Krista M. Goergen

The Impact of Immunosenescence on Humoral Immune Response Variation after Influenza A/H1N1 Vaccination in Older Subjects

Background Although influenza causes significant morbidity and mortality in the elderly, the factors underlying the reduced vaccine immunogenicity and efficacy in this age group are not completely understood. Age and immunosenescence factors, and their impact on humoral immunity after influenza vaccination, are of growing interest for the development of better vaccines for the elderly. Methods We assessed associations between age and immunosenescence markers (T cell receptor rearrangement excision circles – TREC content, peripheral white blood cell telomerase – TERT expression and CD28 expression on T cells) and influenza A/H1N1 vaccine-induced measures of humoral immunity in 106 older subjects at baseline and three timepoints post-vaccination. Results TERT activity (TERT mRNA expression) was significantly positively correlated with the observed increase in the influenza-specific memory B cell ELISPOT response at Day 28 compared to baseline (p-value=0.025). TREC levels were positively correlated with the baseline and early (Day 3) influenza A/H1N1-specific memory B cell ELISPOT response (p-value=0.042 and p-value=0.035, respectively). The expression and/or expression change of CD28 on CD4+ and/or CD8+ T cells at baseline and Day 3 was positively correlated with the influenza A/H1N1-specific memory B cell ELISPOT response at baseline, Day 28 and Day 75 post-vaccination. In a multivariable analysis, the peak antibody response (HAI and/or VNA at Day 28) was negatively associated with age, the percentage of CD8+CD28low T cells, IgD+CD27- naïve B cells, and percentage overall CD20- B cells and plasmablasts, measured at Day 3 post-vaccination. The early change in influenza-specific memory B cell ELISPOT response was positively correlated with the observed increase in influenza A/H1N1-specific HAI antibodies at Day 28 and Day 75 relative to baseline (p-value=0.007 and p-value=0.005, respectively). Conclusion Our data suggest that influenza-specific humoral immunity is significantly influenced by age, and that specific markers of immunosenescence (e.g., the baseline/early expression of CD28 on CD4+ and/or CD8+ T cells and T cell immune abnormalities) are correlated with different humoral immune response outcomes observed after vaccination in older individuals, and thus can be potentially used to predict vaccine immunogenicity.

Inherited variants in regulatory T cell genes and outcome of ovarian cancer.

Although ovarian cancer is the most lethal of gynecologic malignancies, wide variation in outcome following conventional therapy continues to exist. The presence of tumor-infiltrating regulatory T cells (Tregs) has a role in outcome of this disease, and a growing body of data supports the existence of inherited prognostic factors. However, the role of inherited variants in genes encoding Treg-related immune molecules has not been fully explored. We analyzed expression quantitative trait loci (eQTL) and sequence-based tagging single nucleotide polymorphisms (tagSNPs) for 54 genes associated with Tregs in 3,662 invasive ovarian cancer cases. With adjustment for known prognostic factors, suggestive results were observed among rarer histological subtypes; poorer survival was associated with minor alleles at SNPs in RGS1 (clear cell, rs10921202, p = 2.7×10−5), LRRC32 and TNFRSF18/TNFRSF4 (mucinous, rs3781699, p = 4.5×10−4, and rs3753348, p = 9.0×10−4, respectively), and CD80 (endometrioid, rs13071247, p = 8.0×10−4). Fo0r the latter, correlative data support a CD80 rs13071247 genotype association with CD80 tumor RNA expression (p = 0.006). An additional eQTL SNP in CD80 was associated with shorter survival (rs7804190, p = 8.1×10−4) among all cases combined. As the products of these genes are known to affect induction, trafficking, or immunosuppressive function of Tregs, these results suggest the need for follow-up phenotypic studies.

Granzyme B ELISPOT assay to measure influenza-specific cellular immunity.

The immunogenicity and efficacy of influenza vaccination are markedly lower in the elderly. Granzyme B (GrzB), quantified in fresh cell lysates, has been suggested to be a marker of cytotoxic T lymphocyte (CTL) response and a predictor of influenza illness among vaccinated older individuals. We have developed an influenza-specific GrzB ELISPOT assay using cryopreserved PBMCs. This method was tested on 106 healthy older subjects (ages 50-74) at baseline (Day 0) and three additional time points post-vaccination (Day 3, Day 28, Day 75) with influenza A/H1N1-containing vaccine. No significant difference was seen in GrzB response between any of the time points, although influenza-specific GrzB response appears to be elevated at all post-vaccination time points. There was no correlation between GrzB response and hemagglutination inhibition (HAI) titers, indicating no relationship between the cytolytic activity and humoral antibody levels in this cohort. Additionally, a significant negative correlation between GrzB response and age was observed. These results reveal a reduction in influenza-specific GrzB response as one ages. In conclusion, we have developed and optimized an influenza-specific ELISPOT assay for use with frozen cells to quantify the CTL-specific serine protease GrzB, as a measure of cellular immunity after influenza vaccination.

Plasma immune analytes in patients with epithelial ovarian cancer

OBJECTIVES Inflammation is a common feature of epithelial ovarian cancer (EOC), and measurement of plasma markers of inflammation might identify candidate markers for use in screening or presurgical evaluation of patients with adnexal masses. METHODS Plasma specimens from cohorts of 100 patients with advanced EOC (AJCC Stage III and IV), 50 patients with early stage EOC (Stage I and II), and 50 patients with benign surgical conditions were assayed for concentrations of multiple cytokines, toll-like receptor agonists, and vascular growth factors via ELISA and electrochemiluminescence. Immune proteins were then analyzed for association with EOC. Differences in plasma protein levels between benign, early, and advanced EOC patient groups were assessed with and without adjustment for plasma cancer antigen 125 (CA-125) levels. RESULTS Out of 23 proteins tested, six-including interferon gamma (IFNγ), interleukin 6 (IL-6), IL-8, IL-10, tumor necrosis factor alpha (TNFα), and placental growth factor (PlGF)-were univariately associated with EOC (all p<0.005), and one-IL-6-was associated with early stage EOC (p<0.0001). Heat shock protein 90kDa beta member 1 (HSP90B1, gp96) was associated with EOC and early stage EOC with borderline statistical significance (p=0.039 and p=0.026, respectively). However, when adjusted for (CA-125), only HSP90B1 independently predicted EOC (p=0.008), as well as early stage EOC (p=0.014). CONCLUSIONS Multiple plasma cytokines, including IFNγ, IL-6, IL-8, IL-10, TNFα, PlGF, and HSP90B1 are associated with EOC. Of these, HSP90B1 is associated with EOC independent from the biomarker CA-125.

Utility of progranulin and serum leukocyte protease inhibitor as diagnostic and prognostic biomarkers in ovarian cancer

Background: Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer-related death in females and leading gynecologic cause of cancer-related death. Despite the identification of a number of serum biomarkers, methods to identify early-stage disease and predict prognosis remain scarce. We have evaluated two biologically connected serum biomarkers, serum leukocyte protease inhibitor (SLPI) and progranulin (PGRN). Methods: Two-hundred frozen plasma samples were acquired from the Mayo Clinic Biospecimen Repository for Ovarian Cancer Research. Samples were obtained from 50 patients with benign conditions, 50 with American Joint Committee on Cancer (AJCC) stage I and II EOC, and 100 with AJCC stage III and IV EOC. Samples were obtained before surgical resection of a mass and were analyzed for absolute levels of SLPI and PGRN using ELISA assays. Receiver-operator characteristic curves were generated for SLPI and PGRN. Median follow-up was 48 months. Results: Absolute levels of SLPI were significantly elevated in patients with EOC compared with benign disease and predicted the presence of EOC (AUC of 0.812; P = 0.04); SLPI remained elevated in the subset of patients with normal CA-125. PGRN levels were not significantly increased in patients with early-stage or late-stage EOC as a whole, but an increase in PGRN levels was associated with decreased overall survival in advanced EOC. Conclusions: SLPI levels are elevated in EOC, and SLPI shows promise as a diagnostic biomarker for patients with both elevated and normal CA-125 levels. An increase in PGRN is associated with decreased overall survival. Impact: SLPI is elevated in EOC and warrants investigation in a screening study in women at risk for EOC. Cancer Epidemiol Biomarkers Prev; 22(10); 1730–5. ©2013 AACR.

Transthoracic Echocardiography versus Computed Tomography for Ascending Aortic Measurements in Patients with Bicuspid Aortic Valve

Background: Ascending aorta dilatation is common in bicuspid aortic valve (BAV). The aim of this study was to investigate agreement of transthoracic echocardiographic (TTE) measurement of the sinuses of Valsalva and the tubular mid–ascending aorta (Asc‐Ao) compared with electrocardiographically gated computed tomographic angiographic (CTA) assessment in patients with BAV. Methods: Fifty‐three patients with BAV (mean age, 54 ± 14 years; 74% men) who underwent both TTE and CTA imaging for ascending aortic assessment were retrospectively identified. All studies were measured de novo by experts. TTE measurements were obtained at the sinuses and the Asc‐Ao, at both systole and end‐diastole, using both leading edge–to–leading edge (L‐L) and inner edge–to–inner edge (I‐I) methods in the parasternal long‐axis (LAX) view. The sinuses were also measured in the parasternal short‐axis (SAX) view using the same methods plus mid‐diastole. CTA measurements were obtained in diastole using outer wall–to–outer wall (O‐O) and inner wall–to–inner wall (I‐I) methods. Correlation and agreement between the two imaging modalities were assessed using Lin correlation and Bland‐Altman analysis, respectively. Results: Compared with CTA O‐O maximum sinuses diameter, the best correlation and agreement were obtained using the TTE SAX mid‐diastolic L‐L method (&rgr; = 0.89, 2.6 ± 2.3 mm, respectively). Compared with CTA O‐O maximum Asc‐Ao diameter, the TTE LAX systolic L‐L method (&rgr; = 0.93, 1.3 ± 2.5 mm) was best. Compared with CTA I‐I maximum sinuses diameter, the TTE SAX mid‐diastole L‐L method (&rgr; = 0.95, 0.6 ± 2.2 mm) was unbiased. Compared with CTA I‐I maximum Asc‐Ao diameter, the TTE LAX end‐diastolic L‐L method (&rgr; = 0.95, 0.6 ± 2.4 mm) was unbiased. Conclusions: In patients with BAV aortopathy, unbiased agreement between CTA and TTE imaging can be obtained between the CTA I‐I method and TTE SAX mid‐diastolic L‐L method for the sinuses and the TTE LAX end‐diastolic L‐L method for the Asc‐Ao. When using the CTA O‐O method, the best agreement is obtained with the TTE SAX mid‐diastolic L‐L method for the sinuses (bias ˜2 mm) and the TTE LAX systolic L‐L method (bias ˜1 mm) for the Asc‐Ao. HighlightsIn patients with bicuspid aortic valve aortopathy, there is significant systematic underestimation of sinuses of Valsalva by the conventional TTE end‐diastolic L‐L method, as compared to ECG‐gated CTA.In patients with bicuspid aortic valve aortopathy, unbiased agreement between CTA and TTE imaging can be obtained between the CTA I‐I method and the TTE short‐axis mid‐diastolic L‐L method for the sinuses and the conventional TTE method for the Asc‐Ao.In patients with bicuspid aortic valve aortopathy, when using the CTA O‐O method, the best agreement is obtained with the TTE short‐axis mid‐diastolic L‐L method for the sinuses (bias ˜2 mm) and the conventional TTE method for the Asc‐Ao (bias ˜1 mm).

Gene signatures related to HAI response following influenza A/H1N1 vaccine in older individuals

To assess gene signatures related to humoral response among healthy older subjects following seasonal influenza vaccination, we studied 94 healthy adults (50–74 years old) who received one documented dose of licensed trivalent influenza vaccine containing the A/California/7/2009 (H1N1)-like virus strain. Influenza-specific antibody (HAI) titer in serum samples and next-generation sequencing on PBMCs were performed using blood samples collected prior to (Day 0) and at two timepoints after (Days 3 and 28) vaccination. We identified a number of uncharacterized genes (ZNF300, NUP1333, KLK1 and others) and confirmed previous studies demonstrating specific genes/genesets that are important mediators of host immune responses and that displayed associations with antibody response to influenza A/H1N1 vaccine. These included interferon-regulatory transcription factors (IRF1/IRF2/IRF6/IRF7/IRF9), chemokine/chemokine receptors (CCR5/CCR9/CCL5), cytokine/cytokine receptors (IFNG/IL10RA/TNFRSF1A), protein kinases (MAP2K4/MAPK3), growth factor receptor (TGFBR1). The identification of gene signatures associated with antibody response represents an early stage in the science for which further research is needed. Such research may assist in the design of better vaccines to facilitate improved defenses against new influenza virus strains, as well as better understanding the genetic drivers of immune responses.

Assessment of published models and prognostic variables in epithelial ovarian cancer at Mayo Clinic

Objectives Epithelial ovarian cancer (EOC) is an aggressive disease in which first line therapy consists of a surgical staging/debulking procedure and platinum based chemotherapy. There is significant interest in clinically applicable, easy to use prognostic tools to estimate risk of recurrence and overall survival. In this study we used a large prospectively collected cohort of women with EOC to validate currently published models and assess prognostic variables. Methods Women with invasive ovarian, peritoneal, or fallopian tube cancer diagnosed between 2000-2011 and prospectively enrolled into the Mayo Clinic Ovarian Cancer registry were identified. Demographics and known prognostic markers as well as epidemiologic exposure variables were abstracted from the medical record and collected via questionnaire. Six previously published models of overall and recurrence-free survival were assessed for external validity. In addition, predictors of outcome were assessed in our dataset. Results Previously published models validated with a range of c-statistics (0.587-0.827), though application of models containing variables not part of routine practice were somewhat limited by missing data; utilization of all applicable models and comparison of results is suggested. Examination of prognostic variables identified only the presence of ascites and ASA score to be independent predictors of prognosis in our dataset, albeit with marginal gain in prognostic information, after accounting for stage and debulking. Conclusions Existing prognostic models for newly diagnosed EOC showed acceptable calibration in our cohort for clinical application. However, modeling of prospective variables in our dataset reiterates that stage and debulking remain the most important predictors of prognosis in this setting.

Vitamin D, leptin and impact on immune response to seasonal influenza A/H1N1 vaccine in older persons.

ABSTRACT Background: Influenza-related complications are highest in the elderly. Vaccine efficacy is lower due to immunosenescence. Vitamin Ds immunomodulatory role was studied in the context of vaccine response. Methods: We evaluated the effect of baseline 25-(OH) D on vaccine-induced immunological response in a cohort of 159 healthy subjects ages 50–74 in Rochester, MN, who received one dose of seasonal trivalent 2010–2011 influenza vaccine, containing A/California/H1N1- like virus. We examined correlations between 25-(OH) D, leptin, and leptin-related gene SNPs to understand the role of leptin and vitamin Ds effects. Results: The median (IQR) baseline for total 25-(OH) D was 44.4 ng/mL (36.6–52.2 ng/mL). No correlation was observed with age. No correlation between 25-(OH) D levels and humoral immune outcomes existed at any timepoint. There was a weak positive correlation between 25-(OH) D levels and change (Day 75-Day 0) in influenza-specific granzyme-B response (r=0.16, p=0.04). We found significant associations between 3 SNPs in the PPARG gene and 25-(OH) D levels (rs1151996, p=0.01; rs1175540, p= 0.02; rs1175544, p=0.03). Conclusion: Several SNPs in the PPARG gene were significantly associated with baseline 25-(OH) D levels. Understanding the functional and mechanistic relationships between vitamin D and influenza vaccine-induced immunity could assist in directing new influenza vaccine design.

Integration of Immune Cell Populations, mRNA-Seq, and CpG Methylation to Better Predict Humoral Immunity to Influenza Vaccination: Dependence of mRNA-Seq/CpG Methylation on Immune Cell Populations

The development of a humoral immune response to influenza vaccines occurs on a multisystems level. Due to the orchestration required for robust immune responses when multiple genes and their regulatory components across multiple cell types are involved, we examined an influenza vaccination cohort using multiple high-throughput technologies. In this study, we sought a more thorough understanding of how immune cell composition and gene expression relate to each other and contribute to interindividual variation in response to influenza vaccination. We first hypothesized that many of the differentially expressed (DE) genes observed after influenza vaccination result from changes in the composition of participants’ peripheral blood mononuclear cells (PBMCs), which were assessed using flow cytometry. We demonstrated that DE genes in our study are correlated with changes in PBMC composition. We gathered DE genes from 128 other publically available PBMC-based vaccine studies and identified that an average of 57% correlated with specific cell subset levels in our study (permutation used to control false discovery), suggesting that the associations we have identified are likely general features of PBMC-based transcriptomics. Second, we hypothesized that more robust models of vaccine response could be generated by accounting for the interplay between PBMC composition, gene expression, and gene regulation. We employed machine learning to generate predictive models of B-cell ELISPOT response outcomes and hemagglutination inhibition (HAI) antibody titers. The top HAI and B-cell ELISPOT model achieved an area under the receiver operating curve (AUC) of 0.64 and 0.79, respectively, with linear model coefficients of determination of 0.08 and 0.28. For the B-cell ELISPOT outcomes, CpG methylation had the greatest predictive ability, highlighting potentially novel regulatory features important for immune response. B-cell ELISOT models using only PBMC composition had lower performance (AUC = 0.67), but highlighted well-known mechanisms. Our analysis demonstrated that each of the three data sets (cell composition, mRNA-Seq, and DNA methylation) may provide distinct information for the prediction of humoral immune response outcomes. We believe that these findings are important for the interpretation of current omics-based studies and set the stage for a more thorough understanding of interindividual immune responses to influenza vaccination.