Marginal Zone Areas Research Articles

Immunopotentiating effects of herb-partitioned moxibustion on the spleens of cyclophosphamide-induced immunosuppressed rats

BackgroundTo investigate the effec of the herb-partitioned moxibustion on T-lymphocyte activity in immunosuppressed rats through differential modulation of the immune checkpoint molecules CD28 and CTLA-4.MethodsForty-eight Sprague‒Dawley rats were randomly divided into the normal group (NG), the cyclophosphamide model group (CTX), the herb-partitioned moxibustion group (HPM), the CD28 inhibitor + herb-partitioned moxibustion group (aCD28 + HPM), the CTLA-4 inhibitor + herb-partitioned moxibustion group (aCTLA-4 + HPM), and the levamisole group (LEV) (8 rats per group). The immunosuppression model was prepared using cyclophosphamide. HPM treatments was performed via herb-partitioned moxibustion at 4 acupoints, Zhongwan (CV12), Shenque (CV8), Guanyuan (CV4), and Zusanli (ST36). Subsequently, the moxa floss was made into a conical moxa cone, which was then placed on the herbal cake and ignited. Five consecutive moxibustion strokes were performed daily for 10 consecutive days. In addition to the same moxibustion, each rat in the aCD28 + HPM group was injected intraperitoneally with 0.5 mg/kg of CD28 inhibitor per rat on the first day of treatment, and 100 μL of CTLA-4 inhibitor was injected into the aCTLA-4 + HPM group on Days 1, 4, and 7. For the positive control, levamisole (LEV) was administered by gavage at a dose of 2 mg/kg once daily for 10 days.ResultsCompared with those in CTX model rats, the WBC counts in the HPM and other groups were significantly higher. The immobility time of EPM in the HPM group was significantly lower than that of the CTX group. The HE stainin results also showed that after treatment, the the marginal zone area of the spleen tissue in the HPM increased, the number of lymphatic sheath lymphocytes around the small central artery of the spleen increased, and the amount of red pulp containing a small amount of pigmentation was partially reduced. Compared with those in the CTX group, the serum levels of CD28, CTLA-4, B7-1, and B7-2 were significantly lower, and the levels of α-MSH, TrkB, and BDNF were significantly greater in the HPM group. The results of the flow cytometry assay showed a significant increase in the number of CD8 + T lymphocytes after treatment with HPM or other agents compared to that in the CTX group. The immunofluorescence results showed that the levels of CD28 and CTLA-4 lower in spleen tissues than in control tissues, and the binding ability of CD28 to B7-1 and B7-2 was weakened after treatment with HPM and other treatments compared with CTX rats, PCR for CD28, CTLA-4 and B7-1 showed similar results.ConclusionIn the immunosuppressive rat model induced by cyclophosphamide, HPM upregulated the expression of α-MSH, TrkB, and BDNF, and downregulated the expression of CD28 and CTLA-4, thereby enhancing the activity of CD8+ T lymphocytes, restoring spleen function, improving the immunosuppressive state, restoring immune function, and effectively alleviating depressive symptoms.

Interaction of aging with lipoxygenase deficiency initiates hypersplenism, cardiac dysfunction, and profound leukocyte directed non-resolving inflammation.

In the process of physiological cardiac repair, splenic leukocyte-activated lipoxygenases (LOXs) are essential for the biosynthesis of specialized pro-resolving lipid mediators as a segment of an active process of acute inflammation in splenocardiac manner. In contrast, young 12/15LOX-/- mice use a compensatory mechanism that amplifies epoxyeicosatrienoic acid mediators after myocardial infarction, improving cardiac repair, function, and survival. Next, we tested whether deletion of 12/15LOX impacted the genesis of chronic inflammation in progressive aging. To test the risk factor of aging, we used the inter-organ hypothesis and assessed heart and spleen leukocyte population along with the number of inflammation markers in age-related 12/15LOX-/- aging mice (2months, 6months, 13months) and compared with C57BL/6J (WT; wild type) as controls (2months). The 12/15LOX-/- aging mice showed an age-related increase in spleen mass (hypersplenism) and decreased marginal zone area. Results suggest increased interstitial fibrosis in the heart marked with the inflammatory mediator (PGD2) level in 12/15LOX-/- aging mice than WT controls. From a cellular perspective, the quantitative measurement of immune cells indicates that heart and spleen leukocytes (CD11b+ and F4/80+ population) were reduced in 12/15LOX-/- aging mice than WT controls. At the molecular level, analyses of cytokines in the heart and spleen suggest amplified IFN-γ, with reduced COX-1, COX-2, and ALOX5 expression in the absence of 12/15LOX-derived mediators in the spleen. Thus, aging of 12/15LOX-/- mice increased spleen mass and altered spleen and heart structure with activation of multiple molecular and cellular pathways contributing to age-related integrative and inter-organ inflammation.

The Integrin Adaptor Kindlin-3 Is Important for Development and Retention of Marginal Zone B Cells

The Integrin Adaptor Kindlin-3 Is Important for Development and Retention of Marginal Zone B Cells

Periarteriolar stroma cells guide T cells from the red to the white pulp in the spleen.

While both the spleen and lymph nodes are called secondary lymphoid tissues, how lymphocytes enter these tissues are quite different from each other. This is because the architecture of the two types of organs and the mode of lymphocyte migration into these organs are quite distinct. In the spleen, T cells are passively released in the blood flow from the arterioles in the red pulp and marginal zone area. In contrast, T cells in the blood are actively captured on high endothelial venules in lymph nodes by the coordinated actions of CCR7 and several adhesion molecules. A recent finding indicates that T cells, released in the red pulp and marginal zone areas, actively find their way to the white zone by utilizing the migration track created by periarteriolar stromal cells. This finding adds one more piece to our understanding of lymphocyte migration for effective adaptive immune responses in the spleen.

KZF-L162R Mutation Affects Splenic Mature B Cell Development and Alters Expression of Aiolos Target Genes

Large-scale DNA sequencing efforts in chronic lymphocytic leukemia (CLL) have identified a broad array of putative cancer drivers arising from somatic mutations in this disease, but functional understanding of the impact of these genetic events on CLL onset and progression remains to be elucidated. One such example is mutation in the IKZF3 gene, encoding the zinc finger protein AIOLOS, mutated in ~2% of CLLs and associated with fludarabine-refractory disease. AIOLOS is a lymphoid-restricted transcription factor and a chromatin remodeler that plays an essential role in B cell development and maturation. In CLL, the IKZF3 mutation, also reported in few cases of diffuse large B cell lymphoma and mantle cell lymphoma,targets a highly conserved hotspot (L162R, homologous to murine L161R) that is localized in the 2nd zinc finger of the DNA-binding domain, required for DNA sequence recognition. Given the localization of this hotspot mutation, we hypothesized that it impacts the function of AIOLOS to drive CLL.To characterize the effects of the IKZF3-L162R mutation, we generated a knock-in mouse line that conditionally expresses the point mutation in a B cell lineage context through crossing with Cd19-cre mice, generating mouse lines carrying Ikzf3-L161R as either a heterozygous mutation (Ikzf3-L161RHet), homozygous mutation (Ikzf3-L161RHomo) or wild-type Ikzf3(Ikzf3WT).Given the established role of Aiolos in lymphoid differentiation, we first asked how the mutation impacts B cell development. By flow cytometry, using established markers to detect marrow pro-B, pre-B, transitional and mature B cell populations, or peritoneal B1a and B1b cell populations, no differences in the proportion of cells were observed between Ikzf3WT or Ikzf3-L161RHet. In the spleen, however, the average proportion of marginal zone B cells (B220+CD23+CD21high) was markedly reduced in heterozygousmice compared to wild type mice (6 mice/group: 4.9% vs. 11.5%, p=0.006), while the average proportion of follicular B cells (B220+CD23+CD21-) was increased (76% vs. 63%; p=0.003). Immunohistochemical staining of spleen sections confirmed that the marginal zone area was significantly reduced in Ikzf3-L161RHet mice (p=0.01). In addition, we noted a higher proliferation rate of B cells from Ikzf3-L161RHet mice when stimulated with LPS and IL-4 for 3 days (p=0.01), suggesting that the mutation confers a survival advantage to B cells. Similar analyses in Ikzf3-L161RHomo mice are ongoing.By immunofluorescence and immunoprecipitation, neither Aiolos binding with its partners CHD4, SIN3 or HDAC1, nor its cellular distribution were impacted by the mutation. Of note, the total protein level of Aiolos was increased in Ikzf3-L161RHet mice (9 mice/group; p<0.05). Since the mutation localizes to a DNA binding domain, we hypothesized that it modifies the ability of Aiolos to control expression of its target genes. We therefore performed CHIP-seq in Ikzf3WT splenic B cells, and identified Aiolos-associated high confidence peaks (fold change (FC) enrichment compared to input > 20) corresponding to DNA binding sites in the promoters of genes such as Rps19, Ogg1, Dusp2, Phf23 or Brfp1 and confident peaks (FC>10) in the anti-apoptotic gene Mcl1 and in genes involved in BCR signaling (i.e.Syk, Pi3kr1, Nfkbid), suggesting that their expression is under the control of Aiolos.Comparison of the expression by qPCR of these 8 genes in splenic B cells from the 3 mouse lines revealed Dusp2, Mcl1, Syk, Nfkbid and Phf23 to be upregulated in Ikzf3-L161RHomo B cells (p<0.05) but not in Ikzf3-L161RHet B cells. These findings suggest that the mutation directly impacts the expression level of Aiolos target genes. The upregulation of Mcl1 expression is particularly relevant in the context of CLL as dysregulation of anti-apoptotic signaling is characteristic of the disease.In conclusion, these data show that Aiolos mutation affects B cell subpopulation ontogeny, inducing a disproportionate abundance of follicular B cells endowed with high proliferative capacity. The mutation impacts Aiolos transcription capacity leading to upregulation of genes belonging to pathways cardinal to CLL development, including BCR signaling and apoptosis. Ongoing studies focus combining RNA-seq and CHIP-seq in mutant B cells, with the aim of identifying the breadth of differential expressed genes and dysregulated cellular pathways in mutant B cells in an unbiased manner. DisclosuresWu:Neon Therapeutics: Equity Ownership.

α-Galactosylceramide and all trans-retinoic acid work together to promote antigen presentation and antibody production

Abstract α-galactosylceramide (αGalCer) and all trans-retinoic acid (RA) are both known as potent immune regulators on various steps of the immune responses. Our previous studies have shown that αGalCer and RA could regulate B cell differentiation in vitro and increase antibody production in vivo. To investigate the underlying mechanisms related to the antibody response, animal experiments (in 8-wk old female Balb/c mice) were performed to determine the antigen presentation and B cell response during primary immunization. In a one-day experiment, dextran-FITC or KLH-FITC was injected intravenously with or without αGalCer (2 μg in 100 μl PBS with antigen, iv) or RA (37.5 μg in 10 μl canola oil, oral dose); in a 10-day experiment, KLH was injected (100 μg/100 μl PBS) with or without αGalCer/RA treatment. Spleens were analyzed by flow cytometry and immunofluorescent staining. We observed that Dextran and KLH after 24h of injection were localized at the spleen marginal zone (MZ) area, with only KLH colocalized with the metallophilic macrophages; in the presence of αGalCer and RA, the band of metallophilic macrophages became wider. While dextran moved markedly toward the follicular area after treatment, KLH signal was not seen within the follicle, and also became weaker at the MZ area, suggesting the different presentation and processing mechanisms of the antigens. At day 10, substantially more active germinal centers were formed in the presence of αGalCer and RA, with more focused staining of CD23+GL-7+ cells, markers of germinal center B cells. B cell numbers were increased at both time points. Our study suggests that αGalCer and RA together may promote antigen presentation and induce B cell differentiation that ultimately increases antibody production.

Expressionof Sf3b1- K700Ein Murine B Cells Causes Pre-mRNA Splicing and Altered B Cell Differentiation and Function

Mutations in the RNA splicing factor SF3B1 have been identified by large-scale sequencing as putative drivers in chronic lymphocytic leukemia (CLL), but their precise roles in the pathogenesis of CLL remains unknown. Although prior transcriptomic studies using primary CLL samples have led to the appreciation of altered RNA splicing in association with these mutations, understanding of their impact on cellular function has been complicated by their variable mutant allele frequency across samples as well as their common co-occurrence with other heterogeneous gene mutations. We therefore generated a mouse line that conditionally expresses the commonly occurring Sf3b1-K700E mutation at its endogenous murine locus. We obtained B-cell lineage specific expression of the mutant allele by crossing heterozygous floxed Sf3b1-K700E mice with homozygous CD19-Cre knockin mice. We confirmed that expression of the mutant allele was uniquely present as a heterozygous mutation in B cells, but not in other cell lineages.We sought to characterize the impact of Sf3b1-K700E on RNA splicing, B cell function, and CLL in this in vivo model. By unbiased RNA-sequencing of splenic B cells from wildtype and mutant mice (n=3), we profiled the splice isoform changes that were associated with Sf3b1-K700E. Using the tool JuncBASE, we detected, classified and quantified 54 differentially spliced transcripts (P<0.05, absolute delta percent spliced in >10%). Consistent with the altered splicing pattern reported in human CLL samples, the splice variants in our mouse model were highly enriched with altered selection of 3' splice sites (49 of 54 events, P<0.001). Of these, we validated 3 selected splice variants in independent samples by qPCR. Our murine model of Sf3b1-K700E mutation thus recapitulates altered RNA splicing, as per in human disease.We therefore next investigated whether Sf3b1-K700E affects B cell development and function. Splenic B cell numbers were significantly lower in the mutant (n=37) compared to control (n=33, P=0.0027) mice while T cell numbers were equivalent. Flow cytometric analysis of various B cell subpopulations from bone marrow and spleen revealed a significant increase in marginal zone B cells in the mutant mice (n=6, P<0.01). Consistent with this finding, we observed evidence of enlarged marginal zone areas on sections of mutant mouse spleens by visual inspection, with a mean reduction by 25% of proliferating germinal centers (per Ki67+ staining, n=6, average, P<0.05). On average, in vitro culture of splenic B cells with LPS and IL4 revealed mutant B cells to undergo 10-15% less proliferation, with reduced survival upon stimulation. Moreover, serum analysis revealed a 30-45% reduction in production of IgG1 and IgG3 from mutant mice (n=8, P<0.05). Altogether, these results suggest that mutant Sf3b1 induces an intrinsic B cell defect leading preferentially to impaired cellular proliferation.Cellular senescence has been commonly detected in pre-malignant lesions and is related to impaired cell proliferation. Indeed, by quantitative PCR array of 84 genes associated with cellular senescence, we found comparable levels of expression of all genes in T cells from wildtype and mutant splenocytes, but an overall trend of upregulation (range: by 1.5- to 21-fold) in the entire set of 84 genes in mutant B splenocytes, with significant upregulation in 20 genes (n=5, P<0.05). These genes included the critical senescence regulators Cdkn2a (p16) and Cdkn1a (p21), whose elevated expression in Sf3b1 mutated B cells we confirmed at the protein level. Furthermore, we detected increased levels of the cellular senescence mediators Igfbp6 and Igfbp7 in the sera from mutant mice (n=31, P<0.05). Collectively, the data demonstrate that expression of Sf3b1-K700E in B cells leads to the cellular senescent phenotype.While we observed that expression of Sf3b1-K700E results in RNA splicing changes, B cell developmental dysregulation and cellular senescence, expression of this mutation alone did not lead to expansion of CD5+CD19+ cells in vivo over time, despite observations up to 18-months (n=50). Our ongoing studies are now focused on the combined effects of Sf3b1-K700E and other recurrent CLL mutations on evasion of senescence and CLL disease progression. DisclosuresNo relevant conflicts of interest to declare.

Csnk2β, the Regulatory Subunit of Protein Kinase CK2, modulates Peripheral B Cell Development Repressing Notch2 Signaling and Promoting a Proper B-Cell Receptor Signal Transmission

Background. Serine-threonine protein kinase CK2 has been recently involved in the pathogenesis of B-cell tumors, such as B acute lymphoblastic leukemia, B chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma. CK2 acts through a “non-oncogene” addiction mechanism to propel tumor growth, protecting from apoptosis by a phosphorylation-dependent “shielding” mechanism of pro-survival molecules and stimulating oncogenic kinases by helping folding and enzymatic activity. In addition, CK2 has been shown to enhance the transactivation potential of several transcription factors, such as STAT3, NF-κB and c-Myc. The existing data on CK2 function in B cell tumors suggest that this kinase might act as a “hub” downstream signals from surface membrane molecules, like the B-cell (BCR), growth factor and cytokine receptors, as well as from cell-intrinsic pathways – like proteotoxic and DNA-damage-related stress cascades.Aims and methods. To gain insights into the role of CK2 in B-lymphopoiesis and, consequently, in B-cell tumors, we generated CK2β conditional knockout (KO) mice in B-cells by crossing Csnk2β-Flox/Flox mice with CD19-CRE transgenic mice.Results. CK2 kinase activity was decreased in Csnk2β KO B cells. In the bone marrow (BM), Csnk2β KO mice displayed a reduction of B-cells, especially of the B220high IgMint-high recirculating population of transitional and follicular (FO) B cells. Pro-B and pre-B-cell progenitors were slightly reduced in number. In peripheral blood, lymph-nodes, spleen and peritoneal cavity the number of B-cells was markedly reduced. Csnk2β KO mice had lower levels of all the immunoglobulin classes in the serum. The splenic IgDlow IgMhigh B-cell subset was increased whereas the IgDhigh IgMint-low population was decreased. An imbalance between the amount of FO and marginal zone (MZ) B-cells was found with an absolute reduction of FO B cells by approximately 2-folds and an increase of MZ B-cells and MZB cell precursors by up to three folds, on average. Histological and immunofluorescence (IF) analysis revealed a change of size/shape of spleen follicles and a significant expansion of the inter-follicular, marginal zone areas, which appeared to invade the follicle with larger cells. In vitro class-switch recombination assays demonstrated impairment in IgG1 and IgG3 class-switch and a marked reduction of the generation of antibody-producing cells. Anti-IgM stimulation was uncoupled to Ca++ mobilization, indicating a disrupted transmission of the signal from the BCR to the release of Ca++ stores in the endoplasmic reticulum. In vivo sheep red blood cells (SRBC) treatment (T-cell dependent response) showed a conserved up-regulation of GC markers, such as CD38, GL7 and PNA. Nonetheless, the architecture of the reactive follicles was found markedly changed. The analysis of FO, GC and MZ-associated genes showed normal levels of Bcl6, elevated levels of Lrf mRNA and, more significantly, a marked up-regulation of Notch2 target genes, such as Hes1 and Deltex1, in Csnk2β KO B cells. In vivo Notch2 blockage with neutralizing antibodies markedly reduced the MZB cell number in Csnk2β KO mice, indicating a Notch2-dependent MZB expansion associated with Csnk2β loss. High throughput RNAseq analysis was also performed and revealed significant alteration in FOB and MZB-regulating pathways.Conclusions. Here, we found that the β subunit of protein kinase CK2 is a novel regulator of peripheral B cell differentiation. CK2β sustains a proper BCR signal, controls the GC reaction and negatively regulates Notch2 signaling, acting as a master regulator of follicular/marginal zone architecture and terminal homeostasis of FOB and MZB cells. On one side our data enrich the knowledge on the mechanisms regulating B cell development, on the other side they inform about the potential mechanisms altered by CK2 during B-cell tumorigenesis. DisclosuresNo relevant conflicts of interest to declare.

Myeloid cell nuclear differentiation antigen is expressed in a subset of marginal zone lymphomas and is useful in the differential diagnosis with follicular lymphoma.

The diagnosis of marginal zone lymphomas (MZL) is challenged by the lack of specific markers that distinguish them from other low-grade non-Hodgkin B-cell lymphomas. Myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein that labels myelomonocytic cells as well as B lymphocytes that localize to the marginal zone areas of splenic white pulp. We evaluated MNDA expression in a large series of B-cell lymphomas to assess the sensitivity and specificity of this antigen for the characterization of MZL. A total of 440 tissue sections containing extramedullary B-cell lymphomas and 216 bone marrow biopsies containing atypical or neoplastic lymphoid infiltrates were stained for MNDA by immunohistochemistry. Among the extramedullary lymphoma cases, approximately 67% of nodal MZL, 61% of extranodal MZL, and 24% of splenic MZL expressed MNDA. MNDA was also infrequently expressed in other B-cell neoplasms including mantle cell lymphoma (6%), chronic lymphocytic leukemia/small lymphocytic lymphoma (13%), follicular lymphoma (FL) (4%), lymphoplasmacytic lymphoma (25%), and diffuse large B-cell lymphoma (3%). In contrast, MNDA was only expressed in 2.3% of all bone marrow biopsies involved by lymphoid infiltrates, including 2 cases of FL and one case of MZL. Collectively, these data support the inclusion of MNDA in the diagnostic evaluation of extramedullary B-cell lymphomas, particularly those in which the differential diagnosis is between low-grade FL and MZL.

The association of sore throat and psoriasis might be explained by histologically distinctive tonsils and increased expression of skin-homing molecules by tonsil T cells

Recent studies have highlighted the involvement of the palatine tonsils in the pathogenesis of psoriasis, particularly among patients with recurrent throat infections. However, the underlying immunological mechanism is not well understood. In this study we confirm that psoriasis tonsils are infected more frequently by β-haemolytic Streptococci, in particular Group C Streptococcus, compared with recurrently infected tonsils from patients without skin disease. Moreover, we show that tonsils from psoriasis patients contained smaller lymphoid follicles that occupied a smaller tissue area, had a lower germinal centre to marginal zone area ratio and contained fewer tingible body macrophages per unit area compared with recurrently infected tonsils from individuals without skin disease. Psoriasis patients' tonsils had a higher frequency of skin-homing [cutaneous lymphocyte-associated antigen (CLA(+) )] CD4(+) and CD8(+) T cells, and this correlated significantly with their frequency of blood CLA(+) T cells. The psoriasis patients also had a higher frequency of tonsil T cells expressing the interleukin (IL)-23 receptor that was expressed preferentially by the CLA(+) T cell population. In contrast, recurrently infected tonsils of individuals without skin disease had a higher frequency of tonsil T cells expressing the activation marker CD69 and a number of chemokine receptors with unknown relevance to psoriasis. These findings suggest that immune responses in the palatine tonsils of psoriasis patients are dysregulated. The elevated expression of CLA and IL-23 receptor by tonsil T cells may promote the egression of effector T cells from tonsils to the epidermis, suggesting that there may be functional changes within the tonsils, which promote triggering or exacerbation of psoriasis.

The Irf4 Gene, a Susceptibility Locus for Chronic Lymphocytic Leukemia (CLL), Controls Establishment of Follicular and Marginal Zone B Cell Compartments in Mice

Abstract 285Chronic lymphocytic leukemia (CLL) originates from the malignant transformation of mature B cells. Recently, single nucleotide polymorphisms (SNPs) in the 3'UTR region of the Irf4 gene have been associated with an increased risk of developing CLL in independent patient cohorts. IRF4 is a member of the interferon regulatory factor (IRF) family of transcription factors, and in the B lineage is essential for plasma cell differentiation in T-dependent immune responses. Irf4 has been demonstrated to act as an oncogene in multiple myeloma. Conversely, evidence suggests that the corresponding SNPs in the Irf4 gene lead to a reduction of IRF4 mRNA expression, and it was reported that tumor cells in half of CLL cases show reduced IRF4 protein expression compared to normal B cells. Together, these observations suggest that aberrant downregulation of IRF4 expression in mature B cells may contribute to CLL development. However, the normal function of IRF4 in mature B cells is incompletely understood. In order to investigate how IRF4 deficiency affects the biology of mature B cells, we investigated the consequences of deleting Irf4 specifically in B cells in vivo using a conditional Irf4 knockout mouse line.We and others had previously observed that Irf4−/− mice develop an expansion of B cells with a marginal zone (MZ) phenotype (IgMhiIgDloCD23–CD21+). By demarcating the MZ with the MOMA-1 marker, we here show that B cells in Irf4−/− mice localized preferentially in the MZ area, causing MZ hyperplasia. In contrast, the area where follicular B cells normally home contained few B cells. B cell autonomy of the observed phenotype was ascertained by crossing a “floxed” Irf4 allele with CD19-Cre mice to achieve B cell conditional deletion. We then crossed the floxed Irf4 allele with a transgenic mouse that allows inducible deletion of Irf4 specifically in B cells. Whereas flow-cytometric analysis revealed an unchanged ratio between cells with a follicular (IgMloIgDhiCD23+CD21int) vs. a MZ B cell surface phenotype upon Irf4 deletion, immunohistochemical (IHC) stainings of spleen sections for a marker protein (eGFP) that signals gene deletion demonstrated that the Irf4-deleted cells localized preferentially in the MZ, leading to MZ hyperplasia. Together, these results suggest that deletion of Irf4 in B cells alters chemokine responsiveness and migratory capacity. In agreement, global gene expression profile analysis of B cells purified from Irf4−/− and Irf4+/+ mice identified a set of differentially expressed genes with known functions in cell migration and homing. Notably, PLXND1 and the chemokine receptor CXCR7 showed 4.5 and 6.5-fold upregulation, respectively, while the G protein coupled receptor RGS13 and the adhesion molecule ALCAM (CD166) showed 3.5 and 5-fold downregulation in Irf4−/− vs. Irf4+/+ B cells.Unexpectedly, we observed in the profiling analysis that expression of the known NOTCH target genes Deltex1 and Hes5 was significantly upregulated (3 and 5-fold) in Irf4−/− vs. Irf4+/+ B cells. Despite unchanged NOTCH1 and NOTCH2 mRNA levels, Western blot and immunofluorescence analysis showed that Notch2, a gene known to be indispensible for MZ B cell development, was strongly upregulated in Irf4−/− B cells, suggesting that IRF4 is indirectly involved in NOTCH2 repression at a post-transcriptional level. Together with the altered migratory properties of Irf4−/− vs. Irf4+/+ B cells, these findings indicate that a balanced expression of IRF4 and NOTCH2 in B cells is required for establishing the follicular and MZ B cell compartments in mice, and suggest that IRF4 maintains the cellular identity of follicular B cells.The results may have implications for understanding CLL pathogenesis, as both NOTCH1 and NOTCH2 transmembrane receptors were reported to be expressed and activated in CLL B cells, and since NOTCH1 was recently found to be aberrantly activated in a fraction of CLL cases due to genetic mutations. Alterations in the balance of the transcriptional network established by NOTCH and IRF4 either through mutations, polymorphisms, or microenvironmental factors may disrupt normal B cell physiology and thereby contribute to tumorigenesis by an as yet unknown mechanism. Recently, a small fraction of CLL patients were identified that have a recurrent heterozygous somatic mutation in exon 2 of Irf4, providing additional rationale for determining how alterations in IRF4 function may promote CLL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

2.15 The Irf4 Gene, a Susceptibility Locus for Chronic Lymphocytic Leukemia, Controls Establishment of Follicular and Marginal Zone B-Cell Compartments in Mice

Abstract 285 Chronic lymphocytic leukemia (CLL) originates from the malignant transformation of mature B cells. Recently, single nucleotide polymorphisms (SNPs) in the 39UTR region of the Irf4 gene have been associated with an increased risk of developing CLL in independent patient cohorts. IRF4 is a member of the interferon regulatory factor (IRF) family of transcription factors, and in the B lineage is essential for plasma cell differentiation in T-dependent immune responses. Irf4 has been demonstrated to act as an oncogene in multiple myeloma. Conversely, evidence suggests that the corresponding SNPs in the Irf4 gene lead to a reduction of IRF4 mRNA expression, and it was reported that tumor cells in half of CLL cases show reduced IRF4 protein expression compared to normal B cells. Together, these observations suggest that aberrant downregulation of IRF4 expression in mature B cells may contribute to CLL development. However, the normal function of IRF4 in mature B cells is incompletely understood. In order to investigate how IRF4 deficiency affects the biology of mature B cells, we investigated the consequences of deleting Irf4 specifically in B cells in vivo using a conditional Irf4 knockout mouse line. We and others had previously observed that Irf4 −/− mice develop an expansion of B cells with a marginal zone (MZ) phenotype (IgM hi IgD lo CD23 – CD21 + ). By demarcating the MZ with the MOMA-1 marker, we here show that B cells in Irf4 −/− mice localized preferentially in the MZ area, causing MZ hyperplasia. In contrast, the area where follicular B cells normally home contained few B cells. B cell autonomy of the observed phenotype was ascertained by crossing a “floxed” Irf4 allele with CD19-Cre mice to achieve B cell conditional deletion. We then crossed the floxed Irf4 allele with a transgenic mouse that allows inducible deletion of Irf4 specifically in B cells. Whereas flow-cytometric analysis revealed an unchanged ratio between cells with a follicular (IgM lo IgD hi CD23 + CD21 int ) vs. a MZ B cell surface phenotype upon Irf4 deletion, immunohistochemical (IHC) stainings of spleen sections for a marker protein (eGFP) that signals gene deletion demonstrated that the Irf4 -deleted cells localized preferentially in the MZ, leading to MZ hyperplasia. Together, these results suggest that deletion of Irf4 in B cells alters chemokine responsiveness and migratory capacity. In agreement, global gene expression profile analysis of B cells purified from Irf4 −/− and Irf4 +/+ mice identified a set of differentially expressed genes with known functions in cell migration and homing. Notably, PLXND1 and the chemokine receptor CXCR7 showed 4.5 and 6.5-fold upregulation, respectively, while the G protein coupled receptor RGS13 and the adhesion molecule ALCAM (CD166) showed 3.5 and 5-fold downregulation in Irf4 −/− vs. Irf4 +/+ B cells. Unexpectedly, we observed in the profiling analysis that expression of the known NOTCH target genes Deltex1 and Hes5 was significantly upregulated (3 and 5-fold) in Irf4 −/− vs. Irf4 +/+ B cells. Despite unchanged NOTCH1 and NOTCH2 mRNA levels, Western blot and immunofluorescence analysis showed that Notch2 , a gene known to be indispensible for MZ B cell development, was strongly upregulated in Irf4 −/− B cells, suggesting that IRF4 is indirectly involved in NOTCH2 repression at a post-transcriptional level. Together with the altered migratory properties of Irf4 −/− vs. Irf4 +/+ B cells, these findings indicate that a balanced expression of IRF4 and NOTCH2 in B cells is required for establishing the follicular and MZ B cell compartments in mice, and suggest that IRF4 maintains the cellular identity of follicular B cells. The results may have implications for understanding CLL pathogenesis, as both NOTCH1 and NOTCH2 transmembrane receptors were reported to be expressed and activated in CLL B cells, and since NOTCH1 was recently found to be aberrantly activated in a fraction of CLL cases due to genetic mutations. Alterations in the balance of the transcriptional network established by NOTCH and IRF4 either through mutations, polymorphisms, or microenvironmental factors may disrupt normal B cell physiology and thereby contribute to tumorigenesis by an as yet unknown mechanism. Recently, a small fraction of CLL patients were identified that have a recurrent heterozygous somatic mutation in exon 2 of Irf4 , providing additional rationale for determining how alterations in IRF4 function may promote CLL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

IL-7 Is Required for the Development of the Intrinsic Function of Marginal Zone B Cells and the Marginal Zone Microenvironment

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7-deficient mice, although severely lymphopenic, retain small numbers of CD21(high)CD23(low) B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7(-/-) MZB cells and the IL-7(-/-) microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7(-/-) mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7(-/-) or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7(-/-) B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7(-/-) B cells formed B cell follicles with clearly defined IgM(+), MOMA-1(+), and MAdCAM-1(+) MZ structures. Conversely, adoptively transferred GFP(+) C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7(-/-) environment. Naive IL-7(-/-) mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.

CXCR3 expression affects the balance of effector and memory CD8 T cell generation (46.6)

Abstract In response to infection, activated CD8 T cells differentiate in vivo into a heterogeneous population of cells consisting of short-lived, terminally differentiated effector cells and memory precursor cells that give rise to long-lived memory cells. The strength and duration of antigenic stimulation and inflammatory signals influence CD8 T cell differentiation and the balance between these two fates. However, cell extrinsic cues, such as chemokines, that affect the localization and hence the exposure of CD8 T cells to antigenic and inflammatory signals are not completely understood. Here, we find that the inflammatory chemokine receptor CXCR3 is upregulated on activated CD8 T cells and influences their differentiation. CXCR3 guides activated CD8 T cells towards marginal zone areas where they are exposed to antigenic and potentially inflammatory stimuli. In the absence of CXCR3 expression, activated CD8 T cells are localized to the T zones of the splenic white pulp where they are less likely to receive antigenic stimulation. The increased exposure to antigen is associated with an increase in the generation of short-lived effector cells while the decreased exposure to antigen is associated with a quantitative and qualitative enhancement of memory cells. Our findings demonstrate that chemokine receptors and T cell positioning in secondary lymphoid organs can influence the balance of effector and memory T cell generation.

Moderate Physical Training Attenuates the Effects of Perinatal Undernutrition on the Morphometry of the Splenic Lymphoid Follicles in Endotoxemic Adult Rats

Background/Aims: Physical training is a well-known inducer of positive physiological adaptations. The effects of moderate physical training on the morphometry of splenic lymphoid follicles of endotoxemic rats submitted to a perinatal low-protein (LP) diet were evaluated. Methods: Male Wistar rats were divided into two groups according to their mother’s diet (17% casein, control, C) and, undernourished (8% casein, LP diet). On postnatal day 63, the animals were submitted to moderate physical training (8 weeks, 5 days·week^–1, 60 min·day^–1, at 70% of VO_2max). After the physical training period, half of each group received an injection of either lipopolysaccharide (LPS) or saline. Plasma corticosterone concentration, blood differential leukocyte counts and splenic morphometric parameters were analyzed. Results: In undernourished toxemic (LP + LPS), LPS increased plasma corticosterone concentrations, but not in previously trained (LP + T + LPS) animals. Neutrophilia and lymphopenia in response to LPS was more pronounced in pups from undernourished mothers (LP + LPS). LP + LPS animals showed a higher increment (47.4%) in the number of lymphoid follicles, a reduction in the number and size of the splenic follicles, and in the marginal zone area. Those alterations were attenuated in trained animals (LP + T + LPS). Conclusions: Physical training attenuates the effects of nutritional programming on the splenic microanatomy by a mechanism that involves the hypothalamic-pituitary-adrenal axis.

Diversity of genome profiles in malignant lymphoma

Characteristic chromosome translocations are associated with specific disease entities, and are known to play a pivotal role in lymphoma development. Chromosome translocation alone, however, is not sufficient to produce tumors. Factors including the microenvironment and epigenetic and genetic alterations other than chromosome translocations have been shown to play a role in lymphoma development. Follicular lymphoma cells proliferate in close contact with follicular dendritic cells. Mucosa-associated lymphoid tissue (MALT) lymphoma cells proliferate at the marginal zone area of reactive follicles which are formed by preceding chronic inflammation. The importance of genetic alterations other than chromosome translocation has been recognized since the introduction of array comparative genomic hybridization (array CGH). Variations in the genomic profile among patients with the same disease entity have been found by array CGH analyses. These variations indicate that multiple genetic pathways leading to the development of lymphomas may exist and hence result in the variable clinicopathological features observed.

Analysis of Marginal Zone B Cell Development in the Mouse with Limited B Cell Diversity: Role of the Antigen Receptor Signals in the Recruitment of B Cells to the Marginal Zone

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had approximately 2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, V(H)T/lambda2 B cells significantly predominated over V(H)T/lambda1 B cells in MZ-(V(H)T/lambda1:V(H)T/lambda2 approximately 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the V(H)T/lambda2 B cells were further augmented by doubling the V(H)T gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between V(H)T/lambda1 and V(H)T/lambda2 IgM mAbs revealed a polyreactive nature of the V(H)T/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.

Influence of Lymphocytes on the Presence and Organization of Dendritic Cell Subsets in the Spleen

Studies were undertaken to clarify the roles of individual leukocyte populations in maintaining the presence and organization of splenic dendritic cells (DCs). Using Abs specific for DC subsets, we found that the distinct types of DC maintained appropriate compartmentalization within the white pulp of lymphocyte-deficient mice despite an unusual overall distribution of DCs. Even in mice lacking both B and T lymphocytes, the central arteriole remained the structure around which T area DCs were organized. Marginal zone area DCs remained in a peripheral sheath excluded from the T area DCs. Additionally, we revealed an important role for splenic B cells in the presence and organization of marginal zone cells. B-deficient or B- and T-deficient mice lacked sialoadhesin+ marginal zone macrophages and lacked MAdCAM-1 expression in marginal zone reticular endothelial cells. Adoptive transfer of B lymphocytes induced MAdCAM-1 expression but failed to recruit marginal zone macrophages. Taken together, our results demonstrate that the arrival, localization, and persistence of DCs in spleen are events not solely dependent upon signals from the mature B and T cells or marginal zone macrophages. We suggest that specific stromal elements in the vicinity of the central arteriole are primarily responsible for providing directional cues to the DC.

Regulation and Functional Involvement of Macrophage Scavenger Receptor MARCO in Clearance of Bacteria In Vivo

AbstractThe scavenger receptors expressed by macrophages are thought to play an important role in the immune response against bacteria by mediating binding and phagocytosis. A novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (MARCO), has recently been identified. In this study we have generated a panel of mAbs with specificities for different domains of this receptor. Two of those reacting with the C-terminal cysteine-rich domain block ligand binding of MARCO. The in vivo expression of this murine receptor is normally restricted to distinct populations of macrophages in the spleen and lymph nodes. During bacillus Calmette-Guérin (BCG) infection, during bacterial sepsis, or after the injection of purified LPS, however, the expression of MARCO is rapidly induced on macrophages in other tissues, including Kupffer cells in the liver. Using the mouse macrophage cell line J774.2, it was shown that LPS stimulation up-regulates surface expression of MARCO in a dose- and time-dependent fashion. The proinflammatory cytokines IL-1, IL-6, TNF-α, and IFN-γ had little or no effect. Using inhibitory mAbs, the relevance of MARCO for the clearance of circulating bacteria in vivo was determined. Although the overall elimination of live Escherichia coli and Staphylococcus aureus from the blood did not appear to be affected by treatment with these Abs, the capturing of heat-killed bacteria by macrophages in the marginal zone areas of the spleen was clearly inhibited. This study suggests a role for MARCO in the host antibacterial defense.

Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses.

Mouse splenic B cells can be separated based on their distinctive expression of cell surface antigens. Marginal zone (MZ) B cells are CD38high CD21high CD23low/-, while follicular (FO) B cells are CD21int CD23int and newly formed (NF) B cells are CD21dim/- CD23-. Exploiting these phenotypic distinctions, we isolated the three B cell subsets and assessed their other phenotypic differences and functional capabilities in vitro. FO B cells proliferate more than the other B cell subsets in response to either IgM or CD38 cross-linking. MZ B cells proliferate better than FO B cells when stimulated with lipopolysaccharide (LPS), sub-optimal levels of LPS and CD38 cross-linking or CD40 ligation. When NF, FO and MZ B cells were stimulated with either LPS or LPS and interleukin-4, MZ B cells secreted more IgM and IgG3 than the other two subsets. Similarly, calcium fluxes measured within MZ B cells are larger in amplitude and more sustained after B cell receptor cross-linking than those found in the other two subsets. Collectively, these results indicate that CD38high CD21high CD23low/- MZ B cells are specially suited to play a unique role in response to antigens delivered to the MZ area.