Co-culture of primary human T cells with leukemia cells to measure regulatory T cell expansion

Abstract

The expansion of regulatory T cells (Tregs) is known to be mediated by cytokines including IL-10 and TGFβ but has additionally been shown to depend on the interaction of the immune receptors ICOSLG and ICOS. Here, we describe a co-culture system which enables quantification of the ability of leukemia cells to induce Treg expansion through secreted cytokines and direct receptor interactions. The protocol is applicable for MHC-matched and -unmatched experiments and allows assessment of Treg expansion without using a mouse model. For complete details on the use and execution of this protocol, please refer to Külp et al. (2022).

Keywords: Cancer; Cell Biology; Cell culture; Cell isolation; Cell-based Assays; Flow Cytometry/Mass Cytometry; Health Sciences; Immunology.

Graphical abstract

Figure 1

Operational workflow of T cell co-culture PBMC = peripheral blood mononuclear cells. G = granulocytes. E = erythrocytes.

Figure 2

Phase separation of PB after Ficoll-Paque density gradient centrifugation PBMC = Peripheral blood mononuclear cells.

Figure 3

Exemplary dot plots visualize purity assessment of isolated T cells

Figure 4

Flow cytometry gating strategy First, low granularity cells were gated and CD3⁺ T cells selected to separate CD4⁺ and CD8⁺ cells. Following this, viability of these populations was assessed using Annexin V and 7-AAD staining to ensure survival of T cells throughout co-culture. CD25⁺FOXP3⁺ Tregs were gated and their percentage among the CD4⁺ population was quantified. Figure taken from Külp et al. (2022).

Figure 5

Experimental outcomes (A) Percentages of CD25⁺FOXP3⁺ Tregs among CD4⁺ cells for healthy donors 1–6 (HD1 – HD6) following co-culture with SEM::mock (red) or SEM::EGR3 (blue), respectively. (B) Replication of co-culture experiment under addition of IgG control antibody (red) or neutralizing α-ICOSLG antibody exemplary for HD2. (C and D) Cytokine quantification of IL-2 and IL-10 using ELISA for HD1, HD2, and HD3 revealed that ICOSLG antibody blockade strongly decreased IL-10 levels of SEM::mock (p=0.0424) and SEM::EGR3 (p=0.0002) co-cultures (multiple t-tests). Error bars represent the standard error of mean of HD1, HD2, and HD3. Figures taken from Külp et al. (2022).