Juehua Gao

A Dual Role for Interferon-γ in the Pathogenesis of Sjögren's Syndrome-Like Autoimmune Exocrinopathy in the Nonobese Diabetic Mouse

Sjögrens syndrome‐like autoimmune exocrinopathy (AEC) in the nonobese diabetic (NOD) mouse progresses from a preimmune phase to an immune phase, resulting in dry mouth and/or dry eyes. In the present study, the impact of the prototypical T‐helper type 1 cytokine, interferon‐gamma (IFN‐γ), on the onset of AEC was investigated using both the IFN‐γ and the IFN‐γ receptor gene knockout mice, NOD.IFN‐γ–/– and NOD.IFN‐γR–/–, respectively. Neither the NOD.IFN‐γ–/– nor the NOD.IFN‐γR–/– mice exhibited increased acinar cell apoptosis and abnormal salivary protein expression, typically observed in parental NOD mice prior to disease. Without these preimmune phase abnormalities, NOD.IFN‐γ–/– and NOD.IFN‐γR–/– mice showed no subsequent autoimmune responses against the salivary glands at 20 weeks. Interestingly, real‐time polymerase chain reaction and electrophoretic gel mobility shift assays suggested that IFN‐γ and STAT1, as well as the transcriptional activity of STAT1 in NOD glands, were increased at birth. Unlike the neonatal submandibular glands of NOD or NOD‐scid mice that show abnormal glandular morphogenesis at birth, the submandibular glands of the newly constructed congenic strain, NOD‐scid.IFN‐γ–/–, were found to be normal. Taken together, IFN‐γ appears to play a critical role not only during the later immune phase of AEC, but also the early preimmune phase, independent of effector functions of immune cells. How exactly IFN‐γ functions during this period remains speculative.

Nuclear expression of sox11 is highly associated with mantle cell lymphoma but is independent of t(11;14)(q13;q32) in non-mantle cell B-cell neoplasms

Sox11 is a transcription factor involved in embryonic neurogenesis and tissue remodeling. Its role in lymphopoiesis is presently unknown. Recent studies have shown the nuclear expression of sox11 in mantle cell lymphoma, which raises the question about its possible association with t(11;14)(q13;q32), the genetic hallmark of mantle cell lymphoma leading to the overexpression of cyclin D1. In this study, we examined sox11 expression in 211 cases of B-cell neoplasms by immunohistochemistry, and evaluated its association with t(11;14) and overexpression of cyclin D1. Nuclear staining of sox11 was observed in 54 of 57 (95%) mantle cell lymphomas, including 52 of 53 (98%) classical and 2 of 4 variant types. Two of the three mantle cell lymphomas negative for nuclear sox11 staining were analyzed and were positive for t(11;14). All other B-cell lymphomas (114 cases) showed variable positive staining in the cytoplasm, but no nuclear positivity. Sox11 was then examined in plasma cell myeloma and hairy cell leukemia as a subset of plasma cell myeloma carry t(11;14) and overexpress cyclin D1, and cyclin D1 is overexpressed in a subset of hairy cell leukemia independent of t(11;14). We found no nuclear staining of sox11 in 30 plasma cell myelomas examined, including 12 cases with t(11;14)(q13;q32). It is interesting that intense nuclear staining of sox11 was present in a subset of hairy cell leukemias (5 of 10), and was associated with the overexpression of cyclin D1. Our results indicate that the nuclear expression of sox11 is highly associated with mantle cell lymphoma, but is independent of t(11;14)(q13;q32) in non-mantle cell B-cell neoplasms. Its association with the overexpression of cyclin D1 in hairy cell leukemia suggests that sox11 may be involved in the upregulation of cyclin D1 in this leukemia.

Nuclear overexpression of lymphoid-enhancer-binding factor 1 identifies chronic lymphocytic leukemia/small lymphocytic lymphoma in small B-cell lymphomas

Lymphoid-enhancer-binding factor 1 (LEF1), coupling with β-catenin, functions as a key nuclear mediator of WNT/β-catenin signaling, which regulates cell proliferation and survival. LEF1 has an important role in lymphopoiesis, and is normally expressed in T and pro-B cells but not mature B cells. However, gene expression profiling demonstrates overexpression of LEF1 in chronic lymphocytic leukemia, and knockdown of LEF1 decreases the survival of the leukemic cells. So far, the data on LEF1 expression in B-cell lymphomas are limited. This study represents the first attempt to assess LEF1 by immunohistochemistry in a large series (290 cases) of B-cell lymphomas. Strong nuclear staining of LEF1 was observed in virtually all neoplastic cells in 92 of 92 (100%) chronic lymphocytic leukemia/small lymphocytic lymphomas including two CD5− cases, with strongest staining in cells with Richters transformation. LEF1 also highlighted the morphologically inconspicuous small lymphocytic lymphoma component in three composite lymphomas. All 53 mantle cell lymphomas, 31 low-grade follicular lymphomas and 31 marginal zone lymphomas, including 3 CD5+ cases, were negative. In 12 grade 3 follicular lymphomas, LEF1 was positive in a small subset (5–15%) of cells. Diffuse large B-cell lymphoma, however, demonstrated significant variability in LEF1 expression with overall positivity in 27 of 71 (38%) cases. Our results demonstrate that nuclear overexpression of LEF1 is highly associated with chronic lymphocytic leukemia/small lymphocytic lymphoma, and may serve as a convenient marker for differential diagnosis of small B-cell lymphomas. The expression of β-catenin, the coactivator of LEF1 in WNT signaling, was examined in 50 chronic lymphocytic leukemia/small lymphocytic lymphomas, of which 44 (88%) showed negative nuclear staining. The findings of universal nuclear overexpression of LEF1 but lack of nuclear β-catenin in the majority of chronic lymphocytic leukemia/small lymphocytic lymphoma suggest that the pro-survival function of LEF1 in this disease may be independent of WNT/β-catenin signaling.

Immunophenotypic variations in mantle cell lymphoma.

Mantle cell lymphoma (MCL) expresses pan-B-cell antigens and is usually CD5+/CD10-/CD23-/FMC7+. In this study, we evaluated 52 patients with confirmed diagnoses of MCL and identified variant immunophenotypes in 21 patients (19/48 classical and 2/4 variant MCLs), including CD5- in 6 (12%) of 52, CD10+ in 4 (8%) of 50, CD23+ in 10 (21%) of 48, and FMC7- in 4 (11%) of 37 cases. Three cases showed variations in 2 antigens, including CD5-/CD23+, CD10+/FMC7-, and CD23+/FMC7-; they were all classical MCLs. One blastoid variant MCL was CD23+, and one was FMC7-. Evaluation for proliferation index by immunohistochemical analysis for Ki-67 demonstrated no significant difference between MCLs with variant immunophenotypes and MCLs with typical immunophenotypes. The high proliferation index (>60%) was exclusively seen in the blastoid and pleomorphic variants. Our results indicate that immunophenotypic variations are common in MCL, and recognizing the variability is important for accurate subclassification of B-cell lymphoma.

IL-4-STAT6 Signal Transduction-Dependent Induction of the Clinical Phase of Sjögren’s Syndrome-Like Disease of the Nonobese Diabetic Mouse

NOD.B10-H2b and NOD/LtJ mice manifest, respectively, many features of primary and secondary Sjögren’s syndrome (SjS), an autoimmune disease affecting primarily the salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). B lymphocytes play a central role in the onset of SjS with clinical manifestations dependent on the appearance of autoantibodies reactive to multiple components of acinar cells. Previous studies with NOD.IL4−/− and NOD.B10-H2b.IL4−/− mice suggest that the Th2 cytokine, IL-4, plays a vital role in the development and onset of SjS-like disease in the NOD mouse model. To investigate the molecular mechanisms by which IL-4 controls SjS development, a Stat6 gene knockout mouse, NOD.B10-H2b.C-Stat6−/−, was constructed and its disease profile was defined and compared with that of NOD.B10-H2b.C-Stat6+/+ mice. As the NOD.B10-H2b.C-Stat6−/− mice aged from 4 to 24 wk, they exhibited leukocyte infiltration of the exocrine glands, produced anti-nuclear autoantibodies, and showed loss and gain of saliva-associated proteolytic enzymes, similar to NOD.B10-H2b.C-Stat6+/+ mice. In contrast, NOD.B10-H2b.C-Stat6−/− mice failed to develop glandular dysfunction, maintaining normal saliva flow rates. NOD.B10-H2b.C-Stat6−/− mice were found to lack IgG1 isotype-specific anti-muscarinic acetylcholine type-3 receptor autoantibodies. Furthermore, the IgG fractions from NOD.B10-H2b.C-Stat6−/− sera were unable to induce glandular dysfunction when injected into naive recipient C57BL/6 mice. NOD.B10-H2b.C-Stat6−/− mice, like NOD.B10-H2b.IL4−/− mice, are unable to synthesize IgG1 Abs, an observation that correlates with an inability to develop end-stage clinical SjS-like disease. These data imply a requirement for the IL-4/STAT6-pathway for onset of the clinical phase of SjS-like disease in the NOD mouse model.

GATA family transcriptional factors: emerging suspects in hematologic disorders

GATA transcription factors are zinc finger DNA binding proteins that regulate transcription during development and cell differentiation. The three important GATA transcription factors GATA1, GATA2 and GATA3 play essential roles in the development and maintenance of hematopoietic systems. GATA1 is required for the erythroid and megakaryocytic commitment during hematopoiesis. GATA2 is crucial for the proliferation and survival of early hematopoietic cells, and is also involved in lineage specific transcriptional regulation as the dynamic partner of GATA1. GATA3 plays an essential role in T lymphoid cell development and immune regulation. As a result, mutations in genes encoding the GATA transcription factors or alteration in the protein expression level or their function have been linked to a variety of human hematologic disorders. In this review, we summarized the current knowledge regarding the disrupted biologic function of GATA in various hematologic disorders.

Aberrant overexpression of CD14 on granulocytes sensitizes the innate immune response in mDia1 heterozygous del(5q) MDS

The myelodysplastic syndromes (MDSs) include a spectrum of stem cell malignancies characterized by an increased risk of developing acute myeloid leukemia. Heterozygous loss of chromosome 5q (del[5q]) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin proteins, mDia1, which is involved in linear actin polymerization. Mice with mDia1 deficiency develop hematologic features with age mimicking human myeloid neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 heterozygous and knockout mice develop MDS phenotypes with age. In these mice, CD14 was aberrantly overexpressed on granulocytes in a cell-autonomous manner, leading to a hypersensitive innate immune response to lipopolysaccharide (LPS) stimuli through CD14/Toll-like receptor 4 signaling. Chronic stimulation with LPS accelerated the development of MDS in mDia1 heterozygous and knockout mice that can be rescued by lenalidomide. Similar findings of CD14 overexpression were observed on the bone marrow granulocytes of del(5q) MDS patients. Mechanistically, mDia1 deficiency led to a downregulation of membrane-associated genes and a specific upregulation of CD14 messenger RNA in granulocytes, but not in other lineages. These results underscore the significance of mDia1 heterozygosity in deregulated innate immune responses in del(5q) MDS.

Strong expression of EZH2 and accumulation of trimethylated H3K27 in diffuse large B-cell lymphoma independent of cell of origin and EZH2 codon 641 mutation

Gain-of-function EZH2 mutation promotes H3K27 trimethylation (H3K27me3) and lymphoid transformation of germinal center (GC) derived B-cell lymphoma, such as GCB diffuse large B-cell lymphoma (DLBCL), but not activated B-cell (ABC) DLBCL. It is unclear whether expression levels of EZH2 and consequential H3K27me3 vary by EZH2 mutation and/or cell-of-origin in DLBCL. Ninety lymphoma samples including 40 DLBCLs were studied by immunohistochemistry. EZH2 Y641 mutations were detected in three of 20 (15%) GCB and none of 20 ABC types. All 40 DLBCLs showed strong EZH2, expression with high-level H3K27me3 in 90% GCBs and 95% ABCs. In 50 other B-cell lymphomas except for follicular lymphoma, strong EZH2 expression correlated with high-grade features. Immunoblot of DLBCL cell lines and microarray gene expression study of EZH2 in B-cell lymphomas were consistent with the immunohistochemistry findings. High-level EZH2 and H3K27me3 were common in DLBCL independent of cell-of-origin and EZH2 mutation. High-level EZH2 in lymphoma of aggressive features suggests additional therapeutic targets.

EBV-negative aggressive NK-cell leukemia/ lymphoma: A clinical and pathological study from a single institution

Aggressive natural killer (NK)-cell leukemia/lymphoma is a systemic NK-cell neoplasm that preferentially affects Asians with a fulminant clinical course and is almost always associated with Epstein-Barr virus (EBV). The data on EBV-negative aggressive NK-cell leukemia/lymphoma are limited. Here we report a series of three patients (two Caucasians, one African-American) with EBV-negative aggressive NK-cell leukemia/lymphoma from a single institution, including a case diagnosed on post-mortem examination. Similar to EBV-positive aggressive NK-cell leukemia/lymphoma, our patients presented with constitutional symptoms and hepatosplenomegaly, and followed a highly aggressive clinical course. The disease involved peripheral blood, bone marrow, liver, spleen, and lymph node, and the neoplastic cells were pleomorphic with prominent azurophilic granules and demonstrated an atypical NK-cell phenotype. Lack of blood lymphocytosis (3 of 3), bone marrow interstitial infiltration (2 of 3), EBER negativity (3 of 3), and atypical phenotype including CD3 negativity by immunohistochemistry make an early recognition of the disease difficult. Ancillary studies revealed a complex karyotype (1 of 2), overexpression (3 of 3), and amplification (1 of 1) of c-MYC. The polycomb repressive complex 2 pathway-associated proteins EZH2 and H3K27me3 and immune checkpoint protein PD-L1 were overexpressed in three of three and two of three cases, respectively. Our findings indicate that the EBV-negative aggressive NK-cell leukemia/lymphoma shares similar clinicopathological features to the EBV-positive counterpart except for the high prevalence of Asian seen in EBV-positive cases. Overexpression of polycomb repressive complex 2 pathway-associated proteins and PD-L1 suggest potential therapeutic targets for this aggressive disease. Next-generation sequencing on two of three cases identified multiple genetic alterations but were negative for JAK–STAT pathway-associated gene mutations previously reported in EBV-positive NK/T-cell lymphoma, suggesting alternative molecular pathogenic mechanisms for EBV-negative aggressive NK-cell leukemia/lymphoma.

Ineffective erythropoiesis caused by binucleated late-stage erythroblasts in mDia2 hematopoietic specific knockout mice.

The generation of mature red blood cells is initiated from the commitment of hematopoietic stem cells to erythroid progenitors, which is followed by their differentiation to a series of morphologically recognizable erythroblasts.1 At the end of terminal erythropoiesis, the highly condensed nucleus migrates to one side of the cytoplasm of orthochromatic erythroblast, which is followed by the unique enucleation process producing reticulocytes and mature red blood cells.2–4 Our previous study demonstrated that mDia2, which belongs to the mDia formin protein family,5 is a downstream effector protein of Rac GTPases regulating late-stage terminal erythropoiesis, especially enucleation.6 Herein we generated conditional mDia2 knockout mouse models to reveal the roles of mDia2 in adult erythropoiesis. The conditional mDia2 knockout mouse models utilized an mDia2 targeting allele with exons 10 and 11 floxed. The LacZ and neomycin cassettes were flanked by FRT to be removed by FLP recombinase (Online Supplementary Figure S1A). We first crossed mDia2fl/+ mice with E2A-Cre transgenic mice to generate whole body mDia2 knockout mice. The depletion of mDia2 mRNA and protein were confirmed by real-time PCR and Western blot assays (Online Supplementary Figure S1B and S1C, respectively). As previously reported,7 mDia2fl/fl E2A-Cre mice were never generated alive (Online Supplementary Figure S1D). We found that the mDia2 knockout embryos die in uterus at approximately embryonic day 12.5 (E12.5) (Online Supplementary Figure S1E). This demonstrates that mDia2 is essential for embryonic development, which compromises the study of the roles of mDia2 in vivo in adults.