Despite more than 15 years of research, the mechanism(s) through which HIV induces progressive qualitative and quantitative abnormalities in the immune system, ultimately leading to the clinical manifestations of AIDS, remains unclear. The relative contributions to immune destruction by direct lysis of HIV-infected cells and indirect pathogenic mechanisms, mediated by noninfectious virions or viral proteins, remain controversial. Past in vitro studies intended to clarify mechanisms of HIV immunopathogenesis have been confounded by an inability to distinguish effects due to infection and noninfectious virions and/or the use of high concentrations of recombinant viral proteins that do not accurately simulate likely in vivo conditions. In the present study, using HIV virions rendered noninfectious by a novel method that preserves the structure and function of virion surface proteins, we have conclusively shown that concentrations of noninfectious virions within the range found in the plasma of infected individuals can have dramatic pleiotropic effects on T lymphocytes.
The noninfectious virions triggered both CD4+
T lymphocytes to die via an apoptotic mechanism (Fig. and ). Triggering of apoptosis required the conformational, oligomeric integrity of the virions, as heat-denatured virions or equivalent concentrations of soluble rgp120 did not induce activation or trigger apoptosis in the responding T cells (Fig. ). In fact, the concentration of rgp120 required to trigger apoptosis was 100,000- to 1,000,000-fold higher than the gp120 levels present on the virions that induced apoptosis (Fig. A and B). At least one other group has examined the effect of noninfectious, nondenatured HIV particles on unstimulated PBMC-T cells. Protease-defective HIV particles, termed L-2 particles, derived from a chronically HIV-1 infected MOLT-4 cell line, form syncytia in T-cell lines (52
) and trigger apoptosis in CD4+
T lymphocytes (37
). However, these particles have multiple genetic, morphologic, and biochemical abnormalities relative to wild-type particles, including much higher levels of gp120, and lack MHC class II (9
), making it difficult to compare directly to the present results. Nevertheless, these findings and our data strongly suggest that the structural integrity of the virion and the conformation of the gp120 on the virus were significant in triggering apoptosis.
Surprisingly, the conformationally authentic noninfectious virions partially activated and triggered apoptosis not only in CD4+
T cells, but also in the CD8+
T lymphocytes (Fig. ). Thus, the activation and the apoptosis of the responding T lymphocytes cannot be simply a consequence of the binding of gp120 to CD4. One possible mechanism to explain how the CD8+
T cells were dying is that MHC class I present on the virus was interacting with CD8 on the T cell to trigger apoptosis. Another possibility is that noninfectious virions were binding to CD4-positive T cells or macrophages that became activated to secrete tumor necrosis factor alpha (TNF-α) or other cytotoxic cytokines that induced apoptosis in the CD8+
T cells, as suggested by Herbein et al. (33
). Regardless of the exact mechanisms employed, the current results underscore the fact that the virus has evolved strategies to effectively disable the key immunoregulatory (CD4+
T cells) and effector (CD8+
T cells) T cells used by the host to control infection (39
The propensity of the virions to induce apoptosis and deplete the T cells did not correlate with the levels of gp120 on virions (Fig. A and B), but correlated better with whether the virus was propagated from an MHC-expressing cell line or not (Fig. B). The maximum apoptotic effect induced by the noninfectious virions was MHC dependent (Fig. and ) but not HLA-DR restricted (Table ). On average, the combination of gp120 and MHC on the virion had a net effect of cell death on CD4+ T cells, whereas treatment of PBMC with MHC-negative virions resulted in a net increase in CD4+ T cells despite extensive apoptosis (Fig. A and Table ). Although both MHC-positive and -negative virions triggered high levels of apoptosis (Fig. and Table ), the absolute number of CD4+ T lymphocytes is the difference between the growth of CD4+ T cells and the death of the CD4+ T cells. Thus, the MHC-negative virions stimulated expansion of CD4+ T cells in excess of the concomitant loss through apoptosis, with the expansion being particularly notable in certain donors. In contrast, for the MHC-positive virions, the net effect was apoptotic depletion (Fig. and Table ). The exact biochemical mechanisms that MHC-positive and -negative virions use to trigger apoptosis and whether virion-associated MHC class I or class II can present antigen to responding T cells remain to be determined.
The regions of gp120 (CD4 binding, coreceptor binding, or both) that are important for triggering apoptosis are also not known. Clearly, virion-associated gp120 is important, as MHC-negative virions stimulated proliferation and triggered apoptosis and MHC-positive microvesicles did not (Fig. and Table ). Preliminary studies with virions of the HIV isolate HIV-1IIIBx
, which has reduced CD4 dependency (40
), suggest that the CD4 binding domain is important for triggering apoptosis in CD4+
T cells. We are currently producing and characterizing AT-2-inactivated virions from CCR5-tropic isolates to determine whether coreceptor binding is involved in triggering cell death of T lymphocytes.
We are also investigating both the cellular and biochemical mechanisms that the AT-2-inactivated virions use to trigger cell death in CD4+
T lymphocytes. We did not observe formation of syncytia in the PBMC cultures incubated with either the infectious or noninfectious HIV-1 virions, indicating that cell death occurred at the single-cell level. Several different cellular mechanisms have been proposed to account for the increased apoptosis and cell death observed in T lymphocytes from HIV-infected individuals. These include the direct lytic effect of replicating virus (26
), soluble gp120 binding to CD4 to trigger an apoptotic or anergic pathway (10
), activated CD4+
T cells killing both CD4+
T cells (37
), HIV-induced CD8+
lymphokine-activated killers killing CD4+
T cells (76
), infected macrophages expressing FasL killing Fas-positive CD4+
T cells (8
), and soluble factors such as TNF-α (33
) or TRAIL (38
) inducing apoptosis in uninfected bystander T cells. We are currently performing lymphocyte subset fractionation experiments, transwell experiments, and cytokine blocking experiments to determine the cellular mechanism(s) responsible for the apoptosis triggered by the noninfectious virions.
The biochemical signaling pathways activated by the AT-2-inactivated virions to trigger cell death are also not known. The TNF family of death receptors are implicated in the apoptosis of HIV-infected T lymphocytes, but there is controversy regarding whether FasL/Fas-mediated apoptosis or other mechanisms are responsible for the cell death observed in vitro or in vivo. Increased FasL and Fas expression on T lymphocytes has been reported after HIV infection and correlates with increased cell death (3
), but it is not clear whether apoptosis of the T cells is FasL/Fas dependent (26
) or is mediated by other factors such as TRAIL (38
), TNF family members (33
), or ICE/caspase 1-dependent signaling pathways (28
). We observed that the AT-2-inactivated virions dramatically induced both FasL and Fas expression (Fig. G and H) and TNF-α secretion (data not shown). Surprisingly, in preliminary studies, neutralizing antibodies to FasL/Fas or TNF-α/β did not prevent apoptosis, suggesting an alternative mechanism. In all of these studies, AT-2-inactivated virions should prove a useful tool for resolving the cellular and biochemical mechanisms that HIV uses to kill T lymphocytes.
Several lines of evidence suggest that the incorporation of MHC molecules into HIV virions is not random, but rather represents an adaptation developed by the virus to modulate host immune responses. It is well documented that high levels of MHC class I and MHC class II are incorporated into HIV-1 virions grown in vitro (5
) or isolated from patient plasma (42
) and that the incorporation of envelope glycoproteins and MHC molecules into virions is linked (62
). The hypothesis that HIV is capable of evading the immune response by incorporating MHC molecules into its envelope is not unprecedented. MHC class II is present on the virions of the feline leukemia virus (7
), Friend murine leukemia virus (13
), human tumor leukemia virus (1
), and simian retrovirus and simian immunodeficiency virus (54
). Furthermore, it has been shown that soluble MHC class I or MHC class II proteins can induce anergy or apoptosis in CD8+
T lymphocytes, respectively (49
). Previous work has shown that the MHC class II molecules on the HIV virion can present a superantigen to responding CD4 T cells (66
). We have also observed that the MHC-positive, noninfectious virions suppressed tetanus antibody production in an ex vivo tonsil histoculture system whereas MHC-negative, AT-2-inactivated virions did not (A. Sylwester, unpublished data). It is worth noting that virus isolated from HIV-infected individuals early in infection appears to be MHC class II negative, whereas virus isolated late in infection or in AIDS has high levels of MHC class II (41
). Taken together, these data suggest that HIV and perhaps other retroviruses may incorporate HLA molecules as a mechanism to foil the immune response and that virion-associated MHC plays a role either directly or indirectly in HIV pathogenesis.
In summary, we have shown that conformationally authentic, noninfectious HIV-1 virions can partially activate both CD4+ and CD8+ T lymphocytes and trigger an apoptotic form of cell death. T-cell activation and apoptosis required the conformational integrity of the virus, as heat-denatured or equivalent amounts of rgp120 did not induce apoptosis. Virion-associated MHC molecules potentiated cell death. These results suggest that MHC-containing noninfectious virions produced during HIV infection could impact immune responsiveness. Inactivation of HIV with AT-2 is a simple, general method for inactivating any HIV isolate. AT-2-inactivated virions of HIV isolates with envelope glycoproteins having defined genotypic and phenotypic properties will provide the research community with a powerful tool to further dissect the underlying mechanisms of HIV immunopathogenesis.