The Loss of Kdm6a in B-Cell Development Causes Germinal Center Hyperplasia and Impedes the B-Cell Immune Response in a Specific Manner

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been defined. To address this issue, we generated a conditional knockout mouse of the Kdm6a gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate Kdm6a in hematopoietic stem/progenitor cells. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female Kdm6a-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female Kdm6a-null mice bone marrow. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

Clinical analysis of Kimura’s disease in 24 cases from China

Background We reviewed details of Chinese Kimura’s disease (KD) cases. A full clinical analysis was subsequently performed to improve the accuracy of clinical diagnosis and treatment of KD. Methods A total of 24 patients with pathologically confirmed KD treated between March 2008 and March 2018 were reviewed retrospectively for clinical and histopathological analysis. Results In the 24 KD cases, 20 were male and 4 were female with the age of onset ranging from 5 to 65 years. Lesion diameter ranged from 0.6 cm to 7 cm with unilateral involvement being more popular (79%). Imaging examination had a high detection rate for KD involving the parotid gland and subcutaneous but had low specificity. Microscopic analysis indicated that KD mainly involved subcutaneous soft tissue and lymph nodes. The prominent feature of lymphoid tissue was germinal center hyperplasia surrounded by several lobules associated with hyperplastic vascular structures. Out of the 24 patients, 11 experienced recurrence of disease after treatment (surgical resection: 46.2%, surgical resection followed by oral corticosteroids: 71.4% and surgical resection combined with radiotherapy: 0%). Conclusions Our analysis revealed clinical, imaging, and histological characteristics of KD. A better understanding of the disease will help clinicians reduce misdiagnosis and improve the diagnostic rate upon patient first clinical visit.

Clinical analysis of Kimura’s disease in 24 cases from China

Background: We reviewed details of Chinese Kimura’s disease (KD) cases. A full clinical analysis was subsequently performed to improve the accuracy of clinical diagnosis and treatment of KD. Methods: A total of 24 patients with pathologically confirmed KD treated between March 2008 and March 2018 were reviewed retrospectively for clinical and histopathological analysis. Results: In the 24 KD cases, 20 were male and 4 were female with the age of onset ranging from 5 to 65 years. Lesion diameter ranged from 0.6 cm to 7 cm with unilateral involvement being more popular (79%). Imaging examination had a high detection rate for KD involving the parotid gland and subcutaneous but had low specificity. Microscopic analysis indicated that KD mainly involved subcutaneous soft tissue and lymph nodes. The prominent feature of lymphoid tissue was germinal center hyperplasia surrounded by several lobules associated with hyperplastic vascular structures. Out of the 24 patients, 11 experienced recurrence of disease after treatment (surgical resection: 46.2%, surgical resection followed by oral corticosteroids: 71.4% and surgical resection combined with radiotherapy: 0%). Conclusions: Our analysis revealed clinical, imaging, and histological characteristics of KD. A better understanding of the disease will help clinicians reduce misdiagnosis and improve the diagnostic rate upon patient first clinical visit.

Clinical analysis of Kimura’s disease in 24 cases from China

Background We reviewed details of Chinese Kimura’s disease (KD) cases. A full clinical analysis was subsequently performed to improve the accuracy of clinical diagnosis and treatment of KD. Methods A total of 24 patients with pathologically confirmed KD treated between March 2008 and March 2018 were reviewed retrospectively for clinical and histopathological analysis. Results In the 24 KD cases, 20 were male and 4 were female with the age of onset ranging from 5 to 65 years. Lesion diameter ranged from 0.6 cm to 7 cm with unilateral involvement being more popular (79%). Imaging examination had a high detection rate for KD involving the parotid gland and subcutaneous but had low specificity. Microscopic analysis indicated that KD mainly involved subcutaneous soft tissue and lymph nodes. The prominent feature of lymphoid tissue was germinal center hyperplasia surrounded by several lobules associated with hyperplastic vascular structures. Out of the 24 patients, 11 experienced recurrence of disease after treatment (surgical resection: 46.2%, surgical resection followed by oral corticosteroids: 71.4% and surgical resection combined with radiotherapy: 0%). Conclusions Our analysis revealed clinical, imaging, and histological characteristics of KD. A better understanding of the disease will help clinicians reduce misdiagnosis and improve the diagnostic rate upon patient first clinical visit.

VavP-Bcl2 transgenic mice develop follicular lymphoma preceded by germinal center hyperplasia

In human follicular lymphoma the t(14; 18) chromosome translocation activates the antiapoptotic oncogene Bcl2 by linking it to the immunoglobulin heavy chain (IGH) locus. Transgenic mice expressing Bcl2 controlled by an Igh enhancer (Eμ) do not develop follicular lymphoma, although they do have an increased incidence of other B-lymphoid neoplasms. We have now analyzed tumorigenesis in mice bearing a Bcl2 transgene controlled by Vav gene regulatory sequences (VavP), which confer expression in multiple hematopoietic lineages. Unlike Eμ-Bcl2 mice, many VavP-Bcl2 mice older than 10 months developed follicular lymphoma. Young VavP-Bcl2 mice had an overabundance of enlarged germinal centers and greatly elevated numbers of cycling B cells that had undergone IgH class switching and V-gene hypermutation. The peripheral T-cell compartment was larger in the VavP-Bcl2 mice than in Eμ-Bcl2 strains and, notably, CD4 T cells were 5-fold increased over normal. The germinal center hyperplasia required CD4 T cells, because it could be abolished by anti-CD4 antibody in vivo. VavP-Bcl2 mice also had a propensity to develop kidney disease of the autoimmune type. We suggest that the increased survival capacity of B and T cells fosters prolonged germinal center reactions, and that autoreactivity and hypermutation conspire to generate follicular lymphoma.

Expression of Cell-Homologous Genes of Human Herpesvirus-8 in Human Immunodeficiency Virus-Negative Lymphoproliferative Diseases

Human herpesvirus-8 (HHV-8) genome encodes for genes homologous to human cellular genes such as interleukin-6 (IL-6), Cyclin-D, BCL-2, and IL-8 receptor (G-protein–coupled receptor [GCR]). We used reverse transcriptase-polymerase chain reaction to study the expression of these viral genes in lymphoproliferative disorders associated with HHV-8 infection. None of these genes was expressed in 1 case of benign, localized Castleman’s disease (CD), and only viral IL-6 and viral Cyclin-D were transcribed in 2 cases of benign lymphadenopathies with giant germinal center hyperplasia and increased vascularity. In contrast, all 4 genes were transcribed in 1 case of multicentric CD of plasma cell type with aggressive clinical course and in 1 primary effusion lymphoma cell line. Our study provides the evidence that various HHV-8 genes, homologous to cellular genes involved in control of proliferation and apoptosis, may be differently expressed in different lymphoid disorders in vivo.