Eur. J. Immunol. 1992. 22: 1429-1435
Natalie J. Davidson, Chen-lo H. ChenA and Richard L. Boyd Department of Pathology and Immunology, Monash University Medical School, Prahran and Division of Developmental and Clinical ImmunologyA, Department of Pediatrics, Medicine and Microbiology, University of Alabama at Birmingham, Birmingham
Chicken embryonic thymocyte differentiation
1429
Kinetics of chicken embryonic thymocyte development in ovo and in organ culture* Thymocyte development was monitored in an embryonic thymus organ culture system to establish a model in the chicken in which the functional nature of the thymic microenvironment could be assessed. Thymus lobes were removed from 10-day-old embryos and cultured for 2-10 days. Cell yield increased to a maximum in 4-8 days of culture with a corresponding decrease in average cell size. An initial thymocyte population of predominantly CD3-CD4-CDS- cells gave rise to all CD3/CD4/CDS-defined subpopulations in vitro, maintaining high levels of CD3-CD4-CD8+ and CD3+CD4-CD8+ cells and a low representation of CD3-CD4+CD8-, CD3+CD4+CD8-, CD3-CD4+CDS+ and CD3+CD4+CDS+ thymocytes. This is the first observation of a CD3-CD4TDS- population in the chicken. Developmental kinetics of CD3+ cells were similar to that in the embryo, suggesting that the in vitro environment is sufficient to promote and maintain thymocyte maturation. Thymocytes of both the y6 and aP T cell receptor (TcR) lineages developed in that order, confirming in ovo data and the lineage potential of the first wave of thymocyte precursors. One unusual finding was a relative accumulation of y6 TcR+ thymocytes in culture, incorporating all CD4/CD8 subsets, including a previously undetected population, CD4+CD8-. This may indicate a favorable developmental environment or simply a lack of normal cellular emigration. A detailed comparison with T cell development in the embryo demonstrated that the chicken thymus organ culture system reflects thymic events in ovo during a limited time period and thus should prove useful in the identification of functionally relevant thymic molecules.
1 Introduction The thymus provides an environment conducive to thymocyte proliferation and differentiation, an essential feature of which are stromal cell-thymocyte interactions that determine selection processes which shape the TcR repertoire (reviewed in [ l , 2]).To understand thymopoiesis fully requires the identification of cell types and molecules participating in lymphostromal interactions. One approach has involved a system of fetal thymus organ culture (FTOC) which has been successfully used to investigate early events in murine Tcell differentiation (reviewed in [3]). In addition, the effects of modulating the thymic environment by addition of cytokines [4] or mAb, directed towards stromal or lymphoid determinants, to FTOC have been examined (reviewed in [3]) to decipher the mechanisms of thymocyte development. Chicken embryonic thymus organ culture (ETOC) has previously been attempted although, due to the then lack of fully characterized avian T cell markers, the model was unable to be utilized to its full potential [5-71. [I 95561
* This work was supported by grants from the Australian National Health and Medical Research Council (860396), the AntiCancer Council of Victoria (131658), the Monash University Special Research Grant Scheme and National Institute of Health (USA) grants CA13148 and AI30879. Correspondence: Natalie J. Davidson, Department of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, Victoria 3181, Australia Abbreviations: E: Embryonic age (days) ETOC: Embryonic thymus organ culture (chicken) FTOC: Fetal thymus organ culture (mouse)
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
The recent development of mAb specific for chicken T cell differentiation antigens (homologous to mammalian CD2, CD5, CD3, CD4, CD8 and both aP and y6 TcR; reviewed in [S, 91) has facilitated a more detailed assessment of avian thymopoiesis than previously possible, revealing a marked degree of conservation in thymocyte ontogeny between the avian and mammalian classes [8-101. Furthermore, mAb raised against thymic stromal elements, including MHC class I1 reagents [ll-131, indicate a striking similarity in thymic microanatomy. These mAb have also defined novel stromal molecules, not yet described in mammals, specific for the epithelium lining the subcapsule, subtrabeculae and perivascular regions [ l l ] . Of particular interest, these determinants are selectively deficient in L200 chickens which develop autoimmune scleroderma [141. This study establishes the chicken ETOC system, by comparison with in ovo development, as a valid model with which to investigate the functional nature of the thymic microenvironment with thymocytes of both y6 and af3 TcR sublineages developing, including all subsets defined by CD3, CD4 and CDS. Furthermore, by modulating stromal antigen expression, particularly that of non-MHC antigens, through addition of mAb to ETOC, stromal subsets directly associated with distinct phases of thymocyte differentiation may be identified.
2 Materials and methods 2.1 Chickens
Australorp/White Leghorn F1 hybrid embryos and chickens were obtained from Research Poultry Farm (Research, Australia). Eggs were maintained in a humidified incubator 0014-2980/92/0606-1429$3.50+ .25/0
1430
N. J. Davidson, C. H. Chen and R. L. Boyd
at 39°C and embryonic ages estimated by the duration of incubation.
2.2 ETOC Individual thymic lobes were isolated from 10-day-old embryos (10 E), under sterile conditions, and placed on 0.45-pm polysulfone filter membranes (Gelman Sciences, Ann Arbor, MI; 5-8 lobes per filter) supported by blocks of gelatin sponge (Upjohn Co., Kalamazoo, MI), based on the method developed in the murine system [15]. Lobes were cultured in 9-cm plastic petri dishes containing 10 ml RPMI 1640 tissue culture medium (supplemented with 10 % heat-inactivated FCS and 2 mM L-glutamine) for 2-10 days in a humidified, 42°C incubator at 10% CO2-in-air. Medium was replaced after 6-day culture. In some cases, medium was supplemented with fresh, heat inactivated (56 “C) chicken serum, collected by wing-vein bleeding.
Eur. J. Immunol. 1992. 22: 1429-1435
single labeling with culture supernatant mAb, revealed using DDAF or B-conjugated mAb revealed with Tandem. All incubations were of 20-min duration on ice and cells were washed (500 x g, 5 min) in cold PBS/FCS. Background-staining levels were determined using cells incubated with an irrelevant, isotype-matched mAb or second stage reagents alone. FCM was performed using a FACScan (Becton Dickinson, Mountain View, CA) equipped with a single argon laser emitting at 488 nm. FITC, PE and Tandem emission was detected at 530, 585 and 650nm, respectively, using logarithmic amplification. Dead cells were excluded on the basis of fonvard- and side-light scatter measurements, made with linear amplification. Data from 5 x lo4 or 1.5 X lo4 cells were acquired for multi- and single-color labeling, respectively, and analyzed using Lysys I software (Becton Dickinson). An unpaired Student’s t-test was used to determine statistical significance.
3 Results
2.3 Preparation of cell suspensions
3.1 Cell growth in chicken embryonic thymus
Suspensions of cultured thymocytes were prepared by mechanically disrupting the thymus by gently pressing under a glass coverslip in 1 ml cold PBS with 1% FCS and 0.02% NaN3 (PBS/FCS) in a small petri dish. Adult and embryonic thymocyte suspensions were prepared by pushing the tissue through a stainless steel sieve (pore size 200 pm) using a syringe plunger, A single-cell suspension was obtained by passage through fine nylon mesh (pore size 100 pm). Cell counts were determined using a hemocytometer and viability by acridine orange/ethidium bromide exclusion.
Embryonic thymi ranging from 9-12E were compared for suitability in ETOC. Thymus at 10E was the most appropriate; 9E lobes were not yet entirely separate structures, the chicken thymus developing initially as an epithelial cord [17], and thymi from embryos older than 10E reached a critical size more rapidly in vitro, with necrosis evident by 8 days.
2.4 Antibodies and conjugates Reagents included FITC-conjugated anti-CD3 , biotin (B)conjugated anti-CD4, PE-conjugated anti-CD8, TcRl culture supernatant (recognizing yS TcR), B-conjugated TcR2 (a@TcR) and PE-conjugated TcR3 (subfamily a@TcR) (reviewed in [9, 101). Biotinylated antibodies were revealed with Tandem (conjugate of Texas Red and PE coupled to streptavidin, Southern Biotechnology, Birmingham, AL), while an FITC-conjugated F(ab’)2 fragment of affinity-purified sheep anti-mouse (DDAF, Silenus Laboratories, Melbourne, Australia) was employed to reveal supernatant-derived mAb.
Thymus lobes contained approximately 3 x lo4 cells at 10E, which rapidly proliferated, increasing to an average of 2 x lo5 cells per lobe by 6-day ETOC (Fig. 1) when maximal lobe size was achieved, although statistical analysis indicated that there was no significant difference in cell yields from 4-8-day ETOC. By 10-12-day ETOC, lymphoid cell number had decreased dramatically and in some lobes centrally located necrotic regions were evident in
w m
0
2.5 Cell surface staining and multiparameter FCM FCM was based on a protocol described elsewhere [16]. Briefly, thymocytes were labeled simultaneously for detection of CD3, CD4 and CD8 antigens using a two-step protocol: (a) anti-CD3-FITC, anti-CD4-B and anti-CD8PE followed by (b) Tandem. Labeling for yS TcR, CD4 and CD8 antigens involved a five-step protocol: (a) TcRl supernatant; (b) DDAF; (c) a blocking step using normal mouse serum; (d) anti-CD4-B and anti-CD8-PE and (e) Tandem. All mAb and conjugates were previously titrated to give appropriate profiles on adult thymocytes, which were also routinely used to control for labeling efficiency. An indirect immunofluorescence technique was used for
T 10
12
14
16
18
20
INCUBATION TIME (DAYS)@)
Figurel. Viable lymphoid cell yield per lobe for 10E thymus cultured 0-10 days (W) and embryonic thymus (0).Results are presented as mean f SD of 4-20 independent experiments. (a) Horizontal axis refers to embryonic age beginning at 10E,while for cultured thymocytes values correspond to 0-, 2-, 4-, 6-, 8-, 10-day ETOC. Asterisks refer to statistical significance of the comparison between percentages of cultured and embryonic thymocytes, * p 10.05,**p 10.01, ***p 50.001.
Chicken embryonic thymocyte differentiation
Eur. J. Immunol. 1992. 22: 1429-1435
I
*I
CD4+CDS-
1431
CD4+CDS+
35 ;32;33
I35 ! s o ! 5
CD4-CDS140 IW
it
14
CD8
C
ih
I
FORWARD LIGHT SCATTER
Figure 2. The forward light-scatter profiles of thymocytes at (a) 10E; (b) 14E (......) and 4-day cultured thyrnocytes (-); and (c) 18E (.....) and 8-day cultured cells (-) demonstrating a decrease in average cell size with increasing developmental age.
CD3 Figure 3. Three-color irnmunofluorescence analysis of adult (6-8 weeks) thymocytes. Cell were simultaneously labeled for the CD3, CD4 and CD8 antigens as described in Sect. 2.5.The lower panel shows total thymocyte CD3 fluorescence and that for CD4/CD8 populations in lefthight panels.Vertica1 scales cannot be compared as the scale was modified to allow visualization of small populations. The data, obtained by analysis of 5 X lo4 cells in a single sample, represent 25 independent experiments. Numerical values indicate the percentages from one representative experiment.
3.3 Chicken embryonic thymocyte development in vitro and in ovo
tissue sections (data not shown). Comparison with a chronologically equivalent thymus indicated that cell growth in culture was significantly slower than that in ovo (Fig. 1). Contrary to a previous report [7], chicken serum was not required for lymphoid development in ETOC. Addition of chicken serum to the culture medium increased cell yield in some cases, but to highly variable degrees, and only when added in conjunction with FCS; hence it was omitted from this experimental system. Developing thymocytes showed a decrease in relative cell size during the culture period (Fig. 2). An initial population of predominantly large cells developed into a heterogeneous population of varying cell size at 4-day ETOC. By 8-day ETOC the lobes consisted primarily of small thymocytes. This reflected the trend in the embryo on comparison with thymocytes taken at 14 and 18E. 3.2 Expression of CD3, CD4 and CD8 on adult
thymocytes Three-color labeling of adult (6-8 weeks) thymocyte suspensions for CD3, CD4 and CD8 antigens established a standard profile for this strain of chicken (Fig. 3). Thymocytes could be subdivided by expression of CD3 into negative, low and high staining subsets, being reflected in the CD4+CD8+,CD4+CD8- and CD4-CD8+ populations, while the CD4-CD8- cells were CD3- or CD3hi. CD3-CD4+CD8- and CD3-CD4-CD8+ cells were evident with, to our knowledge, only the latter previously described in the chicken.
Thymus lobes excised at 10E were maintained in culture for 0-10 days and analyzed by multicolor FCM, demonstrating the development of CD3, CD4 and CD8-defined subsets in a manner similar to embryonic development. At the onset of culture, 10E, the majority of cells (90%) were CD3-CD4-CD8- and lacked CD3/TcR expression (Figs. 4 and 5). Precursor phenotypes: CD3-CD4+CD8-, CD3-CD4-CDP and CD3-CD4+CD8+ [16, 18-29] were evident, the corresponding CD3+ mature phenotypes being barely detectable (
Natalie J. Davidson, Chen-lo H. ChenA and Richard L. Boyd Department of Pathology and Immunology, Monash University Medical School, Prahran and Division of Developmental and Clinical ImmunologyA, Department of Pediatrics, Medicine and Microbiology, University of Alabama at Birmingham, Birmingham
Chicken embryonic thymocyte differentiation
1429
Kinetics of chicken embryonic thymocyte development in ovo and in organ culture* Thymocyte development was monitored in an embryonic thymus organ culture system to establish a model in the chicken in which the functional nature of the thymic microenvironment could be assessed. Thymus lobes were removed from 10-day-old embryos and cultured for 2-10 days. Cell yield increased to a maximum in 4-8 days of culture with a corresponding decrease in average cell size. An initial thymocyte population of predominantly CD3-CD4-CDS- cells gave rise to all CD3/CD4/CDS-defined subpopulations in vitro, maintaining high levels of CD3-CD4-CD8+ and CD3+CD4-CD8+ cells and a low representation of CD3-CD4+CD8-, CD3+CD4+CD8-, CD3-CD4+CDS+ and CD3+CD4+CDS+ thymocytes. This is the first observation of a CD3-CD4TDS- population in the chicken. Developmental kinetics of CD3+ cells were similar to that in the embryo, suggesting that the in vitro environment is sufficient to promote and maintain thymocyte maturation. Thymocytes of both the y6 and aP T cell receptor (TcR) lineages developed in that order, confirming in ovo data and the lineage potential of the first wave of thymocyte precursors. One unusual finding was a relative accumulation of y6 TcR+ thymocytes in culture, incorporating all CD4/CD8 subsets, including a previously undetected population, CD4+CD8-. This may indicate a favorable developmental environment or simply a lack of normal cellular emigration. A detailed comparison with T cell development in the embryo demonstrated that the chicken thymus organ culture system reflects thymic events in ovo during a limited time period and thus should prove useful in the identification of functionally relevant thymic molecules.
1 Introduction The thymus provides an environment conducive to thymocyte proliferation and differentiation, an essential feature of which are stromal cell-thymocyte interactions that determine selection processes which shape the TcR repertoire (reviewed in [ l , 2]).To understand thymopoiesis fully requires the identification of cell types and molecules participating in lymphostromal interactions. One approach has involved a system of fetal thymus organ culture (FTOC) which has been successfully used to investigate early events in murine Tcell differentiation (reviewed in [3]). In addition, the effects of modulating the thymic environment by addition of cytokines [4] or mAb, directed towards stromal or lymphoid determinants, to FTOC have been examined (reviewed in [3]) to decipher the mechanisms of thymocyte development. Chicken embryonic thymus organ culture (ETOC) has previously been attempted although, due to the then lack of fully characterized avian T cell markers, the model was unable to be utilized to its full potential [5-71. [I 95561
* This work was supported by grants from the Australian National Health and Medical Research Council (860396), the AntiCancer Council of Victoria (131658), the Monash University Special Research Grant Scheme and National Institute of Health (USA) grants CA13148 and AI30879. Correspondence: Natalie J. Davidson, Department of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, Victoria 3181, Australia Abbreviations: E: Embryonic age (days) ETOC: Embryonic thymus organ culture (chicken) FTOC: Fetal thymus organ culture (mouse)
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
The recent development of mAb specific for chicken T cell differentiation antigens (homologous to mammalian CD2, CD5, CD3, CD4, CD8 and both aP and y6 TcR; reviewed in [S, 91) has facilitated a more detailed assessment of avian thymopoiesis than previously possible, revealing a marked degree of conservation in thymocyte ontogeny between the avian and mammalian classes [8-101. Furthermore, mAb raised against thymic stromal elements, including MHC class I1 reagents [ll-131, indicate a striking similarity in thymic microanatomy. These mAb have also defined novel stromal molecules, not yet described in mammals, specific for the epithelium lining the subcapsule, subtrabeculae and perivascular regions [ l l ] . Of particular interest, these determinants are selectively deficient in L200 chickens which develop autoimmune scleroderma [141. This study establishes the chicken ETOC system, by comparison with in ovo development, as a valid model with which to investigate the functional nature of the thymic microenvironment with thymocytes of both y6 and af3 TcR sublineages developing, including all subsets defined by CD3, CD4 and CDS. Furthermore, by modulating stromal antigen expression, particularly that of non-MHC antigens, through addition of mAb to ETOC, stromal subsets directly associated with distinct phases of thymocyte differentiation may be identified.
2 Materials and methods 2.1 Chickens
Australorp/White Leghorn F1 hybrid embryos and chickens were obtained from Research Poultry Farm (Research, Australia). Eggs were maintained in a humidified incubator 0014-2980/92/0606-1429$3.50+ .25/0
1430
N. J. Davidson, C. H. Chen and R. L. Boyd
at 39°C and embryonic ages estimated by the duration of incubation.
2.2 ETOC Individual thymic lobes were isolated from 10-day-old embryos (10 E), under sterile conditions, and placed on 0.45-pm polysulfone filter membranes (Gelman Sciences, Ann Arbor, MI; 5-8 lobes per filter) supported by blocks of gelatin sponge (Upjohn Co., Kalamazoo, MI), based on the method developed in the murine system [15]. Lobes were cultured in 9-cm plastic petri dishes containing 10 ml RPMI 1640 tissue culture medium (supplemented with 10 % heat-inactivated FCS and 2 mM L-glutamine) for 2-10 days in a humidified, 42°C incubator at 10% CO2-in-air. Medium was replaced after 6-day culture. In some cases, medium was supplemented with fresh, heat inactivated (56 “C) chicken serum, collected by wing-vein bleeding.
Eur. J. Immunol. 1992. 22: 1429-1435
single labeling with culture supernatant mAb, revealed using DDAF or B-conjugated mAb revealed with Tandem. All incubations were of 20-min duration on ice and cells were washed (500 x g, 5 min) in cold PBS/FCS. Background-staining levels were determined using cells incubated with an irrelevant, isotype-matched mAb or second stage reagents alone. FCM was performed using a FACScan (Becton Dickinson, Mountain View, CA) equipped with a single argon laser emitting at 488 nm. FITC, PE and Tandem emission was detected at 530, 585 and 650nm, respectively, using logarithmic amplification. Dead cells were excluded on the basis of fonvard- and side-light scatter measurements, made with linear amplification. Data from 5 x lo4 or 1.5 X lo4 cells were acquired for multi- and single-color labeling, respectively, and analyzed using Lysys I software (Becton Dickinson). An unpaired Student’s t-test was used to determine statistical significance.
3 Results
2.3 Preparation of cell suspensions
3.1 Cell growth in chicken embryonic thymus
Suspensions of cultured thymocytes were prepared by mechanically disrupting the thymus by gently pressing under a glass coverslip in 1 ml cold PBS with 1% FCS and 0.02% NaN3 (PBS/FCS) in a small petri dish. Adult and embryonic thymocyte suspensions were prepared by pushing the tissue through a stainless steel sieve (pore size 200 pm) using a syringe plunger, A single-cell suspension was obtained by passage through fine nylon mesh (pore size 100 pm). Cell counts were determined using a hemocytometer and viability by acridine orange/ethidium bromide exclusion.
Embryonic thymi ranging from 9-12E were compared for suitability in ETOC. Thymus at 10E was the most appropriate; 9E lobes were not yet entirely separate structures, the chicken thymus developing initially as an epithelial cord [17], and thymi from embryos older than 10E reached a critical size more rapidly in vitro, with necrosis evident by 8 days.
2.4 Antibodies and conjugates Reagents included FITC-conjugated anti-CD3 , biotin (B)conjugated anti-CD4, PE-conjugated anti-CD8, TcRl culture supernatant (recognizing yS TcR), B-conjugated TcR2 (a@TcR) and PE-conjugated TcR3 (subfamily a@TcR) (reviewed in [9, 101). Biotinylated antibodies were revealed with Tandem (conjugate of Texas Red and PE coupled to streptavidin, Southern Biotechnology, Birmingham, AL), while an FITC-conjugated F(ab’)2 fragment of affinity-purified sheep anti-mouse (DDAF, Silenus Laboratories, Melbourne, Australia) was employed to reveal supernatant-derived mAb.
Thymus lobes contained approximately 3 x lo4 cells at 10E, which rapidly proliferated, increasing to an average of 2 x lo5 cells per lobe by 6-day ETOC (Fig. 1) when maximal lobe size was achieved, although statistical analysis indicated that there was no significant difference in cell yields from 4-8-day ETOC. By 10-12-day ETOC, lymphoid cell number had decreased dramatically and in some lobes centrally located necrotic regions were evident in
w m
0
2.5 Cell surface staining and multiparameter FCM FCM was based on a protocol described elsewhere [16]. Briefly, thymocytes were labeled simultaneously for detection of CD3, CD4 and CD8 antigens using a two-step protocol: (a) anti-CD3-FITC, anti-CD4-B and anti-CD8PE followed by (b) Tandem. Labeling for yS TcR, CD4 and CD8 antigens involved a five-step protocol: (a) TcRl supernatant; (b) DDAF; (c) a blocking step using normal mouse serum; (d) anti-CD4-B and anti-CD8-PE and (e) Tandem. All mAb and conjugates were previously titrated to give appropriate profiles on adult thymocytes, which were also routinely used to control for labeling efficiency. An indirect immunofluorescence technique was used for
T 10
12
14
16
18
20
INCUBATION TIME (DAYS)@)
Figurel. Viable lymphoid cell yield per lobe for 10E thymus cultured 0-10 days (W) and embryonic thymus (0).Results are presented as mean f SD of 4-20 independent experiments. (a) Horizontal axis refers to embryonic age beginning at 10E,while for cultured thymocytes values correspond to 0-, 2-, 4-, 6-, 8-, 10-day ETOC. Asterisks refer to statistical significance of the comparison between percentages of cultured and embryonic thymocytes, * p 10.05,**p 10.01, ***p 50.001.
Chicken embryonic thymocyte differentiation
Eur. J. Immunol. 1992. 22: 1429-1435
I
*I
CD4+CDS-
1431
CD4+CDS+
35 ;32;33
I35 ! s o ! 5
CD4-CDS140 IW
it
14
CD8
C
ih
I
FORWARD LIGHT SCATTER
Figure 2. The forward light-scatter profiles of thymocytes at (a) 10E; (b) 14E (......) and 4-day cultured thyrnocytes (-); and (c) 18E (.....) and 8-day cultured cells (-) demonstrating a decrease in average cell size with increasing developmental age.
CD3 Figure 3. Three-color irnmunofluorescence analysis of adult (6-8 weeks) thymocytes. Cell were simultaneously labeled for the CD3, CD4 and CD8 antigens as described in Sect. 2.5.The lower panel shows total thymocyte CD3 fluorescence and that for CD4/CD8 populations in lefthight panels.Vertica1 scales cannot be compared as the scale was modified to allow visualization of small populations. The data, obtained by analysis of 5 X lo4 cells in a single sample, represent 25 independent experiments. Numerical values indicate the percentages from one representative experiment.
3.3 Chicken embryonic thymocyte development in vitro and in ovo
tissue sections (data not shown). Comparison with a chronologically equivalent thymus indicated that cell growth in culture was significantly slower than that in ovo (Fig. 1). Contrary to a previous report [7], chicken serum was not required for lymphoid development in ETOC. Addition of chicken serum to the culture medium increased cell yield in some cases, but to highly variable degrees, and only when added in conjunction with FCS; hence it was omitted from this experimental system. Developing thymocytes showed a decrease in relative cell size during the culture period (Fig. 2). An initial population of predominantly large cells developed into a heterogeneous population of varying cell size at 4-day ETOC. By 8-day ETOC the lobes consisted primarily of small thymocytes. This reflected the trend in the embryo on comparison with thymocytes taken at 14 and 18E. 3.2 Expression of CD3, CD4 and CD8 on adult
thymocytes Three-color labeling of adult (6-8 weeks) thymocyte suspensions for CD3, CD4 and CD8 antigens established a standard profile for this strain of chicken (Fig. 3). Thymocytes could be subdivided by expression of CD3 into negative, low and high staining subsets, being reflected in the CD4+CD8+,CD4+CD8- and CD4-CD8+ populations, while the CD4-CD8- cells were CD3- or CD3hi. CD3-CD4+CD8- and CD3-CD4-CD8+ cells were evident with, to our knowledge, only the latter previously described in the chicken.
Thymus lobes excised at 10E were maintained in culture for 0-10 days and analyzed by multicolor FCM, demonstrating the development of CD3, CD4 and CD8-defined subsets in a manner similar to embryonic development. At the onset of culture, 10E, the majority of cells (90%) were CD3-CD4-CD8- and lacked CD3/TcR expression (Figs. 4 and 5). Precursor phenotypes: CD3-CD4+CD8-, CD3-CD4-CDP and CD3-CD4+CD8+ [16, 18-29] were evident, the corresponding CD3+ mature phenotypes being barely detectable (
