Atypical B cells (CD21-CD27-IgD-) correlate with lack of response to checkpoint inhibitor therapy in NSCLC.

INTRODUCTION: Checkpoint inhibitor (CI) therapy has revolutionized treatment for non-small cell lung cancer (NSCLC). However, a proportion of patients do not respond to CI therapy for unknown reasons. Although the current paradigm in anti-tumor immunity evolves around T cells, the presence of tertiary lymphoid structures and memory B cells has been positively correlated with response to CI therapy in NSCLC. In addition, double negative (DN) (CD27- IgD-) B cells have been shown to be abundant in NSCLC compared to healthy lung tissue and inversely correlate with the intratumoral presence of memory B cells. Nonetheless, no study has correlated DN B cells to survival in NSCLC. METHODS: In this study, we evaluated the presence and phenotype of B cells in peripheral blood with flow cytometry of patients with NSCLC and mesothelioma before receiving CI therapy and correlated these with clinical outcome. RESULTS: Non-responding patients showed decreased frequencies of B cells, yet increased frequencies of antigen-experienced CD21- DN (Atypical) B cells compared to responding patients and HC, which was confirmed in patients with mesothelioma treated with CI therapy. CONCLUSIONS: These data show that the frequency of CD21- DN B cells correlates with lack of response to CI therapy in thoracic malignancies. The mechanism by which CD21- DN B cells hamper CI therapy remains unknown. Our findings support the hypothesis that CD21- DN B cells resemble phenotypically identical exhausted B cells that are seen in chronic infection or function as antigen presenting cells that induce regulatory T cells.

Plasma markers in pulmonary hypertension subgroups correlate with patient survival.

BACKGROUND: Recent studies have provided evidence for an important contribution of the immune system in the pathophysiology of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). In this report, we investigated whether the inflammatory profile of pulmonary hypertension patients changes over time and correlates with patient WHO subgroups or survival. METHODS: 50 PAH patients (16 idiopathic (I)PAH, 24 Connective Tissue Disease (CTD)-PAH and 10 Congenital Heart Disease (CHD)-PAH), 37 CTEPH patients and 18 healthy controls (HCs) were included in the study. Plasma inflammatory markers at baseline and after 1-year follow-up were measured using ELISAs. Subsequently, correlations with hemodynamic parameters and survival were explored and data sets were subjected to unbiased multivariate analyses. RESULTS: At diagnosis, we found that plasma levels of interleukin-6 (IL-6) and the chemokines (C-X3-C) motif legend CXCL9 and CXCL13 in CTD-PAH patients were significantly increased, compared with HCs. In idiopathic PAH patients the levels of tumor growth factor-ß (TGFß), IL-10 and CXCL9 were elevated, compared with HCs. The increased CXCL9 and IL-8 concentrations in CETPH patients correlated significantly with decreased survival, suggesting that CXCL9 and IL-8 may be prognostic markers. After one year of treatment, IL-10, CXCL13 and TGFß levels changed significantly in the PAH subgroups and CTEPH patients. Unbiased multivariate analysis revealed clustering of PH patients based on inflammatory mediators and clinical parameters, but did not separate the WHO subgroups. Importantly, these multivariate analyses separated patients with < 3 years and > 3 years survival, in particular when inflammatory mediators were combined with clinical parameters. DISCUSSION: Our study revealed elevated plasma levels of inflammatory mediators in different PAH subgroups and CTEPH at baseline and at 1-year follow-up, whereby CXCL9 and IL-8 may prove to be prognostic markers for CTEPH patients. While this study is exploratory and hypothesis generating, our data indicate an important role for IL-8 and CXCL9 in CHD and CTEPH patients considering the increased plasma levels and the observed correlation with survival. CONCLUSION: In conclusion, our studies identified an inflammatory signature that clustered PH patients into WHO classification-independent subgroups that correlated with patient survival.

Fibrocytes are increased in lung and peripheral blood of patients with idiopathic pulmonary fibrosis.

BACKGROUND: Fibrocytes are implicated in Idiopathic Pulmonary Fibrosis (IPF) pathogenesis and increased proportions in the circulation are associated with poor prognosis. Upon tissue injury, fibrocytes migrate to the affected organ. In IPF patients, circulating fibrocytes are increased especially during exacerbations, however fibrocytes in the lungs have not been examined. Therefore, we sought to evaluate if fibrocytes can be detected in IPF lungs and we compare percentages and phenotypic characteristics of lung fibrocytes with circulating fibrocytes in IPF. METHODS: First we optimized flow cytometric detection circulating fibrocytes using a unique combination of intra- and extra-cellular markers to establish a solid gating strategy. Next we analyzed lung fibrocytes in single cell suspensions of explanted IPF and control lungs and compared characteristics and numbers with circulating fibrocytes of IPF. RESULTS: Using a gating strategy for both circulating and lung fibrocytes, which excludes potentially contaminating cell populations (e.g. neutrophils and different leukocyte subsets), we show that patients with IPF have increased proportions of fibrocytes, not only in the circulation, but also in explanted end-stage IPF lungs. These lung fibrocytes have increased surface expression of HLA-DR, increased intracellular collagen-1 expression, and also altered forward and side scatter characteristics compared with their circulating counterparts. CONCLUSIONS: These findings demonstrate that lung fibrocytes in IPF patients can be quantified and characterized by flow cytometry. Lung fibrocytes have different characteristics than circulating fibrocytes and represent an intermediate cell population between circulating fibrocytes and lung fibroblast. Therefore, more insight in their phenotype might lead to specific therapeutic targeting in fibrotic lung diseases.

Emerging roles of innate lymphoid cells in inflammatory diseases: Clinical implications.

Innate lymphoid cells (ILC) represent a group of lymphocytes that lack specific antigen receptors and are relatively rare as compared to adaptive lymphocytes. ILCs play important roles in allergic and nonallergic inflammatory diseases due to their location at barrier surfaces within the airways, gut, and skin, and they respond to cytokines produced by activated cells in their local environment. Innate lymphoid cells contribute to the immune response by the release of cytokines and other mediators, forming a link between innate and adaptive immunity. In recent years, these cells have been extensively characterized and their role in animal models of disease has been investigated. Data to translate the relevance of ILCs in human pathology, and the potential role of ILCs in diagnosis, as biomarkers and/or as future treatment targets are also emerging. This review, produced by a task force of the Immunology Section of the European Academy of Allergy and Clinical Immunology (EAACI), encompassing clinicians and researchers, highlights the role of ILCs in human allergic and nonallergic diseases in the airways, gastrointestinal tract, and skin, with a focus on new insights into clinical implications, therapeutic options, and future research opportunities.

The Notch pathway inhibitor stapled α-helical peptide derived from mastermind-like 1 (SAHM1) abrogates the hallmarks of allergic asthma.

BACKGROUND: The Notch signaling pathway has been implicated in the pathogenesis of allergic airway inflammation. Targeting the active Notch transactivation complex by using the cell-permeable, hydrocarbon-stapled synthetic peptide stapled α-helical peptide derived from mastermind-like 1 (SAHM1) resulted in genome-wide suppression of Notch-activated genes in leukemic cells and other models. However, the efficacy of SAHM1 in allergic asthma models has remained unexplored. OBJECTIVE: We aimed to investigate the therapeutic efficacy of SAHM1 in a house dust mite (HDM)-driven asthma model. METHODS: Topical therapeutic intervention with SAHM1 or a control peptide was performed during sensitization, challenge, or both with HDM in mice. Airway inflammation was assessed by using multicolor flow cytometry, and bronchial hyperreactivity was studied. Additionally, SAHM1 therapy was investigated in mice with established allergic airway inflammation and in a model in which we neutralized IFN-γ during HDM challenge to support the TH2 response and exacerbate asthma. RESULTS: SAHM1 treatment during the challenge phase led to a marked reduction of eosinophil and T cell numbers in bronchoalveolar lavage fluid compared with those in diluent-treated or control peptide-treated mice. Likewise, T-cell cytokine content and bronchial hyperreactivity were reduced. SAHM1 treatment dampened TH2 inflammation during ongoing HDM challenge and enhanced recovery after established asthma. Additionally, in the presence of anti-IFN-γ antibodies, SAHM1 downregulated expression of the key TH2 transcription factor GATA3 and intracellular IL-4 in bronchoalveolar lavage fluid T cells, but expression of the TH17 transcription factor retinoic acid-related orphan receptor γt or intracellular IL-17 was not affected. SAHM1 therapy also reduced serum IgE levels. CONCLUSIONS: Therapeutic intervention of Notch signaling by SAHM1 inhibits allergic airway inflammation in mice and is therefore an interesting new topical treatment opportunity in asthmatic patients.

Development of eosinophilic inflammation is independent of B-T cell interaction in a chronic house dust mite-driven asthma model.

BACKGROUND: Chronic exposure to environmental triggers, such as house dust mite (HDM), drives T helper 2 (Th2) cell-mediated asthma. Recent evidence has shown that B-T cell interaction, and in particular germinal centre reactions and follicular T helper (Tfh) cells are required for the development of eosinophilic airway inflammation in HDM-driven models containing a sensitization and challenge phase. Whether B-T cell interactions are essential for pulmonary eosinophilic inflammation following chronic allergen provocation remains unknown. AIMS: In this study, we investigated the importance of B-T cell interaction in the development of eosinophilic airway inflammation and pulmonary remodelling in a chronic HDM-driven asthma model. METHODS: We exposed C57BL/6, Cd40l-/- , and Mb1-/- mice to HDM three times a week for five consecutive weeks. RESULTS: Chronic HDM exposure induced a pronounced eosinophilic allergic airway inflammation in broncho-alveolar lavage fluid (BALf) and lung tissue, associated with the formation of immunologically active inducible bronchus-associated lymphoid tissue (iBALT) in the lungs. The absence of B cells or lack of CD40L signalling did not hamper eosinophilic inflammation in the airways, although the number of Tfh and Th2 cells was substantially reduced in the lungs. Importantly, type 2 innate lymphoid cell (ILC2) numbers in BALf and lung were not affected by the absence of B cells or B-T cell interaction. Furthermore, eosinophilic airway inflammation is not sufficient to induce pulmonary remodelling and airway hyperresponsiveness. CONCLUSION AND CLINICAL RELEVANCE: From these findings, we conclude that B-T cell interaction is required for robust Tfh and Th2 cell induction, but not essential for eosinophilic airway inflammation during a chronic HDM-driven asthma model.

Basal Ca(2+) signaling is particularly increased in mutated chronic lymphocytic leukemia.

On the basis of somatic hypermutation status of their B-cell antigen receptor (BCR) genes, chronic lymphocytic leukemia (CLL) patients can be divided into unmutated CLL (U-CLL) or mutated CLL (M-CLL). Approximately 30% of CLL patients express a stereotypic BCR, which may indicate that specific antigenic stimulation is driving CLL pathogenesis. Recently, it was reported that BCRs from CLL cells are capable of antigen-independent, cell-autonomous signaling, through recognition of an internal framework 2 (FR2) BCR epitope. We hypothesized that the level of cell-autonomous signaling may differ between CLL subgroups. Therefore, we analyzed Ca(2+) signaling in a series of primary stereotypic or heterogeneous U-CLL and M-CLL (n=68) and healthy controls (n=14). We confirmed that basal Ca(2+) signaling in CLL cells is higher than in normal B cells. Interestingly, we found that basal signaling was particularly increased in M-CLL. The degree of basal signaling did not correlate with membrane immunoglobulin levels, HCDR3 characteristics or FR2/FR3 sequence. We conclude that the level of basal Ca(2+) signaling is not uniformly enhanced in CLL B cells, but is associated with CLL immunoglobulin heavy chain V mutational status, reflecting a distinct cellular origin and possibly a different anergic state induced by repetitive or continuous antigen binding in vivo.

New roads open up for implementing immunotherapy in mesothelioma.

Treatment options for malignant mesothelioma are limited, and the results with conventional therapies have been rather disappointing to this date. Chemotherapy is the only evidence-based treatment for mesothelioma patients in good clinical condition, with an increase in median survival of only 2 months. Therefore, there is urgent need for a different approach to battle this malignancy. As chronic inflammation precedes mesothelioma, the immune system plays a key role in the initiation of this type of tumour. Also, many immunological cell types can be found within the tumour at different stages of the disease. However, mesothelioma cells can evade the surveillance capacity of the immune system. They build a protective tumour microenvironment to harness themselves against the immune system's attacks, in which they even abuse immune cells to act against the antitumour immune response. In our opinion, modulating the immune system simultaneously with the targeting of mesothelioma tumour cells might prove to be a superior treatment. However, this strategy is challenging since the tumour microenvironment possesses numerous forms of defence strategies. In this paper, we will discuss the interplay between immunological cells that can either inhibit or stimulate tumour growth and the challenges associated with immunotherapy. We will provide possible strategies and discuss opportunities to overcome these problems.

Pre-B-cell leukemias in Btk/Slp65-deficient mice arise independently of ongoing V(D)J recombination activity.

The adapter protein Slp65 and Bruton's tyrosine kinase (Btk) are key components of the precursor-B (pre-B) cell receptor (pre-BCR) signaling pathway. Slp65-deficient mice spontaneously develop pre-B-cell leukemia, expressing high levels of the pre-BCR on their cell surface. As leukemic Slp65-deficient pre-B cells express the recombination activating genes (Rag)1 and Rag2, and manifest ongoing immunoglobulin (Ig) light-chain rearrangement, it has been hypothesized that deregulated recombinase activity contributes to malignant transformation. In this report, we investigated whether Rag-induced DNA damage is involved in oncogenic transformation of Slp65-deficient B cells. We employed Btk/Slp65 double-deficient mice carrying an autoreactive 3-83µÎ´ BCR transgene. When developing B cells in their bone marrow express this BCR, the V(D)J recombination machinery will be activated, allowing for secondary Ig light-chain gene rearrangements to occur. This phenomenon, called receptor editing, will rescue autoreactive B cells from apoptosis. We observed that 3-83µÎ´ transgenic Btk/Slp65 double-deficient mice developed B-cell leukemias expressing both the 3-83µÎ´ BCR and the pre-BCR components λ5/VpreB. Importantly, such leukemias were found at similar frequencies in mice concomitantly deficient for Rag1 or the non-homologous end-joining factor DNA-PKcs. We therefore conclude that malignant transformation of Btk/Slp65 double-deficient pre-B cells is independent of deregulated V(D)J recombination activity.

Zoledronic acid impairs myeloid differentiation to tumour-associated macrophages in mesothelioma.

BACKGROUND: Suppressive immune cells present in tumour microenvironments are known to augment tumour growth and hamper efficacy of antitumour therapies. The amino-bisphosphonate Zoledronic acid (ZA) is considered as an antitumour agent, as recent studies showed that ZA prolongs disease-free survival in cancer patients. The exact mechanism is a topic of debate; it has been suggested that ZA targets tumour-associated macrophages (TAMs). METHODS: We investigate the role of ZA on the myeloid differentiation to TAMs in murine mesothelioma in vivo and in vitro. Mice were intraperitoneally inoculated with a lethal dose of mesothelioma tumour cells and treated with ZA to determine the effects on myeloid differentiation and survival. RESULTS: We show that ZA impaired myeloid differentiation. Inhibition of myeloid differentiation led to a reduction in TAMs, but the number of immature myeloid cells with myeloid-derived suppressor cell (MDSC) characteristics was increased. In addition, ZA affects the phenotype of macrophages leading to reduced level of TAM-associated cytokines in the tumour microenvironment. No improvement of survival was observed. CONCLUSION: We conclude that ZA leads to a reduction in macrophages and impairs polarisation towards an M2 phenotype, but this was associated with an increase in the number of immature myeloid cells, which might diminish the effects of ZA on survival.

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK.

X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk-/- mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk-/- recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk-/- mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.

United airways: circulating Th2 effector cells in an allergic rhinitis model are responsible for promoting lower airways inflammation.

BACKGROUND: Allergic rhinitis (AR) and asthma often coexist and are referred to as 'united airways' disease. However, the molecular and cellular pathways that are crucially involved in the interaction between upper and lower airways remain to be identified. OBJECTIVE: We sought to assess whether and how AR exacerbates lower airway inflammation upon allergen challenge in mice. METHODS: We previously developed an intranasal ovalbumin (OVA)-driven AR model, characterized by nasal eosinophilic inflammation, enhanced serum levels of OVA-specific IgE and Th2 cytokine production in cervical lymph nodes. In OVA-sensitized mice with or without AR, a lower airway challenge was given, and after 24 h, lower airway inflammation was analysed. RESULTS: We found that AR mice were more susceptible to eosinophilic inflammation following a lower airway OVA challenge than OVA-sensitized controls. AR mice manifested increased numbers of eosinophils in bronchoalveolar lavage fluid and increased inter-cellular adhesion molecule-1 (ICAM-1) expression on lung endothelium, when compared with OVA-sensitized controls. Depletion of T cells in OVA-challenged AR mice completely abrogated all hallmarks of lower airway inflammation, including enhanced IL-5 and tissue eosinophilia. Conversely, adoptive transfer of Th2 effector cells in naïve animals induced lower airway eosinophilic inflammation after challenge with OVA. Blocking T cell recirculation during AR development by the spingosine-1 analogue FTY720 also prevented lower airway inflammation including ICAM-1 expression in AR mice upon a single lower airway challenge. CONCLUSION: Our mouse model of 'united airways' disease supports epidemiological and clinical data that AR has a significant impact on lower airway inflammation. Circulating Th2 effector cells are responsible for lung priming in AR mice, most likely through up-regulation of ICAM-1.

Cyclooxygenase-2 in mucosal DC mediates induction of regulatory T cells in the intestine through suppression of IL-4.

Oral intake of protein leads to tolerance through the induction of regulatory T cells (Tr cells) in mesenteric lymph nodes (MLNs). Here we show that the inhibition of cyclooxygenase-2 (COX-2) in vivo suppressed oral tolerance and was associated with enhanced differentiation of interleukin (IL)-4-producing T cells and reduced Foxp3(+) Tr-cell differentiation in MLN. As a result, the functional suppressive capacity of these differentiated mucosal T cells was lost. IL-4 was causally related to loss of tolerance as treatment of mice with anti-IL-4 antibodies during COX-2 inhibition restored tolerance. Dendritic cells (DCs) in the MLN differentially expressed COX-2 and reductionist experiments revealed that selective inhibition of the enzyme in these cells inhibited Foxp3(+) Tr-cell differentiation in vitro. Importantly, the inhibition of COX-2 in MLN-DC caused increased GATA-3 expression and enhanced IL-4 release by T cells, which was directly related to impaired Tr-cell differentiation. These data provide crucial insights into the mechanisms driving de novo Tr-cell induction and tolerance in the intestine.

Enforced expression of GATA-3 during T cell development inhibits maturation of CD8 single-positive cells and induces thymic lymphoma in transgenic mice.

The zinc finger transcription factor GATA-3 is of critical importance for early T cell development and commitment of Th2 cells. To study the role of GATA-3 in early T cell development, we analyzed and modified GATA-3 expression in vivo. In mice carrying a targeted insertion of a lacZ reporter on one allele, we found that GATA-3 transcription in CD4(+)CD8(+) double-positive thymocytes correlated with the onset of positive selection events, i.e., TCRalphabeta up-regulation and CD69 expression. LacZ expression remained high ( approximately 80% of cells) during maturation of CD4 single-positive (SP) cells in the thymus, but in developing CD8 SP cells the fraction of lacZ-expressing cells decreased to <20%. We modified this pattern by enforced GATA-3 expression driven by the CD2 locus control region, which provides transcription of GATA-3 throughout T cell development. In two independent CD2-GATA3-transgenic lines, approximately 50% of the mice developed thymic lymphoblastoid tumors that were CD4(+)CD8(+/low) and mostly CD3(+). In tumor-free CD2-GATA3-transgenic mice, the total numbers of CD8 SP cells in the thymus were within normal ranges, but their maturation was hampered, as indicated by increased apoptosis of CD8 SP cells and a selective deficiency of mature CD69(low)HSA(low) CD8 SP cells. In the spleen and lymph nodes, the numbers of CD8(+) T cells were significantly reduced. These findings indicate that GATA-3 supports development of the CD4 lineage and inhibits maturation of CD8 SP cells in the thymus.

Enforced expression of GATA-3 in transgenic mice inhibits Th1 differentiation and induces the formation of a T1/ST2-expressing Th2-committed T cell compartment in vivo.

The transcription factor GATA-3 is essential for early T cell development and differentiation of naive CD4(+) T cells into Th2 effector cells. To study the function of GATA-3 during T cell-mediated immune responses in vivo, we investigated CD2-GATA3-transgenic mice in which GATA-3 expression is driven by the CD2 locus control region. Both in the CD4(+) and the CD8(+) T cell population the proportion of cells exhibiting a CD44(high)CD45RB(low)CD62L(low) Ag-experienced phenotype was increased. In CD2-GATA3-transgenic mice, large fractions of peripheral CD4(+) T cells expressed the IL-1 receptor family member T1/ST2, indicative of advanced Th2 commitment. Upon in vitro T cell stimulation, the ability to produce IL-2 and IFN-gamma was decreased. Moreover, CD4(+) T cells manifested rapid secretion of the Th2 cytokines IL-4, IL-5, and IL-10, reminiscent of Th2 memory cells. In contrast to wild-type CD4(+) cells, which lost GATA-3 expression when cultured under Th1-polarizing conditions, CD2-GATA3-transgenic CD4(+) cells maintained expression of GATA-3 protein. Under Th1 conditions, cellular proliferation of CD2-GATA3-transgenic CD4(+) cells was severely hampered, IFN-gamma production was decreased and Th2 cytokine production was increased. Enforced GATA-3 expression inhibited Th1-mediated in vivo responses, such as Ag-specific IgG2a production or a delayed-type hypersensitivity response to keyhole limpet hemocyanin. Collectively, these observations indicate that enforced GATA-3 expression selectively inhibits Th1 differentiation and induces Th2 differentiation. The increased functional capacity to secrete Th2 cytokines, along with the increased expression of surface markers for Ag-experienced Th2-committed cells, would argue for a role of GATA-3 in Th2 memory formation.

Bruton's tyrosine kinase regulates the activation of gene rearrangements at the lambda light chain locus in precursor B cells in the mouse.

Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an approximately 50% reduction in the frequency of immunoglobulin (Ig) lambda light chain expression, already at the immature B cell stage in the bone marrow. Conversely, transgenic mice expressing the activated mutant Btk(E41K) showed increased lambda usage. As the kappa/lambda ratio is dependent on (a) the level and kinetics of kappa and lambda locus activation, (b) the life span of pre-B cells, and (c) the extent of receptor editing, we analyzed the role of Btk in these processes. Enforced expression of the Bcl-2 apoptosis inhibitor did not alter the Btk dependence of lambda usage. Crossing 3-83mudelta autoantibody transgenic mice into Btk-deficient mice showed that Btk is not essential for receptor editing. Also, Btk-deficient surface Ig(+) B cells that were generated in vitro in interleukin 7-driven bone marrow cultures manifested reduced lambda usage. An intrinsic defect in lambda locus recombination was further supported by the finding in Btk-deficient mice of reduced lambda usage in the fraction of pre-B cells that express light chains in their cytoplasm. These results implicate Btk in the regulation of the activation of the lambda locus for V(D)J recombination in pre-B cells.

Transcriptional control of t lymphocyte differentiation.

Initiation of gene transcription by transcription factors (TFs) is an important regulatory step in many developmental processes. The differentiation of T cell progenitors in the thymus is tightly controlled by signaling molecules, ultimately activating nuclear TFs that regulate the expression of T lineage-specific genes. During the last 2 years, significant progress has been made in our understanding of the signaling routes and TFs operating during the earliest stages of thymic differentiation at the CD4(-)CD8(-) double negative stage. Here we will review the TF families that play an important role in differentiation of thymocytes, particularly focusing on recent new information with respect to the Tcf, bHLH, GATA, and CBF/HES TF families.

Role of Bruton's tyrosine kinase in B cell development.

X-linked agammaglobulinemia (XLA) is one of the most frequent inherited immunodeficiency diseases in man and is characterized by an almost complete arrest of B cell differentiation at the pre-B cell stage. The gene defective in XLA encodes the cytoplasmic signaling molecule Bruton's tyrosine kinase (Btk). Next to the CBA/N strain of mice, carrying a single amino acid substitution mutation in the Btk gene, which results in the X-linked immunodeficiency (xid) phenotype, additional mouse models have been developed to study the role of Btk in vivo. This review discusses the analyses of Btk null-mutants, obtained by gene targeting in embryonic stem cells, and transgenic mice that express wild-type or mutated forms of the Btk gene. These studies provided information on the function of Btk at several important checkpoints throughout B cell development. Analyses of the mouse models indicated that Btk is not essential for pre-B cell receptor signaling in the mouse. By contrast, Btk-mediated B cell receptor signaling appears to be required for the survival of immature B cells in the bone marrow, that have performed a successful immunoglobulin (Ig) L chain locus rearrangement, resulting in the expression of a non-autoreactive Ig on the membrane. Btk is also shown to be involved in signaling pathways that govern the development of peripheral B cells, including follicular entry, follicular maturation and plasma cell differentiation.

Expression of the transcription factor GATA-3 is required for the development of the earliest T cell progenitors and correlates with stages of cellular proliferation in the thymus.

GATA-3 is a zinc-finger transcription factor that is essential for both early T cell development and Th2 cell differentiation. To quantify GATA-3 expression during T cell development in vivo in the mouse, the GATA-3 gene was targeted by insertion of a lacZ reporter by homologous recombination in embryonic stem (ES) cells. Although we could detect GATA-3+ cells throughout T cell development in the thymus, the proportions of GATA-3+ cells varied considerably between the distinct differentiation stages. The two periods of TCR alpha and beta gene recombination, which occur in quiescent or slowly dividing cells, were associated with low proportions of GATA-3+ cells. Conversely, the stage of rapidly proliferating cells, which insulates these two waves of TCR rearrangement, was characterized by a large proportion of GATA-3+ cells. In addition, we generated chimeric mice by injection of GATA-3-deficient, lacZ-expressing ES cells into wild-type blastocysts. In this in vivo competition analysis, no contribution of GATA-3-deficient cells to the T cell lineage was detected, not even in the earliest CD44+CD25- double-negative (CD4-CD8-) cell stage in the thymus. These results parallel data implicating other GATA family members as key regulators of proliferation and survival of early hematopoietic cells. We therefore propose that GATA-3 is required for the expansion of T cell progenitors, and for the control of subsequent proliferation steps, which alternate periods of TCR recombination in the thymus.