Germinal Center B Research Articles

Phosphatidic Acid Promoting the Generation of Interleukin-17A Producing Double-Negative T Cells by Enhancing mTORC1 Signaling in Lupus.

The goal was to investigate the role and intracellular regulatory mechanisms of double-negative T (DNT) cells in the pathogenesis of systemic lupus erythematosus (SLE). DNT cells were assessed in murine models, patients with SLE, and controls using flow cytometry (FCM). DNT cells from either resiquimod (R848) or vehicle-treated C57BL/6 (B6) mice were cultured with B cells from R848-treated mice to explore functions. Differential mechanistic target of rapamycin (mTOR) pathway signaling in DNT cells measured using FCM and quantitative polymerase chain reaction was validated by rapamycin inhibition. Candidate lipid metabolites detected using liquid chromatography with electrospray ionization mass spectrometry/mass spectrometry were functionally assessed in DNT cell cultures. DNT cells were markedly increased in both spontaneous and induced mouse lupus models and in patients with SLE. Expanded DNT cells from R848-treated B6 mice produced elevated interleukin (IL)-17A and IgG with increased germinal center B (GCB) cells. Expansion of DNT cells associated with activation of mTORC1 pathway that both IL-17A levels and the number of DNT cells exhibited dose-dependent reduction with rapamycin treatment. Lipidomics studies revealed differential patterns of lipid metabolites in T cells of R848-treated mice. Among candidate metabolites, elevated phosphatidic acid (PA) that was partially controlled by phospholipase D2 increased the expression of the mTORC1 downstream target p-S6 and positively expanded IL-17A-producing DNT cells. Similarly, elevated proportions of circulating DNT cells in patients with SLE correlated with disease activity and proteinuria, and IL-17A secretion was elevated after in vitro PA stimulation. The accumulation of PA in T cells could activate the mTORC1 pathway, promoting DNT cell expansion and IL-17A secretion, resulting in GCB cell abnormalities in lupus.

Research progress of SREBP and its role in the pathogenesis of autoimmune rheumatic diseases.

Autoimmune rheumatic diseases comprise a group of immune-related disorders characterized by non-organ-specific inflammation. These diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), gout, among others. Typically involving the hematologic system, these diseases may also affect multiple organs and systems. The pathogenesis of autoimmune rheumatic immune diseases is complex, with diverse etiologies, all associated with immune dysfunction. The current treatment options for this type of disease are relatively limited and come with certain side effects. Therefore, the urgent challenge remains to identify novel therapeutic targets for these diseases. Sterol regulatory element-binding proteins (SREBPs) are basic helix-loop-helix-leucine zipper transcription factors that regulate the expression of genes involved in lipid and cholesterol biosynthesis. The expression and transcriptional activity of SREBPs can be modulated by extracellular stimuli such as polyunsaturated fatty acids, amino acids, glucose, and energy pathways including AKT-mTORC and AMP-activated protein kinase (AMPK). Studies have shown that SREBPs play roles in regulating lipid metabolism, cytokine production, inflammation, and the proliferation of germinal center B (GCB) cells. These functions are significant in the pathogenesis of rheumatic and immune diseases (Graphical abstract). Therefore, this paper reviews the potential mechanisms of SREBPs in the development of SLE, RA, and gout, based on an exploration of their functions.

Raman Spectroscopy Imaging As a Tool to Classify Diffused Large B-Cell Lymphoma Subtypes

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogenous, aggressive disease. The ‘cell-of-origin’ classification (COO) distinguishes two major DLBCL subtypes, GCB (germinal center B cell-like) and ABC (activated B cell-like) that are derived from distinct putative cell of origin, differ in prognosis and transcriptional pattern. Comprehensive cluster classification (CCC) based on unbiased “consensus clustering” of transcriptomic microarray data has led to the identification of subtypes that differ in energy source utilization and bioenergetic profiles The oxPhos DLBCL subtype, unlike non-oxPhos, is characterized by increased mitochondrial oxidative phosphorylation and energy transduction. Although COO class assignment can be achieved with multiple clinical diagnostic tools (including transcript-based or immunohistochemical approaches), there is no clinical diagnostic tool appropriate for COO classification. In Raman effect, laser light, as a result of internal oscillations of molecules, is scattered with a change in wavelength, giving a spectrum characteristic for a given substance. This allows the chemical composition of mixtures and even cells to be determined on the basis of the obtained spectra. Thus, it also allows to determine differences in metabolism, including energy metabolism of lymphoma cells. The aim of the study was to use the RS images to recognize and classify DLBCL cells. MATERIALS AND METHODS: DLBCL cell lines (OCI-Ly4, Pfeiffer, Toledo, DHL4, DHL6 , OCI-Ly1 and OCI-Ly7) with known COO and CCC subtype assignments, and B cells isolated from the blood of healthy donors were used. For Raman analysis cells were fixed in 0.5% glutaraldehyde and imaged at 533 and 633 nm emission wavelengths under a WITec Raman microscope. The collected spectral images were subjected to chemometric analyses: characteristic bands analysis, k-means cluster analysis (KMCA), principal component analysis (PCA) and least squares discriminant analysis (PLS-DA) using dedicated software. RESULTS: The analysis of Raman spectra revealed clear changes in the structure and composition of cells after neoplastic transformation. Chemometric analysis of spectral bands showed greater proportion of proteins (1008, 1041 and 1660 cm-1) and lipids (1440, 1240 and 2850 cm-1) in DLBCL cells then B cells from healthy donors. More nucleic acids were observed (733, 790 , 1583 cm-1) in normal B cells that at least partially resulted from the greater ratio of the nucleus to the cytoplasm. The differences resulted in clear separation of malignant and healthy cells in PCA and PLS-DA. Similar approach allowed to characterize differences between ABC vs GCB subtype cell lines and oxPhos vs non-oxPhos subtype. Based on the specific differences between healthy B cells and DLBCL cells a predictive model was built using PLS-DA that accurately determines the chance of the analyzed cell, first, being a lymphoma cell, and second, belonging to the ABC vs GCB or oxPhos vs non-oxPhos DLBCL subtype. The good performance of the constructed predictive model was evaluated by prediction on unknown samples with sensitivity and specificity of 98%. CONCLUSIONS: We present a model of a classification of B cell lymphomas based on label-free Raman spectral imagines, capable of automatic and efficient identification of DLBCL cells spectra. In addition, we describe each lymphoma subtype by its unique spectral profile, linking it to biochemical features. Acknowledgements. The studies were performed as a part of the “ Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Glucose oxidation-dependent survival of activated B cells provides a putative novel therapeutic target for lupus treatment

Aberrant metabolic demand is observed in immune/inflammatory disorders, yet the role in pathogenesis remains unclear. Here, we discover that in lupus, activated B cells, including germinal center B (GCB) cells, have remarkably high glycolytic requirement for survival over Tcell populations, as demonstrated by increased metabolic activity in lupus-activated B cells compared to immunization-induced cells. The augmented reliance on glucose oxidation makes GCB cells vulnerable to mitochondrial ROS-induced oxidative stress and apoptosis. Short-term glycolysis inhibition selectively reduces pathogenic activated B in lupus-prone mice, extending their lifespan, without affecting T follicular helper cells. Particularly, BCMA-expressing GCB cells rely heavily on glucose oxidation. Depleting BCMA-expressing activated B cells with APRIL-based CAR-T cells significantly prolongs the lifespan of mice with severe autoimmune disease. These results reveal that glycolysis-dependent activated B and GCB cells, especially those expressing BCMA, are potentially key lupus mediators, and could be targeted to improve disease outcomes.

A-910823, a squalene-based emulsion adjuvant, induces T follicular helper cells and humoral immune responses via α-tocopherol component.

Adjuvants are chemical or biological materials that enhance the efficacy of vaccines. A-910823 is a squalene-based emulsion adjuvant used for S-268019-b, a novel vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is currently in clinical development. Published evidence has demonstrated that A-910823 can enhance the induction of neutralizing antibodies against SARS-CoV-2 in humans and animal models. However, the characteristics and mechanisms of the immune responses induced by A-910823 are not yet known. To characterize A-910823, we compared the adaptive immune response profile enhanced by A-910823 with that of other adjuvants (AddaVax, QS21, aluminum salt-based adjuvants, and empty lipid nanoparticle [eLNP]) in a murine model. Compared with other adjuvants, A-910823 enhanced humoral immune responses to an equal or greater extent following potent T follicular helper (Tfh) and germinal center B (GCB) cell induction, without inducing a strong systemic inflammatory cytokine response. Furthermore, S-268019-b containing A-910823 adjuvant produced similar results even when given as a booster dose following primary administration of a lipid nanoparticle-encapsulated messenger RNA (mRNA-LNP) vaccine. Preparation of modified A-910823 adjuvants to identify which components of A-910823 play a role in driving the adjuvant effect and detailed evaluation of the immunological characteristics induced by each adjuvant showed that the induction of humoral immunity and Tfh and GCB cell induction in A-910823 were dependent on α-tocopherol. Finally, we revealed that the recruitment of inflammatory cells to the draining lymph nodes and induction of serum cytokines and chemokines by A-910823 were also dependent on the α-tocopherol component. This study demonstrates that the novel adjuvant A-910823 is capable of robust Tfh cell induction and humoral immune responses, even when given as a booster dose. The findings also emphasize that α-tocopherol drives the potent Tfh-inducing adjuvant function of A-910823. Overall, our data provide key information that may inform the future production of improved adjuvants.

A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

Plasma cells (PC) are antibody-secreting cells and terminal effectors in humoral responses. PCs differentiate directly from activated B cells in response to T cell-independent (TI) antigens or from germinal center B (GCB) cells in T cell-dependent (TD) antigen-induced humoral responses, both of which pathways are essentially regulated by the transcription factor BLIMP1. The p38 mitogen-activated protein kinase isoforms have already been implicated in B cell development, but the precise role of p38α in B cell differentiation is still largely unknown. Here we show that PC differentiation and antibody responses are severely impaired in mice with B cell-specific deletion of p38α, while B cell development and the GCB cell response are spared. By utilizing a Blimp1 reporter mouse model, we show that p38α-deficiency results in decreased BLIMP1 expression. p38α-driven BLIMP1 up-regulation is required for both TI and TD PCs differentiation. By combining CRISPR/Cas9 screening and other approaches, we identify TCF3, TCF4 and IRF4 as downstream effectors of p38α to control PC differentiation via Blimp1 transcription. This study thus identifies an important signalling pathway underpinning PC differentiation upstream of BLIMP1, and points to a highly specialized and non-redundant role for p38α among p38 isoforms.

DTX1 Controls Germinal Center B-Cell Development and Lymphomagenesis

DTX1 is highly expressed in germinal center B (GCB)-cells and is targeted by coding mutations in 17% of DLBCL, including 50% of the BN2 genetic subtype that is enriched for the ABC-like cell of origin subtype (Wright et al., Cancer Cell 2020). DTX1 has also been recognized as a prominent target of AID-mediated mutagenesis, with a high rate of non-coding regulatory element (RE) mutations being observed across DLBCL subtypes. The DTX1 gene encodes a ring finger ubiquitin ligase that regulates NOTCH1 recycling. However, the role of DTX1 in GC B-cell development and lymphomagenesis has not been thoroughly investigated. We characterized mutations within the regulatory element of DTX1 using whole genome sequencing data from ICGC and TCGA, together with H3K27Ac ChIP-sequencing data from tonsillar GCB-cells to define active regulatory elements (REs). We observed non-coding mutations of DTX1-proximal REs in 59% of DLBCL tumors. We characterized these putative REs by knocking GFP into the start codon of the endogenous DTX1 gene and performing CRISPR/Cas9 tiling, using reductions of GFP expression as a surrogate for reduced DTX1 RE activity. We observed a significant loss of GFP expression with DTX1 RE-targeted gRNAs compared control gRNAs, confirming these recurrently mutated regions as active REs. We therefore generated a novel floxed Dtx1 genetically engineered mouse model and crossed with Aid-cre for GCB-specific conditional knock-out (cKO). Immunized mice were euthanized at the peak of the GC reaction and analyzed by flow cytometry and multispectral immunofluorescence (mIF) imaging. Mice with monoalleleic (Dtx1+/-) or bialleleic (Dtx1-/-) cKO of Dtx1 had a significant increase in GCB cell frequency compared to wild-type (Dtx1+/+) littermates, with a significant skew of polarization towards the light zone. This was confirmed by mIF analysis, which showed significantly larger and more abundant GCs, and a significantly higher fraction of Ki67+ cells within the GCs, in Dtx1+/- compared to Dtx1+/+ mice. We therefore investigated whether Dtx1 loss may confer a competitive advantage within the GC reaction by generating Dtx1+/+ / Dtx1+/- mixed chimera models in Rag-deficient recipients. Dtx1+/- cells were significantly over-represented within the GCB compartment and showed a significant light-zone skew, and significantly higher frequencies of proliferating (Edu+) GCB cells within the light-zone relative to Dtx1+/+ cells. We therefore investigated changes in GC development using scRNA-sequencing PNA-enriched GCB cells from SRBC-immunized Dtx1+/+ (n=4) and Dtx1+/- littermates (n=4). This confirmed a significant light-zone polarization within GCB cells, and highlighted a dramatic reduction in pre-memory cells within Dtx1+/- mice. Furthermore, differential gene expression analysis identified significant alterations in light-zone gene expression programs within Dtx1+/- GCB cells. To test whether Dtx1 loss may participate in the early stages of lymphomagenesis, we performed serial re-challenge with SRBCs monthly for 3 months and 10 days later euthanized mice. Histological analysis of major organs revealed a significant increase in the frequency of lymphoid aggregates within the lungs, liver and kidneys, indicating extranodal dissemination. In conclusion, these data demonstrate for the first time that highly-recurrent mutations in DLBCL target an active RE for DTX1, resulting in inhibition of DTX1 expression. Dtx1 haploinsufficiency confers a selective advantage to GCB cells that promotes GC hyperplasia with light-zone polarization and alters GCB cell differentiation trajectories, and can result in extranodal dissemination of Dtx1+/- GCB cells following chronic antigen stimulation.

Smarca4 (Brg1) Controls Germinal Center B-Cell Development and Is a Haploinsufficient Tumor Suppressor in Lymphoma

SMARCA4 encodes one of two mutually exclusive ATPases present in BAF (aka SWI/SNF) chromatin remodeling complexes. SMARCA4 is dynamically expressed during normal B-cell development, with high expression in germinal center B (GCB)-cells, and is targeted by heterozygous inactivating mutations in GCB-derived lymphomas including 34% of pediatric Burkitt lymphoma (BL), 17% of adult BL, 4% of GCB-DLBCL. However, the functional role of SMARCA4 in GCB cell development and malignant transformation has not been thoroughly investigated. We generated a novel floxed Smarca4 transgenic mouse model and crossed to the Aid-Cre line for GC-specific conditional knock-out (cKO). Following immunization with sheep red blood cells (SRBCs) to induce a GC reaction, Smarca4+/- mice had a significant increase in GCB-cell abundance compared to Smarca4+/+ littermates (p<0.001) due to both an increased average GC size and number. Notably, Smarca4-/- mice displayed a significantly reduced frequency of GC B-cells (p<0.001) and sorting of residual cells revealed retention of 1 or more copies of the floxed allele, suggesting that biallelic cKO of Smarca4 is lethal to GCB cells. We therefore focused subsequent studies on comparisons between Smarca4+/- and Smarca4+/+ mice. Further analysis revealed significant polarization towards a centroblastic phenotype in Smarca4+/- mice (p<0.001), in line with the centroblastic morphology of human BL. Analysis of competitive advantage with the same GC using bone marrow chimera models showed a significant advantage of Smarca4+/- GCB cells compared to Smarca4+/+, associated with a significant increase in proliferation measured by Edu incorporation and reduction of apoptosis measured by Annexin-B. We extended upon this using single cell RNA-seq (scRNA-seq) of GCB cells from Smarca4+/- and Smarca4+/+ mice, which confirmed an increased centroblastic polarization of Smarca4+/- GCB cells and identified a significant increase in light zone to dark zone recycling cells that may underly this polarization. We validated this using the Myc-GFP allele, which confirmed increased Myc+ recycling cells in GCs from Smarca4+/- mice compared to Smarca4-/- mice. BAF complexes drive ATP-dependent mobilization and eviction of nucleosomes to convert chromatin from inactive to active states. Brg1 deficiency may therefore alter distinct BAF complexes and transcription factor programs. We therefore explored the role of Smarca4 in regulating chromatin accessibility and gene expression using ATAC-seq and RNA-seq in Smarca4+/- and Smarca4+/+ murine GCB-cells. Supervised analysis revealed predominant loss of chromatin accessibility associated with repression of nearby genes. Smarca4+/- cells showed significant loss of accessibility of genes upregulated in centrocytes, and a preferential loss of NF-ĸB target genes (GSEA FDR<0.01). MYC translocations are pathognomonic for BL, in which SMARCA4 mutations are most frequent. We therefore crossed our Smarca4 cKO mice with a cre-inducible Myc knock-in (Myc-KI) allele to mimic combined Smarca4 loss-of-function and Myc activation. The addition of Myc-KI did not lead to a significant increase in GCB cell frequency following a single SRBC immunization. However, serial re-challenge of mice with SRBCs led to lymphadenopathy, splenomegaly and infiltration of highly proliferative B-cells into the major organs (lung, liver, and kidney) in Smarca4+/- + Myc-KI mice following 3 immunizations that was not observed with Smarca4+/- or Myc-KI alleles alone. Furthermore, long term follow-up of serially-challenged mice showed a significant reduction in overall survival in Smarca4+/- + Myc-KI mice compared to Smarca4+/- or Myc-KI mice (p=0.010). Collectively, these data demonstrate a competitive advantage of GCB cells with monoallelic Smarca4 cKO, and a centroblastic GC polarization driven in part by increased recycling. Mechanistically, this is associated with loss of chromatin accessibility and gene expression of genes that are expressed in centrocytes, including NFkB target genes. Monoallelic Smarca4 cKO cooperates with Myc over-expression to drive expansion and extranodal dissemination of GCB cells following serial re-challenge, leading to reduced survival. These data provide the first functional and mechanistic insight into the role of SMARCA4 in GCB cell development and lymphomagenesis.

Untargeted Metabolomics of Cerebrospinal Fluid in Patients with Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) is characterized by a high degree of malignancy and poor prognosis. The current diagnostic techniques include cerebrospinal fluid (CSF) metabolomics examination, imaging examination, needle biopsy, etc., but each examination has certain limitations, and it is difficult for the early identification and diagnosis of the disease. In terms of treatment, relapse and refractory, neurotoxicity and drug resistance are still the main problems currently faced. Metabolomics can measure the composition of metabolites and the dynamic changes of metabolites in cell models, tissues, organs or the whole organism, and has been widely used in disease diagnosis, efficacy evaluation, nutrition and other fields. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS/ technology) has been be used to observe the metabolites changes in CSF. Here, we aimed to explore the application value of metabolomics in the diagnosis and prognosis evaluation of PCNSL. From January 2021 to December 2021, CSF samples from 37 DLBCL patients were collected in Shengli Oilfield Central Hospital and Shandong Cancer Hospital, including 20 male patients and 17 female patients. These patients fall into 4 groups based on clinical presentations: 12 patients in PCNSL group (CNS group), 5 patients in primary testicular diffuse large B-cell lymphoma (DLBCL) group (TE group), 10 patients in Germinal center B (GCB) cell-like DLBCL without central nervous system infiltration (GCB group), and 10 patients in non-GCB DLBCL group without central nervous system infiltration (non-GCB group). The CSF samples were collected before clinical treatment, and the samples were analyzed with UPLC-HRMS/technology. For data analysis, principal component analysis (PCA) was carried out on the obtained components, and the partial least squares discriminant analysis model (PLS-DA) was used for diversification analysis. Analytes with P<0.05 and VIP>1 were considered differential metabolites. The Venn diagram was used to narrow the range of metabolites. By using MetaboAnalyst 5.0 software, pathway enrichment analysis was performed on the identified differential metabolites in CSF, and the corresponding P value was obtained. The metabolic pathways that have a significant effect in CNS group vs TE group, CNS group vs GCB group and GCB group vs non-GCB group were glycerophospholipid metabolism and sphingolipid metabolism. The metabolic pathway that has a significant effect in CNS group vs non-GCB group and TE group vs non-GCB group was glycerophospholipid metabolism. Fatty acid degradation and fatty acid elongation were the significant effects metabolic pathway in TE group vs non-GCB group. We applied Venn diagrams to narrow the range of differential metabolites. There were 47 differential metabolites in the CSF of patients in the PCNSL group vs the non-PCNSL group (Figure 1). Compared with the non-PCNSL group, 33 metabolites such as 3-Hydroxyisovalerylcarnitine, Hesperetin 3'-O-sulfate, and Arteether were up-regulated and 14 metabolites such as Pentaglutamyl folate, Calcipotriol and N-Hexadecanoylpyrrolidine were down-regulated in the CSF of patients in the PCNSL group. In the CNS infiltration high-risk group and the CNS infiltration low-risk group, there were 16 differential metabolites (Figure 2). Compared with the patients in the CNS infiltration low-risk group, 8 metabolites such as 3-Allylphenol sulfate, p-Aminobenzoic acid and 2,3,4-Trichlorophenol were up-regulated and 8 metabolites such as Palmitaldehyde, Carboxamide and Rioprostil were down-regulated in the CNS infiltration high-risk group. In conclusion, glycerophospholipid, sphingolipid, and fatty acid metabolites were the three most important abnormal lipid metabolites existed in PCNSL. This finding may have some value in the development of diagnosis and prognostic tools for evaluating CSF in patients with PCNSL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal germinal center B cells function as a niche for T-cell lymphoma

AbstractAngioimmunoblastic T-cell lymphoma (AITL) is proposed to be initiated by age-related clonal hematopoiesis (ACH) with TET2 mutations, whereas the G17V RHOA mutation in immature cells with TET2 mutations promotes the development of T follicular helper (TFH)-like tumor cells. Here, we investigated the mechanism by which TET2-mutant immune cells enable AITL development using mouse models and human samples. Among the 2 mouse models, mice lacking Tet2 in all the blood cells (Mx-Cre × Tet2flox/flox × G17V RHOA transgenic mice) spontaneously developed AITL for approximately up to a year, while mice lacking Tet2 only in the T cells (Cd4-Cre × Tet2flox/flox × G17V RHOA transgenic mice) did not. Therefore, Tet2-deficient immune cells function as a niche for AITL development. Single-cell RNA-sequencing (scRNA-seq) of >50 000 cells from mouse and human AITL samples revealed significant expansion of aberrant B cells, exhibiting properties of activating light zone (LZ)-like and proliferative dark zone (DZ)-like germinal center B (GCB) cells. The GCB cells in AITL clonally evolved with recurrent mutations in genes related to core histones. In silico network analysis using scRNA-seq data identified Cd40–Cd40lg as a possible mediator of GCB and tumor cell cluster interactions. Treatment of AITL model mice with anti-Cd40lg inhibitory antibody prolonged survival. The genes expressed in aberrantly expanded GCB cells in murine tumors were also broadly expressed in the B-lineage cells of TET2-mutant human AITL. Therefore, ACH-derived GCB cells could undergo independent clonal evolution and support the tumorigenesis in AITL via the CD40–CD40LG axis.

Deletion of microRNA-183-96-182 Cluster in Lymphocytes Suppresses Anti-DsDNA Autoantibody Production and IgG Deposition in the Kidneys in C57BL/6-Faslpr/lpr Mice.

Dysregulated miRNAs have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Our previous study reported a substantial increase in three miRNAs located at the miR-183-96-182 cluster (miR-183C) in several autoimmune lupus-prone mice, including MRL/lpr and C57BL/6-lpr (B6/lpr). This study reports that in vitro inhibition of miR-182 alone or miR-183C by specific antagomirs in activated splenocytes from autoimmune-prone MRL/lpr and control MRL mice significantly reduced lupus-related inflammatory cytokines, interferon-gamma (IFNγ), and IL-6 production. To further characterize the role of miR-182 and miR-183C cluster in vivo in lupus-like disease and lymphocyte phenotypes, we used hCD2-iCre to generate B6/lpr mice with conditional deletion of miR-182 or miR-183C in CD2+ lymphocytes (miR-182−/−B6/lpr and miR-183C−/-B6/lpr). The miR-182−/−B6/lpr and miR-183C−/−B6/lpr mice had significantly reduced deposition of IgG immunocomplexes in the kidney when compared to their respective littermate controls, although there appeared to be no remarkable changes in renal pathology. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C−/−B6/lpr mice after reaching 24 weeks-of age compared to age-matched miR-183Cfl/flB6/lpr controls. In vitro activated splenocytes from miR-182−/−B6/lpr mice and miR-183C−/−B6/lpr mice showed reduced ability to produce lupus-associated IFNγ. Forkhead box O1(Foxo1), a previously validated miR-183C miRNAs target, was increased in the splenic CD4+ cells of miR-182−/−B6/lpr and miR-183C−/−B6/lpr mice. Furthermore, in vitro inhibition of Foxo1 with siRNA in splenocytes from miR-182−/−B6/lpr and miR-183C−/-B6/lpr mice significantly increased IFNγ expression following anti-CD3/CD28 stimulation, suggesting that miR-182 and miR-183C miRNAs regulate the inflammatory IFNγ in splenocytes via targeting Foxo1. The deletion of either miR-182 alone or the whole miR-183C cluster, however, had no marked effect on the composition of T and B cell subsets in the spleens of B6/lpr mice. There were similar percentages of CD4+, CD8+, CD19+, as well as Tregs, follicular helper T (TFH), germinal center B (GCB), and plasma cells in the miR-183C−/−B6/lpr and miR-182−/−B6/lpr mice and their respective littermate controls, miR-183Cfl/flB6/lpr and miR-182fl/flB6/lpr mice. Together, our data demonstrate a role of miR-183C in the regulation of anti-dsDNA autoantibody production in vivo in B6/lpr mice and the induction of IFNγ in in vitro activated splenocytes from B6/lpr mice.

Decreased CD11c-positive dendritic cells in the tumor microenvironment predict double-hit/triple-hit genotype and survival in diffuse large B-cell lymphoma.

Diffuse large B‐cell lymphoma (DLBCL) is the most common type of non‐Hodgkin lymphoma and is a potentially curable disease. However, it is heterogenous, and the prognosis is poor if the tumor cells harbor fusions involving MYC and BCL2 or MYC and BCL6 (double‐hit [DH] lymphoma), or fusions involving all three genes (triple‐hit [TH] lymphoma). Fluorescence in situ hybridization is currently the gold standard for confirming the presence of DH/TH genotypes. However, the test is laborious and not readily available in some laboratories. Germinal center B (GCB) signatures and dual expression of MYC and BCL2 are commonly used as initial screening markers (traditional model) in clinical practice. Our study proposes immunohistochemical markers for more conveniently and accessibly screening DH/TH genotypes in DLBCL. We retrospectively reviewed the clinical and pathological parameters of patients with DLBCL. We assessed the proliferative index, apoptotic index, and tumor microenvironment (TME), with regard to T cells and CD11c(+) dendritic cells, in formalin‐fixed paraffin‐embedded tissue. We then generated a decision tree as a screening algorithm to predict DH/TH genotypes and employed decision curve analysis to demonstrate the superiority of this new model in prediction. We also assessed the prognostic significance of related parameters. Our study revealed that GCB subtypes, a Ki67 proliferative index higher than 70%, and BCL2 expression were significantly associated with DH/TH genotypes. Decreased CD11c(+) dendritic cells in the TME indicated additional risk. Our proposed screening algorithm outperformed a traditional model in screening for the DH/TH genotypes. In addition, decreased CD11c(+) dendritic cells in the DLBCL TME were an independent unfavorable prognosticator. In conclusion, we provide a convenient, well‐performing model that predicts DH/TH genotypes in DLBCL. The prognostic significance of CD11c(+) dendritic cells in the TME might influence the classification and development of immunotherapy for DLBCL in the future.

Targeting temporal metabolic needs of T follicular helper cells to treat cGVHD utilizing a mitochondrial pyruvate carrier inhibitor

Abstract The curative potential of allogeneic hematopoietic stem cell transplantation is limited by development of chronic graft versus host disease (cGVHD) and metabolic regulation of immune cells is a promising therapeutic target. During cGVHD, interactions between T follicular helper (TFH) and germinal center B (GCB) cells result in production of alloreactive class-switched antibodies that attack host tissues, causing multi-organ damage and tissue fibrosis. We show the metabolic dependencies of TFH change throughout cGVHD progression and targeting mitochondrial pyruvate transport is an effective strategy to mitigate disease. Pyruvate, the end product of glycolysis, is transported by the mitochondrial pyruvate carrier (MPC) for use in the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS). TFH require glycolysis in order to induce a GC reaction, both in an immunization model and in an in vivo systemic cGVHD model with bronchiolitis obliterans (BO). Furthermore, Seahorse data from TFH extracted early in disease development in the BO model demonstrate increased dependency on glycolysis. But Seahorse data also indicates TFH undergo a shift away from glycolysis and toward OXPHOS as cGVHD continues. Based on the central role of MPC in tying pyruvate to multiple metabolic pathways, we examined an inhibitor of MPC (7ACC1) in reducing TFH activity. Administration of 7ACC1 resulted in inhibition of GCB production of class switched antibody in an in vitro assay. These findings were additionally supported in vivo, whereby daily treatment after establishment of cGVHD resulted in a decrease in disease severity. These data provide evidence that inhibiting mitochondrial pyruvate transport is a viable therapeutic strategy for cGVHD. This work was supported by grants from National Institutes of Health, National Institute of Allergy, and Infectious Diseases R37 AI34495, T32 AI007313; National Heart, Lung, and Blood Institute R01 HL56067, R01 HL11879, R01 HL115114 ; National Cancer Institute P01 CA065493

EGR2 deletion displays similar and differential effects on immunological development and function in autoinflammation-prone B6/lpr and normal B6 mice

Abstract Transcription factor early growth response protein 2 (EGR2) prevents the development of lupus-like autoimmune disease in C57BL/6 (B6) mice by negatively regulating T cell activation, inflammatory cytokine IFNγ and IL-17 production and by suppressing humoral responses. However, increased EGR2 expression has been observed in lupus and also other autoimmune disorders, which contradicts the autoimmune suppressive role of EGR2. Here, we derived conditional EGR2−/−B6/lpr and EGR2−/−B6 mice to investigate and compare the immune regulatory role of EGR2 in autoinflammation (B6/lpr) versus normal physiological (B6) conditions. Significantly, we found that in B6/lpr mice, but not in B6 mice, EGR2 deletion dramatically suppressed serum levels of anti-dsDNA autoantibodies, total IgG, IgG1, IgG2a, and IgM. We further demonstrated that while EGR2 deletion promoted germinal center B (GCB) cell development in both B6 and B6/lpr mice, it significantly reduced the differentiation of plasma and plasmablasts cells in B6/lpr mice. EGR2 deletion promoted splenomegaly and T cell activation in both B6 and B6/lpr mice, but it differentially regulated the profiles of immune cell subpopulations in the spleens of B6 and B6/lpr mice. Especially, EGR2 deletion dramatically suppressed the development of double negative T cells in B6/lpr mice. Moreover, we found that EGR2 had an opposite role in regulating IL-17 production in in vitro activated splenocytes from B6/lpr and B6 mice, although EGR2 had a similar suppressive role for IFNγ production. Together, our data strongly suggest that the immune and autoimmune regulatory roles of EGR2 are context dependent, and should not be generalized in normal physiology and different pathological conditions. Supported by VMCVM-VCOM Center for One Heath Research Seed Grant Program

Germinal Center B Cells Derived from TET2-Mutated Clonal Hematopoiesis Provide a Microenviromental Niche for Tumor Cells in Angioimmunoblastic T-Cell Lymphoma

Germinal Center B Cells Derived from TET2-Mutated Clonal Hematopoiesis Provide a Microenviromental Niche for Tumor Cells in Angioimmunoblastic T-Cell Lymphoma

Molecular Definition of Activated B Cell-like (ABC) DLBCL Identifies Patients Who May Benefit from Front-Line Intensive R-HDS Chemotherapy with ASCT

BackgroundDiffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in adult patients with respect to clinical presentation, histopathology and molecular-genetic features. While the long-term cure rate after rituximab-containing conventional chemotherapy programs exceeds 80% in young patients with low and low-intermediate risk according to International Prognostic Index (IPI), similar treatments still remain unsatisfactory in patients belonging to high and high-intermediate risk (Pfreundschuh M Lancet Oncol 12:1013-1022, 2011; Coiffier B Blood 116:2040-2045, 2010; Pfreundschuh M Lancet Oncol 7: 379-391, 2006).For these patients the optimization of front-line therapy remains an important objective. Advances in molecular genetics have vastly improved the understanding of the biological diversity in DLBCLs, leading to the discovery of key oncogenic pathways and novel therapeutic targets.In a recently published randomized phase III trial we recruited 246 untreated DLBCL patients with a high-intermediate (56%) or high (44%) IPI score, in order to compare the R-CHOP-14 to the R-HDS plus ASCT therapeutic protocol. In this study the definition of the cell of origin (COO) by immunohistochemistry was unable to discriminate the clinical outcome according to the allocated treatment (Cortelazzo S J Clin Oncol. 2016 33:4015-4022). Herein, we show that a molecular classification of DLBCL may lead to identify patients who most likely benefit from treatment intensification.Methods and PatientsWe were able to run digital multiplex gene expression profiling (Nanostring technology) in 62 of the 246 patients, for whom formalin-fixed paraffin-embedded (FFPE) material was available, to classify them into the molecular COO types: ABC (Activated B Cell), GCB (Germinal Center B Cell) or Unclassified.The Nanostring panel contains 28 genes: 20 genes are used to assign COO subtype according to LST algorithm (Scott D Blood 123: 1214-1217, 2014), the remaining 8 genes are employed to assess MYC and BCL2 expression, to quantify TP53 level, to detect potential therapeutic targets (STAT3, NFKB, PTEN and PKI3CA) and to evaluate macrophages infiltrate (CD68).ResultsFirst of all, we confirmed that the distinction of DLBCL into COO categories by gene expression was more accurate than immunohistochemistry (IHC) determination, which failed especially in classifying non-GCB cases (86% of concordance in GCB cases, 53,6% in ABC plus Unclassified group, 22% considering only ABC). Indeed, considering the entire cohort, the Overall Survival (OS) was significantly different comparing GCB to ABC patients, while IHC failed to identify prognostic subgroups (Figure 1a and 1b).Regarding the effects of the two therapeutic treatments on OS, when we analyzed the 62 patients together or only the GCB group, we observed no significant difference between the two arms. Instead, in ABC group we noticed a clear difference in OS between R-HDS, with 83% of cases that survived, and R-CHOP14, after which only 25% of cases survived.Furthermore we investigated a possible correlation between MYC and BCL2 IHC and Nanostring mRNA expression data, we found no linear relationship but we noticed a positive trend. Moreover BCL2 and MYC expression showed a higher predictive OS value than IHC.ConclusionsOur results suggest that the diagnostic FFPE gene expression assay by Nanostring robustly identifies DLBCL subgroups according to the COO. Moreover, yet preliminary, the molecular definition of the COO is able to identify patients at high risk of poor outcome when treated with R-CHOP and who may benefit by intensified high dose chemotherapy programs or experimental new treatments. [Display omitted] DisclosuresRambaldi:Novartis, Amgen, Celgene, Sanofi: Other: Travel, Accomodations, Expenses; Novartis, Roche/Genentech, Amgen, Italfarmaco: Consultancy. Cesano:NanoString Technologies: Employment, Equity Ownership. Pileri:Takeda Pharmaceuticals: Speakers Bureau.

Efficacy of Salvage Chemotherapy in Diffuse Large B Cell Lymphoma with Primary Treatment Failure According to Putative Cell of Origin

Efficacy of Salvage Chemotherapy in Diffuse Large B Cell Lymphoma with Primary Treatment Failure According to Putative Cell of Origin

EGR2 plays a differential immune regulatory role in normal C57BL/6 mice and autoimmune-prone B6/lpr mice

Abstract Early growth response protein 2 (EGR2) is a zinc-finger transcription factor that belongs to the early growth response (Egr) gene family. Recent studies have shown that EGR2 is required to induce T cell anergy, and it plays an important role in T cell immunity and autoimmunity. Conditional depletion of EGR2 in T lymphocytes in C57BL/6 (B6) mice (EGR2−/−B6) was able to break immune tolerance and induce T cell-driven, a lupus-like autoimmune disease. In EGR2−/−B6 mice, there was also enhanced GC response and humoral responses. However, increased EGR2 expression has been observed in lupus and autoimmune disorder systemic sclerosis. These observations seem to contradict the autoimmune suppressive role of EGR2 in B6 mice. To investigate further the immune regulatory role of EGR2 in an autoimmune context, we derived EGR2−/−B6/lpr mice with EGR2 deficiency in both T and B cells. We found that EGR2 depletion promoted splenomegaly and increased IFNγ, IL-10, IL-2 in both B6 and B6/lpr mice. Significantly, we found that in B6/lpr mice, EGR2 depletion reduced serum levels of anti-dsDNA autoantibodies, total IgG, IgG1, IgG2a, and IgM. Furthermore, in B6/lpr mice, depletion of EGR2 promoted germinal center B (GCB) cell development, but suppressed plasma cell (PC) differentiation. However, in wild-type B6 mice, EGR2 depletion significantly increased anti-dsDNA autoantibodies, accompanied by a significant increase of GCB cell development and a trend of increase in PC differentiation. Together, we demonstrated that EGR2 has a differential role in the normal and autoimmune state, particularly in regulating GCB transition into plasma cell during late B cell development.

ICOS is critical for T follicular regulatory cell differentiation

Abstract The Inducible Costimulator (ICOS) is a T cell costimulatory receptor critical for humoral immunity. ICOS-deficient patients suffer from recurrent infections due to lack of protective antibodies. However, some patients also display signs of antibody-mediated autoimmunity. These findings may reflect a dual role of ICOS: facilitating the differentiation and function of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. While Tfh cells are known to provide help to B cells to produce high affinity antibodies, the main role of Tfr cells seems to be preventing autoantibody generation. Using Foxp3-cre-mediated ICOS knockout (ICOS FC) mice, we show that T regulatory (Treg)-specific ICOS deletion drastically reduces the number of Tfr cells without altering Treg cell numbers. Single cell RNA sequencing further revealed shifts in transitory Tfr precursor populations in immunized ICOS FC mice. Importantly, we observed a lowered ratio of antigen-specific germinal center B (GCB) cells and increased anti-nuclear antibodies in ICOS FC mice, suggesting a rise in autoreactive GCB cells. We also noted variations in isotype composition of total and virus-specific antibodies in infected ICOS FC mice. Mechanistically, our data suggests that ICOS could promote the Treg-to-Tfr transition by regulating CXCR5 expression. Thus, our study demonstrates that ICOS is critical for Tfr cell generation and supports the role of Tfr cells in preventing generation of autoantibodies during germinal center reactions.

Depletion of microRNA-183-96-182 miRNA cluster in lymphocytes suppresses anti-dsDNA autoantibody production and IgG deposition in kidney in C57BL/6-Faslpr/lpr mice

Abstract The miR-183-96-182 (miR-183C) is a highly conserved miRNA cluster among species. Our previous work found a significant upregulation of miR-183C in the splenic cells of three different murine models of systemic lupus erythematosus (SLE). Current studies revealed that miR-183C miRNAs are critically involved in immunity and autoimmunity. In this study, we found that inhibition of miR-182 alone or miR-183C in vitro with antagomirs significantly reduced lupus-related inflammatory cytokine IFN-γ and IL-6 in activated splenocytes from MRL or MRL/lpr mice. To further characterize the pathogenic role of miR-182 and miR-183C in lupus in vivo, we developed B6-lpr mice with conditional depletion of miR-182 or miR-183C in CD2+ lymphocyte. We found that depletion of either miR-182 or miR-183C in the lymphocytes of B6/lpr mice had no obvious effect on T and B cell development as similar percentage of CD4+. CD8+, CD19+, as well as Tregs, follicular helper T (TFH), germinal center B (GCB), and plasma cells were observed in the miR-182−/− and miR-183C−/− and their respective control. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C−/− mice when compared to age-matched controls and the B6/lpr mice with miR-182 or miR-183C deficiency have significantly reduced IgG deposition in the kidneys. Meanwhile, there was reduced IFN production in ex vivo activated splenocytes from the knockout mice. Furthermore, we demonstrated that miR-182 and miR-183C regulated the inflammatory response in splenocytes via targeting forkhead box O1 (Foxo1). Together, our data suggest a potential therapeutic effect of targeting miR-183C in lupus.