Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines.

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.

A Simple and Reliable Method for Early Pregnancy Detection in Inbred Mice.

The study of normal and abnormal development typically requires precise embryonic staging. In mice, this task is accomplished through timed matings and the detection of a copulation plug. However, the presence of a plug is not a definitive indicator of true pregnancy, particularly in inbred mice, in which false-pregnancy rates have been reported to be 50% or higher, depending on the strain. This high rate poses considerable financial and animal use burdens because manipulation of the putative dam is often required before pregnancy can be confirmed by palpation or visual inspection. To address this problem, we examined weight gain in a population of 275 wildtype C57BL/6J mice (age, 12 wk or older) between the time of plug detection and during early embryogenesis (gestational days 7 to 10). In this population, assessing pregnancy according to the presence of a plug alone yielded a 37.1% false-positive rate. Pregnant mice gained an average of 3.49 g, whereas non-pregnant mice gained only 1.15 g. Beginning at gestational day 7.75, implementing an optimal weight-gain discrimination threshold of 1.75 g reduced the false-positive rate to 10.5%, without excluding any pregnant mice. These results were consistent with those from younger (age, 8 wk) wildtype C57BL/6J and FVB/NTac female mice, suggesting broad applicability of this method across age and strain. Our findings provide a simple and effective method for reducing animal use and study costs.

DNA methylation of E-cadherin is a priming mechanism for prostate development.

In prostate and other epithelial cancers, E-cadherin (CDH1) is downregulated inappropriately by DNA methylation to promote an invasive phenotype. Though cancer frequently involves a reawakening of developmental signaling pathways, whether DNA methylation of Cdh1 occurs during organogenesis has not been determined. Here we show that DNA methylation of Cdh1 mediates outgrowth of developing prostate ducts. During the three-day gestational window leading up to and including prostate ductal initiation, Cdh1 promoter methylation increases and its mRNA and protein abundance decreases in epithelium giving rise to prostatic buds. DNA methylation is required for prostate specification, ductal outgrowth, and branching morphogenesis. All three endpoints are impaired by a DNA methylation inhibitor, which also decreases Cdh1 promoter methylation and increases Cdh1 mRNA and protein abundance. A CDH1 function-blocking antibody restores prostatic identity, bud outgrowth, and potentiates epithelial differentiation in the presence of the DNA methylation inhibitor. This is the first study to mechanistically link acquired changes in DNA methylation to the normal process of prostate organogenesis. We propose a novel mechanism whereby Cdh1 promoter methylation restricts Cdh1 abundance in developing prostate epithelium to create a permissive environment for prostatic bud outgrowth. Thus, DNA methylation primes the prostate primordium to respond to developmental cues mediating outgrowth, differentiation and maturation of the ductal network.

FoxO1 controls effector-to-memory transition and maintenance of functional CD8 T cell memory.

During a T cell response, naive CD8 T cells differentiate into effector cells. Subsequently, a subset of effector cells termed memory precursor effector cells further differentiates into functionally mature memory CD8 T cells. The transcriptional network underlying this carefully scripted process is not well understood. In this study, 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 memory precursor effector cells 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.

T-cell intrinsic and extrinsic mechanisms of p27Kip1 in the regulation of CD8 T-cell memory.

CD8 T cells exhibit dynamic alterations in proliferation and apoptosis during various phases of the CD8 T-cell response, but the mechanisms that regulate cellular proliferation from the standpoint of CD8 T-cell memory are not well defined. The cyclin-dependent kinase inhibitor p27(Kip1) functions as a negative regulator of the cell cycle in T cells, and it has been implicated in regulating cellular processes, including differentiation, transcription and migration. Here, we investigated whether p27(Kip1) regulates CD8 T-cell memory by T-cell-intrinsic or T-cell-extrinsic mechanisms, by conditional ablation of p27(Kip1) in T cells or non-T cells. Studies of T-cell responses to an acute viral infection show that p27(Kip1) negatively regulates the proliferation of CD8 T cells by T-cell-intrinsic mechanisms. However, the enhanced proliferation of CD8 T cells induced by T-cell-specific p27(Kip1) deficiency minimally affects the primary expansion or the magnitude of CD8 T-cell memory. Unexpectedly, p27(Kip1) ablation in non-T cells markedly augmented the number of high-quality memory CD8 T cells by enhancing the accumulation of memory precursor effector cells without increasing their proliferation. Further studies show that p27(Kip1) deficiency in immunizing dendritic cells fail to enhance CD8 T-cell memory. Nevertheless, we have delineated the T-cell-intrinsic, anti-proliferative activities of p27(Kip1) in CD8 T cells from its role as a factor in non-T cells that restricts the development of CD8 T-cell memory. These findings have implications in vaccine development and understanding the mechanisms that maintain T-cell homeostasis.

p27(Kip1) negatively regulates the magnitude and persistence of CD4 T cell memory.

Much is known about the differentiation of naive T cells into distinct lineages of effector cells, but the molecular mechanisms underlying the generation and maintenance of CD4 T cell memory are poorly characterized. Our studies ascribe a novel role for the cell cycle regulator p27(Kip1) as a prominent negative regulator of the establishment and long-term maintenance of Th1 CD4 T cell memory. We demonstrate that p27(Kip1) might restrict the differentiation and survival of memory precursors by increasing the T-bet/Bcl-6 ratio in effector CD4 T cells. By promoting apoptosis and contraction of effector CD4 T cells by mechanisms that are at least in part T cell intrinsic, p27(Kip1) markedly limits the abundance of memory CD4 T cells. Furthermore, we causally link p27(Kip1)-dependent apoptosis to the decay of CD4 T cell memory, possibly by repressing the expression of γ-chain receptors and the downstream effector of the Wnt/ß-catenin signaling pathway, Tcf-1. We extend these findings by showing that the antagonistic effects of p27(Kip1) on CD4 T cell memory require its cyclin-dependent kinase-binding domain. Collectively, these findings provide key insights into the mechanisms underlying the governance of peripheral CD4 T cell homeostasis and identify p27(Kip1) as a target to enhance vaccine-induced CD4 T cell memory.

FOXO3 regulates the CD8 T cell response to a chronic viral infection.

Chronic infections with viruses such as hepatitis B virus, hepatitis C virus, and HIV constitute a major global public health problem. Studies of chronic viral infections in humans and mice show that persistent antigenic stimulation induces dysregulation of T cell responses; virus-specific T cells either undergo clonal deletion or lose their ability to display the full spectrum of effector functions, a condition termed functional exhaustion. The ability to generate and retain sufficient numbers of functionally competent T cells, therefore, becomes vitally important in controlling chronic viral infections. Our understanding of the mechanisms governing T cell homeostasis during chronic viral infections, however, is poor. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway controls cell fate decisions in many cell types by modulating the activity of downstream effectors, including the FOXO family of transcription factors. We have observed dynamic, in vivo alterations in the phosphorylation levels of three key proteins (Akt, FOXO1/FOXO3 [FOXO1/3], and mammalian target of rapamycin [mTOR]) involved in this signaling cascade and have identified the transcription factor FOXO3 as a negative regulator of the magnitude and effector function of CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. We report that ablation of FOXO3 in T cells reduced apoptosis, increased the abundance of polyfunctional virus-specific CD8 T cells, and improved viral control. Thus, FOXO3 is a promising candidate target for immunotherapies of chronic viral infection.

Signal integration by Akt regulates CD8 T cell effector and memory differentiation.

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, which 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-12, influence differentiation of SLECs and memory precursor effector cells. 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/ß-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. Whereas 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.

FOXO3 regulates CD8 T cell memory by T cell-intrinsic mechanisms.

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.

Role of PKR and Type I IFNs in viral control during primary and secondary infection.

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.