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In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+ CD25+ T cells in chickens
In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+CD25+ T cells in chickens Revathi Shanmugasundaram and Ramesh K. Selvaraj1 Department of Animal Sciences, Ohio Agricultural Research and Development Center, Wooster 44691 control group. At 16 d posthatch, the anti-chicken CD25 antibody injection decreased CD4+CD25+ cell percentages in the thymus, spleen, and cecal tonsils. Chickens hatched from anti-CD25-antibody-injected eggs had approximately 25% of CD4+CD25+ cells in the cecal tonsils and thymus compared with those in the cecal tonsils and thymus of the control group. The CD4+CD25− cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of interferon-γ and interleukin-2 mRNA than CD4+CD25− cells from the control group. It could be concluded that injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted the CD4+CD25+ cells until 25 d posthatch.
Key words: regulatory T cell, in ovo, anti-CD25, depletion, suppressor 2013 Poultry Science 92:138–142 http://dx.doi.org/10.3382/ps.2012-02593
INTRODUCTION
anti-CD25 antibodies depletes Treg in mice (Onizuka et al., 1999), cats (Smithberg et al., 2008), and chickens (Shanmugasundaram and Selvaraj, 2012b). In chickens, a single intraperitoneal injection of anti-chicken CD25 antibodies depletes Treg at 12 d after CD25 injection. The Treg depletion was maximal at 12 d postinjection. The depletion of Treg was transient, as the Treg population increased to preinjection levels at 20 d after anti-CD25-injection. The CD4+CD25− cells from Tregdepleted birds produced increased amounts of IL-2 and IFNγ mRNA and proliferated more than CD4+CD25− cells from non-Treg-depleted birds (Shanmugasundaram and Selvaraj, 2012b). In chickens, Treg start to appear in the embryonic thymus at 15 d of incubation at low percentages and remain relatively low until hatch. At hatch, the percentage of CD4+CD25+ cells in the thymus increases (Shanmugasundaram and Selvaraj, 2012a). Hence, in ovo injection of anti-chicken CD25 monoclonal antibodies can be expected to deplete Treg in chicks. In chickens, in ovo delivery is a commonly available technique applied to deliver vaccines (Johnston et al., 1997), adjuvants (Ding et al., 2004), vitamins, and several other compounds (Bakyaraj et al., 2012). Identifying whether in ovo injection of anti-chicken CD25 antibod-
The CD4+CD25+ FoxP3+ cells are recognized as regulatory T cells (Treg) in several species. In chickens, thymic CD4+CD25+ cells produce high amounts of interleukin (IL)-10, transforming growth factor-β, cytotoxic T-lymphocyte antigen 4, and lymphocyteactivation gene 3 mRNA; suppress the proliferation of naïve T cells in vitro; and are likely the counterparts of Treg in chickens (Shanmugasundaram and Selvaraj, 2011). In chickens, Treg suppress the activity and proliferation of several immune cells through both contact-dependent pathways that include cytotoxic Tlymphocyte antigen 4, and lymphocyte-activation gene 3 and contact-independent pathways through immunosuppressive cytokines. In vivo depletion of CD4+CD25+ cells during viral infections relieves the in vivo suppression of Treg and improves viral clearance (Zelinskyy et al., 2006; Kottke et al., 2008; Dietze et al., 2011). In vivo injection of ©2013 Poultry Science Association Inc. Received July 6, 2012. Accepted September 8, 2012. 1 Corresponding author: [email protected]
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ABSTRACT The CD4+CD25+ cells have T regulatory cell properties in chickens. This study investigated the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies (0.5 mg/egg) on CD4+CD25+ cell depletion and on amounts of interleukin-2 mRNA and interferon-γ mRNA in CD4+CD25− cells posthatch. Anti-chicken CD25 or PBS (control) was injected into 16-d-old embryos. Chicks hatched from eggs injected with anti-chicken CD25 antibodies had a lower CD4+CD25+ cell percentage in the blood until 25 d posthatch. The anti-chicken CD25 antibody injection nearly depleted CD4+CD25+ cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4+CD25+ cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the
RESEARCH NOTE
MATERIALS AND METHODS All animal protocols were approved by the Institutional Animal Care and Use Committee at The Ohio State University.
Anti-Chicken CD25 Monoclonal Antibodies Production of mouse anti-chicken CD25 monoclonal antibodies is described earlier (Shanmugasundaram and Selvaraj, 2011). Briefly, the extracellular part of chicken CD25 protein was expressed. Mice were immunized against the partial chicken CD25 protein, and the splenic hybridomas were screened for anti-chicken CD25. The positive hybridoma clone was grown in Megacell Dulbecco’s modified Eagle’s medium (#M3942, SigmaAldrich, St. Louis, MO) supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA), 50 µg/mL of gentamycin (Invitrogen, Carlsbad, CA), and 1 mM glutamine (Invitrogen), and concentrated using a protein A column and eluted using 100 mM sodium citrate buffer as described earlier (Shanmugasundaram and Selvaraj, 2012b). The antibody concentration in the final solution was determined in a spectrophotometer to be 0.9 mg/mL.
Experiment I In Ovo Injection of Anti-CD25 Antibodies. Antichicken CD25 monoclonal antibodies (0.5 mg/egg) or 550 µL of PBS was injected into 16-d-old fertile chicken eggs through the amniotic route as described previously (Annamalai and Selvaraj, 2012). Briefly, the large end of the egg was prepared aseptically by swabbing with 70% ethanol, and a small hole sufficient to insert a 20-ga needle was drilled in the middle of the large
end. The test material was injected into the egg using a 1-inch (2.54 cm) 20-ga needle. The eggs were sealed and incubated under standard hatchery conditions. Hatchability was approximately 50%. Chicks were hatched from the eggs. Each treatment group had a total of 12 birds. Approximately 125 µL of blood was drawn from the alternate wing web vein at 0, 8, 16, 25, and 30 d posthatch from one bird. Blood from 4 birds were pooled to obtain 3 replications (n = 3) and analyzed for CD4+CD25+ cells as described below. CD4+CD25+ Cell Percentages in Peripheral Blood After Antibody Injection. Single cell suspensions of the blood lymphocyte were concentrated for peripheral blood mononuclear cells by density centrifugation over Histopaque (1.077 g/mL, Sigma Aldrich, St. Louis, MO) as described earlier (Shanmugasundaram and Selvaraj, 2010). The phycoerythrin (PE) linking of mouse anti-chicken CD25+ was conducted as described earlier (Shanmugasundaram and Selvaraj, 2010). Approximately 1 × 106 cells were incubated with 10 µg/ mL of PE-linked mouse anti-chicken CD25, 1:200 fluorescein isothiocyanate -conjugated mouse anti-chicken CD4 (Southern Biotech, Birmingham, AL) or 1:200 fluorescein isothiocyanate conjugated mouse antichicken CD8 (Southern Biotech), and 1:200 dilution of unlabeled mouse IgG for 45 min. Unbound antibodies were removed by centrifugation. The percentages of CD4+ and CD4+CD25+ cells were analyzed in a flow cytometer (Guava Eascyte, Millipore, Billerica, MA). The CD4+CD25+ cell percentage in the blood was expressed as a percentage of CD4+ cells to facilitate comparison between samples.
Experiment II CD4+CD25+ Cell Percentages in the Thymus, Spleen, and Cecal Tonsils at 16 d Posthatch. Chicks were hatched from eggs injected with 500 µg of antichicken CD25 or 550 µL of PBS (control) in 3 replicates per treatment (n = 3). At 16 d of age, the thymus, spleen, and cecal tonsils were collected. Single cell suspensions of thymus, spleen, and cecal tonsils were concentrated for peripheral blood mononuclear cells by density centrifugation over Histopaque, stained for CD4 and CD25, and analyzed for CD4+ and CD4+CD25+ cells as described earlier (Shanmugasundaram and Selvaraj, 2010). The CD4+CD25+ cell percentage was expressed as a percentage of CD4+ peripheral blood monocytes to facilitate comparison between samples. IFNγ, IL-2, and IL-10 mRNA Amounts in CD4+CD25− Cells from the Spleen and Cecal Tonsils After Antibody Injection. The CD4+CD25− cells were collected using positive selection for CD4+ cells and negative selection for CD25− cells using magnetic beads as described earlier (Shanmugasundaram and Selvaraj, 2012b). Total RNA was extracted from CD4+CD25- cells collected from the spleen and cecal tonsils of birds at 16 d posthatch using TRIzol reagent (Molecular Research Center, Cincinnati, OH) following
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ies depletes a Treg population can have several practical applications. For example, Treg depletion increases vaccine responses in mice (Klages et al., 2010); hence, injecting anti-chicken CD25 antibodies with in ovo vaccines can be expected to improve vaccination efficiency. Two experiments were conducted to study the effect of in ovo injection of anti-chicken CD25 antibodies on Treg depletion. The first experiment studied the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies on CD4+CD25+ cell percentage in the blood. Though CD4+CD25+ cell percentage in the blood can vary widely, blood CD4+CD25+ cell percentage will provide an estimate of the timeline of CD4+CD25+ depletion in chicks hatched from anti-CD25-injected eggs without having to sacrifice the bird. The second experiment studied the effect of anti-chicken CD25 monoclonal antibody injection on CD4+CD25+ percentages in the thymus, spleen, and cecal tonsils and the properties of CD4+CD25− cells from the spleen and cecal tonsils of chicks hatched from CD25-injected eggs at one time point selected based on the results of the first experiment.
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manufacturer’s directions. The mRNA was extracted from CD4+CD25− cells and analyzed for the relative expressions of IFNγ and IL-2 by RT-PCR (iCycler, BioRad, Hercules, CA) using SyBr green after normalizing for β-actin mRNA. The RT-PCR analysis has been described previously for IL-2, IFNγ, and β-actin (Shanmugasundaram and Selvaraj, 2012b). Fold change from the reference was calculated as 2(Ct Sample-housekeeping)/2(Ct Reference-housekeeping), where Ct is the threshold cycle (Schmittgen and Livak, 2008). The Ct was determined by iQ5 software (BioRad) when the fluorescence rises exponentially 2-fold above background. The reference group was the CD4+CD25− cells from chicks hatched from no-injection control eggs.
One way ANOVA (JMP software, Cary, NC) was used to examine the effects of an anti-chicken CD25 injection on the dependent variables. When main effects were significant (P < 0.05), differences between means were analyzed by Student’s t-test means comparison.
RESULTS AND DISCUSSION Experiment I Anti-chicken CD25 antibody injection decreased the CD4+CD25+ cell percentage until 25 d posthatch (Figure 1). The mean CD4+CD25+ cell percentage in the blood was approximately 0.5% until 25 d posthatch. Anti-chicken CD25 antibody injection nearly depleted CD4+CD25+ cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4+CD25+ cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the control group. The CD4+CD25+ cells are produced continuously in the thymus. Injected anti-CD25 antibodies were most likely depleted at 16 d posthatch, and CD4+CD25+ cells from the thymus started to repopulate the bird at 16 d posthatch. Future experiments will identify whether a second anti-CD25 injection at 16 d posthatch will increase the duration of CD4+CD25+ cells depletion beyond 16 d posthatch.
tonsils and thymus compared with the cecal tonsils and thymus of the control group. Injecting anti-CD25 depletes Treg through FcγRIIImediated phagocytosis in mice. Macrophages were critical in depleting the Treg following anti-CD25 injection (Setiady et al., 2010). Injecting anti-CD25 in chickens likely decreased CD4+CD25+ cell percentages through this mechanism in the blood, spleen, and cecal tonsils. We earlier reported that a single intraperitoneal injection of anti-CD25 depletes CD4+CD25+ cells in the blood, spleen, and cecal tonsils but not in the thymus of chickens (Shanmugasundaram and Selvaraj, 2012b). In the current experiment, in ovo injection of anti-CD25
Experiment II CD4+CD25+ Cell Percentages in the Thymus, Spleen, and Cecal Tonsils at 16 d Posthatch. Similar to the near CD4+CD25+ cell depletion observed in blood at 16 d posthatch, anti-chicken CD25 antibody injection decreased the CD4+CD25+ cell percentage in the thymus, spleen, and cecal tonsils at 16 d posthatch (Figure 2). The CD4+CD25+ cell depletion was almost complete in the spleen of chicks hatched from anti-CD25-antibody-injected eggs. Chickens hatched from eggs injected with anti-CD25-antibodies had approximately 25% of the CD4+CD25+ cells in the cecal
Figure 2. The CD4+CD25+ cell percentages in the thymus, spleen, and cecal tonsils posthatch. Chicks were hatched from eggs injected with either 0.5 mg of anti-chicken CD25 monoclonal antibodies or 550 µL of PBS. At 16 d posthatch, the thymus, spleen, and cecal tonsils were analyzed for CD4+CD25+ cells. The CD4+CD25+ cells were expressed as a percentage of CD4+ cells. * indicates significant difference between treatments in that particular organ (P < 0.05). Means + SEM. n = 3.
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Statistical Analysis
Figure 1. The CD4+CD25+ cell percentages in peripheral blood posthatch. Chicks were hatched from eggs injected with either 0.5 mg of anti-chicken CD25 monoclonal antibodies or 550 µL of PBS. Blood was analyzed for CD4+ and CD4+CD25+ cells after concentrating for peripheral blood monocytes by density centrifugation. The CD4+CD25+ cells were expressed as a percentage of CD4+ peripheral blood monocytes. Means (±SEM). * indicates significant difference between treatments in that particular day (P < 0.05). n = 3.
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CD4+CD25+
Figure 3. Relative IL-2 and IFNγ mRNA amounts in CD4+CD25− cells from the spleen and cecal tonsils at 16 d after antibody injection. Chicks were hatched from eggs injected with either 0.5 mg of anti-chicken CD25 monoclonal antibodies or 550 µL of PBS. The CD4+CD25− cells from the spleen and cecal tonsils were collected at 16 d posthatch. The mRNA contents were corrected for β-actin mRNA amounts and normalized to the mRNA contents of the PBS group so that all bars represent fold change compared with the PBS group. * indicates significant difference between treatments in that particular organ (P < 0.05). Means + SEM. n = 3.
cecal tonsils. A single injection of anti-chicken CD25 antibodies depleted CD4+CD25+ cells until 25 d posthatch. Anti-CD25 antibody-mediated Treg depletion has been applied to study the role of Treg in several bacterial infections in mammals. Similarly anti-chicken CD25-induced Treg depletion is a probable model to study the role of Treg in chickens during infections.
REFERENCES Annamalai, T., and R. K. Selvaraj. 2012. Effects of in ovo IL4-plasmid injection on anti-coccidia immune response in a coccidia infection model of chickens. Vet. Immunol. Immunopathol. 145:257–263. Bakyaraj, S., S. K. Bhanja, S. Majumdar, and B. Dash. 2012. Modulation of post-hatch growth and immunity through in ovo supplemented nutrients in broiler chickens. J. Sci. Food Agric. 92:313–320. D’Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin, and G. Trinchieri. 1993. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041–1048. Dietze, K. K., G. Zelinskyy, K. Gibbert, S. Schimmer, S. Francois, L. Myers, T. Sparwasser, K. J. Hasenkrug, and U. Dittmer. 2011. Transient depletion of regulatory T cells in transgenic mice reactivates virus-specific CD8+ T cells and reduces chronic retroviral set points. Proc. Natl. Acad. Sci. USA 108:2420–2425. Ding, X., H. S. Lillehoj, M. A. Quiroz, E. Bevensee, and E. P. Lillehoj. 2004. Protective immunity against Eimeria acervulina following in ovo immunization with a recombinant subunit vaccine and cytokine genes. Infect. Immun. 72:6939–6944. Feinerman, O., G. Jentsch, K. E. Tkach, J. W. Coward, M. M. Hathorn, M. W. Sneddon, T. Emonet, K. A. Smith, and G. AltanBonnet. 2010. Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response. Mol. Syst. Biol. 6:437. Johnston, P. A., H. Liu, T. O’Connell, P. Phelps, M. Bland, J. Tyczkowski, A. Kemper, T. Harding, A. Avakian, E. Haddad, C. Whitfill, R. Gildersleeve, and C. A. Ricks. 1997. Applications in in ovo technology. Poult. Sci. 76:165–178. Klages, K., C. T. Mayer, K. Lahl, C. Loddenkemper, M. W. Teng, S. F. Ngiow, M. J. Smyth, A. Hamann, J. Huehn, and T. Sparwasser. 2010. Selective depletion of Foxp3+ regulatory T cells improves effective therapeutic vaccination against established melanoma. Cancer Res. 70:7788–7799. Kohm, A. P., J. S. McMahon, J. R. Podojil, W. S. Begolka, M. DeGutes, D. J. Kasprowicz, S. F. Ziegler, and S. D. Miller. 2006. Cutting Edge: Anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells. J. Immunol. 176:3301–3305. Kottke, T., F. Galivo, P. Wongthida, R. M. Diaz, J. Thompson, D. Jevremovic, G. N. Barber, G. Hall, J. Chester, P. Selby, K. Harrington, A. Melcher, and R. G. Vile. 2008. Treg depletionenhanced IL-2 treatment facilitates therapy of established tumors using systemically delivered oncolytic virus. Mol. Ther. 16:1217–1226. Liu, F., J. Liu, D. Weng, Y. Chen, L. Song, Q. He, and J. Chen. 2010. CD4+CD25+Foxp3+ regulatory T cells depletion may attenuate the development of silica-induced lung fibrosis in mice. PLoS ONE 5:e15404. Onizuka, S., I. Tawara, J. Shimizu, S. Sakaguchi, T. Fujita, and E. Nakayama. 1999. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res. 59:3128–3133. Schmittgen, T. D., and K. J. Livak. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3:1101– 1108. Setiady, Y. Y., J. A. Coccia, and P. U. Park. 2010. In vivo depletion of CD4+FOXP3+ Treg cells by the PC61 anti-CD25 monoclonal antibody is mediated by FcgammaRIII+ phagocytes. Eur. J. Immunol. 40:780–786.
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decreased the number of cells in the thymus. In mice, in vivo injection of anti-CD25 antibodies downregulates CD25 expression in CD4+CD25+ cells, induces shedding of CD25 from the surface of CD4+CD25+ cells, or both (Kohm et al., 2006). It is possible that in ovo injection of anti-CD25 antibodies decreased the CD25 expression in CD4+CD25+ cells in the thymus. Relative IFNγ and IL-2 mRNA Amounts in CD4+CD25− Cells from the Spleen and Cecal Tonsils After Antibody Injection. The CD4+CD25- cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of IFNγ and IL-2 mRNA than CD4+CD25- cells from the control group (Figure 3). Interleukin-2 increases the proliferation of T cells. We earlier reported that CD4+CD25− cells from birds depleted of CD4+CD25+ cells proliferated more than cells from the control group (Shanmugasundaram and Selvaraj, 2012b). The presence of Tregs reduces the IL-2 mRNA amount in T cells of mice, and a Treg-mediated decrease in the IL-2 mRNA amount of T cells suppresses T cell proliferation (Feinerman et al., 2010). Depleting CD4+CD25+ cells, thus, can be expected to increase the IL-2 mRNA amount in CD4+CD25− cells. The IFNγ is an inflammatory cytokine, and Treg ablation enhances IFNγ production (Liu et al., 2010). IL10 suppresses IFNγ production in humans (D’Andrea et al., 1993). Depleting CD4+CD25+ cells, a source of IL-10 (Shanmugasundaram and Selvaraj, 2011), can be expected to increase the amount of IFNγ mRNA in the Treg-depleted group. In conclusion, injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted CD4+CD25+ cell percentages in the blood, spleen, and
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Shanmugasundaram, R., and R. K. Selvaraj. 2010. In vitro human TGF-beta treatment converts CD4(+)CD25(−) T cells into induced T regulatory like cells. Vet. Immunol. Immunopathol. 137:161–165. Shanmugasundaram, R., and R. K. Selvaraj. 2011. Regulatory T cell properties of chicken CD4+CD25+ cells. J. Immunol. 186:1997– 2002. Shanmugasundaram, R., and R. K. Selvaraj. 2012a. CD4+CD25+ regulatory T cell ontogeny and preferential migration to the cecal tonsils in chickens. PLoS ONE 7:e33970. Shanmugasundaram, R., and R. K. Selvaraj. 2012b. Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on reg-
ulatory T cell depletion and CD4+CD25− T cell properties in chickens. Dev. Comp. Immunol. 36:578–583. Smithberg, S. R., J. E. Fogle, A. M. Mexas, S. K. Reckling, S. M. Lankford, M. B. Tompkins, and G. A. Dean. 2008. In vivo depletion of CD4+CD25+ regulatory T cells in cats. J. Immunol. Methods 329:81–91. Zelinskyy, G., A. R. Kraft, S. Schimmer, T. Arndt, and U. Dittmer. 2006. Kinetics of CD8+ effector T cell responses and induced CD4+ regulatory T cell responses during Friend retrovirus infection. Eur. J. Immunol. 36:2658–2670.
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Key words: regulatory T cell, in ovo, anti-CD25, depletion, suppressor 2013 Poultry Science 92:138–142 http://dx.doi.org/10.3382/ps.2012-02593
INTRODUCTION
anti-CD25 antibodies depletes Treg in mice (Onizuka et al., 1999), cats (Smithberg et al., 2008), and chickens (Shanmugasundaram and Selvaraj, 2012b). In chickens, a single intraperitoneal injection of anti-chicken CD25 antibodies depletes Treg at 12 d after CD25 injection. The Treg depletion was maximal at 12 d postinjection. The depletion of Treg was transient, as the Treg population increased to preinjection levels at 20 d after anti-CD25-injection. The CD4+CD25− cells from Tregdepleted birds produced increased amounts of IL-2 and IFNγ mRNA and proliferated more than CD4+CD25− cells from non-Treg-depleted birds (Shanmugasundaram and Selvaraj, 2012b). In chickens, Treg start to appear in the embryonic thymus at 15 d of incubation at low percentages and remain relatively low until hatch. At hatch, the percentage of CD4+CD25+ cells in the thymus increases (Shanmugasundaram and Selvaraj, 2012a). Hence, in ovo injection of anti-chicken CD25 monoclonal antibodies can be expected to deplete Treg in chicks. In chickens, in ovo delivery is a commonly available technique applied to deliver vaccines (Johnston et al., 1997), adjuvants (Ding et al., 2004), vitamins, and several other compounds (Bakyaraj et al., 2012). Identifying whether in ovo injection of anti-chicken CD25 antibod-
The CD4+CD25+ FoxP3+ cells are recognized as regulatory T cells (Treg) in several species. In chickens, thymic CD4+CD25+ cells produce high amounts of interleukin (IL)-10, transforming growth factor-β, cytotoxic T-lymphocyte antigen 4, and lymphocyteactivation gene 3 mRNA; suppress the proliferation of naïve T cells in vitro; and are likely the counterparts of Treg in chickens (Shanmugasundaram and Selvaraj, 2011). In chickens, Treg suppress the activity and proliferation of several immune cells through both contact-dependent pathways that include cytotoxic Tlymphocyte antigen 4, and lymphocyte-activation gene 3 and contact-independent pathways through immunosuppressive cytokines. In vivo depletion of CD4+CD25+ cells during viral infections relieves the in vivo suppression of Treg and improves viral clearance (Zelinskyy et al., 2006; Kottke et al., 2008; Dietze et al., 2011). In vivo injection of ©2013 Poultry Science Association Inc. Received July 6, 2012. Accepted September 8, 2012. 1 Corresponding author: [email protected]
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ABSTRACT The CD4+CD25+ cells have T regulatory cell properties in chickens. This study investigated the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies (0.5 mg/egg) on CD4+CD25+ cell depletion and on amounts of interleukin-2 mRNA and interferon-γ mRNA in CD4+CD25− cells posthatch. Anti-chicken CD25 or PBS (control) was injected into 16-d-old embryos. Chicks hatched from eggs injected with anti-chicken CD25 antibodies had a lower CD4+CD25+ cell percentage in the blood until 25 d posthatch. The anti-chicken CD25 antibody injection nearly depleted CD4+CD25+ cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4+CD25+ cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the
RESEARCH NOTE
MATERIALS AND METHODS All animal protocols were approved by the Institutional Animal Care and Use Committee at The Ohio State University.
Anti-Chicken CD25 Monoclonal Antibodies Production of mouse anti-chicken CD25 monoclonal antibodies is described earlier (Shanmugasundaram and Selvaraj, 2011). Briefly, the extracellular part of chicken CD25 protein was expressed. Mice were immunized against the partial chicken CD25 protein, and the splenic hybridomas were screened for anti-chicken CD25. The positive hybridoma clone was grown in Megacell Dulbecco’s modified Eagle’s medium (#M3942, SigmaAldrich, St. Louis, MO) supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA), 50 µg/mL of gentamycin (Invitrogen, Carlsbad, CA), and 1 mM glutamine (Invitrogen), and concentrated using a protein A column and eluted using 100 mM sodium citrate buffer as described earlier (Shanmugasundaram and Selvaraj, 2012b). The antibody concentration in the final solution was determined in a spectrophotometer to be 0.9 mg/mL.
Experiment I In Ovo Injection of Anti-CD25 Antibodies. Antichicken CD25 monoclonal antibodies (0.5 mg/egg) or 550 µL of PBS was injected into 16-d-old fertile chicken eggs through the amniotic route as described previously (Annamalai and Selvaraj, 2012). Briefly, the large end of the egg was prepared aseptically by swabbing with 70% ethanol, and a small hole sufficient to insert a 20-ga needle was drilled in the middle of the large
end. The test material was injected into the egg using a 1-inch (2.54 cm) 20-ga needle. The eggs were sealed and incubated under standard hatchery conditions. Hatchability was approximately 50%. Chicks were hatched from the eggs. Each treatment group had a total of 12 birds. Approximately 125 µL of blood was drawn from the alternate wing web vein at 0, 8, 16, 25, and 30 d posthatch from one bird. Blood from 4 birds were pooled to obtain 3 replications (n = 3) and analyzed for CD4+CD25+ cells as described below. CD4+CD25+ Cell Percentages in Peripheral Blood After Antibody Injection. Single cell suspensions of the blood lymphocyte were concentrated for peripheral blood mononuclear cells by density centrifugation over Histopaque (1.077 g/mL, Sigma Aldrich, St. Louis, MO) as described earlier (Shanmugasundaram and Selvaraj, 2010). The phycoerythrin (PE) linking of mouse anti-chicken CD25+ was conducted as described earlier (Shanmugasundaram and Selvaraj, 2010). Approximately 1 × 106 cells were incubated with 10 µg/ mL of PE-linked mouse anti-chicken CD25, 1:200 fluorescein isothiocyanate -conjugated mouse anti-chicken CD4 (Southern Biotech, Birmingham, AL) or 1:200 fluorescein isothiocyanate conjugated mouse antichicken CD8 (Southern Biotech), and 1:200 dilution of unlabeled mouse IgG for 45 min. Unbound antibodies were removed by centrifugation. The percentages of CD4+ and CD4+CD25+ cells were analyzed in a flow cytometer (Guava Eascyte, Millipore, Billerica, MA). The CD4+CD25+ cell percentage in the blood was expressed as a percentage of CD4+ cells to facilitate comparison between samples.
Experiment II CD4+CD25+ Cell Percentages in the Thymus, Spleen, and Cecal Tonsils at 16 d Posthatch. Chicks were hatched from eggs injected with 500 µg of antichicken CD25 or 550 µL of PBS (control) in 3 replicates per treatment (n = 3). At 16 d of age, the thymus, spleen, and cecal tonsils were collected. Single cell suspensions of thymus, spleen, and cecal tonsils were concentrated for peripheral blood mononuclear cells by density centrifugation over Histopaque, stained for CD4 and CD25, and analyzed for CD4+ and CD4+CD25+ cells as described earlier (Shanmugasundaram and Selvaraj, 2010). The CD4+CD25+ cell percentage was expressed as a percentage of CD4+ peripheral blood monocytes to facilitate comparison between samples. IFNγ, IL-2, and IL-10 mRNA Amounts in CD4+CD25− Cells from the Spleen and Cecal Tonsils After Antibody Injection. The CD4+CD25− cells were collected using positive selection for CD4+ cells and negative selection for CD25− cells using magnetic beads as described earlier (Shanmugasundaram and Selvaraj, 2012b). Total RNA was extracted from CD4+CD25- cells collected from the spleen and cecal tonsils of birds at 16 d posthatch using TRIzol reagent (Molecular Research Center, Cincinnati, OH) following
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ies depletes a Treg population can have several practical applications. For example, Treg depletion increases vaccine responses in mice (Klages et al., 2010); hence, injecting anti-chicken CD25 antibodies with in ovo vaccines can be expected to improve vaccination efficiency. Two experiments were conducted to study the effect of in ovo injection of anti-chicken CD25 antibodies on Treg depletion. The first experiment studied the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies on CD4+CD25+ cell percentage in the blood. Though CD4+CD25+ cell percentage in the blood can vary widely, blood CD4+CD25+ cell percentage will provide an estimate of the timeline of CD4+CD25+ depletion in chicks hatched from anti-CD25-injected eggs without having to sacrifice the bird. The second experiment studied the effect of anti-chicken CD25 monoclonal antibody injection on CD4+CD25+ percentages in the thymus, spleen, and cecal tonsils and the properties of CD4+CD25− cells from the spleen and cecal tonsils of chicks hatched from CD25-injected eggs at one time point selected based on the results of the first experiment.
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manufacturer’s directions. The mRNA was extracted from CD4+CD25− cells and analyzed for the relative expressions of IFNγ and IL-2 by RT-PCR (iCycler, BioRad, Hercules, CA) using SyBr green after normalizing for β-actin mRNA. The RT-PCR analysis has been described previously for IL-2, IFNγ, and β-actin (Shanmugasundaram and Selvaraj, 2012b). Fold change from the reference was calculated as 2(Ct Sample-housekeeping)/2(Ct Reference-housekeeping), where Ct is the threshold cycle (Schmittgen and Livak, 2008). The Ct was determined by iQ5 software (BioRad) when the fluorescence rises exponentially 2-fold above background. The reference group was the CD4+CD25− cells from chicks hatched from no-injection control eggs.
One way ANOVA (JMP software, Cary, NC) was used to examine the effects of an anti-chicken CD25 injection on the dependent variables. When main effects were significant (P < 0.05), differences between means were analyzed by Student’s t-test means comparison.
RESULTS AND DISCUSSION Experiment I Anti-chicken CD25 antibody injection decreased the CD4+CD25+ cell percentage until 25 d posthatch (Figure 1). The mean CD4+CD25+ cell percentage in the blood was approximately 0.5% until 25 d posthatch. Anti-chicken CD25 antibody injection nearly depleted CD4+CD25+ cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4+CD25+ cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the control group. The CD4+CD25+ cells are produced continuously in the thymus. Injected anti-CD25 antibodies were most likely depleted at 16 d posthatch, and CD4+CD25+ cells from the thymus started to repopulate the bird at 16 d posthatch. Future experiments will identify whether a second anti-CD25 injection at 16 d posthatch will increase the duration of CD4+CD25+ cells depletion beyond 16 d posthatch.
tonsils and thymus compared with the cecal tonsils and thymus of the control group. Injecting anti-CD25 depletes Treg through FcγRIIImediated phagocytosis in mice. Macrophages were critical in depleting the Treg following anti-CD25 injection (Setiady et al., 2010). Injecting anti-CD25 in chickens likely decreased CD4+CD25+ cell percentages through this mechanism in the blood, spleen, and cecal tonsils. We earlier reported that a single intraperitoneal injection of anti-CD25 depletes CD4+CD25+ cells in the blood, spleen, and cecal tonsils but not in the thymus of chickens (Shanmugasundaram and Selvaraj, 2012b). In the current experiment, in ovo injection of anti-CD25
Experiment II CD4+CD25+ Cell Percentages in the Thymus, Spleen, and Cecal Tonsils at 16 d Posthatch. Similar to the near CD4+CD25+ cell depletion observed in blood at 16 d posthatch, anti-chicken CD25 antibody injection decreased the CD4+CD25+ cell percentage in the thymus, spleen, and cecal tonsils at 16 d posthatch (Figure 2). The CD4+CD25+ cell depletion was almost complete in the spleen of chicks hatched from anti-CD25-antibody-injected eggs. Chickens hatched from eggs injected with anti-CD25-antibodies had approximately 25% of the CD4+CD25+ cells in the cecal
Figure 2. The CD4+CD25+ cell percentages in the thymus, spleen, and cecal tonsils posthatch. Chicks were hatched from eggs injected with either 0.5 mg of anti-chicken CD25 monoclonal antibodies or 550 µL of PBS. At 16 d posthatch, the thymus, spleen, and cecal tonsils were analyzed for CD4+CD25+ cells. The CD4+CD25+ cells were expressed as a percentage of CD4+ cells. * indicates significant difference between treatments in that particular organ (P < 0.05). Means + SEM. n = 3.
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Statistical Analysis
Figure 1. The CD4+CD25+ cell percentages in peripheral blood posthatch. Chicks were hatched from eggs injected with either 0.5 mg of anti-chicken CD25 monoclonal antibodies or 550 µL of PBS. Blood was analyzed for CD4+ and CD4+CD25+ cells after concentrating for peripheral blood monocytes by density centrifugation. The CD4+CD25+ cells were expressed as a percentage of CD4+ peripheral blood monocytes. Means (±SEM). * indicates significant difference between treatments in that particular day (P < 0.05). n = 3.
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Figure 3. Relative IL-2 and IFNγ mRNA amounts in CD4+CD25− cells from the spleen and cecal tonsils at 16 d after antibody injection. Chicks were hatched from eggs injected with either 0.5 mg of anti-chicken CD25 monoclonal antibodies or 550 µL of PBS. The CD4+CD25− cells from the spleen and cecal tonsils were collected at 16 d posthatch. The mRNA contents were corrected for β-actin mRNA amounts and normalized to the mRNA contents of the PBS group so that all bars represent fold change compared with the PBS group. * indicates significant difference between treatments in that particular organ (P < 0.05). Means + SEM. n = 3.
cecal tonsils. A single injection of anti-chicken CD25 antibodies depleted CD4+CD25+ cells until 25 d posthatch. Anti-CD25 antibody-mediated Treg depletion has been applied to study the role of Treg in several bacterial infections in mammals. Similarly anti-chicken CD25-induced Treg depletion is a probable model to study the role of Treg in chickens during infections.
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decreased the number of cells in the thymus. In mice, in vivo injection of anti-CD25 antibodies downregulates CD25 expression in CD4+CD25+ cells, induces shedding of CD25 from the surface of CD4+CD25+ cells, or both (Kohm et al., 2006). It is possible that in ovo injection of anti-CD25 antibodies decreased the CD25 expression in CD4+CD25+ cells in the thymus. Relative IFNγ and IL-2 mRNA Amounts in CD4+CD25− Cells from the Spleen and Cecal Tonsils After Antibody Injection. The CD4+CD25- cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of IFNγ and IL-2 mRNA than CD4+CD25- cells from the control group (Figure 3). Interleukin-2 increases the proliferation of T cells. We earlier reported that CD4+CD25− cells from birds depleted of CD4+CD25+ cells proliferated more than cells from the control group (Shanmugasundaram and Selvaraj, 2012b). The presence of Tregs reduces the IL-2 mRNA amount in T cells of mice, and a Treg-mediated decrease in the IL-2 mRNA amount of T cells suppresses T cell proliferation (Feinerman et al., 2010). Depleting CD4+CD25+ cells, thus, can be expected to increase the IL-2 mRNA amount in CD4+CD25− cells. The IFNγ is an inflammatory cytokine, and Treg ablation enhances IFNγ production (Liu et al., 2010). IL10 suppresses IFNγ production in humans (D’Andrea et al., 1993). Depleting CD4+CD25+ cells, a source of IL-10 (Shanmugasundaram and Selvaraj, 2011), can be expected to increase the amount of IFNγ mRNA in the Treg-depleted group. In conclusion, injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted CD4+CD25+ cell percentages in the blood, spleen, and
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