Background
Immunophenotyping of peripheral blood lymphocytes with monoclonal antibodies via flow cytometry has proven to be a useful tool to evaluate the immunological function of patients with immunodeficiency, autoimmunity, transplantation, tumor, or infection, and is also valuable to monitor treatment responses and disease progression [
1‐
4]. Currently, flow cytometry is the most precise and reliable tool to assess the immunological status, and technical advances in this field have provided new opportunities to better determine the function and improve cell phenotyping of peripheral lymphocytes.
As the predominant lymphocyte subset, CD4
+T cells play crucial roles in numerous conditions such as infection, autoimmunity, transplantation, and tumor. Upon antigenic stimulation, CD4
+T cells adopt one of two opposing fates: a helper T (Th) cell specialized in supporting the clearance of infections or a regulatory T (Treg) cell that functions to attenuate immune responses [
5]. Th1 cells play a critical role in host defense against intracellular pathogens and in autoimmune diseases by producing a key inflammatory cytokine interferon (IFN)-γ [
6]. Th2 cells are important in humoral immunity and protection from helminth infection and are central to the pathogenesis of several allergic inflammatory diseases [
7]. Th17 cells provide critical support for immunity against extracellular bacteria and fungi and are the leading actors in autoimmunity [
5], whereas Treg cells suppress the autoreactive activities of effector CD4
+T cells and thus maintain immune tolerance [
8].
Currently, reference values of lymphocyte subsets are utilized to increase the accuracy of data interpretation in clinical and research settings. Region-specific reference ranges for adult peripheral blood T cells, B cells, and NK cells have also been determined in certain countries [
9‐
16]. However, other than one study from Italy in 2016 [
17], no other study has investigated the characteristics and distributions of circulating CD4
+T cell subpopulations in a wide range of healthy subjects from the Han Chinese population. To fill this knowledge gap, we determined the percentages and absolute counts of peripheral blood CD4
+T cell subpopulations in an age- and sex-balanced population of healthy adults of Han Chinese ethnicity to establish monocentric reference values and also analyzed the characteristics of CD4
+T cell subsets based on sex and age.
Discussion
Clinical diagnosis and research outcomes become more meaningful and accurate when reliable reference ranges of lymphocyte subtypes are available for comparisons with respect to the local population. Flow cytometry is currently considered the “gold standard” for the identification of lymphocyte subsets and evaluation of immune function [
18]. In the present study, we analyzed the distributions and established the reference ranges of lymphocyte subsets and extended our datasets of CD4
+T subpopulations using flow cytometry based on a healthy Han Chinese population in Taiyuan, Shanxi Province, North China.
Although various methodologies and gating strategies have been established to define lymphocyte subsets, the CD45 gating strategy is considered the most appropriate approach to accurately and reliably identify lymphocyte populations [
14]. Moreover, distribution and reference ranges of circulating Th1, Th2, Th17, and Treg cells were assessed using a CD4 gating strategy. Immunofluorescence staining of specific surface markers in combination with intracellular transcription factor and/or cytokine staining was used to identify CD4
+T cell subsets [
18,
19]. These CD4
+T cells were further labeled with functional intracellular molecules (IFN-γ for Th1, IL-4 for Th2, IL-17 for Th17, and Foxp3 for Tregs) or specific extracellular markers (CD25 for Tregs).
To the best of our knowledge, only one previous study from Northeast Italy [
17] reported the reference ranges of Th1, Th2, Th17, and Treg cells. Obvious differences in the reference values of CD4
+T cell subpopulations were observed between the present study and the published data from Northeast Italy [
17], which could be associated with race and sample sizes. Of note, among the primarily identified CD4
+T cell subsets, Th1, Th2, Th17, and Treg cells were associated with different tagging strategies. The Italy study adopted chemokine receptors and other surface molecules for CD4
+T cell immunophenotyping; for example, CD195 (CCR5) was used to label Th1 cells, whereas CD194 (CCR4) and CD161 were used to mark Th2 and Th17 cells, respectively. Therefore, the immunofluorescence staining of Th1, Th2, and Th17 cells in the Italian study differed from that adopted in our study, which could also contribute to the observed differences in reference ranges. Treg cells are an immunosuppressive subset of CD4
+T cells characterized by expression of the master transcription factor Foxp3 [
20]. However, similar to the present study, Treg cells were stained with anti-CD4 FITC, anti-CD25 PE, and anti-Foxp3 PerCP in the Italian study [
17], and yet different values were obtained. One study from Philadelphia, USA, reported the overall frequency of the polyclonal Treg cell population to be approximately 5–15% of total CD4
+T cells [
20], which is higher than that obtained in the present study. This difference could also result from different racial factors and experimental conditions.
We further demonstrated clear differences in the values of CD4
+T cell subpopulations according to the age of the participants, with higher values for Treg cells (both percentages and absolute counts) in the older group (> 40 years old). In addition, Togashi et al. [
21] reported that the abundance of Treg cells in the peripheral blood tends to increase with aging, which supports our data. Moreover, a significant positive correlation was observed between age and Treg cell numbers (both percentages and absolute counts). Sex also has an influence on the lymphocyte subset distribution [
13,
14]. Indeed, we found lower values of Th1 and Treg cells in females than in males, but the differences were not significant. Moreover, a higher percentage of CD4
+T cells in females was also observed. These differences are likely related to different hormonal effects [
14] and confirm the influence of sex on peripheral lymphocyte subsets.
These significant differences above clarify the impact of age and sex on the distribution of certain lymphocyte subpopulations. Thus, the distribution and reference ranges of lymphocyte subsets in healthy participants were evaluated based on age and sex groupings, demonstrating the highest percentages and absolute counts of Treg cells in the group of older males and the lowest values in younger females.
Treg cells are an immunosuppressive subset of CD4
+T cells and play pivotal roles in the prevention of autoimmunity and maintaining immune tolerance. Deficiency in either the number or the function of Treg cells may lead to the breakdown of immune homeostasis and development of autoimmune diseases [
22]. Most autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, have a strong female predilection and preferably occur in young women [
23]. In the present study, Treg cells displayed age- and sex-biased expression, with younger females showing a lower expression than older males, a finding that may explain the susceptibility of young females to autoimmune diseases. Further studies are required to explore the exact underlying mechanism.
Indeed, differences in other lymphocyte subsets, including B cells and NK cells, have also been confirmed in the present study via comparisons of reference values obtained from populations of different parts of the world. These inconsistencies could be attributed to technical advances in flow cytometry, different sample sizes, ethnic variations, and other factors affecting the immune function of individuals. B cells discriminate pathogens from self, eliminate infections, ‘encode’ a memory of pathogen encounters and provide life-long immunity, or produce auto-antibodies to induce autoimmune response [
24]; NK cells play important roles in the innate immune response against tumors [
25]. Furthermore, our study demonstrated lower NK cell values in older subjects, which is consistent with the findings of a prior study [
14]. This phenomenon may be associated with the decreased function of immune surveillance in the older population. However, the sex-wise distribution involved in the number of peripheral NK cells observed in the present study remains to be confirmed and investigated in future studies.
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