Department of Immunology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100083, China
Correspondence to: W.-F. Chen.
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Abstract |
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Keywords: heterogeneity, medullary-type thymocytes, phenotypes
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Introduction |
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In fact, CD4/CD8 SP thymocytes in the thymic medulla are not quiescentthey undergo limited cell divisions and apoptosis (9,10). Medullary-type thymocytes are phenotypically and functionally heterogeneous (4,1115). During the 14-day residence in the thymic medulla, the medullary-type thymocytes must undergo phenotypic and functional modulations. The fully functional mature CD4 SP thymocytes are Qa-2+ cells, which compose only a small proportion of total medullary CD4 SP thymocytes (4,16). Also, the acquisition of granzyme A proteolytic activity occurs in the relatively more mature heat-stable antigen (HSA), but not in HSA+, medullary CD8 SP thymocytes (17,18). It appears that SP thymocytes are undergoing functional maturation within the thymic medulla. A systemic study is necessary to reveal the functional differentiation pathway of medullary-type SP thymocytes and the mechanism controlling this process. As the cell differentiation process is often accompanied by phenotypic changes, analysis of phenotypic heterogeneity should allow studies of differentiation pathways within a particular cell population. In the present study, we analyzed the expression of a set of cell-surface markers in CD8 SP medullary-type thymocytes and identified six subpopulations within the TCRß+CD8 SP cell subset. Accordingly, a linear precursorprogeny relationship among these subpopulations in TCR
ß+CD8 SP thymocytes is proposed.
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Methods |
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mAb
A number of mAb were produced as described below. Anti-CD4 (RL172), anti-3G11 (SM3G11), anti-6C10 (SM6C10) and anti-HSA (M1/69) were produced in our laboratory as ascites or tissue culture supernatants. FITC-labeled anti-CD8 (53.6), anti-ß7 integrin, streptavidin, phycoerythrin (PE)-labeled anti-CD4 (192.19), anti-TCRß (H57-597), anti-L-selectin, anti-HSA (M1/69); CyChrome 5-labeled anti-CD8, anti-CD3 (1452C11), allophycocyanin (APC)-labeled anti-TCRß (H57-597), biotin-labeled anti-3G11, anti-CD69 (H1.2.F3) and anti-Qa-2 (H1-1-2) were purchased from PharMingen (San Diego, CA). FITC-labeled sheep anti-rat IgG was from Sigma (St Louis, MO).
Cell preparation
Fresh adult thymi were homogenized to a suspension of single cells in cold 2% newborn calf serum/RPMI 1640 medium using a steel mesh. CD4+ cells were depleted by two cycles of treatment with an optimal concentration of anti-CD4 (RL172) at 4°C, 40 min, followed by incubation with guinea pig complement for 35 min at 37°C. Viable cells were recovered by centrifugation over a Ficoll/sodium diatrizoate cushion. The viable cells were analyzed by two-color flow cytometry for the expression of CD4 and CD8 molecules. This procedure removed >98% of CD4+ thymocytes. The remaining viable cells were composed of a roughly equal mixture of CD8 SP and DN thymocytes. The same protocol was used to obtain a mixture of 6C10CD8 SP and 6C10 DN cells after treatment with RL172 and anti-6C10 mAb plus complement. CD4CD8+ thymocytes were purified with a MidiMACS cell sorter (Miltenyi Biotec. Auburn, CA). Briefly, CD4 thymocytes were suspended in balanced salt solution buffer supplemented with 2% FBS and incubated for 15 min at 4°C with anti-CD8 microbeads. Positively selected CD8+ cells were then isolated, with >97% purity of CD4CD8+ thymocytes being routinely observed, based on FACS analysis.
Immunofluorescent staining
Thymocytes (1x106/ml) isolated from each individual subpopulation were prepared for fluorescent antibody staining. For two-color analysis, staining was performed directly with anti-CD8FITC and either with anti-CD4PE or anti-TCRßPE. For three-color analysis, anti-CD8CyChrome or anti-CD3CyChrome was used with PE- and FITC-labeled mAb. For four-color analysis, the cells were first stained with anti-6C10, followed by incubation with FITCgoat anti-rat Ig; the cells were then stained with PE-labeled anti-HSA, anti-3G11 or anti-CD69 mAb; the third staining was with anti-CD8CyChrome and the last staining with anti-TCRßAPC. Electronic compensation was performed for multi-color flow cytometry analysis based on the isotype and positive controls of the cells stained the each respective fluorescent mAb. The expression of cell-surface molecules was analyzed by flow cytometry equipped with two lasers (FACSCalibur; Becton Dickinson). Since FL3 and FL4 have cross-beam compensation, CyChrome and APC were used simultaneously. In all cases, gains were adjusted to exclude dead cells. A second gate was placed to analyze CyChrome-stained CD8+ or CD3+ cells. In four-color analysis, logical gates were used to define TCR
ß+CD8+ medullary-type thymocytes. A number of gated files were collected for the analysis of cell-surface markers expressed on CD8 SP cells. Data acquisition and analysis were performed using CellQuest Software (Becton Dickinson).
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Results |
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Relationship between the expression of HSA and CD69.
HSA is a hematopoietic differentiation antigen expressed on virtually all immature DN and DP thymocytes. Maturation from DN to SP is accompanied by down-regulation of HSA (13,18). 6C10CD8 SP medullary-type thymocytes contained three subpopulations of cells based on the expression of CD69 and HSA: CD69+HSAhi (35%), CD69HSAint (40%) and CD69HSA/lo (21%) (Fig. 4B). It seemed that down-regulation of CD69 was observed earlier than the down-regulation of HSA. The cells of CD69+HSAhi might give rise to the CD69HSAint subgroup, which might in turn give rise to CD69HSA/lo CD8 SP medullary thymocytes.
Relationship between the expression of 3G11 and HSA.
As shown previously, the earliest TCRß+CD8 SP thymocytes are 6C10+CD69+3G11+HSAhi. Within 6C10CD8 SP, while HSA was being down-regulated to a low level, TCR
ß+CD8 SP thymocytes still remained 3G11+ (Fig. 4C
). Thus, the expression of HSA was down-regulated earlier than 3G11. However, while HSA was being shut off, 3G11 was also being decreased (Fig. 4C
). Conceivably, 3G11 expression was kinetically first turned on, then off during differentiation among TCR
ß+ CD8 SP thymocytes.
Sequential expression of Qa-2 and 3G11 on 6C10TCRß+CD8 SP thymocytes.
Qa-2, a marker expressed on functional SP cells, was used in combination with other markers to identify phenotypic features of Qa-2+ medullary-type CD8 SP thymocytes. As shown in Fig. 4(DF), the TCR+CD8 SP thymocytes that express high levels of Qa-2 were negative for CD69, HSA and 3G11. By contrast, cells in the Qa-2 population were 30% CD69+, 67% HSAint/hi and 60% 3G11+. Thus, Qa-2CD8 SP medullary thymocytes are composed of cells at various stages of phenotypic differentiation. Among the stages, 6C10+CD69+HSAhi3G11+Qa-2 might be the early, with 6C10CD69+/HSAhi/int 3G11+Qa-2 being an intermediate and 6C10CD69HSA3G11Qa-2/+ being the most mature cells. Notably, 3G11 expression was turned off earlier than the initiation of Qa-2 expression. It is hence conceivable that the 6C10CD69HSAlo3G11+Qa-2 cells may give rise to 6C10CD69HSA3G11Qa-2 cells, which then give rise to 6C10CD69HSA3G11Qa-2+.
CD8 SP thymocytes with the recent thymic emigrants (RTE) phenotype
It is reported that RTE are distinct from most of the medullary-type thymocytes (21). In contrast to medullary-type SP thymocytes, most RTE were L-selectinhigh and CD69. In addition, CD4+CD8 and CD4CD8+ RTE were phenotypically distinct from each other in that the former were ß7 integrin/low, CD45RBint and CD45RC, while the latter were ß7 integrinhigh, CD45RBhigh and CD45RClow. Also, the majority of RTE were HSAQa-2+, implying they are phenotypically more mature (21). In our assays, cross-analysis of HSA and ß7 integrin showed that 22.1% of medullary-type CD8 SP thymocytes had a phenotype that corresponded to RTE. Based on the phenotype of CD4CD8+ RTE that are CD69 ß7 integrinhigh and CD69 L-selectinhigh, cells with the corresponding phenotype constituted 21 and 19% of CD8 SP medullary-type thymocytes, respectively. Approximately 88% of Qa-2+ CD8 SP medullary-type thymocytes expressed high levels of both ß7 integrin and L-selectin (Fig. 5). Unexpectedly, we found that L-selectin was expressed at high levels throughout the development of medullary-type CD8 SP thymocytes. At CD69+ stages, the proportion of L-selectin+ cells was 83%; HSAint/lo/Qa-2 stages, 67%; and Qa-2+ stage, 88%. In terms of ß7 integrin expression, at CD69+ stages, 50% were positive; at CD69HSAint/lo/ stages, 5966% positive, and at Qa-2+ stage, 88% positive. These results suggest that cells at early and intermediate stages have already expressed L-selectin and ß7 integrin. In addition, once these two markers started to express, the expression continued throughout the rest of the developmental stages.
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Discussion |
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Unlike CD4 SP thymocytes, most of which are TCRß+CD3+ and hence are medullary-type cells (23), CD8 SP thymocytes are composed of both TCR
ß+CD3+ (60 ± 5%) and TCR
ßCD3 subsets, with the latter being immature CD8 SP cells and the former being medullary-type CD8 SP thymocytes (24). Immature CD8 SP cells needed to be excluded from the analysis. While immature CD3CD8 SP thymocytes are larger in size than mature CD3+CD8 SP cells, the separation between these cells by size using forward scatter parameter is not very effective (21). Therefore, we need multi-color staining to distinguish the mature CD3+CD8 SP thymocytes.
After depletion of CD4+ cells, the subpopulations among TCRß+CD8 SP thymocytes were identified based on the expression patterns of the TCR
ß, CD3, CD8 and other markers in CD4 cells, then the phenotypic differentiation pathway of TCR
ß+CD8 SP thymocytes was predicted. According to the sequential down-regulation of the markers, such as 6C10, CD69 and HSA, expressed on functionally immature cells, and up-regulation of the markers, such as Qa-2, expressed on functionally mature cells, the medullary-type CD8 SP thymocytes could be phenotypically divided into discrete subpopulations. Thus, we are able to predict the earliest and latest stages of these subpopulations. Cells with phenotypes intermediate to these two extremes are regarded as being at intermediate developmental stages.
CD69 is generally regarded as the earliest marker expressed on the DP thymocytes undergoing positive selection and CD69+ SP thymocytes are thereby regarded as the earliest virgins of medullary cells (19,20). We have adopted the markers of both 6C10 and 3G11 molecules to define CD8 SP medullary-type thymocytes. Four-color staining was used to analyze the expression sequences of 6C10 and other surface markers on CD4 thymocytes. In the TCRß+CD8 SP medullary-type thymocytes, 6C10+ cells expressed high levels of both HSA and CD69. To our surprise, 6C10 was down-regulated earlier than was CD69. It is noted that DP thymocytes are 6C10+3G11. The direct descendants of DP cells that have undergone positive selection within CD8 SP thymocytes would be the 6C10+CD69+3G11+HSAhiCD8 SP thymocytes. These cells are probably the earliest medullary-type CD8 SP thymocytes, which may give rise to 6C10CD69+3G11+HSAhi subpopulations.
Figure 3(C) shows a subpopulation of 6C10+CD69+3G11 cells constituting 5% of total CD8 SP medullary-type thymocytes. These cells were not reported by Hayakawa and Hardy (11,12). Instead, they suggest that 3G11+ and 3G11 CD8 SP thymocytes may have precursorprogeny relationship with respect to the expression of CD45RC and the expression level of CD3. They did not analyze the phenotype by combination of 6C10 and 3G11, and thus were unable to reveal the presence of the 6C10+CD69+3G11CD8 SP phenotype. However, the possibility that it is merely created by quadrant placement in our case cannot be excluded. An additional experiment was performed by depletion of 3G11+CD4+ cell and then analyzing the expression of 6C10 and CD69 in surviving cells. The 3G11TCR+CD8 SP survivors contained both 6C10+CD69+ (18%) and 6C10CD69 (82%) CD8 SP medullary-type thymocytes (data not shown). At present we cannot provide valid evidence for the true presence of this subpopulation by FACS sorting for the lack of the facility in our laboratory. Therefore, this subpopulation is not presented in this paper.
We found that immature CD8 SP thymocytes also express 6C10. Deletion of both 6C10+ and CD4+ thymocytes resulted in a mixture of mature 6C10TCRß+CD8 SP and DN thymocytes without contamination of immature TCR
ßCD8 SP thymocytes. Using three-color FACS analysis, medullary-type 6C10CD8 SP cells were further characterized for the co-expression of CD69, HSA, 3G11 and Qa-2 markers. We found that the molecules expressed on TCR
ß+CD8 SP cells are sequentially down-regulated in the following order: 6C10
CD69
HSA
3G11. As 6C10 was shut off, the CD69+ cells were still present (Fig. 3A
), Thus, the earliest medullary-type CD8 SP thymocytes would be 6C10+CD69+3G11+, which give rise to 6C10CD69+3G11+ thymocytes. Down-regulation of CD69 occurred earlier than HSA as demonstrated by the presence of CD69HSAhi/int cells, which were apparently the progeny of CD69+HSAhi cells. During the stage when HSA was down-regulated from intermediate to low levels, 3G11 remained positive, implying that HSAint3G11+ cells could be the precursors of HSAlo3G11+ cells. When the expression of 3G11 was turned from positive to negative, the cells were CD69HSAlo/, thus the CD69HSAlo3G11+ cells should be the precursors of CD69HSAlo/3G11 cells. Qa-2 was expressed at the final stage when 6C10, CD69, HSA and 3G11 were all down-regulated. Therefore 3G11Qa-2 cells should appear earlier than 3G11Qa-2+ cells. All these phenotypic characteristics allow us to separate medullary-type CD8 SP thymocytes into six subpopulations and their phenotypic maturation progress is suggested as follows:
It is argued that some of the mature CD8 SP subpopulations might represent recirculating mature cells. It seems unlikely as the re-entry of periphery resting T cells to the thymus was extremely limited (<0.02%) in normal adult mice, in contrast to the S phase T blasts which migrated to the thymus in substantial numbers (0.4%) (25). We have also tested the thymic homing of purified CD8+ T cells pooled from mouse lymph nodes (26). Of these purified CD8+ T cells, 99% were small size (forward light scatter versus side scatter analysis) and 98.6% were resting cells (G0/G1 phase). Twenty-four hours after i.v. injection of FITC-labeled purified CD8+ T cells, cell suspensions from different immune organs were prepared and FACS analysis performed. The results indicated that localization of these cells was ~3% in spleen, 1.5% in lymph nodes and virtually undetectable in thymus. Thus, all the subpopulations of CD8 SP medullary-type thymocytes represent the natural residence in thymus.
CD4CD8+ RTE were reported to be CD69ß7 integrinhiL-selectinhi (21). We tried to identify cells with the RTE phenotype amongst medullary-type CD8 SP thymocytes. We found 88% of Qa-2+ cells expressing high levels of ß7 integrin and L-selectin. This implies that a small portion (12%) of Qa-2+ cells might not be able to emigrate into the periphery even though they represent the latest group of mature thymocytes. On the other hand, 83% of CD69+ cells, an early stage of medullary-type CD8 SP thymocytes, expressed high levels of L-selectin. It seems that L-selectin was expressed throughout the major developmental stages of CD8 SP medullary thymocytes. Similarly, ß7 integrin was also expressed during early stages (CD69+) of CD8 SP medullary-type thymocytes. Along the developmental process of medullary-type CD8 SP thymocytes, however, the percentage of ß7 integrin+ cells steadily increased (from 48 to 88%), implying that once ß7 integrin was expressed it was not down-regulated during the remaining differentiation stages. Thus, ß7 integrin and L-selectin can be used as reference markers for RTE, but not as stage-specific markers. They also should not be used as maturation markers for CD8 SP medullary thymocytes.
The fact that 40% Qa-2 cells of mature CD8 SP thymocytes are CD69ß7 integrinhiL-selectinhi, similar to the phenotype of RTE, suggests that there may have a subset of Qa-2 cells that are eligible to emigrate to the periphery. At present, there is no unique marker available to identify this cell subset.
In thymus, cortisone-sensitive cells are phenotypically and functionally immature. After cortisone treatment, virtually all 6C10+ cells in medullary-type CD8 SP thymocytes were deleted, whereas the other subpopulations were unaffected. Thus, the 6C10+TCRß+CD8 SP thymocytes were immature cells.
The identification of subpopulations within CD8 SP medullary-type thymocytes by phenotypic analysis sheds light upon the study of the functional maturation with these cells. In the CD4 SP medullary thymocytes, we found that the functional differentiation pathway of CD4 SP medullary thymocytes is in close correlation with their phenotypic differentiation pathway (23,27).
Medullary thymocytes do undergo limited proliferation (20). In our analysis, the subpopulations of 6C10+CD69+HSA+3G11+Qa-2 and 6C10CD69+HSA+3G11+Qa-2 comprise 55% of total medullary-type CD8 SP thymocytes, and, conceivably, the cell proliferation rate may be higher in these two subpopulations than in the other four subpopulations. As cell differentiation is usually inversely related to their proliferation, the functional acquisition probably occurs at late subpopulations of medullary CD8 SP thymocytes. The validity of this predicted precursorprogeny relationship among medullary-type CD8 SP thymocytes requires further study by ex vivo and in vivo assays. Kinetic generation of SP thymocytes in fetal thymus organ culture is ongoing in our laboratory. Nonetheless, our phenotypic analyses will allow the separation of these cell subpopulations, the estimation of their functional status and allow an understanding of the thymocyte functional maturation process.
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Acknowledgments |
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Abbreviations |
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APC allophycocyanin |
BSS balanced salt solution |
DN double negative |
DP double positive |
HSA heat-stable antigen |
SP single positive |
RTE recent thymic emigrant |
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Notes |
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Received 31 July 2000, accepted 1 December 2000.
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References |
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