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The Journal of Immunology, 1999, 162: 5685-5694.
Copyright © 1999 by The American Association of Immunologists

ChT1, an Ig Superfamily Molecule Required for T Cell Differentiation1 ,2

Kaisa Katevuo3,*, Beat A. Imhof{dagger}, Richard Boyd{ddagger}, Ann Chidgey{ddagger}, Andrew Bean{ddagger}, Dominique Dunon§, Thomas W. F. Göbel and Olli Vainio*

* Turku Immunology Center, Department of Medical Microbiology, University of Turku, Turku, Finland; {dagger} Department of Pathology, Centre Medical Universitaire, University of Geneva, Geneva, Switzerland; {ddagger} Department of Pathology and Immunology, Monash University Medical School, Prahran, Melbourne, Victoria, Australia; § Unité de Recherche Associée-Centre National de la Recherche Scientifique 1135, University of Pierre and Marie Curie, Paris, France; and Basel Institute for Immunology, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The thymus is colonized by circulating progenitor cells that differentiate into mature T cells under the influence of the thymic microenvironment. We report here the cloning and function of the avian thymocyte Ag ChT1, a member of the Ig superfamily with one V-like and one C2-like domain. ChT1-positive embryonic bone marrow cells coexpressing c-kit give rise to mature T cells upon intrathymic cell transfer. ChT1-specific Ab inhibits T cell differentiation in embryonic thymic organ cultures and in thymocyte precursor cocultures on stromal cells. Thus, we provide clear evidence that ChT1 is a novel Ag on early T cell progenitors that plays an important role in the early stages of T cell development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcell differentiation follows immediately after colonization of the thymus by pluripotential hemopoietic progenitor cells and is driven by the interaction of cell surface molecules and cytokines. Most immature cells found in the thymus may not yet be committed to the T lineage, since they can still differentiate into T, B, NK, and dendritic cells (1, 2, 3, 4, 5). The ensuing differentiation pathways are complex, and although multiple stages of development can be identified, it is a continuous process rather than a series of precise shifts. Each level of maturation is defined not by unique stage-specific markers but by a phenotypic profile based on a constellation of membrane molecules (1, 6, 7, 8, 9). Even the earliest intrathymic precursors lacking TCR, CD4 and CD8, which represent <5% of the total thymocyte pool, are divisible into at least four subsets based on differential expression of CD117 (c-kit), CD44 (Pgp-1), and CD25 (IL-2R{alpha}) (2, 8, 10). None of these markers, however, is T-lineage specific. In fact, no cell surface molecule has yet been identified that exclusively distinguishes T-lineage cells from the earliest precursors.

Although many studies have been performed in mice, the chicken offers an exceptional model for analyzing the events of stem cell differentiation and T cell development because of the easy access to embryos at exact stages. A further advantage of the chicken is that T and B cells develop in separate tissues, thymus and bursa, respectively. Lymphoid precursors colonize the embryonic thymus in three distinct waves, beginning on embryonic days (E)4 6.5, 12, and 18 (11, 12, 13, 14). It has been shown that T cell progenitors seeding the thymus during the second period of colonization on E12 stem from the embryonic bone marrow (15) and express the receptor-type tyrosine kinase c-kit and adhesion molecules HEMCAM and BEN (16, 17). These three marker molecules are useful in defining T cell progenitors and immature thymocytes, but their distribution is again not restricted to the T lineage. We therefore searched for new molecules that would allow further characterization of hemopoietic progenitors and lead to better definition of discrete stages of thymocyte differentiation occurring before TCR expression. ChT1 is a T cell Ag that has been shown to be expressed on the surface of thymocytes by E10. In young chickens about 90% of the thymocytes and a subpopulation of peripheral lymphocytes, representing the recent thymic emigrants, are ChT1 positive (18, 19, 20). Expression in the periphery declines with age and also in correlation with partial thymectomy, indicating that ChT1 can be used as an accurate marker for studying thymic function (20).

In the present work we cloned ChT1 cDNA from two thymus cDNA libraries. The Ag was identified as a member of the Ig superfamily (IgSF) with two extracellular Ig domains. We also showed that ChT1 is a close homologue of CTX, a recently cloned Xenopus Ag. Intrathymic cell transfer of ChT1+c-kit+ bone marrow cells into congenic animals defined a T cell progenitor population in embryonic bone marrow. In embryonic thymus organ cultures (ETOC) and in in vitro thymic stromal cell and thymocyte precursor cocultures the differentiation of T cells was inhibited at the CD3-CD4-CD8- stage. These data provide distinct evidence that ChT1 is a novel IgSF molecule that has a functional role in the differentiation of T cells from embryonic bone marrow precursors.


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

H.B2 and H.B15 chickens and chicken embryos from the colonies at the Department of Medical Microbiology, Turku University (Turku, Finland), two ov-Ag congenic H.B19 strains at the Basel Institute for Immunology (Gipf-Oberfrick, Switzerland), and the Australorp x White Leghorn hybrids at the Research Poultry Farm (Research, Australia) were used. The two congenic lines, H.B19ov+ and H.B19ov-, differ in their expression of the ov-Ag on thymocytes and T cells. The ov-Ag is recognized by mAb 11A9 (15, 21).

Abs and flow cytometry

Abs detecting the ChT1 Ag were T10A6 (IgG1) (19), CT1 (IgG1) and CT1a (IgG3) (18), RR5-89 (IgG2b), cF3c210 (IgM), MUI-83 (IgG1) (22), and 2-1 (IgG1). Other Abs were kit2c75 (IgG2a) against c-kit (16), 11A9 (IgM) against ov-Ag, 2-6 (IgG1) and 2-35 (IgG2b) against CD4 (23), 11-39 (IgG1), 3-298 (IgG2b) against CD8{alpha} (24), MUI-78 (IgG2a) against MHC class II (22, 25), 10-2.16 (IgG1) against mouse I-Ak (26), MUI-36 (IgG2a), MUI-53 (IgM), MUI-54 (IgM), MUI-70 (IgM), MUI-71 (IgM), and MUI-82 (IgG1). TCR1 (IgG1) against {gamma}{delta}TCR, TCR2 (IgG1) against {alpha}ß1TCR, TCR3 (IgG1) against {alpha}ß2TCR, and CT3 (IgG1) against CD3 were purchased from Southern Biotechnology Associates (Birmingham, AL). Abs were used as ascites, hybridoma supernatant, or purified Ab. Ab purification and conjugation to biotin (biotin-N-hydroxysuccinimidester, NHS-D-biotin, Calbiochem, La Jolla, CA) and PE (R-PE, Molecular Probes, Leiden, The Netherlands) were performed using standard procedures.

For one-, two-, and three-color immunofluorescence, cell suspensions from thymus, spleen, PBL, and bone marrow cells were prepared as described previously (16, 27). Cells (1 x 106) were incubated with primary mAb followed by incubation with FITC-conjugated anti-mouse Ig (Silenus Laboratories, Hawthorn, Australia) or anti-mouse Ig isotype-specific Abs (Southern Biotechnology Associates). Thereafter, the cells were incubated with secondary mAb, which were used as hybridoma culture supernatants or PE conjugates. The unconjugated mAb were detected with PE-conjugated anti-mouse Ig isotype-specific Abs (Southern Biotechnology Associates). For three-color analysis cells were blocked with 1% normal mouse serum and further incubated with biotinylated tertiary mAb followed by streptavidin Tri-color reagent (Caltag, South San Francisco, CA). After each step the cells were washed twice with PBS containing 2% FCS and 0.01% NaN3. Immunofluorescence analysis was performed with a FACScan instrument (Becton Dickinson, Mountain View, CA).

Intrathymic injection and differentiation of embryonic bone marrow cells

Intrathymic injections were performed as described previously (16, 28). In brief, bone marrow cells from congenic ov+ E13.5 donor animals of the H.B19 strain were flushed from the cavity of the femurs and tibiae, washed twice in PBS, counted, and adjusted to the required cell concentration (15). Before receiving the bone marrow cells, the recipients, 14-day-old ov- congenic H.B19 chicks, were irradiated with 600 rad (Gamma Cell irradiator, Atomic Energy of Canada, Ottawa, Canada). For the intrathymic injection the recipients were anesthetized with an injection of 0.4 ml of ketamine (Imalgene 500, Rhone Merieux, France) diluted 1/10 in PBS i.m. followed by a short inhalation of halotane (Hoechst, Frankfurt, Germany). The donor bone marrow cells were injected into two upper lobes on each side through a dorsal incision in the skin. Ten microliters of cell suspension (1000 or 100 cells/thymus lobe) in PBS was injected. Donor-derived cells from the injected thymus lobes were identified by immunofluorescence 2 wk after chimera construction with the anti-ov mAb 11A9.

Immunoprecipitation and peptide mapping

The lactoperoxidase-catalyzed method (29) was used to surface label 5 x 107 thymocytes (viability, >95%) with 1 mCi of Na125I (Amersham, Aylesbury, U.K.). The cells were lysed in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.1% NaN3, 1 mM Pefabloc (Boehringer Mannheim, Indianapolis, IN), 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 100 µg/ml trypsin inhibitor, 20 mM {epsilon}-amino-n-caproic-acid, 2 µg/ml antipain, 20 mM iodoacetamide (all reagents from Sigma, St. Louis, IL), and 1% Nonidet P-40 (Calbiochem, La Jolla, CA) for 45 min at 4°C. Insoluble particles were removed by centrifugation (10,000 x g, 30 min, 4°C). The lysate was then immunoprecipitated using a solid phase method (30). The absorbed molecules were eluted with Laemmli sample buffer for 45 min at 37°C or with 0.5% SDS/0.1 M 2-ME for 20 min at 80°C for glycolytic enzyme digestions. N-linked carbohydrates were removed by incubating the samples in the presence of 50 U/ml N-glycanase at 37°C overnight. Sialic acids were digested with 3 U/ml neuraminidase at 37°C for 2 h after setting the pH to 6.3 with 1 M CH3COOH; after this treatment the O-linked sugars were removed by incubation with 82 mU/ml of O-glycanase (all enzymes from Genzyme, Boston, MA) at 37°C overnight. Undigested controls for each enzyme were treated similarly but without adding the enzyme. For electrophoresis, 6x Laemmli sample buffer was added, and samples were boiled for 5 min. Reduced conditions were obtained by adding 5% 2-ME before boiling. Electrophoresis was performed on 5–15% gradient SDS-PAGE.

Peptide mapping by proteolysis with endoproteinase Glu-C (protease V8, Boehringer Mannheim) in SDS was performed basically as described by Cleveland (31). Briefly, after exposing the immunoprecipitates to an autoradiography film (Kodak X-OMAT AR, Eastman Kodak, Rochester, NY) the bands were cut out from the dried gel with a sharp scalpel. The gel slices were rehydrated in a sample buffer with 1 mM EDTA for 20 min, placed in the sample wells of a second 5–15% SDS gel, and overlaid with 10 µl of 20% and 10 µl of 10% glycerol in sample buffer and finally with 1 µg (in H2O) of the protease. Electrophoresis was started, and after the samples had reached the stacking gel the run was stopped, and the gel was incubated at 37°C for 60 min to allow protein digestion to proceed. Thereafter electrophoresis was completed.

Ag purification

The ChT1 Ag was purified from chicken thymus Nonidet P-40 lysates by an immunoaffinity column as described previously (32). Samples were run on a 12.5% SDS-PAGE, and the proteins were visualized by silver staining. The relatively pure protein was sequenced directly on a 475A Protein Sequencer (Applied Biosystems, Foster City, CA). Half the material was digested with endoproteinase Lys-C (Boehringer Mannheim) at a ratio of 1/100 at 37°C overnight, and the resulting fragments were separated by reverse phase HPLC and analyzed by microsequencing to obtain internal peptide sequences.

cDNA cloning and sequencing

The COS cell expression screening method was used to clone the cDNA encoding for ChT1 Ag from two different chicken cDNA libraries. The thymus cDNA library in pCDM8 vector from an adult RPRL Line 0 animal was a gift from Dr. J. R. Young. The other library was constructed from mRNA of strain H.B19 E13 thymus into vector pcDNA3 (16). The transfection and staining were performed as described by Tregaskes and Young (33). Positively stained cells were picked up from the slide with a Drummond sequencing pipette (Drummond Scientific, Broomall, PA). Proteins were precipitated, after which the DNA was extracted. The plasmid DNA containing the desired cDNA insert was then introduced to Escherichia coli (MC1061/p3 or TOP10F') by electroporation. For sequencing of the cDNA, the plasmid DNA was extracted using Qiagen spin columns (Qiagen, Chatsworth, CA) and was sequenced with either dideoxy chain termination (Sequenase version 2.0, U.S. Biochemical Corp., Cleveland, OH) or automated cycle sequencing (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin-Elmer, Norwalk, CT) with an ABI 373A DNA Sequencer (Applied Biosystems). The cDNA sequences have been submitted to the EMBL database with accession numbers Y14063 and Y14064. Sequence data analysis and sequence comparisons were performed using the Wisconsin University software package GCG (Genetics Computer Group, Madison, WI), Lasergene (DNAStar, Madison, WI), and Blast (National Center for Biotechnology Information, Bethesda, MD).

Analysis of mRNA expression

Northern blot was performed with mRNA isolated from H.B2 chicken thymus, spleen, liver, bursa, ileum, colon, brain, lung, kidney, testis, and oviduct as well as with Marek’s disease virus-transformed MDCC-CU32 (CU32), MDCC-CU36 (CU36) and reticuloendothelial virus-transformed REVCC-RP13 (RP13) chicken cell lines using RNeasy and Oligotex mRNA Spin column kits (Qiagen). After electrophoretic separation on a 1.2% formaldehyde agarose gel the samples were transferred overnight to a nylon membrane (Hybond-N+, Amersham). The membrane was prehybridized in 1% SDS, 5x Denhardt’s solution, 2x SSPE, 10% dextran sulfate, and 20 µg/ml ssDNA at 60°C for 6 h. For hybridization the membrane was incubated at 60°C overnight in fresh hybridization solution with total cDNA of ChT1 or a PCR product that encodes for a 1030-bp part of the chicken ß-actin cDNA (34) as probes. These were labeled with [{alpha}-32P]dCTP (3000 Ci/mM; Amersham) using a Rediprime labeling kit (Amersham). Membrane was washed in 2x SSC/1% SDS at 60°C for 30 min followed by washing in 0.5x SSC/1% SDS at 60°C for 15 min. Signals were visualized by autoradiography.

To study the mRNA expression of ChT1 further by RT-PCR, 0.1 µg of total RNA from spleen, bursa, liver, small intestine, skeletal muscle, and Marek’s disease virus-transformed MDCC-CU20 (CU20), CU36, and RP13 cells was used in 50 µl of RT-PCR reaction (Titan One Tube RT-PCR Kit, Boehringer Mannheim, Mannheim, Germany). The SMART PCR cDNA Library Construction Kit (Clontech, Palo Alto, CA) was used according to the manufacturer’s instructions to prepare cDNA from E13 bone marrow mRNA for analysis of ChT1 expression in the bone marrow. One microliter of amplified cDNA was used for PCR in a 50-µl reaction, and the PCR conditions were as follows: 96°C for 2 min; then 35 cycles of 96°C for 30 s, 52°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 5 min. ChT1-specific oligonucleotides were V5'-GTG ACC GTT CCT GAG AAG- and C3'-GTT GCT GGC TAT GCA TCG-, which amplify a 572-bp part of the extracellular region of ChT1. ß-Actin was used as a positive control, and H2O was used as a negative control in PCR reactions. After PCR, 15 µl of each sample was run on a 1.2% agarose gel, transferred to a nylon membrane, and hybridized at 65°C overnight in 2x SSC/1% SDS, 10% dextran sulfate, 0.5% pyrophosphate, 0.5 mg/ml ssDNA, and 0.05% low fat powdered milk. Oligonucleotides, V3'-CAG CAC ATT GAC AAT CAC TG- and 4611-TAC CAC AAT GTA CCC TGG C- (35), labeled with [{gamma}-32P]ATP (10 mCi/ml; Amersham) by T4 kinase (Life Technologies, Gaithersburg, MD) were used as probes for ChT1 and ß-actin, respectively. Washes were performed at 65°C in 2x SSC. Radioactive signal was exposed to film.

Embryonic thymus organ culture

For ETOC, thymus lobes were isolated on E10 and cultured for 6 days as previously described (36) in medium that contained purified ChT1 in a concentration of 100 µg/ml specific mAb MUI-83 (dialyzed against RPMI 1640 culture medium). Control cultures lacked mAb, contained an irrelevant, isotype-matched control mAb (10-2.16) that was not reactive with chicken tissues, or contained other chicken thymus- or stromal cell-specific mAb (MUI-36, MUI-53, MUI-54, MUI-70, MUI-71, MUI-78, MUI-82). The results were identical for all control conditions; hence, only the one with isotype-matched control mAb has been included herein. The frequency and cell yield of the various thymocyte subsets present in control vs mAb-treated ETOC were statistically compared using unpaired Student’s t test (*, p < 0.05; {ddagger}, p <= 0.01; §, p <= 0.001).

In vitro thymic stromal cell and thymocyte precursor coculture

In vitro thymic stromal cell and thymocyte precursor coculture was performed as described previously (37). E13 T cell precursors were obtained from ov+ congenic chicks. The thymocyte suspensions were labeled with anti-CD4 and anti-CD8 mAb and then sorted for CD4-CD8- lymphoid cells on a FACStar Plus (Becton Dickinson). The sorted CD4-CD8- cells were prepared at a concentration of 3.4 x 106 cells/ml. Where relevant, purified anti-ChT1 (MUI-83) and anti-c-kit (kit2c75) Abs were added at a concentration of 100 µg/ml. Several control mAbs that were reactive with either chicken thymocytes or stromal cells were also used. Stromal cells from adult chickens (ov-) were prepared by enzymatic digestion of lymphocyte-depleted thymi using 0.15% collagenase/0.1% DNase (Boehringer Mannheim). Stromal cells were enriched by elutriation and resuspended at a concentration of 6.7 x 105 cells/ml (37, 38). T cell precursors were mixed with the stromal cells at a ratio of 5:1 and cocultured as hanging drops in inverted Terasaki plates at 40°C in 10% CO2. Cells were harvested on day 5 and were stained with anti-ov mAb 11A9 followed by staining with anti-CD4 and anti-CD8 mAb. Cells were analyzed by flow cytometry using a FACScan (Becton Dickinson).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue distribution and ontogeny of ChT1

Flow cytometric analysis of adult lymphoid tissues showed that approximately 90% of the thymocytes expressed ChT1; in the spleen 5–20% (n = 12; mean ± SD, 11.3 ± 4.0%) of cells were positive, and in the peripheral blood 5–15% (n = 13; mean ± SD, 8.3 ± 5.3%) of cells were positive (Fig. 1GoA). In the embryo, ChT1+ cells are already present in the thymus on E10 (mean ± SD in three experiments, 4.7 ± 1.9%; data not shown). By E14 expression had reached its adult level, demonstrating that this Ag appears very early on differentiating T-lineage cells (18, 19, 20). ChT1-specific mAb 2-1 and MUI-83 also stained a subpopulation (n = 7; mean ± SD, 4.0 ± 3.2%) of embryonic bone marrow cells (Fig. 1GoB). These cells did not express CD3/TCR complex (data not shown). As expected from the large number of ChT1-expressing cells in the thymus, the Ag was found on thymocytes at all stages of differentiation, defined by the expression of CD4 and CD8 (20). ChT1+ thymocytes and peripheral T cells included {gamma}{delta} T cells as well as both Vß1- and Vß2-expressing {alpha}ß T cells (Fig. 1GoC). Together these results show that ChT1 is highly expressed on most thymocytes and prethymically on a subpopulation of embryonic bone marrow cells as well as on a subset of peripheral T cells.



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  		FIGURE 1. Tissue distribution of ChT1. A, Thymocytes, PBL, and spleen lymphocytes from a young adult chicken were stained with anti-ChT1-specific mAb RR5-89 followed by FITC-conjugated anti-mouse-Ig. The figure shows a representative example of each tissue. The closed histogram shows the expression of ChT1; the open histogram is the negative control for each sample. The percentage of positive cells is shown in the upper right corner and is marked by a vertical bar. B, E13 thymocytes and bone marrow cells were stained with anti-ChT1 mAb 2-1 and were detected by anti-mouse IgG1-FITC. The closed histogram shows the expression of ChT1; the open histogram is the negative control for each sample. The percentage of positive cells is shown in the upper right corner and is marked by a vertical bar. C, Spleen lymphocytes from a young animal were stained with anti-ChT1 mAb RR5-89 and with mAb detecting different TCR subpopulations followed by FITC- and PE-conjugated anti-isotype Ab. The percentage of positive cells in each quadrant is shown in the upper corner.

 
A ChT1-positive population of embryonic bone marrow cells contains T cell progenitors

The E13.5 bone marrow cells can be divided into ChT1+c-kit+ and ChT1-c-kit+ populations (Fig. 2GoA). To determine whether ChT1 is expressed on cells that have a capacity to differentiate into T cells in vivo, we sorted these two cell populations from E13.5 bone marrow of congenic H.B19ov+ animals and injected the cells intrathymically into 14-day-old H.B19ov- recipients. Thymus reconstitution was measured by flow cytometry with the ov-alloantigen-specific mAb 11A9 at 2 wk after injection. A clear thymus reconstitution was obtained by 1000 or even 100 ChT1+ c-kit+ cells with mean chimerism of 13.7 and 6.8%, respectively. In contrast, the injection of ChT1-c-kit+ cells resulted in only 2.9 and 0.9% chimerism, respectively (Table IGo and Fig. 2GoB). The chimeric cells were analyzed for their capacity for thymus reconstitution by the surface expression of CD4, CD8, and TCR. The majority of the donor cells expressed both CD4 and CD8, and only a small fraction of the injected cells had remained at the CD4-CD8- stage. The bone marrow progenitors also developed into CD4 and CD8 single-positive thymocytes (Fig. 2GoC). Differentiated thymocytes expressed both {gamma}{delta}- and {alpha}ß-TCR, indicating that the ChT1+c-kit+ embryonic bone marrow precursors have a capacity to mature along all pathways of T cell development (data not shown).



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  		FIGURE 2. Thymus reconstitution capacity of ChT1+c-kit+ embryonic bone marrow cells. A, E13.5 bone marrow cells from ov+ strain were stained with anti-ChT1 and anti-c-kit mAb. The cells were sorted into ChT1+c-kit+ (region 1) and ChT1-c-kit+ (region 2) populations for intrathymic injections into ov- congenic chickens. B, Two weeks after the intrathymic injection of E13.5 bone marrow cells, the donor cells in the recipient thymi were analyzed by staining with anti-ov-specific mAb 11A9. The histogram shows a representative example of the reconstitution capacity of 1000 ChT1+c-kit+ cells from three experiments. The horizontal bar marks the ov+ population. C, The ov+ cells from B were gated and analyzed for CD4 and CD8 expression to study the T cell differentiation of the injected cells.

 

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  		Table I. Thymus reconstitution capacity of ChT1+c-kit+ and ChT1-c-kit+ cells1

 
ChT1 cDNA cloning and protein characterization

To clone the cDNA encoding the ChT1 Ag, we screened COS cells transiently transfected with an adult RPRL Line 0 thymus cDNA library with various mAb. Clone p10.6 (1114 bp) was isolated with mAb T10A6. The specificity of other mAb (e.g., 2-1, RR5-89, MUI-83, CT1, CT1a) for ChT1 was confirmed by their reactivity with p10.6-transfected COS cells. Another independent clone, pc210 (1087 bp), was isolated with mAb cF3c210 from a cDNA library made from E13 H.B19 thymus mRNA. The complete nucleotide sequence from both strands of these two clones was then determined (Fig. 3Go). The cDNA clones p10.6 and pc210 consist of a 28-bp and an 8-bp, respectively, 5' untranslated region, a 1008-bp open reading frame encoding a 21-aa leader peptide, a 212-aa extracellular region, a 24-aa transmembrane region, and a 78-aa cytoplasmic part followed by a 78-bp and a 71-bp 3' untranslated region that does not contain a poly(A) tail. The coding region sequences of the two clones are identical except for one nucleotide difference in the transmembrane region at position 765, where an A is replaced by a C in the pc210 clone. This changes a charged aspartic acid residue to an aliphatic alanine residue in the deduced protein sequence. The predicted molecular mass of ChT1 is 36.5 kDa.



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  		FIGURE 3. The cDNA sequence of ChT1. The figure shows the cDNA sequence of clone p10.6. Numbers on the left correspond to the position of the first nucleotide, and the numbers on right correspond to the position of the last amino acid in each line. The beginning and end of Ig domains are indicated by < and >, respectively. The cysteines and tryptophans that characterize the Ig domains are in bold. The transmembrane region is underlined. The difference in clone pc210 is marked in the transmembrane region.

 
To further confirm the specificity of mAb and to clarify the start of the mature polypeptide, the ChT1 protein was purified from thymus lysates by an immunoaffinity column using mAb RR5-89. N-terminal microsequencing of the purified protein of 46 kDa resulted in a 30-residue sequence, VVVTVPEKTVNVKTGGxATLLxTYTSSQPL, which completely matched the predicted amino acid sequence. Internal peptides were obtained by partial digestion with an endoproteinase Lys-C. These peptides were sequenced, and two sequences, residues 87–96 (DRITAATSPG) and residues 130–150 (xVIVNVLVKPSKPFxKIEGTP), were assembled. Both internal sequences showed 100% identity to the deduced amino acid sequence and spanned the first half of the extracellular region.

The anti-ChT1-specific mAb immunoprecipitated four bands from a surface-labeled thymocyte lysate as analyzed by SDS-PAGE: a major band of 63 kDa and its putative dimer of 138 kDa as well as minor bands of 46 and 121 kDa, which are probably degradation products of the monomer and putative dimer bands, respectively (Fig. 4Go). A similar pattern was present under reducing, nonreducing, and mild detergent (digitonin) conditions (data not shown). The material that was precipitated with mAb RR5-89 was digested with V8 protease, and SDS-PAGE analysis of the digestion products revealed a similar peptide pattern for each band (data not shown). This suggests that the complex immunoprecipitation pattern is composed of a single polypeptide. The glycosylation of ChT1 was studied by removing N- and O-linked carbohydrates. After treatment with N-glycanase, the core protein of the major band measured about 46 kDa. Additional treatments with neuraminidase and O-glycanase did not change the migration of the protein (data not shown).



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  		FIGURE 4. Immunoprecipitation of ChT1. ChT1 was immunoprecipitated with mAb RR5-89 from the lysate of surface iodinated thymocytes. The precipitated proteins were run on 5–15% SDS-PAGE, which was exposed to an autoradiography film. The m.w. markers are shown on the left.

 
Analysis of the mRNA expression of ChT1

Northern blot analysis with a probe containing the total cDNA of ChT1 revealed two mRNA species, one major band of 4.7 kb and a fainter band of 4.0 kb from the thymus, but no ChT1-specific mRNA was detected in any other organ or cell line tested (Fig. 5GoA and data not shown).



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  		FIGURE 5. Analysis of mRNA expression. A, mRNA of indicated tissues and chicken cell lines was probed with total cDNA of ChT1 on a Northern blot. A PCR product of chicken ß-actin was used as a control probe for the quality of mRNA. The m.w. markers are shown on the left. B, RT-PCR products from total RNA of chicken tissues and cell lines. On the left is the 572-bp PCR product obtained with ChT1-specific primers, in the middle is ChT1 expression after hybridization with an internal ChT1-specific oligonucleotide probe, and on the right is the 1030-bp control PCR product obtained with ß-actin-specific primers. BM, E13 bone marrow.

 
Using a more sensitive RT-PCR method we were also able to show ChT1 mRNA in 4-wk-old chick spleen and E13 bone marrow (Fig. 5GoB). After hybridization of the PCR products with a ChT1-specific oligonucleotide probe, bursa, liver, and small intestine also showed a very low amount of ChT1 mRNA, most likely indicating the presence of a few ChT1+ T-cells in these tissues (Fig. 5GoB).

ChT1 is an IgSF member consisting of two Ig domains

Structural analysis of the deduced ChT1 protein sequence identifies the molecule as a member of the IgSF consisting of two Ig domains: an N-terminal V-like domain and a membrane-proximal C2-like domain. Both represent typical Ig domains, as all cysteines forming intradomain disulfide bridges are conserved in the B and F ß strands, and the tryptophans were conserved at positions 58 and 173 in the C strands. The cysteines at positions 144 and 221 are located in the A and G strands in the C2-like domain, so that during protein tertiary structure formation they may come close enough to form an additional intradomain disulfide bridge. At the end of the V-like domain the sequence AGQSQKSVIVNVLV resembles a J-like segment characterized by features of a diglycine bulge (39, 40) (Fig. 3Go). However, the J-like segment in ChT1 is modified so that the second glycine is replaced by a serine. The extracellular part of ChT1 contains four possible N-glycosylation sites at aa positions 38, 97, 199, and 218 (Fig. 6Go). The putative hydrophobic transmembrane region is followed by a long cytoplasmic tail rich in glutamic acids. The carboxyl-terminal end of the molecule (residues 309–333) probably forms an {alpha}-helical structure. In the cytoplasmic region, Ser265 and Ser266 belong to consensus motif R/KX2S/T for cAMP phosphorylation. Ser266, Ser278, and Ser307 are potential casein kinase II phosphorylation sites (motif SX2E; Fig. 3Go).



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  		FIGURE 6. The alignment of ChT1 and CTX amino acid sequences. Identical amino acids are boxed in black, and the cysteines forming the domain-specific disulfide bridges are marked with gray boxes. Conserved N-glycosylation sites are marked with an asterisk. The leader peptide is marked with a dashed line, and the transmembrane region is indicated with a solid line. The amino acids are aligned according to the Clustal method.

 
ChT1 is homologous to CTX

Comparative sequence analysis indicated that ChT1 is most homologous to the Xenopus thymocyte Ag CTX (41). Overall identity to CTX is 41% (identical amino acids/total amino acids in ChT1), and similarity is 60%. Homologies between the V- and C2-like domains are 48 and 47%, respectively. The transmembrane region shows the highest identity at 54%. In contrast, homology of the cytoplasmic portion is lower, only 23%. All N-glycosylation sites as well as the two extra cysteines in the C2-like domain forming a putative disulfide bridge within the domain are fully conserved between ChT1 and CTX (Fig. 6Go).

Other proteins that were closely related to ChT1 included HCAR/MCAR (24% identity; human and mouse coxsackie and adenovirus receptors) (42), human A33 Ag (23% identity) (43), myelin P0 protein, and Ig heavy and {lambda}-chains of different species. In contrast to the extracellular regions the cytoplasmic part did not show significant homology to any known proteins in the databases.

Anti-ChT1 mAb inhibits thymocyte differentiation

ChT1 is one of the earliest markers expressed during T cell development; therefore, its potential functional role was investigated by the addition of purified anti-ChT1 mAb to ETOC. Chicken ETOC provides a structurally intact thymic microenvironment that allows both lymphoid and stromal cells to develop much as they would in the intact embryo. As for mammals, it thus represents a valid model for functional studies (36, 44). Thymic lobes were carefully removed on E10 and were cultured in the presence (100 µg/ml) of purified anti-ChT1-specific mAb MUI-83, isotype-matched control mAb, or no mAb. Penetration of cultured lobes by MUI-83 and its binding to the target thymocytes was verified by immunohistology and flow cytometry using anti-mouse Ig-FITC (data not shown). In cultures treated with isotype-matched control mAb and a panel of other mAbs against lymphoid and thymic stromal cells, there was no effect on T cell development. The treatment with anti-ChT1 mAb for 6 days, however, had multiple effects on thymopoiesis. It caused a significant (p <= 0.001) decrease in ChT1high cells from >60% in control cultures to approximately 5% (data not shown). The total number of viable thymocytes per lobe was also decreased to 39% of that in control cultures, from 2.6 x 105 to 1 x 105 (p <= 0.001). Such a marked decrease in cell number was reminiscent of mouse fetal thymic organ cultures treated with anti-CD3, which induced apoptosis of immature thymocytes (45). However, electron microscopic examination of MUI-83-treated thymus lobes showed no evidence of extensive cell death, in contrast to lobes that were cultured with either anti-CD3 or ionomycin (data not shown).

The effect of anti-ChT1 on thymocyte subsets, defined by the expression of CD3, CD4, and CD8, was also examined (Table IIGo). The major effect was a block in development downstream from the precursor CD3-CD4-CD8- cells (which were proportionally increased relative to the control cultures), involving markedly reduced CD3-CD4-CD8+ immature intermediates (25% of control cultures), CD3-CD4+CD8+ cells (13% of control cultures), and CD3+CD4+CD8+ cells (31% of control cultures). The effect was not absolute, however, as some phenotypically mature T cells did develop, presumably through positive selection of the low proportion of CD3+CD4+CD8+ cells. One explanation for the incomplete inhibition could have been the difficulty in saturating the ChT1 determinant that is expressed in high levels on the surface of the thymocytes. Flow cytometry of the MUI-83-treated thymocytes with directly conjugated exogenous MUI-83 showed a slight increase in staining over that of anti-mouse Ig-FITC-stained cells, which revealed in situ bound Ab, supporting this possibility (data not shown).


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  		Table II. Effect of anti-ChT1 treatment on ETOC1

 
To investigate the functional role of ChT1 further, we used a recently developed in vitro model for T cell differentiation involving coculture of freshly isolated thymic stromal cells with intrathymic precursor cells (37, 38). In this system mAb can be added directly to the precursor cells from the very onset of coculture, saturating the determinants before contact with the stroma and hence differentiation induction. Sorted CD4-CD8- E13 thymocytes from ov-alloantigen-positive embryos and isolated adult thymic stromal cells (ov-) were cocultured for 4 days in the presence of anti-ChT1, anti-c-kit, no mAb, or a variety of control mAb to thymic stromal or lymphoid molecules. After 4 days the cultures were harvested, the total cell yield was counted, and the expression of CD4 and CD8 was analyzed on ov+ thymocytes by flow cytometry. Treatment with anti-ChT1 was comparable to that with anti-c-kit, so that in both cases the frequency of CD4+CD8+ cells was decreased from 33.7% in control cultures to approximately 3.5 and 3.1%, respectively (Fig. 7Go). A substantial proportion (38.4 and 54.8%, respectively) of double-negative cells partially differentiated to CD4-CD8+, presumably immature thymocytes in the presence of both anti-ChT1 and anti-c-kit mAb, but no CD4+CD8- cells were detectable. The total cell recovery slightly increased in cocultures with either anti-ChT1 or anti-c-kit as a result of most cells staying at an undifferentiated CD4-CD8- or CD4-CD8+ stage (Table IIIGo). There was no alteration numerically or proportionally in the control cocultures. These in vitro data emphasize the functional role of ChT1 during the proximal stages of thymocyte differentiation.



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  		FIGURE 7. T cell differentiation in in vitro thymocyte precursor and stromal cell coculture. E13 thymocyte precursors (sorted CD4-CD8- population from the ov+ congenic strain) were cultured for 4 days with adult thymus stromal cells (from the ov- strain). Anti-ChT1 mAb MUI-83 and anti-c-kit mAb kit2c75 were added to the cultures. After culture the number of ov+ thymocytes was counted, then cells were stained with anti-CD4 and anti-CD8 mAb to analyze T cell differentiation during culture. The percentage of positive cells in each quadrant is shown in the upper corner. The effect of anti-ChT1 mAb is compared with T cell differentiation when no Ab or anti-c-kit mAb was added to the culture. Results shown are representative of three separate experiments.

 

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  		Table III. In vitro coculture of embryonic CD4-CD8- ov+ thymocytes on ov- stromal cells1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Searching for molecules involved in early T cell differentiation, we isolated a cDNA encoding the avian thymocyte Ag ChT1, a member of the IgSF with two extracellular domains, V and C2. The expression of ChT1 on peripheral T cells defines recent thymic emigrants (20). We show here that the ChT1+c-kit+ subpopulation of embryonic bone marrow cells is enriched for T cell progenitors, as assayed by intrathymic cell transfer to congenic animals. Anti-ChT1 mAb also blocks T cell differentiation in vitro in thymic organ cultures and thymocyte precursor cultures on stromal cells. These results demonstrate clearly that ChT1 plays an important role in the early stages of T cell development.

The earliest intrathymic T cell precursors in the mouse have been defined as CD3-CD4-CD8-CD117+CD44+CD25- cells in which the TCR loci are still in germline configuration (8, 46). In addition, several other markers, e.g., Sca-1/2, HSA, Thy-1, and IL-7R, have been used to characterize early T cell precursors either in the thymus or fetal bone marrow (1, 3, 47, 48). However, none of these markers is T-lineage specific. Since ChT1 is the earliest T-lineage marker to be defined to date, being present on thymocytes on E10 of embryogenesis, well before CD4, CD8, and the CD3/TCR complex, and since its expression is developmentally regulated, we explored the functional role of ChT1 in embryonic thymocyte differentiation. In ETOC the treatment with anti-ChT1-specific mAb resulted in a significant decrease in total cell yield. The Ab treatment most significantly perturbed the differentiation step from the CD4-CD8- to the CD4+CD8+ stage. The immature CD3- thymocyte subpopulations were affected to a much larger extent than the CD3+ populations. ChT1 mAb also inhibited T cell differentiation in in vitro coculture of precursors with thymic stromal cells. Thus, these in vitro assays convincingly show that ChT1 has an important function in the early stages of T cell differentiation. A similar effect on T cell development has been reported in reaggregate fetal thymic organ cultures with an anti-E-cadherin-specific mAb that blocks homotypic E-cadherin interactions between T cell precursors and thymic stromal cells (49).

ChT1 was found to be a close homologue to a recently cloned Xenopus thymocyte Ag, CTX (41, 50). The structures of these two molecules are highly conserved, as both are composed of two Ig domains with fully conserved putative N-linked glycosylation sites. In addition to the conserved features of their Ig domain structures, ChT1 and CTX share other interesting characteristics. First, there is a J-like segment with a diglycine bulge at the end of the V domain (39). Most molecules with a J-like segment bind their ligands as dimers and are thought to serve as receptors (39, 40). Although in ChT1 the second glycine of the diglycine bulge is replaced by a serine residue, similar modification has been observed in other molecules, such as human CD28 (51), mouse B cell specific glycoprotein B29 (52), and Xenopus {varsigma} locus light chain (40, 53), without affecting the dimerization function of the motif. Even though CTX does not form dimers spontaneously, it can be induced to dimerize by cross-linking (54). Indeed, earlier reports of ChT1 or of an Ag that most likely is ChT1 have proposed that ChT1 is able to form spontaneous homodimers (18, 55), and our results in this study support this idea. Because the migration of all bands was increased equally after N-deglycosylation, we suggest that the bands are composed of similar proteins and carbohydrates. However, as we were not able to dissociate the proposed dimers by reduction there are other possibilities, in addition to dimerization, to interpret the immunoprecipitation pattern obtained. It may be that the subunits of the protein complex are linked by forces that are not breakable by the reducing agent used. Cross-reactivity of anti-ChT1 mAb with an unrelated protein is still another possibility to produce this result, but since we have used many different anti-ChT1 mAb with identical results this seems to be an unlikely solution. In addition to diglycine bulge, the ChT1 sequence contains an extra cysteine residue, approximately in the B/C loop of the C2-like domain, which could mediate dimerization by forming an intermolecular disulfide bridge. Thus, there is a possibility that ChT1 functions as a lymphocyte-specific receptor during T cell differentiation.

The second common feature with CTX is that the C2-like domain of the ChT1 harbors a conserved extra disulfide bridge, which has been found in the same position in other molecules that are obviously ChT1/CTX relatives, e.g., A33 (43) and HCAR/MCAR (42). Third, the intracellular tail is highly polar, containing several glutamic acid residues. The carboxyl-terminal end of both ChT1 and CTX can be predicted to form an {alpha}-helical structure where the negatively charged amino acids would gather close to one side of the helix and thus possibly interact with other polar intracellular molecules. A similar cytoplasmic tail, rich in glutamic acids, has been found in A33 and HCAR/MCAR. However, even though ChT1 and CTX are clearly homologues, their cytoplasmic regions are quite different. There are several potential serine phosphorylation sites in the ChT1 sequence, none of which is present in CTX. Thus, in addition to a possible extracellular receptor function, ChT1 may function in signal transduction. Determining whether ChT1, CTX, and other products of the ChT1/CTX family of genes mediate similar functions awaits additional experiments in different species.

In addition, to being a T-lineage marker in embryonic thymus, ChT1 expression together with c-kit expression define a prethymic T cell precursor population in embryonic bone marrow. After adoptive intrathymic cell transfer, the ChT1+c-kit+ cell population gave rise to {gamma}{delta} as well as CD4+ and CD8+ mature {alpha}ß T cells. Recently, the existence of a common lymphoid progenitor population, Lin-IL-7R+Thy-1-Sca-1lowc-kitlow, has been described in the mouse (9). Whether the ChT1+c-kit+ bone marrow subpopulation contains T-lineage-restricted, common lymphoid, or multipotent hemopoietic progenitors remains to be clarified. In vivo ChT1 interaction with its putative ligand might result in a maintenance or viability signal that would allow thymic selection events to occur. Interestingly, CTX has been shown to mediate a cellular growth inhibition signal in Xenopus T cell tumors in agreement with the idea that ChT1/CTX is involved in the regulation of cell proliferation (54). According to our data from ETOC and precursor coculture with thymic stromal cells, we suggest that ChT1 is required for thymocyte precursors to develop to the CD4+CD8+ stage; again, this could operate at the level of cell proliferation. It may be that when the cells have matured to the CD3+CD4+CD8+ stage, ChT1 would no longer be crucial for T cell differentiation. However, ChT1 expression would continue for some time after the cell has emigrated from the thymus, thus marking the recent thymic emigrants (20). We hypothesize that the signal received from ChT1 Ag interaction with its ligand is necessary for developing thymocytes to proceed to the stage of positive (and negative) selection. The results presented in this study demonstrate that ChT1 Ag is an early T-lineage-specific marker on embryonic thymus and bone marrow cells and emphasize its importance in the first phases of T cell development.


   Acknowledgments
 
We thank John Young for the RPRL line 0 thymus cDNA library, Riitta Koskinen for her help with cDNA cloning, Natalie Davidson for ETOC subset analysis, Tatsuya Uchida for E13 bone marrow cDNA, Elisabeth Houssaint for the mAb T10A6, and Max D. Cooper and Chen-lo H. Chen for mAb CT1 and CT1a. Ann Sofie Hakulinen, Raija Raulimo, Jean-Pierre Dangy, and Barbara Ecabert provided expert technical assistance, and David Avila performed protein sequencing. We also thank Louis Du Pasquier, Isabelle Chretien, Olli Lassila, and Jan Salomonsen for their critical reading and improvement of the manuscript.


   Footnotes
 
1 This work was supported by the Turku University Foundation, the Academy of Finland, the Human Frontier Science Program (RG 366/96), the Swiss National Science Foundation (Grant 31-49241.96), the National Health and Medical Research Council of Australia, and the Australian Chicken Meat Research and Development Council. The Basel Institute for Immunology was founded and is fully supported by F. Hoffmann-La Roche (Basel, Switzerland). Back

2 The following cDNA sequences have been submitted to the EMBL database with accession numbers Y14063 and Y14064. Back

3 Address correspondence and reprint requests to Dr. Kaisa Katevuo, Turku Immunology Center, Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, 20520 Turku, Finland. E-mail address: Back

4 Abbreviations used in this paper: E, day of embryonic development; IgSF, Ig super family; ETOC, embryonic thymus organ culture; CU20, CU32, and CU36, Marek’s disease virus-transformed MDCC-CU20, MDCC-CU32, and MDCC-CU36 cell lines, respectively; RP13, reticuloendothelial virus-transformed REVCC-RP13 chicken cell line. Back

Received for publication June 22, 1998. Accepted for publication February 26, 1999.


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 Results
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