This application protocol describes how to expand antigen-specific T cells which have been isolated using the MACS® Cytokine Secretion Assay after restimulation with antigen. These T cells are therefore of a memory phenotype. The memory phenotype, particularly for CD8+ cells, is heterogeneous. It contains cells, which divide rapidly in response to antigen-“central memory cells” and cells, which exhibit recall effector function, such as cytotoxicity-“effector memory cells”. These subsets are not mutually exclusive, e.g., cells can divide rapidly but still lyse antigen-loaded targets. The mixture of memory cells isolated with the MACS Cytokine Secretion Assay is entirely dependent on the antigen the T cells responded to, e.g., immune responses against chronic virus infections are often characterized by a pre-dominance of effector memory T cells. As a general rule, central memory cells have a higher proliferation rate in vitro, compared to effector memory cells. Therefore, the rate of proliferation of T cells expanded with this method will depend on the memory phenotype of the cells that have been isolated. This protocol has been optimized to expand both central and effector memory T cells very efficiently. However, the individual results obtained may vary depending on the antigen and the Cytokine Secretion Assay used to isolate the cells. This protocol has been established for the expansion of CMV-specific, IFN-γ–secreting T cells. It may require optimization when working with other antigens or expanding cells which were isolated according to the secretion of cytokines other than IFN-γ.
This expansion system is designed for in vitro expansion of cytokine-secreting, antigen-specific T cells. The antigen-specific T cells in peripheral blood mononuclear cells (PBMCs) or leukapheresis are restimulated with antigen. The responding cells are magnetically isolated, according to their secretion of cytokines, using the MACS Cytokine Secretion Assay. The non-cytokine secreting cell fraction, i.e., the unlabeled flow through, is used as a source of antigen-presenting cells (referred to as “feeder cells” throughout the protocol) after irradiation or treatment with mitomycin C. The cytokine-secreting cells and the feeder cells are co-cultured in vitro in the presence of interleukin 2 (IL-2).
▲ All steps in the following protocol have to be performed under sterile conditions.
Prepare and restimulate cells with antigen and perform the Cytokine Secretion Assay including magnetic labeling referring to the respective data sheet (refer to 4. Protocol for the IFN-γ Secretion Assay, steps 4.1–4.4).
▲ Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of IFN-γ–secreting cells. For details refer to the table in tab Material.
▲ Always wait until the column reservoir is empty before proceeding to the next step.
▲ When enriching antigen-specific T cells, always perform two consecutive column runs to achieve best results.
▲ Refer to the respective user manual for instructions on how to use the autoMACS Pro Separator.
▲ Buffers used for operating the autoMACS Pro Separator should have a temperature of ≥10 °C.
▲ For sample preparation refer to the protocol in the data sheet of the Large Scale IFN-γ Secretion Assay – Enrichment Kit.
▲ For set-up of the CliniMACS Plus Instrument, selection of the separation program, and installation of the CliniMACS Tubing Set follow the detailed instructions given in the CliniMACS Plus User Manual.
The protocol is based on standard T cell expansion procedures where specific T cells are cultured with autologous feeder cells, antigen, and IL-2. Using the cells collected from the unlabeled fraction, provides a convenient source of antigen pre-loaded feeder cells. As the target T cells are already highly activated and enriched they have high co-stimulation requirements to grow properly and require more feeder cells than in standard expansion procedure, where the initial frequency of antigen-specific T cells is low. Therefore, in the first phase of expansion, 100 feeder cells are required per antigen-specific T cell in the isolated positive fraction. After two weeks of expansion the T cells require to be restimulated with feeder cells (which still contain the antigen), at a ratio of 10 feeder cells per T cell. This cycle can be repeated every 2 weeks.
As there will be only a limited amount of cells from the unlabeled fraction, long term culture or quickly growing cells will require a new source of feeder cells. For this purpose, fresh PBMCs from the same donor may be used. The cells should be used in exactly the same way as for the unlabeled fraction, with the addition of antigen at the same concentration as used to originally stimulate the T cells. It is also possible that restimulation of T cells with frozen cells from the unlabeled fraction can be enhanced by addition of fresh antigen.
In order to have feeder cells for later restimulation of the T cells, cells of the unlabeled fraction (or PBMCs from the same donor) can be frozen and later resuscitated.
To prevent the feeder cells growing out during culture, these cells need to be immobilised using irradiation or mitomycin C treatment.
▲ Note: Cells should not be frozen after irradiation or mitomycin C treatment.
For example, irradiate cells with 35–50 Gy (3500–5000 rads) using a caesium 137 source. Individual irradiators may vary in performance, and the optimum degree of irradiation has to be determined.
▲ Mitomycin C is extremely toxic. Observe all manufacturer’s safety warnings.
The following are guidelines as to how cells should grow; the rates of growth will depend on the phenotype of the cells generated. After one or two days it is normal that large clumps of cells appear in the culture. Cells should be observed daily to ensure that medium is not depleted of nutrition. A basic schedule is to feed the T cells every 3–4 days with fresh medium supplemented with 20 IU IL-2 at day 3, 7, and 11 (start is day 0). By day 14, cells need to be restimulated with feeder cells and antigen, followed by a repeated cycle of feeding with medium and IL-2.
▲ If at any point the indicator of the medium turns yellow, remove about 70% of the medium with a pipette without resuspending the cells. Replace volume with medium supplemented with 20 IU/mL of IL-2, and transfer half of the culture to a new identical culture dish.
Start (refer to section Expansion protocol, Initiation of T cell expansion)
Gently resuspend the cells using a pipette and transfer half of the culture to a new identical culture dish. Add an equivalent volume of medium supplemented with 20 IU/mL of IL-2 to each well.
Gently resuspend the cells using a pipette and add 20 IU/mL IL-2 (no fresh medium).
Repeat procedure from Day 3.
▲ Long term (> 4 weeks) culture of T cells selects the cells that grow best in culture – the longer the cells are cultured, the less they will reflect the original effector phenotype of the isolated cells (antigen-specificity is unaltered).
▲ Some T cells may require to be restimulated with feeder cells on a weekly basis after the initial 14-day period.
For T cell expansion the cells should be resuspended in culture medium, containing 5% of human serum and 20 IU of IL-2 at 5×10⁶ cells/mL. The cells should be plated at a density of 2.5×10⁶ cells/cm2. Both the dilution and the cell density are important to assure optimal stimulation and cell growth.
The following table lists culture plate, dish and flask sizes suitable for different cell numbers. It also indicates the appropriate amount of medium to add. For culture in other formats (e.g. culture bags) please scale up accordingly.
|Total cell number||Medium volume to add||Culture plate||Well diameter|
|0.75×106||0.15 mL||96 well||0.64 cm|
|2. 5×106||0.5 mL||48 well||1.13 cm|
|5×106||1 mL||24 well||1.60 cm|
|1×107||2 mL||12 well||2.26 cm|
|2.5×10⁷||5 mL||6 well||3.50 cm|
|Total cell number||Medium volume to add||Culture dish||Dish diameter|
|2.5×10⁷||5 mL||small||3.5 cm|
|7.5×10⁷||15 mL||medium||6 cm|
|20×10⁷||40 mL||large||10 cm|
|45×10⁷||90 mL||extra large||15 cm|
|Total cell number||Medium volume to add||Culture flask||Growth area|
|6×10⁷||12 mL||50 mL||25 cm²|
|20×10⁷||40 mL||250 mL||75 cm²|
|40×10⁷||80 mL _||720 mL||162 cm²|
|55×10⁷||110 mL||900 mL||225 cm²|
|Column||Max. number of labeled cells||Max. number of total cells||Separator|
|MS||10⁷||2×10⁸||MiniMACS, OctoMACS, SuperMACS II|
|LS||10⁸||2×10⁹||MidiMACS, QuadroMACS, SuperMACS II|
|CliniMACS Tubing Set||4×1010||CliniMACS Plus Instrument|
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