Crosstalk between Platelets and SARS-CoV-2: Implications in Thrombo-Inflammatory Complications in COVID-19.

The SARS-CoV-2 virus, causing the devastating COVID-19 pandemic, has been reported to affect platelets and cause increased thrombotic events, hinting at the possible bidirectional interactions between platelets and the virus. In this review, we discuss the potential mechanisms underlying the increased thrombotic events as well as altered platelet count and activity in COVID-19. Inspired by existing knowledge on platelet-pathogen interactions, we propose several potential antiviral strategies that platelets might undertake to combat SARS-CoV-2, including their abilities to internalize the virus, release bioactive molecules to interfere with viral infection, and modulate the functions of immune cells. Moreover, we discuss current and potential platelet-targeted therapeutic strategies in controlling COVID-19, including antiplatelet drugs, anticoagulants, and inflammation-targeting treatments. These strategies have shown promise in clinical settings to alleviate the severity of thrombo-inflammatory complications and reduce the mortality rate among COVID-19 patients. In conclusion, an in-depth understanding of platelet-SARS-CoV-2 interactions may uncover novel mechanisms underlying severe COVID-19 complications and could provide new therapeutic avenues for managing this disease.

Preparation and Characterization of a Polyetherketoneketone/Hydroxyapatite Hybrid for Dental Applications.

Here, we developed a new synthetic method for the production of a new class of polymeric inorganic hybrid biomaterial that has potential for dental implant applications and, in general, other orthopedic applications owing to its excellent mechanical properties and biomechanical compatibility. The new hybrid biomaterial is a composite consisting of polyetherketoneketone (PEKK) and hydroxyapatite (HA). This hybrid material boasts several unique features, including its high HA loading (up to 50 wt%), which is close to that of natural human bone; the homogeneous HA distribution in the PEKK matrix without phase separation; and the fact that the addition of HA has no effect on the molecular weight of PEKK. Nanoindentation analysis was used to investigate the mechanical properties of the composite, and its nano/microstructure variations were investigated through a structural model developed here. Through nanoindentation technology, the newly developed PEKK/HA hybrid biomaterial has an indentation modulus of 12.1 ± 2.5 GPa and a hardness of 0.42 ± 0.09 GPa, which are comparable with those of human bone. Overall, the new PEKK/HA biomaterial exhibits excellent biomechanical compatibility and shows great promise for application to dental and orthopedic devices.

Neonatal LTßR signaling is required for the accumulation of eosinophils in the inflamed adult mesenteric lymph node.

Although eosinophils are important contributors to mucosal immune responses, mechanisms that regulate their accumulation in mucosal-associated lymphoid tissues remain ill-defined. Combining bone marrow chimeras and pharmacological inhibition approaches, here we find that lymphotoxin-beta receptor (LTßR) signaling during the neonatal period is required for the accumulation of eosinophils in the mesenteric lymph nodes (MLN) during an enteric viral infection in adult male and female mice. We demonstrate that MLN stromal cells express genes that are important for eosinophil migration and survival, such as Ccl-11 (eotaxin-1), Ccl7, Ccl9, and Cxcl2, and that expression of most of these genes is downregulated as a consequence of neonatal LTßR blockade. We also find that neonatal LTßR signaling is required for the generation of a rotavirus-specific IgA antibody response in the adult MLN, but eosinophils are dispensable for this response. Collectively, our studies reveal a role for neonatal LTßR signaling in regulating eosinophil numbers in the adult MLN.

Hepatic arterial infusion chemotherapy following simultaneous metallic stent placement and iodine-125 seed strands for advanced cholangiocarcinoma causing malignant obstructive jaundice: a propensity score matching study.

OBJECTIVE: This study aims to evaluate the effectiveness and safety of hepatic arterial infusion chemotherapy (HAIC) following the simultaneous placement of self-expandable metallic stent (SEMS) and iodine-125 (125I) seed strands for the management of advanced cholangiocarcinoma (CCA) patients presenting with malignant obstructive jaundice (MOJ). METHODS: Data from 74 patients with MOJ caused by advanced CCA treated with stent placement with 125I seed strands with or without HAIC between November 2015 and October 2020 were analysed retrospectively. Eighteen patients received 5 sessions of HAIC after SEMS placement with 125I seed strands (HAIC group), and 56 patients only underwent SEMS placement with 125I seed strands and served as controls (control group). HAIC consisted of infusions of gemcitabine (600-1000 mg/m2 given over 30 min) followed by oxaliplatin (60-100 mg/m2 given over 2 h), with an interval of 4 weeks. Propensity score matching (PSM) analysis was used to adjust for differences in the baseline characteristics of the groups (including age, total bilirubin, and serum alanine aminotransferase level). Overall survival (OS), stent patency, and adverse events were compared between the two groups. RESULTS: OS and stent patency were significantly better in patients in the HAIC group than in those in the control group (median survival time: before PSM, 362 vs. 185 days, p = 0.005; after PSM, 357 vs. 183 days, p = 0.012; median duration of stent patency: before PSM, 294 vs. 156 days, p = 0.001; after PSM, 287 vs. 183 days, p = 0.039). All adverse reactions were controllable by temporary symptomatic treatment. Serious complications and treatment-related deaths were not observed. CONCLUSION: Our preliminary study showed that HAIC following SEMS placement with 125I seed strands is effective and safe for the management of advanced CCA patients presenting with MOJ and could improve stent patency and patient survival.

FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

Crosstalk Between Platelets and Microbial Pathogens.

Platelets, small anucleate cells circulating in the blood, are critical mediators in haemostasis and thrombosis. Interestingly, recent studies demonstrated that platelets contain both pro-inflammatory and anti-inflammatory molecules, equipping platelets with immunoregulatory function in both innate and adaptive immunity. In the context of infectious diseases, platelets are involved in early detection of invading microorganisms and are actively recruited to sites of infection. Platelets exert their effects on microbial pathogens either by direct binding to eliminate or restrict dissemination, or by shaping the subsequent host immune response. Reciprocally, many invading microbial pathogens can directly or indirectly target host platelets, altering platelet count or/and function. In addition, microbial pathogens can impact the host auto- and alloimmune responses to platelet antigens in several immune-mediated diseases, such as immune thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia. In this review, we discuss the mechanisms that contribute to the bidirectional interactions between platelets and various microbial pathogens, and how these interactions hold relevant implications in the pathogenesis of many infectious diseases. The knowledge obtained from "well-studied" microbes may also help us understand the pathogenesis of emerging microbes, such as SARS-CoV-2 coronavirus.

SHLD2 promotes class switch recombination by preventing inactivating deletions within the Igh locus.

The newly identified shieldin complex, composed of SHLD1, SHLD2, SHLD3, and REV7, lies downstream of 53BP1 and acts to inhibit DNA resection and promote NHEJ. Here, we show that Shld2-/- mice have defective class switch recombination (CSR) and that loss of SHLD2 can suppress the embryonic lethality of a Brca1Δ11 mutation, highlighting its role as a key effector of 53BP1. Lymphocyte development and RAG1/2-mediated recombination were unaffected by SHLD2 deficiency. Interestingly, a significant fraction of Shld2-/- primary B-cells and 53BP1- and shieldin-deficient CH12F3-2 B-cells permanently lose expression of immunoglobulin upon induction of CSR; this population of Ig-negative cells is also seen in other NHEJ-deficient cells and to a much lesser extent in WT cells. This loss of Ig is due to recombination coupled with overactive resection and loss of coding exons in the downstream acceptor constant region. Collectively, these data show that SHLD2 is the key effector of 53BP1 and critical for CSR in vivo by suppressing large deletions within the Igh locus.

Early-life programming of mesenteric lymph node stromal cell identity by the lymphotoxin pathway regulates adult mucosal immunity.

Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-ß receptor (LTßR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTßR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTßR signaling affects mucosal immune responses during adulthood.

Fibroblastic reticular cells initiate immune responses in visceral adipose tissues and secure peritoneal immunity.

Immune protection of the body cavities depends on the swift activation of innate and adaptive immune responses in nonclassical secondary lymphoid organs known as fat-associated lymphoid clusters (FALCs). Compared with classical secondary lymphoid organs such as lymph nodes and Peyer's patches, FALCs develop along distinct differentiation trajectories and display a reduced structural complexity. Although it is well established that fibroblastic reticular cells (FRCs) are an integral component of the immune-stimulating infrastructure of classical secondary lymphoid organs, the role of FRCs in FALC-dependent peritoneal immunity remains unclear. Using FRC-specific gene targeting, we found that FRCs play an essential role in FALC-driven immune responses. Specifically, we report that initiation of peritoneal immunity was governed through FRC activation in a myeloid differentiation primary response 88 (MYD88)-dependent manner. FRC-specific ablation of MYD88 blocked recruitment of inflammatory monocytes into FALCs and subsequent CD4+ T cell-dependent B-cell activation and IgG class switching. Moreover, containment of Salmonella infection was compromised in mice lacking MYD88 expression in FRCs, indicating that FRCs in FALCs function as an initial checkpoint in the orchestration of protective immune responses in the peritoneal cavity.

The H2B deubiquitinase Usp22 promotes antibody class switch recombination by facilitating non-homologous end joining.

Class switch recombination (CSR) has a fundamental function during humoral immune response and involves the induction and subsequent repair of DNA breaks in the immunoglobulin (Ig) switch regions. Here we show the role of Usp22, the SAGA complex deubiquitinase that removes ubiquitin from H2B-K120, in the repair of programmed DNA breaks in vivo. Ablation of Usp22 in primary B cells results in defects in γH2AX and impairs the classical non-homologous end joining (c-NHEJ), affecting both V(D)J recombination and CSR. Surprisingly, Usp22 depletion causes defects in CSR to various Ig isotypes, but not IgA. We further demonstrate that IgG CSR primarily relies on c-NHEJ, whereas CSR to IgA is more reliant on the alternative end joining pathway, indicating that CSR to different isotypes involves distinct DNA repair pathways. Hence, Usp22 is the first deubiquitinase reported to regulate both V(D)J recombination and CSR in vivo by facilitating c-NHEJ.

The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity.

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.

Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching.

Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.

The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation.

Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR.

Fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is an alloimmune disorder resulting from platelet opsonization by maternal antibodies that destroy fetal platelets. The major risk of FNAIT is severe bleeding, particularly intracranial hemorrhage. Miscarriage has also been reported but the incidence requires further study. Analogous to adult autoimmune thrombocytopenia (ITP), the major target antigen in FNAIT is the platelet membrane glycoprotein (GP)IIbIIIa. FNAIT caused by antibodies against platelet GPIbα or other antigens has also been reported, but the reported incidence of the anti-GPIbα-mediated FNAIT is far lower than in ITP. To date, the maternal immune response to fetal platelet antigens is still not well understood and it is unclear why bleeding is more severe in FNAIT than in ITP. In this review, we introduce the pathogenesis of FNAIT, particularly those new discoveries from animal models, and discuss possible improvements for the diagnosis, therapy, and prevention of this devastating disease.

Hematopoietic LTßR deficiency results in skewed T cell cytokine profiles during a mucosal viral infection.

The lymphotoxin signaling pathway plays an important role in the homeostasis and function of peripheral and mucosal dendritic cells, and dendritic cell-intrinsic lymphotoxin ß receptor expression is required for optimal responses to opportunistic intestinal bacteria. However, it is unknown whether dendritic cell-intrinsic lymphotoxin ß receptor signaling is required for responses to intestinal viral infections. We explored this question by orally administrating murine rotavirus to chimeric mice that lack lymphotoxin ß receptor signaling in the myeloid compartment but retain lymphoid tissues. We found that although clearance of rotavirus was unimpaired in the lymphotoxin ß receptor(-/-) → wild-type chimeric mice compared with wild-type → wild-type chimeric mice, IFN-γ-producing CD8(+) and CD4(+) T cells were significantly increased in the small intestinal lamina propria of lymphotoxin ß receptor(-/-) → wild-type chimeric mice. In contrast, IL-17-producing CD4(+) T cells were reduced in lymphotoxin ß receptor(-/-) → wild-type chimeric mice in the steady state, and this reduction persisted after rotavirus inoculation. In spite of this altered cytokine profile in the small intestinal lamina propria of lymphotoxin ß receptor(-/-) → wild-type chimeric mice, the local production of rotavirus-specific IgA was unperturbed. Collectively, our results demonstrate that lymphotoxin ß receptor signaling in radio-sensitive myeloid cells regulates the balance of IFN-γ and IL-17 cytokine production within the small intestinal lamina propria; however, these perturbations do not affect mucosal antiviral IgA responses.

Maternal anti-platelet ß3 integrins impair angiogenesis and cause intracranial hemorrhage.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against ß3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-ß3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-ß3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-ß3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-ß3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

Platelets and platelet alloantigens: Lessons from human patients and animal models of fetal and neonatal alloimmune thrombocytopenia.

Platelets play critical roles in hemostasis and thrombosis. Emerging evidence indicates that they are versatile cells and also involved in many other physiological processes and disease states. Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life threatening bleeding disorder caused by fetal platelet destruction by maternal alloantibodies developed during pregnancy. Gene polymorphisms cause platelet surface protein incompatibilities between mother and fetus, and ultimately lead to maternal alloimmunization. FNAIT is the most common cause of intracranial hemorrhage in full-term infants and can also lead to intrauterine growth retardation and miscarriage. Proper diagnosis, prevention and treatment of FNAIT is challenging due to insufficient knowledge of the disease and a lack of routine screening as well as its frequent occurrence in first pregnancies. Given the ethical difficulties in performing basic research on human fetuses and neonates, animal models are essential to improve our understanding of the pathogenesis and treatment of FNAIT. The aim of this review is to provide an overview on platelets, hemostasis and thrombocytopenia with a focus on the advancements made in FNAIT by utilizing animal models.

AID and caspase 8 shape the germinal center response through apoptosis.

Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.

Co-stimulation with LPS or Poly I:C markedly enhances the anti-platelet immune response and severity of fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies against fetal/neonatal platelets. FNAIT is also linked with miscarriages, although the incidence and mechanisms of fetal death have not been well studied. IntegrinαIIbß3 (GPIIbIIIa) and the GPIbα complex are major glycoproteins expressed on platelets and are also major antigens targeted in autoimmune thrombocytopenia (ITP), but reported cases of anti-GPIb-mediated FNAIT are rare. Bacterial and viral infections have been causally linked with the pathogenesis of immune-mediated thrombocytopenia (ITP); however, it is unknown whether these infections contribute to the severity of FNAIT. Here, immune responses against platelet antigens were examined by transfusing wild-type (WT) mouse platelets into ß3-/- or GPIbα-/- mice. To mimic bacterial or viral infections, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) were injected intraperitoneally following platelet transfusions. The FNAIT model was established by breeding the immunised female mice with WT male mice. We demonstrated for the first time that the platelet GPIbα has lower immunogenicity compared to ß3 integrin. Interestingly, co-stimulation with LPS or Poly I:C markedly enhanced the immune response against platelet GPIbα and caused severe pathology of FNAIT (i.e. miscarriages). LPS or Poly I:C also enhanced the immune response against platelet ß3 integrin. Our data suggest that bacterial and viral infections facilitate the anti-platelet GPIbα response, which may lead to a severe non-classical FNAIT (i.e. miscarriage but not neonatal bleeding) that has not been adequately reported in humans.

AID-expressing germinal center B cells cluster normally within lymph node follicles in the absence of FDC-M1+ CD35+ follicular dendritic cells but dissipate prematurely.

Upon activation with T-dependent Ag, B cells enter germinal centers (GC) and upregulate activation-induced deaminase (AID). AID(+) GC B cells then undergo class-switch recombination and somatic hypermutation. Follicular dendritic cells (FDC) are stromal cells that underpin GC and require constitutive signaling through the lymphotoxin (LT) ß receptor to be maintained in a fully mature, differentiated state. Although it was shown that FDC can be dispensable for the generation of affinity-matured Ab, in the absence of FDC it is unclear where AID expression occurs. In a mouse model that lacks mature FDC, as well as other LT-sensitive cells, we show that clusters of AID(+)PNA(+)GL7(+) Ag-specific GC B cells form within the B cell follicles of draining lymph nodes, suggesting that FDC are not strictly required for GC formation. However, later in the primary response, FDC-less GC dissipated prematurely, correlating with impaired affinity maturation. We examined whether GC dissipation was due to a lack of FDC or other LTß receptor-dependent accessory cells and found that, in response to nonreplicating protein Ag, FDC proved to be more critical for long-term GC maintenance. Our study provides a spatial-temporal analysis of Ag-specific B cell activation and AID expression in the context of a peripheral lymph node that lacks FDC-M1(+) CD35(+) FDC and other LT-sensitive cell types, and reveals that FDC are not strictly required for the induction of AID within an organized GC-like environment.