CD4+CXCR5+ follicular helper T cells in salivary gland promote B cells maturation in patients with primary Sjogren’s syndrome

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⁺CD27^high plasma cells. Also increase of salivary CD19⁺CD27^high 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⁺CD27^high plasma cells, suggesting that CD4⁺CXCR5⁺Tfh cells may contribute to the pathogenesis of pSS by promoting the maturation of B cells.     

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.     

Ligation of TLR7 on CD19+CD1dhi B cells suppresses allergic lung inflammation via regulatory T cells

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B‐cell population is defined as IL‐10‐producing CD19⁺CD1d^hi cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19⁺CD1d^hi B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19⁺CD1d^hi B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL‐10 reporter, Il10^?/? and Tlr7^?/‐ mice, we formally demonstrate that TLR7 ligation of CD19⁺CD1d^hi B cells increases their capacity to produce IL‐10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7‐elicited CD19⁺CD1d^hi B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3⁺ T regulatory cells in allergen‐sensitized mice, we show that that TLR7‐elicited CD19⁺CD1d^hi B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL‐10‐producing CD19⁺CD1d^hi B cells, which can suppress allergic lung inflammation via T regulatory cells.     

T follicular‐like helper cells in the peripheral blood of patients with primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by exocrine gland dysfunction, mainly causing sicca symptoms. B cells have a prominent role in SS, and the T follicular helper (T_FH) cells provide B cells with survival and specialization signals in germinal centres. Here, we investigate peripheral T_FH cells in pSS. Sixteen pSS patients and healthy controls were enrolled in the study, with 13 women and 3 men in each group. Whole blood was collected and separated into PBMC and plasma, followed by cryopreservation. Plasma samples were analysed for Ro52, Ro60 and La48 autoantibodies by indirect ELISA. For flow cytometric analysis, we defined 4 subsets of TFH‐like cells within the CD3⁺CD4⁺CXCR5⁺ population, namely the ICOS⁻PD‐1⁻, ICOS⁻PD‐1⁺, ICOS⁺PD‐1⁻ and ICOS⁺PD‐1⁺ (“TFH”) cells. We also investigated 4 CD19⁺ B cell subsets, the CD20⁺CD27⁺CD38⁻ memory B cells, CD20⁺CD27⁺CD38⁺ memory B cells, CD20⁻CD27⁺CD38⁺⁺CD138⁻ plasmablasts and CD20⁻CD27⁺CD38⁺⁺CD138⁺ plasma cells. We observed higher fractions of ICOS⁺PD‐1⁻ cells, ICOS⁺PD‐1⁺ (“T_FH”) cells and plasmablasts in pSS patients compared to controls, and lower frequencies of both types of memory B cells. The number of T_FH cells correlated positively with the levels of plasmablasts and plasma cells in the pSS patients, but not in the controls. The pSS patients were stratified according to Ro52/Ro60/La48 serology, and a positive association was found between autoantibody levels and increased level of T_FH cells, plasmablasts and plasma cells and lowered levels of memory B cells. We observed a higher response to Ro/La stimulation in pSS patients compared to controls of the memory B cells, although only significantly for the CD38⁻ memory B cells. Overall, a pathological relation between the ICOS⁺ T follicular‐like helper cells and B cells in pSS was observed, but further work should be conducted to explore their overall impact upon disease progression.     

Evidence that cathelicidin peptide LL‐37 may act as a functional ligand for CXCR2 on human neutrophils

LL‐37, derived from human cathelicidin, stimulates immune responses in neutrophils. Although FPR2 and P2X7 were proposed as LL‐37 receptors, we have shown that among 21 neutrophil receptors only CXCR2 was down‐regulated by LL‐37. LL‐37 functions similarly to CXCR2‐specific chemokines CXCL1 and CXCL7 in terms of receptor down‐regulation and intracellular calcium mobilization on freshly isolated neutrophils. Neutrophils pretreated with CXCL8, a chemokine that binds both CXCR1/2, completely blocked the calcium mobilization in response to LL‐37, while LL‐37 also partially inhibited ¹²⁵I‐CXCL8 binding to neutrophils. SB225002, a selective CXCR2 antagonist, blocked LL‐37‐induced calcium mobilization and migration of neutrophils. LL‐37 stimulates calcium mobilization in CXCR2‐transfected HEK293 cells, CXCR2⁺ THP‐1 cells and monocytes, but not in CXCR1‐transfected HEK293 cells. WKYMVm peptide (ligand for FPR2) does not block LL‐37‐stimulated calcium flux in either THP‐1 (FPR2^?) or monocytes (FPR2^high), further confirming the specificity of LL‐37 for CXCR2 and not FPR2. Among all ligands tested (ATP, BzATP, WKYMVm, CXCL1, and LL‐37), only LL‐37 stimulated migration of monocytes (CXCR2⁺ and FPR2⁺) and migration was inhibited by the CXCR2 inhibitor SB225002. Moreover, CXCR2 but not CXCR1 was internalized in LL‐37‐treated neutrophils. Thus, our data provide evidence that LL‐37 may act as a functional ligand for CXCR2 on human neutrophils.     

CXCR4, but not CXCR3, drives CD8+ T‐cell entry into and migration through the murine bone marrow

The BM serves as a blood‐forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T‐cell migration to and localization inside the BM. As BM accumulates many CXCR3‐expressing memory CD8⁺ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8⁺ T cells in BM, is critically important for homing of all CD8⁺ T‐cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8⁺ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM‐1+VCAM‐1+BP‐1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL‐7 and IL‐15. We therefore conclude that CXCR4 is not only crucial for entry of CD8⁺ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.     

CXCR3high CD8+ T cells with naïve phenotype and high capacity for IFN‐γ production are generated during homeostatic T‐cell proliferation

Naïve phenotype (NP) T cells spontaneously initiate homeostatic proliferation (HP) as T‐cell output is reduced because of physiologic thymic involution with age. However, the effects of sustained HP on overall immune function are poorly understood. We demonstrated that the NP CD8⁺ T cell population in adult thymectomized mice showing accelerated HP has an increased capacity for TCR‐mediated interferon‐γ and tumor necrosis factor α production, which is attributed to an increase in CXCR3⁺ cells in the NP CD8⁺ T cell population. The CXCR3⁺ NP CD8⁺ T cells developed during persistent HP with a slow cell division rate, but rarely during robust antigen‐driven proliferation with a fast cell division rate. In ontogeny, the proportions of CXCR3⁺ cells in the NP CD8⁺ T cell population showed a biphasic profile, which was high at the newborn and aged stages. Upon transfer, CXCR3⁺ NP CD8⁺ T cells, but not CXCR3^? NP CD8⁺ T cells, potently enhanced Th17‐mediated inflammatory tissue reactions in vivo. Furthermore, CXCR3^high NP CD8⁺ T cells with similar features were also detected at variable levels in healthy human blood. These results suggest that CXCR3⁺ NP CD8⁺ T cells generated during physiological HP significantly impact overall immunity at the immunologically vulnerable neonatal and aged stages.     

Phenotypes and distribution of mucosal memory B-cell populations in the SIV/SHIV rhesus macaque model

As vaccine-elicited antibodies have now been associated with HIV protective efficacy, a thorough understanding of mucosal and systemic B-cell development and maturation is needed. We phenotyped mucosal memory B-cells, investigated isotype expression and homing patterns, and defined plasmablasts and plasma cells at three mucosal sites (duodenum, jejunum and rectum) in rhesus macaques, the commonly used animal model for pre-clinical vaccine studies. Unlike humans, macaque mucosal memory B-cells lacked CD27 expression; only two sub-populations were present: naïve (CD21⁺CD27⁻) and tissue-like (CD21⁻CD27⁻) memory. Similar to humans, IgA was the dominant isotype expressed. The homing markers CXCR4, CCR6, CCR9 and α4β7 were differentially expressed between naïve and tissue-like memory B-cells. Mucosal plasmablasts were identified as CD19⁺CD20^+/−HLA-DR⁺Ki-67⁺IRF4⁺CD138^+/− and mucosal plasma cells as CD19⁺CD20⁻HLA-DR⁻Ki-67⁻IRF4⁺CD138⁺. Both populations were CD39^+/−CD27⁻. Plasma cell phenotype was confirmed by spontaneous IgA secretion by ELISpot of positively-selected cells and J-chain expression by real-time PCR. Duodenal, jejunal and rectal samples were similar in B-cell memory phenotype, isotype expression, homing receptors and plasmablast/plasma cell distribution among the three tissues. Thus rectal biopsies adequately monitor B-cell dynamics in the gut mucosa, and provide a critical view of mucosal B-cell events associated with development of vaccine-elicited protective immune responses and SIV/SHIV pathogenesis and disease control.     

Generation of precursor,immature, and mature murine B1‐cell lines from c‐myc/bcl‐xL‐overexpressing pre‐BI cells

Deregulated expression of c‐myc and bcl‐xL is long known to generate transformed B cells in humans and mice. We overexpressed these genes to induce in vitro and in vivo differentiation of fetal liver‐derived mouse pre‐BI cells to B1‐lineage pre‐BII‐like, immature and mature B‐cell lines, and to Ig‐secreting cells. In vitro, doxycycline‐controlled c‐myc/bcl‐xL‐overexpressing CD19⁺CD93⁺c‐kikt⁺IgM^? pre‐BI cells differentiate to and survive as CD19⁺CD93⁺c‐kit^?IgM⁺ immature B1 cells. Timed CpG stimulation of these oncogene‐overexpressing pre‐B or immature B1 cells generates either CD19⁺CD93^lowc‐kit^?IgM^?SLC^? pre‐BII‐like or IgM⁺MHCII⁺CD73⁺CD80⁺CD40⁺ mature B1‐cell lines and IgM‐secreting B1 cells in vitro and fixes their state of differentiation. All cell lines are clonable, but a majority of immature and mature B1‐cell clones eventually reach a nonproliferating, surviving G₀‐state. Transplanted in vivo, c‐myc/bcl‐xL‐overexpressing pre‐B cells expand to mature B1 cells, and to IgM‐ and IgA‐secreting plasmablasts and plasma cells. Within 2 months, plasmablasts have expanded most prominently in BM and spleen, indicating that the host selectively expanded development of these transformed plasma cells. The sIgM⁺ B1‐cell lines and clones offer the possibility to study their roles in the development of B1‐Ab repertoires, of B1‐cell‐mediated autoimmune diseases and of B1‐cell malignancies.     

CD19 as a therapeutic target in a spontaneous autoimmune polyneuropathy

Spontaneous autoimmune polyneuropathy (SAP) in B7‐2 knock‐out non‐obese diabetic (NOD) mice is mediated by myelin protein zero (P0)‐reactive T helper type 1 (Th1) cells. In this study, we investigated the role of B cells in SAP, focusing on CD19 as a potential therapeutic target. We found that P0‐specific plasmablasts and B cells were increased in spleens of SAP mice compared to wild‐type NOD mice. Depletion of B cells and plasmablasts with anti‐CD19 monoclonal antibody (mAb) led to attenuation of disease severity when administered at 5 months of age. This was accompanied by decreased serum immunoglobulin (Ig)G and IgM levels, depletion of P0‐specific plasmablasts and B cells, down‐regulation/internalization of surface CD19 and increased frequency of CD4⁺ regulatory T cells in spleens. We conclude that B cells are crucial to the pathogenesis of SAP, and that CD19 is a promising B cell target for the development of disease‐modifying agents in autoimmune neuropathies.     

Age-related differences in human palatine tonsillar B cell subsets and immunoglobulin isotypes

The tonsils provide defense of the upper aerodigestive tract against pathogens. Although long known to undergo functional changes with age, the precise changes occurring within tonsillar B cell populations remain undefined. In the present study, we investigated age-related changes in palatine tonsillar B cell subsets and immunoglobulin (Ig) isotypes. Palatine tonsils were obtained from forty-two tonsillectomy patients without tonsillitis who were divided into three groups: young children (4–9 years), adolescents (10–19 years), and adults (20–60 years). Tonsillar B cells were then analyzed by flow cytometry. Using expression of CD38 and IgD to define B cell subsets, we found that the frequency of germinal center (GC) B cells in the tonsils was significantly higher, and the frequency of memory B cells lower, in young children as compared to adolescents and adults. Within the GC B cell subsets, adults had a higher frequency of IgA⁺ cells and a lower frequency of IgM⁺ cells as compared to individuals in the younger age groups. Moreover, young children had a higher frequency of IgG⁺ cells in the GC B cell subsets than did individuals in the older age groups. We also observed an abundance of IgM⁺ cells among memory B cells and plasmablasts in young children and IgA⁺ cells in adults. In summary, the proportion of GC B cells in palatine tonsillar B cells decreases with age, while the proportion of memory B cells increases with age. In addition, Ig isotypes in tonsils preferentially switch from IgM to IgA as individuals age.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

Higher frequencies of circulating ICOS+, IL-21+ T follicular helper cells and plasma cells in patients with new-onset membranous nephropathy

Background: T follicular helper (TFH) cells and B cells are known to regulate humoral immune responses. This study is aimed at examining the putative contribution of different subsets of circulating of TFH cells and B cells to membranous nephropathy (MN).

Methods: A total of 45?MN patients and 19 healthy controls (HCs) were examined for the number of TFH cells and B cells by flow cytometry. The level of 24-h urinary protein and eGFR were calculated, and the level of serum cytokines was examined. The potential association among these measures was analyzed.

Results: Compared to the HCs, MN patients had significantly higher numbers of circulating CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, and CD4⁺CXCR5⁺CD28⁺ TFH cells, as well as IgD⁺CD27^?CD19⁺ and CD138⁺CD19⁺ B cells. However, the number of IgD⁺CD27⁺CD19⁺ B cells was significantly lower in MN patients than in the HC. The levels of serum IL-21, IL-2, IL-4, IL-10, IL-17A, and IFN-γ were significantly higher in MN patients than in the HC. Furthermore, the numbers of CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, CD4⁺CXCR5⁺CD28⁺ TFH cells, CD138⁺CD19⁺ B cells, and the level of sera IL-21 were negatively correlated with the values of eGFR, but positively correlated with the levels of 24-h urinary proteins. Following treatment, the numbers of CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, CD4⁺CXCR5⁺CD28⁺ TFH cells, CD138⁺CD19⁺ B cells, and the levels of IL-21 were significantly reduced. In contrast, IL-4 and IL-10 levels were noticeably increased after treatment.

Conclusions: Data suggest that activated TFH and plasma cells may contribute to the pathogenesis of MN.     

A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation

Toll‐like receptors (TLRs) recognize specific pathogen‐associated molecular patterns (PAMPs), which subsequently trigger innate immunity. Recent data also suggest a role for TLRs in the direct activation of adaptive immune cells. In the present study, the expression and function of TLR1–TLR10 were characterized in purified human tonsillar B cells, focusing on differences among CD19⁺ CD38^– CD27^– (naïve B cells), CD19⁺ IgD^– CD27^–[germinal centre (GC) B cells] and CD19⁺ CD38^– CD27⁺ (memory B cells) cells. The study was also designed to compare the TLR expression in B cells obtained from infected and hyperplastic tonsils that served as controls. The results demonstrated a distinct repertoire of TLRs, in which TLR1, TLR2, TLR7, TLR9 and TLR10 predominated. No differences were found among naïve, GC and memory B cells. Tonsillar infection did not substantially alter the TLR expression profile in ex vivo‐isolated B‐cell subsets. Purified CD19⁺ B cells stimulated with Pam₃CSK₄, R‐837 and CpG oligodeoxynucleotide (ODN) 2006, via TLR1/TLR2, TLR7 and TLR9, respectively, showed an induction of interleukin‐6 secretion and an up‐regulated expression of human leucocyte antigen (HLA)‐DR. Collectively, the present study demonstrates that B cells exhibit constitutively high levels of specific TLRs, which are not developmentally regulated during the B‐cell differentiation process. Ongoing microbial infections, such as chronic tonsillitis, do not appear to affect the TLR profile in B cells. Furthermore, the distinct expression of TLRs allows B cells to respond directly to the cognate PAMPs. This further emphasizes the role of TLRs in directly activating adaptive immune cells.     

Endothelial expression of CXCR7 and the regulation of systemic CXCL12 levels

The concentration of CXCL12/SDF‐1 in the bloodstream is tightly regulated, given its central role in leucocyte and stem/progenitor cell egress from bone marrow and recruitment to sites of inflammation or injury. The mechanism responsible for this regulation is unknown. Here we show that both genetic deletion and pharmacological inhibition of CXCR7, a high‐affinity CXCL12 receptor, caused pronounced increases in plasma CXCL12 levels. The rise in plasma CXCL12 levels was associated with an impairment in the ability of leucocytes to migrate to a local source of CXCL12. Using a set of complementary and highly sensitive techniques, we found that CXCR7 protein is expressed at low levels in multiple organs in both humans and mice. In humans, CXCR7 was detected primarily on venule endothelium and arteriole smooth muscle cells. CXCR7 expression on venule endothelium was also documented in immunodeficient mice and CXCR7^+/lacZ mice. The vascular expression of CXCR7 therefore gives it immediate access to circulating CXCL12. These studies suggest that endothelial CXCR7 regulates circulating CXCL12 levels and that CXCR7 inhibitors might be used to block CXCL12‐mediated cell migration for therapeutic purposes.     

Increased CD4+CXCR5+T follicular helper cells in diabetic nephropathy

Background: T follicular helper (Tfh) cells are known to regulate humoral immune response. In this study we examined the correlation of different subsets of peripheral blood Tfh cells in patients with diabetic nephropathy (DN). Methods: A total of 23 DN patients and 15 healthy controls (HC) were investigated for various subsets of Tfh cells by flow cytometry. The molecules ICOS⁺, PD-1⁺, CD28⁺, CD154⁺, IL-21⁺, IFN-γ⁺, IL-4⁺, IL-17⁺ Tfh cells were examined. The subsets of B cells were investigated by flow cytometry. The levels of 24?h urinary protein and estimated glomerular filtration rate (eGFR) were calculated. A potential correlation between the number of different subsets of Tfh cells, B cells and DN, was assessed. Results: The circulating CD4⁺CXCR5⁺PD-1⁺, PD-1⁺CD154⁺, PD-1⁺CD28⁺, PD-1⁺IL-21⁺, PD-1⁺IL-4⁺, PD-1⁺-IL-17⁺-Tfh cell counts, CD38⁺CD19⁺, CD38⁺CD19⁺CD40⁺ B cells and plasma levels of IL-21 were significantly increased in DN patients (p?+CXCR5⁺PD-1⁺ Tfh cell counts negatively correlated with eGFR; Tfh cell counts positively correlated with 24?h urinary protein concentration in DN patients. Post-treatment, there was a significant reduction in the CD4⁺CXCR5⁺PD-1⁺ Tfh cell counts and its subsets, with a corresponding decrease in plasma levels of IL-6 and IL-17A (p?Conclusion: An increased number of CD4⁺CXCR5⁺PD-1⁺ Tfh cells were observed in DN patients, which may be new targets for intervention in DN.     

CXCR5 is critically involved in progression of lupus through regulation of B cell and double‐negative T cell trafficking

The recruitment of immune cells to sites of tissue inflammation is orchestrated by chemokine/chemokine receptor networks. Among these, the CXCL13/CXCR5 axis is thought to be involved critically in systemic lupus erythematosus (SLE) and lupus nephritis pathogenesis. Beyond B cell abnormalities, another hallmark of SLE disease is the occurrence of aberrant T cell responses. In particular, double‐negative (DN) T cells are expanded in the peripheral blood of patients with SLE and in lupus‐prone mice. DN T cells induce immunoglobulin production, secrete proinflammatory cytokines and infiltrate inflamed tissue, including kidneys. We aimed to investigate how CXCR5 deficiency changes immune cell trafficking in murine lupus. We therefore crossed CXCR5^–/– mice with B6/lpr mice, a well‐established murine lupus model. B cell numbers and B cellular immune responses were diminished in CXCR5‐deficient B6/lpr mice. In addition, we observed reduced accumulation of DN T cells in spleen and lymph nodes, paralleled by reduced splenomegaly and lymphadenopathy. In‐vivo migration assays revealed reduced migration of CXCR5‐deficient DN T cells into lymph nodes, and ex‐vivo‐activated CXCR5‐deficient DN T cells failed to infiltrate kidneys of recipients. Moreover, DN T cells and B cells of CXCR5‐deficient B6/lpr mice failed to migrate towards CXCL13 in vitro. We propose that CXCR5 is involved critically in B cell trafficking and germinal cell (GC) formation in murine lupus and in guiding pathogenic DN T cells into lymphoid organs and kidneys, and we therefore describe new pathomechanisms for the CXCL13/CXCR5 axis in SLE.     

CD5-positive B cell subsets in secondary progressive multiple sclerosis

Previous studies have demonstrated that CD5⁺ B cells produce more interleukin (IL)-10 than CD5⁻ B cells and that CD5⁺ B cells confer significant protection against experimental autoimmune encephalomyelitis (EAE). The objective of the present study was to determine whether CD5-positive B cell populations are associated with secondary progressive multiple sclerosis (SPMS) and to explore which subsets on CD5⁺ B cells are associated with SPMS. A total of 26 patients with SPMS, of whom 11 were treated with IFNβ (IFN-SPMS) and 15 were not treated (non-IFN-SPMS), and 19 healthy control (HC) subjects were included in the study. Expression levels of CD11a, CD23, CD25, CD38, CD49d, CD80, CD86, CD138, CCR5, and CXCR5 on CD5⁺ B cells in blood samples were examined by flow cytometry. The percentage of CD5⁺ B cells in the SPMS group was significantly lower than in the HC group. Within the subsets of CD5⁺ B cells, the expression of CD11a in the non-IFN-SPMS group was significantly decreased compared to the HC subjects. Patients with SPMS showed lower CCR5, CD25, and CD138 positivity on CD5⁺ B cells than HC subjects. Our results indicate that CD5⁺ B cell subsets might be associated with pathogenesis of SPMS.     

PEG10 Activation by Co-Stimulation of CXCR5 and CCR7 Essentially Contributes to Resistance to Apoptosis in CD19^＋CD34^＋ B Cells from Patients with B Cell Lineage Acute and Chronic Lymphocytic Leukemia

We investigated CD19~+CD34~+ and CD19~+CD34 B cells from cord blood (CB) and typical patients with B celllineage acute and chronic lymphocytic leukemia (B-ALL and B-CLL) in terms of expression and functions ofCXCR5/CXCL13 and CCR7/CCL19.CXCR5 and CCR7 were selectively frequent expressed on B-ALL,B-CLLand CB CD19~+CD34~+ B cells,but not on CD19~+CD34 B cells.Instead of induction of impressive chemotacticresponsiveness,CXCL13 and CCL19 together induced significant resistance to TNF-α-mediated apoptosis inB-ALL and B-CLL but not CB CD19~+CD34~+ B cells.B-ALL and B-CLL CD19~+CD34~+ B cells expressed elevatedlevel of Paternally Expressed Gene 10 (PEG10),and CXCL13 and CCL19 together significantly up-regulatedPEG10 expression in the cells.We found that CXCL13 and CCL19 together by means of activation of CXCR5and CCR7 up-regulated PEG10 expression and function,subsequent stabilized caspase-3 and caspase-8 inB-ALL and B-CLL CD19~+CD34~+ B cells,and rescued the cells from TNF-α-mediated apoptosis.We suggestedthat normal lymphocytes,especially naive B and T cells,utilized CXCR5/CXCL13 and CCR7/CCL19 formigration,homing,maturation,and cell homeostasis as well as secondary lymphoid tissues organogenesis.Meanwhile certain malignant cells took advantages of CXCR5/CXCL13 and CCR7/CCL19 for infiltration,resistance to apoptosis,and inappropriate proliferation.Cellular & Molecular Immunology.2004;1(4):280-294.