CD154 expression is associated with neutralizing antibody titer levels postinfluenza vaccination in stem cell transplant patients and healthy adults

Abstract

We undertook a prospective longitudinal study to examine humoral and cellular immune responses to influenza vaccination in hematopoietic cell transplant (HCT) patients and healthy adults. Healthy volunteers and HCT patients had blood samples taken prior to influenza vaccination and 30, 90, and 180 days postvaccination. Serum from pre- and postvaccination time points were tested for influenza A IgG and IgM by ELISA as well as tested for neutralizing antibody (NAb) titers via hemagglutination inhibition assay. Polychromatic flow cytometry was used to examine CD4(+) T cells for levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and CD154 (CD40 ligand) expression after stimulation with inactivated flu virus. In healthy subjects, we found a significant increase in Influenza A IgG and IgM levels as well as an increase in NAb titers pre- and post-influenza vaccination. Notably, NAb titers of most HCT patients did not rise to a protective level postvaccination. CD4(+) T cell expression of CD154 and cytokine responses were significantly reduced in HCT recipients compared to healthy adults. A lack of B cell reconstitution and dysfunctional CD4 T cell costimulation (as marked by low CD154 expression) is associated with low NAb levels postvaccination in HCT patients.

Figure 1. Influenza A IgG and IgM levels in hematopoietic cell transplant (HCT) patients (A) and healthy adults (B)

Serum from day 0 (d0) pre-vaccination or day 180 (d180) post-vaccination for healthy adults or d0 and day 30 (d30) and d180 post-vaccination for HCT patients was tested by ELISA for IgG and IgM specific for Influenza A. Black error bars represent the standard deviation from the mean (dashed bar). The p values are calculated using a two-tailed Mann-Whitney test. Healthy subjects showed sustained increases in both IgG and IgM levels at d180 post-vaccination. In HCT patients IgG and IgM levels were elevated at d30 post-vaccination, but for IgM the Ab level had returned to baseline by d180 post-vaccination. * = p<.05, ***=p<.001

Figure 2. Hemagglutination inhibition assay (HAI) measure of neutralizing antibody (NAb) levels in healthy subjects pre- and post-vaccination for the 2007–2008 flu season

Healthy subject sera was tested for NAb titer at d0 and at d180 post-vaccination. NAb titers measured were against the 2007–2008 flu vaccine antigens (A)H1N1, (A)H3N2 and (B)Malaysia flu strains. Black error bars represent the standard deviation from the mean (dashed bar). The dotted black line represents a titer of 40 which is considered protective. ***=p<.001, p = two-tailed Mann-Whitney test.

Figure 3. HAI assay measure of NAb levels in HCT patients pre- and post- vaccination during the 2008–2009 flu season

NAb titer from serum was measured at d0 and at d30 and d180 post-vaccination. HCT patient sera was measured against the 08–09 flu vaccine antigens for (A)H1N1, (A)H3N2, and (B)Florida flu strains. Black error bars represent the standard deviation from the mean (dashed bar). The dotted black line represents a titer of 40 which is considered protective. * = p<.05, **=p<.01, p = two tailed Mann-Whitney test.

Figure 4. CD154 expression on CD4 T cells after stimulation with inactivated trivalent flu vaccine in healthy (A) and HCT (B) subjects

CD154 expression was measured by flow cytometry after 18 hours of stimulation with mock diluent or inactivated flu virus (matched annual vaccine). CD154 expression was then calculated in terms of the absolute number of CD4 T cells at each time point. Black error bars represent the standard deviation from the mean. The p values were determined using a 2 way ANOVA. * = p<.05, **=p<.01, ***=p<.001.

Figure 5. Comparison of Median Fluorescence Intensity (MFI) of CD154 on healthy subjects vs. HCT recipients

(A) The MFI of CD154 on the cell surface after overnight stimulation with flu vaccine is shown. The p values represent two tailed Mann-Whitney test where **=p<.01, ***=p<.001. HCT patients show an attenuated CD154 expression level. (B) The absolute CD4 T cell count of the patients tested is graphed to show CD4 T cell reconstitution over time. There was no statistically significant difference in the mean absolute CD4 T cell count after vaccination.

Figure 6. IFN & TNF double expression on CD4 T cells after stimulation with inactivated flu virus in healthy (A) and HCT (B) subjects

Cytokine expression was measured by flow cytometry after 18 hours of stimulation with mock diluent or inactivated flu virus. Black error bars represent the standard deviation from the mean. The p values were determined using a 2 way ANOVA. * = p<0.05, **=p<0.01.