Fig. 5: KSHV-specific T cells are primed in KSHV-infected humanized mice.

A IFNγ ELISpot of CD19-negative cells isolated from humanized mice after overnight culture with medium (R10), E-CL or KE-CL. Shown as spot forming units per one million CD19-negative cells; data are presented as individual values for R10 and mean values from duplicates for E-CL and KE-CL, with mean and SD. 1 experiment with N = 6 (Mock), N = 4 (EBVzko) and N = 5 (EBVzko+KSHV) animals. B. Experimental outline of the T cell isolation protocol from KSHV/EBV co-infected huNSG mice. CD19-negative splenocytes from KSHV⁺/EBVzko⁺ mice were co-cultured with autologous KE-CLs for 24 h, IFNγ producing cells were isolated by IFNγ capture assay and cultured in limiting dilution. C IFNγ levels (pg/ml) in supernatant of isolated CD4⁺ and CD8⁺ T cell subpopulations after co-culture with E-CL or KE-CL were determined by ELISA upon growth after single cell dilution. N = 1. D Degranulation measured by % CD107a positivity on the isolated T cell subpopulations after co-culture with R10, E-CL or KE-CL. N = 2 (10-1, 10-14 and 1-9), N = 4 (01-1) independent experiments, means of technical duplicates and SD are shown. Two sided unpaired t-test, *p < 0.05; Significant p values 10-1 0.0393; 10-14 0.0235; 01-01 0.0369. E Specific killing (% dead cells compared to target cell without T cell condition) measured for CD8⁺ T cell subpopulation 01-1 after co-culture with E-CL or KE-CL at different effector to target cell ratios. 1 independent experiment, Shown are means of 2 technical replicates with SD. F Specific killing measured for CD4⁺ and CD8⁺ T cell subpopulations after co-culture with E-CL or KE-CL at an effector-target ratio of 2.5 to 1. 2 (10-1, 10-14, 1-9) or 3 (01-01) independent experiments, individual values represent means of technical replicates, bars show means, error bars represent SD. Two sided MWU, **p < 0.01. exact p-value 01-01 0.0043  (G) Number of spot forming units per one million cells measured via IFNγ KSHV proteome wide ELISpot of KSHV-reactive T cells (isolated from the IFNγ capture assay) after stimulation with overlapping peptide pools for the indicated KSHV or control antigens. Figure shows the individual values from all wells with a signal (all data can be found in Supplementary Table 2) with mean and SD. H Relative IFNγ production of the T cell subpopulation 01-1 after co-culture with KE-CL or KE-CL pulsed with the K6 peptide pool. N = 2 independent experiments, n = 3 (KE-CL + T cells) and n = 6 (KE-CL + T cells + K6 peptide mix) replicates per experiment. IFNγ production relative to the mean of the T cell only condition is shown for each replicate, SD is displayed. Two sided Unpaired t-test, **p < 0.01. exact p-value 0.0033. Source data are provided as a Source data file.