During a T cell response, the effector CTL pool contains two cellular subsets: short-lived effector cells (SLECs), a majority of which are destined for apoptosis, and the memory precursor effector cells (MPECs) that differentiate into memory cells. Understanding the mechanisms that govern the differentiation of memory CD8 T cells is of fundamental importance in the development of effective CD8 T cell-based vaccines. The strength and nature of TCR signaling along with signals delivered by cytokines like IL-2 and IL-12influence differentiation of SLECs and MPECs. A central question is, how are signals emanating from multiple receptors integrated and interpreted to define the fate of effector CTLs? Using genetic and pharmacological tools, we have identified Akt as a signal integrator that links distinct facets of CTL differentiation to the specific signaling pathways of FOXO, mTOR, and Wnt/β-catenin. Sustained Akt activation triggered by convergent extracellular signals evokes a transcription program that enhances effector functions, drives differentiation of terminal effectors, and diminishes the CTLs’ potential to survive and differentiate into memory cells. While sustained Akt activation severely impaired CD8 T cell memory and protective immunity, in vivo inhibition of Akt rescued SLECs from deletion and increased the number of memory CD8 T cells. Thus, the cumulative strength of convergent signals from signaling molecules such as TCR, costimulatory molecules, and cytokine receptors governs the magnitude of Akt activation, which in turn controls the generation of long-lived memory cells. These findings suggest that therapeutic modulation of Akt might be a strategy to augment vaccine-induced immunity.

Variants of the Bach2 gene are linked to vitiligo, celiac disease and type I diabetes, but the underlying immunological mechanisms are unknown. Here, we demonstrate that Bach2 plays crucial roles in maintaining T cell quiescence, and governing the differentiation, activation, and survival of foxp3+ Treg cells. Bach2-deficient T cells display spontaneous activation and produce elevated levels of TH1/TH2 type cytokines. Without Bach2, Treg cells exhibit diminished foxp3 expression, depleted numbers, hyper-activation, enhanced proliferation and profound loss of competitive fitness in vivo. Mechanistically, reduced survival of Bach2-deficient Treg cells was associated with reduced Bcl-2 and Mcl-1 levels and elevated Bim:Bcl-2 ratio. Additionally, Bach2 deficiency induced selective loss of Helios− foxp3+ Treg cells and a Treg cell transcriptome skewed towards the TH1/TH2 effector program at the expense of the Treg program. In vitro experiments confirmed that Bach2: (1) is indispensable for TCR/TGF-β-induced foxp3 expression and (2) mitigates aberrant differentiation of Treg cells by repression of the competing Gata3-driven TH2 effector program. Importantly, perturbations in the differentiation of induced Treg cells was linked to a fatal TH2 type chronic inflammatory lung disease in Bach2-deficient mice. Thus, Bach2 enforces T cell quiescence, promotes the development and survival of Treg lineage, restrains aberrant differentiation of Treg cells and protects against immune -mediated diseases.

CD8 T cell responses have three phases: expansion, contraction, and memory. Dynamic alterations in proliferation and apoptotic rates control CD8 T cell numbers at each phase, which in turn dictate the magnitude of CD8 T cell memory. Identification of signaling pathways that control CD8 T cell memory is incomplete. The PI3K/Akt signaling pathway controls cell growth in many cell types by modulating the activity of FOXO transcription factors. But the role of FOXOs in regulating CD8 T cell memory remains unknown. We show that phosphorylation of Akt, FOXO and mTOR in CD8 T cells occurs in a dynamic fashion in vivo during an acute viral infection. To elucidate the potentially dynamic role for FOXO3 in regulating homeostasis of activated CD8 T cells in lymphoid and non-lymphoid organs, we infected global and T cell-specific FOXO3-deficient mice with Lymphocytic Choriomeningitis Virus (LCMV). We found that FOXO3 deficiency induced a marked increase in the expansion of effector CD8 T cells, preferentially in the spleen, by T cell-intrinsic mechanisms. Mechanistically, the enhanced accumulation of proliferating CD8 T cells in FOXO3-deficient mice was not attributed to an augmented rate of cell division, but instead was linked to a reduction in cellular apoptosis. These data suggested that FOXO3 might inhibit accumulation of growth factor-deprived proliferating CD8 T cells by reducing their viability. By virtue of greater accumulation of memory precursor effector cells during expansion, the numbers of memory CD8 T cells were strikingly increased in the spleens of both global and T cell-specific FOXO3-deficient mice. The augmented CD8 T cell memory was durable, and FOXO3 deficiency did not perturb any of the qualitative attributes of memory T cells. In summary, we have identified FOXO3 as a critical regulator of CD8 T cell memory, and therapeutic modulation of FOXO3 might enhance vaccine-induced protective immunity against intracellular pathogens.

During a T cell response, naïve CD8 T cells differentiate into effector cells. Subsequently, a subset of effector cells termed memory precursor effector cells (MPECs) further differentiates into functionally mature memory CD8 T cells. The transcriptional network underlying this carefully scripted process is not well understood. Here, we report that the transcription factor FoxO1 plays an integral role in facilitating effector to memory transition and functional maturation of memory CD4 and CD8 T cells. We find that FoxO1 is not required for differentiation of effector cells, but in the absence of FoxO1, memory CD8 T cells displayed features of senescence and progressive attrition in polyfunctionality, which in turn led to impaired recall responses and poor protective immunity. These data suggest that FoxO1 is essential for maintenance of functional CD8 T cell memory and protective immunity. Under competing conditions in bone marrow chimeric mice, FoxO1-deficiency did not perturb clonal expansion or effector differentiation. Instead, FoxO1-deficient MPECs failed to survive and form memory CD8 T cells. Mechanistically, FoxO1 deficiency perturbed the memory CD8 T-cell transcriptome, characterized by pronounced alterations in the expression of genes that encode transcription factors (including Tcf7), effector molecules, cell cycle regulators and proteins that regulate fatty acid, purine and pyramidine metabolism and mitochondrial functions. We propose that FoxO1 is a key regulator that reprograms and steers the differentiation of effector cells to functionally competent memory cells. These findings have provided fundamental insights into the mechanisms that regulate the quality of CD8 T-cell memory to intracellular pathogens.

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.

The E3 ubiquitin ligase Cbl-b is a negative regulator of TCR signaling that: 1) sets the activation threshold for T cells; 2) is induced in anergic T cells; and 3) protects against autoimmunity. However, the role of Cbl-b in regulating CD8 T cell activation and functions during physiological T cell responses has not been systematically examined. Using the lymphocytic choriomeningitis virus infection model, we show that Cbl-b deficiency did not significantly affect the clonal expansion of virus-specific CD8 T cells. However, Cbl-b deficiency not only increased the steady-state cell surface expression levels of TCR and CD8 but also reduced Ag-induced down-modulation of cell surface TCR expression by effector CD8 T cells. Diminished Ag-stimulated TCR down-modulation and sustained Ag receptor signaling induced by Cbl-b deficiency markedly augmented IFN-γ production, which is known to require substantial TCR occupancy. By contrast, Cbl-b deficiency minimally affected cell-mediated cytotoxicity, which requires limited engagement of TCRs. Surprisingly, despite elevated expression of CD8 and reduced Ag-induced TCR down-modulation, the functional avidity of Cbl-b-deficient effector CD8 T cells was comparable to that of wild-type effectors. Collectively, these data not only show that Cbl-b-imposed constraint on TCR signaling has differential effects on various facets of CD8 T cell response but also suggest that Cbl-b might mitigate tissue injury induced by the overproduction of IFN-γ by CD8 T cells. These findings have implications in the development of therapies to bolster CD8 T cell function during viral infections or suppress T cell-mediated immunopathology.

More than 500 million people in the world are currently afflicted with chronic infections with viruses such as hepatitis B virus, hepatitis C virus, and HIV (24, 37, 41). A common denominator underlying viral persistence in these chronic viral infections (CVIs) is the dysregulation of virus-specific T cell responses; virus-specific T cells either undergo clonal deletion or lose their ability to express the full spectrum of effector functions, a condition termed functional exhaustion (8,22,29,33,41,53,56,59). During CVIs, there is a continuum of T cell proliferation and apoptosis, and the balance between these cellular processes controls the abundance of virus-specific CD8 T cells (18,48,49,53,60). However, the homeostatic mechanisms that control the number of virus-specific T cells under conditions of protracted antigenic stimulation are poorly defined. This is an important issue, because the magnitude of the T cell response is a critical factor in determining viral control.The FOXO family (FOXO1, FOXO3) of transcription factors plays a crucial role in regulating multiple facets of T cell homeostasis, including apoptosis, proliferation, trafficking, and differentiation (16,20,21,34,39,47). In particular, FOXO3 is known to promote the apoptosis of T cells and to limit the clonal expansion of CD8 T cells during an acute viral infection (14, 47). Additionally, FOXO3 has been implicated in vitro as a target for modulating T cell dysregulation in human T cells during HIV infection (52). The principal objective of this study was to gain insight into the role of FOXO3 in orchestrating the dynamics of CD8 T cell responses during a chronic lymphocytic choriomeningitis virus (LCMV) (clone 13) infection in mice. MATERIALS AND METHODSMice and viral infection. The derivation of mice carrying floxed FOXO3 alleles has been described elsewhere (36,50). Mice carrying floxed FOXO3 alleles were bred with CD4-Cre mice at the University of WisconsinMadison (UW-Madison) to generate T-cell-specific FOXO3-deficient (FOXO3L) mice. Wild-type (WT) littermates were used as controls with the FOXO3L mice. Mice used in experiments were between the ages of 6 and 8 weeks, and all experiments were performed in accordance with the protocols approved by the University of Wisconsin School of Veterinary Medicine Institutional Animal Care and Use Committee (IACUC). Mice were infected with 2 ϫ 10 6 PFU of the LCMV clone 13 strain by intravenous (i.v.) injection. Tissue viral titers were quantified by plaque assays with Vero cell monolayers (2).General flow cytometry. Single-cell suspensions of splenocytes were stained with antibodies for surface markers, including CD8, CD44, CD122, CD127, CD62L, LFA-1, and KLRG-1 (BD Biosciences, Franklin Lakes, NJ; eBioscience, San Diego, CA; or Southern Biotech, Birmingham, AL) in conjunction with major histocompatibility complex class I (MHC-I) tetramers (D b ) specific for the class I-restricted LCMV epitopes, NP396, GP33, and GP276 (30). Cells were fixed in 2% paraformaldehyde (PFA) and were acquired in a FACSCal...

Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8+ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8+ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8+ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8+ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8+ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8+ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8+ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8+ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.