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The Journal of Immunology, 2000, 165: 1832-1839.
Copyright © 00 by The American Association of Immunologists

Early Ontogeny of Thymocytes in Pigs: Sequential Colonization of the Thymus by T Cell Progenitors1

Marek Sinkora2,3,*, Jirí Sinkora3,*, Zuzana Reháková* and John E. Butler{dagger}

* Department of Immunology and Gnotobiology, Institute of Microbiology, Czech Academy of Sciences, Novy Hrádek, Czech Republic; and {dagger} Department of Microbiology, University of Iowa, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Successive colonization of the thymus by waves of thymocyte progenitors has been described in chicken-quail chimeras and suggested from studies in mice. In swine, we show that the first CD3{epsilon}-bearing thymocytes appear on day 40 of gestation (DG40). These early thymocytes were CD3{epsilon}high and belonged to the {gamma}{delta} T cell lineage. Mature CD3{epsilon}high {alpha}ß thymocytes were observed 15 days later (DG55), and their occurrence was preceded by the appearance of CD3{epsilon}low thymocytes (DG45). Thereafter, we observed transient changes in thymocyte subset composition (DG56-DG74), which can be explained by a gap in pro-T cell delivery to the thymus. This delivery gap corresponds with the expression of the pan-leukocyte CD45 and pan-myelomonocytic SWC3a markers in fetal liver and bone marrow and is probably caused by shifting of primary lymphopoiesis between these organs. Therefore, we conclude that the embryonic thymus is colonized by at least two successive waves of hemopoietic progenitors during embryogenesis and that the influx of thymocyte progenitors is discontinuous. Surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to compare thymocyte differentiation in pigs with that described for rodents and humans and to propose a model for T cell lymphopoiesis in swine. We also observed that the porcine IL-2R{alpha} (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, we observed a subset of TCR{gamma}{delta}+ thymocytes that were cycling late during their development in the thymus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In comparison to other species, swine express a number of unpredicted differences in the T cell peripheral pool that may reflect differences in T lymphocyte ontogeny and differentiation. Swine, together with other ungulates and birds, have an abundance of {gamma}{delta} T lymphocytes in their peripheral lymphoid pool (1, 2, 3, 4). Furthermore, a substantial number of resting {alpha}ß T lymphocytes in porcine periphery is CD4+CD8+ double positive (DP)4 (3, 4, 5, 6, 7, 8, 9). TCR{gamma}{delta}+CD2- lymphocytes are numerous in pigs (3, 4), and there is a large number of CD8{alpha}{alpha}-bearing cells that includes NK cells, {gamma}{delta} T cells, and at least two subsets of {alpha}ß T cells (10). Despite these unique features of peripheral T cells, thymocytes in young and adult pigs resemble those in other species, and their characteristics can be summarized in the following manner. The majority of thymocytes is DP, while double negative (DN, CD4-CD8-) and single positive (SP, CD4+CD8- or CD4-CD8+) subsets are less frequent (3, 8, 9, 10, 11, 12). The majority of DP thymocytes bears no or very little CD3, and although some DP cells can be found among large, blast-like cells, most of them belong to the well-known population of small cortical DP thymocytes (2, 3). CD3+ porcine thymocytes differ in their staining intensity such that small TCR{gamma}{delta}- thymocytes express CD3 at low to medium density and belong to the {alpha}ß T cell lineage (3). In contrast, medium-sized thymocytes express CD3 at medium to high density and may belong to either the TCR{alpha}ß or the TCR{gamma}{delta} lineage (2, 3). Finally, the expression of other T cell markers is also consistent with findings in other mammalian species, i.e., all DP and SP thymocytes are positive for CD5 and CD6 (9, 12), and almost all are positive for CD1 and CD2 (7, 8). In contrast, the majority of DN thymocytes expresses CD1 at low density (7, 8), while a minor subset is negative for CD2, CD5, and CD6 (8, 9, 12). All DP and DN thymocytes are believed to represent less mature phenotypes. This is supported by the findings that they express lower amounts of both CD5 and CD6 than more differentiated SP thymocytes (9, 12). It has also been shown that almost all CD8-positive cells in the porcine thymus express the CD8{alpha}ß heterodimers, while a substantial proportion of the CD8-bearing cells in the periphery expresses the CD8{alpha}{alpha} homodimers (10).

In this report, we describe the use of various leukocyte surface markers to analyze the development of thymocytes in pig fetuses. Our results suggest that the embryonic thymus is colonized by progenitors in at least two successive waves during embryogenesis. Moreover, we show that the expression of CD25 does not appear to be required for successful generation of normal T cells in pigs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Animals used in the study were derived from Minnesota miniature pigs by repeated crossing with outbred black Vietnam-Asian and white Malaysian-derived pigs and selected for high fecundity and small body size. All sows were serum negative for common swine pathogens. Fetuses were obtained by hysterectomy under systemic halothane-oxygen anesthesia. Gestation age was calculated from the day of mating. All experiments were approved by the Ethical Committee of the Institute of Microbiology, Czech Academy of Science, according to guidelines in the Animal Protection Act.

Preparation of cells

Cell suspensions from thymus and fetal liver were prepared in RPMI 1640 medium supplemented with 2% FCS (2% FCS-RPMI; Sigma, St. Louis, MO) by careful teasing of tissues using two forceps. Bone marrow cells were flushed from tibiae and/or femur with cold PBS. Cells from early bone marrow were isolated by collagenase digestion. Briefly, the excised organs were cut into small pieces, transferred into a digestion medium (2% FCS-RPMI with 100 U/ml collagenase type V; Sigma), and incubated for 30–60 min on an orbital shaker at 37°C. All cell suspensions were filtered through a fine nylon mesh and washed twice in cold PBS containing 0.1% sodium azide and 0.2% gelatin (PBS-GEL). Erythrocytes were removed from the pelleted cells using hypotonic lysis by treating them with 25 ml of distilled water for 30 s followed by osmotic reconstitution with 2x PBS. Finally, the cells were washed twice in cold PBS-GEL, counted, and adjusted to a density of 5 x 106 to 1 x 107 cells per ml.

Immunoreagents

The following mouse anti-pig mAbs were used as primary immunoreagents: anti-CD3{epsilon} (PPT3, IgG1 or PPT6, IgG2b), anti-TCR{gamma}{delta} (PPT17, IgG1 or PPT16, IgG2b), anti-CD4 (10.2H2, IgG2b), anti-CD8 (76-2-11, IgG2a), anti-CD25 (K231.3B2, IgG1), anti-CD45 (K252.1E4, IgG1, pan-leukocyte Ag), anti-IgM (LIG4, IgG1), and anti-SWC3a (74-22-15, IgG2b; pan-myelomonocytic Ag) (13). Anti-CD4, anti-CD8, and anti-CD45 mAb were also labeled with biotin N-hydroxysuccinimide ester (Vector Laboratories, Burlingham, CA) according to a protocol recommended by the manufacturer.

F(ab')2 of goat polyclonal Abs (pAb) specific for mouse Ig subclasses (Southern Biotechnologies Associates, Birmingham, AL) labeled with FITC or R-PE were used as secondary immunoreagents. Biotinylated primary mAb were visualized using a streptavidin-PE conjugate or using a streptavidin-Cy5-Chrome complex (St-Cy5); both streptavidin reagents were purchased from Immunotech (Westbrook, ME).

All immunoreagents were titrated to achieve an optimal signal/noise ratio. Subisotype-matched mouse anti-rat mAb were used as negative controls.

Staining of cells

All staining and washing steps were performed in cold PBS-GEL. Two-color staining was done using 5 x 105 to 1 x 106 cells that had been incubated with a combination of primary mAb of different subisotypes for 30 min and subsequently washed twice. Mixtures of subisotype-specific FITC- and PE-conjugated goat anti-mouse pAb were then added to the cell pellets in appropriate combinations. After 30 min, cells were washed three times and analyzed by flow cytometry or were further used for three-color staining. Simultaneous two-color detection of subisotype-matched mAb involved visualization of the first mAb using a FITC-conjugated goat anti-mouse secondary pAb. The stained cells were then incubated for 10 min with PBS containing 10% heat inactivated, nonimmune normal mouse serum and 0.1% sodium azide (PBS-NMS) to block the free binding sites on the secondary pAb. The second mAb was biotin-labeled and was visualized using a streptavidin-PE conjugate.

Three-color staining involved staining of double-stained cells with a third chromofore. Double-stained cells were incubated for 10 min with PBS-NMS to block free binding sites on previously bound secondary pAb. After washing, the cells were incubated for 30 min with a third biotinylated primary mAb for 30 min and subsequently washed twice. Finally, St-Cy5 was added for 30 min, and the cells were then washed three times before flow cytometric analysis. Alternatively, unstained cells were incubated with a mixture of three primary mouse mAb of different subisotypes, of which one was labeled with biotin. After incubation and washing, mixtures of FITC- and PE-conjugated goat anti-mouse subisotype-specific pAb, plus St-Cy5, were added to the cell pellets.

The DNA content of single- or double-stained cells was determined using the DNA intercalating probe 7-aminoactinomycin D (7-AAD). Surface-stained cells were washed in cold PBS containing 0.1% sodium azide (PBS-Az), centrifuged, and fixed with cold (-20°C) 70% ethanol for 1 h at 4°C, centrifuged again (2000 x g, 10 min, 4°C), and washed in PBS-Az. The pellets were then incubated with 50 µl of 7-AAD in PBS-Az (40 µg/ml) for at least 20 min at 4°C in dark until measured by flow cytometry.

Flow cytometry

Samples were measured on a standard FACSort flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA). A total of 50,000–300,000 events were collected in each measurement. Electronic compensation was used to eliminate spectral overlaps between individual fluorochromes in two- and three-color staining experiments. Damaged and dead cells were excluded from analysis using propidium iodide fluorescence. A FACSort doublet discrimination module was used in DNA content analysis that allowed single-cell events to be discriminated from doublets and higher multiplets.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thymocyte subsets in pigs

Thymic cell subsets in pigs were characterized by mAb specific for porcine CD45, SWC3a, CD4, CD8, CD3{epsilon}, TCR{gamma}{delta}, and IgM. Two-color staining for the pan-leukocyte CD45 and the pan-myelomonocytic SWC3a markers allowed discrimination of lymphoid cells (CD45+SWC3a-) from nonlymphoid ones, i.e., myelomonocytic (CD45+SWC3a+) cells and nonleukocytes (CD45-SWC3a-). Thymic B cells represented a minor population (<1%) during the fetal ages studied (data not shown). These were identified by surface IgM expression. All IgM-CD45+SWC3- leukocytes were hypothesized to belong to the T cell lineage, their number was normalized to 100%, and they are hereafter referred to as thymocytes.

Analysis of CD4, CD8, and CD3{epsilon} expression on porcine thymocytes identified at least: 1) three subsets of large-sized CD3{epsilon}- (CD4-CD8-, CD4-CD8+, and CD4+CD8+; Fig. 1GoB), 2) two subsets of small-sized CD3{epsilon}- (CD4-CD8- and CD4+CD8+; Fig. 1GoC), 3) two subsets of small-sized CD3{epsilon}low (CD4+CD8+ and CD4lowCD8+; Fig. 1GoD), 4) two subsets of small-sized CD3{epsilon}med (CD4+CD8+ and CD4+CD8low; Fig. 1GoE), and 5) four subsets of large-sized CD3{epsilon}high (CD4+CD8+, CD4-CD8-, CD4+CD8-, and CD4-CD8+; Fig. 1GoF) thymocytes. We also observed that {gamma}{delta} thymocytes were medium-sized (Fig. 1GoG), and most of them did not express both CD4 and CD8 (Fig. 1GoH). However, a small number of {gamma}{delta} thymocytes with the CD4-CD8+ or CD4+CD8+ surface phenotype was always present (Fig. 1GoH). It is also important to note that TCR{gamma}{delta} expression appeared with no observable CD3{epsilon}low or TCR{gamma}{delta}low transitional stage (Fig. 1GoG). Because the proportion of CD3{epsilon}highCD4-CD8- thymocytes (Fig. 1GoF, lower left quadrant) corresponded to the number of TCR{gamma}{delta}highCD4-CD8- cells (Fig. 1GoH, lower left quadrant), we have concluded that all thymic DN cells expressing high levels of the CD3/TCR complex are {gamma}{delta} thymocytes. Because of the current unavailability of a mAb specific for TCR{alpha}ß, putative {alpha}ß thymocytes in this work were detected as CD3{epsilon}highTCR{gamma}{delta}- cells. These are hereafter referred to as {alpha}ß thymocytes. Because TCR{gamma}{delta}high DN cells represent {gamma}{delta} thymocytes, the majority of {alpha}ß thymocytes was SP cells, which is typical for mature {alpha}ß T cells (Fig. 1GoF).



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  		FIGURE 1. Analysis of CD3{epsilon}, TCR{gamma}{delta}, CD4, and CD8 expression on thymic cells isolated from a newborn piglet. In late-term fetuses and after birth, nonthymocytes represent <1% of isolated cells and are not considered in this representative analysis. The cells were triple stained with anti-CD8, anti-CD4, and either anti-CD3{epsilon} (A–F) or anti-TCR{gamma}{delta} (G–H) mAb. Based on size (forward scatter) and CD3{epsilon} fluorescence intensity (A), six thymocyte subsets were distinguished: large-sized CD3{epsilon}- (R1, 21%), small-sized CD3{epsilon}- (R2, 26%), small-sized CD3{epsilon}low (R3, 30%), small-sized CD3{epsilon}med (R4, 4%), large-sized CD3{epsilon}high (R5, 14%), and medium-sized CD3{epsilon}low (R6, 5%). The CD8/CD4 profile for each subset is indicated (B–F). The events in the R6 region were shown by DNA analysis to be doublets (see Fig. 9GoG) and were not analyzed. Forward scatter vs TCR{gamma}{delta} expression profile (G) shows that TCR{gamma}{delta}+ thymocytes mostly belong to the large-sized cells (R7, 4%). The CD8/CD4 profile for the latter is also shown (H).

 
As in other species, small thymocytes were a rather homogenous population, most of them being DP cells (Fig. 1Go, C–E). By contrast, a population of large cells differed markedly in surface phenotype (Fig. 1Go, B and F). Fig. 2Go shows the analysis of large CD4- and/or CD8- thymocytes in terms of their expression of CD3{epsilon} and TCR{gamma}{delta}. This analysis suggests that, among CD3{epsilon}- precursors, CD8+ SP cells (Fig. 2GoC, upper left quadrant) greatly outnumbered CD4+ SP cells (Fig. 2GoD, upper left quadrant). Moreover, only low CD4 expression was found on CD3{epsilon}-CD4+CD8- precursor cells (Fig. 2GoD, upper left quadrant). Once again, when the number of cells in the lower right quadrants of Fig. 2Go, C–F was compared, results suggested that CD3{epsilon}+ DN thymocytes belong to the {gamma}{delta} T cell lineage.



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  		FIGURE 2. Representative analysis of CD3{epsilon}, TCR{gamma}{delta}, CD4, and CD8 expression on large thymocytes isolated from 80-day-old fetuses. Thymocytes were triple stained with combinations of anti-CD8, anti-CD4, and either anti-CD3{epsilon} or anti-TCR{gamma}{delta} mAb. CD4- (A) and CD8- (B) large thymocytes were gated using region R1 and region R2, respectively, and analyzed for additional surface phenotypic markers. Dot plots C and E shows gated CD4- cells, while dot plots D and F shows gated CD8- cells.

 
Thymocyte subsets during prenatal ontogeny in pigs

The first leukocytes in the thymic rudiment appeared at the end of the first trimester of intrauterine life (day 38 of gestation, DG38). These were triple negative (TN), and the majority expressed the pan-myelomonocytic Ag SWC3a (Fig. 3Go). One fourth of all leukocytes, however, were SWC3a- cells; these may have represented early thymocyte progenitors (Fig. 3GoA).



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  		FIGURE 3. The phenotype of the first thymic leukocytes recovered. Cells isolated from the rudimentary thymus of 38-day-old fetuses were double-stained with combinations of anti-CD45 and anti-SWC3a (A), anti-CD3{epsilon} and anti-TCR{gamma}{delta} (B), or anti-CD8 and anti-CD4 (C) mAb. The majority of leukocytes (CD45+ cells) in the early thymus expressed the pan-myelomonocytic marker SWC3a (A). While virtually no expression of CD4, CD8, and CD3{epsilon} could be observed (B and C), some leukocytes had the lymphoid phenotype CD45+SWC3a- (A).

 
We first found CD3{epsilon} expression on DG40 in the fetal thymus (Fig. 4GoA). At this time, TCR{gamma}{delta}+ thymocytes accounted for all CD3{epsilon}high cells, and CD3{epsilon}low thymocytes were almost absent (Fig. 4GoB). Until DG50, TCR{gamma}{delta}+ thymocytes represented almost exclusively all CD3{epsilon}high thymocytes (Fig. 4GoB). Thereafter, the proportion of TCR{gamma}{delta}+ thymocytes decreased as TCR{alpha}ß+ cells became the predominant CD3{epsilon}high thymocyte subset (DG55; Fig. 4GoB). Fig. 4Go, A and B indicates that CD3{epsilon}low cells appeared ~10 days before the first TCR{alpha}ß+ cells. The majority of the earliest thymocytes was large CD3{epsilon}- cells (Fig. 4GoC) bearing neither CD4 nor CD8 (Fig. 5GoD). CD3{epsilon}- DP cells were detected soon thereafter (DG45; Fig. 5GoA and Fig. 4GoC). CD45/SWC3a immunophenotyping suggested that the proportion of myelomonocytic cells (SWC3a+) decreased sharply between DG40 and DG45 as the frequency of CD45low and especially CD45high lymphoid cells (SWC3a-) increased (Fig. 6GoB).



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  		FIGURE 4. Expression of CD3{epsilon} and TCR{gamma}{delta} on fetal pig thymocytes. A shows the frequency of CD3{epsilon}+ thymocytes as a percentage of all thymocytes (solid line). The contribution to this value of large CD3{epsilon}high (dashed line) and small CD3{epsilon}low (dotted line) thymocytes is also indicated. B shows the frequency of TCR{gamma}{delta}high (solid line) and TCR{alpha}ßhigh (dotted line) thymocytes. TCR{gamma}{delta}-CD3{epsilon}high cells are considered to be TCR{alpha}ßhigh thymocytes. C shows the frequency of small (solid line) and large (dotted line) CD3{epsilon}- thymocytes. Data are mean values ± SEM from at least three animals for each stage of gestation.

 


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  		FIGURE 5. Expression of CD4 and CD8 on fetal pig thymocytes. The proportion of CD4+CD8+ (A), CD4+CD8- (B), CD4-CD8+ (C), and CD4-CD8- (D) subsets expressed as a percentage of total thymocytes is shown. In each graph, the proportions of total (solid line), small (dashed line), and large (dotted line) thymocytes with selected phenotypes is presented. Data are mean values ± SEM from at least three animals for each stage of gestation.

 


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  		FIGURE 6. Cell size analysis and CD45/SWC3a expression on fetal pig thymocytes. A shows the frequency of small (solid line) and medium plus large (dotted line) thymocytes as determined by forward scatter. B shows the frequency of CD45highSWC3a- (dashed line) and CD45lowSWC3a- (dotted line) thymocytes. Myelomonocytic CD45+SWC3a+ cells are also shown (solid line). Data are mean values ± SEM from at least three animals for each stage of gestation.

 
After DG56–58, we observed major changes in the composition of both leukocyte and lymphoid subsets. First, the proportion of large CD3{epsilon}+ thymocytes increased, and CD3{epsilon}high cells became the dominant thymocyte subset (Fig. 4GoA). This temporary dominance persisted between DG56 and DG74 and included both the TCR{alpha}ß+ and the TCR{gamma}{delta}+ thymocytes (Fig. 4GoB). Consistent with this observation, the proportion of CD3{epsilon}low and CD3{epsilon}- thymocytes decreased (Fig. 4Go, A and C, respectively). Second, the percentage of SP and DN thymocytes increased (Fig. 5Go, B–D), and the number of DP cells decreased (Fig. 5GoA). Finally, large thymocytes (Fig. 6GoA) and leukocytes with a typical myelomonocytic phenotype (CD45+SWC3a+) became more apparent during this period (Fig. 6GoB). The change in the number of fetal thymic leukocytes was most pronounced between DG58 and DG67 (10 days in total). Thereafter, the frequency of individual subsets slowly returned to the levels observed on DG55 ( Figs. 4–6GoGoGo).

After DG74, only minor changes in the composition of thymocyte subsets were observed. The majority of CD3{epsilon}-positive thymocytes was small CD3{epsilon}low cells while a minor proportion was CD3{epsilon}high cells (Fig. 4GoA). The proportion of TCR{alpha}ß+ thymocytes was always slightly higher than that of TCR{gamma}{delta}+ thymocytes. As regards the expression of CD4 and CD8, the majority of thymocytes was DP cells while the minority were DN or SP cells (Fig. 5Go). The number of myelomonocytic cells and B cells was negligible (Fig. 6Go).

Characterization of leukocyte subsets in early primary hemopoietic centers

Precursor and progenitor stages of lymphocyte differentiation in pigs have not been completely phenotyped. While mature lymphocytes and myelomonocytic cells can be characterized as CD45highSWC3- and CD45highSWC3high leukocytes, respectively (13, 14), precursor stages of myelomonocytic lineages have been suggested to represent the majority of the SWC3low population in bone marrow in young pigs (14). Moreover, late pre-B II cells in pigs were defined as CD45lowSWC3- lymphoid cells dominating among small mononuclear leukocytes in the fetal bone marrow (15). Finally, our unpublished results show that all pre-B II stages in pigs have also the CD45lowSWC3- surface phenotype. Thus monitoring the presence of CD45lowSWC3- leukocytes may represent a mean of estimating primary lymphopoietic activity. To address this issue, we studied the frequency of this population among leukocytes during fetal ontogeny. Cells with the CD45lowSWC3- phenotype were first detected in the fetal liver at DG21 (Fig. 7GoA). The proportion of these cells in the fetal liver gradually increased until DG40 and decreased thereafter (Fig. 7GoA). Before DG40, the bone marrow was rudimentary and only few leukocytes, most of them (>96%) being SWC3+, could be obtained. Higher numbers of leukocytes were isolated from the bone marrow on DG45, and CD45lowSWC3a- cells constituted a small minority of the CD45+ cells at this developmental stage (Fig. 7GoB). Nonetheless, the frequency of CD45lowSWC3a- leukocytes recovered from the bone marrow progressively increased and reached its maximum at DG67. The proportion of these cells then remained essentially invariable until DG100 before declining at birth. While our analysis showed relatively numerous CD45high lymphocytes in fetal liver after DG58, the frequency of this population remained low in bone marrow until birth (Fig. 7Go).



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  		FIGURE 7. Expression of CD45 and SWC3a on mononuclear cells (defined by low side scatter values) isolated from fetal liver (A) and bone marrow (B). The proportions of CD45lowSWC3a- (solid line) and CD45highSWC3a- (dotted line) lymphoid subsets are presented as a percentage of total CD45+ cells. The combined proportions of CD45lowSWC3a- and CD45highSWC3a- subsets do not equal 100% because data on the myelomonocytic CD45+SWC3a+ subset are not shown to reduce clutter. Data are mean values ± SEM for at least three animals at each stage of development.

 
CD25 expression on porcine thymocytes

Analysis of CD25 expression on thymocytes in pigs revealed that essentially all cells expressing CD25 were also CD3{epsilon}high and therefore represented more mature stages of thymocyte development (Fig. 8GoA). Virtually no CD3{epsilon}-CD25+ cells were found in fetal thymus since DG38. Staining of thymocytes for TCR{gamma}{delta} vs CD25 showed that the majority of CD25+ cells was TCR{gamma}{delta}- and thus presumed to belong to the {alpha}ß T cell lineage (Fig. 8GoB). Further support for the argument that CD25 is present predominantly on mature stages of thymocytes comes from the finding that the CD25+ cells displayed mostly a SP phenotype (Fig. 8GoE). A small proportion of TCR{gamma}{delta}+ thymocytes also expressed CD25, but the expression was lower than on {alpha}ß thymocytes (Fig. 8GoB). Both CD8+ and CD8- {gamma}{delta} T cells expressing CD25+ were observed (Fig. 8GoF). Based on forward scatter, CD25+ thymocytes represented a homogeneous population of medium-sized cells (Fig. 8GoC). The proportion of CD25+ thymocytes during ontogeny increased as mature CD3{epsilon}high thymocytes became prominent between DG56 and DG74 (compare Fig. 8GoG with Fig. 5Go and Fig. 4Go). While the number of CD25+ cells decreased after DG74, their proportion gradually increased after birth, reaching a relatively high frequency in the adult thymus (Fig. 8GoG).



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  		FIGURE 8. Analysis of CD25 expression on pig thymocytes. Thymocytes isolated from 3-day-old piglets were double-stained with combinations of anti-CD25 and either anti-CD3{epsilon} (A) or anti-TCR{gamma}{delta} (B) mAb or were triple stained with combination of anti-CD25, anti-CD8, and either anti-CD4 or anti-TCR{gamma}{delta} mAb (D–F). The size of the thymocytes expressing IL-2R{alpha} (CD25) is also demonstrated (C). When CD25/CD8/CD4 triple-stained thymocytes were gated for CD25+ cells (D, R1) and analyzed, the majority of gated cells was found within the CD4+CD8- or CD4-CD8+ SP thymocyte subsets (E). The same type of analysis for CD25/CD8/TCR{gamma}{delta} triple-stained thymocytes showed that both CD8- and CD8+ cells are present within the TCR{gamma}{delta}+CD25+ subset (F). G shows the frequency of CD25+ thymocytes during prenatal and postnatal development. Data are mean values ± SEM from at least three animals for each stage of gestation.

 
Cell cycle analysis of thymocytes in pigs

Identification of cycling thymocyte subsets (i.e., cells in S+G2/M cell cycle phase) was performed by simultaneous surface immunophenotyping and staining of DNA with 7-AAD (Fig. 9Go). All cycling thymocytes were large (Fig. 9GoB), and the majority of them was found among DN and DP cells, while relatively few SP thymocytes synthesized DNA (Fig. 9GoF). We also observed that all cycling DP thymocytes were medium-sized, whereas cycling DN cells were generally larger (data not shown). CD3{epsilon}- thymocytes prevailed among proliferating cells (Fig. 9GoD). While most dividing CD3{epsilon}high thymocytes expressed TCR{gamma}{delta}, a small but significant subset of CD3{epsilon}high cycling thymocytes was found to be TCR{gamma}{delta}- cells and presumed to be a mature stage of the {alpha}ß lineage (Fig. 9GoD). No cycling CD3{epsilon}low thymocytes were detected (Fig. 9GoD).



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  		FIGURE 9. Simultaneous analysis of DNA content and CD4/CD8 or CD3{epsilon}/TCR{gamma}{delta} expression on thymocytes from pigs. Thymocytes isolated from 112-day-old fetuses were double-stained with a combination of anti-CD3{epsilon} and anti-TCR{gamma}{delta} or anti-CD8 and anti-CD4 mAb. These were fixed in 70% ethanol, and their DNA was visualized using 7-AAD. The histogram of 7-AAD fluorescence for all thymocytes illustrates the gating strategy that was used to examine thymocytes in G0/G1 or S+G2/M phase of the cell cycle (A). Scatter characteristics (forward vs side scatter profile) for thymocytes in G0/G1 phase (gray dots) and thymocytes in S+G2/M phases (black dots) show that all cycling thymocytes were large (B). When CD3{epsilon}/TCR{gamma}{delta}-stained thymocytes (C) were gated for cells within the S+G2/M phases, the majority of cycling thymocytes was found to be within the CD3{epsilon}- subset although there were also cycling CD3{epsilon}highTCR{gamma}{delta}+ (i.e., {gamma}{delta} cells) and CD3{epsilon}highTCR{gamma}{delta}- (i.e., TCR{alpha}ß+) thymocytes (D). Essentially no CD3{epsilon}low thymocytes were cycling (D). Staining for CD4/CD8 (E) showed that the majority of cycling cells was within the CD4-CD8- and CD4+CD8+ subsets (F). However, consistent with CD3{epsilon}/TCR{gamma}{delta} staining (C and D), some of the cycling thymocytes were also CD4-CD8+ or CD4+CD8- (F). Surface and DNA staining in pig fetuses at different ages resulted in similar profiles. Medium-sized CD3{epsilon}low thymocytes must be doublets because no cells events with such a phenotype were present after gating out all multiple events (G).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most detailed studies on early thymocyte development have been performed in rodents (16, 17, 18, 19, 20, 21) and chickens (22, 23, 24, 25). Data from other species are fragmentary. Early T cell progenitors, i.e., pro-T cells, are derived from stem cells in primary hemopoietic centers and migrate to the thymus where further differentiation takes place (19, 20, 22, 23). It is well established that there are different hemopoietic centers during prenatal and early postnatal ontogeny (19, 22, 23). Although the activity of these primary centers overlap, the gradual shift of primary hemopoiesis from one anatomical site to another during ontogeny has been hypothesized to account for the waves of thymic colonization by pro-T cells that have been reported in chicken/quail chimeras and suggested from studies in mice (16, 17, 18, 19, 20, 21, 22, 23, 24, 25). However, none of these studies provides a direct proof for a discontinuous influx of thymocyte progenitors in an intact system. In chicken, the microsurgical techniques used to introduce xenogeneic cells may have interfered with normal immune system development. The disadvantage of mice is their very short gestation so hypothetical waves of thymic colonization overlap and are difficult to observe. The fetal pigs are more convenient for studies on prenatal ontogeny because of their much longer gestation.

We have used CD3/CD4/CD8 immunophenotyping to correlate thymocyte maturation in pigs with the generally accepted model of intrathymic T cell differentiation developed from studies in mice (26). In pig embryos, the first TN lymphoid elements, probably pro-T cells, could be observed in the thymic rudiment on DG38. The precursor nature of these early nonmyelomonocytic leukocytes is supported by their low expression of CD45. CD3{epsilon}+ thymocytes appeared 2 days later, all of which remained brightly TCR{gamma}{delta}+. Differentiating thymocytes belonging to the {alpha}ß lineage followed a progression from less-differentiated, large TN precursors to small CD3{epsilon}- DP and CD3{epsilon}low DP cells and finally to SP thymocytes (Fig. 10Go). This scenario is also consistent with our cell cycle studies where the majority of cycling thymocytes was large TN and CD3{epsilon}- DP cells, while small thymocytes, either CD3{epsilon}- or CD3{epsilon}low, were not dividing. Moreover, CD3{epsilon}low thymocytes appeared ~10 days before the first mature {alpha}ß thymocytes. Our data suggest that porcine {alpha}ß thymocytes require about 15 days to fully differentiate, while {gamma}{delta} thymocytes do so in <3 days. Combined with our previous findings (4), the data presented here also suggest that {alpha}ß and {gamma}{delta} T cells migrate asynchronously from the thymus to the periphery with {gamma}{delta} T cells populating the periphery before {alpha}ß T cells. This is consistent with studies in other species demonstrating that {gamma}{delta} T cells require a shorter time period for maturation than {alpha}ß T cells, perhaps because the latter are subjected to more rigorous positive and negative selection (16, 17, 18, 24, 25, 27). Interestingly, a large numbers of proliferating TCR{gamma}{delta}+ cells were always observed. This is in a sharp contrast to mice, where only a few {gamma}{delta} thymocytes have the capacity to divide (28). This finding together with the higher frequency of {gamma}{delta} thymocytes in the porcine thymus compared with its murine counterpart may explain why {gamma}{delta} T cells are so prominent a T cell population in the peripheral blood of young piglets (3, 4).



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  		FIGURE 10. Proposed model of {alpha}ß T cell development in the porcine thymus. This schematic representation is deducted from: 1) the appearance of individual thymocyte subsets during early prenatal ontogeny, 2) the finding that the level of surface CD3{epsilon} expression increased during thymocyte development, and 3) staining of DNA using 7-AAD. Large circles demonstrate differentiation stages of large, mitotically active thymocytes, while small circles represent small, nondividing populations. The phenotype of individual subsets is given. An analysis of subsets A–C, D, E–F, G, and H is presented in Fig. 1Go, B, C, D, E, and F, respectively.

 
Once the major differentiation stages of thymocyte development had been identified, we tested the hypothesis of discontinuous colonization of the rudimentary thymus with pro-T cells. In our previous work (4), we observed no dramatic changes in the relative proportion of thymocytes in different stages of prenatal development. In the current study, we tested additional time points and focused on the interval between DG56 and DG76, which represents the period between the first half and the first two-thirds of gestation. During this period, we identified noteworthy changes in thymocyte subset composition. In particular, the proportion of both CD4+ and CD8+ mature {alpha}ß thymocytes as well as {gamma}{delta} cells increased while the proportion of CD3{epsilon}- and CD3{epsilon}low DP thymocytes decreased. Cell recovery from the thymus during this time was unexpectedly low (data not shown), and the proportion of myelomonocytic cells (CD45+SWC3a+) was high. Altogether these findings suggest that the influx of pro-T cells is temporarily interrupted after midgestation because there is a decline in the absolute numbers of early TN precursors and the otherwise predominant CD3{epsilon}-/low DP thymocytes. In accordance with studies in mice and chickens, this can be explained by the shift of hemopoiesis from one primary center (fetal liver) to another (bone marrow). While CD3{epsilon}+ thymocytes were detected on DG40 and peripheral T cells on DG45 (4), almost no leukocytes could be recovered from the bone marrow before DG45. This indicates that the fetal pig bone marrow cannot function as a significant source of blood-forming cells before approximately DG45, and other organs must play the role of primary hemopoietic centers during the early period of prenatal development. The recovery of CD45lowSWC3a- mononuclear cells in different organs implies that the fetal liver is the primary site of lymphopoiesis before DG45. This conclusion is also supported by the finding that the fetal liver contains cells of the B cell lineage at DG30 (29). After DG45, the proportion of CD45lowSWC3a- cells progressively increased among cells recovered from the bone marrow, while this subset gradually disappeared from the fetal liver with increasing fetal age.

In attempt to distinguish between early and late pre-T cells (30, 31) we stained thymocytes from fetal, neonatal, and young piglets for the presence of the IL-2R{alpha}-chain (CD25). Surprisingly, only mature CD3{epsilon}high stages expressed CD25 on their surface. As dividing mature {alpha}ß thymocytes are clearly present from DG55, CD25 expression might be confined to this proliferating terminal stage of thymopoiesis. In rodents and the chicken, the expression of CD25 begins at the pro-T cell level, and the down-regulation of this marker is typical for late pre-T cells (30, 31, 32). However, the need for CD25 expression in thymocyte development is challenged by the finding that CD25 knockout mice have normal thymocyte development (33, 34). The lack of CD25 on T cell precursors and its presence on developing B cells (15) indicates that IL-2R is a B cell rather than a T cell differentiation marker in pigs.

The DP thymocytes described in this study represent a transitional stage of intrathymic development of the {alpha}ß T cell lineage (Fig. 10Go). These DP thymocytes should not be confused with peripheral CD4+CD8low {alpha}ß T cells in adult pigs (3, 4, 5, 6, 7, 8, 9). The latter are CD1- (7, 8), CD29+ (35), MHC class II+ (8), express the CD8{alpha}{alpha} homodimer (10), and are absent before birth and in newborns (4). Such peripheral DP T cells can be generated from CD4+CD8- Th lymphocytes upon stimulation with a recall viral Ag (35). Thus, we and others have hypothesized that these peripheral DP T cells consist of resting effector/memory Th lymphocytes that acquire CD8{alpha}{alpha} as a result of Ag challenge and are not directly related to the DP thymocytes described in this study.


   Acknowledgments
 
We gratefully acknowledge M. Smolová and M. Stojková for excellent technical assistance and the following researchers for the gifts of mAbs: Dr. H. Yang (Institute of Animal Health, Pirbright, U.K.) for anti-CD3{epsilon} (PPT3 and PPT6) and anti-TCR{gamma}{delta} (PPT16 and PPT17), Dr. J. K. Lunney (Animal Parasitology Institute, Beltsville, MD) for anti-CD4 (10.2H2) and anti-SWC3a (74-22-15), Dr. M. D. Pescovitz (Indiana University, Indianapolis, IN) for anti-CD8 (76-2-11), and Dr. C. R. Stokes (University of Bristol, Bristol, U.K.) for anti-CD25 (K231.3B2) and anti-CD45 (K252.1E4).


   Footnotes
 
1 This work was supported by Ministry of Education, Youth, and Sport of the Czech Republic Grant KONTAKT ME 339, National Science Foundation Grant MCB-9723721, U.S. Department of Agriculture-National Research Institute Grant 97-35204-4858, and Grant Agency of the Czech Republic Grant 303/99/0197. Back

2 Address correspondence and reprint request to Dr. Marek Sinkora, University of Iowa, College of Medicine, Department of Microbiology, 3–501L Bowen Science Building, Iowa City, IA 52242. Back

3 M.S. and J.S. contributed equally to this work. Back

4 Abbreviations used in this paper: DP, double positive (CD4+CD8+); DN, double negative (CD4-CD8-); SP, single positive (CD4-CD8+ or CD4+CD8-); pAb, polyclonal Ab; 7-AAD, 7-aminoactinomycin D; DG, day of gestation; TN, triple negative (CD3{epsilon}-CD4-CD8-); SWC, swine workshop cluster. Back

Received for publication December 7, 1999. Accepted for publication May 30, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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