Polyglutamine binding protein 1 (PQBP1) inhibits innate immune responses to cytosolic DNA.

Recent studies have highlighted the importance of immune sensing of cytosolic DNA of both pathogen and host origin. We aimed to examine the role of DNA sensors interferon-γ-inducible protein 16 (IFI16) and cyclic GMP-AMP synthase (cGAS) in responding to cytosolic DNA. We show IFI16 and cGAS can synergistically induce IFNb transcriptional activity in response to cytoplasmic DNA. We also examined the role of polyglutamine binding protein 1 (PQBP1), a protein predominantly expressed in lymphoid and myeloid cells that has been shown to lead to type I interferon production in response to retroviral infection. We show PQBP1 associates with cGAS and IFI16 in THP-1 cells. Unexpectedly, knockout of PQBP1 in THP-1 cells causes significantly increased type I IFN production in response to transfected cytosolic nucleic acids or DNA damage, unlike what is seen in response to retroviral infection. Overexpression of PQBP1 in HEK293 T cells impairs IFI16/cGAS-induced IFNb transcriptional activity. In human cancer patients, low expression of PQBP1 is correlated with improved survival, the opposite correlation of that seen with cGAS or IFI16 expression. Our findings suggest that PQBP1 inhibits IFI16/cGAS-induced signaling in response to cytosolic DNA, in contrast to the role of this protein in response to retroviral infection.

Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt.

The inflammasome activates caspase-1 and the release of interleukin-1ß (IL-1ß) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2(-/-)/Apc(Min/+) than in APC(Min/+) mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1ß and were primarily mediated by a non-bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK-mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2(-/-) mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers.

Evaluation of T cell function in allergic disease.

T lymphocytes play positive and negative roles in the pathogenesis of allergic disease. Isolation and functional characterization of T lymphocyte subpopulations is an important aspect of understanding allergy models and allergy therapies. Measurement of the T cell surface proteins and T cell proliferation can provide insight into T cell activation. T cell function and the identities of T cell subsets can be determined by measuring cytokine production, either via intracellular cytokine staining or ELISPOT. This chapter outlines protocols for T cell isolation as well as the evaluation of surface protein expression, proliferation, intracellular cytokine staining, and ELISPOT.

Measuring T cell function in innate immune models.

Innate immune responses often result in the activation and modulation of T lymphocyte function. Analysis of T lymphocytes in mouse models of innate immunity can allow understanding of the links between the innate and adaptive immune systems. Other T lymphocyte populations display innate-like functions. Isolation of T cells and evaluation of their surface proteins can provide data on T cell activation, as can an analysis of T cell proliferation. Further insight may be obtained by examining cytokine production via intracellular cytokine staining or ELISPOT to determine T cell function. This chapter describes methods for T cell isolation, measurement of surface protein expression, T cell proliferation, intracellular cytokine staining, and ELISPOT.

Cross-regulation between the IL-1ß/IL-18 processing inflammasome and other inflammatory cytokines.

The inflammasome-forming NLRs are well characterized members of a protein complex mediating the activation of caspase-1 and the cleavage of pro-IL-1ß and pro-IL-18 into their active, secreted forms. New data suggest that components of the inflammasome cascade may have roles in influencing inflammasome-independent pathways of cytokine production. These influences on other immune cytokine pathways are complemented by data suggesting that non-inflammasome cytokines can influence the activation of the inflammasome, either directly or by influencing transcription of inflammasome components. The crosstalk between these cytokine cascades may lead to increased abilities for the cell to respond to diverse pathogen threats.

Cutting edge: NLRC5-dependent activation of the inflammasome.

The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1ß and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function. RNA interference-mediated knockdown of NLRC5 nearly eliminated caspase 1, IL-1ß, and IL-18 processing in response to bacterial infection, pathogen-associated molecular patterns, and damage-associated molecular patterns. This was confirmed in primary human monocytic cells. NLRC5, together with procaspase 1, pro-IL-1ß, and the inflammasome adaptor ASC, reconstituted inflammasome activity that showed cooperativity with NLRP3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in a nucleotide-binding domain-dependent but leucine-rich repeat-inhibitory fashion. These results invoke a model in which NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome.

Critical role for IL-21 in both primary and memory anti-viral CD8+ T-cell responses.

While it is well established that CD8(+) T cells generated in the absence of CD4(+) T cells mediate defective recall responses, the mechanism by which CD4(+) T cells confer help in the generation of CD8(+) T-cell responses remains poorly understood. To determine whether CD4(+) T-cell-derived IL-21 is an important regulator of CD8(+) T-cell responses in help-dependent and -independent viral infections, we examined these responses in the IL-21Rα(-/-) mouse model. We show that IL-21 has a role in primary CD8(+) T-cell responses and in recall CD8(+) T-cell responses in help-dependent viral infections. This effect is due to a direct action of IL-21 in enhancing the proliferation of virus-specific CD8(+) T cells and reducing their TRAIL expression. These findings indicate that IL-21 is an important mediator of CD4(+) T-cell help to CD8(+) T cells.

IL-21 induces apoptosis of antigen-specific CD8+ T lymphocytes.

IL-21, a member of the common gamma-chain family of cytokines, has pleiotropic effects on T, B, and NK cells. We found that IL-21 and the prototype common gamma-chain cytokine IL-2 can stimulate proliferation and cytokine secretion by Ag-specific rhesus monkey CD8+ T cells. However, unique among the members of this family of cytokines, we found that IL-21 drives these cells to apoptosis by down-regulation of Bcl-2. These findings suggest that IL-21 may play an important role in the contraction of CD8+ T cell responses.

Virus-specific cellular immune correlates of survival in vaccinated monkeys after simian immunodeficiency virus challenge.

Understanding the characteristics of the virus-specific T-lymphocyte response that will confer optimal protection against the clinical progression of AIDS will inform the development of an effective cellular immunity-based human immunodeficiency virus vaccine. We have recently shown that survival in plasmid DNA-primed/recombinant adenovirus-boosted rhesus monkeys that are challenged with the simian immunodeficiency virus SIVmac251 is associated with the preservation postchallenge of central memory CD4(+) T lymphocytes and robust gamma interferon (IFN-gamma)-producing SIV-specific CD8(+) and CD4(+) T-lymphocyte responses. The present studies were initiated to extend these observations to determine which virus-specific T-lymphocyte subpopulations play a primary role in controlling disease progression and to characterize the functional repertoire of these cells. We show that the preservation of the SIV-specific central memory CD8(+) T-lymphocyte population and a linked SIV-specific CD4(+) T-lymphocyte response are associated with prolonged survival in vaccinated monkeys following challenge. Furthermore, we demonstrate that SIV-specific IFN-gamma-, tumor necrosis factor alpha-, and interleukin-2-producing T lymphocytes are all comparably associated with protection against disease progression. These findings underscore the contribution of virus-specific central memory T lymphocytes to controlling clinical progression in vaccinated individuals following a primate immunodeficiency virus infection.

Preserved CD4+ central memory T cells and survival in vaccinated SIV-challenged monkeys.

Vaccine-induced cellular immunity controls virus replication in simian immunodeficiency virus (SIV)-infected monkeys only transiently, leading to the question of whether such vaccines for AIDS will be effective. We immunized monkeys with plasmid DNA and replication-defective adenoviral vectors encoding SIV proteins and then challenged them with pathogenic SIV. Although these monkeys demonstrated a reduction in viremia restricted to the early phase of SIV infection, they showed a prolonged survival. This survival was associated with preserved central memory CD4+ T lymphocytes and could be predicted by the magnitude of the vaccine-induced cellular immune response. These immune correlates of vaccine efficacy should guide the evaluation of AIDS vaccines in humans.