Sean Murray

Memory CD8+ T Cells Provide Innate Immune Protection against Listeria monocytogenes in the Absence of Cognate Antigen

Interferon (IFN)-γ 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-γ 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-γ 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-γ–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-γ in response to IL-12 and IL-18.

Relative Contributions of NK and CD8 T Cells to IFN-γ Mediated Innate Immune Protection against Listeria monocytogenes

During the innate immune response to Listeria monocytogenes (LM), the secretion of IFN-γ is crucial in controlling bacterial numbers. We have shown recently that CD8 T cells have the ability to rapidly secrete IFN-γ 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-γ-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-γ. 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, colocalizing 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.

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.

The role of TCR stimulation and TGF‐β 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 ∼8‐fold increase in CD94 and ∼125‐fold increase in NKG2A mRNA. In contrast, only a twofold increase in NKG2C mRNA is noted. The addition of TGF‐β, 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 ∼6‐fold increase in mRNA for both chains. TGF‐β also increases CD94/NKG2A expression on memory CD8 T cells that are re‐exposed to antigen. The effect of TGF‐β 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‐β does not increase expression of CD94/NKG2A on resting or activated NK cells. We also show by using purified CD8 T cells, that TGF‐β acts directly on these cells. These results implicate a role for both antigen and TGF‐β in increasing expression of inhibitory CD94/NKG2A receptors on CD8 T cells.

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

Background: MicroRNAs have critical roles in T cell development under normal and stress conditions. Results: Increasing miR-185 levels attenuate T cell development via the targeting of Mzb1, Nfatc3, and Camk4. Conclusion: Elevations in miR-185 impair thymopoiesis at several developmental stages. Significance: miR-185 levels are reduced in 22q11.2 deletion syndrome patients and the identification of its gene targets is clinically informative. miR-185 is a microRNA (miR) that targets Brutons 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 β (TCRβ) 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.

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 CD8αβ T cells in their periphery. However, these non-class Ia-selected CD8αβ T cells are able to mediate immune responses to a number of pathogens. Approximately 60% of the CD8αβ 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 CD8αβCD44high 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 CD8αβCD44high cells develop in a thymus-independent pathway. Functionally, CD8αβCD44high cells from DKO mice are able to secrete IFN-γ 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-γ within 3 h. When purified CD8αβCD44high 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 CD8αβCD44high 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.

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.