The effect of aging on the frequency, phenotype and cytokine production of human blood CD4 + CXCR5 + T follicular helper cells: comparison of aged and young subjects
© Zhou et al.; licensee BioMed Central Ltd. 2014
Received: 14 May 2014
Accepted: 18 August 2014
Published: 23 August 2014
The Erratum to this article has been published in Immunity & Ageing 2015 12:13
T cell-dependent B-cell responses decline with age, indicating declined cognate helper activity of aged CD4 + T cells for B cells. However, the mechanisms remain unclear. T follicular helper (Tfh) cells, a novel T helper subset, play an essential role in helping B cells differentiation into long-lived plasma cells in germinal center (GC) or short-lived plasma cells. In the present study, we proposed that there might existe changes of proportion, phenotype or cytokine production of blood Tfh cells in healthy elderly individuals compared with healthy young individuals.
The results showed that frequencies of aged blood CXCR5 + CD4 + Tfh cells increased compared with young subjects. Both aged and young blood CXCR5 + CD4 + Tfh cells constitutively expressed CD45RO, CCR7 and CD28, and few of these cells expressed CD69 or HLA-DR, which indicated that they were resting memory cells. There was no significant difference of IL-21 frequency production by aged blood CXCR5 + CD4 + Tfh determined by FACS compared with young individuals, however, aged PBMCs produced significantly higher levels of IL-21 evaluated by ELISA. Furthermore, there were no significant differences of percentages of IFN-γ, IL-4, IL-17 or IL-22 production by aged Tfh cells compared with their counterparts of young individuals respectively. However, frequencies of IL-17+ cells within aged CD4 + CXCR5-T cells were markedly lower than in the young individuals. Furthermore we observed different frequencies of IFN-γ, IL-17, IL-4 or IL-22 production by Tfh or by CD4 + CXCR5- cells in aged and young subjects respectively.
Our data demonstrated that the frequencies of blood memory CXCR5 + CD4 + Tfh cells increased in the elderly population. There were similar frequencies of Th characterized cytokine production such as IL-21, IFN-γ, IL-4, IL-17 or IL-22 in aged and young Tfh cells. However, aged PBMCs produced a significantly higher amount of IL-21 compare to young subjects.
The immune system undergoes significant changes with aging, which likely partly contributes to increased susceptibility to infections, malignancies, inflammatory diseases, and reduced response to vaccination of elderly individuals [1, 2]. Significant decline in immune function during aging is termed immunosenescence , and higher frequencies of inflammatory diseases of elderly people indicate that aging could be regarded as a condition of dys-regulated inflammation .
Age-associated alterations in immune dysfunction are found in both innate and adaptive immunity in humans. For example, age-associated changes in NK cell phenotype have been previously reported that can be responsible of functional NK cell deficiency [5, 6]. DCs from elderly individuals are compromised in response to microorganisms but display increased reactivity to self-antigens associated with chronic inflammation and autoimmunity [7–9]. Most importantly, profound changes in T cell function and subsets are observed in elder individuals [10, 11].
Elderly population suffers increased morbidity and mortality from infectious diseases, therefore, the elderly are highly encouraged to receive vaccinations for these infections such as influenza and pneumococcal pneumonia. However, the efficacy of vaccination reduces with aging [12–14]. The mechanisms of increased infections and declined response to vaccination partially due to declined humoral response which are mainly due to function defects in cognate helper function of CD4 + T cells and B cells. Accumulating evidence demonstrated that the genesis, numbers and function of B cell altered with aging- [15–19]. Declined cognate helper activity of aged CD4 + T cells for B cells in mice were observed [20–22]. However, this phenomenon and mechanisms are not well understoodin human beings.
A distinct subset of CD4+ helper T (Th) cells, Tfh cell (Tfh) specialize in providing cognate help to antigen-specific B cells in germinal centers (GCs). Tfh cells play a key role in the formation of GCs and differentiation of GC B cells into plasma cells and memory B cells [23, 24]. In GCs, Tfh cells express master regulator transcription factor Bcl6 for their generation and surface marker CXCR5 which facilitates Tfh cells homing to B cell follicles [25, 26]. IL-21 is an important effector cytokine secreted by Tfh cells and directly regulates B cell proliferation and class switching . However, it is extremely challenging to obtain lymph node samples from humans to define the role of Tfh cells in normal or diseases conditions. Therefore, one study published on immunity evaluated the relationship of blood CXCR5 + CD4 + T cells with Tfh cells, and demonstrated that human blood CXCR5 + CD4 + T cells shared functional properties with Tfh cells, and appeared to represent their circulating memory compartment .
Therefore, in this study we determined whether there were alterations in aged Tfh cells by detecting blood CXCR5 + CD4 + T cells which represented Tfh cells in blood. And we observed CD69 and HLA-DR expression on CXCR5 + CD4 + Tfh cells from young or aged individuals respectively. Furthermore, Th characterized cytokine IL-21, IFN-γ, IL-4, IL-17 or IL-22 secreted by age or young Tfh cells were determined and compared.
Increased proportion of peripheral blood CXCR5 + CD4 + Tfh cells from the elderly individuals
Memory phenotype CD45RO and CCR7, and co-stimulatory CD28 expression of aged blood CXCR5 + CD4 + Tfh cells
Both aged and young blood Tfh cells are in resting state
Similar frequency of IL-21 produced by aged and young blood CXCR5 + CD4 + Tfh cells
Th1, Th2, Th17 and Th22 characterized cytokine production within CD4 + CXCR5 + Tfh or CD4 + CXCR5-T cells from healthy aged and young individuals
Tfh cells have been recently established as a novel Th subset specialized for providing help to B cells in GCs to differentiate into long-lived memory B and plasma cells [23, 24]. Age-related declines in humoral responses are mainly due to defects in the cognate helper function of CD4 + T cells and in the B cells from aged individuals [15–19]. However, this phenomenon and the mechanisms of declined cognate helper activity of aged CD4 + T cells for B cells remain not well understood in human beings. In the present study, we determined whether there are alterations of Tfh cells in aging of human beings.
First, we determined peripheral blood CXCR5 + CD4 + Tfh cells from healthy young and elderly individuals. It is extremely challenging to determine GCs’ Tfh cells through obtaining lymph node samples from healthy humans routinely. As demonstrated by one study published on immunity, human blood CXCR5 + CD4 + T cells shared functional properties with Tfh cells and appeared to represent their circulating memory compartment . Therefore, we defined blood CXCR5 + CD4 + T cells as peripheral blood Tfh by FACS. The results demonstrated that frequencies of aged CXCR5 + CD4 + Tfh cells were higher than those of young Tfh cells, and correlated with age (Figure 1). Consistent with our results, aged mice have increased numbers of isolated lymphoid follicles (ILFs), an increased T-lymphocyte population within aged ILFs . We observed that there were no significant differences of percentages or cell counts of CD4 + T cells. Different literatures reported different data about change of CD4 + T number and percentage in the elderly. Hirokawa et al. observed an age-related increase in the number of CD4+ T cells in the Japanese population . Provinciali et al’s results showed the absolute number of total CD4+ T cells was progressively reduced with increasing age in Italian population . Lower cell counts not percentages of CD4 + T cells in the elderly in Hongkong were observed . The different data might be due to race, sex, CMV infection, and other factors impact on subpopulation of CD4 + T cells [32, 33].
Then, we evaluated and compared the memory phenotype CD45RO and CCR7and co-stimulatory molecule CD28 expression on CXCR5 + CD4 + Tfh cells. The results showed that both aged and young blood CXCR5 + CD4 + Tfh cells constitutively expressed CD45RO and CCR7, and there was no difference between young and aged subjects (Figures 2 and 3). As we know, both CD45RO and CCR7 are expressed by central memory T cells . Consistent with previous reports [28, 35–37], blood CXCR5 + CD4 + Tfh cells co-expressing CD45RO and CCR7 belong to memory T cells. In addition, similar with other studies, we observed that aged memory CD4+ T cells accumulated increased [10, 11, 38]. Moreover, we demonstrated that blood CXCR5 + CD4 + Tfh cells constitutively expressed co-stimulatory molecule CD28 (Figures 2 and 3). CD28, a major co-stimulatory receptor, was responsible for the optimal antigen-mediated T-cell activation, proliferation and survival of T cells. Previous reports demonstrated that CD28 was essential for Tfh development . Furthermore, decreased CD28 expression on aged CD4 + T, CD3 + CD4-T or CXCR5-CD4 + T cells was observed in this study. Consistent with our results, the accumulation of CD28 (-) T cells, particularly within the CD8 subset, is one of the most prominent changes during in the age-associated decline of immune function in humans . Blood CXCR5 + CD4 + Tfh cells constitutively expressed CD28, which indicated that these cells could be fully activated and play a key role in development and function of CXCR5 + CD4 + Tfh cells from both aged and young subjects.
IL-21 is the most potent cytokine known for driving B cells differentiation into plasma cells in both mice and humans, and is highly secreted by Tfh cells, although not exclusively secreted by Tfh cells [27, 41, 42]. In this study, we demonstrated aged PBMCs produced significantly higher levels of IL-21compared with young subjects (Figure 5). Consistent with our observation, there has been recently reported that aged CD4 + T cells from healthy elderly individuals secreted significantly higher levels of IL-21 on priming with dendritic cells (DC) under anti-CD3 plus anti-CD28 mAbs , and IL-21 was mainly produced by memory CD4 + T cells . There were higher memory CD4 + T cells in aged subjects, therefore there were higher levels of IL-21 produced by aged PBMCs. In contrast, there has been reported that aged memory CD4 + T cells expressed lower levels of IL-21 after stimulated with anti-CD3 plus anti-CD28 evaluated by real time RT-PCR . The different results might be due to different experimental condition. IL-21 is a cytokine that has broad effects on immune and non-immune cells, and more and more data suggest this cytokine is critically involved in the initiation and/or progression of inflammatory reactions where self-reactive immune cells or antibodies cause damage in tissue . Increased IL-21 production by aged PBMCs may significantly impact immune functions in elderly individuals, and contribute to aging associated inflammatory condition or diseases.
Recently, there was one literature which reported that human blood CXCR5 + CD4 + Tfh cells comprised three subsets: Th1, Th2 and Th17 cells with different capacity to help B cells. Th2 and Th17 cells within CXCR5+, but not within CXCR5-, compartment efficiently induced naïve B cells to produce Abs. In contrast, Th1 cells from both CXCR5+ and CXCR5- compartments lacked the capacity to help B cells . Our results demonstrated that there were no significant differences of percentages of IFN-γ, or IL-4 production by aged blood CD4 + CXCR5 + Tfh or CD4 + CXCR5-T cells compared with their counterparts of young individuals (Figure 6). Previous studies about age-associated change in IFN-γ or IL-4 production by T cells were largely inconsistent . The frequencies of IL-17+ cells produced by aged CD4 + CXCR5-T cells were markedly lower, and frequencies of IL-17 production by aged Tfh cells didn’t decrease significantly compared with young subjects (Figure 6B).Consistent with our results, previous study demonstrated that purified memory CD4 + T cells from aged people produced lower levels of frequency of Th17 cells compared with the young .
In conclusion, in the present study we observed that frequencies of aged blood CXCR5 + CD4 + Tfh cells increased. Aged or young blood CXCR5 + CD4 + Tfh cells constitutively expressed CD28, and were resting T memory cells. There were similar frequencies of Th characterized cytokine production such as IL-21, IFN-γ, IL-4, IL-17 or IL-22 by aged and young Tfh cells. However, aged PBMCs produced significantly higher amount of IL-21. Future studies should observe whether there are defects of aged Tfh cells in helping B cells to produce Abs.
Materials and methods
This study was conducted in compliance with the Declaration of Helsinki and applicable national laws and regulations, and was approved by the ethics committee of Guangzhou Medical University (Guangzhou, China) and the ethics committee of Guangdong General Hospital, Guangdong Academy of Medical Sciences (Guangzhou, China). And written informed consent was obtained from all healthy aged and young donors or the parents of young children who participated in this study.
Healthy young (age range: 22–39 years old, average age: 29.5, male: n = 25, female: n = 20) and elderly (age range: 60–86 years old, averaged age: 71.0, male: n = 19, female: n = 24,) volunteers were critically selected on the basis of clinical records and laboratory examinations from Guangdong General Hospital. Healthy young children (age: 0.8-2.8 years old, average age: 2.1 years old, 4 girls, 4 boys) were recruited from Guangdong General Hospital. We regarded subjects as being healthy if they had no neoplastic or autoimmune diseases, EBV, HBV, HCV, TB infections, or serious prior illnesses, were receiving on no medication other than anti-hypertensive medication. Routine laboratory examinations of the serum of these individuals were performed to examine the liver and kidney functions. We did not investigate whether the subjects had previous infection with cytomegalovirus (CMV) (Prevalence of CMV infection is 90-100% in developing countries) .
Anti-CD4 PerCP, anti-CD4 PerCP-cy5.5, anti-CD3 APC, anti-CXCR5-Alexafluor 488, anti-CD45RO PE, anti-CCR7 PE, anti-CD28 PE, anti-CD69 PE, anti-HLA-DR PE, anti-IL-21 PE, anti-IL-4 PE, anti-IFN-γ APC and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-17 APC and ELISA for determining IL-21 was purchased from eBioscience (San Diego, CA, USA). Anti-IL-22 PE was purchased from R & D Systems (Abingdon, UK). PMA, ionomycin (INO), saponin and Brefeldin A were purchased from Sigma-Aldrich (Fluka, Sigma, USA).
Cell surface staining
Heparinized blood was collected, then 50 ul blood per tube was added, and anti-CD4 PerCP, anti-CD3 APC, anti-CXCR5-Alexafluor488, anti-CD45RO PE, anti-CCR7 PE, anti-CD28 PE, anti-CD69 PE, anti-HLA-DR PE, and isotype-matched control mAbs were added into tube and mixed, incubated 15–20 minutes at room temperature. Erythrocytes were lysed with ammonium chloride (NH4Cl) for 5–8 minutes. Cells were washed, re-suspended with staining buffer, and detected by FACS Calibur (BD Biosciences, San Jose, CA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Leukocyte count and differential were determined with a routine hematology analyzer (COULTER LH780, Beckman Coulter Inc., USA). The absolute counts of each lymphocyte subpopulation were calculated by multiplying the relative size of the lymphocyte subpopulation and the absolute lymphocyte count.
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood of healthy donors using Ficoll-Hypaque density gradient centrifugation, and washed twice in Hank’s balanced salt solution. These cells were finally adjusted to a final concentration of 2 × 106 /ml in complete RPMI 1640 medium (GIBCO, Grand Island, NY, USA) supplemented with 10% FCS (Sijiqing, China), 100 U/mL penicillin, 100 mg/mL streptomycin, 50 mM 2-mercaptoethanol, and 2 mM L-glutamine (all from GIBCO).
Intracellular cytokine staining
PBMCs were stimulated with PMA (20 ng/ml) and ionomycin (1ug/ml) for 4–6 hours at 37°C in a 5% CO2 humidified atmosphere. At the end of first hour during the incubation, brefeldin A (BFA, 10 μg/ml) was added into the culture. The cells were collected, washed twice with cold PBS, and cells surface staining ofanti-CD4 PerCP-cy5.5, anti-CXCR5-Alexafluor 488 was added for 20-30 min. Cells were washed with PBS, fixed with 4% paraformaldehyde and re-suspended in permeabilization buffer (PBS containing 0.1% saponin and 0.5% BSA). After incubation at 4°C for 2 hours or overnight, intracellular cytokine staining of Anti-IL-21 PE, Anti-IL-22 PE, Anti-IL-4 PE, Anti-IL-17 APC, anti-IFN-γ APC, and isotype-matched control mAbs were added and incubated at 4°C for 25–30 min. Cells were washed with PBS, resuspended in cold staining buffer, and detected by FACSCalibur (BD Biosciences, San Jose, CA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Cell culture and ELISA for IL-21
PBMCs were prepared, adjusted to a final concentration of 2 × 106 /ml, stimulated with PMA and inomycin or medium, and cultured for 3 days. Cell free culture supernatants were harvested and IL-21 was quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instruction. The sensitive of ELISA kit was 31 pg/ml for IL-21.
Comparison between two groups was assessed by Mann Whitney test for two-tails (except Figure 7, performed by paired t test for two tails). P value of < 0.05 was considered to be statistically significant. Data were expressed with mean with SEM in results. All statistical analyses were performed using GraphPad Prism (version 5.0 Software Inc, San Diego, CA, USA).
This work was supported by The Natural Science Foundation of Guangdong province (No. S2011040003741, S2012040006672), The Science and Technology Planned Project of Bureau of Education of Guangzhou (No. 10A159), Distinguished Young Talents in Higher Education of Guangdong province (No. LYM10111), the Guangzhou city-level key disciplines and specialties of Immunology (No. B127007), and Doctoral Fund of Ministry of Education of China (No. 20124423120003), Science and Technology Planning Project of Guangdong Province (2013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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