Cytotoxic T lymphocyte and natural killer cell responses to non-typeable Haemophilus influenzae

Abstract

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have a key role in host defence against infectious pathogens, but their response to bacteria is not well characterized. Non-typeable Haemophilus influenzae is a major cause of respiratory tract infection including otitis media, sinusitis, tonsillitis and chronic bronchitis (especially in chronic obstructive pulmonary disease and bronchiectasis). This bacterium is also present in the pharynx of most healthy adults. The primary factor that may determine whether clinical disease occurs or not is the nature of the lymphocyte response. Here we examined the CTL cell and NK cell responses to nontypeable H. influenzae in healthy control subjects and in subjects who had bronchiectasis and recurrent bronchial infection with this bacterium. Cells were stimulated with live H. influenzae and intracellular cytokine production and release of cytotoxic granules measured. Control subjects had significantly higher levels of interferon gamma production by both CTL and NK cells, while levels of cytotoxic granule release were similar in both groups. The main lymphocyte subsets that proliferated in response to H. influenzae stimulation were the CTL and NK cells. The results suggest that CTL and NK cell responses may be important in preventing disease from nontypeable H. influenzae infection.

Fig. 5

Response of cytotoxic T cells and natural killer (NK) cells to stimulation with four different isolates of non-typeable Haemophilus influenzae in controls (n = 5). All four different isolates produced measurable interferon (IFN)-γ and CD107a expression, but there were some differences between strains. (a) Cytotoxic lymphocyte (CTL) cell production of IFN-γ with strain 1 (MU/MMC 1) producing higher levels of IFN-γ than strain 4 (*P = 0·021). (b) NK cell production of IFN-γ with strain 1 producing significantly higher levels of IFN-γ than strain 3 (**P = 0·004) and strain 4 (**P = 0·003). (c) CTL production of CD107a. (d) NK cell production of CD107. Differences between different groups were analysed using analysis of variance.

Fig. 1

Cytokine production of lymphocyte subsets stimulated using live non-typeable Haemophilus influenzae in control (n = 15) and bronchiectasis (n = 14) subjects. Responses were measured using intracellular cytokine staining and flow cytometry. (a) Cytokine production by cytotoxic T cells. Levels of interferon (IFN)-γ were significantly higher in the control group (*P = 0·023), but levels of other cytokines interleukin (IL)-2, IL-4 and IL-13 were similar in both groups. (b) Cytokine production by natural killer (NK) cells. Levels of IFN-γ were significantly higher in the control group (**P = 0·005), but levels of IL-4 were similar in both groups. (c) Cytokine production by T helper cells. As with the cytotoxic lymphocyte (CTL) and NK cells, the levels of IFN-γ were significantly higher in the control group (*P = 0·025). (d) Example of cytokine staining for IFN-γ and IL-4 in a control subject.

Fig. 2

Cytotoxic granule release by lymphocytes stimulated using live non-typeable Haemophilus influenzae (NTHi) in control (n = 15) and bronchiectasis (n = 14) subjects. CD107a expression was measured using flow cytometry. (a) Production of CD107 and interferon (IFN)-γ by cytotoxic T cells. Levels of CD107a expression were similar in both groups. Only a small proportion (< 10%) of cytotoxic lymphocyte (CTL) cells stained for both CD107a and IFN-γ. (b) Production of CD107a and IFN-γ by natural killer (NK) cells. Levels of CD107a and CD107a/IFN-γ production were similar in both groups. (c) Shows an example of CD107a production by CTL cells in a control subject in response to stimulation with Staphylococcus enterotoxin-B and to NTHi and NK cell response to NTHi.

Fig. 3

Responses of CD56^bright and CD56^dim natural killer (NK) cell subsets to stimulation with non-typeable Haemophilus influenzae in control (n = 15) and bronchiectasis (n = 14) subjects. (a) Dot plot of cytokine staining of lymphocytes for CD3 and CD56. NK cells were negative for CD3 and could be divided into bright (R5) and dim (R4) populations on the basis of their intensity of staining for CD56. (b) In both control (**P = 0·004) and bronchiectasis subjects (*P = 0·046) the CD56^bright NK subset produced significantly higher levels of interferon (IFN)-γ than the CD56^dim NK cell subset. (c) In both control and bronchiectasis subjects there was no significant difference in CD107a production between the two NK subsets. (d) Cytokine staining for CD107 and IFN-γ in CD56^bright and CD56^dim NK cell subsets in a control subject.

Fig. 4

Effect of blockade of major histocompatibility complex (MHC)-I on interferon (IFN)-γ production by cytotoxic T cells; in control subjects (n = 5). (a) MHC-I blockage in controls produced a 70% reduction in expression of IFN-γ by cytotoxic lymphocyte cells that had been stimulated with non-typeable Haemophilus influenzae (NTHi) (*P = 0·033). (b) Cytokine staining for MHC-I block in subject. IL, interleukin; CTL, cytotoxic T lymphocytes.

Fig. 6

Lymphocyte subset proliferation measured by carboxyfluorescein succinimidyl ester in control subject. (a) Lymphocyte subset proliferation with control, Staphylococcus enterotoxin-B (SEB) and non-typeable Haemophilus influenzae (NTHi) in a typical patient. In the unstimulated cells there was some proliferation of the natural killer (NK) cell population. The SEB produced strong stimulation of the T cell populations, but no obvious response could be measured from the other lymphocyte subsets. The NTHi produces proliferation predominantly in the NK cells and with some proliferation in cytotoxic lymphocyte and B cell populations. (b) Example of CD56^bright NK cell proliferation, showing proliferation at baseline which was increased with the addition of NTHi. CTL, cytotoxic T lymphocytes; CFSE, carboxyfluorescein succinimidyl ester.