HLA-E binding peptide specific T-cell lines have immuno-regulatory activity.

(A) Peptide specific T-cell lines were generated by stimulation with peptide in the presence of rIL-7 and further expanded with rIL-2. To investigate their potential capacity to inhibit CD4+ T-cell activity, they were co-cultured with a well characterized CD4 Th1 clone (Rp15 1-1 (1×10e4 cells per well)) proliferating to its cognate peptide (0.5 µg/ml) presented by HLA-DR3+ cells. Dose-dependent addition of peptide stimulated cells (ranging from 0.6–5×10e4 added T-cells) reduced proliferation of the Th1 clone as measured by [3H] TdR incorporation at day 3. T-cell lines generated from 3 donors directed against 3 different peptides are shown (donor 2 against peptide 62, donor 4 against peptide 55 and donor 6 against peptide 68), the T cell line from donor 2 against peptide 54 had a similar suppressive capacity (not shown) CPM = counts per minute. (B) To exclude that suppression was merely the consequence of lysis of the responder T-cells, we analysed cell survival in a CFSE labeling experiment. The responder T-cell clone Rp15 1-1 was labeled with a low dose of CFSE (0.005 µM), whereas a second, isogenic T-cell clone with a different peptide specificity and HLA-DR2 restriction (R2F10), was labeled with a high concentration of CFSE (5 µM). Both responder and irrelevant T-cell clones were HLA-E negative. They were then co-cultured with the HLA-E binding Mtb peptide specific T-cell lines (“Treg”) in the presence of the peptide (0.5 µg/ml) recognized by the responder clone Rp15 1-1 and HLA-DR3+ APCs. After 16 hours CFSE intensity was measured by flowcytometry. In the absence of added Tregs, similar numbers of responder and irrelevant T-cell clones were retrieved (ratio of 1). The addition of “Tregs” to peptide activated or control cultures also resulted in similar numbers of both responder and irrelevant T-cells, thus indicating that the responder clone is not lysed by the Tregs. Simultaneously a 3 day co-culture was performed and analyzed by [3H] TdR uptake. This experimental set-up revealed inhibition of proliferation of the responder clone. Addition of different numbers of Tregs did inhibit proliferation in a suppression assay but not the ratio of responder over irrelevant T-cell numbers in a CFSE intensity assay. (C) Clonal populations were obtained by limiting dilution of the T-cell line of donor 2 against peptide 62, all derived from 0.1 cells/well cultures. Clones were co-cultured in different ratios to Rp15 1-1 as described in (a) and 3H TdR incorporation measured. Three out of the 5 tested clones inhibited proliferation of the indicator clone in a dose dependent manner. (D) K562 target cells selectively expressing HLA-E were loaded with peptide 62 (10 µg/ml) before 51Cr labeling, followed by 5 hour co-incubation with T-cell clones and determination of 51Cr release. Clone 4G10 strongly lysed peptide loaded target cells, whereas 3E11 and 3C1 had moderate lysing capacity.