Following thymic maturation, T cells egress as recent thymic emigrants to peripheral lymphoid organs where they undergo an additional maturation step to mature naive T cells that circulate through secondary lymphoid organs ready to be activated upon pathogenic challenges. Thymic maturation and peripheral T cell survival depend on several signaling cascades, but whether a dedicated mechanism exists that exclusively regulates homeostasis of mature naive T cells without affecting thymocytes and/or recent thymic emigrants remains unknown. In this article, we provide evidence for a specific and exclusive role of the WD repeat containing protein coronin 1 in the maintenance of naive T cells in peripheral lymphoid organs. We show that coronin 1 is dispensable for thymocyte survival and development, egress from the thymus, and survival of recent thymic emigrants. Importantly, coronin 1–deficient mice possessed comparable levels of peripheral T cells within the first 2 wk after birth but failed to populate the peripheral T cell compartment at later stages. Furthermore, dendritic cell– and IL-2/7–dependent T cell survival was found to be independent of coronin 1. Together, these results suggest the existence of a hitherto unrecognized coronin 1–dependent decision switch early during life that is responsible for peripheral naive T cell survival and homeostasis.
T cells originate in the bone marrow as common lymphoid progenitor cells from where they migrate to the thymus. There they undergo three distinct maturation stages, named after the expression of CD4 and CD8 surface molecules (1). During these three stages (double-negative, double-positive, and CD4 or CD8 single-positive [SP] stages), the cells rearrange their TCR and complete two selection processes (2, 3). The two selection processes, negative and positive selection, are needed to ensure that mature thymocytes egressing from the thymus are capable of mounting an immune response based on TCR stimuli and do not mount immune responses against self-molecules, resulting in autoimmunity (4, 5). The maturation and survival of thymocytes are linked to the stimulation via the TCR; depending on the signal strength mediated, the cells survive, are killed, or die by neglect (6, 7). Following egress from the thymus, cells are considered recent thymic emigrants. Recent thymic emigrants are a functionally and phenotypically distinct subset that shows differential expression of surface markers compared with mature naive T cells (reviewed in Ref. 8). Recent thymic emigrants have been shown to express lower levels of IL-7Rα and higher levels of TCR–CD3 complexes, and they produce fewer cytokines (especially IL-2 and IL-4) under stimulating conditions (8–11). What drives the maturation of recent thymic emigrants to mature naive T cells and how their survival is regulated are not fully elucidated. For mature naive T cells, the survival has been linked to IL-7 signaling and interaction of the TCR with MHC (12). For recent thymic emigrants, the survival appears to be independent of MHC–TCR interactions (13), although signaling via IL-7 has recently been linked to an increase in the levels of the prosurvival molecule Bcl-2 (14).
During the first 2 wk of life in mice, the majority of the peripheral T cell pool consists of recent thymic emigrants, which is reduced to ∼20% in 6-wk-old animals, but recent thymic emigrants can also be found in aged animals (15). However, the cells considered recent thymic emigrants in a newborn mouse are functionally different from those observed in an adult mouse. Most striking is their response to IL-7, which results in the proliferation of neonatal, but not adult, recent thymic emigrants. This has been postulated to be of importance in the filling of the T cell compartment in newborn animals (16).
Other than the MHC–TCR interaction and IL-7 signaling, not much is known regarding the molecular requirements for naive T cell homeostasis. We and other investigators have shown that the absence of coronin 1, a member of the WD repeat protein family of coronin proteins, results in severe depletion of naive T cells from peripheral immune organs in mice and human (17–22). However, it remains unknown which precise thymocyte and/or T cell subsets require coronin 1 for their development and/or maintenance. Although in one study, coronin 1 has been suggested to be important for T cell egress (19), no such thymocyte accumulation was observed in other studies (17, 18, 23, 24).
We show that, although coronin 1 is indispensable for the survival of mature naive T cells, mice deficient for coronin 1 have similar thymocyte and recent thymic emigrant numbers compared with wild-type mice. We also found that the role of coronin 1 in peripheral T cell survival is dispensable at early stages of life but becomes essential after 2 wk of age. Additionally, we show that the cells egressing from the thymus respond equally in terms of survival to stimuli provided by dendritic cells or the cytokines IL-2 and IL-7. Taken together, our results suggest the existence of a T cell survival decision switch at 2–3 wk of age that leads to the dependency of T cells on coronin 1 expression.
Materials and Methods
All animals were bred, according to cantonal ethic and husbandry standards, at the animal facility of the Biozentrum, University of Basel, and experiments were performed according to the governing cantonal veterinary rules (license numbers 1893 and 2326). Coronin 1–deficient animals have been described earlier (17, 25, 26) and were from The Jackson Laboratory (JAX stock no. 030203). C57BL/6-Ly5.1 mice were also obtained from the Jackson Laboratory (JAX stock no. 002014). Single-cell suspensions were prepared by mashing organs through a polyamide mesh (150 μm; catalog number [cat. no.] 03-150-38; Sefar Nitex) with a plunger. All adult mice used were matched for age (6–8 wk or >10 wk) and sex; newborn animals were littermates but were not matched for sex.
For genotyping, toes of animals were digested overnight in digestion buffer (100 mM Tris-HCl [pH 8.5], 5 mM EDTA, 0.2% SDS, and 200 mM NaCl supplemented with 100 μg/ml proteinase K). Genotyping was performed by PCR (35 cycles of 1 min at 95°C, 30 s at 55°C, and 40 s at 72°C on an Eppendorf Mastercycler) with the following primers: Coro1aLoxSA1, 5′-GAGACAGGACTCTCTTTG-3′; Coro1aLoxLA1, 5′-GTCCTCAGTAGCTGACTG-3′; and Coro1aLoxMA1: 5′-TAGCAGAAAACCCCAAGC-3′.
The following Abs were obtained from BioLegend: anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53.6-7), anti-CD24 (M1/69), anti-CD45.1 Ly5.1 (A20), anti-CD62L (MEL-14), anti-CD127 IL-7Rα (A7R34), anti-Qa2 (695H1-9-9), and anti-Ter-119 (Ter119). Sphingosine-1-phosphate receptor 1 (S1P1) Ab was from R&D Systems (MAB7086), and LIVE/DEAD marker was from Thermo Fisher Scientific (cat. no. L10120). FACS acquisition was performed on a BD Fortessa with Diva software (v10) and analyzed using FlowJo v10 (TreeStar). Cells were counted, and 2 × 106 cells were stained in 96-well U-bottom plates, with the Ab combinations indicated, in FACS buffer (PBS supplemented with 2% FCS and 2 mM EDTA). Cells were incubated for 30 min on ice; all Abs were used at 1:100. LIVE/DEAD marker was used at 1:1000. S1P1 receptor staining was performed essentially as described by Tang et al. (27). In brief, the Ab was used at a dilution of 1:10, and EDTA was excluded in the FACS buffer. Following a 30-min incubation on ice, cells were incubated with 1:100 biotinylated anti-rat IgG2a (MRG2a-83) for 30 min on ice. After blocking with 1:100 rat IgG (cat. no. I8015; Sigma) for 10 min, the remaining Ab mixture, as well as streptavidin coupled to allophycocyanin or PE, was added. After staining, cells were pelleted (5 min at 4°C, 350 × g), resuspended in 200 μl of FACS buffer, transferred to polystyrene tubes, and analyzed within 2 h.
CD8 depletion was performed based on negative-selection protocols from STEMCELL Technologies. In brief, thymocytes were prepared as described, counted, and resuspended at 100 × 106 per milliliter in PBS/2% FCS. Cell suspensions were kept in 4-ml polystyrene tubes or 13-ml round-bottom tubes, as recommended by STEMCELL Technologies. Cells were incubated with biotinylated Abs for CD8 and TER119 (12.5 μg/ml) and rat sera (50 μl/ml; from a STEMCELL Technologies negative isolation kit; cat. no. 19852). After a 10-min incubation at room temperature, 75 μl/ml EasySep Streptavidin RapidSpheres 50001 (cat. no. 19860; STEMCELL Technologies) were added for 3 min at room temperature. PBS/2% FCS was added to a total volume of 2.5 or 5 ml, depending on the tube size, and transferred to a magnet (EasySep Magnet) for 3 min at room temperature. The supernatant was decanted into fresh tubes, sedimented (10 min 4°C at 350 × g), counted, and stained for CD3, CD4, CD8, CD69, CD62L, and LIVE/DEAD marker, as described above. FACS was performed on a BD FACSAria III, and purity was always >98%. After cell sorting, cells were pelleted (10 min at 4°C at 350 × g) and resuspended directly in TRI Reagent (Sigma; cat. no. T9424) for RNA isolation or in the media indicated at a concentration of 0.75 × 106 cells per milliliter.
After cell sorting, thymocytes (1.5 × 106) were resuspended in 400 μl of TRI Reagent and stored at −80°C. RNA was isolated according to the manufacturer’s protocol (Zymo Research Direct-zol RNA Isolation Kit; cat. no. R2073), followed by transcription of 250 ng into cDNA (using the high-capacity cDNA reverse transcription kit; cat. no. 4368814; Thermo Fisher Scientific). Ten nanograms of cDNA were used for each quantitative RT-PCR (qPCR) reaction, which were performed in triplicates on MicroAmp Optical 96-Well Reaction Plates (cat. no. N8010560; Thermo Fisher Scientific). Amplicon detection was based on SYBR Green reaction mix (cat. no. 4367659; Thermo Fisher Scientific), and PCR was performed on a StepOnePlus Real-Time PCR System using StepOne v2.2 software (both from Applied Biosystems). Results were analyzed using the comparative CT method. Primers were synthesized by Microsynth (Balgach, Switzerland). The following primer sequences were used: NF-κB activation protein (NKAP) forward, 5′-GCGTATCCCAAGAAGAGGTG-3′ and reverse, 5′-GAAGTCGAACAGCCTCCATT-3′; Foxp1 forward, 5′-CAGCCACGAAAGAAACAGAAG-3′ and reverse, 5′-GGTCCTGGTCACCTGATTATA-3′; Krüppel-like factor 2 (KLF2) forward, 5′-ATGGCGCTCAGCGAGCCTAT-3′ and reverse, 5′-AGCAGCTCTGTTCCCAGGCT-3′; and GAPDH forward, 5′-GAGCCAAACGGGTCATCATC-3′ and reverse: 5′-GAGGGGCCATCCACAGTCTT-3′.
Signal joint TCR excision circle analysis
For signal joint TCR excision circle (sjTREC) analysis (28, 29), genomic DNA was isolated from single-cell suspensions of 5 × 106 cells by digestion of 107 cells per milliliter in 10 mM Tris-HCl (pH 7.4) supplemented with 100 μg/ml proteinase K for 1 h at 56°C and 1200 rpm. Proteinase K was inactivated by heating samples to 95°C for 10 min, and 5 μl of sample was used per reaction in subsequent assays. Primers for C57BL/6 mouse sjTREC analysis were based on Broers et al. (30), and DNA input was normalized using a probe for the transferrin receptor (cat. no. 4458366; Thermo Fisher Scientific). Results were analyzed using the comparative CT method.
Thymocyte adoptive transfer
Total thymocytes from 2-wk-old mice were harvested, and 9 × 106 cells were injected i.v. into Ly5.1 recipient mice (31). The thymus, spleen, and mesenteric lymph nodes were harvested 4–24 h later and stained with T cell maturation markers (CD4, CD8, CD24, Qa2 and CD62L), together with CD45.1, as described above.
In vivo proliferation assay by BrdU incorporation
Mice (3 wk old) were injected i.p. with 0.5 mg of BrdU in sterile PBS. Two to four hours later, thymus, spleen, and mesenteric lymph nodes were harvested and stained using anti-BrdU Ab (cat. no. 557891; BD Pharmingen), according to the manufacturer’s protocol, followed by surface T cell markers using anti-CD4 and anti-CD8 Abs.
Cell culture and survival assays
For preparation of dendritic cells, bone marrow of Ly5.1 mice was flushed from the femur and tibia with a 24G syringe. Bone marrow was frozen in 10% DMSO/90% FCS (low endotoxin fetal bovine serum; cat. no. A15-102; lot no. A10209-2936; PAA) and used for subsequent rounds of dendritic cell preparations. For dendritic cell preparation, 6 × 106 cells were seeded into a 10-cm petri dish (cat. no. 351029; Falcon) in 8 ml of RPMI 1640 with 10% FCS, 1 mM sodium pyruvate (cat. no. S8636; Sigma), nonessential amino acids (cat. no. M7145; Sigma), 100 U/ml penicillin 100 μg/ml streptomycin (cat. no. 4333; Sigma), 2 mM l-glutamine (cat. no. 25030; Life Technologies), and 10 μM 2-ME (M7522; Sigma) containing 10 ng/ml GM-CSF (cat. no. 576302; BioLegend). Cells were kept for 7 d with 2 ml of media, and fresh cytokines were replenished every second day. For dendritic cell supernatants, cells were harvested on day 6, washed once in thymocyte media (see below), and cultured in thymocyte media without cytokines for 24 h before the experiment. For coculture experiments, dendritic cells were assessed on the experimental day by microscopy for general morphology; 100,000 cells were seeded into 96-well U-bottom plates (cat. no. 351177; Falcon) or Transwell plates (Corning HTS Transwell, 3 μm; cat. no. 3385) prior to the transfer of thymocytes.
For thymocyte-survival assays, cells were adjusted to 0.75 × 106 cells per milliliter, and 100 μl was added per well. In some experiments, cells were labeled with CellTrace Violet (CTV) dye (CellTrace Violet Cell Proliferation Kit, cat. no. C34557; Thermo Fisher Scientific). Media formulation was RPMI 1640, 10% FCS (cat. no. A15-101; lot no. A10109-1886; PAA), 1 mM sodium pyruvate, nonessential amino acids, penicillin-streptomycin, 2 mM l-glutamine, and 50 μM 2-ME. In general, medium was replenished after 2 d of culture by removal of 75 μl of medium and addition of the same volume. Culture volume was 200 μl in normal cultures and 250 μl for Transwell cultures to ensure proper diffusion between compartments. For dendritic cell supernatant experiments, the supernatant from the day of seeding was filtered (0.2 μm), and 100 μl of supernatant was added to each well. Aliquots of supernatant were stored at 4°C for media replenishment on day 2 of culture. Cytokines (IL-2 [cat. no. 575402] and IL-7 [cat. no. 577802, both from BioLegend]) were added at a concentration of 20 ng/ml. To assess survival at defined time points, cells were pelleted (10 min, 4°C at 350 × g) and stained for LIVE/DEAD marker and CD45.1, as described above. In some experiments, cells were also stained with Alexa Fluor 647–conjugated Bcl-2 Ab (C-2; cat. no. sc-7832; Santa Cruz Biotechnology) and Pacific Blue–conjugated Ki-67 Ab (16A8; cat. no. 652422; BioLegend) using intracellular staining reagent (FOXP3 Fix/Perm Buffer Set; cat. no. 421403; BioLegend).
Graphs were prepared and statistical analysis was performed using GraphPad Prism software (version 5 or 7) and Microsoft Excel.
Thymocyte development in wild-type and coronin 1–deficient animals
Coronin 1–deficient animals have been reported to show a severe lack of peripheral T cells, but whether coronin 1 is important for the development and maturation of thymocytes is unclear (17, 18). To analyze a potential role for coronin 1 in thymocyte development and/or survival, thymi of wild-type or coronin 1–deficient animals were isolated, and single-cell suspensions were prepared. Subsequently, the cells were stained for CD3, CD4, and CD8, and viable cells were assessed by flow cytometry. As shown in Fig. 1A, no difference in the percentage or the number of double-negative, double-positive, or SP cells could be found in the thymus of wild-type versus coronin 1–deficient animals. Because TCR rearrangements occur during the double-positive stages of thymocyte development, TCR usage was analyzed by flow cytometry. Total T cells were isolated via negative selection from the thymus of wild-type or coronin 1–deficient animals. The resulting cells were stained for CD3, CD4, and CD8, as well as with Abs detecting specific mouse Vβ T cells receptor isoforms, followed by FACS analysis. No difference was observed in CD3+CD4+ thymocytes with regard to their TCR repertoire (Fig. 1B). Together with the normal percentages of thymic populations, this indicates that coronin 1 is dispensable for thymocyte maturation up to the SP stage.
Following maturation, thymocytes exist in the thymus as late-stage thymocytes before they egress the thymus as recent thymic emigrants (32). To assess a possible role for coronin 1 at later developmental stages, the levels of cell surface markers that define this cell population, in particular CD62L, Qa2, and CD24, were assessed. Thymocytes exiting the thymus upregulate CD62L and Qa2, whereas they downregulate the expression of CD24. This allows the differentiation into thymocytes preparing to egress (CD62L+CD24+/CD62L+Qa2−) and thymocytes ready to egress (CD62L+CD24lo/CD62L+Qa2+) [see Ref. (8) and Supplemental Fig. 1A for gating strategy]. In peripheral organs, such as the spleen, the same gates can be used to identify cells that have just egressed (CD62L+CD24+/CD62L+Qa2lo) or have already seeded the peripheral T cell pool (CD62L+CD24−/CD62L+Qa2+) (Supplemental Fig. 1A). Analysis of surface expression for CD62L/CD24 in CD4+ thymocytes revealed no difference in the total numbers of thymocytes ready to egress or in the numbers of T cells that recently egressed (Fig. 1C, Supplemental Fig. 1B). The same analysis performed for the surface expression of CD62L and Qa2, which are both upregulated prior to thymic egress, again revealed no difference in the total numbers of recently egressed T cells (Fig. 1D, Supplemental Fig. 1B). The most striking difference, consistent with previous observations, occurs at the mature stages of naive T cells, where coronin 1–deficient animals showed a clear reduction in numbers and percentages (Fig. 1C, 1D, Supplemental Fig. 1B). Together, these data suggest that coronin 1 is dispensable for thymocyte survival and development.
Thymic egress and peripheral migration
Thymocytes that are about to egress do not only upregulate CD62L, they also downregulate the expression of CD69 and are then considered late-stage thymocytes [based on nomenclature described in (8)]. Because coronin 1–deficient animals show no thymic egress phenotype (i.e., accumulation of SP thymocytes, Fig. 1A, 1C, 1D), the expression of genes known to be important for thymic egress, prosurvival signals, and homing/migration signals was measured (Fig. 2A). One of these is KLF2, which is needed for CD62L expression. KLF2 was found to be expressed equally in wild-type and coronin 1–deficient thymocytes sorted for CD62L+CD69− expression. Another important transcription factor, not only for thymic egress but also for the generation of mature naive T cells, is Foxp1. Thymocytes in mice deficient for this factor develop an activated phenotype, resulting in increased apoptosis. However, this is not the case in coronin 1–deficient animals, because the thymocytes have no increased CD44 expression (data no shown), and the gene expression of Foxp1 is unchanged. Furthermore, the expression of the transcriptional repressor NKAP was analyzed. NKAP is mandatory for the maturation of T cells, because lack of this repressor leads to functionally immature recent thymic emigrants. Consistent with the presence of similar numbers of late-stage thymocytes, NKAP expression was not significantly modulated upon deletion of coronin 1. For successful migration from the medulla to the cortical region of the thymus, thymocytes need to regulate the surface expression of the chemokine receptor CCR7. In line with previous data (18), no differences in surface expression were observed between wild-type and coronin 1–deficient thymocytes (Fig. 2B, Supplemental Fig. 2A). To further analyze potential differences in thymocyte or recent thymic emigrant populations, sjTRECs were assessed as a marker for successful thymocyte maturation. sjTRECs originate during TCR rearrangement in the thymus, where small circular episomal DNA circles are produced, and persist in T cells. The DNA circles are diluted with every division and, therefore, can be used as a proxy to assess the age of a peripheral T cell (28–30). As shown in Fig. 2C, levels of sjTRECs were comparable in wild-type and coronin 1–deficient animals, consistent with a similar TCR repertoire in wild-type and coronin 1–deficient mice. Similarly, equal sjTREC levels were observed in splenocytes of both genotypes (Fig. 2C), further indicating a normal recent thymic emigrant compartment in coronin 1–deficient animals. Finally, similar surface expression levels of the receptor for S1P1, which is obligatory for thymic egress (33), were found on wild-type and coronin 1–deficient thymocytes (Fig. 2D, Supplemental Fig. 2B).
Accumulation of mature thymocytes can be observed in most mouse models of thymic egress deficiency (31). To further address whether thymocytes accumulate in the absence of coronin 1, in vivo BrdU labeling was performed, followed by thymocyte analysis 2 or 4 h later to analyze the increase in the BrdU+ population in the thymus. Although there was a slight increase in the CD8 SP BrdU+ percentage between 2 and 4 h, no accumulation of CD4 SP BrdU+ cells was observed in wild-type or coronin 1–deficient mice (Fig. 3, Supplemental Fig. 3).
Next, to analyze the peripheral migration capacity of wild-type and coronin 1–deficient thymocytes in vivo, total thymocytes from 2-wk-old wild-type and coronin 1–deficient mice were adoptively transferred to Ly5.1 recipients. After 4 h, both wild-type and coronin 1–deficient donor cells were detected at similar frequencies in the recipients’ spleens (Fig. 4A). Of note, wild-type and coronin 1–deficient donor thymocytes showed development from a CD62L+Qa2− to a CD62L+Qa2+ status during the 24 h after adoptive transfer (Fig. 4B). Donor cells were also detected in the mesenteric lymph nodes 24 h later. These results strongly suggest that peripheral migration of mature thymocytes is intact in the absence of coronin 1.
Establishment of the peripheral T cell pool in neonatal wild-type and coronin 1–deficient mice
The above data suggest that coronin 1 is dispensable for thymocyte development and egress and is exclusively required for naive T cell survival in the secondary lymphoid organs. However, whether coronin 1 is needed for the transition of recent thymic emigrants into mature naive T cells or for the survival of mature naive T cells per se remains unclear. To further address the stage at which coronin 1 is required, the composition of the secondary lymphoid organs in neonatal wild-type and coronin 1–deficient mice was analyzed during the first 3 wk of age. Because the peripheral T cell pool is expanded through lymphopenia-induced proliferation in neonatal mice (34, 35), an analysis of the T cell compartment in such mice would also reveal an involvement of coronin 1 in T cell proliferation upon entry into a lymphodepleted environment. To do so, spleen and thymus from wild-type and coronin 1–deficient neonatal mice were analyzed for the presence of T cells. As shown in Fig. 5A, during the first 2 wk after birth, peripheral CD3+CD4+ T cell numbers increased to similar numbers (∼2 × 106) in wild-type and coronin 1–deficient mice. Interestingly, although T cell numbers steadily increased to ∼12 × 106 in wild-type mice, numbers remained low (∼2 × 106) in the absence of coronin 1. In contrast to this, thymocyte populations were found to be equal at all ages (Fig. 5A). Additionally, no difference in total cellularity of spleen or thymus was observed (Fig. 5B).
Analysis of the subsets of peripheral T cells in newborn animals showed that, similar to what was observed in total peripheral T cells, recent thymic emigrants (CD62L+Qa2lo) were comparable in wild-type and coronin 1–deficient animals at all ages, whereas the levels of CD62L+Qa2+ wild-type T cells were significantly increased after day 19 compared with coronin 1–deficient T cells (Figs. 5C, 6, Supplemental Fig. 4A–C). Furthermore, analysis of annexin V+ cells revealed the appearance of apoptotic cells only at 6 and 10 wk, whereas there were no differences in thymocytes from wild-type or coronin 1–deficient mice up to 6 wk of age (Supplemental Fig. 4D–F). To analyze whether the difference in T cell numbers after day 19 correlates with an upregulation of coronin 1 at that time, splenocytes and thymocytes from mice expressing GFP under the coronin 1 promoter were analyzed. Fig. 5D shows a representative line graph of CD3+ splenocytes and total thymocytes of these mice, showing equal activity of the coronin 1 promoter during development.
Response of late-stage thymocytes to potential peripheral survival signals
Because coronin 1 appears to be dispensable for the production and survival of peripheral T cells during the first 2 wk after birth, and adult animals show no reduction in recent thymic emigrant cell numbers, we asked whether the paucity of T cells in the periphery of adult coronin 1–deficient mice could be related to a defect in the ability to respond to different signals. To assess the survival capacity of egressing cells, thymocytes were sorted for CD3+/CD4+/CD62L+/CD69− status to ensure that they are proliferation competent and protected from death receptor signaling. As mentioned above, the proliferation capacity was comparable in CD4+ and CD8+ T cells of peripheral organs in wild-type and coronin 1–deficient mice (Fig. 3, Supplemental Fig. 3). To analyze late-stage thymocyte survival in vitro, sorted thymocytes were cultured in medium or in the presence of bone marrow–derived dendritic cells, which are known to provide stimuli needed for peripheral T cell survival (36). As shown in Fig. 7, wild-type and coronin 1–deficient late-stage thymocytes failed to survive in culture when not receiving any additional survival signals. In coculture with immature dendritic cells, wild-type and coronin 1–deficient thymocytes survived over a period of 5 d (Fig. 7B). To assess whether this rescue was due to factors secreted by the dendritic cells or to cell–cell contact, thymocytes were cultured in the filtered supernatant of dendritic cells or in a Transwell coculture system. The thymocytes failed to survive in both settings, not replicating the rescuing phenotype observed in the coculture (compare Fig. 7C with Fig. 7B) and suggesting the importance of cell–cell contact for the observed in vitro survival.
Additional factors to consider in peripheral T cell survival are cytokines (37). Therefore, the capacity of coronin 1–deficient thymocytes to convey prosurvival signals induced by known prosurvival cytokines was investigated. Because it is known that IL-7 and IL-2 are important for the survival of recent thymic emigrants, as well as later stages of T cells, a combination of both cytokines was used as a positive survival control. When supplied with IL-2 and IL-7, wild-type and coronin 1–deficient thymocytes survived equally (Fig. 8A). Culturing the thymocytes with these cytokines individually revealed that the rescue is not due to IL-2 (Fig. 8B) but primarily is due to the presence of IL-7 (Fig. 8C). Consistent with wild-type and coronin 1–deficient thymocytes being rescued by IL-7, surface expression of the IL-7α receptor on CD62L+CD69− thymocytes was found to be similar in wild-type and coronin 1–deficient thymocytes (Fig. 8D). This is in line with recent thymic emigrants depending on IL-7 for their survival, whereas IL-2 is needed for later stages (38). The proliferation of thymocytes supported by IL-7 was also assessed by CTV labeling of the cells (Fig. 8E) and Ki-67 staining (Fig. 8F), which also showed similar outcomes between wild-type and coronin 1–deficient cells, although IL-7 did not stimulate proliferation at this stage (Fig. 8E, 8F). Additionally, Bcl-2 expression was assessed as a prosurvival marker induced by IL-7 (Fig. 8G). No impaired Bcl-2 induction by IL-7 was observed in coronin 1–deficient thymocytes. Thus, the severe T cell lymphocytopenia observed in coronin 1–deficient animals is not due to the inability of thymocytes to convey the overall survival signals provided by IL-7 or dendritic cells.
Maturation of thymocytes and egress from the thymus are tightly regulated mechanisms to ensure correct population of the peripheral T cell niche. Although the factors that control egress are known, the mechanisms involved in the survival and maturation of recent thymic emigrants are less clear. In this study, we have analyzed the early processes involved in mature naive T cell production in a mouse model of naive T cell deficiency: the coronin 1–deficient mouse. We found that thymocyte maturation and egress, as well as seeding of the peripheral T cell pool, were independent of coronin 1 until 2 wk of age; strikingly, after 2 wk, T cell numbers increased dramatically in a strictly coronin 1–dependent manner. Together, this work suggests the existence of a coronin 1–dependent decision switch at 2 wk of age that is crucial for seeding of the peripheral T cell pool.
Thymocytes have to undergo a set of defined maturation stages before they are ready to egress from the thymus (39). Positive- and negative-selection critically depends on TCR signaling; during positive selection the thymocyte TCR is selected to recognize self-MHC molecules, whereas during negative selection the TCR is used to select those thymocytes reacting only weakly to self-antigens, thereby eliminating potentially self-reacting T cells (2). In addition to regulating survival of the different thymocyte subsets, TCR signaling is involved in thymocyte migration, slowing down migration before the cells undergo apoptosis (40). Coronin 1, which is a member of the WD repeat-containing protein family of coronins predominantly expressed in cells of the hematopoietic lineage, is expressed throughout thymocyte development. Although its expression is upregulated after cells reach SP stages (41), the presence and normal numbers of the different thymocyte populations in mice lacking coronin 1 argue strongly for it being dispensable for thymocyte TCR signaling and selection. Although these data are consistent with a recent analysis of a model in which coronin 1 was specifically deleted in the T cell lineage (24), other studies have reported that coronin 1 deletion and/or mutation results in altered thymocyte subpopulations and numbers, ranging from decreased numbers of total SP thymocytes (23) to a partial reduction (18) or even an increase in thymocytes, thereby also linking coronin 1 to T cell egress from the thymus (19). However, it should be noted that thymic accumulation of T cells was exclusively observed in a model in which mutant coronin 1 induced aberrant T cell morphology (Ptcd) (19), unlike what is observed in mice lacking coronin 1 (24, 42).
The data presented in this article are consistent with an absence of an in vivo migratory defect of thymocytes lacking coronin 1, providing further evidence that thymocyte migration defects, as observed in studies analyzing coronin 1–deficient mouse models, may be a result of ex vivo differences between wild-type and coronin 1–deficient cells (18, 19, 42). This is also supported by the similar levels of transcription factors (KLF2, Foxp1, NKAP) reported in this article, as well as cell surface markers (Qa2, CD62L, CD24, CCR7, and S1P1) required for thymic egress in thymocytes from wild-type and coronin 1–deficient mice (43–45). Additionally, the presence of similar numbers and percentages of recent thymic emigrants in wild-type and coronin 1–deficient mice is fully consistent with coronin 1 being dispensable for thymic egress because, upon impaired egress, late-stage thymocytes accumulate in the thymus (46), which is not seen upon coronin 1 deletion. Finally, the adoptive transfer of thymocytes from 2-wk-old mice, an age at which the thymus is still free of circulating mature T cells (data not shown), resulted in similar population of the periphery after 24 h, suggesting a normal capacity to migrate into peripheral organs in the absence of coronin 1. One difference between these results and the previously reported apparent decrease in thymic egress may be the use of juvenile thymocytes in this study versus adult cells in the previous study (18). Collectively, the data presented in this study suggest that coronin 1 is dispensable for thymic egress and peripheral migration.
Once thymocytes have egressed from the thymus, the cells are considered recent thymic emigrants, and their survival appears to be IL-7 dependent (14). Thus far, the only factors known to be important for recent thymic emigrant maturation are the requirement for these cells to enter secondary lymphoid organs and the presence of an intact dendritic cell compartment (47, 48); however, dendritic cell function appears to be independent of coronin 1 (49). Therefore, one explanation for the lack of peripheral T cells in coronin 1–deficient animals could be their inability to enter secondary lymphoid organs. If this is the case, one would expect a higher number of recent thymic emigrants in the blood, because they would be unable to enter secondary lymphoid organs, and a reduction in recent thymic emigrants in the lymphoid organs. Because in the absence of coronin 1, there is not an accumulation of peripheral T cells in the blood or a reduction in recent thymic emigrants in the lymphoid organs, the defect for peripheral T cell survival observed following coronin 1 deficiency is presumably not due to an impaired entry into secondary lymphoid organs. Therefore, the data presented in this article suggest that, similar to thymocytes, recent thymic emigrant survival occurs independently of coronin 1.
To further delineate the step at which coronin 1 is required for population of the secondary lymphoid organs, we studied the population dynamics of newborn mice, which represents a lymphodepleted system (35). Strikingly, as shown in this article, although coronin 1 was found to be fully dispensable for T cell seeding of secondary lymphoid organs for up to 2 wk, T cells failed to populate the periphery after that time point. It should be noted that, in mice, the periphery is seeded with recent thymic emigrants rather than mature naive T cells for the first 2 wk after birth (15). Furthermore, analysis of apoptotic thymocytes, as reported in this article, revealed no differences in 2-wk-old mice, whereas an increase in apoptotic cell percentages was observed in older mice, in agreement with earlier observations (18). Together with our findings that dendritic cell– and IL-2/7–based proliferation and survival appear to be unperturbed upon coronin 1 deletion, this indicates that coronin 1 plays an essential role in the maturation of recent thymic emigrants to mature naive T cells or even later in the survival of mature naive T cells.
The exact pathway in which coronin 1 is involved to mediate mature naive T cell survival remains to be elucidated. Although coronin 1 has been suggested to modulate the F-actin cytoskeleton, thereby regulating T cell survival (18), altered F-actin levels do not correlate with T cell viability nor are other actin-dependent leukocyte functions affected by coronin 1 deletion (25, 42). Furthermore, in contrast to mice lacking coronin 1, deletion of the cytoskeletal regulators Vav and mDia results in altered thymic subsets indicative of defective egress (50–55), although it cannot be excluded that other coronin family members may compensate for the loss of coronin 1. Whether T cells are being maintained in adult mice via a noncanonical pathway, such as recognition of lymphopenia independent of IL-7 involving coronin 1, remains to be analyzed.
In summary, this study shows an exclusive role for coronin 1 in the survival of peripheral T cells at 2 wk, which is at or right after the transition point from recent thymic emigrants to mature naive T cells. It remains unknown whether coronin 1 is required for recent thymic emigrant maturation or, rather, for the survival of mature naive T cells. Furthermore, given the responsiveness of coronin 1–deficient T cells to dendritic cell– and cytokine-mediated signals, it remains to be established in which signal transduction pathway coronin 1 is involved.
We thank Thomas Barthlott for help with S1P1 staining and Katja Fromm for assistance with FACS staining. Furthermore, we thank Rajesh Jayachandran for critical reading of the manuscript and members of the Pieters Laboratory for critical input. We also thank the staff of the mouse facility and the FACS core facility of the Biozentrum, University of Basel.
This work was supported by the Swiss National Science Foundation, the Gebert Rueff Foundation, and the Cantons of Basel.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.