Human small intestinal mucosa harbours a small population of cytolytically active CD8+ alphabeta T lymphocytes

Abstract

Intraepithelial lymphocytes (IEL) in normal human small intestine exhibit cytotoxicity. This study was undertaken to characterize the effector cells and their mode of action. Freshly isolated jejunal IEL and lamina propria lymphocytes (LPL), as well as IEL and LPL depleted of CD4+, CD8+ and T-cell receptor (TCR)-gammadelta+ cells were used as effector cells in anti-CD3-mediated redirected cytotoxicity against a murine FcgammaR-expressing cell line. Effector cell frequencies were estimated by effector to target cell titration and limiting dilution. The capacity of IEL and LPL to kill a Fas-expressing human T-cell line was also analysed. T-cell subsets were analysed for perforin, granzyme B, Fas-ligand (FasL), tumour necrosis factor-alpha (TNF-alpha) and TNF-related apoptosis inducing ligand (TRAIL) mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). Frequencies of IEL expressing the perforin and FasL proteins were determined by immunomorphometry. Both IEL and LPL exhibited significant Ca2+-dependent, anti-CD3-mediated cytotoxicity, approximately 30% specific lysis at the effector to target cell ratio 100. The cytotoxic cells constituted, however, only a small fraction of IEL and LPL ( approximately 0.01%). CD8+ TCR-alphabeta+ cells accounted for virtually all the cytotoxicity and expressed mRNA for all five cytotoxic proteins. The frequency of granzyme B-expressing samples was higher in CD8+ cells than in CD4+ cells (P<0.05 and <0.01 for IEL and LPL, respectively). In addition, both IEL and LPL exhibited significant spontaneous anti-CD3-independent cytotoxicity against Fas-expressing human T cells. This killing was mediated by Fas-FasL interaction. On average, 2-3% of the IEL expressed perforin and FasL. We speculate that CD8+ memory cells accumulate in the jejunal mucosa and that the CD8+ TCR-alphabeta+ lymphocytes executing TCR/CD3-mediated, Ca2+-dependent cytotoxicity are classical cytotoxic T lymphocytes 'caught in the act' of eliminating infected epithelial cells through perforin/granzyme exocytosis. The observed Fas/FasL-mediated cytotoxicity may be a reflection of ongoing down-regulation of local immune responses by 'activation-induced cell death'.

Figure 1

(a) Cytolytic capabilities of freshly isolated human jejunal IEL and subpopulations thereof, in anti-CD3-mediated redirected cytotoxicity assay. Total IEL (black bars), IEL depleted of TCRγδ⁺ cells (hatched bars), IEL depleted of CD4⁺ cells (dark grey bars) and IEL depleted of CD8⁺ cells (light grey bars) were treated with anti-CD3 mAb and used as effector cells in a 4-hr cytotoxicity assay with P815 cells as targets. (b) Anti-CD3-mediated redirected cytotoxicity is inhibited by EGTA. Freshly isolated LPL of one jejunal sample were treated as described above and analysed for cytotoxicity against ⁵¹Cr-labelled P815 cells at different E:T cell ratios in the absence (black bars) or presence of EGTA (hatched bars). Percentage specific lysis was calculated as the mean±1 SD per cent ⁵¹Cr-release of triplicates at the different E:T cell ratios and corrected for the spontaneous ⁵¹Cr-release of target cells.

Figure 2

Frequency of jejunal effector cells in anti-CD3-mediated redirected cytotoxicity as determined by limiting dilution. Freshly isolated jejunal IEL were treated with anti-CD3 mAb as described in the legend to Figure 1. The results are from one experiment in which 1000 P815 target cells and from 400 to 13 000 anti-CD3 mAb-treated IEL were added per well with 30 replicas for each amount of IEL.

Figure 3

Lymphocytes in normal intestine exhibit significant spontaneous cytotoxicity that is dependent on Fas/FasL interaction and not enhanced by anti-CD3 mAb treatment or PMA/Ionomycin activation. (a) Freshly isolated jejunal IEL were analysed for cytotoxicity against ⁵¹Cr-labelled Jurkat cells at different E:T cell ratios. (b) Freshly isolated jejunal IEL were treated either with anti-CD3 mAb for 1 hr at room temperature or with PMA/Ionomycin for 3 hr at 37° or sham treated and thereafter used as effector cells against ⁵¹Cr-labelled Jurkat cells at an E:T cell ratio of 100. (c) Freshly isolated jejunal IEL were either treated with PMA/Ionomycin for 3 hr at 37° or sham treated and thereafter used as effector cells against ⁵¹Cr-labelled Jurkat cells at an E:T cell ratio of 100. Anti-FasL mAb was added at the beginning of the assay as indicated (anti-FasL). Cytotoxicity is calculated as described in the legend to Fig. 1.

Figure 4

Expression of mRNA for proteins involved in cytotoxicity in intraepithelial T-cell subsets. Expression of mRNA for perforin, Granzyme B, Fas ligand, TNF-α, TRAIL, CD45 (CD45RA-RC and CD45RO) and β-actin was determined by RT-PCR of RNA extracted from freshly isolated CD4⁺, CD8⁺ and TCR-γδ⁺ IEL of one jejunal sample. Control=a pool of RNA extracted from PBMC activated with anti-CD3 mAb OKT3 for 4, 7, 20, 48 and 72 hr. Standard=MW markers, 1 kb DNA-ladder.

Figure 5

Perforin- and FasL-expressing cells are present in the mucosa of human normal jejunum. One human jejunal sample stained with anti-perforin mAb δG9 (a,b) and anti-FasL mAb G247-4 (c,d). (a) Two perforin⁺ cells are seen at the top of a villus (arrows). (b) One perforin⁺ cell is seen in the crypt epithelium (arrow) and one positive cell in the lamina propria (arrowhead). (c) One FasL⁺ cell at the top of the villus (arrow) and one positive cell in the lamina propria (arrowhead) are seen. (d) Two FasL⁺ cells are seen in the crypt epithelium (arrows) and three positive cells are seen in the lamina propria (arrowheads). Original magnification ×220.