Metabolite and thymocyte development defects in ADA-SCID mice receiving enzyme replacement therapy

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme intrinsic to the purine salvage pathway, leads to severe combined immunodeficiency (SCID) both in humans and mice. Lack of ADA results in the intracellular accumulation of toxic metabolites which have effects on T cell development and function. While untreated ADA-SCID is a fatal disorder, there are different therapeutic options available to restore ADA activity and reconstitute a functioning immune system, including enzyme replacement therapy (ERT). Administration of ERT in the form of pegylated bovine ADA (PEG-ADA) has proved a life-saving though non-curative treatment for ADA-SCID patients. However, in many patients treated with PEG-ADA, there is suboptimal immune recovery with low T and B cell numbers. Here, we show reduced thymus cellularity in ADA-SCID mice despite weekly PEG-ADA treatment. This was associated with lack of effective adenosine (Ado) detoxification in the thymus. We also show that thymocyte development in ADA-deficient thymi is arrested at the DN3-to-DN4 stage transition with thymocytes undergoing dATP-induced apoptosis rather than defective TCRβ rearrangement or β-selection. Our studies demonstrate at a detailed level that exogenous once-a-week enzyme replacement does not fully correct intra-thymic metabolic or immunological abnormalities associated with ADA deficiency.

Zinc finger protein Zfp335 controls early T cell development and survival through beta-selection-dependent and -independent mechanisms

T cell development in the thymus undergoes the process of differentiation, selective proliferation and survival from CD4-CD8- double negative (DN) stage to CD4+CD8+ double positive (DP) stage prior to the formation of CD4+ helper and CD8+ cytolytic T cells ready for circulation. Each developmental stage is tightly regulated by sequentially-operating molecular networks, of which only limited numbers of transcription regulators have been deciphered. Here we identified Zfp335 transcription factor as a new player in the regulatory network controlling thymocyte development. We demonstrate that Zfp335 intrinsically controls DN to DP transition, as T cell-specific deficiency in Zfp335 leads to a substantial accumulation of DN3 along with reduction of DP, CD4+ and CD8+ thymocytes. This developmental blockade at DN stage results from the impaired intracellular TCRβ expression as well as increased susceptibility to apoptosis in thymocytes. Transcriptomic and ChIP-seq analyses revealed a direct regulation of transcription factors Bcl6 and Rorc by Zfp335. Importantly, enhanced expression of TCRβ and Bcl6/RorγT restores the developmental defect during DN3 to DN4 transition and improves thymocytes survival, respectively. These findings identify a critical role of Zfp335 in controlling T cell development by maintaining intracellular TCRβ expression-mediated β-selection and independently activating cell survival signaling.

Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by compacting super-enhancers

CD4+ and CD8+ double-positive (DP) thymocytes are at a crucial stage during the T cell development in the thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCRβ. Then DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, Regulatory T cells, or invariant nature kill T cells (iNKT) according to the TCR signal. Chromatin organizer SATB1 is highly expressed in DP cells and plays an essential role in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing assay of SATB1-deficient thymocytes showed that the cell identity of DP thymocytes was changed, and the genes specifically highly expressed in DP cells were down-regulated. The super-enhancers regulate the expressions of the DP-specific genes, and the SATB1 deficiency reduced the super-enhancer activity. Hi-C data showed that interactions in super-enhancers and between super-enhancers and promoters decreased in SATB1 deficient thymocytes. We further explored the regulation mechanism of two SATB1-regulating genes, Ets2 and Bcl6, in DP cells and found that the knockout of the super-enhancers of these two genes impaired the development of DP cells. Our research reveals that SATB1 globally regulates super-enhancers of DP cells and promotes the establishment of DP cell identity, which helps understand the role of SATB1 in thymocyte development.

A novel, ataxic mouse model of Ataxia Telangiectasia caused by a clinically relevant nonsense mutation

Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated (ATM) and Aprataxin (APTX), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders is severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons are significantly perturbed (e.g., reduced membrane capacitance, lower action potential thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter Purkinje neuron neural activity, including a progressive reduction in spontaneous action potential firing frequency that correlates with both cerebellar atrophy and ataxia over the animal’s first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Lastly, by inserting a clinically relevant nonsense-type null mutation in Atm, we demonstrate that Small Molecule Read-Through (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic.

T-Cell Acute Lymphoblastic Leukemia: Biomarkers and Their Clinical Usefulness

T-cell acute lymphoblastic leukemias (T-ALL) are immature lymphoid tumors localizing in the bone marrow, mediastinum, central nervous system, and lymphoid organs. They account for 10–15% of pediatric and about 25% of adult acute lymphoblastic leukemia (ALL) cases. It is a widely heterogeneous disease that is caused by the co-occurrence of multiple genetic abnormalities, which are acquired over time, and once accumulated, lead to full-blown leukemia. Recurrently affected genes deregulate pivotal cell processes, such as cycling (CDKN1B, RB1, TP53), signaling transduction (RAS pathway, IL7R/JAK/STAT, PI3K/AKT), epigenetics (PRC2 members, PHF6), and protein translation (RPL10, CNOT3). A remarkable role is played by NOTCH1 and CDKN2A, as they are altered in more than half of the cases. The activation of the NOTCH1 signaling affects thymocyte specification and development, while CDKN2A haploinsufficiency/inactivation, promotes cell cycle progression. Among recurrently involved oncogenes, a major role is exerted by T-cell-specific transcription factors, whose deregulated expression interferes with normal thymocyte development and causes a stage-specific differentiation arrest. Hence, TAL and/or LMO deregulation is typical of T-ALL with a mature phenotype (sCD3 positive) that of TLX1, NKX2-1, or TLX3, of cortical T-ALL (CD1a positive); HOXA and MEF2C are instead over-expressed in subsets of Early T-cell Precursor (ETP; immature phenotype) and early T-ALL. Among immature T-ALL, genomic alterations, that cause BCL11B transcriptional deregulation, identify a specific genetic subgroup. Although comprehensive cytogenetic and molecular studies have shed light on the genetic background of T-ALL, biomarkers are not currently adopted in the diagnostic workup of T-ALL, and only a limited number of studies have assessed their clinical implications. In this review, we will focus on recurrent T-ALL abnormalities that define specific leukemogenic pathways and on oncogenes/oncosuppressors that can serve as diagnostic biomarkers. Moreover, we will discuss how the complex genomic profile of T-ALL can be used to address and test innovative/targeted therapeutic options.

Single-cell multi-omic analysis of thymocyte development reveals NFAT as a driver of CD4/CD8 lineage commitment

CD4 and CD8 T cells play critical roles in the mammalian immune system. While their development within the thymus from the CD4+CD8+ stage has been widely studied as a model of lineage commitment, the underlying mechanism remains unclear. To deconstruct this process, we apply CITE-seq, measuring the transcriptome and over 100 surface proteins in thymocytes from wild-type and lineage-restricted mice. We jointly analyze the paired measurements to build a comprehensive timeline of RNA and protein expression in each lineage, supporting a sequential model of lineage determination in which both lineages go through an initial phase of CD4 lineage audition, which is followed by divergence and specification of CD8 lineage cells. We identify early differences implicating T cell receptor signaling via calcineurin-NFAT in driving CD4 lineage commitment. Pharmacological inhibition validates the requirement of calcineurin- NFAT for CD4, but not CD8, lineage development, providing insight into the CD4/CD8 commitment mechanism.

NFAT transcription factors are essential and redundant actors for leukemia initiating potential in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with few available targeted therapies. We previously reported that the phosphatase calcineurin (Cn) is required for LIC (leukemia Initiating Capacity) potential of T-ALL pointing to Cn as an interesting therapeutic target. Calcineurin inhibitors have however unwanted side effect. NFAT transcription factors play crucial roles downstream of calcineurin during thymocyte development, T cell differentiation, activation and anergy. Here we elucidate NFAT functional relevance in T-ALL. Using murine T-ALL models in which Nfat genes can be inactivated either singly or in combination, we show that NFATs are required for T-ALL LIC potential and essential to survival, proliferation and migration of T-ALL cells. We also demonstrate that Nfat genes are functionally redundant in T-ALL and identified a node of genes commonly deregulated upon Cn or NFAT inactivation, which may serve as future candidate targets for T-ALL.