Human Peripheral Blood Gamma Delta T Cells: Report on a Series of Healthy Caucasian Portuguese Adults and Comprehensive Review of the Literature

Abstract

Gamma delta T cells (Tc) are divided according to the type of Vδ and Vγ chains they express, with two major γδ Tc subsets being recognized in humans: Vδ2Vγ9 and Vδ1. Despite many studies in pathological conditions, only a few have quantified the γδ Tc subsets in healthy adults, and a comprehensive review of the factors influencing its representation in the blood is missing. Here we quantified the total γδ Tc and the Vδ2/Vγ9 and Vδ1 Tc subsets in the blood from 30 healthy, Caucasian, Portuguese adults, we characterized their immunophenotype by 8-color flow cytometry, focusing in a few relevant Tc markers (CD3/TCR-γδ, CD5, CD8), and costimulatory (CD28), cytotoxic (CD16) and adhesion (CD56) molecules, and we examined the impacts of age and gender. Additionally, we reviewed the literature on the influences of race/ethnicity, age, gender, special periods of life, past infections, diet, medications and concomitant diseases on γδ Tc and their subsets. Given the multitude of factors influencing the γδ Tc repertoire and immunophenotype and the high variation observed, caution should be taken in interpreting "abnormal" γδ Tc values and repertoire deviations, and the clinical significance of small populations of "phenotypically abnormal" γδ Tc in the blood.

Keywords: Vdelta1; Vdelta2; Vgamma9; gamma delta T cell repertoire; gamma delta T cells; human cytomegalovirus; human herpes viruses; immune response; normal reference values.

Figure 1

Dot plots obtained from the peripheral blood of a healthy adult stained with an eight-color panel of mAbs, which includes a pan anti-TCR-γδ mAb, anti-Vδ1 (A) or an anti-Vδ2 (B) mAbs, and mAbs against CD3, CD5, CD8, CD28, CD16 and CD56. Vδ1 Tc (A, red dots) represented 5.6% of γδ Tc, 0.9% of Tc and 0.2% of WBC, whereas Vδ2 Tc (B, red dots) were 94.4% of γδ Tc, 14.5% of Tc and 2.7% of WBC. Strategy used for γδ Tc gating and analysis: After removing debris and cell aggregates using the SSC-A/FSC-A and the FSC-H/FSC-A dot plots, Tc were identified based on the expression of CD3 (first dot plot in the first row of each panel, black dots), and observed for the staining with anti-TCR γδ and the mAb specific for each of the Vγ or Vδ regions (second dot plot in the first row of each panel, black dots). Afterwards, γδ Tc expressing the putative V chain were painted in red; other γδ Tc were painted in green; and TCR-γδ negative Tc (i.e., αβ Tc) were painted in blue (third dot plot in the first row of each panel). Finally, γδ Tc were analyzed for their FSC and SSC (second row of each panel) and for the expression of cell surface molecules (third and fourth rows of each panel).

Figure 2

Median fluorescence intensities observed for CD3, CD5, CD8, CD16, CD28 and CD56 molecules on blood αβ (gray boxes) and γδ Tc (white boxes) (A), and percentages of CD5+, CD8+, CD16+, CD28+ and CD56+ cells among peripheral blood γδ Tc (B) in the study population of healthy adults. p values (Mann–Whitney U test) are indicated inside the graphic.

Figure 3

Correlations between the percentages of CD5-, CD8+, CD16+ and CD56+ cells, and the percentages of CD28- γδ Tc in in the study population of healthy adults. The Spearman’s rank correlation coefficients and p values are indicated inside the graphics.

Figure 4

Percentages of Vδ1+, Vδ2+, Vδ1-Vδ2-, Vγ9+ and Vγ9- Tc among peripheral blood γδ Tc in the whole study population (A) and in each of the healthy adults studied, numbered by order of decreasing percentage of Vδ2+ Tc, followed by order of decreasing percentage of Vδ1+ Tc (B).

Figure 5

Median fluorescence intensity (A) and percentages of positive cells (B) observed for CD3, TCR- γδ, CD5, CD8, CD16, CD28 and CD56 molecules in the peripheral blood Vδ1 (gray boxes) and Vδ2 (white boxes) Tc from the study population of healthy adults.

Figure 6

Correlations between age and the percentages and absolute numbers of γδ Tc in the study population of healthy adults. The Spearman’s rank correlation coefficients and p values are indicated inside the graphics.

Figure 7

Percentages and absolute numbers of total γδ T cells and γδ T cells expressing Vδ1, Vδ2, and Vγ9, in the peripheral blood from healthy adult individuals 20–45 years old, compared to those 46–70 years old. p values (Mann-Whitney U test) are indicated inside the graphics.

Figure 8

Correlations between age and the percentages and absolute numbers of CD28- αβ Tc, CD8+CD28- αβ Tc, CD28- γδ Tc, CD16+ γδ Tc, CD56+ γδ Tc and CD16/CD56+ γδ Tc in the study population of healthy adults. The Spearman’s rank correlation coefficients and p values are indicated inside the graphics.

Figure 9

Percentages and absolute numbers of total γδ T cells and γδ T cells expressing Vδ1, Vδ2 and Vγ9, in the peripheral blood of healthy adult females, compared to healthy adult males. p values (Mann-Whitney U test) are indicated inside the graphics.

Figure 10

Relations between age and the percentages and absolute numbers of γδ Tc, and the absolute numbers of the γδ Tc subsets in the study population of healthy adult females (A) and males (B). The Spearman’s rank correlation coefficients and p values are indicated inside the graphics.