The interplay between HIV replication immediately after infection and the host immune response, both innate and adaptive, determines the course of HIV disease. If indeed HIV-specific CD8+ T cells are responsible for the initial resolution of peak viremia during the first few weeks of infection, it follows that during this timeframe, the CD8+ T cells must be most antiviral. A detailed characterization of HIV-specific CD8+ T cells during acute HIV infection, therefore, affords the greatest likelihood of discovering how CD8+ T cells eliminate HIV-infected host cells before a thorough seeding of HIV reservoirs, CTL escape mutations, CD4 depletion, and T-cell exhaustion manifest themselves and confuse the assessment of CD8+ T-cell control over HIV.
What is the best way to characterize HIV-specific CD8+ T cells? The prevailing tactic in the field is to correlate the absolute number of functions an antigen-specific CD8+ T cell may perform simultaneously to its antiviral capacity. However, ‘polyfunctionality’ is an ideology that developed as a result of our inability to identify a specific functional correlate of protection from HIV. Since no single function emerged as a leading correlate, we surmised that multiple functions must work in concert if CD8+ T cells actually control HIV replication. Henceforth, a detailed characterization of the antiviral nature of HIV-specific CD8+ T cells must include an assessment of their capacity to be polyfunctional. However, this approach poses a conundrum: isn’t the polyfunctional nature of a CD8+ T cell simply a product of the number of functions for which are assayed? Are we not biasing our interpretation of the data by screening for 5, 6, or 7 functions? For example, just as a CD8+ T cell will never be considered polyfunctional if it is only tested in an IFN-γ ELISpot assay, surely a cell will be multi- functional if we assay it for 10 functions? Does that mean it is necessarily antiviral?
Consider the functional profile below (). IFN-γ, TNF-α, and IL-2 are the most commonly measured T cell functions. Judging by the lack of polyfunctional subsets, one might conclude that these CD8+ T cells were not particularly antiviral. However, including CD107a and MIP-1β in our analysis alters the distribution of responses, resulting in an increase in functionality (), thereby improving their perceived antiviral nature. It is not difficult to imagine that the more functions for which we screen the better the odds for polyfunctionality. As technology advances and the number of parameters we are able to quantify simultaneously expands, the concept of CD8+ T-cell polyfunctionality as an immune correlate of protection, defined simply as being able to perform more than 2 functions simultaneously, becomes increasingly ambiguous.
The degree of polyfunctionality of a population of CD8+ T cells correlates to the number of functions for which are assayed
That brings us back to square one: trying to identify specific CD8+
T-cell functions that truly have a negative impact on HIV replication. The temporal association between the appearance of HIV-specific CTLs in the periphery and a sharp decline in HIV viremia resulted from an assessment of cytotoxic potential in vitro
and formed the first premise for CD8+
T cells as a correlate of HIV control (4
). Since then, researchers have assayed for HIV-specific CD8+
T-cell responses by techniques that require much less labor, time, and manipulation of the cells. The ELISpot assay was popular for a long period of time (and still is), largely because it is amenable to strict validation procedures, but polychromatic flow cytometry has clearly become the assay-of-choice for measuring antigen-specific immune responses, because of its multi-dimensional nature. These techniques quantify mostly cytokines and other non-cytolytic proteins, which have dramatically expanded our characterization of antigen-specific T-cell responses in unprecedented detail.
In the case of HIV, however, it can be argued that the measurement of cytokines, such as IFN-γ and IL-2, has obfuscated our understanding of the host anti-HIV response. Whereas measurement of HIV-specific CD8+
T-cell frequency by MHC tetramers initially showed an inverse relationship to viral load (46
), the measurement of HIV- specific IFN-γ production determined a proportional relationship between HIV-specific CD8+
T cells and HIV viral load (47
). IFN-γ was presumed to be an antiviral marker, because CD8+
T-cell clones that produced IFN-γ early after stimulation were shown to develop into CTLs after further long-term culture (48
). Moreover, most research on HIV-specific CD8+
T cells during acute HIV infection reported weak responses in frequency, magnitude, and breadth, based on IFN-γ production (50
), suggesting an immediate deficiency in anti-HIV-specific effector activity.
IL-2 secretion is considered by some to be indispensible for protection, since HIV-specific CD8+
T cells from chronically infected HIV progressors that upregulate IFN-γ failed to simultaneously produce IL-2, whereas this dual capability was preserved among CMV- and EBV-specific CD8+
T cells (53
). Furthermore, IL-2 secretion by CD8+
T cells is correlated to proliferation (53
), which is preserved in HIV subjects with non-progressive infections (33
). While IL-2 is certainly critical for the preservation of HIV-specific CD8+
T-cell function during chronic infection (31
), its relevance during acute HIV infection is debatable. IL-2 does not itself have antiviral activity (although it may promote it in other cells) and, in the case of HIV, may be a driver of viral replication by augmenting the availability of activated target CD4+
T cells. IL-2 production is taken for granted as a necessary component of a protective immune response because of studies on CMV, EBV, and influenza, in which virus-specific CD8+
T cells often secrete IL-2 (38
). Comparison of IL-2 production from CD8+
T cells specific for influenza, adenovirus, EBV, and CMV indicates differential production is likely linked to viral load (55
). EBV and influenza infections generally do not result in significant chronic antigenic burdens. CMV remains active and establishes a constant antigenic presence; however, depending on the cycle, the viral burden may be high or low. Adenovirus-specific CD8+
T cells very likely undergo intermittent stimulation due to re-infection with alternate adenovirus serotypes (56
) or adenovirus persistence (57
), thus serving as a good model for CD8+
T-cell responses in the setting of frequent antigenic burden. Indeed, IL-2 production from EBV- and influenza-specific CD8+
T cells is typically high compared to that of CMV and adenovirus (55
). Thus, when there is no circulating antigen or the level is low, IL-2 production by virus-specific CD8+
T cells is high, whereas in the presence of a constant viral burden IL-2 production is low or absent. This agrees with the murine LCMV model of CD8+
T-cell responsiveness, in which IL-2 producing CD8+
T cells are lost when clone 13 establishes chronic infection but not after resolution of acute infection by the Armstrong clone (58
). A formal role for IL-2 in clearing primary viral infection has not been demonstrated. Thus, IL-2 should not be considered an important correlate of protection against viral infection and should rather be viewed as an indicator of response longevity in the absence of antigen.