Follicular Dendritic Cell Networks Research Articles

Localization of DC-SIGN Expressing Cells in Follicular Lymphoma

The high incidence of unusual mannose-rich glycans on the expressed surface immunoglobulins of follicular lymphoma (FL) suggests a pathogenic role for this modification. While lectin-mediated signaling has been proposed to provide trophic support to FL cells, the localization of the lectins has not been extensively characterized. Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) is a mannose-selective lectin named for its expression on classic marrow-derived dendritic cells. In vitro, recombinant DC-SIGN induces signaling in primary FL cells. The nodules of Follicular lymphoma (nFL) are the neoplastic counterparts of normal germinal centers (GC). Both GC and nFL contain variable numbers of centrocytic and/or centroblastic B cells, T-cells, and follicular dendritic cells (FDCs), but macrophages are typically found only in GC. Although termed “follicular dendritic cells”, FDCs are thought to be derived from non-blood stem-cells, potentially pericytes. Therefore, nFL lack classic dendritic cells and macrophages so it is unclear what cells in FL could express DC-SIGN. Therefore, we systematically characterized FDC and DC- SIGN localization in sixteen FL and nine Follicular hyperplasia (FH) lymph nodes. Using flow cytometry of disaggregated FL nodes, we were not able to identify a specific cell population expressing DC-SIGN. However, using immunohistochemistry and Immunofluorescence methods, we identified nine cases of FH and five cases of FL (n=5/16) with DC-SIGN and FDC co-localization. Immunohistochemical stains of the FDCs using CD23 and CD21 showed that the follicular dendritic structures were disrupted in almost all the FL cases assessed showing either a peripheral crescentic staining or broken FDC meshwork within the follicle. We identified five cases of FL with an average of more than four well visualized and complete FDC structures staining per low power field (4X) while the remaining cases had an average of less than two complete FDC network staining. There was near complete absence of CD21 positive FDCs meshwork identified in 50% of the FL cases compared to only one case lacking CD23 positive FDCs structure staining. In contrast, FH cases (n=9) showed complete staining of the FDC meshwork within the follicles with both CD21/CD23. For FH, the average number of complete FDC staining with CD23 was 18 while an average of 10 complete FDC meshworks stained with CD21. Furthermore, immunofluorescence imaging analysis showed different pattern of localization of CD23 positive FDC with DC-SIGN with between the two conditions. In FH, DC-SIGN and CD23 colocalization was identified mainly in the light zone. For FL, we observed that DC-SIGN overlap with CD23 localized in the intra-follicular areas and co-localized with CD163 (M2 macrophages) in the inter-follicular zones. Confocal imaging confirmed colocalization of CD23 and DC-SIGN on FDCs with close association to MUM1-positive B cells in both GC and nFL. Additional analysis also showed that 80% (n=5) of the cases which had more than four complete FDC structures staining with CD23 co-localized with DC-SIGN in the intra-follicular areas and the cases with complete FDC network disruption did not. There was no significant relationship identified between the extent of FDC networks and the fraction of non-neoplastic B cells in the FL cases. In summary, our data show that DC-SIGN is expressed on FDCs in FL and FH even though FDCs are not the marrow-derived DCs for which DC-SIGN is named. Furthermore, localization of DC-SIGN to the follicle suggests a potential role for the microenvironment in the pathogenesis of FL and possible therapeutic target.

Long-term retention of antigens in germinal centers is controlled by the spatial organization of the follicular dendritic cell network.

Germinal centers (GCs) require sustained availability of antigens to promote antibody affinity maturation against pathogens and vaccines. A key source of antigens for GC B cells are immune complexes (ICs) displayed on follicular dendritic cells (FDCs). Here we show that FDC spatial organization regulates antigen dynamics in the GC. We identify heterogeneity within the FDC network. While the entire light zone (LZ) FDC network captures ICs initially, only the central cells of the network function as the antigen reservoir, where different antigens arriving from subsequent immunizations colocalize. Mechanistically, central LZ FDCs constitutively express subtly higher CR2 membrane densities than peripheral LZ FDCs, which strongly increases the IC retention half-life. Even though repeated immunizations gradually saturate central FDCs, B cell responses remain efficient because new antigens partially displace old ones. These results reveal the principles shaping antigen display on FDCs during the GC reaction.

Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging

The magnitude and quality of the germinal center (GC) response decline with age, resulting in poor vaccine-induced immunity in older individuals. A functional GC requires the co-ordination of multiple cell types across time and space, in particular across its two functionally distinct compartments: the light and dark zones. In aged mice, there is CXCR4-mediated mislocalization of T follicular helper (TFH) cells to the dark zone and a compressed network of follicular dendritic cells (FDCs) in the light zone. Here we show that TFH cell localization is critical for the quality of the antibody response and for the expansion of the FDC network upon immunization. The smaller GC and compressed FDC network in aged mice were corrected by provision of TFH cells that colocalize with FDCs using CXCR5. This demonstrates that the age-dependent defects in the GC response are reversible and shows that TFH cells support stromal cell responses to vaccines.

Targeting antigens to phosphatidylserine triggers rapid T- and B-cell responses

Abstract Phosphatidylserine (PS) is a phospholipid that is present in each cell membrane. In intact cells, PS is kept intracellularly, in dying cells, extracellular vesicles (EVs) and activated platelets, PS flips outside where it can be accessed by PS-binding reagents, such as Milk fat globule-EGF factor 8 (MFG-E8). In previous studies, we have established in vivouse of fluorescent MFG-E8 to label and characterize EVs. Here, we measured the antigen-specific immune responses elicited by targeting antigen (Ag) to PS +structures, presumably EVs via MFG-E8 as a novel vaccination strategy. In contrast to non-targeted conventional Ag, PS-targeting by fusing Ag to MFG-E8 caused accumulation in the marginal zone of lymphoid follicles soon after injection. Hours later, Ag-MFG-E8 was deposited on the follicular dendritic cell network without the need of previous primary immunization. Mice immunized with PS-targeted Ag/Alum induced long-lasting germinal centers (GCs) and significantly more GC B cells. Furthermore, it resulted in an approx. 13,000-fold increase in Ag-specific IgG-levels and strongly accelerated antibody class switch recombination as compared to immunization with conventional Ag/Alum. Ag targeted to PS also induced more efficient CD8 T cell activation and IFN-gamma production post immunization. Therefore, targeting Ag to EVs by harnessing the PS-binding properties of MFG-E8 might turn out to be of advantage for the induction of very rapid and long-lasting immune responses. This project was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research foundation) – Project-ID 210592381 - SFB 1054 B03 to Thomas Brocker and Z02 to Jan Kranich.

Lymph Node Follicle-Targeting STING Agonist Nanoshells Enable Single-Shot M2e Vaccination for Broad and Durable Influenza Protection.

The highly conserved matrix protein 2 ectodomain (M2e) of influenza viruses presents a compelling vaccine antigen candidate for stemming the pandemic threat of the mutation-prone pathogen, yet the low immunogenicity of the diminutive M2e peptide renders vaccine development challenging. A highly potent M2e nanoshell vaccine that confers broad and durable influenza protectivity under a single vaccination is shown. Prepared via asymmetric ionic stabilization for nanoscopic curvature formation, polymeric nanoshells co-encapsulating high densities of M2e peptides and stimulator of interferon genes (STING) agonists are prepared. Robust and long-lasting protectivity against heterotypic influenza viruses is achieved with a single administration of the M2e nanoshells in mice. Mechanistically, molecular adjuvancy by the STING agonist and nanoshell-mediated prolongation of M2e antigen exposure in the lymph node follicles synergistically contribute to the heightened anti-M2e humoral responses. STING agonist-triggered T cell helper functions and extended residence of M2e peptides in the follicular dendritic cell network provide a favorable microenvironment that induces Th1-biased antibody production against the diminutive antigen. These findings highlight a versatile nanoparticulate design that leverages innate immune pathways for enhancing the immunogenicity of weak immunogens. The single-shot nanovaccine further provides a translationally viable platform for pandemic preparedness.

Deciphering the spectrum of cutaneous lymphomas expressing TFH markers

T-follicular helper (TFH) markers are expressed in the microenvironnement of marginal zone B-cell lymphoma (MZL), and in lymphomas arising from TFH-cells, sometimes making the differential diagnosis difficult. In the skin, the “TFH-spectrum” is poorly defined, going from primary cutaneous lymphoproliferative disorder with small/medium CD4+ T-cells (SMLPD) to cutaneous localizations of systemic angioimmunoblastic T-cell lymphoma (cAITL), and may pass through intermediate forms (primary cutaneous T-follicular helper derived lymphoma, not otherwise specified (PCTFHL,NOS)). We retrospectively analyzed 20 MZL, 13 SMLPD, 5 PCTFHL, and 11 cAITL clinically, histologically, and molecularly, to define tools to differentiate them. Characteristics that might favor the diagnosis of MZL over SMLPD are: multiple skin nodules (p < 0.001), nodular architecture (p < 0.01), residual germinal centers with follicular dendritic cell network (p < 0.001), monotypic plasma cells (p < 0.001), and few staining with PD1 (p = 0.016) or CXCL13 (p = 0.03). PCTFHL and cAITL presented as multiple (p < 0.01) lesions, in older patients (p < 0.01), with systemic symptoms and/or biological alterations (p < 0.01). Immunophenotypic loss of T-cell markers (p < 0.001), BCL6 (p = 0.023) and/or CD10 staining (p = 0.08), and a higher proliferative index (≥ 30%, p = 0.039) favoured these diagnoses over SMLPD. Pathogenic variants were observed by genomic sequencing in 47% of MZL (TNFAIP3 (32%), EP300 (21%), NOTCH2 (16%), KMT2D (16%), CARD11 (10.5%)), 8% of SMLPD (TET2), 40% of PCTFHL (SOCS1 (20%), ARID1A (20%)) and 64% of cAITL (TET2 (63.6%), RHOA (36.4%), NOTCH1 (9%)). This study characterizes the various clinical and histological features between cutaneous lymphomas expressing TFH markers and highlights the value of the interest of screening for genomic mutations in difficult cases.

Salmonella infection induces the reorganization of follicular dendritic cell networks concomitant with the failure to generate germinal centers.

Germinal centers (GCs) are sites where plasma and memory B cells form to generate high-affinity, Ig class-switched antibodies. Specialized stromal cells called follicular dendritic cells (FDCs) are essential for GC formation. During systemic Salmonella Typhimurium (STm) infection GCs are absent, whereas extensive extrafollicular and switched antibody responses are maintained. The mechanisms that underpin the absence of GC formation are incompletely understood. Here, we demonstrate that STm induces a reversible disruption of niches within the splenic microenvironment, including the T and B cell compartments and the marginal zone. Alongside these effects after infection, mature FDC networks are strikingly absent, whereas immature FDC precursors, including marginal sinus pre-FDCs (MadCAM-1+) and perivascular pre-FDCs (PDGFRβ+) are enriched. As normal FDC networks re-establish, extensive GCs become detectable throughout the spleen. Therefore, the reorganization of FDC networks and the loss of GC responses are key, parallel features of systemic STm infections.

Tumor Microenvironment Composition Correlates with Quantitative 18F-FDG PET-CT Features and Serum TARC in Patients with Relapsed or Refractory Hodgkin Lymphoma

Background Classical Hodgkin lymphoma (cHL) is a heterogeneous disease characterized by a limited number of Hodgkin Reed-Sternberg (HRS) cells in a complex tumor microenvironment (TME). Presence of cHL can be visualized by 18F-FDG PET-CT and can be detected in serum by measuring the HRS cell-derived chemokine TARC. While both these methods have shown prognostic value, a clear link between HRS/TME composition, serum TARC levels, and glucose metabolism measured by PET is lacking. The aim of this study was to assess correlations between gene expression based cell composition in tissue, quantitative PET features and serum TARC in patients with relapsed or refractory (R/R) cHL. Methods Lymph node biopsies, PET scans and serum samples at time of relapse were analyzed from 30 patients with R/R cHL who were treated within a clinical trial (NCT02280993). Immunohistochemical (IHC) staining was done for TARC. Soluble serum TARC was measured by ELISA. RNA was extracted from paraffin embedded lymph node biopsies and the expression of 235 genes related to the TME was measured using NanoString probes. The data were normalized on the geometric mean of 10 housekeeping genes. Genes were assigned to various TME signatures according to the literature (Scott et al., JCO 2012). Signature scores were calculated by taking the geometric mean of its constituent genes. PET scans were analyzed using a threshold of standard uptake value (SUV)4.0, which we have validated previously (Driessen et al., JNM 2021). Metabolic tumor volume (MTV), intensity (e.g. SUVpeak, SUVmean) and distance (D) features (e.g. number of lesions, Dmax, the largest distance between two lesions, and spread, the sum of all distances between lesions) were calculated at patient-level. Correlations were assessed using Spearman's rank correlation coefficients. Unsupervised K-means clustering with 1000 repeats was performed on gene signatures that showed a significant correlation with at least one PET feature. Results As expected, samples with positive TARC IHC staining showed higher TARC (CCL17) expression compared to samples with weak or negative TARC IHC (mean log2 expression 10.2 (95%CI: 9.5-10.8) vs 6.9 (95%CI: 6.2-7.5); p<0.001). The HRS cell signature correlated significantly with serum TARC levels (R=0.54; p=0.002), but not with PET features [Figure 1A]. The NK cell and eosinophil signatures also showed significant correlations with serum TARC levels (R=0.54; p=0.002 and R=0.49; p=0.006, respectively). The macrophage signature correlated significantly with SUVpeak (R=0.39; p=0.039). The antigen presenting cell (APC) signature correlated significantly with serum TARC (R=0.43; p=0.018), SUVpeak (R=0.50; p=0.006) and MTV (R=0.57; p=0.001). Within the APC signature, CD80 and CD40 showed the highest correlations for SUVpeak and MTV. Interestingly, the follicular dendritic cell (FDC) network signature showed significant negative correlations with SUVpeak (R=-0.38; p=0.044), SUVmean (R=-0.43; p=0.019) and MTV (R=-0.37; p=0.046). Unsupervised clustering based on the three gene signatures that showed significant correlations with at least one PET feature (i.e. APC, macrophage and FDC network) resulted in two distinct clusters with a significant difference in MTV and SUVpeak [Figure 1B]. Cluster 1 was associated with low MTV and SUVpeak, showed significant enrichment for the FDC network signature, and showed a trend for enrichment of B cells (p=0.15) and Th2 cells (p=0.15). Cluster 2 was associated with higher MTV and SUVpeak and showed significant enrichment for APCs, macrophages, Th1 cells, resting NK cells, myeloid lineage 1 and 2, neutrophils and viral genes. Conclusions We showed significant correlations between several quantitative PET features such as SUVpeak and MTV with gene signatures of macrophages, APCs and FDC networks, but not with the HRS cell signature. This suggests that TME cell types confer a high glucose metabolism which may explain differences in PET features between patients better than the abundance of HRS cells. These findings can aid in the understanding of the biological correlation of PET-CT scan findings in clinical practice and their relationship to cHL pathophysiology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Angioimmunoblastic T-cell lymphoma with extensive follicular dendritic cell and fibroblastic reticular cell network proliferation mimicking follicular dendritic cell sarcoma: A case report with pathologic, immunophenotypic, and molecular findings

Angioimmunoblastic T-cell lymphoma (AITL) is a common type of nodal peripheral T-cell lymphoma, which always presents with extensive follicular dendritic cell (FDC) meshwork. Here, we report a case of AITL combined with extensive spindle cell meshwork. Spindle cells occupied were positive for the FDC markers CD21, CD23, and CD35. Furthermore, some cells were positive for desmin and smooth muscle actin (SMA), suggesting the differentiation of fibroblastic reticular cell (FRC). Interestingly, the proliferation of spindle cells was so extensive that was easily misdiagnosed as FDC sarcoma (FDCS). Next-generation sequencing showed that the common mutations reported in AITL, including RHOA, TET2, and IDH2, were also detected in this case, while the genes that are recurrently mutated in FDCS were not detected. Regrettably, the patient died 19 months later. Overall, we highlight the unusual morphologic features in an AITL patient with extensive FDC and FRC network that may be misdiagnosed as FDCS, and careful morphological observation and immunochemical and molecular examinations are crucial for an accurate diagnosis.

CD169+ subcapsular sinus macrophage-derived microvesicles are associated with light zone follicular dendritic cells.

Follicular dendritic cells (FDCs) are a specialized type of stromal cells that exclusively reside in B-cell follicles. When inflammation occurs, the FDC network is reorganized to support germinal center (GC) polarization into the light zone (LZ) and dark zone (DZ). Despite the indispensable role of FDCs in supporting humoral responses, the FDC regulatory requirements remain incompletely defined. In this study, we unexpectedly observed an accumulation of CD169+ subcapsular sinus macrophage (SSM)-derived microvesicles (MVs) in the B-cell zone, which were tightly associated with the FDC network. Interestingly, a selective deposition of CD169+ MVs was detected in both GC LZ FDCs in secondary follicles and on predetermined LZ FDCs in primary follicles. The ablation of CD169+ MVs, resulting from SSM depletion, resulted in significantly decreased expression of LZ-related genes in FDCs. In addition, we found that CD169+ MVs could colocalize with fluorescently tagged antigen-containing immune complexes (ICs), supporting a possible role of CD169+ MVs in transporting antigens to the FDC network. Thus, our data reveal intimate crosstalk between FDCs and SSMs located outside B-cell follicles via SSM-released MVs, providing a novel perspective on the mechanisms underlying the regulation of FDC maturation and polarization.

Investigating the Mechanism of Germinal Center Shutdown.

Germinal centers (GCs) are transient structures where affinity maturation of B cells gives rise to high affinity plasma and memory cells. The mechanism of GC shutdown is unclear, despite being an important phenomenon maintaining immune homeostasis. In this study, we used a mathematical model to identify mechanisms that can independently promote contraction of GCs leading to shutdown. We show that GC shutdown can be promoted by antigen consumption by B cells, antigen masking by soluble antibodies, alterations in follicular dendritic cell (FDC) network area, modulation of immune complex cycling rate constants, alterations in T follicular helper signaling, increased terminal differentiation and reduced B cell division capacity. Proposed mechanisms promoted GC contraction by ultimately decreasing the number of B cell divisions and recycling cells. Based on the in-silico predictions, we suggest a combination of experiments that can be potentially employed by future studies to unravel the mechanistic basis of GC shutdown such as measurements of the density of pMHC presentation of B cells, FDC network size per B cell, fraction of cells expressing differentiation markers. We also show that the identified mechanisms differentially affect the efficiency of GC reaction estimated based on the quantity and quality of resulting antibodies.

Lymphotoxin-alpha expression in the meninges causes lymphoid tissue formation and neurodegeneration.

Organized meningeal immune cell infiltrates are suggested to play an important role in cortical grey matter pathology in the multiple sclerosis brain, but the mechanisms involved are as yet unresolved. Lymphotoxin-alpha plays a key role in lymphoid organ development and cellular cytotoxicity in the immune system and its expression is increased in the CSF of naïve and progressive multiple sclerosis patients and post-mortem meningeal tissue. Here we show that persistently increased levels of lymphotoxin-alpha in the cerebral meninges can give rise to lymphoid-like structures and underlying multiple sclerosis-like cortical pathology. Stereotaxic injections of recombinant lymphotoxin-alpha into the rat meninges led to acute meningeal inflammation and subpial demyelination that resolved after 28 days, with demyelination being dependent on prior subclinical immunization with myelin oligodendrocyte glycoprotein. Injection of a lymphotoxin-alpha lentiviral vector into the cortical meningeal space, to produce chronic localized overexpression of the cytokine, induced extensive lymphoid-like immune cell aggregates, maintained over 3 months, including T-cell rich zones containing podoplanin + fibroblastic reticular stromal cells and B-cell rich zones with a network of follicular dendritic cells, together with expression of lymphoid chemokines and their receptors. Extensive microglial and astroglial activation, subpial demyelination and marked neuronal loss occurred in the underlying cortical parenchyma. Whereas subpial demyelination was partially dependent on previous myelin oligodendrocyte glycoprotein immunization, the neuronal loss was present irrespective of immunization. Conditioned medium from LTα treated microglia was able to induce a reactive phenotype in astrocytes. Our results show that chronic lymphotoxin-alpha overexpression alone is sufficient to induce formation of meningeal lymphoid-like structures and subsequent neurodegeneration, similar to that seen in the progressive multiple sclerosis brain.

POS0145 CLINICAL RESPONSE TO RITUXIMAB IS ASSOCIATED WITH PREVENTION OF B-CELL DRIVEN SALIVARY GLAND INFLAMMATION AND EPITHELIAL RESTORATION AS REVEALED BY MOLECULAR PATHOLOGY: RESULTS FROM THE TRACTISS TRIAL IN PRIMARY SJOGREN’S SYNDROME

BackgroundThe TRial for Anti-B-Cell Therapy In patients with pSS (TRACTISS) is the largest multi-centre, placebo-controlled, phase-III trial with the administration of 2 cycles of Rituximab (RTX) or placebo at week 0 and 24, with trial clinical endpoints at week 48. Despite the primary endpoints (30% reduction in fatigue or oral dryness) were not met, RTX treated patients showed an improvement in secondary endpoints, such as unstimulated whole salivary flow (UWSF), and salivary gland (SG) total ultrasound score1,2. Additionally, recent post-hoc analysis of TRACTISS using novel CRESS composite endpoints3, highlighted a significantly increased response rate in the RTX vs placebo arm.ObjectivesTo perform the first longitudinal analysis of matched transcriptomic and histological data of SG biopsies of pSS patients treated with RTX vs placebo at 3 time points, over 48 weeks, from the TRACTISS cohort, in order to identify mechanisms of response/resistance to B cell depletion.Methods29 pSS patients randomised to RTX or placebo arm consented for labial SG biopsies at week 0, 16 and 48. Patients received two 1000mg cycles of RTX or placebo at week 0 and 24. SG focus score, inflammatory aggregate area fraction, B-cells (CD20+), T-cells (CD3+), follicular dendritic cells (FDCs) (CD21+) and plasma cells (CD138+) density were assessed using quantitative digital image analysis. RNA sequencing with deconvolution and pathway analysis was performed to identify genes signatures and consensus gene modules as biomarkers of disease evolution and response/resistance to therapy.ResultsPlacebo-treated SGs showed worsening of SG inflammation highlighted by the increment of aggregate size, B-cell density, development of new FDC networks, and a higher ectopic GC prevalence over 48 weeks, compared to RTX-treated patients. No difference in focus score, total T-cell and plasma cell infiltration was observed. RTX downregulated genes involved in immune cell recruitment and inflammatory aggregate organisation (e.g. CXCL13, CCR7 and PDCD1). Gene signature-based analysis of 35 immune cell types using XCell highlighted how RTX blocked class-switched and memory-B-cells accumulation in SGs over 48 weeks. Pathway analyses confirmed the downregulation of leukocyte migration, MHC-II antigen presentation, and T-cell co-stimulation immunological pathways, such as the CD40 receptor complex pathway. Among RTX-treated patients, only CRESS-responders demonstrated prevention of worsening B cell-driven molecular pathology signatures over time and a significant improvement in UWSF, in parallel with the upregulation of molecular pathways associated to SG restoration of the glandular epithelium. None of the above effects were observed at week 16 after the first RTX cycle.ConclusionTwo RTX infusions repeated at week 24 exerted beneficial effects on labial SG inflammatory infiltration in pSS by downregulating genes involved in immune cell recruitment, activation and organisation in ectopic GCs. Conversely, all the above parameters showed significant evolution in placebo treated patients over 48 weeks demonstrating progression of SG immunopathology. Clinical responders to RTX based on CRESS response criteria were characterised by preservation of exocrine function which appear driven by SG epithelial restoration.

B cells in pancreatic cancer stroma.

Pancreatic cancer is a disease with high unmet clinical need. Pancreatic cancer is also characterised by an intense fibrotic stroma, which harbours many immune cells. Studies in both human and animal models have demonstrated that the immune system plays a crucial role in modulating tumour onset and progression. In human pancreatic ductal adenocarcinoma, high B-cell infiltration correlates with better patient survival. Hence, B cells have received recent interest in pancreatic cancer as potential therapeutic targets. However, the data on the role of B cells in murine models is unclear as it is dependent on the pancreatic cancer model used to study. Nevertheless, it appears that B cells do organise along with other immune cells such as a network of follicular dendritic cells (DCs), surrounded by T cells and DCs to form tertiary lymphoid structures (TLS). TLS are increasingly recognised as sites for antigen presentation, T-cell activation, B-cell maturation and differentiation in plasma cells. In this review we dissect the role of B cells and provide directions for future studies to harness the role of B cells in treatment of human pancreatic cancer.

Evolution of the Tumor Microenvironment throughout Progression and Transformation of EZH2 Mutant Follicular Lymphoma

The genetic hallmarks of follicular lymphomas include BCL2 translocations and somatic mutations of epigenetic modifier genes such as EZH2. Histologically, FLs typically feature a rich microenvironment, most notably featuring extensive follicular dendritic cell (FDC) networks with dendrites making extensive contact with lymphoma cells. In recent work we showed that the main effect of EZH2 gain-of-function mutations in GC B-cells is to enable them to become less dependent of T-cell help and strengthen their immune synapse formation with FDCs, which induces aberrant proliferation and survival of GC centrocytes and hence formation of a unique lymphoma-permissive immune niche. However, it is still unknown how interactions between EZH2 mutant GC B-cells and other immune cells change throughout the progression of the disease.To evaluate the evolution of the tumor microenvironment (TME) throughout EZH2 mutant lymphoma progression, we developed a genetically engineered mouse model designed for conditional expression of gain-of-function Ezh2 mutant and BCL2 in GC B-cells, Rosa26 LSL.BCL2.IRES.GFP;Ezh2 Y641F;Cγ1Cre (hereafter BCL2/Ezh2 Y641F), in which Cγ1-driven Cre excision of a STOP cassette in the Rosa26 locus leads to overexpression of BCL2 and GFP, and expression of the endogenous Ezh2 mutant Y641F. This mouse model develops low-grade follicular-like lymphoma, characterized by expanded follicles composed largely of centrocyte neoplastic GC B-cells and extensive FDC meshwork, along with presence of CD4 +, TFH and regulatory T cells (FOXP3 +), as depicted by multiparametric in situ imaging and multiparametric flow cytometry. At cytological level, cells are predominantly small with condensed chromatin, irregular nuclei, scant cytoplasm, and inconspicuous nucleoli, without sheets of large cells. Over time, these low grade FLs progress to advanced grade, characterized by disruption of follicle structures, expansion of centroblast-like large tumor cells and reduced CD4 + T cell infiltration and FDC meshwork.Furthermore, we have developed a murine transformed FL cell line by sequential passages of an original BCL2/Ezh2 Y641F low-grade FL into immunodeficient Rag1KO mice. We have transduced this BCL2/Ezh2 Y641F cell line with luciferase, and it successfully engrafted and homed to lymphoid organs when injected i.v. into immunocompetent C57BL6 mice. The cell line consists of high proliferative GC B-cells with multiple and irregular nuclei, open chromatin and prominent nucleoli that resemble DLBCL. The microenvironment of tumors developed in C57BL6 mice is characterized by disruption of follicle structures, severe reduction or absence of FDC meshwork, decreased CD4 +, CD8 + and Tregs, downregulation of MHC-I and MHC-II. Therefore, our mouse model also recapitulates progression and transformation stages.Since MYC translocations are frequent events that occur during histological transformation of FL, we modeled this more aggressive EZB MYC subtype of DLBCL. For that, we further crossed our BCL2/Ezh2 Y641F mice to Rosa26 LSL.MYC.IRES.hCD2. We generated cohorts of transplanted mice by injecting bone marrow cells from BCL2, BCL2/Ezh2 Y641F, BCL2/MYC, and BCL2/Ezh2 Y641F/MYC mice into lethally irradiated C57BL6 recipients. Recipient mice (n=5 per group) were immunized with SRBC to induce formation of GCs and sacrificed 5.5 months post bone marrow transplant. All genotypes showed expansion of FAS +CD38 -BCL2 GFP+ GC B-cells in spleen and lymph nodes; however, the proportion of GC B-cells in Ezh2 Y641F was significantly increased compared with Ezh2 WT, in both presence and absence of MYC. In contrast, FAS -CD38 +BCL2 GFP+ and CD138 +BCL2 GFP+ cells were significantly increased in MYC + mice and decreased in Ezh2 Y641F, suggesting that Ezh2 mutation is required to maintain the GC phenotype. The acquisition of Ezh2 Y641F in BCL2/MYC mice induced a reduction of CD4 + and CD8 + in the TME, and decreased TFH without changes in TFR proportions in GCs. Strikingly, FDC meshwork was significantly shrank in MYC + cases, and partially restored by acquisition of Ezh2 Y641F, indicating that MYC overexpression may contribute to acquire FDC independency for the survival of lymphoma cells.Our results indicate that progression of FL critically affects the TME and acquisition of additional mutations such as MYC alters the interactions between lymphoma cells and tumor supportive immune cells in TME. DisclosuresMelnick: Epizyme: Consultancy; Janssen: Research Funding; KDAC Therapeutics: Current holder of individual stocks in a privately-held company; Celgene Corporation: Consultancy; Constellation Pharmaceuticals: Consultancy; Astra Zeneca: Consultancy; Daiichi Sankyo: Consultancy; Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sanofi US Services: Research Funding.

Development of follicular dendritic cells in lymph nodes depends on retinoic acid-mediated signaling.

Specialized stromal cells occupy and help define B- and T-cell domains, which are crucial for proper functioning of our immune system. Signaling through lymphotoxin and TNF receptors is crucial for the development of different stromal subsets, which are thought to arise from a common precursor. However, mechanisms that control the selective generation of the different stromal phenotypes are not known. Using in vitro cultures of embryonic mouse stromal cells, we show that retinoic acid-mediated signaling is important for the differentiation of precursors towards the Cxcl13pos follicular dendritic cell (FDC) lineage, and also blocks lymphotoxin-mediated Ccl19pos fibroblastic reticular cell lineage differentiation. Accordingly, at the day of birth we observe the presence of Cxcl13posCcl19neg/low and Cxcl13neg/lowCcl19pos cells within neonatal lymph nodes. Furthermore, ablation of retinoic acid receptor signaling in stromal precursors early after birth reduces Cxcl13 expression, and complete blockade of retinoic acid signaling prevents the formation of FDC networks in lymph nodes.

Tre-P-28 - Primary cutaneous B-cell lymphoma leg-type related to an anti-TNF-alpha therapy

<h3>Introduction:</h3> Drug-induced primary cutaneous B-cell lymphomas are rare. <h3>Observation:</h3> A 63 year old woman was referred for a lumbar cutaneous lesion which had been rapidly growing for 1 month. Her past medical history included multiple allergies and a rheumatoid arthritis which was treated with oral methotrexate 10 mg per week and adalimumab. Methotrexate had been taken for 8 years, while adalimumab had been initiated 6 months earlier. Physical examination revealed a solitary thick, violaceous plaque of 6 X 4 cm in diameters, without enlarged lymph nodes. No fatigue, fever or pruritus were present. The first clinical hypothesis was a cutaneous B-cell lymphoma. The histological examination of a skin biopsy specimen showed a monomorphous dermal proliferation of large centroblast-like cells with large round, sometimes irregular, nuclei. Only rare reactive small cells were present. The infiltrate spared the epidermis, with a Grenz zone. The tumor cells were positive for CD45, CD20, bcl2, bcl6, MUM1, highly proliferating (Ki67 95%), and negative for CD3, CD30, CD10 and EBV/EBER. No CD21 or CD23 positive follicular dendritic cell network was identified. Fluorescence in situ hybridization showed no BCL2, BCL6 or CMYC rearrangement, while high resolution melting identified the MYD88 L265P mutation. A B-cell rearrangement was detected in the lesion. Whole body FDG-PET/CT showed a single hypermetabolic activity corresponding to the cutaneous lombar lesion, without distant tumours. Although the Borrelia burgdorferi serology was negative, a 15 days course of doxycyline 200 mg daily was given. Once the diagnosis of primary cutaneous large B-cell lymphoma, leg type (PCLBCL-LT) was made, adamimumab (but not methotrexate) was discontinued. The tumor completely disappeared within 4 months. No other TNF inhibitors were later given. No recurrence was observed after a follow-up time of 4 years. <h3>Discussion:</h3> PCDLBCL-LT are aggressive B-cell lymphomas mainly (but not exclusively) located on the leg, characterized by a monomorphous proliferation of large CD20+, Bcl2+, Mum1+, CD10 - B cells, frequently exhibiting the MYD88 L265P mutation, as observed in our patient. In contrast with indolent subtypes of primary cutaneous B-cell lymphomas, spontaneous regressions in PCLBCL-LT are exceptional, and constantly followed by recurrences. Progression of undiagnosed cutaneous lymphomas after anti-TNF-alpha therapy (mainly cutaneous T-cell lymphomas misdiagnosed as psoriasis or eczema) have been reported [1]. However, the role of anti-TNF-alpha in the occurrence of new lymphoproliferative disorders remains controversial [2,3]. To our knowledge, no PCLBCL-LT related to TNF-alpha inhibitors has been reported to date. Although methotrexate may play a role as a cofactor for the development of this B-cell malignancy, the negative EBV staining and the long-term complete remission after adalimumab withdrawal while methotrexate was continued, strongly argue for a predominant role of the anti-TNF-alpha in the occurrence of this lymphoma.

Emerging roles for IL-6 family cytokines as positive and negative regulators of ectopic lymphoid structures

IL-6 family cytokines display broad effects in haematopoietic and non-haematopoietic cells that regulate immune homeostasis, host defence, haematopoiesis, development, reproduction and wound healing. Dysregulation of these activities places this cytokine family as important mediators of autoimmunity, chronic inflammation and cancer. In this regard, ectopic lymphoid structures (ELS) are a pathological hallmark of many tissues affected by chronic disease. These inducible lymphoid aggregates form compartmentalised T cell and B cell zones, germinal centres, follicular dendritic cell networks and high endothelial venules, which are defining qualities of peripheral lymphoid organs. Accordingly, ELS can support local antigen-specific responses to self-antigens, alloantigens, pathogens and tumours. ELS often correlate with severe disease progression in autoimmune conditions, while tumour-associated ELS are associated with enhanced anti-tumour immunity and a favourable prognosis in cancer. Here, we discuss emerging roles for IL-6 family cytokines as regulators of ELS development, maintenance and activity and consider how modulation of these activities has the potential to aid the successful treatment of autoimmune conditions and cancers where ELS feature.

P0383 - Ectopic germinal centres with B and T cells and follicular dendritic cell networks in urethral stricture tissue: Possible avenue for immunological treatments

P0383 - Ectopic germinal centres with B and T cells and follicular dendritic cell networks in urethral stricture tissue: Possible avenue for immunological treatments

POS0782 FOLLICULAR DENDRITIC CELL NETWORKS IN MINOR SALIVARY GLAND BIOPSIES: A MARKER OF DISEASE ACTIVITY AT BASELINE AND DURING THE FOLLOW-UP IN SJÖGREN’S SYNDROME

Background:Minor salivary gland biopsy (MSGB) has been increasingly recognized as an important tool for the stratification of patients with primary Sjögren’s syndrome (pSS). Recently, the presence of follicular dendritic cell (FDC) networks in the inflammatory infiltrate has been associated with more severe biological and serological abnormalities compared with less organized infiltrates.Objectives:To investigate the associations between the presence and number of FDC networks in foci and pSS glandular and extra-glandular disease activity at baseline and during the follow-up.Methods:Consecutive MSGBs performed in daily practice for clinically suspected pSS from January 2017 to October 2020 were reviewed. Patients’ demographic, clinical, biological and serological data were obtained from medical records. The ESSDAI was used to measure disease activity at baseline and at the end of the follow-up in pSS patients. For histopathology, 3 µm sections were cut from each formalin-fixed paraffin-embedded block of MSGBs and stained with haematoxylin and eosin (H&amp;E). Immunohistochemical stainings were performed on additional 3 μm sections in order to detect T lymphocytes (CD3), B lymphocytes (CD20) and follicular dendritic cells (CD21). Glandular tissue areas, number of foci, focus score (FS) and the presence and number of FDC networks were assessed.Results:We reviewed 330 consecutive MSGBs from patients with suspected pSS: out of them 146/330 (44%) were classified as nonspecific chronic sialadenitis (NSCS) whereas 184/330 (56%) as focal lymphocytic sialadenitis (FLS). According to the ACR/EULAR criteria the diagnosis of pSS was confirmed in 130 patients (117 F:13 M, age 56±13 yrs). The mean (S.D) surface area of the MSGBs was 7.8 (3.9) mm2. The number of foci in the FLS samples ranged from 1 to 12 (mean (S.D)=3.1 (2.6)), whereas the FS ranged from 0.3 to 9.0 (mean (S.D) = 1.4 (1.2)).The presence of FDC networks was documented in 106/330 (32%) of the samples; in 54/106 (51%) of these MSGBs the number of FDC networks ranged from 2 to 8 (mean (S.D)=3.1 (1.4)). The number of FDC networks significantly correlated with the number of foci (r=0.721**) and FS (r=0.707**). Patients with FDC networks in the inflammatory infiltrate presented more serological abnormalities (i.e anti-Ro/SSA, anti-LA/SSB, Rheumatoid factor) and elevated IgG levels (p&lt;0.001). In pSS patients, the number of FDC networks slightly correlated also with C4 levels (r=-0.216*), peripheral lymphocyte count (r=-0.274**) and with glandular (r=0.213*), and biological (r=0.230**) domains of the ESSDAI at baseline. After a mean (S.D) follow-up of 21(13) months, the number of FDC networks still correlated with the final total ESSDAI (r=0.312**).Conclusion:The presence and number of FDC networks in foci represent a useful histopathological parameter able to reflect disease activity at baseline and during the follow-up, thus allowing more personalized interventions.Disclosure of Interests:None declared