Protective role of gamma interferon during the recall response to influenza virus

Abstract

During secondary immune responses to influenza virus, virus-specific T memory cells are a major source of gamma interferon (IFN-gamma). We assessed the contribution of IFN-gamma to heterologous protection against the A/WSN/33 (H1N1) virus of wild-type and IFN-gamma-/- mice previously immunized with the A/HK/68 (H3N2) virus. The IFN-gamma-/- mice displayed significantly reduced survival rates subsequent to a challenge with various doses of the A/WSN/33 virus. This was associated with an impaired ability of the IFN-gamma-/- mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers was noted. However, the IFN-gamma-/- mice developed type A influenza virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the wild-type mice, as demonstrated by both cytotoxicity and a limiting-dilution assay for the estimation of CTL precursor frequency. The pulmonary recruitment of T cells in IFN-gamma-/- mice was not dramatically affected, and the percentage of CD4(+) and CD8(+) T cells was similar to that of wild-type mice. The T cells from IFN-gamma-/- mice did not display a significant switch toward a Th2 profile. Furthermore, the IFN-gamma-/- mice retained the ability to mount significant titers of WSN and HK virus-specific hemagglutination-inhibiting antibodies. Together, these results are consistent with a protective role of IFN-gamma during the heterologous response against influenza virus independently of the generation and local recruitment of cross-reactive CTLs.

FIG. 1

Survival profiles of wild-type (wt) and IFN-γ−/− (ko) mice infected with various doses of the WSN virus. Mice were immunized with the live HK (H3N2) virus and challenged 1 month later with the WSN virus (A to C). As controls, we included naive mice and mice immunized with the WSN virus (D). The mice were observed during a period of 20 days following the challenge, and the recovery of survivors was documented by the absence of infectious virus in the lungs. Daily results are expressed as percent survival.

FIG. 2

Body weight loss of IFN-γ−/− (A) and wild-type (B) mice infected with the WSN virus. Seven mice in each group were immunized with the live HK virus and challenged 1 month later with a dose of 2.25 × 10⁷ TCID₅₀ of the WSN virus. Body weight was individually recorded every 2 days. The results are expressed separately for mice that recovered or did not survive as mean percent body weight loss ± SEM.

FIG. 3

Cytotoxicity of splenocytes from wild-type and IFN-γ−/− mice immunized with the WSN virus. Mice were immunized with the live WSN virus 7 days before sacrifice. Splenocytes were harvested and tested in a standard ⁵¹Cr release assay against infected and noninfected target cells. The results are expressed as mean percent specific lysis at various effector-to-target cell (E/T) ratios. The experiment was carried out in triplicate wells. SEs were less than 25% of the means. Four mice in each group were included in the experiment.

FIG. 4

Recall responses of CTLs from IFN-γ−/− (ko) and wild-type (wt) mice previously immunized with the live HK virus. As controls, we included IFN-γ−/− and wild-type mice not immunized with the HK virus. Seven days after infection with the WSN virus, freshly harvested splenocytes pooled from three mice in each group were tested in a standard ⁵¹Cr release assay against target cells coated with a control NP-D^b peptide (A) or an NP-K^d peptide that is a dominant CTL epitope (B) or infected with the WSN virus (C). Results are expressed as mean percent specific lysis (after subtraction of percent lysis against noninfected, noncoated target cells) at various effector-to-target cell (E/T) ratios. The experiment was carried out in triplicate wells, and the SDs were less than 25% of the means.

FIG. 5

Estimation of pCTL frequency in IFN-γ−/− (ko) and wild-type (wt) mice by limiting-dilution analysis. Mice were immunized with the live HK virus and, after 1 month, infected with the WSN virus. Seven days after infection, the splenocytes from three mice in each group were pooled and restimulated in a limiting-dilution manner. The results of a standard ⁵¹Cr release assay are expressed as percent negative wells versus number of responder cells per well. The pCTL frequency was estimated after linear interpolation as the number of responder cells per well corresponding to 37% negative wells.

FIG. 6

Local recruitment of CTLs in lungs of IFN-γ−/− (ko) and wild-type (wt) mice immunized with the live HK virus and then infected with the WSN virus. Lymphocytes from lung tissue pooled from three mice in each group were isolated and stimulated in vitro with the PR8 (A) or HK (B) virus. Cytotoxicity was tested by a standard ⁵¹Cr release assay against WSN virus-infected target cells. Results are expressed as mean percent specific lysis ± SE at various responder/stimulator (R/S) ratios.