Expansion of Vgamma9 Vdelta2 T cells is triggered by Francisella tularensis-derived phosphoantigens in tularemia but not after tularemia vaccination

Abstract

Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. Here we demonstrate that during the first weeks of infection, a significant increase in levels of Vgamma9 Vdelta2 cells occurred in peripheral blood: in 13 patients analyzed 7 to 18 days after the onset of disease, these lymphocytes represented, on average, 30.5% of CD3+ cells and nearly 100% of gammadelta+ T cells. By contrast, after vaccination with the live vaccine strain (LVS) of F. tularensis, only a minor increase occurred. Eleven days after vaccination, gammadelta T cells represented an average of 6.7% and Vgamma9 Vdelta2 cells represented an average of 5.3% of T cells, as in control subjects. Since derivatives of nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vgamma9 Vdelta2 cells, this observation prompted an investigation of phosphoantigens in F. tularensis strains. The F. tularensis phosphoantigens triggered in vitro a proliferative response of human Vgamma9 Vdelta2 peripheral blood leukocytes as well as a cytotoxic response and tumor necrosis factor release from a Vgamma9 Vdelta2 T-cell clone. Quantitatively similar phosphoantigenic activity was detected in acellular extracts from two clinical isolates (FSC171 and Schu) and from LVS. Taken together, the chemical nature of the stimulus from the clinical isolates and the significant increase in levels of Vgamma9 Vdelta2 cells in peripheral blood of tularemia patients indicate that phosphoantigens produced by virulent strains of F. tularensis trigger in vivo expansion of gammadelta T cells in tularemia.

FIG. 1

Flow cytometric analysis of lymphocytes in the peripheral blood of a control individual (left) and a patient 12 days after the onset of tularemia (right). Lymphocytes were gated according to their morphological parameters. The percentages of gated cells staining positively for CD3 and Vγ9 were determined.

FIG. 2

Evolution of the Vγ9 T-cell subset during tularemia infection. (A) Percentage of Vγ9 T cells in peripheral blood at various times following the onset of tularemia (triangles). The filled dot indicates the average number of Vγ9 T cells in a control group denying exposure to F. tularensis. (B) Representative flow cytometric analysis of successive peripheral blood samples from one patient (at 8, 12, and 27 days after onset). The numbers represent the percentages of Vγ9⁺ cells among CD3⁺ cells.

FIG. 2

Evolution of the Vγ9 T-cell subset during tularemia infection. (A) Percentage of Vγ9 T cells in peripheral blood at various times following the onset of tularemia (triangles). The filled dot indicates the average number of Vγ9 T cells in a control group denying exposure to F. tularensis. (B) Representative flow cytometric analysis of successive peripheral blood samples from one patient (at 8, 12, and 27 days after onset). The numbers represent the percentages of Vγ9⁺ cells among CD3⁺ cells.

FIG. 3

Phosphatases degrade the F. tularensis stimulus for the Vγ9Vδ2 clone G12. (A) TNF production by a Vγ9Vδ2 T-cell clone (G12) was estimated after 3 h of incubation in medium supplemented with crude mycobacterial extract (ME) (1 mg/ml), TUBag1 (phosphoantigen from M. tuberculosis; 0.1 μM), or F. tularensis cell extract (200 μg/ml) from the virulent strain (FSC171) or the vaccine strain (LVS) before and after treatment with calf intestinal alkaline phosphatase (1 U/ml) plus nucleotide pyrophosphatase (0.1 U/ml). Spontaneous release was subtracted to yield the data presented. (B) Vγ9Vδ2 T-cell clone (G12) autocytotoxicity was estimated after incubation in medium supplemented with F. tularensis cell extract (1,000 μg/ml) from the virulent strain (FSC171) or the vaccine strain (LVS) before and after treatment for 30 min at 37°C with calf intestinal alkaline phosphatase plus nucleotide pyrophosphatase as in panel A. Results are expressed as percent ⁵¹Cr release, calculated by dividing experimental minus spontaneous release by maximal release (i.e., from cells incubated with detergent).

FIG. 4

Comparative titration of phosphoantigenic loads in extracts from F. tularensis LVS and virulent strains. Comparison of TNF release by the G12 clone incubated with various concentrations of bacterial extracts (numbered bars): virulent strains of F. tularensis, FSC171 (bars 2) and Schu (bars 3), F. tularensis LVS (bars 4), and M. fortuitum extract (bars 1), all of which were used at a maximum concentration of 3 × 10³ μg/ml. TNF release of 1 pg/ml resulted from culture medium alone.

FIG. 5

Evolution of the Vγ9Vδ2 T-cell subset during vaccination with F. tularensis LVS. (A) Percentage of Vγ9Vδ2 T cells in peripheral blood at various times following vaccination with F. tularensis LVS (triangles). (B) Flow cytometric analysis of γδ T lymphocytes in the peripheral blood of a vaccinee (a total of seven individuals were vaccinated). Analysis was performed before (left) and 11 days after (right) vaccination. Lymphocytes were gated according to their morphological parameters. The percentages of CD3 cells staining positively for Vγ9 are indicated in the upper right quadrants.

FIG. 5

Evolution of the Vγ9Vδ2 T-cell subset during vaccination with F. tularensis LVS. (A) Percentage of Vγ9Vδ2 T cells in peripheral blood at various times following vaccination with F. tularensis LVS (triangles). (B) Flow cytometric analysis of γδ T lymphocytes in the peripheral blood of a vaccinee (a total of seven individuals were vaccinated). Analysis was performed before (left) and 11 days after (right) vaccination. Lymphocytes were gated according to their morphological parameters. The percentages of CD3 cells staining positively for Vγ9 are indicated in the upper right quadrants.