Altered Expression of CXCL13 and Its Chemokine Receptor CXCR5 on B Lymphocytes during Active Graves' Orbitopathy.

PURPOSE: To characterize the phenotypic abnormalities of peripheral B cells in patients with Graves' orbitopathy (GO) and explore the role of chemokine CXC ligand 13 and its receptor type 5 (CXCL13/CXCR5) in relation to B-cell homeostasis using specific neutralizing antibodies. METHODS: Adults with active GO (n = 22), inactive GO (n = 28), and healthy control subjects (n = 28) were included in the study. Peripheral B cells and B-cell subsets were quantified and analyzed for CXCR5 expression by flow cytometry. The serum CXCL13 concentration was measured by enzyme-linked immunosorbent assays. For chemotactic experiments, Transwell plates were used, and migrating B cells were further analyzed by flow cytometry. RESULTS: Compared to healthy subjects, patients with active GO had a significantly higher number of CD19+ B cells and the CD19+CD27+ memory B-cell subset (P = .041 and P = .019, respectively), whereas a marginal increase in the number of these cells was found in patients with inactive GO (P = .062 and P = .087, respectively). Serum CXCL13 levels were significantly higher in patients with active GO (86.9 ± 30.4 pg/mL) than in those with inactive GO (41.7 ± 18.1 pg/mL; P < .001) and in healthy subjects (36.2 ± 7.8 pg/mL; P < .001). The increased CXCL13 concentration was positively and significantly correlated with the clinical activity score (r = 0.757, P < .001). Finally, serum from patients with active GO exerted a stronger chemotactic activity towards B cells and the CD19+CD27+ memory B-cell subset. Blocking CXCL13 or CXCR5 with neutralizing antibodies reduced B-cell migration by a mean of 20%. CONCLUSIONS: Our data suggest that aberrant CXCL13/CXCR5 expression may contribute to the deficits in B-lymphocyte homeostasis observed in active GO.

Chemokine CXCL13 acts via CXCR5-ERK signaling in hippocampus to induce perioperative neurocognitive disorders in surgically treated mice.

BACKGROUND: Perioperative neurocognitive disorders (PNDs) occur frequently after surgery and worsen patient outcome. How C-X-C motif chemokine (CXCL) 13 and its sole receptor CXCR5 contribute to PNDs remains poorly understood. METHODS: A PND model was created in adult male C57BL/6J and CXCR5-/- mice by exploratory laparotomy. Mice were pretreated via intracerebroventricular injection with recombinant CXCL13, short hairpin RNA against CXCL13 or a scrambled control RNA, or ERK inhibitor PD98059. Then surgery was performed to induce PNDs, and animals were assessed in the Barnes maze trial followed by a fear-conditioning test. Expression of CXCL13, CXCR5, and ERK in hippocampus was examined using Western blot, quantitative PCR, and immunohistochemistry. Levels of interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) in hippocampus were assessed by Western blot. RESULTS: Surgery impaired learning and memory, and it increased expression of CXCL13 and CXCR5 in the hippocampus. CXCL13 knockdown partially reversed the effects of surgery on CXCR5 and cognitive dysfunction. CXCR5 knockout led to similar cognitive outcomes as CXCL13 knockdown, and it repressed surgery-induced activation of ERK and production of IL-1ß and TNF-α in hippocampus. Recombinant CXCL13 induced cognitive deficits and increased the expression of phospho-ERK as well as IL-1ß and TNF-α in hippocampus of wild-type mice, but not CXCR5-/- mice. PD98059 partially blocked CXCL13-induced cognitive dysfunction as well as production of IL-1ß and TNF-α. CONCLUSIONS: CXCL13-induced activation of CXCR5 may contribute to PNDs by triggering ERK-mediated production of pro-inflammatory cytokines in hippocampus.

CXCR5 and ICOS expression identifies a CD8 T-cell subset with TFH features in Hodgkin lymphomas.

A better characterization of T-cell subsets in the microenvironment of classical Hodgkin lymphoma (cHL) would help to develop immunotherapies. Using multicolor flow cytometry, we identified in 6 of 43 cHL tissue samples a previously unrecognized subset of CD8 T cells coexpressing CXCR5 and inducible T-cell costimulator (ICOS) molecules (CD8CXCR5+ICOS+). These cells shared phenotypic features with follicular helper T (TFH) cells including low CCR7 expression together with high expression of B-cell lymphoma-6, programmed cell death 1, B and T lymphocyte attenuator, CD200, and OX40. They had deficient cytotoxicity, low interferon-γ secretion, and common functional properties with intratumoral CD4+ TFH cells, such as production of interleukin-4 (IL-4), IL-21, CXCL13, and capacity to sustain B cells. Gene profiling analysis showed a significant similarity between the signatures of CD8CXCR5+ICOS+ T cells and CD4+ TFH cells. Benign lymphadenitis tissues (n = 8) were devoid of CD8CXCR5+ICOS+ cells. Among the 35 B-cell lymphoma tissues analyzed, including follicular lymphomas (n = 13), diffuse large cell lymphomas (n = 12), marginal zone lymphomas (MZLs; n = 3), mantle cell lymphomas (n = 3), and chronic lymphocytic leukemias (n = 4), only 1 MZL sample contained CD8CXCR5+ICOS+ cells. Lymphoma tumors with CD8CXCR5+ICOS+ cells shared common histopathological features including residual germinal centers, and contained high amounts of activated CD8CXCR5-ICOS+ cells. These data demonstrate a CD8 T-cell differentiation pathway leading to the acquisition of some TFH similarities. They suggest a particular immunoediting process with global CD8 activation acting mainly, but not exclusively, in HL tumors.

Diminished CXCR5 expression in peripheral blood of patients with Sjögren's syndrome may relate to both genotype and salivary gland homing.

Genetic investigations of Sjögren's syndrome (SS) have identified a susceptibility locus at p23.3 of chromosome 11, which contains the CXCR5 gene. C-X-C motif chemokine receptor 5 (CXCR5) is a chemokine receptor expressed on B and T cell subsets, and binds the chemotactic ligand C-X-C motif chemokine ligand 13 (CXCL13). In this study we aimed to link the genetic association with functional effects and explore the CXCR5/CXCL13 axis in SS. Expression quantitative trait loci analysis of the 11q23.3 locus was performed using B cell mRNA expression data from genotyped individuals. Lymphocyte surface markers were assessed by flow cytometry, and CXCL13 levels by a proximity extension assay. CXCR5+ and CXCL13+ cells in minor salivary glands were detected using immunohistochemistry. Our results demonstrated that SS-associated genetic polymorphisms affected the expression of CXCR5 (P < 0·01). Notably, a decreased percentage of CXCR5+ cells, with lower CXCR5 expression, was observed for most circulating B and T cell subsets in SS patients, reaching statistical significance in CD19+ CD27+ immunoglobulin (Ig)D+ marginal zone (P < 0·001), CD19+ CD27+ IgD- memory (P < 0·05) and CD27-IgD double-negative (P < 0·01) B cells and CD4+ CXCR3- CCR6+ Th17 cells (P < 0·05). CXCL13 levels were increased in patient plasma (P < 0·001), and immunohistochemical staining revealed expression of CXCL13 and higher numbers of CXCR5+ cells (P < 0·0001) within focal infiltrates and interstitially in salivary glands of SS patients. In conclusion, we link a genetic susceptibility allele for SS to a functional phenotype in terms of decreased CXCR5 expression. The decrease of CXCR5+ cells in circulation was also related to homing of B and T cells to the autoimmune target organ. Therapeutic drugs targeting the CXCR5/CXCL13 axis may be useful in SS.

A distinct subpopulation of CD25- T-follicular regulatory cells localizes in the germinal centers.

T-follicular helper (Tfh) cells differentiate through a multistep process, culminating in germinal center (GC) localized GC-Tfh cells that provide support to GC-B cells. T-follicular regulatory (Tfr) cells have critical roles in the control of Tfh cells and GC formation. Although Tfh-cell differentiation is inhibited by IL-2, regulatory T (Treg) cell differentiation and survival depend on it. Here, we describe a CD25- subpopulation within both murine and human PD1+CXCR5+Foxp3+ Tfr cells. It is preferentially located in the GC and can be clearly differentiated from CD25+ non-GC-Tfr, Tfh, and effector Treg (eTreg) cells by the expression of a wide range of molecules. In comparison to CD25+ Tfr and eTreg cells, CD25- Tfr cells partially down-regulate IL-2-dependent canonical Treg features, but retain suppressive function, while simultaneously up-regulating genes associated with Tfh and GC-Tfh cells. We suggest that, similar to Tfh cells, Tfr cells follow a differentiation pathway generating a mature GC-localized subpopulation, CD25- Tfr cells.

Protein Kinase C Epsilon Cooperates with PTEN Loss for Prostate Tumorigenesis through the CXCL13-CXCR5 Pathway.

PKCε, an oncogenic member of the PKC family, is aberrantly overexpressed in epithelial cancers. To date, little is known about functional interactions of PKCε with other genetic alterations, as well as the effectors contributing to its tumorigenic and metastatic phenotype. Here, we demonstrate that PKCε cooperates with the loss of the tumor suppressor Pten for the development of prostate cancer in a mouse model. Mechanistic analysis revealed that PKCε overexpression and Pten loss individually and synergistically upregulate the production of the chemokine CXCL13, which involves the transcriptional activation of the CXCL13 gene via the non-canonical nuclear factor κB (NF-κB) pathway. Notably, targeted disruption of CXCL13 or its receptor, CXCR5, in prostate cancer cells impaired their migratory and tumorigenic properties. In addition to providing evidence for an autonomous vicious cycle driven by PKCε, our studies identified a compelling rationale for targeting the CXCL13-CXCR5 axis for prostate cancer treatment.

Increased circulating CXCR5+ CD4+ T follicular helper-like cells in oral lichen planus.

BACKGROUND: CD4+ T-helper cell is crucial for the inflammatory autoimmune condition of oral lichen planus (OLP). Recently, the pathogenetic functions of T follicular helper (Tfh) cells, a subtype of CD4+ T-helper cells, have been revealed in autoimmune diseases for their pivotal regulation on humoral immunity. To explore the potential pathophysiological role of Tfh cells in OLP, the expression of circulating Tfh-like cells and its correlations with IL-21 and B cells were investigated. METHODS: The frequencies of CXCR5+ CD4+ Tfh-like cells and CD19+ B cells were analyzed in peripheral blood of patients with OLP and controls by flow cytometry, respectively. Besides, the serum IL-21 concentration was measured using ELISA technology. Furthermore, the correlations of CXCR5+ CD4+ Tfh-like cells with CD19+ B cells and serum IL-21 expression levels were evaluated. RESULTS: This study showed significant increased circulating Tfh-like cells (P < 0.05) and B cells (P < 0.0001), as well as decreased serum IL-21 expression (P < 0.001) in OLP. Besides, the frequency of Tfh-like cells exhibited negative correlation with B cells in OLP (r = -0.435, P < 0.05). In particular, the proportion of CXCR5+ CD4+ Tfh-like cells in peripheral blood mononuclear cells of erosive OLP was higher than non-erosive OLP and controls (P = 0.012 and 0.021, respectively). CONCLUSIONS: Increased circulating Tfh-like cells may be involved in the pathogenesis of OLP through abnormal modulation on B-cell proliferation and IL-21 production, and associated with different clinical forms of OLP.

Altered Expression of CXCL13 and CXCR5 in Intractable Temporal Lobe Epilepsy Patients and Pilocarpine-Induced Epileptic Rats.

The mechanisms that underlie the pathogenesis of epilepsy are still unclear. Recent studies have indicated that inflammatory processes occurring in the brain are involved in a common and crucial mechanism in epileptogenesis. C-X-C motif chemokine ligand 13 (CXCL13) and its only receptor, C-X-C motif chemokine receptor 5 (CXCR5), are highly expressed in the central nervous system (CNS) and participate in inflammatory responses. The present study aimed to assess the expression of CXCL13 and CXCR5 in the brain tissues of both patients with intractable epilepsy (IE) and a rat model (lithium-pilocarpine) of temporal lobe epilepsy (TLE) to identify possible roles of the CXCL13-CXCR5 signaling pathway in epileptogenesis. Real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemical, double-labeled immunofluorescence and Western blot analyses were performed in this study. CXCL13 and CXCR5 mRNA expression and protein levels were found to be significantly up-regulated in the TLE patients and TLE rats. Further, CXCL13 and CXCR5 protein levels were altered during the different epileptic phases after onset of status epilepticus (SE) in the pilocarpine model rats, including the acute phase (6, 24, and 72 h), latent phase (7 and 14 days) and chronic phase (30 and 60 days groups). Moreover, double-labeled immunofluorescence analysis revealed that CXCL13 was mainly expressed in the cytomembranes and cytoplasm of neurons and astrocytes, while CXCR5 was mainly expressed in the cytomembranes and cytoplasm of neurons. Thus, the CXCL13-CXCR5 signaling pathway may play a possible pathogenic role in IE. CXCL13 and CXCR5 may represent potential biomarkers of brain inflammation in epileptic patients.

Regulatory and Helper Follicular T Cells and Antibody Avidity to Simian Immunodeficiency Virus Glycoprotein 120.

T follicular regulatory cells (TFR) are a suppressive CD4(+) T cell subset that migrates to germinal centers (GC) during Ag presentation by upregulating the chemokine receptor CXCR5. In the GC, TFR control T follicular helper cell (TFH) expansion and modulate the development of high-affinity Ag-specific responses. In this study, we identified and characterized TFR as CXCR5(+)CCR7(-) "follicular" T regulatory cells in lymphoid tissues of healthy rhesus macaques, and we studied their dynamics throughout infection in a well-defined animal model of HIV pathogenesis. TFR were infected by SIVmac251 and had comparable levels of SIV DNA to CXCR5(-)CCR7(+) "T zone" T regulatory cells and TFH. Contrary to the SIV-associated TFH expansion in the chronic phase of infection, we observed an apparent reduction of TFR frequency in cell suspension, as well as a decrease of CD3(+)Foxp3(+) cells in the GC of intact lymph nodes. TFR frequency was inversely associated with the percentage of TFH and, interestingly, with the avidity of the Abs that recognize the SIV gp120 envelope protein. Our findings show changes in the TFH/TFR ratio during chronic infection and suggest possible mechanisms for the unchecked expansion of TFH cells in HIV/SIV infection.

Codelivery of Envelope Protein in Alum with MVA Vaccine Induces CXCR3-Biased CXCR5+ and CXCR5- CD4 T Cell Responses in Rhesus Macaques.

The goal of an HIV vaccine is to generate robust and durable protective Ab. Vital to this goal is the induction of CD4(+) T follicular helper (TFH) cells. However, very little is known about the TFH response to HIV vaccination and its relative contribution to magnitude and quality of vaccine-elicited Ab titers. In this study, we investigated these questions in the context of a DNA/modified vaccinia virus Ankara SIV vaccine with and without gp140 boost in aluminum hydroxide in rhesus macaques. In addition, we determined the frequency of vaccine-induced CD4(+) T cells coexpressing chemokine receptor, CXCR5 (facilitates migration to B cell follicles) in blood and whether these responses were representative of lymph node TFH responses. We show that booster modified vaccinia virus Ankara immunization induced a distinct and transient accumulation of proliferating CXCR5(+) and CXCR5(-) CD4 T cells in blood at day 7 postimmunization, and the frequency of the former but not the latter correlated with TFH and B cell responses in germinal centers of the lymph node. Interestingly, gp140 boost induced a skewing toward CXCR3 expression on germinal center TFH cells, which was strongly associated with longevity, avidity, and neutralization potential of vaccine-elicited Ab response. However, CXCR3(+) cells preferentially expressed the HIV coreceptor CCR5, and vaccine-induced CXCR3(+)CXCR5(+) cells showed a moderate positive association with peak viremia following SIV251 infection. Taken together, our findings demonstrate that vaccine regimens that elicit CXCR3-biased TFH cell responses favor Ab persistence and avidity but may predispose to higher acute viremia in the event of breakthrough infections.

Targeting Antigen to Clec9A Primes Follicular Th Cell Memory Responses Capable of Robust Recall.

Targeting Ags to dendritic cell (DC) surface receptors can induce a variety of responses depending on the DC type targeted, the receptor targeted, and the adjuvant used. Clec9A (DNGR-1), which is expressed by CD8(+) DCs, has been shown to bind F-actin exposed on damaged cells. Targeting Ag to this receptor in mice and nonhuman primates induces strong humoral immunity even in the absence of adjuvant, a process seen for a few select DC receptors. In contrast with other receptors, however, targeting Clec9A induces long-lived, affinity-matured Ab responses that are associated with efficient CD4(+) T cell responses shown to possess properties of follicular Th cells (TFH). In this article, we provide definitive evidence that Clec9A targeting promotes the development of TFH by showing that responding CD4 T cells express CXCR5, PD1, the TFH transcription factor Bcl6, and the cytokine IL-21, and that these cells localize to germinal centers. Furthermore, we extend studies from the model Ag OVA to the viral Ag glycoprotein D of HSV-1 and examine the capacity of primed TFH to form functional memory. We show that targeting glycoprotein D to Clec9A even in the absence of adjuvant induced long-lived memory CXCR5(+) PD1(hi) CD4(+) T cells that proliferated extensively upon secondary challenge and rapidly developed into effector TFH. This was associated with enhanced germinal center B cell responses and accelerated Ab production. Our study indicates that targeting Ags to Clec9A in the absence of adjuvant routinely generates TFH responses that form long-lived memory capable of robust secondary TFH responses.

Abundance and specificity of influenza reactive circulating memory follicular helper and non-follicular helper CD4 T cells in healthy adults.

CD4 T-cell responses are functionally complex and regulate many aspects of innate and adaptive immunity. Follicular helper (Tfh) cells are CD4 T cells specialized to support B-cell production of isotype-switched, high-affinity antibody. So far, studies of Tfh cells in humans have focused on their differentiation requirements, with little research devoted to their antigen specificity. Here, after separating circulating human memory CD4 T cells based on expression of CXCR5, a signature marker of Tfh, we have quantified and assayed the influenza protein antigen specificity of blood Tfh cells and CD4 T cells lacking this marker. Through the use of peptide pools derived from nucleoprotein (NP) or haemagglutinin (HA) and a panel of human donors, we have discovered that circulating Tfh cells preferentially recognize peptide epitopes from HA while cells lacking CXCR5 are enriched for specificity toward NP. These studies suggest that reactive CD4 T cells specific for distinct viral antigens may have generalized differences in their functional potential due to their previous stimulation history.

T follicular helper, but not Th1, cell differentiation in the absence of conventional dendritic cells.

Development of long-lived humoral immunity is dependent on CXCR5-expressing T follicular helper (Tfh) cells, which develop concomitantly to effector Th cells that support cellular immunity. Conventional dendritic cells (cDCs) are critical APCs for initial priming of naive CD4(+) T cells but, importantly, also provide accessory signals that govern effector Th cell commitment. To define the accessory role of cDCs during the concurrent development of Tfh and effector Th1 cells, we performed high-dose Ag immunization in conjunction with the Th1-biased adjuvant polyinosinic:polycytidylic acid (pI:C). In the absence of cDCs, pI:C failed to induce Th1 cell commitment and IgG2c production. However, cDC depletion did not impair Tfh cell differentiation or germinal center formation, and long-lived IgG1 responses of unaltered affinity developed in mice lacking cDCs at the time point for immunization. Thus, cDCs are required for the pI:C-driven Th1 cell fate commitment but have no crucial accessory function in relation to Tfh cell differentiation.

CXCL13-CXCR5 axis promotes the growth and invasion of colon cancer cells via PI3K/AKT pathway.

CXCL13, an inflammatory factor in the microenvironment, plays a vital role in the progression of inflammatory diseases and tumors. CXCL13 and its receptor CXCR5 have been reported to be associated with poor prognosis of advanced colon cancer. However, the molecular mechanisms of CXCL13-CXCR5 axis in colon cancer remain elusive. The aim of this study was to investigate the role of CXCR5-CXCL13 axis in the growth and invasion of colon cancer cells. Our results showed that CXCL13 promoted the growth, migration, and matrigel invasion of colon cancer cells. Furthermore, CXCL13 increased the expression and secretion of MMP-13, and stimulated the activation of PI3K/AKT pathway. After knockdown of CXCR5 by siRNA, the biological functions of colon cancer cells regulated by CXCL13 were significantly inhibited. In addition, inhibition of PI3K/AKT pathway by specific inhibitor LY294002 suppressed the CXCL13-mediated growth, migration, and invasion of colon cancer cells. Together, our findings suggest that CXCL13-CXCR5 axis promotes the growth, migration, and invasion of colon cancer cells, probably via PI3K/AKT pathway. Thus, CXCL13 may be a useful biomarker for the detection and treatment of colon cancer.

C-type lectin-like receptor LOX-1 promotes dendritic cell-mediated class-switched B cell responses.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses.

Expression and clinical significance of CXCR5/CXCL13 in human non­small cell lung carcinoma.

CXCR5 and/or CXCL13 expression is elevated in certain carcinomas and lymphomas. To determine if these factors are involved in progression of non-small cell lung cancer (LuCa), we evaluated their expression in patients with various forms of this disease. Lung biopsies from patients with non-neoplastic cells (n=8), squamous cell carcinoma (SCC; n=24), or adenocarcinoma (AC; n=54) were stained for CXCR5. Histopathological analysis of these samples showed significantly higher expression of CXCR5 (p<0.001) in carcinomas (i.e., SCCs and ACs) relative to non­neoplastic lung tissue. Nuclear and membrane CXCR5 intensities were highest in ACs, with median values of 185 and 130, respectively, followed by SCCs with median values of 170 and 110, respectively. The lowest nuclear and membrane expressions of CXCR5 were found in non-neoplastic tissues, having median values of 142 and 90, respectively. Sera from SCC patients (n=17), AC patients (n=14), and healthy controls (n=9) were tested for the presence of CXCL13. Serum CXCL13 levels in LuCa patients were higher than in healthy controls. CXCR5 expression in cell lines of human non-small cell lung carcinoma (NCI-H1915) and small cell lung carcinoma (SW-1271) were evaluated by flow cytometry. CXCR5 expression was higher in NCI-H1915 cells relative to SW-1271 cells. The functional significance of CXCR5 expression was tested in a migration assay. In response to CXCL13, more NCI-H1915 cells migrated than SW-1271 cells. These findings suggest that the CXCR5­CXCL13 axis influences LuCa progression. After validation in larger patient groups, CXCR5 and CXCL13 may prove useful as biomarkers for LuCa. Correspondingly, blockade of this axis could serve as an effective therapy for LuCa.

Id2 and Id3 maintain the regulatory T cell pool to suppress inflammatory disease.

Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell-specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells.

Binding of HLA-G to ITIM-bearing Ig-like transcript 2 receptor suppresses B cell responses.

Inhibition of B cells constitutes a rational approach for treating B cell-mediated disorders. We demonstrate in this article that the engagement of the surface Ig-like transcript 2 (ILT2) inhibitory receptor with its preferential ligand HLA-G is critical to inhibit B cell functions. Indeed, ILT2-HLA-G interaction impedes both naive and memory B cell functions in vitro and in vivo. Particularly, HLA-G inhibits B cell proliferation, differentiation, and Ig secretion in both T cell-dependent and -independent models of B cell activation. HLA-G mediates phenotypic and functional downregulation of CXCR4 and CXCR5 chemokine receptors on germinal center B cells. In-depth analysis of the molecular mechanisms mediated by ILT2-HLA-G interaction showed a G0/G1 cell cycle arrest through dephosphorylation of AKT, GSK-3ß, c-Raf, and Foxo proteins. Crucially, we provide in vivo evidence that HLA-G acts as a negative B cell regulator in modulating B cell Ab secretion in a xenograft mouse model. This B cell regulatory mechanism involving ILT2-HLA-G interaction brings important insight to design future B cell-targeted therapies aimed at reducing inappropriate immune reaction in allotransplantation and autoimmune diseases.

CXCL13-CXCR5 co-expression regulates epithelial to mesenchymal transition of breast cancer cells during lymph node metastasis.

We investigated the expression of -CXC chemokine ligand 13 (CXCL13) and its receptor -CXC chemokine receptor 5 (CXCR5) in 98 breast cancer (BC) patients with infiltrating duct carcinoma, out of which 56 were found lymph node metastasis (LNM) positive. Interestingly, co-expression of CXCL13 and CXCR5 showed a significant correlation with LNM. Since, epithelial to mesenchymal transition (EMT) is highly associated with metastasis we investigated EMT-inducing potential of CXCL13 in BC cell lines. In CXCL13-stimulated BC cells, expression of various mesenchymal markers (Vimentin, N-cadherin), EMT regulators (Snail, Slug), and matrix metalloproteinase-9 (MMP9) was increased, whereas the expression of epithelial marker E-cadherin was found to be decreased. In addition, expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which is known to regulate MMP9 expression via Src activation, was also significantly increased after CXCL13 stimulation. Using specific protein kinase inhibitors, we confirmed that CXCL13 stimulated EMT and MMP9 expression via RANKL-Src axis in BC cell lines. To further validate this observation, we examined gene expression patterns in primary breast tumors and detected significantly higher expression of various mesenchymal markers and regulators in CXCL13-CXCR5 co-expressing patients. Therefore, this study showed the EMT-inducing potential of CXCL13 as well as demonstrated the prognostic value of CXCL13-CXCR5 co-expression in primary BC. Moreover, CXCL13-CXCR5-RANKL-Src axis may present a therapeutic target in LNM positive BC patients.

Identification of novel markers for mouse CD4(+) T follicular helper cells.

CD4(+) T follicular helper (TFH) cells are central for generation of long-term B-cell immunity. A defining phenotypic attribute of TFH cells is the expression of the chemokine R CXCR5, and TFH cells are typically identified by co-expression of CXCR5 together with other markers such as PD-1, ICOS, and Bcl-6. Herein, we report high-level expression of the nutrient transporter folate R 4 (FR4) on TFH cells in acute viral infection. Distinct from the expression profile of conventional TFH markers, FR4 was highly expressed by naive CD4(+) T cells, was downregulated after activation and subsequently re-expressed on TFH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory TFH cells. Comparative gene expression profiling of FR4(hi) versus FR4(lo) Ag-specific CD4(+) effector T cells revealed a molecular signature consistent with TFH and TH1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in TFH cells compared with TH1 cells, and phenotypic analysis confirmed expression of CD73 on TFH cells. As there is now considerable interest in developing vaccines that would induce optimal TFH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of TFH cells following vaccination and infection.