Germinal Center-like Structures Research Articles

Tertiary lymphoid structures in autoimmune diseases.

Tertiary lymphoid structures (TLSs) are organized lymphoid-like aggregations in non-lymphoid tissues. Tissues with chronic and persistent inflammation infiltration may drive and form ectopic germinal center-like structures, which are very common in autoimmune diseases, chronic infections, and tumor microenvironments. However, the mechanisms governing the formation of TLSs are still being explored. At present, it is not clear whether the formation of TLSs is associated with local uncontrolled immune inflammatory responses. While TLSs suggest a good prognosis in tumors, the opposite is true in autoimmune diseases. This review article will discuss the current views on initiating and maintaining TLSs and the potential therapeutic target in autoimmune diseases.

The chilling origin of germinal centers.

Germinal center-like structures have been identified in ectothermic vertebrates, establishing germinal centers as a universal component of humoral immunity (see related Research Article by Shibasaki et al.).

Cold-blooded vertebrates evolved organized germinal center-like structures.

Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)-specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non-M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA-associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igμ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.

Intrinsic B cell TLR-BCR linked coengagement induces mature and protective antibody responses in the absence of T cells

Abstract Maturation of the antibody response is critical for the generation of virus and bacteria neutralizing antibodies. It entails CSR and SHM together with plasma cell and memory B cell differentiation, and it is generally dependent on T cell help. Prompted by our finding that CSR and plasma cell differentiation can be induced in vitro by a TLR ligand together with BCR crosslinking, we hypothesized that TLR-BCR coengagement can induce a fully mature antibody response in vivo in the absence of T cells, and such coengagement must entail physical linkage of TLR and BCR ligands. To test this hypothesis, we vaccinated Tcrβ−/−Tcrδ−/− mice with NP-LPS or Salmonella Typhimurium flagellin (coengaging B-cell TLR4 with NP-specific BCR or TLR5 with flagellin-specific BCR). Vaccination of Tcrβ−/−Tcrδ−/− mice with NP-LPS or flagellin induced activation of the B cell NF-κB pathway to elicit NP or flagellin-specific class-switched IgG and IgA antibodies and plasma cells. It also induced formation of germinal center-like (GC) structures, B cell SHM, selective clonal expansion and intraclonal diversification, and generation of memory B cells. Similar antibody responses to NP and flagellin were reproduced in NSG/B mice (by grafting immunodeficient NSG mice with purified C57BL/6 mouse B cells), thereby showing that such responses could be generated in the absence of not only T cells but all other immune cells. Finally, LPS and flagellin induced class-switched and specific antibodies which neutralized E. coli and S. Typhimurium, respectively, and protected mice from lethal infection. Thus, TLR-BCR coengagement can induce a mature and protective antibody response, complete with CSR/SHM, plasma cell and memory B cell differentiation in the absence of T cells. Supported by NIH grants AI 079705, AI 105813/105813S1, AI138944, AI 167416 and Lupus Research Alliance grant LRA 641363 to Paolo Casali

Ectopic lymphoid follicles in progressive multiple sclerosis: From patients to animal models.

Ectopic lymphoid follicles (ELFs), resembling germinal centre‐like structures, emerge in a variety of infectious and autoimmune and neoplastic diseases. ELFs can be found in the meninges of around 40% of the investigated progressive multiple sclerosis (MS) post‐mortem brain tissues and are associated with the severity of cortical degeneration and clinical disease progression. Of predominant importance for progressive neuronal damage during the progressive MS phase appears to be meningeal inflammation, comprising diffuse meningeal infiltrates, B‐cell aggregates and compartmentalized ELFs. However, the absence of a uniform definition of ELFs impedes reproducible and comparable neuropathological research in this field. In this review article, we will first highlight historical aspects and milestones around the discovery of ELFs in the meninges of progressive MS patients. In the next step, we discuss how animal models may contribute to an understanding of the mechanisms underlying ELF formation. Finally, we summarize challenges in investigating ELFs and propose potential directions for future research.

Inflammatory Stratification in Primary Sjögren's Syndrome Reveals Novel Immune Cell Alterations in Patients' Minor Salivary Glands.

There is a critical need to deconvolute the heterogeneity displayed by the minor salivary glands of primary Sjögren’s syndrome (pSS) patients. This is challenging primarily because the disease etiology remains unknown. The hypothesis includes that initial events in the disease pathogenesis target the salivary glands, thereby triggering the development of focal infiltrates (≥50 mononuclear cells) and finally germinal center-like structures. However, the proportion of key mononuclear immune cells residing at these sites, in combination with the overall ratio of morphometric tissue atrophy and adipose infiltration within the minor salivary glands (MSG) parenchyma at distinct phases of inflammatory disease establishment and progression have not been quantified in detail. In this cross-sectional study, we intended to address this problem by stratifying 85 patients into mild (S1), moderate (S2), and severe (S3) stages using the Inflammatory severity index. We found that mild (<3%) and marked (≥3%) levels of atrophy were accompanied by the respective levels of adipose infiltration in the non-SS sicca controls (p <0.01), but not in pSS patients. The percentage of adipose infiltration significantly correlated with the age of patients (r = 0.458, p <0.0001) and controls (r = 0.515, p <0.0001). The CD4+ T helper cell incidence was reduced in the focal infiltrates of the MSG of S2 patients compared to S1 (p <0.01), and in S2 compared to S1 and S3 combined (p <0.05). CD20+ B cells increased from S1 to S3 (p <0.01) and S2 to S3 (p <0.01), meanwhile CD138+ plasma cells diminished in S3 patients compared to both S1 and S2 groups combined (p <0.01). The proportion of patients with anti-Ro/SSA+, anti-La/SSB+, and RF+ increased over the course of inflammatory disease progression and they were significantly more common in the S3 group relative to S1 (p <0.05). On the other hand, S2 patients measured a higher mean salivary flow relative to S1 and S3 patients combined (p <0.05). Our results demonstrate how the proposed Inflammatory severity index stratification revealed pathological cell and tissue-associated aberrations in the salivary component over the course of inflammatory progression, and their correlations to clinical outcomes. This could be directly transferred to the optimization of available diagnostic strategies applied for pSS patients.

Intratumoral T cell clonality and survival in a randomized phase II study of preoperative autophagy inhibition in combination with gemcitabine and nab-paclitaxel treatment in patients with resectable pancreatic cancer.

e16001 Background: Autophagy is a cell survival mechanism that is upregulated in pancreatic ductal adenocarcinoma (PDAC). PDAC autophagy results in an altered metabolic phenotype that promotes tumor progression, chemotherapeutic resistance, and immune evasion. Methods: We previously completed a randomized phase II clinical trial of preoperative gemcitabine-nab-paclitaxel with (PGH n = 34) and without (PG, n = 30) autophagy inhibition in patients with resectable and borderline resectable PDAC, which demonstrated increased Evans Grade histopathologic and serum CA 19-9 response with autophagy inhibition (IRB 13-074, NCT01128296 ). Utilizing the resected FFPE tumor specimens from evaluable patients, we completed paired multiplex immunohistochemistry (CD4, CD8, FOXP3, CD20, CD68, Pan-CK) and T &amp; B cell receptor RNA sequencing to assess the intratumoral adaptive immune response and correlates of outcome. Results: Autophagy inhibition increased the number of infiltrating CD8 T cells (1133±490 vs 712±460 average cells per high power field, p = 0.01), CD8:CD20 ratio (2.22±3.1 vs 0.96±1.1, p = 0.02) and reduced the CD4:CD8 ratio (2.04±0.87 vs 3.01±2.09, p = 0.03). No effect was observed on the number of immature or mature germinal center-like tertiary lymphoid structures (TLS), though the number of TLS correlated with increased infiltration of CD4 T cells (r = 0.40, p &lt; 0.001), T-regulatory cells (r = 0.26, p = 0.03) and CD20 B cells (r = 0.65, p &lt; 0.001). Although the total number of productive T and B cell receptors increased with autophagy inhibition (167217±105961 vs 97339±5,1628, p = 0.02), no apparent effects were observed on Vαβ TCR or BCR IgH, Igκ, Igλ clonality. Independent of treatment, intratumoral CD8 counts were associated with an improved CA 19-9 response (r = 0.32, p = 0.04) and in a subset of short term ( &lt; 2 years, n = 17) and long term ( &gt; 4 years, n = 10) survivors (LTS), a lowered CD4:CD8 ratio was identified in LTS (1.83±0.63 vs 2.8±0.90, p = 0.01). Dominance of B cell receptors was a prominent feature of the immune repertoire in all patients (average expression: Vα 0.6%, Vβ 0.8%, IgH 18.9%, Igκ 32.3%, Igλ 47.2%) with an IgA skewed immunoglobulin class switching (mean 63% of all BCRs). Increased αβ T cell receptor clonality above the median level was associated with a CA 19-9 response (r = 0.37, p = 0.06) and greater overall survival (median OS 38.3 vs 19.3 months, p = 0.02), indicative of possible tumor specific clonal expansion. Conclusions: Preoperative autophagy inhibition increased the number of tumor infiltrating CD8 T cells in patients with localized pancreatic cancer. Intratumoral αβ T cell receptor clonality was associated with CA 19-9 response and improved overall survival. Combination treatment regimens increasing PDAC specific CD8 responses are warranted. Clinical trial information: NCT01978184.

Expression of BAFF and BAFF receptors in primary Sjögren's syndrome patients with ectopic germinal center-like structures.

B cell-activating factor (BAFF) is an essential cytokine in primary Sjögren's syndrome (pSS) physiopathology. It has been reported that pSS patients develop germinal center-like (GC-like) structures in their minor salivary glands (MSGs). BAFF, BAFF-R, TACI, and BCMA expression was analyzed in MSGs from 29 subjects (nonspecific chronic sialadenitis and focal lymphocytic sialadenitis with the presence [pSS-GC(+)] or absence [pSS-GC(-)] of GC-like structures). Twenty-four percent of patients showed ectopic GC-like structures and a high focus score [p < 0.001 vs pSS-GC(-)]. BAFF serum levels (sBAFF) were high in pSS patients (p = 0.025 vs healthy subjects). However, the pSS-GC(-) group showed higher sBAFF levels than pSS-GC(+) patients. BAFF and BAFF-R glandular expression levels were higher in pSS-GC(+) patients, without significant differences compared to pSS-GC(-) patients. Soluble levels of BAFF correlated with anti-La/SSB antibodies and disease duration. Our results showed that BAFF could contribute to focal lymphocytic infiltration. The role of BAFF-binding receptors in MSGs is proposed as a mechanism for the possible establishment of ectopic GC-like structures and disease progression in some patients. In conclusion, this study supports previous evidence that considers the active BAFF system role in the pathogenesis of pSS and the need for strong biomarkers in this disease.

A humanized mouse platform supporting in vivo maturation of human antibody responses

Abstract Mice are widely used models for research and drug discovery, yet provide limited predictive value due to their divergence from humans in immune elements and functions. This underscores the need for more faithful models of the in vivo human immune response. To address this shortcoming, we have generated a HuMice platform by grafting NSG KitW-41J mice with fresh human cord blood CD34+ stem cells via intracardiac injection. This yielded up to 98% human CD45+ cell reconstitution within all major lymphoid compartments, including GALT. Human leukocyte development and long-term persistence in CD34+ cell-engrafted HuMice revealed robust T and B cell maturation, leading to emergence of IgM, IgG, IgA and IgE forming cells. B cells from HuMice underwent in vitro CSR and plasma cell differentiation as efficiently as B cells isolated from healthy humans. Once conditioned with our immune potentiation treatment, HuMice supported T-dependent and T-independent antibody responses involving formation of germinal center-like structures, class-switching, somatic hypermutation, as well as plasma cell and memory B cell differentiation, this entailed further maturation of the B cell compartment resulting in high B2/B1 cell ratio. HuMice were amenable to hydrocarbon-induced autoimmunity leading to lupus-like disease and were susceptible to infection by and respond to human pathogens such as vaccinia virus and EBV. Thus, we have devised a novel in vivo HuMice platform, which allows for generation and maturation of human antibody responses to microbial agents and lends itself to in vivo modeling of autoimmunity, thereby offering opportunities for identification of therapeutic targets and vaccine development.

Thymic B Cells Promote Germinal Center-Like Structures and the Expansion of Follicular Helper T Cells in Lupus-Prone Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive T and B cells, autoantibody production, and immune complex deposition in various organs. Previous evidence showed abnormal accumulation of B cells in the thymus of lupus-prone mice, but the role of this population in the progression of the disease remains mostly undefined. Here we analyzed the spatial distribution, function, and properties of this thymic B cell population in the BWF1 murine model of SLE. We found that in diseased animals, thymic B cells proliferate, and cluster in structures that resemble ectopic germinal centers. Moreover, we detected antibody-secreting cells in the thymus of diseased-BWF1 mice that produce anti-dsDNA IgG autoantibodies. We also found that thymic B cells from diseased-BWF1 mice induced the differentiation of thymocytes to follicular helper T cells (TFH). These data suggest that the accumulation of B cells in the thymus of BWF1 mice results in the formation of germinal center-like structures and the expansion of a TFH population, which may, in turn, activate and differentiate B cells into autoreactive plasma cells. Therefore, the thymus emerges as an important niche that supports the maintenance of the pathogenic humoral response in the development of murine SLE.

Subclinical Atherosclerosis in Primary Sjögren's Syndrome: Does Inflammation Matter?

Sjögren's syndrome (SS) is a systemic autoimmune disease mainly characterized by inflammatory involvement of exocrine gland. Atherosclerosis is a complex process leading to plaque formation in arterial wall with subsequent cardiovascular (CV) events. Recently, numerous studies demonstrated that SS patients bear an increased CV risk. Since activation of immune system is a key element in atherosclerosis, it is interesting to analyze whether and how the autoimmune and inflammatory events characterizing SS pathogenesis directly or indirectly contribute to atherosclerosis risk in these patients. An increase in circulating endothelial microparticles and integrins, which may be a consequence of endothelial damage and impaired repair mechanisms, has been demonstrated in SS. Increased endothelial expression of adhesion molecules with subsequent infiltration of inflammatory cells into arterial wall is also a critical event in atherosclerosis. The early inflammatory events taking place in the atherosclerotic plaque cause an increase in alarmins, such as S100A8/A9, which seems to be associated with SS disease activity and, in turn, induce up-regulation of interleukin (IL)-1β and other pro-atherogenic cytokines. Interestingly, increased IL-1β levels were also detected in tertiary lymphoid structures developing in vessel adventitia adjacent to the atherosclerotic plaque, suggesting a direct role of IL-1β in this process. Similar to these structures, germinal center-like structures arising in SS exocrine glands are also tertiary lymphoid systems where T-helper (Th) cell subsets govern the adaptive immune response. Th1 cells are the most prevalent subtype and have been shown to be strongly involved in both SS pathogenesis and atherosclerosis. Th17 cells are attracting great interest and few studies showed its importance in SS development. Albeit in low amounts, a Th17 signature was also detected in atherosclerotic plaques and some animal models demonstrated a significant pro-atherogenic role and positive effects of IL-17A blockade. Despite the fact that T cells have a pivotal role in the inflammatory process that ultimately leads to atherosclerosis, B cells have also been detected in atherosclerotic plaques, although their exact role is still mostly unknown with studies showing contrasting results. In this scenario, the role of inflammation in atherosclerosis pathogenesis in patients with SS needs to be further explored.

Different operators and histologic techniques in the assessment of germinal center-like structures in primary Sjo¨gren's syndrome minor salivary glands

Different operators and histologic techniques in the assessment of germinal center-like structures in primary Sjo¨gren's syndrome minor salivary glands

Different operators and histologic techniques in the assessment of germinal center-like structures in primary Sjögren's syndrome minor salivary glands.

ObjectiveA standardization of minor salivary gland (MSG) histopathology in primary Sjögren’s syndrome (pSS) has been recently proposed. Although there is strong agreement that germinal center (GC)-like structures should be routinely identified, due to their prognostic value, a consensus regarding the best protocol is still lacking. Aim of this study was to compare the performance of different histological techniques and operators to identify GC-like structures in pSS MSGs. MSG biopsies from 50 pSS patients were studied.MethodsThree blinded operators (one pathologist and two rheumatologists with different years of experience in pSS MSG assessment) assessed 50 MSGs of which one slide was stained with haematoxylin and eosin (H&E) and consecutive slides were processed to investigate CD3/CD20, CD21 and Bcl-6 expression.ResultsBy assessing 225 foci, the best agreement was between H&E-stained sections evaluated by the rheumatologist with more years of experience in pSS MSG assessment and CD3/CD20 segregation. In the foci with CD21 positivity, the agreement further increased. Bcl-6- foci could display a GC, detected with other staining, but not vice versa.ConclusionGC assessment on H&E-stained sections should be performed with caution, being operator-dependent. The combination of H&E with CD3/CD20 and CD21 staining should be recommended as it is reliable, feasible, able to overcome the bias of operator experience and easily transferrable into routine practice.

Optimizing conventional DMARD therapy for Sjögren's syndrome.

Abstract Primary Sjogren's syndrome (pSS) is an auto immune disorder characterized by exocrine dysfunction as a result of chronic inflammation of the glands. Part of the patients also develops inflammation in other organs. In a complex interplay of different cell types such as T-cells, B-cells, dendritic cells, monocytes/macrophages and NK cells and their effector molecules, all contribute to one of the ultimate hallmarks of pSS: B-cell hyperactivity, subsequent autoantibody production and eventually formation of germinal center-like structures in the salivary gland. Effective treatment options for this disease are currently lacking. Biological DMARDs (bDMARDs) including those targeting B-cells or B-cell activation (directly or indirectly) have been studied, so far with limited efficacy. Besides that, their high costs provide a major drawback for implementation. Relatively inexpensive conventional DMARDs (cDMARDs) with well-known safety profiles have been shown efficacious in numerous clinical studies in multiple (rheumatic) diseases. cDMARDs target several pathways that are crucial in pSS immunopathology and some have proven to effectively inhibit B-cell hyperactivity and immune activation when given to patients. However, strong conclusions about potential efficacy are hampered by lack of standardization of inclusion criteria and outcome measures, dosing and validated biomarkers for patient selection. Proper implementation of these could help to optimize the use of cDMARDs in pSS treatment. In analogy with effective treatment strategies in for example rheumatoid arthritis, combination of two cDMARDs targeting different dysregulated pathways might result in additive or synergistic inhibition of immune activation. In view of this and the unique and potent mechanisms of action to target immunopathology in pSS, optimizing cDMARDs for treatment of pSS is worthwhile.

Ectopic lymphoid tissues support local immunoglobulin production in patients with chronic rhinosinusitis with nasal polyps

The contribution of ectopic lymphoid tissues (eLTs) to local immunoglobulin hyperproduction in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) is unclear. We sought to explore the cellular basis, formation mechanisms, and function of eLTs in patients with CRSwNP. We graded lymphoid aggregations in sinonasal mucosa and histologically studied their structures. The expression of lymphorganogenic factors and molecules required for immunoglobulin production was measured by using real-time PCR, and their localization was analyzed by means of immunohistochemistry and immunofluorescence. The phenotype of follicular helper T cells was analyzed by performing flow cytometry. Immunoglobulin levels were quantified by using the Bio-Plex assay or ImmunoCAP system. Nasal tissue explants were challenged exvivo with Dermatophagoides pteronyssinus group 1 (Der p 1), and the expression of Iε-Cμ and Iε-Cγ circle transcripts was detected by using seminested PCR. Increased formation of eLTs with germinal center-like structures was discovered in patients with eosinophilic (20.69%) and noneosinophilic (17.31%) CRSwNP compared with that in patients with chronic rhinosinusitis without nasal polyps (5.66%) and control subjects (3.70%). The presence of eLTs was associated with increased expression of lymphorganogenic and inflammatory chemokines and cytokines, as well as their receptors. The expression of molecules required for immunoglobulin production, generation of follicular helper T cells, and production of IgE in eosinophilic polyps and IgG and IgA in both eosinophilic and noneosinophilic polyps were predominantly upregulated in patients with eLTs. After Der p 1 challenge exvivo, Iε-Cμ transcript was detected only in eosinophilic polyps with eLTs but not in polyps without eLTs and noneosinophilic polyps. eLTs might support local immunoglobulin production and therefore significantly contribute to the development of CRSwNP.

Tbet and IL-36γ cooperate in therapeutic DC-mediated promotion of ectopic lymphoid organogenesis in the tumor microenvironment

ABSTRACT We have previously reported that direct injection of dendritic cells (DC) engineered to express the Type-1 transactivator Tbet (i.e., DC.Tbet) into murine tumors results in antitumor efficacy in association with the development of structures resembling tertiary lymphoid organs (TLO) in the tumor microenvironment (TME). These TLO contained robust infiltrates of B cells, DC, NK cells, and T cells in proximity to PNAd+ blood vessels; however, they were considered incomplete, since the recruited B cells failed to organize into classic germinal center-like structures. We now report that antitumor efficacy and TLO-inducing capacity of DC.Tbet-based i.t. therapy is operational in peripheral lymph node-deficient LTA−/− mice, and that it is highly dependent upon a direct Tbet target gene product, IL-36γ/IL-1F9. Intratumoral DC.Tbet fails to provide protection to tumor-bearing IL-36R−/− hosts, or to tumor-bearing wild-type recipient mice co-administered rmIL-1F5/IL-36RN, a natural IL-36R antagonist. Remarkably, the injection of tumors with DC engineered to secrete a bioactive form of mIL-36γ (DC.IL36γ) also initiated therapeutic TLO and slowed tumor progression in vivo. Furthermore, DC.IL36γ cells strongly upregulated their expression of Tbet, suggesting that Tbet and IL-36γ cooperate to reinforce each other's expression in DC, rendering them competent to promote TLO formation in an “immunologically normalized,” therapeutic TME.

Characteristics of germinal center-like structures in patients with Sjögren's syndrome.

To analyze the relationship between ectopic germinal centers (GCs) in the salivary glands and the clinical/laboratory characteristics of patients with Sjögren's syndrome (SS). Retrospectively, 126 patients with primary SS (pSS) and 16 patients with secondary SS (sSS) were analyzed. Minor salivary gland biopsies were evaluated for the presence of GC-like morphology by hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining for CD21. Clinical and serological data were obtained from medical records. GC-like structures were observed in 36/126 (28.6%) pSS patients and 4/16 (25.0%) sSS patients. The mean inflammatory focus score of the gland was significantly higher in GC-positive samples than in GC-negative ones in both pSS and sSS patients (P = 0.007 and 0.024, respectively). In pSS, significantly elevated titers of rheumatoid factor (RF)-IgM (P = 0.023) and antinuclear antibodies (ANA) (P = 0.036), increased levels of IgA (P = 0.012) and IgG (P = 0.017) were encountered in GC-positive patients. The GC-positive group also presented higher prevalence of anti-SSA antibodies, lower levels of white blood cells, higher levels of erythrocyte sedimentation rate and γ-globulin, although not statistically significant. In sSS patients with ectopic GC formation, ANA titers were remarkably elevated. The anticyclic citrullinated peptide (anti-CCP)-IgG titers and the prevalence of antikeratin antibody (AKA)-IgG, antiperinuclear factor (APF)-IgG were also increased, yet not significantly. GCs were found to be associated with antibody and immunoglobulin production. This study indicates that SS patients with ectopic GCs have distinct features. Ectopic GC structures were particularly noted in patients with higher focus scores, and might play an essential role in sustaining antibody production as well as B cell activation.

An adaptive immune response driven by mature, antigen-experienced T and B cells within the microenvironment of oral squamous cell carcinoma.

Lymphocyte infiltrates have been observed in the microenvironment of oral cancer; however, little is known about whether the immune response of the lymphocyte infiltrate affects tumor biology. For a deeper understanding of the role of the infiltrating-lymphocytes in oral squamous cell carcinoma (OSCC), we characterized the lymphocyte infiltrate repertoires and defined their features. Immunohistochemistry revealed considerable T and B cell infiltrates and lymphoid follicles with germinal center-like structures within the tumor microenvironment. Flow cytometry demonstrated that populations of antigen-experienced CD4+ and CD8+ cells were present, as well as an enrichment of regulatory T cells; and T cells expressing programmed death-1 (PD-1) and T cell Ig and mucin protein-3 (Tim-3), indicative of exhaustion, within the tumor microenvironment. Characterization of tumor-infiltrating B cells revealed clear evidence of antigen exposure, in that the cardinal features of an antigen-driven B cell response were present, including somatic mutation, clonal expansion, intraclonal variation and isotype switching. Collectively, our results point to an adaptive immune response occurring within the OSCC microenvironment, which may be sustained by the expression of specific antigens in the tumor.

Association of Antibodies to Interferon-Inducible Protein-16 With Markers of More Severe Disease in Primary Sjögren's Syndrome.

Interferon-inducible protein-16 (IFI16) is an intracellular DNA receptor involved in innate immunity. We evaluated the frequency, phenotypic characteristics, and clinical associations of anti-IFI16 antibodies in patients with primary Sjögren's syndrome (SS), and quantitated expression levels of IFI16 in SS and control salivary gland lysates. Anti-IFI16 antibodies were assayed by enzyme-linked immunosorbent assay using sera from patients with primary SS (n = 133) and from healthy controls (n = 47). Sera from systemic lupus erythematosus (SLE) patients (n = 132) were included as disease controls. Immunoprecipitation of in vitro transcription-translated IFI16 was used to determine which portion of IFI16 the antibodies recognized. Expression of IFI16 in salivary gland lysates was quantitated by immunoblotting. Anti-IFI16 antibodies were present in the sera of 38 of 133 SS patients (29%) compared to 1 of 47 healthy controls (2.1%) (SS versus controls; P < 0.0002) and in 31 of 132 SLE controls (24%). In SS, anti-IFI16 antibodies were associated with an abnormal Schirmer's test (P = 0.003), hyperglobulinemia (P = 0.02), antinuclear antibody ≥1:320 (P = 0.01), germinal center-like structures in labial salivary gland lymphoid infiltrates (P = 0.01), and higher focus scores (3.4 versus 2.4; P = 0.005). High-titer IFI16 antibodies were directed against an epitope outside the N-terminus in 9 of 13 SS patients (69%). IFI16 was expressed in 4 of 5 (80%) of SS and 1 of 6 (17%) of control labial salivary glands. Anti-IFI16 antibodies are a prominent specificity in primary SS and are associated with markers of severe disease. IFI16 is expressed at higher levels in SS salivary glands compared to controls. These high levels in disease target tissue may contribute to the ongoing anti-IFI16 immune response.

Novel CXCL13 transgenic mouse: inflammation drives pathogenic effect of CXCL13 in experimental myasthenia gravis.

Abnormal overexpression of CXCL13 is observed in many inflamed tissues and in particular in autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disease mainly mediated by anti-acetylcholine receptor autoantibodies. Thymic hyperplasia characterized by ectopic germinal centers (GCs) is a common feature in MG and is correlated with high levels of anti-AChR antibodies. We previously showed that the B-cell chemoattractant, CXCL13 is overexpressed by thymic epithelial cells in MG patients. We hypothesized that abnormal CXCL13 expression by the thymic epithelium triggered B-cell recruitment in MG. We therefore created a novel transgenic (Tg) mouse with a keratin 5 driven CXCL13 expression.The thymus of Tg mice overexpressed CXCL13 but did not trigger B-cell recruitment. However, in inflammatory conditions, induced by Poly(I:C), B cells strongly migrated to the thymus. Tg mice were also more susceptible to experimental autoimmune MG (EAMG) with stronger clinical signs, higher titers of anti-AChR antibodies, increased thymic B cells, and the development of germinal center-like structures. Consequently, this mouse model finally mimics the thymic pathology observed in human MG.Our data also demonstrated that inflammation is mandatory to reveal CXCL13 ability to recruit B cells and to induce tertiary lymphoid organ development.