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Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment.

To determine events that transpire during the earliest stages of human T cell development, we have studied fetal tissues before (7 wk), during (8.2 wk), and after (9.5 wk to birth) colonization of the fetal thymic rudiment with hematopoietic stem cells. Calculation of the approximate volumes of the 7- and 8.2-wk thymuses revealed a 35-fold increase in thymic volumes during this time, with 7-wk thymus height of 160 microM and volume of 0.008 mm3, and 8.2-wk thymus height of 1044 microM and volume of 0.296 mm3. Human thymocytes in the 8.2-wk thymus were CD4+ CD8 alpha+ and cytoplasmic CD3 epsilon+ cCD3 delta+ CD8 beta- and CD3 zetta-. Only 5% of 8-wk thymocytes were T cell receptor (TCR)-beta+, < 0.1% were TCR-gamma+, and none reacted with monoclonal antibodies against TCR-delta. During the first 16 wk of gestation, we observed developmentally regulated expression of CD2 and CD8 beta (appearing at 9.5 wk), CD1a,b, and c molecules (CD1b, then CD1c, then CD1a), TCR molecules (TCR-beta, then TCR-delta), CD45RA and CD45RO isoforms, CD28 (10 wk), CD3 zeta (12-13 wk), and CD6 (12,75 wk). Whereas CD2 was not expressed at the time of initiation of thymic lymphopoiesis, a second CD58 ligand, CD48, was expressed at 8.2 wk, suggesting a role for CD48 early in thymic development. Taken together, these data define sequential phenotypic and morphologic changes that occur in human thymus coincident with thymus colonization by hematopoietic stem cells and provide insight into the molecules that are involved in the earliest stages of human T cell development.

S tudy of the earliest stages of human T cell development provides important information regarding the molecules involved in the complex series of events that transpire during establishment of the T cell repertoire. Knowledge of the sequence of expression of developmentally regulated T cell functional molecules can provide insight into mechanisms of normal T cell maturation, and can provide information necessary to form hypotheses regarding molecular mechanisms of thymus growth. We must understand the stages of normal T cell development to devise successful strategies for immune reconstitution of a variety of acquired and congenital T cell immune deficiency syndromes.
Whereas enormous progress has been made in our understanding of the earliest stages of murine (for review see reference 1) and avian (for review see reference 2) thymus development, the sequence of events at the time of colonization of the human thymus with hematopoietic stem cells has not been studied. We have previously studied human fetal thymuses at 7 wk, before lymphoid colonization of the thymus (3), and at 9.5 wk of gestation, 1.5 wk after hematopoietic stem cell migration to the thymus (3)(4)(5). Our prior efforts to study lymphoid fetal thymus at earlier than 9.5 wk of gestational age were thwarted by lack of availability of thymus tissues at the time of colonization of the thymus, the small size of the human thymus at 8 wk ('~1 mm • i mm x 0.5 mm), and the frequent contamination of first trimester fetal tissue with placental tissue containing CD7 +, decidual granular lymphocytes of NK lineage (6,7). Thus, the phenotypic and morphologic events that occur at the initiation of lymphopoiesis in the human thymus have not been observed. In this study, we have definitively identified the human thymus in fetal thorax at 8.2 wk of gestation, defined its three-dimensional structure, and determined the reactivity of 64 mAbs with this tissue, as well as with fetal thymuses at 9.5, 10, 12.75, 13, and 16 wk of gestational age. Our data document the extraordinary thymus growth that occurs at the time of stem cell colonization of the thymus and demonstrate developmentally regulated expression of a large series of lymphoid and thymic microenvironment molecules.

Materials and Methods
Thymic Tissue. Fetal tissues were obtained as discarded material from the Pathology Department, Duke University Medical Center, from either elective first trimester abortions or at the time of surgery for ectopic pregnancy as described (3)(4)(5). Gestational age, determined by crown-rump length, menstrual records when available, and fetal part morphology, ranged from 7 to 34 wk of gestation (3)(4)(5). Fetal tissues 50 (7 wk; reference 3) and 58 (8.2 wk) were serially sectioned through the thorax and neck area in 4-#M sections and hematoxylin and eosin (H&E)l-stained sections were prepared every 40 #M (50; reference 3) or 80 #M (58). In these tissues, the thymus was identified as a keratin-containing structure in the neck lateral to the trachea and esophagus (7 wk; reference 3) or in the upper mediastinum anterior to the trachea and great vessels ( Immunofluorescence Assays. Indirect immunofluorescence (IF) assays were performed on acetone-fixed 4-#M tissue sections as described (3,4) by use of FITC-conjugated goat anti-mouse Ig (Kirkegaard and Perry Labs., Inc., Gaithersburg, MD). Indirect IF assays on cell suspensions were performed as described (3)(4)(5) and analyzed on either a FACS | Star Plus (Becton Dickinson & Co., Mountain View, CA) or profile flow cytometer (Coulter Corp., Hialeah, FL). IF assays were usually performed in duplicate, and select mAb IF assays were repeated three times. wk of gestation (before lymphoid colonization) was devoid of CD45 § cells and consisted of only two spherical thymic rudiments, each '~160 x 180 x 200 #M, in the lateral neck areas (3). Only four H&E-stained sections were available for this tissue; thus, a fully interpretable computerized image could not be generated. The calculated total volume (both rudiments) of nonlymphoid thymus 50 at 7 wk was 0.008 mm 3.

Computer-assisted Three-dimensional Reconstruction of Fetal
In contrast, 8.2-wk thymus 58 was filled with CD45 + hematopoietic cells, and was 1,044 #M in length, 1,200 #M wide, and 480 #M deep. The 8.2-wk right and left thymic rudiments were located anterior to the esophagus, trachea, and great vessels in the upper thorax (Fig. 1). Computerassisted reconstruction of the 8.2-wk fetal thymus demonstrated that the lower lobes of the thymus had fused, whereas the right and left upper thymic rudiments remained separate. The right thymic rudiment was a nonlobulated cylinder, whereas the left thymic rudiment was just beginning to become lobulated. Most published images of fetal thymus at this stage of development show two elongated cylindrical tubes 1 Abbreviations used in this paper: c, cytoplasmic; H&E, hematoxylin and eosin; ICAM, intercellular adhesion molecule; IF, immunofluorescence; s, surface; TdT, terminal deoxynucleotidyl transferase; TE, thymic epithelial; VLA, very late antigen of activation. on each side of the neck, meeting in the middle (9). However, analysis of the actual three dimensional structure of the 8.2-wk thymus demonstrated a globular structure, with the right and left lobes beginning to rotate around a central axis and intertwine. The calculated volume of 8.2-wk thymus 58 was 0.296 mm 3. Thus, we estimated that the thymus increases in volume ~35-fold from 7 wk (nonlymphoid stage) to 8.2 wk (after lymphoid colonization).

Expression of Human T Cell Lineage Molecules During Early
Thymus Development. The expression of a panel of T cell lineage molecules (Table 1) was studied in 8.2-, 9.5-, 10-, 12.75-, 13-, and 16-wk fetal thymuses (Table 2). Studies in thymuses 48 (9.5 wk), 22 (10 wk) and 40 (12.75 wk) have been reported previously for some of the molecules studied (3)(4)(5) and are included in Table 2 for completeness. The new and striking findings in this study were (a) lack of expression of CD2 at the time of thymic colonization at 8.2 wk; (b) lack of expression at 8.2 wk of CD8/3 with expression of CD8o~; and (c) sequential expression of CDlb, then CDlc, then CDla molecules from 8.2 to 13 wk. Fig. 2 shows lack of CD2 and CD8B expression at 8.2 wk, with all 8.2-wk thymocytes CD4+CD8ot+CD7+, and CD5 § The finding that most thymocytes at this stage were CD4 § CD8 § (double positive) was also surprising, since most murine thymocytes at the stage analogous to the 8.2-wk human thymus development (11 d) are CD4-CDS- (10,11). By 10 wk, all thymocytes expressed CD8/~, followed by appearance of CDScffj5 + and CDSot/~-subsets in the thymic medulla at 12.75 wk ( Table 2). By 12.75 wk, CDla, -b, and -c expression was similar to that seen in postnatal thymus, with the typical CD1 pattern of all cortex and few medullary cells being CD1 +.

Expression of CD45 Molecule Isoforms During Early Human T Cell Development.
It has been suggested that CD45tLA + human thymocytes represent the generative thymocyte lineage destined for positive selection and thymic export, whereas CD45RO § thymocytes are destined to undergo negative or no selection and die intrathymically (12). We found few CD45RA + cells in 8.2-and 9.5-wk thymuses (Fig. 3, B and E), whereas in 12.75-wk thymus, a distribution of CD45RA § cells similar to postnatal thymus was seen, that is, scattered cortical thymocytes were CD45RA § , with large clusters of CD45RA + thymocytes in the thymic medulla ( Fig. 3 H). Similarly, by 10-12.75 wk, most thymocytes were CD45RO + (Fig. 3 I), raising the possibility of onset of thymocyte-negative selection during this period (13,14).
As previously reported (4), TCR-6 expression peaked (11% of T cells) at 9.5 wk, the time of first appearance of TCR-5 1449 Haynes and Heinly   ( Table 3). In this study with anti-V/~l and ova2 mAbs, we found that the ratio of V/i2/V/~I T cells varied over time of gestation (Fig. 5). There were two general peaks of V#2 predominance over V/il TCR-8 + cells, initially at 9.5 wk and later beginning at 16 wk and continuing through the last trimester. Even though the ratio of V#2/V81 remained high until birth, the precentage of total TCR-/~ + thymocytes remained low during the second and third trimesters.
Because it is often difficult to distinguish between thymic 1450 Early Human T Cell Development    0  I  ,  I  ,  I  ,  I  ,  I  ,  I  ,  I  ,  I  ,  I I Analysis shows an initial peak in the percentage of TCR-~ cells at 9.5 wk of gestation (8) with most of the TCR-5 cells V/~2 + . By week 12, the percentage of TCR-/~ cells decreased to 1-2% of total thymocytes, with V~2 + cells predominating throughout fetal thymus development.

Analysis of Thymic Microenvironment Components of 8.2-wk
Fetal Thymus 58. We have previously reported the analysis of thymic microenvironment components of thymuses of 9.5 wk of gestation and older with many of the reagents listed in Table 1 (3,(22)(23)(24)(25)(26). The key issues with the 8.2-wk fetal thymus were (a) Could separate endodermal (TE-3 +) (early cortical) and ectodermal (TE-4 +, p19 +) (early medullary) zones of TE cells be visualized? (b) Were vessels present within the parenchyma of the right and left 8.2-wk thymus? (c) Were macrophages and dendritic ceUspresent in the thymus at 8. First, all 8.2-wk thymic epithelium was TE-3 +, whereas only the central TE zone was TE-4 +, directly demonstrating that TE-4 medullary epithelium was located in an inner core of the thymus cylinder, and demonstrating that the TE-3 antigen, though preferentially expressed in cortical TE in postnatal thymus (23), was equally expressed at 8.2 wk in both cortical and medullary TE cells (Fig. 8, C and  D). Thus, these data suggested that before thymus lobulation, there was a TE-4-outer cortical (endodermal) epithelial zone surrounding a TE-4 + inner medullary (ectodermal) epithelial core.
There were no vessels revealed by morphology or antivessel mAb reactivity in the nonlobulated right thymus, but in the lobulated left thymus, there were vessels approaching (Fig. Papiernik et al. (27) have suggested that hematopoietic stem cells, upon entering the thymus, are driven to differentiate into thymic macrophages and dendritic cells as well as T cells. Kurtzberg et al. showed CD7+CD3-CD4-CD8 -thymocyte differentiation into myeloid as well as lymphoid lineages in vitro (28). In 8.2-wk thymus 58, no immature myeloid cells were detected with CD33 mAb MY9 (not shown). CD14 + and CDllb + macrophages were present in 8.2-wk thymus, as were CD16 + cells with a similar morphology (not shown). No HB15a + dendritic cells were found in 8.2wk thymus. Whereas intrathymic lymphocytes were CD34-, scattered large cells with long cytoplasmic processes were CD34 +, and similar cells were seen around the spinal cord, aorta, fetal liver, and esophagus (not shown). CD34 + cells around the esophagus were CD4 -CD8 -CD7-CD44 + MO-1+HB15a-CD33-. Thus, cells of monocyte/macrophage lineage were present within human thymus coincident with colonization of the thymus by stem cells, and CD34 + CD44+MO-1 + cells were scattered around the esophagus, aorta, and spinal cord. However, no evidence of active intrathymic myelopoiesis was seen.

Discussion
In this study, we have used a large panel of anti-human mAbs to define phenotypic and morphologic events that transpire during the earliest stages of human thymus development, and have outlined the developmentally regulated expression of a number of functional T cell molecules.
Auerbach (29) has proposed that TE cell-fibroblast interactions occur during thymus development that provide potent stimulatory signals to TE cell growth. Nonlymphoid thymuses in human SCID are dysplastic due to lack of colonization of the thymus by stem cells (30). Early work with bone marrow reconstitution in human SCID suggested an inductive effect of hematopoietic stem cells on thymic epithelial proliferation (31). Recent work with transgenic mice 1454 Early Human T Cell Development has directly shown the dependence of murine medullary TE cell growth on the presence of thymocytes in the thymus (for review see reference 32). Thus, the striking 35-fold increase in volume of the 8.2-wk (colonized) thymus compared with the 7-wk nonlymphoid thymus was expected. What was unexpected was the globular shape and the intertwined upper poles of the 8.2-wk right and left thymus lobes.
We have previously shown that one adhesion ligand pair involved in TE cell ligation of thymocytes is CD2 on thymocytes and CD58 (LFA-3) on TE cells (33,34). Although CD2-LFA-3 interactions have been postulated to be important in triggering TCR-early T cells (35), CD2-homologous recombinant mice show no defect in T cell development (36), and treatment of mice with anti-CD2 mAbs early in life does not interfere with thymopoiesis (37). Nonetheless, we and others (3)(4)(5) have generally regarded CD2 among the earliest T cell molecules expressed on developing T cells in the thymus. Thus, it was surprising that CD2 expression was not detected in 8.2-wk thymus with two CD2 mAbs (35.1 and OKTll) (Fig. 2 F) that reacted well with thymuses from other gestational ages. Importantly, CD2-surface (s)CD3e + fetal thymocytes have been identified in 20-24-wk thymuses, demonstrating that during human fetal thymus development, it is not obligatory for CD2 to be expressed surface sCD3/TCR expression (38). Our study shows that after stem cell colonization of the human thymus, CD3, CD8, CD3e, CD3~, CD48, and TCR-fl expression all precede CD2 expression. Arulanandam et al. (15) demonstrated that human CD48 is a weak ligand for CD58, and suggested that CD48-CD58 interactions in man are vestigial events left over from the primary role CD48 plays as a CD58 ligand in other species such as mice. The demonstration that CD2 was not expressed in human thymus at the initiation of thymopoiesis provides evidence that, if CD58 ligation is important at this stage of thymus development, CD48-CD58 interactions may be involved in thymocyte-thymic stromal interactions.
The sequential acquisition of CDSo~ (8.2 wk) followed by CDSfl (9.5-10 wk) is consistent with the work of Galy et al. (39), who described an early T cell maturation pathway in sCD3-postnatal thymocytes of CD8-to CDSc~ + to CDSodfl + thymocytes. CD8o~ associates with p56-1ck and is critical for CD8 + thymic-positive selection on MHC class I molecules (40), CDSfl expression augments TCR-MHC class I interactions above the effects of CD8ot (41), but the role of CDSfl in thymocyte-positive selection is not known. If CD8fl is required for CD8 + T cell selection, then the acquisition of CD8fl expression at 9.5-10 wk implies that MHC class I-mediated selection does not occur until this time. Similarly, CD28 expression first occured at 10 wk, and CD28 has been postulated to be required for thymocyte-positive selection (42), though not for thymocyte-negative selection (43).
The gradual acquisition of medullary thymocyte CD45RA expression at 10-12.75 wk also implies the gradual onset of positive selection. CD45P, A + thymocytes have been suggested to be those destined to survive intrathymic selection events (12). Recently, studies in transgenic mice demonstrated that CD45RA expression is induced on thymocytes that have undergone positive selection (44). Conversely, expression of CD45RO by thymocytes increases the efficacy of TCR-mediated thymocyte apoptosis and MHC-restricted negative selection (14,45). Thus, CD45P, O expression at 9.5-10 wk suggests that thymocyte negative selection could begin at the same time as positive selection or just before. TCR-mediated signaling not only requires CD45 tyrosine phosphatase expression but also requires expression of CD3~" (46). Acquisition of CD3~" between 10 and 12.75 wk also implies that thymocyte selection does not begin to occur until this time.
The sequence of expression of TCR-fl, TCR-y, and then TCR-6 is similar to that seen in mice, with two important differences. First, in the mouse there is a wave of TCR-'y/8 cell development before TCR-ot/fl cell development (for reviews see references 1, 47). In human thymus, TCR-/~ cells peak in percentage at 9.5 wk of fetal development (4). However, in humans, appearance of TCR-8 cells in thymus coincides with increasing TCR-fl and sTCR-ol/fl expression (3)(4)(5)48). Thus, in humans there is near-coincident appearance of TCR-6 and -cz/fl thymocytes. Our observations of TCR V82 + cell predominance over TCR V/~I + cells throughout fetal thymic development are compatible with the observations of others (49)(50)(51), who found that TCR V/~2 + expression in T cell clones from 11-22-wk fetal thymuses predominated over TCR V81. Parker et al. (52) proposed that TCR V62 + cells develop in the thymus during fetal gestation, seed the periphery, and clonally expand upon contact with antigen.
Second, in the 8.2-wk human thymus, TCR-fl synthesis begins simultaneously with T cell precursor colonization of the thymus, whereas in the mouse, TCR-fl synthesis begins around day 15, 5 d after mouse thymus colonization by stem cells (53). It has recently been demonstrated in mice that TCR-fl can be expressed on the developing thymocyte surface either as a dimer or associated with a novel protein, gp33, as well as expressed in an ot/fl heterodimer (for review see reference 54). Due to the small size of the 8.2-wk thymus (<1 mm3), we were limited to phenotypic analysis of the thymus in situ via IF assays on tissue sections, thus negating our ability to definitively distinguish surface from cytoplasmic(c) mAb reactivity. In the mouse, all thymocytes are sCD4-sCD8-from the time of colonization of the murine thymic rudiment on day 10-11 until day 16-17 (10,11). Levelt et al. (53) demonstrated that cytoplasmic expression of murine TCR-fl begins on day 15, with 3,5% of thymocytes cTCR-fl + before expression of CD4 or CD8. Thus, the 8.2wk human thymus is similar to murine day 15 thymus in that 8.2-wk thymus TCR-3 expression (5%) is likely cytoplasmic, but differs from mouse thymus in that most human thymocytes just after thymus colonization express CD4 and CD8, whereas day 15 mouse thymocytes are CD4-CD8- (10,11,53).
Our previous work has shown that by 16 wk of gestation, CD4,CD8,CD3e and TCR-fl were expressed in fetal thymus in patterns similar to postnatal thymus, and gave rise to the prediction that the T cell repertoire is established in humans by the end of the first trimester (22). Recent molecular anal-yses of the human thymus TCR Vj8 repertoire at 15-17 wk of gestation have shown no difference from the newborn thymus VB repertoire (55)(56)(57).
Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that functions in the absence of a template to act as a somatic mutagen by the insertion of extra random nucleotides (N regions) at the D-J joining region during TCR-~/ rearrangements, contributing to TCR-/3 diversity. TdT expression was not found by immunohistology in human thymocytes until 20 wk of gestation (48). However, George and Schroeder (57) found TCR N region additions as early as 8 wk of gestation with PCR identification of TdT transcripts, and demonstrated progressive increases in N additions in TCR-3, CDR3, VD, and VDJ transcripts during fetal development. Whether the 8-wk tissue in this latter study was as early as tissue 58 in our study (i.e., was a nonlobulated thymic rudiment) is unknown since morphologic data were not reported (57). Nonetheless, all data point to initiation of diversification of the TCR repertoire very early on in human T cell ontogeny.
Investigators have isolated CD4-CD8-sCD3E-cells from human fetal liver and postnatal thymus and shown that they give rise to CD3~ + CD3b + NK cells, suggesting a dose lineage relationship between T and NK calls (58,59). We did not observe a stage of human thymic development wherein a majority of thymocytes expressed NK markers such as CD16 or CD56, although scattered CD16 + cells were present in thymus at 8.2 wk.
The expression of VLA 1-6 in thymus development beginning at 15 wk has been previously reported (21). New in our study is the demonstration of reactivity of VLA-1, -2, -3, -4, -6, and B1 mAbs on 8.2-wk thymus. Sanchez et al. (60) have demonstrated VLA-4 expression on T cell progenitors in human fetal liver, and TE cells may bind certain thymocyte subsets via VLA-4 (Le, P. T., personal communi-cation). A number of integrin molecules have been postulated to be involved in stem cell migration to the human thymic rudiment, but this question is difficult to directly address experimentally.
Our study identified the organization of TE-4 + central medullary thymic epithelium surrounded by TE-4-cortical epithelium in the 8.2-wk thymus. The gradual appearance of CD45RA § cells in central thymic areas, and the expression of CD6 and CD28 in thymocytes in central TE areas, also suggested that the primordial thymic medulla is the central core of the 8.2-wk thymic rudiment that has begun to organize immediately subsequent to thymus colonization. In a separate study, we have found that MHC class I and II allotypic determinants are also expressed brightly in the central 8.2-wk thymus area-a pattern typical of medullary TE cell MHC expression (Haynes, B. F., unpublished observations).
Finally, it was of interest to observe CD34 + cells around the aorta, spinal cord, and esophagus at 8.2 wk. Recently, two groups have suggested that mesoderm around the dorsal aorta, gonads, and mesonephros (AGM) areas of mice, and not yolk sac, provide the source of stem cells before fetal liver development (61). Whereas no functional data are possible from our study, one explanation for the presence of CD34 + cells in the dorsal thorax of 8.2-wk fetal tissue is that this area may be analogous to the AGM mesoderm in mice (61).
Thus, our study has defined phenotypic and morphologic changes that occur in and around the human thymus at the time of colonization of the thymus by hematopoietic stem cells. This study raises a number of important questions regarding human thymus function, including the nature of the role of CD48-CD58 interactions in early T cell development, the significance of developmentally regulated thymocyte CDlb, -c, -a expression, the regulatory mechanisms involved in CD83, CD28, and CD6 expression, and the functional roles integrins play in early intrathymic maturation.