Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

Table 4 Real time RT-PCR spike and recovery and normal distribution: IFNγ response from TIL cells spiked in normal PBMC

(A) TIL1520 response to gp100 peptides
	Flu		gp100₂₀₉		gp100_pool		PHA
TIL1520/PBMC	Mean	SD	Mean	SD	Mean	SD	Mean	SD
PBMC only	40.1	95.0	0.9	0.4	1.1	0.4	337.4	316.4
1/50000	56.7	148.0	4.0	4.2	2.8	1.6	261.9	238.9
1/20000	33.5	76.4	16.1	18.3	8.9	8.2	347.3	439.2
1/10000	40.0	100.4	14.1	8.6	34.5	65.7	168.4	163.8
1/5000	40.9	99.9	24.2	21.4	21.4	15.0	258.8	227.3
1/1000	25.9	63.4	55.2	30.3	49.8	36.9	126.1	95.8
(B) TIL1235 response to MART-1 peptide
	Flu		MART-1		PHA
TIL1235/PBMC	Mean	SD	Mean	SD	Mean	SD
PBMC only	28.2	51.7	1.1	0.5	366.3	516.5
1/50000	36.1	101.3	1.8	1.1	162.2	142.6
1/20000	56.8	113.8	3.1	1.8	161.1	145.1
1/10000	58.7	125.1	3.7	1.8	183.7	197.9
1/5000	41.3	86.3	5.2	2.2	163.6	199.1
1/1000	46.1	98.0	17.8	12.3	168.1	221.7

TIL cells at different numbers were spiked into PBMC from individual healthy donors and IFNγ response examined. Response (average from 10 different donors) is shown as fold increase. All donors are HLA-A2 positive screened and confirmed to be HIV negative. Peptide stimulation is HLA-A2 restricted and specific for CD8⁺ T cells. The SD is high due to variability in individual response among 10 healthy subjects. This finding is expected. Fold increase is calculated as follows using CD8 as internal controls: (IFNγ from peptide and mitogen stimulation/CD8)/(IFNγ from HIV stimulation/CD8).

ISSN: 1479-5876