Despite abundant literature on both the memory differentiation profiles and polyfunctional capacities of HIV-specific CD8+ T cells at different stages of infection (9
), less is known about how these T cell characteristics are associated with each other. In this study, we examined the relationship between the memory phenotype of CD8+ T cells and the polyfunctional responses of these cells, as measured by IFNγ, TNFα, MIP1β and CD107a expression. We studied HIV-specific CD8+ T cell responses in 20 HIV-1 infected individuals at approximately 34 weeks post infection, and compared them to CMV-specific T responses. We detected a higher proportion of polyfunctional CD8+ T cells specific for CMV compared to HIV, which agrees with previous observations (34
). HIV-specific responses were enriched for cells with an early-differentiated phenotype, whilst CMV-specific cells were mainly of a terminally-differentiated phenotype, consistent with earlier findings (14
). Our novel observation was a distinct pattern of polyfunctionality between HIV− and CMV-specific CD8+ T cell memory subsets. For HIV-specific CD8+ T cells, early-differentiated memory cells (CD45RO+CD27+) were enriched for a polyfunctional response, and there was a significant reduction in the number of functions in terminally-differentiated cells (CD45RO−CD27−). In contrast, CMV-specific CD8+ T cells exhibited a polyfunctional profile that was similar across early, late or terminally-differentiated memory subsets. Moreover, the CMV response was highly polyfunctional (80–90% of cells expressing 3 or 4 functions) in both HIV-infected and uninfected individuals, compared to HIV-specific CD8+ T cells (40–50%). This implies that there is not a global decrease in CD8+ T cell polyfunctionality in the background of HIV infection. Differences in antigen load or recurrence or CD4+ help may account for differential (poly)function between HIV-specific CD8+ T cells and cells of other specificities. Overall, this suggests that the hierarchical loss in the number of functions by an antigen-specific CD8+ T cell as memory differentiation proceeds is dependent on the infecting pathogen, and that memory phenotype and polyfunctional characteristics of CD8+ T cells can differ significantly.
Seminal work from Sallusto and colleagues (20
) introduced the concept of antigen-specific cells being divided into memory subsets expressing different phenotypic markers with distinct homing and survival abilities, and also alluded to a functional distinction between different memory subsets. A number of factors have been shown to shape this functional and phenotypic heterogeneity of antigen-specific T cells. Co-stimulatory signals engaged during T cell priming, as well as cytokines such as IL-2 and IL-21, can modulate T cell functionality (38
). Major determinants of CD8+ T cell polyfunctionality are antigen concentration (26
) and TCR affinity (44
). Work from murine models has proposed that the clonal expansion process can modulate the profile of secreted cytokines (45
). With regard to memory differentiation of CD8+ T cells, the degree of cell maturation is dependent on similar factors, namely antigen load, co-stimulation signals and the cytokine environment (reviewed in (47
)). Hence, being regulated by similar factors, it can be speculated that cell differentiation and polyfunctional potential could be co-dependent phenomena, where the generation and maintenance of late-stage differentiated effector cells, endowed with a rapid response to pathogens and a high degree of polyfunction, would be favorable to ensure viral control. Indeed, in certain well-controlled infections or successful vaccinations (such as CMV, vaccinia or yellow fever vaccine), antigen-specific CD8+ T cells that are generated are highly differentiated and highly polyfunctional (23
). In contrast, during uncontrolled HIV infection, HIV-specific CD8+ T cells exhibit mainly an early-differentiated memory phenotype, often regarded as an immature stage (14
). However, the relationship between maturation and polyfunction appears to be more complex, as antigen-experienced cells exhibit heterogeneous memory and cytokine secretion profiles dependent upon different antigen specificities (reviewed in (21
)). Our data are in agreement with the latter observation, where we show that differentiation towards late memory for HIV-specific CD8+ T cells is accompanied by a progressive loss of polyfunctional capacities, whilst CMV-specific CD8+ T cells retain their polyfunctional capacities regardless of memory differentiation.
Although sustained HIV replication appears to play a predominant role in driving CD8+ T cells towards a late stage of memory maturation (15
), we found no difference in the memory-polyfunction association between those with high and low HIV viral loads, i.e. those controlling viral replication did not resemble the stable ‘polyfunctionality regardless of memory phenotype’ pattern of CMV-specific CD8+ T cells. It would be of interest to determine whether this is also the case for HIV-specific cells from individuals on long-term, successful ART, where antigen load is reduced.
Our starting point for this study was that polyfunctional T cells, capable of carrying out a range of functions simultaneously, exhibit superior protective immunity (9
). We can speculate that measuring multiple functions on a per cell basis may more closely reflect the ability of CD8+ T cells to impart antiviral effects and may be more relevant than focusing only on one function, such as IFNγ. However, the combination of specific functions rather the number of functions per se
may offer a more refined and accurate assessment of protective immunity (50
). Upregulation of perforin has recently been highlighted as an important indicator of cytotoxic potential for control of HIV (51
). This is also a consideration in the question of how maturation profiles of CD8+ T cells and functional abilities are linked, since specific functions such as perforin and IL-2 may indeed be associated with particular memory phenotypes, whilst other functions may not be as tightly regulated. Loss of CD28 expression appears to be coupled to loss of IL-2 production, whilst T-bet expression correlates with perforin upregulation (53
). In our study, we did not measure perforin and nor could we detect IL-2 responses. Indeed, CMV-specific CD8+ T cells rarely produce IL-2 (53
) and IL-2 production in the context of HIV infection is confined to long-term non-progressors (54
Excessive activation of the immune system can influence both the function and phenotype of CD8+ T cells. Several studies have described the upregulation of PD-1 and other inhibitory receptors such as CD160, 2B4 and LAG3 on CD8+ T cells in chronic viral infections, including HIV, which results in defective cytokine production and lack of polyfunctional responses. This can be partially reversed by blocking the interaction of these receptors with their ligands (56
). Yamamoto and colleagues (37
) recently described elevated levels and co-expression of several negative regulators on HIV-specific CD8+ T cells compared to CMV-specific cells. Co-expression of these inhibitory receptors correlated inversely with polyfunctionality, and PD-1 blockade restored cytokine production. It would thus be of interest to determine whether, in HIV infection, the memory maturation profile of HIV-specific CD8+ T cells coincides with their degree of functional inhibition.
In conclusion, our data show that the polyfunctional abilities of HIV-specific CD8+ T cells are influenced by the stage of memory differentiation, which is not the case for CMV-specific responses. This emphasizes that different pathogens generate CD8+ T cell responses with distinct polyfunctional-memory subset profiles; this may reflect the distinct life histories of pathogens and their interactions with the immune system. A better understanding of which memory-function combinations lead to superior and durable protective immunity against HIV is needed.