Expansion of circulatory Vγ9Vδ2 T cells in tularemia and Pontiac fever, two intracellular bacterial diseases with widely different clinical expression

Abstract: Although well established that human Vγ9Vδ2 T cells may expand in circulation during intracellular bacterial infections, most underlying studies included only a few cases and only some diseases had been studied so far. In tularemia, a severe invasive disease, only one patient had been described. Legionellosis, including the mild flue-like Pontiac disease caused by Legionella micdadei, had not been studied at all. The aim of the present thesis was to study the circulatory Vγ9Vδ2-T cell response in these two intracellular bacterial diseases. The number of cases included was large enough to draw general conclusions. At various intervals, Vγ9Vδ2-T-cell counts and the capability of the cells to produce proinflammatory cytokines were assayed. Finally, the nature of the stimulating antigens was determined.In the acute phase of tularemia, we showed a marked increase of circulatory Vγ9Vδ2 T cells. When 181 samples from 108 patients with ulceroglandular tularemia were assayed, the percentage of Vγ9Vδ2 T cells was found to increase from ~5 to > 20% after the first week of disease. During the ensuing 24 months, levels were normalized. Vaccination with the live attenuated vaccine strain Francisella tularensis LVS, on the other hand, did not cause an increase in numbers of Vγ9Vδ2 T cells.Within an outbreak of Pontiac fever, 14 cases were well defined with regard to incubation time and onset of disease. In samples obtained 4 to 6 days after onset of disease, the mean percentage of Vγ9Vδ2 T cells was ~ 1%, i.e., 20% of normal values. Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset of disease, values were ~ 15%. Later, values slowly decreased. In both tularemia and Pontiac fever, the capacity of Vγ9Vδ2 T cells to produce TNF-α in response to phorbol myristate acetate in vitro was transiently decreased, in tularemia up to 6 weeks after onset of disease and in Pontiac fever in samples obtained 5-7 weeks after onset of disease.Nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 T cells. Various strains of F. tularensis, including LVS, and a strain of L. micdadei were shown to produce Vγ9Vδ2 T-cell stimulating phosphoantigen. Notably, stimulation with an extract from each agent caused a similar degree of expansion of cells from subjects infected with the homologous and heterologous agent and also of cells from healthy subjects. Thus no immunospecific memory was detected in the Vγ9Vδ2-T cell response.Since it had been suggested that homologs of the conserved heat shock protein, chaperon-60, may be recognized by human Vγ9Vδ2 T cells, we determined the subpopulation of T cells responding to this protein as well as to DnaK, another heat-shock protein. Under in vitro conditions allowing a vigorous expansion of Vγ9Vδ2 T in response to a phosphoantigen, no expansion of γδ T cells in response to Cpn60 or DnaK of F. tularensis occurred. αβ T cells of tularemia-primed subjects, on the other hand, responded vigorously to the heat-shock proteins.In conclusion, two intracellular bacterial diseases with widely varying clinical expression were both associated with expansion of circulating Vγ9Vδ2 T cells. The expansion was prominent, long-lasting, and consistent within large numbers of individuals tested. In Pontiac fever, the expansion of Vγ9Vδ2 T cells was preceded by a depletion of the cells in circulation, implicating a possible extravasal migration into an infected site before the occurrence of rapid expansion and reentrance to blood. Both in tularemia and Pontiac fever, a modulation of the cytokine expression of Vγ9Vδ2 T cells was demonstrated in vitro, suggesting the presence of modulation of the inflammatory response. In extracts from in vitro culture of F. tularensis and L. micdadei, Vγ9Vδ2 T-cell stimulating phosphoantigens were identified and according to cross stimulation experiments, they induced expansion in vitro of Vγ9Vδ2 T cells without regard to immunospecific memory.