1iB), but not CD94 expression (Fig. 1iC),
by SF CD8+CD28− Treg. Neither RA(MTX) PB nor SF CD8+CD28− Treg expressed alternative co-stimulatory molecules, 4-1BB, PD-1 or ICOS ex vivo. However, following anti-CD3 stimulation, 4-1BB (Fig. 1hD), PD-1 (Fig. 1hE) and ICOS (Fig. 1hF) were up-regulated on CD8+CD28− Treg and a significantly higher this website expression by SF Treg was observed. Functional studies of CD8+CD28− Treg showed that HC CD8+CD28− Treg could suppress autologous PBMC proliferation significantly at a 1:1 ratio of CD8+CD28− Treg : PBMC (Fig. 2a). Suppression was dose-dependent, as determined by initial assays. Responder PBMC proliferation was suppressed significantly at 1:1 (PBMC responder, 13 347 ± 2417 cpm versus 1:1, 7164 ± 3535 cpm, P = 0·04) and 0·2:1 (10 759 ± 1496 cpm, P = 0·03). No suppression was observed at the ratio of: 0·1:1 [13 606 ± 1905 cpm, P = not significant (n.s.)]. In contrast, RA(MTX) CD8+CD28− Treg were unable to suppress autologous responder PBMC proliferation (Fig. 2b), although RA(TNFi) CD8+CD28− Treg showed limited but significant suppressor function (Fig. 2c). To ensure that suppression at 1:1 was not due to competition for nutrients or space, two PBMC controls were included: PBMC 1 (2·105 cells/well) and PBMC 2
(1·105 cells/well). To determine if natural killer (NK) cell activity was part of the suppressor mechanism we compared purified subpopulations of CD8+CD28− Treg, free of NK cells and CD8+CD28−CD56+ Treg compared with CD8+CD28− Treg. No Non-specific serine/threonine protein kinase significant differences were found between the groups. For example, responder see more PBMC proliferation (13 347 ± 1209 cpm) was suppressed significantly at a ratio of 1:1 by CD8+CD28−CD16− Treg (9017 ± 854 cpm P = 0·04) and CD8+CD28−CD56+Treg (7164 ± 3535 cpm, P = 0·04). In addition, total cell counts and viability were investigated and no reduction was observed. The relative importance of soluble mediators and/or direct cell-contact as a mechanism for the suppressive function of CD8+CD28− Treg was investigated by co-culture of CD8+CD28− Treg separated from the autologous responder PBMC by a semi-permeable membrane TW. The TW
contained autologous CD14+ monocytes (MO) to ensured full stimulation of CD8+CD28− T cells by anti-CD3. Parallel cultures contained cells in direct contact. HC (Fig. 2d) and RA(TNFi) (Fig. 2f) CD8+CD28− Treg suppressed responder PBMC proliferation in the presence and absence of a TW; however, no suppression was seen in experiments with RA(MTX) CD8+CD28− Treg (Fig. 2e). It was noted that the degree of suppression in HC and RA(TNFi) cultures tended to be greater in the presence of TW, suggesting that direct cell contact did not enhance the suppressive function of these cells and that soluble mediators were involved. To establish whether the improved regulatory function of RA(TNFi) CD8+CD28− Treg ex vivo was a result of TNF-α blockade, anti-TNF antibody was added to RA(MTX) cultures.