Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Lymphocyte migration regulation related proteins in urine exosomes may serve as a potential biomarker for lung cancer diagnosis.

BACKGROUND: The migration of lymphocytes shares many similarities in mode and mechanism with the metastasis of lung cancer tumor cells. But changes in the expression of lymphocyte migration regulation related proteins in urine exosomes remain unclear. This study is to investigate the expression changes of lymphocyte migration regulation related proteins in urine exosomes of lung cancer patients, and further verify their correlation with the development and progression of lung cancer. METHODS: Urine exosomes were collected from lung cancer patients and healthy people aged 15-79 years. Mass spectrometry was used to screen and explore the expression changes of lymphocyte migration regulation related proteins in healthy people of different ages. Enzyme-linked immunosorbent assay and western blotting were used to detect the expression changes of lymphocyte migration regulation related proteins in lung cancer patients. RESULTS: Analyzing the data of urine exosome proteomics, a total of 12 lymphocyte related proteins were identified, 5 of which were lymphocyte migration regulation related proteins. Among these proteins, WASL and STK10 proteins showed a gradual decrease in expression with age, and WNK1 protein showed a gradual increase. Lung cancer patients had reduced expression of WASL and increased expression of STK10 and WNK1 proteins in urine exosomes compared to normal people. Urine exosome WASL, STK10, and WNK1 were diagnosed with lung cancer, with a combined AUC of 0.760. CONCLUSIONS: Lymphocyte migration regulation related proteins were differentially expressed in the urine exosome of lung cancer patients, and WASL, STK10 and WNK1 may serve as potential biomarkers for lung cancer diagnosis.

Chemokines and other mediators in the development and functional organization of lymph nodes.

Secondary lymphoid organs like lymph nodes (LNs) are the main inductive sites for adaptive immune responses. Lymphocytes are constantly entering LNs, scanning the environment for their cognate antigen and get replenished by incoming cells after a certain period of time. As only a minor percentage of lymphocytes recognizes cognate antigen, this mechanism of permanent recirculation ensures fast and effective immune responses when necessary. Thus, homing, positioning, and activation as well as egress require precise regulation within LNs. In this review we discuss the mediators, including chemokines, cytokines, growth factors, and others that are involved in the formation of the LN anlage and subsequent functional organization of LNs. We highlight very recent findings in the fields of LN development, steady-state migration in LNs, and the intranodal processes during an adaptive immune response.

Deoxycholic acid enhancement of lymphocyte migration through direct interaction with the intestinal vascular endothelium.

BACKGROUND AND AIM: The small intestine plays a central role in gut immunity, and enhanced lymphocyte migration is involved in the pathophysiology of various enteropathy. Bile acid (BA) is closely related to lipid metabolism and gut microbiota and essential for gut homeostasis. However, the effects of BA on gut immunity have not been studied in detail, especially on the small intestine and lymphocyte migration. Therefore, we aimed to investigate the effect of BA on small intestinal lymphocyte microcirculation. METHODS: The effect of deoxycholic acid (DCA), taurocholic acid (tCA), or cholic acid (CA) on the indomethacin (IND)-induced small intestinal enteropathy in mice was investigated. Lymphocyte movements were evaluated after exposure to BA using intravital microscopy. The effects of BA on surface expression of adhesion molecules on the vascular endothelium and lymphocytes through BA receptors were examined in vitro. RESULTS: IND-induced small intestinal enteropathy was histologically aggravated by DCA treatment alone. The expression of adhesion molecules ICAM-1 and VCAM-1 was significantly enhanced by DCA. Exposure to DCA increased lymphocyte adhesion in the microvessels of the ileum, which was partially blocked by anti-α4ß1 integrin antibody in vivo. The expression of ICAM-1 and VCAM-1 was significantly enhanced by DCA in vitro, which was partially suppressed by the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist. The S1PR2 antagonist significantly ameliorated IND-induced and DCA-exaggerated small intestinal injury. CONCLUSION: DCA exacerbated IND-induced small intestinal enteropathy. DCA directly acts on the vascular endothelium and enhances the expression levels of adhesion molecules partially via S1PR2, leading to enhanced small intestinal lymphocyte migration.

Dynamic Imaging of IEL-IEC Co-Cultures Allows for Quantification of CD103-Dependent T Cell Migration.

Intraepithelial lymphocytes (IEL) are widely distributed within the small intestinal epithelial cell (IEC) layer and represent one of the largest T cell pools of the body. While implicated in the pathogenesis of intestinal inflammation, detailed insight especially into the cellular cross-talk between IELs and IECs is largely missing in part due to lacking methodologies to monitor this interaction. To overcome this shortcoming, we employed and validated a murine IEL-IEC (organoids) ex vivo co-culture model system. Using livecell imaging we established a protocol to visualize and quantify the spatio-temporal migratory behavior of IELs within organoids over time. Applying this methodology, we found that IELs lacking CD103 (i.e., integrin alpha E, ITGAE) surface expression usually functioning as a retention receptor for IELs through binding to E-cadherin (CD324) expressing IECs displayed aberrant mobility and migration patterns. Specifically, CD103 deficiency affected the ability of IELs to migrate and reduced their speed during crawling within organoids. In summary, we report a new technology to monitor and quantitatively assess especially migratory characteristics of IELs communicating with IEC ex vivo. This approach is hence readily applicable to study the effects of targeted therapeutic interventions on IEL-IEC cross-talk.

High-resolution imaging of living gut mucosa: lymphocyte clusters beneath intestinal M cells are highly dynamic structures.

In the Peyer's patches of the small intestine, specialized epithelial cells, the membranous (M) cells, sample antigenic matter from the gut lumen and bring it into contact with cells of the immune system, which are then capable of initiating specific immune reactions. Using autofluorescence 2-photon (A2P) microscopy, we imaged living intestinal mucosa at a 0.5-µm resolution. We identified individual M cells without the aid of a marker and in vivo analyzed their sampling function over hours. Time-lapse recordings revealed that lymphocytes associated with M cells display a remarkable degree of motility with average speed rates of 8.2 µm/min, to form new M cell-associated lymphocyte clusters within less than 15 min. The lymphocytes drastically deform the M cells' cytoplasm and laterally move from one lymphocyte cluster to the next. This implies that the micro-compartment beneath M cells is a highly efficient container to bring potentially harmful antigens into contact with large numbers of immunocompetent cells. Our setup opens a new window for high-resolution 3D imaging of functional processes occurring in lymphoid and mucosal tissues.

Ephrin-Eph signaling usage by a variety of viruses.

Ephrin-Eph signaling is a receptor tyrosine kinase signaling pathway involved in a variety of cellular mechanisms, of which many are related to the adhesion or migration of cells. Both the Eph receptor and ephrin ligand are abundantly present on a wide variety of cell types, and strongly evolutionary conserved. This review provides an overview of how 18 genetically diverse viruses utilize the Eph receptor (Eph), ephrin ligand (ephrin) or ephrin-Eph signaling to their advantage in their viral life cycle. Both Ephs and ephrins have been shown to serve as entry receptors for a variety of viruses, via both membrane fusion and endocytosis. Ephs and ephrins are also involved in viral transmission by vectors, associated with viral replication or persistence and lastly to neurological damage caused by viral infection. Although therapeutic opportunities targeting Ephs or ephrins do not seem feasible yet, the current research does propose two models for the viral usage of ephrin-Eph signaling. Firstly, the viral entry model, in which membrane molecules are used for viral entry, leading to cells being used for replication or as a transporter. Secondly, the advantageous expression ephrin-Eph signaling model, where viruses adapt the expression of Ephs or ephrins to change cell-cell interaction to their advantage. These models can guide future research questions on the usage of Ephs or ephrins by viruses and therapeutic opportunities.

Amelioration of colitis through blocking lymphocytes entry to Peyer's patches by sphingosine-1-phosphate lyase inhibitor.

BACKGROUND AND AIM: Sphingosine-1-phosphate (S1P) receptor 1, a therapeutic target of the S1P1 agonist FTY720, plays a crucial role in lymphocyte migration and is expressed in several cells including naïve T lymphocytes and endothelial cells. 2-Acetyl-4-tetrahydroxybutyl imidazole (THI), an inhibitor of S1P lyase, exhibits immunomodulatory activity through increasing the S1P concentration in the secondary lymphoid organs, but its effects on colitis remain unclear. This study aimed to clarify how THI affects colitis and migration of naïve T lymphocytes in Peyer's patches (PPs). METHODS: The effect of THI on gut immunity was investigated by analyzing the dextran sulfate sodium (DSS)-induced murine colitis model, lymphocyte components in thoracic duct lymphocytes (TDLs), and microscopic movement of TDLs in PPs. RESULTS: 2-Acetyl-4-tetrahydroxybutyl imidazole ameliorated DSS-induced colitis histologically by causing a significant decrease in colonic lymphocyte infiltration and expression of mucosal pro-inflammatory cytokines. THI suppressed the inflow of naïve T lymphocytes into the thoracic duct. Microscopic observation of PPs in control animals revealed that many TDLs egressed to the stroma and migrated to lymph capillaries after attaching to the high endothelial venules (HEVs). THI or FTY720 treatment in recipient animals blocked lymphocyte egression from the HEVs to the stroma. CONCLUSIONS: This is the first study to clarify the ameliorating effects of THI on DSS-induced colitis. Microscopic observations demonstrated the involvement of HEVs in the egression of S1P-dependent gut-tropic T lymphocytes to lymph capillaries. This S1P lyase inhibitor might become a novel immunosuppressant for inflammatory bowel disease therapy by blocking infiltration of lymphocytes through HEVs into the stroma in PPs.

Clathrin regulates lymphocyte migration by driving actin accumulation at the cellular leading edge.

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.

Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis.

Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune-mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1-deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)35-55 -induced EAE, a model of human MS. The lack of PLD1 reduced chemokine-mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1-deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1-dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.

Selective Forms of Therapy in the Treatment of Inflammatory Bowel Diseases.

Selective interference with the functioning of the immune system consisting of the selective blockade of pro-inflammatory factors is a modern, promising, and developing strategy for the treatment of diseases resulting from dysregulation of the immune system, including inflammatory bowel disease. Inhibition of the TNF alpha pathway, group 12/23 cytokines, and lymphocyte migration is used in the treatment of severe or moderate ulcerative colitis and Crohn's disease. Intracellular signal transduction by influencing the phosphorylation of SAT (signal transducer and activator of transcription) proteins remains in clinical trials.

CTLs From Patients With Atherosclerosis Show Elevated Adhesiveness and Distinct Integrin Expression Patterns on 2D Substrates.

Atherosclerosis is the major cause of cardiovascular disease that is characterized by plaque formation in the blood vessel wall. Atherosclerotic plaques represent sites of chronic inflammation with diverse cell content that is shifted toward the prevalence of cytotoxic T-lymphocytes (CTLs) upon plaque progression. The studies of CTL recruitment to atherosclerotic plaques require adequate in vitro models accounting for CTL interactions with chemokine-ligands and extracellular matrix fibers via surface chemokine receptors and integrins. Here we applied such a model by investigating CTL adhesion and migration on six types of coated surfaces. We assessed adhesion and motility metrics, the expression of chemokine receptors, and integrins in CTLs of patients with atherosclerosis and healthy donors. Using fibronectin, platelet-poor plasma from patients with atherosclerosis, and conditioned medium from atherosclerotic plaques we revealed the role of substrate in CTL adhesiveness: fibronectin alone and fibronectin combined with platelet-poor plasma and conditioned medium elevated the CTL adhesiveness - in patients the elevation was significantly higher than in healthy donors (p = 0.02, mixed 2-way ANOVA model). This was in line with our finding that the expression levels of integrin-coding mRNAs were elevated in the presence of fibronectin (p < 0.05) and ITGB1, ITGA1, and ITGA4 were specifically upregulated in patients compared to healthy donors (p < 0.01). Our experimental model did not affect the expression levels of mRNAs CCR4, CCR5, and CX3CR1 coding the chemokine receptors that drive T-lymphocyte migration to plaques. Thus, we demonstrated the substrate-dependence of integrin expression and discriminated CTLs from patients and healthy donors by adhesion parameters and integrin expression levels.

Cell polarity regulators, multifunctional organizers of lymphocyte activation and function.

Cell polarity regulators are ubiquitous, evolutionary conserved multifunctional proteins. They contain a variety of protein-protein interaction domains endowing them the capacity to interact with cytoskeleton structures, membrane components and multiple regulatory proteins. In this way, they act in complexes and are pivotal for cell growth and differentiation, tissue formation, stability and turnover, cell migration, wound healing, and others. Hence some of these proteins are tumor suppressors. These cellular processes rely on the establishment of cell polarity characterized by the asymmetric localization of proteins, RNAs, membrane domains, or organelles that together condition cell shape and function. Whether apparently stable, as in epithelia or neurons, or very dynamic, as in immune cells, cell polarity is an active process. It involves cytoskeleton reorganization and targeted intracellular traffic, and results in cellular events such as protein synthesis, secretion and assembly taking place at defined cell poles. Multiple polarity regulators orchestrate these processes. Immune cells are particularly versatile in rapidly polarizing and assuming different shapes, so to swiftly adopt specialized behaviors and functions. Polarity regulators act in various ways in different immune cell types and at their distinct differentiation states. Here we review how cell polarity regulators control different processes and functions along T lymphocyte physiology, including cell migration through different tissues, immunological synapse formation and effector functions.

Pathobiont-responsive Th17 cells in gut-mouth axis provoke inflammatory oral disease and are modulated by intestinal microbiome.

Host immune response via Th17 cells against oral pathobionts is a key mediator in periodontitis development. However, where and how the Th17-type immune response is induced during the development of periodontitis is not well understood. Here, we demonstrate that gut translocation of the oral pathobiont Porphyromonas gingivalis (Pg) exacerbates oral pathobiont-induced periodontitis with enhanced Th17 cell differentiation. The oral pathobiont-responsive Th17 cells are differentiated in Peyer's patches and translocated systemically in the peripheral immune tissues. They are also capable of migrating to and accumulating in the mouth upon oral infection. Development of periodontitis via the oral pathobiont-responsive Th17 cells is regulated by the intestinal microbiome, and altering the intestinal microbiome composition with antibiotics affects the development of periodontitis. Our study highlights that pathobiont-responsive Th17 cells in the gut-mouth axis and the intestinal microbiome work together to provoke inflammatory oral diseases, including periodontitis.

A Requirement of Protein Geranylgeranylation for Chemokine Receptor Signaling and Th17 Cell Function in an Animal Model of Multiple Sclerosis.

Precisely controlled lymphocyte migration is critically required for immune surveillance and successful immune responses. Lymphocyte migration is strictly regulated by chemokines and chemokine receptors. Here we show that protein geranylgeranylation, a form of post-translational protein lipid modification, is required for chemokine receptor-proximal signaling. Mature thymocytes deficient for protein geranylgeranylation are impaired for thymus egress. Circulating mature T cells lacking protein geranylgeranylation fail to home to secondary lymphoid organs or to transmigrate in response to chemokines in vitro. Mechanistically, protein geranylgeranylation modifies the γ-subunits of the heterotrimeric small GTPases that are essential for chemokine receptor signaling. In addition, protein geranylgeranylation also promotes the differentiation of IL-17-producing T helper cells while inhibiting the differentiation of Foxp3+ regulatory T cells. Finally, mice with T cell lineage-specific deficiency of protein geranylgeranylation are resistant to experimental autoimmune encephalomyelitis induction. This study elucidated a critical role of protein geranylgeranylation in regulating T lymphocyte migration and function.

Corrigendum: A Requirement of Protein Geranylgeranylation for Chemokine Receptor Signaling and Th17 Cell Function in an Animal Model of Multiple Sclerosis.

[This corrects the article DOI: 10.3389/fimmu.2021.641188.].

Enhanced Migratory Capacity of T Lymphocytes in Severe Chagasic Patients Is Correlated With VLA-4 and TNF-α Expression.

Trypanosoma cruzi infection in humans leads to progression to chronic chagasic myocarditis (CCM) in 30% of infected individuals, paralleling T cell inflammatory infiltrates in the heart tissue. T-cell trafficking into the hearts of CCM patients may be modulated by in situ expression of chemotactic or haptotactic molecules, as the chemokine CXCL12, the cytokine tumor necrosis factor-alpha (TNF-α), and extracellular matrix proteins (ECM), such as fibronectin. Herein we evaluated the expression of fibronectin, CXCL12, and TNF-α in the myocardial tissue of T. cruzi seropositive (asymptomatic or with CCM), as well as seronegative individuals as healthy controls. Hearts from CCM patients exhibited enhanced expression of these three molecules. CXCL12 and TNF-α serum levels were also increased in CCM individuals. We then evaluated T lymphocytes from chronic chagasic patients by cytofluorometry, in terms of membrane expression levels of molecules involved in cell activation and cell migration, respectively, HLA-DR and the VLA-4 (very late antigen-4, being one integrin-type fibronectin receptor). Indeed, the expression of HLA-DR and VLA-4 was enhanced on T lymphocytes from chagasic patients, especially in the CCM group. To further approach the dynamics of T cell migratory events, we performed fibronectin-, TNF-α-, and CXCL12-driven migration. Peripheral blood mononuclear cells (PBMCs) and T cells from CCM patients presented an ex vivo enhanced migratory capacity driven by fibronectin alone when this ECM protein was placed in the membrane of transwell migration chambers. When TNF-α was previously placed upon fibronectin, we observed a further and significant increase in the migratory response of both PBMCs and T lymphocytes. Overall, these data suggest the existence in patients with chronic Chagas disease of a cardiac inflammatory infiltrate vector that promotes the recruitment and accumulation of activated T cells, driven in part by enhanced tissue expression of fibronectin and TNF-α, as well as the respective corresponding VLA-4 and TNF receptors.

Targeting Autophagy Facilitates T Lymphocyte Migration by Inducing the Expression of CXCL10 in Gastric Cancer Cell Lines.

Autophagy is a type of cellular catabolic degradation process that occurs in response to nutrient starvation or metabolic stress, and is a valuable resource for highly proliferating cancer cells. Autophagy also facilitates the resistance of cancer cells to antitumor therapies. However, the involvement of autophagy in regulating CXCL10 expression in gastric cancer (GC) cells and T lymphocyte migration remains unclear. In this study, we aimed to investigate the effect of autophagy inhibition on CXCL10 expression and T lymphocyte infiltration in GC and elucidate the underlying mechanism. Analysis of public databases revealed a positive correlation between CXCL10 expression and both prognosis of patients with GC and the expression profile of T lymphocyte markers in the GCs. Chemotaxis and spheroid infiltration assays revealed that CXCL10 induced T lymphocyte migration and infiltration into GC spheroids, an in vitro three-dimensional cell culture model. In addition, in vitro autophagy inhibition in GC cells increased CXCL10 expression under both normal and hypoxic culture conditions. Further investigation on the underlying mechanism showed that in vitro autophagy inhibition suppressed the JNK signaling pathway and further enhanced CXCL10 expression in GC cells. Collectively, our results provide novel insights for understanding the role of autophagy in regulation of intra-tumor immunity.

Tumor inhibition or tumor promotion? The duplicity of CXCR3 in cancer.

Tumor tissue includes cancer cells and normal stromal cells such as vascular endothelial cells, connective tissue cells (cancer associated fibroblast, mesenchymal stem cell), and immune cells (tumor-infiltrating lymphocytes or TIL, dendritic cells, eosinophils, basophils, mast cells, tumor-associated macrophages or TAM, myeloid-derived suppressor cells or MDSC). Anti-tumor activity is mainly mediated by infiltration of NK cells, Th1 and CD8+ T cells, and correlates with expression of NK cell and T cell attracting chemokines. Nevertheless, cancer cells hijack tissue homeostasis through secretion of cytokines and chemokines that mediate not only the induction of an inflamed status that supports cancer cell survival and growth, but also the recruitment and/or activation of immune suppressive cells. CXCL9, CXCL10, and CXCL11 are known for their tumor-inhibiting properties, but their overexpression in several hematologic and solid tumors correlates with disease severity, suggesting a role in tumor promotion. The dichotomous nature of CXCR3 ligands activity mainly depends on several molecular mechanisms induced by cancer cells themselves able to divert immune responses and to alter the whole local environment. A deep understanding of the nature of such phenomenon may provide a rationale to build up a CXCR3/ligand axis targeting strategy. In this review, we will discuss the role of CXCR3 in cancer progression and in regulation of anti-tumor immune response and immunotherapy.

Effects of sleep on the splenic milieu in mice and the T cell receptor repertoire recruited into a T cell dependent B cell response.

Sleep is known to improve immune function ranging from cell distribution in the naïve state to elevated antibody titers after an immune challenge. The underlying mechanisms still remain unclear, partially because most studies have focused on the analysis of blood only. Hence, we investigated the effects of sleep within the spleen in female C57BL/6J mice with normal sleep compared to short-term sleep-deprived animals both in the naïve state and after an antigen challenge. Lack of sleep decreased the expression of genes associated with immune cell recruitment into and antigen presentation within the spleen both in the naïve state and during a T cell dependent B cell response directed against sheep red blood cells (SRBC). However, neither T cell proliferation nor formation of SRBC-specific antibodies was affected. In addition, the T cell receptor repertoire recruited into the immune response within seven days was not influenced by sleep deprivation. Thus, sleep modulated the molecular milieu within the spleen whereas we could not detect corresponding changes in the primary immune response against SRBC. Further studies will show whether sleep influences the secondary immune response against SRBC or the development of the B cell receptor repertoire, and how this can be compared to other antigens.