Characterization and TCR variable region gene use of mouse resident nasal gammadelta T lymphocytes

Abstract

Tissue-resident gammadelta T lymphocytes, such as dendritic epidermal T cells, intestinal intraepithelial lymphocytes (IEL), and resident pulmonary lymphocytes, are known to support local tissue homeostasis and host defense. Inhaled antigens, toxins, and microorganisms first interact with the immune system through contact with the nasal mucosa. Herein, we characterized two populations of resident nasal lymphocytes (RNL) that are present in the nasal mucosa: nasal IEL (nIEL) and nasal lamina propria lymphocytes (nLPL). gammadelta TCR+ and alphabeta TCR+ nIEL and nLPL were detected by immunofluorescent staining. Mononuclear cells (5-15%) were CD3+ RNL by FACS analysis. Among the CD3+ RNL, 20-30% were GL3+ gammadelta T cells, which were double-negative for CD4 and CD8 and predominantly expressed a Vgamma4/Vdelta1 TCR. These results demonstrate that RNL might be crucial for the host defense and tissue homeostasis in the nasal mucosa.

Fig. 1.

Resident γδ and αβ T lymphocytes in mouse and human nasal mucosa. Frozen sections of mouse nasal mucosa (C57BL/6, female, 6–8 weeks of age) were stained with mAb to the γδ TCR (GL3; PE-labeled) and CD3 (2C11; FITC-labeled). (A) The orange-colored cell (arrow) is the γδ T lymphocyte, which is positive for CD3 and GL3, and the green-colored cell (arrowhead) is the αβ T lymphocyte, which is positive only for CD3. The small figure is for negative control. (B) Yellow-to-orange-colored cells are γδ T lymphocytes, which are positive for CD3 and GL3. (C) Green-colored cells are αβ T lymphocytes, which are positive only for CD3. (D) Frozen sections of human nasal mucosa were stained with a FITC-conjugated mAb to the γδ TCR (B1). We observed the nIEL and nLPL, which expressed γδ TCR. Dotted lines depict the basement membrane of the skin. Original magnification, ×400.

Fig. 2.

γδ T lymphocyte population in RNL and nIEL versus nLPL. We harvested mononuclear cells (up to 5×10⁵ cells/mouse) with collagenase. (A) Flow cytometric analysis with mAb to the γδ TCR (GL3) and CD3 (2C11) was performed on RNL. Cells were gated on live mononuclear cells (5–15%), which were CD3⁺ RNL. Among the CD3⁺ RNL, 20–30% (26.4±5.9%) were GL3⁺ γδ T cells. (B) Flow cytometric analysis with mAb to the γδ TCR and CD3 was performed after isolation of nIEL and nLPL. γδ T cells were 20.7 ± 7.2% in nIEL and 25.5 ± 6.4% in nLPL. (C) Following LPS treatment (25 μg/day at a concentration of 250 μg/ml; from Escherichia coli serotype 0565:B5) via intranasal instillation for 3 days, γδ TCR⁺ cells increased 1.33 ± 0.27 times, and αβ TCR⁺ cells increased 2.35 ± 0.58 times. These differences were statistically significant (*, P<0.05). Data represent four to six independent experiments with four mice per experiment.

Fig. 3.

Flow cytometric analysis of CD4 and CD8 expression in nIEL versus nLPL. To analyze the CD4 and CD8 coreceptor expression in nasal resident αβ T lymphocytes, we gated the mononuclear cells with TCR-β (Α and Β). (A) The CD4:CD8 ratio was 1.38:1 in nIEL (relatively large CD8⁺ population) and 2.75:1 in nLPL. (B) The proportion of CD8⁺ αβ T lymphocytes was much higher in nIEL (25.9±3.7%) than nLPL (13.3±4.2%). (C) GL3⁺ γδ T cells were double-negative for CD4 and CD8 in nIEL and nLPL. Data represent four to six independent experiments with four mice per experiment.

Fig. 4.

Variable region gene use of RNL by RT-PCR. The sequences for primers for each of the Vγ/Jγ1 and Vδ/Jδ1 genes were used as published previously [12, 18, 19]. (A) RNL predominantly expressed Vγ4 and also expressed Vγ2 and Vγ3 TCR. (B) RNL predominantly expressed Vδ1 TCR and also expressed Vδ4 and Vδ7 TCR. Splenocytes, 7-17 cells (DETC cell line), intestinal IEL (iIEL), PAM 2-12 keratinocyte cell line, and 3T3 fibroblast cell line were used for positive and negative controls. Data represent four to six independent experiments with four mice per experiment.

Fig. 5.

Variable region gene use of RNL by flow cytometry. (A) RNL infrequently expressed variant Vγ2 and Vγ3 TCR, which is compatible with PCR data. (B) Flow cytometric analysis was performed with 17D1 antibody in GL3⁺ RNL. 17D1 antibody is known to bind to invariant Vγ3/Vδ1 TCR and also bind to invariant Vγ4/Vδ1 TCR only after incubation with a mAb to the γδ TCR (GL3). Sixty-one percent of γδ TCR⁺ RNL bound to 17D1 antibody only after incubation with GL3 antibody. RNL barely bound to 17D1 without any preincubation or after incubation with irrelevant antibody (TCR-β). These results indicate that 17D1-reactive cells were mostly Vγ4/Vδ1 TCR⁺ cells, not Vγ3/Vδ1 TCR⁺ cells. Spleen cells were used as a control. (C) To confirm the Vγ/Vδ gene use of the 17D1-positive cell population in our RNL system, RT-PCR was conducted on FACS-sorted, 17D1-positive RNL after GL3 preincubation. In this experiment, we found that this 17D1-positive population predominantly expressed Vγ4 and Vδ1 TCR. Data represent four to six independent experiments with four mice per experiment.