Skip to main content
Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Impaired Antibody Response to Influenza Vaccine in HIV-Infected and Uninfected Aging Women Is Associated with Immune Activation and Inflammation

  • Anita Parmigiani ,

    Contributed equally to this work with: Anita Parmigiani, Maria L. Alcaide

    Affiliation Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, Florida, United States of America

  • Maria L. Alcaide ,

    Contributed equally to this work with: Anita Parmigiani, Maria L. Alcaide

    Affiliation Division of Infectious Diseases, University of Miami Miller School of Medicine, Miami, Florida, United States of America

  • Ricardo Freguja,

    Affiliation Department of Surgery, Oncology and Gastroenterology, Oncology and Immunology Section, Unit of Viral Oncology and AIDS Reference Centre, University of Padua, Padua, Italy

  • Suresh Pallikkuth,

    Affiliation Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, Florida, United States of America

  • Daniela Frasca,

    Affiliation Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, Florida, United States of America

  • Margaret A. Fischl,

    Affiliation UM AIDS Clinical Research Unit, Division of Infectious Diseases, University of Miami Miller School of Medicine, Miami, Florida, United States of America

  • Savita Pahwa

    Spahwa@med.miami.edu

    Affiliation Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, Florida, United States of America

Abstract

Background

Aging and HIV infection are independently associated with excessive immune activation and impaired immune responses to vaccines, but their relationships have not been examined.

Methods

For selecting an aging population we enrolled 28 post-menopausal women including 12 healthy volunteers and 16 HIV-infected women on antiretroviral treatment with <100 HIV RNA copies/ml. Antibody titers to trivalent influenza vaccination given during the 2011-2012 season were determined before and 4 weeks after vaccination.

Results

Seroprotective influenza antibody titers (≥1:40) were observed in 31% HIV+ and 58% HIV-uninfected women pre-vaccination. Following vaccination, magnitude of antibody responses and frequency of seroprotection were lower in HIV+ (75%) than in HIV (91%) women. Plasma IL-21, the signature cytokine of T follicular helper cells (Tfh), and CD4 T cell IL-21R were upregulated with seroconversion (≥4 fold increase in antibody titer). Post-vaccine antibody responses were inversely correlated with pre-vaccination plasma TNFα levels and with activated CD4 T cells, including activated peripheral (p)Tfh. Plasma TNFα levels were correlated with activated pTfh cells (r=0.48, p=0.02), and inversely with the post-vaccination levels of plasma IL-21 (r=-0.53, p=0.02). In vitro TNFα blockade improved the ability of CD4 T cells to produce IL-21 and of B cells to secrete immunoglobulins, and addition of exogenous IL-21 to cell cultures enhanced B cell function. Higher frequencies of activated and exhausted CD8 T and B cells were noted in HIV+ women, but these markers did not show a correlation with antibody responses.

Conclusions

In aging HIV-infected and uninfected women, activated CD4 and pTfh cells may compromise influenza vaccine-induced antibody response, for which a mechanism of TNFα-mediated impairment of pTfh-induced IL-21 secretion is postulated. Interventions aimed at reducing chronic inflammation and immune activation in aging, HIV-infected patients may improve their response to vaccines.

Introduction

Infectious diseases take a massive toll on the well being of both the elderly and the HIV-infected population [1,2]. The situation is particularly concerning with regard to vaccine-preventable diseases, with up to 1,000 times greater risk of death in older adults compared to vaccine-aged children [3]. Indeed, the elderly (>65 yrs) account for 90% of the >35,000 affected by annual influenza epidemics [4,5]. HIV-infected people are at a significantly higher risk than the general population at all ages for acquiring seasonal influenza infection, despite vaccination and virologic control with combination antiretroviral therapy (cART) [6-8]. Seasonal influenza vaccination is recommended for elderly as well as HIV-infected individuals to reduce influenza-related morbidity and mortality [9], but immune response to influenza vaccination is frequently impaired in both these high-risk populations [10-13]. With the considerable increase in life expectancy of HIV-infected persons with cART, and the increasing incidence of new HIV infections at older ages, it is estimated that by 2015, 50% of HIV-infected population will be ≥50 years of age [14]. Given the independent detrimental effects of aging and of HIV infection on the immune system [15-17], it is important to investigate the cumulative effects of HIV and aging on immunity, e.g. as assessed by responsiveness to seasonal influenza vaccines.

Although influenza vaccines elicit both cellular and humoral responses [18], immune protection is largely correlated with post-vaccination serum antibody (Ab) titers [19]. An essential step in the generation of vaccine induced Ab-secreting B cells is the interaction of antigen-primed B cells and T follicular helper cells (Tfh) in the germinal center reaction, where Tfh cells provide essential helper function for B cells to undergo proliferation, isotype switching and somatic hypermutation (reviewed in [20]). Tfh cells are characterized by surface expression of the CXC chemokine receptor 5 (CXCR5), that promotes their homing to lymphoid germinal centers [21], and by abundant production of the cytokine interleukin (IL)-21, that plays a major role in inducing B cell differentiation and proliferation [22] and in preserving plasma cells [23,24]. Recently, a CD4 T memory cell subset in peripheral blood bearing functional and partial phenotypic similarity to lymph node Tfh has been identified and has been designated as peripheral (p)Tfh [25-27]. The pTfh cells represent approximately 15% of circulating CD4 T cells in humans [27], express CXCR5 and provide critical help to B cells for antibody secretion in an IL-21-dependent manner [26]. In a study of vaccine responses to the pandemic H1N1/09 influenza vaccine in HIV infected young patients, impaired vaccine responses were associated with defective function of pTfh and in the IL-21/IL-21R system [27,28]. In physiologic aging, impaired Ab responses to seasonal influenza vaccination have been largely attributed to intrinsic B and T cell defects [29-33], but pTfh have not been investigated.

A common feature of both HIV/AIDS and aging is the associated inflammation and immune activation of varying degrees [34,35], which is nevertheless greater in HIV-infected individuals than in age-matched uninfected controls, as we recently demonstrated in a cohort of post-menopausal women [36]. Tumor necrosis factor (TNF)α, one of the first pro-inflammatory factors that was described in HIV infection [37], is elevated also in the elderly [38,39], and its increased levels have been associated with disease progression and treatment failure in HIV-infected patients [40-42], and with increased risk for atherosclerosis in the aging population [43]. Intriguingly, this cytokine has been reported to decrease both T and B cell responses in vitro [44-46]. Excessive immune activation is known to favor disease progression and to hinder CD4 T cell immune reconstitution in HIV-infected persons [47-49]. In this study we have demonstrated that immune activation and the proinflammatory cytokine TNFα can have a detrimental effect on influenza vaccine responses in aging HIV-infected post-menopausal women.

Materials and Methods

Study population

Women were considered eligible if they were older than 45 years of age, had been amenorrheic for ≥12 months, were not receiving hormonal replacement therapy, steroids, immunosuppressant medications, and did not have active malignancies. HIV infection was documented by licensed EIA and, if needed, confirmatory western blotting prior to enrollment. The study protocol and the informed consent forms were approved by the University of Miami Institutional Review Board and participants were enrolled after written informed consent was obtained. The study population included 12 HIV-uninfected (HIV) and 16 HIV-infected (HIV+) women on ART with virologic suppression (HIV RNA <100 copies/ml) for ≥6 months prior, median CD4 count of 519 cells/mm3 (190-1,046 cells/mm3), and median nadir CD4 count of 124 cells/mm3 (4-249 cells/mm3). ART included two nucleoside reverse transcriptase inhibitors with either a Ritonavir boosted protease inhibitor, the non-nucleoside reverse transcriptase inhibitor Efavirenz or the integrase inhibitor Raltegravir. HIV+ and HIV women were matched for age and time to menopause. Characteristics of the study population are summarized in Table S1.

Study participants were given a single non-adjuvanted dose of the 2011-2012 recommended influenza vaccine, containing 15 μg hemagglutinin of each of the following strains: A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2) and B/Brisbane/60/2008 (FLUARIX® from GSK, UK). Vaccine preparation included the same viruses that were used for the 2010-2011 influenza season. Eighteen participants (5 HIV and 11 HIV+) had been vaccinated in the previous season whereas vaccination history was unavailable in the remainder. None had influenza symptoms at enrollment or follow up.

Processing of blood samples

Peripheral venous blood was collected by venipuncture into heparinized tubes at pre-vaccination (t0) and four weeks post-vaccination (wk4). Blood samples were processed immediately after collection. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-hypaque density sedimentation, cryopreserved in FBS + 10% DMSO and stored in liquid nitrogen; plasma was stored at -80°C.

Serological assessments

Hemagglutination inhibition assay (HIA).

Antibody titers specific for the whole vaccine were determined in t0 and wk4 plasma samples by HIA [50]. Whole vaccine was used instead of the individual antigens because the Ab titers were comparable to those seen against the individual viral strains [32]. Briefly, receptor-destroying enzyme (Denka Seiken, Campbell, CA) was added to the plasma samples to remove nonspecific inhibitors, incubated overnight at 37°C, and heat inactivated at 56°C for 1 hour. For each sample, a two-fold serial dilution was incubated with an equal volume (25 μl) of 4 hemagglutinin units of the same vaccine that was given to study participants (FLUARIX®, GSK) for 1 hour at room temperature. Fifty μl of 1% suspension of chicken red blood cells (Rockland™, Gilbertsville, PA) were added to each well, and antibody titers were determined 2 hours later. Paired pre- and post-vaccination samples from the same donor were tested simultaneously; each serial dilution was performed in triplicate. Seroprotection was defined as a vaccine-specific Ab titer ≥1:40, seroconversion was defined as a ≥4-fold increase in post-vaccination titers.

Multiplex plasma cytokine measurement.

TNFα, IL-6 and IL-8 plasma levels were measured using a customized MILLIPLEX™ Cytokine Human Ultrasensitive magnetic bead panel (EMD Millipore, Billerica, MA). Undiluted samples were incubated overnight with cytokine-specific beads at 4°C with shaking. After washing, biotinylated detection antibodies and streptavidin-PE were subsequently added. The beads were washed and acquired on a MAGPIX instrument (Luminex Corporation, Austin, TX). Mean fluorescent intensity (MFI) data were collected and analyzed with MILLIPLEX™ Analyst Software (EMD Millipore).

Plasma IL-21 measurement.

IL-21 levels were measured in undiluted plasma samples by ELISA (eBioscience, San Diego, CA), following manufacturer’s protocol.

Multicolor flow cytometry

Monoclonal antibodies.

The following anti-human monoclonal antibodies (MoAbs) were utilized for flow cytometry studies: CD3 AmCyan, Ki-67 PerCPCy5.5, CXCR5 AF647, IL-21R APC, CD20 APCCy7, CD21 PeCy5, CD27 AF700 and PD-1 PECy5 from BD Biosciences (San Jose, CA); CD4 QDot655, CD8 QDot605, CD10 QDot605 and CD45RO ECD from Invitrogen (Eugene, OR); HLA-DR AF488, CD38 PerCP, CD28 FITC, FcRL4 PE and CD57 APC from BioLegend (San Diego, CA). Live/Dead® Fixable Violet Dead Cell Stain (ViViD) Kit (Invitrogen) was used for exclusion of dead cells. Appropriate isotype control MoAbs were used for gating.

Immunophenotyping.

Cryopreserved PBMC were thawed and rested overnight. One million cells were incubated with ViViD stain and MoAbs for surface markers for 15 minutes, washed, fixed, permeabilized with Cytofix/Cytoperm Buffer (BD Biosciences), and stained for intracellular marker Ki-67 for 30 minutes. Cells were then washed, resuspended in PBS + 1% paraformaldehyde and acquired on a BD Biosciences LSRFortessa analyzer after proper instrument setting and compensation [51,52]. At least 500,000 events in the lymphocyte gate were acquired per sample. Data analysis was performed using FlowJo platform (TreeStar, San Carlo, CA). Frequencies of desired markers were determined in gated live (ViViD) cells. T cell populations were defined as follows: CD4 (CD3+CD4+), pTfh (CD3+CD4+CD45RO+CXCR5+, as described in [26]) and CD8 (CD3+CD8+) cells. B cell differentiation subsets, determined based on phenotypic characteristics described by Moir and Fauci (reviewed in [53]) and also by us [28], were defined as: early transitional (CD21lo/negCD27CD10+), late transitional (CD21hiCD27CD10+), naïve (CD21hiCD27CD10), resting memory (CD21hiCD27+CD10), activated memory (CD21lo/negCD27+CD10), and exhausted tissue-like (CD21lo/negCD27CD10).

Memory CD4+B cell co-cultures

Cell enrichment and culture.

B cells were isolated from healthy donor PBMC by positive selection using the EasySep™ Human CD19 Positive Selection Kit (Stemcell Technologies, Vancouver, Canada). From the remaining cells, memory (CD45RA) CD4 T cells were obtained by negative selection with the EasySep™ Human Memory CD4 T Cell Enrichment Kit (Stemcell Technologies). Purity of cell populations was 95-98%. B and memory CD4 T cells were co-cultured at a 1:1 ratio in duplicate wells. The following reagents were added to the cultures: 200 ng/ml staphylococcal enterotoxin B (SEB, Sigma-Aldrich), 50 ng/ml recombinant human (rh)TNFα, 2 μg/ml anti-human TNFα blocking antibody, 50 ng/ml rhIL-21 (Invitrogen), 20 μg/ml rhIL-21R-Fc chimera (R&D Systems, Minneapolis, MN). Appropriate isotype Abs were used as controls. Culture supernatants were tested for levels of IL-21 (day 3), IgM, IgG and IgA (day 7) using commercially available ELISA kits (from eBioscience and Bethyl Laboratories, respectively). Cells were phenotyped as described above.

Statistical analysis

Differences between groups were analyzed by Student’s t-test, Mann-Whitney test, or one-way ANOVA according to data distribution. Correlations between two variables were evaluated by Pearson correlation and linear regression. Analyses were performed using GraphPad Prism (GraphPad Software Inc, La Jolla, CA). P values <0.05 were considered significant. Results are presented as mean ± standard deviation.

Results

Response to the influenza vaccine

The reverse cumulative distribution curves in Figure 1A and B show anti-influenza antibody titers before and 4 weeks after vaccination, respectively. Immune responses to the influenza vaccine were overall higher in the HIV group as compared to the HIV-infected group. Before vaccination (t0), 58% of the HIV and 31% of the HIV+ women had a protective Ab titer (≥1:40), likely due to the persistence of antibodies generated during previous influenza seasons [54] (Figure 1A). Four weeks after vaccination (wk4), 92% of the HIV women were seroprotected, while only 75% of the HIV+ women had a protective Ab titer (Figure 1B). The increase in vaccine-specific Ab titer from baseline to wk4 post-vaccination was significant in both HIV and HIV+ women, but wk4 Ab titers were higher in the HIV group as compared to the HIV+ group (Figure 1C). The seroconversion rate did not differ between HIV and HIV+ women, with 50% of the HIV and 37.5% of the HIV+ displaying a ≥4 fold increase of Ab titer at wk4. However, the magnitude of Ab response was significantly greater in the HIV than in the HIV+ women (Figure 1D).

thumbnail
Figure 1. Antibody response to influenza vaccination.

Antibody titers specific for the whole 2011-2012 seasonal influenza vaccine were determined before (t0) and 4 weeks (wk4) after vaccination by hemagglutination inhibition assay in the plasma of 12 HIV and 16 HIV+ elderly women. Reverse cumulative distribution curves for the antibody titers prior to (A) and 4 weeks after (B) vaccination for HIV-uninfected (open circles) and HIV-infected (filled circles) donors were derived to illustrate immune responses. (C) Reciprocal of Ab titers before and after vaccination. (D) Ratio between the wk4 and the baseline reciprocal of Ab titer. P values were calculated with Student’s t-test or Mann-Whitney test as appropriate.

https://doi.org/10.1371/journal.pone.0079816.g001

Association between CD4 T cell activation and humoral response to the influenza vaccine

The immune phenotype of HIV+ aging women consisted of higher frequency of activated (CD38+HLA-DR+), replicating (Ki-67+) CD4 and pTfh cells, and higher levels of activated and senescent (CD28CD57+) CD8 T cells, as compared to the HIV controls (Table S2). There was an inverse correlation between the frequency of activated CD4 and pTfh cells and the Ab titer at wk4 post-vaccination, both in the HIV and in the HIV+ women (Figure 2A and B). An inverse correlation was observed between wk4 Ab titers and the frequency of senescent CD4 T lymphocytes in the uninfected group (Figure 2A). No association was observed between CD8 T cell activation or senescence and post-vaccination Ab titers (Figure 2C). There was no association between CD4 or pTfh activation and pre-vaccination Ab titers (not shown).

thumbnail
Figure 2. T cell activation and senescence are associated with decreased humoral response to influenza vaccination.

Frozen PBMC obtained before influenza vaccination were thawed, rested over night and stained with monoclonal antibodies for immunophenotyping of CD4 (ViViD-CD3+CD4+), pTfh (ViViD-CD3+CD4+CD45RO+CXCR5+) and CD8 (ViViD-CD3+CD8+) T cell subsets. Correlations were established between the reciprocal of influenza Ab titer at week 4 and the frequency of activated (CD38+HLA-DR+), dividing (Ki-67+) and senescent (CD28-CD57+) CD4 (A), pTfh (B) and CD8 (C) T cells for 10 HIV (open dots) and 15 HIV+ (filled dots) post-menopausal women. Pearson analysis was utilized to establish statistical correlation between variables.

https://doi.org/10.1371/journal.pone.0079816.g002

B cell alterations in HIV+ women are not associated with Ab response to the influenza vaccine

In HIV infection, B cell abnormalities in both subsets distribution and phenotype markers have been associated with altered effector function [55]. Pre-vaccination frequencies of B cell differentiation subsets determined in HIV-uninfected and infected aging women are depicted in Figure 3A. B cell distribution into differentiation subsets in HIV+ patients differed from uninfected controls as previously described [28,56]. Expression of the inhibitory receptor FcRL4 was found to be higher in several B cell subsets in the HIV+ group as compared to the HIV controls (Figure 3B). No significant changes were observed at wk4 post vaccination in relation to t0 (not shown). In contrast to what shown for CD4 and pTfh activation status (Figure 2A and B), no association between B cell alterations and wk4 influenza Ab titers were observed in HIV-uninfected or infected donors (Figure 3C).

thumbnail
Figure 3. Phenotypic alterations in B cell subsets in HIV-infected aging women are not associated with antibody response to influenza vaccination.

PBMC were stained with surface MoAbs to CD20, CD21, CD10, CD27 and FcRL4. Lymphocytes were gated based on forward and side scatter. CD3-CD20+ cells were gated into CD21hi and CD21lo/neg cells and further divided based on the expression of CD27 and CD10 as: early transitional (CD21lo/negCD27-CD10+), late transitional (CD21hiCD27-CD10+), naïve (CD21hiCD27-CD10), resting memory (CD21hiCD27+CD10), activated memory (CD21lo/negCD27+CD10), and exhausted tissue-like (CD21lo/negCD27-CD10) subsets. (A) The percentage of cells in each B cell subpopulation was determined in 10 healthy controls (open dots) and 15 HIV-infected women (filled dots) before vaccination. (B) Expression of the exhaustion marker FcRL4 was evaluated in each B cell subset. (C) Correlations were established between the B cell populations that resulted significantly different between HIV- and HIV+ in A and B and the reciprocal of influenza Ab titer at week 4. Correlation between variables was established with Pearson analysis.

https://doi.org/10.1371/journal.pone.0079816.g003

TNFα plasma levels inversely correlate with Ab response to the influenza vaccine, and are associated with CD4 T and pTfh cell activation

We sought to investigate the relation between levels of proinflammatory cytokines TNFα, IL-6 and IL-8 (see Table S2 for pre-vaccination levels of these cytokines) and magnitude of antibody response to the influenza vaccine. In both HIV and HIV+ donors, pre-vaccination plasma TNFα levels correlated inversely with wk4 Ab titers (Figure 4A), directly with the frequency of activated and dividing CD4 and pTfh cells (Figure 4B and C), and did not show an association with frequencies of senescent CD4 and CD8 T cells (Figure 4B and D), or with CD8 T cell activation (Figure 4D). No association was observed between Ab titer and IL-6 or IL-8 plasma levels, or with altered B cell subsets observed in Figure 3A and B (not shown).

thumbnail
Figure 4. Association between circulating levels of TNFα, Ab response to influenza vaccination and T cell activation and senescence.

(A) Correlation between TNFα plasma levels at t0 and the reciprocal of vaccine-specific Ab titers at week 4 was established for 12 HIV and 14 HIV+ aging women. Plasma TNFα levels were measured using a customized MILLIPLEX™ Cytokine Human Ultrasensitive magnetic bead panel (EMD Millipore). (B-D) T cells were immunophenotyped using MoAbs specific for activation (CD38, HLA-DR), proliferation (Ki-67) and senescence (CD28, CD57) markers. Frequencies of CD4, pTfh and CD8 T cell subsets expressing these markers were correlated with pre-vaccination TNFα plasma levels. Statistical analysis was performed using Pearson correlation.

https://doi.org/10.1371/journal.pone.0079816.g004

TNFα promotes T cell activation and impairs the secretion of IL-21 and immunoglobulins in vitro

Since high pre-vaccination plasma levels of TNFα were associated with pTfh activation status and low Ab response to the influenza vaccine (Figure 4A and C), we sought to investigate the effect of TNFα on T and B cells in vitro. We isolated memory CD4 T cells (as surrogates of pTfh cells) from leukopacks of 4 young healthy donors [frequency of pTfh within the purified memory CD4 T cells was comparable across all donors (22.3±2.48%)] and co-cultured them with autologous B cells in the presence of SEB. As expected, the addition of exogenous TNFα increased the expression of activation markers in both CD4 and pTfh cells (Figure 5A). Importantly, the addition of exogenous TNFα to SEB-stimulated cultures resulted in decreased IL-21 production, and maximum secretion of IL-21 was detected in the presence of blocking TNFα antibody (Figure 5B). Moreover, in the presence of SEB, exogenous TNFα resulted in decreased immunoglobulin levels, while TNFα blockade significantly increased immunoglobulin production as compared to SEB alone (Figure 5C). Immunoglobulin production increased in IL-21-supplemented cultures, and it was drastically reduced in the presence of IL-21R-Fc blocking Ab (Figure 5D). When IL-21 and TNFα were simultaneously blocked, no differences in immunoglobulin levels were observed as compared to the samples in which only IL-21 was blocked (not shown).

thumbnail
Figure 5. TNFα blockade promotes IL-21 secretion and IgG production in CD4-B cell co-cultures.

Memory CD4 T cells obtained from 4 healthy donors were cultured with autologous B cells at a 1:1 ratio in the presence of SEB and/or TNFα, IL-21, anti-anti-TNFα or anti-IL-21R blocking antibodies or the appropriate isotype control. (A) Phenotyping was performed on day 3 of co-culture in gated live CD4 and pTfh cells. (B) IL-21 levels measured by ELISA in day 3 culture supernatants. (C, D) IgM, IgG and IgA levels were evaluated by ELISA after 7 days of co-culture. In (D), IgG levels in samples where IL-21R-Fc was added were not measurable because of the cross-reactivity to the Fc portion of IL-21R-Fc. P values were calculated using paired Student’s t-test for comparisons between two groups, and repeated measures one-way ANOVA for comparisons between larger groups.

https://doi.org/10.1371/journal.pone.0079816.g005

Changes in the IL-21/IL-21R system associated with antibody response to the influenza vaccine

We previously observed that a protective humoral response to the influenza vaccine is associated with increased levels of IL-21 and increased expression of IL-21R in B cells post-vaccination [28]. Investigation of the IL-21/IL-21R system revealed an increase in IL-21 plasma levels from t0 to wk4 in those HIV and HIV+ aging women who were seroprotected at wk4, but not in the HIV+ non-seroprotected women (Figure 6A). The post-vaccination plasma levels of IL-21 correlated with the wk4 Ab titers (not shown). The increase in IL-21 plasma levels from t0 to wk4 was inversely associated to the pre-vaccination circulating levels of TNFα (Figure 6B). IL-21R MFI in CD4 T cells pre-vaccination was significantly higher in HIV as compared to the HIV+ women, both in seroprotected and non-seroprotected participants (Figure 6C). An increase in IL-21R MFI from t0 to wk4 was detected only in CD4 T cells of seroprotected women, while no changes in IL-21R MFI were observed in CD4 T cells from non-seroprotected donors, or in the CD8 T cell compartment of either SP or NSP donors (Figure 6D). IL-21R expression was also increased post-vaccination in B cells of seroprotected women, although not significantly (not shown).

thumbnail
Figure 6. Increased plasma IL-21 and IL-21R expression in CD4 T cells is associated with humoral response to the influenza vaccine.

(A) Circulating levels of IL-21 were measured by ELISA at t0 and wk4 in the plasma of 11 HIV seroprotected (HIV SP), 12 HIV+ seroprotected (HIV+ SP) and 4 HIV+ non seroprotected (HIV+ NSP) post-menopausal women. (B) Correlation between TNFα plasma levels at baseline and the ratio between wk4 and baseline plasma IL-21. (C, D) IL-21R mean fluorescence intensity (MFI) was established in live CD4 (C) and CD8 (D) T cells of 8 HIV SP, 11 HIV+ SP and 4 HIV+ NSP aging women. Frozen cells obtained before and four weeks after vaccine administration were thawed, rested overnight, stained for surface markers and acquired by flow cytometry. IL-21R MFI was established for ViViD-CD3+CD4+ or ViViD-CD3+CD8+ events. Statistical analysis was performed using paired Student’s t-test for comparisons within groups, unpaired Student’s t-test for comparisons between groups, and Pearson correlation as appropriate.

https://doi.org/10.1371/journal.pone.0079816.g006

Discussion

Influenza infection is associated with significant morbidity and mortality in the elderly and HIV-infected individuals, underscoring the importance of effective vaccination-induced protection for these populations. It is well established however that both these high risk groups can have impaired influenza vaccine responses [10,13,29-33]. Our present study in post-menopausal women implies that the burden of HIV infection on top of aging is likely to increase the risk for deficient influenza vaccine responses in comparison to HIV uninfected aging women. We describe the novel association of impaired vaccine response to ongoing immune activation. In particular, we demonstrate that an activated state of peripheral T follicular helper cells and decreased vaccine-induced plasma IL-21, the signature cytokine of Tfh, are associated with impaired influenza-vaccine induced antibodies. We provide novel experimental data implicating TNFα as a major contributor for these deficiencies.

For selecting a clear functional state of aging, we performed this study in post-menopausal women and thus avoided any influence of perimenopausal hormonal fluctuations on inflammation and immune activation. The study participants were recipients of the 2011-2012 seasonal influenza vaccine, which contained the same strains that had been included in the 2010-2011 vaccine composition [containing an A/California/7/2009 (H1N1)-like virus, an A/Perth/16/2009 (H3N2)-like virus and a B/Brisbane/60/2008-like virus]. In our study cohort, the pre-vaccination influenza Ab titers were in the protective range (≥1:40) in 58% of HIV and 31% of HIV+ aging women, presumably due to persisting or cross-reactive antibodies resulting from previous infection or vaccination. Although seroconversion rates were similar between HIV-infected and uninfected women, they differed in the response to vaccination. In the HIV+ women, antibody titers at wk4 and the fold increase in titers from t0 were lower, with several showing <4 fold increase in comparison to the HIV control group, pointing to less immune competence in the HIV-infected women.

To elucidate factors contributing to immune impairment, we investigated the relationship between Ab responses and cellular immune activation and inflammatory cytokines. Several studies have previously shown that activated CD8 T cells are associated with disease progression in HIV infection [48,49,57]. Interestingly, for the influenza vaccine-induced Ab response, it was CD4 T cell activation rather than CD8 T cell activation that showed this inverse relationship, suggesting that immune activation may have different outcomes that are specific for each cell type. Since among CD4 T cells, the pTfh subset promotes B cell proliferation and maturation [25-27], we investigated this subset and noted that the presence of activated pTfh cells prior to vaccination had a strong negative correlation with magnitude of post-vaccination Ab titers, both in HIV-uninfected and HIV-infected donors. This observation suggests that in an activated state, pTfh cells are less competent in providing the needed help to B cells to differentiate into Ab-producing cells, ultimately leading to impaired Ab response to influenza vaccine.

Both aging and HIV infection can lead to excessive inflammation. The inflammation that accompanies physiologic aging has disparate etiologies including, but not limited to, hormonal changes, diet, environmental and genetic causes. We have recently reported that in the post-menopause state several measures of immune activation and inflammation, notably T cell activation and exhaustion, monocyte/macrophage activation, and proinflammatory cytokines IL-6, IL-8 and TNFα, were higher in HIV+ women as compared to HIV-uninfected controls [36]. The drivers of immune activation in HIV disease in virally suppressed individuals are not well understood, but may include microbial translocation due to persistent virus-induced injury in gut-associated lymphoid tissue [58]. However, no association between markers of microbial translocation (circulating levels of LPS, monocyte/macrophage activation marker sCD14) and magnitude of antibody response to the influenza vaccine was observed in our study cohort (data not shown). Levels of plasma TNFα had a significant negative relationship with vaccine-specific Ab titers at week 4 post-vaccination in both HIV and HIV+ aging women. Prolonged TNFα exposure is known to impair T cell responses (namely TCR-induced proliferation and cytokine production) by attenuating T cell receptor signaling [44,45], and anti-TNF therapy in patients with psoriasis and inflammatory bowel disease results in improved responses to TCR stimulation ex vivo [59]. We investigated the influence of TNFα experimentally by adding it to cell cultures and noted that it could lead to upregulation of activation markers on CD4 T cells including pTfh cells. TNFα blockade in co-cultures of purified memory CD4 T cells and B cells enhanced Ab production supporting the contention that TNFα has a negative influence on Ab responses. Additionally, TNFα can potentially inhibit B cells directly as suggested by studies in mice [46] and by the observation that B cells of aging individuals can secrete TNFα with intrinsic inhibition of their function [60].

The helper function of Tfh and pTfh cells is mediated by cognate interaction with B cells and IL-21 secretion [25-27,55,61]. We previously demonstrated in young HIV+ adults who responded to influenza vaccine that their plasma IL-21 levels increased with upregulation of IL-21R in B cells, while vaccine non-responders failed to do either [28]. In the present study as well, we observed higher plasma IL-21 levels only in participants who were seroprotected at week 4, and frequency of IL-21R expression in CD4 T cells was also higher in these individuals. These findings expand the role of the IL-21/IL-21R system in regulating vaccine responses by activities not only in the B- but also in the CD4 T cell compartment. A trend of IL-21R upregulation in B cells was also observed, even though it did not reach statistical significance likely due to small sample size. Interestingly, IL-21R levels in CD8 T lymphocytes remained unaltered, implying that mechanisms other than IL-21 are involved in the generation of influenza-specific CD8 T cells. We have previously shown that the pTfh CD4 T cell subset resides almost exclusively in the memory CD4 T cell pool and that IL-21 production by pTfh is critical for influenza Ab response [27]. Here we demonstrated that addition of TNFα resulted in decreased IL-21 secretion in CD4 cell cultures, whereas IL-21 addition in our experimental model resulted in increased Ig production. As expected, IL-21 blockade resulted in suppressed Ig production based on the known critical role of IL-21 in supporting B cell function [22-24]. Taken together, our findings imply that excessive TNFα can impair pTfh function and IL-21 secretion, resulting in deficient help to B cells and poor Ab responses. Lack of clinical samples precluded us from doing experiments to investigate the mechanism of TNF alpha on adaptive immune responses in co-culture studies. We cannot exclude the possibility that in vivo, the decreases in IL-21 and antibody secretion iresulted from death of activated cells or a direct inhibitory effect of TNF-alpha on B cells.

Previous studies have implicated immune senescence for failure to respond to influenza vaccination [62,63]. Accumulation of senescent lymphocytes has also been reported for HIV-infected individuals [64,65], possibly as consequence of chronic antigenic stimulation. We observed that senescent CD4 and CD8 T cells in our cohort were in higher frequency in HIV+ women. Since senescent T lymphocytes are characterized by lack of the costimulatory signal transduced by CD28, decreased ability to secrete IL-2 upon activation and poor proliferative capacity (reviewed in [66]), it is not surprising that accumulation of these cells is associated with decline of the immune function and poor humoral response to influenza vaccination. B cell exhaustion and loss of memory B cells in HIV-infected adults [67] have been associated with impaired humoral response to influenza vaccination. In our cohort alterations in B cell subsets distribution and increased B cell exhaustion (FcRL4) were observed in HIV+ as compared to the uninfected participants. However, no significant association between vaccine-specific Ab titers and these B cell abnormalities was observed in this cohort. Moreover, the fact that no correlation between altered B cell subsets and TNFα levels was observed suggests that the overriding defect is that of TNFα-derived excessive T cell activation.

Limitations of this pilot study are the small sample size, absence of data for other causes of immune activation such as chronic co-infections e.g. with CMV, HCV [68,69], and possibility of residual HIV replication despite virus suppression [70], and the lack of a study group composed of young HIV-infected and uninfected donors, that would have allowed to dissect the effects of aging versus HIV infection on the immune response to influenza vaccine. Moreover, Ab titers were established for the whole vaccine rather than for its three individual components, possibly underestimating the contribution of the specific strains to the seroprotection levels of the donors. Nevertheless, this is the first study to focus on influenza vaccine-induced Ab responses in the context of aging and HIV infection and immune activation. The novel finding reported herein is the association of activated pTfh and impaired IL-21 production, with negative correlation of TNFα with poor Ab responses. Elevated plasma TNFα levels are present in HIV aging population as well as in young HIV+ individuals [35,71]. Our results provide a rationale to further evaluate the role of pTfh in vaccine response in elderly HIV+ individuals and in other proinflammatory conditions where abnormal antibody responses are observed upon vaccination. Interventions aimed to reduce chronic inflammation and activation in HIV-infected patients may improve their response to vaccination. Studies in larger cohorts may help to define predictive immunologic signatures of vaccine responses.

Supporting Information

Table S1.

Characteristics of the study population. Demographic characteristics and HIV disease markers of the study population. Menopause was defined as lack of menstruation for more than 12 months. Median values are shown for age, time to menopause and CD4 counts. Lower limit for plasma HIV RNA detection was 20 copies/ml. Statistical differences between groups were analyzed by Student t-test.

https://doi.org/10.1371/journal.pone.0079816.s001

(PDF)

Table S2.

Cellular and soluble markers of immune activation, exhaustion and senescence in HIV- and HIV+ aging women. Cryopreserved PBMC were thawed and rested overnight, stained with ViViD and monoclonal antibodies and acquired on a flow cytometer. Lymphocytes were gated based on forward and side scatter, and gates for exclusion of singlets and dead cells (ViViD+ events) were drawn. Expression of activation (CD38, HLA-DR, Ki-67), exhaustion (PD-1) and senescence (CD28, CD57) markers was evaluated in live CD4, pTfh and CD8 T cells. Plasma levels of cytokines were measured using a customized MILLIPLEXTM Cytokine Human Ultrasensitive magnetic bead panel (EMD Millipore). Statistical differences between groups were analyzed by Student t-test. Significant P values are shown in bold.

https://doi.org/10.1371/journal.pone.0079816.s002

(PDF)

Acknowledgments

We thank the Laboratory Sciences Core of the CFAR and Sylvester Comprehensive Cancer Center for facilitating conduct of the flow cytometry studies, Maria Romero for helping with the hemagglutination inhibition assay and the patients for participation in this study.

Author Contributions

Performed the experiments: AP RF S. Pallikkuth. Analyzed the data: AP MA. Wrote the manuscript: AP MA DF MF S. Pahwa. Supervised the project: S. Pahwa.

References

  1. 1. Bender BS (2003) Infectious disease risk in the elderly. Immunol Allergy Clin North Am 23: 57-64, vi doi:https://doi.org/10.1016/S0889-8561(02)00078-4. PubMed: 12645878.
  2. 2. Gardner ID (1980) The effect of aging on susceptibility to infection. Rev Infect Dis 2: 801-810. doi:https://doi.org/10.1093/clinids/2.5.801. PubMed: 6763306.
  3. 3. Gardner P, Pabbatireddy S (2004) Vaccines for women age 50 and older. Emerg Infect Dis 10: 1990-1995. doi:https://doi.org/10.3201/eid0703.040469. PubMed: 15550213.
  4. 4. Kogut I, Scholz JL, Cancro MP, Cambier JC (2012) B cell maintenance and function in aging. Semin Immunol 24: 342-349. doi:https://doi.org/10.1016/j.smim.2012.04.004. PubMed: 22560930.
  5. 5. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N et al. (2003) Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 289: 179-186. doi:https://doi.org/10.1001/jama.289.2.179. PubMed: 12517228.
  6. 6. Cohen C, Simonsen L, Sample J, Kang JW, Miller M et al. (2012) Influenza-related mortality among adults aged 25-54 years with AIDS in South Africa and the United States of America. Clin Infect Dis 55: 996-1003. doi:https://doi.org/10.1093/cid/cis549. PubMed: 22715173.
  7. 7. Sheth AN, Althoff KN, Brooks JT (2011) Influenza susceptibility, severity, and shedding in HIV-infected adults: a review of the literature. Clin Infect Dis 52: 219-227. doi:https://doi.org/10.1093/cid/ciq110. PubMed: 21288848.
  8. 8. Sheth AN, Patel P, Peters PJ (2011) Influenza and HIV: lessons from the 2009 H1N1 influenza pandemic. Curr HIV/AIDS Rep 8: 181-191. doi:https://doi.org/10.1007/s11904-011-0086-4. PubMed: 21710214.
  9. 9. website CDC (2011). Available: . http://www.cdc.gov/flu/protect/hiv-flu.htm. Accessed 2013, October 10.
  10. 10. Tebas P, Frank I, Lewis M, Quinn J, Zifchak L et al. (2010) Poor immunogenicity of the H1N1 2009 vaccine in well controlled HIV-infected individuals. AIDS 24: 2187-2192. doi:https://doi.org/10.1097/QAD.0b013e32833c6d5c. PubMed: 20616698.
  11. 11. Goodwin K, Viboud C, Simonsen L (2006) Antibody response to influenza vaccination in the elderly: a quantitative review. Vaccine 24: 1159-1169. doi:https://doi.org/10.1016/j.vaccine.2005.08.105. PubMed: 16213065.
  12. 12. Jackson LA, Chen WH, Stapleton JT, Dekker CL, Wald A et al. (2012) Immunogenicity and safety of varying dosages of a monovalent 2009 H1N1 influenza vaccine given with and without AS03 adjuvant system in healthy adults and older persons. J Infect Dis 206: 811-820. doi:https://doi.org/10.1093/infdis/jis427. PubMed: 22782949.
  13. 13. Malaspina A, Moir S, Orsega SM, Vasquez J, Miller NJ et al. (2005) Compromised B cell responses to influenza vaccination in HIV-infected individuals. J Infect Dis 191: 1442-1450. doi:https://doi.org/10.1086/429298. PubMed: 15809902.
  14. 14. High KP, Brennan-Ing M, Clifford DB, Cohen MH, Currier J et al. (2012) HIV and aging: state of knowledge and areas of critical need for research. A report to the NIH Office of AIDS Research by the HIV and Aging Working Group. J Acquir Immune Defic Syndr 60(Suppl 1): S1-18. doi:https://doi.org/10.1097/QAI.0b013e318246538f. PubMed: 22688010.
  15. 15. Gruver AL, Hudson LL, Sempowski GD (2007) Immunosenescence of ageing. J Pathol 211: 144-156. doi:https://doi.org/10.1002/path.2104. PubMed: 17200946.
  16. 16. Deeks SG, Verdin E, McCune JM (2012) Immunosenescence and HIV. Curr Opin Immunol 24: 501-506. doi:https://doi.org/10.1016/j.coi.2012.05.004. PubMed: 22658763.
  17. 17. Desai S, Landay A (2010) Early immune senescence in HIV disease. Curr HIV/AIDS Rep 7: 4-10. doi:https://doi.org/10.1007/s11904-009-0038-4. PubMed: 20425052.
  18. 18. Murasko DM, Bernstein ED, Gardner EM, Gross P, Munk G et al. (2002) Role of humoral and cell-mediated immunity in protection from influenza disease after immunization of healthy elderly. Exp Gerontol 37: 427-439. doi:https://doi.org/10.1016/S0531-5565(01)00210-8. PubMed: 11772530.
  19. 19. Hobson D, Curry RL, Beare AS, Ward-Gardner A (1972) The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. J Hyg (Lond) 70: 767-777. doi:https://doi.org/10.1017/S0022172400022610.
  20. 20. Crotty S (2011) Follicular helper CD4 T cells (TFH). Annu Rev Immunol 29: 621-663. doi:https://doi.org/10.1146/annurev-immunol-031210-101400. PubMed: 21314428.
  21. 21. Ansel KM, Ngo VN, Hyman PL, Luther SA, Förster R et al. (2000) A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 406: 309-314. doi:https://doi.org/10.1038/35018581. PubMed: 10917533.
  22. 22. Zotos D, Coquet JM, Zhang Y, Light A, D'Costa K et al. (2010) IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism. J Exp Med 207: 365-378. doi:https://doi.org/10.1084/jem.20091777. PubMed: 20142430.
  23. 23. Ozaki K, Spolski R, Ettinger R, Kim HP, Wang G et al. (2004) Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6. J Immunol 173: 5361-5371. PubMed: 15494482.
  24. 24. Ettinger R, Sims GP, Fairhurst AM, Robbins R, da Silva YS et al. (2005) IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. J Immunol 175: 7867-7879. PubMed: 16339522.
  25. 25. Chevalier N, Jarrossay D, Ho E, Avery DT, Ma CS et al. (2011) CXCR5 expressing human central memory CD4 T cells and their relevance for humoral immune responses. J Immunol 186: 5556-5568. doi:https://doi.org/10.4049/jimmunol.1002828. PubMed: 21471443.
  26. 26. Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L et al. (2011) Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity 34: 108-121. doi:https://doi.org/10.1016/j.immuni.2010.12.012. PubMed: 21215658.
  27. 27. Pallikkuth S, Parmigiani A, Silva SY, George VK, Fischl M et al. (2012) Impaired peripheral blood T-follicular helper cell function in HIV-infected nonresponders to the 2009 H1N1/09 vaccine. Blood 120: 985-993. doi:https://doi.org/10.1182/blood-2011-12-396648. PubMed: 22692510.
  28. 28. Pallikkuth S, Pilakka Kanthikeel S, Silva SY, Fischl M, Pahwa R et al. (2011) Upregulation of IL-21 receptor on B cells and IL-21 secretion distinguishes novel 2009 H1N1 vaccine responders from nonresponders among HIV-infected persons on combination antiretroviral therapy. J Immunol 186: 6173-6181. doi:https://doi.org/10.4049/jimmunol.1100264. PubMed: 21531891.
  29. 29. Weinberger B, Herndler-Brandstetter D, Schwanninger A, Weiskopf D, Grubeck-Loebenstein B (2008) Biology of immune responses to vaccines in elderly persons. Clin Infect Dis 46: 1078-1084. doi:https://doi.org/10.1086/529197. PubMed: 18444828.
  30. 30. Lang PO, Govind S, Mitchell WA, Siegrist CA, Aspinall R (2011) Vaccine effectiveness in older individuals: what has been learned from the influenza-vaccine experience. Ageing Res Rev 10: 389-395. doi:https://doi.org/10.1016/j.arr.2010.09.005. PubMed: 20888439.
  31. 31. Frasca D, Blomberg BB (2011) Aging affects human B cell responses. J Clin Immunol 31: 430-435. doi:https://doi.org/10.1007/s10875-010-9501-7. PubMed: 21318330.
  32. 32. Frasca D, Diaz A, Romero M, Landin AM, Phillips M et al. (2010) Intrinsic defects in B cell response to seasonal influenza vaccination in elderly humans. Vaccine 28: 8077-8084. doi:https://doi.org/10.1016/j.vaccine.2010.10.023. PubMed: 20974306.
  33. 33. Frasca D, Diaz A, Romero M, Phillips M, Mendez NV et al. (2012) Unique biomarkers for B-cell function predict the serum response to pandemic H1N1 influenza vaccine. Int Immunol 24: 175-182. doi:https://doi.org/10.1093/intimm/dxr123. PubMed: 22281510.
  34. 34. Klatt NR, Funderburg NT, Brenchley JM (2013) Microbial translocation, immune activation, and HIV disease. Trends Microbiol 21: 6-13. doi:https://doi.org/10.1016/j.tim.2012.09.001. PubMed: 23062765.
  35. 35. Franceschi C, Bonafè M, Valensin S, Olivieri F, De Luca M et al. (2000) Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 908: 244-254. PubMed: 10911963.
  36. 36. Alcaide ML, Parmigiani A, Pallikkuth S, Roach M, Freguja R et al. (2013) Immune Activation in HIV-Infected Aging Women on Antiretrovirals—Implications for Age-Associated Comorbidities: A Cross-Sectional Pilot Study. PLOS ONE 8: e63804. doi:https://doi.org/10.1371/journal.pone.0063804. PubMed: 23724003.
  37. 37. Lähdevirta J, Maury CP, Teppo AM, Repo H (1988) Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome. Am J Med 85: 289-291. doi:https://doi.org/10.1016/0002-9343(88)90576-1. PubMed: 3414726.
  38. 38. Bruunsgaard H, Andersen-Ranberg K, Jeune B, Pedersen AN, Skinhøj P et al. (1999) A high plasma concentration of TNF-alpha is associated with dementia in centenarians. J Gerontol A Biol Sci Med Sci 54: M357-M364. doi:https://doi.org/10.1093/gerona/54.7.M357. PubMed: 10462168.
  39. 39. Paolisso G, Rizzo MR, Mazziotti G, Tagliamonte MR, Gambardella A et al. (1998) Advancing age and insulin resistance: role of plasma tumor necrosis factor-alpha. Am J Physiol 275: E294-E299. PubMed: 9688632.
  40. 40. Aukrust P, Liabakk NB, Müller F, Lien E, Espevik T et al. (1994) Serum levels of tumor necrosis factor-alpha (TNF alpha) and soluble TNF receptors in human immunodeficiency virus type 1 infection--correlations to clinical, immunologic, and virologic parameters. J Infect Dis 169: 420-424. doi:https://doi.org/10.1093/infdis/169.2.420. PubMed: 7906293.
  41. 41. Godfried MH, van der Poll T, Weverling GJ, Mulder JW, Jansen J et al. (1994) Soluble receptors for tumor necrosis factor as predictors of progression to AIDS in asymptomatic human immunodeficiency virus type 1 infection. J Infect Dis 169: 739-745. doi:https://doi.org/10.1093/infdis/169.4.739. PubMed: 7907641.
  42. 42. Aukrust P, Müller F, Lien E, Nordoy I, Liabakk NB et al. (1999) Tumor necrosis factor (TNF) system levels in human immunodeficiency virus-infected patients during highly active antiretroviral therapy: persistent TNF activation is associated with virologic and immunologic treatment failure. J Infect Dis 179: 74-82. doi:https://doi.org/10.1086/314572. PubMed: 9841825.
  43. 43. Bruunsgaard H, Skinhøj P, Pedersen AN, Schroll M, Pedersen BK (2000) Ageing, tumour necrosis factor-alpha (TNF-alpha) and atherosclerosis. Clin Exp Immunol 121: 255-260. doi:https://doi.org/10.1046/j.1365-2249.2000.01281.x. PubMed: 10931139.
  44. 44. Cope AP, Liblau RS, Yang XD, Congia M, Laudanna C et al. (1997) Chronic tumor necrosis factor alters T cell responses by attenuating T cell receptor signaling. J Exp Med 185: 1573-1584. doi:https://doi.org/10.1084/jem.185.9.1573. PubMed: 9151895.
  45. 45. Isomäki P, Panesar M, Annenkov A, Clark JM, Foxwell BM et al. (2001) Prolonged exposure of T cells to TNF down-regulates TCR zeta and expression of the TCR/CD3 complex at the cell surface. J Immunol 166: 5495-5507. PubMed: 11313388.
  46. 46. Frasca D, Romero M, Diaz A, Alter-Wolf S, Ratliff M et al. (2012) A molecular mechanism for TNF-alpha-mediated downregulation of B cell responses. J Immunol 188: 279-286. doi:https://doi.org/10.4049/jimmunol.1003964. PubMed: 22116831.
  47. 47. Hazenberg MD, Otto SA, van Benthem BH, Roos MT, Coutinho RA et al. (2003) Persistent immune activation in HIV-1 infection is associated with progression to AIDS. AIDS 17: 1881-1888. doi:https://doi.org/10.1097/00002030-200309050-00006. PubMed: 12960820.
  48. 48. Liu Z, Cumberland WG, Hultin LE, Prince HE, Detels R et al. (1997) Elevated CD38 antigen expression on CD8+ T cells is a stronger marker for the risk of chronic HIV disease progression to AIDS and death in the Multicenter AIDS Cohort Study than CD4+ cell count, soluble immune activation markers, or combinations of HLA-DR and CD38 expression. J Acquir Immune Defic Syndr Hum Retrovirol 16: 83-92. doi:https://doi.org/10.1097/00042560-199710010-00003. PubMed: 9358102.
  49. 49. Mocroft A, Bofill M, Lipman M, Medina E, Borthwick N et al. (1997) CD8+,CD38+ lymphocyte percent: a useful immunological marker for monitoring HIV-1-infected patients. J Acquir Immune Defic Syndr Hum Retrovirol 14: 158-162. doi:https://doi.org/10.1097/00042560-199702010-00009. PubMed: 9052725.
  50. 50. Hsiung GD, Fong CKY, Landry ML (1994) Hsiung's Diagnostic Virology: As Illustrated by Light and Electron Microscopy. Yale University Press. p. 404.
  51. 51. Maecker HT, Trotter J (2006) Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69: 1037-1042. PubMed: 16888771.
  52. 52. Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay P, Roederer M (2006) Quality assurance for polychromatic flow cytometry. Nat Protoc 1: 1522-1530. doi:https://doi.org/10.1038/nprot.2006.250. PubMed: 17406444.
  53. 53. Moir S, Fauci AS (2009) B cells in HIV infection and disease. Nat Rev Immunol 9: 235-245. doi:https://doi.org/10.1038/nri2524. PubMed: 19319142.
  54. 54. Skowronski DM, Tweed SA, De Serres G (2008) Rapid decline of influenza vaccine-induced antibody in the elderly: is it real, or is it relevant? J Infect Dis 197: 490-502. doi:https://doi.org/10.1086/524146. PubMed: 18275271.
  55. 55. Lane HC, Masur H, Edgar LC, Whalen G, Rook AH et al. (1983) Abnormalities of B-cell activation and immunoregulation in patients with the acquired immunodeficiency syndrome. N Engl J Med 309: 453-458. doi:https://doi.org/10.1056/NEJM198308253090803. PubMed: 6224088.
  56. 56. Moir S, Malaspina A, Ho J, Wang W, Dipoto AC et al. (2008) Normalization of B cell counts and subpopulations after antiretroviral therapy in chronic HIV disease. J Infect Dis 197: 572-579. doi:https://doi.org/10.1086/526789. PubMed: 18240953.
  57. 57. Levacher M, Hulstaert F, Tallet S, Ullery S, Pocidalo JJ et al. (1992) The significance of activation markers on CD8 lymphocytes in human immunodeficiency syndrome: staging and prognostic value. Clin Exp Immunol 90: 376-382. PubMed: 1458674.
  58. 58. Douek D (2007) HIV disease progression: immune activation, microbes, and a leaky gut. Top HIV Med 15: 114-117. PubMed: 17720995.
  59. 59. Bosè F, Raeli L, Garutti C, Frigerio E, Cozzi A et al. (2011) Dual role of anti-TNF therapy: enhancement of TCR-mediated T cell activation in peripheral blood and inhibition of inflammation in target tissues. Clin Immunol 139: 164-176. doi:https://doi.org/10.1016/j.clim.2011.01.015. PubMed: 21334981.
  60. 60. Frasca D, Diaz A, Romero M, Mendez NV, Landin AM et al. (2013) Young and elderly patients with type 2 diabetes have optimal B cell responses to the seasonal influenza vaccine. Vaccine.
  61. 61. Alcaide ML, Parmigiani A, Pallikkuth S, Roach M, Freguja R et al. (2013) Immune Activation in HIV-Infected Aging Women on Antiretrovirals—Implications for Age-Associated Comorbidities: A Cross-Sectional Pilot Study.
  62. 62. Goronzy JJ, Fulbright JW, Crowson CS, Poland GA, O'Fallon WM et al. (2001) Value of immunological markers in predicting responsiveness to influenza vaccination in elderly individuals. J Virol 75: 12182-12187. doi:https://doi.org/10.1128/JVI.75.24.12182-12187.2001. PubMed: 11711609.
  63. 63. Trzonkowski P, Myśliwska J, Szmit E, Wieckiewicz J, Lukaszuk K et al. (2003) Association between cytomegalovirus infection, enhanced proinflammatory response and low level of anti-hemagglutinins during the anti-influenza vaccination--an impact of immunosenescence. Vaccine 21: 3826-3836. doi:https://doi.org/10.1016/S0264-410X(03)00309-8. PubMed: 12922116.
  64. 64. Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ et al. (2003) Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells. Blood 101: 2711-2720. doi:https://doi.org/10.1182/blood-2002-07-2103. PubMed: 12433688.
  65. 65. Effros RB, Allsopp R, Chiu CP, Hausner MA, Hirji K et al. (1996) Shortened telomeres in the expanded CD28-CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis. AIDS 10: F17-F22. doi:https://doi.org/10.1097/00002030-199601000-00003. PubMed: 8828735.
  66. 66. Weng NP, Akbar AN, Goronzy J (2009) CD28(-) T cells: their role in the age-associated decline of immune function. Trends Immunol 30: 306-312. doi:https://doi.org/10.1016/j.it.2009.03.013. PubMed: 19540809.
  67. 67. Moir S, Ho J, Malaspina A, Wang W, DiPoto AC et al. (2008) Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals. J Exp Med 205: 1797-1805. doi:https://doi.org/10.1084/jem.20072683. PubMed: 18625747.
  68. 68. Hunt PW, Martin JN, Sinclair E, Epling L, Teague J et al. (2011) Valganciclovir reduces T cell activation in HIV-infected individuals with incomplete CD4+ T cell recovery on antiretroviral therapy. J Infect Dis 203: 1474-1483. doi:https://doi.org/10.1093/infdis/jir060. PubMed: 21502083.
  69. 69. Yao ZQ, Moorman JP (2013) Immune Exhaustion and Immune Senescence: Two Distinct Pathways for HBV Vaccine Failure During HCV and/or HIV Infection. Arch Immunol Ther Exp (Warsz).
  70. 70. Buzón MJ, Massanella M, Llibre JM, Esteve A, Dahl V et al. (2010) HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med 16: 460-465. doi:https://doi.org/10.1038/nm.2111. PubMed: 20228817.
  71. 71. Salazar-Gonzalez JF, Martinez-Maza O, Aziz N, Kolberg JA, Yeghiazarian T et al. (1997) Relationship of plasma HIV-RNA levels and levels of TNF-alpha and immune activation products in HIV infection. Clin Immunol Immunopathol 84: 36-45. doi:https://doi.org/10.1006/clin.1997.4364. PubMed: 9191882.