Identifying Activated T Cells in Reconstituted RAG Deficient Mice Using Retrovirally Transduced Pax5 Deficient Pro-B Cells

Various methods have been used to identify activated T cells such as binding of MHC tetramers and expression of cell surface markers in addition to cytokine-based assays. In contrast to these published methods, we here describe a strategy to identify T cells that respond to any antigen and track the fate of these activated T cells. We constructed a retroviral double-reporter construct with enhanced green fluorescence protein (EGFP) and a far-red fluorescent protein from Heteractis crispa (HcRed). LTR-driven EGFP expression was used to enrich and identify transduced cells, while HcRed expression is driven by the CD40Ligand (CD40L) promoter, which is inducible and enables the identification and cell fate tracing of T cells that have responded to infection/inflammation. Pax5 deficient pro-B cells that can give rise to different hematopoietic cells like T cells, were retrovirally transduced with this double-reporter cassette and were used to reconstitute the T cell pool in RAG1 deifcient mice that lack T and B cells. By using flow cytometry and histology, we identified activated T cells that had developed from Pax5 deficient pro-B cells and responded to infection with the bacterial pathogen Listeria monocytogenes. Microscopic examination of organ sections allowed visual identification of HcRed-expressing cells. To further characterize the immune response to a given stimuli, this strategy can be easily adapted to identify other cells of the hematopoietic system that respond to infection/inflammation. This can be achieved by using an inducible reporter, choosing the appropriate promoter, and reconstituting mice lacking cells of interest by injecting gene-modified Pax5 deficient pro-B cells

Crucial Role for BAFF-BAFF-R Signaling in the Survival and Maintenance of Mature B Cells

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools

Enforced Expression of the Transcriptional Coactivator OBF1 Impairs B Cell Differentiation at the Earliest Stage of Development

OBF1, also known as Bob.1 or OCA-B, is a B lymphocyte-specific transcription factor which coactivates Oct1 and Oct2 on B cell specific promoters. So far, the function of OBF1 has been mainly identified in late stage B cell populations. The central defect of OBF1 deficient mice is a severely reduced immune response to T cell-dependent antigens and a lack of germinal center formation in the spleen. Relatively little is known about a potential function of OBF1 in developing B cells. Here we have generated transgenic mice overexpressing OBF1 in B cells under the control of the immunoglobulin heavy chain promoter and enhancer. Surprisingly, these mice have greatly reduced numbers of follicular B cells in the periphery and have a compromised immune response. Furthermore, B cell differentiation is impaired at an early stage in the bone marrow: a first block is observed during B cell commitment and a second differentiation block is seen at the large preB2 cell stage. The cells that succeed to escape the block and to differentiate into mature B cells have post-translationally downregulated the expression of transgene, indicating that expression of OBF1 beyond the normal level early in B cell development is deleterious. Transcriptome analysis identified genes deregulated in these mice and Id2 and Id3, two known negative regulators of B cell differentiation, were found to be upregulated in the EPLM and preB cells of the transgenic mice. Furthermore, the Id2 and Id3 promoters contain octamer-like sites, to which OBF1 can bind. These results provide evidence that tight regulation of OBF1 expression in early B cells is essential to allow efficient B lymphocyte differentiation

Silencing and Nuclear Repositioning of the λ5 Gene Locus at the Pre-B Cell Stage Requires Aiolos and OBF-1

The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes λ5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the λ5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function

Pathway of Toll-Like Receptor 7/B Cell Activating Factor/B Cell Activating Factor Receptor Plays a Role in Immune Thrombocytopenia In Vivo

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by anti-platelet autoantibody-mediated platelet destruction. Antigen-presenting cell (APC) dysfunction is considered to play crucial roles in ITP. However, how APC affects autoreactive B cells in ITP is still unknown. Using a mouse model of immune thrombocytopenia, we demonstrated an increase in levels of TLR7 in splenic mononuclear cells (SMCs). Using both TLR7 agonist and TLR7 silencing lentivirus, we found stimulation of TLR7 decreased platelet counts and increased levels of platelet-associated IgG (PAIgG) in ITP mice, which correlates TLR7 with platelet destruction by autoantibodies. Levels of serum BAFF increased significantly in ITP mice and stimulation of TLR7 promoted secretion of BAFF. Among the three BAFF receptors, only BAFF receptor (BAFF-R) increased in ITP mice. However, activation of TLR7 showed no effect on the expression of BAFF receptors. These findings indicate that upregulation of TLR7 may augment BAFF secretion by APC and through ligation of BAFF-R promote autoreactive B cell survival and thus anti-platelet autoantibody production. The pathway of TLR7/BAFF/BAFF-R provides us with an explanation of how activation of APC affects autoantibody production by B cells in ITP and thus might provide a reasonable therapeutic strategy for ITP

Profiling of the Tetraspanin CD151 Web and Conspiracy of CD151/Integrin β1 Complex in the Progression of Hepatocellular Carcinoma

Tetraspanin CD151 has been implicated in metastasis through forming complexes with different molecular partners. In this study, we mapped tetraspanin web proteins centered on CD151, in order to explore the role of CD151 complexes in the progression of hepatocellular carcinoma (HCC). Immunoprecipitation was used to isolate tetraspanin complexes from HCCLM3 cells using a CD151 antibody, and associated proteins were identified by mass spectrometry. The interaction of CD151 and its molecular partners, and their roles in invasiveness and metastasis of HCC cells were assayed through disruption of the CD151 network. Finally, the clinical implication of CD151 complexes in HCC patients was also examined. In this study, we identified 58 proteins, characterized the tetraspanin CD151 web, and chose integrin β1 as a main partner to further investigate. When the CD151/integrin β1 complex in HCC cells was disrupted, migration, invasiveness, secretion of matrix metalloproteinase 9, and metastasis were markedly influenced. However, both CD151 and integrin β1 expression were untouched. HCC patients with high expression of CD151/integrin β1 complex had the poorest prognosis of the whole cohort of patients. Together, our data show that CD151 acts as an important player in the progression of HCC in an integrin β1-dependent manner

All-Trans Retinoic Acid Promotes TGF-β-Induced Tregs via Histone Modification but Not DNA Demethylation on Foxp3 Gene Locus

It has been documented all-trans retinoic acid (atRA) promotes the development of TGF-β-induced CD4(+)Foxp3(+) regulatory T cells (iTreg) that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs.Addition of atRA to naïve CD4(+)CD25(-) cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-β not only increased Foxp3(+) iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-β-treated CD4(+) cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-β-treated CD4(+) cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4(+) cells treated with TGF-β alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4(+) cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS) elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered.We have identified the cellular and molecular mechanism(s) by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation

BAFF Promotes Th17 Cells and Aggravates Experimental Autoimmune Encephalomyelitis

BAFF, in addition to promoting B cell survival and differentiation, may affect T cells. The objective of this study was to determine the effect of BAFF on Th17 cell generation and its ramifications for the Th17 cell-driven disease, EAE.Th17 cells were increased in BAFF-Tg B6 (B6.BTg) mice and decreased in B6.Baff(-/-) mice. Th17 cells in B6.Baff(-/-) mice bearing a BAFF Tg (B6.Baff(-/-).BTg mice) were identical to those in B6.BTg mice, indicating that membrane BAFF is dispensable for Th17 cell generation as long as soluble BAFF is plentiful. In T + non-T cell criss-cross co-cultures, Th17 cell generation was greatest in cultures containing B6.BTg T cells and lowest in cultures containing B6.Baff(-/-) T cells, regardless of the source of non-T cells. In cultures containing only T cells, Th17 cell generation followed an identical pattern. CD4(+) cell expression of CD126 (IL-6R α chain) was increased in B6.BTg mice and decreased in B6.Baff(-/-) mice, and activation of STAT3 following stimulation with IL-6 + TGF-β was also greatest in B6.BTg cells and lowest in B6.Baff(-/-) cells. EAE was clinically and pathologically most severe in B6.BTg mice and least severe in B6.Baff(-/-) mice and correlated with MOG(35-55) peptide-induced Th17 cell responses.Collectively, these findings document a contribution of BAFF to pathogenic Th17 cell responses and suggest that BAFF antagonism may be efficacious in Th17 cell-driven diseases

CD10 is a marker for cycling cells with propensity to apoptosis in childhood ALL

CD10 constitutes a favourable prognostic marker for childhood acute lymphoblastic leukaemia. Since correlations between CD10, cell cycle and apoptotic abilities were demonstrated in various cell types, we investigated whether differences existed in the cycling/apoptotic abilities of CD10-positive and CD10-negative B acute lymphoblastic leukaemia cells. Twenty-eight cases of childhood acute lymphoblastic leukaemia (mean age of 6.8 years) were subdivided into two groups according to high (17 cases, 93.2±4.5%, MRFI 211±82 CD10-positive cells) or low (11 cases, 11.5±6.2%, MRFI 10±7 CD10-negative cells) expression of CD10. CD10-positive acute lymphoblastic leukaemia cells were cycling cells with elevated c-myc levels and propensity to apoptosis, whereas CD10-negative acute lymphoblastic leukaemia cells had lower cycling capacities and c-myc levels, and were resistant to apoptosis in vitro. A close correlation between all these properties was demonstrated by the observations that the few CD10-positive cells found in the CD10-negative acute lymphoblastic leukaemia group displayed elevated c-myc and cycling capacities and were apoptosis prone. Moreover, exposure of CD10-positive acute lymphoblastic leukaemia B cells to a peptide nucleic acid anti-gene specific for the second exon of c-myc caused inhibition of c-myc expression and reduced cell cycling and apoptotic abilities as well as decreased CD10 expression