A new mode for the diagnosis of angioimmunoblastic T-cell lymphoma.

In order to explore a new mode for the diagnosis of angioimmunoblastic T-cell lymphoma (AITL), 31 cases of AITL and 28 cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) were used as the study subjects. Identifying T follicular helper (TFH) cells with CD4, CD10, Bcl-6, and PD-1, identifying proliferative B cells with CD20 and EZH2, identifying proliferative follicular dendritic cells (FDCs) with CD21 and CD23, and analyzing the value of TFH/B/FDC proliferation and immunolocalization in the diagnosis of AITL. (1) Outside the inherent lymphoid follicles, simultaneous proliferation of TFH/B/FDC (a new diagnostic mode) were observed in AITL [83.87%; 26/31], with their immunolocalizations in the same site [83.87%; 26/31], while this phenomenon was not observed in 28 cases of PTCL-NOS (P<0.05). (2) The sensitivity and specificity of using this new mode to diagnose AITL were both high (83.87%, 100%), which was superior to CD2 (100%, 0%), CD3 (100%, 0%), CD4 (100%, 32.14%), CD5 (100%, 25%), CD10 (61.9%, 100%), Bcl-6 (42.86%, 100%), PD-1 (83.87%, 96.43%), and its Youden Index (0.84) was the highest. The areas under the curve (AUC) of CD10, Bcl-6, PD-1, and new mode to diagnosis AITL were 0.81, 0.71, 0.90, and 0.92, respectively, while the new mode had the highest AUC. The simultaneous proliferation of TFH/B/FDC cells outside the inherent lymphoid follicles can be used to assist in the diagnosis of AITL, and the simultaneous spatiotemporal proliferation of TFH/B/FDC cells is a specific immunomorphology of AITL.

Vagus nerve stimulation modulates distinct acetylcholine receptors on B cells and limits the germinal center response.

Acetylcholine is produced in the spleen in response to vagus nerve activation; however, the effects on antibody production have been largely unexplored. Here, we use a chronic vagus nerve stimulation (VNS) mouse model to study the effect of VNS on T-dependent B cell responses. We observed lower titers of high-affinity IgG and fewer antigen-specific germinal center (GC) B cells. GC B cells from chronic VNS mice exhibited altered mRNA and protein expression suggesting increased apoptosis and impaired plasma cell differentiation. Follicular dendritic cell (FDC) cluster dispersal and altered gene expression suggested poor function. The absence of acetylcholine-producing CD4+ T cells diminished these alterations. In vitro studies revealed that α7 and α9 nicotinic acetylcholine receptors (nAChRs) directly regulated B cell production of TNF, a cytokine crucial to FDC clustering. α4 nAChR inhibited coligation of CD19 to the B cell receptor, presumably decreasing B cell survival. Thus, VNS-induced GC impairment can be attributed to distinct effects of nAChRs on B cells.

EBV-Positive Inflammatory Follicular Dendritic Cell Sarcoma of the Spleen: Report of an Aggressive Form With Molecular Characterization.

EBV-positive inflammatory follicular dendritic cell sarcoma (EBV+ inflammatory FDCS) is a rare neoplasm almost exclusively located in the spleen or liver. It is characterized by a proliferation of EBV-positive spindle-shaped cells bearing follicular dendritic cell markers, associated with an abundant lymphoplasmacytic infiltrate. EBV+ inflammatory FDCS is often asymptomatic or responsible for mild symptoms. It usually displays an indolent course and its prognosis is excellent after tumor removal, although relapsing and metastatic forms exist. Herein, we describe an aggressive form of splenic EBV+ inflammatory FDCS in a 79-year-old woman presenting with abdominal pain, deterioration of general health status, major inflammatory syndrome, and symptomatic hypercalcemia. A splenectomy was performed leading to a rapid improvement in her clinical condition and normalization of laboratory abnormalities. Unfortunately, her symptoms and laboratory abnormalities reappeared 4 months later. Computed tomography showed a mass in the splenectomy site and multiple liver and peritoneal nodules. Further analyses were performed on tumor tissue and showed positive phospho-ERK staining of tumoral cells indicating activation of MAPK pathway. Inactivating mutations were found on CDKN2A and NF1 genes. Subsequently, the patient's condition deteriorated rapidly. Since interleukin-6 levels were dramatically increased, tocilizumab was used but only had a transient effect on the patient's symptoms and inflammatory syndrome. Antitumor agent gemcitabine was initiated but her clinical condition continued to deteriorate and the patient died 2 weeks later. The management of aggressive forms of EBV+ inflammatory FDCS remains challenging. However, since these tumors seem to display genetic alterations, better characterization could lead to molecular targeted therapies.

Akt, IL-4, and STAT Proteins Play Distinct Roles in Prostaglandin Production in Human Follicular Dendritic Cell-like Cells.

This study aimed to explore the role of Akt protein in the induction and inhibition of prostaglandin (PG) in human follicular dendritic cell (FDC)-like cells. FDC-like cells and B cells were isolated from human tonsils. PG production was assessed using enzyme immunoassay, while the upstream cyclooxygenase-2 (COX-2) protein levels were measured using immunoblotting with FDC-like cells transfected with Akt siRNA to analyze the impact of Akt knockdown. The COX-2 expression and PG production induced with IL-1ß were significantly increased by Akt knockdown. However, IL-1ß did not significantly alter either total or phosphorylated Akt protein levels. Akt knockdown resulted in the augmentation of COX-2 expression induced by B cells, although the addition of B cells did not significantly modulate both total and phosphorylated Akt proteins. In contrast, IL-4 specifically exhibited a potent inhibitory effect on COX-2 protein induction and PG production via STAT6. The inhibitory activity of IL-4 was not hampered by Akt knockdown. Interestingly, COX-2 expression levels induced with IL-1ß were markedly modulated with STAT1 and STAT3 knockdown. STAT1 silencing resulted in further augmentation of COX-2, whereas STAT3 silencing prohibited IL-1ß from stimulating COX-2 expression. The current results suggest that Akt, IL-4, and STAT1 play inhibitory roles in PG production in FDC-like cells and expand our knowledge of the immune inflammatory milieu.

Three-dimensional human germinal centers of different sizes in patients diagnosed with lymphadenitis show comparative constant relative volumes of B cells, T cells, follicular dendritic cells, and macrophages.

Germinal centers (GCs) are some of the most important structures in the human immune system. As such, their cell types and functions have been thoroughly investigated. B cells, T cells, follicular dendritic cells (FDCs), and macrophages have widely been found to typically be aggregated in GCs. However, the amount of space occupied by each of these cell types has yet to be investigated. In this study, we conducted confocal laser-based 3D cell-volume quantification of typical GC cells under reactive conditions in lymphadenitis and investigated how volume proportions change during GC development. For this investigation, we used anti-CD3 (T cells), anti-CD20 and anti-Pax5 (B cells), anti-CD23 (FDCs), anti-CD68 (macrophages), and DAPI (nuclear staining). We detected average proportions of about 11% CD3, 9% CD20, 6% CD23, and 2% CD68 in the largest possible regions of interest within GCs. Interestingly, these values remained steady relatively independent of GC size. The remarkably low B cell proportion can be attributed to technical constraints given the use of the CD20 antibody in 3D. Applying the B cell marker Pax5, we found that about 44% of the volume was occupied by B cells after extrapolating the volume of B cell nuclei to that of whole B cells. We concluded that Pax5 is more suitable than anti-CD20 for 3D B cell quantification in GCs. The substantial unstained volume in GCs raises the question of whether other cell types fill these open spaces. Our 3D investigation enabled a unique morphological and volumetric evaluation of GC cells that balance their overall volumes in GCs.

The Follicular Dendritic Cells and HPV 18 Interrelation in Head and Neck Squamous Cell Carcinomas of the Larynx.

Background and Objectives: Even if they are cells of controversial origin (mesenchymal, perivascular, or fibroblastic), follicular dendritic cells (FDC) are present in all organs. The aim of this study was to establish the FDC expression pattern and its interrelation with HPV 18 expression in laryngeal squamous cell carcinoma (LSCC). Materials and Methods: Fifty-six cases of LSCC were evaluated by simple and double immunostaining. The following score was used: 0 (negative or few positive cells), 1 (10-30% of positive cells), 2 (30-50% of cells), and 3 (over 50% of cells). Results: The expression of CD 21-positive cells with dendritic morphology (CDM) was noticed in the intratumoral area of conventional (well and poorly differentiated types and HPV 18 positive cases with a value of 2 for the score) and papillary types (HPV-18 negative cases with a score of 1). The highest value of 2 for the score of CDM in HPV-18 positive cases was found in the peritumoral area of well- and poorly-differentiated conventional LSCCs. A significant correlation was found between scores of CDM from the intratumoral area and those of the peritumoral area (p = 0.001), between CDM and non-dendritic morphology cells (NDM) of the intratumoral area (p = 0.001), and between HPV-18 status and peritumoral NDM cells (p = 0.044). Conclusions: The FDC and NDM cell score values of intratumoral and peritumoral areas may represent important parameters of LSCCs. This may contribute to a better stratification of laryngeal carcinoma cases and the individualized selection of clinical treatment protocols.

FDC-SP as a diagnostic and prognostic biomarker and modulates immune infiltrates in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC), one of the top 10 causes of cancer death, is responsible for more than 90% of all cases of primary renal cancer worldwide. Follicular dendritic cell-secreted protein (FDC-SP) specifically binds to activated B cells and regulates the generation of antibodies. It is also thought to promote cancer cell invasion and migration, which could help with tumor metastases. This study aimed to assess the efficacy of FDC-SP in the diagnosis and prognosis of RCC and to investigate the relationship between immune infiltration in RCC and these outcomes. RESULTS: RCC tissues had significantly higher levels of FDC-SP protein and mRNA than normal tissues. The high level of FDC-SP expression was linked to the T stage, histological grade, pathological stage, N stage, M stage, and OS event. Functional enrichment analysis identified the major pathways that were enriched as immune response regulation, complement, and coagulation. Immunological checkpoints and immune cell infiltration were observed to substantially correlate with the levels of FDC-SP expression. FDC-SP expression levels showed the ability to precisely distinguish high-grade or high-stage renal cancer (area under the curve (AUC) = 0.830, 0.722), and RCC patients with higher FDC-SP expression levels had worse prognoses. The AUC values for one-, two-, and five-year survival rates were all greater than 0.600. Moreover, the FDC-SP expression is an independent predictive biomarker of OS in RCC patients. CONCLUSION: FDC-SP may be a prospective therapeutic target in RCC as well as a possible diagnostic and prognostic biomarker associated with immune infiltration.

Extranodal Mesenteric Follicular Dendritic Cell Sarcoma Expressing Keratin Antigens: What Pitfalls Initiate Diagnostic Clues.

Extranodal follicular dendritic cell sarcomas are infrequent diagnostically challenging tumors. Because of their rarity, heterogeneous histomorphologic features and variable histologic grades a significant number of extranodal lesions are prone to be misdiagnosed. Even though they have a characteristic immunoprofile, expression of a range of nonspecific markers is well documented. Even though they are typically negative for keratins, few authors have reported lesions expressing keratin. Keratin expressing tumors are more likely to be misinterpreted by pathologists further deterring their inclusion in the differential diagnosis. We report an intraabdominal mesenteric follicular dendritic cell sarcoma in a 44-year-old male that immunophenotypically expressed keratin antigens. The lesion showed a high-grade pleomorphic epithelioid appearance and the initial differential diagnosis included lymphoma, sarcomas, melanoma, and carcinomas. Follicular dendritic cell sarcoma was not considered. Expression of epithelial membrane antigen and keratin further deterred the diagnosis which was reached only after extensive use of immunomarkers. The tumor cells expressed CD21, CD23, and D2-40. Morphologically, the tumor showed some thymoma-like features with occasional TDT-expressing background T-lymphocytes. These features were hints to reconsider our differential diagnosis to include follicular dendritic cell tumors. Awareness of this aberrant staining of epithelial immunomarkers and attention to certain clues should encourage pathologists to consider this entity. Speculative assumptions may explain this unusual keratin expression in some lesions. The histomorphologic and immunohistochemical heterogeneity may suggest different variants and grades of follicular dendritic cell sarcomas. The prevalence, importance, and histogenesis of keratin expression in follicular dendritic cell sarcomas warrant further studies.

New definitions of human lymphoid and follicular cell entities in lymphatic tissue by machine learning.

Histological sections of the lymphatic system are usually the basis of static (2D) morphological investigations. Here, we performed a dynamic (4D) analysis of human reactive lymphoid tissue using confocal fluorescent laser microscopy in combination with machine learning. Based on tracks for T-cells (CD3), B-cells (CD20), follicular T-helper cells (PD1) and optical flow of follicular dendritic cells (CD35), we put forward the first quantitative analysis of movement-related and morphological parameters within human lymphoid tissue. We identified correlations of follicular dendritic cell movement and the behavior of lymphocytes in the microenvironment. In addition, we investigated the value of movement and/or morphological parameters for a precise definition of cell types (CD clusters). CD-clusters could be determined based on movement and/or morphology. Differentiating between CD3- and CD20 positive cells is most challenging and long term-movement characteristics are indispensable. We propose morphological and movement-related prototypes of cell entities applying machine learning models. Finally, we define beyond CD clusters new subgroups within lymphocyte entities based on long term movement characteristics. In conclusion, we showed that the combination of 4D imaging and machine learning is able to define characteristics of lymphocytes not visible in 2D histology.

FDCSP Is an Immune-Associated Prognostic Biomarker in HPV-Positive Head and Neck Squamous Carcinoma.

Background: Head and neck squamous carcinoma (HNSC) poses a major threat to human life. The role of human papillomavirus (HPV) infection in the initiation and progression of HNSC is becoming more widely accepted. HPV-positive (HPV+) HNSC has shown unique responses to cancer therapies, which may be due to differences in immune cell infiltration. It is critical to determine how the immune responses to HPV in HNSC are regulated. Methods: Transcriptome data of HNSC from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database were analyzed. Then, the CIBERSORT algorithm was used to calculate immune cell infiltration in HNSC. FDCSP expression level was detected by qPCR in the HNSC tissues collected from the Nanfang Hospital. Results: Follicular dendritic cell secreted protein (FDCSP) was highly expressed in HPV+ HNSC, and higher expression of FDSCP was associated with a favorable prognosis. In HPV+ HNSC samples, FDCSP significantly increased the proportion of T follicular helper cells (TFHs). FDCSP expression was also found to be associated with TP53 mutation status in HPV+ HNSC. The function of FDCSP was intimately connected to chemokine pathways, particularly with the C-X-C motif chemokine ligand 13 (CXCL13). We verified that the high expression of FDCSP in HPV+ HNSC and higher FDCSP is closely related to prognosis in HNSC samples we collected by qPCR. Conclusions: Collectively, these findings may provide fresh evidence that FDCSP is a potential chemokine-associated prognostic biomarker in HPV+ HNSC.

The immunoreceptor SLAMF8 promotes the differentiation of follicular dendritic cell-dependent monocytic cells with B cell-activating ability.

Follicular dendritic cells (FDCs) play a crucial role in generating high-affinity antibody-producing B cells during the germinal center (GC) reaction. Herein, we analysed the altered gene expression profile of a mouse FDC line, FL-Y, following lymphotoxin ß receptor stimulation, and observed increased Slam-family member 8 (Slamf8) mRNA expression. Forced Slamf8 expression and SLAMF8-Fc addition enhanced the ability of FL-Y cells to induce FDC-induced monocytic cell (FDMC) differentiation. FDMCs accelerated GC-phenotype proliferation in cultured B cells, suggesting that they are capable of promoting GC responses. Furthermore, a pulldown assay showed that SLAMF8-Fc could bind to SLAMF8-His. Overall, the homophilic interaction of SLAMF8 promotes FDMC differentiation and SLAMF8 might act as a novel regulator of GC responses by regulating FDMC differentiation.

Foot-and-mouth disease virus localisation on follicular dendritic cells and sustained induction of neutralising antibodies is dependent on binding to complement receptors (CR2/CR1).

Previous studies have shown after the resolution of acute infection and viraemia, foot-and-mouth disease virus (FMDV) capsid proteins and/or genome are localised in the light zone of germinal centres of lymphoid tissue in cattle and African buffalo. The pattern of staining for FMDV proteins was consistent with the virus binding to follicular dendritic cells (FDCs). We have now demonstrated a similar pattern of FMDV protein staining in mouse spleens after acute infection and showed FMDV proteins are colocalised with FDCs. Blocking antigen binding to complement receptor type 2 and 1 (CR2/CR1) prior to infection with FMDV significantly reduced the detection of viral proteins on FDCs and FMDV genomic RNA in spleen samples. Blocking the receptors prior to infection also significantly reduced neutralising antibody titres, through significant reduction in their avidity to the FMDV capsid. Therefore, the binding of FMDV to FDCs and sustained induction of neutralising antibody responses are dependent on FMDV binding to CR2/CR1 in mice.

Role of the CXCL13/CXCR5 Axis in Autoimmune Diseases.

CXCL13 is a B-cell chemokine produced mainly by mesenchymal lymphoid tissue organizer cells, follicular dendritic cells, and human T follicular helper cells. By binding to its receptor, CXCR5, CXCL13 plays an important role in lymphoid neogenesis, lymphoid organization, and immune responses. Recent studies have found that CXCL13 and its receptor CXCR5 are implicated in the pathogenesis of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, primary Sjögren's syndrome, myasthenia gravis, and inflammatory bowel disease. In this review, we discuss the biological features of CXCL13 and CXCR5 and the recent findings on the pathogenic roles of the CXCL13/CXCR5 axis in autoimmune diseases. Furthermore, we discuss the potential role of CXCL13 as a disease biomarker and therapeutic target in autoimmune diseases.

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.

Establishment of an ATLL cell line (YG-PLL) dependent on IL-2 and IL-4, which are replaced by OX40-ligand+ HK with poly-L-histidine and dermatan sulfate.

We established an IL-2 and IL-4 (IL2/4) - dependent adult T-cell leukemia/lymphoma (ATLL) cell line (YG-PLL) by adding poly-L-lysine (PLL) to the culture medium. YG-PLL originates from lymphoma cells and contains a defective HTLV-I proviral genome. Although YG-PLL cannot survive without IL-2/4, the follicular dendritic cell (FDC)-like cell line HK expressing OX40-ligand gene (OX40L+HK) inhibited their death in the presence of soluble neutral polymers. After the prevention of cell death, YG-PLL proliferated on OX40L+HK without IL2/4 in the presence of two kinds of positively or negatively charged polymers. In particular, dermatan sulfate and poly-L-histidine supported growth for more than 4 months. Therefore, the original lymphoma cells proliferated transiently in the presence of IL2/4, and their growth arrest was inhibited by the addition of PLL. Furthermore, YG-PLL lost IL2/4 dependency by the following 3-step procedure: preculture with IL2/4 and neutral polymers, 3-day culture with neutral polymer on OX40L+HK to inhibit cell death, and co-culture with OX40L+HK in the presence of the positively and negatively charged polymers. The extracellular environment made by soluble polymers plays a role in the growth of ATLL in vitro.

The Migration of Human Follicular Dendritic Cell-Like Cell Is Facilitated by Matrix Metalloproteinase 3 Expression That Is Mediated through TNFα-ERK1/2-AP1 Signaling.

Follicular dendritic cells are important stromal components of the germinal center (GC) and have pivotal roles in maintaining the GC microenvironment for high-affinity antibody production. Tumor necrosis factor-α (TNFα) is essential for the development and functions of follicular dendritic cells. Despite the importance of follicular dendritic cells in humoral immunity, their molecular control mechanisms have yet to be fully elucidated due to the lack of an adequate investigation system. Here, we have used a unique human primary follicular dendritic cell-like cell (FDCLC) to demonstrate that the migration of these cells is enhanced by TNFα-mediated metalloproteinase 3 (MMP3) expression. MMP3 was found to be highly expressed in normal human GCs and markedly upregulated in human primary FDCLCs by TNFα. TNFα induced ERK1/2 phosphorylation and the transcription of MMP3 through AP1. TNFα treatment increased FDCLC migration, and a knockdown of MMP3 significantly reduced the TNFα-induced migration of FDCLCs. Overall, we have newly identified a control mechanism for the expression of MMP3 in FDCLCs that modulates their migration and may indicate an important role in GC biology. Since GCs are observed in the lesions of autoimmune diseases and lymphomas, targeting the MMP3/TNFα-mediated migration of stromal cells in the B cell follicle may have great potential as a future therapeutic modality against aberrant GC-associated disorders.

CD169+ Subcapsular Macrophage Role in Antigen Adjuvant Activity.

Vaccines have played a pivotal role in improving public health, however, many infectious diseases lack an effective vaccine. Controlling the spread of infectious diseases requires continuing studies to develop new and improved vaccines. Our laboratory has been investigating the immune enhancing mechanisms of Toll-like receptor (TLR) ligand-based adjuvants, including the TLR2 ligand Neisseria meningitidis outer membrane protein, PorB. Adjuvant use of PorB increases costimulatory factors on antigen presenting cells (APC), increases antigen specific antibody production, and cytokine producing T cells. We have demonstrated that macrophage expression of MyD88 (required for TLR2 signaling) is an absolute requirement for the improved antibody response induced by PorB. Here-in, we specifically investigated the role of subcapsular CD169+ marginal zone macrophages in antibody production induced by the use of TLR-ligand based adjuvants (PorB and CpG) and non-TLR-ligand adjuvants (aluminum salts). CD169 knockout mice and mice treated with low dose clodronate treated animals (which only remove marginal zone macrophages), were used to investigate the role of these macrophages in adjuvant-dependent antibody production. In both sets of mice, total antigen specific immunoglobulins (IgGs) were diminished regardless of adjuvant used. However, the greatest reduction was seen with the use of TLR ligands as adjuvants. In addition, the effect of the absence of CD169+ macrophages on adjuvant induced antigen and antigen presenting cell trafficking to the lymph nodes was examined using immunofluorescence by determining the relative extent of antigen loading on dendritic cells (DCs) and antigen deposition on follicular dendritic cells (FDC). Interestingly, only vaccine preparations containing PorB had significant decreases in antigen deposition in lymphoid follicles and germinal centers in CD169 knockout mice or mice treated with low dose clodronate as compared to wildtype controls. Mice immunized with CpG containing preparations demonstrated decreased FDC networks in the mice treated with low dose clodronate. Conversely, alum containing preparations only demonstrated significant decreases in IgG in CD169 knockout mice. These studies stress that importance of subcapsular macrophages and their unique role in adjuvant-mediated antibody production, potentially due to an effect of these adjuvants on antigen trafficking to the lymph node and deposition on follicular dendritic cells.

A Novel Image Analysis Approach Reveals a Role for Complement Receptors 1 and 2 in Follicular Dendritic Cell Organization in Germinal Centers.

Follicular dendritic cells (FDCs) are rare and enigmatic cells that mainly reside in germinal centers (GCs). They are capable of capturing immune complexes, via their Fc (FcRs) and complement receptors (CRs) and storing them for long periods in non-degradative vesicles. Presentation of ICs on FDCs to B cells is believed to drive affinity maturation. CR1 and CR2 are expressed on B cells and FDCs. Cr2 knock out (KO) mice, lacking both receptors, have impaired antibody and GC responses. Utilizing a novel ImageJ macro to analyze confocal fluorescence microscopy images of spleen sections, we here investigate how FDCs in wild type (WT) and Cr2 KO mice behave during the first two weeks after immunization with sheep red blood cells (SRBC). Mice were immunized with SRBC i.v. and spleen and serum samples harvested at various time points. As expected, antibody and GC responses in Cr2 KO mice were impaired in comparison to WT mice. Fewer FDCs were identified in Cr2 KO mice, and these exhibited differential localization and organization in comparison to WT mice. WT FDCs were primarily located within GCs at the light zone/dark zone border. FDCs from WT but not Cr2 KO mice were actively dispersed in GCs, i.e. tended to move away from each other, presumably to increase their surface area for B cell interaction. FDCs from Cr2 KO mice were more often found on follicles outside of the GCs and those within the GCs were closer to the periphery in comparison to WT FDCs. Expression of CR1 and CR2, FcγRIIB, and FcµR increased in FDCs from WT mice during the course of immunization. The results suggest that decreased ability to capture ICs by FDCs lacking CR1 and CR2 may not be the only explanation for the impaired GC and antibody responses in Cr2 KO mice. Poor FDC organization in GCs and failure to increase receptor expression after immunization may further contribute to the inefficient immune responses observed.

Single-cell sequencing of human white adipose tissue identifies new cell states in health and obesity.

White adipose tissue (WAT) is an essential regulator of energy storage and systemic metabolic homeostasis. Regulatory networks consisting of immune and structural cells are necessary to maintain WAT metabolism, which can become impaired during obesity in mammals. Using single-cell transcriptomics and flow cytometry, we unveil a large-scale comprehensive cellular census of the stromal vascular fraction of healthy lean and obese human WAT. We report new subsets and developmental trajectories of adipose-resident innate lymphoid cells, dendritic cells and monocyte-derived macrophage populations that accumulate in obese WAT. Analysis of cell-cell ligand-receptor interactions and obesity-enriched signaling pathways revealed a switch from immunoregulatory mechanisms in lean WAT to inflammatory networks in obese WAT. These results provide a detailed and unbiased cellular landscape of homeostatic and inflammatory circuits in healthy human WAT.