Circulating gamma delta T cells in response to Salmonella enterica serovar enteritidis exposure in chickens

Abstract

gammadelta T cells are considered crucial to the outcome of various infectious diseases. The present study was undertaken to characterize gammadelta (T-cell receptor 1(+) [TCR1(+)]) T cells phenotypically and functionally in avian immune response. Day-old chicks were orally immunized with Salmonella enterica serovar Enteritidis live vaccine or S. enterica serovar Enteritidis wild-type strain and infected using the S. enterica serovar Enteritidis wild-type strain on day 44 of life. Between days 3 and 71, peripheral blood was examined flow cytometrically for the occurrence of gammadelta T-cell subpopulations differentiated by the expression of T-cell antigens. Three different TCR1(+) cell populations were found to display considerable variation regarding CD8alpha antigen expression: (i) CD8alpha(+high) TCR1(+) cells, (ii) CD8alpha(+dim) TCR1(+) cells, and (iii) CD8alpha(-) TCR1(+) cells. While most of the CD8alpha(+high) TCR1(+) cells expressed the CD8alphabeta heterodimeric antigen, the majority of the CD8alpha(+dim) TCR1(+) cells were found to express the CD8alphaalpha homodimeric form. After immunization, a significant increase of CD8alphaalpha(+high) gammadelta T cells was observed within the CD8alpha(+high) TCR1(+) cell population. Quantitative reverse transcription-PCR revealed reduced interleukin-7 receptor alpha (IL-7Ralpha) and Bcl-x expression and elevated IL-2Ralpha mRNA expression of the CD8alphaalpha(+high) gammadelta T cells. Immunohistochemical analysis demonstrated a significant increase of CD8alpha(+) and TCR1(+) cells in the cecum and spleen and a decreased percentage of CD8beta(+) T cells in the spleen after Salmonella immunization. After infection of immunized animals, immune reactions were restricted to intestinal tissue. The study showed that Salmonella immunization of very young chicks is accompanied by an increase of CD8alphaalpha(+high) gammadelta T cells in peripheral blood, which are probably activated, and thus represent an important factor for the development of a protective immune response to Salmonella organisms in chickens.

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.

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).

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).

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).

FIG. 5.

Expression of cytokine receptor chains (IL-2Rα and IL-7Rα) and apoptotic-related proteins (Fas, Fas-ligand, and Bcl-x) of peripheral γδ 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.