Parallel analysis of multiple human memory CD4+ T-cell subsets within antigen-specific responses using cell proliferation dyes

Abstract

Activation induced marker (AIM) assays are being used increasingly to measure antigen-specific T-cell responses, but this activation can alter cell lineage defining phenotypic markers. We aimed to extend the utility of AIM assays to enable pre-activation defined cell populations to be tracked and quantified within T-cell memory responses. We sorted three ex vivo CD4⁺ T-cell populations prior to any activation using well defined ex vivo lineage surface marker combinations. These populations were memory non-Tregs, CD39⁺ Tregs and CD39^neg Tregs, although any three memory CD4⁺ T-cell populations able to be isolated by cell surface markers could potentially be tracked. These cells were labeled with three distinct fluorescent cell proliferation dyes (CFSE, CellTrace Violet and Cell Proliferation Dye eF670) and then all autologous PBMCs were reconstituted maintaining ex vivo cell ratios and CD25/OX40 AIM assays performed with CMV and HSV antigens. This approach enabled tracking of pre-defined cell populations within antigen stimulated responses using both activation marker and cell proliferation readouts. We confirmed that although CD39⁺ Tregs comprise a substantial proportion of AIM assay responses, they do not make substantial contributions to the proliferative response. This extends the utility of AIM assays to enable parallel analysis of the relative contribution of several CD4⁺ memory T-cell subsets to recall responses.

Keywords: AIM assay; Tregs; antigen-specific response; cell proliferation; memory.

Figure 1

Comparative proliferation of CFSE, CTV and CPDeF670 labeled cells. (a) Unstimulated ex vivo CD4⁺CD45RO⁺CD25^highCD127^low Tconv cells were sorted and stained with either CTV, CFSE or CPDeF670 and stimulated in separate wells for 4 days with soluble anti‐CD3 in the presence of autologous CD3‐depleted PBMCs, representative plots for n = 2 donors from independent experiments. (b) Labeled Tconv cells from a CMV seropositive donor were combined and stimulated with CMV lysate in the presence of autologous CD3‐depleted PBMCs for 5 days. Gating strategy for analyzing cell proliferation within individual dye‐labeled populations. Data are representative of n = 2 donors from independent experiments. (c) Schematic of assay setup and analysis timeline.

Figure 2

Isolation and proliferation tracking of ex vivo memory T‐cell populations. (a) Gating strategy for isolating unstimulated ex vivo CD4⁺ naïve T cells (CD45RO^neg), memory Tconvs (CD45RO⁺CD25^lowCD127⁺, labeled with CTV); memory Tregs (CD45RO⁺CD25^highCD127^low) that were CD39^neg (labeled with CFSE); and CD39⁺ (labeled with CPDeF670). (b) Representative responses for CMV lysate stimulated or unstimulated wells with labeled, sorted PBMCs or unsorted PBMCs. Proportions of dye‐labeled memory populations within total CD4⁺ T cells and antigen‐specific CD25⁺OX40⁺ cells for assays stimulated with HSV lysate (n = 1, square symbols), CMV‐lysate (n = 4, circle symbols) or CMV‐P1 (n = 2, triangle symbols), all independent experiments. Due to low cell numbers, CFSE⁺ CD39^neg Tregs were quantified for n = 1 HSV lysate and n = 2 CMV lysate. (c) Representative FACS plots showing how, after gating as per Figure 1b, proliferating cells were defined as CD25⁺dye^dim, with quadrants set using unstimulated cells. (d) Cell proliferation from assays stimulated with either HSV lysate (n = 1), CMV lysate (n = 3) or CMV‐P1 (n = 2), all independent experiments.