Senescence-associated ß-galactosidase reveals the abundance of senescent CD8+ T cells in aging humans.

Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to disease and infection. The degree to which immune cell senescence contributes to this decline remains unclear, however, since markers that label immune cells with classical features of cellular senescence accurately and comprehensively have not been identified. Using a second-generation fluorogenic substrate for ß-galactosidase and multi-parameter flow cytometry, we demonstrate here that peripheral blood mononuclear cells (PBMCs) isolated from healthy humans increasingly display cells with high senescence-associated ß-galactosidase (SA-ßGal) activity with advancing donor age. The greatest age-associated increases were observed in CD8+ T-cell populations, in which the fraction of cells with high SA-ßGal activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high SA-ßGal activity, but not those with low SA-ßGal activity, were found to exhibit features of telomere dysfunction-induced senescence and p16-mediated senescence, were impaired in their ability to proliferate, developed in various T-cell differentiation states, and had a gene expression signature consistent with the senescence state previously observed in human fibroblasts. Based on these results, we propose that senescent CD8+ T cells with classical features of cellular senescence accumulate to levels that are significantly higher than previously reported and additionally provide a simple yet robust method for the isolation and characterization of senescent CD8+ T cells with predictive potential for biological age.

Triggering of the cGAS-STING Pathway in Human Plasmacytoid Dendritic Cells Inhibits TLR9-Mediated IFN Production.

Plasmacytoid dendritic cells (pDCs) are potent producers of type I and type III IFNs and play a major role in antiviral immunity and autoimmune disorders. The innate sensing of nucleic acids remains the major initiating factor for IFN production by pDCs. TLR-mediated sensing of nucleic acids via endosomal pathways has been studied and documented in detail, whereas the sensing of DNA in cytosolic compartment in human pDCs remains relatively unexplored. We now demonstrate the existence and functionality of the components of cytosolic DNA-sensing pathway comprising cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of IFN gene (STING) in human pDCs. cGAS was initially located in the cytosolic compartment of pDCs and time-dependently colocalized with non-CpG double-stranded immunostimulatory DNA (ISD). Following the colocalization of ISD with cGAS, the downstream pathway was triggered as STING disassociated from its location at the endoplasmic reticulum. Upon direct stimulation of pDCs by STING agonist 2'3' cGAMP or dsDNA, pDC-s produced type I, and type III IFN. Moreover, we documented that cGAS-STING-mediated IFN production is mediated by nuclear translocation of IRF3 whereas TLR9-mediated activation occurs through IRF7. Our data also indicate that pDC prestimulation of cGAS-STING dampened the TLR9-mediated IFN production. Furthermore, triggering of cGAS-STING induced expression of SOCS1 and SOCS3 in pDCs, indicating a possible autoinhibitory loop that impedes IFN production by pDCs. Thus, our study indicates that the cGAS-STING pathway exists in parallel to the TLR9-mediated DNA recognition in human pDCs with cross-talk between these two pathways.

Regulation of Transcription Factor E2-2 in Human Plasmacytoid Dendritic Cells by Monocyte-Derived TNFα.

Plasmacytoid dendritic cells (pDCs) are innate immune cells and potent producers of interferon alpha (IFNα). Regulation of pDCs is crucial for prevention of aberrant IFN production. Transcription factor E2-2 (TCF4) regulates pDC development and function, but mechanisms of E2-2 control have not been investigated. We used freshly-isolated human peripheral blood mononuclear cells stimulated with toll-like receptor 7, 9, and 4 agonists to determine which factors regulate E2-2. After activation, pDCs decreased E2-2 expression. E2-2 downregulation occurred during the upregulation of costimulatory markers, after maximal IFN production. In congruence with previous reports in mice, we found that primary human pDCs that maintained high E2-2 levels produced more IFN, and had less expression of costimulatory markers. Stimulation of purified pDCs did not lead to E2-2 downregulation; therefore, we investigated if cytokine signaling regulates E2-2 expression. We found that tumor necrosis factor alpha (TNFα) produced by monocytes caused decreased E2-2 expression. All together, we established that primary human pDCs decrease E2-2 in response to TNFα and E2-2 low pDCs produce less IFN but exhibit more costimulatory molecules. Altered expression of E2-2 may represent a mechanism to attenuate IFN production and increase activation of the adaptive immune compartment.

Self-Renewal and Toll-like Receptor Signaling Sustain Exhausted Plasmacytoid Dendritic Cells during Chronic Viral Infection.

Although characterization of T cell exhaustion has unlocked powerful immunotherapies, the mechanisms sustaining adaptations of short-lived innate cells to chronic inflammatory settings remain unknown. During murine chronic viral infection, we found that concerted events in bone marrow and spleen mediated by type I interferon (IFN-I) and Toll-like receptor 7 (TLR7) maintained a pool of functionally exhausted plasmacytoid dendritic cells (pDCs). In the bone marrow, IFN-I compromised the number and the developmental capacity of pDC progenitors, which generated dysfunctional pDCs. Concurrently, exhausted pDCs in the periphery were maintained by self-renewal via IFN-I- and TLR7-induced proliferation of CD4- subsets. On the other hand, pDC functional loss was mediated by TLR7, leading to compromised IFN-I production and resistance to secondary infection. These findings unveil the mechanisms sustaining a self-perpetuating pool of functionally exhausted pDCs and provide a framework for deciphering long-term exhaustion of other short-lived innate cells during chronic inflammation.

Toxoplasma gondii Inactivates Human Plasmacytoid Dendritic Cells by Functional Mimicry of IL-10.

Plasmacytoid dendritic cells (pDCs) are the major producers of IFN-α, an antiviral cytokine involved in immunomodulation and control of HIV type 1 replication, whereas Toxoplasma gondii is a life-threatening opportunistic infection in AIDS patients. During infection with HIV type 1, human pDCs decrease in circulation and remaining pDC produce lower amounts of IFN-α in response to viral stimulation. In this study, we investigated the impact of coinfection with T. gondii on the innate virus-directed responses of human pDCs. Using intracellular flow cytometry and fluorescence microscopy, we determined that T. gondii invaded but did not induce IFN-α or TNF-α in human pDC. However, T. gondii inhibited IFN-α and TNF-α produced in response to HSV and HIV, thus functionally inactivating pDC. IFN-α production was inhibited only in cells infected by T. gondii, which inhibited neither uptake of GFP-HSV nor localization of TLR9 in CD71+ endosomes, directing us to investigate downstream events. Using imaging flow cytometry, we found that both T. gondii and IL-10 inhibited virus-induced nuclear translocation, but not phosphorylation, of IFN response factor 7. Blockade of IFN response factor 7 nuclear translocation and inhibition of the IFN-α response was partially reversed by a deficiency in the T. gondii-derived ROP16 kinase, known to directly phosphorylate STAT3, a critical mediator of IL-10's anti-inflammatory effects. Taken together, our results indicate that T. gondii suppresses pDC activation by mimicking IL-10's regulatory effects through an ROP16 kinase-dependent mechanism. Our findings further imply a convergent mechanism of inhibition of TLR signaling by T. gondii and IL-10 and suggest potential negative consequences of HIV/T. gondii coinfection.

Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDC) are rare cells found in peripheral blood and lymphoid tissues. pDC are considered to be "professional" type I IFN-producing cells and produce 10- to 100-fold more IFN-α than other cell types in response to enveloped viruses or synthetic TLR7 and TLR9 agonists. In this study, purified pDC were found to express high levels of IFN-λ receptor mRNA, as well as cell-surface IFN-λ receptor. We have developed intracellular flow cytometry assays using Abs to IFN-λ1/3 or -λ2 to assess the expression of IFN-λ proteins by pDC. We observed that a subset of human pDC expresses only intracellular IFN-α, whereas another subset produces both IFN-α and IFN-λ after stimulation with virus or the TLR9 agonist, CpG A; the cells that coexpressed IFN-α and IFN-λ were the cells with the highest levels of IFN-α expression. Ab cross-linking of CD4 or CD303 molecules on pDC inhibited both HSV-induced IFN-λ and IFN-α production. Like the production of IFN-α, the HSV-induced IFN-λ production in pDC was mediated through TLR9 and independent of virus replication. Exogenous IFN-λ treatment of pDC resulted in increased virus-induced expression of both IFN-α and IFN-λ. In addition, both exogenous IFN-λ and -α inhibited dexamethasone-induced apoptosis of pDC. We conclude that pDC are major producers of IFN-λ1 and -λ2 in response to viral stimulation and also express functional receptors for this cytokine. Thus, IFN-λ can serve as an autocrine signal to strengthen the antiviral response of pDC by increasing IFN-α and IFN-λ production, resulting in prolonged pDC survival.

Interferon regulatory factor 5 activation in monocytes of systemic lupus erythematosus patients is triggered by circulating autoantigens independent of type I interferons.

OBJECTIVE: Genetic variants of interferon regulatory factor 5 (IRF-5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF-5 regulates the expression of proinflammatory cytokines and type I interferons (IFNs) believed to be involved in the pathogenesis of SLE. The aim of this study was to determine the activation status of IRF-5 by assessing its nuclear localization in the immune cells of SLE patients and healthy donors, and to identify SLE-associated triggers of IRF-5 activation. METHODS: IRF-5 nuclear localization in subpopulations of peripheral blood mononuclear cells from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. The activation and function of IRF-5 were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream flow cytometry, and cytokine expression was analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: IRF-5 was activated in a cell type-specific manner; monocytes from SLE patients had constitutively elevated levels of nuclear IRF-5, as compared to natural killer cells and T cells. SLE serum was identified as a trigger for IRF-5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens composed of apoptotic/necrotic material triggered IRF-5 nuclear accumulation in monocytes. Production of the cytokines IFNα, tumor necrosis factor α, and interleukin-6 in monocytes stimulated with SLE serum or autoantigens was distinct, yet showed a correlation with the kinetics of IRF-5 nuclear localization. CONCLUSION: This study provides the first formal proof that IRF-5 activation is altered in the monocytes of SLE patients, which can be attributed, in part, to the SLE blood environment.

Modulation of human beta-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC), monocytes, and epithelial cells by influenza virus, Herpes simplex virus, and Sendai virus and its possible role in innate immunity.

hBD comprise a family of antimicrobial peptides that plays a role in bridging the innate and adaptive immune responses to infection. The expression of hBD-2 increases upon stimulation of numerous cell types with LPS and proinflammatory cytokines. In contrast, hBD-1 remains constitutively expressed in most cells in spite of cytokine or LPS stimulation; however, its presence in human PDC suggests it plays a role in viral host defense. To examine this, we characterized the expression of hBD-1 in innate immune cells in response to viral challenge. PDC and monocytes increased production of hBD-1 peptide and mRNA as early as 2 h following infection of purified cells and PBMCs with PR8, HSV-1, and Sendai virus. However, treatment of primary NHBE cells with influenza resulted in a 50% decrease in hBD-1 mRNA levels, as measured by qRT-PCR at 3 h following infection. A similar inhibition occurred with HSV-1 challenge of human gingival epithelial cells. Studies with HSV-1 showed that replication occurred in epithelial cells but not in PDC. Together, these results suggest that hBD-1 may play a role in preventing viral replication in immune cells. To test this, we infected C57BL/6 WT mice and mBD-1((-/-)) mice with mouse-adapted HK18 (300 PFU/mouse). mBD-1((-/-)) mice lost weight earlier and died sooner than WT mice (P=0.0276), suggesting that BD-1 plays a role in early innate immune responses against influenza in vivo. However, lung virus titers were equal between the two mouse strains. Histopathology showed a greater inflammatory influx in the lungs of mBD-1((-/-)) mice at Day 3 postinfection compared with WT C57BL/6 mice. The results suggest that BD-1 protects mice from influenza pathogenesis with a mechanism other than inhibition of viral replication.

Characterization of the NOD/scid-[Tg]DR1 mouse expressing HLA-DRB1*01 transgene: a model of SCID-hu mouse for vaccine development.

OBJECTIVE: We previously showed enhanced engraftment of human T cells in the transgenic NonObese Diabetic/severe combined immunodeficient (NOD/scid)-DR1 mice, compared to NOD/scid mice. We now characterize their immunobiology, innate immunity, and intrahepatic neonatal engraftment of cord blood mononuclear cells (CBMNC), and test immune responses of these chimeric mice to an experimental cancer vaccine. METHODS: Fluorescence in situ hybridization analysis, blood biochemistry, hematology, and fluorescein-activated cell sorting analyses of cellular subsets were performed on NOD/scid-DR1 mice, in comparison to parental NOD/scid mice. Innate immunity and lifespan were examined. Histology of engrafted tissues and short-term intrahepatic engraftment of CBMNC were performed. Intracellular interferon-gamma (IFN-gamma) production was assessed in mice immunized with cancer vaccine. RESULTS: The DR1 transgene was located on chromosome 5 and no significant changes were observed in blood chemistry, peripheral blood counts, lymphoid subsets, natural killer cell and lipopolysaccharide response, and antigen presentation in the NOD/scid-DR1 mice, compared to NOD/scid mice. Interestingly, NOD/scid-DR1 mice had a significantly longer lifespan (approximately 14 months) than NOD/scid mice (approximately 8.5 months). Engraftment with human cord blood cells resulted in slight changes in the architecture/structure of spleens. No correlation was found between DR1 genotype of the donor CBMNC and extent of engraftment of human T cells. Enhanced engraftment of human cells was observed with intrahepatic injections of CBMNC in neonatal NOD/scid DR1 mice. Intracellular IFN-gamma was detected in human cells, when chimeric mice were immunized with a cancer vaccine. CONCLUSION: NOD/scid-DR1 mice were similar in most of the physiological parameters as the NOD/scid mice, with the exception of longer lifespan. Intrahepatic engraftment of neonatal mice is the preferred protocol of xenotransplantation in this model and the engrafted human cells can respond to a cancer vaccine.

Two discrete promoters regulate the alternatively spliced human interferon regulatory factor-5 isoforms. Multiple isoforms with distinct cell type-specific expression, localization, regulation, and function.

Interferon regulatory factor-5 (IRF-5) is a mediator of virus-induced immune activation and type I interferon (IFN) gene regulation. In human primary plasmacytoid dendritic cells (PDC), IRF-5 is transcribed into four distinct alternatively spliced isoforms (V1, V2, V3, and V4), whereas in human primary peripheral blood mononuclear cells two additional new isoforms (V5 and V6) were identified. The IRF-5 V1, V2, and V3 transcripts have different noncoding first exons and distinct insertion/deletion patterns in exon 6. Here we showed that V1 and V3 have distinct transcription start sites and are regulated by two discrete promoters. The V1 promoter (P-V1) is constitutively active, contains an IRF-E consensus-binding site, and is further stimulated in virus-infected cells by IRF family members. In contrast, endogenous V3 transcripts were up-regulated by type I IFNs, and the V3 promoter (P-V3) contains an IFN-stimulated responsive element-binding site that confers responsiveness to IFN through binding of the ISGF3 complex. In addition to V5 and V6, we have identified three more alternatively spliced IRF-5 isoforms (V7, V8, and V9); V5 and V6 were expressed in peripheral blood mononuclear cells from healthy donors and in immortalized B and T cell malignancies, whereas expression of V7, V8, and V9 transcripts were detected only in human cancers. The results of this study demonstrated the existence of multiple IRF-5 spliced isoforms with distinct cell type-specific expression, cellular localization, differential regulation, and dissimilar functions in virus-mediated type I IFN gene induction.

Characterization of virus-responsive plasmacytoid dendritic cells in the rhesus macaque.

Plasmacytoid dendritic cells (PDC) are potent producers of alpha interferon (IFN-alpha) in response to enveloped viruses and provide a critical link between the innate and adaptive immune responses. Although the loss of peripheral blood PDC function and numbers has been linked to human immunodeficiency virus (HIV) progression in humans, a suitable animal model is needed to study the effects of immunodeficiency virus infection on PDC function. The rhesus macaque SIV model closely mimics human HIV infection, and recent studies have identified macaque PDC, potentially making the macaque a good model to study PDC regulation. In this study, we demonstrate that peripheral blood PDC from healthy macaques are both phenotypically and functionally similar to human PDC and that reagents used for human studies can be used to study macaque PDC. Both human and macaque PBMC expressed IFN-alpha in response to herpes simplex virus (HSV), the prototypical activator of PDC, as measured by using an IFN bioassay and IFN-alpha-specific enzyme-linked immunospot assays. Similar to human PDC, macaque PDC were identified by using flow cytometry as CD123+ HLA-DR+ lineage- cells. In addition, like human PDC, macaque PDC expressed intracellular IFN-alpha, tumor necrosis factor alpha, macrophage inflammatory protein 1beta/CCL4, and IFN-inducible protein 10/CXCL10 upon stimulation with HSV, all as determined by intracellular flow cytometry. We found that IFN regulatory factor 7, which is required for the expression of IFN-alpha genes, was, similar to human PDC, expressed at high levels in macaque PDC compared to monocytes and CD8+ T cells. These findings establish the phenotypic and functional similarity of human and macaque PDC and confirm the utility of tools developed for studying human PDC in this animal model.

Intra-thymic/splenic engraftment of human T cells in HLA-DR1 transgenic NOD/scid mice.

A HLA-DR1 transgenic mouse (NOD/scid-DR1) was derived by breeding the existing B10.M/J-[Tg]DR1 mouse with the NOD/scid mouse. The intention was to enhance engraftment of human T cells by providing human class II elements in the tissues. Thymus and spleen fragments from adult NOD/scid-DR1 mice were transplanted under the syngeneic kidney capsules, followed by injection of human cord blood mononuclear cells (CBMNC) into transplanted tissues. FACS analyses showed that human T and B cells were consistently detected in the peripheral blood and spleen, of the chimeric mice. An average of 20% of human cells was found in the spleen and the engrafted thymus/spleen tissues. Furthermore, human cells from these tissues could proliferate with anti-human CD3 antibody and these mice could generate humoral and cellular responses to allogeneic human cells. Cytokines, such as IL-10, GMCSF, IFN-gamma, and TNF-alpha were also detected in the supernatants of the cultured human cells from the chimeric mice, when they were stimulated with allogeneic cells. Therefore, a novel mouse model with functional circulating human T and B cells was established that would facilitate the exploration of vaccine, the disease processes of autoimmunity, HIV infection, and human cancer.

Detection of HBD1 peptide in peripheral blood mononuclear cell subpopulations by intracellular flow cytometry.

Production of human beta-defensin1 (HBD1) in response to LPS in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells (PDC) was examined. Since PDC make up only 0.1-0.5% of the peripheral blood mononuclear cell population, we developed a method to determine HBD1 peptide levels using four-color flow cytometry, which can examine several cell surface or intracellular markers at once. Coupled with intracellular flow cytometry, we determined that PDC and monocytes only made significant amounts of HBD1 when exposed to >50ng/ml LPS for 2h. This response was limited to monocytes when ultrapure LPS was used, and was inhibited in PDC by chloroquine treatment.

Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod.

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I interferon in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary interferon-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-alpha and IFN-omega from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-alpha and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-3 and IFN-alpha; resiquimod more effectively matures pDC than either IL-3 or IFN-alpha alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.

Natural interferon-alpha producing cells: the plasmacytoid dendritic cells.

NIPCs have had a long history of study, with the first observations of peripheral blood cells with IFN-producing capacity being made approximately 20 years ago. Over the past three years with the identification of NIPCs as plasmacytoid dendritic cells, an enormous amount has been learned about their function in both innate and adaptive immunity and their dysregulation in certain viral infections such as HIV infection and in autoimmunity. Further studies of their mechanisms of IFN-alpha induction, of their distribution and in vivo function are required. It is anticipated that a further understanding of the PDC will lead to the ability to specifically manipulate these cells in human disease.

Phenotypic variation in Actinobacillus actinomycetemcomitans during laboratory growth: implications for virulence.

This study examined alteration of specific virulence traits associated with phenotypic changes seen when a low-passage disease-associated and well maintained parent strain of Actinobacillus actinomycetemcomitans was compared to a laboratory-grown spontaneous variant/mutant. Clinical isolates of A. actinomycetemcomitans recovered from periodontitis patients typically grow as rough, adherent colonies on primary culture but undergo transformation to smooth, non-adherent colonies following repeated passage in vitro. The relationship of these phenotypic changes to the virulence of the organism or to the processes that underlie this transformation are not understood. A fresh clinical isolate, designated strain CU1000, was obtained from the first molar site of a patient with classical signs of localized juvenile periodontitis and used as the parent strain to study virulence-related phenotypes. Following several passages of CU1000 on selective agar, a spontaneous variant that demonstrated smooth, opaque, non-adherent colonies was isolated and designated strain CU1060. This study compared the properties of these two strains with respect to colony morphology, autoaggregation, surface appendages, adherence to saliva-coated hydroxyapatite (SHA), LPS chemotype and activity, induction of fibroblast proteinase activity and antigenic properties. CU1000 demonstrated rough, raised, star-positive colonies which upon electron microscopic examination revealed the presence of large, flexible, bundled fibrils. In addition, CU1000 showed adherence to SHA, several unique protein antigens and elevated endotoxin and fibroblast proteinase activity. CU1060, on the other hand, showed minimal adherence to SHA and fewer reactive proteins compared to the fresh clinical isolates. This strain formed smooth, opaque colonies on agar, showed minimal fibril formation and limited endotoxin and fibroblast-proteinase-inducing activity. These findings demonstrate that clinical isolates of A. actinomycetemcomitans undergo significant virulence-reducing phenotypic alterations during in vitro passage and support the need to study this organism in its clinical form.