Perforin and IL-2 Upregulation Define Qualitative Differences among Highly Functional Virus-Specific Human CD8+ T Cells

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Abstract
The prevailing paradigm of T lymphocyte control of viral replication is that the protective capacity of virus-specific CD8+ T cells is directly proportional to the number of functions they can perform, with IL-2 production capacity considered critical. Having recently defined rapid perforin upregulation as a novel effector function of antigen-specific CD8+ T cells, here we sought to determine whether new perforin production is a component of polyfunctional CD8+ T cell responses that contributes to the control of several human viral infections: cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza (flu), and adenovirus (Ad). We stimulated normal human donor PBMC with synthetic peptides whose amino acid sequences correspond to defined CTL epitopes in the aforementioned viruses, and then used polychromatic flow cytometry to measure the functional capacity and the phenotype of the responding CD8+ T cells. While EBV and flu-specific CD8+ T cells rarely upregulate perforin, CMV-specific cells often do and Ad stimulates an exceptionally strong perforin response. The differential propensity of CD8+ T cells to produce either IL-2 or perforin is in part related to levels of CD28 and the transcription factor T-bet, as CD8+ T cells that rapidly upregulate perforin harbor high levels of T-bet and those producing IL-2 express high amounts of CD28. Thus, “polyfunctional” profiling of antigen-specific CD8+ T cells must not be limited to simply the number of functions the cell can perform, or one particular memory phenotype, but should actually define which combinations of memory markers and functions are relevant in each pathogenic context. Although CD8+ T cells are thought to be largely responsible for the control of viral infections, exactly how they mediate protection is uncertain. One approach to assessing their protective capacity is to measure several of their functions simultaneously. Generally, it is believed the more functions a cell can perform, the better its potential to control viral replication. A multi-functional response including interleukin-2 (IL-2) production is currently valued as the key correlate of protection. We recently characterized a novel CD8+ T cell function: rapid perforin upregulation, which serves to contribute to and sustain the killing of virally infected host cells. In this study, we show that new perforin is abundant during adenovirus and cytomegalovirus infections, but scarcely detected in the context of influenza and Epstein-Barr virus. Importantly, perforin and IL-2 are rarely co-expressed. The significance of this relationship is that we can no longer assume the more functions a CD8+ T cell performs in response to a virus the better. Thus, when considering vaccine design, no single functional profile will likely be protective across all pathogens. Rather, vaccine-induced T cell responses may need to be “pathogen-specific”, as different T cell functional responses will be important for controlling different viral infections.