Methods for the production of TCR gamma delta + T cells
The present invention relates to novel methods for the isolation and the selective ex vivo expansion of V32′ TCRy6+ T cells and to their clinical application.
1. A method for expanding Vδ2-TCRγδ+ T cells in a sample comprising:
culturing cells in the sample in a culture medium comprising a T cell mitogen which is an antibody or fragment thereof and interleukin-15, in the absence of interleukin-4 and in the absence of feeder cells;
or
culturing cells in the sample in a culture medium comprising a T cell mitogen which is an antibody or fragment thereof and interleukin-4; in the absence of interleukin-15 and in the absence of feeder cells.
2. The method according to claim 1 , wherein the antibody or fragment thereof is an anti-CD3 antibody.
3. The method according to claim 1 , wherein the antibody or fragment thereof is an anti-TCRγδ antibody.
4. The method according to claim 3 , wherein the antibody or fragment thereof is TS8.2 or TS-1.
5. The method according to claim 4 , comprising culturing cells in the sample in a first culture medium comprising a T cell mitogen which is an antibody or fragment thereof and IL-4 in the absence of IL-15.
6. The method according to claim 5 , further comprising culturing cells in a second culture medium comprising a T cell mitogen which is an antibody or fragment thereof and IL-15 in the absence of IL-4.
7. The method according to claim 1 , wherein the culture medium further comprises a growth factor selected from interferon-γ, interleukin-21 or interleukin-1β.
8. The method according to claim 1 , wherein the culture medium further contains serum or plasma.
9. The method according to claim 1 , wherein prior to culturing the cells, the cells in the sample are enriched for TCRγδ+ T cells.
10. The method according to claim 1 , wherein the sample is blood or tissue or fractions thereof.
11. The method according to claim 10 , wherein the sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, epithelia, thymus, bone marrow, spleen, liver, cancerous tissue, infected tissue, lymph node tissue or fractions thereof.
12. The method according to claim 1 , wherein the sample is human peripheral blood or a fraction thereof.
13. The method according to claim 1 , wherein in the culture medium the T cell mitogen is present in an amount from about 10 to about 5000 ng/ml and interleukin-4 is present in an amount from about 1 to about 1000 ng/ml, preferably wherein in the culture medium the T cell mitogen is present in an amount from about 20 to about 2000 ng/ml and interleukin-4 is present in an amount from about 5 to about 500 ng/ml, more preferably wherein in the culture medium the T cell mitogen is present in an amount from about 50 to about 1000 ng/ml; and interleukin-4 is present in an amount from about 20 to about 200 ng/ml, still more preferably wherein the culture medium comprises 70 ng/mL of a T cell mitogen and 100 ng/mL of interleukin-4.
14. The method according to claim 1 , wherein in the culture medium the T cell mitogen is present in an amount from about 0.1 to about 50 μg/ml and interleukin-15 is present in an amount from about 1 to about 1000 ng/ml, preferably wherein in the culture medium the T cell mitogen is present in an amount from about 0.3 to about 10 μg/ml and interleukin-15 is present in an amount from about 2 to about 500 ng/ml, more preferably wherein in the culture medium the T cell mitogen is present in an amount from about 0.5 to about 5 μg/ml and interleukin-15 is present in an amount from about 20 to about 200 ng/ml, still more preferably wherein the culture medium comprises 1 μg/mL of a T cell mitogen and 70 ng/mL of interleukin-15.
15. The method according to claim 7 , wherein in the culture medium interferon-γ is present in an amount from about 1 to about 1000 ng/ml, and interleukin-21 and interleukin-1β are present in an amount from 1 to about 500 ng/ml; preferably wherein in the culture medium interferon-γ is present in an amount from about 2 to about 500 ng/ml, and interleukin-21 and interleukin-1β are present in an amount from 2 to about 200 ng/ml; more preferably wherein in the culture medium interferon-γ is present in an amount from about 20 to about 200 ng/ml, and interleukin-21 and interleukin-1β are present in an amount from 5 to about 100 ng/ml; still more preferably wherein the culture medium comprises 70 ng/ml of interferon-γ, 15 ng/ml of interleukin-21, and 15 ng/ml of interleukin-1l.
16. The method according to claim 1 , wherein the antibody or fragment thereof is immobilised.
17. The method according to claim 1 , wherein the antibody or fragment thereof is soluble.