CELLULAR

137, 175-188 (1991)

lMMUNOLOGY

Production of a Monoclonal Antibody Strongly Reacting with Immature Thymic T Lymphocytes and Its lmmunohistological Application CHIERI KURASHIMA, MASANORI UTSUYAMA, MICHIYUKI

EIKO MORIIZUMI, Department

KASAI, AIKO KONNO, AND KATSUIKU HIROKAWA

of Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173, Japan Received

January

17, 1991; accepted May

16, 1991

A mon’oclonal antibody Th-5 has been produced against mouse immature thymic lymphocytes and emplsoyedto study the process of T cell differentiation in the thymus. Immunohistologically, Th-5 positive thymic T lymphocytes were first found at Day 12 of gestation. They increased in number as well as staining intensity until Day 18 of gestation and decreased thereafter. Th-5 antigen expression was not seen in lymphoid cells in the fetal liver. In the newborn thymus, lymphocytes in the subcapsular layer were still strongly positive, while other cortical lymphocytes became moderately positive for Th-5. Th-5 positiveness was more pronounced in the medulla than in the cortex in the thymus of young adult mice. The staining pattern of Th-5 in the thymus was apparently different from those with other T cell markers (Thy-l, CD3, CD4, CD5, CD8) including J 1 Id, Pgp-1, IL-2R, and 3AlO (TCRy8). Plow cytometric analyses showed that the expression of Th-5 was mostly associated with the Thy-l antigen. However, the fluorescent intensity of Th-5 gradually declined with ontogenic development of the thymus, and the molecular size of the antigen was approximately 100 kDa, which is different from Thy-l antigen (25-30 kDa). Considering these findings, the strong expression of Th-5 could be one of the markers of immature thymic T lymphocytes in the early phase of the ontogenic development. o 1991 Academic press, I~C.

INTRODUCTION The thymus is the major site for T cell maturation; i.e., precursor T cells derived from the fetal liver or the bone marrow migrate into the thymus, proliferate, differentiate, and give rise to mature T cells (17, 27-29). It is well known that mature murine T lymphocytes express the Thy- 1 antigen (3, 17,30) as well as either the CD4 (L3T4) or the CD8 (Lyt-2) antigen, referred to as single positive (SP) T lymphocytes, while immature T lymphocytes in the thymus are recognized within a subpopulation of lymphocytes lacking both CD4 and CD8 markers, referred to as double negative (DN) lymphocytes (18). These immature DN cells are supposed to develop through transient CI>4+CD8+ double positive (DP) cells giving rise to mature CD4+CD8- or CD4-CD8+ single positive T lymphocytes (11, 12). During this process, T lymphocytes are positively selected, having TCR repertoires which can recognize antigens restricted to the host MHC molecule ( 17), and those T lymphocytes responding to self-antigens are negatively selected for establishment of tolerance (20-26). However, the immature DN cells in the thymus are now known to show a marked heterogeneity in their expression of other surface markers (14) although a marker(s) for real immature 175 0008-8749/91

$3.00

Copy-i& 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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thymic lymphocytes migrating into the thymus has not yet been obtained. Based upon the above-mentioned information, the present study was designed to produce a new marker(s) for immature cells committed to migrate into the thymus or to proliferate in the fetal thymus, and to examine the process of T cell differentiation within the thymic microenvironment. MATERIALS

AND

METHODS

Animals C57BL/6CrSlc mice and Wistar rats were purchased from Shizuoka (Japan) Agricultural Cooperative Association for Laboratory Animals and reared in a specific pathogen-free colony at the Tokyo Metropolitan Institute of Gerontology. Immunization

of Rats

The homogenate of thymic tissues obtained from newborn C57BL/6 mice was emulsified in equal amounts of Freund’s complete adjuvant and injected subcutaneously into the foot pads of 3-month-old male Wistar rats. Two weeks later, the rats were immunized again in the same way and the spleens were removed 4 days after the last treatment. Cell Fusion The cell fusion was performed as usually done elsewhere (1). Briefly, 5 X 10’ immune spleen cells were fused to 5 X lo6 myeloma cells (P3-X63-Ag8.653) in 0.5 ml of mixture solution (polyethylene glycol-3000; 1 g + Eagle’s MEM; 1.0 ml + DMSO; 0.35 ml) for 1 min, diluted with 10 ml of prewarmed MEM (37°C) sedimented at 2008 for 10 min, and resuspended in RPM1 1640 with 10% fetal bovine serum (FBS). The resultant cell suspension was adjusted to contain 5 X lo5 myeloma cells per millileter and distributed to microplates (96 wells, Corning 25860), 0.2 ml per well. The next day the cells were first cultured in selective HAT medium containing hypoxanthine, aminopterin, and thymidine for 10 to 14 days, then in HT medium, and finally in regular RPM1 1640 medium with 10% FBS. Detection of Specific Antibody and Cloning of Hybridoma

Cells

The reactivity of culture supernatant to EL-4 cells was first examined in all wells by ELISA and those wells showing a positive reaction were abandoned. Then, the supematant of residual wells was reacted with frozen sections of liver and thymus from C57BL/6 mice and immunohistologically examined by indirect immunoperoxidase method for the presence of any specific antibodies to thymic tissues. Frozen sections were prepared from the liver and thymus at 16 fetal days as well as at the newborn stage. The air-dried sections were fixed with ice-cooled acetone, washed in PBS, reacted with supematant of each well, washed again with PBS, and reacted with peroxidase-labeled rabbit anti-rat immunoglobulins (Dakopatts, Denmark). The sections were then washed in PBS and incubated in Kamovsky’s solution (0.02% 3,3’diaminobenzidine tetrahydrochloride and 0.0 1% hydrogen peroxide in Tris-HCl buffer, 0.05 M, pH 7.6). One well showing negative reaction to EL-4 and containing specific antibody to lymphocytes of fetal thymus was selected for cloning and designated as Th-5. The hybridoma cell line was cloned according to the limiting dilution method, placed in a larger bottle, and injected intraperitoneally into BALB/c nude mice pre-

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treated with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, Aldrich, WI) in order to obtain an ascitic form of the antibody solution. The isotype of the antibody was examined by Ouchterlony’s double diffusion method, Immunoblotting

Analysis

Thymic lymphocytes, 3 X 1O’, obtained from a fetus at Day 18 of gestation were washed with PBS and exposed to 0.5 ml of extraction buffer (2% digitonin, 0.5 M sucrose, 2 nti EDTA, 0.02% azide in 20 mA4 PBS, pH 7.2) (15) for 30 min at 4°C incubation to obtain cell lysates. Digitonin was obtained from Wako (Japan). After the incubation, the mixture of fetal thymic lymphocytes and extraction buffer was centrifuged ;at 16,OOOgfor 30 min. The supernatants were analyzed by 10% polyacrylamide gel electrophoresis (16). TIGN-cloned T cells, 1 X 106, (obtained by low-dose irradiation, Th-S+Thy- I+, CD4-CD8-, maintained without IL-2; kindly provided by Dr. M. Muto, NIRS, Chiba, Japan) were also processed in the same manner and were employed in this assay. Lymphocyte samples were separated by polyacrylamide gel electrophoresis and transferred to a sheet of nitrocellulose membrane ( 19). The bands of antigen recognized by Th-5 antibody were examined by the indirect immunoperoxidase method using POD immu:nostain set (Wako, Japan). Immunohistology The tissue distribution of the antigen recognized by Th-5 monoclonal antibody was immunohistologically examined by the indirect immunoperoxidase method. Frozen sections of &al mice ( 12, 14, 16, 17, 18, and 19 days of gestation) and various organs of newborn and adult mice were reacted with the monoclonal antibody and visualized as mentioned above. For comparison, the frozen sections were also stained by Thy1, CD3 (2Cll), CD5 (Lyt-1), CD8 (Lyt-2) CD4 (L3T4), Pgp-1, Jlld, 3AlO (TCR yS), and interleukin-2 receptor (IL-2R). Biotinylated anti-Thy-l, CD5, CD8, and CD4 antibodies ‘were purchased from Becton-Dickinson Immunocytometry System (Mountain View, CA). Peroxidase-labeled avidin D was obtained from Vector (Burlingame, CA). Labeling

of .FITC

Th-5 monoclonal antibody was labeled with fluorescein isothiocyanate cording to the method described elsewhere (2).

(FITC) ac-

Flow Cytometry Cell suspension was prepared from thymus, spleen, bone marrow, and peripheral blood of C57BL/6 mice at various ages. Two-color staining (FITC and phycoerythrin, PE) of these cell suspensions was performed using the monoclonal antibody (Th-5) and other antibodies such as Thy- 1, CD8, and CD4. The stained cell suspensions were analyzed by a flow cytometer (FCM- 1, JASCO, Tokyo). RESULTS Immunohist8010gical

Staining Pattern of Th-5 as Compared with Other T Cell Markers

Frozen se’ctions of thymic tissues obtained from fetal, newborn, and adult mice were reacted with Th-5 antibody and various other reagents. Table 1 summarizes the

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1

The Intensity of Immunohistological Staining of Th-5 and Other T Cell Markets in the Thvmus According to Age Th-5

Thy- 1

CD3

CD4

CD8

CD5

Jl Id

&P-l

+ ++ +++ +++ ++ ++ + ++ +/++ ++ +/++ +/++ ++ + ++

-I+++ +++ +++ +++ + +++ + +++ + +++ + +++ +

-/+ + + -/+ + + ++ + ++ + ++ ++ +++

-/+ + +/++ +++ +++ ++ +++ ++ +++ ++ +++ ++ +++ ++

+ + ++ ++ + +++ + +++ + +++ + +++ +

+ + ++ +/++ ++ +/++ ++ + ++ + +++ + +++

-/+ -/+ -I+ + ++ ++ + ++ + ++ + ++ + ++ +

-t + + +/++ +/++ + +/++ + +/++ + +/++ + +/++ + +/++

+

+++

++

+++

+++

+

++

+

+

-/+

+++ + ++ + ++ + +/++

++ +++ ++ +++ ++ +++ ++

+ +++ + +++ + +++ +

+++ + +++ +

+/++ + + +

+/++

-/+

-I-+

-ft -/+

-

-I+

-/+

-

-‘i’+ +/++

-‘It

-/+

-/+

-

-I+

-I+

-/+

-

Age fl2d f14d f16d f17d f18d f19d C M Id C M 2d C M lw c M

2w

c M

3wc M 5mC M 23m C M 33m C M

++ + +/++ -/+ + -/+ -/+

Note. +++, ++;

-I+,

Strongly very weakly,

+ +++ + ++ + ++ +

positive; ++, moderately positive; range between - and +. C, cortex;

f, weakly positive; M, medulla.

IL-2R

-

++ + -‘i+ -I+ -;+ -‘if -‘i+ -‘i+

-I+ -f+

+/++,

range between

3AlO

-

++ ++

-;+ -I+ -I+ -/+ -I+ -I+ -I+ -I+ -

+ and

intensity of immunohistological staining of these markers in the thymus according to age. At Day 12 of gestation, a very weak positive reaction for Th-5 was seen in the thymic lymphocytes (Fig. 4a) which were weakly positive with Pgp-1 , faintly positive with J 1 Id, but still negative with Thy-l and 3AlO (TCR yS). At Day 14 of gestation,

FIG. 1. Immunohistological staining of thymus tissues of mice at day 14 ofgestation with various reagents. Thymic T lymphocytes were moderately to strongly positive with Th-5 (a), weakly positive with Thy-l (b) and J 1 Id (c), and weakly to moderately positive with Pgpl (d) and IL-2R (e). Magnification: X65.

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the thymic lymphocytes were moderately positive with Th-5 (Fig. la), weakly positive with Pgp-1 (Fig. Id) and IG2R (Fig. le), and very weakly positive with Thy-l (Fig. lb), Jl Id (Fig. lc), but still negative with 3AlO. Thus, the onset of Th-5 expression on embryonal thymic lymphocytes was apparently earlier than that of Thy- 1 antigen. At Day 16 of gestation, almost all thymic lymphocytes were Th-5 strongly positive and there were no positive cells in the liver (Fig. 2a). The staining pattern of Th-5, positive in the thymus and negative in the liver, was also the casefor the Thy- 1 antigen (Fig. 2b). IL-2R positive cells were also found in the thymus but not in the liver (Fig. 2e). Pgp-1 antigen, one of markers for immature T cells, was seen not only in some of thymic lymphocytes but also in many lymphoid cells in the fetal liver (Fig. 2d). J 1Id antigen was weakly positive for almost all lymphocytes of fetal thymus and strongly positive for many lymphoid cells in the fetal liver (Fig. 2~). In contrast with these two markers (Pgp-1 and Jl Id), the expression of Th-5 was clearly specific to thymic lymphocytes. The expression of both J 11d and Pgp-1 was not specific to thymic lymphocytes, as they were expressednot only in the thymus and liver but also in other organs; i.e., fetal brain and cartilage were Jl Id+ and skin and connective tissues were Pgp-l+. At Day 16 of gestation 3AlO positive lymphocytes were first detected in the thymus with a tendency to localize in the inner portion of the thymus and the pattern of distribution was quite different from that of Th-5 (Fig. 2f). In the newborn thymus, 1 day after the birth, most thymic lymphocytes which had been Th-5 strongly positive at Day 16 of gestation became moderately positive for Th-5 (Fig. 3a.).In other words, strongly positive cells were seenmainly in the subcapsular area and scattered in the cortex. Most of medullary lymphocytes were weakly positive for both Th-5 and Thy- 1, although all cortical thymic lymphocytes were strongly positive with Thy-l (Fig. 3b). Jl Id was moderately positive for most of cortical and medullary lymphocytes (Fig. 3c) and a number of Pgp-1 positive cells were seen in both cortex and medulla (Fig. 3d). Some of the thymic lymphocytes, mainly in the subcapsular layer, expressed IL-2R antigen (Fig. 3e). In l-day-old newborn thymus 3AlO (TCR yS) positive cells were rare. These staining patterns of Jl Id, Pgp-1, IL2R, and 3AlO in the newborn thymus were apparently different from that of Th-5. Figure 4 summarizes the change in the immunohistological staining intensity of Th-5 positivenessin the thymus with advanced development. Th-5 positive cells started to appear in the thymus at Day 12 of gestation (Fig. 4a), increased in number and in intensity of the expression until Day 18 of gestation (Figs. 4b, 4c), and declined thereafter with the advance of ontogenic development (Figs. 4d-4h). The number of Th-5 strongly posjtive cells progressively declined in the cortex after the birth (Figs. 4e-4h) and Th-5 m’oderately positive cells gradually increased in the medulla (Figs. 4h and 5). In the old atrophic thymus obtained from mouse at 33 months of age,Th-5 obviously positive cells were seldom found (Table 1). With reference to other T cell markers such as CD3, CD5, CD4, and CD8, these cell markers became detectable in the thymus at Day 16 of gestation, 4 days after the first appearance of Th-5 antigen in the thymus at Day 12 of gestation. In l-week-old thymus, CD5 strongly positive thymic lymphocytes were mainly seen in medulla, CD8 strongly positive cells mainly in the cortex, and CD4 positive cells widely distributed throughout the thymus. The immunohistological staining intensities of CD5, CD8, and CD4 peaked at 1 week of age and were maintained until 33 months (Table 1). In contrast, CD3 moderately positive cells were scatteredin the thymus of newborn mice, followed by accumulation of strongly positive cells in thymic medulla of 2-

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iLiver

FIG. 2. Immunohistology of the thymus (lower half of each figure) and the fetal liver (upper half of each figure) at Day 16 of gestation. Almost all thymic lymphocytes were Th-5 strongly positive and there were no Th-5 positive cells in the liver (a); this was also the case for the Thy-l antigen (b). Jl Id antigen was weakly positive for thymic lymphocytes and strongly positive for lymphoid cells in the liver(c). Pgp- 1 antigen was seen not only in some of thymic lymphocytes but also in many lymphoid cells in the liver (d). IL-2R positive cells were found in the thymus but not in the liver(e). The 3AlO positive lymphocytes were detected in the thymus with a tendency to localize in the inner portion of the thymus, but not in the liver (f). Magnification: X 154.

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FIG. 3. Immunohistological staining of thymus of newborn mouse by various reagents. Th-5 strongly positive cells were seen in the subcapsular area and scattered in the cortex (a) and weakly positive cells in the medulla. All cortical cells were strongly positive with Thy-l (b). Jl Id antigen was weakly to moderately expressedon most of the cells (c). Pgp-1 (d) and IL-2R (e) positive cells were occasionally seen.Magnification: X262.

week-old mice. Th-5 was thus quite different in the immunohistological staining pattern in the thymus from those cell markers (CD3, CD5, CD4, and CD8). Flow Cytometric Profiles of Th-5 Positive Cells in the Thymus and Lymphoid Organs as Compared with Other T Cell Markers Flow cytometric data (Fig. 6) show that more than 75% of thymic lymphocytes at Day 14 of gestation were Th-5 strongly positive and Thy-l positive, simultaneously. Th-S+Thy- II+ (both positive) cells gradually increased thereafter comprising approximately 89 a.nd 93% of the total lymphoid cells at Day 16 of gestation and 3 weeks of age, respectively, although the fluorescent intensity of Th-5 gradually declined, from strong to moderate positiveness.There were a small number of the thymic lymphocytes which were Th-5 strongly positive and Thy-l negative, and the cell number of this fraction declined with age, i.e., 9.4, 4.0, 1.3, and 0.4% of total lymphoid cells at Day 14, Day 16, at the time of birth, and 3 weeks old, respectively. Those Th-5 positive thymic lymphocytes at Day 16 of gestation were mostly double negative (CD4-CD8-, 70.7%).With the advance of ontogenic development, the double negative (CD4-CD8-) Th-5+ population decreased and the double positive (CD4+CD8~+)Th-5+ population increased,together with a gradual decline in the staining intensity of Th-5 (Fig. 7). Thus, almost all the Thy-l+, CD4+CD8+ cells were Th-5 moderately positive in the thymus of 9-week-old young adult mice, with a small percentage (4.9%) of double negative Th-5 strongly positive cells. In the spleen, peripheral blood (PBL), and bone marrow obtained from 6-week-old and 18-month-old mice, most of the T cells (Thy-l positive) were Th-5 moderately positive (Fig. 8). However, there were also a small number of Th-S+Thy-l- cells in these tissues,i.e., 2.1,4.2, and 1.2%in the spleen, PBL, and bone marrow, respectively. A similar reactivity of Th-5 with T cells was observed in other strains of mice (BALB/c, C3H), but not in rats (Wistar).

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FIG. 4. Change in the Th-5 staining intensity in the thymus at various developmental stages. Th-5 weakly positive cells were scattered in the thymus at Day 12 of gestation (a). Positive cells increased in number and intensity of expression at Day 14 (b) and Day 16 (c), and peaked at Day 18 of gestation showing numerous strongly positive cells. In the newborn thymus (e), most of cortical lymphocytes became moderately positive with scattered presence of strongly positive cells. At 1 week of age, most of cortical and medullary lymphocytes were moderately positive (f). At 2 weeks of age, the staining intensity became weaker in the cortex, but still moderate in the medulla (g). At 4 weeks of age, the staining intensity was apparently stronger in the medulla than in the cortex (h). Magnification: a-g, X 173; and h, X87.

Irnmunochemical Analysis of Antigen Recognized by Th-5 Th-5 monoclonal antibody was determined to be of rat IgG-2a by using Ouchterlony’s immunodiffusion technique. On immunoblotting, Th-5 antibody reacted with a relatively broad band in the sample of fetal thymic lymphocytes as well as two distinct bands in the sample of TIGN (a T cell clone, Th-S+Thy-l+, CD4-CD8-) at approximately 100 kDa in both cases(Fig. 9). Thus the antigens recognized by Th-5 antibody have been detected at around 100 kDa in molecular weight, which was apparently different from the molecular weight of Thy- 1 antigen. DISCUSSION Th-5 strongly positive cells in the embryonic thymus are very interesting, since they might contain the precursor cells of more mature thymic lymphocytes and peripheral T cells. Th-5 antibody immunostained not only immature thymic lymphocytes but also mature T cells, although the staining intensity was apparently stronger in the former than in the latter. In this context, we have to consider two questions about the

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FIG. 5. Comparison of immunohistological patterns of Th-5 and Thy-l in l-week-old thymus. The number of Th-5 strongly positive cells progressivelydeclined in the cortex and Th-5 moderately positive cells gradually increased in the medulla (a), while all cortical thymic lymphocytes were strongly positive with Thy-l (b). Magnification: X89.

character of Th-5 antigen. One question is whether Th-5 is similar to Thy-l or not and the other is whether Th-5 is different from other markers already known for immature thymic lymphocytes. As to the: first question, Th-5 is distinct from Thy-l for the following reasons. (1) Immunohistologically, the onset of expression of Th-5 antigen is earlier than that of Thy- 1 antigen during the ontogeny of the thymus; i.e., Th-5 positive thymic lymphocytes were first found in the thymus at Day 12 of gestation, while Thy- 1 positive thymic lymphocytes were first seen at Day 14 of gestation. (2) Th-5 strongly positive cells began to decline in number after the end stage of gestation, although some of them remained in the cortex until 1 week after birth, and this immunohistological pattern was apparently different from Thy-l. (3) The distribution of Th-5 positive cells in the young adult thymus was quite different from that of Thy-l positive cells; i.e., Th-5 staining was stronger in the medulla than in the cortex, while Thy-l staining was stronger in the cortex than in the medulla. (4) Th-5 antigen molecule was detected at approximately 100 kDa in molecular weight by immunoblotting analysis, which was distinct from Thy-l molecules found at 25-30 kDa in molecular weight (3). Regarding the second question, Jl Id and Pgp-1, both of which are well-known markers to identify immature T lymphocytes, should be considered. J 11d reacts with more than 90% of thymic lymphocytes, while it has no detectable activity for extrathymic mature T cells (4-6). At Day 12 of gestation, the number of J 1Id positive thymic lymphocytes was more numerous than that of Th-5 positive cells. However, J 11d immunostains not only thymic lymphocytes but also erythrocytes, neutrophils, most B cells, and epitopes in many other organs. In the present study, it was shown that the strongest reaction of Jl Id was not seen in fetal thymic lymphocytes but in

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Thymus

f14d

f16d

Ib

0.4%

3w

Th-5 Green fluorescent

intensity

FIG. 6. Flow cytometric profiles of Th-5 in thymic lymphocytes at 14 and 16 days of gestation and 3 weeks of age. More than 75% of thymic lymphocytes at Day 14 of gestation were Th-YThy- I+. These cells gradually increasedthereafter, showing 88.8 and 92.9%at Day 16 of gestationand 3 weeksof age,respectively, although the fluorescent intensity of Th-5 gradually declined. There was a small number of Thy- 1+Th-5cells in the thymus. This population decreasedin percentagewith ontogenic development, i.e., 9.4,4.0, and 0.4% at Days 14 and 16 and 3 weeks of age, respectively.

lymphoid cells in fetal liver at Day 16 of gestation. In contrast, Th-5 was exclusively specific to cells of T cell lineage and did not immunostain the lymphoid cells in the fetal liver. Becauseof the reasonsmentioned above, it can be said that Th-5 is different from Jl Id. Pgp-1 (equivalent to CD44), a cell-surface glycoprotein of 80-90 kDa, plays a major role in cellular adhesion for both hematolymphoid and nonhematolymphoid cell types (3 1,32). In mouse T cells, Pgp-1 acts as a differentiation alloantigen and its expression is seen before the onset of expression of CD4 and CD8 (7). Furthermore, the most thymus-homing progenitor cells in the adult mouse thymus bear Pgp- 1 (B- 10). Budd

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f17d

NB

9w Green fluorescent

intensity

FIG. 7. Flow cytometric profiles of thymic lymphocytes stained with Th-5, L3T4 (CD4), and Lyt-2 (CDS) during the ontogenic development. From top to bottom, Days 16, 17, and 18 of gestation, newborn, and 9 weeks of age. Th-5 strongly positive cells mostly belonged to double negative (L3T4-Lyt-2-, CD4-8-) cells (70.7%) at Day 16 of gestation (f16d). Double negative and Th-5 strongly positive cells gradually decreased, while double positive Th-5 positive cells gradually increased thereafter with ontogenic development. Fluorescent intensity of Th-5 was weaker in the latter population than in the former, and gradually decreased in the latter population with the advance of ontogenic development.

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ET AL.

PBL

Bone marrow 7

Th-5 Green fluorescent

intensity

FIG. 8. Flow cytometric profiles of lymphocytes in the spleen, peripheral blood (PBL), and bone marrow obtained from 6-week-old and 18-month-old mice, stained with Thy- I and Th-5. Most of the Thy- 1 positive cells were Th-5 moderately positive. There were a small number of cells showing Thy-l- and Th-SC in all samples examined.

et al. (33) reported that Pgp-1 was expressedby a minor subpopulation of peripheral CD4+ or CD8+ T cells but not mature medullary-type thymic lymphocytes. In the present study, however, Pgp-1 positive thymic lymphocytes were also found in the medulla. Indeed, Haynes et al. (34) described Pgp-1 as a medullary thymocyte antigen and they suggestedthat Pgp-1 appeared to be absolutely acquired by medullary lymphocytes during T cell maturation. The observations obtained in the present study indicate the difference between Th-5 and Pgp-1; i.e., Pgp-1 positive cells were found not only in the thymus but also in other tissues and were especially prominent in the hematopoietic system of the fetal liver, while Th-5 antigen was exclusively seen in cells of T cell lineage, but not in the fetal liver. In addition to these markers (Jl Id and Pgp-1), IL-2R (6, 7) and 3AlO (TCR yS) ( 13,23, 35, 36) are also considered as antigens expressedon fetal thymic lymphocytes. However, the immunohistological pattern of Th-5 staining was quite different from those of IL-2R and 3A 10. The most predominant point of the difference was that the number of IL-2R+ or 3A 1O+cells was much lower than that of Th-5+ cells in the fetal thymus. It is interesting to note that the magnitude of Th-5 expression in the flow cytometric analysis was more intense in double negative (CD4-8-) cells than in double positive (CD4+8+) cells as well as in mature peripheral T cells. Moreover, the Th-5 positiveness staining of the thymic double positive population became weaker with advance of ontogenic development; i.e., the staining intensity was distinctly weaker in 9-weekold thymus than in newborn thymus. It is generally accepted that double negative

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kDa

72-

46-

29-j

1 2 Th-5

3 4 Control

FIG. 9. The result of immunoblotting analysisby Th-5 antibody. Th-5 antibody immunostained a relatively broad band in the sample of fetal thymic lymphocytes (lane 1) as well as two distinct bands in the sample of TIGN (lane 2) at approximately 100kDa in both cases.TIGN is CD4-CD8- and Thy-l+ cell line, obtained by low doseirmdiation. No visible bands were observed at around 100kDa when Th-5 antibody was omitted during the incubation in fetal thymic lymphocytes (lane 3) and in TIGN (lane 4).

cells (CD4-8-) give rise to mature single positive CD4+ and CD8+ T cells, through or without the transitional step at CD4+8+ T cells (11, 12). Taken together, the magnitude of Th-5 expression appears to be dependent on the maturation stage of thymic lymphocytes and the strong expression of Th-5 could be one of the markers for immature thymic T lymphocytes which are rapidly proliferating in the early phaseof the ontogenic development. Double negative (CD4-8-) thymic lymphocytes are quite heterogenous and composed of many subpopulations (14). In this context, Th-5 was not useful for identification of any subpopulation within double negative cells. However, it is interesting to note that there was a small fraction of Thy- 1- and Th-Sf cells in the thymus, spleen, and even in the bone marrow. In any event, further investigations will be required to characterize these Thy-llTh-5+ cells and to determine the functional role of the Th5 molecule as it is one of the common surface antigens in T cell lineage. REFERENCES 1. Hirokawa, K., Utsuyama, M., Moriizumi, E., and Handa, S., Thymus 8, 349, 1986. 2. Wofsy, L.., Henry, C., Kimura, J., and North, J., In “Selected Methods in Cellular Immunology” (B. B. hdishell and S. M. Shiigi, Eds.), pp. 287-306. Freeman, San Francisco, 1980. 3. Ledbetter, J. A., and Herzenber& L. A., Immunol. Rev. 47, 63, 1979. 4. Bruce, J., Symington, F. W., McKeam, T. J., and Sprent, J., J. Zmmunol. 127, 2496, 1981. 5. Crispe, I. N., and Bevan, M. J., J. Immunol. 138, 20 13, 1987.

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