CD8+ T cell-mediated suppressive activity inhibits HIV-1 after virus entry with kinetics indicating effects on virus gene expression

Abstract

Individuals infected with HIV-1 have varying rates of progression to AIDS. Cellular immune responses, comprised of cytolytic and noncytolytic CD8(+) T cell effector functions, are considered important for controlling viremia and maintaining the clinically asymptomatic state. Although there is general agreement regarding CD8(+) T lymphocyte cytotoxic functions, considerable controversy exists over the nature of the noncytolytic antiviral activity of CD8(+) cells. The discovery that RANTES (regulated on activation, normal T cell expressed and secreted), MIP-1alpha, and MIP-1beta (macrophage inflammatory protein 1 alpha and beta) could inhibit HIV-1 replication by blocking viral entry processes led to the notion that these molecules are responsible for the CD8(+) cell suppressive activity. However, T tropic HIV isolates requiring the CXCR4 coreceptor for entry are insensitive to the antiviral effects of these beta-chemokines. Using a CXCR4-dependent virus, we determined that the mechanism of CD8(+) T cell-mediated activity did act after viral entry into the host cell. We also define the kinetics of the HIV life cycle in primary activated human CD4(+)-enriched T cells by using an HIV-1 reporter virus system pseudotyped with the CXCR4-dependent HIV-1 envelope gene of NL4-3. Analysis of these kinetic data indicates that CD8(+) T cell-mediated suppressive activity acts at a stage in the viral life cycle after entry and independently of the HIV envelope. Additionally, we show that the antiviral activity targets stages of the virus life cycle correlating with transcription and early proviral gene expression. These findings not only provide a range of possible targets for the CD8(+) T cell-mediated activity but also support the notion that this antiviral activity is multifactorial in nature.

Figure 1

(A) Transformed CD8⁺ T cells from an asymptomatic HIV⁺ individual suppress CCR5-dependent HIV-1 (QZ4734) and CXCR4-dependent HIV-1 (IIIB) in a quantitative CD8⁺ suppression assay. Closed symbols, transformed CD8⁺ T cells from an asymptomatic HIV⁺ individual; open symbols, CD8⁺ T cells from a pool of HIV⁻ donors; circles, R5 virus; squares, X4 virus. The vertical axis indicates log reduction in virus titer, defined as virus infectivity in the presence (V_n) divided by the virus infectivity in the absence (V_o) of CD8⁺ lymphocytes. Asterisks (*) indicate actual tissue culture 50% infective dose per ml below detection limits at <25. These results (day 13) are representative of three experiments performed in duplicate. (B) CD8⁺ T cells from an HIV⁺ asymptomatic individual suppress X4 HIV-1 in a single-cycle infection assay. CD4⁺-enriched T lymphocytes were infected with a reporter virus pseudotyped with the CXCR4-dependent envelope NL4-3. Either transformed CD8⁺ T cells from an asymptomatic HIV⁺ individual (hatched bars) or CD8⁺ T cells from a pool of HIV⁻ donors (solid bars) were added at increasing E:T. The results are representative of three experiments performed in duplicate. RLU, relative light units; JR-HVS, a patient-derived herpesvirus saimiri-transformed cell line.

Figure 2

Kinetics of X4 HIV-1 infection in primary CD4⁺ T lymphocytes. Inhibitors of the different stages of the HIV life cycle were added at particular time intervals after HIV-1 infection. Infection in the presence of these inhibitors was compared with the control infection (absence of inhibitors) to determine the relative time of action for each inhibitor. The results shown are an average of two to six experiments performed in duplicate.

Figure 3

(A) Kinetics of CD8⁺ T cell-mediated suppression and X4 HIV-1 entry in primary CD4⁺ T lymphocytes. CD4⁺-enriched T lymphocytes were infected with X4 HIV pseudotyped virus (NL4–3). Two agents that inhibit viral entry were added at different times after infection. DP178 is an HR2 peptide mimetic that blocks gp41-mediated fusion. Anti-CD4⁺ mAb #19 blocks binding of gp120 to CD4⁺. Transformed CD8⁺ T cells from an asymptomatic HIV⁺ individual (hatched bars) or CD8⁺ T cells from a pool of HIV⁻ donors (solid bars) were added at a 2:1 E:T at various times after infection. At 24 h, the CD8⁺ effectors had a 94.3% inhibition of control infection that was limited to 77% at 48 h. The results shown are the average of two or three experiments performed in duplicate. RLU, relative light units. (B) CD8⁺ suppression is independent of HIV-1 envelope-mediated entry. CD4⁺-enriched T lymphocytes were infected with a reporter virus pseudotyped with the A-MLV envelope for 2 h, and then the virus was removed. HVS-transformed CD8⁺ T cells with X4 suppressive activity (JR-HVS and HS-HVS, hatched bars) or HVS-transformed CD8⁺ T cells without X4 suppressive activity (WS-HVS, solid bars), were added at a 2:1 E:T after removal of the virus. Percentage of infection is calculated based on infection of the CD4⁺ targets without CD8⁺ T cells (100%). Results are an average of three experiments for JR-HVS (5.2%) and HS-HVS CD8⁺ effectors (9%) and two experiments for the WS-HVS CD8⁺ cells (95%).

Figure 4

Late time course of CD8⁺ T cell-mediated suppression and tat-mediated transactivation. CD4⁺-enriched T lymphocytes were infected with X4 HIV pseudotyped virus (NL4-3). Either a tat inhibitor or JR-HVS CD8⁺ effectors were added up to 60 h after infection. The results shown are an average of three experiments performed in duplicate.

Figure 5

Kinetic model of the targets of CD8⁺ T cell-mediated anti-HIV activity. In primary CD4⁺-enriched T lymphocytes, entry occurs within 2–6 h, and reverse transcription (RT) occurs up to about 14 h. At 24–72 h, transcription from the integrated provirus and expression of gene products take place. CD8⁺ T cell-mediated suppression of HIV-1 targets those stages of the virus life cycle involving transcription and early gene expression.