Immunology 1978 34 9
Lymphoid antigenic determinants of the chicken CELLULAR REPRESENTATION AND TISSUE LOCALIZATION
R. L. BOYD & H. A. WARD Department of Pathology and Immunology, Monash Medical School, Melbourne, Victoria, Australia, 3181
Received 14 March 1977; accepted for publication 12 April 1977
Summary. Antisera were prepared against a variety of chicken tissues with a view to detecting antigens specific for subpopulations of T and B cells at different maturation stages. By appropriate absorption analysis, the following antigens were defined: (a) thymus organ-specific antigen (CTOA); (b) T lymphocyte-specific antigen (CTLA); (c) B lymphocyte-specific antigen (CBLA); (d) mature B lymphocyte-specific antigen (CMBLA); (e) a foetalassociated antigen (CFAA) present on embryonic haemopoietic cells, and adult bone marrow and immature bursa cells, suggesting the cell types concerned may be at an early stage of development, possibly including precursors. For comparison with the above antigens, cells were also examined for surface Ig and IgG. T cells were found in periarteriolar sheaths in the spleen, and predominantly in the sub-epithelium and sub-mucosa of the caecal tonsil of the gut-associated lymphoid tissue (GALT). B cells were localized in periellipsoidal sheaths and germinal centres in the spleen, and in primary follicles or germinal centres in the GALT. The thymic and bursal medullas contained the mature populations of T and B lymphocytes respectively.
INTRODUCTION
Specific antigens on T and B lymphocytes have been identified by use of hetero- or alloantisera in a number of mammalian species (review: Greaves, Owen & Raff, 1973) and also in chickens (Forget, Potworowski, Richer & Borduas, 1970; McArthur, Chapman & Thorbecke, 1971; Ivanyi & Lydyard, 1972; Malchow, Droege & Strominger, 1972; Potworowski, 1972; Hudson & Roitt, 1973; Wick, Albini & Milgrom, 1973). In mice, the use of alloantisera has enabled identification of several stages in T cell development (review: Schlesinger, 1972), but in other species such studies have been limited by the lack of readily available, highly inbred strains. Alterations in the class of membrane Ig have been used for studying the differentiation of B cells (e.g. Kincade & Cooper, 1971; Albini & Wick, 1973; Pierce, Asofsky & Solliday, 1973) but this determinant may not be specific for B lymphocytes (Bankhurst, Warner & Sprent, 1971; Marchalonis, Cone & Atwell, 1972; Santana, Wedderburn & Turk, 1974). Because of their easier preparation, heteroantisera greatly facilitate the study of T and B lymphocyte developmental pathways. The chicken, with a clearly defined dichotomy of the immune system, is invaluable for such studies. As the basis for studying T and B cell differentiation we prepared rabbit antisera to various chicken lymphoid cells in order to define antigens specific for
Correspondence: Dr H.A. Ward, Department of Pathology and Immunology, Monash Medical School, Melbourne, Victoria, Australia 3181. Dr R.L. Boyd present address: Institut Fur Allgemeine und Experimentelle Pathologie, Universitat Innsbruck, Austria.
9
10
R. L. Boyd & H. A. Ward
different steps in lymphocyte pathways, and for the lymphoid micro-environment, particularly in the bursa. One aspect of the latter antigens, the specificity of the cortical reticulin framework of the bursa, has been reported (Boyd, Ward & Muller, 1976). In this paper we present data on the cellular representation and tissue localization of lymphoid antigenic determinants in the chicken.
MATERIALS AND METHODS Cell suspensions Tissues were obtained from freshly killed 3-12week-old, or embryonic, Australorp/White Leghorn F1 hybrids (Musgrove Chicks Pty. Ltd., Croydon, Australia), immediately teased out, and the cells washed and resuspended in Hanks' balanced salt solution, pH 7-6 (HBSS). Blood leucocytes were separated from a 1: 1 dilution of whole blood in Alsever's solution by collection of the buffy coat after centrifuging at 170 gay for 5 min. The buffy coat cells were resuspended in HBSS and the leucocytes purified by four sequential centrifugations at 170 gay for 5 min. Cell viability (trypan blue exclusion) was in excess of 90 % for all suspensions. In experiments requiring purified lymphocyte populations, phagocytic cells were removed from the suspensions by incubation of 108 cells in 5 ml HBSS with 0 3 g carbonyl iron (B.D.H.) for 1 h at 370 with occasional shaking. Non-phagocytic cells were decanted, phagocytes being retained by a magnet. Such a procedure completely removed cells capable of non-specifically binding aggregates of fluorescent
conjugates. Tissue sections
Freshly removed tissues were rinsed in HBSS, snap-frozen in a liquid nitrogen-isopentane slurry at approximately - 1600 and stored at -70°. Cryostat sections (6,pm) were mounted on chemically cleaned slides and air-dried at 40 for a minimum of 2 h. Alteration of immune status For stimulation of the lymphoid system, adult birds were injected i.p. with 3 ml heat-killed Brucella abortus suspension (Diagnostic Agent, Batch No. 612-1, Commonwealth Serum Laboratories, Melbourne, Australia) on days 1 and 14, and killed 7 days later. The agglutinating antibody titre of the resultant serum was 1: 128. Production of antisera Pre-immune sera for use in control preparations
were collected from rabbits 1-3 days before immuni-
zation. Rabbit anti-chicken sera were produced using the 'two-pulse' method (Levey & Medawar, 1966). Suspensions of 5 x 108 to 109 adult thymus or bursa cells or 6 day embryonic yolk sac cells were injected i.v. on days 1 and 14 and the rabbits bled out 1 week later. The blood was held at 370 for 1 h and then at 40 overnight. Serum was separated, centrifuged at 900 gay for 10 min, incubated at 56° for 30 min to inactivate complement and stored in aliquots at -200.
Immunological testing of antisera Serum or bile Ig were separated by precipitation with 40% saturated ammonium sulphate. Glutaraldehyde-insolubilized Ig for immunoadsorption was prepared according to Avrameas & Ternynck (1969). Unless otherwise stated, serum absorptions were performed according to Nairn (1976) with adult chicken tissues. All sera were absorbed initially with red blood cells to remove any haemagglutinating activity. Conjugates Goat anti-rabbit globulin was prepared using the procedure of Ironside (1968); the y-globulin fraction was conjugated with fluorescein isothiocyanate (Nairn, 1976)-fluorescein to protein molar ratio 4-8 before absorption-and was used at a protein concentration of approximately 0-66 mg/ml. After appropriate absorption, it gave no staining of fowl tissues by itself. y-globulin fractions of rabbit antisera to chicken Ig and IgG (Pentex, Kankakee, U.S.A.) were conjugated with fluorescein isothiocyanate; the conjugates had fluorochrome to protein molar ratios, before absorption, of 5 2 and 4-1 respectively, and were used at a protein concentration of approximately 0-35 mg/ml. After absorption with chicken kidney and thymus homogenates and thymus cell suspensions, these two conjugates showed specificity for Ig or IgG, as demonstrated by immuno-electrophoresis.
Immunofluorescence Standard membrane and section immunofluorescence tests with specific antisera were as described by Nairn (1976). Parallel control preparations were treated with phosphate buffered saline, pH 7-2, HBSS or pre-immune rabbit serum. Specimens were examined by narrow-band blue or darkground
Chicken lymphoid antigens ultraviolet fluorescence microscopy using a yellow or colourless barrier filter respectively (Nairn, 1976). Cell suspension counts of positive cells were performed on a minimum of 200 cells per slide; each result is the mean from at least three experiments, in each of which cells from a minimum of two birds were pooled. The antiserum titres against various cell types were taken as the highest serum dilution giving positive staining in suspensions of 6x 106 cells. Tissue section staining was done in triplicate
using different frozen blocks each time.
11
100F
U-
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0
an0_ CA
404 201 ". \
4
RESULTS Anti-thymus
serum
(i) Chicken T-lymphocyte specific antigen (CTLA). After nine absorptions of the initial anti-thymus serum with bursa cells, this antiserum stained by membrane immunofluorescence (Table 1) approximately 93% of thymus, 61 % of spleen, 46% of blood leucocytes, 7 % of bone marrow and less than 1 % of adult bursa cells in suspension. Three absorptions of the original anti-thymus serum with thymus cells completely neutralized the activity. Although anti-CTLA membrane immunofluorescence staining intensities of thymus and peripheral T lymphocytes were similar, the titres for these populations differed (Fig. 1). Splenic T cells gave a titre of 1: 32 but for thymocytes, dilutions above 1: 32 still stained approximately 40 % of the cells, suggesting the presence of two populations of thymus cells, with high or low CTLA concentration. On tissue sections the anti-CTLA serum showed Table 1. Organ distribution of lymphocyte antigens (% positive cells)
Organ Antigen
Thymus
CTLA CTOA CBLA CMBLA CFAA Ig IgG
93 79 5 1 0-25 6 1
Bursa
Lymphoid antigenic determinants of the chicken CELLULAR REPRESENTATION AND TISSUE LOCALIZATION
R. L. BOYD & H. A. WARD Department of Pathology and Immunology, Monash Medical School, Melbourne, Victoria, Australia, 3181
Received 14 March 1977; accepted for publication 12 April 1977
Summary. Antisera were prepared against a variety of chicken tissues with a view to detecting antigens specific for subpopulations of T and B cells at different maturation stages. By appropriate absorption analysis, the following antigens were defined: (a) thymus organ-specific antigen (CTOA); (b) T lymphocyte-specific antigen (CTLA); (c) B lymphocyte-specific antigen (CBLA); (d) mature B lymphocyte-specific antigen (CMBLA); (e) a foetalassociated antigen (CFAA) present on embryonic haemopoietic cells, and adult bone marrow and immature bursa cells, suggesting the cell types concerned may be at an early stage of development, possibly including precursors. For comparison with the above antigens, cells were also examined for surface Ig and IgG. T cells were found in periarteriolar sheaths in the spleen, and predominantly in the sub-epithelium and sub-mucosa of the caecal tonsil of the gut-associated lymphoid tissue (GALT). B cells were localized in periellipsoidal sheaths and germinal centres in the spleen, and in primary follicles or germinal centres in the GALT. The thymic and bursal medullas contained the mature populations of T and B lymphocytes respectively.
INTRODUCTION
Specific antigens on T and B lymphocytes have been identified by use of hetero- or alloantisera in a number of mammalian species (review: Greaves, Owen & Raff, 1973) and also in chickens (Forget, Potworowski, Richer & Borduas, 1970; McArthur, Chapman & Thorbecke, 1971; Ivanyi & Lydyard, 1972; Malchow, Droege & Strominger, 1972; Potworowski, 1972; Hudson & Roitt, 1973; Wick, Albini & Milgrom, 1973). In mice, the use of alloantisera has enabled identification of several stages in T cell development (review: Schlesinger, 1972), but in other species such studies have been limited by the lack of readily available, highly inbred strains. Alterations in the class of membrane Ig have been used for studying the differentiation of B cells (e.g. Kincade & Cooper, 1971; Albini & Wick, 1973; Pierce, Asofsky & Solliday, 1973) but this determinant may not be specific for B lymphocytes (Bankhurst, Warner & Sprent, 1971; Marchalonis, Cone & Atwell, 1972; Santana, Wedderburn & Turk, 1974). Because of their easier preparation, heteroantisera greatly facilitate the study of T and B lymphocyte developmental pathways. The chicken, with a clearly defined dichotomy of the immune system, is invaluable for such studies. As the basis for studying T and B cell differentiation we prepared rabbit antisera to various chicken lymphoid cells in order to define antigens specific for
Correspondence: Dr H.A. Ward, Department of Pathology and Immunology, Monash Medical School, Melbourne, Victoria, Australia 3181. Dr R.L. Boyd present address: Institut Fur Allgemeine und Experimentelle Pathologie, Universitat Innsbruck, Austria.
9
10
R. L. Boyd & H. A. Ward
different steps in lymphocyte pathways, and for the lymphoid micro-environment, particularly in the bursa. One aspect of the latter antigens, the specificity of the cortical reticulin framework of the bursa, has been reported (Boyd, Ward & Muller, 1976). In this paper we present data on the cellular representation and tissue localization of lymphoid antigenic determinants in the chicken.
MATERIALS AND METHODS Cell suspensions Tissues were obtained from freshly killed 3-12week-old, or embryonic, Australorp/White Leghorn F1 hybrids (Musgrove Chicks Pty. Ltd., Croydon, Australia), immediately teased out, and the cells washed and resuspended in Hanks' balanced salt solution, pH 7-6 (HBSS). Blood leucocytes were separated from a 1: 1 dilution of whole blood in Alsever's solution by collection of the buffy coat after centrifuging at 170 gay for 5 min. The buffy coat cells were resuspended in HBSS and the leucocytes purified by four sequential centrifugations at 170 gay for 5 min. Cell viability (trypan blue exclusion) was in excess of 90 % for all suspensions. In experiments requiring purified lymphocyte populations, phagocytic cells were removed from the suspensions by incubation of 108 cells in 5 ml HBSS with 0 3 g carbonyl iron (B.D.H.) for 1 h at 370 with occasional shaking. Non-phagocytic cells were decanted, phagocytes being retained by a magnet. Such a procedure completely removed cells capable of non-specifically binding aggregates of fluorescent
conjugates. Tissue sections
Freshly removed tissues were rinsed in HBSS, snap-frozen in a liquid nitrogen-isopentane slurry at approximately - 1600 and stored at -70°. Cryostat sections (6,pm) were mounted on chemically cleaned slides and air-dried at 40 for a minimum of 2 h. Alteration of immune status For stimulation of the lymphoid system, adult birds were injected i.p. with 3 ml heat-killed Brucella abortus suspension (Diagnostic Agent, Batch No. 612-1, Commonwealth Serum Laboratories, Melbourne, Australia) on days 1 and 14, and killed 7 days later. The agglutinating antibody titre of the resultant serum was 1: 128. Production of antisera Pre-immune sera for use in control preparations
were collected from rabbits 1-3 days before immuni-
zation. Rabbit anti-chicken sera were produced using the 'two-pulse' method (Levey & Medawar, 1966). Suspensions of 5 x 108 to 109 adult thymus or bursa cells or 6 day embryonic yolk sac cells were injected i.v. on days 1 and 14 and the rabbits bled out 1 week later. The blood was held at 370 for 1 h and then at 40 overnight. Serum was separated, centrifuged at 900 gay for 10 min, incubated at 56° for 30 min to inactivate complement and stored in aliquots at -200.
Immunological testing of antisera Serum or bile Ig were separated by precipitation with 40% saturated ammonium sulphate. Glutaraldehyde-insolubilized Ig for immunoadsorption was prepared according to Avrameas & Ternynck (1969). Unless otherwise stated, serum absorptions were performed according to Nairn (1976) with adult chicken tissues. All sera were absorbed initially with red blood cells to remove any haemagglutinating activity. Conjugates Goat anti-rabbit globulin was prepared using the procedure of Ironside (1968); the y-globulin fraction was conjugated with fluorescein isothiocyanate (Nairn, 1976)-fluorescein to protein molar ratio 4-8 before absorption-and was used at a protein concentration of approximately 0-66 mg/ml. After appropriate absorption, it gave no staining of fowl tissues by itself. y-globulin fractions of rabbit antisera to chicken Ig and IgG (Pentex, Kankakee, U.S.A.) were conjugated with fluorescein isothiocyanate; the conjugates had fluorochrome to protein molar ratios, before absorption, of 5 2 and 4-1 respectively, and were used at a protein concentration of approximately 0-35 mg/ml. After absorption with chicken kidney and thymus homogenates and thymus cell suspensions, these two conjugates showed specificity for Ig or IgG, as demonstrated by immuno-electrophoresis.
Immunofluorescence Standard membrane and section immunofluorescence tests with specific antisera were as described by Nairn (1976). Parallel control preparations were treated with phosphate buffered saline, pH 7-2, HBSS or pre-immune rabbit serum. Specimens were examined by narrow-band blue or darkground
Chicken lymphoid antigens ultraviolet fluorescence microscopy using a yellow or colourless barrier filter respectively (Nairn, 1976). Cell suspension counts of positive cells were performed on a minimum of 200 cells per slide; each result is the mean from at least three experiments, in each of which cells from a minimum of two birds were pooled. The antiserum titres against various cell types were taken as the highest serum dilution giving positive staining in suspensions of 6x 106 cells. Tissue section staining was done in triplicate
using different frozen blocks each time.
11
100F
U-
\
0
an0_ CA
404 201 ". \
4
RESULTS Anti-thymus
serum
(i) Chicken T-lymphocyte specific antigen (CTLA). After nine absorptions of the initial anti-thymus serum with bursa cells, this antiserum stained by membrane immunofluorescence (Table 1) approximately 93% of thymus, 61 % of spleen, 46% of blood leucocytes, 7 % of bone marrow and less than 1 % of adult bursa cells in suspension. Three absorptions of the original anti-thymus serum with thymus cells completely neutralized the activity. Although anti-CTLA membrane immunofluorescence staining intensities of thymus and peripheral T lymphocytes were similar, the titres for these populations differed (Fig. 1). Splenic T cells gave a titre of 1: 32 but for thymocytes, dilutions above 1: 32 still stained approximately 40 % of the cells, suggesting the presence of two populations of thymus cells, with high or low CTLA concentration. On tissue sections the anti-CTLA serum showed Table 1. Organ distribution of lymphocyte antigens (% positive cells)
Organ Antigen
Thymus
CTLA CTOA CBLA CMBLA CFAA Ig IgG
93 79 5 1 0-25 6 1
Bursa
