HIV-1-specific CTL exert potent antiviral effects that are mediated by distinct cytotoxic and noncytotoxic mechanisms. Whereas the role of β-chemokines in inhibiting R5 strains of HIV-1 is well established, soluble factors produced by CD8+ cells that inhibit X4 strains of virus are less well defined. In addition, there are few studies that address the relationship between these two effector mechanisms in the inhibition of X4 strains of the virus. Here we show that the noncytolytic, X4 virus-specific antiviral properties of HIV-1-specific CTL and CD8+ cells from seropositive persons have similar characteristics. Both exist in a preformed state within the cells, and both have similar initial kinetics of release following stimulation of cells. In addition, antiviral suppression mediated by both appears to be due to a secreted protein, since it can be inhibited by proteinase K treatment and is heat labile. The fact that the magnitude and kinetics of suppression are significantly different from those observed with CD8+ cells from uninfected persons underscores that this noncytolytic suppression is induced by HIV infection.
Our data indicate that the lower amounts of naive CD8+
cells are responsible for the detected higher release of X4-suppressive factors of HIV-1-seropositive individuals and HIV-specific CTL compared to CD8+
cells of seronegative individuals (Fig. ; Table ) and that the suppressive factor(s) suppresses HIV-1 replication in highly infected CD4+
cells (Fig. ). Additionally, our data directly examine the properties of the inhibitory activity and indicate that the suppressive factor(s) is not likely a cytokine, for a number of reasons. The kinetics of release of the antiviral activity is distinct from the pattern of secretion of cytokines, chemokines, and interleukins. In our in vitro system we found that none of the known X4-suppressive factors (IL-16, MDC, I-309, and SDF-1) (1
) displayed a pattern of release similar to that of the X4-suppressive factor(s) here described. Significant differences in release were observed at both 4 and 16 h after stimulation, comparing CD8+
cells of seronegative individuals with CD8+
cells of seropositive individuals and HIV-1-specific CTL clones (Fig. ). We also tested for cytokines (IFN-γ, TNF-α, and GM-CSF), interleukins (IL-13 and IL-16), and suppressive chemokines (MIP-1α, MIP-1β, and RANTES) known to inhibit R5 viruses (9
), and we found that none of these factors showed a pattern of secretion similar to that of these suppressive factors. Nevertheless, we do show that HIV-1-specific CTL release IL-16, MDC, and I-309, and this can occur in picogram to nanogram amounts (Fig. A). Additionally, these molecules used as recombinant proteins were not able to significantly suppress X4 HIV-1 even in high concentrations (Fig. ). Here we show that of the chemokines tested, only SDF-1 was able to suppress X4 HIV-1 (Fig. ), but this molecule was not detectable by ELISA. Additionally, SDF-1 RNA expression was not found with an SDF-1-specific DNA probe (data not shown), which is consistent with findings of others (17
). Not only was the pattern of secretion of the tested cytokines and chemokines different, but the amounts produced for IL-16, MDC, and I-309 were not substantial enough to explain the measured suppressive activity. To exclude biologically active molecules of these chemokines not distinguished by the ELISA used but possibly responsible for the inhibition measured, we performed studies with neutralizing antibodies against IL-16, MDC, and I-309. None of these antibodies alone or in combination decreased the suppressive activity in the supernatants used, indicating that these molecules are not responsible for the suppressive activity (Fig. ). Additionally, Western blots of the heparin-bound fraction and the 40-kDa heparin-bound Superdex fraction with anti-IL-16, anti-MDC, and anti-I-309 antibodies showed no evidence of these chemokines (Fig. C and Fig. C).
Although the above data suggest that the factor is not a known cytokine or chemokine, a number of experiments support the conclusion that the suppressive factor is a preformed secreted protein. The suppressive activity was found to be 100% degradable by proteinase K and heat (Fig. ). In this respect, it appeared distinct from the 30 to 40-kDa CD8+
; J. A. Levy, personal communication), which has been reported to be heat stable. Additionally, the secretion of the soluble factor(s) was not significantly suppressed with cycloheximide in HIV-seropositive bulk CD8+
= 0.062) and CTL clones (P
= 0.882), whereas it was totally abolished from CD8+
cells of seronegative individuals (P
= 0.010). Thus, de novo
synthesis was necessary to achieve measurable inhibition in supernatants from seronegative CD8+
cells after 4 h of stimulation, but this was not characteristic for seropositive persons (Fig. ). Monensin and brefeldin A treatment decreased the suppression activity, indicating that factor release involves the exocytotic pathway. Additionally, monensin and brefeldin A treatment showed that the suppressive activity was not part of the RANTES-glycoaminoglycan complex (5
) because RANTES was not blocked by monensin and brefeldin A treatment (Table ).
Although we have not precisely identified the active fraction mediating antiviral suppressive activity, our data should facilitate future studies to further elucidate the contributing components. Our data indicate that there is at least one factor with two distinct configurations which differ in size and heparin binding properties. Approximately half of the total suppressive activity is within a heparin-bound fraction that contains proteins with molecular sizes of >50 kDa as determined by Centricon centrifugation. Additionally, the 350 mM heparin-bound fraction could not down-regulate CXCR4 (Fig. ). This excludes a mechanism of inhibition for X4 viruses seen for the chemokines (3
). Also, the 350 mM heparin-bound fraction and the 40-kDa heparin-bound Superdex fraction did not induce a Ca2+
flux (Fig. ). Additionally, the correlation between inhibition seen from the Superdex eluates and the prevalence of a 43-kDa main protein as measured by SDS-PAGE indicates that the chemokines are not responsible for the inhibition. Chemokines are typically much smaller (<10 kDa) and would be expected to bind to heparin and to induce a Ca2+
). A second fraction with suppressive activity did not bind to heparin and had proteins smaller than 50 kDa but larger than 3 kDa, also as determined by Centricon centrifugation (Fig. ). Other studies of CD8+
cell noncytotoxic suppression have not examined the ability to bind to heparin, and so this finding cannot be compared to other published studies. Additionally, the suppressive factor(s) described here may be different from others described in the literature because a different stimulation approach was used compared to the conventional methods with anti-CD3/anti-CD28 and/or PHA and IL-2 stimulation and collection of supernatants 3 to 8 days later. Further experiments will be required to fully define the factors described here, determine their mechanism of inhibition, and establish at which step in the viral life cycle the CD8+
cell factor(s) is active (6
). These data also need to be examined in the context of other studies of non cytolytic inhibition where a CD8+
CAF was reported to be released by baboon CD8+
) or Epstein-Barr virus-specific CD8+
In summary, our data provide a functional link between CTL and CD8+ cell-derived virus-suppressive factors. We hypothesize that the noncytotoxic activity may be particularly important at the level of the local microenvironment, where it may serve an important function in inhibiting the spread of infectious virus.