Table 3.

Validation of ex vivo assay specificity and sensitivity in the context of known immune-dominant T cell epitopes

(n) log2 IFN-γ/β-act
	A*0201		A*0201;07;03		A*0201;03;05; 07		Non A2
	N = 7		N = 10		N = 11		N = 9
	AVE	p2-value	AVE	p2-value	AVE	p2-value	AVE	p2-value
HLA-A2 controls
PP65:495-503 (A2)	1.58	0.118	1.51	0.045	1.49	0.036	0.99	0.945
FLU M1:58–66 (A2)	1.36	0.003	1.33	0.000	1.30	0.001	0.96	0.620
GP100:209–217 (A2)	0.85	0.181	0.88	0.212	0.91	0.265	0.92	0.105
FLU (A3)	1.04	0.936	1.00	0.955	1.03	0.924	1.00	0.707
PWM	2.48	0.007	2.43	0.001	2.46	0.000	1.58	0.000
OKT-3	1.81	0.011	1.77	0.003	1.83	0.001	1.30	0.005

Normalized (see statistical section) (n) log₂ IFN-γ/β-actin values reflecting the transcriptional response of PBMC to stimulation with immune dominant epitopes from CMV (pp65:495–503) and Flu (Flu M1:58–66) known to be associated with HLA-A*02 alleles are shown. As negative controls for epitope-specificity associated with HLA-A*02 presentation, gp100:209–217 was used as a HLA-A*02-associated epitope known to induce IFN-γ transcripts detectable ex vivo only in HLA-A*0201-bearing individuals with metastatic melanoma who had been previously exposed to the same epitope for anti-cancer immunization purposes [17, 18]. As negative control for HLA-specificity the HLA-A*03 restricted Flu matrix peptide RLEDVFAGK was used [44]. To assess maximum expression of IFN-γ the general T cell stimulators PWM and OKT-3 were used that stimulate T cells (OKT-3) and immune cells (PWM) independently of epitope/HLA restriction; p ₂-values refer to paired two-tailed t test between the individual log₂ IFN-γ/β-actin values compared to the normalization factor (NF) for each autologous PBMC. NF = average of the IFN-γ/β-actin values for all pools tested in each individual’s PBMC (see statistical section). Significant (≤ 0.05) values are shown underlined with the associated (n) log₂ IFN-γ/β-actin values are shown in italics