of replication with T-cell tropic strains of HIV-1, though viral production was lower than that observed in CD4+ T-cells. CD8+ T-cell infection was found to be productive through ELISA, RT-PCR and flow cytometric analyses. In addition, the CD8+CD45RO+ memory T-cell population supported higher levels of HIV-1 replication than CD8+CD45RA+ naïve T-cells. However, infection of CD8+CD45RO+ T-cells did not affect their proliferative response to the majority of mitogens tested. We conclude, with numerous lines of evidence detecting and measuring infection of CD8+ T-cells and their subsets, that this cellular target and potential reservoir may be central to HIV-1 pathogenesis.

CD8+ T cell-mediated suppression of HIV long terminal repeat-driven gene expression is not associated with improved clinical status

Objectives:To determine the associations between the suppression of HIV-1 long terminal repeat (LTR)-mediated gene expression by CD8+ T-cell supernatants and clinical correlates of well-being, including CD4+ and CD8+ T-cell counts, β-chemokine production and clinical stage of disease. Methods:Culture supernatants of activated CD8+ T cells derived from a panel of HIV-1-infected subjects were assessed for their ability to suppress HIV-1 LTR-mediated chloramphenicol acetyl transferase (CAT) expression. The percentage suppression of gene expression was correlated with CD4+ and CD8+ T-cell counts and clinical stage of infection. Some individuals within this group were followed at 2–3 month intervals over time to assess the consistency of the suppression. Selected CD8+ T-cell culture supernatants of diverse suppressive ability were screened for the levels of the β-chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β and RANTES. Results:The ability of CD8+ T cells of HIV-1-infected subjects to suppress HIV-1 LTR-mediated gene expression did not show a dependence upon high CD4+ T-cell counts or on the clinical stage or duration of infection. The ability to suppress gene expression did show a relationship with higher CD8+ T-cell counts and correlated with the levels of β-chemokines in the culture supernatants. In contrast, strong suppression was mediated by CD8+ T-cell supernatants from some subjects with very low CD8+ T-cell counts and relatively low chemokine levels. Conclusions:Although the suppression of gene expression by CD8+ T-cell culture supernatants showed statistical correlation with β-chemokine levels and with higher CD8+ T-cell count, no correlation could be found with correlates of clinical well-being.

Calcium-mediated inhibition of phorbol ester and Tax trans-activation of the human T cell leukaemia virus type 1

Human Jurkat T cells containing a stably integrated human T cell leukaemia virus type 1 (HTLV-1) long terminal repeat (LTR) reporter gene construct were used to study the role of calcium-dependent cellular activation pathways in LTR trans-activation. Treatment of these cells with the calcium ionophore ionomycin resulted in a reduced basal response of the LTR and reduced responses to 12-O-tetradecanoylphorbol-13-acetate-and Tax-mediated trans-activation. This effect was also observed for virus production in the HTLV-1-producing T cell line MT-2. Experiments designed to determine the events underlying this inhibition, using inhibitors of calcium-related events, revealed that the ionomycin-induced repression of the LTR was alleviated in all cases by cyclosporin A. This compound was also effective in preventing the ionomycin-induced reduction in virus production in MT-2 cells. These results suggest a role for calcium-related events in the down-regulation of HTLV-1 expression.

Human immunodeficiency virus-1 infection protects against a Tc1-to-Tc2 shift in CD8(+) T cells.

Despite the reports of dysfunction of the lytic abilities of CD8(+) T cells during human immunodeficiency virus-1 (HIV-1) disease progression, the effects of infection on the noncytolytic functions of CD8(+) T cells have not been well characterized to date. We examined the effect of HIV-1 infection on the cytokine and chemokine responses of peripheral blood-derived CD8(+) T cells in an in vitro system. Activation of HIV-1-infected CD8(+) T cells with phytohemagglutinin resulted in a 4- to 8-fold increase in the production of macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation normal T-cell expressed and secreted, and interleukin (IL)-16. Treatment of activated HIV-1-infected CD8(+) T cells with anti-CD3 monoclonal (M) antibody (Ab) and IL-15 induced strong production of interferon-γ (IFN-γ). Treatment of cells with anti-IL-12 MAb and IL-4 to induce a Tc1-to-Tc2 shift resulted in no change in viral production levels or IFN-γ production within the HIV-1-infected CD8(+) T cell population. Initiation of a Tc2-to-Tc1 shift resulted in a 6-fold increase in HIV-1 replication and 2- to 3-fold higher levels of IFN-γ, demonstrating that infection can protect against a Tc1-to-Tc2 shift in CD8(+) T cells.

Production of CD8+ T cell nonlytic suppressive factors by CD28, CD38, and HLA-DR subpopulations.

HIV infection may be modified by CD8(+) T cells by the production of nonlytic antiviral factors. To determine subpopulations that mediate nonlytic, antiviral activity, we examined the production of beta chemokines and of CD8 antiviral factor (CAF) by different subsets, using CD8(+) cells derived from 24 HIV-1-infected and 25 uninfected individuals. Subjects with CD8(+) cell counts greater than 200/microl produced increased levels of MIP-1alpha by CD8(+)CD28(+), CD8(+)CD38(-), and CD8(+)HLA-DR(+) subsets as compared with uninfected controls. CD8(+)CD38(-) cells produced higher levels of MIP-1beta and RANTES. CAF production was increased by CD8(+)CD38(+) and CD8(+)HLA-DR(+) cells of HIV-infected individuals as compared with uninfected controls. Chemokine production was increased by cells that do not express activation markers, whereas CAF activity was increased by cells expressing CD38 or HLA-DR. These findings shed light on CD8(+) T cell noncytotoxic antiviral factor production during HIV infection.