Although HIV-specific CD8+ T cells have long been suspected of mediating immunologic control in LTNPs, qualitative features of the CD8+ T cell response that clearly segregate with LTNPs and might mediate effective control of HIV have remained poorly understood. The efficient elimination of an HIV-infected CD4+ T cell via the granule-exocytosis pathway involves a series of events that include T cell activation, expansion, upregulation of effector molecules, degranulation, and delivery of lytic granule contents to the target. In the present study, we examined these functions in the HIV-specific CD8+ T cells of patients with and without immunologic control of HIV. To control for differences in proliferation or death of effector or target cells or effects not mediated by CD8+ T cells or lytic granules, we applied assays that measured effector cell frequency, infected target frequency, delivery of functional GrB into targets, and HIV-infected CD4+ T cell elimination. The HIV-specific CD8+ T cells of LTNPs were found to have extraordinary cytotoxic capacity on a per-cell basis. The potent ability of HIV-specific CD8+ T cells of LTNPs to eliminate HIV-infected CD4+ T cells was mediated by delivery of GrB to target cells. These data provide an effector function and mechanism that clearly segregate with LTNPs.
Perhaps as importantly, these results have implications that may extend to other chronic viral infections of experimental animals or humans. It has been the accepted paradigm for some time that memory cells maintain full cytotoxic capacity (reviewed in Seder and Ahmed, 2003
; Trambas and Griffiths, 2003
). On the basis of this model, re-expansion does not involve qualitative changes in cytotoxic capacity but rather is primarily quantitative, merely reflecting increases in the actual numbers of effector memory cells. However, a determination of whether increases in lytic granule content translate into qualitative changes has been limited by the lack of antibodies for some lytic granule proteins in mice and a lack of assays that measure cytotoxicity on a per-cell basis. In the present study, there were dramatic increases in per-cell killing capacity over 6 days associated with increases in perforin and GrB. This observation strongly suggests that restimulation of memory cells involves not only quantitative expansion, but also increases in lytic granule content that provide large qualitative increases in cytotoxic capacity.
At first inspection, it was surprising that the greatest differences in cytotoxic function between patient groups were observed after several days of exposure to autologous HIV-infected CD4+ T cells. However, the function of CD8+ T cells should probably be interpreted in the context of levels of antigen and likelihood of recent stimulation in vivo. The measurement of the CD8+ T cell response in LTNPs, treated progressors, or vaccinees is performed under conditions of low or absent antigen. Thus, an examination of such a recall response involves measurements of activation and cytokine secretion over several hours, cell-cycle progression and loading of effector molecules over several days, and target cell recognition and degranulation over 1–4 hr. Our results suggest that, under conditions of low amounts of antigen, measurements of virus-specific cell frequency and direct ex vivo cytotoxicity may not provide the best measure of the ability to restrict virus replication. Rather, proliferative capacity, upregulation of effector molecules, and target cell elimination are all important functions of the recall response of memory cells that should be considered.
This study also documents qualitative defects of the HIV-specific CD8+
T cells of progressors. We observed that the HIV-specific CD8+
T cells of Rx < 50, unlike those of LTNPs, require ongoing exposure to antigen for persistence, reminiscent of some recent findings in mice acutely or chronically infected with LCMV (Shin et al., 2007
). In the present study and prior work, we did not detect defects in early events of HIV-specific CD8+
T cell activation in progressors, such as recognition of autologous virus-infected cells and cytokine production. Diminished HIV-specific cytotoxicity of viremic progressor or Rx < 50 CD8+
T cells relative to those of LTNPs was also not caused by defects in degranulation. HIV-infected CD4+
T cell elimination by progressors’ cells did not exceed 60%, even at very high measured E:T ratios. Diminished killing by progressors’ CD8+
T cells was not due to death of cytotoxic cells because they produced IFN-γ 6 hr later. Taken together, these data suggest that diminished killing capacity of HIV-specific CD8+
T cells of progressors is due to deficient loading of lytic granules and is likely to be an important mechanism accounting for the loss of immunologic control of HIV in these patients.
We also observed that considerable expansion and cytotoxic potential can be restimulated from the PBMCs of progressors, suggesting that these properties are not permanently lost by deletion, anergy, or replicative senescence. The recovery of proliferation by PMA+Io described here and the previously reported decreases in IL-2 production by HIV-specific CD8+
T cells of progressors (Betts et al., 2006
; Zimmerli et al., 2005
) suggested that there might be some disruption of calcium flux or the calcineurin-NFAT pathway. Nuclear translocation of NFAT is an important early event in cell proliferation and IL-2 transcription (reviewed in Sundrud and Rao, 2007
). Although we observed that a significantly greater proportion of HIV-specific CD8+
T cells of LTNPs translocated NFAT upon peptide stimulation, there was considerable overlap between patient groups. It is possible that larger differences in NFAT translocation would be observed between patient groups, similar to differences observed with the ICE assay, if a larger number of specificities were sampled in assays using infected cells instead of peptide stimuli.
These results begin to address some of the cause-and-effect relationships between the cellular immune response and HIV viremia. Lowering of viral antigen levels by antiretroviral therapy is sufficient to restore HIV-specific CD4+
T cell proliferation, IL-2 production, and surface phenotype to levels observed in LTNPs (Tilton et al., 2007
). It is also sufficient to restore PD-1, CD127, or IL-2 expression by HIV-specific CD8+
T cells in results presented here and some recent studies (Rehr et al., 2008
; Streeck et al., 2008
). Because immunologic control of HIV replication is not restored in treated-progressor cohorts, these results suggest changes in these parameters are a consequence of viremia and not a cause of the loss of immunologic control. However, we have not observed a similar restoration of HIV-specific CD8+
T cell proliferative capacity, lytic granule loading, or cytotoxic potential here or in prior work. It remains possible that one or several host or viral factors may increase peak viremia during acute infection, resulting in disruption of proliferative or cytotoxic capacity. However, this is a separate issue because the subject of the present study is to determine those functions that are disrupted during the chronic phase of infection. The results of the present study suggest that proliferative and cytotoxic capacity are disrupted functions of the HIV-specific CD8+
T cell response. A formal demonstration that cells with these functions intact cause immunologic control of lentiviral replication would require passive-transfer studies in humans or experimental animals.
Perhaps most importantly, data from the present study suggest that induction of cells able to undergo rapid expansion and mediate cytotoxicity upon antigen encounter are possible goals for HIV vaccines. Most vaccine trials and studies of chronic infection have relied heavily upon detection of HIV-specific cells by cytokine secretion in response to high concentrations of non-optimal peptides with excess costimulatory antibodies and varying degrees of intercell contact. The HIV-infected cell assay used in the present study permits measurement of the response in the context of maintained major histocompatibility complex (MHC)-peptide stoichiometry, Nef-mediated MHC downregulation, and naturally processed peptides. The findings of the present study demonstrate the qualitative features of an effective virus-specific CD8+ T cell response that should be considered in the testing of the next generation of prophylactic and therapeutic vaccines for HIV.