In Vitro T Cell Expansion and Function - Astarte Bio · 2019. 12. 13. · Mixed Lymphocyte...
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Comparison of Culture Media for In Vitro T Cell Expansion and Function Ponni Anandakumar, Benjamin A. Tjoa, and P. Anne Lodge, Astarte Biologics, Bothell, WA astartebio.com Getting on with discovery. Background Identification of a reliable culture media to support in vitro T cell studies has become an important link in the chain of various Immuno-Oncology strategies. While many labs have chosen one favorite media for their T cell culture needs, it may be prudent to identify alternatives that can perform suitably, whether one works in the development of cell-based assays to screen potential drug candidates or generates and expands antigen-specific T cells. To address this issue, we have conducted a series of studies comparing the performance of several culture media. Methods A list of culture media (including several classic media + supplements as well as several new media) was compared to several commercially available T cell media in the generation of primary MLR (mixed-lymphocyte reaction), antigen-recall assay (e.g., CMV, tetanus), antigen-specific T cell proliferation assay, as well as in anti-CD3/CD28 driven T cell expansion culture. Cells PBMC were obtained from normal donors in our study protocol. The protocol for collection of PBMC has been reviewed and approved by an accredited independent review board (IRB) and participants have given their informed consent for use of their cells in research. B lymphoblastoid cells used in some experiments were derived from these PBMC as were antigen-specific T cells. Recall Antigen Assay PBMC are added to U-bottom 96 well plates at 250,000 cells per well in a 100 μL volume. The antigens are prepared at 2x the desired final concentration and added to the wells at 100 μL. Tetanus toxoid and CMV antigens are from our catalog and used at 1 μg/mL, PHA was purchased from Sigma and used at 1 μg/mL and the lipopolysaccharide (LPS) was also purchased from Sigma and used at a final concentration of 100 ng/mL. Cultures of PBMC and antigen were incubated at 37˚C, 5% CO 2 and after 4 days culture medium was collected for cytokine analysis. Cytokine Analysis Cytokines were analyzed using a U-Plex kit from Meso Scale Discovery. Interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) were routinely measured in the recall antigen assay. Antigen-Specific Proliferation Assay Tetanus toxoid-specific T cells were used to evaluate the effect of media formulations on antigen specific proliferation. Autologous B-LCL were inactivated by incubation with 50 μg/mL of mitomycin C for 45 minutes at 37˚C. After this incubation, mitomycin C was removed by 3 washes with PBS. The inactivated cells were plated at 20,000 cells per well in a U-bottom 96 well plate. Peptide antigen was added to indicated concentrations and tetanus toxoid-specific T cells were added at 20,000 cells per well. The cultures were incubated for 4 days and proliferation was measured by addition of CellTiter Glo™. Mixed Lymphocyte Reaction B-lymphoblastoid cells were used to stimulate PBMC in a mixed lymphocyte reaction. The batch of PBMC and B-LCL were kept constant for this series of experiments. Proliferation of the B-LCL was prevented by incubation of the cells with 50 μg/mL of mitomycin C (Sigma) for 45 minutes at 37˚C. After this incubation, mitomycin was removed by 3 washes with PBS and following the last wash the cells were suspended in the desired medium. These APC were added at a range of cell numbers per well in a 100 μL volume. PBMC were added at 20,000 cells per well in a volume of 100 μL of the appropriate medium. The culture was incubated for 5 days and proliferation was measured by the addition of CellTiter Glo™. Stimulation with Anti-CD3 and Anti-CD28 PBMC were stimulated with ImmunoCult™, an anti-CD3, anti-CD28 conjugate from Stem Cell Technology. This reagent was used at a range of concentrations in test medium. PBMC were added at 100,000 cells per well in 96 well plates or cultured in bulk for flow cytometric analysis. Flow Cytometric Analysis Fluorescent antibodies were purchased from BioLegend and used at the volumes recommended by the manufacturer. Antibodies were incubated with cells for 15 minutes in the dark, washed once and data was acquired using a FACScan cytometer. Further analysis was performed using FCS Express (DeNovo Software). Results Classical media supplemented with several defined components can support primary in vitro responses as measured by cytokine production. Sustained T cell proliferation demanded additional supplementation and revealed greater differences between media. One representative data from these studies is included in this abstract. This experiment demonstrates the effect of human AB serum (HS) or fetal bovine serum (FBS) added to the culture medium X-VIVO™ 15 (Lonza, Walkersville, MD). At low peptide concentrations (3 and 10 ng/mL), the presence of HS and FBS inhibits T cell proliferation compared to X-VIVO™ 15 alone. Conclusions • Supplementation of media with human serum does not support optimal cytokine production in the recall antigen assay. • Minimal supplementation of DME/F12 is sufficient to support cytokine production and proliferation in short-term (5-day) assays. • Supplementation of culture medium with serum supports growth but requires higher concentrations of antigen. • Choice of culture medium influences not only growth rate but phenotype and function of resulting T cells. Figure 1 Each panel shows a different donor PBMC tested for antigen response and the IFNγ produced in DME/F12 supplemented with either fetal bovine serum or human AB serum at 10% or using X-VIVO™ 15. Figure 2 Each panel depicts data from a different PBMC donor. Cells were tested for recall antigen response using either DME/F12 supplemented with ITS+Premix and human serum albumin or X-VIVO™ 15. Figure 3 An MLR conducted in the presence of three media formulations. X-VIVO™ 15 supplemented with human serum and DME/F12 with ITS+Premix and human albumin supported greater cell growth at low concentrations of stimulating B-LCL. Figure 4 Tetanus toxoid-specific T cells were incubated with APC and the indicated concentrations of peptide antigen. After 4 days incubation at 37˚C the proliferation was measured by addition of CellTiter Glo™. Figure 6 Proliferation stimulated by anti-CD3+anti-CD28 using a second donor’s PBMC. ImmunoCult™ was added to culture medium at 6.25 μL per mL and a series of 1:2 dilutions as well as a control without ImmunoCult™. after 4 days culture the proliferation was measured using CellTiter Glo™ Figure 7 Phenotypic analysis of T cells following stimulation with anti-CD3+anti-CD28. PBMC were stimulated using ImmunoCult™ in 3 different serum-free medium formulations. IL-2 was added after the initial 24 hours and after 6 more days the cells were collected and analyzed for expression of CD4, CD8, CD45RA, CD45RO, and CD62L. X-VIVO™ 15 in row 1, Medium A in row 2 and Medium B in row 3. Figure 5 Proliferation stimulated by anti-CD3+anti-CD28. ImmunoCult™ was added to culture medium at 6.25 μL per mL and a series of 1:2 dilutions as well as a control without ImmunoCult™. After 4 days culture the proliferation was measured using CellTiter Glo™.
Transcript of In Vitro T Cell Expansion and Function - Astarte Bio · 2019. 12. 13. · Mixed Lymphocyte...
Comparison of Culture Media for In Vitro T Cell Expansion and FunctionPonni Anandakumar, Benjamin A. Tjoa, and P. Anne Lodge, Astarte Biologics, Bothell, WA
astartebio.com Getting on with discovery.
BackgroundIdenti� cation of a reliable culture media to support in vitro T cell studies has become an important link in the chain of various Immuno-Oncology strategies. While many labs have chosen one favorite media for their T cell culture needs, it may be prudent to identify alternatives that can perform suitably, whether one works in the development of cell-based assays to screen potential drug candidates or generates and expands antigen-speci� c T cells. To address this issue, we have conducted a series of studies comparing the performance of several culture media.
MethodsA list of culture media (including several classic media + supplements as well as several new media) was compared to several commercially available T cell media in the generation of primary MLR (mixed-lymphocyte reaction), antigen-recall assay (e.g., CMV, tetanus), antigen-speci� c T cell proliferation assay, as well as in anti-CD3/CD28 driven T cell expansion culture.
CellsPBMC were obtained from normal donors in our study protocol. The protocol for collection of PBMC has been reviewed and approved by an accredited independent review board (IRB) and participants have given their informed consent for use of their cells in research. B lymphoblastoid cells used in some experiments were derived from these PBMC as were antigen-speci� c T cells.
Recall Antigen AssayPBMC are added to U-bottom 96 well plates at 250,000 cells per well in a 100 μL volume. The antigens are prepared at 2x the desired � nal concentration and added to the wells at 100 μL. Tetanus toxoid and CMV antigens are from our catalog and used at 1 μg/mL, PHA was purchased from Sigma and used at 1 μg/mL and the lipopolysaccharide (LPS) was also purchased from Sigma and used at a � nal concentration of 100 ng/mL. Cultures of PBMC and antigen were incubated at 37˚C, 5% CO2 and after 4 days culture medium was collected for cytokine analysis.
Cytokine AnalysisCytokines were analyzed using a U-Plex kit from Meso Scale Discovery. Interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) were routinely measured in the recall antigen assay.
Antigen-Speci� c Proliferation AssayTetanus toxoid-speci� c T cells were used to evaluate the e� ect of media formulations on antigen speci� c proliferation. Autologous B-LCL were inactivated by incubation with 50 μg/mL of mitomycin C for 45 minutes at 37˚C. After this incubation, mitomycin C was removed by 3 washes with PBS. The inactivated cells were plated at 20,000 cells per well in a U-bottom 96 well plate. Peptide antigen was added to indicated concentrations and tetanus toxoid-speci� c T cells were added at 20,000 cells per well. The cultures were incubated for 4 days and proliferation was measured by addition of CellTiter Glo™.
Mixed Lymphocyte ReactionB-lymphoblastoid cells were used to stimulate PBMC in a mixed lymphocyte reaction. The batch of PBMC and B-LCL were kept constant for this series of experiments. Proliferation of the B-LCL was prevented by incubation of the cells with 50 μg/mL of mitomycin C (Sigma) for 45 minutes at 37˚C. After this incubation, mitomycin was removed by 3 washes with PBS and following the last wash the cells were suspended in the desired medium. These APC were added at a range of cell numbers per well in a 100 μL volume. PBMC were added at 20,000 cells per well in a volume of 100 μL of the appropriate medium. The culture was incubated for 5 days and proliferation was measured by the addition of CellTiter Glo™.
Stimulation with Anti-CD3 and Anti-CD28PBMC were stimulated with ImmunoCult™, an anti-CD3, anti-CD28 conjugate from Stem Cell Technology. This reagent was used at a range of concentrations in test medium. PBMC were added at 100,000 cells per well in 96 well plates or cultured in bulk for � ow cytometric analysis.
Flow Cytometric AnalysisFluorescent antibodies were purchased from BioLegend and used at the volumes recommended by the manufacturer. Antibodies were incubated with cells for 15 minutes in the dark, washed once and data was acquired using a FACScan cytometer. Further analysis was performed using FCS Express (DeNovo Software).
ResultsClassical media supplemented with several de� ned components can support primary in vitro responses as measured by cytokine production. Sustained T cell proliferation demanded additional supplementation and revealed greater di� erences between media. One representative data from these studies is included in this abstract. This experiment demonstrates the e� ect of human AB serum (HS) or fetal bovine serum (FBS) added to the culture medium X-VIVO™ 15 (Lonza, Walkersville, MD). At low peptide concentrations (3 and 10 ng/mL), the presence of HS and FBS inhibits T cell proliferation compared to X-VIVO™ 15 alone.
Conclusions• Supplementation of media with human serum does not support optimal
cytokine production in the recall antigen assay.• Minimal supplementation of DME/F12 is su� cient to support cytokine
production and proliferation in short-term (5-day) assays.• Supplementation of culture medium with serum supports growth but
requires higher concentrations of antigen.• Choice of culture medium in� uences not only growth rate but phenotype
and function of resulting T cells.
Figure 1Each panel shows a di� erent donor PBMC tested for antigen response and the IFNγ produced in DME/F12
supplemented with either fetal bovine serum or human AB serum at 10% or using X-VIVO™ 15.
Figure 2Each panel depicts data from a di� erent PBMC donor. Cells were tested for recall antigen response using either DME/F12 supplemented with ITS+Premix and human serum albumin or X-VIVO™ 15.
Figure 3An MLR conducted in the presence of three media formulations. X-VIVO™ 15 supplemented with
human serum and DME/F12 with ITS+Premix and human albumin supported greater cell growth at low concentrations of stimulating B-LCL.
Figure 4Tetanus toxoid-speci� c T cells were incubated with APC and the indicated concentrations of peptide antigen.
After 4 days incubation at 37˚C the proliferation was measured by addition of CellTiter Glo™.
Figure 6Proliferation stimulated by anti-CD3+anti-CD28 using a second donor’s PBMC. ImmunoCult™
was added to culture medium at 6.25 μL per mL and a series of 1:2 dilutions as well as a control without ImmunoCult™. after 4 days culture the proliferation was measured using CellTiter Glo™
Figure 7Phenotypic analysis of T cells following stimulation with anti-CD3+anti-CD28. PBMC were stimulated using ImmunoCult™ in 3 di� erent
serum-free medium formulations. IL-2 was added after the initial 24 hours and after 6 more days the cells were collected and analyzed for expression of CD4, CD8, CD45RA, CD45RO, and CD62L. X-VIVO™ 15 in row 1, Medium A in row 2 and Medium B in row 3.
Figure 5Proliferation stimulated by anti-CD3+anti-CD28. ImmunoCult™ was added to culture
medium at 6.25 μL per mL and a series of 1:2 dilutions as well as a control without ImmunoCult™. After 4 days culture the proliferation was measured using CellTiter Glo™.
