Integrity of IKK/NF‐κB Shields Thymic Stroma That Suppresses Susceptibility to Autoimmunity,Fungal Infection,and Carcinogenesis

A pathogenic connection between autoreactive T cells, fungal infection, and carcinogenesis has been demonstrated in studies of human autoimmune polyendocrinopathy‐candidiasis‐ectodermal dystrophy (APECED) as well as in a mouse model in which kinase‐dead Ikkα knock‐in mice develop impaired central tolerance, autoreactive T cell–mediated autoimmunity, chronic fungal infection, and esophageal squamous cell carcinoma, which recapitulates APECED. IκB kinase α (IKKα) is one subunit of the IKK complex required for NF‐κB activation. IKK/NF‐κB is essential for central tolerance establishment by regulating the development of medullary thymic epithelial cells (mTECs) that facilitate the deletion of autoreactive T cells in the thymus. In this review, we extensively discuss the pathogenic roles of inborn errors in the IKK/NF‐κB loci in the phenotypically related diseases APECED, immune deficiency syndrome, and severe combined immunodeficiency; differentiate how IKK/NF‐κB components, through mTEC (stroma), T cells/leukocytes, or epithelial cells, contribute to the pathogenesis of infectious diseases, autoimmunity, and cancer; and highlight the medical significance of IKK/NF‐κB in these diseases.     

Age‐associated alterations in CD8α+ dendritic cells impair CD8 T‐cell expansion in response to an intracellular bacterium

Age‐associated decline in immunity to infection has been documented across multiple pathogens, yet the relative contributions of the aged priming environment and of lymphocyte‐intrinsic defects remain unclear. To address the impact of the aging environment on T‐cell priming, adult naïve OT‐I TCR transgenic CD8 T cells, specific for the H‐2K^b‐restricted immunodominant OVA_257‐264 epitope, were transferred into adult or old recipient mice infected with the recombinant intracellular bacterium Listeria monocytogenes carrying the chicken ovalbumin protein (Lm‐OVA). We consistently found that adult OT‐I CD8 expansion was reduced in aged recipient mice, and this correlated with numeric, phenotypic, and functional defects selectively affecting CD8α+ dendritic cells (DC). Following Lm‐OVA infection, aged mice failed to accumulate CD8α+ DC in the spleen, and these cells expressed much lower levels of critical costimulatory molecules in the first three days following infection. Further, aged CD8α+ DC showed impaired uptake of the bacteria at very early time points following infection. Treatment of aged mice with Flt3 ligand (Flt3L) improved the number of DC present in the spleen prior to Lm‐OVA infection, and improved, but did not reconstitute, OT‐I expansion to Lm‐OVA infection. These results suggest that age‐associated changes in antigen uptake, pathogen sensing, and/or antigen presentation contribute to impaired adaptive immune responses to microbial pathogens with aging.     

Dendritic cells,chemokine receptors and autoimmune inflammatory diseases

Dendritic cells (DC) have been implicated in the induction of autoimmune diseases and have been identified in lesions associated with several autoimmune inflammatory diseases. Since DC are regarded as the professional antigen-presenting cell (APC) of the immune system and the only APC capable of activating na?ve T cells, they are likely to play a significant role in breaking tolerance of self-reactive lymphocytes and in supporting autoimmune responses in these diseases. A number of studies have revealed that small molecular weight chemotactic proteins known as chemokines are present within the autoimmune lesions and may contribute to the recruitment not only of DC populations, but also of immune cells such as T cells, B cells, neutrophils and monocytes into the site, and to the formation of organized lymphoid tissue structures within the target organ. The focus of this review will be a discussion of the role of chemokines in the recruitment of DC in human autoimmune inflammatory disorders, specifically the trafficking of DC into the inflammatory sites and the subsequent migration of differentiated DC from the inflammatory sites into the draining lymph nodes. Once DC are properly positioned within the lymph nodes, circulating antigen specific na?ve T cells can interact with DC and become activated, clonally expanded and stimulated to undergo differentiation into antigen-experienced memory T cells. Subsequent reactivation of memory T cells that enter the autoimmune lesions by DC present in the inflammatory lesion is thought to play a central role in tissue inflammation.     

Immunity to Salmonella from a dendritic point of view

Dendritic cells (DC) are the key link between innate and adaptive immunity. Features of DC, including their presence at sites of antigen entry, their ability to migrate from peripheral sites to secondary lymphoid organs, and their superior capacity to stimulate naïve T cells places them in this pivotal role in the immune system. DC also produce cytokines, particularly IL‐12, upon antigen encounter and can thus influence the ensuing adaptive immune response. As DC are phagocytic antigen‐presenting cells located at sites exposed to bacterial invaders, studies have been performed to gain insight into the role of DC in combating bacterial infections. Indeed, studies with Salmonella have shown that DC can internalize and process this bacterium for peptide presentation on MHC‐II as well as MHC‐I. DC can also act as bystander antigen‐­presenting cells by presenting Salmonella antigens after internalizing neighbouring cells that have undergone Salmonella‐induced apoptotic death. DC also produce IL‐12 and TNF‐α upon Salmonella encounter. Moreover, studies in a murine infection model have shown that splenic DC increase surface expression of co‐stimulatory molecules during infection, and DC contain intracellular bacteria. In addition, quantitative changes occur in splenic DC numbers in the early stages of oral Salmonella infection, and this is accompanied by redistribution of the defined DC subsets in the spleen of infected mice. DC from Salmonella‐infected mice also produce cytokines and can stimulate bacteria‐specific T cells upon ex vivo co‐culture. In addition, DC may play a role in the traversal of bacteria from the intestinal lumen. Studying the function of DC during Salmonella infection provides insight into the capacity of this sophisticated antigen‐presenting cell to initiate and modulate the immune response to bacteria.     

Activation of human dendritic cells by the PorA protein of Neisseria meningitidis

The major porin proteins present in the outer membrane of Neisseria meningitidis, the causative agent of life‐threatening meningitis and septicaemia, are believed to have potent immunostimulatory effects. In this study, the interactions between human monocyte‐derived dendritic cells (mo‐DC) and the PorA porin were investigated, in order to reveal the role of this protein in promoting innate and adaptive immune responses. Recombinant (r)PorA induced mo‐DC maturation, as reflected by reduced receptor‐mediated endocytosis, increased production of the chemokines IL‐8, RANTES, MIP‐1α and MIP‐1β and augmented expression of the surface markers CD40, CD54, CD80, CD86 and major histocompatibility complex class II molecules. However, rPorA induced either low level or no significant secretion of pro‐inflammatory cytokines from mo‐DC. The protein potently augmented the capacity of mo‐DC to activate both allogeneic CD4⁺ memory T‐cells and CD4⁺RA⁺ naïve T‐cells. In addition, rPorA appeared to inhibit the production of IL‐12p70 that follows from the interaction between CD40 on the mo‐DC and CD40‐ligand on T‐cells, thereby directing T‐cell differentiation towards a Th2 type response. These data demonstrate that PorA is involved in DC activation and in influencing the nature of the T‐helper immune response, which are important properties for generating antibody responses required for protective immunity against meningococci and for determining the immuno‐adjuvant effects of this protein.     

TCR‐β repertoire analysis of antigen‐specific single T cells using a high‐density microcavity array

The antigen specificity of cytotoxic T cells, provided by T‐cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single‐cell level is required. In this study, we applied a high‐density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR‐β gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent‐labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T‐cell population. The isolated single cells were then directly utilized for RT‐PCR. By sequencing the amplified PCR products, antigen‐specific TCR‐β repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single‐cell level. This simple, accurate, and cost‐effective technique for single‐cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells. Biotechnol. Bioeng. 2010;106: 311–318. © 2010 Wiley Periodicals, Inc.     

Degenerate self-reactive human T-cell receptor causes spontaneous autoimmune disease in mice

Thyroid autoimmune disorders comprise more than 30% of all organ-specific autoimmune diseases and are characterized by autoantibodies and infiltrating T cells. The pathologic role of infiltrating T cells is not well defined. To address this issue, we generated transgenic mice expressing a human T-cell receptor derived from the thyroid-infiltrating T cell of a patient with thyroiditis and specific for a cryptic thyroid-peroxidase epitope. Here we show that mouse major histocompatibility complex molecules sustain selection and activation of the transgenic T cells, as coexpression of histocompatibility leukocyte antigen molecules was not needed. Furthermore, the transgenic T cells had an activated phenotype in vivo, and mice spontaneously developed destructive thyroiditis with histological, clinical and hormonal signs comparable with human autoimmune hypothyroidism. These results highlight the pathogenic role of human T cells specific for cryptic self epitopes. This new 'humanized' model will provide a unique tool to investigate how human pathogenic self-reactive T cells initiate autoimmune diseases and to determine how autoimmunity can be modulated in vivo.     

Antigen specificity acquisition of adoptive CD4+ regulatory T cells via acquired peptide-MHC class I complexes

The Ag-specific CD4(+) regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4(+) Tr cells is unclear. In this study, we generated IL-10- and IFN-gamma-expressing type 1 CD4(+) Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4(+) T cells with IL-10-expressing adenovirus (AdV(IL-10))-transfected and OVA-pulsed dendritic cells (DC(OVA/IL-10)). We demonstrated that both in vitro and in vivo DC(OVA/IL-10)-stimulated CD4(+) Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4(+) Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8(+) T cells, leading to an enhanced suppression of DC(OVA)-induced CD8(+) T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by approximately 700% relative to analogous CD4(+) Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4(+)25(+) Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8(+) T cell responses and antitumor immunity after uptake of DC(OVA)-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4(+) Tr cells via acquiring DC's pMHC I may be an important mean in augmenting CD4(+) Tr cell suppression.     

Reduced naïve CD8+ T‐cell priming efficacy in elderly adults

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8⁺ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8⁺ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8⁺ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8⁺ T‐cell responses specific for a model antigen. Reduced CD8⁺ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8⁺ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.     

Stromal cell‐derived factor‐1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF‐κB‐dependent pathways

Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal‐derived factor‐1α (SDF‐1α) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF‐1 and CXCR4 (SDF‐1 receptor). Treatment of osteosarcoma cells with SDF‐1α increased the migration and cell surface expression of αvβ3 integrin. CXCR4‐neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF‐1α‐induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF‐1α‐mediated migration and integrin expression. Stimulation of cells with SDF‐1α increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited SDF‐1α‐mediated cell migration and integrin up‐regulation. Stimulation of cells with SDF‐1α induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. Furthermore, the SDF‐1α‐mediated increasing κB‐luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKα and IKKβ mutants. Taken together, these results suggest that the SDF‐1α acts through CXCR4 to activate MEK and ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrins and contributing the migration of human osteosarcoma cells. J. Cell. Physiol. 221: 204–212, 2009. © 2009 Wiley‐Liss, Inc     

Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Dendritic cells in the skin – potential use for melanoma treatment

Melanoma is an aggressive malignancy with poor prognosis. Eradication of tumor cells requires an effective interaction between melanoma cells and different players of the immune system. As the most potent professional antigen‐presenting cells, dendritic cells (DCs) play a pivotal role in mounting a specific immune response where their intratumoral and peritumoral density as well as their functional status are correlated with clinical staging of the disease and with patients’ survival. Under steady‐state conditions, internalization of apoptotic cells by immature DCs designates a state of tolerance to self‐antigens. Nevertheless, pathogens and necrotic cells interacting with pattern recognition receptors trigger downstream signaling pathways that evoke maturation of DCs, leading to the production of pro‐inflammatory cytokines. These mature DCs are essential for T‐cell priming and subsequent development of a specific immune response. Altered functions of DCs have an impact on the development of various disorders including autoimmune diseases and cancers. Herein, we focus on the checkpoints created throughout DCs antigen capturing and presentation to T cells, with subsequent development of either tolerance or immune response, with an emphasis on the role played by DCs in melanoma tumorigenesis and their therapeutic potential.     

Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis

Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC) we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs) present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity.     

Shigella effector IpaH4.5 targets 19S regulatory particle subunit RPN13 in the 26S proteasome to dampen cytotoxic T lymphocyte activation

Subversion of antigen‐specific immune responses by intracellular pathogens is pivotal for successful colonisation. Bacterial pathogens, including Shigella, deliver effectors into host cells via the type III secretion system (T3SS) in order to manipulate host innate and adaptive immune responses, thereby promoting infection. However, the strategy for subverting antigen‐specific immunity is not well understood. Here, we show that Shigella flexneri invasion plasmid antigen H (IpaH) 4.5, a member of the E3 ubiquitin ligase effector family, targets the proteasome regulatory particle non‐ATPase 13 (RPN13) and induces its degradation via the ubiquitin–proteasome system (UPS). IpaH4.5‐mediated RPN13 degradation causes dysfunction of the 19S regulatory particle (RP) in the 26S proteasome, inhibiting guidance of ubiquitinated proteins to the proteolytically active 20S core particle (CP) of 26S proteasome and thereby suppressing proteasome‐catalysed peptide splicing. This, in turn, reduces antigen cross‐presentation to CD8+ T cells via major histocompatibility complex (MHC) class I in vitro. In RPN13 knockout mouse embryonic fibroblasts (MEFs), loss of RPN13 suppressed CD8+ T cell priming during Shigella infection. Our results uncover the unique tactics employed by Shigella to dampen the antigen‐specific cytotoxic T lymphocyte (CTL) response.     

Thymus‐derived regulatory T cells restrain pro‐inflammatory Th1 responses by downregulating CD70 on dendritic cells

The severity and intensity of autoimmune disease in immune dysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX) patients and in scurfy mice emphasize the critical role played by thymus‐derived regulatory T cells (tTregs) in maintaining peripheral immune tolerance. However, although tTregs are critical to prevent lethal autoimmunity and excessive inflammatory responses, their suppressive mechanism remains elusive. Here, we demonstrate that tTregs selectively inhibit CD27/CD70‐dependent Th1 priming, while leaving the IL‐12‐dependent pathway unaffected. Immunized mice depleted of tTregs showed an increased response of IFN‐γ‐secreting CD4⁺ T cells that was strictly reliant on a functional CD27/CD70 pathway. In vitro studies revealed that tTregs downregulate CD70 from the plasma membrane of dendritic cells (DCs) in a CD27‐dependent manner. CD70 downregulation required contact between Tregs and DCs and resulted in endocytosis of CD27 and CD70 into the DC. These findings reveal a novel mechanism by which tTregs can maintain tolerance or prevent excessive, proinflammatory Th1 responses.     

The role of B cells in regulating the magnitude of immune response

Recently, accumulating evidence has suggested that B cell depletion therapy with rituximab is effective not only in autoantibody‐associated, but also in T cell‐mediated, autoimmune diseases. It is likely that B cells play an important role in regulating the extent of immune response in both physiological and pathological conditions. When a severe infection occurs, pathogens spread throughout the bloodstream. B cells in the blood capture the pathogens, via their specific antigen receptors (surface immunoglobulins), then present the specific antigen to T cells in the spleen, thus increasing the degree of T‐cell immune responses to systemic infection. Similarly, in the exacerbation stage of autoimmunity, a large amount of autoantigens may be released into the blood and be captured by autoantigen specific B cells, and this may be followed by presentation of the antigen to CD4 positive autoreactive T cells resulting in extensive activation and proliferation of autoreactive T cells. Thus, it has been suggested that B‐cell depletion therapy for autoimmune diseases is most useful for the “vicious cycle” phase of autoreactive immune response. The recognition of this paradigm for the role of B cells in regulating the magnitude of immune response will help to facilitate both basic and clinical research on the regulation of immune responses.     

Hyper‐reactive cloned mice generated by direct nuclear transfer of antigen‐specific CD4+ T cells

T‐cell receptor (TCR)‐transgenic mice have been employed for evaluating antigen‐response mechanisms, but their non‐endogenous TCR might induce immune response differently than the physiologically expressed TCR. Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen‐specific CD4⁺ T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre‐rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic‐type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter‐regulated antigen‐specific TCR are a unique animal model with allergic predisposition for investigating CD4⁺ T‐cell‐mediated pathogenesis and cellular commitment in immune diseases.