A. Joosten, Simone E. van Meijgaarden, Krista C. van Weeren, Pascale Kazi, Fatima Geluk, Annemieke D. L. Savage, Nigel Drijfhout, Jan W. Flower, Darren R. A. Hanekom, Willem Klein, Michèl R. H. M. Ottenhoff, Tom HLA-E binding peptide specific T-cell lines have immuno-regulatory activity. <p>(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<sup>+</sup> 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<sup>+</sup> 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 [<sup>3</sup>H] 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<sup>+</sup> 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 [<sup>3</sup>H] 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 <sup>51</sup>Cr labeling, followed by 5 hour co-incubation with T-cell clones and determination of <sup>51</sup>Cr release. Clone 4G10 strongly lysed peptide loaded target cells, whereas 3E11 and 3C1 had moderate lysing capacity.</p> binding;peptide;t-cell;lines;immuno-regulatory 2013-02-21
    https://plos.figshare.com/articles/figure/_HLA_E_binding_peptide_specific_T_cell_lines_have_immuno_regulatory_activity_/532106
10.1371/journal.ppat.1000782.g005