17β-Estradiol Enhances the Response of Plasmacytoid Dendritic Cell to CpG

Gender differences in immune capabilities suggest that sex hormones such as estrogens were involved in the regulation of the immunocompetence. Numerous studies also suggest that plasmacytoid dendritic cells (PDCs) play a pathogenic role in SLE. However, it is unclear whether estrogen can modulate the function of PDCs to influence the development of SLE. In the present study, PDCs from murine spleens were treated with 17β-estradiol (E2) and CpG respectively or both in vitro, then cell viability, costimulatory molecule expression, cytokine secretion of PDCs, as well as stimulatory capacity of PDCs to B cells were analyzed. Results showed that E2 and CpG increased the cell viability and costimulatory molecule expression on PDCs synergistically. Moreover, the intracellular and extracellular secretion of IFN-α was increased by E2 or E2 plus CpG. In addition, E2 and CpG also increased the stimulatory capacity of PDCs to B cells, and the viability of B cells was decreased after neutralizing IFN-α significantly. In the experiments in vivo, mice received daily s.c. injections of E2 and CpG respectively or both, then we found that the plasma concentration of IgM were elevated by E2 and CpG synergistically and the expression of IFN-α/β in spleens were noticeably increased by CpG plus E2 compared with the treatment of E2 or CpG only. This study indicates that E2 could exacerbate PDCs'' activation with CpG, which further activates B cells to upregulate susceptibility to autoantigens. IFN-α plays an important role in the stimulatory effect of PDCs on B cells. E2 stimulation of IFN-α production may result in female prevalence in autoimmune diseases such as SLE through activation of PDCs. This study provides novel evidence of relationship between estrogen and SLE and also sheds light on gender biases among SLE patients.     

NK Cells Regulate CD8+ T Cell Priming and Dendritic Cell Migration during Influenza A Infection by IFN-γ and Perforin-Dependent Mechanisms

An effective immune response against influenza A infection depends on the generation of virus-specific T cells. NK cells are one of the first-line defenses against influenza A infection. We set out to delineate the role of NK cells in T cell immunity using a murine model of influenza A infection with A/PR/8/34. We show that early T cell recruitment mainly occurs in the posterior mediastinal lymph node (pMLN). Depletion of NK cells significantly impaired both dendritic cell (DC) and T cell recruitment into the pMLN. A similar reduction of T cell recruitment was observed when migration was blocked by pertussis toxin, suggesting that migration of pulmonary NK cells and DCs regulates cell recruitment to the pMLN. T cell recruitment was dependent on IFN-γ, and transfer of IFN-γ-competent naive NK cells into IFN-γ(-/-) mice restored T cell recruitment, whereas IFN-γ-deficient NK cells failed to do so. In addition, NK cell depletion reduced the uptake and transport of influenza A virus by DCs, and significantly impaired the virus-specific T cell response. Both IFN-γ(-/-) and perforin(-/-) mice showed reduced viral Ag transport by DCs, suggesting that the ability of NK cells to influence virus transport depends on IFN-γ and perforin. In summary, our data suggest that NK cells play a critical role in the initiation and shaping of the T cell response after influenza A infection.     

Identification of Cytolytic CD161?CD56+ Regulatory CD8 T Cells in Human Peripheral Blood

We previously developed methods for establishing CD8 regulatory T cell (Treg) clones from normal human peripheral blood and demonstrated that these clones were capable of killing T cell receptor (TCR)-activated autologous CD4 T cells. Based on phenotypic and functional characterization of the CD8 Treg clones, we have identified a corresponding population of endogenous CD8 Treg in normal human peripheral blood. These cells appear morphologically as large lymphocytes with abundant cytoplasm and have the following unique phenotype: CD3⁺CD8⁺CD161⁻CD56⁺. The majority of CD8 Treg express CD45RA and CD62L with low or negative expression of CD45RO, CD25, CD27, CD28 and CCR7. The expression of CD94 and NKG2a on CD8 Treg was elevated compared to conventional CD8 T cells. Following in vitro activation, this T cell subset is capable of killing TCR-activated CD4 T cells. These studies identify an endogenous CD8 Treg population in humans and it will now be possible to characterize these cells in a variety of clinical conditions.     

Lymph-Node Resident CD8α+ Dendritic Cells Capture Antigens from Migratory Malaria Sporozoites and Induce CD8+ T Cell Responses

Malaria infection begins when a female Anopheles mosquito injects Plasmodium sporozoites into the skin of its host during blood feeding. Skin-deposited sporozoites may enter the bloodstream and infect the liver, reside and develop in the skin, or migrate to the draining lymph nodes (DLNs). Importantly, the DLN is where protective CD8⁺ T cell responses against malaria liver stages are induced after a dermal route of infection. However, the significance of parasites in the skin and DLN to CD8⁺ T cell activation is largely unknown. In this study, we used genetically modified parasites, as well as antibody-mediated immobilization of sporozoites, to determine that active sporozoite migration to the DLNs is required for robust CD8⁺ T cell responses. Through dynamic in vivo and static imaging, we show the direct uptake of parasites by lymph-node resident DCs followed by CD8⁺ T cell-DC cluster formation, a surrogate for antigen presentation, in the DLNs. A few hours after sporozoite arrival to the DLNs, CD8⁺ T cells are primed by resident CD8α⁺ DCs with no apparent role for skin-derived DCs. Together, these results establish a critical role for lymph node resident CD8α⁺ DCs in CD8⁺ T cell priming to sporozoite antigens while emphasizing a requirement for motile sporozoites in the induction of CD8⁺ T cell-mediated immunity.     

Cytomegalovirus Expresses the Chemokine Homologue vXCL1 Capable of Attracting XCR1+ CD4? Dendritic Cells

Cytomegaloviruses (CMV) have developed various strategies to escape the immune system of the host. One strategy involves the expression of virus-encoded chemokines to modulate the host chemokine network. We have identified in the English isolate of rat CMV (murid herpesvirus 8 [MuHV8]) an open reading frame encoding a protein homologous to the chemokine XCL1, the only known C chemokine. Viral XCL1 (vXCL1), a glycosylated protein of 96 amino acids, can be detected 13 h postinfection in the supernatant of MuHV8-infected rat embryo fibroblasts. vXCL1 exclusively binds to CD4⁻ rat dendritic cells (DC), a subset of DC that express the corresponding chemokine receptor XCR1. Like endogenous rat XCL1, vXCL1 selectively chemoattracts XCR1⁺ CD4⁻ DC. Since XCR1⁺ DC in mice and humans have been shown to excel in antigen cross-presentation and thus in the induction of cytotoxic CD8⁺ T lymphocytes, the virus has apparently hijacked this gene to subvert cytotoxic immune responses. The biology of vXCL1 offers an interesting opportunity to study the role of XCL1 and XCR1⁺ DC in the cross-presentation of viral antigens.     

Cell Budding from Normal Appearing Epithelia: A Predictor of Colorectal Cancer Metastasis?

Background: Colorectal carcinogenesis is believed to be a multi-stage process that originates with a localized adenoma, which linearly progresses to an intra-mucosal carcinoma, to an invasive lesion, and finally to metastatic cancer. This progression model is supported by tissue culture and animal model studies, but it is difficult to reconcile with several well-established observations, principally among these are that up to 25% of early stage (Stage I/II), node-negative colorectal cancer (CRC) develop distant metastasis, and that circulating CRC cells are undetectable in peripheral blood samples of up to 50% of patients with confirmed metastasis, but more than 30% of patients with no detectable metastasis exhibit such cells. The mechanism responsible for this diverse behavior is unknown, and there are no effective means to identify patients with pending, or who are at high risk for, developing metastatic CRC.Novel findings: Our previous studies of human breast and prostate cancer have shown that cancer invasion arises from the convergence of a tissue injury, the innate immune response to that injury, and the presence of tumor stem cells within tumor capsules at the site of the injury. Focal degeneration of a capsule due to age or disease attracts lymphocyte infiltration that degrades the degenerating capsules resulting in the formation of a focal disruption in the capsule, which selectively favors proliferating or “budding” of the underlying tumor stem cells. Our recent studies suggest that lymphocyte infiltration also triggers metastasis by disrupting the intercellular junctions and surface adhesion molecules within the proliferating cell buds causing their dissociation. Then, lymphocytes and tumor cells are conjoined through membrane fusion to form tumor-lymphocyte chimeras (TLCs) that allows the tumor stem cell to avail itself of the lymphocyte''s natural ability to migrate and breach cell barriers in order to intravasate and to travel to distant organs. Our most recent studies of human CRC have detected nearly identical focal capsule disruptions, lymphocyte infiltration, budding cells, and the formation of TLCs. Our studies have further shown that age- and type-matched node-positive and -negative CRC have a significantly different morphological and immunohistochemical profile and that the majority of lymphatic ducts with disseminated cells are located within the mucosa adjacent to morphologically normal appearing epithelial structures that express a stem cell-related marker.New hypothesis: Based on these findings and the growth patterns of budding cells revealed by double immunohistochemistry, we further hypothesize that metastatic spread is an early event of carcinogenesis and that budding cells overlying focal capsule disruptions represent invasion- and metastasis-initiating cells that follow one of four pathways to progress: (1) to undergo extensive in situ proliferation leading to the formation of tumor nests that subsequently invade the submucosa, (2) to migrate with associated lymphocytes functioning as “seeds” to grow in new sites, (3) to migrate and intravasate into pre-existing vascular structures by forming TLCs, or (4) to intravasate into vascular structures that are generated by the budding cells themselves. We also propose that only node-positive cases harbor stem cells with the potential for multi-lineage differentiation and unique surface markers that permit intravasation.     

The Gene Expression Profile of CD11c+CD8α? Dendritic Cells in the Pre-Diabetic Pancreas of the NOD Mouse

Two major dendritic cell (DC) subsets have been described in the pancreas of mice: The CD11c⁺CD8α⁻ DCs (strong CD4+ T cell proliferation inducers) and the CD8α⁺CD103⁺ DCs (T cell apoptosis inducers). Here we analyzed the larger subset of CD11c⁺CD8α⁻ DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR) to elucidate abnormalities in underlying gene expression networks. CD11c⁺CD8α⁻ DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c⁺CD8α⁻ DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24) was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c⁺CD8α⁻ DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c⁺CD8α⁻ DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.     

CD8α Dendritic Cells Drive Establishment of HSV-1 Latency

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α^−/− (lack functional CD8 T cells and CD8α⁺ DCs), CD8β^−/− (have functional CD8α⁺ T cells and CD8α⁺ DCs), and β2m^−/− (lack functional CD8 T cells but have CD8α⁺ DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α⁺ DCs) versus WT C3H (have functional CD8α T cells and CD8α⁺ DCs) mice. We also determined whether the phenotype of CD8α^−/− and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8⁺ T cells or bone marrow (BM) derived CD8α⁺ DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences.     

Minireview: Characterization of the Yeast Mitochondria Unselective Channel: A Counterpart to the Mammalian Permeability Transition Pore?

Large and unselective permeabilities through the inner membrane of yeast mitochondria have been observed for more than 20 years, but the characterization of these permeabilities, leading to hypothesize the existence of a large-conductance unselective channel in yeast inner mitochondrial membrane, was done only recently by several groups. This channel has been tentatively identified as a yeast counterpart to the mammalian permeability transition pore, the crucial role of which is now well-documented in physiopathological phenomena, such as Ca²⁺ homeostasis, ischemic damages, or programmed cell death. The aim of this review is to make a point on the known characteristics of this yeast mitochondrial unselective channel (YMUC) and to analyze whether or not it can be considered as a yeast permeability transition pore.     

CD117-positive Cells of the Heart: Progenitor Cells or Mast Cells?

Human cardiac stem/progenitor cells and their potential for repair of heart injury are a current hot topic of research. CD117 has been used frequently as a marker for identification of stem/progenitor cells in the heart. However, cardiac mast cells, which are also CD117⁺, have not been excluded by credible means when selecting putative cardiac progenitors by using CD117 as a marker. We evaluated the relationship between CD117⁺ cells and mast cells in the left ventricle of human hearts (n=5 patients, ages 1 week–75 years) with the well-established mast cell markers tryptase, toluidine blue, and thionine. A large number (85–100%) of CD117⁺ cells in the human heart were specifically identified as mast cells. In addition, mast cells showed weak or moderate CD45 immunostaining signals. These results indicate that the majority of CD117⁺ cells in the heart are mast cells and that these cells are distinctly positive for CD45, although staining was weak or moderate. These results strongly suggest that the newly reported CD117⁺/CD45^dim/moderate putative cardiac progenitor cells are mast cells. The significance of this observation in stem cell research of the heart is discussed. (J Histochem Cytochem 58:309–316, 2010)     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

Reduction of the CD16?CD56bright NK Cell Subset Precedes NK Cell Dysfunction in Prostate Cancer

Background

Natural cytotoxicity, mediated by natural killer (NK) cells plays an important role in the inhibition and elimination of malignant tumor cells. To investigate the immunoregulatory role of NK cells and their potential as diagnostic markers, NK cell activity (NKA) was analyzed in prostate cancer (PCa) patients with particular focus on NK cell subset distribution.

Methods

Prospective data of NKA and NK cell subset distribution patterns were measured from 51 patients initially diagnosed with PCa and 54 healthy controls. NKA was represented by IFN-γ levels after stimulation of the peripheral blood with Promoca®. To determine the distribution of NK cell subsets, PBMCs were stained with fluorochrome-conjugated monoclonal antibodies. Then, CD16⁺CD56^dim and CD16⁻CD56^bright cells gated on CD56⁺CD3⁻ cells were analyzed using a flow-cytometer.

Results

NKA and the proportion of CD56^bright cells were significantly lower in PCa patients compared to controls (430.9 pg/ml vs. 975.2 pg/ml and 2.3% vs. 3.8%, respectively; p<0.001). Both tended to gradually decrease according to cancer stage progression (p for trend = 0.001). A significantly higher CD56^dim-to-CD56^bright cell ratio was observed in PCa patients (41.8 vs. 30.3; p<0.001) along with a gradual increase according to cancer stage progression (p for trend = 0.001), implying a significant reduction of CD56^bright cells in relation to the alteration of CD56^dim cells. The sensitivity and the specificity of NKA regarding PCa detection were 72% and 74%, respectively (best cut-off value at 530.9 pg/ml, AUC = 0.786).

Conclusions

Reduction of CD56^bright cells may precede NK cell dysfunction, leading to impaired cytotoxicity against PCa cells. These observations may explain one of the mechanisms behind NK cell dysfunction observed in PCa microenvironment and lend support to the development of future cancer immunotherapeutic strategies.     

Memory CD8+ T Cells: Orchestrators and Key Players of Innate Immunity?

Over the past decades, the dichotomy between innate and adaptive immune responses has largely dominated our understanding of immunology. Upon primary encounter with microbial pathogens, differentiation of adaptive immune cells into functional effectors usually takes several days or even longer, making them contribute to host protection only late during primary infection. However, once generated, antigen-experienced T lymphocytes can persist in the organism and constitute a pool of memory cells that mediate fast and effective protection to a recall infection with the same microbial pathogen. Herein, we challenge this classical paradigm by highlighting the “innate nature” of memory CD8⁺ T cells. First, within the thymus or in the periphery, naïve CD8⁺ T cells may acquire phenotypic and functional characteristics of memory CD8⁺ T cells independently of challenge with foreign antigens. Second, both the “unconventional” and the “conventional” memory cells can rapidly express protective effector functions in response to sets of inflammatory cytokines and chemokines signals, independent of cognate antigen triggering. Third, memory CD8⁺ T cells can act by orchestrating the recruitment, activation, and licensing of innate cells, leading to broad antimicrobial states. Thus, collectively, memory CD8⁺ T cells may represent important actors of innate immune defenses.     

Characterization of Spontaneous and TGF-β-Induced Cell Motility of Primary Human Normal and Neoplastic Mammary Cells In Vitro Using Novel Real-Time Technology

The clinical complications derived from metastatic disease are responsible for the majority of all breast cancer related deaths. Since cell migration and invasion are a prerequisite for metastasis their assessment in patient cancer cells in vitro may have prognostic value for the tumor''s metastatic capacity. We employed real-time cell analysis (RTCA) on the xCELLigence DP system to determine in vitro motility of patient-derived primary human breast cancer epithelial cells (HBCEC). Initially, the RTCA assay was validated using established human breast cancer cell lines with either an invasive (MDA-MB-231, MDA-MB-435s) or a non-invasive phenotype (MCF-7, MDA-MB-468), and primary NSCLC cells (Tu459). Previous standard assays of cell migration/invasion revealed that only MDA-MB-231, −435s, and Tu459 cells exhibited spontaneous and TGF-β1-stimulated migration and invasion through a Matrigel barrier. In the present study, the TGF-β1-stimulated activities could be blocked by SB431542, a potent kinase inhibitor of the TGF-β type I receptor ALK5. Application of the RTCA assay to patient-derived tumor cells showed that 4/4 primary HBCEC and primary NSCLC cells, but not normal human mammary epithelial cells (HMEC), displayed high spontaneous migratory and invasive activity which correlated with higher MMP-2 expression and uPA protein levels in HBCEC compared to HMEC. Upon treatment with TGF-β1, HBCEC exhibited morphologic and gene regulatory alterations indicative of epithelial-to-mesenchymal transition. However, exclusively the invasive but not the migratory activity of HBCEC was further enhanced by TGF-β1. This indicates the requirement for molecular, e.g. integrin interactions with Matrigel components in HBCEC in order to become responsive to pro-invasive TGF-β effects. Together, these results show for the first time that tumorigenic HBCEC but not normal HMEC possess a strong basal migratory as well as a basal and TGF-β1-inducible invasive potential. These findings qualify the RTCA assay as an in vitro migration/invasion testing system for patient-specific primary breast cancer cells.     

BDCA2/Fc?RIγ Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells

Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses. Human plasmacytoid dendritic cells (pDCs) uniquely express blood dendritic cell antigen 2 (BDCA2) protein, a C-type lectin lacking an identifiable signaling motif. We demonstrate here that BDCA2 forms a complex with the transmembrane adapter FcɛRIγ. Through pathway analysis, we identified a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of the B cell receptor (BCR), which is distinct from the system involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of the BCR-like cascade, which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands. Therefore, by associating with FcɛRIγ, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.     

Why Don't CD8+ T Cells Reduce the Lifespan of SIV-Infected Cells In Vivo?

In January 2010 two groups independently published the observation that the depletion of CD8+ cells in SIV-infected macaques had no detectable impact on the lifespan of productively infected cells. This unexpected observation led the authors to suggest that CD8+ T cells control SIV viraemia via non-lytic mechanisms. However, a number of alternative plausible explanations, compatible with a lytic model of CD8+ T cell control, were proposed. This left the field with no consensus on how to interpret these experiments and no clear indication whether CD8+ T cells operated primarily via a lytic or a non-lytic mechanism. The aim of this work was to investigate why CD8+ T cells do not appear to reduce the lifespan of SIV-infected cells in vivo.