CD40 Drives Central Nervous System Autoimmune Disease by Inducing Complementary Effector Programs via B Cells and Dendritic Cells.

Costimulatory CD40 plays an essential role in autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis (MS). However, how CD40 drives autoimmune disease pathogenesis is not well defined. Here, we used a conditional knockout approach to determine how CD40 orchestrates a CNS autoimmune disease induced by recombinant human myelin oligodendrocyte glycoprotein (rhMOG). We found that deletion of CD40 in either dendritic cells (DCs) or B cells profoundly reduced EAE disease pathogenesis. Mechanistically, CD40 expression on DCs was required for priming pathogenic Th cells in peripheral draining lymph nodes and promoting their appearance in the CNS. By contrast, B cell CD40 was essential for class-switched MOG-specific Ab production, which played a crucial role in disease pathogenesis. In fact, passive transfer of MOG-immune serum or IgG into mice lacking CD40 on B cells but not DCs reconstituted autoimmune disease, which was associated with inundation of the spinal cord parenchyma by Ig and complement. These data demonstrate that CD40 supports distinct effector programs in B cells and DCs that converge to drive a CNS autoimmune disease and identify targets for intervention.

A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations.

Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.

Antigen targeting to dendritic cells combined with transient regulatory T cell inhibition results in long-term tumor regression.

Therapeutic vaccinations against cancer are still largely ineffective. Major caveats are inefficient delivery of tumor antigens to dendritic cells (DCs) and excessive immune suppression by Foxp3+ regulatory T cells (Tregs), resulting in defective T cell priming and failure to induce tumor regression. To circumvent these problems we evaluated a novel combinatorial therapeutic strategy. We show that tumor antigen targeting to DC-SIGN in humanized hSIGN mice via glycans or specific antibodies induces superior T cell priming. Next, this targeted therapy was combined with transient Foxp3+ Treg depletion employing hSIGNxDEREG mice. While Treg depletion alone slightly delayed B16-OVA melanoma growth, only the combination therapy instigated long-term tumor regression in a substantial fraction of mice. This novel strategy resulted in optimal generation of antigen-specific activated CD8+ T cells which accumulated in regressing tumors. Notably, Treg depletion also allowed the local appearance of effector T cells specific for endogenous B16 antigens. This indicates that antitumor immune responses can be broadened by therapies aimed at controlling Tregs in tumor environments. Thus, transient inhibition of Treg-mediated immune suppression potentiates DC targeted antigen vaccination and tumor-specific immunity.

Epigenetic targeting of activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation (SHM) by deaminating cytosine residues in immunoglobulin genes (Igh, Igκ, and Igλ). At a lower frequency, AID also causes collateral DNA damage at non-Ig loci, including genes that are rearranged or mutated in B-cell lymphoma. Precisely how AID is recruited to these off-target sites is not entirely understood. To gain further insight into how AID selects its targets, we compared AID-mediated translocations in two different cell types, B cells and mouse embryonic fibroblasts (MEFs). AID targets a distinct set of hotspots in the two cell types. In both cases, hotspots are concentrated in highly transcribed but stalled genes. However, transcription alone is insufficient to recruit AID activity. Comparison of genes similarly transcribed in B cells and MEFs but targeted in only one of the two cell types reveals a common set of epigenetic features associated with AID recruitment in both cells. AID target genes are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac) and marks of active transcription (such as H3K36me3) in both fibroblasts and B cells, indicating that these features are universal mediators of AID recruitment.

Advantages of Foxp3(+) regulatory T cell depletion using DEREG mice.

Several mechanisms enable immunological self-tolerance. Regulatory T cells (Tregs) are a specialized T cell subset that prevents autoimmunity and excessive immune responses, but can also mediate detrimental tolerance to tumors and pathogens in a Foxp3-dependent manner. Genetic tools exploiting the foxp3 locus including bacterial artificial chromosome (BAC)-transgenic DEREG mice have provided essential information on Treg biology and the potential therapeutic modulation of tolerance. In DEREG mice, Foxp3(+) Tregs selectively express eGFP and diphtheria toxin (DT) receptor, allowing for the specific depletion of Tregs through DT administration. We here provide a detailed overview about important considerations such as DT toxicity, which affects any mouse strain treated with DT, and Treg rebound after depletion. Additionally, we point out the specific advantages of BAC-transgenic DEREG mice including their suitability to study organ-specific autoimmunity such as type I diabetes. Moreover, we discuss recent insights into the role of Tregs in viral infections. In summary, DEREG mice are an important tool to study Treg-mediated tolerance and its therapeutic circumvention.

Few Foxp3⁺ regulatory T cells are sufficient to protect adult mice from lethal autoimmunity.

Foxp3 specifies the Treg cell lineage and is indispensable for immune tolerance. Accordingly, rare Foxp3 mutations cause lethal autoimmunity. The mechanisms precipitating more prevalent human autoimmune diseases are poorly understood, but involve a combination of genetic and environmental factors. Many autoimmune diseases associate with a partial Treg-cell dysfunction, yet mouse models reflecting such complex pathophysiological processes are rare. Around 95% of Foxp3(+) Treg cells can be specifically depleted in bacterial artifical chromosome (BAC)-transgenic Depletion of REGulatory T cells (DEREG) mice through diphtheria toxin (DT) treatment. However, Treg-cell depletion fails to cause autoimmunity in adult DEREG mice for unclear reasons. By crossing Foxp3(GFP) knock-in mice to DEREG mice, we introduced additional genetic susceptibility that does not affect untreated mice. Strikingly, DT treatment of DEREG × Foxp3(GFP) mice rapidly causes autoimmunity characterized by blepharitis, tissue damage, and autoantibody production. This inflammatory disease is associated with augmented T-cell activation, increased Th2 cytokine production and myeloproliferation, and is caused by defective Treg-cell homeostasis, preventing few DT-insensitive Treg cells from repopulating the niche after Treg-cell depletion. Our study provides important insights into self-tolerance. We further highlight DEREG × Foxp3(GFP) mice as a model to investigate the role of environmental factors in precipitating autoimmunity. This may help to better understand and treat human autoimmunity.

Amyloid arthropathy associated with multiple myeloma: polyarthritis without synovial infiltration of CD20+ or CD38+ cells.

OBJECTIVES: To describe histological, immunohistochemical and ultrastructural features of synovial biopsies of amyloid arthropathy associated with multiple myeloma (MM). METHODS: Synovial biopsies from affected joints of two patients with MM and amyloid arthropathy were examined with light and electron microscopy, and immunohistochemically for expression of CD3, CD8, CD20, CD38, CD68, Ki-67 and vWF. Results were compared to values from osteoarthritis (OA, n = 26), rheumatoid arthritis (RA, n = 24) and normal (n = 15) synovial membranes. RESULTS: There was no or only mild lining hyperplasia. Vascular density was not elevated, and there were few Ki-67+ proliferating cells in the stroma. The Krenn synovitis score classified one specimen as "low-grade" and one as "high-grade" synovitis. CD68+ and CD3+ cells were the predominant mononuclear inflammatory cells, whereas CD20+ and CD38+ cells were absent from both synovial membrane and synovial fluid sediment. Electron microscopy demonstrated amyloid phagocytosis by synovial macrophages. In hierarchical clustering the two amyloid arthropathy specimens were more closely related to OA than to RA or normal synovium. CONCLUSIONS: This first detailed immunohistological analysis of MM-associated amyloid arthropathy suggests that it is a chronic synovitis that evolves despite the loss of humoral immunity seen in advanced MM. Instead, amyloid phagocytosis by synovial macrophages likely triggers and perpetuates local disease.

Amyloid arthropathy associated with multiple myeloma: a systematic analysis of 101 reported cases.

OBJECTIVE: Amyloid deposition in multiple myeloma (MM) may lead to an arthropathy resembling rheumatoid arthritis (RA). Since a systematic description of its natural history is lacking, we have performed a systematic analysis of all published cases. METHODS: Literature review featuring backward and forward database searches and direct inspection of reference lists. Inclusion criteria were as follows: publication between 1931 and 2012, diagnosis of multiple myeloma, and demonstration of light chain amyloid (AL) in any organ or in synovial fluid, arthritis, or synovitis. RESULTS: Overall, 101 cases were identified. Median age was 59 years and the male-to-female ratio was 1:1. A systemic manifestation of MM was reported in 88 cases. In 53 of these, characteristic physical findings (carpal tunnel syndrome, macroglossia, shoulder pad, and soft tissue swelling/masses) were present. Arthritis manifested before the diagnosis of MM in 63 cases, with 33 cases initially misdiagnosed as RA. There were 72 cases of poly-, 17 of oligo-, and three of monoarthritis. The shoulder joint was most commonly affected, followed by knees and small hand joints. Median synovial fluid leukocyte count was 2460 cells/mm(3), and was normal in seven cases. Synovial histopathology often featured mild synovitis without plasma cell infiltration. Imaging revealed articular or periarticular inflammation in many cases and bone lesions near 22% of affected joints. Treatments varied but led to some improvement in the majority of cases. CONCLUSIONS: These results solidify previous experience that MM arthropathy tends to feature a symmetric RF-negative nonerosive polyarthritis. However, the results also highlight the diversity of its presentations and stress the importance of arthropathy as a potentially under-recognized presenting manifestation of MM.

In vivo targeting of human DC-SIGN drastically enhances CD8⁺ T-cell-mediated protective immunity.

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⁺ T-cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC-SIGN (DC-specific-ICAM3-grabbing-nonintegrin/CD209) is a C-type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC-SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC-SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti-DC-SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti-DC-SIGN antibodies conjugated to OVA induced strong and persistent antigen-specific CD4⁺ and CD8⁺ T-cell responses, which efficiently protected from infection with OVA-expressing Listeria monocytogenes. Thus, we propose DC targeting via DC-SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.

Layers of dendritic cell-mediated T cell tolerance, their regulation and the prevention of autoimmunity.

The last decades of Nobel prize-honored research have unequivocally proven a key role of dendritic cells (DCs) at controlling both T cell immunity and tolerance. A tight balance between these opposing DC functions ensures immune homeostasis and host integrity. Its perturbation could explain pathological conditions such as the attack of self tissues, chronic infections, and tumor immune evasion. While recent insights into the complex DC network help to understand the contribution of individual DC subsets to immunity, the tolerogenic functions of DCs only begin to emerge. As these consist of many different layers, the definition of a "tolerogenic DC" is subjected to variation. Moreover, the implication of DCs and DC subsets in the suppression of autoimmunity are incompletely resolved. In this review, we point out conceptual controversies and dissect the various layers of DC-mediated T cell tolerance. These layers include central tolerance, Foxp3(+) regulatory T cells (Tregs), anergy/deletion and negative feedback regulation. The mode and kinetics of antigen presentation is highlighted as an additional factor shaping tolerance. Special emphasis is given to the interaction between layers of tolerance as well as their differential regulation during inflammation. Furthermore, potential technical caveats of DC depletion models are considered. Finally, we summarize our current understanding of DC-mediated tolerance and its role for the suppression of autoimmunity. Understanding the mechanisms of DC-mediated tolerance and their complex interplay is fundamental for the development of selective therapeutic strategies, e.g., for the modulation of autoimmune responses or for the immunotherapy of cancer.

Foxp3-mediated suppression of CD95L expression confers resistance to activation-induced cell death in regulatory T cells.

CD4(+)CD25(++)Foxp3(+) regulatory T cells (Tregs) control self-reactive cells to maintain peripheral tolerance. Treg homeostasis has to be controlled tightly to ensure balanced Treg-mediated suppression. One mechanism that regulates the CD4(+) T cell pool is activation-induced cell death (AICD). This is mimicked in vitro by TCR restimulation-induced expression of the death ligand CD95L (FasL/APO-1L/CD178) in expanded T cells. These cells express the death receptor CD95 (Fas/APO-1), and binding of CD95L to CD95 results in AICD. In contrast, Tregs do not undergo AICD upon TCR (re)stimulation in vitro despite a functional CD95 cell death pathway. In this study, we show that human and murine Tregs express low levels of CD95L upon stimulation. Knockdown of the transcriptional repressor Foxp3 partially rescues CD95L expression and AICD in human Tregs. Moreover, upon stimulation Foxp3-mutant Tregs from Scurfy mice express CD95L similar to conventional T cells. We further addressed whether exogenous CD95 stimulation provides a mechanism of Treg homeostatic control in vivo in mice. Triggering of CD95 reduced Treg numbers systemically as reflected by in vivo imaging and decreased GFP(+) Treg numbers ex vivo. Our study reveals that Foxp3 negatively regulates CD95L expression in Tregs and demonstrates that Tregs are susceptible to homeostatic control by CD95 stimulation.

Selective depletion of Foxp3+ regulatory T cells improves effective therapeutic vaccination against established melanoma.

Tumor-bearing individuals have been reported to harbor increased numbers of Foxp3(+) regulatory T cells (Treg), which prevent the development of efficient antitumor immune responses. Thus, Treg depletion has already been tested as a promising therapeutic approach in various animal models and entered clinical trials. However, the use of nonspecific Treg targeting agents such as CD25 depleting antibodies, which in addition to CD25(+) Tregs also deplete recently activated CD25(+) effector T cells, potentially masked the tremendous potential of this therapeutic strategy. To avoid such nonspecific effects, we used transgenic DEREG (depletion of regulatory T cells) mice, which express a diphtheria toxin receptor under control of the Foxp3 locus, allowing selective depletion of Foxp3(+) Tregs even during ongoing immune responses. We showed that Foxp3(+) Treg depletion induced partial regression of established ovalbumin (OVA)-expressing B16 melanoma, which was associated with an increased intratumoral accumulation of activated CD8(+) cytotoxic T cells. The antitumor effect could be significantly enhanced when Treg depletion was combined with vaccination against OVA. To further assess whether this therapeutic approach would break self-tolerance, we crossed DEREG mice with RipOVA(low) mice, expressing OVA as neo-self-antigen under control of the rat insulin promoter. In these mice, combined Treg depletion and vaccination also induced tumor regression without the onset of diabetes. Together, our data suggest that selective Treg targeting strategies combined with vaccinations against tumor-associated (self) antigens have the potential to evoke efficient antitumor responses without inducing overt autoimmunity. These findings might have implications for future therapeutic interventions in cancer patients.

Nonfunctional regulatory T cells and defective control of Th2 cytokine production in natural scurfy mutant mice.

Foxp3(+) regulatory T cells (Tregs) are crucial for preventing autoimmunity. We have demonstrated that depletion of Foxp3(+) Tregs results in the development of a scurfy-like disease, indicating that Foxp3(-) effector T cells are sufficient to induce autoimmunity. It has been postulated that nonfunctional Tregs carrying potentially self-reactive T cell receptors may contribute to scurfy (sf) pathogenesis due to enhanced recognition of self. Those cells, however, could not be identified in sf mutants due to the lack of Foxp3 protein expression. To address this issue, we crossed the natural sf mouse mutant with bacterial artificial chromosome transgenic DEREG (depletion of regulatory T cells) mice. Since DEREG mice express GFP under the control of an additional Foxp3 promoter, those crossings allowed proving the existence of "would-be" Tregs, which are characterized by GFP expression in the absence of functional Foxp3. Sf Tregs lost their in vitro suppressive capacity. This correlated with a substantial reduction of intracellular cAMP levels, whereas surface expression of Treg markers was unaffected. Both GFP(+) and GFP(-) sf cells produced high amounts of Th2-type cytokines, reflected also by enhanced Gata-3 expression, when tested in vitro. Nevertheless, sf Tregs could be induced in vitro, although with lower efficiency than DEREG Tregs. Transfer of GFP(+) sf Tregs, in contrast to GFP(-) sf T cells, into RAG1-deficient animals did not cause the sf phenotype. Taken together, natural and induced Tregs develop in the absence of Foxp3 in sf mice, which lack both suppressive activity and autoreactive potential, but rather display a Th2-biased phenotype.

Identification of novel monosodium urate crystal regulated mRNAs by transcript profiling of dissected murine air pouch membranes.

INTRODUCTION: The murine air pouch is a bursa-like space that resembles the human synovial membrane. Injection of monosodium urate (MSU) crystals into the pouch elicits an acute inflammatory response similar to human gout. We conducted the present study to identify mRNAs that were highly regulated by MSU crystals in the pouch membrane. METHODS: Air pouch membranes were meticulously dissected away from the overlying skin. Gene expression differences between MSU crystal stimulated and control membranes were determined by oligonucleotide microarray analysis 9 hours after injection of MSU crystals or buffer only. Differential regulation of selected targets was validated by relative quantitative PCR in time course experiments with dissected air pouch membranes and murine peritoneal macrophages. RESULTS: Eleven of the 12 most highly upregulated mRNAs were related to innate immunity and inflammation. They included mRNAs encoding histidine decarboxylase (the enzyme that synthesizes histamine), IL-6, the cell surface receptors PUMA-g and TREM-1, and the polypeptides Irg1 and PROK-2. IL-6 mRNA rose 108-fold 1 hour after crystal injection, coinciding with a surge in mRNAs encoding IL-1beta, tumour necrosis factor-alpha and the immediate early transcription factor Egr-1. The other mRNAs rose up to 200-fold within the subsequent 3 to 8 hours. MSU crystals induced these mRNAs in a dose-dependent manner in cultured macrophages, with similar kinetics but lower fold changes. Among the downregulated mRNAs, quantitative PCR confirmed significant decreases in mRNAs encoding TREM-2 (an inhibitor of macrophage activation) and granzyme D (a constituent of natural killer and cytotoxic T cells) within 50 hours after crystal injection. CONCLUSION: This analysis identified several genes that were previously not implicated in MSU crystal inflammation. The marked rise of the upregulated mRNAs after the early surge in cytokine and Egr-1 mRNAs suggests that they may be part of a 'second wave' of factors that amplify or perpetuate inflammation. Transcript profiling of the isolated air pouch membrane promises to be a powerful tool for identifying genes that act at different stages of inflammation.