The zinc-finger protein MAZR is part of the transcription factor network that controls the CD4 versus CD8 lineage fate of double-positive thymocytes.

The CD4 versus CD8 lineage specification of thymocytes is linked to coreceptor expression. The transcription factor MAZR has been identified as an important regulator of Cd8 expression. Here we show that variegated CD8 expression by loss of Cd8 enhancers was reverted in MAZR-deficient mice, which confirms that MAZR negatively regulates the Cd8 loci during the transition to the double-positive (DP) stage. Moreover, loss of MAZR led to partial redirection of major histocompatibility complex (MHC) class I-restricted thymocytes into CD4(+) helper-like T cells, which correlated with derepression of Th-POK, a central transcription factor for helper-lineage development. MAZR bound the silencer of the gene encoding Th-POK, which indicated direct regulation of this locus by MAZR. Thus, MAZR is part of the transcription factor network that regulates the CD8 lineage differentiation of DP thymocytes.

The guanine nucleotide exchange factor Rin-like controls Tfh cell differentiation via CD28 signaling.

T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody-producing B cells in humans and mice. Here, we identify the guanine nucleotide exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4+ T cell in vitro cultures. Mechanistically, adoptive transfer experiments using WT and Rinl-KO naïve CD4+ T cells unraveled T cell-intrinsic GEF-dependent functions of Rinl. Further, Rinl regulates CD28 internalization and signaling, thereby shaping CD4+ T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore, connect Rinl with CD28 internalization and signaling pathways in CD4+ T cells, demonstrating for the first time the importance of endocytic processes for Tfh differentiation.

The protein tyrosine kinase Tec regulates a CD44highCD62L- Th17 subset.

The generation of Th17 cells has to be tightly controlled during an immune response. In this study, we report an increase in a CD44(high)CD62L(-) Th17 subset in mice deficient for the protein tyrosine kinase Tec. CD44(high)CD62L(-) Tec(-/-) CD4(+) T cells produced enhanced IL-17 upon activation, showed increased expression levels of IL-23R and RORγt, and IL-23-mediated expansion of Tec(-/-) CD4(+) T cells led to an increased production of IL-17. Tec(-/-) mice immunized with heat-killed Streptococcus pneumoniae displayed increased IL-17 expression levels in the lung postinfection with S. pneumoniae, and this correlated with enhanced pneumococcal clearance and reduced lung inflammation compared with Tec(+/+) mice. Moreover, naive Tec(-/-) OT-II CD4(+) T cells produced higher levels of IL-17 when cultured with OVA peptide-loaded bone marrow-derived dendritic cells that have been previously activated with heat-killed S. pneumoniae. Taken together, our data indicated a critical role for Tec in T cell-intrinsic signaling pathways that regulate the in vivo generation of CD44(high)CD62L(-) effector/memory Th17 populations.

The transcriptional regulator PLZF induces the development of CD44 high memory phenotype T cells.

Transcriptional pathways controlling the development of CD44(hi) memory phenotype (MP) T cells with "innate-like" functions are not well understood. Here we show that the BTB (bric-a-brac, tramtrack, broad complex) domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44(hi), but not in CD44(lo), CD4(+) T cells. Transgenic expression of PLZF during T cell development and in CD4(+) and CD8(+) T cells induced a T cell intrinsic program leading to an increase in peripheral CD44(hi) MP CD4(+) and CD8(+) T cells and a corresponding decrease of naïve CD44(lo) T cells. The MP CD4(+) and CD8(+) T cells produced IFNgamma upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in single-positive thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted natural killer T cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4(+) transgenic T cells showed reduced IL-2 and IFNgamma production but increased IL-4 secretion as a result of enhanced IL-4 production of the CD44(hi)CD62L(+) subset. Our data indicate that PLZF is a novel regulator of the development of CD44(hi) MP T cells with a characteristic partial innate-like phenotype.

Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

The protein tyrosine kinase Tec regulates mast cell function.

Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon Fc epsilon RI stimulation of BM-derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C(4) levels were reduced in Tec(-) (/) (-) BMMC. Furthermore, the production of IL-4 was severely impaired, and GM-CSF, TNF-alpha and IL-13 levels were also diminished. Finally, a comparison of WT, Tec(-) (/) (-), Btk(-) (/) (-) and Tec(-) (/) (-)Btk(-) (/) (-) BMMC revealed a negative role for Btk in the regulation of IL-4 production, while for the efficient production of TNF-alpha, IL-13 and GM-CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well-characterized family member Btk.

The Transcription Factor MAZR/PATZ1 Regulates the Development of FOXP3+ Regulatory T Cells.

Forkhead box protein P3+ (FOXP3+) regulatory T cells (Treg cells) play a key role in maintaining tolerance and immune homeostasis. Here, we report that a T cell-specific deletion of the transcription factor MAZR (also known as PATZ1) leads to an increased frequency of Treg cells, while enforced MAZR expression impairs Treg cell differentiation. Further, MAZR expression levels are progressively downregulated during thymic Treg cell development and during in-vitro-induced human Treg cell differentiation, suggesting that MAZR protein levels are critical for controlling Treg cell development. However, MAZR-deficient Treg cells show only minor transcriptional changes ex vivo, indicating that MAZR is not essential for establishing the transcriptional program of peripheral Treg cells. Finally, the loss of MAZR reduces the clinical score in dextran-sodium sulfate (DSS)-induced colitis, suggesting that MAZR activity in T cells controls the extent of intestinal inflammation. Together, these data indicate that MAZR is part of a Treg cell-intrinsic transcriptional network that modulates Treg cell development.

The transcription factor MAZR preferentially acts as a transcriptional repressor in mast cells and plays a minor role in the regulation of effector functions in response to FcεRI stimulation.

Mast cells are key players in type I hypersensitivity reactions in humans and mice and their activity has to be tightly controlled. Previous studies implicated the transcription factor MAZR in the regulation of mast cell function. To study the role of MAZR in mast cells, we generated a conditional Mazr allele and crossed Mazr (F/F) mice with the Vav-iCre deleter strain, which is active in all hematopoietic cells. MAZR-null BM-derived mast cells (BMMC) were phenotypically indistinguishable from wild-type BMMCs, although the numbers of IL-3 generated Mazr (F/F) Vav-iCre BMMCs were reduced in comparison to Mazr (F/F) BMMCs, showing that MAZR is required for the efficient generation of BMMC in vitro. A gene expression analysis revealed that MAZR-deficiency resulted in the dysregulation of 128 genes, with more genes up- than down-regulated in the absence of MAZR, indicating that MAZR acts as a transcriptional repressor in mast cells. Among the up-regulated genes were the chemokines Ccl5, Cxcl10, Cxcl12, the chemokine receptor Ccr5 and the cytokine IL18, suggesting an immunoregulatory role for MAZR in mast cells. Enforced expression of MAZR in mature Mazr-deficient BMMCs rescued the altered expression pattern of some genes tested, suggesting direct regulation of these genes by MAZR. Upon FcεRI stimulation, Mazr expression was transiently down-regulated in BMMCs. However, early and late effector functions in response to FcεRI-mediated stimulation were not impaired in the absence of MAZR, with the exception of IL-6, which was slightly decreased. Taken together, out data indicate that MAZR preferentially acts as a transcriptional repressor in mast cells, however MAZR plays only a minor role in the transcriptional networks that regulate early and late effector functions in mast cells in response to FcεRI stimulation.

Tec family kinases: regulation of FcεRI-mediated mast-cell activation.

Mast cells express the high-affinity receptor for IgE (FcεRI) and are key players in type I hypersensitivity reactions. They are critically involved in the development of allergic rhinitis, allergic asthma and systemic anaphylaxis, however, they also regulate normal physiological processes that link innate and adaptive immune responses. Thus, their activation has to be tightly controlled. One group of signaling molecules that are activated upon FcεRI stimulation is formed by Tec family kinases, and three members of this kinase family (Btk, Itk and Tec) are expressed in mast cells. Many studies have revealed important functions of Tec kinases in signaling pathways downstream of the antigen receptors in lymphocytes. This review summarizes the current knowledge about the function of Tec family kinases in FcεRI-mediated signaling pathways in mast cell.

The effects of dasatinib on IgE receptor-dependent activation and histamine release in human basophils.

Dasatinib is a multitargeted drug that blocks several tyrosine kinases. Apart from its well-known antileukemic activity, the drug has attracted attention because of potential immunosuppressive and anti-inflammatory effects. We report that dasatinib at 1 microM completely blocks anti-IgE-induced histamine release in blood basophils in healthy donors, and allergen-induced release of histamine in sensitized individuals. In addition, dasatinib inhibited FcepsilonRI-mediated release of IL-4 and IgE-mediated up-regulation of CD13, CD63, CD164, and CD203c in basophils. The effects of dasatinib were dose-dependent (IC(50): 50-500 nM) and specific for FcepsilonRI activation in that the drug failed to inhibit C5a-induced or Ca-ionophore-induced histamine release. Interestingly, at lower concentrations, dasatinib even promoted FcepsilonRI-dependent histamine release in basophils in allergic subjects. In consecutive studies, dasatinib was found to interact with and block several FcepsilonRI downstream targets in basophils, including Btk. Correspondingly, FcepsilonRI-mediated histamine secretion in basophils was markedly reduced in Btk knockout mice and in a patient with Btk deficiency. However, the remaining "low-level" mediator secretion in Btk-deficient cells was fully blocked down again by 1 muM dasatinib. Together, these data suggest that dasatinib inhibits FcepsilonRI-mediated activation of basophils through multiple signaling molecules including Btk. Dasatinib may be an interesting agent for immunologic disorders involving Btk-dependent responses or/and FcepsilonRI activation of basophils.

Pax5: the guardian of B cell identity and function.

The transcription factor Pax5 is essential for commitment of lymphoid progenitors to the B lymphocyte lineage. Pax5 fulfils a dual role by repressing B lineage 'inappropriate' genes and simultaneously activating B lineage-specific genes. This transcriptional reprogramming restricts the broad signaling capacity of uncommitted progenitors to the B cell pathway, regulates cell adhesion and migration, induces V(H)-DJ(H) recombination, facilitates (pre-)B cell receptor signaling and promotes development to the mature B cell stage. Conditional Pax5 inactivation in early and late B lymphocytes revealed an essential role for Pax5 in controlling the identity and function of B cells throughout B lymphopoiesis. PAX5 has also been implicated in human B cell malignancies, as it is deregulated by chromosomal translocations in a subset of acute lymphoblastic leukemias and non-Hodgkin lymphomas.

Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function.

The transcription factor Pax5 represses B lineage-inappropriate genes and activates B cell-specific genes in B lymphocytes. Here we have identified 170 Pax5-activated genes. Conditional mutagenesis demonstrated that the Pax5-regulated genes require continuous Pax5 activity for normal expression in pro-B and mature B cells. Expression of half of the Pax5-activated genes is either absent or substantially reduced upon Pax5 loss in plasma cells. Direct Pax5 target genes were identified based on their protein synthesis-independent activation by a Pax5-estrogen receptor fusion protein. Chromatin immunoprecipitation (ChIP) of Pax5 together with chromatin profiling by ChIP-on-chip analysis demonstrated that Pax5 directly activates the chromatin at promoters or putative enhancers of Pax5 target genes. The Pax5-activated genes code for key regulatory and structural proteins involved in B cell signaling, adhesion, migration, antigen presentation, and germinal-center B cell formation, thus revealing a complex regulatory network that is activated by Pax5 to control B cell development and function.

Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells.

The transcription factor Pax5 represses lineage-inappropriate genes and activates B cell-specific genes in B lymphocytes. By identifying 110 Pax5-repressed genes, we now demonstrate that Pax5 downregulates diverse biological activities including receptor signaling, cell adhesion, migration, transcriptional control, and cellular metabolism at B cell commitment. The T lymphoid or myeloid expression of these genes demonstrates that Pax5(-/-) pro-B cells and common lymphoid progenitors display lymphoid and myeloid promiscuity of gene expression. These lineage-inappropriate genes require continuous Pax5 activity for their repression, as they are reactivated in committed pro-B cells and mature B cells following conditional Pax5 deletion. Pax5-repressed genes are also reexpressed in plasma cells, which depend for normal function on Cd28 and Ccr2 reactivation. The loss of Pax5 during terminal differentiation thus contributes to the plasma cell transcription program. Finally, ectopic expression of the Pax5-repressed chemokine gene Ccl3 in B cells results in increased osteoclast formation and bone loss, demonstrating that Pax5-mediated gene repression is essential for normal homeostasis of hematopoietic development.

Epigenetic silencing of the c-fms locus during B-lymphopoiesis occurs in discrete steps and is reversible.

The murine c-fms (Csf1r) gene encodes the macrophage colony-stimulating factor receptor, which is essential for macrophage development. It is expressed at a low level in haematopoietic stem cells and is switched off in all non-macrophage cell types. To examine the role of chromatin structure in this process we studied epigenetic silencing of c-fms during B-lymphopoiesis. c-fms chromatin in stem cells and multipotent progenitors is in the active conformation and bound by transcription factors. A similar result was obtained with specified common myeloid and lymphoid progenitor cells. In developing B cells, c-fms chromatin is silenced in distinct steps, whereby first the binding of transcription factors and RNA expression is lost, followed by a loss of nuclease accessibility. Interestingly, regions of de novo DNA methylation in B cells overlap with an intronic antisense transcription unit that is differently regulated during lymphopoiesis. However, even at mature B cell stages, c-fms chromatin is still in a poised conformation and c-fms expression can be re-activated by conditional deletion of the transcription factor Pax5.