Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells

Tischer, Sabine; Priesner, Christoph; Heuft, Hans-Gert; Goudeva, Lilia; Mende, Wolfgang; Barthold, Marc; Kloeß, Stephan; Arseniev, Lubomir; Aleksandrova, Krasimira; Maecker-Kolhoff, Britta; Blasczyk, Rainer; Koehl, Ulrike; Eiz-Vesper, Britta

doi:10.1186/s12967-014-0336-5

Table 5 Outcome of CMVpp65-specific T-cell separation by MiniMACS CSA

From: Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells

A	1. Validation run
	Positive control		CMVpp65_pp		Negative control
	OF	TCF	OF	TCF	OF	TCF
volume [ml]	0.6	1.0	0.6	1.0	0.6	1.0
viability [%]	98.81	33.19	98.70	15.63	98.67	12.21
WBCs_(CD45+) [x10⁶/ml]	42.70	0.07	47.50	0.01	39.50	0.00
WBCs_abs [x10⁶]	25.62	0.07	28.50	0.01	23.70	0.00
T cells_(CD3+CD56-) [% of WBCs]	27.26	35.72	27.56	43.33	31.66	15.62
T cells [/μl]	11650.00	24.90	13100.00	6.23	12500.00	0.70
T cells_abs [x10⁶]	6.99	0.02	7.86	0.01	7.50	0.00
T cells_(CD3+CD4+) [% of CD3⁺]	50.34	33.51	50.25	25.85	49.05	63.35
T cells_(CD3+CD8+) [% of CD3⁺]	49.67	66.51	49.77	74.15	50.97	36.71
IFN-γ ⁺ T cells [% of CD3 ⁺ ]	0.69	82.33	0.23	69.27	0.01	2.89
IFN-γ⁺ T cells [/μl]	80.33	20.50	30.17	4.32	1.25	0.02
IFN-γ ⁺ T cells [x10 ⁴ ]	4.74	1.21	1.79	0.15	0.07	0.00
IFN-γ⁻ T cells [% of CD3⁺]	99.31	17.67	99.77	30.73	99.99	97.11
IFN-γ⁻ T cells [/μl]	11566.67	4.40	13066.67	1.92	12500.00	0.68
IFN-γ⁻ T cells [x10⁴]	682	026	778	0.07	724	0.02
IFN-γ⁺ T cells_(CD3+) [% of CD4]	0.18	23.44	0.04	9.06	0.01	1.72
IFN-γ ⁺ T cells _(CD3+CD4+) [% of CD4]	0.37	70.45	0.09	35.32	0.01	2.82
IFN-γ⁺ T cells_(CD3+CD4+) [/μl]	21.67	5.88	5.93	0.57	0.61	0.01
IFN-γ ⁺ T cells _{abs (CD3+CD4+)} [x10 ⁴ ]	1.28	0.35	0.35	0.02	0.04	0.00
IFN-γ⁺ T cells_(CD3+) [% of CD8]	0.49	58.79	0.18	59.14	0.00	0.52
IFN-γ ⁺ T cells _(CD3+CD8+) [% of CD8]	0.95	87.52	0.37	78.94	0.00	1.42
IFN-γ⁺ T cells_(CD3+CD8+) [/μl]	55.00	14.50	24.17	3.65	0.00	0.00
IFN-γ ⁺ T cells _{abs (CD3+CD8+)} [x10 ⁴ ]	3.24	0.86	1.44	0.13	0.00	0.01
B	*2. Validation run*
	Positive control		CMVpp65 _pp		Negative control
	OF	TCF	OF	TCF	OF	TCF
volume [ml]	0.6	1.0	0.6	1.0	0.6	1.0
viability [%]	98.72	89.04	98.92	85.16	98.87	29.95
WBCs_(CD45+) [x10⁶/ml]	2.38	0.18	2.74	0.00	2.78	0.00
WBCs_abs [x10⁶]	1.43	0.18	1.64	0.00	1.67	0.00
T cells_(CD3+CD56-) [% of WBCs]	95.94	88.43	89.37	68.87	88.83	55.91
T cells [/μl]	2283.33	162.00	2450.00	1.42	2466.67	11.10
T cells_abs [x10⁶]	1.37	0.16	1.47	0.00	1.48	0.00
T cells_(CD3+CD4+) [% of CD3⁺]	71.87	75.54	71.00	43.38	69.45	71.15
T cells_(CD3+CD8+) [% of CD3⁺]	28.36	24.51	29.06	56.73	30.59	28.85
IFN-γ ⁺ T cells [% of CD3 ⁺ ]	5.74	60.77	0.13	59.62	0.02	3.85
IFN-γ⁺ T cells [/μl]	131.00	98.30	3.18	0.85	0.49	0.43
IFN-γ ⁺ T cells [x10 ⁴ ]	7.86	9.83	0.19	0.08	0.03	0.00
IFN-γ⁻ T cells [% of CD3⁺]	94.26	39.23	99.87	40.38	99.98	96.15
IFN-γ⁻ T cells [/μl]	2150.00	63.50	2450.00	0.57	2466.67	10.70
IFN-γ⁻ T cells [x10⁴]	129	6.35	147	0.06	148	0.11
IFN-γ⁺ T cells_(CD3+) [% of CD4]	3.83	43.77	0.07	14.53	0.02	1.92
IFN-γ ⁺ T cells _(CD3+CD4+) [% of CD4]	4.99	56.07	0.06	32.76	0.03	2.70
IFN-γ⁺ T cells_(CD3+CD4+) [/μl]	81.83	68.50	1.04	0.20	0.52	0.21
IFN-γ ⁺ T cells _{abs (CD3+CD4+)} [x10 ⁴ ]	4.91	6.85	0.06	0.02	0.03	0.00
IFN-γ⁺ T cells_(CD3+) [% of CD8]	1.80	16.45	0.06	45.09	0.00	1.92
IFN-γ ⁺ T cells _(CD3+CD8+) [% of CD8]	5.75	64.53	0.21	78.34	0.00	6.67
IFN-γ⁺ T cells_(CD3+CD8+) [/μl]	37.17	25.60	1.50	0.63	0.00	0.21
IFN-γ ⁺ T cells _{abs (CD3+CD8+)} [x10 ⁴ ]	2.23	2.56	0.09	0.06	0.00	0.00
C	*3. Validation run*
	Positive control		CMVpp65 _pp		Negative control
	OF	TCF	OF	OF	TCF	OF
volume [ml]	0.6	1.0	0.6	1.0	0.6	1.0
viability [%]	98.57	69.24	98.87	45.75	98.63	26.57
WBCs_(CD45+) [x10⁶/ml]	4.23	0.02	3.33	0.01	3.66	0.00
WBCs_abs [x10⁶]	2.54	0.02	2.00	0.01	2.20	0.00
T cells_(CD3+CD56-) [% of WBCs]	98.13	94.39	99.99	95.45	95.87	73.00
T cells [/μl]	4150.00	21.20	3333.33	11.90	3516.67	0.00
T cells_abs [x10⁶]	2.49	0.02	2.00	0.01	2.11	0.00
T cells_(CD3+CD4+) [% of CD3⁺]	72.20	66.59	72.07	88.51	68.63	84.38
T cells_(CD3+CD8+) [% of CD3⁺]	27.80	33.41	27.93	11.49	31.37	15.62
IFN-γ ⁺ T cells [% of CD3 ⁺ ]	2.73	70.70	1.49	76.41	0.00	1.56
IFN-γ⁺ T cells [/μl]	113.33	15.00	49.50	9.06	0.00	0.00
IFN-γ ⁺ T cells [x10 ⁴ ]	6.80	1.50	2.97	0.91	0.00	0.00
IFN-γ⁻ T cells [% of CD3⁺]	97.27	29.30	98.51	23.59	100.00	98.44
IFN-γ⁻ T cells [/μl]	4033.33	6.22	3283.33	2.80	3516.67	0.00
IFN-γ⁻ T cells [x10⁴]	242	0.62	197	0.28	211	0.00
IFN-γ⁺ T cells_(CD3+) [% of CD4]	1.55	45.27	1.35	68.14	0.00	0.76
IFN-γ ⁺ T cells _(CD3+CD4+) [% of CD4]	2.17	67.26	1.83	76.52	0.00	1.04
IFN-γ⁺ T cells_(CD3+CD4+) [/μl]	65.00	9.50	43.83	8.03	0.00	0.00
IFN-γ ⁺ T cells _{abs (CD3+CD4+)} [x10 ⁴ ]	3.90	0.95	2.63	0.80	0.00	0.00
IFN-γ⁺ T cells_(CD3+) [% of CD8]	1.19	26.20	0.18	8.80	0.00	0.52
IFN-γ ⁺ T cells _(CD3+CD8+) [% of CD8]	4.33	81.11	0.72	78.15	0.00	3.33
IFN-γ⁺ T cells_(CD3+CD8+) [/μl]	50.00	5.75	6.68	1.06	0.00	0.00
IFN-γ ⁺ T cells _{abs (CD3+CD8+)} [x10 ⁴ ]	3.00	0.58	0.40	0.11	0.00	0.00

The small-scale MiniMACS CSA was performed as a control for the large-scale CliniMACS CCS process from the same leukapheresis. Detailed information for all three MiniMACS CSA processes (A-B) including viability [%], viable cell number [x106/ml; x106; /μl] and specific cell frequencies [%] for all CSA fractions was determined. The results for the representative analysis of the cells from the original fraction (OF) and T-cell fraction (TCF) are shown. As a positive control, cells were stimulated with SEB, while cells cultured in medium alone served as the negative control; CMVpp65pp = CMVpp65 peptide pool. Bold data reflected the results obtained for the antigen-specific IFN-γ-positive T cells.

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