Nef interference with HIV-1-specific CTL antiviral activity is epitope specific

Abstract

HIV-1 Nef and HIV-1-specific cytotoxic T lymphocytes (CTLs) have important and opposing roles in the immunopathogenesis of HIV-1 infection. Nef-mediated down-modulation of HLA class I on infected cells can confer resistance to CTL clearance, but the factors determining the efficiency of this process are unknown. This study examines the impact of Nef on the antiviral activity of several CTL clones recognizing epitopes from early and late HIV-1 proteins, restricted by HLA-A, -B, and -C molecules. CTL-targeting epitopes in early proteins remained susceptible to the effects of Nef, although possibly to a lesser degree than CTL-targeting late protein epitopes, indicating that significant Nef-mediated HLA down-regulation can precede even the presentation of early protein-derived epitopes. However, HLA-C-restricted CTLs were unaffected by Nef, consistent with down-regulation of cell-surface HLA-A and -B but not HLA-C molecules. Thus, CTLs vary dramatically in their susceptibility to Nef interference, suggesting differences in the relative importance of HLA-A- and HLA-B- versus HLA-C-restricted CTLs in vivo. The data thus indicate that HLA-C-restricted CTLs may have an under-appreciated antiviral role in the setting of Nef in vivo and suggest a benefit of promoting HLA-C-restricted CTLs for immunotherapy or vaccine development.

Figure 1.

Measurement of Nef effects on the antiviral activities of CTL clones. CTL clones 42758-RL10-3.22 (Rev specific, B*07 restricted) and S16-RY11-10.41 (Nef specific, C*07 restricted) were tested for inhibition of NL4-3–based viruses containing wild-type Nef or Nef with the M20A mutation (rendering it unable to down-regulate HLA class I) in parallel. The target cells were acutely infected Jurkat cells (expressing both B*07 and C*07). Replication as assessed by measuring supernatant p24 antigen (log units picograms/milliliter) is plotted over time. (A) For clone 42758-RL10-3.22, inhibition of the wild-type virus at day 6 was 0.8 log₁₀ units (6.3-5.5) and inhibition efficiency was 0.13 (0.8 ÷ 6.3). Inhibition of the M20A virus at day 6 was 5.4 log₁₀ units (6.3-0.9) and inhibition efficiency was 0.86 (5.4 ÷ 6.3). Thus, the Nef effect ratio of wild-type to defective Nef virus inhibition was 0.15 (0.13 ÷ 0.86). (B) Similarly for clone S16-RY11-10.41, inhibition efficiency of the wild-type and M20A viruses were 0.49 and 0.59, respectively, yielding a Nef effect ratio of 0.83.

Figure 2.

Reproducibility of Nef effects on the antiviral activities of HIV-1–specific CTL clones on repeat testing. Five CTL clones were tested as described in Figure 1. The means (± SEM) are plotted for multiple independent experiments with each clone. Comparisons revealed that 68A62, S1-SL9-3.23T, 18030D23, and 42758-RL10-3.22 were statistically indistinguishable from each other (2-tailed t test P ≥ .26 for all comparisons between these clones), but clone S16-RY11-10.41 was statistically distinct from the others (P ≤ .01 for all comparisons to the other clones).

Figure 3.

Comparisons of Nef effects on CTL-recognizing late protein versus early protein epitopes. For the 8 epitopes shown (5 from the late proteins RT, Gag, and Env, and 3 from the early proteins Nef and Rev), CTL clones were tested for Nef effects as described in Figure 1. The bar plots represent average results for each epitope. In the case that there were multiple repeats of individual clones (as described in Figure 2 and Table 1), these were averaged; if there were multiple clones from the same HIV-1–infected person, these were then again averaged (to apply equal weight to CTL clones from different persons). Finally, results across all persons were averaged (if clones recognizing the same epitope from multiple persons were tested) to generate the single values plotted for each epitope. MEAN LATE indicates the average for the 5 late protein-derived epitopes (± 1 SD) and MEAN EARLY indicates the average for the 3 early protein-derived epitopes.

Figure 4.

C-restricted CTLs have potent antiviral activity against HIV-1 and are unaffected by Nef-mediated HLA down-regulation. (A) CTL clones KS7 (Env specific, B*07 restricted) and SE7/126E (Env specific, C*15 restricted) were compared for their abilities to inhibit HIV-1 with wild-type or deleted Nef. The target cells were primary CD4⁺ T lymphocytes from a seronegative B*07⁺/C*15⁺ donor, and the assay was otherwise performed as in Figure 1. The potent level of inhibition of replication seen here for clone SE7/126E (and clone S16-RY11-10.41 in Figure 1) was typical of multiple experiments with HLA-C–restricted CTL clones. (B) Nef effects on the A- and B-restricted CTL epitopes shown in Figure 3 were compared to several C-restricted clones (listed in Table 1) that were tested and summarized in the same manner. The values for the A- and B-restricted CTLs versus the C-restricted CTLs were significantly different (2-tailed t test, P < .001). MEAN A/B indicates the average of results from HLA-A– and -B–restricted epitopes, and MEAN C indicates the average of results from HLA-C–restricted epitopes. Error bars represent the standard deviation for all A/B- or C-restricted CTLs.