BACKGROUND

Experimental atherosclerosis is characterized by the formation of tertiary lymphoid structures (TLOs) within the adventitial layer, which involves the chemokine-expressing aortic smooth muscle cells (SMCs). TLOs have also been described around human atherothrombotic arteries but the mechanisms of their formation remain poorly investigated. Herein, we tested whether human vascular SMCs play the role of chemokine-expressing cells that would trigger the formation of TLOs in atherothrombotic arteries.

RESULTS

We first characterized, by flow cytometry and immunofluorescence analysis, the prevalence and cell composition of TLOs in human abdominal aneurysms of the aorta (AAAs), an evolutive form of atherothrombosis. Chemotaxis experiments revealed that the conditioned medium from AAA tissues recruited significantly more B and T lymphocytes than the conditioned medium from control (N-AAA) tissues. This was associated with an increase in the concentration of CXCL13, CXCL16, CCL19, CCL20, and CCL21 chemokines in the conditioned medium from AAA tissues. Immunofluorescence analysis of AAA cryosections revealed that α-SMA-positive SMCs were the main contributors to the chemokine production. These results were confirmed by RT-qPCR assays where we found that primary vascular SMCs from AAA tissues expressed significantly more chemokines than SMCs from N-AAA. Finally, in vitro experiments demonstrated that the inflammatory cytokines found to be increased in the conditioned medium from AAA were able to trigger the production of chemokines by primary SMCs.

CONCLUSIONS

Together, these results suggest that human vascular SMCs in atherothrombotic arteries, in response to inflammatory signals, are converted into chemokine-expressing cells that trigger the recruitment of immune cells and the formation of aortic TLOs.

Peyer's patches (PPs) are the major mucosal immune-inductive site, and germinal centers (GCs) in PPs determine the quality of the Abs produced. PP GCs are continuously induced by the gut microbiota, and their maintenance contributes to the induction of strong IgA responses to Ags. In this study, we investigated the role of formyl peptide receptor (FPR)-mediated signaling in the maintenance of PP GCs, because FPRs recognize the microbiota and initiate an innate immune response by chemotaxis. We found that follicular dendritic cells (FDCs), a key organizer of B cell follicles and GCs in mucosal immunity, express Fpr2. Additionally, Fpr2-mediated signaling in PP FDCs promoted Cxcl13 and B cell activating factor expression, as well as B cell proliferation and activation. Therefore, we suggest that Fpr2-mediated signaling in FDCs plays a key role in GC maintenance in PPs and results in an Ag-specific IgA response in the gut mucosal immune compartment.

Bacterial, parasitic, and viral infections are well-known causes of lymphoid tissue disorganization, although the factors, both host and/or pathogen derived, that mediate these changes are largely unknown. Ehrlichia muris infection in mice causes a loss of germinal center (GC) B cells that is accompanied by the generation of extrafollicular T-bet⁺ CD11c⁺ plasmablasts and IgM memory B cells. We addressed a possible role for TNF-α in this process because this cytokine has been shown to regulate GC development. Ablation of TNF-α during infection resulted in an 8-fold expansion of GL7⁺ CD38^lo CD95⁺ GC B cells, and a 2.5- and 5-fold expansion of CD138⁺ plasmablasts and T-bet⁺ memory cells, respectively. These changes were accompanied by a reduction in splenomegaly, more organized T and B cell zones, and an improved response to Ag challenge. CXCL13, the ligand for CXCR5, was detected at 6-fold higher levels following infection but was much reduced following TNF-α ablation, suggesting that CXCL13 dysregulation also contributes to loss of lymphoid tissue organization. T follicular helper cells, which also underwent expansion in infected TNF-α--deficient mice, may also have contributed to the expansion of T-bet⁺ B cells, as the latter are known to require T cell help. Our findings contrast with previously described roles for TNF-α in GCs and reveal how host-pathogen interactions can induce profound changes in cytokine and chemokine production that can alter lymphoid tissue organization, GC B cell development, and extrafollicular T-bet⁺ B cell generation.

Defective adaptive humoral immune responses to mucosal immunogens, but intact systemic responses, are increasingly recognized in patients with IgA nephropathy (IgAN). Reduced expression of IgA+, J chain+ cells in the gut lamina propria, with collateral increases in these cells in the marrow, is also documented. Thus, there seems to be a derangement in a 'mucosa-marrow axis' in IgAN patients. Recent evidence indicates that chemokines regulate the localization of B cells and their progeny into respiratory and intestinal lamina propria, and into other lymphoid organs as well. Particularly, secretory epithelial cells express the chemokine CCL28, whereas small bowel cells uniquely express CCL25. Extramucosal sites preferentially express CXCL12, CXCL13 and/or CXCL16. Reciprocally, plasmablasts committed to IgA synthesis ubiquitously express the receptor (CCR10) for CCL28, and a subset also express the receptor (CCR9) for CCL25; neither of these is present on cells committed to IgG or IgM synthesis. Herein, the potential contributions of virally induced innate responses to defective mucosal immunity and overproduction of oligomeric IgA in the marrow and tonsils will be reviewed, particularly with respect to the influence that viral infection exerts upon the expression of selected chemokine and receptor pairs. The ramifications for pathogenesis of IgAN will be considered.

We have previously reported that the accumulation of IL-17-producing cells could mediate tumor protective immunity by promoting the migration of NK cells, T cells and dendritic cells in esophageal squamous cell carcinoma (ESCC) patients. However, there were no reports concerning the effect of IL-17A on tumor infiltrating B cells. In this study, we investigated the accumulation of CD20+ B cells in the ESCC tumor nests and further addressed the effect of IL-17A on the migration and cytotoxicity of B cells. There was positive correlation between the levels of CD20+ B cells and IL-17+ cells. IL-17A could promote the ESCC tumor cells to produce more chemokines CCL2, CCL20 and CXCL13, which were associated with the migration of B cells. In addition, IL-17A enhanced the IgG-mediated antibody and complement mediated cytotoxicity of B cells against tumor cells. IL-17A-stimulated B cells gained more effective direct killing capability through enhanced expression of Granzyme B and FasL. The effect of IL-17A on the migration and cytotoxicity of B cells was IL-17A pathway dependent, which could be inhibited by IL-17A inhibitor. This study provides further understanding of the roles of IL-17A in humoral response, which may contribute to the development of novel tumor immunotherapy strategy.

A long-standing paradigm in B cell immunology is that effective somatic hypermutation and affinity maturation require cycling between the dark zone and light zone of the germinal center. The cyclic re-entry hypothesis was first proposed based on considerations of the efficiency of affinity maturation using an ordinary differential equations model for B cell population dynamics. More recently, two-photon microscopy studies of B cell motility within lymph nodes in situ have revealed the complex migration patterns of B lymphocytes both in the preactivation follicle and post-activation germinal center. There is strong evidence that chemokines secreted by stromal cells and the regulation of cognate G-protein coupled receptors by these chemokines are necessary for the observed spatial cell distributions. For example, the distribution of B cells within the light and dark zones of the germinal center appears to be determined by the reciprocal interaction between the level of the CXCR4 and CXCR5 receptors and the spatial distribution of their respective chemokines CXCL12 and CXCL13. Computer simulations of individual-based models have been used to study the complex biophysical and mechanistic processes at the individual cell level, but such simulations can be challenging to parameterize and analyze. In contrast, ordinary differential equations are more tractable, but traditional compartment model formalizations ignore the spatial chemokine distribution that drives B cell redistribution. Motivated by the desire to understand the motility patterns observed in an individual-based simulation of B cell migration in the lymph node, we propose and analyze the dynamics of an ordinary differential equation model incorporating explicit chemokine spatial distributions. While there is experimental evidence that B cell migration patterns in the germinal center are driven by extrinsically regulated differentiation programs, the model shows, perhaps surprisingly, that feedback from receptor down-regulation induced by external chemokine fields can give rise to spontaneous interzonal and intrazonal oscillations in the absence of any extrinsic regulation. While the extent to which such simple feedback mechanisms contributes to B cell migration patterns in the germinal center is unknown, the model provides an alternative hypothesis for how complex B cell migration patterns might arise from very simple mechanisms.

Chemokines are a family of cytokines inducing cell migration and inflammation. Recent reports have implicated the roles of chemokines in cell differentiation. However, little is known about the functional roles of chemokines in adipocytes. Here, we explored gene expression levels of chemokines and chemokine receptors during adipogenic differentiation. We have found that two chemokines, chemokine (C-X-C motif) ligand 3 (CXCL3) and CXCL13, as well as CXC chemokine receptor 2 (CXCR2), a CXCL3 receptor, are highly expressed in mature adipocytes. When 3T3-L1 cells and ST2 cells were induced to differentiate, both the number of lipid droplets and the expression levels of adipogenic markers were significantly promoted by the addition of CXCL3, but not CXCL13. Conversely, gene knockdown of either CXCL3 or CXCR2 by specific siRNA effectively inhibited the course of adipogenic differentiation. CXCL3 treatment of 3T3-L1 cells significantly induced the phosphorylation of ERK and c-jun N-terminal kinase (JNK). Furthermore, CXCL3-induced CCAAT-enhancer binding protein (C/EBP)β and δ expression was suppressed by both ERK and JNK-specific inhibitors. Furthermore, chromatin immunoprecipitation assay revealed functional binding of PPARγ2 within the cxcl3 promoter region. Taken together, these results have indicated that CXCL3 is a novel adipokine that facilitates adipogenesis in an autocrine and/or a paracrine manner through induction of c/ebpb and c/ebpd.

<AbstractText>SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres' formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of <em>CXCL13</em> correlate with germinal centres' activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of <em>CXCL13</em> as a biomarker of SG pathology in two independent SS cohorts.</AbstractText><AbstractText>109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects' (n = 19) sera were used as control.</AbstractText><AbstractText><em>CXCL13</em> serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed.</AbstractText><AbstractText>Our data foster the use of <em>CXCL13</em> to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.</AbstractText>

BACKGROUND

The use of TNF-inhibitors and/or the IL-6 receptor antagonist, tocilizumab, in rheumatoid arthritis (RA) have pleiotropic effects that also involve circulating B-cells. The main goal of this study was to assess the effect of TNF-inhibitors and tocilizumab on B-cell phenotype and gene expression in RA.

METHODS

Blood samples were collected from untreated early RA (ERA) patients, established RA patients under methotrexate treatment, established RA patients before and after treatment with TNF-inhibitors and tocilizumab, and healthy donors. B-cell subpopulations were characterized by flow cytometry and B-cell gene expression was analyzed by real-time PCR on isolated B-cells. Serum levels of BAFF, CXCL13 and sCD23 were determined by ELISA.

RESULTS

The frequency of total CD19+ B cells in circulation was similar between controls and all RA groups, irrespective of treatment, but double negative (DN) IgD-CD27- memory B cells were significantly increased in ERA and established RA when compared to controls. Treatment with TNF-inhibitors and tocilizumab restored the frequency of IgD-CD27- B-cells to normal levels, but did not affect other B cell subpopulations. TACI, CD95, CD5, HLA-DR and TLR9 expression on B-cells significantly increased after treatment with either TNF-inhibitors and/ or tocilizumab, but no significant changes were observed in BAFF-R, BCMA, CD69, CD86, CXCR5, CD23, CD38 and IgM expression on B-cells when comparing baseline with post-treatment follow-ups. Alterations in B-cell gene expression of BAFF-R, TACI, TLR9, FcγRIIB, BCL-2, BLIMP-1 and β2M were found in ERA and established RA patients, but no significant differences were observed after TNF-inhibitors and tocilizumab treatment when comparing baseline and follow-ups. Serum levels of CXCL13, sCD23 and BAFF were not significantly affected by treatment with TNF-inhibitors and tocilizumab.

CONCLUSIONS

In RA patients, the use of TNF-inhibitors and/ or tocilizumab treatment affects B-cell phenotype and IgD-CD27- memory B cells in circulation, but not B-cell gene expression levels.

The interactions of CD4⁺ T cells and B cells are fundamental for the generation of protective antibody responses, as well as for the development of harmful autoimmune diseases. Recent studies of human tissues and blood samples have established a new subset of CD4⁺ B helper T cells named peripheral helper T (Tph) cells. Unlike T follicular helper (Tfh) cells, which interact with B cells within lymphoid organs, Tph cells provide help to B cells within inflamed tissues. Tph cells share many B helper-associated functions with Tfh cells and induce B cell differentiation toward antibody-producing cells. The differentiation mechanism is also partly shared between Tph and Tfh cells in humans, and both Tfh and Tph cells can be found within the same tissues, including cancer tissues. However, Tph cells display features distinct from those of Tfh cells, such as the expression of chemokine receptors associated with Tph cell localization within inflamed tissues and a low Bcl-6/Blimp1 ratio. Unlike that of Tfh cells, current evidence shows that the target of Tph cells is limited to memory B cells. In this review, we first summarize recent findings on human Tph cells and discuss how Tph and Tfh cells play shared and distinct roles in human diseases.

Keywords: CXCL13, Autoantibodies; T follicular helper cells; autoimmune diseases; peripheral helper T cells.

Intestinal mononuclear phagocytes (MPs) are composed of heterogeneous dendritic cell (DC) and macrophage subsets necessary for the initiation of immune response and control of inflammation. Although MPs in the normal intestine have been extensively studied, the heterogeneity and function of inflammatory MPs remain poorly defined. We performed phenotypical, transcriptional, and functional analyses of inflammatory MPs in infectious Salmonella colitis and identified CX3CR1⁺ MPs as the most prevalent inflammatory cell type. CX3CR1⁺ MPs were further divided into three distinct populations, namely, Nos2⁺CX3CR1^lo, Ccr7⁺CX3CR1^int (lymph migratory), and Cxcl13⁺CX3CR1^hi (mucosa resident), all of which were transcriptionally aligned with macrophages and derived from monocytes. In follow-up experiments in vivo, intestinal CX3CR1⁺ macrophages were superior to conventional DC1 (cDC1) and cDC2 in inducing Salmonella-specific mucosal IgA. We next examined spatial organization of the immune response induced by CX3CR1⁺ macrophage subsets and identified mucosa-resident Cxcl13⁺CX3CR1^hi macrophages as the antigen-presenting cells responsible for recruitment and activation of CD4⁺ T and B cells to the sites of Salmonella invasion, followed by tertiary lymphoid structure formation and the local pathogen-specific IgA response. Using mice we developed with a floxed Ccr7 allele, we showed that this local IgA response developed independently of migration of the Ccr7⁺CX3CR1^int population to the mesenteric lymph nodes and contributed to the total mucosal IgA response to infection. The differential activity of intestinal macrophage subsets in promoting mucosal IgA responses should be considered in the development of vaccines to prevent Salmonella infection and in the design of anti-inflammatory therapies aimed at modulating macrophage function in inflammatory bowel disease.

OBJECTIVE

Clinical trials of IV-rituximab have proved successful. It is unclear whether intrathecal (IT)-rituximab is more efficacious at lower doses. We examine its effects on B-cell biomarkers.

METHODS

MS patients received IT-rituximab at 3 time-points. CSF and serum samples were obtained at up to 5 time-points (days 0, 7, 14, 56 and 112). Serum and CSF BAFF and CXCL13, and CSF kappa and lambda free light chains (FLC) were measured. Flow cytometry was performed, examining effects on lymphocytes, CD3-19+ and CD3-20+ cells.

RESULTS

CSF BAFF fell following rituximab (p=0.0091 absolute values, p=0.0284 change from baseline) whilst serum BAFF increased across time-points 1-4 (p=0.0005 absolute values, p=0.0017 change from baseline). There were significant reductions in CD20+ and CD19+ cells in blood from baseline (p<0.0001) but not in CSF. CSF kappa FLC levels significantly increased (p=0.0480).

CONCLUSIONS

BAFF levels fall in CSF but increase in serum following IT-rituximab. Rituximab appears to act peripherally with dramatic decreases in peripheral CD20+ and CD19+ cells. It is likely that CSF B-cell counts were too low to enable differences to be seen. The rapid reduction in B-cells suggests rituximab has immediate effects. The profound depletion of B-cells, despite low doses of rituximab, underlines rituximab's efficacy.

Introduction: Immune checkpoint inhibitors (ICIs) have proved to be an effective treatment for up to 40% of muscle-invasive bladder cancer (MIBC), but there is still a need for better performing biomarkers allowing to improve prediction of response to ICI. Response to immunotherapy in soft-tissue sarcoma, melanoma and renal cell carcinoma have been recently linked to the presence of tertiary lymphoid structures (TLS) in the tumour. TLS are organised aggregates of T, B and dendritic cells, participating in adaptive antitumor immune response. The chemokine CXCL13 is involved in the formation of TLS, and is reported as a reliable transcriptomic marker of TLS.

Objectives: In this study, we sought to assess whether CXCL13 transcript expression can be a prognostic biomarker for ICI-treated MIBC patients and also investigated whether it can serve a biomarker of TLS in MIBC.

Methods: We analysed transcriptomic data from three publicly available MIBC cohorts and evaluated pathological slides from the TCGA-BLCA cohort for TLS presence and stage of maturation.

Results: We showed that CXCL13 was independently associated with both prolonged survival (HR = 0.8, 95% CI [0.68-0.94]) and objective response (p < 0.0001) in patients treated with ICI, at the difference of others immunological signatures. However, it was not a predictor for non-ICI-treated MIBC, suggesting a predictive effect of ICI efficacy. Finally, we validated that CXCL13 expression was correlated with tumour TLS in TCGA data set (p < 0.001), and can serve as a marker of TLS in bladder cancer.

Conclusion: These results support that CXCL13 expression, as a surrogate for tumour TLS, is a relevant candidate predictive biomarker of response to ICI for patients with advanced-stage bladder cancer.

Keywords: Bladder cancer; CXCL13; Immunotherapy; Response to immunotherapy; Tertiary lymphoid structures.

Purpose: Lung adenocarcinoma (LUAD) is the leading cause of cancer-related deaths worldwide. Although tumor cell-T cell interactions are known to play a fundamental role in promoting tumor progression, these interactions have not been explored in LUAD. Methods: The 10x genomics single-cell RNA sequencing (scRNA-seq) and gene expression data of LUAD patients were obtained from ArrayExpress, TCGA, and GEO databases. scRNA-seq data were analyzed and infiltrating tumor cells, epithelial cells, and T cells were identified in the tumor microenvironment. Differentially expressed ligand-receptor pairs were identified in tumor cells/normal epithelial cells and tumor T cells/non-tumor T cells based on corresponding scRNA-seq and gene expression data, respectively. These important interactions inside/across cancer cells and T cells in LUAD were systematically analyzed. Furthermore, a valid prognostic machine-learning model based on ligand-receptor interactions was built to predict the prognosis of LUAD patients. Flow cytometry and qRT-PCR were performed to validate the significantly differently expressed ligand-receptor pairs. Results: Overall, 39,692 cells in scRNA-seq data were included in our study after quality filtering. A total of 65 ligand-receptor pairs (17 upregulated and 48 downregulated), including LAMB1-ITGB1, CD70-CD27, and HLA-B-LILRB2, and 96 ligand-receptor pairs (41 upregulated and 55 downregulated), including CCL5-CCR5, SELPLG-ITGB2, and CXCL13-CXCR5, were identified in LUAD cancer cells and T cells, respectively. To explore the crosstalk between cancer cells and T cells, 114 ligand-receptor pairs, including 11 ligand-receptor pair genes that could significantly affect survival outcomes, were identified in our research. A machine-learning model was established to accurately predict the prognosis of LUAD patients and ITGB4, CXCR5, and MET were found to play an important role in prognosis in our model. Flow cytometry and qRT-PCR analyses indicated the reliability of our study. Conclusion: Our study revealed functionally significant interactions within and between cancer cells and T cells. We believe these observations will improve our understanding of potential mechanisms of tumor microenvironment contributions to cancer progression and help identify potential targets for immunotherapy in the future.

Keywords: Cell-to-cell interactions; Lung adenocarcinoma; Machine learning; Single-cell RNA-seq; Survival.

OBJECTIVE

B lymphocyte is the dominant infiltrating cell type in periodontitis lesions. CXCL13, produced by follicular dendritic cells, endothelial cells and fibroblasts, is crucial for B-cell trafficking. An association between chronic inflammation and lymphoid organogenesis has been reported in infection and in autoimmune responses, in which T-cell/B-cell follicles with a follicular dendritic cell network are formed. The aim of this study was to examine CXCL13 expression and follicular dendritic cell distribution in relation to B-cell infiltration in chronic inflammatory periodontal lesions.

METHODS

Fifty-eight gingival tissue biopsies from patients with periodontitis and 25 samples from subjects with gingivitis were analyzed. Gene expression for CXCL13 and for the CD21 long isoform was analyzed using the reverse transcription-polymerase chain reaction. Immunohistochemical analysis was performed using antibodies to CXCL13, CXCR5, follicular dendritic cells, CD3 and CD19 on serial cryostat sections.

RESULTS

mRNA for CXCL13 was expressed in both periodontitis and gingivitis tissues. The number of CXCL13+ cells was significantly higher in periodontitis than in gingivitis in connective tissues subjacent to the pocket epithelium and positively correlated with the number of CD19+ cells. CXCL13+ cells were distributed in B-cell-dominant areas both with and without follicular dendritic cells. Although obvious reticular networks of follicular dendritic cells were not found in periodontitis and gingivitis, the accumulation of follicular dendritic cells in B-cell-dominant areas in periodontitis was observed in some patients.

CONCLUSIONS

These findings suggested that CXCL13 and follicular dendritic cells were involved in B-cell recruitment to, and B-cell distribution in, chronic inflammatory periodontal lesions.

The random amino acid copolymer poly(Y,E,A,K)(n) (Copaxone®) is widely used in multiple sclerosis treatment and a second generation copolymer poly(Y,F,A,K)(n) with enhanced efficacy in experimental autoimmune encephalomyelitis in mice has been described. A major mechanism through which copolymers function to ameliorate disease is the generation of immunosuppressive IL-10-secreting regulatory T cells entering the CNS. In addition, the antigen presenting cell to which these copolymers bind through MHC Class II proteins may have an important role. Here, both CCL22 (a Th2 cell chemoattractant) in large amounts and CXCL13 in much smaller amounts are shown to be secreted after administration of YFAK to mice and to a smaller extent by YEAK parallel to their serum concentrations. Moreover, bone marrow-derived macrophages secrete CCL22 in vitro in response to YFAK and to higher concentrations of YEAK. Strikingly, these chemokines are also secreted into serum of MHC Class II -/- mice, indicating that an innate immune receptor on these cells also has an important role. Thus, both the innate and the adaptive immune systems are involved in the mechanism of EAE amelioration by YFAK. The enhanced ability of YFAK to stimulate the innate immune system may account for its enhanced efficacy in EAE treatment.

Differential expressions of immune molecules have been shown in the thymi with pathological results, including myasthenia gravis (MG). CD25 is an activation marker expressed on T cells. CXCL13 mediates the homing and motility of B cells in secondary lymphoid tissues. Herein, we investigated the expressions of CD25 and CXCL13 in the thymi of thymic hyperplasia patients with MG or with non-MG. A total of 34 thymic hyperplasia patients with MG (20 generalized MG (GMG) and 14 ocular MG (OMG) and six thymic hyperplasia patients without MG were enrolled and analyzed using immunohistochemical staining and real-time polymerase chain reaction for CD25 and CXCL13. Our study demonstrated a higher expression of both CD25 and CXCL13 in hyperplastic thymi with OMG or GMG compared to those with non-MG. According to the immunohistochemical results, we observed that CD25 expression was significantly lower in atrophic thymi and non-MG hyperplastic thymi, compared with that in infant thymi (P = 0.002 and 0.005, respectively). In contrast to CD25 expression, we did not observe differential expression of CXCL13 among three control groups. And a similar CD25 mRNA expression was found in real-time polymerase chain reaction (PCR) results. We observed that both hyperplastic thymi with OMG or GMG expressed significantly higher levels of CD25 than those with non-MG (P = 0.007 and 0.001, respectively). And an increase of CD25 expression was observed in hyperplastic thymi with GMG compared to those with OMG (P = 0.030). Similarly, CXCL13 expression was significantly higher in hyperplastic thymi with GMG or with OMG than those with non-MG (P = 0.001 and 0.050, respectively). No significant CXCL13 expression difference was found between hyperplastic thymi with GMG and those with OMG (P>> 0.05). The real-time PCR results showed a similar tendency of CD25 mRNA expression among the thymi of non-MG, OMG, and GMG patients, but the difference did not reach significance (P>> 0.05). An obvious increased expression of CXCL13 was found in hyperplastic thymi with GMG patients, compared to those with non-MG and OMG patients (P = 0.003 and 0.071, respectively). There was no difference found between hyperplastic thymi with non-MG and with OMG. Regression analysis showed a positive correlation between thymic CD25 level and MG symptom severity (F = 28.240; P = 0.000, r = 0.523). Similarly, a positive correlation was found between thymic CXCL13 expression and MG disease severity (F = 36.093; P = 0.000, r = 0.671). Taken together, our findings suggest CD25 and CXCL13 participate in the pathogenesis of MG and may influence the clinical symptoms of MG.

Activation of the noncanonical pathway through the interaction of lymphotoxin (LT)-alpha(1)beta(2) and LT-betaR is essential for the development of secondary lymphoid organs including lymph nodes (LN) and Peyer's patches (PP). Although TNFR-associated factor (TRAF) 2 and TRAF5 were identified as signal transducers for the LT-betaR, roles for TRAF2 and TRAF5 in the development of secondary lymphoid organs remain obscure. In this study, we show that PP but not mesenteric LN development is severely impaired in traf2(-/-) and traf2(-/-)traf5(-/-) mice. Development of VCAM-1(+) and ICAM-1(+) mesenchymal cells and expression of CXCL13, a crucial chemokine for the development of PP, are severely impaired in PP anlagen in the intestines of traf2(-/-) mice. Surprisingly, TNF-alpha stimulation potently up-regulates cxcl13 mRNA expression in wild-type murine embryonic fibroblasts, which is impaired in traf2(-/-) and relA(-/-) murine embryonic fibroblasts. Moreover, RelA is recruited to the promoter of cxcl13 gene upon TNF-alpha stimulation and PP development is impaired in TNFR type 1 (tnfr1)(-/-) mice. These results underscore a crucial role for the TNFR1-TRAF2-RelA-dependent canonical pathway in the development of PP through up-regulation of cxcl13 mRNA.

Chemokines are key players in the elicitation of immune response, by selectively attracting subpopulations of immune cells to the site of antigen presentation. Therefore, they are natural candidates for modulating immune responses to antigens qualitatively and quantitatively. We have selected chemokines associated with different arms of the immune response, i.e. RANTES/CCL5, B-lymphocyte chemoattractant/CXCL13, and monocyte chemoattractant protein-1/CCL2, and co-injected DNA expression constructs encoding these chemokines with constructs encoding two HIV antigens, gp120 and gp160, in mice. We subsequently measured markers of both cellular and humoral immune responses, and found that these chemokines qualitatively influenced the outcome of immune responses to both antigens, essentially according to their predicted association to Th profiles. These results are relevant towards the engineering of novel vaccine and immune-based therapies, and point to chemokines as candidate adjuvant and immunomodulatory molecules.

The mouse Pactolus gene is an evolutionary paralogue to the CD18/beta2 integrin subunit and is preferentially expressed by neutrophils. When first identified, it was assumed Pactolus would function as an adhesion receptor similar to other beta integrin subunits. The analysis of mice genetically deficient in Pactolus, however, did not define any lesion in neutrophil migration, adhesion or phagocytosis. Therefore a wider analysis of the Pactolus deficiency was initiated using transcriptional profiling during an inflammatory insult. This screen identified a single transcript, CXCL13, that was elevated in cells from a peritoneal lavage of the wild type animal compared to the Pactolus-deficient animal. Our analyses confirmed resident macrophages as being responsible for the chemokine using intracellular CXCL13 staining and additional cell markers to phenotypically characterize such cells. The resident CXCL13-expressing cells (which do not express Pactolus) are functionally distinct from the macrophages recruited into the peritoneal cavity following the inflammatory stimulation since the recruited macrophages do not express detectable levels of the chemokine. The numbers and expression patterns of these resident CXCL13-expressing cells do not vary in naïve animals of wild type or Pactolus-deficient origin. Additionally, Pactolus-deficient neutrophils do not preferentially kill (compared to wild type) CXC13-expressing macrophages. These data suggest that during an inflammatory response, Pactolus may help retain CXCL13-expressing cells within the peritoneal environment.

BACKGROUND

B-cell dysregulation has been implicated but not fully characterized in pediatric opsoclonus-myoclonus syndrome (OMS), a neuroblastoma-associated neuroinflammatory disorder.

OBJECTIVE

To assess the role of B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), two critical B cell-modulating cytokines, as potential biomarkers of disease activity and treatment biomarkers in OMS.

METHODS

Soluble BAFF and APRIL were measured in cerebrospinal fluid (CSF) and serum by ELISA in 433 children (296 OMS, 109 controls, 28 other inflammatory neurological disorders (OIND)). BAFF-R receptors on circulating CD19+ B cells were measured by flow cytometry. A blinded scorer rated motor severity on the OMS Evaluation Scale. Immunotherapies were evaluated cross-sectionally and longitudinally.

RESULTS

The mean CSF BAFF concentration, which was elevated in untreated OMS and OIND, correlated with OMS severity category (P = 0.006), and reduction by adrenocorticotropic hormone or corticotropin (ACTH) (-61%) or corticosteroids (-38%) was seen at each level of severity. In contrast, CSF APRIL was normal in OMS and OIND and unaffected by immunotherapy. When the entire OMS dataset was dichotomized into 'high' versus 'normal' CSF BAFF concentration, the phenotype of the high group included greater motor severity and number of CSF oligoclonal bands, and a higher concentration of inflammatory chemokines CXCL13 and CXCL10 in CSF and CXCL9 and CCL21 in serum. Serum APRIL was 6.7-fold higher in the intravenous immunoglobulins (IVIg) group, whereas serum BAFF was 2.6-fold higher in the rituximab group. The frequency of B cell BAFF-R expression was similar in untreated and treated OMS. Longitudinal studies of CSF BAFF revealed a significant decline in ACTH-treated patients (with or without rituximab) (P < 0.0001). Longitudinal studies of serum APRIL showed a 2.9-fold increase after 1 to 2 g/kg IVIg monotherapy (P = 0.0003).

CONCLUSIONS

Striking distinctions in BAFF/APRIL signaling were found. OMS displayed heterogeneity in CSF BAFF expression, which met many but not all criteria as a potential biomarker of disease activity. We speculate that CSF BAFF may have more utility in a biomarker panel than as a stand-alone biomarker, and that the selective upregulation of both serum APRIL by IVIg and BAFF by rituximab, as well as downregulation of CSF BAFF by ACTH/steroids, may have utility as treatment biomarkers.

We investigated CD4(+)CD34(+), CD8(+)CD34(+), CD4(+)CD34(-), and CD8(+)CD34(-) T cells from cord blood and from typical patients with T-cell-lineage acute lymphocytic leukemia and T-cell-lineage chronic lymphocytic leukemia in terms of expression and functions of CXCR5/CXCL13. We found that CXCR5 was selectively frequently expressed on T-cell-lineage acute (chronic) lymphocytic leukemia (T-ALL) CD8(+)CD34(+) T cells, but not on T-ALL CD4(+)CD34(+), CD4(+)CD34(-), and CD8(+)CD34(-) T cells. CXCR5 was rarely expressed on all types of CD34(+) and CD34(-) CB or T-CLL T cells. CXCL13/B cells attracting chemokine 1 induced significant resistance to TNF-alpha-mediated apoptosis in T-ALL CD8(+)CD34(+) T cells, instead of induction of chemotactic and adhesive responsiveness. A proliferation-inducing ligand expression in T-ALL CD8(+)CD34(+) T cells was upregulated by CXCL13/BCA-1 (B-cell attracting chemokine 1). The CXCR5/CXCL13 pair by means of activation of APRIL (A proliferation-inducing ligand) induced resistance to apoptosis in T-ALL CD8(+)CD34(+) T cells in livin-dependent manner. In this process, cell-cell contact in culture was necessary. Based on our findings, we suggested that there were differential functions of CXCR5/CXCL13 in distinct types of cells. Normal lymphocytes, especially naive B and T cells, utilized CXCR5/CXCL13 for migration, homing, maturation, and cell homeostasis, as well as secondary lymphoid tissue organogenesis. Meanwhile, certain malignant cells took advantages of CXCR5/CXCL13 for infiltration, resistance to apoptosis, and inappropriate proliferation.

Infection of mice with Sindbis virus (SINV) produces encephalomyelitis and provides a model for examination of the central nervous system (CNS) immune response to alphavirus infection. Clearance of infectious virus is accomplished through a cooperative effort between SINV-specific antibody and IFN-γ, but the regulatory interactions are poorly understood. To determine the effects of IFN-γ on clinical disease and the antiviral immune response, C57BL/6 mice lacking IFN-γ (Ifng-/-) or IFN-γ receptor (Ifngr1-/-) were studied in comparison to WT mice. Maximum production of Ifng mRNA and IFN-γ protein in the CNS of WT and Ifngr1-/- mice occurred 5-7 days after infection, with higher levels of IFN-γ in Ifngr1-/- mice. Onset of clinical disease was earlier in mice with impaired IFN-γ signalling, although Ifngr1-/- mice recovered more rapidly. Ifng-/- and Ifngr1-/- mice maintained body weight better than WT mice, associated with better food intake and lower brain levels of inflammatory cytokines. Clearance of infectious virus from the spinal cords was slower, and CNS, but not serum, levels of SINV-specific IgM, IgG2a and IgG2b were lower in Ifngr1-/- and Ifng-/- mice compared to WT mice. Decreased CNS antiviral antibody was associated with lower expression of mRNAs for B-cell attracting chemokines CXCL9, CXCL10 and CXCL13 and fewer B cells in the CNS. Therefore, IFN-γ signalling increases levels of CNS pro-inflammatory cytokines, leading to clinical disease, but synergistically clears virus with SINV-specific antibody at least in part by increasing chemokine production important for infiltration of antibody-secreting B cells into the CNS.