Circulating γδ T Cells in Response to Salmonellaenterica Serovar Enteritidis Exposure in Chickens

Chickens.Specific-pathogen-free White Leghorn chickens were hatched at the facilities of the Friedrich-Loeffler-Institute, Jena, Germany, from eggs obtained from Charles River Deutschland GmbH, Extertal, Germany. Experimental and control groups were kept in separate rooms; commercial feed (in powder form without antibiotics or other additives) and drinking water were both available ad libitum. Cleaning and feeding regimens were organized which prevented cross-contamination effectively throughout the course of the experiment.

Experimental design and bacteriology. Salmonella vac E (TAD Pharmazeutisches Werk GmbH, Cuxhaven, Germany), a commercial live Salmonella enterica serovar Enteritidis (SE-LV) vaccine strain, and a nonattenuated Salmonellaenterica serovar Enteritidis 147 (SE 147) wild-type strain (33) were used for oral immunization of the chicks on their first day of life. The viable count of the attenuated SE-LV was 1 × 10⁸ to 2 × 10⁸ CFU per bird, and that of the nonattenuated SE 147 was 1× 10⁷ to 2 × 10⁷ CFU/bird. On day 44 of life, all immunized and nonimmunized chickens were infected orally using a nalidixic acid-resistant variant of the wild-type strain Salmonella enterica serovar Enteritidis 147 (SE 147 N) at a dose of 2 × 10⁸ CFU/bird. Oral administration of 0.1 ml bacterial suspension/bird was performed by instillation into the crop using a syringe with an attached flexible tube. Another control group was neither immunized nor infected. In summary, the following animal groups were investigated: group 1 (SE-LV/SE 147 N), immunization with SE-LV on day 1 of life and infection with SE 147 N on day 44 of life; group 2 (SE 147/SE 147 N), immunization with SE 147 on day 1 of life and infection with SE 147 N on day 44 of life; control group 3 (nothing/SE 147 N), no immunization, infection with SE 147 N on day 44 of life; control group 4 (nothing/nothing), no immunization, no infection.

All Salmonella strains used in this study were stored in the Microbank system (PRO-LAB Diagnostics, Ontario, Canada) at −20°C. The bacterial suspensions for immunization and infection were cultivated by shaking (18 h at 37°C) in nutrient broth (SIFIN, Berlin, Germany). Doses were estimated by measuring extinction at 600 nm against a calibration graph determined for the strains used and subsequently confirmed by plate counting on nutrient agar (SIFIN).

Bacterial counts in spleens were estimated after infection using a standard plating method as described previously (33). Briefly, homogenized organ samples were diluted in phosphate-buffered saline, plated on deoxycholate-citrate agar (SIFIN) supplemented with sodium nalidixate (50 μg/ml) to detect the challenge organisms (SE 147 N), and incubated at 37°C for 18 to 24 h.

Peripheral blood leukocyte isolation.To study the dynamic of γδ T-cell subsets after Salmonella exposure, peripheral blood leukocytes of five animals per day of examination (days 4, 7, 11, 14, 21, 24, 28, 32, 43, 45, 46, 49, 50, 52, 53, 56, 63, and 71 of life) and group were analyzed. For comparison of avian peripheral TCR1⁺ with TCR2⁺ and TCR3⁺ T cells, peripheral blood of chickens was analyzed between days 49 and 56 of life (in total, 15 nontreated animals). For that, the animals were sacrificed and completely bled to death. Lymphocytes of peripheral blood were isolated as described previously (8). Briefly, heparinized blood was mixed with 3% hetastarch (Sigma Immuno Chemicals, St. Louis, Mo.) and centrifuged at 65 × g for 10 min to allow erythrocytes to sediment. The leukocytes of the supernatant were washed twice in phosphate-buffered saline and subsequently used for flow cytometry.

Flow cytometry and cell sorting.For cell sorting, T-cell characterization, and analysis of the dynamic ofγδ T-cell subsets, fluorescein isothiocyanate-labeled mouse anti-avian TCR1 (γδ), TCR2 (αβ Vβ₁), and TCR3 (αβ Vβ₂) antibodies, an R-phycoerythrin-labeled mouse anti-avian CD8α monoclonal antibody, and biotin-conjugated mouse anti-avian CD4, CD8β, CD28 MHC class II antibodies (all from Southern Biotechnology Associates, Eching, Germany) were used. The biotin-conjugated antibodies were detected by Alexa Fluor 633-linked streptavidin (Molecular Probes, Leiden, The Netherlands). All antibody concentrations and dilutions were tested prior to starting the animal experiment. For every test, 2 × 10⁵ leukocytes were incubated in parallel with the appropriate monoclonal antibodies (30 min in the dark). Aliquots of 10,000 to 20,000 cells per sample were analyzed using a FACSCalibur (Becton Dickinson, Heidelberg, Germany) equipped with a 15-mW, 488-nm argon ion laser and a 633diode laser.

Additionally, γδ T-cell subpopulations (CD8α^+high CD8β⁺ TCR1⁺, CD8α^+high CD8β⁻ TCR1⁺, and CD8α⁻ CD8β⁻ TCR1⁺) of three SE 147-infected animals each on days 7, 11, and 14 of life were sorted by means of the FACSVantage SE instrument (Becton Dickinson, Heidelberg, Germany) for quantification of cytokine mRNA expression. For everyγδ T-cell subset, at least 50,000 cells were collected and stored at −80°C until use.

Quantitative real-time RT-PCR.Total RNA was extracted from cell samples using the RNeasy mini kit (QIAGEN, Hilden, Germany) by following the manufacturer's instructions, eluted in 50μ l RNase-free water per 50,000 cells, and stored at− 80°C. For functional characterization ofγδ T-cell subsets, mRNA expression of avian cytokine receptor chains (IL-2Rα and IL-7Rα) and apoptotic related proteins (Fas, Fas ligand, and Bcl-x) was determined by the QuantiTect SYBR green one-step reverse transcription (RT)-PCR kit (QIAGEN) according to the instructions of the manufacturer. Amplification and detection of specific products were performed using the Mx3000P real-time PCR system (Stratagene, La Jolla, CA) using the following cycle profile: one cycle of 50°C for 30 min and 96°C for 15 min, 45 cycles of 94°C for 30 s and appropriate annealing temperature for 30 s, followed by 72°C for 30 s. Primer sequences and annealing temperatures are given in Table 1. To avoid amplification of cellular DNA of each primer pair, at least one primer spans an intron-exon boundary. The threshold method was used for quantification of the mRNA level.Δ C_T (cycle threshold change) values were calculated on the basis of internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results were expressed as 40-ΔC_T values.

Immunohistochemistry.Samples from the cecum and spleen of animals (days 1 to 56 of life) were examined by immunohistochemistry to determine the invasion of different T-cell subsets. Cryostat sections of 7-μm thickness were prepared, and detection of cells was done using unlabeled primary monoclonal antibodies against the TCR1, CD8α, and CD8β antigens (Southern Biotechnology Associates, Eching, Germany) and a commercially available staining kit (PAP, ChemMate Detection kit, peroxidase antiperoxidase, rabbit/mouse; DakoCytomation, Hamburg, Germany). As a negative control, slides were incubated with preimmune mouse serum (dilution, 1:500) instead of the monoclonal antibodies. Sections were counterstained with hematoxylin and mounted with Canada balsam (Riedel de Haen AG, Seelze-Hannover, Germany).

Image analysis.Immunohistological tissue preparations were examined by light-microscopic image analysis (analySIS 3.0; Soft Imaging System GmbH). All measurements were performed at a magnification of ×20. Using the computer software, the regions of interest were drawn interactively as a polygon (cecum) or rectangle (spleen) on the screen. At least 5 regions of interest of each tissue section and antibody were scanned, the percentage of antibody-stained areas was determined, and the mean values were calculated.

Statistical analysis.The data of flow cytometric analyses and image analyses were evaluated statistically. The Student t test for comparison of two independent samples was used for statistical evaluation of differences between the groups (each immunized group against the control group). P values of≤ 0.05 were considered significant.

Viable counts of bacteria were converted into logarithmic form. For the purposes of statistical analysis, a viable log₁₀ count of <1.47 (limit for direct plate detection) obtained from a sample becoming positive only after enrichment was rated a log₁₀ value of 1.0. A sample which yielded no Salmonella growth after enrichment was rated a log₁₀ value of 0. Data were evaluated by multifactorial variance analysis. The factors considered were group and time. P values of <0.05 were regarded as statistically significant (software from Statgraphics Plus, Inc., Rockville, MD).

Clinical signs after Salmonella immunization and infection.In the present study, morphological and functional aspects of the γδ T-cell response in chickens after immunization and infection with S. enterica serovar Enteritidis were examined in blood and tissue.

The oral administration of attenuated live SE-LV did not result in morbidity or symptoms of intestinal inflammation. When using a dose of 1 × 10⁷ to 2 × 10⁷ CFU/bird of SE 147 in day-old chicks, strong colonization of the intestine and spread to liver was observed, but severe clinical signs were absent (34). Only a few birds showed signs of intestinal inflammation in the first week after administration. Infection with SE 147 N failed to generate any signs of morbidity or clinical symptoms in immunized and control birds.

Bacteriology.Oral immunization of chickens produced protective effects compared to nonimmunized controls (Table 2). Immunization using the nonattenuated S. enterica serovar Enteritidis wild-type strain 147 led to the most efficient protection, and no challenge organisms were detected in the spleen in the course of the experiment.

Birds immunized using an attenuated live Salmonella enterica serovar Enteritidis vaccine (SE-LV) revealed a considerably diminished number of the challenge strain in the spleen (reduction of 1.0 to 1.5 log₁₀ units) compared to nonimmunized controls.

Comparison of TCR1⁺ with TCR2⁺ and TCR3⁺ T lymphocytes in nontreated chickens.To compare avianγδ with αβ T lymphocytes in peripheral blood, double- and triple-staining experiments were performed. Flow cytometric analysis proved the presence of three different TCR1⁺, TCR2⁺, and TCR3⁺ T-cell subsets in the blood of nonimmunized animals (Fig. 1). These subpopulations could be discriminated by their variable intensity of CD8α antigen expression as follows: (i) CD8α^+high TCR1⁺, CD8α^+high TCR2⁺, and CD8α^+high TCR3⁺ cells (TCR⁺ cells expressing high concentrations of CD8α antigen); (ii) CD8α^+dim TCR1⁺, CD8α^+dim TCR2⁺, or CD8α^+dim TCR3⁺ cells (TCR⁺ cells expressing diminished concentrations of the CD8α antigen); (iii) CD8α⁻ TCR1⁺, CD8α⁻ TCR2⁺, or CD8α⁻ TCR3⁺ cells (TCR⁺ cells expressing no CD8α antigen).

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FIG. 1.

(A) Flow cytometric analysis of avian peripheral lymphocytes of a representative animal (56 days old). Gated cells shown in the forward scatter/side scatter dot plot diagram represent the lymphocyte population subjected to analysis of T-cell subpopulations (R1). The other dot plot diagrams show the two-color fluorescence analyses of lymphocytes for TCR1 and CD8α expression, TCR2 and CD8α expression, and TCR3 and CD8α expression of the gated lymphocytes. FITC, fluorescein isothiocyanate; RPE, R-phycoerythrin. (B) Percentages of cells positively stained for CD4, CD8β, CD28, or MHC class II antigen among the circulating TCR1⁺, TCR2⁺, and TCR3⁺ T-cell subsets defined in dependence on their CD8α antigen expression in nonimmunized animals (49 to 56 days old; in total, 15 animals). MHC class II staining of TCR3⁺ cells was not done.

The majority of γδ T cells were not found to express CD8α antigen on their surface. The CD8α^+high TCR1⁺ and CD8α^+dim TCR1⁺ cell populations occurred in similar proportions. In comparison, most of the TCR2⁺ and TCR3⁺ cells displayed diminished CD8α expression. A smaller amount of the TCR2⁺ and TCR3⁺ cells either expressed the CD8α antigen at a high concentration or showed no CD8α staining. Compared to TCR1⁺ and TCR2⁺ T cells, TCR3⁺ cells were only found in small quantities in blood (Fig. 1A).

For further characterization of αβ and γδ T cells defined above, the expression of CD4, CD8β, CD28, and MHC class II antigen was examined by flow cytometry (Fig. 1B). Most of the CD8α^+high TCR1⁺, CD8α^+high TCR2⁺, and CD8α^+high TCR3⁺ T cells additionally expressed the CD8β antigen, which means that these cells bear the CD8αβ heterodimeric form of the CD8 antigen. In contrast, most of the CD8α^+dim TCR1⁺, CD8α^+dim TCR2⁺, and CD8α^+dim TCR3⁺ T cells showed no CD8β expression, indicating the sole occurrence of the CD8αα homodimeric form of the CD8 antigen on the plasma membrane of these cells.

The CD28 antigen was found on approximately 30% of CD8α^+high TCR1⁺, CD8α^+high TCR2⁺, and CD8α^+high TCR3⁺ T-cell subsets. CD8α^+dim TCR1⁺, CD8α^+dim TCR2⁺, and CD8α⁻ TCR2⁺ T cells showed hardly any CD28 antigen expression. A high number (approximately 70%) of CD8α^+dim TCR3⁺ and CD8α⁻ TCR3⁺ T cells was CD28 positive.

The vast majority of CD8α^+dim TCR2⁺ and CD8α^+dim TCR3⁺ T cells, as well as CD8α⁻ TCR2⁺ and CD8α⁻ TCR3⁺ T cells, additionally expressed the CD4 antigen.

Approximately 40% of CD8α⁻ TCR2⁺ T cells also showed a distinct MHC class II expression. MHC class II expression of CD8⁺ TCR3⁺ cells was not investigated.

Dynamics of TCR1⁺ cell subsets after Salmonella immunization in blood.To examine the possible induction of a γδ T-cell response to Salmonella organisms, the percentage of TCR1⁺ T cells characterized by their CD8α antigen expression was analyzed within the circulating lymphocyte population of immunized chicks compared to nonimmunized control animals. Additionally, the expression of CD8β and CD28 on the defined TCR1⁺ cell subsets was investigated (Fig. 2).

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FIG. 2.

Dynamic of the percentages of CD8α^+high TCR1⁺ (A) and CD8α⁻ TCR1⁺ (B) T-cell subsets in peripheral blood of chicks immunized with Salmonella enterica serovar Enteritidis (SE-LV) vaccine strain or nonattenuated Salmonella enterica serovar Enteritidis 147 wild-type strain (SE 147) on day 1 of life and infected with Salmonella enterica serovar Enteritidis 147 N on day 44 of life compared to nonimmunized controls. Results are shown between days 1 and 71 (A) or 1 and 43 (B) of life. Data represent means ± standard deviations. Asterisks indicate a significant difference between the treated and control groups (P ≥ 0.05). (C) Percentages of CD8β⁺ cells among the CD8α^+high TCR1⁺ and CD8α^+dim TCR1⁺ T-cell subsets in peripheral blood of chickens immunized with the two different Salmonella enterica serovar Enteritidis (SE-LV or SE 147) strains on day 1 of life and infected with Salmonella enterica serovar Enteritidis 147 N on day 44 of life in comparison to nonimmunized controls. Results are shown between day 1 and day 71 of life. Data represent means ± standard deviations. Asterisks indicate a significant difference between the treated and the control group (P ≥ 0.05). (D) Percentages of CD28⁺ cells among the CD8α^+high TCR1⁺ subset in peripheral blood of chickens immunized with the two different Salmonella enterica serovar Enteritidis strains (SE-LV or SE 147) on day 1 of life and infected with Salmonella enterica serovar Enteritidis 147 N on day 44 of life in comparison to nonimmunized controls. Results are shown between day 1 and day 71 of life. Data represent means ± standard deviations. Asterisks indicate a significant difference between the treated and control groups (P ≥ 0.05).

CD8α^+high TCR1⁺ cells were generally found in very low numbers in the peripheral blood of immunized and nonimmunized chicks on day 4 of life. After immunization with SE-LV or SE 147 wild type, significantly (P < 0.05) higher numbers of theseγδ T cells were observed (Fig. 2A). The peak in the number of CD8α^+high TCR1⁺ cells was reached on days 7 (SE-LV) and 11 (SE 147) of life. Subsequently, the percentages decreased to the level of nonimmunized animals. The percentages of CD8α^+dim TCR1⁺ cells in peripheral blood were significantly increased (P < 0.05) in animals immunized with SE 147 and/or SE-LV compared to nonimmunized controls (data not shown). A peak was seen on days 7 and 11 (SE 147) and on day 14 (SE-LV) of life. Afterwards, the number of these cells dropped to the level of the control birds until day 21 of life. From day 21 onward, a moderate but significant decrease (P < 0.05) of these cells was detected in immunized chicks compared to controls (data not shown).

CD8α⁻ TCR1⁺ cells were significantly reduced (P < 0.05) in immunized animals compared to control birds. This phenomenon was observed from day 21 onward and was most conspicuous in chickens immunized with SE 147. On day 43 of life, the percentages of the CD8α⁻ TCR1⁺ T cells within the lymphocyte population of the immunized birds were nearly the same as found in controls (Fig. 2B).

As a consequence of immunization of day-old chicks, the CD8α^+high TCR1⁺-to-CD8α⁻ TCR1⁺ cell ratio within the γδ T-cell population of immunized animals shifted significantly in favor of the CD8α^+high TCR1⁺ cell subset between days 7 and 14 of life (data not shown).

In immunized birds, the number of CD8α^+high TCR1⁺ cells expressing the β chain of the CD8 antigen within the CD8α^+high TCR1⁺ cell population was significantly decreased between days 4 and 32 (Fig. 2C). This indicated an enormous increase of CD8αα^+high TCR1⁺ cells in this γδ T-cell subset.

Concerning the expression of the CD28 antigen on TCR1⁺ cells, an increase of CD28⁺ cells among the CD8α^+high TCR1⁺ T lymphocytes could be observed in immunized animals between day 4 and day 11 of age (Fig. 2D).

Dynamics of TCR1⁺ cell subsets after Salmonella infection in blood.To evaluate a possible second or memory immune response to Salmonella infection on day 44 of life, the percentages of TCR1⁺ T cells characterized by their CD8α antigen expression were analyzed in the previously immunized and nonimmunized chickens. Furthermore, the expression of CD8β and CD28 antigens on the defined TCR1⁺ cell subsets was studied after infection between days 44 and 71 (Fig. 2).

In peripheral blood, the nonimmunized but infected group showed a significant (P < 0.05) increase of CD8α^+high TCR1⁺ cells 5 and 6 days after infection (days 49 and 50 of life) compared with birds neither immunized nor infected with SE 147 N. In animals immunized on the first day of life, the percentages of the CD8α^+high TCR1⁺ cells remained unchanged after Salmonella infection compared to the nonimmunized group. The percentages of the CD8α^+dim TCR1⁺ and CD8⁻ TCR1⁺ cells were not significantly changed after infection.

Regarding CD28 antigen expression of the three TCR1⁺ T cell subsets, no changes were observed after infection of chickens immunized on the first day of life.

Dynamics of TCR1⁺, CD8α⁺, and CD8β⁺ cells after Salmonella infection in cecum and spleen.The local immune response after Salmonella immunization and infection has been examined by immunohistochemistry using monoclonal antibodies to CD8α, CD8β, and TCR1 antigens in the cecum and spleen.

In the cecum, significantly more CD8α⁺, CD8β⁺, and TCR1⁺ cells were detected in SE-LV- and SE 147-immunized chickens than in the controls between days 4 and 21 of life (Fig. 3). In the ceca of previously immunized chickens, a second significant increase of CD8α⁺, CD8β⁺, and at lower degree, TCR1⁺ cells were detected after challenge on day 44 of life. Whereas the SE 147-immunized animals showed the second increase very rapidly (CD8α, peak on day 45 of life; CD8β, peak on day 46 of life), in the SE-LV-immunized animals, this phenomenon appeared later after challenge (CD8α, peak on day 52 of life; CD8β, peak on day 50 of life). The percentages of cecal TCR1⁺ cells were also increased after challenge in SE 147- and SE-LV-immunized birds but showed high standard deviations.

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FIG. 3.

Percent positive area of TCR1⁺, CD8α⁺, and CD8β⁺ cells in ceca of chickens immunized with the two different Salmonella enterica serovar Enteritidis strains (SE-LV or SE 147) on day 1 of life and infected with Salmonella enterica serovar Enteritidis 147 N on day 44 of life in comparison to nonimmunized controls. Asterisks indicate a significant difference between the treated and control groups (P ≥ 0.05).

In spleens of SE-LV- and SE 147-immunized animals, the percentages of CD8α⁺ and TCR1⁺ T cells were increased between days 8 and 21 of life. In contrast, the number of CD8β⁺ cells was significantly decreased between days 7 and 21 (Fig. 4). After challenge on day 44 of the already immunized animals, no changes concerning the number of the investigated T-cell populations were detectable. Only the nonimmunized but challenged control animals showed a decrease of CD8β⁺ cells, which was observed on days 45 and 46.

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FIG. 4.

Percent positive area of TCR1⁺, CD8α⁺, and CD8β⁺ cells in spleens of chickens immunized with the two different Salmonella enterica serovar Enteritidis strains (SE-LV or SE 147) on day 1 of life and infected with Salmonella enterica serovar Enteritidis 147 N on day 44 of life in comparison to nonimmunized controls. Asterisks indicate a significant difference between the treated and control groups (P ≥ 0.05).

Quantification of cytokine mRNA expression of peripheral γδ T-cell subsets.The levels of mRNA expression of the γδ T-cell subpopulation (CD8α^+high CD8β⁺ TCR1⁺, CD8α^+high CD8β⁻ TCR1⁺, and CD8α⁻ CD8β⁻ TCR1⁺ cells) after SE 147 immunization are shown in Fig. 5. Higher IL-2Rα mRNA expression was evident in CD8α^+high CD8β⁻ TCR1⁺ (CD8αα^+highγδ T cells) cells than in CD8α^+high CD8β⁺ TCR1⁺ (CD8α^+highβ ⁺ γδ T cells) and CD8⁻ TCR1⁺ (CD8α⁻ γδ T cells) cells. Additionally, the CD8αα^+highγδ T cells showed lower expression of IL-7Rα and Bcl-x mRNA than both CD8α^+highβ ⁺ γδ cells and CD8α⁻ γδ T cells. There was an increase of IL-7Rα mRNA expression of the CD8αα^+high γδ T cells from days 7 to 14 after immunization.

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FIG. 5.

Expression of cytokine receptor chains (IL-2Rα and IL-7Rα) and apoptotic-related proteins (Fas, Fas-ligand, and Bcl-x) of peripheralγδ T-cell subsets (CD8α^+highβ⁺ TCR1⁺, CD8αα^+high TCR1⁺, and CD8α⁻ TCR1⁺ cells) after SE 147 immunization on day 1 of life. Values are given as corrected mean 40-ΔC_T (40-delta ct) values.

Hardly any difference of the Fas and Fas-L mRNA expression was found between the CD8αα^+high and CD8α^+highβ⁺γδ T-cell subsets. However, differences between CD8α^+high γδ cells and CD8α⁻ γδ cells were evident. Both CD8α^+high γδ T-cell subsets expressed more Fas and Fas-L mRNA than found in CD8α⁻ γδ T cells.