Development of the primary and secondary lymphoid organs is a tightly controlled process. These tissues are highly organized to maximize efficiency of the immune response. Spontaneous and targeted mutations in laboratory mice have led to better understanding of the molecular interactions and signaling pathways essential to the development and organization of lymphoid tissues, and the functional consequences of loss or disruption of the normal structures. On the basis of studies of mutations in mice and other species, it has been determined that a wild-type allele of the Foxn1 gene is required for normal thymic development and function. The Tlx1, Bapx1, Tcf21, Wt1 and Dh genes are essential for development of the spleen, while mutations of Nkx2-3, Lta, Ltb, Ltbr, Map3k14, Relb, Tnf, Tnfrsf1a, Cxcl13, Blr1 (Cxcr5), or cpdm genes result in disruption of normal splenic microarchitecture. The requirements for organized lymph nodes vary according to anatomic location, but most rely on Id2 (Idb2) and Rorc, in addition to lymphotoxins and Tnfrsf11a, Tnfsf11, Relb, Map3k14, Cxcl13, and Blr1 genes. Development of Peyer's patches is dependent on Id2 and Rorc genes, lymphotoxins, and Relb, Map3k14, Il7r, and cpdm genes. Less is known about the requirements for nasal-associated lymphoid tissues (NALT), but Id2 is a requirement. Here we review abnormalities of lymphoid organ development in immunodeficient mutant mice, including spontaneous and targeted mutations of Id2, Rorc, Tnf, Tnfrsf1a, Lta, Ltb, Ltbr, Tnfrsf11a, Tnfsf11, Relb, Map3k14, IL7r, Blr1, and Cxcl13 genes.

BACKGROUND

New approaches for non-invasive embryo-quality assessment are among the major goals in Reproductive Medicine. We hypothesize that the detection of changes in the protein profile of the culture media in which blastocysts are cultured could be a potential indicator of the viability of the embryo and, thus, a useful tool for selecting the more appropriate blastocysts to be transferred.

METHODS

Using protein-array technology, we analysed the protein profile corresponding to 24 h conditioned media of blastocysts that implanted versus those that did not implant. A statistical approach was followed to compare each of these media versus a medium in the absence of blastocysts (control medium). In addition, a gene ontology functional analysis-including those proteins showing a statistical difference among conditions-was performed, and a network with the predicted functional partners and corresponding relationships was obtained.

RESULTS

The soluble TNF receptor 1 and IL-10 increased significantly and MSP-alpha, SCF, CXCL13, TRAILR3 and MIP-1beta decreased significantly when the protein profile of the blastocyst culture medium was compared with the control medium. CXCL13 (BLC) and granulocyte-macrophage colony-stimulating factor was also decreased significantly in the implanted blastocyst media compared with that in media from the non-implanted counterparts with a similar morphology. None of the proteins included in the array was increased significantly in the implanted blastocyst-conditioned media.

CONCLUSIONS

The differences identified in the protein profile of the culture media in the presence of implanted versus non-implanted blastocysts can be considered a new non-invasive approach in the search for new tools to diagnose blastocyst viability.

OBJECTIVE

Graves' disease (GD) and Hashimoto's thyroiditis (HT) are characterized by lymphocytic infiltrates partly resembling secondary lymphoid follicles in the thyroid. CXCR5 and its ligand CXCL13 regulate compartmentalization of B- and T-cells in secondary lymphoid organs. The aim of the study was to elucidate the role of this chemokine receptor-ligand pair in thyroid autoimmunity.

METHODS

Peripheral blood and thyroid-derived lymphocyte subpopulations were examined by flow cytometry for CXCR5. CXCR5 and CXCL13 cDNA were quantified in thyroid tissues by real-time RT-PCR.

RESULTS

We found no differences between the percentages of peripheral blood CXCR5+ T- and B-cells in GD patients (n=10) and healthy controls (n=10). In GD patients, the number of memory CD4+ cells expressing CXCR5 which are functionally characterized as follicular B helper T-cells is higher in thyroid-derived (18+/-3%) compared with peripheral blood T-lymphocytes (8+/-2%). The highest CXCL13 mRNA levels were found in HT (n=2, 86.1+/-1.2 zmol (10(-21) mol) cDNA/PCR) followed by GD tissues (n=16, 9.6+/-3.5). Only low amounts were determined in thyroid autonomy (TA) (n=11) thyroid tissues, irrespective of whether the autonomous nodule (0.5+/-0.1) or the surrounding normal tissue (1.8+/-0.7) had been analyzed. The same differences were found for CXCR5 (HT: 179.1+/-6.8; GD: 17.4+/-10.6; TA(nodule): 0.8+/-0.5; TA(normal): 4.4+/-3.6). In GD, there is a correlation between CXCL13 and CXCR5 mRNA levels and the number of focal lymphocytic infiltrates and germinal centers as well as anti-thyroperoxidase but not anti-TSH receptor autoantibodies.

CONCLUSIONS

CXCR5 and CXCL13 play an essential role in maintaining B- and T-cells in lymphocytic infiltrates and ectopic follicles in thyroid tissue from patients affected by autoimmunity.

BACKGROUND

Immunosenescence is the age-related decline and dysfunction of protective immunity leading to a marked increase in the risk of infections, autoimmune disease, and cancer. The majority of studies have focused on immunosenescence in the systemic immune system; information concerning the effect of aging on intestinal immunity is limited. Isolated lymphoid follicles (ILFs) are newly appreciated dynamic intestinal lymphoid structures that arise from nascent lymphoid tissues, or cryptopatches (CP), in response to local inflammatory stimuli. ILFs promote "homeostatic" responses including the production of antigen-specific IgA, thus playing a key role in mucosal immune protection. ILF dysfunction with aging could contribute to immunosenescence of the mucosal system, and accordingly we examined phenotypic and functional aspects of ILFs from young (2 month old) and aged (2 year old) mice.

RESULTS

We observed that aged mice have increased numbers of ILFs and increased numbers of structures corresponding to an early stage of CPs transforming into ILFs. The cellular composition of ILFs in aged mice is altered with a smaller B-lymphocyte population and an increased T-lymphocyte population. The ILF T-lymphocyte population is notable by the presence of CD4+ CD8αα+ T-lymphocytes, which are absent from the systemic compartment. The smaller B-lymphocyte population in ILFs from aged mice is directly correlated with decreased mRNA and protein expression of CCL20 and CXCL13, two chemokines that play crucial roles in recruiting B-lymphocytes into ILFs. Aged mice had elevated levels of serum and fecal immunoglobulins and despite the decreased B-lymphocyte population, ILFs from aged mice displayed increased IgA production. The immunoglobulin repertoire was skewed in aged mice, and ILFs demonstrated a repertoire usage similar to that of the systemic pool in both young and aged mice.

CONCLUSIONS

Here we observed that ILF development, cellular composition, and immunoglobulin production are altered with aging suggesting that ILF dysfunction contributes to mucosal immunosenescence.

The expression pattern of a subset of chemokines and their corresponding receptors was investigated in primary central nervous system lymphomas (PCNSL). The tumor cells consistently expressed CXCR4, CXCL12, CXCR5, and CXCL13, both at mRNA and protein levels. Cerebral endothelial cells were positive for CXCL12 and CXCL13, while reactive astrocytes and microglial cells expressed CXCL12, CCR5, and CCR6. Inflammatory T cells in PCNSL were characterized by CCR5 and CCR6 positivity. Taken together, our data indicate a cell type-specific repertoire of chemokine and chemokine receptor expression in PCNSL suggesting that chemokine-mediated interactions facilitate crossing of the blood-brain barrier as well as intracerebral dissemination of PCNSL cells. In addition, chemokines expressed by tumor cells may contribute to induction of reactive glial changes and influence the composition of inflammatory infiltrates in PCNSL. Therefore, cell type specific expression of distinct chemokine profiles likely plays a role in the pathogenesis of PCNSL and may contribute to their characteristic histological appearance.

Elevated levels of the chemokine CXCL13 have been observed in the plasma of chronically HIV-1-infected subjects and have been correlated with plasma viremia, which in turn has been linked to progressive dysregulation of humoral responses. In this study we sought to identify mechanisms of CXCL13 induction in response to HIV-1 infection. Plasma levels of CXCL13 in HIV-1-infected antiretroviral therapy-naive subjects correlated with viral load and were higher compared with antiretroviral therapy-treated HIV-1-infected and HIV-1-uninfected subjects. To elucidate the relationship between HIV-1 viremia and CXCL13 plasma levels, PBMCs from uninfected donors were stimulated with HIV-1 infectious virions, HIV-1 ssRNA, TLR 7 and 8 agonists, or IFN-α. The cellular sources of CXCL13 were determined by intracellular cytokine staining of cell populations. CXCL13 was produced by monocytes after stimulation with TLR 7 and 8 ligands or HIV-1-derived ssRNA. CXCL13 production by monocytes required TLR7 activation of plasmacytoid dendritic cells and secretion of type I IFN. IFN-α alone was sufficient to induce CXCL13 expression in human monocytes. In sum, we identified a novel mechanism of HIV-1-induced CXCL13 secretion-one caused by TLR7 induction of type I IFN by plasmacytoid dendritic cells and subsequent IFN stimulation of monocytes. Our findings are relevant in understanding how HIV-1 infection leads to immune dysregulation and provide the opportunity to develop and test potential therapeutic interventions.

Human immunodeficiency virus (HIV)-induced inflammation and immune activation persist after initiation of combination antiretroviral therapy (cART) and HIV suppression and may contribute to mortality risks that exceed those in HIV-uninfected populations, though associations are unclear.

In the prospective Multicenter AIDS Cohort Study, comprising men who have sex with men from Baltimore, Chicago, Los Angeles, and Pittsburgh, concentrations of 24 biomarkers of inflammation and immune activation were measured in stored serum from HIV-positive men obtained after cART-induced HIV suppression between 1996 and 2009. The outcome was nonaccidental death, with follow-up until 2014. We used Cox proportional hazards models to test whether biomarker concentrations predict time from HIV suppression to death and adjusted for multiple tests. Exploratory factor analysis (EFA) was employed to identify groupings of biomarkers that predict mortality risk.

Of 670 men followed up from HIV suppression, 54 died by the end of 2013. After adjustment for age, CD4(+) cell count, hepatitis B or C virus infection, and smoking, concentrations in the highest quartile of 4 biomarkers were significantly associated with mortality risk after controlling the false discovery rate at 5%: interleukin (IL) 6 (hazard ratio, 3.54; 95% confidence interval, 2.06-6.10), soluble IL 2Rα (3.29, 1.85-5.85), soluble CD14 (2.67, 1.55-4.61), and chemokine (CXC motif) ligand 13 (CXCL13; 2.26; 1.29-3.95). EFA yielded 2 biomarker groupings that were independent predictors of mortality risk.

Despite having undetectable HIV RNA levels during cART, men with higher concentrations of several biomarkers (particularly IL 6, soluble IL 2Rα, soluble CD14, and CXCL13) had higher hazards of long-term mortality. Correlations observed among biomarker concentrations may represent underlying inflammatory processes that contribute to mortality risk.

Cerebrospinal fluid (CSF) biomarkers can reflect different aspects of the pathophysiology of relapsing-remitting multiple sclerosis (RRMS). Understanding the impact of different disease modifying therapies on the CSF biomarker profile may increase their implementation in clinical practice and their appropriateness for monitoring treatment efficacy. This study investigated the influence of first-line (interferon beta) and second-line (natalizumab) therapies on seven CSF biomarkers in RRMS and their correlation with clinical and radiological outcomes. We included 59 RRMS patients and 39 healthy controls. The concentrations of C-X-C motif chemokine 13 (CXCL13), C-C motif chemokine ligand 2 (CCL2), chitinase-3-like protein 1 (CHI3L1), glial fibrillary acidic protein, neurofilament light protein (NFL), and neurogranin were determined by ELISA, and chitotriosidase (CHIT1) was analyzed by spectrofluorometry. RRMS patients had higher levels of NFL, CXCL13, CHI3L1, and CHIT1 than controls (p < 0.001). Subgroup analysis revealed higher NFL, CXCL13 and CHIT1 levels in patients treated with first-line therapy compared to second-line therapy (p = 0.008, p = 0.001 and p = 0.026, respectively). NFL and CHIT1 levels correlated with relapse status, and NFL and CXCL13 levels correlated with the formation of new magnetic resonance imaging lesions. Furthermore, we found an association between inflammatory and degenerative biomarkers. The results indicate that CSF levels of NFL, CXCL13, CHI3L1, and CHIT1 correlate with the clinical and/or radiological disease activity, providing additional dimensions in the assessment of treatment efficacy.

OBJECTIVE

To examine amount of CD4+CXCR5+Tfh cells and B cells subsets in salivary gland and peripheral blood from patients with primary Sjogren's syndrome (pSS) and to analyze whether the frequency of CD4+CXCR5+Tfh cells is associated with pSS pathologic process.

METHODS

The percentages of CD4+CXCR5+Tfh cells and B cell subsets were examined by flow cytometry. B-lymphocyte chemoattraetant (BLC; also called CXCL13), IL-21, IL-6 from the serum of pSS patients was assessed by polymerase chain reaction-enzyme-linked immunosorbent assay (ELISA).

RESULTS

The percentages of CD4+CXCR5+Tfh cells in peripheral blood were increased in pSS patients, but decreased after treatment with glucocorticoid and/or immunosuppressive drugs. Abnormal B cell subsets appeared in salivary and peripheral blood of pSS patients. The frequency of salivary CD4+CXCR5+Tfh cells was positively correlated with CD19+CD27+ memory B cells and CD19+CD27high plasma cells. Also increase of salivary CD19+CD27high plasma cells was positively associated with serum ANA titer of pSS patients.

CONCLUSIONS

CD4+CXCR5+Tfh cells are significantly increased in salivary and peripheral blood in pSS patients with aberrant CD19+CD27+ memory B cells and CD19+CD27high plasma cells, suggesting that CD4+CXCR5+Tfh cells may contribute to the pathogenesis of pSS by promoting the maturation of B cells.

In multiple sclerosis (MS), the presence of B cells, plasma cells and excess immunoglobulins in central nervous system lesions and in the cerebrospinal fluid implicate the humoral immune system in disease pathogenesis. However, until the advent of specific B-cell-depleting therapies, the critical role of B cells and their products in MS was unproven. Rituximab, a monoclonal antibody that depletes B cells by targeting the CD20 molecule, has been shown to effectively reduce disease activity in patients with relapsing MS as a single agent. Our investigator-initiated phase II study is the only published clinical trial in which rituximab was used as an add-on therapy in patients with relapsing MS who had an inadequate response to standard injectable disease-modifying therapies (DMTs). The primary endpoint, magnetic resonance imaging (MRI) gadolinium-enhanced (GdE) lesion number before versus after rituximab, showed significant benefit of rituximab (74% of post-treatment MRI scans being free of GdE lesions compared with 26% free of GdE lesions at baseline; p < 0.0001). No differences were noted comparing patients on different DMTs. Several secondary clinical endpoints, safety and laboratory measurements (including B- and T-cell numbers in the blood and cerebrospinal fluid (CSF), serum and CSF chemokine levels, antibodies to myelin proteins) were assessed. Surprisingly, the decline in B-cell number was accompanied by a significant reduction in the number of T cells in both the peripheral blood and CSF. Rituximab therapy was associated with a significant decline of two lymphoid chemokines, CXCL13 and CCL19. No significant changes were observed in serum antibody levels against myelin proteins [myelin basic protein (MBP) and myelin/oligodendrocyte glycoprotein (MOG)] after treatment. These results suggest that B cells play a role in MS independent from antibody production and possibly related to their role in antigen presentation to T cells or to their chemokine/cytokine production.

BACKGROUND

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune disorder of the central nervous system (CNS). Its immunopathogenesis has been proposed to include early cerebrospinal fluid (CSF) lymphocytosis, subsequent CNS disease restriction and B cell mechanism predominance. There are limited data regarding T cell involvement in the disease. To contribute to the current knowledge, we investigated the complex system of chemokines and cytokines related to B and T cell functions in CSF and sera samples from anti-NMDAR encephalitis patients at different time-points of the disease. One patient in our study group had a long-persisting coma and underwent extraordinary immunosuppressive therapy.

METHODS

Twenty-seven paired CSF/serum samples were collected from nine patients during the follow-up period (median 12 months, range 1-26 months). The patient samples were stratified into three periods after the onset of the first disease symptom and compared with the controls. Modified Rankin score (mRS) defined the clinical status. The concentrations of the chemokines (C-X-C motif ligand (CXCL)10, CXCL8 and C-C motif ligand 2 (CCL2)) and the cytokines (interferon (IFN)γ, interleukin (IL)4, IL7, IL15, IL17A and tumour necrosis factor (TNF)α) were measured with Luminex multiple bead technology. The B cell-activating factor (BAFF) and CXCL13 concentrations were determined via enzyme-linked immunosorbent assay. We correlated the disease period with the mRS, pleocytosis and the levels of all of the investigated chemokines and cytokines. Non-parametric tests were used, a P value <0.05 was considered to be significant.

RESULTS

The increased CXCL10 and CXCL13 CSF levels accompanied early-stage disease progression and pleocytosis. The CSF CXCL10 and CXCL13 levels were the highest in the most complicated patient. The CSF BAFF levels remained unchanged through the periods. In contrast, the CSF levels of T cell-related cytokines (INFγ, TNFα and IL17A) and IL15 were slightly increased at all of the periods examined. No dynamic changes in chemokine and cytokine levels were observed in the peripheral blood.

CONCLUSIONS

Our data support the hypothesis that anti-NMDAR encephalitis is restricted to the CNS and that chemoattraction of immune cells dominates at its early stage. Furthermore, our findings raise the question of whether T cells are involved in this disease.

OBJECTIVE

The aim of the study was to assess the concentration of chemokines: CXCL10, XCL11, CXCL12, CXCL13 in serum and cerebrospinal fluid (CSF) in patients with tick-borne encephalitis (TBE) before and after treatment. We evaluated also the usefulness of these molecules in diagnosis and monitoring of inflammation in TBE.

METHODS

Twenty three patients hospitalized in The Department of Infectious Diseases and Neuroinfections of Medical University in Białystok, Poland were included in the study. Patients were divided into 2 groups: TBE group-patients with confirmed TBE and control group (CG): patients with excluded TBE and other inflammatory diseases of CNS. Concentration of CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1α, CXCL13/BLC/BCA-1 in serum and CSF were measured with ELISA kits (R&D Systems, USA) according to the protocols.

RESULTS

The analysis of chemokines concentration in TBE patients before treatment and control group using ROC showed that serum CXCL10 and CXCL13 and CSF CXCL10, CXCL11, CXCL12 and CXCL13 differentiate both groups (p<0.05). The analysis of CXCL10, CXCL11, CXCL12 and CXCL13 before and after treatment showed that CXCL10 and CXCL11 in CSF and CXCL13 in serum differentiates both groups with p<0.05.

CONCLUSIONS

Concentration of CSF CXCL10, CXCL11, CXCL12, CXCL13 and serum CXCL10, CXCL13 may be good biomarkers of CNS inflammation caused by TBEV. Moreover concentration of CXCL10 in CSF and CXCL13 in serum may be used as indicators of patients recovery.

BACKGROUND

Recent advances in the immunotherapeutic treatment of cancer have led to the development of multiple new directed therapies including monoclonal antibodies that block the immune checkpoint T-cell receptor programmed death 1 (PD-1) and the PD-1 ligand, programmed death ligand 1 (PD-L1). Various immune-related toxicities have been associated with these drugs including, most commonly, skin rashes.

METHODS

Five cases of lichenoid dermatitis, including one case of lichenoid mucositis and one case of lichen sclerosus, associated with anti-PD-L1 and anti-PD1 therapy were compared with three biopsies of non-drug-related lichen planus (LP) and three lichen planus-like keratoses (LPLK) used as controls.

RESULTS

Histopathologic and immunophenotypic analysis of these lichenoid lesions demonstrated significantly greater histiocytic infiltrates than observed in control lichenoid reactions (p = 0.0134). We also observed increased spongiosis and epidermal necrosis. No significant differences were seen in expression of CD3, CD4:CD8, CD20, PD-1, CD25, Foxp3, CXCL13 and PD-L1 expression.

CONCLUSIONS

These findings expand the literature of immune-related toxicities of PD-L1 and PD-1 blockade to include lichenoid dermatitis and lichenoid mucositis. Of note, these cutaneous side effects were amenable to topical treatment, without the need for medication dose reduction or discontinuation.

OBJECTIVE

Transformation of follicular lymphoma is a critical event associated with a poor prognosis. The role of the tumor microenvironment in previous transformation studies has yielded conflicting results.

METHODS

To define cell subtypes associated with transformation, we examined tissue specimens at diagnosis from patients with follicular lymphoma that later transformed and, using immunohistochemistry (IHC), stained for CD68, CD11c, CD21, CXCL13, FOXP3, PD1, and CD14. Cell content and the pattern of expression were evaluated. Those identified as significantly associated with time to transformation (TTT) and overall survival (OS) were further characterized by flow cytometry and multicolor IHC.

RESULTS

Of note, 58 patients were analyzed with median TTT of 4.7 years. The pattern of PD1(+) and CD14(+) cells rather than the quantity of cells was predictive of clinical outcomes. On multivariate analysis, including the follicular lymphoma international prognostic index score, CD14(+) cells localized in the follicle were associated with a shorter TTT (HR, 3.0; P = 0.004). PD1(+) cells with diffuse staining were associated with a shorter TTT (HR, 1.9; P = 0.045) and inferior OS (HR, 2.5; P = 0.012). Multicolor IHC and flow cytometry identified CD14(+) cells as follicular dendritic cells (FDC), whereas PD1(+) cells represented two separate populations, TFH and exhausted T cells.

CONCLUSIONS

These results identify the presence of PD1(+) T cells and CD14(+) FDC as independent predictors of transformation in follicular lymphoma. Clin Cancer Res; 20(11); 2862-72. ©2014 AACR.

The tumor microenvironment comprises stromal and tumor cells which interact with each other through complex cross-talks that are mediated by a variety of growth factors, cytokines, and chemokines. The chemokine ligand 13 (CXCL13) and its chemokine receptor 5 (CXCR5) are among the key chemotactic factors which play crucial roles in deriving cancer cell biology. CXCL13/CXCR5 signaling axis makes pivotal contributions to the development and progression of several human cancers. In this review, we discuss how CXCL13/CXCR5 signaling modulates cancer cell ability to grow, proliferate, invade, and metastasize. Furthermore, we also discuss the preliminary evidence on context-dependent functioning of this axis within the tumor-immune microenvironment, thus, highlighting its potential dichotomy with respect to anticancer immunity and cancer immune-evasion mechanisms. At the end, we briefly shed light on the therapeutic potential or implications of targeting CXCL13/CXCR5 axis within the tumor microenvironment.

Myasthenia gravis (MG) is an autoimmune disease mainly caused by antiacetylcholine receptor autoantibodies (seropositive (SP) disease) or by Abs against unknown autoantigenic target(s) (seronegative (SN) disease). Thymectomy is usually beneficial although thymic hyperplasia with ectopic germinal centers is mainly observed in SP MG. To understand the role of thymus in the disease process, we compared the thymic transcriptome of non-MG adults to those of SP patients with a low or high degree of hyperplasia or SN patients. Surprisingly, an overexpression of MHC class II, Ig, and B cell marker genes is observed in SP but also SN MG patients. Moreover, we demonstrate an overexpression of CXCL13 in all MG thymuses leading probably to the generalized B cell infiltration. However, we find different chemotactic properties for MG subgroups and, especially, a specific overexpression of CCL21 in hyperplastic thymuses triggering most likely ectopic germinal center development. Besides, SN patients present a peculiar signature with an abnormal expression of genes involved in muscle development and synaptic transmission, but also genes implicated in host response, suggesting that viral infection might be related to SN MG. Altogether, these results underline differential pathogenic mechanisms in the thymus of SP and SN MG and propose new research areas.

A cohort based study has been undertaken to investigate the possible association of genetic polymorphisms in genes functionally related to follicular T helper (TfH) cells with non-responsiveness to hepatitis B virus (HBV) vaccination. A total of 24 single nucleotide polymorphisms (SNPs) in 6 TfH related genes (CXCR5, ICOS, CXCL13, IL-21, BCL6 and CD40L) were investigated in 20 non-responders and 45 responders to HBV vaccination. Genetic association analysis revealed that three SNPs (rs497916, rs3922, rs676925) in CXCR5 and one SNP (rs355687) in CXCL13 were associated with hepatitis B vaccine efficacy. In addition, significantly unbalanced distributions of two haplotypes, defined by three SNPs (rs497916, rs3922, rs676925) within CXCR5, were also seen between non-responders and responders. Furthermore, we demonstrated that the rs3922 "GG" genotype was associated with higher levels of CXCR5 than the "AG" and "AA" genotype in a group of healthy volunteers. A dual luciferase report assay was used to confirm that the "G" allele in rs3922 may lead to higher gene expression than the "A" allele, implicating that rs3922 might be a functional SNP affecting CXCR5 expression. These results indicated that polymorphism associated changes in CXCR5 expression in TfH cells may be associated with non-responsiveness to hepatitis B vaccination.

Marginal zone (MZ) B cells contain a large number of autoreactive clones and the expansion of this compartment has been associated with autoimmunity. MZ B cells also efficiently transport blood-borne antigen to the follicles where they activate T cells and differentiate into plasma cells. Using the B6.NZM2410.Sle1.Sle2.Sle3 (B6.TC) model of lupus, we show that the IgM+ CD1d(hi)/MZ B-cell compartment is expanded, and a large number of them reside inside the follicles. Contrary to the peripheral B-cell subset distribution and their activation status, the intrafollicular location of B6.TC IgM+ CD1d(hi)/MZ B cells depends on both bone marrow- and stromal-derived factors. Among the factors responsible for this intrafollicular location, we have identified an increased response to CXCL13 by B6.TC MZ B cells and a decreased expression of VCAM-1 on stromal cells in the B6.TC MZ. However, the reduced number of MZ macrophages observed in B6.TC MZs was independent of the IgM+ CD1d(hi)/B-cell location. B7-2 but not B7-1 deficiency restored IgM+ CD1d(hi)/MZ B-cell follicular exclusion in B6.TC mice, and it correlated with tolerance to dsDNA and a significant reduction of autoimmune pathology. These results suggest that follicular exclusion of IgM+ CD1d(hi)/MZ B cells is an important B-cell tolerance mechanism, and that B7-2 signaling is involved in breaching this tolerance checkpoint.

The CXC chemokine receptor (CXCR)5 is rapidly induced on activated CD4(+) T cells, allowing migration toward secondary lymphoid tissue follicles, where the CXCR5 ligand CXCL13/BCA-1 is produced. Such CXCR5(+) T cells provide efficient help for B cell immunoglobulin production and are termed follicular B helper T (T(FH)) cells. However, the molecular mechanisms by which T(FH) cells provide B cell help are unknown. Here, we demonstrate that newly generated (antigen-primed) T(FH) cells express a phenotype consistent with induction of B cell proliferation, but co-culture with primed B cells resulted in a switch to a plasma cell-inducing phenotype, characterized by loss of CD154, induction of CD70 and an increase in IL-10 production capacity. The ability to produce IL-10 could be maintained as a stable phenotype, but its secretion was strictly dependent on inducible costimulator (ICOS) signaling. Furthermore, B cells preserved a lymph node migration phenotype in proliferating T(FH) cells by preventing the loss of CC chemokine receptor (CCR)7 and the induction of CCR5. Thus, B cells directly modulate the B cell helper phenotype in T(FH) cells and actively promote their prolonged co-localization with these cells.

Multiple elements are known to participate in ischemia/reperfusion (I/R)-mediated tissue injury. Amongst them, B cells have been shown to contribute by the production of antibodies that bind to ischemic cells and fix complement. It is currently unknown whether B cells participate through antibody-independent mechanisms in the pathogenesis of I/R. In a mesenteric I/R model we found that B cells infiltrate the injured intestine of normal and autoimmune mice 2h after reperfusion is established. B cell depletion protected mice from the development of I/R-mediated intestinal damage. The protection conferred by B cell depletion was significantly greater in MRL/lpr mice. Finally, we show that ischemic tissue expressed the B cell-attractant CXCL13 and infiltrating B cells expressed the corresponding receptor CXCR5. Our data grant B cells an antibody-independent role in the pathogenesis of intestinal I/R and suggest that B cells accumulate in the injured tissue in response to the chemokine CXCL13.

Studies in aged mice show that the architecture of B-cell areas appears disrupted and that newly made B cells fail to incorporate into the spleen. These observations may reflect altered migration of immature and mature B cells. Using adoptive transfer, we tested the effect of the aged microenvironment and the intrinsic ability of donor B cells from aged mice to migrate to spleens of intact hosts. Spleens of aged recipients were deficient in attracting young or old donor immature B cells. In contrast, immature and mature B cells maintained an intrinsic ability to migrate to young recipient spleens, except that as the aged immature B cells matured, fewer appeared to enter the recirculating pool. CXCL13 protein, which is necessary for the organization of B-cell compartments, was elevated with age and differences in CXCL13 distribution were apparent. In aged spleens, CXCL13 appeared less reticular, concentrated in patches throughout the follicles, and notably reduced in the MAdCAM-1(+) marginal reticular cells located at the follicular edge. Despite these differences, the migration of young donor follicular B cells into the spleens of old mice was not impacted; whereas, migration of young donor marginal zone B cells was reduced in aged recipients. Finally, the aged bone marrow microenvironment attracted more donor mature B cells than did the young marrow. Message for CXCL13 was not elevated in the marrow of aged mice. These results suggest that the aged splenic microenvironment affects the migration of immature B cells more than mature follicular B cells.

It has been recently shown that systemically injected neural precursor cells (NPCs) could cross brain endothelium and favor functional recovery in animal models of multiple sclerosis (MS). Here we show that human NPCs express receptors of the chemokines IL8 and CXCL13 (CXCR1 and CXCR5, respectively) and migrate across brain endothelial cells in vitro, in response to these chemokines. Considering that these chemokines have been found overexpressed in MS in active, but not inactive areas of demyelination, our data suggest that systemically injected human NPCs may be considered for targeting active areas of demyelination in therapeutic approaches of MS.

Integrin-mediated adhesion is a crucial step in lymphocyte extravasation and homing. We show here that not only the chemokines CXCL12 and CXCL13 but also the lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) enhance adhesion of murine follicular and marginal zone B cells to ICAM-1 in vitro. This process involves clustering of integrin LFA-1 and is blocked by pertussis toxin, suggesting that G(i) family G-proteins are involved. In addition, lysophospholipid-induced adhesion on ICAM-1 depends on Rho and Rhokinase, indicative of an involvement of G(12)/G(13), possibly also G(q)/G(11) family G-proteins. We used G(12)/G(13)- or G(q)/G(11)-deficient B cells to study the role of these G-protein families in lysophospholipid-induced adhesion and found that the pro-adhesive effects of LPA and S1P are completely abrogated in G(12)/G(13)-deficient marginal zone B cells, reduced in G(12)/G(13)-deficient follicular B cells, and normal in G(q)/G(11)-deficient B cells. We also show that loss of lysophospholipid-induced adhesion results in disinhibition of migration in response to the follicular chemokine CXCL13, which might contribute to the abnormal localization of splenic B cell populations observed in B cell-specific G(12)/G(13)-deficient mice in vivo. Taken together, this study shows that lysophospholipids regulate integrin-mediated adhesion of splenic B cells to ICAM-1 through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11).

B cell development is exquisitely sensitive to location within specialized niches in the bone marrow and spleen. Location within these niches is carefully orchestrated through chemotactic and adhesive cues. In this article, we demonstrate the requirement for the actin-bundling protein L-plastin (LPL) in B cell motility toward the chemokines CXCL12 and CXCL13 and the lipid chemoattractant sphingosine-1-phosphate, which guide normal B cell development. Impaired motility of B cells in LPL(-/-) mice correlated with diminished splenic maturation of B cells, with a moderate (40%) loss of follicular B cells and a profound (>80%) loss of marginal zone B cells. Entry of LPL(-/-) B cells into the lymph nodes and bone marrow of mice was also impaired. Furthermore, LPL was required for the integrin-mediated enhancement of Transwell migration but was dispensable for integrin-mediated lymphocyte adhesion. These results suggest that LPL may participate in signaling that enables lymphocyte transmigration. In support of this hypothesis, the phosphorylation of Pyk-2, a tyrosine kinase that integrates chemotactic and adhesive cues, is diminished in LPL(-/-) B cells stimulated with chemokine. Finally, a well-characterized role of marginal zone B cells is the generation of a rapid humoral response to polysaccharide Ags. LPL(-/-) mice exhibited a defective Ab response to Streptococcus pneumoniae, indicating a functional consequence of defective marginal zone B cell development in LPL(-/-) mice.