Increase In B220 Research Articles

Abstract A87: Characterization of the involvement of transcriptional repressor IRF2BP2 on T-cell proliferative response

Abstract IRF2BP2 (Interferon Regulatory Factor-2 Binding Protein-2) is a transcriptional repressor involved in the regulation of gene expression in several different biologic contexts. It has been shown that IRF2BP2 expression is induced by the tumor suppressor protein p53 during genotoxic stress in osteosarcoma cell lines, which leads to a prevention of cell death. At least two translocations generating fusion genes involving IRF2BP2 were described distinct types of neoplasia as the IRF2BP2-CDX1 in chondrosarcoma and the IRF2BP2-RARA in acute promyelocytic leukemia. Recently, our group demostrated that IRF2BP2 represses NFAT1 (Nuclear factor of activated T-cells) transcriptional activity in CD4 T lymphocytes. In the present work we investigate the involvement of IRF2BP2 in the biology of CD4 T lymphocytes activation. We showed that ectopic expression of IRF2BP2 repressing CD4 T cell proliferation induced upon activation. We did not observe any differences in cell death of transduced cell cultures overexpressing IRF2BP2; however, we did observe a deleterious role in cell survival in the presence of an apoptotic stimulus by making these cells more prone to die after irradiation. In vivo, transferred IRF2BP2-overexpressing transduced CD4 T cells displayed an impaired expansion capacity compared to controls. Next, we generated a genetic model of conditional transgenic mice that overexpresses IRF2BP2 on T lymphocytes (IRF2BP2fl/flLck-cre+). Analysis by qRT-PCR demonstrated that CD4 T lymphocytes from IRF2BP2fl/flLck-cre+ mice presented an 8- to 10-fold increase of IRF2BP2 mRNA in comparison to wild-type animals (IRF2BP2+/+Lck-cre-). To evaluate the involvement of IRF2BP2 overexpression in T cell homeostasis we characterized the lymphocyte population in the thymus, spleen, and peripheral lymph nodes. In fact, IRF2BP2fl/flLck-cre+ animals showed a decrease in the total T-cell population in lymph nodes and thymus compared with controls. Furthermore, our results showed a reduction on CD3+ cells and a compensatory increase in B220+ compartment in the spleen and lymph nodes. IRF2BP2fl/flLck-cre+ animals also showed fewer counts of CD4+ and CD8+ cells in the thymus, spleen, and lymph nodes. Taken together, our data suggest a role for IRF2BP2 in controlling different parameters of CD4 T-cell function such as proliferation and cell survival, and may act as a regulator of immunologic checkpoint. Financial Support: FAPERJ, CNPq, INCT-Cancer. Citation Format: Barbara L. M. O Vieira, Cristiane S. Secca, Giuliana Mognol, Anjana Rao, João P. B. Viola. Characterization of the involvement of transcriptional repressor IRF2BP2 on T-cell proliferative response [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A87.

Mirn23a Inhibits B Lymphopoiesis through Antagonism of the B Cell Transcription Factor Network

Transcription factors (TFs) regulate genetic networks to direct the differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature blood lineages. Historically studies have focused on identifying genes activated by TFs that act as master hematopoietic regulators. Recently, it has become appreciated that these regulatory TFs direct one distinct differentiation program while simultaneously directing the repression of genes associated with alternative blood lineages. MicroRNAs (miRNAs) are short (~22nt) non-coding RNA molecules that negatively regulate gene expression at the post-transcriptional level and are hypothesized to act downstream of lineage specific TFs to contribute to the silencing of genes associated with alternative cell fate adoption. MiRNAs are observed to direct cell fate choice in assays utilizing overexpression and knockdown approaches; however, experiments from mouse gene targeting have yet to confirm these results. Specifically no miRNA has been shown to direct the differentiation of multipotent progenitors (MPPs) to granulocyte monocyte progenitors (GMPs) or common lymphoid progenitors (CLPs). We previously observed that overexpression of the mirn23a gene (Codes for three mature miRNAs: miR-23a, miR-24-2, and miR-27a) in hematopoietic progenitors increased myelopoiesis at the expense of B lymphopoiesis both in vitro and in vivo. To follow up this observation we generated a mirn23a germline knockout mouse. Flow cytometry characterization of hematopoiesis in mirn23a-/- mice reveals a significant increase in B220+ B-lymphocytes in both the bone marrow (BM) and the spleen. Furthermore, this increase in B-lymphocytes was shown to be at the expense of myeloid cells, as CD11b+ cells were significantly decreased in both the BM and spleen. Analysis of the BM progenitor populations revealed a significant increase in the CLP population, due to an increase in the B-cell specific lymphoid progenitor (BLP, CLP+ Ly6D+) CLP subset. Consistent with decreased mature myeloid cells, we also observed a decrease in BM GMPs. The phenotype appears to be cell intrinsic as ex vivo culture of mirn23a-/- progenitor cells on OP9 stroma results in a significant increase in B220+ B-cells, and a decrease in CD11b+ myeloid cells compared to wild type cultures. Consistent with mirn23a promoting myeloid development, we observe that expression of mirn23a in 70z/3 pre-B cells increases myeloid specific gene expression programs. The upregulation of the myeloid genes may be due to the insufficient repression by B cell transcription factors Ebf1 and Pax5, which are downregulated in mirn23a expressing cells. Interestingly Ebf1 associates with the mirn23a promoter as shown by chromatin immunoprecipitation (ChIP) assay, and Ebf1 represses mirn23a promoter activity in luciferase reporter assays. Consistent with a role in repressing mirn23a, Ebf1 knockdown in A20 B Lymphoma cells increases miR-23a expression while overexpression of Ebf1 in NIH/3t3 cells decreases miR-23a expression. Previously we observed that the myeloid transcription factor PU.1 binds to the mirn23a promoter to positively regulate transcription. Additionally we observe that myeloid transcription factor C/EBP alpha associates with the mirn23a locus in ChIP assays. Data from our knockout mouse supports a critical role for mirn23a in determining immune cells fates. Our current results support the conclusion that myeloid transcription factors PU.1 and C/EBP alpha activate mirn23a in order to antagonize B cell gene regulation and promote myeloid cell fate adoption, whereas the essential B cell transcription factor Ebf1 represses mirn23a expression to avoid this repression during B cell specification. DisclosuresNo relevant conflicts of interest to declare.

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220+ CD4–CD8– (DN) T lymphocytes. The precise ontogeny of B220+ DN T cells is unknown. B220+ DN T lymphocytes could be derived from effector CD4+ and CD8+ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220+ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220+ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220+ T cells observed in normal MRL+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220+ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.

A Methyl-Deficient Diet Modifies Early B Cell Development

A functional methyl group donor is essential for the epigenetic regulation of all biological events due to the importance of DNA methylation and histone methylation as an epigenetic marker. However, the epigenetic alterations in the immune system due to methyl donor deficiency are not well known. In this study, we tried to address this question by studying the lymphocyte development and DNA methylation changes caused by a methyl-deficient diet (MDD). We fed one group of C57BL/6J mice with a methyl-sufficient diet (MSD) and the other group with an MDD for 5 months. Flow cytometry analyses of their immune systems showed a decrease in B220+ IgM+ (immature B) cells and an increase in B220+ IgM– (pro/pre-B) cells in the bone marrow of mice fed an MDD. By means of an in vitro OP9 coculture system, we recognized that this B220+ IgM– cell fraction from the MDD has an intrinsic developmental defect. When we quantitatively measured the mRNA expression levels of transcription factors and recombination machinery related to B cell development in the B220+ IgM– cell fraction of their bone marrow, we found that ADA, EBF1, DNTT and Pax5 mRNA expression levels were significantly downregulated in mice fed with an MDD. In addition, there was a drastic decrease in histone methylation profile H3K4me3 in the Pax5 and EBF1 promoters in these B220+ IgM– B cells. However, CpG-DNA methylation profiles had not changed and this revealed that these two promoters are demethylated even under an MSD condition. We also found changed expression levels of the Polycomb group genes (mel18, bmi1, Pc1, Pc2, Ring1A, Ring1B, Ph1) on semi-quantitative RT-PCR. These results indicate that under an MDD condition, early B cell development in bone marrow is easily affected by epigenetic alterations.

Loss of P2X7 receptor membrane expression in B220+ double-negative T lymphocytes of autoimmune MRL/lpr mice (143.32)

Abstract Lupus is an autoimmune disease influenced by multiple genetic loci. Fas-deficient MRL/lpr mice exhibit lupus and lymphoproliferative syndromes with massive accumulation of B220+CD4-CD8- (DN) T lymphocytes. Pathogenic B220+ DN T lymphocytes are derived from normal activated T lymphocytes which have not undergone activation-induced cell death due to inactivation of Fas. Normal activated peripheral T lymphocytes up-regulate the membrane phosphatase B220 before undergoing apoptosis. In Fas-deficient mice, activated T cells down-regulate CD4 or CD8 molecules but retain the expression of B220. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of inflammatory cytokines and cell death. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP-mediated stimulation of P2X7R. This decrease in P2X7R activity parallels the increase in B220+ DN T-cell numbers in lymphoid organs. The rare B220+ T cells observed in normal mice are also resistant to ATP treatment. Moreover, B cells, which express B220 as a developmental marker, are also dramatically less sensitive to ATP treatment. Using rabbit anti-P2X7R sera, we found a strong reduction in P2X7R levels on B220+ cells. Our results prompt the conclusion that B220 expression strongly correlates with the negative regulation of ATP/P2X7R pathway on both T and B cells.

Identification of Phenolic Dermal Sensitizers in a Wound Closure Tape

A latex-allergic patient presented with a severe local reaction to a non-latex wound closure bandage following surgery. Extracts of the bandage were analyzed by gas chromatograph-electron impact-mass spectrometry (GC EI-MS) in the total ion monitoring mode. Components were identified by their ion mass fingerprint and elution time as a corresponding standard from the GC column. The chemicals identified were 4,4′-thiobis-(6-tert-butyl-m-cresol) (TBBC), 6-tert-Butyl-m-cresol (BC), 2,4-di-tert-butylphenol (BP) and erucamide (EA). Sensitization potential of these chemicals was evaluated using two quantitative structure-activity relationship (QSAR) programs. The phenol 2,6-di-tert-butyl-4-(hydroxymethyl)phenol (BHP) was also included in the test series. It was initially thought to be present in the bandage but detectable levels could not be confirmed. The potential for TBBC to induce a sensitization response was predicted by both Derek for Windows and TOPKAT 6.2. The potential for BC and BP to induce a sensitization response was predicted by Derek for Windows, but not TOPKAT. BHP and EA were not predicted to be sensitizers by either QSAR program. Local lymph node assay (LLNA) analysis of the chemicals identified TBBC, BP, and BC as potential sensitizers with EC3 values between 0.2 and 4.5%. None of the animals exhibited body weight loss or skin irritation at the concentrations tested. In agreement with the toxicological modeling, BHP did not induce a sensitization response in the LLNA. Following a positive LLNA response, TBBC, BP, and BC were further characterized by phenotypic analysis of the draining lymph nodes. A positive LLNA result coupled with a lack of increase in B220+IgE+ cell and serum IgE characterize these chemicals as Type IV sensitizers. These studies used a multidisciplinary approach combining clinical observation, GC-EI-MS for chemical identification, QSAR modeling of chemicals prior to animal testing, and the LLNA for determination of the sensitization potential of chemicals in a manufactured product.

Multilineage Expansion of Lymphoid and Myeloid Cells in Mice Expressing PML-RARα under the Control of the Murine Cathepsin G Locus.

In one of our laboratory’s models of acute promyelocytic leukemia (APL), the PML-RARα fusion cDNA is knocked into the 5′ UT of the azurophil granule protease, cathepsin G. Nearly all mCG-PML-RARα mice develop lethal leukemia with promyelocytic features following a 150–400 day latent period. We originally chose the cathepsin G gene for the targeting locus because its expression was believed to be restricted to promyelocytes. However, we have now observed high levels of cathepsin G expression with gene expression profiling of leukemic bone marrow samples from patients with AML FAB M1 or M2, i.e. blasts with minimal or no differentiation (M1: n=21, mean raw expression value for cathepsin G=23,885 ± 31,737; M2: n=22, mean=24,088 ± 26,585; M3: n=14, mean 116,029 ± 47,017). To examine whether cathepsin G is normally expressed in murine hematopoietic progenitor cells, we performed gene expression arrays with highly purified Sca-1+/lin− bone marrow cells; cathepsin G mRNA was easily detected in these cells (n=4, mean cathepsin G raw expression value=9,324 ± 5,082; array average normalized to 1,500). We therefore decided to determine whether the early progenitor compartment in mCG-PML-RARα mice was expanded due to unexpected expression of the transgene in KLS (c-kit+, Lin−, Sca-1+) cells; however, no difference was detected in the frequency of marrow-derived KLS cells between mCG-PML-RARα and WT mice that were age, strain, and gender matched (0.074% vs 0.071%, p=0.89). The GMP compartment (Lin−, c-kit+, Sca1−, CD34+, FcRγ+) showed a tendency towards expansion in mCG-PML-RARα mice (0.10% vs 0.032%) but the difference was not significant (p=0.067). To better assess stem cell function in these mice, we performed a competitive repopulation study using marrow derived from Ly 5.2/mCG-PML-RARα mice mixed with Ly5.1/WT marrow cells at various ratios (1:1, 9:1, and 1:9) that were transplanted into genetically compatible hosts. Lineage markers in the peripheral blood were tested at 6, 12, and 24 weeks to assess the contribution of mCG-PML-RARα-derived progenitor and stem cells to the B, T, and myeloid lineages. As expected, we observed a highly reproducible increase in the proportion of Gr-1+ cells derived from mCG-PML-RARα donors at all time points (78.8% ± 11% donor-derived cells at a 1:1 donor: recipient ratio at 6 months, p=0.0006). Surprisingly, we also observed a reproducible increase in B220+/CD19+ cells (66.7% ± 4%, p<0.0001) and CD3+ cells from the mCG-PML-RARα donors (60.4% ± 3% p=0.0010) at 6 months, suggesting that mCG-PML-RARα also confers a growth or survival advantage to B and T cells. To determine whether cathepsin G was expressed in these compartments, we purified CD19+ B and CD3+ T cells by flow cytometry (>99% purity) and did not detect cathepsin G mRNA using a sensitive, knockout-proven qRT-PCR analysis. These data strongly suggest that both the WT cathepsin G gene and the mutant mCG-PML-RARα allele are unexpectedly expressed in the stem cell compartment, with both myeloid and lymphoid progeny of these cells displaying a growth advantage in vivo. However, lymphoid malignancies have not been detected in these mice (n>400), suggesting that PML-RARα is unable to initiate transformation in lymphocytes. Our data imply that the PML-RARα gene is activated in a multipotent compartment in this mouse model, raising the possibility that PML-RARα may not actually ‘reprogram’ progenitor cells to undergo self-renewal, but may rather initiate transformation in pluripotent cells with intrinsic self-renewal capabilities.

Rap1 Signal Controls B Cell Receptor Repertoire and Generation of Self-Reactive B1a Cells

We previously reported that the mice deficient for SPA-1, a Rap1 GTPase-activating protein, developed hematopoietic stem cell disorders. Here, we demonstrate that SPA-1(-/-) mice show an age-dependent increase in B220(high) B1a cells producing anti-dsDNA antibody and lupus-like nephritis. SPA-1(-/-) peritoneal B1 cells revealed the altered Vkappa gene repertoire, including skewed Vkappa4 usage and the significant Igkappa/Iglambda isotype inclusion indicative of extensive receptor editing. Rap1GTP induced OcaB gene activation via p38MAPK-dependent Creb phosphorylation, and consistently, SPA-1(-/-) immature BM B cells showing high Rap1GTP exhibited the augmented expression of OcaB and Vkappa4 genes. SPA-1(-/-) BM cells could transfer the autoimmunity in association with the generation of peritoneal B220(high) B1a cells in Rag-2(-/-) recipients. Finally, a portion of SPA-1(-/-) mice developed B1 cell leukemia with hemolytic autoantibody. Present results suggest that the regulated Rap1 signal in the immature B cells plays a role in modifying the B cell receptor repertoire and in maintaining the self-tolerance.

Selective Rapid B-Cell Depletion In Vivo by Pharmacologic IKK2 Inhibition.

Studies using genetically deficient mice have revealed that members of the NF-kB family play key roles in B-cell development. IKK2 which activates NF-kB by targeting degradation of IkB, is also required for B-cell development. We have studied the role of IKK2 in hematopoiesis using a chemical specific inhibitor (ML120B). Mice given daily oral dosing of ML120B for 4 days had severe B-cell depletion in spleen and bone marrow. B-cells at all stages (pro-B, pre-B, immature and mature B) were depleted 10 fold in the bone marrow while granulocyte numbers were largely unaffected. IKK2 inhibition in vivo showed selective sensitivity of B-cell progenitors (4 fold decrease) in the marrow compared to myeloid progenitors which were unaffected at an equivalent dose. Foci of cells with an apoptotic morphology were visible in bone marrow and spleen within 6 hours of a single oral dose. Apoptotic cells detected by labeling fragmented DNA were increased within splenic follicles (6 fold) and bone marrow. Also an increase in B220+ / annexin V+ cells and a decrease in pre-B (B220+/IgM−) cells in the marrow were observed. RNA expression studies in the marrow 6 hours after a single oral dose revealed a decrease in IL-7 and increased GM-CSF expression. Image analysis of B220 in spleens within 18 hours of a single dose of an IKK inhibitor revealed decreased follicle size. In order to evaluate hematopoietic progenitor sensitivity to NF-kB inhibition, dose responses to ML120B, panepoxydone (PPD) and proteasome inhibitor Lactacystin (Lcyst) were evaluated in B-cell and myeloid bone marrow colony assays. Inhibitors PPD and Lcyst were more effective at inhibiting B-cell colony growth than myeloid colony growth. In summary, pharmacologic inhibition of IKK2 results in a rapid induction of apoptosis with preferential depletion of B-cells and retention of myeloid cells and progenitors within the bone marrow.

Generation and Analysis of Transgenic Mice Reveal a Role for CRE Binding Proteins in Multiple Stages of B Cell Development, Functional Maturation, Proliferation and Apoptosis.

Cyclic AMP response element binding protein-1 (CREB-1) is expressed in various stages of B cell development in the bone marrow. Antigen receptor induced DNA binding activity and activation dependent phosphorylation suggests a role for CREB-1 in early B cell development and functional maturation in-vivo. This was directly tested in transgenic mice over expressing a dominant negative Ser119-ala phosphomutant CREB-1 in bone marrow (BM). Northern blot analysis of total RNA and Western blot analysis of the protein isolated from 4 independent transgenic lines revealed expression of the transgene in the BM and peripheral B cells. Flow cytometric analysis of BM cells from transgenic (Tg) and non-transgenic (NTg) littermates revealed ~65% reduction in B220+/IgM− pro-B and pre-B population (7.56±0.89% NTg vs. 3.5±1.1% Tg mice) and B220+/IgM+ immature and mature B cells (4.4±0.98 in NTg vs 1.4± 0.19% in Tg mice). Detailed analysis of BM cells from Tg and NTg littermates by multiparameter flow cytometry revealed ~70% increase in B220+CD43+CD24+(int) pre-BI cells (39% in NTg vs 67% in Tg mice), and ~60% decrease in B220+CD43+CD24++(high) pre-BII B cells (46% in Ntg vs 20% in Tg mice), indicating a developmental block in pre-BI to pre-BII transition in mCREB-1 mice. Adoptive transfer of BM cells from Tg or NTg mice into sub-lethally irradiated RAG-2−/− mice, revealed cell intrinsic defect in Tg bone marrow B cells. RT-PCR analysis of RNA from Tg pre-BI B cells revealed increased c-jun and decreased jun-B transcripts with minimal changes in c-fos, PCNA, mb-1 and vpreB. In contrast to pre-BI cells, increased c-jun and junB transcripts were observed in pre-BII B cells. Cell cycle analysis exhibited a consistent decrease in S phase entry in pre-BII B cells from Tg compared to NTg mice at 48 hours without stimulation (41% in NTg vs 11% in Tg) or in response to IL-7 (48% in NTg vs 27% in Tg). Consistent with the BM defect, mCREB-1 Tg mice revealed ~ 40% decrease in the IgM+B220+ cells (29.3±0.96% in NTg vs. 17.7±2.9% Tg) in the spleen. This is further reflected in significant reduction in the absolute number of mature B cells in the spleen [(36±3.6 in NTg vs. 22 ±1.9% in Tg mice, p<0.01]. Interestingly when compared to NTg mice, mCREB-1 Tg mice revealed about 45% decrease in CD21dimCD23high follicular B cells with minimal change in the CD21highCD23dim marginal zone B cell (16.5±1.4x106 in NTg vs 9.5±0.92 x106 cells in Tg mice P<0.01). In contrast to the bone marrow and spleen, analysis of peritoneal cells revealed significant increase in the total cell number and B220+IgM+ population in Tg mice compared to NTg littermates [22±3% in NTg vs 33±2.6% in Tg mice (p<0.01)]. The increased cell number and the abnormal distribution reflected a 3–4 fold increase in IgMhighIgDlowMac-1Int B1 B cells. This increase is reflected in both CD5+ B-1a and CD5− B-1b B cells in the Tg mice and is attributed to resistance to apoptosis as evidenced by Annexin V/PI staining. These studies provide the first evidence for a role for CRE binding proteins in multiple stages of B cell development, functional maturation, proliferation and apoptosis. Ongoing studies are aimed to define the role of CREB-1 transcription factor in human B cell malignancies including chronic lymphocytic leukemia and acute lymphocytic leukemia.

Evaluation of lymphocyte subpopulations in draining lymph node cells following allergen and irritant

The murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for the assessment of the contact sensitization potential. However, there is a need to develop a non-radioisotopic endpoint for the LLNA, because of the radioisotopic method’s requiring the use of special facilities. In this study, we investigated to evaluate the lymphocyte subpopulations in the lymph node cells following allergen and irritant treatment. Female Balb/c mice were treated by the topical application on the dorsum of both ears with sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI), and α-hexylcinnamaldehyde (HCA), and an irritant, sodium lauryl sulfate (SLS), once daily for three consecutive days. The lymph node (LN) cells were harvested 72 h after the final treatment. Phenotypic analysis of lymphocytes subsets was performed with a flow cytometry. The allergens DNCB, TDI, and HCA and an irritant, SLS increased cell number compared to the vehicle. Mice were treated with DNCB, HCA, and TDI showed a preferential increase in the percentage of B220+CD40+ cells compared with vehicle and irritant-treated mice. There was an increase in B220+CD86+ cells of mice treated with DNCB, TDI, and HCA, but no significant increases were observed in mice treated with SLS. Mice were treated with DNCB and TDI showed an increase in the percentage of B220+CD23+ cells compared with vehicle and irritant-treated mice. These results suggest that analysis of B cell activation marker, CD40 on B cells may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice.

Impaired B cell development and function in mice with a targeted disruption of the homeobox gene Hex.

Hex is a homeobox gene that is expressed in all stages of B cell development except plasma cells. We studied lymphocyte development in the absence of Hex by using the RAG1-deficient blastocyst complementation system because homozygous disruption of Hex is embryonic lethal. Hex(-/-);RAG1(-/-) chimeric mice had severely reduced numbers of mature B cells, pre-B cells, and CD5(+) B cells with a striking 15-fold increase in the percentage of B220(-)CD19(+) cells in the bone marrow. Hex(-/-);RAG1(-/-) chimeric mice failed to generate IgG antibodies to T cell-independent antigens, although their serum IgM levels and antibody responses to T cell-dependent antigens were intact. Therefore, Hex is necessary for B cell development and function and its absence results in a dramatic increase in B220(-)CD19(+) cells.

Disorders in the immune responses of T- and B-cells in mice administered intravenous verotoxin 2

To obtain a better insight into the pathogenesis of verotoxin-producing Escherichia coli (VTEC)-associated diseases, we explored the effect of verotoxin 2 (VT2) on the immune response in mice. The distribution of lymphocyte phenotypes and the lymphocyte immune response were examined after intravenous administration of VT2 to mice. Among the peripheral lymphocytes and splenocytes of 4-week-old C57BL/6 mice, there was first of all a decrease in T-cells, which began 24 h after intravenous administration of VT2 (50 ng/kg, lethal dose). The CD4 + cell subpopulations of the peripheral blood and spleen were significantly decreased at 24 h, while the B220 + splenocyte subpopulation was markedly decreased at 45 h after VT2 administration. In the thymus, a decrease in CD4 +CD8 + cells was predominantly observed near death. Interestingly, in E. coli lipopolysaccharide (LPS)-responder mouse strains (C57BL/6 and C3H/HeN) cotreated with LPS, the susceptibility to VT2 was enhanced, and the increase in B220 + cells induced by LPS alone was suppressed. Furthermore, splenocytes from C57BL/6 mice treated with VT2 (50 ng/kg) 6–24 h earlier reduced LPS-induced proliferative responses to 50–52% of that in control cells, indicating that the effect of VT2 on the immunoresponse seen in vivo may be negatively exerted on the proliferation of the cells. In addition, the number of splenocytes that produced anti-sheep red blood cell antibody was decreased in mice treated with VT2. These results suggest that VTEC infection may eliminate CD4 + and CD8 + T-cells and B-cells by affecting their survival and proliferative responses, leading to reduced antibody production.

Changes in B and T lymphocytes associated with mycobacteria-induced protection of NOD mice from diabetes.

Most female NOD mice spontaneously develop insulin-dependent diabetes mellitus (IDDM) after the 4th month of age. We have recently reported that infection of 2-month-old NOD mice with Mycobacterium avium prevents IDDM expression in these mice. We have searched here for changes in splenic lymphocytes that are associated with the effect of M. avium vaccination. Three experimental groups of female NOD mice were studied: (i) animals infected with 10B viable M. avium bacteria (mice that become protected from IDDM); (ii) mice inoculated with 10B heat-killed (HK) M. avium bacilli, and (iii) untreated age-matched NOD mice. Similar treatments were given to mice of the NON strain which are related to NOD mice but do not develop IDDM. Flow cytometry was used to compare M. avium-infected, HK M. avium inoculated and untreated NOD and NON mice with regard to subpopulations of splenic lymphocytes bearing the surface antigens CD3, CD4, CD8, IgM and B220. We found that M. avium infection of NOD mice caused a sustained enhancement in T cells that was due to an early and transient increase in CD8+ T cells (detected at day 7 of infection). This was followed by marked augmentation in the number of CD4+ T cells at days 14 and 30. There was also elevation in B220+ B cells at days 14 and 30, and of IgM+ B cells at day 30 of infection. Inoculation of NOD mice with HK mycobacteria, which did not prevent IDDM, failed to produce significant changes in the number of T and B cells. No significant enhancement in T and B cells was observed in NON mice that were injected with either viable or HK M. avium bacilli. In NOD mice that reached 16 months of age because of being protected from IDDM (due to the M. avium infection) there was an increase in B220+ B cells. We conclude that: (i) M. avium-induced protection of NOD mice from diabetes depends on the viability of the bacteria; (ii) the protective effect of the infection is associated with an early and marked increase in helper T cells and with a smaller elevation in B cells; (iii) elevation in B cells, but not in T cells, is associated with long term mycobacteria-induced protection of NOD mice from IDDM.