Interleukin-2 and loss of immunity in experimental Mycobacterium avium infection

Abstract

Experimental infection of mice with a virulent strain of Mycobacterium avium leads to a slowly progressive disease, which we have previously shown culminates in loss of gamma interferon (IFN-gamma) production by T lymphocytes and death of the animals approximately 40 weeks after infection. Here we investigated the changes in T-cell activation, the production of interleukin-2 (IL-2), and the response to IL-2 throughout M. avium infection as a possible explanation for this loss. We found that there is a steady increase in the percentage of T cells expressing activation markers right to the end of infection. However, in vivo T-cell proliferation, measured as a percentage of CD4(+) and CD8(+) cells incorporating 5-bromo-2'-deoxyuridine, initially increased but then remained constant. In the final stages of infection there was a decline in proliferation of activated (CD62L(-)) T cells. Since IL-2 is a major driver of T-cell proliferation, we asked whether this was due to loss of IL-2 responsiveness or production. However, CD25 (IL-2Ralpha) continued to be highly expressed in the terminal stages of infection, and although IL-2 production declined, addition of recombinant IL-2 to cultures could not rescue the final loss of IFN-gamma production.

FIG. 1.

Bacterial burden and CD4⁺ and CD8⁺ T-cell decline. (A) Numbers of bacteria in the lungs (▪), spleens (⧫), and livers (•) of mice at different times after intranasal infection with 1 × 10⁵ CFU of M. avium per mouse. The means and standard deviations based on the data for five mice per group are shown. (B and C) Changes in the percentages (B) and numbers (C) of CD4⁺ and CD8⁺ T cells in the spleens of mice at different times after infection, assayed on the same day. The values are means and standard deviations based on the data for three mice per group. The results of one representative experiment of 10 similar experiments are shown. Symbols: ▪, CD4⁺, infected; □, CD4⁺, old uninfected; •, CD8⁺, infected; ○, CD8⁺, old uninfected.

FIG. 2.

Changes in expression of activation markers after M. avium infection. (A and B) Representative staining of CD4⁺ T cells with CD44 (A) or CD62L (B) after 25 weeks of infection. The dotted lines show the results obtained for the negative controls. The bars show what was considered to be CD44^bright or CD62L⁻ and the percentage of CD4⁺ T cells that were CD44^bright or CD62L⁻ in each case. FITC, fluorescein isothiocyanate. (C to F) Percentages (C and E) and numbers (D and F) of CD4⁺ (▪) or CD8⁺ (•) cells that were CD44^bright (C and D) or negative for CD62L (E and F). The number of cells was calculated by multiplying the percentage of activated T cells by the number of spleen cells recovered. All points represent means based on the data for three mice, and the error bars indicate standard deviations. Data for young normal control mice, age matched for 6 weeks after infection, are plotted at zero time. Data for the old normal control mice (CD4⁺ [□] and CD8⁺[○]) are plotted as if the mice were infected when they were 6 weeks old, the age when all mice were infected. The results of one representative experiment of three independent experiments are shown.

FIG. 3.

In vivo proliferation after infection: percentages (A) and numbers (B) of CD4⁺ or CD8⁺ BrdU⁺ T cells. Mice were fed BrdU in their drinking water for 7 days, and this was followed by staining of spleen cells for CD4 or CD8 and BrdU. The mice had been infected for different times, but their cells were assayed on the same day. The values are means based on the data obtained for three mice at different times after infection. The error bars indicate standard deviations. The results of one representative experiment of four similar experiments are shown. Symbols: ▪, CD4⁺, infected; □, CD4⁺, old uninfected; •, CD8⁺, infected; ○, CD8⁺, old uninfected.

FIG. 4.

Scatter plots of BrdU and CD62L staining. Spleen cells were stained with monoclonal antibodies specific for CD4, CD62L, and BrdU. CD4⁺ cells were gated, and the results of staining for CD62L (Mel-14) (y axis) and BrdU (x axis) are shown. The percentage of cells falling in each quadrant is shown for each time point after infection. Similar results were obtained when CD8⁺ T cells were stained and gated in the same manner. (A) Negative control; (B) uninfected; (C) 7 weeks after infection; (D) 21 weeks after infection; (E) 36 weeks after infection. FITC, fluorescein isothiocyanate.

FIG. 5.

In vivo proliferation of activated T cells after M. avium infection. Mice were fed BrdU in their drinking water for 7 days. Spleen cells were stained for CD4 or CD8, CD62L, and BrdU. Percentages (A) and numbers (C) of BrdU⁺ cells gated on CD4⁺ CD62L⁻ or CD4⁺ CD62L⁺ T cells and percentages (B) and numbers (D) of BrdU⁺ gated on CD8⁺, CD62L⁻, or CD62L⁺ T cells are shown. The values are means and standard deviations based on the data for three mice per group. The mice had been infected for different times, but their cells were assayed on the same day. The results of one representative experiment of two similar experiments are shown. Symbols: ▪, infected, CD62L⁻; □, old uninfected, CD62L⁻; •, infected, CD62L⁺; ○, old uninfected, CD62L⁺.

FIG. 6.

Changes in expression of CD25 after M. avium infection. Expression of CD25 was determined by flow cytometry. The mean percentages (A) and numbers (B) of CD4⁺ CD25⁺ (▪) and CD8⁺ CD25⁺ (bull;) T cells in three mice are shown. Data for young normal control mice, age matched for 6 weeks after infection, were plotted at zero time. Data for old normal control mice (CD4⁺[□] and CD8⁺[○]) were plotted as if the mice were infected when they were 6 weeks old, the age when all mice were infected. The results of one representative experiment of three similar experiments are shown.

FIG. 7.

Changes in the numbers of cytokine-producing cells during M. avium infection. (A) ELISPOT assays were used to determine the numbers of IL-2- and IFN-γ-producing cells after stimulation in vitro with M. avium. Cells cultured without M. avium had ≤5 cytokine-producing cells/10⁶ spleen cells (data not shown). The values are means and standard deviations based on the data for groups of three mice. The results of one representative experiment of three independent experiments are shown. Symbols: ▪, IL-2-producing cells; □, IL-2 normal cells; •, IFN-γ-producing cells; ○, IFN-γ normal cells. (B) Scatter plot of the number of IFN-γ- and IL-2-producing cells from the same mouse at different times after M. avium infection. Data pooled from three independent experiments are shown.