Qingsheng Li

The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin.

The mechanisms underpinning integration of instructions that program naive CD8+ T cells for effector and/or memory differentiation are not well understood. Herein, we demonstrate that interleukin-12 (IL-12) enhanced and sustained antigen and costimulatory molecule (B7.1)-induced mTOR kinase activity in naive CD8+ (OT-I) T cells via phosphoinositide 3-kinase and STAT4 transcription factor pathways. Blocking mTOR activity by rapamycin reversed IL-12-induced effector functions because of loss of persistent expression of the transcription factor T-bet. Rapamycin treatment of IL-12-conditioned OT-I cells promoted persistent Eomesodermin expression and produced memory cell precursors that demonstrated enhanced sustenance and antigen-recall responses upon adoptive transfer. The memory cell precursors showed greater tumor efficacy than IL-12-conditioned effector OT-I cells. These results identify mTOR as the central regulator of transcriptional programs that determine effector and/or memory cell fates in CD8+ T cells. Targeting mTOR activity offers new opportunities to regulate CD8+ T cell-mediated immunity.

IL-21 Enhances and Sustains CD8+ T Cell Responses to Achieve Durable Tumor Immunity: Comparative Evaluation of IL-2, IL-15, and IL-21

Cytokines that use the common receptor γ-chain for regulating CD8+ T cell responses to Ag include IL-2, IL-15, and the recently identified IL-21. The ability of these cytokines to regulate antitumor activity in mice has generated considerable interest in understanding their mode of action. In this study we compare the abilities of IL-2, IL-15, and IL-21 to stimulate immunity against tumors in a syngeneic thymoma model. Durable cures were only achieved in IL-21-treated mice. By monitoring both endogenous and adoptively transferred tumor Ag-specific CD8+ T cells, it was determined that IL-21 activities overlap with those of IL-2 and IL-15. Similar to IL-2, IL-21 enhanced Ag activation and clonal expansion. However, unlike IL-2 treatment, which induces activation-induced cell death, IL-21 sustained CD8+ T cell numbers long term as a result of increased survival, an effect often attributed to IL-15. These findings indicate that the mechanisms used by IL-21 to promote CD8+ T cell responses offer unique opportunities for its use in malignant diseases and infections.

Transcription Factor Foxo1 Represses T-bet-Mediated Effector Functions and Promotes Memory CD8+ T Cell Differentiation

The evolutionary conserved Foxo transcription factors are important regulators of quiescence and longevity. Although, Foxo1 is known to be important in regulating CD8(+) T cell trafficking and homeostasis, its role in functional differentiation of antigen-stimulated CD8(+) T cells is unclear. Herein, we demonstrate that inactivation of Foxo1 was essential for instructing T-bet transcription factor-mediated effector differentiation of CD8(+) T cells. The Foxo1 inactivation was dependent on mTORC1 kinase, given that blockade of mTORC1 abrogated mTORC2-mediated Akt (Ser473) kinase phosphorylation, resulting in Foxo1-dependent switch from T-bet to Eomesodermin transcription factor activation and increase in memory precursors. Silencing Foxo1 ablated interleukin-12- and rapamycin-enhanced CD8(+) T cell memory responses and restored T-bet-mediated effector functions. These results demonstrate an essential role of Foxo1 in actively repressing effector or terminal differentiation processes to promote memory CD8(+) T cell development and identify the functionally diverse mechanisms utilized by Foxo1 to promote quiescence and longevity.

A Central Role for mTOR Kinase in Homeostatic Proliferation Induced CD8+ T cell Memory and Tumor Immunity

The cell-intrinsic mechanisms guiding naive CD8+ T cells for clonal expansion and memory generation via homeostatic proliferation (HP) are unclear. Here, we have shown that HP of naive CD8+ T cells requires IL-7-, but not IL-15-induced mTOR kinase activation. HP-induced mTOR enhances transcription factor T-bet for functional maturation and CD122 expression, which sensitizes for an IL-15-dependent memory transition by favoring transcription factor Eomesodermin over T-bet. Inhibition of mTOR blocks T-bet and CD122 expression but preserves memory in an IL-15-independent manner by promoting Eomesodermin expression. The ability of rapamycin to augment HP-induced memory was cell-intrinsic given that silencing mTOR in CD8+ T cells generated identical outcomes. Strikingly, HP-induced CD8+ T cell memory generated by IL-15-dependent or -independent mechanisms demonstrated identical tumor efficacy. These results indicate a central role for mTOR in HP-induced CD8+ T cell responses and demonstrate the importance for CD8+ memory in HP-induced tumor efficacy.

Tc17 Cells Are Capable of Mediating Immunity to Vaccinia Virus by Acquisition of a Cytotoxic Phenotype

CD8 T cells can acquire cytokine-secreting phenotypes paralleling cytokine production from Th cells. IL-17–secreting CD8 T cells, termed Tc17 cells, were shown to promote inflammation and mediate immunity to influenza. However, most reports observed a lack of cytotoxic activity by Tc17 cells. In this study, we explored the anti-viral activity of Tc17 cells using a vaccinia virus (VV) infection model. Tc17 cells expanded during VV infection, and TCR transgenic Tc17 cells were capable of clearing recombinant VV infection. In vivo, adoptively transferred Tc17 cells lost the IL-17–secreting phenotype, even in the absence of stimulation, but they did not acquire IFN-γ–secreting potential unless stimulated with a virus-encoded Ag. However, examination of cells following infection demonstrated that these cells acquired cytotoxic potential in vivo, even in the absence of IFN-γ. Cytotoxic potential correlated with Fasl expression, and the cytotoxic activity of postinfection Tc17 cells was partially blocked by the addition of anti-FasL. Thus, Tc17 cells mediate VV clearance through expression of specific molecules associated with cytotoxicity but independent of an acquired Tc1 phenotype.

Regulating Mammalian Target of Rapamycin To Tune Vaccination-Induced CD8+ T Cell Responses for Tumor Immunity

Vaccine strategies aimed at generating CD8+ T cell memory responses are likely to show augmented efficacy against chronic challenges like tumor. The abundance in variety of memory CD8+ T cells behooves development of vaccine strategies that generate distinct memory responses and evaluate them for tumor efficacy. In this study, we demonstrate the ability of a variety of rapamycin treatment regimens to regulate virus vaccination-induced CD8+ T cell memory responses and tumor efficacy. Strikingly, a short course of high-dose, but not low-dose, rapamycin treatment transiently blocks viral vaccination-induced mammalian target of rapamycin activity in CD8+ T cells favoring persistence and Ag-recall responses over type 1 effector maturation; however, prolonged high-dose rapamycin administration abrogated memory responses. Furthermore, a short course of high-dose rapamycin treatment generated CD8+ T cell memory responses that were independent of IL-15 and IL-7 and were programmed early for sustenance and greater tumor efficacy. These results demonstrate the impact a regimen of rapamycin treatment has on vaccine-induced CD8+ T cell responses and indicates that judicious application of rapamycin can augment vaccine efficacy for chronic challenges.

IL-12-Programmed Long-Term CD8+ T Cell Responses Require STAT4

Immunological adjuvants activate innate immune cells for Ag presentation and elicitation of cytokines like IL-12 that promote T cell expansion and effector differentiation. An important but elusive aim for most immunization strategies is to produce memory T cells that provide durable immunity. Recent evidence demonstrates that the context of Ag presentation instructionally programs T cells for short- and long-term responses. However, the role and mechanisms by which cytokines like IL-12 condition CD8 T cells for long-term responses remain relatively uncharacterized. In this study, we show that brief exposure (20 h) of naive TCR-transgenic CD8 cells to IL-12 during Ag stimulation leads to transient phosphorylation of STAT4 for robust effector differentiation. Moreover, the IL-12-induced STAT4 engenders greater clonal expansion of the Ag-activated CD8 cells by regulating the expression of the transcriptional factor Bcl3- and Bcl2-related genes that promote survival of Ag-activated CD8 cells. Remarkably, the IL-12-conditioned CD8 T cells demonstrate increased sensitivity to IL-7 and IL-15, whereby they are rendered “fit” for homeostatic self-renewal as well as augmented CD4-dependent recall responses that are effective at controlling Salmonella infection in vivo. This information provides new insights into mechanisms by which IL-12 conditions CD8 T cells for long-term immunity, which is likely to benefit development of new strategies for the use of IL-12 in infectious diseases and cancer.

Fine-tuning CD8(+) T cell functional responses: mTOR acts as a rheostat for regulating CD8(+) T cell proliferation, survival and differentiation?

Naïve CD8+ T cells are instructed by antigen, co-stimulatory molecules and cytokines to undergo proliferation, clonal expansion and differentiation for effector and memory functions. The integration of extracellular instructions induces coordinated signaling cascades and transcriptional programs to determine CD8+ T cell functional fate. Although, an important role for the energy sensitive kinase-mammalian target of rapamycin (mTOR) in regulating T cell proliferation and cell survival in-step with cellular metabolic status has been demonstrated, emerging information indicates that mTOR also acts as a critical regulator of effector and memory functional fates in CD8+ T cells. Herein, we discuss the pathways by which cellular metabolism regulates proliferation, survival and functional differentiation of CD8+ T cells and focus on the role of mTOR as the rheostat that strikes a balance between effector and memory differentiation of CD8+ T cells for enabling adaptive host immunity.

Antigen-induced Erk1/2 activation regulates Ets-1-mediated sensitization of CD8+ T cells for IL-12 responses

The presence of IL‐12 during antigen stimulation instructs naive CD8+ T cells for long‐term effector responses, but their mechanisms of collaboration are not understood completely. Herein, we report that CD8+ T cells (OT‐I T cells) stimulated with antigen for a longer duration show enhanced sensitization to IL‐12 as a result of Erk1/2‐dependent, increased Ets‐1 phosphorylation and subsequent increases in IL‐12Rβ2 expression. Correspondingly, naive OT‐I T cells stimulated by antigen for a longer duration in the presence of IL‐12, irrespective of frequency of APCs, show robust effector maturation and mount long‐term antigen‐recall responses upon adoptive transfer. These results identify the role of antigen strength‐dependent Erk1/2 activation for Ets‐1‐mediated collaboration with IL‐12 in CD8+ T cells.

Aspergillus fumigatus extract differentially regulates antigen-specific CD4+ and CD8+ T cell responses to promote host immunity

Invasive aspergillosis is a major cause of morbidity and mortality in the severely immunocompromised. The paucity of information about the mechanisms by which Aspergillus‐derived factors regulate antigen‐specific T cell responses in vivo poses a significant hurdle for devising effective immunization strategies to treat or prevent aspergillosis. By monitoring adoptively transferred T cell receptor transgenic, naive CD4+ (OT‐II) and CD8+ (OT‐I) T cells specific for distinct peptides of a nominal antigen, chicken ovalbumin (OVA), we demonstrate that sensitization with Aspergillus fumigatus (Af) extract plus OVA protein considerably enhances OT‐I and OT‐II T cell activation, which results in clonal expansion, primarily as a result of increased proliferation. The sensitization provided by Af extract promotes OT‐I expansion accompanied by differentiation into interferon‐γ‐producing cytotoxic cells. It is surprising that no effector differentiation of the induced OT‐II response was observed. Moreover, the Af extract‐induced OT‐I and OT‐II T cell expansion was transient, as considerable contraction in the numbers of detectable OT‐I and OT‐II T cells was evidenced by Day 10. In agreement with these observations, sensitization with Af extract plus OVA marginally promoted host immunity against an OVA‐expressing thymoma (E.G7) challenge, and the protection was enhanced by resensitization with Af extract and OVA. Our results demonstrate the ability of Af extract to differentially regulate antigen‐specific CD4+ and CD8+ T cell responses, resulting in limited augmentation of host immunity. This information suggests that strategies to target CD4+ T cell effector maturation may promote host immunity to Aspergillus and unexpectedly demonstrates the use for Af extract as a CD8+ T cell adjuvant.