The T-box transcription factors T-bet and Eomesodermin (Eomes) have been well defined as key drivers of immune cell development and cytolytic function. While the majority of studies have defined the roles of these factors in the context of murine T-cells, recent results have revealed that T-bet, and possibly Eomes, are expressed in other immune cell subsets. To date, the expression patterns of these factors in subsets of human peripheral blood mononuclear cells beyond T-cells remain relatively uncharacterized. In this study, we used multiparametric flow cytometry to characterize T-bet and Eomes expression in major human blood cell subsets, including total CD4+ and CD8+ T-cells, γδ T-cells, invariant NKT cells, natural killer cells, B-cells, and dendritic cells. Our studies identified novel cell subsets that express T-bet and Eomes and raise implications for their possible functions in the context of other human immune cell subsets besides their well-known roles in T-cells.

Recent data suggest that CD8+ T-cell effector activity is an important component in the control of HIV replication in elite controllers (ECs). One critical element of CD8+ T-cell effector function and differentiation is the T-box transcription factor T-bet. In the present study, we assessed T-bet expression, together with the effector proteins perforin, granzyme A (Grz A), granzyme B (Grz B), and granulysin, in HIV-specific CD8+ T cells from ECs (n = 20), chronically infected progressors (CPs; n = 18), and highly active antiretroviral therapy (HAART)–suppressed individuals (n = 19). Compared with the other cohort groups, HIV-specific CD8+ T cells among ECs demonstrated a superior ability to express perforin and Grz B, but with no detectable difference in the levels of Grz A or granulysin. We also observed higher levels of T-bet in HIV-specific CD8+ T cells from ECs, with an ensuing positive correlation between T-bet and levels of both perforin and Grz B. Moreover, HIV-specific CD8+ T cells in ECs up-regulated T-bet to a greater extent than CPs after in vitro expansion, with concomitant up-regulation of perforin and Grz B. These results suggest that T-bet may play an important role in driving effector function, and its modulation may lead to enhanced effector activity against HIV.

In mice, two T-box transcription factors, T-bet and Eomes, drive the differentiation of CD8 T-cell lineages; however, little is known regarding their role in human CD8 T-cell differentiation. Here, we characterized T-bet and Eomes expression and localization within human CD8 memory T-cell populations. We find T-bet and Eomes are broadly expressed in human memory CD8 T cells, with increasing levels of T-bet and Eomes strongly correlating with differentiation from central memory to effector memory and effector subpopulations. In resting T-cells, T-bet levels directly correlate to subcellular localization, with a higher propensity for nuclear expression of T-bet within T-bethi cells and predominately cytoplasmic expression in T-betlo cells. Additionally, Eomes is also localized to either the nucleus or cytoplasm. Upon T-cell receptor stimulation, the percentage of T-cells that express T-bet dramatically increases, while the percentage of cells expressing Eomes remains largely unchanged across all memory populations. Interestingly, T-bet, but not Eomes, relocalizes to the nucleus in the majority of cells across all populations within 24 hours post-stimulation. These data indicate that T-bet and Eomes are likely regulated at the level of subcellular localization, potentially via different mechanisms. Together, these findings suggest a novel model for CD8 T-cell differentiation in humans based on the localization of T-bet and Eomes.

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.

SUMMARY Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.