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HLA-DR expression in human fetal thymocytes
HLA-DR Expression in Human Fetal Thymocytes Seong Hoe Park, Young Mee Bae, Tae Jin Kim, Il Soo Ha, Sunyoung Kim, Je G. Chi, and Sang Kook Lee
A B S T R A C T : We analyzed the expression of MHC class I (W6/32) and class II (HLA-DR) antigens on human fetal and postnatal thymocytes by fluorescence-activated cell sorting. Less than 5% of prenatal thymocytes expressed HLA-DR before week 12 of gestation. However, the number of HLA-DR-positive cells significantly increased during the late second and third trimester of gestation, when > 50% of prenatal thymocytes expressed HLA-DR. Such high-level expressions of HLA-DR in
fetal thymocytes were also demonstrated by Northernblot analysis and immunohistochemistry. After birth, the percentage of HLA-DR-positive cells in thymocytes decreased gradually. A high-level expression of class I antigen was also observed in thymocytes from the early stages of gestation, but, in contrast to MHC class II, a majority of postnatal thymocytes maintained high levels of class I antigen after birth. Human Immunology 33,294298 (1992)
ABBREVIATIONS
FACS MHC
fluorescence-activated cell sorter major histocompatibility complex
TCR
T-cell receptor
INTRODUCTION The essential role of the thymus in the differentiation and functional maturation of bone-marrow-derived precursors into immunocompetent T-lymphocytes is well established [1, 2]. The thymic gland consists of two major components; the lymphoid cells and the epithelial cells (with additional mesenchymal elements). The stem cells of the lymphoid population originate from hematopoietic precursors [3]. In addition to the acquisition or loss of thymocyte differentiation antigens, thymocytes are subject to positive and negative selections. Prothymocytes undergo T-cell receptor (TCR) gene rearrangements, which generate tremendous diversity from a limited gene pool [4]. Clonally variable TCRs, made up of two different polypeptide chains, a and fl, interact with antigen-derived peptide fragments bound to products of From the Departments of Pathology tS.H.P.; Y.M.B.; T.J.K.; j.G.C.; S.K.L.), and Pediatrics (1.S.H.), and Cancer Research Institute (S.H.P.; Y.M.B.) College of Medicine; and the Institute for Molecular Biology and Genetics (S.K.), Seoul National University, Seoul, South Korea. Address reprint requests to Dr. Seong Hoe Park, Department of Pathology, Medtcal College, Yongon-dong Chongno-gu, Seoul, Korea. Received October 7, 1991," accepted February 21, 1992. 294 0198-8859/92/$5.00
major histocompatibility complex (MHC) on the surface of target cells [5]. It is thus clear that the repertoire of mature T cells bearing TCRs is under strict control. During the selection process, T-lymphoid cells learn to recognize antigens in the context of the M H C antigens within the thymus; T cells, after having arrived at the peripheral lymphoid organs, recognize foreign antigens in conjunction with M H C antigens according to the pattern of this previous thymic influence [6]. Therefore, the status of expression and distribution pattern of major histocompatibility antigens on various types of thymic constituents in the human thymus has become an area of major interest in immunology, due to their important role in thymic education [7-11]. M H C class II antigens, unlike class I antigens, are quite restricted in their distribution. These molecules are found mainly on antigen-presenting cells (monocytes/macrophages and dendritic cells), B cells, and some activated T cells. Although they are known to be present on thymic epithelial cells, they have been reported to be absent or expressed at very low levels in thymocytes themselves [ 1 2 - 1 6 ] . We have analyzed the Human Immunology 33, 294-298 (1992) © American Society for Histocompatibility and Immunogenetics, 1992
HLA-DR Expression in Human Fetal Thymocytes
expression of HLA class I and HLA class II (DR) on human fetal and postnatal thymocytes by flow-cytometric study. Unexpectedly, we found that MHC class II antigens were expressed on fetal thymocytes at high levels after 14 weeks of gestation and were progressively lost after birth. These findings were confirmed by Northern-blot analysis. MATERIALS AND METHODS
Thymic tissues. Fetal thymic tissues were obtained from fragments of thymuses that were removed from fetuses of various gestational age after legal terminations of pregnancies. (The reasons for legal termination include rape, traffic accident, and various types of diseases that might seriously threaten the mother's life.) The gestational age of each fetus was deduced from the crownrump length or maternal records and was represented by the actual or fertilization age for the embryo or fetus. Postnatal thymic tissues were taken from donors undergoing cardiac surgery at the Seoul National University Children's Hospital. Prior permission was granted from parents of the patients, in the case of partial thymectomy for clear exposure of the heart. Indirect immunofluorescence and immunohistochemica/ analysis. Fresh thymic tissues, within 4 hours after delivery, were minced with sharp scissors in RPMI media. The viability of the thymocytes was confirmed to be > 9 0 % by trypan blue staining. Cells were resuspended in phosphate-buffered saline (PBS) containing 0.05% sodium azide and 0.2% bovine serum albumin; 1 million cells were incubated with 80 ~zl of 1 : 100 dilution of purified monoclonal antibody (L243) at 4°C for 1 hour, washed twice, and then incubated for 30 minutes with a 1:30 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (IgG) (Zymed Laboratories, CA) at 4°C. Control samples were incubated with isotype matched control antibody and subsequently with FITC-conjugated secondary antibody. After the cells were washed in PBS containing 0.05 % sodium azide, they were resuspended in 1 ml of PBS containing 1% formaldehyde. Expressions of class I and class II antigens were studied by fluorescence-activated cell sorter (FACS) analysis using FACScan (Becton-Dickinson, Mountain View, CA), and a percentage of fluorescence-positive cells in 1 x 104 cells was obtained. For two-color flow-cytometric analysis, cells were first stained with anti-HLA-DR, washed, and then incubated with FITC-conjugated goat anti-mouse IgG as described above. Subsequently, cells were further stained with phycoerythrin-conjugated anti-CD4 or anti-CD8. The samples were gated on forward light scatter to exclude dead cells. For immunohis-
295
tochemical study, cryostat sections (4 /~m) were airdried, fixed with acetone, and immunostained with avidin-biotin-peroxidase methods as previously described [17]. Monoclonal antibodies L243 (IgG2aK, HLA-DR), B7/21 (IgG1K, HLA-DP), SK10 (IgGIK, HLA-DQ), SK3 (IgG1K, CD4), and SK1 (IgG1K, CD8) were purchased from Becton-Dickinson. W6/32 (IgG2a) is a monomorphic antibody detecting a common epitope on HLA class I antigen (HLA-A, -B, and -C). Ascites from L243 hybridoma clone was purified with protein-A Sepharose column.
Northern-blot analysis. Total thymocytes were size-fractionated on a 50% single-step Percoll density gradient and subsequently purified with a panning procedure using monoclonal antibodies specific for thymic epithelial cells (SHE-l) and monocytes (3C10) to eliminate the contaminating thymic epithelial cells and monocytes. Total cellular RNAs were prepared from human fetal thymocytes by the guanidine thiocyanate-cesium chloride method [19]. RNAs were separated by electrophoresis through a 1.3% agarose gel containing 2.2 M formaldehyde and then transferred to nitrocellulose filters. The filters were hybridized at 42°C overnight with appropriate D N A probes described by Tonnelle et al. [19]. The final wash of the filters was at 55°-65°C in 0.2 x SSC (1 x SSC = 0.15 M NaCl-15 mM sodium citrate, pH 7.0/0.01% SDS).
RESULTS A N D D ISCU SSIO N
FACS analysis of MHC class I and class II antigens. The expression of MHC class I and class II antigenes was analyzed on fetal (from 9 to 34 weeks) and postnatal (8 days to 5 years) by FACS (Table 1). The amount of thymocytes positive to HLA-DR was < 5 % prior to 12 weeks of gestation, but significantly increased thereafter. During the third trimester of gestation, the average percentage of positive cells was 50%, and in all samples studied, > 4 0 % cells expressed HLA-DR with increase in mean fluorescence intensity. After birth, the number of HLA-DR ÷ thymocytes decreased gradually. It was also found that 7 % - 1 1 % of thymocytes expressed HLA-DP and HLA-DQ at 10-18 weeks of gestation (data not shown). We have also determined the percentage of thymocytes expressing class I antigen by using the antibody W6/32 (Table 1). As expected, the number ofW6/32 ÷ thymocytes rapidly increased with gestational age. In the majority of thymus samples obtained after 16 weeks gestation, > 7 5 % of thymocytes expressed class I antigen. In contrast to class II, postnatal thymocytes maintained high levels of class I expression; for example,
296
S . H . P a r k et al.
TABLE 1
FACS analysis of human fetal and postnatal thymocytes Percentage of positive cells
Mean fluorescence intensity
Thymocyte
No. of samples
HLA-DR
W 6/32
HLA-DR
W 6/32
Prenatal (weeks) 9-10 14-26 29-33
4 16 4
2.8 -+ 2. F 33.2 + 15.3 52.3 -+ 21.8
5.6 -+ 4.8 67.7 ± 22.3 86.8 -+ 11.9
14.5 -+ 5.5 81.4 ± 23.4 60.3 ± 18.1
25 -+ 16.5 159,3 -+ 68.5 279.8 -+ 102.9
Postnatal (years) 1 2 3 4 5
12 3 1 1 1
32.3 -+ 11.2 19.1 + 8.6 1 32 1.1
84.1 -+ 10.8 97.1 ± 1.2 78.5 98.3 92.8
60.2 ± 17.9 26.7 ± 4.2 7.0 19.6 9.5
180.1 -+ 60.6 169.9 ± 43.8 350.2 421.4 374.7
Mean -+ SD.
>90% of thymocytes prepared from thymus obtained from 1 to 5 years after birth were positive for class I. We also tested whether the high frequency of HLADR + thymocytes in fetal thymus was due to contamination by other cells bearing HLA-DR. Two-color flowcytometric analyses of early postnatal thymocytes using
F I G U R E 1 T w o - c o l o r i m m u n o f l u o r e s c e n c e analysis o f the d i s t r i b u t i o n o f C D 4 ( a ) / C D 8 (b) and H L A - D R o n early postnatal t h y m o c y t e s . T h y m o c y t e s w e r e stained according to Materiak and Methods D R , H L A - D R .
HLA-DR and CD4/CD8 showed that about 26.5% of CD4 + cells were HLA-DR positive and - 3 1 . 4 % of CD8 + cells had HLA-DR, confirming that thymocytes definitely express HLA-DR antigen on their surface (Fig. 1). Furthermore, macrophages or thymic epithelial cells can be readily distinguished microscopically, and immunofluorescence studies using appropriate monoclonal antibodies clearly demonstrated the minimal contamination of < 3 % with these types of cells• The expression of Ia on mouse thymocytes and peripheral T cells has been reported [12-14], but the percentage of Ia + cells was much lower than that of DR-
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HLA-DR Expression in Human Fetal Thymocytes
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FIGURE 2 Hybridization analysis of mRNA from human thymocytes. F and PN indicate RNA samples from fetal and postnatal thymocytes, respectively. Control indicates RNA isolated from the human monocytic cell line U937 infected with human immunodeficiency virus type 1. Numbers above the lanes are weeks of gestation or months after birth. The DNA probes used for hybridization are as described in Materials and Methods. ~-Tubulin RNA was used as RNA-loading controls.
positive cells in human fetal thymocytes as reported here. It has also previously been reported that M H C class II antigens were not expressed in any gestational period [15, 16]. T h e r e f o r e our results contrast with those of these previous analyses. It is not clear what causes such discrepancies. However, it is worth noting that our study involved a much larger number of prenatal thymuses than previous reports, which would render our analysis statistically more reliable.
Tissue sections. T o confirm a high frequency of HLADR + thymocytes in fetal thymus, we performed immunohistochemical analysis by staining 4-/~m-thick frozen sections of the fetal and postnatal thymic tissues using the immunoperoxidase method with anti-HLAD R (data not shown). In fetal thymuses (18 weeks of gestational age), the great majority of the cells in the cortex (cortical thymocytes) were stained with antiHLA-DR. Positive staining was seen along the periphery of the cells, suggesting the immunoreactivity of the
cell membrane. H L A - D R antigens were also detectable on medullary thymocytes with less intensity. The similar staining patterns were found with HLA-DP and -DQ antigens (data not shown). In the thymus of a 5 year old, weak staining was seen in the cortex and medulla, which probably represents thymic epithelial cells expressing M H C class II antigens. These results confirmed the data obtained by the above FACS analysis.
R N A hybridization analysis. To confirm high level of class II expression in fetal thymocytes, we also performed Northern-blot analysis using RNAs prepared from various stages of human fetal and postnatal thymocytes. As a control, total cellular R N A isolated from U937 cells (a human monocytic cell line) infected with human immunodeficiency virus type I was analyzed on the same gel. These monocytic cells expressed almost no or very low levels of class II genes (lane 1, Fig. 2), but a high level of class I (lane 1, Fig. 2). As an RNAloading control, c~-tubulin R N A was also measured. The mRNAs of the class II genes were readily detectable 17 weeks after gestation. Their levels remained significant until 22 months after birth. In the case of class I m R N A , high-level expressions were observed in fetal thymocytes throughout gestation, consistent with the data by FACS analysis shown in Table 1. It must be noted that the R N A samples in each lane were prepared from different individuals and also that the fetal thymocyte samples used in this analysis were different from those used in the FACS analysis. However, the pattern of class II expression is still comparable between these two analyses, confirming the high level of expression of class II antigens in human fetal thymocytes. It is very unlikely that class II m R N A in fetal thymocytes might be from contaminating thymic epithelial cells and monocytes since these cells were removed by size fractionation and panning procedure prior to R N A isolation. It has recently been reported that activated peripheral T cells can present peptide fragments through their cell surface H L A - D R [20]. We are still not aware of whether it is exactly the same case in the H L A - D R + thymocytes. It is our assumption, however, that these H L A - D R + thymocyte might act as a antigen-presenting cells, presenting an enormous peptide repertoire, namely, endogeneous T C R fragments to adjacent HLADR-positive or negative thymocytes during positive selection events within the developing thymus. Another supportive finding for this idea is from the recent report that M H C class II molecules can present some antigenic determinants derived from endogenous proteins [21]. In summary, our results indicate that a significant fraction of fetal thymocytes express class II antigens throughout their developmental progression to mature T cells. These H L A - D R + thymocytes might represent a
298
developmental stage of T-cell maturation and may be involved in thymic education, providing a large repertoire of endogenous protein during thymic selection.
ACKNOWLEDGMENTS We thank Drs. Edmond J. Yunis and Cox Terhorst (DanaFaber Cancer Institute) for their critical reading of the manuscript, and Yong Jin Kim (Department of Cardiac Surgery, Seoul National University Children's Hospital) for providing the thymus used in this study. This study was supported in part by a clinical research grant from Seoul National University Hospital (1990) and a grant (KOSEH-SRC-56-CRC-17) from Korean Science and Engineering Foundation (1991).
REFERENCES 1. Cantor H, Weissman I: Development and function of subpopulations of thymocytes and T lymphocytes. Prog Allergy 20:1, 1976. 2. Stutman O: Intrathymic and extrathymic T cell maturation. Immunol Rev 42:138, 1978. 3. Kronenberg M, Siu G, Hood LE, Shastri N: The molecular genetics of the T cell antigen receptor and T cell antigen recognition. Ann Rev Immunol 4:529, 1986. 4. Le Douarin NM, Jotereau FV: Tracing of cells of the avian thymus through embryonic life in interspecific chimeras. J Exp Med 142:23, 1975. 5. Bjorkman PJ, Saper MA, Samraoi B, Bennett WS, Strominger JL, Wiley DC: Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329:506, 1987. 6. McMichael A: HLA restriction of human cytotoxic T lymphocytes specific for influenza virus. J Exp Med 148:1458, 1978. 7. Janossy G, Thomas JA, Bollum FJ, Granger S, Pizzolo G, Bradstock KF, Wong L, Michael A, Ganeshaguru K, Hoffbrand AV: The human thymic microenvironment: an immunohistologic study. J Immunol 125:202, 1980. 8. Abo T, Miller CA, Gartland GL, Balch CM: Differentiation stages of human natural killer cells in lymphoid tissues from fetal to adult life. J Exp Med 157:273, 1983. 9. Rosenthal P, Rimm IJ, Umiel T, GriffinJD, Osathanondh R, Schlossman SF, Nadler LM: Ontogeny of human he-
S.H. Park et al.
matopoietic cells: Analysis utilizing monoclonal antibodies. J Immunol 31:232, 1983. 10. Royo C, Touraine J-L, De Bouteiller O: Ontogeny o f T lymphocytes differentiation in the human fetus: Acquisition of phenotype and functions. Thymus 10:57, 1987. 11. Bradstock KF, Janossy G, Pizzolo G, Hoffbrand AV, McMichael A, Pilch JR, Milstein C, Beverley P, Bollum FJ: Subpopulation of normal and leukemic human thymocytes: An analysis with the use of monoclonal antibodies. J Natl Cancer Inst 65:33, 1980. 12. Scollay R, Jacobs S, Jerabek L, Butcher E, Weissman I: T cell maturation: thymocytes and thymus migrant subpopulations defined with monoclonal antibodies to MHC region antigens. J Immunol 124:2845, 1980. 13. Fathman CG, Cone JL, Sharrow SD, Tyrer H, Sachs DH: In alloantigen(s) detected on thymocytes by use of a fluorescence-activated cell sorter. J Immunol 115:584, 1975. 14. Hammerling GJ, Hammerling U, Lemke H: Isolation of twelve monoclonal antibodies and reactivity with B and T lymphocytes. Immunogenetics 8:433, 1979. 15. Janossy G, Tidman N, Papageorgiou ES, Kung PC, Goldstein G: Distribution o f T lymphocyte subsets in the human bone marrow and thymus: an analysis with monoclonal antibodies. J Immunol 126:1608, 1981. 16. Richard Y, Boumsell L, Coppin H, Mischall Z, LemerleJ, Bernard A: Correspondence between lectin-defined and surface antigen-defined subpopulations in the human thymus: its variation during ontogeny. J Immunol 127:252, 1981. 17. Hsu HM, Raine L, Fanger H: Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled (PAP) procedures. J Histochem 29:577, 1981. 18. Chirgwin JM, Przybyla AE, McDonald RE, Rutter WJ: Isolation of biologically active ribonucleic acid from source enriched in ribonuclease. Biochemistry 18:5294, 1979. 19. Tonnelle C, Demars R, Long EO: DOB: A new B chain gene in HLA-D with a distinct regulation of regulation. EMBO J 4:2839, 1985. 20. LaSalle JM, Kohei O, Hailer DA: Presentation of autoantigen by human T cells. J Immunol 147:774, 1991. 21. Brooks A, Hartley S, Kjer-Nielsen L, PereraJ, Goodnow CC, Basten A, McCluskey J: Class II-restricted presentation of an endogenously derived immunodominant T-cell determinant of hen egg lysozyme. PNAS 88:3290, 1991.
A B S T R A C T : We analyzed the expression of MHC class I (W6/32) and class II (HLA-DR) antigens on human fetal and postnatal thymocytes by fluorescence-activated cell sorting. Less than 5% of prenatal thymocytes expressed HLA-DR before week 12 of gestation. However, the number of HLA-DR-positive cells significantly increased during the late second and third trimester of gestation, when > 50% of prenatal thymocytes expressed HLA-DR. Such high-level expressions of HLA-DR in
fetal thymocytes were also demonstrated by Northernblot analysis and immunohistochemistry. After birth, the percentage of HLA-DR-positive cells in thymocytes decreased gradually. A high-level expression of class I antigen was also observed in thymocytes from the early stages of gestation, but, in contrast to MHC class II, a majority of postnatal thymocytes maintained high levels of class I antigen after birth. Human Immunology 33,294298 (1992)
ABBREVIATIONS
FACS MHC
fluorescence-activated cell sorter major histocompatibility complex
TCR
T-cell receptor
INTRODUCTION The essential role of the thymus in the differentiation and functional maturation of bone-marrow-derived precursors into immunocompetent T-lymphocytes is well established [1, 2]. The thymic gland consists of two major components; the lymphoid cells and the epithelial cells (with additional mesenchymal elements). The stem cells of the lymphoid population originate from hematopoietic precursors [3]. In addition to the acquisition or loss of thymocyte differentiation antigens, thymocytes are subject to positive and negative selections. Prothymocytes undergo T-cell receptor (TCR) gene rearrangements, which generate tremendous diversity from a limited gene pool [4]. Clonally variable TCRs, made up of two different polypeptide chains, a and fl, interact with antigen-derived peptide fragments bound to products of From the Departments of Pathology tS.H.P.; Y.M.B.; T.J.K.; j.G.C.; S.K.L.), and Pediatrics (1.S.H.), and Cancer Research Institute (S.H.P.; Y.M.B.) College of Medicine; and the Institute for Molecular Biology and Genetics (S.K.), Seoul National University, Seoul, South Korea. Address reprint requests to Dr. Seong Hoe Park, Department of Pathology, Medtcal College, Yongon-dong Chongno-gu, Seoul, Korea. Received October 7, 1991," accepted February 21, 1992. 294 0198-8859/92/$5.00
major histocompatibility complex (MHC) on the surface of target cells [5]. It is thus clear that the repertoire of mature T cells bearing TCRs is under strict control. During the selection process, T-lymphoid cells learn to recognize antigens in the context of the M H C antigens within the thymus; T cells, after having arrived at the peripheral lymphoid organs, recognize foreign antigens in conjunction with M H C antigens according to the pattern of this previous thymic influence [6]. Therefore, the status of expression and distribution pattern of major histocompatibility antigens on various types of thymic constituents in the human thymus has become an area of major interest in immunology, due to their important role in thymic education [7-11]. M H C class II antigens, unlike class I antigens, are quite restricted in their distribution. These molecules are found mainly on antigen-presenting cells (monocytes/macrophages and dendritic cells), B cells, and some activated T cells. Although they are known to be present on thymic epithelial cells, they have been reported to be absent or expressed at very low levels in thymocytes themselves [ 1 2 - 1 6 ] . We have analyzed the Human Immunology 33, 294-298 (1992) © American Society for Histocompatibility and Immunogenetics, 1992
HLA-DR Expression in Human Fetal Thymocytes
expression of HLA class I and HLA class II (DR) on human fetal and postnatal thymocytes by flow-cytometric study. Unexpectedly, we found that MHC class II antigens were expressed on fetal thymocytes at high levels after 14 weeks of gestation and were progressively lost after birth. These findings were confirmed by Northern-blot analysis. MATERIALS AND METHODS
Thymic tissues. Fetal thymic tissues were obtained from fragments of thymuses that were removed from fetuses of various gestational age after legal terminations of pregnancies. (The reasons for legal termination include rape, traffic accident, and various types of diseases that might seriously threaten the mother's life.) The gestational age of each fetus was deduced from the crownrump length or maternal records and was represented by the actual or fertilization age for the embryo or fetus. Postnatal thymic tissues were taken from donors undergoing cardiac surgery at the Seoul National University Children's Hospital. Prior permission was granted from parents of the patients, in the case of partial thymectomy for clear exposure of the heart. Indirect immunofluorescence and immunohistochemica/ analysis. Fresh thymic tissues, within 4 hours after delivery, were minced with sharp scissors in RPMI media. The viability of the thymocytes was confirmed to be > 9 0 % by trypan blue staining. Cells were resuspended in phosphate-buffered saline (PBS) containing 0.05% sodium azide and 0.2% bovine serum albumin; 1 million cells were incubated with 80 ~zl of 1 : 100 dilution of purified monoclonal antibody (L243) at 4°C for 1 hour, washed twice, and then incubated for 30 minutes with a 1:30 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (IgG) (Zymed Laboratories, CA) at 4°C. Control samples were incubated with isotype matched control antibody and subsequently with FITC-conjugated secondary antibody. After the cells were washed in PBS containing 0.05 % sodium azide, they were resuspended in 1 ml of PBS containing 1% formaldehyde. Expressions of class I and class II antigens were studied by fluorescence-activated cell sorter (FACS) analysis using FACScan (Becton-Dickinson, Mountain View, CA), and a percentage of fluorescence-positive cells in 1 x 104 cells was obtained. For two-color flow-cytometric analysis, cells were first stained with anti-HLA-DR, washed, and then incubated with FITC-conjugated goat anti-mouse IgG as described above. Subsequently, cells were further stained with phycoerythrin-conjugated anti-CD4 or anti-CD8. The samples were gated on forward light scatter to exclude dead cells. For immunohis-
295
tochemical study, cryostat sections (4 /~m) were airdried, fixed with acetone, and immunostained with avidin-biotin-peroxidase methods as previously described [17]. Monoclonal antibodies L243 (IgG2aK, HLA-DR), B7/21 (IgG1K, HLA-DP), SK10 (IgGIK, HLA-DQ), SK3 (IgG1K, CD4), and SK1 (IgG1K, CD8) were purchased from Becton-Dickinson. W6/32 (IgG2a) is a monomorphic antibody detecting a common epitope on HLA class I antigen (HLA-A, -B, and -C). Ascites from L243 hybridoma clone was purified with protein-A Sepharose column.
Northern-blot analysis. Total thymocytes were size-fractionated on a 50% single-step Percoll density gradient and subsequently purified with a panning procedure using monoclonal antibodies specific for thymic epithelial cells (SHE-l) and monocytes (3C10) to eliminate the contaminating thymic epithelial cells and monocytes. Total cellular RNAs were prepared from human fetal thymocytes by the guanidine thiocyanate-cesium chloride method [19]. RNAs were separated by electrophoresis through a 1.3% agarose gel containing 2.2 M formaldehyde and then transferred to nitrocellulose filters. The filters were hybridized at 42°C overnight with appropriate D N A probes described by Tonnelle et al. [19]. The final wash of the filters was at 55°-65°C in 0.2 x SSC (1 x SSC = 0.15 M NaCl-15 mM sodium citrate, pH 7.0/0.01% SDS).
RESULTS A N D D ISCU SSIO N
FACS analysis of MHC class I and class II antigens. The expression of MHC class I and class II antigenes was analyzed on fetal (from 9 to 34 weeks) and postnatal (8 days to 5 years) by FACS (Table 1). The amount of thymocytes positive to HLA-DR was < 5 % prior to 12 weeks of gestation, but significantly increased thereafter. During the third trimester of gestation, the average percentage of positive cells was 50%, and in all samples studied, > 4 0 % cells expressed HLA-DR with increase in mean fluorescence intensity. After birth, the number of HLA-DR ÷ thymocytes decreased gradually. It was also found that 7 % - 1 1 % of thymocytes expressed HLA-DP and HLA-DQ at 10-18 weeks of gestation (data not shown). We have also determined the percentage of thymocytes expressing class I antigen by using the antibody W6/32 (Table 1). As expected, the number ofW6/32 ÷ thymocytes rapidly increased with gestational age. In the majority of thymus samples obtained after 16 weeks gestation, > 7 5 % of thymocytes expressed class I antigen. In contrast to class II, postnatal thymocytes maintained high levels of class I expression; for example,
296
S . H . P a r k et al.
TABLE 1
FACS analysis of human fetal and postnatal thymocytes Percentage of positive cells
Mean fluorescence intensity
Thymocyte
No. of samples
HLA-DR
W 6/32
HLA-DR
W 6/32
Prenatal (weeks) 9-10 14-26 29-33
4 16 4
2.8 -+ 2. F 33.2 + 15.3 52.3 -+ 21.8
5.6 -+ 4.8 67.7 ± 22.3 86.8 -+ 11.9
14.5 -+ 5.5 81.4 ± 23.4 60.3 ± 18.1
25 -+ 16.5 159,3 -+ 68.5 279.8 -+ 102.9
Postnatal (years) 1 2 3 4 5
12 3 1 1 1
32.3 -+ 11.2 19.1 + 8.6 1 32 1.1
84.1 -+ 10.8 97.1 ± 1.2 78.5 98.3 92.8
60.2 ± 17.9 26.7 ± 4.2 7.0 19.6 9.5
180.1 -+ 60.6 169.9 ± 43.8 350.2 421.4 374.7
Mean -+ SD.
>90% of thymocytes prepared from thymus obtained from 1 to 5 years after birth were positive for class I. We also tested whether the high frequency of HLADR + thymocytes in fetal thymus was due to contamination by other cells bearing HLA-DR. Two-color flowcytometric analyses of early postnatal thymocytes using
F I G U R E 1 T w o - c o l o r i m m u n o f l u o r e s c e n c e analysis o f the d i s t r i b u t i o n o f C D 4 ( a ) / C D 8 (b) and H L A - D R o n early postnatal t h y m o c y t e s . T h y m o c y t e s w e r e stained according to Materiak and Methods D R , H L A - D R .
HLA-DR and CD4/CD8 showed that about 26.5% of CD4 + cells were HLA-DR positive and - 3 1 . 4 % of CD8 + cells had HLA-DR, confirming that thymocytes definitely express HLA-DR antigen on their surface (Fig. 1). Furthermore, macrophages or thymic epithelial cells can be readily distinguished microscopically, and immunofluorescence studies using appropriate monoclonal antibodies clearly demonstrated the minimal contamination of < 3 % with these types of cells• The expression of Ia on mouse thymocytes and peripheral T cells has been reported [12-14], but the percentage of Ia + cells was much lower than that of DR-
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FIGURE 2 Hybridization analysis of mRNA from human thymocytes. F and PN indicate RNA samples from fetal and postnatal thymocytes, respectively. Control indicates RNA isolated from the human monocytic cell line U937 infected with human immunodeficiency virus type 1. Numbers above the lanes are weeks of gestation or months after birth. The DNA probes used for hybridization are as described in Materials and Methods. ~-Tubulin RNA was used as RNA-loading controls.
positive cells in human fetal thymocytes as reported here. It has also previously been reported that M H C class II antigens were not expressed in any gestational period [15, 16]. T h e r e f o r e our results contrast with those of these previous analyses. It is not clear what causes such discrepancies. However, it is worth noting that our study involved a much larger number of prenatal thymuses than previous reports, which would render our analysis statistically more reliable.
Tissue sections. T o confirm a high frequency of HLADR + thymocytes in fetal thymus, we performed immunohistochemical analysis by staining 4-/~m-thick frozen sections of the fetal and postnatal thymic tissues using the immunoperoxidase method with anti-HLAD R (data not shown). In fetal thymuses (18 weeks of gestational age), the great majority of the cells in the cortex (cortical thymocytes) were stained with antiHLA-DR. Positive staining was seen along the periphery of the cells, suggesting the immunoreactivity of the
cell membrane. H L A - D R antigens were also detectable on medullary thymocytes with less intensity. The similar staining patterns were found with HLA-DP and -DQ antigens (data not shown). In the thymus of a 5 year old, weak staining was seen in the cortex and medulla, which probably represents thymic epithelial cells expressing M H C class II antigens. These results confirmed the data obtained by the above FACS analysis.
R N A hybridization analysis. To confirm high level of class II expression in fetal thymocytes, we also performed Northern-blot analysis using RNAs prepared from various stages of human fetal and postnatal thymocytes. As a control, total cellular R N A isolated from U937 cells (a human monocytic cell line) infected with human immunodeficiency virus type I was analyzed on the same gel. These monocytic cells expressed almost no or very low levels of class II genes (lane 1, Fig. 2), but a high level of class I (lane 1, Fig. 2). As an RNAloading control, c~-tubulin R N A was also measured. The mRNAs of the class II genes were readily detectable 17 weeks after gestation. Their levels remained significant until 22 months after birth. In the case of class I m R N A , high-level expressions were observed in fetal thymocytes throughout gestation, consistent with the data by FACS analysis shown in Table 1. It must be noted that the R N A samples in each lane were prepared from different individuals and also that the fetal thymocyte samples used in this analysis were different from those used in the FACS analysis. However, the pattern of class II expression is still comparable between these two analyses, confirming the high level of expression of class II antigens in human fetal thymocytes. It is very unlikely that class II m R N A in fetal thymocytes might be from contaminating thymic epithelial cells and monocytes since these cells were removed by size fractionation and panning procedure prior to R N A isolation. It has recently been reported that activated peripheral T cells can present peptide fragments through their cell surface H L A - D R [20]. We are still not aware of whether it is exactly the same case in the H L A - D R + thymocytes. It is our assumption, however, that these H L A - D R + thymocyte might act as a antigen-presenting cells, presenting an enormous peptide repertoire, namely, endogeneous T C R fragments to adjacent HLADR-positive or negative thymocytes during positive selection events within the developing thymus. Another supportive finding for this idea is from the recent report that M H C class II molecules can present some antigenic determinants derived from endogenous proteins [21]. In summary, our results indicate that a significant fraction of fetal thymocytes express class II antigens throughout their developmental progression to mature T cells. These H L A - D R + thymocytes might represent a
298
developmental stage of T-cell maturation and may be involved in thymic education, providing a large repertoire of endogenous protein during thymic selection.
ACKNOWLEDGMENTS We thank Drs. Edmond J. Yunis and Cox Terhorst (DanaFaber Cancer Institute) for their critical reading of the manuscript, and Yong Jin Kim (Department of Cardiac Surgery, Seoul National University Children's Hospital) for providing the thymus used in this study. This study was supported in part by a clinical research grant from Seoul National University Hospital (1990) and a grant (KOSEH-SRC-56-CRC-17) from Korean Science and Engineering Foundation (1991).
REFERENCES 1. Cantor H, Weissman I: Development and function of subpopulations of thymocytes and T lymphocytes. Prog Allergy 20:1, 1976. 2. Stutman O: Intrathymic and extrathymic T cell maturation. Immunol Rev 42:138, 1978. 3. Kronenberg M, Siu G, Hood LE, Shastri N: The molecular genetics of the T cell antigen receptor and T cell antigen recognition. Ann Rev Immunol 4:529, 1986. 4. Le Douarin NM, Jotereau FV: Tracing of cells of the avian thymus through embryonic life in interspecific chimeras. J Exp Med 142:23, 1975. 5. Bjorkman PJ, Saper MA, Samraoi B, Bennett WS, Strominger JL, Wiley DC: Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329:506, 1987. 6. McMichael A: HLA restriction of human cytotoxic T lymphocytes specific for influenza virus. J Exp Med 148:1458, 1978. 7. Janossy G, Thomas JA, Bollum FJ, Granger S, Pizzolo G, Bradstock KF, Wong L, Michael A, Ganeshaguru K, Hoffbrand AV: The human thymic microenvironment: an immunohistologic study. J Immunol 125:202, 1980. 8. Abo T, Miller CA, Gartland GL, Balch CM: Differentiation stages of human natural killer cells in lymphoid tissues from fetal to adult life. J Exp Med 157:273, 1983. 9. Rosenthal P, Rimm IJ, Umiel T, GriffinJD, Osathanondh R, Schlossman SF, Nadler LM: Ontogeny of human he-
S.H. Park et al.
matopoietic cells: Analysis utilizing monoclonal antibodies. J Immunol 31:232, 1983. 10. Royo C, Touraine J-L, De Bouteiller O: Ontogeny o f T lymphocytes differentiation in the human fetus: Acquisition of phenotype and functions. Thymus 10:57, 1987. 11. Bradstock KF, Janossy G, Pizzolo G, Hoffbrand AV, McMichael A, Pilch JR, Milstein C, Beverley P, Bollum FJ: Subpopulation of normal and leukemic human thymocytes: An analysis with the use of monoclonal antibodies. J Natl Cancer Inst 65:33, 1980. 12. Scollay R, Jacobs S, Jerabek L, Butcher E, Weissman I: T cell maturation: thymocytes and thymus migrant subpopulations defined with monoclonal antibodies to MHC region antigens. J Immunol 124:2845, 1980. 13. Fathman CG, Cone JL, Sharrow SD, Tyrer H, Sachs DH: In alloantigen(s) detected on thymocytes by use of a fluorescence-activated cell sorter. J Immunol 115:584, 1975. 14. Hammerling GJ, Hammerling U, Lemke H: Isolation of twelve monoclonal antibodies and reactivity with B and T lymphocytes. Immunogenetics 8:433, 1979. 15. Janossy G, Tidman N, Papageorgiou ES, Kung PC, Goldstein G: Distribution o f T lymphocyte subsets in the human bone marrow and thymus: an analysis with monoclonal antibodies. J Immunol 126:1608, 1981. 16. Richard Y, Boumsell L, Coppin H, Mischall Z, LemerleJ, Bernard A: Correspondence between lectin-defined and surface antigen-defined subpopulations in the human thymus: its variation during ontogeny. J Immunol 127:252, 1981. 17. Hsu HM, Raine L, Fanger H: Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled (PAP) procedures. J Histochem 29:577, 1981. 18. Chirgwin JM, Przybyla AE, McDonald RE, Rutter WJ: Isolation of biologically active ribonucleic acid from source enriched in ribonuclease. Biochemistry 18:5294, 1979. 19. Tonnelle C, Demars R, Long EO: DOB: A new B chain gene in HLA-D with a distinct regulation of regulation. EMBO J 4:2839, 1985. 20. LaSalle JM, Kohei O, Hailer DA: Presentation of autoantigen by human T cells. J Immunol 147:774, 1991. 21. Brooks A, Hartley S, Kjer-Nielsen L, PereraJ, Goodnow CC, Basten A, McCluskey J: Class II-restricted presentation of an endogenously derived immunodominant T-cell determinant of hen egg lysozyme. PNAS 88:3290, 1991.