Interferon-gamma acts directly on CD8+ T cells to increase their abundance during virus infection

Abstract

Interferon-gamma (IFNgamma) is important in regulating the adaptive immune response, and most current evidence suggests that it exerts a negative (proapoptotic) effect on CD8(+) T cell responses. We have developed a novel technique of dual adoptive transfer, which allowed us to precisely compare, in normal mice, the in vivo antiviral responses of two T cell populations that differ only in their expression of the IFNgamma receptor. We use this technique to show that, contrary to expectations, IFNgamma strongly stimulates the development of CD8(+) T cell responses during an acute viral infection. The stimulatory effect is abrogated in T cells lacking the IFNgamma receptor, indicating that the cytokine acts directly upon CD8(+) T cells to increase their abundance during acute viral infection.

Figure 1.

CD8⁺ T cell abundance is markedly reduced in the absence of IFNγ or IFNγR. (A) The CTL response induced in IFNγ^2/−, IFNγR^−/−, and control C57BL/6 mice 8 d after infection was measured in vivo by injecting GP33-coated CFSE^hi and uncoated CFSE^lo target cells into mice. The numbers indicate the percentage of peptide-coated cells deleted in the recipient mice. (B) Dot plots show CD44 expression by splenocytes at 8 d after infection. An uninfected C57BL/6 mouse is shown for comparison. (C) Bar graphs depict the number of activated cells that are CD44^hi, CD62L^lo, or Ly6A/E^hi in infected mice. (D) The virus-specific responses in wild-type and IFNγR⁻ mice were quantified at day 8 after infection by ICCS. GP_33–41-specific cells are identified by ovals, and the numbers indicate the percentage of specific cells among all CD8⁺ T cells. (E) The numbers of epitope-specific CD8⁺ T cells, based on the spleen cell count and intracellular staining, are shown, along with the fold reductions between ^+/+ and γR1^−/− mice. The bars represent the average ± SD from five or six mice per group, from four independent experiments. For each group, the p-value compared with ^+/+ mice is <0.001 (unpaired Student's t test).

Figure 2.

Expression of the IFNγR on CD8⁺ T cells varies over the course of viral infection. (A) Representative dot plots show changes in expression of IFNγR1 on T cells after infection. The percentages of CD8⁺ T cells expressing elevated amounts of the receptor are shown. (B) IFNγ^2/− CD8⁺ T cells express normal amounts of IFNγR1 before infection (unshaded histogram), and up-regulate expression 8 d after infection (shaded histogram). Dotted line shows an isotype control antibody. (C) Changes over time in the geometric mean fluorescence of IFNγR1 on wild-type splenic CD8⁺ T cells (○) and the number of IFNγR1⁺ cells per spleen (•). (D) Resting (CD44^lo) and activated (CD44^hi) CD8⁺ T cells are identified by a dot plot (left) and expression of IFNγR1 among these cells is shown by the histograms (right). Dotted line shows an isotype control antibody. (E) IFNγR1 expression on CD8⁺ T cells in the indicated tissues of uninfected mice (unshaded) or of day 8 mice (shaded). The dotted histogram shows the decreased expression in spleen at one day after infection; in the other tissues, the change in fluorescence at this time was minimal.

Figure 3.

The stimulatory effect of IFNγ on CD8⁺ T cells requires that they express IFNγR. Equivalent numbers of wild-type (Thy1.2⁺, Ly5a⁺) and IFNγR^−/− (Thy1.2⁺, Ly5a⁻) CD8⁺ T cells were mixed and adoptively transferred intravenously into unirradiated, Thy1.1 mice. The recipient mice were infected and the responses of the donor (Thy1.2⁺) cells were measured 8 d after infection. (A) Representative dot plot shows the expanded donor cell populations (Thy1.2⁺), and these cells were gated, and are analyzed in B–G. (B) IFNγR⁺ CD8⁺ T cells outnumber their IFNγR⁻ counterparts by ∼4:1. (C) CD44 expression on donor cells; the percentage of cells within each quadrant is shown. (D) The antigen-specific response of the donor cells was measured by ICCS. The numbers represent the responding donor cells as percentage of all donor cells. (E) The proportion of wild-type and IFNγR1⁻ donor CD8⁺ T cells in these same mice at day 0 (measured in the blood before infection) is shown. (F) The percentage of donor cells that are Ly5a⁺ or Ly5a⁻ in an uninfected recipient mouse at day 0 (measured in blood) and 8 d later (measured in spleen). (G) The ratio of ^+/+ to γR^−/− donor cells is shown for four epitopes, based on ICCS. Each point represents an individual animal, and the means ± SD are shown as horizontal lines. For each of four epitopes, the responses in ^+/+ and ^−/− cell populations were compared using unpaired Student's t test. The p-values are: 0.0039 (GP₃₃); 0.0001 (NP₃₉₆); 0.021 (GP₂₇₆); and 0.038 (NP₂₀₅).

Figure 4.

The stimulatory actions of IFNγ on CD8⁺ T cells occur during the course of virus infection. TcR-transgenic mice were bred as described in the text, to provide a source of IFNγR1⁺ TcR-transgenic CD8⁺ T cells (Thy1.2⁺, Ly5a⁺), and IFNγR1^−/− TcR-transgenic CD8⁺ T cells (Thy1.2⁺, Ly5a⁻). (A) Equal numbers of these IFNγR1⁺ (shaded)- and IFNγR1^−/− (unshaded)-transgenic CD8⁺ T cells were mixed and are shown (dotted line = isotype control). This 1:1 mix of IFNγR1⁺ and IFNγR1⁻ cells was injected into Thy1.1 recipients (each host mouse received a total of 6 × 10⁴ TcR-transgenic CD8⁺ T cells), and the following day the recipient mice were infected. 8 d later, the responses of the two donor cell populations were analyzed by flow cytometry. (B) The expanded Thy1.2 TcR-transgenic donor cells are enclosed in an oval, and only these cells are analyzed in C–J. (C) There are threefold more IFNγR⁺ (Ly5a⁺) cells than IFNγR⁻ cells. (D) The higher number of IFNγR1⁺ cells is confirmed by Db33-41 tetramer staining. (E–G) The activation status of the day 8 donor cells is similar, regardless of their expression of IFNγR. The cells are CD44^hi, and at day 8, both donor populations show similar reductions in CD62L and CD127. (H) A similar proportion of each donor cell population produces IFNγ upon GP₃₃ peptide stimulation. (I) The ratio of ^+/+ to IFNγR^−/−-transgenic donor cells is summarized from 12 mice analyzed from three independent experiments (left); 11 of the 12 animals showed a ratio >1 (dotted line). The numbers of IFNγR1⁺ and γR^−/−-transgenic cells were calculated from 12 mice (right). The averages ± SD are indicated. The differences between the two groups was statistically significant, as indicated by a p-value of 0.0025, derived from a two-tailed paired Student's t test. (J) The antiapoptosis marker bcl-2 is reduced at day 8, to the same extent in the IFNγR⁺ and the IFNγR⁻ populations.