The CD8+ cell noncytotoxic anti-HIV response can be blocked by protease inhibitors

Abstract

CD8+ cells from healthy HIV-infected individuals can suppress HIV replication in infected CD4(+) cells without killing the cells. This CD8+ cell noncytotoxic antiviral response (CNAR), observed by coculture of CD8+ cells with infected CD4+ cells, is associated with secretion of a CD8+ cell antiviral factor (CAF). In attempts to identify CAF, we discovered that certain protease inhibitors, particularly leupeptin, can block, by up to 95%, the anti-HIV activity in CD8+ cell culture fluids as well as inhibit CNAR. The effect is dose-dependent and is observed in up to 70% of the CAF and CNAR assays by using fluids and cells from several different subjects. Pretreatment of CD8+ cells with leupeptin reduces CNAR, further supporting an inhibitory effect on a CD8+ cell product. This inhibitory activity of protease inhibitors does not affect cell growth, expression of activation antigens, or viability of either CD8+ cells or the infected CD4+ cells. The results suggest that a part of the CD8+ cell noncytotoxic response involves the activity of a protease or a protein that interacts with protease inhibitors. Proteolysis of a CD8+ cell product(s) may be involved. This observation offers a promising approach for identifying the mechanism of CNARCAF activity.

Figure 1

Inhibition of CAF activity by the protease inhibitor leupeptin. CD4⁺ T cells, acutely infected with the β-chemokine-insensitive isolate HIV-1_SF2C, were cultured in the presence of a 50% dilution of control medium (■), a CAF-negative CD8⁺ cell supernatant (), or a CAF-positive CD8⁺ cell supernatant (), each pretreated for 30 min with the indicated concentration of leupeptin. The data represent the peak RT activity in fluids from the respective cultures ± 1 SD (28). A representative example of six separate experiments is shown.

Figure 2

Leupeptin inhibition of the CD8⁺ cell-mediated suppression of HIV replication. The CNAR was measured against HIV-1_SF33 acutely infected CD4⁺ T cells in the continued presence of the protease inhibitor leupeptin (A–E) or antipain (C) at the indicated CD8⁺ cell/CD4⁺ cell input ratios. The infected CD4⁺ target cells used were either heterologous (A–C) or autologous (D and E) with respect to the effector CD8⁺ cells, which were either PHA-stimulated (A and D) or nonstimulated (B, C, and E). The concentrations (in micrograms per milliliter) of the protease inhibitor used were 0 (■), 0.1 (), 1 (), and 10 (□) (see Tables 1 and 2 for molar equivalents). HIV replication was monitored every 3 d by measuring RT activity in culture fluid samples (28). The percent suppression of HIV replication was calculated by using RT values in the coculture fluids at the time of peak virus replication, typically on d 6 postinfection (see Materials and Methods). The level of RT activity in the fluids of untreated infected CD4⁺ cell control cultures always reached >200,000 cpm/ml at peak virus replication. The data are representative of three to eight separate experiments for each condition.

Figure 3

The effect of pretreatment of CD8⁺ and CD4⁺ T cells with leupeptin. (A) Nonexogenously stimulated antiviral CD8⁺ effector cells () and infected heterologous CD4⁺ target cells () were separately cultured in the absence (■) or presence of 20 μg/ml (43 μM) leupeptin for 2 d before washing and using in CNAR assays at the indicated CD8⁺ cell/CD4⁺ cell input ratios. The CD4⁺ cells cultured in the absence of leupeptin (■) served as cell targets for the control cocultures. The extent of suppression of HIV replication was determined by using RT levels in culture fluids at the peak of HIV replication, typically 4 d after the initiation of the coculture. The level of RT activity in the culture fluids of treated and untreated CD4⁺ target cells at this time point was 732,000 and 688,000 cpm/ml, respectively. The data are representative of two to three separate studies. (B) Nonexogenously stimulated antiviral CD8⁺ cells were cultured in the presence of various concentrations of leupeptin (micrograms per milliliter) for 2 d before washing and using in CNAR assays with autologous infected CD4⁺ T cells at a CD8⁺ cell/CD4⁺ cell input ratio of 0.5:1; 0 (■), 0.2 (), 2 (), 20 (□). The extent of suppression of HIV replication was determined at the indicated days after initiation of the CNAR coculture assays. After 2 d of culturing the infected CD4⁺ target cells alone (during the CD8⁺ cell pretreatment period), the level of RT activity in the culture fluid was 5,000. Two and 4 d after initiating the CD4⁺/CD8⁺ cocultures, the level of RT activity in the culture fluids of the CD4⁺ cell control wells was 67,000 and 228,000, respectively. The data are representative of two separate studies.