pre-TCR Signaling Research Articles

Caught in a Wnt storm: Complexities of Wnt signaling in hematopoiesis.

The Wnt signaling pathway is an evolutionary conserved pathway that is involved in the development of almost every organ system in the body and provides self-renewal signals for most, if not all, adult stem cell systems. In recent years, this pathway has been studied by various research groups working on hematopoietic stem cells, resulting in contradicting conclusions. Here, we discuss and interpret the results of these studies and propose that Wnt dosage, the source of hematopoietic stem cells, and interactions with other pathways explain these disparate results.

Role of P53 on T Lymphopoisis from Human Embryonic Stem Cells

Tumor suppressor P53 regulates multiple signaling pathways triggered by diverse cellular stresses including DNA damages, oncogenic stimulations, and hypoxic stress, resulting in cell-cycle arrest, apoptosis, and senescence. P53 signaling is also important for double-stranded DNA breaks (DSBs) induced during physiologic events, i.e., rearrangement of antigen-specific receptors. It has been reported that P53-mediated DSB checkpoint contribute to normal murine T lymphopoiesis, especially at the double-negative (DN) stage which is defined as CD4-CD8- fraction in thymus and requires rearrangements of the T cell receptor (TCR) b locus and successful pre-TCR signaling (Guidos CJ et al., Genes Dev, 1996; Jiang D et al., J Exp Med 2006; HaksMC et al., Immunity, 1999). Here we defined the role of P53 on hematopoietic development, especially lymphopoiesis, from human embryonic stem cells (ESCs).Firstly we modified P53 gene of human ESC H1 by utilizing genome editing tool of zinc finger nuclease (ZFN) targeting the 5th exon of the P53 gene, kindly provided by Sangamo BioSciences. Sequencing analysis of the P53 knockout (KO) ES cells showed the successful deletion at the 5th exon which induced the frame shift of the downstream sequence in both of its alleles. qRT-PCR showed no stable expression of full length P53 mRNA and western blot analysis of P53 phosphorylation status in P53 KO ESCs showed undetectable levels of phosphorylated or non-phosphorylated P53 proteins when cultured in the presence or absence of apoptotic signal triggered by mitomycin C (MMC). In consistent with this, P53 KO ESCs showed significant resistance to MMC-induced cell death. In addition, P53 KO ESCs lacked apoptotic stimulation-induced upregulation of P53 downstream target genes including P53 up-regulated modulator of apoptosis (PUMA). On the other hand induction of P53 target gene P21 was not observed both in H1 and P53 KO ESCs, as reported previously by other groups (Ginis I et al., Dev Biol, 2004; Barta T et al., Stem Cells, 2010; Garc'a CP et al., Stem Cell Res, 2014; World J et al., Stem Cells, 2014).We then induced hematopoietic differentiation of P53 KO ESCs through embryoid body formation. Erythroid lineage cells developed from human ESCs were significantly suppressed in the absence of P53 signaling during embryoid body maturation. Pharmacological inhibition of P53 had the same effect as genetic disruption of P53 gene. CD34+ hematopoietic precursors were isolated from embryoid bodies originated from H1 and P53 KO ECSs, plated on OP9-DL1 stromal cells, and cultured in the presence of stem cell factor (SCF), FLT3 ligand, and interleukin (IL)-7. After 3-4 weeks of culture, CD45+CD3+ T lineage cells were induced from both H1 and P53 KO ECSs-derived CD34+ cells. Among these cells, most of the cells were in CD4+CD8+ double-positive (DP) stage, with increase in the yield of DP cells in the absence of P53 signaling (H1: 343 cells/1 x 106 input CD34+ cells; P53 KO: 2476 cells/1 x 106 input CD34+ cells; Figure). Whether pharmacological inhibition of P53 had the similar effect on T lymphopoiesis as genetic disruption of P53 gene needs to be investigated furthermore.Our data indicate that P53 mediated signaling regulate in vitro early T lymphopoiesis from human pluripotent stem cells, especially at the transition from double negative into DP stage. These observations promoted us to perform high throughput transcriptome analysis including cDNA microarray analysis between early T lineage cells derived from H1 and P53 KO ESCs. Genes associated with the early T lymphopoiesis from human ESCs were identified and currently under further characterization. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A genome wide transcriptional model of the complex response to pre-TCR signalling during thymocyte differentiation.

Developing thymocytes require pre-TCR signalling to differentiate from CD4-CD8- double negative to CD4+CD8+ double positive cell. Here we followed the transcriptional response to pre-TCR signalling in a synchronised population of differentiating double negative thymocytes. This time series analysis revealed a complex transcriptional response, in which thousands of genes were up and down-regulated before changes in cell surface phenotype were detected. Genome-wide measurement of RNA degradation of individual genes showed great heterogeneity in the rate of degradation between different genes. We therefore used time course expression and degradation data and a genome wide transcriptional modelling (GWTM) strategy to model the transcriptional response of genes up-regulated on pre-TCR signal transduction. This analysis revealed five major temporally distinct transcriptional activities that up regulate transcription through time, whereas down-regulation of expression occurred in three waves. Our model thus placed known regulators in a temporal perspective, and in addition identified novel candidate regulators of thymocyte differentiation.

BTB-ZF Protein Znf131 Regulates Cell Growth of Developing and Mature T Cells.

Many members of the BTB-ZF family have been shown to play important roles in lymphocyte development and function. The role of zinc finger Znf131 (also known as Zbtb35) in T cell lineage was elucidated through the production of mice with floxed allele to disrupt at different stages of development. In this article, we present that Znf131 is critical for T cell development during double-negative to double-positive stage, with which significant cell expansion triggered by the pre-TCR signal is coupled. In mature T cells, Znf131 is required for the activation of effector genes, as well as robust proliferation induced upon TCR signal. One of the cyclin-dependent kinase inhibitors, p21(Cip1) encoded by cdkn1a gene, is one of the targets of Znf131. The regulation of T cell proliferation by Znf131 is in part attributed to its suppression on the expression of p21(Cip1).

Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection.

pre-T cell receptor (TCR) and Notch signaling induce transient self-renewal or “β-selection” of TCRβ+ CD4 CD8 double-negative-3 (DN3) and DN4 progenitors that differentiate into CD4 CD8 double positive (DP) thymocytes which then rearrange Tcra. Interleukin-7 (IL-7) promotes Bcl2-dependent survival of TCRβ− DN thymocytes, but IL-7 functions during β-selection remain unclear. Here, we show that IL-7 signals TCRβ+ DN3 and DN4 thymocytes to upregulate genes involved in cell growth and represses Bcl6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate, but rearranged Tcra prematurely and differentiated rapidly. Bcl6 deletion, but not BCL2 over-expression, partially restored DN4 self-renewal in the absence of IL-7. Thus, IL-7 critically collaborates with pre-TCR and Notch signaling to coordinate proliferation, differentiation and Tcra recombination during β-selection.

Transcription factors and target genes of pre-TCR signaling.

Almost 30 years ago pioneering work by the laboratories of Harald von Boehmer and Susumo Tonegawa provided the first indications that developing thymocytes could assemble a functional TCRβ chain-containing receptor complex, the pre-TCR, before TCRα expression. The discovery and study of the pre-TCR complex revealed paradigms of signaling pathways in control of cell survival and proliferation, and culminated in the recognition of the multifunctional nature of this receptor. As a receptor integrated in a dynamic developmental process, the pre-TCR must be viewed not only in the light of the biological outcomes it promotes, but also in context with those molecular processes that drive its expression in thymocytes. This review article focuses on transcription factors and target genes activated by the pre-TCR to drive its different outcomes.

P57 regulates T-cell development and prevents lymphomagenesis by balancing p53 activity and pre-TCR signaling

AbstractT cells are key components of the immune system, playing a central role in cell-mediated immunity. The sequential differentiation of T cells is associated with strict regulation of the cell cycle at each developmental stage. A balance between p53 activity and pre–T cell receptor (TCR) signaling regulates proliferation and differentiation decisions made by these cells. The relation between maintenance of this balance and the function of cell cycle regulators has remained largely unknown, however. We now show that mice with T cell–specific deficiency of the cyclin-dependent kinase inhibitor p57 manifest a differentiation block at the early stage of T cell maturation. Further genetic analysis showed that this defect is attributable to an imbalance between p53 activity and pre-TCR signaling caused by hyperactivation of the E2F-p53 pathway. Moreover, ablation of both p57 and p53 in T cells led to the development of aggressive thymic lymphomas with a reduced latency compared with that apparent for p53-deficient mice, whereas ablation of p57 alone did not confer susceptibility to this hematologic malignancy. Our results thus show that the p57-E2F-p53 axis plays a pivotal role in the proper development of T cells as well as in the prevention of lymphomagenesis.

Protein phosphatase 4 is an essential positive regulator for Treg development, function, and protective gut immunity.

BackgroundProtein phosphates 4 (PP4), encoded by the ppp4c gene, is a ubiquitously expressed phosphatase that has been implicated in the regulation of cytokine signaling and lymphocyte survival; recent reports suggest that PP4 may be involved in pre-TCR signaling and B cell development. However, whether PP4 also modulates the functions of peripheral T cells has not been investigated due to the lack of a suitable in vivo model. Treg cells are a specialized subset of CD4 helper T cells that can suppress the proliferation of activated effector T cells. In the absence of this negative regulation, autoimmune syndromes and inflammatory diseases, such as human Crohn’s disease, will arise.ResultsIn this report, we generated mice with T cell-specific ablation of the ppp4c gene (CD4cre:PP4f/f) and a Foxp3-GFP reporter gene to examine the roles of PP4 in Treg development and function. Characterizations of the CD4cre:PP4f/f mice showed that PP4 deficiency induced partial αβ T lymphopenia and T cell hypo-proliferation. Further analyses revealed significant reductions in the numbers of thymic and peripheral Treg cells, as well as in the efficiency of in vitro Treg polarization. In addition, PP4-deficient Treg cells exhibited reduced suppressor functions that were associated with decreased IL-10, CTLA4, GITR and CD103 expression. More interestingly, the CD4cre:PP4f/f mice developed spontaneous rectal prolapse and colitis with symptoms similar to human Crohn’s disease. The pathogenesis of colitis required the presence of commensal bacteria, and was correlated with reduced Treg cells in the gut. Nevertheless, PP4-deficient Treg cells were still capable of suppressing experimental colitis, suggesting that multiple factors contributed to the onset of the spontaneous colitis.ConclusionsWhile the molecular mechanisms remain to be investigated, our results clearly show that PP4 plays a non-redundant role for the differentiation, suppressor activity and gut homeostasis of Treg cells. The onset of spontaneous colitis in the CD4cre:PP4f/f mice further suggests that PP4 is essential for the maintenance of protective gut immunity. The CD4cre:PP4f/f mice thus may serve as a good model for studying the interactions between Treg cells and gut commensal bacteria for the regulation of mucosal immunity.

Successful β-selection depends on TCF-1 (HEM4P.228)

Abstract The transcription factor TCF-1 is expressed very early during thymic development and is important for early T cell specification. TCF-1 expression further increases after b-selection and is maintained at high levels in differentiated T cells, but its function at these later stages has not been examined. We used mice with conditional alleles of TCF-1 (TCF-1f/f) to delete TCF-1 at two time points of T cell development. Whereas deletion of TCF-1 in double-positive thymocytes (CD4-cre TCF-1f/f) had little impact on later T cell populations, the effect of the deletion at double-negative 3 (DN3) stage (CD2-cre TCF-1f/f) was dramatic. Thymic cellularity of CD2-cre TCF-1f/f mice was decreased more than 10-fold compared to littermates control, and competitive BM chimeras clearly revealed a sharp defect at the b-selection checkpoint in these mice. Analysis of TCF-1-deleted DN3 thymocytes did not reveal any defects in T cell gene expression and pre-TCR expression and signaling. However, post b-selection DN3 and DN4 thymocytes failed to display the increase in cell size and proliferation characteristic of successful b-selection. Because these two phenomena have been previously attributed to c-Myc, we examined its expression and found that c-Myc protein is reduced in TCF-1-deleted post b-selection DN3. We are currently examining the mechanism by which TCF-1 controls c-Myc. Our work to date implicates TCF-1 downstream of the pre-TCR during the development of αβ T cells.

Gene targeting of RhoA reveals its essential role in thymocyte development and metabolism (LYM7P.718)

Abstract RhoA GTPase is an intracellular signal transducer capable of regulating a wide range of cell functions including cytoskeleton dynamics, proliferation, and survival. In T lymphocytes, studies by using dominant negative mutant or C3 transferase expressing transgenic mice suggest that RhoA is involved in TCR signaling and related T cell functions such as polarization, migration, survival, and proliferation. To date, the physiological role of RhoA in T cell development remains unclear. In this study, we have achieved T cell-specific deletion of RhoA and found that RhoA gene disruption caused (1) a drastic decrease in the numbers of all thymocyte subsets including CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), CD4+CD8- single positive (SP), and CD4-CD8+ SP T cells, (2) an impaired β-selection at DN stage and positive selection at DP stage associated with altered pre-TCR and TCR signaling transduction, respectively, (3) a diminished thymocyte proliferation which could be due to impaired cytokinesis, (4) an increased apoptosis, and (5) an enhanced mitochondrial function as manifested by increased oxidative phosphorylation, glycolysis, mitochondrial membrane potential, mitochondrial numbers, mitochondrial DNA and ROS production. Thus, RhoA critically regulates T cell development by impacting on β-selection, positive selection, cell proliferation and survival through mediating pre-TCR signaling, TCR signaling, cytokinesis, and metabolism.

Pontin is required for pre-TCR signaling at the β-selection checkpoint in T cell development

Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Based on high expression in lymphoid tissues, we examined whether Pontin has a T cell-specific function. We generated Pontinf/f;Lck-Cre mice, in which Pontin can be conditionally deleted in T cells and then explored T cell-specific function of Pontin in vivo. Here, we show that specific abrogation of Pontin expression in T cells almost completely blocked development of αβ T cells at the β-selection checkpoint by inducing cell apoptosis indicating that Pontin is essential for early T cell development. Pontin-deficient thymocytes show a comparable expression level of T cell receptor (TCR)β chain, but have enhanced activation of p53 and Notch signaling compared to wild-type thymocytes. Intriguingly, the developmental block of αβ T cells can be partially rescued by loss of p53. Together, our data demonstrate a novel role of Pontin as a crucial regulator in pre-TCR signaling during T cell development.

Nck Recruitment to the TCR Required for ZAP70 Activation during Thymic Development

The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3ε after TCR engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4(+)CD8(+) thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired tyrosine phosphorylation of CD3ζ, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic differences in both knockin mice.

CD81 interacts with the T cell receptor to suppress signaling.

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling.

Towards a Purely Oncogenetic Risk Classification of Adult T-ALL: a GRAALL Study

AbstractAbstract 881T-cell acute lymphoblastic leukemia (T-ALL) represents a heterogeneous group of acute leukemias, which account for 25% of adult ALL. The GRAALL group recently reported a significant improvement in the outcome of BCR-ABL negative adult ALL using an intensified treatment protocol and a significantly better outcome in T-ALL harbouring NOTCH1 and/or FBXW7 (N/F) mutations compared to unmutated cases. Despite this, a third of N/F mutated T-ALL patients relapse and the identification of a T-ALL subgroup with very favorable outcome remains desirable.In a series of 212 adult T-ALLs included in the multicenter randomized GRAALL-2003 and 2005 trials, we searched for N/K-RAS (exon 1) mutations and PTEN (exon 7 mutations and gene deletion by CGH-array SNP-6 Affymetrix®) defects, which are considered as “type B3” mutations involved in pre-TCR signalling. Overall survival (OS) and event-free survival (EFS) were estimated by the Kaplan-Meier method, and then compared by the log-rank test.NOTCH1 and/or FBXW7 mutations were identified in 143 (67%) of the 212 patients and lack of N/F mutation was associated with a poor prognostic. N-RAS, K-RAS and PTEN mutations were identified in 3/191 (1.6%), 17/191 (8.9%) and 17/175 (9.7%) patients, respectively. PTEN genomic deletions/mutations and N/K-RAS activating mutations were virtually mutually exclusive. N/K-RAS mutations were more frequent in TCR negative phenotype and CNS positive T-ALLs, but did not correlate with other classical parameters, EGIL phenotype, N/F status, or cortico- or chemo-sensitivity. PTEN alterations were more frequent in mature TCR expressing, SIL-TAL+, N/F unmutated cases with high leukemic bulk tumors, but did not significantly differed with respect to age, gender, CNS involvement, cortico- or chemo-sensitivity. When analyzed separately, N/K-RAS mutations or PTEN genomic abnormalities demonstrated trends to a worse outcome.We then analyzed the effect of N/K-RAS mutations and/or PTEN genomic abnormalities on the good prognosis associated with N/F mutations by a multivariate Cox model for EFS and OS, entering the two N/F and RAS/PTEN covariates, as well as an interaction term. The prognostic significance of N/F mutations was still observed (HR, 0.26 [95% CI, 0.15–0.46] and 0.26 [95% CI, 0.14–0.49] with P<0.0001 for EFS and OS, respectively), with a significant interaction between N/F and RAS/PTEN mutations (P=0.03 and 0.05 for EFS and OS, respectively. In other terms, the favorable impact of N/F mutation was still observed in, and was restricted to patients without RAS/PTEN abnormalities. These observations led us to propose a new T-ALL oncogenetic classifier defining low-risk patients as those with N/F mutation but no RAS/PTEN mutation (97 out of 189 [51%] patients in the present cohort) and all other patients (49%, including 13% N/F and RAS/PTEN mutated cases) as high-risk patients (Figures 1A and 1B). Comparing this refined oncogenetic classifier to the simple N/F classification, hazard ratios for high-risk patients increased from 2.6 (95% CI, 1.7–4.0) to 3.25 (95% CI, 2.0–5.3) for EFS and from 2.5 (95% CI, 1.5–4.0) to 3.3 (95% CI, 1.9–5.8) for OS. When adjusting the effect of the new N/F-RAS-PTEN classifier to age (using the 35-year cutoff) and WBC (using the 100.109/L cut-off), the oncogenetic classifier remained the only significant prognostic covariate (HR= 3.2 (95% CI, 1.9–5.15) and 3.2 (95% CI, 1.9–5.6); P<0.0001 and <0.0001, for EFS and OS, respectively). The prognostic impact was maintained when GRAALL-2003 and GRAALL-2005 patients were analysed separately.Taken together, these data demonstrate that detection of RAS and PTEN mutations add significant prognostic value to assessment of N/F status, allowing identification of nearly 50% very good prognosis T-ALL adults. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Synergistic Effects of Interleukin-7 and Pre-T Cell Receptor Signaling in Human T Cell Development

The role of IL-7 in pre-T cell receptor (TCR) signaling during human T cell development is poorly understood. To study this, we engineered Molt3, a T cell progenitor T-acute lymphoblastic leukemia cell line, using lentiviral IL-7 receptor α (IL-7Rα) to serve as a model system. IL-7 promoted pre-TCR activation in IL-7Rα(hi) Molt3 as illustrated by CD25 up-regulation after anti-CD3 stimulation. Anti-CD3 treatment activated Akt and Erk1/2 signaling pathways as proven using specific inhibitors, and IL-7 further enhanced both signaling pathways. The close association of IL-7Rα with CD3ζ in the pre-TCR complex was illustrated through live imaging confocal fluorescence microscopy. These results demonstrate a direct and cooperative role of IL-7 in pre-TCR signaling.

Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation

BackgroundThe role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system.ResultsIn this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRαβ+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vβ repertoire.ConclusionsOur study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.

Anti-CD3ε mAb improves thymic architecture and prevents autoimmune manifestations in a mouse model of Omenn syndrome: therapeutic implications

AbstractOmenn syndrome (OS) is an atypical primary immunodeficiency characterized by severe autoimmunity because of activated T cells infiltrating target organs. The impaired recombinase activity in OS severely affects expression of the pre-T-cell receptor complex in immature thymocytes, which is crucial for an efficient development of the thymic epithelial component. Anti-CD3ε monoclonal antibody (mAb) treatment in RAG2−/− mice was previously shown to mimic pre-TCR signaling promoting thymic expansion. Here we show the effect of anti-CD3ε mAb administration in the RAG2R229Q mouse model, which closely recapitulates human OS. These animals, in spite of the inability to induce the autoimmune regulator, displayed a significant amelioration in thymic epithelial compartment and an important reduction of peripheral T-cell activation and tissue infiltration. Furthermore, by injecting a high number of RAG2R229Q progenitors into RAG2−/− animals previously conditioned with anti-CD3ε mAb, we detected autoimmune regulator expression together with the absence of peripheral immunopathology. These observations indicate that improving epithelial thymic function might ameliorate the detrimental behavior of the cell-autonomous RAG defect. Our data provide important therapeutic proof of concept for future clinical applications of anti-CD3ε mAb treatment in severe combined immunodeficiency forms characterized by poor thymus function and autoimmunity.

Alternative Splicing Controlled by Heterogeneous Nuclear Ribonucleoprotein L Regulates Development, Proliferation, and Migration of Thymic Pre-T Cells

The regulation of posttranscriptional modifications of pre-mRNA by alternative splicing is important for cellular function, development, and immunity. The receptor tyrosine phosphatase CD45, which is expressed on all hematopoietic cells, is known for its role in the development and activation of T cells. CD45 is known to be alternatively spliced, a process that is partially regulated by heterogeneous nuclear ribonucleoprotein (hnRNP) L. To investigate the role of hnRNP L further, we have generated conditional hnRNP L knockout mice and found that LckCre-mediated deletion of hnRNP L results in a decreased thymic cellularity caused by a partial block at the transition stage between double-negative 4 and double-positive cells. In addition, hnRNP L(-/-) thymocytes express aberrant levels of the CD45RA splice isoforms and show high levels of phosphorylated Lck at the activator tyrosine Y394, but lack phosphorylation of the inhibitory tyrosine Y505. This indicated an increased basal Lck activity and correlated with higher proliferation rates of double-negative 4 cells in hnRNP L(-/-) mice. Deletion of hnRNP L also blocked the migration and egress of single-positive thymocytes to peripheral lymphoid organs in response to sphingosine-1-phosphate and the chemokines CCL21 and CXCL12 very likely as a result of aberrant splicing of genes encoding GTPase regulators and proteins affecting cytoskeletal organization. Our results indicate that hnRNP L regulates T cell differentiation and migration by regulating pre-TCR and chemokine receptor signaling.

Double knockout of Id2 &amp; Id3 in developing T cells promotes the development of CD4-CD8- αβ T cell (63.3)

Abstract αβ T cell development is regulated by sequential rearrangement and expression of TCRβ and TCRα genes at the DN (CD4-CD8-) and DP (CD4+CD8+) stage, respectively. Expression of TCRβ paired with preTα at the DN stage leads to αβ lineage commitment and differentiation into the DP stage. DP cells undergo TCRα gene rearrangement, resulting in expression and the selection of functional αβTCR. Id3 has been shown to be an important sensor downstream of both preTCR and TCR signals. However, Id3 knockout only leads to a partial block in αβ lineage development, suggesting functional compensation from other Id proteins. To further investigate functions of Id proteins at the preTCR and TCR checkpoints, we simultaneously deleted Id3 and the structurally and functionally related Id2 gene at the early DN stage of T cell development. We find that removal of Id2 and Id3 failed to block T cell development at the preTCR checkpoint although imposed a strong block at the TCR checkpoint. Furthermore, deletion of both Id2 and Id3 promoted development of an aberrant population of αβ T cells that bear DN (CD4-CD8-) phenotype. This genetic system revealed a novel function of Id proteins in regulation of alternative αβ lineages during T cell development. We will present the latest information on characterization of this CD4-CD8- αβ T cell lineage associated with Id2 and Id3 disruption in developing T cells.