Functional glycosylation of dystroglycan is crucial for thymocyte development in the mouse.

BACKGROUND: Alpha-dystroglycan (alpha-DG) is a cell surface receptor providing a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton. During its biosynthesis, alpha-DG undergoes specific and unusual O-glycosylation crucial for its function as a high-affinity cellular receptor for ECM proteins. METHODOLOGY/PRINCIPAL FINDINGS: We report that expression of functionally glycosylated alpha-DG during thymic development is tightly regulated in developing T cells and largely confined to CD4(-)CD8(-) double negative (DN) thymocytes. Ablation of DG in T cells had no effect on proliferation, migration or effector function but did reduce the size of the thymus due to a significant loss in absolute numbers of thymocytes. While numbers of DN thymocytes appeared normal, a marked reduction in CD4(+)CD8(+) double positive (DP) thymocytes occurred. In the periphery mature naïve T cells deficient in DG showed both normal proliferation in response to allogeneic cells and normal migration, effector and memory T cell function when tested in acute infection of mice with either lymphocytic choriomeningitis virus (LCMV) or influenza virus. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that DG function is modulated by glycosylation during T cell development in vivo and that DG is essential for normal development and differentiation of T cells.

Germ cell-specific targeting of DICER or DGCR8 reveals a novel role for endo-siRNAs in the progression of mammalian spermatogenesis and male fertility.

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.

Programming of marginal zone B-cell fate by basic Kruppel-like factor (BKLF/KLF3).

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.

Development and selection of Valpha l4i NKT cells.

Valpha14 invariant natural killer T (Valpha14i NKT) cells are a unique lineage of mouse T cells that share properties with both NK cells and memory T cells. Valpha14i NKT cells recognize CDld-associated glycolipids via a semi-invariant T cell receptor (TCR) composed of an invariant Valpha14-Jalpha 18 chain paired preferentially with a restricted set of TCRbeta chains. During development in the thymus, rare CD4+ CD8+ (DP) cortical thymocytes that successfully rearrange the semi-invariant TCR are directed to the Valpha14i NKT cell lineage via interactions with CD d-associated endogenous glycolipids expressed by other DP thymocytes. As they mature, Valphal4i NKT lineage cells upregulate activation markers such as CD44 and subsequently express NK-related molecules such as NKI.1 and members of the Ly-49 inhibitory receptor family. The developmental program of Valpha l4i NKT cells is critically regulated by a number of signaling cues that have little or no effect on conventional T cell development, such as the Fyn/SAP/SLAM pathway, NFkappaB and T-bet transcription factors, and the cytokine IL-15. The unique developmental requirements of Valphal4i NKT cells may represent a paradigm for other unconventional T cell subsets that are positively selected by agonist ligands expressed on hematopoietic cells.

Expression of genes encoding the pre-TCR and CD3 complex during thymus development.

The mature TCR is composed of a clonotypic heterodimer (alpha beta or gamma delta) associated with the invariant CD3 components (gamma, delta, epsilon and zeta). There is now considerable evidence that more immature forms of the TCR-CD3 complex (consisting of either CD3 alone or CD3 associated with a heterodimer of TCR beta and pre-T alpha) can be expressed at the cell surface on early thymocytes. These pre-TCR complexes are believed to be necessary for the ordered progression of early T cell development. We have analyzed in detail the expression of both the pre-TCR and CD3 complex at various stages of adult thymus development. Our data indicate that all CD3 components are already expressed at the mRNA level by the earliest identifiable (CD4lo) thymic precursor. In contrast, genes encoding the pre-TCR complex (pre-T alpha and fully rearranged TCR beta) are first expressed at the CD44loCD25+CD4-CD8- stage. Detectable surface expression of both CD3 and TCR beta are delayed relative to expression of the corresponding genes, suggesting the existence of other (as yet unidentified) components of the pre-TCR complex.

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells

Introduction: Cancer stem cells (CSC) display plasticity and self renewal properties reminiscent of normal tissue stem cells but the events responsible for their emergence remain obscure. We have recently identified CSC in Ewing sarcoma family tumors (ESFT) and shown that they arise from mesenchymal stem cells from the bone marrow. Objective of the study: To analyze the mechanisms underlying cancer stem cell development in ESFT. Methods: Primary human mesenchymal stem cells (MSC) isolation from adult and pediatric bone marrow. Retroviral delivery of fusion protein (EWS-FLI1) to primary MSC, and transcriptional and phenotypical analysis. Results: We show that the EWS-FLI-1 fusion gene, associated wit 85-90% of ESFT and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2 and NANOG in human pediatric MSC (hpMSC) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSC expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWSFLI- 1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Conclusion: Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a cancer stem cell phenotype.

Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1.

Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.

Formation of virus-like clusters is an intrinsic property of the tumor necrosis factor family member BAFF (B cell activating factor).

The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH >or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.

Cooperating pre-T-cell receptor and TCF-1-dependent signals ensure thymocyte survival.

Intrathymic T-cell maturation critically depends on the selective expansion of thymocytes expressing a functionally rearranged T-cell receptor (TCR) beta chain. In addition, TCR-independent signals also contribute to normal T-cell development. It is unclear whether and how signals from the 2 types of pathways are integrated. Here, we show that T-cell factor-1 (TCF-1), a nuclear effector of the canonical wingless/int (wnt)/catenin signaling pathway, ensures the survival of proliferating, pre-TCR(+) thymocytes. The survival of pre-TCR(+) thymocytes requires the presence of the N-terminal catenin-binding domain in TCF-1. This domain can bind the transcriptional coactivator beta-catenin and may also bind gamma-catenin (plakoglobin). However, in the absence of gamma-catenin, T-cell development is normal, supporting a role for beta-catenin. Signaling competent beta-catenin is present prior to and thus arises independently from pre-TCR signaling and does not substantially increase on pre-TCR signaling. In contrast, pre-TCR signaling significantly induces TCF-1 expression. This coincides with the activation of a wnt/catenin/TCF reporter transgene in vivo. Collectively, these data suggest that efficient TCF-dependent transcription requires that pre-TCR signaling induces TCF-1 expression, whereas wnt signals may provide the coactivator such as beta-catenin. The 2 pathways thus have to cooperate to ensure thymocyte survival at the pre-TCR stage.

T cell receptor V beta repertoire in mice lacking endogenous mouse mammary tumor provirus.

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.

Cytotoxic T-cell and NK-cell Lymphomas: Current Questions and Controversies.

The cytotoxic T-cell and natural killer (NK)-cell lymphomas and related disorders are important but relatively rare lymphoid neoplasms that frequently are a challenge for practicing pathologists. This selective review, based on a meeting of the International Lymphoma Study Group, briefly reviews T-cell and NK-cell development and addresses questions related to the importance of precise cell lineage (αβ-type T cell, γδ T cell, or NK cell), the implications of Epstein-Barr virus infection, the significance of anatomic location including nodal disease, and the question of further categorization of enteropathy-associated T-cell lymphomas. Finally, developments subsequent to the 2008 World Health Organization Classification, including the recognition of indolent NK-cell and T-cell disorders of the gastrointestinal tract are presented.

Functional education of invariant NKT cells by dendritic cell tuning of SHP-1.

Invariant NKT (iNKT) cells play key roles in host defense by recognizing lipid Ags presented by CD1d. iNKT cells are activated by bacterial-derived lipids and are also strongly autoreactive toward self-lipids. iNKT cell responsiveness must be regulated to maintain effective host defense while preventing uncontrolled stimulation and potential autoimmunity. CD1d-expressing thymocytes support iNKT cell development, but thymocyte-restricted expression of CD1d gives rise to Ag hyperresponsive iNKT cells. We hypothesized that iNKT cells require functional education by CD1d(+) cells other than thymocytes to set their correct responsiveness. In mice that expressed CD1d only on thymocytes, hyperresponsive iNKT cells in the periphery expressed significantly reduced levels of tyrosine phosphatase SHP-1, a negative regulator of TCR signaling. Accordingly, heterozygous SHP-1 mutant mice displaying reduced SHP-1 expression developed a comparable population of Ag hyperresponsive iNKT cells. Restoring nonthymocyte CD1d expression in transgenic mice normalized SHP-1 expression and iNKT cell reactivity. Radiation chimeras revealed that CD1d(+) dendritic cells supported iNKT cell upregulation of SHP-1 and decreased responsiveness after thymic emigration. Hence, dendritic cells functionally educate iNKT cells by tuning SHP-1 expression to limit reactivity.

Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus.

We have recently reported that Notch 1, a member of the Notch multigene family, is essential for the development of murine T cells. Using a mouse model in which Notch 1 is inactivated in bone marrow (BM) precursors we have shown that B cells instead of T cells are found in the thymus of BM chimeras. However, it is not clear whether these B cells develop by default from a common lymphoid precursor due to the absence of Notch 1 signaling, or whether they arise as a result of perturbed migration of BM-derived B cells and/or altered homeostasis of normal resident thymic B cells. In this report we show that Notch 1-deficient thymic B cells resemble BM B cells in phenotype and turnover kinetics and are located predominantly in the medulla and corticomedullary junction. Peripheral blood lymphocyte analysis shows no evidence of recirculating Notch1(-/)- BM B cells. Furthermore, lack of T cell development is not due to a failure of Notch1(-/)- precursors to home to the thymus, as even after intrathymic reconstitution with BM cells, B cells instead of T cells develop from Notch 1-deficient precursors. Taken together, these results provide evidence for de novo ectopic B cell development in the thymus, and support the hypothesis that in the absence of Notch 1 common lymphoid precursors adopt the default cell fate and develop into B cells instead.

Environmental and T cell-intrinsic factors limit the expansion of neonatal follicular T helper cells but may be circumvented by specific adjuvants.

Follicular Th (T(FH)) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5(high)PD-1(high) CD4(+) T(FH) cells that exhibit T(FH) features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal T(FH) cells fail to expand and to acquire a full-blown GC T(FH) phenotype, as reflected by a higher ratio of GC T(FH)/non-GC CD4(+) T cells in immunized adults than neonates (3.8 × 10(-3) versus 2.2 × 10(-3), p = 0.01). Following the adoptive transfer of naive adult OT-II CD4(+) T cells, OT-II T(FH) cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4(+) OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell-intrinsic factors. Postponing immunization to later in life increases the number of T(FH) cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC T(FH) and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the T(FH) cell development limits early life GC responses and that adjuvants/delivery systems supporting T(FH) differentiation may restore adultlike early life GC B cell responses.

Regulation of B Cell Gene Expression and Function by Ikaros, Helios and Bcl6

B lymphocytes constitute a key branch of adaptive immunity by providing specificity to recognize a vast variety of antigens by B cell antigen receptors (BCR) and secreted antibodies. Antigen recognition activates the cells and can produce antibody secreting plasma cells via germinal center reaction that leads to the maturation of antigen recognition affinity and switching of antibody effector class. The specificity of antigen recognition is achieved through a multistep developmental pathway that is organized by interplay of transcription factors and signals through BCR. Lymphoid malignancies arise from different stages of development in abnormal function of transcriptional regulation. To understand the B cell development and the function of B cells, a thorough understanding of the regulation of gene expression is important. The transcription factors of the Ikaros family and Bcl6 are frequently associated with lymphoma generation. The aim of this study was to reveal the targets of Ikaros, Helios and Bcl6 mediated gene regulation and to find out the function of Ikaros and Helios in B cells. This study uses gene targeted DT40 B cell lines and establishes a role for Ikaros family factors Ikaros and Helios in the regulation of BCR signaling that is important at developmental checkpoints, for cell survival and in activation. Ikaros and Helios had opposing roles in the regulation of BCR signals. Ikaros was found to directly repress the SHIP gene that encodes a signaling lipid-metabolizing enzyme, whereas Helios had activating effect on SHIP expression. The findings demonstrate a balancing function for these two Ikaros family transcription factors in the regulation of BCR signaling as well as in the regulation of gene expression. Bcl6 was found to repress plasma cell gene expression program while maintaining gene expression profile of B cells. Analysis of direct Bcl6 target genes suggested novel mechanisms for Bcl6-mediated suppression of plasma cell differentiation and promoting germinal center phenotype.