CD4 mRNA Expression Research Articles

Uncoupling of Natural IgE Production and CD23 Surface Expression Levels

CD23, the low affinity receptor for immunoglobulin E (IgE), has been proposed to play a critical role in the regulation of IgE production, based on altered IgE levels in CD23-deficient mice and transgenic mouse models, as well as in mouse strains with mutations in the CD23 gene, e.g. 129 substrains. Here, we have investigated a mouse line termed LxT1 that expresses reduced CD23 surface levels on B cells, and its influence on natural IgE production. Extensive phenotypic analysis showed that CD23 surface expression was reduced in LxT1 compared to the control, without affecting B cell development in general. This CD23low surface level in LxT1 mice is not as a result of reduced CD23 mRNA expression levels or intracellular accumulation, but linked to a recessive locus, a 129-derived region spanning 28 Mb on chromosome 8, which includes the CD23 gene. Sequence analysis confirmed five mutations within the CD23 coding region in LxT1 mice, the same as those present in New Zealand Black (NZB) and 129 mice. However, this CD23low phenotype was not observed in all 129 substrains despite carrying these same CD23 mutations in the coding region. Moreover, serum IgE levels in LxT1 mice are as low as those in the C57BL/6 (B6) strain, and much lower than those in 129 substrains. These data indicate that the CD23 surface level and serum IgE level are uncoupled and that neither is directly regulated by the mutations within the CD23 coding region. This study suggests that caution should be taken when interpreting the immunological data derived from mice with different genetic background, especially if the gene of interest is thought to influence CD23 surface expression or serum IgE level.

BAFF Receptor mAb Treatment Ameliorates Development and Progression of Atherosclerosis in Hyperlipidemic ApoE−/− Mice

AimsOption to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE−/− mouse.Methods and ResultsTo determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE−/− mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93− CD19+ B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19+ B cells, CD4+ and CD8+ T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE−/− mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93− CD19+ B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment.ConclusionAnti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE−/− mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.

Molecular cloning, characterization and tissue expression of CD4 in Chinese goose

CD4 protein is a single chain transmembrane glycoprotein belonging to the immunoglobulin superfamily that recognizes the major histocompatibility complex (MHC) class II molecules. It plays an important role in cell-mediated immunity. Here, the full-length cDNA of CD4 in Chinese goose (Anser cygnoides) was cloned and identified. The goose CD4 is 1940bp in length and encodes a single open-reading frame of 480 amino acids. The putative amino acid sequence of goose CD4 consisted of a signal peptide, four potential N-glycosylation sites, a transmembrane region and a cytoplasmic tail. The multiple sequence alignment and phylogenetic analysis suggested that goose CD4 shared a higher similarity with avian than other vertebrates. Semi-quantitative RT-PCR analysis showed that the highest level of CD4 mRNA transcripts was presented in the thymus, and relatively lower in the spleen, small intestine, brain and trachea. Low expression was seen in the bursa of fabricius, cecal tonsil, cecum, skin, lung, kidney and liver. In gosling, the CD4 transcript was expressed with high abundance in the thymus, and relatively lower in the spleen, cecal tonsil and small intestine. However, in adult goose, high expression was seen in the thymus, spleen and cecum, and relatively lower in the cecal tonsil and small intestine. During the course of NGVEV infection, the obvious increase in CD4 gene expression was observed in the spleen, bursa of fabricius and harderian gland. Interestingly, a notable decrease in CD4 mRNA expression in the small intestine at 5d PI and followed by an increase of that at 19d PI were shown.

CD30 Expression in Diffuse Large B-Cell Lymphoma

AbstractAbstract 1558▪▪This icon denotes a clinically relevant abstract Background:Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with a variable clinical course. The addition of rituximab (R) to CHOP combination chemotherapy has improved overall (OS) and progression free survival (PFS) but some patients progress despite treatment and alternative therapies are needed. Brentuximab vedotin is an antibody-drug conjugate that targets CD30. It is efficacious in the treatment of relapsed Hodgkin lymphoma and relapsed systemic anaplastic large cell lymphoma, two lymphomas associated with CD30 expression. The association between DLBCL and CD30 expression has not been well described. The aim of this study was to examine CD30 expression in DLBCL. Design:395 cases of formalin-fixed paraffin-embedded DLBCL (excluding PMBCL) in a tissue microarray were independently evaluated by two pathologists for expression of CD30, CD10, BCL6, and MUM1 by immunohistochemistry (IHC) and EBV RNA (EBER) by in situ hybridization. CD30 expression was correlated with cell of origin (COO) phenotype, OS and PFS, EBV infection, International Prognostic Index (IPI) score and CD30 mRNA expression. The COO phenotype, germinal center B-cell like (GCB) or non-GCB, was determined by IHC using the Hans algorithm. CD30 mRNA expression and COO genotype by gene expression profiling (GEP) were determined using Affymetrix U133 2.0 Plus arrays (n=170). Outcome analysis only included patients treated with R-CHOP chemotherapy. CD30 was considered positive by IHC if any malignant cells exhibited membranous staining. The threshold for calling higher CD30 expression by GEP was determined using X-Tile software. Results:25% (95/385) of DLBCL cases expressed CD30 by IHC with excellent concordance between two observers (r = 0.94). CD30 expression trended towards a non-GCB phenotype but was not significantly different (p=0.067). CD30 expression was not associated with PFS or OS in all R-CHOP treated cases (n=313); however, it was associated with a prolonged PFS in GCB-DLBCL (n=147) (p=0.019). In GCB-DLBCL CD30 expression remained an independent predictor of PFS in a multivariate analysis with IPI (p=0.038). CD30 expression by IHC was significantly associated with higher levels of CD30 mRNA (p=0.002). ABC-DLBCL exhibited significantly higher expression levels of CD30 mRNA (p=0.037). Higher CD30 mRNA expression was associated with a prolonged PFS in all R-CHOP treated DLBCL (p=0.012) as well as in DLBCL with a GCB-genotype (p=0.008), but not ABC or U-genotypes. Higher CD30 mRNA expression was also associated with a prolonged OS in the GCB-genotype (p=0.022). In the GCB-genotype higher CD30 mRNA expression remained an independent predictor of PFS, but not OS, in a multivariate analysis with IPI (p=0.037). EBV was identified in 3% of DLBCL (11/391), all of which exhibited a non-GCB phenotype (p=0.001) and were almost exclusively positive for CD30 expression (10/11)(p=<0.001). Conclusions:CD30 is expressed in approximately 25% of DLBCL and brentuximab vedotin could be considered for study in combination with traditional front-line therapies or as an alternative therapy in the relapsed or refractory disease. CD30 immunohistochemistry may be useful as a prognostic marker in R-CHOP treated GCB-DLBCL and the significant association of CD30 with EBV-positive non-GCB DLBCL suggests a distinct pathobiology for these cases. Disclosures:Slack:Seattle Genetics: Research Funding. Steidl:Seattle Genetics: Research Funding. Gascoyne:Seattle Genetics: Research Funding.

Hematopoietic Cell Kinase (HCK) Is Highly Expressed in Myelodysplastic Syndrome (MDS) and Regulates Proliferation, Cell Cycle Progression and Hematopoietic Differentiation

AbstractAbstract 3813The pathogenesis of MDS is complex and remains elusive. The molecular characterization of this disease has been a tool to obtain a better understanding. Our group recently identified new possible target genes involved in MDS pathophysiology through the microarray analysis of CD34+ cells from MDS patients. One of the interesting overexpressed genes found was the HCK gene, a Src kinase family member. This family integrates signaling from a variety of cell-surface receptors, such as RTK, EPO receptor, chemokine receptor, MET and CXCR4, to regulate diverse cellular responses including proliferation, differentiation, apoptosis and migration. HCK is restrictedly expressed in hematopoietic cells and overexpressed in some types of solid cancer and in acute lymphoid leukemia; however, functional role of HCK in MDS and myeloid cells is still unknown. Here, using qPCR we investigated the HCK mRNA expression in CD34+ and total bone marrow (BM) cells from 54 MDS patients (FAB 39 low-risk and 15 high-risk), receiving no treatment, and 24 normal donors. We evaluated the role of HCK, studying its effect on proliferation, cell cycle and apoptosis by silencing this gene in U937 myeloid cell line. To inhibit HCK, specific shRNA expressing lentiviral vectors targeting the HCK gene or no specific sequence were used. Cell growth was measured by MTT colorimetric assay. Annexin-PI was used to evaluate apoptosis and cell cycle was analyzed by flow cytometry. These assays were carried out in lentiviral transduced cells treated, or not, with rapamycin(10 or 100nM) and LY294002(50nM). We further analyzed the HCK expression during granulocytic and erythroid differentiation of established leukemia cell lines models. Granulocytic differentiation was induced in NB4 or HL60 cells by treatment with ATRA(10−6 M) for 4 days. Erythroid differentiation was induced in KU812 cells by treatment with HE(50μM) and HU(100μM) for 4 days. We observed a significant increase in HCK mRNA expression of MDS CD34+ cells (P=0.002) and BM cells (P=0.03) compared to normal cells. HCK mRNA expression in low-risk MDS cells was higher compared to high-risk MDS cells. In U937 cells, HCK inhibition resulted in a significant decrease in proliferation compared to control cells (P<0.0001). Interestingly, the combination of HCK inhibition/rapamycin or LY treatment, inhibitors of PI3K/mTOR pathway, showed no reduction in cell proliferation compared to HCK inhibition alone. HCK inhibition induced cell cycle delay in G0/G1 phase and a consequent decrease in cell percentages in the S phase. The combination of HCK inhibition/rapamycin or LY treatment showed a significant decrease in cell number in G0/G1 phase (P=0.007) and increase in S phase (P=0.01) compared to HCK inhibited alone. Apoptosis was unaffected by HCK knockdown. HCK expression was significantly increased in NB4 or HL60 cells after granulocytic differentiation and in KU812 cells after erythroid differentiation (P<0.01). Furthermore, HCK mRNA was significantly up-regulated during erythroid differentiation of MDS low-risk CD34+ cells but not in normal CD34+ cells. Considering that some transmembrane receptor (CXCR4, MET, chemokine) expressions are altered in MDS patients and their downstream pathway is mediated by Scr family/PI3K, our results suggest that HCK overexpression in MDS may be related to alteration in the expression of some of these cell-surface receptors. Loss of HCK expression may contribute to disease progression by skewing the balance between proliferation and differentiation, perhaps giving malignant cells a proliferative advantage. Further, HCK inhibition in U937 cells potently decreases proliferation through PI3K/mTOR pathway since the combination of HCK inhibition/rapamycin or LY treatment showed no alteration in the proliferation. In addition, HCK protein might have role in cell cycle control by modulating cell cycle gene expression in G0/G1 phase check point. Moreover, granulocyte and erythroid differentiation results in an HCK overexpression probably because this gene is an adaptor protein that is essential for intermediating downstream signaling of activated cell-surface receptors. Our findings also indicate that HCK plays a role in myeloid cell abnormalities, through different cell activities, including proliferation, cell cycle and differentiation, suggesting that HCK represents a possible therapeutic target in MDS. Support: FAPESP, CNPq. Disclosures:No relevant conflicts of interest to declare.

Deregulation of TLR2-JMJD3 Innate Immunity Signaling, Including a Rare TLR2 SNP As a Potential Somatic Mutation, in Myelodysplastic Syndromes (MDS)

Abstract 1700To better understand molecular bases of MDS pathogenesis, we performed a genome-wide CHIP-Seq analysis of H3K4me3, a histone mark associated with gene activation. In MDS CD34+ cells (N=4), 36 genes showed higher levels of promoter-H3K4me3 compared to controls. 10 of 11 randomly selected genes from these 36 showed increased mRNA expression in MDS CD34+ cells (N=100), supporting the positive correlation between expression and increased promoter H3K4me3. Pathway Analysis indicated that a majority of these genes are involved in innate immunity signal and NF-kB activation. This was validated by increased phospho-p65 in MDS bone marrow CD34+ specimens (N=15). Knock-down of 4 of these genes (C5AR1, FPR2, PTAFR and TYROBP) in OCI-AML3 cells resulted in reduction of p-p65. The observation of innate immunity signal activation and epigenetic deregulation led us screen key innate immunity activators, the Toll-like Receptor (TLR) family genes, and histone methylation regulators in MDS CD34+ cells. Among 8 TLRs and 24 histone methylation regulators, TLR1, 2 and 6, and Jmjc-domain histone demethylase JMJD3 were found to be significantly overexpressed in MDS CD34+ cells compared to control counterparts. This is of biological significance because TLR1 and 6 form functional hetero-dimmers with TLR2. Also, JMJD3 expression can be activated by TLR-NFkB in macrophages. To study TLR2 activation in HSC, we treated CD34+ cells with TLR2 agonists. This led to increased expression of JMJD3, supporting the biological function of TLR2 signal in HSC. We observed that JMJD3 knockdown in OCI-AML3 cells led to reduced expression of innate immunity genes (N=7), accompanied with changes of promoter H3K27me3 and H3K4me3, suggesting that JMJD3 forms a positive feedback to perpetuate innate immunity pathway. To further study the role of TLR-JMJD3 in MDS, we performed capture deep sequencing in 40 MDS bone marrow mononuclear cells (TLR1, 2, 4 and 6, JMJD3, UTX, UTY and JMJD1A). Seven different rare SNP in the coding regions of JMJD3, UTY, JMJD1A, and TLR2 were identified in 5 patients. Among them, one SNP of TLR2 causes a missense mutation, changing a conserved hydrophobic Phe217 to a hydrophilic Ser. We then analyzed the presence of TLR2 F217S in 150 MDS samples by Sanger sequencing. TLR2 F217S was observed in 17 (11%) patients. To evaluate the somatic nature of this alteration, CD3+ cells from 15 corresponding patients were sequenced and only two (13%) CD3+ cell samples carried TLR2 F217S. In the available 9 CD34+ cDNA samples, TLR2 F217S was observed in 8 (90%). We then expressed wild-type or F217S TLR2 in 293T cells, a cell line without endogenous TLR2 expression. Reporter assays indicated that in the absence of TLR2 agonist wildtype and F217S mutant TLR2 led to similar levels of NF-kB activation, whereas F217S led to an increased NF-kB activation compared to wildtype at the presence of TLR2 agonists. F217S also led to increased activation (phospho- and polyubiquitin-) of IRAK1, a key signal mediator for TLR signaling. These results suggest that TLR2 F217S led to more robust innate immunity signal activation when stimulated by agonists. We further studied the impact of TLR2 activation on hematopoietic differentiation. Colony formation of CD34+ cells indicated that TLR2 agonists led to decreased number of erythroid colonies, which was confirmed by flowcytometry that demonstrated TLR2 agonist treatments could reduce the number of CD71 high/HLADR low featured erythroid precursors. To examine the effect of targeting TLR2-JMJD3 in primary MDS cells, we transduced MDS CD34+ cells with shRNAs. In 4 CD34+ cases isolated from lower risk MDS, 3 transduced with JMJD3 shRNA and 4 transduced with TLR2 shRNA had increased ratio of erythroid colonies. In average, JMJD3 and TLR2 shRNA transduction led to a 50% increase in erythroid colonies. This was accompanied by increased expression of genes positively involved in differentiation of erythroid lineage (GYPA, GATA1 and EPOR). Finally, CD34+ cell mRNA expression levels of four genes in this study (NCF2, AQP9, MEFV and TLR1) were associated with overall survival of patients. Taken together, these studies highlight the implication of the deregulation of TLR2-JMJD3 mediated innate immunity signals in MDS pathogenesis and suggest that intervention of this pathway may have therapeutic potential in MDS. Disclosures:No relevant conflicts of interest to declare.

Epigenetic Modulation of CD30 Gene Expression in Alemtuzumab Resistant T Cell Malignancy:Rationale and Efficacy of Brentuximab Vedotin.

Abstract 2454▪▪This icon denotes a clinically relevant abstract Background:T-Cell prolymphocytic leukemia (T-PLL) is an aggressive hematological disease mainly affecting adults. It is characterized by skin involvement and high lymphocyte counts. It affects mature T-cells mainly of the CD4+/CD8- phenotype. Although treatment with alemtuzumab, an anti-CD52 monoclonal antibody (mAb), induces complete remission (CR) in >50% of patients, it is short lived (median: 6 mos) and resistance is common. Epigenetic therapy with the histone deacetylase inhibitors (HDACi) vorinostat (SAHA) or romidepsin and the hypomethylating agent cladribine (2-CdA), can circumvent lack of response to alemtuzumab. Brentuximab Vedotin (SGN-35) is an antibody drug conjugate ADC) targeted against CD30 (TNFRSF8) and we have show here this ADC has activity in combination with epigenetic agents in T-PLL. Methods:Five T-PLL patients were studied. Diagnosis was made by hematopathology, flow cytometry and TCR rearrangement studies. Alemtuzumab (30 mg SQ d1,3,5) and 2-CdA (5mg/m2 IVd1–5), followed by alemtuzumab, 2-CdA and SAHA (400mg po daily X 5 days) or romidepsin (14mg/m2IV d 1,8,15) was given. After, epigenetic therapy with 2-CdA, SAHA, and romidepsin expression of CD30 in skin and blood was assayed where applicable.SGN-35 was administered at 1.8 mg/kg IV q 3 weeks. Peripheral blood was obtained and assayed under an IRB approved protocol. Genes of interest were analyzed by qRT-PCR. Chromatin Immunoprecipitation (ChIP) assays were used to study epigenetic changes at the CD30 promoter. Results:Of the 5 patients studied, three were alemtuzumab refractory with disease in blood and/or skin. With the addition of 2-CdA and SAHA or romidepsin, all three patients’ peripheral blood counts normalized and flow cytometry and PCR assays were negative for residual disease. One patient at the time of relapse developed skin lesions that were unresponsive to treatment with alemtuzumab, romedpsin, 2-CdA, or palatrexate. Lack of response to alemtuzumab in this patient was circumvented through induction of the cell surface receptor CD30, the target for the potent ADC, brentuximab vedotin. qRT-PCR assays on leukemic blood demonstrated that 4/5 of the T-PLL patients demonstrated increased CD30 mRNA following epigenetic therapy (mean: 14; range: 1.3–69 fold). These increases in CD30 mRNA expression correlated with increases in CD30 protein levels by Western blot analysis. CD30 and CD52 expression in the skin was confirmed by immunohistochemical analysis. In two patients, ChIP analysis of the CD30 promoter showed decreases in histone methylation markers and increases in Pol II binding at the coding sequence after treatment with epigenetic agents, consistent with increased CD30 expression. The patient with skin lesions had a dramatic response of his skin and normalization of his WBC count, with flow and PCR studies of skin and blood negative for residual disease after two cycles of SGN35.In contrast, a second patient with T-PLL who was refractory to up front alemtuzumab (IV) responded and achieved remission with the addition of 2-CdA, Her leukemic cells 2 days post treatment expressed CD30 mRNA in. At relapse 16 months later, her leukemic cells did not express CD30 and treatment with 2-CdA and romedepsin was unable to derepress CD30 gene expression. Correlative studies with ChiP assays differed from the previous patient. Further elaboration and follow up will be presented with both patients. Conclusions:Therapy with HDACi and 2-CdA epigenetically augments clinical activity of alemtuzumab and SGN-35, as evidenced by de-repression of TNFSFR8 (CD30) and increased response to treatment in one patient. While CD30 is a gene that can be epigenetically regulated in T cell malignancies, further complexities in epigenetic regulation of CD30 require additional investigation. Correlative studies are underway to elucidate the regulation of CD30 by epigenetic mechanisms. We present a rationale for the use of epigenetic drugs in combination with mAbs such as alemtuzumab and ADCs in T-PLL. A prospective multicenter clinical trial is warranted. Disclosures:Off Label Use: Brentuximab vedotin for PLL. Epner:Seattle Genetics: Speakers Bureau.

Notch Signalling Inhibits CD4 Expression during Initiation and Differentiation of Human T Cell Lineage

The Delta/Notch signal transduction pathway is central to T cell differentiation from haemopoietic stem cells (HSCs). Although T cell development is well characterized using expression of cell surface markers, the detailed mechanisms driving differentiation have not been established. This issue becomes central with observations that adult HSCs exhibit poor differentiation towards the T cell lineage relative to neonatal or embryonic precursors. This study investigates the contribution of Notch signalling and stromal support cells to differentiation of adult and Cord Blood (CB) human HSCs, using the Notch signalling OP9Delta co-culture system. Co-cultured cells were assayed at weekly intervals during development for phenotype markers using flow cytometry. Cells were also assayed for mRNA expression at critical developmental stages. Expression of the central thymocyte marker CD4 was initiated independently of Notch signalling, while cells grown with Notch signalling had reduced expression of CD4 mRNA and protein. Interruption of Notch signalling in partially differentiated cells increased CD4 mRNA and protein expression, and promoted differentiation to CD4+ CD8+ T cells. We identified a set of genes related to T cell development that were initiated by Notch signalling, and also a set of genes subsequently altered by Notch signal interruption. These results demonstrate that while Notch signalling is essential for establishment of the T cell lineage, at later stages of differentiation, its removal late in differentiation promotes more efficient DP cell generation. Notch signalling adds to signals provided by stromal cells to allow HSCs to differentiate to T cells via initiation of transcription factors such as HES1, GATA3 and TCF7. We also identify gene expression profile differences that may account for low generation of T cells from adult HSCs.

Chronic social stress impairs virus specific adaptive immunity during acute Theiler's virus infection

Prior exposure to social disruption (SDR) stress exacerbates Theiler's murine encephalomyelitis virus (TMEV) infection, a model of multiple sclerosis. Here we examined the impact of SDR on T cell responses to TMEV infection in SJL mice. SDR impaired viral clearance and exacerbated acute disease. Moreover, TMEV infection alone increased CD4 and CD8 mRNA expression in brain and spleen while SDR impaired this response. SDR decreased both CD4+ and CD8+ virus-specific T cells in CNS, but not spleen. These findings suggest that SDR-induced suppression of virus-specific T cell responses contributes to impairments in viral clearance and exacerbation of acute disease.

Organic cation transporters OCT1 and OCT2 determine the accumulation of lamivudine in CD4 cells of HIV-infected patients

Identifying factors that determine concentrations of antiretroviral drugs in CD4 cells are important for improving therapeutic efficacy. Experimental models indicate that the nucleoside reverse transcriptase inhibitor lamivudine is transported by the organic cation transporters 1 and 2 (OCT1 and OCT2, respectively). Here, we tested whether OCT1 and OCT2 contribute to the uptake of lamivudine into native CD4 cells of human immunodeficiency virus (HIV)-infected individuals. CD4 cells obtained by non-activated cell sorting from 35 individuals with HIV-1 infection were incubated with lamivudine (10 μM, 30 min), and intracellular concentrations of lamivudine and its active metabolite lamivudine triphosphate were determined by liquid chromatography tandem mass spectrometry. The expression of OCT1 and OCT2 mRNA was measured by quantitative real-time polymerase chain reaction (PCR). A model of OCT2-transfected CD4 cells was established for mechanistic investigations. Intracellular concentrations of lamivudine and its active metabolite lamivudine triphosphate showed strong linear correlations with each other and with the CD4 mRNA expression of OCT1 and OCT2 (r > 0.80). Coincubation with protease inhibitors (ritonavir, nelfinavir) that inhibit OCT1 and OCT2 yielded decreased intracellular concentrations of lamivudine and lamivudine triphosphate. Incubation of CD4 cells from healthy donors transfected with an OCT2 expression vector yielded increased concentrations of lamivudine and lamivudine triphosphate. Our studies indicate a role of OCT1 and OCT2 for the cellular accumulation of lamivudine in HIV-infected individuals.

MiR-140-3p regulation of TNF-α-induced CD38 expression in human airway smooth muscle cells

CD38, a membrane protein expressed in airway smooth muscle (ASM) cells, plays a role in cellular Ca(2+) dynamics and ASM contractility. In human ASM (HASM) cells, TNF-α induces CD38 expression through activation of MAPKs, NF-κB, and AP-1, and its expression is differentially elevated in cells from asthmatic patients compared with cells from nonasthmatic subjects. The CD38 3'-untranslated region (UTR) has targets for miR-140-3p. We hypothesized that miR-140-3p regulates CD38 expression in HASM cells by altering CD38 mRNA stability. Basal and TNF-α-induced expression of miR-140-3p was determined in nonasthmatic ASM (NAASM) and asthmatic ASM (AASM) cells. NAASM and AASM cells were transfected with control, miR-140-3p mimic, or miR-140-3p antagomirs, and CD38 expression and CD38 mRNA stability were determined. Luciferase reporter assays were used to determine miR-140-3p binding to the CD38 3'-UTR. Activation of p38, ERK, and JNK MAPKs, NF-κB, and AP-1 was determined in miR-140-3p mimic-transfected NAASM. TNF-α attenuated miR-140-3p expression in NAASM and AASM cells, but at a greater magnitude in AASM cells. CD38 mRNA expression was attenuated by miR-140-3p mimic at comparable magnitude in NAASM and AASM cells. Mutated miR-140-3p target on the CD38 3'-UTR reversed the inhibition of luciferase activity by miR-140-3p mimic. CD38 mRNA stability was unaltered by miR-140-3p mimic in NAASM or AASM cells following arrest of transcription. TNF-α-induced activation of p38 MAPK and NF-κB was attenuated by miR-140-3p mimic. The findings indicate that miR-140-3p modulates CD38 expression in HASM cells through direct binding to the CD38 3'-UTR and indirect mechanisms involving activation of p38 MAPK and NF-κB. Furthermore, indirect mechanisms appear to play a major role in the regulation of CD38 expression.

Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.

Lentivirally overexpressed T-bet regulates T-helper cell lineage commitment in chronic hepatitis B patients

Chronic hepatitis B virus (HBV) infection is commonly considered to occur as a result of disturbance of the immune system. T-box expressed in T cells (T-bet) is an essential transcription factor for T helper (Th) cell differentiation and function. The aim of this study was to investigate the effect of T-bet overexpression on Th cell differentiation and the possible mechanism in chronic hepatitis B (CHB) patients. CD4+ T cells from the peripheral blood of 23 CHB patients, 8 acute hepatitis B (AHB) patients and 10 healthy controls were isolated. T-bet mRNA expression of CD4+ T cells was detected by quantitative real-time polymerase chain reaction (PCR). The T-bet DNA fragment was subcloned into the pGC-FU vector containing GFP to generate a recombinant lentiviral vector, pGC-FU-T-bet, while a no-load pGC-FU vector was used as the negative control. After transduction into CD4+ T cells from another 22 CHB patients, the induction of Th1- and Th2-type cytokines was assayed by an enzyme-linked immunosorbent assay (ELISA), and RT-PCR and western blot analysis were used to measure the mRNA and transcription levels of H2.0-like homeobox (HLX1), GATA-3 and STAT-6. T-bet mRNA expression in CD4+ T cells from AHB patients was enhanced compared with CHB patients and healthy controls. Th1-type cytokines and HLX1 expression was upregulated, while Th2-type cytokines and GATA-3 and STAT-6 expression was repressed after lentiviral introduction of T-bet. In conclusion, lentivirally overexpressed T-bet regulates Th cell lineage commitment in CHB patients, which may be mediated by regulating HLX1, GATA-3 and STAT-6 expression.

Abstract 4391: CD20 as a target for therapy in solid tumors

Abstract Using the K14-HPV16 mouse model of squamous carcinogenesis (SCC), we previously reported that B cells foster neoplastic progression through deposition of immunoglobulin complexes in premalignant tissue via Fcα receptor-dependent activation of recruited myeloid cells. Here we evaluated therapeutic interventions targeting these pathways in preclinical trials through administration of depleting αCD20 antibody and small molecule inhibitor of Syk kinase. Both approaches harbored efficacy in preventing premalignant progression to the dysplastic/carcinoma in situ state. Screening a diverse spectrum of human solid tumors revealed SCCs of the vulva, head and neck, as well as pancreatic ductal adenocarcinomas (PDAC) as scoring positively for “signatures” of B cell or plasma cell infiltration, i.e. Ig or CD20 mRNA expression, thereby identifying carcinomas potentially amenable to anti-B cell therapies. Accordingly, B cell-deficient mice failed to support growth of either transplantable orthotopic SCC or PDAC. While administration of αCD20 mAB as a single agent was inefficient in impeding growth of preexisting SCCs, when delivered in combination with cytotoxic chemotherapy, e.g., paclitaxel, carboplatin or cisplatin, αCD20 mAb significantly improved chemotherapeutic response and improved survival by a mechanism dependent on CD8+ T cells. These data reveal that blocking protumorigenic programs regulating by humoral immunity, in combination with chemotherapy, effectively reprograms the tumor immune microenvironment and improves outcome. The authors acknowledge generous support from the NIH/NCI (R01CA130980, R01CA13256, R01CA140943, R01CA15531), the Department of Defense (W81XWH-09-1-0342, W81XWH-10-BCRP-EOHS-EXP) and the Susan G Komen Foundation (KG111084) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4391. doi:1538-7445.AM2012-4391

T-Lymphocyte and Cytokine Expression in Human Inflammatory Periapical Lesions

IntroductionLymphocytes, among many cells, express different sets of cytokines, chemokines, and receptors, which are considered important mediators of periapical immune response to infection. MethodsThe aim of this study was to evaluate the mRNA expression of CD4+CD28+ and CD8+ T genes and the gene expression of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-17A, IL-10, CCL2/MCP-1, CCL4, CCL5, CXCR4, CCR5, and receptor activator for nuclear factor kappa B ligand (RANKL) in periapical interstitial fluid from human root canal infections. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions. ResultsReal-time polymerase chain reaction demonstrated significantly higher levels of CD4+CD28+ and CD8+ T-cell markers in the former root canal condition and an increase of IL-10 and CXCR4, followed by a decrease of proinflammatory cytokines such as RANKL, interferon-γ, IL-1β, and CCL5. ConclusionsAnalyses of T-lymphocyte and cytokine expression in periapical area were able to show that distinct root canal conditions might play regulatory roles in controlling local immune/inflammatory processes.

SDF-1α induces angiogenesis after traumatic brain injury

This study aimed to investigate the effects of SDF-1α on brain angiogenesis and neurological functional recovery in rats after traumatic brain injury (TBI) and the potentially involved mechanisms. Youth male Wistar rats were injured via lateral fluid percussion injury and then randomly divided into one of 3 groups: I. vehicle treated group; II. SDF-1α neutralizing antibody treated group and III. rhSDF-1α treated group. rhSDF-1α and its neutralizing antibody or normal saline were administered to the brain penumbra via stereotactic injection 30min after TBI. Modified neurological severity score (mNSS) and Morris water maze (MWM) test were used to assess the neurologic functional recovery (n=6/group). 14days after injury, animals were euthanized and brain tissues were collected for quantitative real time polymerase chain reaction (qRT-PCR) (n=6/group) and immunohistochemistry (n=6/group) analysis. mNSS and MWM test indicated distinct amelioration of neurological disability in rhSDF-1α group(P<0.05). Microvessel density (MVD) of rhSDF-1α treated animals was remarkably increased around the injured area. On the contrary, MVD of the SDF-1α antibody administrated group was significantly decreased compared to that of vehicle treated animals (P<0.05). The mNSS and MVD had significant negative correlation as tested by Spearman rank correlation coefficient. Immunofluorescence staining showed that CD34 and CXCR4 co-expressed on microvessels. The rhSDF-1α treated animals had greater, contrarily, the SDF-1α antibody treated animals had lesser number of double positive microvessels compared to that of vehicle treated animals. The mRNA expression of CD34 and CXCR4 was obviously elevated in the rhSDF-1α administration group, conversely, declined in SDF-1α antibody treated animals around the injured area compared with that of the vehicle treatment group (P<0.05). These data indicated that SDF-1α could induce angiogenesis after TBI, potentially via SDF-1/CXCR4 axis.

CD20 Protein Immunohistochemistry-Positive/Flow Cytometry-Negative Diffuse Large B-Cell Lymphoma—Analyses of the Molecular Mechanisms and Rituximab Effectiveness

Abstract 2659 Background:Rituximab is a key molecular targeting drug for CD20(+) B-cell lymphoma, and CD20 protein expression is essential in the treatment mechanism of rituximab. We previously reported that the change to the CD20(−) phenotypic after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009; Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009). Recently, we encountered newly diagnosed patients with diffuse large B-cell lymphomas (DLBCL) which are CD20(+) on immunohistochemistry (IHC) but CD20(−) on flow cytometry (FCM). For these patients, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance have been clarified, and the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims:To confirm the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype using primary DLBCL cells, and to confirm the effectiveness of rituximab in vitro. Methods:Genomic DNA, mRNA, and total protein were extracted from primary DLBCL cells showing the CD20 IHC(+)/FCM(−) phenotype, and subjected to DNA sequencing, quantitative RT-PCR, and immunoblotting. L26 and B1 anti-CD20 antibodies were used in IHC and FCM analyses, respectively. Primary DLBCL cells were transplanted into NOD-SCID mice, and the engrafted lymphoma cells were used for in vitro CDC assay using rituximab. Results and Discussion:Ten patients with primary DLBCL had CD79a(+)/L26(+) results on IHC and CD19(+)/CD20(−) results on FCM. Quantitative RT-PCR indicated that CD20 mRNA expression that was significantly lower (almost 10 times) than that of positive control primary DLBCL cells. Immunoblotting analysis showed that CD20 protein expression was relatively lower than that of positive control cells, and no differences in length could be detected. No genetic mutations in the coding sequence of CD20 gene were detected in all samples examined in DNA sequencing analysis, suggesting that CD20 partial deletion is not the main reason for this phenotype. Interestingly, FCM analyses using fluorescent-labeled rituximab indicated that rituximab could partially recognize FCM CD20-B1(−)/CD19(+) B-cells, suggesting that the binding sensitivity of rituximab is much stronger than that of the B1 antibody. Lymphoma cells showing CD20 L26-IHC(+)/B1-FCM(−) phenotype proliferated in NOD-SCID mice and were partially killed by rituximab on in vitro CDC assay. These data suggest that rituximab is effective even for B-lymphoma cells with low CD20 expression if FCM using rituximab can be made to recognize those cell populations. Conclusion:Lower expression of CD20 mRNA may be one of the critical reasons for the CD20 L26-IHC(+)/B1-FCM(−) phenotype. FCM analysis using rituximab may be helpful to detect cell populations that are sensitive to rituximab treatment. Thus, rituximab therapy may be recommended if we can confirm the existence of cell populations recognized by rituximab on FCM. Disclosures:Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo.: Research Funding; Otsuka Pharma.: Research Funding.

Over‐Expression of Sphingosine Kinase‐1 Enhances a Progenitor Phenotype in Human Endothelial Cells

The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de-differentiation of mature human endothelial cells toward a progenitor phenotype. The lipid enzyme sphingosine kinase-1 was lentivirally over-expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. Sphingosine kinase-1 mRNA expression was induced approximately 150-fold with a resultant 20-fold increase in sphingosine kinase-1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de-differentiated phenotype with increased viability, reduced uptake of acetylated low-density lipoprotein and decreased tube formation in Matrigel in the cells over-expressing sphingosine kinase-1. These findings suggest that over-expression of sphingosine kinase-1 in human endothelial cells promotes, in part, their de-differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use.

Induced Interleukin-19 Contributes to Cell-Mediated Immunosuppression in Patients Undergoing Coronary Artery Bypass Grafting With Cardiopulmonary Bypass

Coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass (CPB) promotes immunosuppression, which predisposes patients to infectious complications. We investigated the role of interleukin (IL)-19 in the functions of CD4+ T cells in patients undergoing CABG with CPB. Blood samples were withdrawn from 42 patients undergoing elective CABG with CPB. Serum levels of IL-19 were analyzed by enzyme-linked immunosorbent assay (ELISA). The CD4+/CD25+ T-cell population was determined with flow cytometry. Isolated CD4+ T cells were cultured and assayed for proliferation and cytokine production under phorbol myristate acetate/ionomycin stimulation. Cytokine production and Foxp3 mRNA expression in CD4+ T cells from healthy volunteers with or without IL-19 treatment were determined with ELISA and real-time polymerase chain reaction, respectively. Proliferation percentages were 162%, 48%, 34%, and 39%, and interferon (IFN)-γ production was 1.22 ng/mL, 0.56 ng/mL, 0.33 ng/mL, and 0.35 ng/mL in the CD4+ T cells of patients before CPB and at 24 hours, 48 hours, and 96 hours, respectively, after CPB. Serum levels of IL-19 were higher but negatively correlated with CD4+ T-cell proliferation and IFN-γ production. The populations of CD4+/CD25+ T cells and expression of Foxp3 mRNA in T cells were higher and were positively correlated with IL-19 levels after CPB. Treatment with IL-19 reduced T-cell proliferation and IFN-γ production, increased Foxp3 mRNA expression, and induced the regulatory activity of CD4+ T cells. Interleukin-19 reduces T-cell responses and promotes the regulatory activity of CD4+ T cells. Induced IL-19 in patients undergoing CABG with CPB contributes to cell-mediated immunosuppression.

Electroporation Enhances Immunogenicity of a DNA Vaccine Expressing Woodchuck Hepatitis Virus Surface Antigen in Woodchucks

The development of therapeutic vaccines for chronic hepatitis B virus (HBV) infection has been hampered by host immune tolerance and the generally low magnitude and inconsistent immune responses to conventional vaccines and proposed new delivery methods. Electroporation (EP) for plasmid DNA (pDNA) vaccine delivery has demonstrated the enhanced immunogenicity of HBV antigens in various animal models. In the present study, the efficiency of the EP-based delivery of pDNA expressing various reporter genes first was evaluated in normal woodchucks, and then the immunogenicity of an analog woodchuck hepatitis virus (WHV) surface antigen (WHsAg) pDNA vaccine was studied in this model. The expression of reporter genes was greatly increased when the cellular uptake of pDNA was facilitated by EP. The EP of WHsAg-pDNA resulted in enhanced, dose-dependent antibody and T-cell responses to WHsAg compared to those of the conventional hypodermic needle injection of WHsAg-pDNA. Although subunit WHsAg protein vaccine elicited higher antibody titers than the DNA vaccine delivered with EP, T-cell response rates were comparable. However, in WHsAg-stimulated mononuclear cell cultures, the mRNA expression of CD4 and CD8 leukocyte surface markers and Th1 cytokines was more frequent and was skewed following DNA vaccination compared to that of protein immunization. Thus, the EP-based vaccination of normal woodchucks with pDNA-WHsAg induced a skew in the Th1/Th2 balance toward Th1 immune responses, which may be considered more appropriate for approaches involving therapeutic vaccines to treat chronic HBV infection.