Granzyme B: Correlates with protection and enhanced CTL response to influenza vaccination in older adults

Abstract

This study compared serum antibody titers and granzyme B (GrzB) levels in virus-stimulated peripheral blood mononuclear cells following influenza vaccination. Twelve of 239 older adults who subsequently developed laboratory-diagnosed influenza illness (LDI) had significantly lower GrzB levels compared to subjects without LDI (p=0.004). Eight subjects with LDI in the previous year showed an enhanced GrzB response to vaccination (p=0.02). Serum antibody titers following vaccination did not distinguish those older adults who developed LDI from those who did not. These results suggest that GrzB levels could be combined with antibody titers to more effectively predict vaccine efficacy in older adults.

Figure 1

PBMC stimulated 20 hours with live influenza virus and granzyme B (GrzB) activity measured in PBMC lysates. Geometric mean GrzB levels are shown for each of the study subsets including subjects who did not develop influenza in either influenza season (No flu), subjects who subsequently developed influenza (Flu), and subjects who had had influenza during the previous influenza season (Prior Flu). Results are shown for the pre- (0 wks) and post-vaccination (4- and 10-wks) time points. (A) Results for PBMC stimulated with the A/H3N2 strain of influenza virus. In the Flu subset (n=12), the GrzB response to vaccination was poor and mean GrzB levels were significantly lower post-vaccination compared to the No flu subset (n=210, p=0.004). The effect of a previous influenza A/H3N2 illness on the response to vaccination was demonstrated in the Prior flu subset (n=8), showing an enhanced response with significantly increased post-vaccination levels of GrzB when compared to the No flu subset (p=0.02). (B) Results for PBMC stimulated with the influenza B strain at 4-weeks and 10-weeks post-vaccination (pre-vaccination not tested) in the second year showed no significant difference between the three subsets, consistent with the lack of cross-reactivity between strains of influenza A and B. Error bars represent standard error of the mean.

Figure 2

PBMC from healthy young (HY) and older (HO) adults, and older adults with congestive heart failure (CHF) obtained at 4 weeks post-vaccination (2008–09 season) were cultured for 12 hours in the absence (No virus) or presence (+ Virus) of live influenza virus. The proportion of CD8+ (CD56− or CD56+) and CD8−CD56+ within the peripheral blood lymphocyte (PBL) population, and the proportion expressing intracellular granzyme B (GrzB) and the degranulation marker, CD107a, are shown. (A) Representative dot plots are shown for lymphocytes expressing CD8 and/or CD56 (1^st column), and the expression of GrzB and CD107a in subsets of CD8+CD56− (2^nd column), CD8+CD56+ (3^rd column), and CD8−CD56+ (4^th column) subsets. (B) The graph shows the individual results for the proportions of lymphocytes that were CD8+CD56−, CD8+CD56+ or CD8−CD56+ in virus-stimulated PBMC and controls; the crossbar indicates the median for each group. The CD8+CD56− subset did not increase with virus stimulation and healthy older adults showed significantly lower proportion of PBL in this subset compared to the other two groups (p=0.04). In contrast, there was a significant increase in the proportion of CD8+CD56+ (activated CTL; p=0.03) and CD8−CD56+ (activated NK cells; p=0.003) in response to virus stimulation. (C) The proportion of lymphocytes expressing CD107a and GrzB is shown for each of the three CD8/CD56 subsets. Overall, there was a significant increase in the proportion of CD107a+GrzB+ in the CD8+CD56− (p=0.0003), CD8+CD56+ (p<0.0001) and CD8−CD56+ (p<0.0001) subsets with virus stimulation. (D) Individual results for the overall response to influenza virus within each of the effector subsets of CTL (CD8+CD56+/−CD107a+GrzB+) and activated NK cells (aNK, CD8−CD56+CD107a+GrzB+) are shown as the % of total PBL. There was a statistically significant increase in the proportion of CD8+ and CD8−CD56+ subsets within the total PBL population (p<0.0001) but the response in both subsets was significantly lower for the CHF group compared to the healthy young and older groups (p<0.005 for CTL, p<0.02 for activated NK cells),

Figure 2

PBMC from healthy young (HY) and older (HO) adults, and older adults with congestive heart failure (CHF) obtained at 4 weeks post-vaccination (2008–09 season) were cultured for 12 hours in the absence (No virus) or presence (+ Virus) of live influenza virus. The proportion of CD8+ (CD56− or CD56+) and CD8−CD56+ within the peripheral blood lymphocyte (PBL) population, and the proportion expressing intracellular granzyme B (GrzB) and the degranulation marker, CD107a, are shown. (A) Representative dot plots are shown for lymphocytes expressing CD8 and/or CD56 (1^st column), and the expression of GrzB and CD107a in subsets of CD8+CD56− (2^nd column), CD8+CD56+ (3^rd column), and CD8−CD56+ (4^th column) subsets. (B) The graph shows the individual results for the proportions of lymphocytes that were CD8+CD56−, CD8+CD56+ or CD8−CD56+ in virus-stimulated PBMC and controls; the crossbar indicates the median for each group. The CD8+CD56− subset did not increase with virus stimulation and healthy older adults showed significantly lower proportion of PBL in this subset compared to the other two groups (p=0.04). In contrast, there was a significant increase in the proportion of CD8+CD56+ (activated CTL; p=0.03) and CD8−CD56+ (activated NK cells; p=0.003) in response to virus stimulation. (C) The proportion of lymphocytes expressing CD107a and GrzB is shown for each of the three CD8/CD56 subsets. Overall, there was a significant increase in the proportion of CD107a+GrzB+ in the CD8+CD56− (p=0.0003), CD8+CD56+ (p<0.0001) and CD8−CD56+ (p<0.0001) subsets with virus stimulation. (D) Individual results for the overall response to influenza virus within each of the effector subsets of CTL (CD8+CD56+/−CD107a+GrzB+) and activated NK cells (aNK, CD8−CD56+CD107a+GrzB+) are shown as the % of total PBL. There was a statistically significant increase in the proportion of CD8+ and CD8−CD56+ subsets within the total PBL population (p<0.0001) but the response in both subsets was significantly lower for the CHF group compared to the healthy young and older groups (p<0.005 for CTL, p<0.02 for activated NK cells),

Figure 3

Geometric mean antibody titers measured in serum, pre- (0 wks) and post-vaccination (4- and 10-wks) are shown for the 2004–05 influenza season. In subjects who developed influenza illness (Flu, n=8), antibody titers to the infecting strain, A/Wyoming (A/H3N2), were similar to those who did not develop influenza except at 20-weeks post-vaccination where elevated titers reflect the response to influenza infection in the Flu subset. Prior Flu subjects (n=8) maintained high titers to A/Wyoming due to influenza infection in the previous influenza season but the response to vaccination was similar to the Flu and No Flu subsets. Error bars represent standard error of the mean