Functionality of CD4+ and CD8+ T cells from tonsillar tissue

Abstract

For many years, tonsillectomy has been used routinely in children to treat chronic or recurrent acute tonsillitis. Palatine tonsils are secondary lymphoid organs and the major barrier protecting the digestive and respiratory tracts from potential invasive microorganisms. They have been used as sources of lymphoid tissue; however, despite the hundreds of papers published on tonsillectomy, no studies addressing the functionality of the CD4(+) and CD8(+) T cells from chronically infected tonsils have yet been published. The aim of this study was to analyse the functionality of the CD4(+) and CD8(+) T cells with respect to tonsillar tissue. We used an affordable approach to measure the frequency of antigen-specific CD4(+) T cells, the direct ex-vivo cytotoxicity of CD8(+) T cells, memory T cell phenotype, cytokine profile and DC phenotype. Our results demonstrate that CD4(+) and CD8(+) T cells from tonsillar tissue are totally functional, as shown by their ability to produce cytokines, to degranulate and to differentiate into effector-memory T cells.

Fig. 1

Phenotypical profile of memory CD41 and CD81 T cell subsets using multi-colour flow cytometry. Tonsillar mononuclear cells were stained with directly conjugated monoclonal antibodies against CD3, CD4, CD8, CD45RA, CD62L and CCR7 in a six-colour experiment. Cells were first identified based on side-scatter and CD3 properties, followed by gating on CD4⁺ or CD8⁺ T cells. Differential gating on CD4⁺ or CD8⁺ T cells based on CD62L and CD45RA basal expression revealed four populations: naive T cells (CD45RA⁺CD62L⁺), central memory T cells (CD45RA^-CD62L⁺), effector memory T cells (CD45RA^-CD62L^-) and terminally differentiated effector memory T cells (TEMRA) (CD45RA⁺CD62L^-). At least 10⁵ events were analysed.

Fig. 2

Assessment of antigen-specific CD4⁺ T cells according to intracellular antigen-induced CD154 expression. Comparison of CD154 expression in CD4⁺ T cells cultured for 16 h in the presence of medium alone, monensin alone or Staphylococcal enterotoxin B (SEB) plus monensin. The percentage of CD154-expressing CD4⁺ T cells is shown. The bars show mean ± standard deviation (n = 10). Analysis of variance (anova) with Bonferroni's post-test.

Fig. 3

Acquisition of cell surface CD107 is correlated with a loss of intracellular perforin in CD8⁺ T cells. Tonsillar mononuclear cells were stimulated with Staphylococcal enterotoxin B (SEB) and incubated for up to 4 h in the presence of (a) anti-CD107 and (b) fluorescein isothiocyanate (FITC) and brefeldin. At the time-points designated on the figure, cells were removed, washed and permeabilized, followed by CD3, CD8 and perforin staining. Accumulative data of 10 independent experiments are shown. Dots are mean ± standard deviation (n = 10).

Fig. 4

Polyfunctional cytokine-producing CD4⁺ and CD8⁺ T cells within tonsillar tissue after in-vitro stimulation. Simultaneous analysis of the functional profile of CD4⁺ and CD8⁺ T cells on the basis of interferon (IFN)-γ, interleukin (IL)-2 or tumour necrosis factor (TNF)-α production (a) and (b). Qualitative analysis of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell responses by polychromatic flow cytometry. Shown are representative flow cytometry analyses of the functional profile of CD4⁺ and CD8⁺ T cell response. Profiles are gated on live CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells, and the various combinations of IFN-γ, IL-2 and TNF-α are shown following stimulation with CD3 and CD28 monoclonal antibodies (mAbs) (c). All the possible combinations of the various functions are shown on the x-axis, whereas the percentages of the distinct cytokine-producing cell subsets within CD4⁺ and CD8⁺ T cells are shown on the y-axis. Cells were gated on CD3⁺ lymphocytes, followed by gating on CD4⁺ or CD8⁺ cells and then analysed for cytokine production. The bars show mean ± standard deviation (n = 10). ***P < 0·0001, as calculated by the t-test.

Fig. 5

Expression of dendritic cell (DC) markers on tonsillar mononuclear cells (TMCs). The DC fraction from TMCs was analysed for expression of the DC and activation markers: CD1a, CD1c, CD11c, CD33, CD40, CD80, CD86 and CD123. The pseudo-colour density plots show the electronic gates used to identify double-positive subsets based on human leucocyte D-related (HLA-DR) expression.