We appreciate Dr. Shaw and colleagues’ interest in our work. We agree that an improved understanding of immune responses in heart failure patients is of critical importance.
Regarding our finding of higher IL-10 concentrations in patients with heart failure (HF), while the source of IFN-γ and IL-10 in our culture model is not specific to only cytotoxic T-lymphocytes (CTL), a shift from Th1 (IFN-γ) to Th2 (IL-10) cytokines has been associated with reduced CTL activity.1 Moreover, a previous study has shown that IFN-γ:IL-10 ratios measured in a cell culture model can be predictive of granzyme B levels (Grz B, a key enzyme in the viral clearing process), which also associate with CTL activity and vaccine effectiveness.2, 3 The lower IFN-γ:IL-10 ratios in HF participants compared to controls reported in our study may suggest decreased CTL activity. The ratio of IFN-γ and IL-10 rather than just their sources may provide valuable insight into T-cell protection afforded by influenza vaccination.
We believe the Wilcoxon rank-sum test is a valid statistical approach to compare the continuous outcomes measures between the two treatment groups, and indeed, both the suggested Mann-Whitney U test and the Wilcoxon rank-sum test produce identical statistical results.4 Dr. Shaw and colleagues also question the rationale for performing a log transformation when a non-parametric test was used and suggest that log-transformed data should be analyzed using a T-test. Though it is common for skewed data to be log-transformed in order that a T-test may be used, this is not the only reason to use the log transformation nor does a log transformation preclude use of a non-parametric test. We chose to present changes in log-transformed values from pre- to post-vaccination as this approach is more commonly employed;2 a Wilcoxon rank-sum comparison of the percentage change from pre- to post-vaccination using untransformed values would have produced mathematically identical results.
We reported a trend toward better serologic responses to the A/H3N2 vaccine strain among HF participants taking carvedilol compared to those taking metoprolol (p=0.06). Both T-cells and B-cells express beta-2 adrenergic receptors (β2-AR).5 Norepinephrine stimulation of β2-AR has been shown to reduce the ability of CD4+ T cells to produce cytokines necessary for cell expansion, a response not seen when cells were also cultured with a beta antagonist.6, 7 Our lab also has data in support of reduced cytokine production when PBMCs are cultured with influenza antigens and the beta2-specific agonist salmeterol, and ongoing experiments in our laboratory confirm that adding carvedilol to culture reverses this effect. It is not unreasonable to hypothesize that beta antagonists with varying degrees of beta2 activity may differ in their ability to affect immune function to influenza vaccination in the setting of an overactive sympathetic nervous system. We acknowledge that our hypothesis generating findings require replication in a larger cohort.
While there were age differences between HF participants and controls in our study, and immune responses to influenza vaccine are reduced in older adults, the mean ages in both groups were well below age 65 years. Further, previous studies examining influenza vaccine responses by age compared groups with distinctly disparate ages.8 Although different statistically, we do not feel the age differences in our study are clinically meaningful. Even so, we reanalyzed our data excluding results from the four participants in the HF group over the age of 65 years (ages 75, 77, 79, and 81) and found that the cytokine and serological response differences between the HF group and controls were the same as in the initial analysis (Table 1).
Table 1.
T-cell and Humoral Responses to Influenza Vaccine
| HF | Healthy Controls | P value** | P-value# | |
|---|---|---|---|---|
| Interferon gamma (pg/mL) | ||||
| Pre-immunization | 10.8 | 30.4 | 0.24 | 0.2 |
| Post-immunization | 94.5 | 119.4 | ||
| IL-10 (pg/mL) | ||||
| Pre-immunization | 2.0 | 5.1 | 0.0004 | 0.0007 |
| Post-immunization | 22.5 | 12.2 | ||
|
| ||||
| Seroprotection Ab > 40 HAU* | 24/24 | 17/17 | 1 | |
| Seroprotection 4-fold increase | 14/24 | 14/17 | 0.11 | |
|
| ||||
| Antibody concentrations (HAU) H3N2 (A/Wisconsin) | ||||
| Pre-immunization | 80 | 80 | 0.009 | 0.008 |
| Post-immunization | 160 | 320 | ||
| H1N1 (A/New Caledonia) | ||||
| Pre-immunization | 20 | 80 | 0.9 | 0.95 |
| Post-immunization | 60 | 160 | ||
| B (Malaysia) | ||||
| Pre-immunization | 10 | 10 | 0.08 | 0.05 |
| Post-immunization | 40 | 80 | ||
Seroprotection and seroconversion rate differences analyzed by chi-square
Comparisons analyzed by Wilcoxon rank-sum test
Comparisons analyzed by t-test
In light of the recent pandemic of the A/H1N1 (swine-type) influenza virus, examining vaccine immune responses in patients with HF who are at high risk for influenza-related complications is of paramount importance. Our study shows that patients with HF exhibit a different T-cell response phenotype compared to controls. We also noted a reduced humoral response to the A/H3N2 vaccine strain despite similar rates of overall seroprotection and seroconversion between groups, suggesting that responses by antigens may add important information about differing vaccine efficacy in patients with HF. These data underscore the importance of further study of immune responses in HF patients, and continuous evaluation of optimal vaccination strategies for high risk adults.
Footnotes
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Contributor Information
Orly Vardeny, Assistant Professor, University of Wisconsin, School of Pharmacy, 777 Highland Avenue, Madison, WI 53705-2222, Phone: 608.265.0591, Fax: 608.265.5421
Nancy K. Sweitzer, Assistant Professor, Director, Heart Failure Program, Cardiovascular Section, Department of Medicine, University of Wisconsin School of Medicine and Public Health
Michelle A. Detry, Assistant Scientist, Department of Biostatistics & Medical Informatics, University of Wisconsin - Madison
John J.M. Moran, Research Assistant, University of Wisconsin School of Pharmacy
Maryl R. Johnson, Professor, Director, Heart Failure and Transplant Program, Cardiovascular Section, Department of Medicine, University of Wisconsin School of Medicine and Public Health
Mary S. Hayney, Associate Professor, University of Wisconsin School of Pharmacy
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