IMMUNOLOGY AND MOLECULAR BIOLOGY Flow Cytometric Analysis of T Lymphocyte Subpopulations in Large-Bodied Turkey Lines and a Randombred Control Population1 Z. Li,* K. E. Nestor,*,2 Y. M. Saif,† and M. Luhtala‡,3 *Department of Animal Sciences, and †Food Animal Health Research Program, The Ohio State University, Wooster, Ohio 44691; and ‡Department of Medical Microbiology, Turku University, Turku, Finland ABSTRACT To investigate the effect of BW selection on immune cell populations of turkeys, T lymphocyte subpopulation analyses were conducted using peripheral blood from lines selected for increased BW and a randombred control population. The lines used included an experimental line (F) selected long-term for increased 16wk BW, a randombred control line (RBC2) that served as the base population of the F line, and sire lines (A and B) from each of two major commercial turkey breeders. The peripheral blood lymphocytes were isolated and stained with mouse anti-chicken CD4 and CD8α antibodies in flow cytometric analysis. The polymorphism of CD8α in the F and A lines detected with the CT8 mono-
clonal antibody (mouse anti-chicken CD8α antibody) did not appear to be associated with BW and shank parameters. The present results showed that the F line had a significantly larger CD4+CD8− T cell subpopulation than did the RBC2 line at both ages, and this population proportion in the F line was also larger than that for one commercial sire line at 24 wk of age. There were no differences in other T cell subsets. The BW selection may have resulted in changes in T lymphocyte subpopulations and, therefore, may have affected disease resistance. The increased susceptibility to infectious diseases in the F line may be associated with the higher CD4+CD8− T cell subpopulation and the CD8α polymorphism.
(Key words: turkey, body weight, selection, CD4 T cells, CD8 T cells) 2000 Poultry Science 79:219–223
Research has been conducted on the humoral responses of turkeys from the F and RBC2 lines. The results showed that antibody titers in response to inactivated P. multocida and NDV vaccines detected with ELISA were not positively correlated with the resistance to these specific diseases observed in the lines (Tsai et al., 1992; Sacco et al., 1994). Moreover, the decreased disease resistance in the F line to P. multocida and NDV could not be explained by known changes in the frequency of haplotypes of the MHC Class II (Zhu et al., 1995; Nestor et al., 1996c). However, the F-line turkeys were found to have lower mitogenic responses to concanavalin A and higher frequency of CD8α polymorphism (Li et al., 1998, 1999). The objectives of the present study were to assess the relationship of the turkey CD8α polymorphism with growth parameters at different ages and to compare the proportions of T lymphocyte subsets in the peripheral blood from different turkey lines.
INTRODUCTION Selection for increased BW has been reported to be associated with a decrease in disease resistance in chickens (Han and Smyth, 1972, 1973; Mauldin et al., 1978; Qureshi and Havenstein, 1994) and turkeys (Saif et al., 1984; Sacco et al., 1991; Nestor et al., 1996a,b). Saif et al. (1984) reported that turkeys from a subline (F) selected for increased 16-wk BW had significantly higher mortality than its randombred control line (RBC2) during natural outbreaks of erysipelas and fowl cholera. Previous results have shown that average 16-wk BW of male turkeys in the F line has been increased about 75% (11.8 vs 6.8 kg) as compared with its parent line RBC2 (Nestor et al., 1996b). In addition, the poults in the F line also had higher mortality than the RBC2 line when experimentally challenged with Pasteurella multocida, the causative agent for fowl cholera (Sacco et al., 1991; Nestor et al., 1996b), and Newcastle disease virus (NDV) (Tsai et al., 1992).
MATERIALS AND METHODS Animals
Received for publication January 25, 1999. Accepted for publication October 15, 1999. 1 Salaries and research support provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. 2 To whom correspondence should be addressed:
[email protected]. 3 Current address: Department of Microbiology, Nordland hospital, Bodø, Norway.
Four lines of turkeys were used including two experimental (RBC2 and F lines) and two commercial sire lines Abbreviation Key: mAb = monoclonal antibody; PBL = peripheral blood lymphocytes; PE = phycoerythrin; NDV = Newcastle disease virus; RBC2 = randombred control line.
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(A and B). The RBC2 line was a randombred control line (Nestor, 1977a), whereas the F line was a subline of RBC2 selected for increased 16-wk BW (Nestor, 1977b, 1984; Nestor et al., 1996a). Commercial sire lines A and B were obtained from two different primary turkey breeders. The birds were provided a multiple-ration system with declining protein (Naber and Touchburn, 1970) and water ad libitum. In each experiment, the lines were grown intermingled, sexes separate.
Monoclonal Antibodies Mouse CT4 and CT8 monoclonal antibodies (mAb) (both isotypes: IgG1) recognizing chicken CD4 and CD8α T lymphocytes, respectively, have been described earlier (Chan et al., 1988). The 3-298 mAb (isotype: IgG2b) recognizes the chicken CD8α molecule (Luhtala et al., 1997a). The CT4 mAb can fully cross-react with turkey peripheral blood lymphocytes (PBL), whereas the CT8 mAb can only partially cross-react with turkey PBL with large line differences (Li et al., 1999). About 50% of F-line turkeys failed to cross-react with the CT8 mAb. A panel of mAb specific for chicken CD8 was screened in flow cytometric analysis to test for cross-reactivity with turkey PBL in the study of Li et al. (1999). Among the mAb, the 3-298 mAb was found to fully cross-react with turkey PBL in all lines and to immunoprecipitate a polypeptide with a relative molecular mass of about 34 kDa, which may be the turkey homologue of the CD8 molecule under reducing conditions. The K55 mAb hybridoma supernatant (Chung et al., 1991), recognizing chicken lymphocytes, was kindly provided by H. S. Lillehoj (USDA, Beltsville, MD 20705). The 3-298 and K55 mAb were used as the primary antibody in an indirect immunofluorescence staining procedure.
Flow Cytometric Analysis Experiment 1. This experiment was used to assess the relationship of CD8α polymorphism and growth parameters. Turkeys from Lines F, RBC2, and commercial sire Line A were used. The BW were recorded at 8, 16, and 20 wk of age, whereas shank parameters (including width, depth, and length) were measured only at 16 wk of age. Shank width (lateral) and depth (anterior-posterior) were measured at the dew claw using calipers. The shank length was measured from the hock to the footpad. The turkey CD8+ T cell population expressing CT8+ allotypic CD8α was detected with phycoerythrin (PE) conjugated with the CT8 mAb (CT8-PE).4 The PBL were isolated by low-speed centrifugation (60 × g for 10 min) and Histopaque-10775 density gradient separation and were washed with PAB solution (PBS containing 0.2%
4
Southern Biotechnology Associates, Inc., Birmingham, AL 35209. Sigma Chemical Co., St. Louis, MO 63178-9916. 6 Coulter Corp., Miami, FL 33196. 7 Caltag Laboratory Inc., Burlingame, CA 94010. 5
NaN3 and 0.2% BSA, pH 7.2). In the direct immunofluorescence staining, 100 µL of 1 × 106 cells were incubated with 50 µL of 1:100 diluted CT8-PE for 30 min on ice. After washing with PAB and fixation with 1.0% paraformaldehyde, the fluorescence intensities were measured by an Elite flow cytometer.6 Experiment 2. In this experiment, heparinized blood samples were used to compare the proportions of T lymphocyte subsets from turkeys of Lines F and RBC2 at the ages of 16 and 24 wk and turkeys in Commercial Sire Line B at the age of 24 wk. The CT4 and 3-298 mAb were used in this study to estimate the proportion of turkey T cell subpopulations. The PBL were isolated, washed, and enumerated as above. In the dual color immunofluorescence staining, 3-298 mAb was used in the indirect staining by first incubation for 30 min on ice followed by subsequent incubation with PE:cyanine 5 conjugated with goat anti-mouse IgG2b.7 Fluorescein isothiocyanate conjugated with CT44 was used in the direct staining. The lymphocyte population was estimated with the K55 mAb. The PBL were incubated with a 1:50 dilution of K55 hybridoma supernatant for 30 min on ice and then were incubated with PE conjugated with goat anti-mouse IgG4 for 30 min at 4 C. After washing with PAB and fixation with 1.0% paraformaldehyde, the fluorescence intensities were measured by an Elite flow cytometer. All necessary controls were included.
Statistical Analysis In Experiment 2, the percentages of positive T lymphocyte subsets were adjusted by dividing the percentages of positive T cell subsets by the total lymphocyte percentage estimated with K55 mAb from each individual. In both experiments, line differences were evaluated by one-way ANOVA using general linear models procedure of SAS (SAS Institute, 1988). Differences between lines were compared by the LSD test. A difference with a probability of P < 0.05 was considered statistically significant.
RESULTS Relationship of CD8α Polymorphisms and Growth Parameters The CT8 mAb cross-reacted with PBL from most of the turkeys from the RBC2 and A lines and with PBL from less than half of the turkeys in the F line (Table 1). The results suggested a CD8α polymorphism in the turkey PBL. Based on the cross-reaction of the CT8 mAb with PBL, turkeys in each line were divided into two groups: CT8+ and CT8−. The BW at 8, 16, and 20 wk of age and shank measurements (width, depth, and length) at 16 wk of age for turkeys from the Lines RBC2, F, and commercial sire A are presented in Table 1. Within each line and sex subgroup for the F and A lines, there was no significant difference in BW and shank parameters between the CT8+ and CT8− groups. However, significant differences were observed between the CT8+ and CT8− groups for BW at
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RESEARCH NOTE TABLE 1. Body weights at 8, 16, and 20 wk of age and shank width, depth, and length at 16 wk of age (least squares means ± SEM) in the F, RBC2, and A lines according to T cell staining type1 Body weight 2
Line
Sex
Group
A A F F
Female Female Female Female
CT8+ CT8− CT8+ CT8−
F F RBC2 RBC2
Male Male Female Female
CT8+ CT8− CT8+ CT8−
Birds
8 wk
16 wk
(no.) 14 4 8 11 4 7 18 3
Shank measurements at 16 wk of age 20 wk
Width
(kg) ± ± ± ± ± ± ± ±
3.8 3.9 3.8 3.9 4.4 4.4 1.9 2.1
0.07 0.24 0.11 0.10 0.24 0.11 0.05b 0.04a
11.1 11.2 10.4 10.6 14.7 14.8 5.0 5.3
± ± ± ± ± ± ± ±
Depth
(mm)
0.16 0.44 0.05 0.12 0.30 0.18 0.09b 0.09a
13.3 13.3 12.5 12.6 18.5 18.8 5.8 6.2
± ± ± ± ± ± ± ±
0.21 0.45 0.12 0.14 0.59 0.50 0.09b 0.11a
16.1 16.5 15.1 15.1 17.1 17.6 10.7 11.5
Length
(mm)
± ± ± ± ± ± ± ±
0.27 0.83 0.33 0.26 0.74 0.41 0.17b 0.13a
21.4 21.8 22.0 21.6 26.4 24.8 17.1 17.8
± ± ± ± ± ± ± ±
(cm) 0.27 0.62 0.33 0.26 0.60 1.30 0.14 0.45
17.2 17.0 18.4 18.3 22.7 22.5 16.1 16.2
± ± ± ± ± ± ± ±
0.10 0.41 0.12 0.12 0.22 0.18 0.10 0.46
Within lines and sexes, means for cell types in a column with no common superscript differ significantly (P < 0.05). Turkeys were divided into two groups according to the cross-reaction of the CT8 monoclonal antibody with turkey peripheral blood lymphocytes in flow cytometric analysis: CT8+ (positive reaction) and CT8− (negative reaction). 2 A = a commercial sire line; RBC2 = a randombred control line; and F = a subline of the RBC2 line selected for increased 16-wk body weight. a,b 1
8, 16, and 20 wk of age and for shank width at 16 wk of age in female turkeys from the RBC2 line.
Flow Cytometric Analysis of CD4 and CD8 T Cell Subsets from Turkey PBL
CD8− and CD4+CD8− cell subsets as well as a lower CD4:CD8 ratio. No significant differences, except in the percentage of CD4+CD8+ subsets, were observed between the RBC2 and B lines at 24 wk of age.
DISCUSSION
The proportion of CD4- and CD8-defined cell subsets in turkey PBL from the three lines is given in Table 2. At 16 wk of age, turkeys in the F line had a significantly higher percentage of CD4+CD8− subset and a higher ratio of CD4+CD8− (helper T cells) to CD4−CD8+ (cytotoxic T cells) (CD4:CD8 ratio) than did the RBC2 line (P < 0.05). No significant line difference was observed in the other cell populations. At 24 wk of age, turkeys in the F line still had a higher percentage of CD4+CD8− subpopulation than did the RBC2 line. Compared with Line F, the commercial sire Line B had a significantly higher percentage of the CD4+CD8+ subset but lower percentages of CD4−
The CD4 and CD8 molecules were originally defined as the cellular surface markers for helper T lymphocytes and for cytotoxic-suppressor T cells, respectively (Williams et al., 1977; Reinherz et al., 1979). The structure and function of CD4 and CD8 are well conserved between chickens and mammals (Luhtala, 1997, 1998). The CT8 mAb has been reported to recognize chicken CD8α molecule (Chan et al., 1988). Previous results showed that the CT8 mAb failed to cross-react with some turkey PBL with large line differences (Li et al., 1999). The PBL were found to be CT8− in a few turkeys in the RBC2
TABLE 2. The proportion of CD4- and CD8-defined cell subsets of peripheral blood lymphocytes in turkeys from different lines1 at 16 and 24 wk of age2 Percentage of T lymphocyte subsets −
Age (wk)
Line
CD4 CD8
16
F RBC2 F RBC2 B
14.7 14.4 18.0 18.0 17.3
24
± ± ± ± ±
+
1.3 1.0 1.0 1.7 1.1
CD4+CD8+ 24.1 26.0 27.0 31.3 38.1
± ± ± ± ±
1.9 1.9 1.8b 0.9b 2.5a
CD4−CD8− 47.5 49.7 47.6 45.6 40.7
± ± ± ± ±
4.2 2.3 1.1a 1.9ab 2.5b
CD4+CD8− 13.7 9.9 7.4 5.1 4.0
± ± ± ± ±
1.6a 0.6b 0.8a 0.4b 0.5b
Ratio3 0.94 0.71 0.42 0.30 0.23
± ± ± ± ±
0.08a 0.07b 0.06a 0.04ab 0.03b
a,b For each cell subset and line within the same age, means in a column with no common superscript differ significantly (P < 0.05). 1 B = a commercial sire line; RBC2 = a randombred control line; and F = a subline of the RBC2 line selected for increased 16-wk body weight. 2 Peripheral blood lymphocytes (PBL) were prepared from 10 turkeys from each line of Lines F and RBC2 at 16 wk of age and from 6 turkeys from each of the F, RBC2, and B lines at 24 wk of age. The PBL were incubated with the 3-298 monoclonal antibody (mAb) supernatant and then incubated with fluorescein isothiocyanate conjugated with CT4 mAb and phycoerythrin:Cyanine5 conjugated with goat anti-mouse IgG2b. Immunofluorescence densities were examined by a flow cytometer. All the proportions were percentages of lymphocytes. The percentages of subpopulations were based on the total lymphocytes as measured with the K55 monoclonal antibody using indirect staining. Values represent the least squares mean percentage ± SEM. 3 Ratio was calculated as the ratio of the percentage of the CD4+CD8− subsets to the percentage of the CD4− CD8+ subset. Values represent the least squares mean ± SEM.
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and A lines and in more than half the turkeys in the F line. It was suggested that polymorphisms also exist in the turkey CD8α molecule, as shown in chickens (Luhtala et al., 1997b). Because the F line is a fast-growing turkey line, the present experiment was used to examine whether the polymorphism detected by the CT8 mAb was associated with BW and shank parameters within each line. No difference was observed in these parameters between CT8+ and CT8− groups in the A and F lines. Either the CT8+ or CT8− may be composed of more than one CD8 allelic product. Absence of significant findings, especially in the F and A lines, may be the result of alleles with opposing effects in each group. Several different polymorphisms might exist in the turkey CD8 molecule that may not be detected by using only one antibody, as has been shown in the chicken (Luhtala, 1997, 1998). However, only a few turkeys were found to be CD8− in the RBC2 line. It is possible that the CT8− T cell population found in a lower proportion in the RBC2 line may be also inherited in a Mendelian manner, as was suggested by Luhtala et al.(1997a) for the peripheral CD4+CD8αα T cells. In that study, the genes were reported to accumulate to about 50% in the F line during long-term selection for increased BW. Based on the low proportion of peripheral CT8− T cell population in the A line that exhibits rapid growth, CT8− T cells may not be related to growth, and the difference in growth between two groups in the RBC2 line may have resulted from the limited number of samples in the CT8− group of Line RBC2. Previous reports showed that the F line was more susceptible to infectious diseases than the RBC2 line (Sacco et al., 1991; Tsai et al., 1992; Nestor et al., 1996b) but had higher antibody titers in the responses to sheep red blood cell antigen (Li and Nestor, unpublished data) and to P. multocida and NDV vaccines (Sacco et al., 1994). In addition, the F line had significantly lower numbers of lymphocytes than the RBC2 line (Bayyari et al., 1997). It is not clear how the higher relative percentage of the CD4+CD8− T cell subset may be associated with the susceptibility to specific diseases in the F line. The CD4+ T helper cells give assistance to both T and B cells in initiating the immune responses. Thus, the relative higher percentage of the CD4+CD8− subset (helper T cells) in the F line, under influence of certain kinds of cytokines or hormones beneficial to the growth, may secrete some cytokines that may favorably activate B cell differentiation to produce higher antibody titers but inhibit the cytotoxic T cell activity or activate the suppressor T cells at the same time. The effect of cytokines on the avian growth and immune system was thoroughly reviewed by Klasing and Johnstone (1991). In summary, the turkey CD8α polymorphism was not consistently associated with BW and shank parameters. Compared with the RBC2 line, the F line turkeys had a higher percentage of the CD4+CD8− subset at 16 and 24 wk of age and a higher CD4:CD8 ratio at 16 wk of age. Further studies should be conducted to observe the effect of the CD8α polymorphism and the changes in lympho-
cyte subpopulations on disease resistance in different turkey lines.
ACKNOWLEDGMENTS The authors thank British United Turkeys of America, PO Box 727, Lewisberg, WV 14901, and Hybrid Turkeys Inc., 650 Riverbend Drive, Suite C, Kitchner, Ontario, Canada N2K 3S2, for providing financial support. The authors are grateful to O. Vainio, Turku University, Finland for donating the 3-298 mAb.
REFERENCES Bayyari, G. R., W. E. Huff, N. C. Rath, J. M. Balog, L. A. Newberry, J. D. Villines, J. K. Skeeles, N. B. Anthony, and K. E. Nestor, 1997. Effect of the genetic selection of turkeys for increased body weight and egg production on immune and physiological responses. Poultry Sci. 76:289–296. Chan, M., C. H. Chen, L. L. Ager, and M. D. Cooper, 1988. Identification of the avian homologues of mammalian CD4 and CD8 antigens. J. Immunol. 140:2133–2138. Chung, K. S., H. S. Lillehoj, and M. C. Jenkins, 1991. Avian leukocyte common antigens: Molecular weight determination and flow cytometric analysis using new monoclonal antibodies. Vet. Immunol. Immunopathol. 28:259–273. Han, P.F.-S., and J. R. Smyth, Jr., 1972. The influence of growth rate on the development of Marek’s disease in chickens. Poultry Sci. 51:975–985. Han, P.F.-S., and J. R. Smyth, Jr., 1973. The influence of maternal effects on the response of fast and slow growing chickens to a Marek’s disease virus. Poultry Sci. 52:909–915. Klasing, K. C., and B. J. Johnstone, 1991. Monokines in growth and development. Poultry Sci. 70:1781–1789. Li, Z., K. E. Nestor, and Y. M. Saif, 1998. Effect of selection for increased body weight in turkeys on T lymphocyte subpopulations and mitogenic responses. Poultry Sci. 77(Suppl.1 ):152.(Abstr.) Li, Z., K. E. Nestor, Y. M. Saif, S. Fan, M. Luhtala, and O. Vainio, 1999. Cross-reactive anti-chicken CD4 and CD8 monoclonal antibodies suggest polymorphism of the turkey CD8α molecule. Poultry Sci. 78:1526–1531. Luhtala, M., 1997. Avian CD4, CD8alpha-alpha and CD8alphabeta T cell coreceptor molecules. Pages 1–95 in: Annales Universitatis Vol 274. Painosalama Oy, Turku, Finland. Luhtala, M, 1998. Chicken CD4, CD8αα, and CD8αβ T cell coreceptor molecules. Poultry Sci. 77:1858–1873. Luhtala, M., O. Lassila, P. Toivanen, and O. Vainio, 1997a. A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells. Eur. J. Immunol. 27:189–193. Luhtala, M., C. A. Tregaskes, J. R. Young, and O. Vainio, 1997b. Polymorphism of chicken CD8-alpha, but not CD8-beta. Immunogenetics 46:396–401. Mauldin, J. M., P. B. Siegel, and W. B. Gross, 1978. Dwarfism in diverse genetic backgrounds. 2. Behavior and disease resistance. Poultry Sci. 57:1488–1492. Naber, E. C., and S. P. Touchburn, 1970. Ohio poultry rations. Ohio Coop. Ext. Serv. Bull. 343. The Ohio State University, Columbus, OH. Nestor, K. E., 1977a. The stability of two randombred control populations of turkeys. Poultry Sci. 56:54–57. Nestor, K. E., 1977b. Genetics of growth and reproduction in the turkey. 5. Selection for increased body weight alone and in combination with increased egg production. Poultry Sci. 56:337–347. Nestor, K. E., 1984. Genetics of growth and reproduction in the turkey. 9. Long-term selection for increased 16-week body weight. Poultry Sci. 63:2114–2122.
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Newcastle disease virus and Pasteurella multocida vaccines. Avian Dis. 38:33–36. Sacco, R. E., Y. M. Saif, K. E. Nestor, N. B. Anthony, D. A. Emmerson, and R. N. Dearth, 1991. Genetic variation in resistance of turkeys to experimental challenge with Pasteurella multocida. Avian Dis. 35:950–954. Saif, Y. M., K. E. Nestor, R. N. Dearth, and P. A. Renner, 1984. Case report—Possible genetic variation in resistance of turkeys to erysipelas and fowl cholera. Avian Dis. 28:770–773. SAS Institute, 1988. SAS威/STAT User’s Guide. SAS Institute Inc., Cary, NC. Tsai, H. J., Y. M. Saif, K. E. Nestor, D. A. Emmerson, and R. A. Patterson, 1992. Genetic variation in resistance of turkeys to experimental infection with Newcastle disease virus. Avian Dis. 36:561–565. Williams, A. F., E. G. Galfre, and C. Milstein, 1977. Analysis of cell surface by xenogeneic myeloma hybrid antibodies: Differentiation antigens of rat lymphocytes. Cell 12:663–671. Zhu, J., K. E. Nestor, and S. J. Lamont, 1995. Survey of major histocompatibility complex Class II haplotypes in four turkey lines using restriction fragment length polymorphism analysis with nonradioactive DNA detection. Poultry Sci. 74:1067–1073.
