CD19-Cre Mice Research Articles

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.

Targeting sirtuin-1 alleviates experimental autoimmune colitis by induction of Foxp3+ T-regulatory cells (MUC2P.818)

Abstract Induced Foxp3+ T-regulatory cells (iTreg) are essential to gastrointestinal immune homeostasis and play an important role in preventing autoimmune colitis. We have previously shown that inhibiting sirtuin-1 (Sirt1) can augment Treg function and hypothesized that targeting of Sirt1 might enhance iTreg development and thereby represent a potential therapy for inflammatory bowel disease (IBD). We adoptively transferred CD4+CD25-Foxp3- T effector (TE) cells from wild-type (C57BL/6) or fl-Sirt1/CD4cre mice into B6/Rag1-/- mice and monitored the mice until they lost 10-15% of their weight. Adoptive transfer of TE cells lacking Sirt1 to B6/Rag1-/- mice resulted in increased iTreg formation compared to mice receiving wild-type TE cells (2.3 ±1.6%, vs. 6.2 ±3%, in wild type and fl-Sirt1/CD4cre TE recipients, respectively, p<0.001). This correlated with attenuated colitis and reduced weight loss in fl-Sirt1/CD4cre vs. wild type TE recipients (1.04 ±1.4% vs. 13.97 ±2.2%, respectively, p<0.001). In a second model of IBD, we used pharmacologic Sirt1 targeting of mice receiving multiple cycles of dextran sodium sulfate (DSS) in their drinking water, alternated with fresh water. Likewise, wild-type mice receiving cyclic DSS and a Sirt1 inhibitor, EX-527, had reduced weight loss (5.8 ±5.9% vs. 13.2 ±6.9%, p=0.03) and increased iTreg formation compared to controls. Sirt1 appears a promising target for pharmacologic therapy of IBD as a result of promoting iTreg development.

Targeting sirtuin-1 improves murine renal allograft survival and function (TRAN3P.901)

Abstract Pharmacologic inhibition of sirtuin-1 (Sirt1) augments suppressive functions of Foxp3+ regulatory T cells (Treg) and prolongs murine cardiac allograft survival. We investigated if Sirt1 targeting produced longer allograft function and allograft-dependent survival in a MHC-mismatched renal transplant model. After transplantation of BALB/c kindey allografts into C57Bl/6 wild type or fl-Sirt1/CD4cre recipients, native kidneys were removed three days post-operatively. We followed the mice for survival, and allograft function by weekly serum electrolyte and renal function (BUN, creatinine), as well as hematocrit. We observed, that loss of Sirt1 in T cells led to prolonged median renal allograft-dependent survival from 14 days (IQR: 11-91.25) to 143 days (IQR: 27-147) in wild type controls vs. fl-Sirt1/CD4cre recipients (Mantel-Cox, p<0.0004). In addition, by comparing mice that survived until 13 weeks, loss of Sirt1 in the recipient’s T cells led to improved renal allograft function, with 34.2 ±20 vs. 90.8 ±37.6 mg/dL BUN (p<0.001), and lower creatinine 0.33 ±0.14 vs. 0.61 ±0.3 mg/dL (p<0.001) in fl-Sirt1/CD4cre vs. wild type control mice, respectively. Long-term surviving fl-Sirt1/CD4cre renal transplant recipients (>100 days) became tolerant to antigen re-challenge with a BALB/c, but rejected third-party C3H cardiac allografts. In conclusion, deletion of Sirt1 in T cells is effective at prolonging allograft function and allograft-dependent survival.

The transcriptional repressor NKAP is required for iNKT cell development and maturation (HEM3P.274)

Abstract Invariant Natural Killer T (iNKT) cells are an innate lineage of lymphocytes that can produce copious amount of cytokines within hours of stimulation. In the thymus, positive selection into the iNKT cell lineage occurs at the double positive (DP) stage, before maturation through various stages (stage 0-3) to complete development. NKAP, a transcriptional repressor, is required for conventional T lymphocyte development and maturation. NKAP interacts with Hdac3 for its repressor function. Previously, using CD4-cre NKAP cKO mice, we have shown that NKAP is required for positive selection into the iNKT cell lineage. To study the role of NKAP in later developmental stages, we generated PLZF-cre NKAP cKO mice with NKAP deletion occurring after entry into the iNKT lineage at stage 0. In the PLZF-cre NKAP cKO mice we observed a significant decrease in the absolute number of iNKTs with a 20-fold reduction at stage 2 and 200-fold reduction at stage 3. This defect is cell intrinsic and cannot be rescued by over-expression of Bcl-XL. Our preliminary results indicate that there may be a block in iNKT cell maturation, as expression of transcription factors Tbet, Gata3, and ROR-γt that define NKT1, NKT2 and NKT17 lineages respectively, are decreased in PLZF-cre NKAP cKO. PLZF-cre Hdac3 cKO mice have a similar defect in iNKT development. Thus, NKAP together with Hdac3 may regulate iNKT cell development and maturation.

The epigenetic regulator Uhrf1 facilitates the proliferation and maturation of colonic regulatory T cells.

Intestinal regulatory T cells (Treg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal Treg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in Treg cells. Mice with T cell-specific deficiency in Uhrf1 (Uhrf1(fl/fl)Cd4-Cre mice) showed defective proliferation and functional maturation of colonic Treg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of Treg cells. As a consequence, Uhrf1(fl/fl)Cd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.

Conditional Deletion of Ferritin H in Mice Reduces B and T Lymphocyte Populations

The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fthlox/lox; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fthlox/lox;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation.

Targeting sirtuin-1 alleviates experimental autoimmune colitis by induction of Foxp3+ T-regulatory cells.

Induced Foxp3+ T-regulatory cells (iTreg) are essential to gastrointestinal immune homeostasis and loss of the ability to develop iTregs may lead to autoimmune colitis. We previously showed a role for Sirtuin-1 (Sirt1) in control of Treg function and hypothesized that targeting of Sirt1 might enhance iTreg development and thereby represent a potential therapy for inflammatory bowel disease (IBD). We adoptively transferred CD4+CD25−Foxp3− T effector (TE) cells from wild-type (C57BL/6) or fl-Sirt1/CD4cre mice into B6/Rag1−/− mice and monitored the mice until they lost 10-15% of their weight. Adoptive transfer of TE cells lacking Sirt1 to B6/Rag1−/− mice resulted in a 2.8-fold increase in iTreg formation compared to mice receiving wild-type TE cells and correlated with attenuated colitis and reduced weight loss (1.04±1.4% vs. 13.97±2.2%, respectively, p<0.001). In a second model of IBD, we used pharmacologic Sirt1 targeting of mice receiving multiple cycles of dextran sodium sulfate (DSS) in their drinking water, alternated with fresh water. Likewise, wild-type mice receiving cyclic DSS and a Sirt1 inhibitor, EX-527, had reduced weight loss (5.8±5.9% vs. 13.2±6.9%, p=0.03) and increased iTreg formation compared to controls. Sirt1 appears a promising target for pharmacologic therapy of IBD as a result of promoting iTreg development.

NFAT2 Is a Critical Regulator Of Anergy Induction In CLL

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. Expression of CD38 and ZAP70 define a subgroup of patients with enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggessive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype refering to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells.For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eµ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eµ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 wks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). To identify novel NFAT2 target genes in CLL cells, we performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays.Mice with NFAT2 knock out exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells and a significantly reduced life expectancy (200 vs. 325 days) as compared to control animals. Flow cytometric analysis at distinct time points showed a pronounced infiltration by CD5+ B cells of the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko exhibited significantly higher rates of proliferation than control animals. To identify NFAT2 target genes resonsible for the observed alterations in the disease phenotype, we subsequently peformed a gene expression analysis with CD5+CD19+ CLL cells from TCL1+NFAT2 ko mice with CLL cells from TCL1+ mice serving as controls. Here, we detected a significantly altered expression of 22 genes associated with B cell anergy in the TCL1+NFAT2 ko cohort. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1, Hspa14 and CD166 constituting the strongest hits with up to 10fold reduced gene expression. Downregulation of the identified target genes was subsequently confirmed using RT-PCR and Western Blotting.In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells. In addition, we could show that NFAT2 controls the expression of several anergy-associated genes and that its absence prevents the acquisition of an anergic phenotype by the CLL cells correlating with a significantly more aggressive course of the disease. Taken together, our data demonstrate that NFAT2 plays an essential role in the pathogenesis of CLL and implicate this transcription factor as a potential target in its treatment. Disclosures:No relevant conflicts of interest to declare.

Shp1 regulates T cell homeostasis by limiting IL-4 signals

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell–intrinsic role of Shp1, we characterized mice with a T cell–specific Shp1 deletion (Shp1fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1(fl/fl) CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4⁺ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1- deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.

Cell of origin in radiation‐induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele

Bcl11b is a haploinsufficient tumor suppressor, mutations or deletion of which has been found in 10-16% of T-cell acute lymphoblastic leukemias. Bcl11b(KO) (/+) heterozygous mice are susceptible to thymic lymphomas, a model of T-cell acute lymphoblastic leukemia, when γ-irradiated, and irradiated Bcl11b(KO) (/+) mice generate clonally expanding or premalignant thymocytes before thymic lymphoma development. Cells with radiation-induced DNA damages are assumed to be the cells of origin in tumors; however, which thymocyte is the tumor cell origin remains obscure. In this study we generated Bcl11b(flox/+) ;Lck-Cre and Bcl11b(flox/+) ;CD4-Cre mice; in the former, loss of one Bcl11b allele occurs in thymocytes at the immature CD4(-) CD8(-) stage, whereas in the latter the loss occurs in the more differentiated CD4(+) CD8(+) double-positive stage. We examined clonal expansion and differentiation of thymocytes in mice 60days after 3Gy γ-irradiation. Half (9/18) of the thymuses in the Bcl11b(flox/+) ;Lck-Cre group showed limited rearrangement sites at the T-cell receptor-β (TCRβ) locus, indicating clonal cell expansion, but none in the Bcl11b(flox/+) ;CD4-Cre group did. This indicates that the origin of the premalignant thymocytes is not in double-positive cells but immature thymocytes. Interestingly, those premalignant thymocytes underwent rearrangement at various different sites of the TCRα locus and the majority showed a higher expression of TCRβ and CD8, and more differentiated phenotypes. This suggests the existence of a subpopulation of immature cells within the premalignant cells that is capable of proliferating and continuously producing differentiated thymocytes.

NKAP is required for iNKT cell development (P4450)

Abstract Invariant Natural Killer T cells (iNKT) are innate lineage of lymphocytes with distinct developmental pathway than conventional αβ T cells. The transcriptional repressor NKAP is required for early T cell development and maturation of conventional αβ T cells. Using CD4-cre NKAP cKO mice, we show that NKAP is required for positive selection of thymocytes into the iNKT cells lineage. Deletion of NKAP using CD4-cre leads to an early block in iNKT cell development at the DP stage. This cell intrinsic defect was not due to decreased DP thymocyte survival or inability of NKAP deficient thymocytes to rearrange their Vα14-Jα18 TCR. Moreover, ectopic over-expression of Vα14-Jα18 invariant TCR α chain rescued the development of iNKT cells. This suggests that NKAP plays a role in the positive selection of thymocytes into the iNKT cell lineage. Interestingly, conditional deletion of NKAP associated protein Hdac3 using CD4-cre also leads to a similar block in iNKT cell development, but not conventional T cell development. This is first evidence demonstrating for an Hdac in T cell development. Whether NKAP and Hdac3 work together functionally to regulate the positive selection of DP thymocytes into iNKT cell lineage is currently being investigated.

Constitutive AKT activation impairs B cell homeostasis and class switch recombination (P1443)

Abstract The serine/threonine kinase AKT controls not only cellular metabolism, cell survival and proliferation but is also an important regulator for peripheral B cell maturation especially representing a critical check point in phases of proliferation and differentiation. In this study we investigated the role of constitutive AKT activation in B cell development and maturation through the use of a mouse strain allowing for the conditional expression of a N-terminally myristoylated AKT (ROSA-AKT-C) in cell types that express the Cre recombinase. The myristoylation signal recruits AKT-C to the plasma membrane where AKT becomes constantly phospho-activated. This mouse was crossed to CD19 Cre mice leading to the B cell specific overexpression of AKT-C (AKTBOE). AKTBOE mice displayed splenomegaly compared to control mice as a result of increased absolute numbers of not only B cells but also T cells, neutrophiles, monocytes and mature macrophages. Interestingly, in these spleens, a B cell specific loss of CD23 expression was observed. Of note, B cells isolated from AKTBOE mice did not form germinal centers and were not able to class switch in vitro and in vivo. The investigation of immunoglobulin titers by ELISA revealed reduced IgM, and almost no IgG1, IgG2a, IgG3 and IgA serum levels. Thus, the B cell specific overexpression of AKT-C underlines its importance in B cell maturation, homeostasis and class switch recombination.

CRK/CRKL proteins regulate T cell migration and activation (P5104)

Abstract Crk adaptor proteins include three family members: CrkI and CrkII are from the alternatively spliced crk gene, and CrkL is from the crk-like gene. These ubiquitously expressed proteins play important roles in multiple signaling pathways. However, their functions in T cells are poorly characterized, partly because germline-deficiency for Crk or CrkL leads to lethality in mice. By breeding Crkflox/flox:CrkLflox/flox mice to CD4Cre mice, we generated mice conditionally deficient for both Crk and CrkL in mature T cells. These mice are viable and fertile. We find that T cells lacking both Crk and CrkL develop normally, but are defective in chemotactic responses to multiple chemokines. Further analysis revealed that while Rac1 activation is intact, Rap1 activation is perturbed, as is tyrosine phosphorylation of the Rap1 GEF C3G. Consistent with this, these cells exhibit reduced chemokine-stimulated adhesion to ICAM-1 and fibronectin, and defective diapedesis. Using a cutaneous DTH model, we show that Crk and CrkL are required for efficient T cell migration to sites of inflammation. In addition to regulating T cell migration, Crk proteins also control T cell activation. Double deficient T cells showed significant proliferative defects upon allogeneic antigen stimulation. These results demonstrate a key role for Crk proteins in T cell function. We are currently working on defining Crk/CrkL-dependent pathways and analyzing functions of Crk proteins in singly deficient T cells.

Abstract 3598: Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele.

Abstract Mouse thymic lymphoma (TL) has been used for study of radiation carcinogenesis because of its high incidence. The lymphoma or leukemia is a mouse model of human T-cell acute lymphoblastic leukemias (T-ALL), which is a malignant clonal expansion of thymocytes that accounts for about 15% of ALL cases. Ionizing radiation can damage DNA, and the cells with damaged DNA are assumed to be the cells of origin in tumors. However, which cell in the thymus or the bone marrow leads to TL remains obscure. Bcl11b gene encodes zinc-finger transcription proteins, mutations or deletion of which has been found in 10-16% of T-ALL. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas when γ-irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes within atrophic thymus. Resembling clonal expansion of thymocytes was reported in the T-ALL model overexpressing LMO2 oncogene. Interestingly, LMO2-induced clonally expanding thymocytes persist long-term and comprise cells with the stem cell-like self-renewal property that function for the bone marrow stem cells before TL development. In this study, we developed Bcl11bflox/+;Lck-Cre and Bcl11bflox/+;CD4-Cre mice, in which loss of one Bcl11b allele occurs in thymocytes at the immature CD4−CD8− stage without expression of CD4 and CD8 cell surface markers and at the CD4+CD8+ double-positive (DP) stage, respectively. Those mice were subjected to γ-irradiation of 3Gy at 8 weeks of age and their thymocytes were characterized of clonal expansion, differentiation and cell number. The clonal expansion was observed in only the former mice, suggesting the origin of the premaligant thymocytes not in the DP cells, the cell type seen in a majority of TL. The clonally expanded thymocytes possessed common rearrangement sites at the TCRβ locus but they underwent rearrangement at various different sites at the TCRα locus, and their majority showed mature phenotypes, a higher expression of TCRβ and the CD8 expression. This suggests the presence of a subpopulation of immature thymocytes that is capable to rearrange DNA at the TCRα locus in DP cells to continuously produce further differentiated thymocytes. We also found the decrease in thymocyte number in irradiated Bcl11bflox/+;Lck-Cre mice. This probably reflects the reduced proliferation of bone marrow-derived progenitors, and this reduction may weaken the competition in niche with premaligant thymocytes and help for them to survive and proliferate. Citation Format: Ryo Kominami, Rieka Go, Satoshi Hirose, Yukio Mishima. Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3598. doi:10.1158/1538-7445.AM2013-3598

The transcriptional repressor NKAP is required for the development of iNKT cells

Invariant natural killer T cells have a distinct developmental pathway from conventional αβ T cells. Here we demonstrate that the transcriptional repressor NKAP is required for invariant natural killer T cell but not conventional T cell development. In CD4-cre NKAP conditional knockout mice, invariant natural killer T cell development is blocked at the double-positive stage. This cell-intrinsic block is not due to decreased survival or failure to rearrange the invariant Vα14-Jα18 T cell receptor-α chain, but is rescued by overexpression of a rec-Vα14-Jα18 transgene at the double-positive stage, thus defining a role for NKAP in selection into the invariant natural killer T cell lineage. Importantly, deletion of the NKAP-associated protein histone deacetylase 3 causes a similar block in the invariant natural killer T cell development, indicating that NKAP and histone deacetylase 3 functionally interact to control invariant natural killer T cell development.

Germline TSC1 mutations are permissive for T lymphocyte development and homeostasis in Tuberous Sclerosis individuals.

Event Abstract Back to Event Germline TSC1 mutations are permissive for T lymphocyte development and homeostasis in Tuberous Sclerosis individuals. Karolina Pilipow1, 2*, Veronica Basso2, Nicola Migone3 and Anna Mondino2 1 Vita-Salute San Raffaele University, Italy 2 San Raffaele Scientific Institute, Italy 3 University of Turin, Department of Genetics, Biology and Biochemistry, Italy Antigen recognition on the surface of antigen presenting cells initiate a variety of intracellular signals critical for naïve T cells to commit to proliferate and differentiate. Others and we have shown the TSC/mTOR axis to be critical in coordinating cell quiescence, proliferation and fate determination. Blocking mTOR by Rapamycin, or genetic inactivation of components of the mTOR Complex (mTORC) or TSC1, unbalances mTORC1 and mTORC2 regulation, impacting on cell quiescence, proliferation and differentiation. In humans, germline mutations of TSC1 predispose to Tuberous Sclerosis Complex disease, an autosomal dominant disorder characterized by multifocal hamartomatosis. Whether such mutations impact on T cell functions in these individuals was unknown. To this aim we analyzed subset representation, phenotype and function of T cells from two independent families with characterized mutations of TSC1 in parallel to T cells from healthy volunteers and to mouse T cells with mono- or bilallelic deletion of Tsc1 (from T-lineage restricted Tsc1+/- and Tsc1-/- mice). We found that heterozygosis of Tsc1/TSC1 is sufficient for proper hamartin expression and both homeostatic and TCR-induced mTORC1 and mTORC2-dependent signaling in mice and human T cells. In contrast, loss of Tsc1/TSC1 following biallelic genetic inactivation and shRNA-induced down-regulation unbalanced mTORC-dependent FOXO1/3 regulation, leading to loss of mitochondrial potential apoptotic cell death. Thus, while heterozygosis of Tsc1/TSC1 is sufficient for proper T lymphocyte development and homeostasis, its more severe loss is incompatible with both mouse and human T cell survival. Acknowledgements The authors wish to thank TSC subjects and their family for support, members of the San Raffaele Scientific Institute for suggestions, Dr Rossella Galli for Tsc1 fl/fl mice, Dr Ruggero Pardi for CD4cre mice and Dr Vincenzo Calautti (Dulbecco Telethon Institute, Molecular Biotechnology Center, University of Turin) for the help with TSC1 shRNA. This work was supported by the Associazione Italiana Ricerca sul Cancro. KP benefited of fellowships from the San Raffaele Vita-Salute University and FIRC. Keywords: T lymphocytes, Intracellular signaling, TSC/mTOR, intrinsic apoptosis, Tuberous Sclerosis Complex Disease Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Pilipow K, Basso V, Migone N and Mondino A (2013). Germline TSC1 mutations are permissive for T lymphocyte development and homeostasis in Tuberous Sclerosis individuals.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00976 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Karolina Pilipow, Vita-Salute San Raffaele University, Milan, Italy, kpilipow@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Karolina Pilipow Veronica Basso Nicola Migone Anna Mondino Google Karolina Pilipow Veronica Basso Nicola Migone Anna Mondino Google Scholar Karolina Pilipow Veronica Basso Nicola Migone Anna Mondino PubMed Karolina Pilipow Veronica Basso Nicola Migone Anna Mondino Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

FLVCR, a Heme Exporter, Is Required for Peripheral T Cell Survival.

AbstractAbstract 2162Heme is essential for every mammalian cell, however, free heme can induce free radical formation and cellular damage, therefore cells must carefully regulate heme levels. The feline leukemia virus subgroup C receptor (FLVCR) exports heme from cells. Conditional deletion of Flvcr was shown to cause progressive anemia in neonatal and adult mice (Science 319:825–8, 2008). Using a transplant model, we previously demonstrated that Flvcr-deleted thymocytes were blocked at the CD4+CD8+ double-positive (DP) stage (Blood [ASH Annual Meeting Abstracts] 114: 913, 2009). To characterize the temporal requirement for FLVCR in developing thymocytes, we crossed Flvcrflox/flox mice to thymocyte-specific cre recombinase strains: Lck-cre mice, which express cre in early CD4+CD8+ double-negative (DN) thymocytes, and CD4-cre mice, which turn on cre in late DN/early DP thymocytes. Flvcrflox/flox;Lck-cre mice had similar numbers of DN and DP thymocytes compared to controls, however, CD4+ and CD8+ single-positive (SP) thymocytes and peripheral T cells were nearly absent, similar to what we observed in our previous transplant model. In contrast, Flvcrflox/flox;CD4-cre mice had intact thymic development with normal numbers of SP, but there were few CD4+ and CD8+ T cells in the periphery. When we analyzed deletion efficiency of these T cells, CD8+ T cells showed only 50% Flvcr deletion and were nearly all CD44-high, implying that only incompletely-deleted CD8+ T cells survived and expanded. Taken together, these results show that FLVCR is required not only for T cell development beyond the DP stage, but also for the survival of mature T cells in the periphery.We next adoptively transferred thymocytes from Flvcrflox/flox;CD4-cre mice or controls into sub-lethally irradiated Rag1−/− mice. Normal SP thymocytes undergo homeostatic proliferation when transferred into an “empty” host. At day 12 and 20 post-adoptive transfer, few Flvcrflox/flox;CD4-cre CD4+ or CD8+ T cells were found, in contrast to mice that had received Flvcr+/flox;CD4-cre thymocytes. To determine whether Flvcr-deleted T cells failed to undergo homeostatic proliferation, we used carboxyfluorescein succinimidyl ester (CFSE) to label Flvcrflox/flox;CD4-cre or control thymocytes prior to adoptive transfer. At day 8, similar numbers of Flvcrflox/flox;CD4-cre and control T cell were found in the periphery and both had diluted CFSE equally, thus initial proliferation was not affected. However, by day 20, few Flvcr-deleted T cells were present compared to controls. Experiments are currently underway to understand how and why Flvcr-deleted T cells fail to persist long-term.The finding that FLVCR is required for T cell development and peripheral survival is intriguing because there is no known specific role for heme in T cell development or function. We carried out transcriptional profiling on sorted DP thymocytes from Rag1−/− mice transplanted with Flvcr-deleted or control bone marrow to determine whether FLVCR loss led to gene expression changes that might explain the block in T cell development. Surprisingly, there were few transcriptional changes, suggesting that FLVCR loss has an abrupt impact on T cell development late in the DP stage. This finding, together with the apparent normal development of Flvcr-deleted B lymphocytes and myeloid lineages, leads us to hypothesize that FLVCR plays a specific role in T cell development starting at the DP stage and persisting throughout T cell life. FLVCR is a member of the major facilitator superfamily of secondary active transporters. While FLVCR has been shown to export heme, it is not known whether it can import or export other small molecules or metabolites. We are now using a bioinformatics approach on published datasets to analyze metabolic gene expression during normal thymic development and in various mature T cell subsets to identify metabolic pathways that are specific for the DP-SP transition in thymocytes as well as in mature, peripheral T cells. We will then test whether these pathways are altered in Flvcr-deleted thymocytes and mature T cells. These studies may uncover a new role for heme in T cell metabolism, function, and survival, or a new non-heme role for FLVCR. Disclosures:No relevant conflicts of interest to declare.

Genetic Loss of NFAT2 Induces Profound Acceleration of CLL in the TCL1 Mouse Model

AbstractAbstract 862NFAT 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. NFAT is inactivated and relocated to the cytoplasm by a network of several kinases. Although identified originally as a major transcriptional regulator in T cells, it is now clear that NFAT transcription factors also possess important roles in other cells of the hematopoietic system including dendritic cells, mast cells, megakaryocytes and B cells. Several recent studies have demonstrated that Calcineurin/NFAT signaling is involved in the pathogenesis of a wide array of hematological malignancies including diffuse large B cell lymphoma, CLL as well as Burkitt and Burkitt-like lymphomas.Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific CD19 promoter. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eμ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eμ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 weeks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10).Mice with NFAT2 knock out exhibited a significantly accelerated accumulation of CD5+CD19+ CLL cells as compared to control animals. Flow cytometric analysis at distinct time points showed a tremendous infiltration by CD5+ B cells in the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. At approximately 26 weeks of age, NFAT2 ko mice showed an approximately 40 fold increased lymphocyte count in the peripheral blood than their litter mate controls (1500/μL vs. 60000/μL). Splenomegaly and lymphatic adenopathy was also significantly increased in the NFAT ko population. Furthermore, NFAT2 ko mice showed a dramatically reduced median survival (200 vs. 325 days) and maximum survival (265 vs. 398 days) in comparison to regular TCL1 transgenic mice. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko at an age of approximately 7 months exhibited significantly higher rates of proliferation than control animals.In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells implicating Ca2+/NFAT signaling as a potential target for the treatment of this disease. Disclosures:No relevant conflicts of interest to declare.