CXC chemokine ligand-13 (CXCL13) has been implicated in oral squamous cell carcinoma (OSCC) tumor progression and osteolysis. The tumor necrosis factor family member RANKL (receptor activator of NF-κB ligand), a critical bone resorbing osteoclastogenic factor, has an important role in cancer invasion of bone/osteolysis. Here, we show high-level expression of CXCL13 in primary human OSCC tumor specimens; however, human bone marrow-derived stromal (SAKA-T) and murine preosteoblast (MC3T3-E1) cells produce at very low level. Recombinant CXCL13 (0-15 ng/ml) dose dependently induced CXCR5 expression in SAKA-T and MC3T3-E1 cells. Conditioned media obtained from OSCC cell lines increased the RANKL expression and an antibody against the CXCL13 specific receptor, CXCR5 markedly decreased RANKL expression in these cells. Furthermore, CXCL13 increased hRANKL-Luc promoter activity. Superarray screening identified c-Myc and NFATc3 transcription factors upregulated in CXCL13-stimulated SAKA-T cells. Immunohistochemical analysis of OSCC tumors that developed in athymic mice demonstrated RANKL and NFATc3 expression in tumor and osteoblast cells, however, showed p-c-Myc expression specific to osteoblastic cells at the tumor-bone interface. We further identified NFATc3 expression, but not c-Myc activation in primary human OSCC tumor specimens compared with adjacent normal tissue. Also, CXCL13 significantly increased p-ERK1/2 in SAKA-T and MC3T3-E1 cells. siRNA suppression of c-Myc expression markedly decreased CXCL13-induced RANKL and NFATc3 expression in preosteoblast cells. Chromatin-immuno precipitation assay confirmed p-c-Myc binding to the hRANKL promoter region. In summary, c-Myc activation through CXCL13-CXCR5 signaling axis stimulates RANKL expression in stromal/preosteoblast cells. Thus, our results implicate CXCL13 as a potential therapeutic target to prevent OSCC invasion of bone/osteolysis.

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3-stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083-96. ©2017 AACR.

OBJECTIVE

CXCL13 plays a unique role in the trafficking and homing of B1 cells associated with its cognate receptor, CXCR5. The CXCR5-CXCL13 axis has been previously demonstrated to be a poor prognosis factor in malignancies. However, the clinical significance of the CXCR5-CXCL13 expression in colorectal cancer carcinoma (CRC) remains unclear. The aim of this study was to investigate the CXCR5-CXCL13 expression in CRC and determine its correlation with the progression and prognosis of the tumor.

METHODS

A total of 144 paraffin-embedded specimens with advanced colon cancer were assessed for CXCR5 and CXCL13 by immunohistochemistry. Patients' long-term survival was also monitored. There were significant differences in lymph node metastasis (p = 0.0066), neural invasion (p = 0.0061) and neural invasion (p = 0.0001) between high and low expression of CXCR5.

RESULTS

There were significant differences in distant metastasis (p = 0.0261), TNM stage (p = 0.0409), differentiation (p < 0.0001) and neural invasion of the CXCL13. Both CXCR5 and CXCL13 was associated with poor correlation with the overall survival (OS) and relapse-free survival (RFS).

CONCLUSIONS

Our data suggest that the CXCR5 and CXCL13 may play a crucial role in the development, metastasis and relapse of advanced colon cancer. They can be used as prognostic markers of colon cancer in clinical practice.

OBJECTIVE

The risk of developing metastatic squamous cell carcinoma for patients with head and neck squamous cell carcinoma (HNSCC) is very high. Because these patients are often heavy tobacco users, they are also at risk for developing a second primary cancer, with squamous cell carcinoma of the lung (LSCC) being the most common. The distinction between a lung metastasis and a primary LSCC is currently based on certain clinical and histologic criteria, although the accuracy of this approach remains in question.

METHODS

Gene expression patterns derived from 28 patients with HNSCC or LSCC from a single center were analyzed using penalized discriminant analysis. Validation was done on previously published data for 134 total subjects from four independent Affymetrix data sets.

RESULTS

We identified a panel of 10 genes (CXCL13, COL6A2, SFTPB, KRT14, TSPYL5, TMP3, KLK10, MMP1, GAS1, and MYH2) that accurately distinguished these two tumor types. This 10-gene classifier was validated on 122 subjects derived from four independent data sets and an average accuracy of 96% was shown. Gene expression values were validated by quantitative reverse transcription-PCR derived on 12 independent samples (seven HNSCC and five LSCC). The 10-gene classifier was also used to determine the site of origin of 12 lung lesions from patients with prior HNSCC.

CONCLUSIONS

The results suggest that penalized discriminant analysis using these 10 genes will be highly accurate in determining the origin of squamous cell carcinomas in the lungs of patients with previous head and neck malignancies.

The serological hallmark of primary biliary cirrhosis (PBC) is the presence of high titer and specific antimitochondrial antibodies (AMAs). Although there is no global immune defect in patients with PBC, there is widespread dysregulated B-cell function, including increased sera levels of immunoglobulin M and enhanced B-cell responses to cytosine-phosphate-guanine stimulation. The mechanisms involved in this B-cell dysfunction have remained unknown. To address this issue, we focused on identifying the frequencies of B-cell subsets in patients with PBC and the mechanisms that lead to B-cell dysregulation, including the relationships with chemokine (C-X-C motif) receptor 5 (CXCR5)(+) CD4(+) T cells. Herein, we report that elevations of both serum and intrahepatic interleukin-21 (IL-21) were found in patients with PBC and, in particular, promoted B-cell proliferation, signal transducer and activator of transcription 3 phosphorylation and AMA production in vitro. More important, upon stimulation with recombinant E2 subunit of pyruvate dehydrogenase complex, CXCR5(+) CD4(+) T cells in PBC produced higher levels of IL-21 than healthy controls. Additionally, sorted CXCR5(+) CD4(+) T cells increased production of AMAs by autologous CD19(+) B cells. Indeed, elevated expression of intrahepatic chemokine (C-X-C motif) ligand 13 (CXCL13), a key chemokine of CXCR5(+) cells, was uniquely found within the portal tracts in PBC, accompanied by infiltrates of CD4(+) , CXCR5(+) , CD19(+) , and CD38(+) cells.

CONCLUSIONS

CXCL13 promotes aggregation of CD19(+) B cells and CXCR5(+) CD4(+) T cells, which directs the aberrant AMA response by IL-21. These data have implications for potential immunotherapy and also reflect the unique lymphoid biology in liver of PBC.

The interaction of L-selectin on lymphocytes with sulfated ligands on high endothelial venules (HEVs) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte homing. The MECA-79 monoclonal antibody reports HEV-expressed ligands for L-selectin by recognizing a critical sulfation-dependent determinant on these ligands. HEC-GlcNAc6ST, a HEV-localized sulfotransferase, is essential for the elaboration of functional ligands within lymph nodes, as well as the generation of the MECA-79 epitope. Here, we use an antibody against murine HEC-GlcNAc6ST to study its expression in relationship to the MECA-79 epitope. In lymph nodes, the enzyme is expressed in the Golgi apparatus of high endothelial cells, in close correspondence with luminal staining by MECA-79. In lymph node HEVs of HEC-GlcNAc6ST-null mice, luminal staining by MECA-79 is almost abolished, whereas abluminal staining persists although reduced in intensity. HEV-like vessels in several examples of inflammation-associated lymphoid neogenesis, including nonobese diabetic mice, also exhibit concomitant expression of the sulfotransferase and luminal MECA-79 reactivity. The correlation extends to ectopic lymphoid aggregates within the pancreas of RIP-BLC mice, in which CXCL13 is expressed in islets. Analysis of the progeny of RIP-BLC by HEC-GlcNAc6ST-null mice establishes that the enzyme is responsible for the MECA-79 defined luminal ligands.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation caused by a combination of airways disease (bronchiolitis) and parenchymal destruction (emphysema), whose relative proportion varies from patient to patient.

To explore and contrast the molecular pathogenesis of emphysema and bronchiolitis in COPD.

We used network analysis of lung transcriptomics (Affymetrix arrays) in 70 former smokers with COPD to compare differential expression and gene coexpression in bronchiolitis and emphysema.

We observed that in emphysema (but not in bronchiolitis) (1) up-regulated genes were enriched in ontologies related to B-cell homing and activation; (2) the immune coexpression network had a central core of B cell-related genes; (3) B-cell recruitment and immunoglobulin transcription genes (CXCL13, CCL19, and POU2AF1) correlated with emphysema severity; (4) there were lymphoid follicles (CD20(+)IgM(+)) with active B cells (phosphorylated nuclear factor-κB p65(+)), proliferation markers (Ki-67(+)), and class-switched B cells (IgG(+)); and (5) both TNFRSF17 mRNA and B cell-activating factor protein were up-regulated. These findings were by and large reproduced in a group of patients with incipient emphysema and when patients with emphysema were matched for the severity of airflow limitation of those with bronchiolitis.

Our study identifies enrichment in B cell-related genes in patients with COPD with emphysema that is absent in bronchiolitis. These observations contribute to a better understanding of COPD pathobiology and may open new therapeutic opportunities for patients with COPD.

Long-lived humoral immune responses depend upon the generation of memory B cells and long-lived plasma cells during the germinal center (GC) reaction. These memory compartments, characterized by class-switched IgG and high-affinity Abs, are the basis for successful vaccination. We report that a new member of the plexin family of molecules, plexin-D1, controls the GC reaction and is required for secondary humoral immune responses. Plexin-D1 was not required for B cell maturation, marginal zone precursor development, dark and light zone formation, Igλ(+) and Igκ(+) B cell skewing, B1/B2 development, and the initial extrafollicular response. Plexin-D1 expression was increased following B cell activation, and PlxnD1(-/-) mice exhibited defective GC reactions during T-dependent immune activation. PlxnD1(-/-) B cells showed a defect in migration toward the GC chemokines, CXCL12, CXCL13, and CCL19. Accordingly, PlxnD1(-/-) mice exhibited defective production of IgG1 and IgG2b, but not IgG3 serum Ab, accompanied by reductions in long-lived bone marrow plasmacytes and recall humoral memory responses. These data show a new role for immune plexins in the GC reaction and generation of immunologic memory.

Grb2 (growth-factor receptor-bound protein-2) is a signaling adaptor that interacts with numerous receptors and intracellular signaling molecules. However, its role in B-cell development and function remains unknown. Here we show that ablation of Grb2 in B cells results in enhanced B-cell receptor signaling; however, mutant B cells do not form germinal centers in the spleen after antigen stimulation. Furthermore, mutant mice exhibit defects in splenic architecture resembling that observed in B-cell-specific lymphotoxin-β-deficient mice, including disruption of marginal zone and follicular dendritic cell networks. We find that grb2(-/-) B cells are defective in lymphotoxin-β expression. Although lymphotoxin can be up-regulated by chemokine CXCL13 and CD40 ligand stimulation in wild-type B cells, elevation of lymphotoxin expression in grb2(-/-) B cells is only induced by anti-CD40 but not by CXCL13. Our results thus define Grb2 as a nonredundant regulator that controls lymphoid follicle organization and germinal center reaction. Loss of Grb2 has no effect on B-cell chemotaxis to CXCL13, indicating that Grb2 executes this function by connecting the CXCR5 signaling pathway to lymphotoxin expression but not to chemotaxis.

Dendritic cells (DC) play a pivotal role in regulating immune responses. We previously reported aberrant high production of B lymphocyte chemoattractant (BLC/CXCL13) by DC in aged BWF1 mice, amurine model for systemic lupus erythematosus (SLE). We describe here that CD11b+CD11c+ cells were markedly increased in the peripheral blood (PBL-DC) in aged BWF1, but not in similarly aged NZB or NZW mice. Part of PBL-DC showed a typical dendritic morphology and expressed MHC class II molecules, and had a weak, but significant antigen-presenting ability in mixed lymphocytereaction. PBL-DC were chemoattracted to several chemokines in vitro including secondary lymphoid tissue chemokine (SLC), liver and activation-regulated chemokine (LARC), RANTES, macrophage inflammatory protein-1alpha, whereas splenic mature DC from aged BWF1 mice were preferentially chemoattracted towards SLC. BLC production was induced when PBL-DC were cultured in the presence of TNF-alpha for 3 days. BLC expression was also induced in bone marrow-derived DC when they were differentiated into mature DC in the presence of TNF-alpha and IL-1beta, while both IFN-alpha and IFN-gamma failed to induce BLC expression in bone marrow-derived DC. Since TNF-alpha expression is increased in aged BWF1 mice, DC recruitment in the circulation and maturation into BLC-producing DC by TNF-alpha may play a pivotal role in the development of systemic autoimmune diseases.

BACKGROUND

Intrarenal B cell clusters are associated with poor clinical outcome in acute interstitial rejection. The incidence of B cell aggregates in vascular rejection and the effect of therapy with the monoclonal CD20 antibody rituximab on intrarenal B cells are currently unclear.

METHODS

We analyzed the incidence of B cell clusters in patients with vascular rejection by immunohistochemistry and compared the influence of rituximab treatment plus conventional therapy with that of conventional immunosuppression alone on intrarenal B cells. Furthermore intrarenal expression of the B cell attracting chemokine BCA-1/CXCL13 and the lymphoid chemokine SLC/CCL21 were analyzed.

RESULTS

Nine of 16 patients with vascular rejection displayed intrarenal B cell clusters strictly co-localizing with expression of the B cell attractant chemokine BCA-1/CXCL13. Addition of rituximab to conventional treatment lead to complete depletion of intrarenal B cells (98.3+/-136.4 CD20, 90.7+/-113.2 CD19 vs. 0+/-0 CD20, 0+/-0 CD19 B cells/hpf, P<0.001). Creatinine decreased from 5.0+/-4.1 to 1.9+/-0.4 mg/dl at discharge, and to 1.9+/-0.5 mg/dl after three months (P<0.02). No effect on intrarenal B cells was observed in the patients not treated with rituximab (72.8+/-73.0 vs. 80.3+/-75.3 CD20, 75.6+/-86.6 vs. 85.7+/-82.0 CD19). At discharge, creatinine had improved in this group from 5.1+/-4.1 mg/dl to 1.8+/-0.5 mg/dl and to 1.7+/-0.6 mg/dl after 3 months (P<0.05).

CONCLUSIONS

In summary, our study reports two main findings, namely the previously unrecognized high prevalence of intrarenal B cell clusters in 56% of biopsies with acute vascular rejection and a complete depletion of intrarenal B cells by addition of Rituximab to conventional treatment.

Chronic lymphocytic leukemia (CLL) is an indolent lymphoproliferative disorder characterized by both circulating peripheral disease as well as involvement of the lymph nodes and bone marrow. Increasing evidence suggests that the stromal microenvironment provides anti-apoptotic and pro-survival signals to CLL cells, and may contribute significantly to resistance to a wide variety of treatments. Our understanding of the complex interactions involved in CLL cell trafficking continues to grow. Chemokines and corresponding chemokine receptors are key factors for organizing CLL cell trafficking and homing and the complex cellular interactions between CLL and accessory cells. Important chemokines include CCL3, CCL4, and CCL22, which are released by CLL cells, and CXCL12, CXCL13, CXCL9, 10, 11, CCL 19, and CCL21, which are constitutively secreted by various stromal cells. Integrins such as VLA-4 (CD49d) as well as selectins and CD44 also likely play a role in directing CLL cell migration within the tissue microenvironments. Data are also emerging that other molecules such as MMP-9 and cytoskeletal proteins also contribute to CLL cell trafficking. Though this interplay is complex, it is critical that we improve our understanding of CLL cell trafficking to facilitate the development of novel therapies that target these pathways. Several drugs in clinical development, such as CXCR4 antagonists and PI3K, Btk, and Syk inhibitors appear to modulate CLL cell trafficking and CLL-stroma interactions. Here, we review the current understanding of the molecular interactions that underlie CLL cell trafficking and we highlight some of the promising approaches underway to target these pathways therapeutically in CLL.

BACKGROUND

T follicular helper (T(FH)) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for T(FH) cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified.

METHODS

In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas.

RESULTS

Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed T(FH)-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other T(FH)-associated molecules.

CONCLUSIONS

ICOS is a useful molecule for identifying T(FH) cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a T(FH)-like profile) suggests its inclusion in the antibody panel for diagnosing T(FH)-derived lymphomas. Our findings provide further evidence that the histological spectrum of T(FH)-derived lymphomas is broader than previously assumed.

OBJECTIVE

The B cell chemoattractant chemokine ligand 13 (CXCL13) is emerging as a new biochemical marker in RA. This study was undertaken to dissect the relationship between CXCL13 expression levels in the synovium and clinico-pathological variables relevant to RA pathogenesis and outcome.

METHODS

Synovial tissues from 71 RA patients were evaluated by immunohistochemistry. Thirty paired samples were used for comparative gene expression analysis by quantitative real-time PCR. CXCL13 levels were analysed in relation to cellular, molecular and clinical features of inflammation, lymphocyte activation and joint damage.

RESULTS

In patients with early disease (<12 months duration), CXCL13 expression correlated significantly with synovial markers of local disease activity and systemic inflammation. Such correlation was less evident in established RA. Notably, the association with lymphocyte infiltration and with expression of B/T cell-related activation and proliferation genes, such as activation-induced cytidine deaminase, IFN-γ and IL-2, remained highly significant independent of disease duration and local disease activity. Patients featuring the highest levels of CXCL13 were more frequently ACPA positive and IgG ACPA titres were increased in the high CXCL13 expression group. Furthermore, the frequency of erosive disease on radiographs was significantly higher in the upper tertile of CXCL13 expression (P = 0.01 with adjustment for disease duration and ACPA). Accordingly, synovial CXCL13 and the local receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) ratio significantly co-varied (ρ = 0.52, P < 0.01), independent of the level of local inflammation.

CONCLUSIONS

Synovial CXCL13 appears to be a marker of a more severe pattern of RA disease, characterized by increased lymphocyte activation and bone remodelling beyond the level of conventional markers of inflammation.

The aim of this study is to set up single molecular secreted phosphoprotein 1 (SPP1) upstream invasive network of lung adenocarcinoma. This paper proposed an integrated method based on linear programming and a decomposition procedure with integrated analysis of the significant function cluster using Kappa statistics and fuzzy heuristic clustering. Our study proved that only modules appearing in lung adenocarcinoma include cytokine module (CXCL13, GREM1_2 inhibition), cell adhesion module (COL11A1_2 activation; CDH3 inhibition), and receptor binding module (NMU activation; CXCL13, GREM1_2 inhibition), which increase the invasion of cancer cell. We compared skeletal development, signal, biological regulation, sequence variant modules between human normal adjacent tissues and lung adenocarcinoma. SPP1 skeletal development module appears in human normal adjacent tissues (COL11A1_1 activation; COL10A1 inhibition), whereas in lung adenocarcinoma (COL11A1_2, COL1A2 activation); signal module appears in human normal adjacent tissues (COL11A1_1, CXCL13, MMP11, SPINK1 activation; COL10A1, COL3A1 inhibition), whereas in lung adenocarcinoma (COL11A1_2, COL1A2, MMP12 activation; CDH3, CXCL13, GREM1_2, MMP11, SPINK1 inhibition); biological regulation module appears in human normal adjacent tissues (CXCL13, MKI67, PYCR1 activation; NEK2, SPDEF, TOP2A_2, TOX3_1 inhibition), whereas in lung adenocarcinoma (HMGB3, MKI67, NMU, PYCR1, TOX3_2 activation; CXCL13, SPDEF, TOP2A_2 inhibition); sequence variant module appears in human normal adjacent tissues (COL11A1_1, MKI67, MMP11 activation; ASPM, COL10A1, COL3A1, NEK2, TMPRSS4, TOP2A_2 inhibition), whereas in lung adenocarcinoma (COL11A1_2, COL1A2, HMMR, MKI67, MMP12 activation; ABCC3, ASPM, CDH3, MMP11, TOP2A_2 inhibition). It can be deduced that modules above in human normal adjacent tissues reflect the invasive inhibition of normal cells, whereas in lung adenocarcinoma increase the invasion of cancer cell. Our study of SPP1 upstream invasive network may be useful to identify novel and potentially targets for prognosis and therapy of lung adenocarcinoma.

We investigated thoroughly the effect of lncRNA PART1 on prostate cancer cells proliferation and apoptosis, through regulating toll-like receptor (TLR) pathways. LncRNA PART1 expression was also examined by quantitative real-time polymerase chain reactions (qRT-PCR) in human tissues and the cells lines LNCaP and PC3. After transfection with si-PART1 or control constructs, the cell viability was measured by MTS and colony formation assays. In addition, the apoptosis rate of the prostate cancer cells was validated by TUNEL staining. Relationships between lncRNA PART1 expression and TLR pathway genes were demonstrated by qRT-PCR and Western blotting. High levels of lncRNA PART1 expression were correlated with advanced cancer stage and predication of poor survival. LncRNA PART1 levels was increased in PCa cells treated with 5α-dihydrotestosterone (DHT), confirming PART1 was directly induced by androgen. Moreover, down-regulation of lncRNA PART1 inhibited prostate cancer cell proliferation and accelerated cell apoptosis. In addition, lncRNA PART1 induced downstream genes expression in TLR pathways including TLR3, TNFSF10 and CXCL13 to further influence prostate cancer cells, indicating its carcinogenesis on prostate cancer. LncRNA PART1 promoted cell proliferation ability and apoptosis via the inhibition of TLR pathways in prostate cancer. LncRNA PART1 could hence be considered as a new target in the treatment of prostate cancer.

BACKGROUND

CXC chemokines are induced by inflammatory stimuli in epithelial cells and some, like MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, are antibacterial for Streptococcus pyogenes.

RESULTS

SpeB from S. pyogenes degrades a wide range of chemokines (i.e. IP10/CXCL10, I-TAC/CXCL11, PF4/CXCL4, GROalpha/CXCL1, GRObeta/CXCL2, GROgamma/CXCL3, ENA78/CXCL5, GCP-2/CXCL6, NAP-2/CXCL7, SDF-1/CXCL12, BCA-1/CXCL13, BRAK/CXCL14, SRPSOX/CXCL16, MIP-3alpha/CCL20, Lymphotactin/XCL1, and Fractalkine/CX3CL1), has no activity on IL-8/CXCL8 and RANTES/CCL5, partly degrades SRPSOX/CXCL16 and MIP-3alpha/CCL20, and releases a 6 kDa CXCL9 fragment. CXCL10 and CXCL11 loose receptor activating and antibacterial activities, while the CXCL9 fragment does not activate the receptor CXCR3 but retains its antibacterial activity.

CONCLUSIONS

SpeB destroys most of the signaling and antibacterial properties of chemokines expressed by an inflamed epithelium. The exception is CXCL9 that preserves its antibacterial activity after hydrolysis, emphasizing its role as a major antimicrobial on inflamed epithelium.

T follicular helper cells (Tfh) are important regulators of humoral responses. Human Tfh polarization pathways have been thus far associated with Th1 and Th17 polarization pathways. How human Tfh cells differentiate in Th2-skewed environments is unknown. We show that thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DCs) promote human Tfh differentiation from naive CD4 T cells. We identified a novel population, distinct from Th2 cells, expressing IL-21 and TNF, suggestive of inflammatory cells. TSLP-induced T cells expressed CXCR5, CXCL13, ICOS, PD1, BCL6, BTLA, and SAP, among other Tfh markers. Functionally, TSLP-DC-polarized T cells induced IgE secretion by memory B cells, and this depended on IL-4Rα. TSLP-activated DCs stimulated circulating memory Tfh cells to produce IL-21 and CXCL13. Mechanistically, TSLP-induced Tfh differentiation depended on OX40-ligand, but not on ICOS-ligand. Our results delineate a pathway of human Tfh differentiation in Th2 environments.

Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair.

The chemokine CXCL13 mediates recruitment of B cells to tumors and is essential for the formation of tertiary lymphoid structures (TLSs). TLSs are thought to support antitumor immunity and are associated with improved prognosis. However, it remains unknown whether TLSs are formed in response to the general inflammatory character of the tumor microenvironment, or rather, are induced by (neo)antigen-specific adaptive immunity. We here report on the finding that the TGFβ-dependent CD103⁺CD8⁺ tumor-infiltrating T-cell (TIL) subpopulation expressed and produced CXCL13. Accordingly, CD8⁺ T cells from peripheral blood activated in the presence of TGFβ upregulated CD103 and secreted CXCL13. Conversely, inhibition of TGFβ receptor signaling abrogated CXCL13 production. CXCL13⁺CD103⁺CD8⁺ TILs correlated with B-cell recruitment, TLSs, and neoantigen burden in six cohorts of human tumors. Altogether, our findings indicated that TGFβ plays a noncanonical role in coordinating immune responses against human tumors and suggest a potential role for CXCL13⁺CD103⁺CD8⁺ TILs in mediating B-cell recruitment and TLS formation in human tumors.

The SWI/SNF chromatin remodeling complex plays pivotal roles in mammalian transcriptional regulation. In this study, we identify the human requiem protein (REQ/DPF2) as an adaptor molecule that links the NF-kappaB and SWI/SNF chromatin remodeling factor. Through in vitro binding experiments, REQ was found to bind to several SWI/SNF complex subunits and also to the p52 NF-kappaB subunit through its nuclear localization signal containing the N-terminal region. REQ, together with Brm, a catalytic subunit of the SWI/SNF complex, enhances the NF-kappaB-dependent transcriptional activation that principally involves the RelB/p52 dimer. Both REQ and Brm were further found to be required for the induction of the endogenous BLC (CXCL13) gene in response to lymphotoxin stimulation, an inducer of the noncanonical NF-kappaB pathway. Upon lymphotoxin treatment, REQ and Brm form a larger complex with RelB/p52 and are recruited to the BLC promoter in a ligand-dependent manner. Moreover, a REQ knockdown efficiently suppresses anchorage-independent growth in several cell lines in which the noncanonical NF-kappaB pathway was constitutively activated. From these results, we conclude that REQ functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in noncanonical NF-kappaB transcriptional activation and its associated oncogenic activity.

Relapsing fever (RF) is a multisystemic borrelial infection with frequent neurologic involvement referred to as neuroborreliosis. The absence of an effective antibody response results in persistent infection. To study the consequences to the brain of persistent infection with the RF spirochete Borrelia turicatae, we studied B cell (Igh6-/-) and B and T (Rag1-/-) cell-deficient mice inoculated with isogenic serotypes 1 (Bt1) or 2 (Bt2). We found that Bt1 was more tissue tropic than Bt2, not only for brain but also for heart. Igh6-/- mice developed more severe clinical disease than Rag1-/- mice. Bt1-infected brains had widespread microgliosis/brain macrophage activation despite localization of spirochetes in the leptomeninges rather than the brain parenchyma itself. Oligoarray analysis revealed that CXCL13 was the most upregulated gene in the brain of Bt1-infected Igh6-/- mice. CXCL13 was also the most abundant of the chemokines we measured in infected blood. Persistent infection did not result in injury to the brain. Treatment with exogenous interleukin-10 reduced microgliosis in the brain and production of CXCL13 in the blood. We concluded that brain involvement in B cell-deficient mice persistently infected with B. turicatae is characterized by prominent microgliosis and production of CXCL13 without detectable injury.

BACKGROUND

Prospective cohort studies often quantify serum immune biomarkers at a single time point to determine risk of cancer and other chronic diseases that develop years later. Estimates of the within-person temporal stability of serum markers partly assess the utility of single biomarker measurements and may have important implications for the design of prospective studies of chronic disease risk.

METHODS

Using archived sera collected from 200 HIV-seronegative men at three visits spaced over approximately 2 years, concentrations of 14 biomarkers (ApoA1, sCD14, sgp130, sIL-6R, sIL-2Rα, sTNFR2, BAFF/BLyS, CXCL13, IFN-γ, interleukin [IL]-1β, IL-6, IL-8, IL-10, and TNF-α) were measured in a single laboratory. Age- and ethnicity-adjusted intraclass correlation coefficients (ICC) were calculated for each biomarker, and mixed linear regression models were used to examine the influence of age, ethnicity, season, and study site on biomarker concentrations.

RESULTS

Across all three study visits, most biomarkers had ICC values indicating fair to excellent within-person stability. ApoA1 (ICC = 0.88) and TNF-α (ICC = 0.87) showed the greatest stability; the ICC for IL-8 (ICC = 0.33) was remarkably less stable. The ICCs were similar when calculated between pairs of consecutive visits. The covariables did not influence biomarker levels or their temporal stability. All biomarkers showed moderate to strong pairwise correlations across visits.

CONCLUSIONS

Serum concentrations of most evaluated immune biomarkers displayed acceptable to excellent within-person temporal reliability over a 2-year period. Further investigation may be required to clarify the stability of IL-8.

CONCLUSIONS

These findings lend support to using these serologic immune biomarkers in prospective studies investigating associations with chronic diseases.

Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-β deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.