IL-17RA is essential for optimal localization of follicular Th cells in the germinal center light zone to promote autoantibody-producing B cells.

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Death receptor 5-targeted depletion of interleukin-23-producing macrophages, Th17, and Th1/17 associated with defective tyrosine phosphatase in mice and patients with rheumatoid arthritis.

OBJECTIVE: Bidirectional interactions between granulocyte-macrophage colony-stimulating factor-positive (GM-CSF+) T cells and interferon regulatory factor 5-positive (IRF-5+) macrophages play a major role in autoimmunity. In the absence of SH2 domain-containing phosphatase 1 (SHP-1), GM-CSF-stimulated cells are resistant to death receptor (DR)-mediated apoptosis. The objective of this study was to determine whether TRA-8, an anti-DR5 agonistic antibody, can eliminate inflammatory macrophages and CD4 T cells in the SHP-1-deficient condition. METHODS: Ubiquitous Cre (Ubc.Cre) human/mouse-chimeric DR5-transgenic mice were crossed with viable SHP-1-defective motheaten (mev/mev) mice. TRA-8 was administered weekly for up to 4 weeks. The clinical scores, histopathologic severity, and macrophage and CD4 T cell phenotypes were evaluated. The role of TRA-8 in depleting inflammatory macrophages and CD4 T cells was also evaluated, using synovial fluid obtained from patients with rheumatoid arthritis (RA). RESULTS: The levels of inflammatory macrophages (interleukin-23-positive [IL-23+] IRF-5+) and CD4 T cells (IL-17+ GM-CSF+) were elevated in mev/mev mice. In DR5-transgenic mev/mev mice, DR5 expression was up-regulated in these 2 cell populations. TRA-8 treatment depleted these cell populations and resulted in a significant reduction in inflammation and in the titers of autoantibodies. In synovial cells from patients with RA, the expression of IRF5 and DR5 was negatively correlated with the expression of PTPN6. TRA-8, but not TRAIL, suppressed RA inflammatory macrophages and Th17 cells under conditions in which the expression of SHP-1 is low. CONCLUSION: In contrast to TRAIL, which lacks the capability to counteract the survival signal in the absence of SHP-1, TRA-8 eliminated both IRF-5+ IL-23+ M1 macrophages and pathogenic GM-CSF+ IL-17+ CD4 T cells in a SHP-1-independent manner. The results of the current study suggest that TRA-8 can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF-5 axis.

Extension of the germinal center stage of B cell development promotes autoantibodies in BXD2 mice.

OBJECTIVE: Regulator of G protein signaling (RGS) proteins inhibit chemokine signaling by desensitizing G protein-coupled receptor signals. This study was undertaken to determine the mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in germinal centers (GCs), using BXD2-Rgs13-/- mice. METHODS: Confocal and light microscopy imaging techniques were used to determine the location of cells that express RGS13 and activation-induced cytidine deaminase (AID) in the mouse spleen, and the number of plasmablasts. The levels of GC and plasma cell program transcripts in GC B cells were determined by real-time quantitative polymerase chain reaction (qPCR). Differential interleukin-17 (IL-17)-mediated expression of RGS13 in GC versus non-GC B cells was analyzed using A20 and 70Z/3 B cells. RESULTS: In the spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 up-regulated RGS13 in A20 GC cells but not 70Z/3 non-GC B cells. BXD2- Rgs13-/- mice exhibited smaller GCs and lower AID levels, suggesting lower somatic hypermutation and affinity maturation. However, GC B cells from BXD2- Rgs13-/- mice showed increased levels of IgMbright plasmablasts, up-regulation of the genes encoding plasma program, including interferon regulatory factor 4, B lymphocyte-induced maturation protein 1, and X-box binding protein 1 and the p-CREB target genes Fosb and Obf1, and down-regulation of the GC program genes Aid, Pax5, and Bach2 compared to BXD2 mice. BXD2-Rgs13-/- mice had lower titers of IgG autoantibodies and IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity. CONCLUSION: RGS13 deficiency is associated with a reduction in GC program genes and the exit of fewer pathogenic IgM plasmablasts in BXD2 mice. Our findings indicate that prolonged GC program, mediated by up-regulation of RGS13, enhances AID expression and enables the generation of pathogenic autoantibodies in autoreactive GCs.

Histopathologic Features and Differential Diagnosis in Challenging Cases of Nodular Lymphocyte Predominant B-cell Lymphoma/Nodular Lymphocyte Predominant Hodgkin Lymphoma.

Nodular lymphocyte predominant Hodgkin lymphoma was termed "nodular lymphocyte predominant B-cell lymphoma" in the International Consensus Classification (ICC), to emphasize clinical and biological differences from classic Hodgkin lymphoma (CHL). The abbreviation "NLP" represents both terms in the ICC and World Health Organization classifications. Variations in the growth pattern, originally reported as Fan patterns A-F, are designated as either grade 1 or grade 2 in the ICC. NLP is uncommon, and in some cases an accurate diagnosis is challenging. The objectives of this article were to review the histopathologic features of NLP and the differential diagnosis from other key entities including de novo T-cell/histiocyte-rich large B-cell lymphoma (THRLBL) and lymphocyte-rich classic Hodgkin lymphoma (LRCHL). Histologically, NLP Fan pattern E (THRLBL-like) can be indistinguishable from de novo THRLBL. However, focal nodular areas, clustering of tumor cells, presence of few admixed small B-cells or FDC meshworks, and T-cell rosettes favor NLP Fan pattern E and argue against de novo THRLBL. NLP may also be confused with LRCHL. Patients with NLP are younger than those with LRCHL, and LRCHL may show mediastinal involvement. In LRCHL, the nodular pattern often contains eccentrically located small regressed germinal centers and intact small dense FDC meshworks, in contrast to the expanded, and fragmented FDC meshworks in NLP. Neoplastic cells that are positive for CD30 and CD15 but negative for CD20 and CD79a are characteristic of LRCHL. Additionally, Fascin and Gata3 are commonly positive in LRCHL but usually negative in NLP.

Efficient parameter inference in networked dynamical systems via steady states: A surrogate objective function approach integrating mean-field and nonlinear least squares.

In networked dynamical systems, inferring governing parameters is crucial for predicting nodal dynamics, such as gene expression levels, species abundance, or population density. While many parameter estimation techniques rely on time-series data, particularly systems that converge over extreme time ranges, only noisy steady-state data is available, requiring a new approach to infer dynamical parameters from noisy observations of steady states. However, the traditional optimization process is computationally demanding, requiring repeated simulation of coupled ordinary differential equations. To overcome these limitations, we introduce a surrogate objective function that leverages decoupled equations to compute steady states, significantly reducing computational complexity. Furthermore, by optimizing the surrogate objective function, we obtain steady states that more accurately approximate the ground truth than noisy observations and predict future equilibria when topology changes. We empirically demonstrate the effectiveness of the proposed method across ecological, gene regulatory, and epidemic networks. Our approach provides an efficient and effective way to estimate parameters from steady-state data and has the potential to improve predictions in networked dynamical systems.

Novel high-risk acute myeloid leukemia subgroup with ERG amplification and Biallelic loss of TP53.

ETS-related gene (ERG) amplification, observed in 4-6% of acute myeloid leukemia (AML), is associated with unfavorable prognosis. To determine coincident effects of additional genomic abnormalities in AML with ERG amplification (ERGamp), we examined 11 ERGamp cases of 205 newly diagnosed AML using chromosomal microarray analysis and next generation sequencing. ERGamp cases demonstrated a distinct pattern of high genetic complexity: loss of 5q, chromothripsis and TP53 loss of function variants. Remarkably, allelic TP53 loss or loss of heterozygosity (LOH) co-occurring with TP53 inactivating mutation dramatically effected ERGamp tumor patient outcome. In the presence of homozygous TP53 loss of function, ERGamp patients demonstrated no response to induction chemotherapy with median overall survival (OS) of 3.8 months (N = 9). Two patients with heterozygous loss of TP53 function underwent alloSCT without evidence of relapse at one year. Similarly, a validation TCGA cohort, 6 of the 8 ERGamp cases with TP53 loss of function demonstrated median OS of 2.5 months. This suggests that with TP53 mutant ERGamp AML, successive loss of the second TP53 allele, typically by 17p deletion or LOH identifies a specific high-risk subtype of AML patients who are resistant to standard induction chemotherapy and need novel approaches to avert the very poor prognosis.

Cytologic features of blastic plasmacytoid dendritic cell neoplasm involving liver: A case report and literature review.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare but clinically aggressive hematologic malignancy, believed to originate from plasmacytoid dendritic cells (PDCs) although it possesses multilineage potentials. Due to the dismal prognosis, accurate and rapid diagnosis is critical for early management. The disease usually initially involves skin and bone marrow. Here we report the cytopathologic findings in a case of BPDCN involving the liver in a patient previously diagnosed with BPDCN from skin and lymph node biopsies. The fine-needle aspiration specimen from the liver lesion demonstrates a hypercellular smear of atypical epithelioid cells dispersed singly or in loose groups. These cells have enlarged, eccentric, round to irregular nuclei with fine chromatin. The agranular gray-blue cytoplasm shows delicate wispy cytoplasmic extensions and cytoplasmic microvacuoles. Binucleation is common. The concurrent core biopsy shows that the neoplastic plasmacytoid cells with eccentric nuclei were positive for CD4, CD7, CD43, CD56, and CD68, confirming the diagnosis of BPDCN. Mutations of ASXL1 and TET2, classic for BPDCN, and a complex karyotype were detected in skin, bone marrow, and lymph node specimens. We catalog the heterogeneous pathologic features of this rare disease, emphasizing the clinical and histopathological correlation. The differential diagnoses and review of literature are also included. Awareness of this disease and accurate diagnosis are emphasized to aid early management and potentially produce a better clinical outcome.

MicroRNA-mediated regulation of Ubc9 expression in cancer cells.

PURPOSE: As an E2-conjugating enzyme for sumoylation, Ubc9 plays a critical role in sumoylation-mediated cellular pathways, ultimately impacting cell growth and cancer development. The aim of this study was to investigate the regulation of Ubc9 in cancer cells. EXPERIMENTAL DESIGN: Immunohistochemistry and Western blot were used to determine Ubc9 expression in paraffin-embedded tumor tissue and frozen specimens of the matched tumors from the same patient, respectively. To establish the causal relationship between miR-30e and Ubc9 expression, we overexpressed miR-30e and then determined the resultant effects on Ubc9 expression. To determine whether miR-30e directly targets Ubc9, we did luciferase assays using luciferase reporters carrying the 3'-untranslated region (3'-UTR) of the Ubc9 gene. RESULTS: We found that Ubc9 is up-regulated in breast, head and neck, and lung cancer specimens. In addition, an examination of eight pairs of matched breast tumor specimens by Western blot analysis revealed that, on average, the level of Ubc9 is 5.7-fold higher in tumor than in the matched normal breast tissue. Of interest, we present evidence that Ubc9 is subjected to posttranscriptional regulation by microRNA, and the miR-30 family, such as miR-30e, negatively regulates Ubc9 expression. In contrast to Ubc9, miR-30e is underexpressed in tumors. Moreover, ectopic expression of miR-30e suppresses cell growth, which can be partially reversed by Ubc9. Finally, using luciferase-Ubc9-3'-UTR reporters, we show that Ubc9 is a direct target for miR-30e by interactions with the putative miR-30e binding sites. CONCLUSION: These results provide new insight into regulation of Ubc9 in cancer cells.

First reported case of disseminated Microascus gracilis infection in a lung transplant patient.

Microascus gracilis is a specie of the genus Microascus in the family of Microascaceae and has been isolated from lung. It has never been reported as the cause of disseminated infection in humans. Herein, we report a fatal case of disseminated Microascus gracilis infection in a 65-year-old man with a history of primary idiopathic pulmonary fibrosis, status-post bilateral lung transplant. His course was complicated by donor lung cultures positive for multiple organisms and persistent pleural effusions. Multiple lung biopsy and bronchial lavage specimens were negative for mold. Later, pleural fluid cultures grew M. gracilis confirmed by DNA sequencing. Despite aggressive antifungal treatment, the patient continued to deteriorate with altered mental status. Imaging showed scattered hemorrhagic and hypodense lesions in the brain. The patient eventually succumbed to his infections and a restricted autopsy was performed. Autopsy findings included multiple hemorrhagic foci and abscesses involving the whole brain. Numerous punctuate, tan-white circular lesions were on the endocardium and diffuse tan exudates covered the pericardium and lungs. Histologically, similar fungal organisms with septate branching hyphae and short chains of conidia were identified, along with hemorrhage, neutrophilic inflammation, and necrosis in the brain, pleura, peripheral parenchyma of lungs and heart. This is the first reported case of disseminated M. gracilis infection in an immunosuppressed human, indicating it can cause localized infections and disseminated infections. This case increases our awareness of such fatal opportunistic infections, particularly in lung transplant patients, and urges earlier aggressive prophylaxis, diagnosis, and treatment.

Identification of follicular T helper cells in tissue sections.

Follicular T helper (Tfh) cells are a critical population of CD4 T helper cells that are primarily localized in the germinal centers (GCs) to help B cell maturation and antibody production. Tfh cells can be identified in tissue sections based on the expression of a panel of classical Tfh surface makers, transcription marker(s), and effector-function cytokines, as well as by their unique anatomic proximity to other GC cells, including follicular dendritic cells (FDC) and GC B cells. Here, we describe an immunofluorescence staining method for visualization of GC Tfh cells in frozen spleen tissue sections of the autoimmune BXD2 mouse using a confocal imaging strategy. Tfh cells were characterized based on the expression of CD4, CXCR5, Bcl6, IL-21, and IL-17.

Interleukin-21 promotes germinal center reaction by skewing the follicular regulatory T cell to follicular helper T cell balance in autoimmune BXD2 mice.

OBJECTIVE: Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular helper T (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently showed that interleukin-21 (IL-21) promoted Tfh cell differentiation in autoimmune BXD2 mice that develop spontaneous GCs. This study was undertaken to determine the modulatory effects of IL-21 on Tfr cells and the Tfr cell to Tfh cell balance in BXD2 mice. METHODS: The percentage and phenotype of Tfr cells were determined in BXD2 and BXD2-IL21(-/-) mice. The effects of IL-21 on Tfr cells and the Tfr cell:Tfh cell ratio were evaluated. Sorted Tfr cells from BXD2-IL21(-/-) mice were cocultured with Tfh cells and B cells, or transferred into BXD2 mice to determine their function. RESULTS: The percentages and numbers of GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-IL21(-/-) mice than in wild-type BXD2 mice. Administration of AdIL-21 to BXD2-IL21(-/-) mice decreased the percentages and numbers of Tfr cells and the Tfr cell:Tfh cell ratio but increased the number of GC B cells in the spleen. Recombinant murine IL-21 suppressed FoxP3 and significantly reduced Tgfb1, Il2, and Gitr but enhanced Il21, Il6, Pd1, Cxcr5, and Icos expression in Tfr cells. IL-21 also counteracted Tfr cell-mediated inhibition of antibody secretion in the Tfh cell-B cell coculture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased the numbers of autoantibody-producing B cells. CONCLUSION: Our findings indicate that high levels of IL-21 selectively enhance Tfh cell differentiation but inhibit Tfr cell commitment and the suppressive function of Tfr cells on Tfh cells and B cells, suggesting that IL-21 skews the balance from Tfr cells to Tfh cells to promote autoreactive GC reactions in BXD2 mice.