CD19-Cre Mice Research Articles

T cell deletion of Serum and Glucocorticoid‐Regulated Kinase 1 (SGK1) Attenuates Hypertension and End‐Organ Inflammation

We have previously shown that the T cell‐derived pro‐inflammatory cytokine, interleukin 17A (IL17A), is upregulated by and promotes angiotensin II‐induced hypertension and contributes to renal and vascular dysfunction. It was recently demonstrated that an excess of 40 mM of sodium chloride enhances IL17A production from CD4+ T cells in an SGK1 dependent manner. We confirmed the effect of salt on CD4+ T cell differentiation and extended this finding to CD8+ T cells in which 40mM of excess salt increased the expression of IL17A (4.7 fold, p=0.0003), TonEBP (2.3 fold, p <0.002), and the salt‐sensing kinase SGK1 (2.2 fold, p=.001) in naive CD8+ cells cultured under Th17 polarizing conditions. Since dietary salt intake is associated with hypertension, we hypothesized that T cell SGK1 promotes hypertension and contributes to end‐organ dysfunction. To test this hypothesis, we crossed SGK1fl/fl mice with CD4cre mice to delete SGK1 in T lymphocytes. Loss of T cell SGK1 resulted in a blunted blood pressure response to angiotensin II infusion (24.8 mmHg reduction, p=0.01) and DOCA‐salt treatment (15.51 mmHg reduction, p<0.05). Moreover, renal and vascular inflammation in response to angiotensin II infusion and/or DOCA‐salt treatment were abrogated in these mice compared to SGK1fl/fl control mice. Angiotensin II infusion increased total (CD45+) leukocytes in the kidney from 55.4 to 120.4 x103 (p<0.01) in SGK1fl/fl mice while there was no increase in mice with T cell deletion of SGK1 (48.1 to 47.5×103, p=ns). Similarly, angiotensin II increased total (CD45+) leukocytes in the aorta from 5.7 to 52.4×103 (p<0.01) in SGK1fl/fl mice compared to no increase in mice with T cell deletion of SGK1 (16.1 to 10.1×103, p=ns). DOCA‐salt induction increased total (CD45+) leukocytes in the aorta in SGK1fl/fl mice from 7.4 to 20.6×103 (p<0.05) compared to no increase in mice with T cell deletion of SGK1 (8.7 to 10.8×103, p=ns). Furthermore, vascular reactivity studies demonstrated that vessels from mice with T cell deletion of SGK1 were protected from angiotensin II induced endothelial dysfunction. These studies demonstrate that T cell SGK1 may be a novel therapeutic target for hypertension and the associated end‐organ inflammation.

An Essential Role for the Transcription Factor Runx1 in T Cell Maturation.

The transcription factor Runx1 has essential roles throughout hematopoiesis. Here, we demonstrate that Runx1 is critical for T cell maturation. Peripheral naïve CD4+ T cells from CD4-cre Runx1 cKO mice are phenotypically and functionally immature as shown by decreased production of TNF-α upon TCR stimulation. The loss of peripheral CD4+ T cells in CD4-cre Runx1 cKO mice is not due to defects in homeostasis or decreased expression of IL-7Rα, as transgenic expression of IL-7Rα does not rescue the loss of CD4+ T cells. Rather, immature Runx1-deficient CD4+ T cells are eliminated in the periphery by the activation and fixation of the classical complement pathway. In the thymus, there is a severe block in all aspects of intrathymic T cell maturation, although both positive and negative selection are unaltered. Thus, loss of Runx1 leads to the earliest characterized block in post-positive selection intrathymic maturation of CD4 T cells.

CNS accumulation of regulatory B cells is VLA-4-dependent.

Objective:To investigate the role of very late antigen-4 (VLA-4) on regulatory B cells (Breg) in CNS autoimmune disease.Methods:Experimental autoimmune encephalomyelitis (EAE) was induced in mice selectively deficient for VLA-4 on B cells (CD19cre/α4f/f) by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p)35–55 or recombinant human (rh) MOG protein. B-cell and T-cell populations were examined by flow cytometry and immunohistochemistry. Breg were evaluated by intracellular IL-10 staining of B cells and, secondly, by coexpression of CD1d and CD5.Results:As previously reported, EAE was less severe in B-cell VLA-4-deficient vs control CD19cre mice when induced by rhMOG, a model that is B-cell-dependent and leads to efficient B-cell activation and antibody production. Paradoxically, B-cell VLA-4-deficient mice developed more severe clinical disease than control mice when EAE was induced with MOG p35-55, a B-cell-independent encephalitogen that does not efficiently activate B cells. Peripheral T-cell and humoral immune responses were not altered in B-cell VLA-4-deficient mice. In MOG p35-55-induced EAE, B-cell VLA-4 deficiency reduced CNS accumulation of B but not T cells. Breg were detected in the CNS of control mice with MOG p35-55-induced EAE. However, more severe EAE in B-cell VLA-4-deficient mice was associated with virtual absence of CNS Breg.Conclusions:Our results demonstrate that CNS accumulation of Breg is VLA-4-dependent and suggest that Breg may contribute to regulation of CNS autoimmunity in situ. These observations underscore the need to choose the appropriate encephalitogen when studying how B cells contribute to pathogenesis or regulation of CNS autoimmunity.

Abstract B25: Disruption of KMT2D-dependent histone methylation perturbs GC B cell development and cooperates with BCL2 deregulation in lymphomagenesis

Abstract Modulation of chromatin accessibility through histone modification is a key step in the regulation of gene transcription and its disruption by genetic lesions has been implicated in malignant transformation. Indeed, a consistent theme in recent cancer genome studies has been the discovery of recurrent mutations in multiple histone/chromatin modifier genes, including methyltransferases, acetyltransferases and histone themselves. Among these, KMT2D (MLL2 or MLL4), encoding for a histone H3K4 methyltransferase, emerged as one of the most common targets of genetic lesion in B cell non-Hodgkin lymphoma, being found in ~30% of diffuse large B cell lymphoma (DLBCL) and ~90% of follicular lymphoma (FL), which together account for over 70% of all lymphoma diagnoses (Pasqualucci et al, Nat Genetics 2011; Morin et al, Nature 2011). KMT2D mutations are mostly represented by truncating events that are predicted to remove the protein C-terminal enzymatic domains, thus inactivating its function; however, missense mutations were also found in a subset of cases, suggesting selection for a functional role. These events are biallelically distributed in one third of mutated cases, while the remaining >60% harbor monoallelic mutations, consistent with a role as a tumor suppressor. Interestingly, analysis of the history of clonal evolution during FL transformation to DLBCL suggests that KMT2D mutations may be already present in a common precursor clone before divergent evolution to FL or DLBCL, suggesting an early role during B cell clonal expansion (Pasqualucci et al, Cell Rep, 2014; Green et al, Blood, 2013). To elucidate the functional consequences of KMT2D mutations, we first examined the effects of 16 DLBCL/FL-derived KMT2D missense mutant alleles on its enzymatic activity in vitro. The results showed that all 8 mutants located in the C-terminal portion of the protein were associated with significantly diminished H3K4 mono-, di- and tri-methylation activity. Consistently, a significant reduction in global methylation was observed in Kmt2d deficient murine B-cells, as well as in 4 biallelically truncated cell lines, indicating that this methyltransferase can influence all three H3K4 modifications. To gain further insights into the program regulated by KMT2D in germinal center (GC) B cells (i.e. the normal counterpart of FL/DLBCL) and the mechanism by which its loss contributes to lymphomagenesis, we crossed mice carrying a conditional Kmt2d knockout allele (Lee J et al, Elife, 2013) with either CD19Cre or Cγ1Cre deletor mice, leading to gene inactivation early during B-cell development (Kmt2dCD19KO), thus mimicking the postulated “common precursor model,” or specifically in mature, GC B-cells (Kmt2dCγ1KO). Notably, deletion of Kmt2d before, but not after initiation of the GC reaction led to a significant increase in GC B-cells and enhanced B cell proliferation. These changes were accompanied by the acquisition of distinct transcriptional signatures enriched in cell cycle regulation and apoptosis genes. A cross-species strategy combining gene expression profile analysis of murine GC B-cells and Kmt2d chromatin immunoprecipitation and sequencing of purified human GC B cells identified a core of KMT2D direct targets genes involved in biological programs with critical functions in B cells physiology, including B cell receptor signaling, lymphocyte migration, and chemokine signaling components. Finally, while loss of Kmt2d alone in Kmt2dCγ1KO was not sufficient to induce tumor development, its combination with VavP-BCL2 transgenic mice increased the incidence of GC-derived lymphomas resembling the features of the human tumors. These data support a role for KMT2D as a tumor suppressor gene whose early loss during B cell development facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cell. Citation Format: Jiyuan Zhang, David Dominguez-Sola, Shafinaz Hussein, Ji-Eun Lee, Antony B. Holmes, Mukesh Bansal, Sofija Vlasevska, Tongwei Mo, Hongyan Tang, Katia Basso, Kai Ge, Riccardo Dalla-Favera, Laura Pasqualucci. Disruption of KMT2D-dependent histone methylation perturbs GC B cell development and cooperates with BCL2 deregulation in lymphomagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B25.

Genetic Loss of NFAT2 Leads to CLL Transformation

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. While some patients exhibit enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggressive disease, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype referring to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Up to 10% of CLL patients transform to an aggressive form of lymphoma over time (Richter´s syndrome) and show a significantly worse treatment outcome. Here, we analyzed the role of NFAT2 in CLL with respect to maintenance of the anergic phenotype and the development of Richter´s syndrome.For this purpose, we generated conditional NFAT2fl/fl CD19-Cre knock out mice on the Eµ-TCL1 transgenic background. These mice exhibit NFAT2 deletion limited to the B cell lineage and develop a human-like CLL due to the B cell specific expression of the TCL1 oncogene. Conditional TCL1+NFAT2fl/fl CD19-Cre mice with B cell specific deletion of NFAT2 served as the experimental cohort while TCL1 transgenic mice without an NFAT2 deletion served as controls. To extend our observations do the human disease, we further analyzed primary blood samples from patients with CLL (n=30) and Richter´s syndrome (n=5).Mice with genetic loss of NFAT2 in their B cell compartment showed a significantly more aggressive disease course with accelerated accumulation of CD5+ CD19+ CLL cells in different organs, significantly higher proliferation rates and a dramatically reduced life expectancy (200 vs. 325 days) as compared to TCL1 control animals. Immunohistochemical analysis of spleen and lymph nodes upon necropsy showed a transformed disease phenotype in the NFAT2 ko cohort with diffuse distortion of lymph node and spleen architecture by large B cells with blastic chromatin while animals with intact NFAT2 expression showed small lymphocytic infiltrates with conserved B and T cell areas as expected. Using calcium imaging technology, we were able to show that CLL cells with regular NFAT2 expression were essentially unable to mobilize calcium upon IgM ligation and thus exhibited the typical anergic phenotype. CLL cells with NFAT2 deletion, on the contrary, showed a highly IgM-responsive phenotype with inducible activation of downstream signaling via AKT and ERK. We subsequently defined a genetic signature of anergy in CLL cells employing Affymetrix microarrays consisting of Lck, Pacsin1 and the E3 ligases Cbl and Grail, which were highly expressed in anergic CLL cells with normal NFAT2 expression and virtually absent upon NFAT2 ablation. We could further show by western blotting with phospho-specific antibodies that Lck was constitutively activated in anergic CLL cells while it was essentially inactive in NFAT2 ko cells suggesting an important role for Lck in mediating the anergic phenotype.Primary human CLL samples (n=30) showed a significant overexpression of NFAT2 and Lck, predominantly in its active conformation, as assessed by western blotting and RT-PCR. Tissue samples (lymph nodes, bone marrow) from patients with Richter's Syndrome (n=4) on the other hand demonstrated a significantly reduced expression of NFAT2 and complete loss of Lck expression. Calcium imaging revealed that a subset of human CLL cells showed an anergic phenotype and were unable to flux calcium while tumor cells from Richter patients exhibited an intensive spontaneous calcium flux as well as hyperresponsiveness upon IgM ligation.In summary, our data provide strong evidence that NFAT2 and its target gene Lck are important mediators of anergy induction in CLL and that their loss leads to disease transformation to aggressive B cell lymphoma (Richter´s syndrome). Taken together, our results demonstrate that the NFAT2-LCK axis plays an important role in the pathogenesis of CLL and implicate it as a potential target in its treatment. DisclosuresNo relevant conflicts of interest to declare.

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.

Complete Block of Early B Cell Differentiation in Mice Lacking the Endosomal Adaptor Protein p14

Human primary immunodeficiency caused by a point mutation in the 3' untranslated region of the endosomal adaptor protein p14 (also known as Lamtor2) resulted in severely impaired function of neutrophils, B cells, T cells and melanocytes. However, complexity of the phenotype and scarcity of human material preclude in-depth studies.Therefore, to gain insight into the role of p14 in B cell development and function, we generated loxP conditional knock-out mice. Using mb-1-Cre mice we demonstrated that loss of p14 at the preB1 stage lead to a complete block of B cell development, resulting in the absence of IgM-positive B cells. Further, to test the significance of p14 deficiency in peripheral organs, we took advantage of CD19-Cre mice, which have limited efficiency in deleting target genes in the bone marrow, but reach up to 95% efficiency in spleen. Thus, we could demonstrate that later in B cell development, p14 was essential for the generation and activation of mature B lymphocytes. While B1 cell development was maintained, splenic follicular B cells were massively reduced in the absence of p14. Furthermore, activation of B cell receptor (BCR) resulted in impaired intracellular signalling and proliferation of p14 deficient B cells. In particular, lack of p14 lead to delayed internalization of BCR and endosomal processing associated with impaired mobilization of Ca++ from intracellular stores as well as aberrant phosphorylation of BCR-associated kinases.In conclusion, our data revealed that p14 is a critical regulator of B cell development and function, which acts by modulating BCR signalling. DisclosuresNo relevant conflicts of interest to declare.

Abstract 140: Deficiency of Serum Glucocorticoid Kinase 1 (SGK1) in T Lymphocytes Blunts Hypertension and Abrogates Renal/Vascular Inflammation

We have previously shown that the T cell-derived pro-inflammatory cytokine, interleukin 17A (IL17A), is upregulated by and promotes angiotensin II-induced hypertension and contributes to renal and vascular dysfunction. It was recently demonstrated that an excess of 40 mM of sodium chloride enhances IL17A production from CD4+ T cells in an SGK1 dependent manner. We confirmed the effect of salt on CD4+ T cell differentiation and extended this finding to CD8+ T cells in which 40mM of excess salt increased the expression of IL17A (4.7 fold, p=0.0003), TonEBP (2.3 fold, p=0.002), and the salt-sensing kinase SGK1 (2.2 fold, p=0.001) in naive CD8+ cells cultured under Th17 polarizing conditions. As dietary salt intake is linked to hypertension, we hypothesized that T cell SGK1 is essential to the ability of T cells to mediate hypertension. To test this hypothesis, we crossed SGK1 fl/fl mice with CD4cre mice to delete SGK1 in T lymphocytes. Interestingly, loss of T cell SGK1 resulted in a blunted blood pressure response to angiotensin II infusion (by 31.25 mmHg, p=0.01). Moreover, angiotensin II-induced renal and vascular inflammation was abolished in these mice compared to SGK1 fl/fl control mice. Angiotensin II infusion increased total (CD45+) leukocytes in the kidney from 55.4 to 120.4x10 3 (p&lt;0.01) in SGK1 fl/fl mice while there was no increase in mice with T cell deletion of SGK1 (48.1 to 47.5x10 3 , p=ns). Similarly, angiotensin II increased total (CD45+) leukocytes in the aorta from 5.7 to 52.4x10 3 (p&lt;0.01) in SGK1 fl/fl mice compared to no increase in mice with T cell deletion of SGK1 (16.1 to 10.1x10 3 , p=ns). Further characterization of these mice will lead to a more thorough understanding of how T cells sense salt and promote hypertension. These studies demonstrate that T cell SGK1 may be a novel therapeutic target for hypertension and the associated end-organ inflammation.

Histone Deacetylase 3 Is Required for T Cell Maturation.

Recent thymic emigrants are newly generated T cells that need to undergo postthymic maturation to gain functional competency and enter the long-lived naive T cell pool. The mechanism of T cell maturation remains incompletely understood. Previously, we demonstrated that the transcriptional repressor NKAP is required for T cell maturation. Because NKAP associates with histone deacetylase 3 (HDAC3), we examined whether HDAC3 is also required for T cell maturation. Although thymic populations are similar in CD4-cre HDAC3 conditional knockout mice compared with wild-type mice, the peripheral numbers of CD4(+) and CD8(+) T cells are dramatically decreased. In the periphery, the majority of HDAC3-deficient naive T cells are recent thymic emigrants, indicating a block in T cell maturation. CD55 upregulation during T cell maturation is substantially decreased in HDAC3-deficient T cells. Consistent with a block in functional maturation, HDAC3-deficient peripheral T cells have a defect in TNF licensing after TCR/CD28 stimulation. CD4-cre HDAC3 conditional knockout mice do not have a defect in intrathymic migration, thymic egress, T cell survival, or homeostasis. In the periphery, similar to immature NKAP-deficient peripheral T cells, HDAC3-deficient peripheral T cells were bound by IgM and complement proteins, leading to the elimination of these cells. In addition, HDAC3-deficient T cells display decreases in the sialic acid modifications on the cell surface that recruit natural IgM to initiate the classical complement pathway. Therefore, HDAC3 is required for T cell maturation.

Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells.

Invariant natural killer T cells are a distinct subset of T cells that exert Janus-like functions. Moreover, Notch signaling is known to have critical roles in the development and functions of T cells. However, it is not known whether Notch signaling contributes to the development or functions of invariant natural killer T cells. Here, we found that CD4-specific gene ablation of Notch 1 and Notch 2 (N1N2(-/-)) increased the number of invariant natural killer T cells in the thymus but decreased them in the liver. N1N2(-/-) mice showed impaired thymic maturation of invariant natural killer T cells from the NK1.1(-)CD44(+) to the NK1.1(+)CD44(+) stage, resulting in accumulation of NK1.1(-)CD44(+) invariant natural killer T cells in the thymus. Upon activation, hepatic invariant natural killer T cells from N1N2(-/-) mice produced lower cytokine levels and increased apoptosis versus wild-type invariant natural killer T cells. Furthermore, Notch 1/Notch 2-deficient, but not wild type, invariant natural killer T cells failed to promote antibody-induced arthritis in CD1d(-/-) mice. Unlike N1N2(-/-) mice, RBP-j(lox) (/) (lox) CD4-Cre mice showed similar percentages and numbers of thymic invariant natural killer T cells to wild-type mice but had defects in their homeostasis, maturation, and cytokine production in the liver. Taken together, our data indicate distinct effects of Notch signaling on invariant natural killer T cells in the thymus and liver, which are at least partly independent of RBP-j in the thymus.

Histone deacetylase 3 is necessary for T cell maturation (HEM2P.238)

Abstract Recent thymic emigrants (RTEs) are newly generated T cells that need to undergo post-thymic maturation in the periphery. The mechanism of T cell maturation remains unclear. Histone deacetylase 3 (HDAC3) controls gene regulation, chromatin accessibility and genomic stability. However, the function of HDAC3 in T cell has yet to be elucidated. As we previously demonstrated that HDAC3 is a binding partner to NKAP, we hypothesize that HDAC3, like NKAP, may regulate T cell maturation. We generated CD4-cre HDAC3 cKO mice, which have similar numbers of DN, DP, CD4 SP and CD8 SP thymocytes as compared to WT mice; however, the peripheral numbers of CD4+ and CD8+ T cells are dramatically decreased in CD4-cre HDAC3 cKO mice. When we examined Rag1-GFP CD4-cre HDAC3 cKO mice, the majority of peripheral naïve T cells are Rag1-GFP+ RTEs indicating a block in T cell maturation. There is no defect in the positive selection, negative selection, migration, or homeostasis in CD4-cre HDAC3 cKO mice. However, expression of the complement inhibitor CD55, which is induced during maturation, is severely decreased. In the periphery, similar to NKAP-deficient T cells, HDAC3-deficient T cells are targeted for elimination by complement. In addition, HDAC3-deficient T cells have decreased sialic acid modifications on the cell surface, mainly due to α2-3, and α2-8 linkages, that recruit natural IgM to initiate the classical complement pathway. Thus, HDAC3 is required for T cell maturation.

Runx1 is required for T cell maturation (HEM2P.246)

Abstract T cell development depends on coordinated gene expression changes in order to progress through developmental checkpoints. The loss of transcription factor Runx1 in CD4-cre Runx1 conditional knockout (cKO) mice leads to decreased numbers of CD4 single positive (SP) thymocytes, CD4 peripheral T cells and lower expression of IL-7Rα, a key player of T cell homeostasis, by CD4+ Runx1 deficient T cells . Low IL-7Rα is thought to cause decreased CD4 T cell survival. However, we show that constitutive expression of an IL-7Rα transgene in CD4-cre Runx1 cKO mice does not restore thymic or peripheral CD4 cellularity. We also see similar expression of Bcl-2 and Bcl-xL in WT and Runx1-deficient CD4 T cells, indicating that a Runx1-mediated IL-7Rα-independent mechanism might be responsible. As IL-7Rα expression increases over the course of T cell maturation, we asked if Runx1 is needed for maturation. During thymic maturation, post-positive selection CD4 SP thymocytes migrate from cortex to medulla, changing from CCR7lowCD24high to CCR7highCD24high and finally CCR7highCD24low before thymic egress. We saw a block at the CCR7lowCD24high stage in CD4-cre Runx1 cKO CD4 SP thymocytes. Additionally, other maturation markers including CD55 and Qa2 were also downregulated. Thus, Runx1 is required for thymic maturation of CD4 SP thymocytes.

C-Myb coordinates survival and expression of key signaling components required for the pre-BCR checkpoint (HEM1P.220)

Abstract The c-Myb transcription factor is abundantly expressed during B cell development and is down regulated as the cells mature. We targeted Myb in mice with loxP sites (Mybf/f). By crossing these mice to other strains that direct Cre expression to different stages of B cell development (Rag1-cre, Mb1-cre, CD19-cre) we are able to assess subsequent B-lineage maturation. In Mybf/f Mb1-cre mice, c-Myb is required for B cell differentiation beyond the pre-pro-B cell stage and survival of CD19+ pro-B cells. c-Myb controls survival of pro-B cells by suppression of the proapoptotic factors Bmf and Bim. We find that IL-7 signaling represses the expression of Bmf and Bim. c-Myb controls the expression of CD127 but forced expression of CD127 is not sufficient to repress Bmf and Bim or rescue survival of c-Myb deficient pro-B cells. Examination of negative mediators of IL-7 signaling revealed increased SOCS3 mRNA in c-Myb deficient pro-B cells and forced expression of SOCS3 suppressed proliferation and survival of CD19+ pro-B cells in the presence of IL-7. Surprisingly, Mybf/f CD19-cre Bcl2-Tg mice contain severely reduced large and small pre-B cell compartments. Examination of mRNA expression in these cells revealed that c-Myb controls expression of key regulators of the pre-BCR checkpoint, including CD127, lambda-5, cyclin D3 and Cxcr4. c-Myb coordinates survival in pro-B cells with the expression of key components of the signaling pathways that drive the pro-B to pre-B cell transition.

Immature recent thymic emigrants are eliminated by complement.

Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to become long-lived mature naive T cells. In CD4-cre NKAP conditional knockout mice, NKAP-deficient RTEs fail to complete T cell maturation. In this study, we demonstrate that NKAP-deficient immature RTEs do not undergo apoptosis, but are eliminated by complement. C3, C4, and C1q are bound to NKAP-deficient peripheral T cells, demonstrating activation of the classical arm of the complement pathway. As thymocytes mature and exit to the periphery, they increase sialic acid incorporation into cell surface glycans. This is essential to peripheral lymphocyte survival, as stripping sialic acid with neuraminidase leads to the binding of natural IgM and complement fixation. NKAP-deficient T cells have a defect in sialylation on cell surface glycans, leading to IgM recruitment. We demonstrate that the defect in sialylation is due to aberrant α2,8-linked sialylation, and the expression of three genes (ST8sia1, ST8sia4, and ST8sia6) that mediate α2,8 sialylation are downregulated in NKAP-defcient RTEs. The maturation of peripheral NKAP-deficient T cells is partially rescued in a C3-deficient environment. Thus, sialylation during T cell maturation is critical to protect immature RTEs from complement in the periphery.

Bcl11b prevents the intrathymic development of innate CD8 T cells in a cell intrinsic manner.

If Bcl11b activity is compromised, CD4(+)CD8(+) double-positive (DP) thymocytes produce a greatly increased fraction of innate CD8(+) single-positive (SP) cells highly producing IFN-γ, which are also increased in mice deficient of genes such as Itk, Id3 and NF-κB1 that affect TCR signaling. Of interest, the increase in the former two is due to the bystander effect of IL-4 that is secreted by promyelocytic leukemia zinc finger-expressing NKT and γδT cells whereas the increase in the latter is cell intrinsic. Bcl11b zinc-finger proteins play key roles in T cell development and T cell-mediated immune response likely through TCR signaling. We examined thymocytes at and after the DP stage in Bcl11b (F/S826G) CD4cre, Bcl11b (F/+) CD4cre and Bcl11b (+/S826G) mice, carrying the allele that substituted serine for glycine at the position of 826. Here we show that Bcl11b impairment leads to an increase in the population of TCRαβ(high)CD44(high)CD122(high) innate CD8SP thymocytes, together with two different developmental abnormalities: impaired positive and negative selection accompanying a reduction in the number of CD8SP cells, and developmental arrest of NKT cells at multiple steps. The innate CD8SP thymocytes express Eomes and secrete IFN-γ after stimulation with PMA and ionomycin, and in this case their increase is not due to a bystander effect of IL-4 but cell intrinsic. Those results indicate that Bcl11b regulates development of different thymocyte subsets at multiple stages and prevents an excess of innate CD8SP thymocytes.

DMBA/TPA Treatment Is Necessary for BCC Formation from Patched Deficient Epidermal Cells in Ptchflox/floxCD4Cre+/− Mice

The development of basal cell carcinoma (BCC), the most frequently diagnosed tumor among persons with European ancestry, is closely linked to mutations in the Hedgehog (Hh) receptor and tumor suppressor Patched1 (Ptch). Using Ptch(flox/flox)CD4Cre(+/-) mice, in which Ptch was ablated in CD4Cre-expressing cells, we demonstrate that the targeted cells can give rise to BCC after treatment with DMBA (7,12-dimethylbenz(a)anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate), but not after wounding of the skin. In addition, in this model, BCC are not caused by malfunctioning of Ptch-deficient T cells, as BCC did not develop when bone marrow (BM) of Ptch(flox/flox)CD4Cre(+/-) mice was transplanted into Ptch wild-type mice. Instead, lineage-tracing experiments and flow cytometric analyses suggest that the tumors are initiated from rare Ptch-deficient stem cell-like cells of the epidermis that express CD4. As DMBA/TPA is a prerequisite for BCC development in this model, the initiated cells need a second stimulus for expansion and tumor formation. However, in contrast to papilloma, this stimulus seems to be unrelated to alterations in the Ras signaling cascade. Together, these data suggest that biallelic loss of Ptch in CD4(+) cells does not suffice for BCC formation and that BCC formation requires a second so far unknown event, at least in the Ptch(flox/flox)CD4Cre(+/-) BCC mouse model.

Abstract 3140: Context specific effects of the BRAFV600E mutation on hematopoiesis identifies novel models of BRAF mutant hematopoietic disorders

Abstract BRAFV600E mutations have recently been identified in nearly 100% of patients with the chronic lymphoproliferative disorder hairy cell leukemia (HCL), as well as a small percentage of patients with the plasma cell malignancy multiple myeloma. Despite extensive knowledge regarding the functional effects of BRAFV600E expression in epithelial tissues, very little is understood about the role of the BRAFV600E mutation in hematopoietic transformation. We therefore utilized a conditional BRafV600E murine model crossed with Mx1-cre, Vav-cre, Cd19-cre, and Cγ1-cre transgenic mice to delineate the effects of mutant BRaf expression in pre-natal and post-natal hematopoietic stem and progenitor cells (HSPCs), B-lineage cells, and germinal center B cells respectively. We also investigated the origin of the BRAFV600E mutation in HCL patient bone marrow samples using prospective isolation of sorted HSPC populations followed by quantitative sequencing for the BRAFV600E mutation. Surprisingly, we identified the presence of the BRAFV600E mutation in long-term hematopoietic stem cells of HCL patients, and we also observed marked alterations in HSPC frequencies. Consistent with the human genetic data, expression of BRafV600E in HSPCs of mice resulted in a lethal transplantable hematopoietic disorder characterized by splenomegaly, anemia, thrombocytopenia, increased circulating soluble CD25, and increased clonogenic capacity of B-lineage cells- all classic features of human HCL. In contrast, restricting expression of BRafV600E to B-lineage cells did not result in disease even up to 1.5 years of age. We next assessed the effects of the BRafV600E mutation on HSPC self-renewal and lineage specification. We plated whole BM cells from Mx1-cre BRafV600E mice in methylcellulose containing myeloid/erythroid cytokines or lymphopoietic cytokes (IL-7). BRafV600E cells demonstrated impaired colony formation in myeloid/erythroid conditions. However, BRafV600E HSPCs exhibited limitless replating capacity when plated in the presence of IL-7, indicating that the BRAF mutation induces aberrant B lineage cell self-renewal. A clear competitive advantage was also seen with competitive transplantation of BRafV600E BM cells, identifying an increase in HSPC self-renewal associated with the BRAF mutation. Data from the murine models studied here and characterization of the BM compartment in HCL patients suggest that the cytopenias seen in HCL patients are due in part to HSPC-intrinsic effects of the BRAFV600E mutation on erythropoiesis, megakarypoiesis, and myelopoiesis. Moreover, these data suggest that the use of therapies targeting MAP-kinase signaling in HCL may lead to durable remissions not only through effects on mature leukemic cells, but also through targeted inhibition of signaling and survival in mutant HSPCs. Citation Format: Eunhee Kim, Stephen S. Chung, Jae H. Park, Young Rock Chung, Piro Lito, Julie Feldstein, Wenhuo Hu, Wendy Beguilin, Sebastien Monette, Cihangir Duy, Raajit Rampal, Leon Telis, Minal Patel, Min Kyung Kim, Ari M. Melnick, Neal Rosen, Martin S. Tallman, Christopher Y. Park, Omar Abdel-Wahab. Context specific effects of the BRAFV600E mutation on hematopoiesis identifies novel models of BRAF mutant hematopoietic disorders. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3140. doi:10.1158/1538-7445.AM2014-3140

Regulatory T cells and T-cell-derived IL-10 interfere with effective anti-cytomegalovirus immune response.

Cytomegalovirus (CMV) establishes lifelong chronic infection in its host with mostly asymptomatic or only mild disease, but under immunosuppressive conditions the virus can reactivate and infection can cause life-threatening disease. CMV has evolved several mechanisms to escape from host's immunity, allowing persistence of the virus. Until now, it remains elusive whether regulatory T cells (Tregs) have an impact on insufficient host immune response against the virus in this context. In the present study, we provide evidence that CD4(+)Foxp3(+) naturally occurring Tregs (nTregs) as well as CD4(+)Foxp3(-)IL-10(+)-induced Tregs (iTregs) interfere with an effective anti-mouse CMV (mCMV) immune response. Depletion of Foxp3(+) Tregs by using DEREG mice resulted in enhanced T-cell activation as measured by the expression of CD62L, granzyme B and interferon (IFN)-γ and was associated with reduced viral titers in salivary glands, the organ where mCMV mainly persists. Moreover, we identified CD4(+)Foxp3(-) T cells to produce elevated levels of the immunosuppressive cytokine IL-10 at early time points during mCMV infection. Analysis of T-cell activation and viral replication in mCMV-infected IL-10(flox/flox) × CD4-cre mice and IL-10(flox/flox) × FIC mice revealed that T-cell-specific inactivation of IL-10, but not Foxp3(+) Treg-specific IL-10 ablation alone, resulted in elevated IFN-γ production by T cells associated with a significant decrease in viral loads in salivary glands. Thus, our data illustrate a crucial role for CD4(+)Foxp3(+) nTregs as well as IL-10-producing CD4(+)Foxp3(-) iTregs in the regulation of appropriate T-cell responses and viral clearance during mCMV infection.

Interaction of CD5 and CD72 is involved in regulatory T and B cell homeostasis

Regulatory IL-10-producing CD1dhighCD5+CD19+ B cells and CD4+CD25+Foxp3+ T cells have been found to modulate immune responses in autoimmunity, infection, and cancer, but the interaction between these two cell subsets remains unclear. Through cell culture and flow cytometry (FACS), we analyzed the interaction of regulatory T cells (Tregs) and regulatory B cells (Bregs). A neutralizing antibody was used to determine the role of CD5 and CD72 in maintaining regulatory T and B cell homeostasis. We found that CD19+CD5+CD1dhi Bregs induced expansion of CD4+Foxp3+ Tregs, and CD4+CD25+ Tregs also induced expansion of IL-10-expressing Bregs. Once CD72 or CD5 was blocked, both IL-10-expressing Bregs and CD4+Foxp3+Tregs were reduced in the different cultures. Finally, FACS analysis demonstrated that Foxp3+CD4+Treg cells were reduced in CD19Cre mice defective of CD5 on the surface of B cells. The study suggests that the interaction of CD5 and CD72 plays a critical role in maintaining regulatory T and B cell homeostasis.

FcγRIIb on myeloid cells rather than on B cells protects from collagen-induced arthritis.

Extensive analysis of a variety of arthritis models in germline KO mice has revealed that all four receptors for the Fc part of IgG (FcγR) play a role in the disease process. However, their precise cell type-specific contribution is still unclear. In this study, we analyzed the specific role of the inhibiting FcγRIIb on B lymphocytes (using CD19Cre mice) and in the myeloid cell compartment (using C/EBPαCre mice) in the development of arthritis induced by immunization with either bovine or chicken collagen type II. Despite their comparable anti-mouse collagen autoantibody titers, full FcγRIIb knockout (KO), but not B cell-specific FcγRIIb KO, mice showed a significantly increased incidence and severity of disease compared with wild-type control mice when immunized with bovine collagen. When immunized with chicken collagen, disease incidence was significantly increased in pan-myeloid and full FcγRIIb KO mice, but not in B cell-specific KO mice, whereas disease severity was only significantly increased in full FcγRIIb KO mice compared with incidence and severity in wild-type control mice. We conclude that, although anti-mouse collagen autoantibodies are a prerequisite for the development of collagen-induced arthritis, their presence is insufficient for disease development. FcγRIIb on myeloid effector cells, as a modulator of the threshold for downstream Ab effector pathways, plays a dominant role in the susceptibility to collagen-induced arthritis, whereas FcγRIIb on B cells, as a regulator of Ab production, has a minor effect on disease susceptibility.