Circulating {gamma}{delta} T Cells in Response to Salmonella enterica Serovar Enteritidis Exposure in Chickens

${gamma}$ ${delta}$ T cells are considered crucial to the outcome of various infectious diseases.The present study was undertaken to characterize ${gamma}$ ${delta}$ (T-cell receptor1⁺ [TCR1⁺]) T cells phenotypically and functionally in avianimmune response. Day-old chicks were orally immunized with Salmonellaenterica serovar Enteritidis live vaccine or S. enterica serovarEnteritidis wild-type strain and infected using the S. entericaserovar Enteritidis wild-type strain on day 44 of life. Betweendays 3 and 71, peripheral blood was examined flow cytometricallyfor the occurrence of ${gamma}$ ${delta}$ T-cell subpopulations differentiatedby the expression of T-cell antigens. Three different TCR1⁺cell populations were found to display considerable variationregarding CD8 ${alpha}$ antigen expression: (i) CD8 ${alpha}$ ^+high TCR1⁺ cells,(ii) CD8 ${alpha}$ ^+dim TCR1⁺ cells, and (iii) CD8 ${alpha}$ ^– TCR1⁺ cells. Whilemost of the CD8 ${alpha}$ ^+high TCR1⁺ cells expressed the CD8 ${alpha}$ ß heterodimericantigen, the majority of the CD8 ${alpha}$ ^+dim TCR1⁺ cells were foundto express the CD8 ${alpha}$ ${alpha}$ homodimeric form. After immunization, a significantincrease of CD8 ${alpha}$ ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells was observed within the CD8 ${alpha}$ ^+high TCR1⁺cell population. Quantitative reverse transcription-PCR revealedreduced interleukin-7 receptor ${alpha}$ (IL-7R ${alpha}$ ) and Bcl-x expressionand elevated IL-2R ${alpha}$ mRNA expression of the CD8 ${alpha}$ ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells.Immunohistochemical analysis demonstrated a significant increaseof CD8 ${alpha}$ ⁺ and TCR1⁺ cells in the cecum and spleen and a decreased percentageof CD8ß⁺ T cells in the spleen after Salmonella immunization.After infection of immunized animals, immune reactions wererestricted to intestinal tissue. The study showed that Salmonellaimmunization of very young chicks is accompanied by an increaseof CD8 ${alpha}$ ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells in peripheral blood, which are probablyactivated, and thus represent an important factor for the developmentof a protective immune response to Salmonella organisms in chickens.

INTRODUCTION

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Introduction

Bacteria of the genus Salmonella are responsible for a variety ofacute and chronic diseases in poultry. Moreover, paratyphoid Salmonellaorganisms are among the most important pathogens inducing food-bornezoonoses throughout the world. Poultry products represent asubstantial source of salmonella infection in humans, with Salmonellaenterica serovar Enteritidis and Salmonella enterica serovarTyphimurium being the serovars most frequently associated withhuman food poisoning. The manifestations and consequences ofparatyphoid Salmonella infections may vary markedly dependingon the age of the birds (5). While infections in young chicksare often characterized by considerable morbidity and mortality,birds aged some weeks and adults are far less susceptible to lethaleffects of Salmonella exposure and may experience intestinalcolonization as well as systemic dissemination without signs ofillness.

Vaccination is widely regarded as the most realistic approachto efficiently control the invasive serovars of S. entericaserovar Enteritidis and S. enterica serovar Typhimurium in thepoultry industry. In this context, comprehensive knowledge ofimmunological defense mechanisms of chickens against Salmonellaexposure represents a crucial prerequisite for successful vaccinedevelopment. The immune-stimulating properties of various Salmonellavaccine preparations have already been examined for their potentialto enhance antibody production, cell-mediated immunity, andinduction of invasion of different cell populations in severalorgans or peripheral blood (3, 8, 9, 13, 23, 24, 39). Notably,T-cell reactions and cellular immune responses seem to be ofcentral importance for defense against Salmonella infectionsin chickens (8, 9, 23, 24).

T lymphocytes, which are characterized by their expression ofspecial T cell receptors (TCR), have been classified into twosubgroups: ${alpha}$ ß and ${gamma}$ ${delta}$ T-cell receptor-bearing cells, withthe former comprising the classical CD4⁺ and CD8⁺ cells. Avian ${alpha}$ ß T cells are additionally subdivided into Vß₁and Vß₂ cells. Compared to the Vß₁ population,the Vß₂ ${alpha}$ ß T cells only constitute a smallproportion of the total ${alpha}$ ß T-cell population of avianperipheral blood. The ${gamma}$ ${delta}$ T-cell subgroup was first described inthe middle of the 1980s (4, 11). Meanwhile, a number of interestingdistinctions between ${gamma}$ ${delta}$ and ${alpha}$ ß T cells have been found,such as tissue distribution, repertoire restrictions, and nonclassical majorhistocompatibility complex (MHC) restriction (1, 22). In addition, ${gamma}$ ${delta}$ T cells were shown to possess antimicrobial activity in bacterial,parasitic, and viral infections (15, 19, 45) as well as to perform awide range of functions, such as cytokine production, cytotoxic activity,immunomodulation, granuloma organization, and regulation of inflammation(10, 12, 26, 35, 41). Based on the expression of different V ${gamma}$ or V ${delta}$ elements, tissue localization, TCR diversity, and the expressionof some surface antigens, ${gamma}$ ${delta}$ T cells could be divided into various functionallydifferent subpopulations in mice and humans (14), cattle (25, 46),and rats (42).

In mature chickens, T cells bearing the ${gamma}$ ${delta}$ TCR comprise up to50% of the peripheral T-cell pool (2). Recent studies in our labrevealed up-regulation of the percentage of CD8⁺- ${gamma}$ ${delta}$ -receptor-positiveT cells after S. enterica serovar Typhimurium infection of chicksin peripheral blood and tissues (8). Functional CD8 is a dimericprotein and exists either as a CD8 ${alpha}$ ß heterodimer oras a CD8 ${alpha}$ ${alpha}$ homodimer. These two forms are differentially expressedon a variety of lymphocytes and seem to serve distinct functions (21).Although the occurrence of a relatively large proportion ofCD8⁺ ${gamma}$ ${delta}$ T cells expressing only the ${alpha}$ -chain of the CD8 could beshown in spleens of embryos and young chicks (44), there isno knowledge of possible functions of these cells in chickens.In mice (28, 29) and humans (27), CD8 ${alpha}$ ${alpha}$ is expressed on ${alpha}$ ßT-cell subsets displaying a memory phenotype.

The development of memory CD8⁺ T cells is a dynamic multistageprocess that includes an initial T-cell activation with adjacentT-cell expansion, a contraction phase (deletion, activation-inducedcell death), and the final memory cell development (40). Morerecent work has indicated that cytokine receptors, such as interleukin-2receptor (IL-2R), IL-7R, and IL-15R, all of which include thecommon ${gamma}$ -chain as an integral signaling component, play an important rolein the development, survival, and homeostatic proliferationof lymphocyte populations. This has been most clearly establishedfor CD8⁺ T cells. Moreover, the expression of IL-2, IL-7, andIL-15 cytokines and their receptors have been shown to be crucialfor CD8⁺ T-cell memory generation and maintenance (40). On the otherhand, the accumulation of antiapoptotic factors, such as the Bcl-2family members Bcl-2 and Bcl-x_L, correlate with enhanced lymphocytesurvival (40, 36), and Fas-FasL interaction seems to be essentialfor peripheral T-cell deletion (36).

In the present study, peripheral blood CD8 ${alpha}$ -positive and -negative ${gamma}$ ${delta}$ T cells were characterized in detail with regard to their expressionof several surface antigens (CD4, CD8ß, CD28, andMHC class II) and compared with the Vß1 and Vß2 ${alpha}$ ßT cells of avian peripheral blood. Additionally, morphologicaland functional aspects of the ${gamma}$ ${delta}$ T-cell response in chickens afterimmunization and infection with S. enterica serovar Enteritidiswere examined in blood and tissue. For this purpose, changesof the proportions of ${gamma}$ ${delta}$ T-cell subsets characterized by CD8 andCD28 antigen expression, mRNA expression levels of IL-2R ${alpha}$ and IL-7R ${alpha}$ ,and apoptotic markers (Fas, Fas-L, and Bcl-x) were analyzed.

MATERIALS AND METHODS

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Materials and Methods

Chickens. Specific-pathogen-free White Leghorn chickens were hatched atthe 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 separaterooms; commercial feed (in powder form without antibiotics orother additives) and drinking water were both available ad libitum.Cleaning and feeding regimens were organized which preventedcross-contamination effectively throughout the course of theexperiment.

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 Salmonella entericaserovar Enteritidis 147 (SE 147) wild-type strain (33) wereused for oral immunization of the chicks on their first dayof life. The viable count of the attenuated SE-LV was 1 x 10⁸to 2 x 10⁸ CFU per bird, and that of the nonattenuated SE 147was 1 x 10⁷ to 2 x 10⁷ CFU/bird. On day 44 of life, all immunizedand nonimmunized chickens were infected orally using a nalidixicacid-resistant variant of the wild-type strain Salmonella entericaserovar Enteritidis 147 (SE 147 N) at a dose of 2 x 10⁸ CFU/bird.Oral administration of 0.1 ml bacterial suspension/bird wasperformed by instillation into the crop using a syringe withan attached flexible tube. Another control group was neitherimmunized nor infected. In summary, the following animal groupswere investigated: group 1 (SE-LV/SE 147 N), immunization withSE-LV on day 1 of life and infection with SE 147 N on day 44of life; group 2 (SE 147/SE 147 N), immunization with SE 147on day 1 of life and infection with SE 147 N on day 44 of life;control group 3 (nothing/SE 147 N), no immunization, infectionwith 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 theMicrobank system (PRO-LAB Diagnostics, Ontario, Canada) at –20°C.The bacterial suspensions for immunization and infection werecultivated by shaking (18 h at 37°C) in nutrient broth (SIFIN,Berlin, Germany). Doses were estimated by measuring extinctionat 600 nm against a calibration graph determined for the strainsused and subsequently confirmed by plate counting on nutrientagar (SIFIN).

Bacterial counts in spleens were estimated after infection usinga standard plating method as described previously (33). Briefly, homogenizedorgan samples were diluted in phosphate-buffered saline, platedon 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 ${gamma}$ ${delta}$ 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 comparisonof avian peripheral TCR1⁺ with TCR2⁺ and TCR3⁺ T cells, peripheralblood of chickens was analyzed between days 49 and 56 of life(in total, 15 nontreated animals). For that, the animals weresacrificed and completely bled to death. Lymphocytes of peripheralblood were isolated as described previously (8). Briefly, heparinizedblood was mixed with 3% hetastarch (Sigma Immuno Chemicals, St.Louis, Mo.) and centrifuged at 65 x g for 10 min to allow erythrocytesto sediment. The leukocytes of the supernatant were washed twicein phosphate-buffered saline and subsequently used for flowcytometry.

Flow cytometry and cell sorting. For cell sorting, T-cell characterization, and analysis of thedynamic of ${gamma}$ ${delta}$ T-cell subsets, fluorescein isothiocyanate-labeled mouseanti-avian TCR1 ( ${gamma}$ ${delta}$ ), TCR2 ( ${alpha}$ ß Vß₁), and TCR3( ${alpha}$ ß Vß₂) antibodies, an R-phycoerythrin-labeledmouse anti-avian CD8 ${alpha}$ monoclonal antibody, and biotin-conjugated mouseanti-avian CD4, CD8ß, CD28 MHC class II antibodies(all from Southern Biotechnology Associates, Eching, Germany)were used. The biotin-conjugated antibodies were detected byAlexa Fluor 633-linked streptavidin (Molecular Probes, Leiden,The Netherlands). All antibody concentrations and dilutionswere tested prior to starting the animal experiment. For everytest, 2 x 10⁵ leukocytes were incubated in parallel with theappropriate monoclonal antibodies (30 min in the dark). Aliquotsof 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, ${gamma}$ ${delta}$ T-cell subpopulations (CD8 ${alpha}$ ^+high CD8ß⁺TCR1⁺, CD8 ${alpha}$ ^+high CD8ß^– TCR1⁺, and CD8 ${alpha}$ ^– CD8ß^–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 ofcytokine mRNA expression. For every ${gamma}$ ${delta}$ T-cell subset, at least50,000 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 minikit (QIAGEN, Hilden, Germany) by following the manufacturer'sinstructions, eluted in 50 µl RNase-free water per 50,000cells, and stored at –80°C. For functional characterizationof ${gamma}$ ${delta}$ T-cell subsets, mRNA expression of avian cytokine receptorchains (IL-2R ${alpha}$ and IL-7R ${alpha}$ ) and apoptotic related proteins (Fas,Fas ligand, and Bcl-x) was determined by the QuantiTect SYBRgreen one-step reverse transcription (RT)-PCR kit (QIAGEN) accordingto the instructions of the manufacturer. Amplification and detectionof specific products were performed using the Mx3000P real-timePCR system (Stratagene, La Jolla, CA) using the following cycleprofile: one cycle of 50°C for 30 min and 96°C for 15min, 45 cycles of 94°C for 30 s and appropriate annealingtemperature for 30 s, followed by 72°C for 30 s. Primersequences and annealing temperatures are given in Table 1. Toavoid amplification of cellular DNA of each primer pair, atleast one primer spans an intron-exon boundary. The thresholdmethod was used for quantification of the mRNA level. ${Delta}$ C_T (cyclethreshold change) values were calculated on the basis of internalstandard glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Resultswere expressed as 40- ${Delta}$ C_T values.

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TABLE 1. Primer sequences for real-time RT-PCR

Immunohistochemistry. Samples from the cecum and spleen of animals (days 1 to 56 oflife) were examined by immunohistochemistry to determine theinvasion of different T-cell subsets. Cryostat sections of 7-µmthickness were prepared, and detection of cells was done usingunlabeled primary monoclonal antibodies against the TCR1, CD8 ${alpha}$ ,and CD8ß 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, slideswere incubated with preimmune mouse serum (dilution, 1:500)instead of the monoclonal antibodies. Sections were counterstainedwith hematoxylin and mounted with Canada balsam (Riedel de HaenAG, Seelze-Hannover, Germany).

Image analysis. Immunohistological tissue preparations were examined by light-microscopicimage analysis (analySIS 3.0; Soft Imaging System GmbH). Allmeasurements were performed at a magnification of x20. Usingthe computer software, the regions of interest were drawn interactivelyas a polygon (cecum) or rectangle (spleen) on the screen. Atleast 5 regions of interest of each tissue section and antibodywere 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 wereevaluated statistically. The Student t test for comparison oftwo independent samples was used for statistical evaluationof differences between the groups (each immunized group againstthe 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 countof <1.47 (limit for direct plate detection) obtained froma sample becoming positive only after enrichment was rated alog₁₀ value of 1.0. A sample which yielded no Salmonella growthafter enrichment was rated a log₁₀ value of 0. Data were evaluated bymultifactorial variance analysis. The factors considered weregroup and time. P values of <0.05 were regarded as statisticallysignificant (software from Statgraphics Plus, Inc., Rockville, MD).

RESULTS

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Results

Clinical signs after Salmonella immunization and infection. In the present study, morphological and functional aspects ofthe ${gamma}$ ${delta}$ T-cell response in chickens after immunization and infectionwith S. enterica serovar Enteritidis were examined in bloodand tissue.

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

Bacteriology. Oral immunization of chickens produced protective effects comparedto nonimmunized controls (Table 2). Immunization using the nonattenuatedS. enterica serovar Enteritidis wild-type strain 147 led tothe most efficient protection, and no challenge organisms weredetected in the spleen in the course of the experiment.

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TABLE 2. Number of challenge organisms of Salmonella enterica serovar Enteritidis 147 N in spleens of immunized and control chickens

Birds immunized using an attenuated live Salmonella entericaserovar Enteritidis vaccine (SE-LV) revealed a considerablydiminished number of the challenge strain in the spleen (reductionof 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 avian ${gamma}$ ${delta}$ with ${alpha}$ ß T lymphocytes in peripheral blood,double- and triple-staining experiments were performed. Flow cytometricanalysis 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 theirvariable intensity of CD8 ${alpha}$ antigen expression as follows: (i) CD8 ${alpha}$ ^+highTCR1⁺, CD8 ${alpha}$ ^+high TCR2⁺, and CD8 ${alpha}$ ^+high TCR3⁺ cells (TCR⁺ cellsexpressing high concentrations of CD8 ${alpha}$ antigen); (ii) CD8 ${alpha}$ ^+dim TCR1⁺,CD8 ${alpha}$ ^+dim TCR2⁺, or CD8 ${alpha}$ ^+dim TCR3⁺ cells (TCR⁺ cells expressingdiminished concentrations of the CD8 ${alpha}$ antigen); (iii) CD8 ${alpha}$ ^–TCR1⁺, CD8 ${alpha}$ ^– TCR2⁺, or CD8 ${alpha}$ ^– TCR3⁺ cells (TCR⁺ cellsexpressing no CD8 ${alpha}$ 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 CD8 ${alpha}$ expression, TCR2 and CD8 ${alpha}$ expression, and TCR3 and CD8 ${alpha}$ expression of the gated lymphocytes. FITC, fluorescein isothiocyanate; RPE, R-phycoerythrin. (B) Percentages of cells positively stained for CD4, CD8ß, CD28, or MHC class II antigen among the circulating TCR1⁺, TCR2⁺, and TCR3⁺ T-cell subsets defined in dependence on their CD8 ${alpha}$ 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 ${gamma}$ ${delta}$ T cells were not found to express CD8 ${alpha}$ antigenon their surface. The CD8 ${alpha}$ ^+high TCR1⁺ and CD8 ${alpha}$ ^+dim TCR1⁺ cell populationsoccurred in similar proportions. In comparison, most of the TCR2⁺and TCR3⁺ cells displayed diminished CD8 ${alpha}$ expression. A smalleramount of the TCR2⁺ and TCR3⁺ cells either expressed the CD8 ${alpha}$ antigen at a high concentration or showed no CD8 ${alpha}$ staining. Comparedto TCR1⁺ and TCR2⁺ T cells, TCR3⁺ cells were only found in smallquantities in blood (Fig. 1A).

For further characterization of ${alpha}$ ß and ${gamma}$ ${delta}$ T cells definedabove, the expression of CD4, CD8ß, CD28, and MHCclass II antigen was examined by flow cytometry (Fig. 1B). Mostof the CD8 ${alpha}$ ^+high TCR1⁺, CD8 ${alpha}$ ^+high TCR2⁺, and CD8 ${alpha}$ ^+high TCR3⁺ Tcells additionally expressed the CD8ß antigen, whichmeans that these cells bear the CD8 ${alpha}$ ß heterodimericform of the CD8 antigen. In contrast, most of the CD8 ${alpha}$ ^+dim TCR1⁺,CD8 ${alpha}$ ^+dim TCR2⁺, and CD8 ${alpha}$ ^+dim TCR3⁺ T cells showed no CD8ßexpression, indicating the sole occurrence of the CD8 ${alpha}$ ${alpha}$ homodimeric formof the CD8 antigen on the plasma membrane of these cells.

The CD28 antigen was found on approximately 30% of CD8 ${alpha}$ ^+highTCR1⁺, CD8 ${alpha}$ ^+high TCR2⁺, and CD8 ${alpha}$ ^+high TCR3⁺ T-cell subsets. CD8 ${alpha}$ ^+dimTCR1⁺, CD8 ${alpha}$ ^+dim TCR2⁺, and CD8 ${alpha}$ ^– TCR2⁺ T cells showed hardlyany CD28 antigen expression. A high number (approximately 70%)of CD8 ${alpha}$ ^+dim TCR3⁺ and CD8 ${alpha}$ ^– TCR3⁺ T cells was CD28 positive.

The vast majority of CD8 ${alpha}$ ^+dim TCR2⁺ and CD8 ${alpha}$ ^+dim TCR3⁺ T cells, aswell as CD8 ${alpha}$ ^– TCR2⁺ and CD8 ${alpha}$ ^– TCR3⁺ T cells, additionallyexpressed the CD4 antigen.

Approximately 40% of CD8 ${alpha}$ ^– TCR2⁺ T cells also showed adistinct 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 ${gamma}$ ${delta}$ T-cell response to Salmonellaorganisms, the percentage of TCR1⁺ T cells characterized bytheir CD8 ${alpha}$ antigen expression was analyzed within the circulatinglymphocyte population of immunized chicks compared to nonimmunizedcontrol animals. Additionally, the expression of CD8ßand CD28 on the defined TCR1⁺ cell subsets was investigated(Fig. 2).

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FIG. 2. Dynamic of the percentages of CD8 ${alpha}$ ^+high TCR1⁺ (A) and CD8 ${alpha}$ ^– 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 CD8ß⁺ cells among the CD8 ${alpha}$ ^+high TCR1⁺ and CD8 ${alpha}$ ^+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 CD8 ${alpha}$ ^+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).

CD8 ${alpha}$ ^+high TCR1⁺ cells were generally found in very low numbers inthe peripheral blood of immunized and nonimmunized chicks onday 4 of life. After immunization with SE-LV or SE 147 wildtype, significantly (P < 0.05) higher numbers of these ${gamma}$ ${delta}$ Tcells were observed (Fig. 2A). The peak in the number of CD8 ${alpha}$ ^+highTCR1⁺ cells was reached on days 7 (SE-LV) and 11 (SE 147) oflife. Subsequently, the percentages decreased to the level ofnonimmunized animals. The percentages of CD8 ${alpha}$ ^+dim TCR1⁺ cellsin peripheral blood were significantly increased (P < 0.05)in animals immunized with SE 147 and/or SE-LV compared to nonimmunizedcontrols (data not shown). A peak was seen on days 7 and 11(SE 147) and on day 14 (SE-LV) of life. Afterwards, the numberof these cells dropped to the level of the control birds untilday 21 of life. From day 21 onward, a moderate but significantdecrease (P < 0.05) of these cells was detected in immunizedchicks compared to controls (data not shown).

CD8 ${alpha}$ ^– TCR1⁺ cells were significantly reduced (P < 0.05) inimmunized animals compared to control birds. This phenomenonwas observed from day 21 onward and was most conspicuous in chickensimmunized with SE 147. On day 43 of life, the percentages of theCD8 ${alpha}$ ^– TCR1⁺ T cells within the lymphocyte population ofthe immunized birds were nearly the same as found in controls(Fig. 2B).

As a consequence of immunization of day-old chicks, the CD8 ${alpha}$ ^+high TCR1⁺-to-CD8 ${alpha}$ ^– TCR1⁺cell ratio within the ${gamma}$ ${delta}$ T-cell population of immunized animalsshifted significantly in favor of the CD8 ${alpha}$ ^+high TCR1⁺ cell subsetbetween days 7 and 14 of life (data not shown).

In immunized birds, the number of CD8 ${alpha}$ ^+high TCR1⁺ cells expressingthe ß chain of the CD8 antigen within the CD8 ${alpha}$ ^+highTCR1⁺ cell population was significantly decreased between days4 and 32 (Fig. 2C). This indicated an enormous increase of CD8 ${alpha}$ ${alpha}$ ^+high TCR1⁺cells in this ${gamma}$ ${delta}$ T-cell subset.

Concerning the expression of the CD28 antigen on TCR1⁺ cells,an increase of CD28⁺ cells among the CD8 ${alpha}$ ^+high TCR1⁺ T lymphocytescould be observed in immunized animals between day 4 and day11 of age (Fig. 2D).

Dynamics of TCR1⁺ cell subsets after Salmonella infection in blood. To evaluate a possible second or memory immune response to Salmonellainfection on day 44 of life, the percentages of TCR1⁺ T cells characterizedby their CD8 ${alpha}$ antigen expression were analyzed in the previouslyimmunized and nonimmunized chickens. Furthermore, the expressionof CD8ß and CD28 antigens on the defined TCR1⁺ cellsubsets was studied after infection between days 44 and 71 (Fig. 2).

In peripheral blood, the nonimmunized but infected group showeda significant (P < 0.05) increase of CD8 ${alpha}$ ^+high TCR1⁺ cells5 and 6 days after infection (days 49 and 50 of life) comparedwith birds neither immunized nor infected with SE 147 N. Inanimals immunized on the first day of life, the percentagesof the CD8 ${alpha}$ ^+high TCR1⁺ cells remained unchanged after Salmonellainfection compared to the nonimmunized group. The percentagesof the CD8 ${alpha}$ ^+dim TCR1⁺ and CD8^– TCR1⁺ cells were not significantlychanged after infection.

Regarding CD28 antigen expression of the three TCR1⁺ T cellsubsets, no changes were observed after infection of chickensimmunized on the first day of life.

Dynamics of TCR1⁺, CD8 ${alpha}$ ⁺, and CD8ß⁺ cells after Salmonella infection in cecum and spleen. The local immune response after Salmonella immunization andinfection has been examined by immunohistochemistry using monoclonalantibodies to CD8 ${alpha}$ , CD8ß, and TCR1 antigens in thececum and spleen.

In the cecum, significantly more CD8 ${alpha}$ ⁺, CD8ß⁺, and TCR1⁺cells were detected in SE-LV- and SE 147-immunized chickensthan in the controls between days 4 and 21 of life (Fig. 3).In the ceca of previously immunized chickens, a second significant increaseof CD8 ${alpha}$ ⁺, CD8ß⁺, and at lower degree, TCR1⁺ cells weredetected after challenge on day 44 of life. Whereas the SE 147-immunizedanimals showed the second increase very rapidly (CD8 ${alpha}$ , peak onday 45 of life; CD8ß, peak on day 46 of life), inthe SE-LV-immunized animals, this phenomenon appeared laterafter challenge (CD8 ${alpha}$ , peak on day 52 of life; CD8ß,peak on day 50 of life). The percentages of cecal TCR1⁺ cellswere also increased after challenge in SE 147- and SE-LV-immunizedbirds but showed high standard deviations.

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FIG. 3. Percent positive area of TCR1⁺, CD8 ${alpha}$ ⁺, and CD8ß⁺ 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 percentagesof CD8 ${alpha}$ ⁺ and TCR1⁺ T cells were increased between days 8 and21 of life. In contrast, the number of CD8ß⁺ cellswas 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-cellpopulations were detectable. Only the nonimmunized but challengedcontrol animals showed a decrease of CD8ß⁺ cells,which was observed on days 45 and 46.

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FIG. 4. Percent positive area of TCR1⁺, CD8 ${alpha}$ ⁺, and CD8ß⁺ 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 ${gamma}$ ${delta}$ T-cell subsets. The levels of mRNA expression of the ${gamma}$ ${delta}$ T-cell subpopulation (CD8 ${alpha}$ ^+highCD8ß⁺ TCR1⁺, CD8 ${alpha}$ ^+high CD8ß^– TCR1⁺,and CD8 ${alpha}$ ^– CD8ß^– TCR1⁺ cells) after SE 147immunization are shown in Fig. 5. Higher IL-2R ${alpha}$ mRNA expressionwas evident in CD8 ${alpha}$ ^+high CD8ß^– TCR1⁺ (CD8 ${alpha}$ ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells) cells than in CD8 ${alpha}$ ^+high CD8ß⁺ TCR1⁺ (CD8 ${alpha}$ ^+high ß⁺ ${gamma}$ ${delta}$ T cells) and CD8^– TCR1⁺ (CD8 ${alpha}$ ^– ${gamma}$ ${delta}$ T cells) cells. Additionally,the CD8 ${alpha}$ ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells showed lower expression of IL-7R ${alpha}$ and Bcl-xmRNA than both CD8 ${alpha}$ ^+high ß⁺ ${gamma}$ ${delta}$ cells and CD8 ${alpha}$ ^– ${gamma}$ ${delta}$ T cells. There was an increase of IL-7R ${alpha}$ mRNA expression of the CD8 ${alpha}$ ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells from days 7 to 14 after immunization.

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FIG. 5. Expression of cytokine receptor chains (IL-2R ${alpha}$ and IL-7R ${alpha}$ ) and apoptotic-related proteins (Fas, Fas-ligand, and Bcl-x) of peripheral ${gamma}$ ${delta}$ T-cell subsets (CD8 ${alpha}$ ^+highß⁺ TCR1⁺, CD8 ${alpha}$ ${alpha}$ ^+high TCR1⁺, and CD8 ${alpha}$ ^– TCR1⁺ cells) after SE 147 immunization on day 1 of life. Values are given as corrected mean 40- ${Delta}$ C_T (40-delta ct) values.

Hardly any difference of the Fas and Fas-L mRNA expression wasfound between the CD8 ${alpha}$ ${alpha}$ ^+high and CD8 ${alpha}$ ^+highß⁺ ${gamma}$ ${delta}$ T-cellsubsets. However, differences between CD8 ${alpha}$ ^+high ${gamma}$ ${delta}$ cells and CD8 ${alpha}$ ^– ${gamma}$ ${delta}$ cells were evident. Both CD8 ${alpha}$ ^+high ${gamma}$ ${delta}$ T-cell subsets expressedmore Fas and Fas-L mRNA than found in CD8 ${alpha}$ ^– ${gamma}$ ${delta}$ T cells.

DISCUSSION

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Discussion

In the present study, the phenotype of avian T cells, particularly CD8 ${alpha}$ ⁺ ${gamma}$ ${delta}$ T cells, was specified to gain more insight into the role ofsingle ${gamma}$ ${delta}$ T-cell subsets in defense against Salmonella organismsof chickens. Based on our previous findings (8), the dynamics ofCD8⁺ and ${gamma}$ ${delta}$ T-cell subsets was investigated after immunizationand infection of chickens with attenuated and nonattenuatedSalmonella enterica serovar Enteritidis strains in peripheralblood and organs.

The phenotypic characterization of avian TCR1⁺, TCR2⁺, and TCR3⁺cells demonstrated the presence of completely separate cellpopulations not only concerning the TCR antigen but also becauseof the expression of further antigens. A closer relationshipbetween the TCR2⁺ and the TCR3⁺ subpopulations than with TCR1⁺cells was shown by the relatively high frequency of CD4⁺ antigen expressionof both subpopulations. The detected different expression of theCD28 antigen on TCR2⁺ and TCR3⁺ cells indicates distinct activationlevels of these T-cell populations. It has been already shownthat TCR2⁺ and TCR3⁺ cells are separate sublineages of avianT cells by observation of a compensatory increase in TCR3⁺ cellfrequency when the development of the TCR2⁺ subpopulation wasinhibited (17). Other authors described the ability of TCR2⁺cells, but not of TCR3⁺ cells, to migrate to the chicken intestine, wherethey provide help to mucosal B cells for immunoglobulin A production (16).

Each of the three TCR subsets could be further subdivided inthree single subsets based on their intensive, weak, or absentCD8 ${alpha}$ expression. In contrast to CD8 ${alpha}$ ^+dim TCR1⁺ cells, CD8 ${alpha}$ ^+dim TCR2⁺and CD8 ${alpha}$ ^+dim TCR3⁺ cells of the White Leghorn chickens additionallyexpressed the CD4 antigen. These cells did not express the CD8ßchain, indicating the expression of only the CD8 ${alpha}$ ${alpha}$ homodimericform of the CD8 antigen. Accordingly, other studies demonstratedthat, in some H.B15 chickens, a large subpopulation of peripheralblood CD4⁺ T cells expresses only CD8 ${alpha}$ mRNA but not CD8ß (30).The same authors have shown that these CD4⁺ CD8 ${alpha}$ ${alpha}$ T cells caninduce an in vivo alloreaction comparable to that of normal CD4⁺T cells, which represents direct evidence for peripheral bloodCD4⁺ CD8⁺ T cells functioning as helper CD4 T cells. It seemsthat the expression of CD8 ${alpha}$ does not interfere with the functionof peripheral blood CD4⁺ T cells in chickens.

Based on CD8 ${alpha}$ antigen expression, we demonstrated for the firsttime the existence of three ${gamma}$ ${delta}$ T-cell subsets in avian blood and showedthat most of circulating CD8^+high TCR1⁺ cells express the ${alpha}$ ß heterodimericform of the CD8 antigen, whereas CD8^+dim TCR1⁺ cells presentthe ${alpha}$ ${alpha}$ homodimeric form of the CD8 antigen. It seems that ${gamma}$ ${delta}$ T cellsare able to express both forms of the CD8 antigen dependingon their localization or species investigated. Straube and Herrmann(42) considered rats to be the only species with frequent expressionof the CD8 ${alpha}$ ß antigen by ${gamma}$ ${delta}$ T cells. In spleens of avianembryos and young chicks, a relatively large proportion of CD8⁺ ${gamma}$ ${delta}$ T cells expressing only the ${alpha}$ -chain of CD8 has been found (44).

The three ${gamma}$ ${delta}$ T-cell subsets defined in dependence on their CD8 ${alpha}$ antigen expression showed different reactions after Salmonellaimmunization of day-old chicks. This fact indicates not onlyphenotypic but also functional differences between these subsets.A difference in the overall functional and/or activation statusof human circulating CD8^– versus CD8⁺ ${gamma}$ ${delta}$ T cells has beenpostulated (25). A comparison of avian CD8 ${alpha}$ ^+high and CD8 ${alpha}$ ^+dim ${gamma}$ ${delta}$ T cells in our experiment showed that the response of CD8 ${alpha}$ ^+highT cells was more evident. In swine, possible functional differencesbetween CD8^+dim and CD8^+high ${alpha}$ ß T cells have been discussedin connection with different expression of a second ${alpha}$ or ßchain of the CD8 antigen (38, 43).

The detected decrease of CD8ß expression on CD8 ${alpha}$ ^+highTCR1⁺ cells after Salmonella immunization indicates an increasein the number of CD8 ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells expressing the CD8 ${alpha}$ ${alpha}$ homodimer.Additionally, a decreased number of CD8ß⁺ T cellswas seen in the spleens of immunized chickens. Whether thiswas due to a down-regulation of the CD8ß antigen onthe single cell or an influx of CD8 ${alpha}$ ⁺ homodimeric ${gamma}$ ${delta}$ T cells hasyet to be examined. However, this is the first demonstrationof an increase of CD8 ${alpha}$ ${alpha}$ ^+high circulating ${gamma}$ ${delta}$ T cells after Salmonellaexposure of chicks. Similarly, rat splenic T cells showed persistent down-regulationof the expression of CD8ß but not of CD8 ${alpha}$ on ${gamma}$ ${delta}$ T cellsafter in vitro activation (42). CD8 ${alpha}$ ß is more effectivethan CD8 ${alpha}$ ${alpha}$ in facilitating recognition of the same peptide antigenby TCR in vitro (47). Thus, the CD8ß down-regulationcould presumably be a mechanism for limiting the generationof TCR-mediated signals after Salmonella infection in vivo.Moreover, a requirement of transient CD8 ${alpha}$ ${alpha}$ expression for memoryT-cell generation has been proven by use of E8₁-deficient micethat cannot generate CD8 ${alpha}$ ${alpha}$ homodimers (31). In mice (28, 29) andhumans (27), the CD8 ${alpha}$ ${alpha}$ antigen is expressed on distinct T-cellsubsets that constitutively display a memory phenotype. That ${gamma}$ ${delta}$ T-cell subsets are also able to differentiate into memory Tcells has already been shown in humans (7, 20). However, itis not known whether the rise of CD8 ${alpha}$ ${alpha}$ homodimeric ${gamma}$ ${delta}$ T cells canbe considered a suitable marker for memory T-cell generationor memory T cells themselves in chickens.

In this study, avian CD8 ${alpha}$ ${alpha}$ ^+high homodimeric, CD8 ${alpha}$ ^+highß⁺ heterodimeric,and CD8 ${alpha}$ ^– ${gamma}$ ${delta}$ T-cell subsets were characterized concerningtheir mRNA expression for interleukin receptor chains involvedin the memory generation (IL-2R ${alpha}$ and IL-7R ${alpha}$ ) and the apoptosis-relatedproteins Fas, Fas-ligand, and Bcl-x after Salmonella stimulationfor the first time. It was demonstrated that all three avian ${gamma}$ ${delta}$ T-cell subsets were able to express mRNA of the investigatedproteins. CD8 ${alpha}$ ${alpha}$ ^+high homodimeric ${gamma}$ ${delta}$ T cells showed relatively reducedlevels of IL-7R ${alpha}$ and Bcl-x but an elevated level of IL-2R ${alpha}$ mRNA expressioncompared to the CD8 ${alpha}$ ß heterodimeric and CD8 ${alpha}$ ^– ${gamma}$ ${delta}$ T cells. Interestingly, the expression of the IL-7R ${alpha}$ mRNA increasedover the time of investigation from 6 to 13 days after Salmonella immunization.These results, together with the observed significant increaseof the percentage of CD8 ${alpha}$ ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells and the increased CD28antigen expression on the CD8 ${alpha}$ ^+high ${gamma}$ ${delta}$ T cells after immunization,led to the assumption that the observed CD8 ${alpha}$ ${alpha}$ ^+high homodimeric ${gamma}$ ${delta}$ T-cell subset represented activated ${gamma}$ ${delta}$ T cells after clonal expansionthat underwent the contraction phase of the primary immune responseagainst Salmonella enterica serovar Enteritidis. Recent datashowed that T-cell stimulation induces considerable modificationsof IL-7R ${alpha}$ chain expression (37, 40). While naive T cells areIL-7R ${alpha}$ positive, T-cell activation leads to down-regulation ofIL-7R ${alpha}$ and large-scale apoptotic episodes thereafter, resultingin a substantial reduction in the number of antigen-specific Tcells. Only a subpopulation of T cells escapes death and remainsa stable population of memory T cells. Progression to memoryis accompanied by the reexpression of the IL-7R ${alpha}$ chain (37, 40).

A decrease in the percentage of peripheral CD8^– ${gamma}$ ${delta}$ T cellsin Salmonella enterica serovar Enteritidis-immunized birds wasfound. This might be due to (i) an up-regulation of CD8 moleculeexpression or (ii) a fast migration of CD8^– ${gamma}$ ${delta}$ T cells tosites of infection, such as the intestine or spleen. It hasbeen shown earlier, as well as in this study, that ${gamma}$ ${delta}$ T cellsoccur in higher numbers in the gut and spleen after immunizationof chickens with Salmonella enterica serovar Typhimurium (8)and Salmonella enterica serovar Enteritidis, respectively. Theperipheral blood may be a major source for ${gamma}$ ${delta}$ T cells of the intestineand vice versa. A proliferation and rapid recirculation of intestinal ${gamma}$ ${delta}$ T cells into the blood was seen in swine (43). Additionally,it has been shown that bovine CD8^– ${gamma}$ ${delta}$ T cells of newborncalves express E-selectin ligands, which are responsible foradhesion, and that they are able to migrate to acute and chronicsites of inflammation (18, 32).

After infection, splenic Salmonella clearance was more rapidin immunized animals than in nonimmunized controls. Immunizationusing the nonattenuated S. enterica serovar Enteritidis wild-typestrain 147 produced the strongest protection: no challenge organismswere detected in the spleen during the course of the experiment.The rapid clearance of the Salmonella organisms after infectionof Salmonella-immunized birds did not correlate with changesof T-cell composition in peripheral blood or spleen after infection.In contrast, our data showed a restriction of the secondarycellular immune response to the intestine after Salmonella entericaserovar Enteritidis infection of already immunized birds. Astrong increase of CD8⁺ and TCR1⁺ T cells in the intestinallamina propria could be observed quickly after challenge especiallyof SE 147-immunized animals. Thus, the Salmonella clearancein the spleen correlated well with the observed secondary immuneresponse in the cecum, as higher percentages of CD8⁺ and TCR1⁺T cells in the cecum mean lower numbers of Salmonella organismsin the spleen after challenge. Similarly, Beal et al. (6) founda restriction of the antigen-specific humoral and cellular immune responseto the intestine after Salmonella enterica serovar Typhimuriuminfection of immunized chickens.

Generally speaking, immunization of day-old chicks using liveattenuated or nonattenuated Salmonella strains resulted in variable reactionsof ${gamma}$ ${delta}$ T-cell subsets. The emergence of ${gamma}$ ${delta}$ T-cell populations inthe blood indicates that they are responding to the pathogenand/or providing a protective immune response. The lower ${gamma}$ ${delta}$ T-cellresponse after Salmonella live vaccine immunization than after Salmonellawild-type strain immunization of day-old chicks on the otherhand seems to lead to a weaker protection after Salmonella infection.Distinct reactions of the defined circulating ${gamma}$ ${delta}$ T-cell subsetsafter Salmonella immunization and infection of chickens indicatethat phenotypically characterized ${gamma}$ ${delta}$ T-cell subsets may servedifferent immunological purposes in chickens. The detected down-regulationof the CD8ß antigen on avian circulating CD8 ${alpha}$ ^+highTCR1⁺ cells and the low IL-7R ${alpha}$ mRNA expression of CD8 ${alpha}$ ${alpha}$ ^+high ${gamma}$ ${delta}$ T cellsafter Salmonella immunization of young chicks in this studymight be a consequence of a transient activation or specializationof a single ${gamma}$ ${delta}$ T-cell subset in vivo that eventually may leadto the generation of specific memory T cells.

ACKNOWLEDGMENTS

This study was supported by the European Union, grant FOOD-CT-2003-505523.

We thank Konrad Sachse for helpful assistance in editing the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Friedrich-Loeffler-Institute, Institute of Molecular Pathogenesis, Naumburger Str. 96a, D-07743 Jena, Germany. Phone: 49 3641 804 410. Fax: 49 3641 804 228. E-mail: angela.berndt{at}fli.bund.de.

Editor: F. C. Fang

REFERENCES

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