Background
Methods
Allogeneic cell registry, alloCELL
Selection of a suitable CMV-specific T-cell donor
Detection of IFN-γ secreting CMV-specific T cells by cytokine secretion assay
Generation of clinical-grade CMV-specific T cells by CliniMACS CCS
Quality control: assessment of the final T-cell product by flow cytometry
Statistical analysis
Results
Verification of CMVpp65-specific T-cell repertoire in preselected T-cell donors from alloCELL registry
Donor | HLA-typing |
alloCELL
| Verification and detailed analysis of CMV-specific memory T-cell frequencies | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
TCR-pMHC interaction | EliSpot | Staining of T-cell subsets | TCR-pMHC interaction | CSA | EliSpot | |||||||||
A | B | C | DRB1 | DQB1 | % A02pp65M
| spw | % CD3 | % CD4 | % CD8 | % A02pp65M
| %OF | %TCF | spw | |
[CD19−
CD3+CD8+] | [IFN-γ+] | [CD45+CD19−] | [CD3+] | [CD3+] | [CD19−
CD3+CD8+] | [CD3+IFN-γ+] | [CD3+IFN-γ+] | [IFN-γ+] | ||||||
1 | *02:01 *68:01 | *08:01 *39:01 | *07:01 *12:03 | *01:01 *03:01 | *02:01 *05:01 | 2.45 | 273 | 78.88 | 52.47 | 41.24 | 1.5 | 1.7 | 75.48 | 236 |
2 | *25:01 *32:01 | *08:01 *35:01 | *04:01 *07:01 | *03:01 *14:01 | *02:01 *05:03 | n.a. | 162 | 59.65 | 69.53 | 26.61 | n.a. | 0.21 | 38.39 | 178.4 |
3 | *02:01 *11:01 | *27:02 *55:01 | *02:02 *03:03 | *15:01 *16:01 | *05:02 *06:02 | 0.31 | 142 | 63.79 | 68.41 | 26.7 | 0.34 | 7.6 | 89.63 | 306.5 |
Validation of CMVpp65-specific T-cell enrichment by CliniMACS CCS
A |
1. Validation run
| ||||
---|---|---|---|---|---|
Leukapheresis | OF | TCF | WF | NF | |
volume [ml] | 133 | 100 | 40 | 335 | 276 |
viability [%] | 99.80 | 99.06 | 51.13 | 92.34 | 99.38 |
WBCs(CD45+) [x106/ml] | 6.77 | 1.42 | 0.14 | 0.02 | 0.49 |
WBCsabs [x106] | 900.41 | 142.00 | 5.51 | 8.15 | 136.07 |
T cells(CD3+CD56-) [% of WBCs] | 41.93 | 47.80 | 31.68 | 41.58 | 44.55 |
T cells [/μl] | 2834.59 | 680.00 | 43.50 | 10.12 | 219.57 |
T cellsabs [x106] | 377.00 | 68.00 | 1.74 | 3.39 | 60.60 |
T cells(CD3+CD4+) [% of CD3+] | 53.00 | 50.82 | 26.20 | 49.74 | 51.01 |
T cells(CD3+CD8+) [% of CD3+] | 47.02 | 49.20 | 73.85 | 50.31 | 49.01 |
IFN-γ
+
T cells [% of CD3
+
]
|
0.03
|
1.02
|
81.17
|
27.13
|
0.63
|
IFN-γ+ T cells [/μl] | 0.85 | 6.94 | 35.50 | 2.74 | 1.38 |
IFN-γ
+
T cells [x10
6
]
|
0.11
|
0.69
|
1.42
|
0.92
|
0.38
|
IFN-γ− T cells [% of CD3+] | 99.97 | 98.98 | 18.83 | 72.87 | 99.37 |
IFN-γ− T cells [/μl] | 2834.59 | 673.00 | 8.20 | 7.37 | 218.12 |
IFN-γ− T cells [x106] | 377.00 | 67.30 | 0.33 | 2.47 | 60.20 |
IFN-γ+ T cells(CD3+) [% of CD4] | 0.02 | 0.29 | 12.91 | 6.36 | 0.17 |
IFN-γ
+
T cells
(CD3+CD4+)
[% of CD4]
|
0.04
|
0.58
|
50.63
|
13.28
|
0.41
|
IFN-γ+ T cells(CD3+CD4+) [/μl] | 0.60 | 2.00 | 5.78 | 0.67 | 0.46 |
IFN-γ
+
T cells
abs (CD3+CD4+)
[x10
6
]
|
0.08
|
0.20
|
0.23
|
0.22
|
0.13
|
IFN-γ+ T cells(CD3+) [% of CD8] | 0.01 | 0.71 | 67.96 | 20.49 | 0.40 |
IFN-γ
+
T cells
(CD3+CD8+)
[% of CD8]
|
0.02
|
1.43
|
91.84
|
40.21
|
0.73
|
IFN-γ+ T cells(CD3+CD8+) [/μl] | 0.27 | 4.79 | 29.50 | 2.04 | 0.79 |
IFN-γ
+
T cells
abs (CD3+CD8+)
[x10
6
]
|
0.04
|
0.48
|
1.18
|
0.69
|
0.22
|
B
|
2. Validation run
| ||||
Leukapheresis
|
OF
|
TCF
|
WF
|
NF
| |
volume [ml] | 114 | 100 | 40 | 337 | 288 |
viability [%] | 99.31 | 98.07 | 62.05 | 99.27 | 98.83 |
WBCs(CD45+) [x106/ml] | 18.77 | 2.36 | 0.03 | 0.00 | 0.66 |
WBCsabs [x106] | 2133.91 | 236.00 | 1.06 | 0.69 | 190.94 |
T cells(CD3+CD56-) [% of WBCs] | 16.97 | 36.69 | 27.92 | 25.73 | 41.15 |
T cells [/μl] | 3175.44 | 865.00 | 7.43 | 0.53 | 272.92 |
T cellsabs [x106] | 362.00 | 86.50 | 0.30 | 0.18 | 78.60 |
T cells(CD3+CD4+) [% of CD3+] | 71.57 | 62.28 | 78.45 | 69.19 | 62.36 |
T cells(CD3+CD8+) [% of CD3+] | 28.50 | 37.75 | 21.80 | 30.88 | 37.71 |
IFN-γ
+
T cells [% of CD3
+
]
|
0.11
|
0.07
|
19.18
|
3.80
|
0.01
|
IFN-γ+ T cells [/μl] | 3.50 | 0.61 | 1.42 | 0.02 | 0.03 |
IFN-γ
+
T cells [x10
6
]
|
0.40
|
0.06
|
0.05
|
0.01
|
0.01
|
IFN-γ− T cells [% of CD3+] | 99.89 | 99.93 | 80.82 | 96.20 | 99.99 |
IFN-γ− T cells [/μl] | 3175.44 | 864.00 | 6.00 | 0.51 | 272.92 |
IFN-γ− T cells [x106] | 362.00 | 86.40 | 0.23 | 0.17 | 78.60 |
IFN-γ+ T cells(CD3+) [% of CD4] | 0.07 | 0.02 | 4.95 | 1.51 | 0.01 |
IFN-γ
+
T cells
(CD3+CD4+)
[% of CD4]
|
0.10
|
0.03
|
6.43
|
1.93
|
0.02
|
IFN-γ+ T cells(CD3+CD4+) [/μl] | 2.27 | 0.16 | 0.38 | 0.01 | 0.03 |
IFN-γ
+
T cells
abs (CD3+CD4+)
[x10
6
]
|
0.26
|
0.02
|
0.02
|
0.00
|
0.01
|
IFN-γ+ T cells(CD3+) [% of CD8] | 0.04 | 0.04 | 13.84 | 2.18 | 0.00 |
IFN-γ
+
T cells
(CD3+CD8+)
[% of CD8]
|
0.12
|
0.10
|
62.96
|
6.52
|
0.00
|
IFN-γ+ T cells(CD3+CD8+) [/μl] | 1.09 | 0.32 | 1.02 | 0.01 | 0.00 |
IFN-γ
+
T cells
abs (CD3+CD8+)
[x10
6
]
|
0.12
|
0.03
|
0.04
|
0.00
|
0.00
|
C
|
3. Validation run
| ||||
Leukapheresis
|
OF
|
TCF
|
WF
|
NF
| |
volume [ml] | 109 | 100 | 43 | 330 | 263 |
viability [%] | 99.56 | 97.86 | 58.92 | 90.43 | 98.21 |
WBCs(CD45+) [x106/ml] | 15.73 | 1.62 | 0.12 | 0.01 | 0.53 |
WBCsabs [x106] | 1714.31 | 162.00 | 5.01 | 4.82 | 138.49 |
T cells(CD3+CD56-) [% of WBCs] | 12.76 | 30.94 | 36.52 | 31.07 | 28.56 |
T cells [/μl] | 2009.17 | 500.00 | 42.56 | 4.55 | 150.19 |
T cellsabs [x106] | 219.00 | 50.00 | 1.83 | 1.50 | 39.50 |
T cells(CD3+CD4+) [% of CD3+] | 75.96 | 72.60 | 87.29 | 80.31 | 70.89 |
T cells(CD3+CD8+) [% of CD3+] | 24.08 | 27.43 | 12.81 | 19.71 | 29.13 |
IFN-γ
+
T cells [% of CD3
+
]
|
0.06
|
1.11
|
63.13
|
36.29
|
0.47
|
IFN-γ+ T cells [/μl] | 1.20 | 5.55 | 26.74 | 1.65 | 0.71 |
IFN-γ
+
T cells [x10
6
]
|
0.13
|
0.56
|
1.15
|
0.54
|
0.19
|
IFN-γ− T cells [% of CD3+] | 99.94 | 98.89 | 36.87 | 63.71 | 99.53 |
IFN-γ− T cells [/μl] | 2009.17 | 494.00 | 15.67 | 2.89 | 149.81 |
IFN-γ− T cells [x106] | 219.00 | 49.40 | 0.67 | 0.95 | 39.40 |
IFN-γ+ T cells(CD3+) [% of CD4] | 0.06 | 0.87 | 53.17 | 30.37 | 0.47 |
IFN-γ
+
T cells
(CD3+CD4)
[% of CD4]
|
0.06
|
1.10
|
58.25
|
34.13
|
0.49
|
IFN-γ+ T cells(CD3+CD4+) [/μl] | 0.91 | 3.99 | 21.63 | 1.24 | 0.52 |
IFN-γ
+
T cells
abs (CD3+CD4+)
[x10
6
]
|
0.10
|
0.40
|
0.93
|
0.41
|
0.14
|
IFN-γ+ T cells(CD3+) [% of CD8] | 0.02 | 0.30 | 11.48 | 7.75 | 0.10 |
IFN-γ
+
T cells
(CD3+CD8+)
[% of CD8]
|
0.00
|
0.99
|
88.29
|
37.27
|
0.30
|
IFN-γ+ T cells(CD3+CD8+) [/μl] | 0.00 | 1.36 | 4.81 | 0.33 | 0.13 |
IFN-γ
+
T cells
abs (CD3+CD8+)
[*10
6
]
|
0.00
|
0.14
|
0.21
|
0.11
|
0.03
|
n = 3
|
%CD3
+
IFN-γ
+
recovery
|
%CD4
+
IFN-γ
+
recovery
|
%CD8
+
IFN-γ
+
recovery
|
%CD3
+
IFN-γ
+
purity
|
%CD4
+
IFN-γ
+
purity
|
%CD8
+
IFN-γ
+
purity
|
% total viability
|
---|---|---|---|---|---|---|---|
Mean
| 67.86 | 68.81 | 57.20 | 54.49 | 38.44 | 81.03 | 57.37 |
SD
| 22.66 | 57.20 | 23.42 | 31.88 | 27.98 | 15.75 | 1.61 |
Median
| 77.69 | 69.95 | 70.37 | 63.13 | 50.63 | 88.29 | 58.92 |
Min
| 41.94 | 11.04 | 30.16 | 19.18 | 6.43 | 62.96 | 51.13 |
Max
| 83.94 | 125.43 | 71.08 | 81.17 | 58.25 | 91.84 | 62.05 |
Stability evaluation of CliniMACS CCS-enriched T-cell products
Parameter | Validation run | TCF after enrichment | 48 h | 54 h | 72 h | |||
---|---|---|---|---|---|---|---|---|
value | recovery | value | recovery | value | recovery | |||
WBC | 1. run | 1.43x107
| 9.60x106
| 67.00% | 7.80x106
| 54.50% | 7.60x106
| 53.10% |
2. run | 5.00x106
| 4.40x106
| 88.00% | 4.20x106
| 84.00% | 4.00x106
| 80.00% | |
3. run | 1.25x107
| 8.60x106
| 69.00% | 7.30x106
| 58.60% | 6.50x106
| 51.70% | |
viable WBC | 1. run | 1.01x107
| 8.00x106
| 79.50% | 7.20x106
| 71.60% | 6.20x106
| 61.70% |
2. run | 3.40x106
| 3.20x106
| 94.10% | 3.80x106
| >100% | 3.60x106
| >100% | |
3. run | 6.00x106
| 5.20x106
| 85.70% | 4.30x106
| 71.40% | 3.90x106
| 64.30% | |
viable T cells | 1. run | 1.74x106
| 1.42x106
| 81.61 | 1.32x106
| 75.86% | 1.22x106
| 70.11% |
2. run | 2.97x106
| 2.17x105
| 73.06% | 1.97x105
| 66.33% | 2.49x105
| 83.84% | |
3. run | 1.83x106
| 2.12x106
| >100% | 1.85x106
| >100% | 2.09x106
| >100% |
Enrichment of CMVpp65-specific T cells by non-GMP MiniMACS CSA as a control process
A |
1. Validation run
| |||||
---|---|---|---|---|---|---|
Positive control | CMVpp65pp
| 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 |
WBCsabs [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 cellsabs [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 |
WBCsabs [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 cellsabs [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 |
WBCsabs [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 cellsabs [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
|