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Keywords:

  • reference values;
  • αβ T lymphocytes;
  • γδ T lymphocytes;
  • flow cytometry

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Background:

Normal reference values in healthy subjects for T-lymphocytes for both types of receptors, αβ and γδ, and their subsets are yet to be defined. The aim of this study was to measure peripheral blood αβ and γδ total T-lymphocytes and their subsets in a population of healthy subjects, in order to obtain valid reference values for studies in human pathology.

Methods:

We studied a total of 157 healthy subjects, 78 men and 79 women, establishing their levels of CD3+, CD4+, CD8+, CD56+, αβCD3+, αβCD3+CD4+, αβCD3+CD8+, αβCD3+CD56+, γδCD3+, γδCD3+CD4−CD8−, γδCD3+CD8+, and γδCD3+CD56+ T-cells by flow cytometry. The T-cell subsets were compared for different age and gender groups.

Results:

A significant decrease in CD3+, CD3+CD4+, CD3+CD4+ αβ, and CD3+ γδ T-cells was observed in elderly subjects. CD3+, CD3+ αβ, and CD3+CD4+ αβ T-cells increased in women, while CD3+CD56+ αβ T-cells increased in men.

Conclusions.

These reference values could be useful in further research studies for assessing changes that occur in the different αβ and γδ T subsets in human pathology. © 2012 International Clinical Cytometry Society

Thirty years ago, all specific immune responses were believed to be produced by T-lymphocytes expressing TCR-αβ surface receptor. In 1984, however, a T-lymphocyte-specific cDNA was discovered that coded a protein that is similar to immunoglobulin and contains genes that reorganize themselves in some T-lymphocytes. This cDNA coded a polypeptide designated as γ, which was linked to a second polypeptide called δ to form the TCR-γδ (1). Accordingly, two different subsets of T-lymphocytes are known at present, depending on the type of membrane antigen-receptor they express: αβ T-lymphocytes that express the αβ-TCR antigen receptor and γδ T-lymphocytes (γδ LT) that express the γδ-TCR antigen receptor (2–5). Not much is known about γδ T-lymphocytes, partly due to their more recent discovery, although since then, several immunological studies have tried to differentiate between the two T-lymphocyte subsets, with special emphasis on γδ LT due to their outstanding functions (6).

Most of these studies have been carried out on laboratory animals, but some have investigated various pathologies in human subjects, albeit with a limited number of patients and their corresponding healthy controls. Normal T-lymphocyte values have yet to be established in healthy subjects for both types of receptors, αβ and γδ, as well as their αβ CD4+, αβ CD8+, and γδ CD8+ subsets. Only one study, conducted on 104 healthy patients, has analyzed total γδ-LT, comparing differences between young and elderly subjects, but without studying their subsets (7).

Our aim was to establish total αβ and γδ T-lymphocytes values in peripheral blood in a healthy population and also study these two subsets to obtain reference values for studies in human pathology.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Study Population

The subjects participating in the study (N = 157) were recruited from the relatives of patients and the staff of the Arnau de Villanova Hospital in Valencia (Spain). During the selection period, candidates were asked about their state of health via an oral questionnaire. Patients with any of the following characteristics were excluded from the study: known immunodeficiency, autoimmune disease, genetic factors predisposing to certain pathologies, vaccination within the previous 6 months, current immunosuppressive treatment, significant smoking habit, or alcohol intake or exposure to toxins at work. No deviations were found in the selected subjects with respect to sex, race, or geographical location. Volunteers signed informed consent to participate in the study.

The study was approved by our hospital's Ethics Committee for Clinical Research.

Variables Studied

The following variables were recorded: age and gender, complete blood count, and T-lymphocyte populations: total CD3+, CD3+CD4+, CD3+CD8+, CD3+CD56+, αβCD3+, αβCD3+CD4+, αβCD3+CD8+, αβCD3+CD56+, γδCD3+, γδCD3+CD4−CD8−, γδCD3+CD8+, and γδCD3+CD56+. Double-positive CD4+/CD8+ were excluded from the analysis.

Methods of Analytical Determination

Peripheral blood samples obtained by venipuncture were collected in K3-EDTA anticoagulant and processed within 6 h of collection. Blood cell counts were performed using the Coulter LH750 automated hematology analyzer (Beckman Coulter, Fullerton, CA).

Anticoagulated venous blood was aliquoted in 100 l amounts into 12.75-mm polypropylene tubes (Becton Dickinson) and incubated for 15 min at room temperature with the appropriate fluorochrome-conjugated monoclonal antibodies at the manufacturer's recommended concentration. Using the TQ-Prep Workstation (Beckman Coulter) and the ImmunoPrep Reagent System (Beckman Coulter), stained whole blood samples were subjected to red blood cell lysis with formic acid (ImmunoPrep A), leukocyte stabilization with sodium carbonate (ImmunoPrep B), and cell fixation with paraformaldehyde (ImmunoPrep C).

Whole blood was stained using direct immunofluorescence and simultaneous quadruple labeling with the following monoclonal antibodies: CD45, CD4, CD8, CD56, CD3, CD19, TCRαβ, and TCRγδ for the T αβ and γδ lymphocytes study. The monoclonal antibodies were conjugated with fluorescein isothiocyanate, phycoerythrin, Texas red phycoerythrin, and R-phycoerythrin-cyanine 5 (PC5).

The γ-δ T-lymphocyte populations were analyzed with PC5-conjugated antihuman γδ TCR [Beckman Coulter, Miami (clone: IMMU510)]. This clone recognizes all γδ T cells, regardless of the variable genes or junction regions they express, as assessed by flow immunofluorescence studies on polyclonal γ/δ T-cells lines as well as on γ-δ T-cell clones.

The α-β T-lymphocyte populations were analyzed with PC5-conjugated antihuman αβ TCR (Beckman Coulter [clone: IP26A]). This clone recognizes a monomorphic determinant of the αβ T-cell receptor complex, staining between 89.4 and 98.4% of CD3 positive cells in normal blood.

Fluorescence analysis was performed using a Beckman–Coulter multiparameter flow cytometry analyzer, Cytomics FC 500, Florida (USA) and later analyzed with CXP Software. A minimum of 30,000 events were measured. Absolute counts of circulating cell subsets were calculated using the percentages obtained by flow cytometry, and the leukocyte count was obtained from the hematological analyzer, using a dual-platform counting technology (18).

A range of internal quality assurance procedures were followed, including daily calibration of flow cytometer optical alignment and fluidic stability using Flow-Check (Beckman Coulter) fluorospheres and daily monitoring of whole-blood preparation procedures and monoclonal antibody reactivity using Immuno-Trol (Beckman Coulter) control cells. An external quality assurance procedure was also implemented through participation in a performance-monitoring network operated by the Sociedad Ibérica de Citometría (Iberian Society of Citometry).

Statistical Analysis

Descriptive statistics and their 95% confidence intervals were obtained using standard procedures. The assumption of a normal distribution for continuous variables was verified using graphic examination and the Kolmogorov–Smirnov test with a Lilliefor's significance correction. When normality was assumed, the Student t or ANOVA tests were used to compare the mean values of the quantitative variables. When the hypothesis of normality for the quantitative variable was not accepted, the nonparametric Mann–Whitney U test or the Kruskall–Wallis test was used. Significance was defined as a P-value of less than 0.05. Data were analyzed using the statistical software SPSS, version 18 (SPSS).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Demographic Parameters

Of the 157 subjects studied, 78 were men (49.7%) and 79 women (50.3%). The subjects' average age was 57.4 years (CI 95%: 54.3–60.3), ranging from 18 to 95 years. The average age by gender was of 62.3 (CI 95%: 57.7–66.9) for men and 52.6 (CI 95%: 48.4–56.8) for women, P = 0.002.

The number of subjects included in each group was as follows: aged 18–30, 24 subjects; 31–40, 12 subjects; 41–50, 24 subjects; 51–60, 26 subjects; 61–70, 20 subjects; 71–80, 28 subjects; 81–95, 23 subjects.

Lymphocyte Subsets

The absolute number and percentage of peripheral blood total, helper, and cytotoxic T-cells were estimated, based on the expression of CD3+, CD4+, and CD8+, respectively. Double-positive CD4+/CD8+ were excluded from analysis. These subsets were also analyzed, based on αβ-TCR and γδ-TCR expression.

The absolute mean values of the lymphocyte subsets and their confidence interval at 95% are shown in Table 1. Percentages are shown in Table 2.

Table 1. Peripheral Blood Lymphocyte Subsets (Absolute Values)
 MinimumMaximumMedian (95% CI)
  1. N = 157. CI, confidence interval. Values are expressed as ×109/L means (95% confidence interval).

T lymphocytes1.00005.30002.2407 (2.1193–2.3814)
CD3+0.63543.43651.5925 (1.4966–1.7009)
CD3+CD4+0.22522.93261.0095 (0.9408–1.0875)
CD3+CD8+0.17662.24910.6502 (0.6062–0.7159)
CD3+CD56+0.01101.11650.2859 (0.2530–0.3187)
CD3+ αβ0.42033.32801.4626 (1.3747–1.5634)
CD3+CD4+ αβ0.02022.91070.9607 (0.8794–1.0258)
CD3+CD8+ αβ0.07001.82090.5086 (0.4601–0.5595)
CD3+CD56+ αβ0.00190.91940.0806 (0.0640–0.0972)
CD3+ γδ0.00450.31870.0698 (0.0593–0.0813)
CD3+CD4−CD8− γδ0.00010.27260.0501(0.0418–0.0584)
CD3+CD8+ γδ0.00030.14300.0191 (0.0158–0.0235)
CD3+CD56+ γδ0.00010.12560.0125 (0.0095–0.0156)
Table 2. Peripheral Blood Lymphocyte Subsets (Percentages)
 MinimumMaximumMedian (95% CI)
  1. N = 157. CI, confidence interval. Values are expressed as ×109/L means (95% confidence interval).

T lymphocytes11.8056.2032.90 (31.57–34.48)
CD3+41.5999.8070.50 (69.08–72.38)
CD3+CD4+10.2479.2644.83 (43.24–46.43)
CD3+CD8+9.4168.6828.77 (27.64–30.66)
CD3+CD56+0.6953.8712.87 (11.37–14.37)
CD3+ αβ26.2784.5164.82 (63.48–66.64)
CD3+CD4+ αβ0.7878.6742.26 (40.39–44.00)
CD3+CD8+ αβ4.1463.0022.23 (20.74–23.91)
CD3+CD56+ αβ0.1038.313.61 (2.84–4.38)
CD3+ γδ0.1914.033.13 (2.71–3.60)
CD3+CD4−CD8− γδ0.1212.622.22 (1.87–2.58)
CD3+CD8+ γδ0.025.710.84 (0.71–1.01)
CD3+CD56+ γδ0.014.830.56 (0.44–0.68)

The mean values are grouped according to gender in Table 3. The total T-cell CD3+, CD3+CD4+, CD3+ αβ, and CD3+CD4+ αβ values were significantly higher in women than in men. Only CD3+CD56+ showed significantly higher values in men; the CD3+CD56+ αβ levels were also higher in men, almost reaching significance (P = 0.055).

Table 3. Peripheral Blood Lymphocyte Subsets by Gender
 Male (N = 78); median (95% CI)Female (N = 79); median (95% CI)Sig.
  1. N = 157. CI, confidence interval; Sig, statistical significance (P-value); NS, not significant. Values are expressed in ×109/L.

T lymphocytes2.0841 (1.9289–2.2392)2.4054 (2.2004–2.6104)0.019
CD3+1.4575 (1.3235–1.5914)1.7306 (1.5810–1.8802)0.008
CD3+CD4+0.8668 (0.7794–0.9541)1.1516 (1.0428–1.2603)<0.001
CD3+CD8+0.6709 (0.5838–0.7579)0.6519 (0.5819–0.7219)N.S.
CD3+CD56+0.3141 (0.2611–0.3672)0.2434 (0.2015–0.2853)0.026
CD3+ αβ1.3398 (1.2155–1.4642)1.5895 (1.4519–1.7272)0.010
CD3+CD4+ αβ0.8312 (0.7457–0.9168)1.0658 (0.9532–1.1784)0.002
CD3+CD8+ αβ0.4999 (0.4220–0.5779)0.5189 (0.4545–0.5834)N.S.
CD3+CD56+ αβ0.0995 (0.0666–0.1323)0.0632 (0.0491–0.0774)0.055
CD3+ γδ0.0645 (0.0482–0.0808)0.0759 (0.0607–0.0910)N.S.
CD3+CD4−CD8− γδ0.0459 (0.0339–0.0578)0.0550 (0.0432–0.0669)N.S.
CD3+CD8+ γδ0.0203 (0.0142–0.0264)0.0190 (0.0140–0.0240)N.S.
CD3+CD56+ γδ0.0122 (0.0078–0.0166)0.0129 (0.0086–0.0172)N.S.

Conventional T-cell subsets are shown in Figure 1. Subsets defined by αβ TCR and γδ-TCR expression are shown in Figures 2 and 3, respectively. In elderly subjects, a significant decrease was observed in CD3+, CD3+CD4+, CD3+CD4+ αβ, and CD3+ γδ T cells subsets, although CD3+CD56+ levels increased in this age group.

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Figure 1. Peripheral blood T-lymphocyte subsets by age group. Values are expressed as ×109/L means (95% confidence interval). A: CD3+ T cells; a (P = 0.019), b (P = 0.015), c (P = 0.041). B: CD3+CD4+T cells; a (P < 0.05). C: CD3+CD8+; no significant differences between age groups. D: CD3+CD56+ T cells; a (P < 0.001), b (P = 0.007), c (P = 0.028).

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thumbnail image

Figure 2. Peripheral blood αβ T-lymphocyte subsets by age group. Values are expressed as ×109/L means (95% confidence interval). A: CD3+ αβ T cells; a (P = 0.034). B: CD3+CD4+ αβ T cells; a (P < 0.05). C: CD3+CD8+ αβ T cells; no significant differences between age groups. D: CD3+CD56+ αβ T cells; no significant differences between age groups.

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thumbnail image

Figure 3. Peripheral blood γδ T-lymphocyte subsets by age group. Values are expressed as ×109/L means (95% confidence interval). A: CD3+ γδ T cells; a (P < 0.05). B: CD3+CD4−CD8− γδ T cells; a (P < 0.05). C: CD3+CD8+ γδ T cells; no significant differences between age groups. D: CD3+CD56+ γδ T cells; a (P < 0.05).

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A validation was performed in an independent cohort of individuals (n = 20), following the same selection procedures. We did not find any significant differences in the results with respect to the reference group.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The reference values for T-lymphocyte subsets are presented, including αβ and γδ subsets, grouped by sex and age ranges. This study is valuable and unique in that it assesses a wide variety of lymphocyte subsets including CD3+, CD3+CD4+, CD3+CD8+, CD3+CD56+, αβCD3+, αβCD3+CD4+, αβCD3+CD8+, αβCD3+CD56+, γδCD3+, γδCD3+CD4-CD8-, γδCD3+CD8+, and γδCD3+CD56+ in a large group of healthy adults.

The absolute and proportional levels of peripheral blood CD3+, CD4+, and CD8+ T cells obtained in this study are considerably higher than those observed in previous studies performed in Switzerland, Iran, and India (8–10), and slightly higher than those observed in Germany (11), although they were similar to those obtained in a study undertaken in Italy (12).

To examine age-related changes in lymphocyte subsets, we studied a group of healthy adults stratified by age, with subjects ranging in age from 18 to 95 years. Some previous studies did not analyze age-related changes in lymphocyte populations or limited their attention to ages ranging from 20 to 50 years. We observed a significant decrease in total absolute CD3+ lymphocyte numbers in subjects aged between 60 and 95 years. This decrease involved CD4+, CD4+ αβ, CD3+ αβ, and CD3+ γδ, but CD8+ αβ and γδ T-cells maintained similar values with age. This finding may reflect aging of the immune system, likely related to the process of thymus involution. It would be advisable to investigate why T-helper lymphocytes decrease with age while the levels of cytotoxic lymphocytes remain unchanged. Other studies observed a decrease in both CD4+ and CD8+ T-lymphocyte subsets with age (8, 13), or a decrease in CD3+ and CD8+ but not in CD4+ (11), or even no significant changes at all (10, 12, 14). One possible explanation for the lack of differences in T-lymphocytes with age in these previous studies is the fact that most of the subjects were quite young, with very few aged over 60 years.

Other alterations related to immune-system aging could be the significant increase in the absolute numbers of CD3+CD56+ T-cells from the age of 50 onward, with a particularly large increase between the ages of 75 and 95. These changes in T-cells capable of mediating non-MHC-restricted cytotoxicity have also been described by Sansoni et al. (13) and, together with the increase in natural killer cells, could represent changes designed to compensate for the drop in T-lymphocytes observed in the elderly.

It is worth noting that the percentage of peripheral blood γδ T-lymphocytes is lower than in other studies, which present figures of around 5–10% of T-lymphocytes. The mean value obtained in our study was 3%. γδ T cells have been considered to play a main role in innate immunity for various reasons: they do not need previous antigen processing by MHC; they display a wide variety of antigen recognition and they have a privileged location in the mucosa. γδ T cells tend to decrease around the ages of 70–79, depending on the γδ CD3+CD4−CD8−, which is the most frequent subset of γδ T cells in the peripheral blood. We have found no differences in γδ CD3+CD8+ values with age. This decrease in γδ T-lymphocytes with age has already been described by Argentati et al. (7), who also reported alterations in the cytokine production pattern in elderly patients, with a higher percentage of cells producing TNF-α. These alterations could be related to the poor prognosis of infections and predisposition to tumors in elderly patients as part of the process called immunosenescence (15, 16). αβ T-cells also decrease with age, particularly the CD3+αβ and CD4+αβ lymphocytes, although no differences were found in CD8+αβ T cells with age.

Immune studies examining gender-dependent changes, in both humans and mice, suggest that females produce higher antibody levels than males. In general, females tend to show more intense immune-mediated reactions (17). We observed a statistically significant increase in the number of CD3+ T-lymphocytes in women, at the expense of CD4+ T-lymphocytes. This increase is dependent on the CD3+αβ and CD4+αβ T cells. There was no difference in the CD8+ T-lymphocytes. Regarding the γδ lymphocyte populations, no statistically significant gender-dependent differences were seen in either the CD3+ γδ, CD3+CD4−CD8− γδ, CD3+CD4+CD8+ γδ or the CD3+CD56+ γδ T-cell subsets.

There was a striking and significant increase in CD3+CD56+ T-cells in men, which appears to depend on the CD3+CD56+ αβ T-cells, as no significant differences were observed in the CD3+CD56+ γδ T-cells.

The presence of higher values of CD3+ and CD4+ T-cells and lower values of NK cells in women has previously been described in a population of Italian healthy adults, a country similar to ours in lifestyle and genetics (12). The Mediterranean diet and lifestyle have been associated with lower mortality rates (19) and a decrease in the incidence of infectious diseases (20), suggesting an immunological similarity common to the Mediterranean region.

Among the possible limitations of our study, we should mention that we were unable to establish reference intervals for peripheral blood CD3+CD4+ γδ lymphocyte subsets. This is due to the fact that we have found in most samples less than 15 events in this population, despite acquiring a minimum number of 30,000 events reaching 150,000 acquired events in the majority of cases (∼80% of cases). This suggests that this population is extremely scarce in peripheral blood. We have found a mean absolute value of 0.0052 × 109/l for this subset of lymphocytes.

Little is known about CD3+CD4+ γδ T-lymphocytes in humans. We recently reported a decrease in the overall lymphocyte population in peripheral blood of patients with Crohn's disease compared to healthy controls. This decrease was more evident in γδ T cells, especially CD3+CD8+ γδ T-lymphocytes, and CD3+CD4+ γδ T-lymphocytes (21). Median value of CD3+CD4+ γδ T-lymphocytes in healthy controls was 0.0037 × 109/L. To our knowledge, this was the first report in which this lymphocyte subset was analyzed in human peripheral blood. CD3+CD4+ γδ T cells have been suggested to participate in helping B cells produce IgE. In particular, the presence of CD3+CD4+ γδ T cells in mouse spleen has been correlated with elevated levels of serum IgE (22, 23). Some studies on human fetal liver have suggested that CD3+CD4+ γδ T cells do not produce IL-2 and are devoid of any cytolytic activity (24, 25). Further studies are needed to investigate this T-cell subpopulation in human peripheral blood.

Another possible limitation of our study was that absolute values of the lymphocyte subsets were obtained by dual-platform counting technology. The total lymphocyte counts were determined by the hematology cell counter, and the proportion of lymphocyte subsets was obtained by the flow cytometer. The absolute counts of the lymphocyte subsets were then calculated from these two determinations. This approach has traditionally been considered the reference method for absolute cell counts, but it may have some technical limitations. As a result, attention has recently turned to the development of single-platform technologies in which the absolute counts are determined by the flow cytometer alone, without any need for the results of the hematology counters. This single-platform approach offers fewer sources of variability. Some earlier studies, however, have found a good correlation of these different methods (12, 18).

In conclusion, there is a significant decrease in CD3+, CD3+CD4+, CD3+CD4+ αβ, and CD3+ γδ T-cell values in elderly subjects, although the CD3+CD56+ T-cell values increase in this age group. The CD3+, CD3+ αβ, and CD3+CD4+ αβ T-cells increased in women, while the CD3+CD56+ αβ T-cell counts increased in men. We believe that these reference values are potentially useful in subsequent research studies for assessing changes that may occur in human pathology in the different αβ and γδ T-subsets.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

We are grateful to A.J. Hueso, A. Payá, C. Enguidanos, A. Almiñana, and P. Ferrandis (Hematology and Biological Chemistry Department, Arnau de Vilanova Hospital, Valencia, Spain).

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
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