Differential tumor necrosis factor receptor 2-mediated editing of virus-specific CD8+ effector T cells

Abstract

Much of the CD8(+) T cell response in H2(b) mice with influenza pneumonia is directed at the nucleoprotein(366-374) (NP(366)) and acid polymerase(224-233) (PA(224)) peptides presented by the H2D(b) MHC class I glycoprotein. These D(b)NP(366)- and D(b)PA(224)-specific T cell populations are readily analyzed by staining with tetrameric complexes of MHC(+) peptide (tetramers) or by cytokine production subsequent to in vitro stimulation with the cognate peptides. The D(b)PA(224)-specific CD8(+) effector T cells make more tumor necrosis factor (TNF) alpha than the comparable CD8(+)D(b)NP(366)(+) set, a difference reflected in the greater sensitivity of the CD8(+)D(b)PA(224)(+) population to TNF receptor (TNFR) 2-mediated apoptosis under conditions of in vitro culture. Freshly isolated CD8(+)D(b)NP(366)(+) and CD8(+)D(b)PA(224)(+) T cells from influenza-infected TNFR2(-/-) mice produce higher levels of IFN-gamma and TNF-alpha after in vitro stimulation with peptide, although the avidity of the T cell receptor-epitope interaction does not change. Increased numbers of both CD8(+)D(b)PA(224)(+) and CD8(+)D(b)NP(366)(+) T cells were recovered from the lungs (but not the spleens) of secondarily challenged TNFR2(-/-) mice, a pattern that correlates with the profiles of TNFR expression in the TNFR2(+/+) controls. Thus, it seems that TNFR2-mediated editing of influenza-specific CD8(+) T cells functions to limit the numbers of effectors that have localized to the site of pathology in the lung but does not modify the size of the less activated responder T cell populations in the spleen. Therefore, the massive difference in magnitude for the secondary, although not the primary, response to these D(b)NP(366) and D(b)PA(224) epitopes cannot be considered to reflect differential TNFR2-mediated T cell editing.

Fig. 1.

Prevalence of IFN-γ⁺TNF-α⁺ T cells in TNFR2^+/+ and TNFR2^-/- mice. Lymphocytes were isolated from the spleen and the pneumonic lung (by BAL) on day 8 after primary Hkx31 challenge infection of TNFR2^+/+ and TNFR2^-/- mice, then stained for IFN-γ and TNF-α expression after 5 h of in vitro stimulation with 1 μM peptide. (A) Typical flow cytometry plots for peptide-stimulated D^bNP₃₆₆- and D^bPA₂₂₄-specific CD8⁺ T cells in the BAL. (B and C) IFN-γ⁺TNF-α⁺/IFN-γ⁺ ratios for BAL (B) and spleen (C) populations from the TNFR2^+/+ (filled bars) and TNFR2^-/- (open bars) mice. The data in B and C are expressed as mean ± SD, and statistical significance was determined by using Student's t test (^*, P < 0.005; ^**, P < 0.001; n = 5).

Fig. 2.

TCR-epitope binding characteristics for TNFR2^+/+ and TNFR2^-/- mice. Lymphocyte populations were recovered from the pneumonic lung by BAL (A) or from the spleen (B) of either TNFR2^+/+ (filled symbols) or TNFR2^-/- (open symbols) mice on day 8 of a primary HKx31 response. The populations were labeled with anti-CD8α-FITC and either the D^bNP-PE (▪) or D^bPA-PE (•) tetramers at room temperature for 60 min in medium containing azide, then incubated at 37°C in the presence of anti-H2D^b to block any rebinding of eluted tetramer. The data are expressed as a percentage of the initial percentage of CD8⁺ tetramer⁺ at various times after transfer to 37°C. (C and D) Other cells from the same BAL (C) and spleen (D) population of TNFR2^+/+ (filled bars) or TNFR2^-/- (open bars) mice were incubated with anti-CD8β, then stimulated with 1 μM NP₃₆₆ or PA₂₂₄ peptide. The level of TNF-α production was determined by intracellular cytokine staining, and the data are presented as a percentage of the maximum TNF-α response observed in the absence of anti-CD8β. Statistical differences between the NP₃₆₆- and PA₂₂₄-specific responses were determined by using Student's t test (^*, P < 0.007; ^**, P < 0.0001; †, P < 0.005; n = 5).

Fig. 3.

Differential TNFR2-mediated loss after in vitro stimulation. Enriched CD8⁺ T cells (1 × 10⁵) from TNFR2^+/+ (A and B) or congenic TNFR2^-/- (C and D) H2^b mouse spleen were cultured without peptide (open bars) or with 10 μM (hatched bars) or 0.01 μM (filled bars) peptide. The results are from two independent experiments (Exp. 1 in A and C and Exp. 2 in B and D). Viable lymphocytes (by trypan blue exclusion) were counted after 36-40 h. The histograms show the mean ± SD cell counts for CD8α-allophycocyanin⁺ T cells staining with the D^bNP₃₆₆-PE or D^bPA₂₂₄-PE tetramer. These values were compared by Student's t test (^*, P < 0.01; ^**, P < 0.002). The same populations were stained with annexin V-FITC, and the necrotic cells were gated out by PI staining. Above the histograms are the percentages of tetramer⁺annexin V⁺/PI^-, which can be considered to measure the extent of apoptosis in these still-viable (PI^-) lymphocytes.

Fig. 4.

Cell surface expression of TNFR2 on virus-specific CD8⁺ T cells recovered from the lung but not the spleen. The CD8⁺ set was enriched from cell populations obtained by BAL (A and B) or disruption of the spleen (C and D) on day 7 after secondary HKx31 → PR8 challenge and stained with the D^bNP₃₆₆ (A and C) or D^bPA₂₂₄ (B and D) tetramers, anti-CD8α-FITC, and biotinylated anti-mouse TNFR2 (broken line). The-TNFR2 staining was developed with streptavidin-allophycocyanin. The negative control was an irrelevant primary isotype-specific Ab (bold line). The fluorescence-activated cell sorting profiles are representative of three separate experiments.

Fig. 5.

Virus-specific CD8⁺ T cell numbers in BAL populations from TNFR2^-/- mice. Secondarily challenged (HKx31 → PR8) TNFR2^+/+ (▪) and TNFR2^-/- (•) mice were sampled at various time points, and CD8⁺ T cell populations from five mice were enriched from individual spleens (A and B) or pooled BALs (C and D), then stained with anti-CD8α and the D^bNP₃₆₆ (A and C) or D^bPA₂₂₄ (B and D) tetramers. The numbers of virus-specific CD8⁺ T cells (E and F) were determined from the percentage of cells staining and the total cell counts (data not shown). The analysis of the BAL was repeated on day 7 for the TNFR2^+/+ (filled bars) and TNFR2^-/- (open bars) to give the numbers of CD8⁺D^bNP₃₆₆⁺ (E) and CD8⁺D^bPA₂₂₄⁺ (F) T cells. Statistical significance was determined by Student's t test (^*, P < 0.007; ^**, P < 0.001; n = 5). (G) Lung homogenates from the TNFR2^+/+ and TNFR2^-/- mice were titrated in embryonated hen's eggs, and the 50% egg infectious dose (EID₅₀) was determined by the capacity of infected allantoic fluid to agglutinate chicken erythrocytes.