The generation of high-affinity antibody-secreting plasma cells critically depends on the presence of CD4 T cells during the germinal center (GC) reaction. GC T cells are so far incompletely characterized in terms of phenotype and function. Here, we show that human follicular B helper T (T(FH)) cells are characterized by high expression of the homeostatic chemokine receptor CXCR5 and the costimulatory molecule ICOS, but not CD57 expression. CXCR5(hi)ICOS(hi) CD4 T cells are the most potent inducers of IgG production that also secrete large amounts of the B cell-attracting chemokine CXCL13. CXCR5(hi)ICOS(hi) CD4 T cells differ from other tonsillar CD4 T cell subsets in their stimulatory activity, proliferative capacity and susceptibility to apoptosis. Large-scale gene expression analysis revealed that T(FH) cells are only distantly related to CXCR5(-) and CXCR5(+) central memory T (T(CM)) as well as effector memory T (T(EM)) cells present in the periphery. CXCR5(hi)ICOS(hi) CD4 T cells appear to be terminally differentiated T helper cells that express a unique set of transcription factors related to the Notch signaling pathway and thus differentiate independent of other T helper cell populations.

High expression of CXCR5 is one of the defining hallmarks of T follicular helper cells (T(FH)), a CD4 Th cell subset that promotes germinal center reactions and the selection and affinity maturation of B cells. CXCR5 is also expressed on 20-25% of peripheral blood human central memory CD4 T cells (T(CM)), although the definitive function of these cells is not fully understood. The constitutive expression of CXCR5 on T(FH) cells and a fraction of circulating T(CM) suggests that CXCR5(+) T(CM) may represent a specialized subset of memory-type T(FH) cells programmed for homing to follicles and providing B cell help. To verify this assumption, we analyzed this cell population and show its specialized function in supporting humoral immune responses. Compared with their CXCR5(-) T(CM) counterparts, CXCR5(+) T(CM) expressed high levels of the chemokine CXCL13 and efficiently induced plasma cell differentiation and Ig secretion. We found that the distinct B cell helper qualities of CXCR5(+) T(CM) were mainly due to high ICOS expression and pronounced responsiveness to ICOS ligand costimulation together with large IL-10 secretion. Furthermore, B cell helper attributes of CXCR5(+) T(CM) were almost exclusively acquired on cognate interaction with B cells, but not with dendritic cells. This implies that a preferential recruitment of circulating CXCR5(+) T(CM) to CXCL13-rich B cell follicles is required for the promotion of a quick and efficient protective secondary humoral immune response. Taken together, we propose that CXCR5(+) T(CM) represent a distinct memory cell subset specialized in supporting Ab-mediated immune responses.

Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3+ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR = 2.13, P = 0.0008). An immune response gene expression signature and presence of TILs and CD3+ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.

Evidence from mouse chronic viral infection models suggests that CD8+ T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8+ T lymphocyte populations with high (PD-1T), intermediate (PD-1N) and no PD-1 expression (PD-1-) from non-small-cell lung cancer patients. PD-1T T cells showed a markedly different transcriptional and metabolic profile from PD-1N and PD-1- lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1T lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1T cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention.

Bronchus-associated lymphoid tissue (BALT) was originally described as a mucosal lymphoid organ in the lungs of some species. However, while the lungs of naive mice and humans typically lack BALT, pulmonary infection in mice leads to the development of inducible BALT (iBALT), which is located in peribronchial, perivascular, and interstitial areas throughout the lung. Here we investigated whether iBALT forms in patients with a variety of interstitial lung diseases. We show that while iBALT can be found in the lungs of patients suffering from multiple diseases, well-developed iBALT is most prevalent in patients with pulmonary complications of RA and Sjögren syndrome. In these patients, iBALT consisted of numerous B cell follicles containing germinal centers and follicular dendritic cells. A loosely defined T cell area surrounded the B cell follicles while lymphatics and high endothelial venules were found at the B cell/T cell interface. Increased expression of lymphoid-organizing chemokines, such as CXCL13 and CCL21, as well as molecules involved in the immunopathology of RA, such as B cell-activating factor of the TNF family (BAFF), ICOS ligand, and lymphotoxin, correlated with more well-developed iBALT. Finally, the presence of iBALT correlated with tissue damage in the lungs of RA patients, suggesting that iBALT participates in local RA pathogenesis.

The molecular alterations underlying the pathogenesis of angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, unspecified (PTCL-u) are largely unknown. In order to characterize the ontogeny and molecular differences between both entities, a series of AITLs (n = 18) and PTCLs-u (n = 16) was analyzed using gene expression profiling. Unsupervised clustering correlated with the pathological classification and with CD30 expression in PTCL-u. The molecular profile of AITLs was characterized by a strong microenvironment imprint (overexpression of B-cell- and follicular dendritic cell-related genes, chemokines, and genes related to extracellular matrix and vascular biology), and overexpression of several genes characteristic of normal follicular helper T (T(FH)) cells (CXCL13, BCL6, PDCD1, CD40L, NFATC1). By gene set enrichment analysis, the AITL molecular signature was significantly enriched in published T(FH)-specific genes. The enrichment was higher for sorted AITL cells than for tissue samples. Overexpression of several T(FH) genes was validated by immunohistochemistry in AITLs. A few cases with molecular T(FH)-like features were identified among CD30(-) PTCLs-u. Our findings strongly support that T(FH) cells represent the normal counterpart of AITL, and suggest that the AITL spectrum may be wider than suspected, as a subset of CD30(-) PTCLs-u may derive from or be related to AITL.

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

BACKGROUND

Lyme neuroborreliosis (LNB) is a nervous system infection caused by Borrelia burgdorferi sensu lato (Bb).

OBJECTIVE

To present evidence-based recommendations for diagnosis and treatment.

METHODS

Data were analysed according to levels of evidence as suggested by EFNS.

CONCLUSIONS

The following three criteria should be fulfilled for definite LNB, and two of them for possible LNB: (i) neurological symptoms; (ii) cerebrospinal fluid (CSF) pleocytosis; (iii) Bb-specific antibodies produced intrathecally. PCR and CSF culture may be corroborative if symptom duration is <6 weeks, when Bb antibodies may be absent. PCR is otherwise not recommended. There is also not enough evidence to recommend the following tests for diagnostic purposes: microscope-based assays, chemokine CXCL13, antigen detection, immune complexes, lymphocyte transformation test, cyst formation, lymphocyte markers. Adult patients with definite or possible acute LNB (symptom duration <6 months) should be offered a single 14-day course of antibiotic treatment. Oral doxycycline (200 mg daily) and intravenous (IV) ceftriaxone (2 g daily) are equally effective in patients with symptoms confined to the peripheral nervous system, including meningitis (level A). Patients with CNS manifestations should be treated with IV ceftriaxone (2 g daily) for 14 days and late LNB (symptom duration >6 months) for 3 weeks (good practice points). Children should be treated as adults, except that doxycycline is contraindicated under 8 years of age (nine in some countries). If symptoms persist for more than 6 months after standard treatment, the condition is often termed post-Lyme disease syndrome (PLDS). Antibiotic therapy has no impact on PLDS (level A).

Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8(+) cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFβ receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.

OBJECTIVE

To investigate functional properties of the germinal center (GC)-like structures observed in salivary glands of patients with Sjögren's syndrome (SS) and to determine the frequency with which such structures develop.

METHODS

Hematoxylin and eosin-stained sections from 165 minor salivary gland biopsy samples were screened for GC-like structures. Expression of markers for GCs (CD3, CD20, Ki-67, CD35, CD31), adhesion molecules (intercellular adhesion molecule 1, lymphocyte function-associated antigen 1, vascular cell adhesion molecule 1, very late activation antigen 4), chemokines (CXCL13, CCL21, CXCL12), and production of autoantibodies (anti-Ro/SSA and anti-La/SSB) was investigated by immunohistochemistry. Apoptosis was investigated by TUNEL staining.

RESULTS

GC-like structures were observed in 28 of 165 patients (17%). When GCs were defined as T and B cell aggregates with proliferating cells with a network of follicular dendritic cells and activated endothelial cells, such microenvironments were found in all patients in whom structures with GC-like morphology were observed. The defined microenvironments were not found in patients without apparent GC-like structures. The GCs formed within the target tissue showed functional features with production of autoantibodies (anti-Ro/SSA and anti-La/SSB) and apoptotic events (by TUNEL staining), and the local production of anti-Ro/SSA and anti-La/SSB autoantibodies was significantly increased (P = 0.04) in patients with GC development.

CONCLUSIONS

Lymphoid neogenesis and functional ectopic GC formation take place in salivary glands of a subset of patients with SS. Our data suggest that the ectopic secondary lymphoid follicles contain all elements needed for driving the autoimmune response. Our findings underscore a key role for the target organ in recruitment of inflammatory cells and propagation of the disease process.

The chemokines CCL21 and CXCL13 are immune factors that dictate homing and motility of lymphocytes and dendritic cells in lymphoid tissues. However, the means by which these chemokines are regulated and how they influence cell trafficking during immune responses remain unclear. We show that CCL21 and CXCL13 are transiently down-regulated within lymphoid tissues during immune responses by a mechanism controlled by the cytokine interferon-gamma. This modulation was found to alter the localization of lymphocytes and dendritic cells within responding lymphoid tissues. As a consequence, priming of T cell responses to a second distinct pathogen after chemokine modulation became impaired. We propose that this transient chemokine modulation may help orchestrate local cellularity, thus minimizing competition for space and resources in activated lymphoid tissues.

How Tregs migrate to GCs, and whether they regulate the helper activity of the T cells in GCs (GC-Th cells) remains poorly understood. We found a T cell subset in human tonsils that displays potent suppressive activities toward GC-Th cell-dependent B cell responses. These Tregs with the surface phenotype of CD4+CD25+CD69- migrate well to CCL19, a chemokine expressed in the T cell zone, but poorly to CXCL13, a chemokine expressed in the B cell zone. This migration toward the T cell-rich zone rapidly changes to trafficking toward B cell follicles upon T cell activation. This change in chemotactic behavior upon activation of T cells is consistent with their switch in the expression of the 2 chemokine receptors CXCR5 and CCR7. CD4+CD25+CD69- Tregs suppress GC-Th cells and GC-Th cell-induced B cell responses such as Ig production, survival, and expression of activation-induced cytosine deaminase. Our results have identified a subset of Tregs that is physiologically relevant to GC-Th cell-dependent B cell responses and a potential regulation mechanism for the trafficking of these Tregs to GCs.

Macrophages are either classically (M1) or alternatively-activated (M2). Whereas this nomenclature was generated from monocyte-derived macrophages treated in vitro with defined cytokine stimuli, the phenotype of in vivo-derived macrophages is less understood. We completed Affymetrix-based transcriptomic analysis of macrophages from the resolution phase of a zymosan-induced peritonitis. Compared with macrophages from hyperinflamed mice possessing a pro-inflammatory nature as well as naive macrophages from the uninflamed peritoneum, resolution-phase macrophages (rM) are similar to monocyte-derived dendritic cells (DCs), being CD209a positive but lacking CD11c. They are enriched for antigen processing/presentation (MHC class II [H2-Eb1, H2-Ab1, H2-Ob, H2-Aa], CD74, CD86), secrete T- and B-lymphocyte chemokines (Xcl1, Ccl5, Cxcl13) as well as factors that enhance macrophage/DC development, and promote DC/T cell synapse formation (Clec2i, Tnfsf4, Clcf1). rM are also enriched for cell cycle/proliferation genes as well as Alox15, Timd4, and Tgfb2, key systems in the termination of leukocyte trafficking and clearance of inflammatory cells. Finally, comparison with in vitro-derived M1/M2 shows that rM are neither classically nor alternatively activated but possess aspects of both definitions consistent with an immune regulatory phenotype. We propose that macrophages in situ cannot be rigidly categorized as they can express many shades of the inflammatory spectrum determined by tissue, stimulus, and phase of inflammation.

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

Though much is known about the function of T lymphocytes in the adaptive immune response against Mycobacterium tuberculosis, comparably little is understood regarding the corresponding role of B lymphocytes. Indicating B cells as components of lymphoid neogenesis during pulmonary tuberculosis, we have identified ectopic germinal centers (GCs) in the lungs of infected mice. B cells in these pulmonary lymphoid aggregates express peanut agglutinin and GL7, two markers of GC B cells, as well as CXCR5, and migrate in response to the lymphoid-associated chemokine CXCL13 ex vivo. CXCL13 is negatively regulated by the presence of B cells, as its production is elevated in lungs of B cell-deficient (B cell(-/-)) mice. Upon aerosol with 100 CFU of M. tuberculosis Erdman, B cell(-/-) mice have exacerbated immunopathology corresponding with elevated pulmonary recruitment of neutrophils. Infected B cell(-/-) mice show increased production of IL-10 in the lungs, whereas IFN-gamma, TNF-alpha, and IL-10R remain unchanged from wild type. B cell(-/-) mice have enhanced susceptibility to infection when aerogenically challenged with 300 CFU of M. tuberculosis corresponding with elevated bacterial burden in the lungs but not in the spleen or liver. Adoptive transfer of B cells complements the phenotypes of B cell(-/-) mice, confirming a role for B cells in both modulation of the host response and optimal containment of the tubercle bacillus. As components of ectopic GCs, moderators of inflammatory progression, and enhancers of local immunity against bacterial challenge, B cells may have a greater role in the host defense against M. tuberculosis than previously thought.

OBJECTIVE

To identify gene expression signatures in minor salivary glands (MSGs) from patients with primary Sjogren's syndrome (SS).

METHODS

A 16K complementary DNA microarray was used to generate gene expression profiles in MSGs obtained from 10 patients with primary SS and 10 control subjects. The data were analyzed by 2 different strategies, one strict primary analysis and one subanalysis that allowed for inclusion of genes with no signal in more than 3 samples from each group. The results were validated by quantitative reverse transcriptase-polymerase chain reaction techniques.

RESULTS

We found a distinct difference in gene expression levels in MSGs, enabling a simple class prediction method to correctly classify 19 of the 20 samples as either patient or control, based on the top 5 differentially expressed genes. The 50 most differentially expressed genes in the primary SS group compared with the control group were all up-regulated, and a clear pattern of genes involved in chronic inflammation was found. <em>CXCL13</em> and CD3D were expressed in>>/=90% of primary SS patients and in </=10% of the controls. Lymphotoxin beta, as well as a number of major histocompatibility complex genes, cytokines, and lymphocyte activation factors, manifested its role in the pathogenesis of SS. Numerous type I interferon genes related to virus infection were found among the top 200 genes, with increased expression in primary SS. Interestingly, the expression of carbonic anhydrase II, which is essential in saliva production and secretion, and the apoptosis regulator Bcl-2-like 2 were down-regulated in primary SS patients.

CONCLUSIONS

We have identified distinct gene expression profiles in MSGs from patients with primary SS that provide new knowledge about groups of genes that are up-regulated or down-regulated during disease, constituting an excellent platform for forthcoming functional studies.

Antigen-driven T cell education and subsequent pathogen elimination present particular challenges for the immune system. Pathogens generally enter the body at peripheral sites such as the skin, gastrointestinal tract or lung, areas from which naïve T cells are largely excluded. Instead, naïve T cells constantly recirculate through secondary lymphoid organs, such as lymph nodes and Peyer's patches, in search for antigen brought to these locations by means of afferent lymphatic channels. Here, antigen-loaded dendritic cells present antigen-peptide-MHC complexes to clonotypic T cells and provide appropriate co-stimulatory signals for immune response initiation. As a result, short-lived effector T cells and long-lived memory T cells are generated that reach the peripheral tissue for participation in immune responses and immune surveillance. Effector and memory T cell relocation is non-random, due to tissue-specific "address codes" that allow proper tissue homing. This process involves adhesion molecules, including selectins, integrins, and corresponding vascular ligands as well as the large family of chemokines and their receptors. Here, we discuss the changes in chemokine receptor expression that occur during T cell activation and differentiation, and the ways in which these changes impact on the migration potential of naïve, effector, and memory T cells. We summarize our current understanding of T cell homing to the T zone and B cell follicles within secondary lymphoid tissues and highlight the two chemokine receptors CCR7 and CXCR5 that recognize chemokines constitutively present either in the T zone (CCR7 ligands CCL19/ELC and CCL21/SLC) or follicular compartment (CXCR5 ligand CXCL13/BCA-1). CCR7 is characteristic for naive and central memory T (T(CM)) cells whereas CXCR5 distinguishes follicular B helper T (T(FH)) cells. In addition, we further subdivide long-lived memory T cells into CCR7-negative effector memory T (T(EM)) cells and peripheral immune surveillance T (T(PS)) cells. The latter term designates the extraordinarily large subset of memory T cells with primary residence in normal (healthy) peripheral tissues. Our current understanding of T(PS) cell migration and function is highly fragmentary, but these cells are thought to provide immediate protection locally at the site of pathogen entry. Here, we propose that the tissue distribution of T(PS) cells is determined by a distinct set of chemokines and corresponding receptors that differs from those operating in secondary lymphoid tissues and inflammatory sites.

Antigenic stimulation through the B-cell antigen receptor (BCR) is considered to promote the expansion of chronic lymphocytic leukemia (CLL) B cells. The spleen tyrosine kinase (Syk), a key component of BCR signaling, can be blocked by R406, a small-molecule Syk inhibitor, that displayed activity in CLL patients in a first clinical trial. In this study, we investigated the effects of BCR stimulation and R406 on CLL cell survival and migration. The prosurvival effects promoted by anti-IgM stimulation and nurselike cells were abrogated by R406. BCR triggering up-regulated adhesion molecules, and increased CLL cell migration toward the chemokines CXCL12 and CXCL13. BCR activation also enhanced CLL cell migration beneath marrow stromal cells. These responses were blocked by R406, which furthermore abrogated BCR-dependent secretion of T-cell chemokines (CCL3 and CCL4) by CLL cells. Finally, R406 inhibited constitutive and BCR-induced activation of Syk, extracellular signal-regulated kinases, and AKT, and blocked BCR-induced calcium mobilization. These findings suggest that BCR activation favors CLL cell homing, retention, and survival in tissue microenvironments. R406 effectively blocks these BCR-dependent responses in CLL cells, providing an explanation for the activity of R406 in patients with CLL.

Clonally expanded populations of B cells carrying somatic mutations of Ig variable (V) region genes have been detected in the CNS of subjects with multiple sclerosis (MS), suggesting that a process of B cell affinity maturation with ensuing production of potentially pathogenic autoantibodies may occur inside the CNS. Here, we have characterized the B cell subsets present in the cerebrospinal fluid (CSF) of MS patients and of individuals with other inflammatory neurological disorders by flow cytometry. CD19(+)CD38(high+)CD77(+), Ki67(+), Bcl-2(-) centroblasts, i.e., a B cell subset found exclusively in secondary lymphoid organs, were detected in the CSF but not in paired peripheral blood from both patient groups. CD27(+)IgD(-) memory B cells, i.e., cells with hyper-mutated IgV genes, were significantly increased in the CSF vs. paired peripheral blood and displayed up-regulation of the CD80 and CD86 costimulatory molecules and of CC chemokine receptor (CCR) 1, CCR2, and CCR4 in both patient groups. Lymphotoxin-alpha, CXC ligand (CXCL) 12, and CXCL13, key mediators of lymphoid neogenesis, were present in the CSF from patients with MS and other inflammatory neurological disorders and were expressed in MS brain tissue, with selective localization in the outer layer of the capillary vessel wall. In conclusion, this study suggests that a compartmentalized B cell response occurs within the CNS during an ongoing inflammatory reaction, through a recapitulation of all stages of B cell differentiation observed in secondary lymphoid organs. The presence of lymphotoxin-alpha, CXCL12, and CXCL13 in the CNS may provide favorable microenvironmental conditions for these events.

The location of embryonic lymph node development is determined by the initial clustering of lymphoid tissue-inducer (LTi) cells. Here we demonstrate that both the chemokine CXCL13 and the chemokine CCL21 attracted LTi cells at embryonic days 12.5-14.5 and that initial clustering depended exclusively on CXCL13. Retinoic acid (RA) induced early CXCL13 expression in stromal organizer cells independently of lymphotoxin signaling. Notably, neurons adjacent to the lymph node anlagen expressed enzymes essential for RA synthesis. Furthermore, stimulation of parasymphathetic neural output in adults led to RA receptor (RAR)-dependent induction of CXCL13 in the gut. Therefore, our data show that the initiation of lymph node development is controlled by RA-mediated expression of CXCL13 and suggest that RA may be provided by adjacent neurons.

The chemokine receptor CXCR4 is expressed in B cells at multiple stages of their development. CXCR4 function in humoral immunity has not been fully investigated. We have generated gene-targeted mice in which CXCR4 can be selectively inactivated in B cells and have shown that it is required for retention of B cell precursors in the bone marrow. CXCR4-deficient B cell precursors that migrated prematurely became localized in splenic follicles despite their unresponsiveness to CXCL13. Concomitantly, mature B cell populations were reduced in the splenic marginal zone and primary follicles, and in the peritoneal cavity in the mutant animals, as were T-independent antibody responses. In addition, aberrant B cell follicles formed ectopically in intestinal lamina propria around Peyer's patches. These findings establish an important role for CXCR4 in regulating homeostasis of B cell compartmentalization and humoral immunity.

T cells are key elements of cancer immunotherapy1 but certain fundamental properties, such as the development and migration of T cells within tumours, remain unknown. The enormous T cell receptor (TCR) repertoire, which is required for the recognition of foreign and self-antigens2, could serve as lineage tags to track these T cells in tumours3. Here we obtained transcriptomes of 11,138 single T cells from 12 patients with colorectal cancer, and developed single T cell analysis by RNA sequencing and TCR tracking (STARTRAC) indices to quantitatively analyse the dynamic relationships among 20 identified T cell subsets with distinct functions and clonalities. Although both CD8+ effector and 'exhausted' T cells exhibited high clonal expansion, they were independently connected with tumour-resident CD8+ effector memory cells, implicating a TCR-based fate decision. Of the CD4+ T cells, most tumour-infiltrating T regulatory (Treg) cells showed clonal exclusivity, whereas certain Treg cell clones were developmentally linked to several T helper (TH) cell clones. Notably, we identified two IFNG+ TH1-like cell clusters in tumours that were associated with distinct IFNγ-regulating transcription factors -the GZMK+ effector memory T cells, which were associated with EOMES and RUNX3, and CXCL13+BHLHE40+ TH1-like cell clusters, which were associated with BHLHE40. Only CXCL13+BHLHE40+ TH1-like cells were preferentially enriched in patients with microsatellite-instable tumours, and this might explain their favourable responses to immune-checkpoint blockade. Furthermore, IGFLR1 was highly expressed in both CXCL13+BHLHE40+ TH1-like cells and CD8+ exhausted T cells and possessed co-stimulatory functions. Our integrated STARTRAC analyses provide a powerful approach to dissect the T cell properties in colorectal cancer comprehensively, and could provide insights into the dynamic relationships of T cells in other cancers.

Immunization with a T cell-dependent antigen elicits production of specific memory B cells and antibody-secreting cells (ASCs). The kinetic and developmental relationships between these populations and the phenotypic forms they and their precursors may take remain unclear. Therefore, we examined the early stages of a primary immune response, focusing on the appearance of antigen-specific B cells in blood. Within 1 wk, antigen-specific B cells appear in the blood with either a memory phenotype or as immunoglobulin (Ig)G1 ASCs expressing blimp-1. The memory cells have mutated V(H) genes; respond to the chemokine CXCL13 but not CXCL12, suggesting recirculation to secondary lymphoid organs; uniformly express B220; show limited differentiation potential unless stimulated by antigen; and develop independently of blimp-1 expression. The antigen-specific IgG1 ASCs in blood show affinity maturation paralleling that of bone marrow ASCs, raising the possibility that this compartment is established directly by blood-borne ASCs. We find no evidence for a blimp-1-expressing preplasma memory compartment, suggesting germinal center output is restricted to ASCs and B220(+) memory B cells, and this is sufficient to account for the process of affinity maturation.

In the Peyer's patches (PPs), germinal centers (GCs) are chronically induced by bacteria and are the major sites for generation of gut immunoglobulin A (IgA) immune responses. Whether follicular dendritic cells (FDCs) within the GCs directly contribute to the IgA production in PPs is unknown. We showed here that direct stimulation of FDCs by bacterial products and retinoic acid synergistically enhanced the expression of the chemokine CXCL13, the survival factor BAFF, and molecules that facilitate the secretion and activation of the cytokine TGF-beta1. A reduced production of these molecules by PP FDCs associated with deficiencies in the Toll-like receptor pathway or vitamin A resulted in decreased numbers of GC B cells and defective generation of IgA(+) B cells within PP GCs. Our data indicate that PP FDCs are conditioned by environmental stimuli to express key factors for B cell migration, survival, and preferential generation of IgA in gut.