Figure 3.

Anti-HRS cell activity of CD4⁺ T cells displays an allogeneic reaction. HLA genotype of HL cell line L428 was determined. Thereafter, CD4⁺ T cells were isolated from MHC-II compatible donors and subsequently co-cultured with HRS cells (A–D) or co-injected into tumor-bearing NSG mice (E, F). (A–D) HLA-matched (mCD4⁺) or unmatched (uCD4⁺) CD4⁺ T cells were co-cultured with HRS cells in a ratio of 20:1 for 14 d. Every 2–3 d T cells and HRS cells were counted using flow cytometry after staining for CD3 and CD30. Experiment was performed in triplicates and repeated with two different MHC-II compatible donors showing similar results. (A) Growth curves of HRS cells co-cultured with (w) mCD4⁺ or uCD4⁺ T cells in comparison to a corresponding monoculture. (B) Growth curves of mCD4⁺ or uCD4⁺ T cells co-cultured with HRS cells in comparison to untreated control. (C) Frequency of CD3-positive HRS cells (rosettes) over time. (D) Cluster formation was recorded by assessment of cluster diameters. Experiment was performed twice with n > 50. (E–G) Co-culture experiments were translated into a murine xenograft model. NSG mice were first inoculated with HRS cells s.c. and 7 d later co-injected with mCD4⁺ or uCD4⁺ T cells. Experiment was performed twice with n = 5. (E) Volume of s.c. tumors was measured every 3–4 d. (F) Relative (rel.) weight (% of initial weight) was assed every 3–4 d for 6 weeks. (G) Mice were sacrificed after 6 weeks and tumors (*) were processed by histological analysis. Representative images of s.c. tumors stained for CD3 and CD30 showing tumor-infiltration by mCD4⁺ T cells.