Yevgeniy Yuzefpolskiy

MicroRNA-17~92 regulates effector and memory CD8 T-cell fates by modulating proliferation in response to infections.

The precise microRNAs and their target cellular processes involved in generation of durable T-cell immunity remain undefined. Here we show a dynamic regulation of microRNAs as CD8 T cells differentiate from naïve to effector and memory states, with short-lived effectors transiently expressing higher levels of oncogenic miR-17-92 compared with the relatively less proliferating memory-fated effectors. Conditional CD8 T-cell-intrinsic gain or loss of expression of miR-17-92 in mature cells after activation resulted in striking reciprocal effects compared with wild-type counterparts in the same infection milieu-miR-17-92 deletion resulted in lesser proliferation of antigen-specific cells during primary expansion while favoring enhanced IL-7Rα and Bcl-2 expression and multicytokine polyfunctionality; in contrast, constitutive expression of miR-17-92 promoted terminal effector differentiation, with decreased formation of polyfunctional lymphoid memory cells. Increased proliferation upon miR-17-92 overexpression correlated with decreased expression of tumor suppressor PTEN and increased PI3K-AKT-mTOR signaling. Thus, these studies identify miR17-92 as a critical regulator of CD8 T-cell expansion and effector and memory lineages in the physiological context of acute infection, and present miR-17-92 as a potential target for modulating immunologic outcome after vaccination or immunotherapeutic treatments of cancer, chronic infections, or autoimmune disorders.

Early CD8 T-cell memory precursors and terminal effectors exhibit equipotent in vivo degranulation

Early after priming, effector CD8 T cells are distinguished into memory precursor and short-lived effector cell subsets (MPECs and SLECs). Here, we delineated a distinct in vivo heterogeneity in killer cell lectin-like receptor G1 (KLRG-1) expression, which was strongly associated with diverse MPEC and SLEC fates. These in vivo MPECs and SLECs expressed equivalent levels of cytotoxic molecules and effector cytokines. Using a unique in vivo degranulation assay, we found that the MPECs and SLECs similarly encountered infected target cells and elaborated equivalent levels of cytotoxicity in vivo. These data provide direct in vivo evidence that memory-fated cells pass through a robust effector phase. Additionally, the preferential localization of the MPECs in the lymph nodes, where a lesser degree of cytotoxicity was elaborated, suggests that the MPECs may be protected from excessive stimulation and terminal differentiation by virtue of their differential tissue localization. These data provide novel mechanistic insights into the linear decreasing potential model of memory differentiation.Cellular & Molecular Immunology advance online publication, 28 July 2014; doi:10.1038/cmi.2014.48

Vitamin D Receptor Signals Regulate Effector and Memory CD8 T Cell Responses to Infections in Mice

BACKGROUND Vitamin D insufficiency is associated with broad-ranging human disease sequelae such as bone disease, cancer, cardiovascular disease, allergy, autoimmune disorders, diabetes, and infectious diseases. Disease risk and severity of a large proportion of the nonskeletal disorders heavily involve the cytotoxic cluster of differentiation (CD) 8 T lymphocyte (CTL) arm of cellular adaptive immunity. Considering the importance of vitamin D in CTL-dependent diseases, there is a critical need for systematic in-depth explorations into the role of vitamin D deficiency in generation and maintenance of CTL immunity during infections and vaccinations. OBJECTIVE With the use of wild-type (WT) vitamin D-sufficient mice and the vitamin D receptor knockout (Vdr(-/-)) mouse model of in vivo deficiency of vitamin D signaling, we systematically analyzed the impact of vitamin D deficiency on antigen-specific effector and memory CD8 T cell responses to acute viral and bacterial infections. METHODS WT and Vdr(-/-) mice were infected with lymphocytic choriomeningitis virus, a natural mouse pathogen, and antigen-specific CTL responses were analyzed during priming, expansion, contraction, and memory phases. Magnitude, breadth, cytokine production, and localization of antiviral effector and memory CTLs to lymphoid and nonlymphoid tissues were specifically assessed. RESULTS The absence of vitamin D signals led to 1) aberrant CD8 T cell effector differentiation (∼2-fold lower granzyme B and reduced B cell lymphoma 2; P ≤ 0.05) and enhanced contraction (∼15% increase; P ≤ 0.05) in antigen-specific CTLs; 2) a significantly restricted (P ≤ 0.05) breadth of the antigen-specific CD8 T cell effector and memory repertoire; and 3) preferential localization of effector (∼2.5-fold increase; P ≤ 0.01) and memory (∼5-fold increase; P ≤ 0.001) CD8 T cells to the lymph nodes compared to nonlymphoid tissues. CONCLUSION Our data show a previously unrecognized impact of vitamin D deficiency on the quantity, quality, breadth, and location of CD8 T cell immunity to acute viral and bacterial infections.

Development of a novel Francisella tularensis Live Vaccine Strain expressing ovalbumin provides insight into Francisella tularensis-specific CD8+ T cell responses.

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in a pathogen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, pathogen-specific CD8+ T cells expand after the first week and produce IFN-γ but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, F. tularensis-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more pathogen-specific CD8+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

Development of a novel Francisella tularensis Live Vaccine Strain expressing ovalbumin provides insight into antigen-specific CD8+ T cell responses

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in an antigen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, adoptively transferred OVA-specific CD8+ T cells expand after the first week and produce IFN-γ but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, OVA-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more antigen-specific CD8+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

Dicer Regulates the Balance of Short-Lived Effector and Long-Lived Memory CD8 T Cell Lineages.

MicroRNAs constitute a major post-transcriptional mechanism for controlling protein expression, and are emerging as key regulators during T cell development and function. Recent reports of augmented CD8 T cell activation and effector differentiation, and aberrant migratory properties upon ablation of Dicer/miRNAs in naïve cells have established a regulatory role of miRNAs during priming. Whether miRNAs continue to exert similar functions or are dispensable during later stages of CD8 T cell expansion and memory differentiation remains unclear. Here, we report a critical role of Dicer/miRNAs in regulating the balance of long-lived memory and short-lived terminal effector fates during the post-priming stages when CD8 T cells undergo clonal expansion to generate a large cytotoxic T lymphocyte (CTL) pool and subsequently differentiate into a quiescent memory state. Conditional ablation of Dicer/miRNAs in early effector CD8 T cells following optimal activation and expression of granzyme B, using unique dicerfl/fl gzmb-cre mice, led to a strikingly diminished peak effector size relative to wild-type antigen-specific cells in the same infectious milieu. Diminished expansion of Dicer-ablated CD8 T cells was associated with lack of sustained antigen-driven proliferation and reduced accumulation of short-lived effector cells. Additionally, Dicer-ablated CD8 T cells exhibited more pronounced contraction after pathogen clearance and comprised a significantly smaller proportion of the memory pool, despite significantly higher proportions of CD127Hi memory precursors at the effector peak. Combined with previous reports of dynamic changes in miRNA expression as CD8 T cells differentiate from naïve to effector and memory states, these findings support distinct stage-specific roles of miRNA-dependent gene regulation during CD8 T cell differentiation.