ADAR1 is an RNA-editing enzyme that is abundant in the thymus. We have previously reported that ADAR1 is required for establishing central tolerance during the late stage of thymocyte development by preventing MDA5 sensing of endogenous dsRNA as nonself. However, the role of ADAR1 during the early developmental stage remains unknown. In this study, we demonstrate that early thymocyte-specific deletion of ADAR1 in mice caused severe thymic atrophy with excessive apoptosis and impaired transition to a late stage of development accompanied by the loss of TCR expression. Concurrent MDA5 deletion ameliorated apoptosis but did not restore impaired transition and TCR expression. In addition, forced TCR expression was insufficient to restore the transition. However, simultaneous TCR expression and MDA5 deletion efficiently ameliorated the impaired transition of ADAR1-deficient thymocytes to the late stage. These findings indicate that RNA-editing–dependent and –independent functions of ADAR1 synergistically regulate early thymocyte development.
Posttranscriptional modifications contribute a variety of information to RNAs, which adds to the complexities of RNA regulation. One of these modifications is adenosine-to-inosine RNA editing, which occurs in a dsRNA structure (1–5). In mammals, adenosine-to-inosine RNA editing is catalyzed by adenosine deaminase acting on RNA (ADAR) 1 and ADAR2 (1, 4). Furthermore, ADAR1 is composed of two isoforms (i.e., a short p110 isoform in the nucleus and a long p150 isoform that localizes predominantly in the cytoplasm). These isoforms are transcribed using different promoters, with the p150 isoform augmentatively induced after type I IFN stimulation (6). Although ADAR1 and ADAR2 are ubiquitously expressed, ADAR1 p110 and ADAR2 are abundantly found in the brain, whereas ADAR1 p150 is highly expressed in the thymus (7–10).
Adar1 knockout (KO) (A1−/−), Adar1 p150-specific KO, and Adar1 knock-in mice that harbor an editing-inactive E861A point mutation (A1E861A/E861A) embryonically lethal and show high expression of type I IFN and type I IFN-stimulated genes (ISGs) (11–14). However, this lethality and elevated ISG expression can be rescued by concurrent deletion of the melanoma differentiation-associated protein 5 (MDA5, derived from the Ifih1 gene), a cytosolic exogenous dsRNA sensor, or its downstream target, mitochondrial antiviral-signaling protein (MAVS) (13, 15, 16). Therefore, RNA editing catalyzed by ADAR1, especially the p150 isoform, is essential to prevent the recognition of endogenous dsRNA as nonself by MDA5. Of note, concurrent deletion of MDA5 or MAVS prolongs the survival of A1−/− mice to a few days after birth (15, 16), whereas MDA5-decifient A1E861A/E861A mice (A1E861A/E861A Ifih1−/−) survive until adulthood (13, 17). These data suggest that ADAR1 has RNA-editing–independent functions. However, although some functions have been reported using an in vitro system (18–21), the RNA-editing–independent functions of ADAR1 in vivo remain to be specified.
T cells that originate from progenitor cells from fetal liver and adult bone marrow play a central role in the adaptive immune system (22). After migration to the thymus, progenitor cells become differentiated into CD4−CD8− double-negative (DN) thymocytes. The DN stage is further subdivided into four stages: from DN1 (CD44+CD25−) through DN2 (CD44+CD25+), and from DN3 (CD44−CD25+) to DN4 (CD44−CD25−). At the DN3 stage, thymocytes undergo an essential checkpoint called β-selection by forming a pre-TCR complex, which is composed of a functionally rearranged TCR β-chain, an invariant pre-T α-chain, and CD3 molecule (23, 24). Subsequently, signal transduction mediated by the pre-TCR complex allows DN3 thymocytes to survive, proliferate, and differentiate into the DN4 stage. These eventually become CD4+CD8+ double-positive (DP) thymocytes, which finally mature to either CD4+CD8− single-positive (4SP) or CD4−CD8+ single-positive (8SP) thymocytes (22). Given that rearrangements of DNA at the TCRβ gene loci occur randomly, most of the out-of-frame TCRβ transcripts are not translated but removed by nonsense-mediated decay (NMD) (25, 26). Thus, tight regulation of the clearance of out-of-frame TCRβ transcripts is required for the proper development of thymocytes at early stages (27). In addition, cross-talk between thymocytes and medullary thymic epithelial cells (mTECs) is essential for T cell development (28, 29).
Given that ADAR1, especially the p150 isoform, is highly expressed in the thymus, we recently investigated its role during the late stage of thymocyte development by crossing Adar1flox/flox (A1flox/flox) with Cd4cre mice (Cd4cre A1flox/flox mice) in which Cre recombinase was expressed from the DP stage onward (7). Although we did not observe a significant reduction in the total number of thymocytes without enhancing apoptosis in these mutant mice, the proportions of 4SP and 8SP thymocytes were significantly decreased with the elevated expression of type I ISGs, leading to the accumulation of DN and DP thymocytes. Consequently, the establishment of central self-tolerance was severely impaired (7, 30). Of note, concurrent deletion of MDA5 restored the populations of DN, DP, 4SP, 8SP thymocytes, and ISG expression in Cd4cre A1flox/flox mice, which suggests that ADAR1-mediated RNA editing regulates the late stage of thymocyte development by preventing activation of the MDA5-dependent pathway. However, the role of ADAR1 during the DN stage remains unknown.
In this study, we report that ADAR1 regulates early thymocyte development through MDA5-dependent and -independent pathways. We found that early thymocyte-specific deletion of ADAR1 in mice caused severe thymic atrophy with enhanced apoptosis and the impairment of DN3-to-DN4 transition accompanied by the enhanced expression of type I ISGs and loss of TCR expression. Concurrent deletion of MDA5 partially restored the total number of thymocytes by suppressing apoptosis and the enhanced expression of type I ISGs, whereas transition of ADAR1-deficient DN thymocytes to the DP stage was still severely restricted because of a lack of amelioration of reduced TCR expression upon MDA5 depletion. This was most likely caused by an increase in out-of-frame TCRβ transcripts in an MDA5-independent manner. In contrast, the forced expression of TCR efficiently promoted DN3-to-DN4 transition, whereas ADAR1-deficient DN thymocytes did not transition to the DP stage. In addition, TCR expression did not ameliorate the enhanced apoptosis of ADAR1-deficient DN thymocytes. These results suggest that forced TCR expression was not sufficient to achieve a transition from the DN to the DP stage. Finally, we found that, although it was not fully restored, forced TCR expression and concurrent deletion of MDA5 efficiently ameliorated the impairment of DN-to-DP transition. These findings indicate that RNA-editing–dependent and –independent functions of ADAR1 synergistically regulate early thymocyte development.
Materials and Methods
Mice were bred and maintained in the Institute of Experimental Animal Sciences, Faculty of Medicine, Osaka University at 23 ± 1.5°C under automatic control with a 12 h light/dark cycle and humidity of 45 ± 15%. All animal experimental procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Osaka University.
A1flox/flox, A1E861A/E861A, A2flox/flox, and Rag2 KO mice were kindly provided by Drs. Kazuko Nishikura (The Wistar Institute), Carl R. Walkley (University of Melbourne), Shin Kwak (The University of Tokyo), and Kiyoshi Takeda (Osaka University), respectively. Lckcre, HY-TCR+, and Ifih1−/− mice were obtained from the National Institute of Biomedical Innovation (Osaka, Japan), the European Mouse Mutant Archive repository (Munich, Germany), and the Oriental Bio Service (Kyoto, Japan), respectively. All of the mice used in this study had a C57BL/6J genetic background.
Flow cytometry and cell sorting
To prepare single-cell suspensions of thymocytes or splenocytes for flow cytometry and cell sorting, freshly isolated thymus or spleen was mashed through a 70-μm cell strainer (Greiner Bio-One). The cell suspensions were subsequently treated with RBC lysis buffer (BioLegend) for lysing RBCs. Cells were then counted and stained with fluorescent dye–conjugated Abs against CD4 (RM4-5; BioLegend) and CD8 (53-6.7; BioLegend) with or without Abs against CD1d (1B1; BioLegend), CD11c (N418; BioLegend), CD25 (PC61.5; Tonbo Biosciences), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), MHC class II (MHC II; I-A/I-E: M5/114.15.2; BioLegend), NK1.1 (PK136; BioLegend), TCRβ (H57-597; Tonbo Biosciences), and TCRγδ (GL3; BD Biosciences). After incubating cells with Abs at 4°C for 30 min, the cells were washed with Dulbecco’s PBS and analyzed by flow cytometry or sorted by cell sorter. For intracellular Ki-67 staining, cells stained with anti-CD4, anti-CD8, and anti-TCRβ Abs were fixed and permeabilized by Fix Buffer I (BD Biosciences) and Perm/Wash Buffer I (BD Biosciences), respectively, following the manufacturer’s protocol and then incubated with anti–Ki-67 Ab (16A8; BioLegend) for 30 min. To count the number of cortical thymic epithelial cells (cTECs) and mTECs, suspensions of thymic stromal cells were isolated as described previously (31), with several modifications. In brief, freshly isolated thymus was cut into small pieces and then incubated with digestion buffer containing 0.1 U/ml Liberase (Merck) and 2 μg/ml DNase I (Thermo Fisher Scientific) at 37°C for 30 min. After collecting supernatants, the remaining tissues were incubated with digestion buffer at 37°C for 20 min and then passed through an 18-gauge needle. After collecting supernatants, the remaining tissues were further incubated with digestion buffer at 37°C for 20 min and mechanically broken up using a 26-gauge needle. After complete digestion, the suspension of stromal cells in pooled supernatants was filtered through a 70-μm cell strainer. Cells were stained with anti-CD45 (30-F11; BioLegend), anti–epithelial cell adhesion molecule (EpCAM; CD326: G8.8; BioLegend), and anti–Ly-51 (6C3; BioLegend) Abs at 4°C for 30 min followed by washing with Dulbecco’s PBS before analysis with a flow cytometer. Data were collected using a FACSCanto II flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star).
Annexin V staining was used to evaluate apoptosis levels, as previously described (7). In brief, thymocytes were incubated with allophycocyanin-conjugated annexin V (640919; BioLegend) in Annexin V Binding Buffer (BD Biosciences) for 15 min at 25°C in the dark, according to the manufacturer’s protocol. Stained thymocytes were then analyzed using a FACSCanto II flow cytometer (BD Biosciences).
Total RNA was extracted from DN thymocytes or each DN subpopulation, as previously described (7). In brief, thymocytes were stained with fluorescent dye–conjugated Abs against CD4 (RM4-5; BioLegend) and CD8 (53-6.7; BioLegend). The DN population was then fractionated using an SH800 cell sorter (Sony Biotechnology). To fractionate each DN subpopulation, thymocytes were stained with anti-CD25 (PC61.5; Tonbo Biosciences) and anti-CD44 (IM7; BioLegend) Abs, together with anti-CD4 and anti-CD8 Abs. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. To extract total RNA from thymic stromal cells, the tissue remnants of a macerated thymus on a cell strainer were used.
Quantitative RT-PCR analysis
As previously described (7), cDNA was synthesized from total RNA (50–500 ng) using a ReverTra Ace qPCR RT Master Mix with gDNA remover (TOYOBO). Mixtures of quantitative RT-PCR (qRT-PCR) reaction were prepared by combining target-specific primers and probes with THUNDERBIRD Probe qPCR Mix (TOYOBO). The qRT-PCR was performed using an ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems). Sequences of primers and probes for Adar1 (p150 and p110), Adar1 p150, Ifih1, Ddx58, Irf7, Ifit1, Cxcl10, Rsad2, and GAPDH have been previously described (7). The expression level of each mRNA relative to that of GAPDH mRNA was calculated by the ΔΔCt method.
Treatment with anti-CD3ε Ab
Mice were i.p. injected with 50 μg of anti-mouse CD3ε Ab (145-2C11; BioLegend) (32). Seven days after injection, the thymus of each mouse was removed to prepare single-cell suspensions of thymocytes.
Evaluation of out-of-frame TCRβ transcripts
Total RNA (100 ng) purified from DN thymocytes was incubated with 0.1 U/μl DNase I (Thermo Fisher Scientific) at 37°C for 15 min. Next, cDNA was synthesized using a SuperScript III First-Strand Synthesis System with random hexamers (Thermo Fisher Scientific). The TCRβ gene segments were amplified by Phusion Hot Start High-Fidelity DNA Polymerase (Thermo Fisher Scientific) using the following primers: Vβ2 long (5′-ATCCCTGGATGACGTGGTATC-3′) and Jβ2.2 long (5′-CAGCTTTGAGCCTTCACCAAAGTA-3′) (33). The resultant PCR products were separated by agarose gel electrophoresis, purified with a QIAEX II Gel Extraction Kit (QIAGEN), and then subcloned into a Zero Blunt TOPO vector using a Zero Blunt TOPO PCR Cloning Kit for Sequencing (Thermo Fisher Scientific). The TCRβ gene segment in each plasmid was sequenced with a M13 reverse primer attached to the kit (Thermo Fisher Scientific).
The Mann–Whitney U test was used for statistical analyses. All values are displayed as the mean ± SEM. Statistical significance was set at *p < 0.05 and **p < 0.01.
ADAR1 was required for early T cell development
To investigate the role of ADARs in early T cell development, we crossed A1flox/flox and Adar2flox/flox (A2flox/flox) mice with Lckcre mice, in which Cre recombinase is expressed from the DN stage onward, under the control of the proximal Lck promoter (34). Although no abnormality was observed in the thymus of Lckcre A2flox/flox mice (Fig. 1A), Lckcre A1flox/flox mice manifested a significant reduction in the total number of thymocytes (0.7% of A1 control mice) together with severe thymic atrophy (Fig. 1A, 1B, Supplemental Table I). These results were in contrast to previous results showing that ADAR1 deletion from the DP stage affected the differentiation but not the total number of thymocytes (7). In addition, ADAR2-deficient thymocytes were normally mature, whereas most ADAR1-deficient thymocytes were retained at the DN stage, resulting in a severe reduction in the number of DP, 4SP, and 8SP thymocytes (Fig. 1C, 1D). The number of CD4+ and CD8+ cells was also significantly reduced in the spleen of Lckcre A1flox/flox mice (Fig. 1E, 1F). Taken together, these results suggest that ADAR1 but not ADAR2 is required for the survival and differentiation of thymocytes during early developmental stages.
ADAR1 deficiency induced reduced TCRβ expression but increased expression of type I ISGs in thymocytes
Given that most ADAR1-deficient thymocytes were retained at the DN stage, we further investigated subpopulations of DN thymocytes by dividing these into from DN1 to DN4 stages. qRT-PCR analysis revealed that the expression level of total Adar1 mRNA as well as Adar1 p150 mRNA at the DN1 stage was the highest of all DN stages and was comparable to that in thymic stromal cells (Fig. 2A, 2B). Adar1 expression was decreased ∼2-fold at the DN2 stage and tended to become slightly decreased up to the DN4 stage (Fig. 2A, 2B). During this developmental change in Adar1 expression, we found that Lck promoter-driven Adar1 deletion induced a significant decrease in the relative proportion of DN4 thymocytes among DN subpopulations (Fig. 2C). In addition, TCRβ expression on the surface of DN thymocytes, which is required for survival, proliferation, and differentiation of most thymocytes, was barely detectable in ADAR1-deficient DN thymocytes (Fig. 2D). In contrast, although some thymic progenitor cells can differentiate into non-αβ T cells, such as γδ T cells, dendritic cells, and NK cells (35–37), the expression of these cell markers on DN thymocytes was not significantly changed in Lckcre A1flox/flox mice (Fig. 2E–G). These results suggest that ADAR1 deficiency arrested thymocyte maturation at the DN stage because of a severe reduction in TCRβ expression, at least in part. Therefore, we next investigated TCRβ expression on the surface of splenic CD4+ and CD8+ cells. Although the proportion of TCRβ+ T cells in the spleen of Lckcre A1flox/flox mice was significantly lower than that in A1 control mice, more than 60% of ADAR1-deficient splenic T cells expressed TCRβ (Supplemental Fig. 1A, 1B). Furthermore, the proportion of Ki-67+ proliferative cells among TCRβ-expressing T cells was significantly increased, especially in CD4+ cells in the spleen of Lckcre A1flox/flox mice (Supplemental Fig. 1C, 1D). In addition, Lckcre A1flox/flox mice showed an increase in the proportion of splenic CD44+ effector/memory T cells, resulting in a decreased proportion of CD62L+ naive cells (Supplemental Fig. 1E, 1F). Collectively, these results indicate that ADAR1 deficiency in thymocytes from the early stages may lead to cell expansion in peripheral tissues to compensate for the severely reduced number of thymocytes and stacking at the DN stage, generating a lymphopenic environment.
We further observed enhanced apoptosis of DN thymocytes in Lckcre A1flox/flox mice (Fig. 3A). In addition, Lckcre A1flox/flox mice showed a significant decrease in the number of mTECs but not cTECs (Fig. 3B). These abnormalities most likely contributed to severe thymic atrophy together with a significant reduction in thymocytes (Fig. 1A, 1B). Given that apoptosis is induced by the loss of ADAR1-mediated RNA editing coupled with the increased expression of type I ISGs in some cell types, such as hematopoietic stem cells, erythroid cells, B cell lineage cells, and hepatocytes (11–13, 15, 38–40), we evaluated the expression level of type I ISGs in DN thymocytes. This analysis demonstrated that the expression of all type I ISGs examined increased ∼10–100-fold in ADAR1-deficient DN thymocytes (Fig. 3C). Taken together, the loss of ADAR1 inhibits TCRβ expression but promotes expression of type I ISGs, which leads to enhanced apoptosis and the impaired maturation of DN thymocytes.
Aberrant early development of ADAR1-deficient thymocytes was not fully rescued by blockade of MDA5-dependent pathway
To investigate the contribution of the MDA5-dependent pathway to the abnormalities found in ADAR1-deficient thymocytes, we deleted MDA5 from thymocytes by crossing Lckcre A1flox/flox mice with Ifih1−/− mice. Although the thymus in Lckcre A1flox/flox mice was too small to examine ADAR1 expression, we could quantify the expression level of Adar1 mRNA in DN subpopulations prepared from Lckcre A1flox/flox Ifih1−/− mice. This analysis demonstrated that Lck promoter-driven Adar1 deletion tended to start from the DN3 stage and led to efficient deletion at the DN4 stage (Fig. 4A), which was in accordance with the inhibited transition between DN3 and DN4 stages in Lckcre A1flox/flox mice (Fig. 2C). We then examined the effect of MDA5 deletion on the expression of type I ISGs and found that, although it was not completely ameliorated, concurrent deletion of MDA5 significantly suppressed the aberrant induction of type I ISGs found in ADAR1-deficient DN thymocytes (Fig. 4B). Furthermore, the enhanced apoptosis of ADAR1-deficient DN thymocytes was normalized by the concurrent deletion of MDA5 (Fig. 4C). However, the reduced number of thymocytes was accompanied by thymic atrophy that was only partially restored (29.6% of A1 control mice) in Lckcre A1flox/flox Ifih1−/− mice (Fig. 5A, 5B, Supplemental Table I). In addition, although it was slightly ameliorated, the transition to DP, 4SP, and 8SP stages was still severely inhibited (Fig. 5C). This is attributable to the partial rescue of the transition from the DN3 to DN4 stage (Fig. 5D). Therefore, we examined TCRβ expression on the surface of ADAR1/MDA5–deficient DN thymocytes and found that concurrent deletion of MDA5 could not restore TCRβ expression at all (Fig. 5E). Of note, the proportion of out-of-frame TCRβ transcripts, which are not used for translation, was significantly increased in Lckcre A1flox/flox Ifih1−/− mice (Fig. 5F). Taken together, these results suggest that concurrent deletion of MDA5 suppresses enhanced apoptosis that is coupled with the aberrant induction of type I ISGs found in ADAR1-decifient DN thymocytes, whereas TCRβ expression, which is essential for maturation from the DN stage, is regulated in a MDA5 pathway–independent manner. Importantly, we detected no abnormalities in development during the DN stage in TCRβ expression on DN thymocytes or in apoptosis level in A1E861A/E861A Ifih1−/− mice (Supplemental Fig. 2A–C), which suggests that ADAR1 regulates TCRβ expression in an RNA-editing–independent manner.
Forced expression of TCR transgene rescued DN3-to-DN4 transition but not enhanced apoptosis in Lckcre A1flox/flox mice
Given that inhibition of the MDA5-dependent pathway did not restore TCRβ expression in ADAR1-deficient DN thymocytes, we next investigated the effect of forced expression of the TCR transgene on aberrant thymocyte development caused by ADAR1 deficiency. Thus, we crossed Lckcre A1flox/flox mice with HY-TCR transgenic (Tg) mice (41), which express TCRα and TCRβ Tg genes, to create Lckcre A1flox/flox HY-TCR+ (Lckcre A1flox/flox TCR Tg) mice. Given that HY-TCR–expressing thymocytes are subjected to negative selection in male but not female mice, we used only female mice for subsequent analyses. It is noteworthy that the normal size of the thymus and number of thymocytes in HY-TCR Tg mice (A1 control TCR Tg mice) was ∼60% and ∼50% of A1 control mice, respectively (Fig. 6A, 6B, Supplemental Table I). However, although Lckcre A1flox/flox TCR Tg mice exhibited a 10-fold upregulation of the total thymocyte number with a 3-fold larger thymus as compared with Lckcre A1flox/flox mice, forced TCR expression was not sufficient to promote transition of ADAR1-deficient DN thymocytes to the DP stage (Fig. 6C). Thus, we further investigated subpopulations of DN thymocytes and TCRβ expression on DN thymocytes and showed that transition from the DN3 to DN4 stage was markedly ameliorated in parallel with the increased expression of TCRβ (Fig. 6D, 6E). Nevertheless, although we reconfirmed that the administration of Abs against CD3ε, a component of the TCR complex that mimics TCR stimulation, induced DP thymocytes in Rag2 KO mice in which a pre-TCR complex was not expressed because of defects in TCRβ rearrangement (32, 42), it failed to induce the transition of DN thymocytes to the DP stage in Lckcre A1flox/flox mice (Supplemental Fig. 3A, 3B). These results indicate that not only TCR expression but also the TCR signaling pathway are impaired in ADAR1-deficient DN thymocytes. In addition, forced TCR expression did not suppress the enhanced apoptosis found in ADAR1-deficient DN thymocytes (Fig. 6F), which suggests that enhanced apoptosis is caused by activation of the MDA5 pathway but was independent of reduced TCR expression. Taken together, forced expression of TCR increases the total number of mainly DN ADAR1-deficient thymocytes and promotes a transition from a DN3 to DN4 stage, whereas it is insufficient to achieve transition from a DN to a DP stage.
Forced TCR expression and concurrent KO of MDA5 synergistically ameliorated the aberrant early development of ADAR1-deficient thymocytes
Finally, to investigate the combined effect of TCR expression and inhibition of MDA5 activation on the development of ADAR1-deficient thymocytes, Lckcre A1flox/flox TCR Tg mice were crossed with lfih1−/− mice to generate Lckcre A1flox/flox lfih1−/− TCR Tg mice. Consequently, concurrent deletion of MDA5 increased the total thymocyte number and thymus size ∼3.1-fold and ∼1.4-fold, respectively, as compared with those in Lckcre A1flox/flox TCR Tg mice (Fig. 7A, 7B, Supplemental Table I). Numbers and weights reached ∼40% and ∼75% of those found in A1 control TCR Tg mice (Fig. 7A, 7B, Supplemental Table I). Furthermore, although most thymocytes (>95%) were retained at the DN stage in Lckcre A1flox/flox TCR Tg mice, more than 30% of total thymocytes were differentiated into DP, 4SP, or 8SP stages in Lckcre A1flox/flox lfih1−/− TCR Tg mice, a bigger number than found in Lckcre A1flox/flox lfih1−/− mice (Figs. 5C, 6C, 7C, 7D). Collectively, these data suggest that ADAR1 regulates early thymocyte development via MDA5-dependent and -independent pathways that exert synergistic effects.
ADAR1 deficiency during the early developmental stages of thymocytes inhibited the transition from DN3 to DN4. This was attributable to the abolished expression of TCRβ, which forms a part of the pre-TCR complex. This is consistent with the findings that DN3-to-DN4 transition was severely disrupted in mice deficient in genes that are involved in the pre-TCR complex or its signaling pathway, such as RAG (43, 44), SLP-76 (32, 45), LAT (46), and CD3ε (47). Of note, although enhanced apoptosis and the elevated expression of type I ISGs found in ADAR1-deficient DN thymocytes were rescued by the concurrent deletion of MDA5, the expression of TCRβ was not recovered. In addition, the level of TCRβ was normal in A1E861A/E861A mice under the condition of MDA5 deletion; ADAR1 protein, rather than its RNA-editing activity, is required for TCRβ expression during early T cell development. These findings suggest the presence of an RNA-editing–independent function of ADAR1 in vivo, which is required for T cell development. It is known that the rearrangement of DNA at TCRβ gene loci randomly occurs so that T cell clones have immunological diversity. This resulted in two-thirds of rearranged TCRβ transcripts that were out-of-frame, eventually eliminated by the NMD pathway (25, 26, 48). Indeed, Tg mice that ubiquitously express a dominant-negative form of human Upf1, an effector protein of the NMD pathway, exhibit a reduced number of total thymocytes and show impaired transition from a DN to a DP stage; this is accompanied by the accumulation of out-of-frame TCRβ transcripts and abolished TCRβ expression (49). Furthermore, similar phenotypes can be observed in Lckcre Upf2flox/flox mice (27). Of note, it was reported that ADAR1 binds directly to Upf1 in an RNA-independent manner in the nucleus and participates in mRNA surveillance together with Upf1 (50). Therefore, the accumulation of out-of-frame TCRβ transcripts induced by ADAR1 deficiency may be caused by impaired mRNA surveillance. Accordingly, an in-frame TCR transgene was expressed on the surface of ADAR1-deficient DN thymocytes in Lckcre A1flox/flox TCR Tg mice, and allowed these to differentiate into DN4 thymocytes. Therefore, our findings suggest that ADAR1 may be required for mRNA surveillance, especially for out-of-frame TCRβ transcripts during early T cell development. Further studies are required to determine whether this is an RNA-editing–independent function of ADAR1. Another possible RNA-editing–independent function involved in early T cell development may be related to microRNA processing. It has been demonstrated that ADAR1 promotes microRNA maturation by forming heterodimers with Dicer in an RNA-editing–independent manner using an in vitro system (18). Given that the depletion of microRNAs by Dicer KO in thymocytes at an early developmental stage increases apoptosis, resulting in a reduction in total thymocyte number (51), it is worthwhile investigating whether ADAR1 may contribute to the processing of certain microRNAs required for early T cell development in an RNA-editing–independent manner.
Our studies demonstrated that concurrent deletion of MDA5 restored the DN4-to-DP transition of ADAR1-deficient thymocytes in a very limited manner without rescuing the expression of TCRβ. Therefore, we had initially expected that enforced TCR transgene expression would restore DN4-to-DP transition. However, unexpectedly, although enforced TCR transgene expression successfully resulted in the transition of ADAR1-deficient thymocytes from the DN3 to the DN4 stage, it failed to generate DP thymocytes. This suggests that the transition of ADAR1-deficient thymocytes to the DP stage requires other factors in addition to TCR expression. Indeed, we showed that the administration of anti-CD3ε Ab, which mimics TCR stimulation, failed to induce the transition from the DN to the DP stage in Lckcre A1flox/flox mice, which was in contrast to the efficient transition observed in Rag2 KO mice that express no pre-TCR complex because of defects in TCR rearrangement (32, 42). This data indicates that TCR signaling in DN thymocytes is likely disrupted in Lckcre A1flox/flox as well as Lckcre A1flox/flox TCR Tg mice. To support this idea, in accordance with our previous finding that CD4 promoter-driven Adar1 deletion from DP thymocytes, which normally express TCR, did not respond to TCR stimulation, whereas this impaired TCR signal transduction was rescued by the concurrent deletion of MDA5 (7), enforced expression of the TCR transgene, and concurrent deletion of MDA5 synergistically improved the transition of ADAR1-deficient thymocytes from the DN4 to the DP stage. These findings suggest that early T cell development requires coordinated regulation by ADAR1 via MDA5-dependent and -independent pathways.
We found that the number of mTECs was markedly reduced, which most likely contributed to the severe thymic atrophy observed in Lckcre A1flox/flox mice. During negative selection in the thymus, mTECs present a variety of self-antigens to eliminate thymocytes expressing TCRs that strongly react with these cells (52). However, the maturation of mTECs also requires TCR+ thymocytes, given that mTECs are substantially lost and not organized in discrete medullary areas in TCR-deficient mice (53). This evidence suggests that the severe reduction in the number of mTECs in Lckcre A1flox/flox mice was probably caused by the abolished expression of TCRβ in ADAR1-deficient thymocytes. Intriguingly, it was reported that RNA-editing activity in mTECs, which is likely mediated by ADAR1, is comparable to that in the brain, one of the organs showing the highest RNA-editing frequency (8). These higher editing events may increase the diversity of the self-antigen repertoire required for thymic education of immature thymocytes. Therefore, it is important to elucidate the role of ADAR1 in mTECs, for instance, by creating mTEC-specific Adar1 KO mice, to understand the cross-talk between mTECs and thymocytes, which might, at least in part, be mediated by ADAR1 and its RNA-editing activity.
Mutations in the ADAR1 gene are known to be a cause of the autoimmune disorder Aicardi–Goutières syndrome (AGS), an inflammatory encephalopathy that mimics a congenital viral infection (54, 55). Given that severe thymic atrophy is commonly seen during infections by various pathogens (56–59), severe thymic atrophy may also occur during AGS pathogenesis as observed in Lckcre A1flox/flox mice. Indeed, it has been reported that some patients with AGS exhibit lymphopenia (60, 61). Therefore, it is worth investigating whether early T cell development in the thymus is affected in patients with AGS, which may provide a novel insight into the pathogenesis of AGS.
We thank Drs. Kazuko Nishikura (The Wistar Institute), Carl R. Walkley (University of Melbourne), Shin Kwak (The University of Tokyo), and Kiyoshi Takeda (Osaka University) for providing A1flox/flox, A1E861A/E861A, A2flox/flox, and Rag2 KO mice, respectively. We thank all staff at the Center for Medical Research and Education and the Center of Medical Innovation and Translational Research, Common Fundamental Technology Department, Graduate School of Medicine, Osaka University for technical support.
This work was supported by Japan Society for the Promotion of Science/Ministry of Education, Culture, Sports, Science, and Technology KAKENHI Grants-in-Aid 17K19352 (to Y. Kawahara), 15K19126 and 18K15186 (to T.N.), 18J11733 (to T.V.), and 18K11526 (to Y. Kato) and by grants from the SENSHIN Medical Research Foundation (to Y. Kawahara), the Nagao Memorial Fund, the Naito Foundation, the Astellas Foundation for Research on Metabolic Disorders, Novartis Research Grants (to T.N.), the Takeda Science Foundation, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Y. Kawahara and T.N.). T.V. was supported by scholarships from The Interdisciplinary Program for Biomedical Science of Osaka University and a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science/Ministry of Education, Culture, Sports, Science, and Technology.
The online version of this article contains supplemental material.
Abbreviations used in this article:
Adar1 knock-in mice that harbor an editing-inactive E861A point mutation
adenosine deaminase acting on RNA
cortical thymic epithelial cell
epithelial cell adhesion molecule
melanoma differentiation-associated protein 5
- MHC II
MHC class II
medullary thymic epithelial cell
The authors have no financial conflicts of interest.