Protocol for the isolation and purification of human follicular dendritic cells for functional assays

In this protocol, we detail how to isolate and purify human follicular dendritic cells (FDCs) from lymphoid tissues. FDCs play a vital role in antibody development by presenting antigens to B cells in germinal centers. The assay involves enzymatic digestion and fluorescence-activated cell sorting and is successfully applied to various lymphoid tissues, including tonsils, lymph nodes, and tertiary lymphoid structures. Our robust technique enables the isolation of FDCs and facilitates downstream functional and descriptive assays. For complete details on the use and execution of this protocol, please refer to Heesters et al

Maturing Human CD127+ CCR7+ PDL1+ Dendritic Cells Express AIRE in the Absence of Tissue Restricted Antigens.

Expression of the Autoimmune regulator (AIRE) outside of the thymus has long been suggested in both humans and mice, but the cellular source in humans has remained undefined. Here we identify AIRE expression in human tonsils and extensively analyzed these "extra-thymic AIRE expressing cells" (eTACs) using combinations of flow cytometry, CyTOF and single cell RNA-sequencing. We identified AIRE+ cells as dendritic cells (DCs) with a mature and migratory phenotype including high levels of antigen presenting molecules and costimulatory molecules, and specific expression of CD127, CCR7, and PDL1. These cells also possessed the ability to stimulate and re-stimulate T cells and displayed reduced responses to toll-like receptor (TLR) agonists compared to conventional DCs. While expression of AIRE was enriched within CCR7+CD127+ DCs, single-cell RNA sequencing revealed expression of AIRE to be transient, rather than stable, and associated with the differentiation to a mature phenotype. The role of AIRE in central tolerance induction within the thymus is well-established, however our study shows that AIRE expression within the periphery is not associated with an enriched expression of tissue-restricted antigens (TRAs). This unexpected finding, suggestive of wider functions of AIRE, may provide an explanation for the non-autoimmune symptoms of APECED patients who lack functional AIRE.JF and HS were funded by project ERC-2013-ADG number 341038. MB was funded by EMBO ALTF 786-2013. BH was supported by the Netherlands Organization for Scientific Research (NWO) Veni program (91618032). LH, JpvH, and ST were supported by a grant from the Dutch Arthritis Foundation (2013_2_37). MM was supported by Wellcome Trust (grant105045/Z/14/Z). JM was supported by core funding from the European Molecular Biology Laboratory and from Cancer Research UK (award number 17197)

Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice

Background The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. Methods We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-Associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. Results PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γsignature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. Conclusions By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB

Maturing Human CD127+ CCR7+ PDL1+ Dendritic Cells Express AIRE in the Absence of Tissue Restricted Antigens

Expression of the Autoimmune regulator (AIRE) outside of the thymus has long been suggested in both humans and mice, but the cellular source in humans has remained undefined. Here we identify AIRE expression in human tonsils and extensively analyzed these “extra-thymic AIRE expressing cells” (eTACs) using combinations of flow cytometry, CyTOF and single cell RNA-sequencing. We identified AIRE+ cells as dendritic cells (DCs) with a mature and migratory phenotype including high levels of antigen presenting molecules and costimulatory molecules, and specific expression of CD127, CCR7, and PDL1. These cells also possessed the ability to stimulate and re-stimulate T cells and displayed reduced responses to toll-like receptor (TLR) agonists compared to conventional DCs. While expression of AIRE was enriched within CCR7+CD127+ DCs, single-cell RNA sequencing revealed expression of AIRE to be transient, rather than stable, and associated with the differentiation to a mature phenotype. The role of AIRE in central tolerance induction within the thymus is well-established, however our study shows that AIRE expression within the periphery is not associated with an enriched expression of tissue-restricted antigens (TRAs). This unexpected finding, suggestive of wider functions of AIRE, may provide an explanation for the non-autoimmune symptoms of APECED patients who lack functional AIRE

Correction:How the COVID-19 pandemic highlights the necessity of animal research (vol 30, pg R1014, 2020)

(Current Biology 30, R1014–R1018; September 21, 2020) As a result of an author oversight in the originally published version of this article, a number of errors were introduced in the author list and affiliations. First, the middle initials were omitted from the names of several authors. Second, the surname of Dr. van Dam was mistakenly written as “Dam.” Third, the first name of author Bernhard Englitz was misspelled as “Bernard” and the surname of author B.J.A. Pollux was misspelled as “Pullox.” Finally, Dr. Keijer's first name was abbreviated rather than written in full. These errors, as well as various errors in the author affiliations, have now been corrected online

Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα.

Patients with ectodermal dysplasia with immunodeficiency (ED-ID) caused by mutations in the inhibitor of NF-κB α (IκBα) are susceptible to severe recurrent infections, despite normal T and B cell numbers and intact in vitro lymphocyte function. Moreover, the outcome of hematopoietic stem cell transplantation (HSCT) in these patients is poor despite good engraftment. Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID. Strikingly, the mice lacked lymph nodes, Peyer's patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling. Lymphotoxin β receptor (LTβR)-driven induction of chemokines and adhesion molecules mediated by both canonical and noncanonical NF-κB pathways was impaired, and levels of p100 were markedly diminished in the mutant. IκBα mutant → Rag2(-/-), but not WT→IκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs. Defective architectural cell function explains the immunodeficiency and poor outcome of HSCT in patients with IκBα deficiency and suggests that correction of this niche is critical for reconstituting their immune function

The Role of Dendritic Cells in S. pneumoniae Transport to Follicular Dendritic Cells

Affinity-mature B cells require cognate antigen, retained by follicular dendritic cells (FDCs), for clonal selection within germinal centers. Studies on how FDCs in lymphoid tissues acquire antigen have relied primarily on model protein antigens. To examine delivery of intact bacteria to FDCs, we used inactivated Streptococcus pneumonia (SP). We found that both medullary macrophages and a subset of SIGN-R1-positive dendritic cells (DCs) in the lymph node capture SP from the draining afferent lymphatics. The presence of DCs is required for initial complement activation, opsonization of the bacteria, and efficient transport of SP to FDCs. Moreover, we observed a major role for transport of bacteria to FDCs by naive B cells via a CD21-dependent pathway. We propose a mechanism by which efficient transport of SP to FDCs is dependent on DCs for initial binding and activation of complement and either direct transport to FDCs or transfer to naive B cells

The follicular dendritic cell: At the germinal center of autoimmunity?

Summary: Autoimmune diseases strain healthcare systems worldwide as their incidence rises, and current treatments put patients at risk for infections. An increased understanding of autoimmune diseases is required to develop targeted therapies that do not impair normal immune function. Many autoimmune diseases present with autoantibodies, which drive local or systemic inflammation. This indicates the presence of autoreactive B cells that have escaped tolerance. An important step in the development of autoreactive B cells is the germinal center (GC) reaction, where they undergo affinity maturation toward cognate self-antigen. Follicular dendritic cells (FDCs) perform the essential task of antigen presentation to B cells during the affinity maturation process. However, in recent years, it has become clear that FDCs play a much more active role in regulation of GC processes. Here, we evaluate the biology of FDCs in the context of autoimmune disease, with the goal of informing future therapeutic strategies

Do follicular dendritic cells regulate lupus-specific B cells?

The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. In this review we describe recently published results in which a B cell receptor-knock-in mouse strain specific for nucleolar self-antigens was bred with mice deficient in complement C4 and discuss the implications for the lupus field. Absence of C4 leads to a breakdown in the elimination of autoreactive B cell clones at the transitional stage. This is characterized by a relative increase in their response to a range of stimuli, entrance into follicles and a greater propensity to form self-reactive germinal centers. In this review, a model is proposed in which, in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells and follicular dendritic cells, resulting in secretion of Type I interferon. This allows for the maturation and activation of self-reactive B cell clones leading to increased spontaneous formation of germinal centers and subsequent generation of autoantibodies. (C) 2014 Elsevier Ltd. All rights reserve