Differential ability of surface and endosomal TLRs to induce CD8 T cell responses in vivo.

TLR activation on dendritic cells (DCs) induces DC maturation and secretion of proinflammatory cytokines, both of which are important for activation and differentiation of CD4 T cells. The importance of TLR activation on DCs for CD8 T cell responses is less clear. In this study, we tested the ability of different TLRs to regulate CD8 T cell responses to pathogens. We found that although all TLRs are able to induce CD8 T cell activation in vitro, there are profound differences in their ability to activate CD8 T cells in vivo. The nucleic acid recognizing endosomal TLRs, TLR3 and TLR9, had a potent ability to induce CD8 T cell activation. However, the surface TLRs, TLR2 and TLR4, that recognize bacterial ligands were not only incapable of inducing CD8 T cell priming, but they had a dominant effect of inhibiting CD8 T cell expansion induced by activation of endosomal TLRs. We found that TLR2 and TLR4, acting in a MyD88-dependent manner, influenced CD8 T cell priming by altering the composition of DCs in the draining lymph nodes. Our results have important implications for combined bacterial and viral infections and suggest that bacterial infections could constrain the ability of the host to mount effective antiviral CD8 T cell immunity.

Transgenic expression of microRNA-185 causes a developmental arrest of T cells by targeting multiple genes including Mzb1.

miR-185 is a microRNA (miR) that targets Bruton's tyrosine kinase in B cells, with reductions in miR-185 linked to B cell autoantibody production. In hippocampal neurons, miR-185 targets both sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and a novel Golgi inhibitor. This miR is haploinsufficient in 90-95% of individuals with chromosome 22q11.2 deletion syndrome, patients who can present with immune, cardiac, and parathyroid problems, learning disorders, and a high incidence of schizophrenia in adults. The reduced levels of miR-185 in neurons cause presynaptic neurotransmitter release. Many of the 22q11.2 deletion syndrome patients have a thymic hypoplasia, which results in a peripheral T cell lymphopenia and unusual T helper cell skewing. The molecular targets of miR-185 in thymocytes are unknown. Using an miR-185 T cell transgenic approach, increasing levels of miR-185 attenuated T cell development at the T cell receptor ß (TCRß) selection checkpoint and during positive selection. This caused a peripheral T cell lymphopenia. Mzb1, Nfatc3, and Camk4 were identified as novel miR-185 targets. Elevations in miR-185 enhanced TCR-dependent intracellular calcium levels, whereas a knockdown of miR-185 diminished these calcium responses. These effects concur with reductions in Mzb1, an endoplasmic reticulum calcium regulator. Consistent with their haploinsufficiency of miR-185, Mzb1 levels were elevated in thymocyte extracts from several 22q11.2 deletion syndrome patients. Our findings indicate that miR-185 regulates T cell development through its targeting of several mRNAs including Mzb1.

Microbial infection-induced expansion of effector T cells overcomes the suppressive effects of regulatory T cells via an IL-2 deprivation mechanism.

Foxp3(+) regulatory T (Treg) cells are a critical cell population that suppresses T cell activation in response to microbial and viral pathogens. We identify a cell-intrinsic mechanism by which effector CD4(+) T cells overcome the suppressive effects of Treg cells in the context of three distinct infections: Toxoplasma gondii, Listeria monocytogenes, and vaccinia virus. The acute responses to the parasitic, bacterial, and viral pathogens resulted in a transient reduction in frequency and absolute number of Treg cells. The infection-induced partial loss of Treg cells was essential for the initiation of potent Th1 responses and host protection against the pathogens. The observed disappearance of Treg cells was a result of insufficiency in IL-2 caused by the expansion of pathogen-specific CD4(+) T cells with a limited capacity of IL-2 production. Exogenous IL-2 treatment during the parasitic, bacterial, and viral infections completely prevented the loss of Treg cells, but restoration of Treg cells resulted in a greatly enhanced susceptibility to the pathogens. These results demonstrate that the transient reduction in Treg cells induced by pathogens via IL-2 deprivation is essential for optimal T cell responses and host resistance to microbial and viral pathogens.

IL-12 selectively programs effector pathways that are stably expressed in human CD8+ effector memory T cells in vivo.

CD8(+) cytotoxic T lymphocytes play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. IL-12 and IFN-α/ß are potent "signal 3" cytokines that are involved in both effector and memory cell development. Although the majority of effector cells are eliminated as inflammation resolves, some survive within the pool of memory cells and retain immediate effector function. In this study, we demonstrate that IL-12 instructs a unique program of effector cell differentiation that is distinct from IFN-α/ß. Moreover, effector memory (T(EM)) cells within peripheral blood display many common attributes of cells differentiated in vitro in response to IL-12, including proinflammatory cytokine secretion and lytic activity. A pattern of IL-12-induced genes was identified that demarcate T(EM) from central memory cells, and the ontologies of these genes correlated precisely with their effector functions. Further, we uncovered a unique program of gene expression that was acutely regulated by IL-12 and reflected in stable gene expression patterns within T(EM), but not T central memory cells in vivo. Thus, this study directly links a selective set of IL-12-induced genes to the programming of effector functions within the stable population of human CD8(+) T(EM) cells in vivo.

CD4-CD8- T cells control intracellular bacterial infections both in vitro and in vivo.

Memory T cells, including the well-known CD4(+) and CD8(+) T cells, are central components of the acquired immune system and are the basis for successful vaccination. After infection, CD4(+) and CD8(+) T cells expand into effector cells, and then differentiate into long-lived memory cells. We show that a rare population of CD4(-)CD8(-)CD3(+)alphabeta(+)gammadelta(-)NK1.1(-) T cells has similar functions. These cells potently and specifically inhibit the growth of the intracellular bacteria Mycobacterium tuberculosis (M. tb.) or Francisella tularensis Live Vaccine Strain (LVS) in macrophages in vitro, promote survival of mice infected with these organisms in vivo, and adoptively transfer immunity to F. tularensis LVS. Furthermore, these cells expand in the spleens of mice infected with M. tb. or F. tularensis LVS, and then acquire a memory cell phenotype. Thus, CD4(-)CD8(-) T cells have a role in the control of intracellular infection and may contribute to successful vaccination.

Reciprocal responsiveness to interleukin-12 and interferon-alpha specifies human CD8+ effector versus central memory T-cell fates.

Multiple innate signals regulate the genesis of effector and memory CD8+ T cells. In this study, we demonstrate that the innate cytokines interleukin (IL)-12 and interferon (IFN)-alpha/beta regulate distinct aspects of effector and memory human CD8+ T-cell differentiation. IL-12 exclusively promoted the development of IFN-gamma- and tumor necrosis factor (TNF)-alpha-secreting T effector memory (T(EM)) cells, whereas IFN-alpha drove the development of T central memory (T(CM)) cells. The development of T(EM) and T(CM) was linked to cell division. In rapidly dividing cells, IL-12 programmed T(EM) through induction of the IL-12 receptor beta2. In contrast, IFN-alpha regulated T(CM) development by slowing the progression of cell division in a subpopulation of cells that selectively expressed elevated IFN-alpha/beta receptor-2. The strength of signal delivered through T-cell receptor (TCR) engagement regulated the responsiveness of cells to IL-12 and IFN-alpha. In the presence of both IL-12 and IFN-alpha, these cytokine signals were amplified as the strength of the TCR signal was increased, promoting the simultaneous development of both T(CM) and T(EM). Together, our results support a novel model in which IL-12 and IFN-alpha act in a nonredundant manner to regulate the colinear generation of both effector and memory cells.

Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen.

Interferon (IFN)-gamma plays an important role in the innate immune response against intracellular bacterial pathogens. It is commonly thought that natural killer cells are the primary source of this cytokine that is involved in activating antibacterial effects in infected cells and polarizing CD4+ T cells toward the Th1 subset. However, here we show that both effector and memory CD8+ T cells have the potential to secrete IFN-gamma in response to interleukin (IL)-12 and IL-18 in the absence of cognate antigen. We demonstrate that memory CD8+ T cells specific for the ovalbumin protein secrete IFN-gamma rapidly after infection with wild-type Listeria monocytogenes (LM). Furthermore, small numbers of ovalbumin-specific, memory CD8+ T cells can reduce spleen and liver bacterial counts in IFN-gamma-deficient mice 3 d after LM infection. Up-regulation of the receptors for IL-12 and IL-18 provides a mechanism for the ability of memory CD8+ T cells to respond in this antigen nonspecific manner. Thus, CD8+ T cells play an important role in the innate immune response against intracellular pathogens by rapidly secreting IFN-gamma in response to IL-12 and IL-18.

The role of CD8 T cells in innate immunity and in antigen non-specific protection.

The role of CD8 T cells in adaptive immune responses is well understood. These lymphocytes respond through their T cell receptors to diverse antigens presented by MHC class I molecules by proliferating, secreting cytokines and chemokines, and directly lysing infected cells. Recently, a role for CD8 T cells in the innate immune response has become apparent. Independent of T cell receptor ligation, CD8 T cells can mount a response against pathogens by secreting cytokines and can defend against tumors by directly killing transformed cells. This innate response has been shown to be beneficial in controlling several types of bacterial infections. However, a subset of CD8 T cells that have innate non-antigen-specific capabilities has been implicated in self-reactivity, which could lead to autoimmunity.

Synthesis and SAR of 2-aryloxy-4-alkoxy-pyridines as potent orally active corticotropin-releasing factor 1 receptor antagonists.

A series of 2-aryloxy-4-alkoxy-pyridines ( 1) was identified as novel, selective, and orally active antagonists of the corticotropin-releasing factor 1 (CRF 1) receptor. Among these, compound 2 (CP-316311) is a potent and selective CRF 1 receptor antagonist with an IC 50 value of 6.8 nM in receptor binding and demonstrates oral efficacy in central nervous system (CNS) in vivo models. The regiochemistry of compounds in this series was determined by an X-ray structural analysis. A method to control regioselectivity via pyridine- N-oxides was developed. The synthesis of compounds in series 1 (Figure ) and [ (3)H]- 2 as well as the structure-activity relationship (SAR) are discussed. The in vitro, ex vivo, and in vivo properties of representative compounds are described herein. Compound 2 was advanced to phase II depression trials to test the hypothesis that CRF 1 antagonists could be used clinically as antidepressant drugs.

2-aryloxy-4-alkylaminopyridines: discovery of novel corticotropin-releasing factor 1 antagonists.

An orally active clinical candidate of corticotropin-releasing factor 1 (CRF 1) antagonist 1 showed a significant positive food effect in dog and human after oral administration. Efforts to address the food effect issue led us to explore and discover compounds in series 2 as orally active CRF 1 receptor antagonists, in which some compounds showed improved physicochemical properties while retaining desired pharmacological properties. Compound 3a (CP-376395) was selected for further development, due not only to its reduced food effects but also its greater efficacy in CNS models. Compound 3a was advanced to the clinic. The synthesis of representative potential candidates and their in vitro, ex vivo, and in vivo data are described.

The role of CD94/NKG2 in innate and adaptive immunity.

CD94/NKG2 is a heterodimer expressed on natural killer (NK) and a small subset of T cells. This receptor varies in function as an inhibitor or activator depending on which isoform of NKG2 is expressed. The ligand for CD94/NKG2 is HLA-E in human and its homolog, Qa1 in mouse, which are both nonclassical class I molecules that bind leader peptides from other class I molecules. Although <5% of CD8 T cells express the receptor in a naïve mouse, its expression is upregulated upon specific recognition of antigen. Similar to NK cells, most CD8 T cells that express high levels of CD94 co-express NKG2A, the inhibitory isoform. The engagement of this receptor can lead to a blocking of cytotoxicity. However, these receptors have also been implicated in the cell survival of both NK and CD8 T cells. The level of CD94 expression is inversely correlated with the level of apoptosis in culture. Thus, CD94/NKG2 receptors may regulate effector functions and cell survival of NK cells and CD8 T cells, thereby playing a crucial role in the innate and adaptive immune response to a pathogen.

Genetic analysis of influences on survival following Toxoplasma gondii infection.

Survival of mice during the acute stage of Toxoplasma gondii infection was not influenced by the MHC Class I gene, L(d), but was influenced by the MHC Class II genes, Ia and Ie. As unexplained variability was noted in our initial studies of influence of the L(d) gene on survival, influence of the L(d) gene region on survival in the presence of a number of variables was studied. Although route of administration and dose of parasites, and age and gender of the mice markedly influenced outcome of T. gondii infection, the Class I L(d) gene did not modify survival in any of these circumstances. In separate studies, using mice with a differing genetic background, i.e. H-2(b), C57BL/10 mice, presence of Ia or Ie alone diminished survival even though presence of Ia reduced parasite burden. When neither or both the Ia and Ie genes were present together, survival was greater. In separate analyses of our studies of AxB BxA recombinant inbred mice, similar influences of MHC genes on survival and parasite burden following peroral infection were confirmed. Previously undescribed associations of novel genetic loci and survival and parasite burden also were identified. Genetic loci associated with enhanced survival included D8Mit42, D1Mit3, Iapls1-16, D8Mit14, Hoxb, Mpmv29, Pmv45, and Emv-2; genetic loci associated with reduced parasite burden included H-2, D17Mit62, D17Mit83, D17Mit21, D17Mit34, D17Mit47, D18Mit4, and Gln3-5. These studies demonstrate the importance of MHC region genes (but not L(d)) for survival, and the influence of other novel genes, and endogenous and exogenous variables on survival and parasite burden specified by host genes following T. gondii infection.

Pharmacological inhibition of TPL2/MAP3K8 blocks human cytotoxic T lymphocyte effector functions.

CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens. During development, antigen-presenting cells secrete innate cytokines such as IL-12 and IFN-α, which drive CTL differentiation into diverse populations of effector and long-lived memory cells. Using whole transcriptome analyses, the serine/threonine protein kinase Tpl2/MAP3K8 was found to be induced by IL-12 and selectively expressed by effector memory (TEM) CTLs. Tpl2 regulates various inflammatory pathways by activating the ERK mediated MAP kinase pathway in innate immune cells such as macrophages and dendritic cells. In this study, we found that a specific small molecule Tpl2 inhibitor blocked IFN-γ and TNF-α secretion as well as cytolytic activity of human CTLs. This pathway was specific for human effector CTLs, as the Tpl2 inhibitor did not block IFN-γ and TNF-α secretion from murine effector CTLs. Further, IL-12 failed to induce expression of Tpl2 in murine CTLs, and Tpl2 deficient murine CTLs did not exhibit any functional deficiency either in vitro or in vivo in response to L. monocytogenes infection. In summary, we identified a species-specific role for Tpl2 in effector function of human CTLs, which plays a major role in adaptive immune responses to intracellular pathogens and tumors.

Somatic expression of PyMT or activated ErbB2 induces estrogen-independent mammary tumorigenesis.

Estrogen signaling is required for the proliferation of normal breast epithelial cells. However, prophylactic inhibition of estrogen signaling fails to prevent 56% of human breast cancer cases. The underlying mechanism is not well understood. Aberrant activation of growth factor signaling is known to provide alternative proliferation pathways in breast cells that are fully transformed, but it is not known whether activation of growth factor signaling can substitute for estrogen signaling in causing aberrant proliferation in the normal breast epithelium. Here, we report that in a retrovirus-based somatic mouse model (replication-competent ALV-LTR splice acceptor/tumor virus A) that closely mimics the evolution of sporadic human breast cancers, mammary epithelial cells harboring PyMT or activated ErbB2 evolve into tumors independent of estrogen or other ovarian functions in contrast to previous observations of estrogen-dependent cancer formation in germ line mouse models of ErbB2 activation. Importantly, ErbB2 activation in normal mammary cells causes estrogen-independent proliferation in both estrogen receptor (ER)-negative cells as well as in normally quiescent ER-positive cells. Therefore, aberrant activation of growth factor signaling contributes to estrogen-independent proliferation of both preneoplastic and cancerous mammary cells, and prophylactic therapy against both growth factor signaling and estrogen signaling may need to be considered in women with increased risk of breast cancer.

Imatinib mesylate inhibits antigen-specific memory CD8 T cell responses in vivo.

Imatinib mesylate (IM) is effective at inducing complete cytogenetic remission in patients with chronic myelogenous leukemia. Because its influence on CD8 T cell responsiveness in vivo is unknown, we investigated the effects of IM by analyzing the response of OT-1 CD8 T cells to Listeria monocytogenes (LM) that express the cognate epitope OVA(257-264) (LM-OVA). In vitro, IM had no effect on Ag-specific expansion, cell division, cell cycle progression, or IFN-gamma expression in naive or memory OT-1 T cells. However, IM induced apoptosis of naive and memory OT-1 T cells at doses of >5 microM. At 15 microM IM, OT-1 T cells did not survive in in vitro cultures. The primary response of OT-1 T cells in vivo to LM-OVA infection was unaltered. In contrast, continuous IM treatment resulted in a diminished memory OT-1 response. The expression of IL-7Ralpha, a receptor required for memory cell survival, was lower (on OT-1 cells) in animals receiving IM. These results indicate that IM treatment affects the ability of the CD8 memory pool to respond to Ag and has the potential to increase susceptibility to infection.

The specific and essential role of MAVS in antiviral innate immune responses.

The mitochondrial antiviral signaling protein (MAVS) mediates the activation of NFkappaB and IRFs and the induction of interferons in response to viral infection. In vitro studies have also suggested that MAVS is required for interferon induction by cytosolic DNA, but the in vivo evidence is lacking. By generating MAVS-deficient mice, here we show that loss of MAVS abolished viral induction of interferons and prevented the activation of NFkappaB and IRF3 in multiple cell types, except plasmacytoid dendritic cells (pDCs). However, MAVS was not required for interferon induction by cytosolic DNA or by Listeria monocytogenes. Mice lacking MAVS were viable and fertile, but they failed to induce interferons in response to poly(I:C) stimulation and were severely compromised in immune defense against viral infection. These results provide the in vivo evidence that the cytosolic viral signaling pathway through MAVS is specifically required for innate immune responses against viral infection.

Thymus-dependent memory phenotype CD8 T cells in naive B6.H-2Kb-/-Db-/- animals mediate an antigen-specific response against Listeria monocytogenes.

B6.H-2Kb-/-Db-/- (DKO) mice have greatly reduced numbers of mature CD8alphabeta T cells in their periphery. However, these non-class Ia-selected CD8alphabeta T cells are able to mediate immune responses to a number of pathogens. Approximately 60% of the CD8alphabeta T cells in the spleen and peripheral lymph nodes of naive DKO mice display a memory (CD44high) phenotype. To investigate the origins of these non-class Ia-selected CD8alphabetaCD44high cells, we traced the phenotype of recent thymic emigrants and found that most were CD44low. We also determined whether their appearance was thymus dependent and found that only a small percentage of non-class Ia-selected CD8alphabetaCD44high cells develop in a thymus-independent pathway. Functionally, CD8alphabetaCD44high cells from DKO mice are able to secrete IFN-gamma in response to IL-12 and IL-18 in the absence of cognate Ag. When challenged with anti-CD3 in vivo, nearly half of these cells produce IFN-gamma within 3 h. When purified CD8alphabetaCD44high cells from Thy1.2.DKO mice were transferred into Thy1.1 DKO recipients and then challenged with Listeria monocytogenes, an Ag-specific anti-L. monocytogenes response was observed 6 days later. Our data suggest that non-class Ia-selected CD8alphabetaCD44high cells in naive animals can respond rapidly to Ag and play a role in the innate as well as the early phase of the acquired immune response.

Relative contributions of NK and CD8 T cells to IFN-gamma mediated innate immune protection against Listeria monocytogenes.

During the innate immune response to Listeria monocytogenes (LM), the secretion of IFN-gamma is crucial in controlling bacterial numbers. We have shown recently that CD8 T cells have the ability to rapidly secrete IFN-gamma independent of Ag, in response to IL-12 and IL-18, during a LM infection. In the current study, we compared the relative abilities of NK and CD8 T cells to provide innate immune protection. Upon transfer of either NK or memory OT-I T cells (specific for the OVA protein) into IFN-gamma-deficient hosts that were infected subsequently with wild-type LM, both cell types were found in the spleen and had the ability to secrete IFN-gamma. However, the OT-I T cells were more effective at providing innate immune protection as determined by spleen and liver LM burdens. We used immunocytochemistry to demonstrate that upon infection with LM, marginal zone macrophages were localized to the T cell area of the splenic follicle. Transferred memory OT-I T cells were also found in the T cell area of the spleen, co-localizing with the LM and macrophages. In sharp contrast, NK cells were found predominantly in the red pulp region of the spleen. In addition, memory OT-I T cells were also found to be associated with LM lesions in the liver. These results highlight the importance of CD8 T cells in innate immune responses to LM and suggest that their increased protective ability compared with NK cells is the result of their colocalization with LM and macrophages.

The role of TCR stimulation and TGF-beta in controlling the expression of CD94/NKG2A receptors on CD8 T cells.

Following antigen recognition, murine CD8 T cells express CD94/NKG2A receptors. Our results show that this up-regulation occurs rapidly in vitro and is accompanied by an approximately 8-fold increase in CD94 and approximately 125-fold increase in NKG2A mRNA. In contrast, only a twofold increase in NKG2C mRNA is noted. The addition of TGF-beta, but not IL-10, IL-12 or IL-15, leads to a further increase in cell membrane expression of these receptors, as well as a approximately 6-fold increase in mRNA for both chains. TGF-beta also increases CD94/NKG2A expression on memory CD8 T cells that are re-exposed to antigen. The effect of TGF-beta on increasing CD94/NKG2A expression on both naive and memory CD8 T cells occurs only when there is a concurrent stimulation through the TCR. In contrast, TGF-beta does not increase expression of CD94/NKG2A on resting or activated NK cells. We also show by using purified CD8 T cells, that TGF-beta acts directly on these cells. These results implicate a role for both antigen and TGF-beta in increasing expression of inhibitory CD94/NKG2A receptors on CD8 T cells.