Our previous studies have defined a differentiation program followed by the newly generated single-positive (SP) thymocytes before their emigration to the periphery. In the present study, we further characterize the development of CD4SP cells in the thymic medulla using mainly intrathymic adoptive transfer assays. By analyzing the differentiation kinetics of the donor cells, which were shown to home correctly to the medullary region following adoptive transfer, we established the precursor–progeny relationship among the four subsets of CD4SP thymocytes (SP1–SP4) and demonstrated that the progression from SP1 to SP4 was unidirectional and largely synchronized. Notably, while the phenotypic maturation from SP1 to SP4 was achieved in 2–3 days, a small fraction of donor cells could be retained in the thymus for a longer period, during which they further matured in function. BrdU incorporation indicated that cell expansion occurred at multiple stages except SP1. Nevertheless, CFSE labeling revealed that only a limited number of cells actually divided during their stay in the medulla. As to the thymic emigration, there was a clear bias toward cells with increasing maturity, but no distinction was found between dividing and nondividing thymocytes. Collectively, these data not only provide solid evidence for a highly ordered differentiation program for CD4SP thymocytes, but they also illustrate several important features associated with the developmental process.

The thymus provides a unique microenvironment for the development and generation of functional T cells. After thymic entry, hemopoietic progenitors are first positioned in the cortex, where they proceed through the CD4CD8 (double-negative) and CD4+CD8+ (double-positive, DP)3 stage to differentiate into CD4+CD8 or CD4CD8+ single-positive (SP) cells (1, 2, 3). The SP thymocytes are then relocated to the medulla, where they stay resident for a prolonged period before exiting to the periphery (4). Although the medullary residency accounts for about one-half the lifespan of a thymocyte, it is one of the least studied areas of T cell development, partly because the thymic medulla has long been considered a “waiting room” for thymocytes to be exported. Recent studies, however, have revealed a more dynamic and eventful SP stage in the medulla.

During their residency in the medulla, the newly generated SP thymocytes undergo sequential changes in the expression of several surface markers, including the progressive down-regulation of CD69 and heat-stable Ag and the up-regulation of Qa-2, H-2K, and CD45RB (5, 6, 7). Simultaneously, cells gradually acquire functional competence to respond to a variety of stimuli (5, 8, 9). In addition to the phenotypic transition and functional maturation, evidence is accumulating that negative selection against self-reactive thymocytes mainly takes place in the medulla (10). As such, mice with a defective medulla often demonstrate various signs of autoimmunity. The autoimmune regulator deficiency provides a good example. As a transcriptional regulator primarily expressed by medullary epithelial cells, the autoimmune regulator drives the ectopic expression of a battery of organ-specific genes in the thymus. Its mutation thus leads to the development of autoimmunity because thymocytes reactive to self-Ags fail to be counterselected (11, 12). Finally, the SP stage has been identified to be the potential branchpoint between conventional T cells and regulatory T (Treg) cells or natural killer T (NKT) cells, as the lineage-specific markers, such as Foxp3 and NK1.1, are not acquired or fully manifested until this stage (13, 14, 15).

These findings have resurrected our interest in the differentiation of SP thymocytes in the medulla. However, many aspects of this differentiation step remain either completely elusive or controversial, such as the differentiation kinetics, the exact substage at which negative selection or lineage commitment occurs, and the intrinsic and environmental elements that regulate cell retention and emigration. Solutions to these issues clearly would require the full appreciation of the cellular process. Our previous studies were therefore focused on the understanding of the developmental program followed by medullary thymocytes (16, 17). By careful analysis of the expression pattern of cell-surface molecules, TCR+CD4+CD8 thymocytes were resolved into four subsets: SP1 (6C10+CD69+), SP2 (6C10CD69+), SP3 (CD69Qa-2), and SP4 (CD69Qa-2+). Together, these four subsets define a consecutive multistage program for the development of CD4SP thymocytes. More recently, we closely monitored the generation of the different subsets during mouse ontogeny. The sequential emergence of SP1 to SP4 renders further support for such a program (our manuscript in preparation).

The current study was undertaken to directly test the precursor–progeny relationship of the different subsets of CD4SP thymocytes using an intrathymic adoptive transfer assay. Moreover, some of the longstanding issues concerning medullary thymocyte development were reexamined in light of the new development scheme.

Congenic C57BL/6 Ly-5.1 (CD45.1) and C57BL/6 Ly-5.2 (CD45.2) (6- to 8-wk-old) mice were used as donors and recipients, respectively, in an intrathymic adoptive transfer assay. They were bred in the animal breeding facilities at Peking University Health Science Center (Beijing, China) under specific pathogen-free conditions. The experimental procedures on use and care of animals had been approved by the ethics committee of Peking University Health Science Center.

Anti-CD8 (3.155) and anti-CD4 (GK1.5) were prepared from hybridomas obtained from the American Type Culture Collection. Rat anti-mouse ER-TR5 was a gift from Dr. Yu-fei Jiang (National Institutes of Health/National Cancer Institute). The 6C10 (SM6C10) was from Dr. Linna Ding (National Institutes of Health) and was subsequently labeled with FITC. Purified anti-CD3 (145-2c11) and CD28 (37.51); fluorochrome-conjugated Abs against CD4 (H129.19 or RM4–5), CD8 (53-6.7), TCRβ (H57–597), CD69 (H1.2.F3), CD24 (M1/69), Qa-2 (H1-1-2), CD45.1 (A20), CD45RA (14.8), NK1.1 (PK136), and BrdU; and mouse Ig, streptavidin-PE/APC, and isotype control Abs were purchased from BD Pharmingen. Anti-Qa-2-FITC (H1-1-2) and CD25-PE (PC61) were from eBioscience; IFN-γ-APC (XMG1.2), IL-10-FITC (JES5–16E3), IL-2-FITC (JES6-5H4), and IL-4-APC (11B11) were from BioLegend; goat anti-mouse Ig-FITC was from ZhongShan Biotechnology; and goat anti-hamster Ig-tetramethylrhodamine isothiocyanate was from Santa Cruz Biotechnology.

Thymocytes, splenocytes, and lymph node cells were suspended in 2% newborn calf serum–balanced salt solution and stained with fluorescent Abs of various combinations. Appropriate isotype-matched Abs were included for compensation adjustment. Dead cells were excluded on the basis of low forward-light scatter and propidium iodide (PI) staining. Data acquisition and analysis were performed on FACSCalibur or FACSAria using CellQuest software (BD Biosciences).

Single-cell suspensions of thymocytes were treated with anti-CD8 (3.155) mAb and complement (guinea pig sera) to remove CD8+ cells (including both CD4CD8+ SP and CD4+CD8+ DP cells). After two cycles of killing and removal of dead cells by density centrifugation, the viable cells were stained with TCRβ-PE or APC, CD4-APC or PE-Cy7, CD8-APC-Cy7 (53-6.7), CD69-PerCP-CY5.5, and 6C10-FITC or Qa-2-FITC and then subjected to cell sorting (FACSAria). The purity of individual sorted medullary thymocyte subgroups was 97–99% as reanalyzed on FACS. In some experiments, cells were further stained with anti-CD25-PE and NK1.1-PE before sorting to exclude Treg and NKT cells.

Intrathymic injection was performed as described (18, 19). In brief, 1 × 106 purified SP cells from CD45.1 mice in 10 μl of PBS were injected intrathymically into the left lobes of anesthetized and nonirradiated CD45.2 congenic recipients using a Hamilton syringe. The control mice were injected with 10 μl of PBS. At various time points after transfer, the recipient mice were sacrificed and the thymocytes were collected and stained with 6C10-FITC or Qa-2-biotin + streptavidin-APC and then with CD45.1-PE, CD69-PerCP-CY5.5, and PI. Analysis was focused on CD45.1+PI cells. Donor-derived cells were also collected and analyzed from the spleen and lymph nodes (axillary, inguinal, and mesenteric lymph nodes).

CytoSpot assay was performed with the Cytofix/Cytoperm with GolgiStop kit according to the manufacturer’s instructions (BD Pharmingen). Briefly, thymocytes and peripheral CD4 T cells were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 or 48 h. GolgiStop (brefeldin A 10 μg/ml) was added to the culture in the last 5 h. Cells were fixed and permeabilized by incubating with Cytofix/Cytoperm for 30 min on ice. After washing, cells were stained with anti-IL-2-FITC/IFN-γ-APC or anti-IL-10-FITC/IL-4-APC and subjected to analysis by flow cytometry. Splenocytes treated with PMA/ionomycin were used as a positive control.

To examine the proliferation of different subsets of CD4SP cells after adoptive transfer, purified CD45.1 donor cells were labeled with a fluorescent dye, CFSE (Molecular Probes). Two microliters of CFSE stock solution (5 mM in DMSO) was added to the thymocyte suspension (107 cells in 1 ml). After incubation for 10 min at 37°C, cells were washed immediately and injected intrathymically into nonirradiated CD45.2 recipient mice.

C57BL/6 mice received two intraperitoneal injections of BrdU (1 mg each at 4-h intervals). Thymocytes were harvested 1 h after the second injection. BrdU incorporation was detected using the APC BrdU Flow Kit (BD Pharmingen). After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm buffer for 15–30 min on ice and then treated with DNase to expose incorporated BrdU. Subsequently, cells were stained with APC-conjugated anti-BrdU Ab.

Frozen sections (5 μm) of thymus were prepared by cryostatic sectioning of tissues embedded in OCT compound. Sections were fixed with cold acetone, air dried, washed in PBS, and blocked with 10% normal goat serum in PBS-0.1% BSA. After incubation with optimal dilutions of ER-TR5 and anti-CD45.1 Abs for 30 min at 25°C, the slides were probed with FITC-conjugated goat anti-rat Ig and tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse Ig for 30 min at 25°C. Multicolor images were acquired on a confocal SP2 AOBS (Leica Microsystems). After acquisition, images were merged to generate RGB composite pictures using the software TCS SP2.

RNA was isolated with the TRIzol reagent (Invitrogen) and used as template for cDNA synthesis. Quantitative PCR was performed on an iCycler sequence detection instrument (Bio-Rad) using the EvaGreen qPCR Master Mix (OPE Technology Development Company) with the following primers: 5′-TCG CCG ACA GCA GCA AGA TG-3′ and 5′-AAA GCC AGG TCA GCG AGC AAT C-3′ for sphingosine 1-phosphate receptor-1 (S1P1); and 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′ and 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′ for β-actin.

Student’s t test was performed using GraphPad Prism software (GraphPad Software). Differences were considered significant when p values were <0.05.

Our previous studies have resolved the medullary CD4SP thymocytes into several phenotypically distinct subsets that presumably represent consecutive stages in an ordered differentiation program (16). To validate such a program and establish the precursor–progeny relationship among these subsets, intrathymic adoptive transfer was performed with cells isolated from CD45.1+ donors using a strategy illustrated in Fig. 1 A. In brief, thymocytes were first treated with anti-CD8 and complement to remove CD8SP and DP cells. The viable cells were then stained for TCRβ, CD4, CD8, CD69, and 6C10 or Qa-2, and the TCRβ+CD4+CD8 population was sorted into the four subsets defined earlier (i.e., SP1–SP4). Subsequently, each individual subset was intrathymically injected into CD45.2+-congenic mice and monitored for its differentiation potential.

FIGURE 1.

Cell separation and purity analysis. A, After treatment with anti-CD8 (3.155) mAb and complement, viable cells (pooled from 20 mice each time) were separated by density centrifugation and then stained with mAbs against mouse CD4, CD8 (53-6.7), TCR, CD69, and 6C10 or Qa-2. TCR+CD4+CD8 medullary thymocytes were gated and sorted into the four subsets defined in the text (i.e., SP1–SP4). The numbers in the top-right corners indicate the percentage of cells in each quadrant before sorting (or the purity of the sorted cells, which was 98–99%). B, To further exclude contamination of NKT and Treg cells, PE-conjugated anti-NK1.1 and anti-CD25 Abs were included in the mixture of staining Abs. SP1 cells were then isolated from the TCR+CD4+CD8NK1.1CD25 population.

FIGURE 1.

Cell separation and purity analysis. A, After treatment with anti-CD8 (3.155) mAb and complement, viable cells (pooled from 20 mice each time) were separated by density centrifugation and then stained with mAbs against mouse CD4, CD8 (53-6.7), TCR, CD69, and 6C10 or Qa-2. TCR+CD4+CD8 medullary thymocytes were gated and sorted into the four subsets defined in the text (i.e., SP1–SP4). The numbers in the top-right corners indicate the percentage of cells in each quadrant before sorting (or the purity of the sorted cells, which was 98–99%). B, To further exclude contamination of NKT and Treg cells, PE-conjugated anti-NK1.1 and anti-CD25 Abs were included in the mixture of staining Abs. SP1 cells were then isolated from the TCR+CD4+CD8NK1.1CD25 population.

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SP1 (Fig. 2 A): After adoptive transfer, SP1 cells underwent sequential changes in surface expression of 6C10, CD69, and Qa-2. At day 1, most cells already lost 6C10 expression but still maintained CD69 on their surface, which is characteristic of SP2 cells (6C10CD69+, 79%). At day 2, a large proportion of the cells further down-regulated the expression of CD69, progressing to either the SP3 (44%) or SP4 (15%) stage. Meanwhile, SP2 cells dropped to 41%, and SP1 cells virtually disappeared. At day 3, more cells gained Qa-2 expression to become SP4 cells, whereas SP2 and SP3 cells decreased to 7 and 30%, respectively. At day 4, SP4 cells further increased to 79% in proportion while the rest were retained at the SP3 stage. Thereafter, all donor-derived cells detected in the thymus assumed the SP4 phenotype (data not shown).

FIGURE 2.

Dynamics of the maturation process of different subsets of CD4SP thymocytes following intrathymic adoptive transfer. Each sorted subset of TCR+CD4SP thymocytes from CD45.1+ donors was intrathymically injected into nonirradiated congenic recipients (1 × 106/10 μl/lobe). Control mice were injected with BSS. One to 4 days after adoptive transfer (D1-D4), recipient mice were sacrificed and the thymocytes were analyzed by flow cytometry. The 6C10 vs CD69 and/or CD69 vs Qa-2 expressions were determined in CD45.1+ donor thymocytes. A, SP1; B, SP2; C, SP3; D, SP4. The numbers in the top-right corner indicate the percentage of cells in each quadrant. The experiment was repeated three to five times and similar results were obtained.

FIGURE 2.

Dynamics of the maturation process of different subsets of CD4SP thymocytes following intrathymic adoptive transfer. Each sorted subset of TCR+CD4SP thymocytes from CD45.1+ donors was intrathymically injected into nonirradiated congenic recipients (1 × 106/10 μl/lobe). Control mice were injected with BSS. One to 4 days after adoptive transfer (D1-D4), recipient mice were sacrificed and the thymocytes were analyzed by flow cytometry. The 6C10 vs CD69 and/or CD69 vs Qa-2 expressions were determined in CD45.1+ donor thymocytes. A, SP1; B, SP2; C, SP3; D, SP4. The numbers in the top-right corner indicate the percentage of cells in each quadrant. The experiment was repeated three to five times and similar results were obtained.

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SP2 (Fig. 2 B): As expected, SP2 cells exhibited slightly accelerated maturation kinetics than SP1 in adoptive transfer. At day 1, half of cells lost CD69 expression to differentiate into the SP3 (35%) or SP4 (10%) stage while the other half maintained the SP2 phenotype. SP2 cells gradually decreased thereafter and eventually disappeared at day 4. SP3 cells constituted more than half of the donor population at day 2, but reduced to <10% at day 4. SP4 cells, in contrast, progressively increased in proportion, accounting for 86% of the donor-derived cells by day 4.

SP3 (Fig. 2 C): Within 24 h after transfer, one-third of the cells developed into the SP4 stage, as suggested by Qa-2 expression. By day 3, >90% of the donor cells assumed the SP4 phenotype.

SP4 (Fig. 2 D): In terms of Qa-2 and CD69 expression, cells recovered at various time points after transfer maintained a phenotype identical with that of the input cells.

The data from adoptive transfer clearly demonstrate the precursor–progeny relationship among the four subsets. Together, these four subsets define a unidirectional pathway for the maturation of CD4SP thymcoytes. Interestingly, the progression from SP1 to SP4 could be accomplished in as short as 2–3 days in adoptive transfer. This is in comparison with the estimated 1–2-wk residency of SP thymocytes in the medulla.

In addition to the phenotypic maturation, we monitored the retention of adoptively transferred CD4SP cells in the thymus and their emigration to the periphery. The number of donor-derived cells was found to gradually decrease in the thymus. In general, the decline was more rapid with cells representing later stages (Fig. 3 A). However, more cells were constantly recovered in the first 3 days post-transfer in the SP2 group than in the SP1 group. The reason behind this exception is further discussed below. Note also the difference in the retention time for the different subsets. Although it took 5–7 days for SP1 and SP2 cells to drop to undetectable levels, donor-derived cells disappeared from the thymus within 4 days in the adoptive transfer of SP3 and SP4 cells.

FIGURE 3.

Retention and emigration of adoptively transferred CD4SP thymocytes. After intrathymic adoptive transfer of different subsets of CD4SP thymocytes (SP1–SP4), their retention in the thymus and emigration to the periphery were monitored and compared. Donor-derived cells were traced by CD45.1 expression with a background staining of <0.01%. Dead cells were excluded by using propidium iodide staining. Data were collected from three to four mice at each time point for each subset tested. A, The absolute number of donor cells retained in the thymus over the next 4 days following adoptive transfer. B, The absolute number of donor cells emerging in the spleen. C, The relative number of donor cells detected of 107 cells pooled from mesenteric, inguinal, and axillary lymph nodes. D, The percentage of Qa-2+ donor cells in the lymph nodes from days 2 to 7 following intrathymic transfer of SP1 cells. E, The relative amount of S1P1 mRNA in the four subsets of CD4SP thymocytes from adult mice as determined by real-time PCR.

FIGURE 3.

Retention and emigration of adoptively transferred CD4SP thymocytes. After intrathymic adoptive transfer of different subsets of CD4SP thymocytes (SP1–SP4), their retention in the thymus and emigration to the periphery were monitored and compared. Donor-derived cells were traced by CD45.1 expression with a background staining of <0.01%. Dead cells were excluded by using propidium iodide staining. Data were collected from three to four mice at each time point for each subset tested. A, The absolute number of donor cells retained in the thymus over the next 4 days following adoptive transfer. B, The absolute number of donor cells emerging in the spleen. C, The relative number of donor cells detected of 107 cells pooled from mesenteric, inguinal, and axillary lymph nodes. D, The percentage of Qa-2+ donor cells in the lymph nodes from days 2 to 7 following intrathymic transfer of SP1 cells. E, The relative amount of S1P1 mRNA in the four subsets of CD4SP thymocytes from adult mice as determined by real-time PCR.

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The decrease in the number of donor-derived cells in the thymus was accompanied by an increase in the periphery. Following the intrathymic injection of SP1 thymocytes, donor-derived cells were first detected in the spleen and lymph nodes at day 2, and they steadily increased afterward. Transfer experiments with SP2, SP3, and SP4 cells further demonstrated that the more mature subsets tend to appear and accumulate in the peripheral lymphoid organs in a more rapid manner (Fig. 3, B and C). Significantly more cells (p < 0.05), for example, were seen in the spleen at day 4 after transfer with SP3 and SP4 cells than with SP1 and SP2 cells.

In SP1 transfer assay, the first appearance of donor-derived cells in the periphery correlated with the generation of Qa-2+ SP4 cells in the thymus. To determine whether exit from the thymus is restricted to fully mature cells, we examined Qa-2 expression in thymic emigrants during 7 days after SP1 transfer. As shown in Fig. 3,D, only half of the cells were Qa-2+ at day 2, but the proportion of such cells gradually increased over the next few days, reaching almost 100% at day 5. Therefore, differentiation to the SP4 stage is not a prerequisite for thymic emigration, but there is a tendency for cells to leave toward the late stage. It was recently found that S1P1 was critically involved in the control of thymocyte egress (20). We therefore monitored S1P1 mRNA expression during CD4SP thymocyte maturation. S1P1 expression was low in SP1 and SP2 cells, but there was a 10-fold increase at the transition from SP2 to SP3 and a further up-regulation at the SP4 stage (Fig. 3 E). Such a pattern is consistent with the preferential emigration of more mature cells.

Several studies suggest that NKT and Treg cells are derived from early CD4SP thymocytes and complete their differentiation in the thymic medulla (13, 14, 15). With the developmental scheme introduced here, we sought to determine the representation of these cells in different CD4SP subsets. NK1.1+ cells were found to reside almost exclusively in the SP4 subset, accounting for 7.5% of the whole population. CD25+ cells, in contrast, were widely distributed among all subsets but with substantial enrichment in SP4 (Fig. 4 A). These results support that the specification to the NKT and Treg lineages predominantly takes place at a rather late stage in CD4SP differentiation.

FIGURE 4.

Expression of NK1.1 and CD25 in CD4SP subsets and the intrathymic maturation of NK1.1 and CD25-depleted SP1 cells. A, Thymocytes from adult mice were prepared and stained as described in Fig. 1. NK1.1 and CD25 expression in the four subsets of CD4SP cells were shown. The numbers in the top-right corner indicate the percentage of NK1.1 or CD25+ cells. B, SP1 cells were further depleted of NKT and Treg cells (as described in Fig. 1,B) and intrathymically injected into the recipients. The phenotypic maturation of the donor cells was traced as in Fig. 2. The experiment was repeated three times and similar results were obtained.

FIGURE 4.

Expression of NK1.1 and CD25 in CD4SP subsets and the intrathymic maturation of NK1.1 and CD25-depleted SP1 cells. A, Thymocytes from adult mice were prepared and stained as described in Fig. 1. NK1.1 and CD25 expression in the four subsets of CD4SP cells were shown. The numbers in the top-right corner indicate the percentage of NK1.1 or CD25+ cells. B, SP1 cells were further depleted of NKT and Treg cells (as described in Fig. 1,B) and intrathymically injected into the recipients. The phenotypic maturation of the donor cells was traced as in Fig. 2. The experiment was repeated three times and similar results were obtained.

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Potentially, the presence of NKT and Treg cells in CD4SP thymocytes could complicate the interpretation of the adoptive transfer assays described above. To address this concern, we repeated the assay with SP1 thymocytes that had been deprived of cells expressing NK1.1 and CD25 using a strategy illustrated in Fig. 1,B. The differentiation of these cells in the thymus showed a kinetics virtually identical with that to “conventional” SP1 thymocytes (Figs. 2,A and 4 B).

Given that the injection site is largely random in the adoptive transfer assay, it is instructive to know how the donor cells are distributed within the thymus. To this end, CD4SP thymocytes were isolated and injected into the thymus of recipient mice. At different time points after injection, frozen sections of thymus were prepared and stained with anti-CD45.1 and ER-TR5 Abs to reveal the donor cells and the medullary region, respectively. At 3 h after injection, CD45.1+ donor cells were identified in both cortical and medullary areas adjacent to the injection site. By day 3, however, the injected cells were found to reside almost exclusively in the medulla (Fig. 5). In fact, donor cell enrichment in the medulla was apparent as early as 20 h after transfer (data not shown). Therefore, the developing CD4SP thymocytes are endowed with a capacity to be directed to the medullary region, which provides the appropriate milieu for their further maturation.

FIGURE 5.

Migration to and enrichment in the thymic medulla of adoptively transferred CD4SP thymocytes. Three hours or 3 days after intrathymic injection of SP2 cells, the recipient mice were sacrificed and the frozen sections of the thymus were stained with ER-TR5 (green) and CD45.1 mAb (red) to show the intrathymic localization of the donor cells. A representative of three independent experiments was shown with magnifications of 4× and 10×.

FIGURE 5.

Migration to and enrichment in the thymic medulla of adoptively transferred CD4SP thymocytes. Three hours or 3 days after intrathymic injection of SP2 cells, the recipient mice were sacrificed and the frozen sections of the thymus were stained with ER-TR5 (green) and CD45.1 mAb (red) to show the intrathymic localization of the donor cells. A representative of three independent experiments was shown with magnifications of 4× and 10×.

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Several studies have indicated that a significant number of recently generated SP thymocytes undergo cell division before export to the periphery (21, 22, 23). To further characterize this late intrathymic expansion phase, we investigated the proliferation potential of each individual subset of CD4SP thymocytes. Mice received two intraperitoneal injections of BrdU at a 4-h interval, and the incorporation of BrdU into thymocytes was analyzed 1 h after the second injection. Overall, BrdU+ cells accounted for 2–4% of the CD4SP, which is comparable to a previous report by Penit and Vasseur (21). Among the individual subsets, SP2 contained the highest proportion of BrdU+ cells. A significant number of cycling cells were also seen in SP3 and SP4, whereas such cells were barely detectable in SP1 (Fig. 6). The striking difference in proliferation between SP1 and SP2 cells may be partly responsible for the increased cell number in the thymus after adoptive transfer of SP2 vs SP1 cells as described earlier.

FIGURE 6.

BrdU+ cells among the various subsets of CD4SP thymocytes. Mice received two intraperitoneal injections of BrdU at a 4-h interval. The thymus was removed for analysis 1 h after the second injection. Thymocytes were first stained with mAbs against mouse CD4, CD8, TCR, CD69, and 6C10 or Qa-2. BrdU were then detected with APC-conjugated anti-BrdU Abs. A, The percentage of BrdU+ cells in each subset of CD4SP thymocytes from a representative mouse. B, The average percentage of BrdU+ cells in each CD4SP subsets from three independent experiments with three mice for each. Two-tailed Student’s t test was performed (* p < 0.05, ** p < 0.01).

FIGURE 6.

BrdU+ cells among the various subsets of CD4SP thymocytes. Mice received two intraperitoneal injections of BrdU at a 4-h interval. The thymus was removed for analysis 1 h after the second injection. Thymocytes were first stained with mAbs against mouse CD4, CD8, TCR, CD69, and 6C10 or Qa-2. BrdU were then detected with APC-conjugated anti-BrdU Abs. A, The percentage of BrdU+ cells in each subset of CD4SP thymocytes from a representative mouse. B, The average percentage of BrdU+ cells in each CD4SP subsets from three independent experiments with three mice for each. Two-tailed Student’s t test was performed (* p < 0.05, ** p < 0.01).

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We then asked to what extent CD4SP thymocytes could divide in the thymic medulla. SP2 cells from CD45.1 donors were labeled with CFSE and transferred into the thymus of the recipient mice, together with CFSE-labeled CD45RA+CD4+ splenocytes from CD45.2 mice. At days 2, 4, and 6 post-transfer, donor cells were recovered from the thymus and lymph nodes, and the history of cell division was traced by monitoring the CFSE intensity using CD45.1CFSE+CD45RA+CD4+ cells as nondividing controls. The donor cells were shown to undergo up to four rounds of division in the thymus within 6 days. The percentage of cells that divided at least once increased from 5% at day 2 to 30% at day 4 and 37% at day 6 (Fig. 7). The donor cells detected in lymph nodes displayed a similar history of cell division except that the proportion of divided cells was always slightly lower than that in the thymus at each time point, suggesting that the thymic emigration was not biased toward recently divided cells. Using the ModFit LT program, we calculated the percentage of parent cells that underwent division to give rise to the CFSE profile observed. The 37.2 and 32.1% postmitotic cells detected in the thymus and lymph nodes at day 6 after transfer were actually derived from only 14.2 and 14.0% of parent cells, respectively. Therefore, cell expansion seems to be a relatively rare event in the medulla.

FIGURE 7.

Division of adoptively transferred SP2 cells. SP2 thymocytes (CD45.1) and CD45RA+CD4+ splenocytes (CD45.2) were sorted and mixed at a ratio of 10:1. After labeling with CFSE, the cell mixture was intrathymically injected. Donor cells were recovered from the thymus and lymph nodes of the recipient at days 2, 4, and 6 posttransfer. The intensity of CFSE was analyzed using the software ModFit LT 3.0. The CD45.1CFSE+CD45RA+CD4+ splenocytes (R2 gate) served as nondividing controls at each time point. Results are representative of three to five separate experiments. The numbers indicate the percentage of cells that divided at least once.

FIGURE 7.

Division of adoptively transferred SP2 cells. SP2 thymocytes (CD45.1) and CD45RA+CD4+ splenocytes (CD45.2) were sorted and mixed at a ratio of 10:1. After labeling with CFSE, the cell mixture was intrathymically injected. Donor cells were recovered from the thymus and lymph nodes of the recipient at days 2, 4, and 6 posttransfer. The intensity of CFSE was analyzed using the software ModFit LT 3.0. The CD45.1CFSE+CD45RA+CD4+ splenocytes (R2 gate) served as nondividing controls at each time point. Results are representative of three to five separate experiments. The numbers indicate the percentage of cells that divided at least once.

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Our previous studies have demonstrated that the function of CD4SP thymocytes progressively increase from SP1 to SP4 stage (24). Those data, however, were obtained in bulk cultures under stimulation with Con A. In the present study we reanalyzed cytokine secretion by different subsets following stimulation with anti-CD3 and anti-CD28 using CytoSpot assay so as to estimate their functionality on a per-cell basis. At SP1 stage, low levels of IL-2 production were detected in a small number of cells (∼1%). At SP2 stage, the IL-2-producing cells increased to 2.7%, with a few cells starting to produce IFN-γ or IL-4 as well. At SP4 stage, cells first acquired the capacity for IL-10 production and for simultaneous production of multiple cytokines. Meanwhile, cells competent for IL-2, IFN-γ, or IL-4 production further increased in number, reaching 7.1, 2.2, and 1.0%, respectively (Fig. 8 A). Notably, the functional maturation was mainly manifested as an increase in the number of competent cells rather than enhanced productivity by individual cells.

FIGURE 8.

Functional capacity of freshly isolated CD4SP thymocytes and adoptively transferred SP4 cells with different retention times in the thymus. A, Freshly isolated SP1, SP2, and SP4 cells were stimulated with anti-CD3 and anti-CD28 Abs. Their production of IL-2 and IFN-γ, and IL-4 and IL-10, was analyzed by intracellular staining at 24 and 48 h, respectively. Splenic CD4 T cells were used as a positive control. Results were representative of three to five independent experiments. B, Following adoptive transfer of purified SP4 cells, the CD45.1+ donor cells were recovered by magnetic sorting from the thymus and lymph nodes at 24 and 96 h posttransfer. The cells pooled from six to eight mice were analyzed for IFN-γ and IL-4 production by gating on the CD45.1+ population. To identify RTEs at 96 h, the recipient mice were intrathymically injected with FITC at 72 h posttransfer, and the subsequent analysis was focused on CD45.1+FITC+ cells harvested from lymph nodes 24 h later. a, the percentage of IFN-γ- or IL-4-producing cells in SP4 cells with a thymic retention time of 24 or 96 h or in RTEs isolated from lymph nodes 24 or 96 h posttransfer; b, the level of IFN-γ and IL-4 production as measured by mean fluorescence intensity (MFI). Results are presented as mean ± SD from at least three individual experiments. Statistical analysis was performed using Student’s two-tailed t test (*p < 0.05, **p < 0.01).

FIGURE 8.

Functional capacity of freshly isolated CD4SP thymocytes and adoptively transferred SP4 cells with different retention times in the thymus. A, Freshly isolated SP1, SP2, and SP4 cells were stimulated with anti-CD3 and anti-CD28 Abs. Their production of IL-2 and IFN-γ, and IL-4 and IL-10, was analyzed by intracellular staining at 24 and 48 h, respectively. Splenic CD4 T cells were used as a positive control. Results were representative of three to five independent experiments. B, Following adoptive transfer of purified SP4 cells, the CD45.1+ donor cells were recovered by magnetic sorting from the thymus and lymph nodes at 24 and 96 h posttransfer. The cells pooled from six to eight mice were analyzed for IFN-γ and IL-4 production by gating on the CD45.1+ population. To identify RTEs at 96 h, the recipient mice were intrathymically injected with FITC at 72 h posttransfer, and the subsequent analysis was focused on CD45.1+FITC+ cells harvested from lymph nodes 24 h later. a, the percentage of IFN-γ- or IL-4-producing cells in SP4 cells with a thymic retention time of 24 or 96 h or in RTEs isolated from lymph nodes 24 or 96 h posttransfer; b, the level of IFN-γ and IL-4 production as measured by mean fluorescence intensity (MFI). Results are presented as mean ± SD from at least three individual experiments. Statistical analysis was performed using Student’s two-tailed t test (*p < 0.05, **p < 0.01).

Close modal

As shown above, the phenotypic differentiation from SP1 to SP4 was achieved within 2–3 days after adoptive transfer, but the donor cells could be retained in the thymus for a much longer period. To determine whether cells undergo further functional maturation after phenotypic conversion to the SP4 stage, we directly compared IFN-γ and IL-4 production by SP4 cells from the thymus and RTEs (recent thymic emigrants) from lymph nodes recovered at 24 and 96 h after adoptive transfer of purified SP4 cells. To identify cells emigrating to the periphery between 72 and 96 h, the recipient mice were intrathymically injected with FITC at 72 h posttransfer, and the subsequent analysis was focused on CD45.1+FITC+ cells harvested from lymph nodes 24 h later. CytoSpot assay showed that the SP4 cells recovered at day 4 contained a significantly higher proportion of IL-4-producing cells than did those at day 1 (4.03 ± 0.62% vs 1.56 ± 0.85%; p < 0.05), whereas the change in the number of IFN-γ-producing cells was not statistically significant. Comparison of RTEs harvested at day 4 and day 1 revealed a similar trend, with a significant increase occurring in IL-4-producing cells (5.44 ± 0.35% vs 3.93 ± 0.70%; p < 0.05) but not in IFN-γ-producing cells (Fig. 8,Ba). In addition to the increased cell number, RTEs at day 4 showed elevated mean fluorescence intensity in IL-4 staining, implying enhanced IL-4 production on a per-cell basis (Fig. 8 Bb). Collectively, these results support continuous functional maturation of SP4 cells during their extended residence in the thymus.

In comparison to SP4 cells recovered from the thymus, RTEs harvested at the same time point tended to mount a more potent response to anti-CD3/CD28 stimulation. This was true in terms of the number of IFN-γ- and IL-4-producing cells as well the amount of IL-4 produced by individual cells (Fig. 8 B). Potentially, this may be due to either preferential emigration of more mature cells or postthymic maturation, as suggested by Boursalian et al. (25).

In the present study, we analyzed the differentiation kinetics of CD4SP medullary thymocytes and determined the precursor–progeny relationship of the various subsets using intrathymic adoptive transfer assay. Specifically, each subset of CD4SP thymocytes was isolated from CD45.1+ donors and then intrathymically injected into nonirradiated, CD45.2+ congenic mice. In comparison to the irradiated models of thymic reconstitution, where growth factor production and microenvironmental niches are disturbed by massive cytodepletion, nonirradiated recipients provide a normal thymic architecture for the further maturation of donor cells. To address the concern whether the donor cells would be directed into the right niche, we examined their distribution in the recipient thymus. Immunofluorescent staining revealed marked relocation of donor cells from the injection site to the medullary area following adoptive transfer. Presumably, this may be due to the high level of CCR7 expression by SP thymocytes (26). Furthermore, to illustrate the potential interference of contaminant Treg and NKT cells, a comparative analysis was made with CD25- and NK1.1-deprived and nondeprived CD4SP thymocytes. Similar results were obtained. Taken together, we think that the data presented here should be a close reflection of the normal differentiation process of “conventional” CD4SP thymocytes in situ.

After adoptive transfer, SP1 cells sequentially developed into SP2, SP3, and SP4 cells; SP2 developed into SP3 and SP4; and SP3 developed into SP4, whereas SP4 cells underwent no phenotypic changes. These results provide unequivocal evidence for the precursor–progeny relationship among the four subsets, and they confirm that the differentiation of CD4SP thymocytes in the medulla follows a linear, one-way program. More detailed analysis of the population dynamics indicated that the progression from one stage to the next is grossly synchronized, and that the complete turnover of a given subset is accomplished in a relatively short period, usually 2–3 days as measured by its disappearance in adoptive transfer.

The time course of medullary thymocyte development has been studied by several groups. Continuous labeling with [3H]thymidine showed that the complete replacement of the medullary compartment took ∼2 wk (27). However, pulse labeling with BrdU suggested that the turnover of BrdU+ SP thymcoytes occurred in 5–7 days (7). The latter estimation was supported by another study, in which a time lag of 6–7 days was revealed between the generation of CD4SP cells in the thymus and their emergence in lymph nodes following the rescue of CD4+ T cell compartments in MHC class II-deficient mice by intrathymic injection of adenovirus vectors carrying class II genes (28). In the adoptive transfer assay, we found that, depending on the developmental stages of the cells being transferred, the donor cells persisted in the thymus for 4–7 days. Although this result was in good accord with previous reports, we were surprised to see that SP1 cells could develop into SP4 cells in 2 days and that the complete conversion to SP4 took no more than 4 days. Additionally, we detected a significant number of donor cells in the periphery within 2–3 days after intrathymic injection of SP1 cells. The rapid developmental progression and emigration raise the possibility that the prolonged medullary residency observed in previous studies may simply result from the persistence of a relatively small fraction of SP4 cells. Phenotypic characterization of these “leftover” cells revealed no significant enrichment for NK1.1 and CD25 expression (data not shown), excluding the possibility of selective retention of NKT and Treg cells.

The mechanism regulating thymic retention and emigration is not well defined. Recent studies, however, have indicated a critical role of S1P1 (one of the S1P receptors expressed by mature SP thymocytes) in this process, as its deficiency leads to the accumulation of SP cells in the thymus and the absence of T cells in the periphery (20, 29). In analysis of S1P1 expression by different subsets of CD4SP cells, we saw a strikingly preferential expression in more mature subsets. Such a pattern provides a good explanation for the bias toward the more mature cells in the emigration of thymocytes. However, the question remains as to why some SP4 cells stay in thymus longer than do others. We speculate that, upon reaching maturity, their egress from the thymus is largely a stochastic process.

What are the consequences of the extended retention in the medulla for SP4 cells? Despite the extensive screening of surface markers, we failed to see any further phenotypic change in SP4 cells over the prolonged residency in the medulla. In contrast, we did observe a correlation between the resident time and cell functionality. SP4 cells were intrathymically injected and then recovered from the recipient thymus at day 1 or day 4. The frequency of IL-4-producing cells, and possibly that of IFN-γ-producing cells, was found to be markedly elevated in the latter population. A similar increase was also observed in the comparison of donor-derived RTEs at day 4 versus those at day 1. Thus, the extended residency may be needed for further functional maturation.

Recent studies using BrdU incorporation and CSFE labeling have identified a late phase of intrathymic cell proliferation at the SP stage, both in adult mice and in neonates (but being more prominent in the latter) (21, 22, 23, 30, 31). It is generally assumed that this proliferation may serve to expand the mature T cell repertoire before export to the periphery. Nevertheless, two issues closely related to this assumption remained to be resolved, namely the stage specificity and the extent of the expansion. By pulse labeling with BrdU, we demonstrated that CDSP thymocytes contained 2–4% cycling cells. This number is in good agreement with previous reports. However, while the study by Penit and Vasseur indicated that the cycling cells were largely restricted to the HSAQa-2high population (equivalent to SP4) (21), we found that BrdU incorporation occurred at all stages except SP1, with the highest being found at SP2. Furthermore, in contrast to the substantial enrichment of cycling cells in RTEs that they reported, our study showed no evidence that the recently divided cells are favored for export. Finally, despite the accumulation of mitotic cells over time (30–40% at day 6 after transfer), cell division was overall a relatively rare event, involving only a small fraction of parent cells (12–15%). This is obviously in contrast to the idea that all SP thymocytes divide once before emigration. The signal that drives the proliferation of SP thymocytes remains to be defined. Using reaggregate thymic organ culture, Hare and colleagues demonstrated that the expansion of SP cells requires sustained interaction with thymic epithelial cells (30). As cell division occurs in cocultures with epithelial cells expressing no or mismatched MHC molecules (30, 31), TCR-MHC interaction does not seem to be involved in the proliferation of SP thymocytes. Instead, it is speculated that the supporting role of epithelial cells may result from the production of soluble factors, such as IL-7 (22, 30).

In summary, the CD4SP thymocyte differentiation in the thymic medulla follows a linear and unidirectional pathway, and the progression from one stage to the next is largely synchronized. Although the phenotypic maturation from SP1 to SP4 can be accomplished in 2–3 days, cells may be retained in the thymus for an extended period (up to 7 days), during which they undergo further functional maturation and a wave of proliferation. Understanding of the cellular process should facilitate further inquiry into the molecular mechanisms governing SP thymocyte development.

We thank Jia-Ping Tao and Shi-Liang Ma for the excellent FACS technical assistance.

The authors have no financial conflicts of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from National Natural Sciences Foundation (30330520 and 30525044) and Natural Basic Research Program of China (2006CB504300 and 2006CB910101).

3

Abbreviations used in this paper: DP, double-positive cells; NKT, natural killer T cell; PI, propidium iodide; RTE, recent thymic emigrants; S1P1, sphingosine 1-phosphate receptor-1; SP, single-positive cells; Treg, regulatory T cell.

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