BackgroundThe ability to expand virus- or tumor-specific T cells without damaging their functional capabilities is critical for success adoptive transfer immunotherapy of patients with opportunistic infection or tumor. Careful comparisons can help identify expansion methods better suited for particular clinical settings and identify recurrent deficiencies requiring new innovation. MethodsWe compared the efficacy of magnetic beads coated with anti-CD3 and anti-CD28 (anti-CD3/CD28 beads), and soluble anti-CD3 plus mixed mononuclear cells (designated a rapid expansion protocol or REP) in expanding normal human T cells. ResultsBoth anti-CD3/CD28 beads and soluble anti-CD3 promoted extensive expansion. Beads stimulated greater CD4 cell growth (geometric mean of 56- versus 27-fold (p. With advances in the methods for selecting and manipulating T cells there is increasing interest in the adoptive transfer of bioactive T cells as a treatment for infections and cancer.
- Dynabeads™ Mouse T-activator Cd3/cd28 For T-cell Expansion And Activation
- Dynabeads Human T - Activator Cd3/cd28 Free
This approach has been used successfully to transfer antiviral immunity after stem cell transplantation , and is under active investigation in treating malignancy. Antigen-specific T cells suitable for transfer can only be retrieved from blood or tissue sites in relatively small numbers, consequently they usually are expanded specifically or nonspecifically prior to transfer. Such ex vivo manipulations, however, potentially can damage T cell homing, proliferation, and survival after infusion ,.
Given this risk, the choice of methods may have important implications for clinical efficacy.Antibodies against CD3 are a central element in many T cell proliferation protocols. Immobilized on a surface, anti-CD3 delivers a strong proliferative signal through the T cell receptor complex (signal 1) but in the absence of additional costimulatory signals (signal 2), the resulting proliferation is often followed by premature T cell apoptosis or anergy. By immobilizing anti-CD3 and anti-CD28 to simultaneously deliver signal 1 and a costimulatory signal 2, proliferation can be increased without provoking early cell death. The expanding cells also demonstrate enhanced ability to release cytokines and lyse targets cells in an MHC unrestricted manner. Consequently, magnetic beads coated with anti-CD3 and anti-CD28 (anti-CD3/CD28 beads) have proved a convenient reagent for expansion which has been used experimentally to boost T cell immunity in immunosuppressed cancer patients – and enhance the anti-tumor effect of donor lymphocyte infusions after allotransplantation. These studies have established that beads can be used to expand functional T cells, and that some of these cells can persist in vivo postinfusion.While these results are encouraging, the bead expansion technique has limitations. Ex vivo expansion stimulates the generation of effector T cells with increased lytic and cytokine producing capability , but the capacity of these cells for additional homing and proliferation after infusion is uncertain.
While CD4 cells respond very well to anti-CD3/CD28 stimulation, CD8 cells proliferate less extensively with an increased rate of apoptosis. Given the importance of CD8 T cells in the anti-tumor response, this is a significant concern.One commonly used alternative approach for stimulating proliferation is the incubation of T cells with soluble anti-CD3 antibody in the presence of Fc receptor bearing accessory cells –, an approach designated the 'Rapid Expansion Protocol' (REP). Antibody 'presented' to T cells in this manner clearly generates a more effective proliferative signal than soluble anti-CD3 alone or anti-CD3 immobilized on a plastic surface.
This presumably reflects the dual benefit of more extensive anti-CD3-T cell receptor crosslinking on a surface, and the costimulation provided by cell-cell interaction between T cells and Fc receptor positive accessory cells such as monocytes which constitutively express CD80 , CD86 , and CD137. These complex interactions in some respects mimic events during physiologic antigen presentation.
Given its efficacy, this approach has been used extensively for expansion of T cell clones and lines for in vitro and clinical adoptive transfer studies –,.To gain further insight into the similarities and differences between the T cell responses produced by beads and REP, the current studies critically compare their impact on T cell survival, proliferation, and phenotype. While both beads and anti-CD3 are effective in expanding T cells, our studies demonstrate substantial differences in their impact on CD8 cells that merit consideration in situations where preservation of the CD8 T cell response in important. Antibodies, beads, and chemicalsCD45RA/FITC, CD45RA/PE, CD57/FITC, CD28/PE, CD4/PerCP, CD8/PerCP, CD27/APC, brefeldin A, anti-IFNγ/FITC, anti-TNFα/PE, 7-Amino-Actinomycin D (7-AAD), and appropriate isotype controls were purchased from BD Biosciences. Anti-human CCR7-phycoerythrin was obtained from R&D Systems. Biotinylated anti-CD3 and anti-CD28 antibodies were purchased from eBioscience.
Anti-CD3 (Orthoclone OKT3) was provided by Stephen Migueles (NIAID, Bethesda, MD). Flow-Check Fluorospheres were purchased from Beckman Coulter. Streptavidin-labeled Dynabeads (M280) and CD3/CD28 T cell expander beads were obtained from Invitrogen. Carboxyfluorescein succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR) and recombinant human IL-2 was purchased from PeproTech (Rocky Hill NJ). Preparation of anti-CD3/CD28 beadsTo prepare antibody-coated beads of varying composition, streptavidin-labeled beads were coated with varying mixtures of biotinylated anti-CD3 and anti-CD28 antibodies. To this end, streptavidin-M280 beads were washed once with sterile PBS/BSA and resuspended at 10-50 millions beads/ml. Preliminary dose response studies, using FITC-labeled anti-mouse IgG and flow cytometry to monitor biotinylated antibody binding to beads, established that beads were saturated by 100 ng of biotinylated antibody/million beads.
Consequently this total immunoglobulin/bead ratio was routinely used for bead coating. To vary the ratio of antibody coating on beads equimolar solutions of anti-CD3 and anti-CD28 were mixed at 1:0, 1:5, 1:10, 1:20, 1:40, 1:80, 2 1:160, and 0:1 ratios. Control beads were coated with biotinylated IgG1 isotype. Coating was performed on a rotator stand at room temperature for 2-3 hours. Beads were then washed two times with filtered PBS/BSA, once with complete medium, and then resuspended in RPMI 1640 complete medium.
Antibody coating was performed as needed, but preliminary studies established that beads could be stored 4°C for at least one week without any change in potency. In selected studies, T cells were also stimulated using commercially prepared anti-CD3 and anti-CD28 coated beads (CD3/CD28 T cell Expander, Invitrogen). Flow cytometryFlow cytometry was performed using a 4-color Facscalibur (BD biosciences). The standard phenotypic analysis was performed at different time point using antibody panels as described in the results.
The flow data was analyzed using Flow-Jo software. Human leukocyte acquisition and purificationNormal healthy donors gave informed consent to blood donation or leukapheresis procedures performed as specified in clinical protocols approved by the Institutional Review Board of the Clinical Center of the National Institutes of Health. Mixed mononuclear cells (MNCs) obtained by leukepheresis or prepared from buffy coats using Ficoll-Paque density gradient centrifugation, were cryopreserved, and thawed as previously described.To prepare T cell subsets for selected experiments, MNCs from freshly collected buffy coat cells were purified by negative selection using CD8 + Memory T Cell Isolation and CD8 + Naive T Cell Isolation Kits purchased from Miltenyi Biotech.
Monitoring T cell division and early expansion using CFSE labeled cellsTo monitor cells division and expansion during the early days after stimulation, cells were CFSE-labeled and monitored using methods described by Hawkins, et al. In brief, to label cells, 2-5 × 10 7 mixed mononuclear cells or cultured T cells maintained in RPMI 1640 containing 10% fetal calf serum plus 100 unit/ml penicillin, 100 ug/ml streptomycin, and 2 mM glutamine (RPMI/FCS) were incubated with 2 μM CFSE at 37°C for 10 min. Cells were then washed three times to remove unbound CFSE, resuspended in fresh RPMI/FCS, and incubated overnight. Labeled cells were then distributed (50,000/well) in a 96 well round bottom plate in wells also containing anti-CD3/CD28 coated beads (three beads/cell), anti-CD3 (30 ng/ml), or no additional stimulator. When using anti-CD3 to re-treat previously stimulated cells, 100,000 irradiated MNCs (accessory cell:responder ratio of 2:1) were also added as a source of Fc receptor positive accessory cells suitable for 'presentation' of anti-CD3 to T cells. Fresh cells received IL2 (50 U/ml) on day 2. Restimulated cells were maintained with 50 U/ml of IL2 from day 1.
Dynabeads™ Mouse T-activator Cd3/cd28 For T-cell Expansion And Activation
Wells were fed with additional medium containing IL2 at day 4 or 5 and every 2-3 days thereafter. With continued growth, the contents of wells were diluted 4 fold into new wells with fresh medium and IL2 as needed to prevent overcrowding.To monitor cell growth, at selected time points after stimulation, 10,000 calibrator beads/well (Flow-Check Fluorospheres, Beckman Coulter, CA) were added to wells along with PE labeled anti-CD4 or anti-CD8 antibodies. The contents of the well were mixed, incubated for one half hour, and then washed twice with PBS/BSA (1%) before addition of 7-AAD to exclude dead cells in flow cytometry analysis. All measurements of cell composition and number were performed in quadruplicate.The absolute number of cells per well at each time point was calculated based on the number of calibrator beads and the number of viable cells detected per well by flow cytometry using the formula:The proportion of cells undergoing 0-6 divisions could then be quantitated based on the pattern of CFSE fluorescence using Flo-Jo software, and the total number of viable cells per well. Bulk stimulation of T cells in vitroTo monitor cell phenotype and cell expansion over a 3 week period, fresh MNC or cells expanded previously using anti-CD3/CD28 beads or anti-CD3 were cultured in 12 well plates (5 × 10 6 cells in 2 ml/well) with anti-CD3/CD28 beads (three beads/cell), anti-CD3 (30 ng/ml), or medium alone. Previously expanded cells restimulated with anti-CD3 routinely also received irradiated autologous MNC (2 cells/responder) as a source of Fc receptor positive accessory cells.
Medium containing recombinant human IL2 (50 units/ml) was added to freshly cultured cells at day 2 and to restimulated cells throughout the process. Beads were removed using a magnet on day 7 post stimulation. Cell counts of freshly stimulated cells were monitored at least twice weekly and cultures were fed every other day with fresh RPMI/FCS and IL2, and transferred to flasks or frozen as needed to maintain cell numbers between 0.75 and 2 × 10 6/ml. Because of the presence of irradiated autologous feeder cells in REP treated cells, viable cell counts were not used to monitor cell growth in restimulated cultures until after day 7 by which time no more viable irradiated cells were present. Measurement of Intracytoplasmic cytokine ProductionT cells harvested 14 days after stimulation with anti-CD3/CD28 beads or soluble anti-CD3 were treated for 4 hours with phorbol myristate acetate (PMA, 35 nM) and the calcium ionophore A23187 (0.5 μM) or with medium alone in the presence of brefeldin (Golgiplug, BD Bioscience). Cells were then incubated with anti-CD8 PerCP and CD27 APC for 30 minutes, fixed and permeabilized using Cytofix/cytoperm solution (BD Bioscience) as recommended by the manufacturer, and stained intracellularly using anti-IFNγ FITC and antiTNFα PE.
Dynabeads Human T - Activator Cd3/cd28 Free
Duplicate samples were stained with an appropriate isotype control. Cytokine expression in treated and control cells was then assessed using flow cytometry. StatisticsPaired t-tests and nonparametric 2-tail Wilcoxon matched pairs tests were performed using Graphpad Prism Software. Time course for T cell response to stimulationThe CFSE-labeled CD4 and CD8 T cells in MNCs began dividing 40-50 hours after stimulation with anti-CD3/CD28 beads or soluble anti-CD3. CD8 cells divided slightly more rapidly than CD4 cells, and there were no consistent differences in early response to the two stimuli (Figure and ).
The number of viable cells at hour 40 (just before proliferation began) was similar in unstimulated, bead-stimulated, and anti-CD3-stimulated wells indicating neither stimulus caused extensive early activation-induced cell death (AICD) (Figure and ). Consistent with the timing of cell division, expansion in cell number in response to either stimulus was delayed until 50-60 hours after stimulation. To compare the expansion produced by anti-CD3/CD28 beads and anti-CD3, we monitored changes in cell number in bulk cultures over a 21-day period in 11 separate studies (Figure ) and noted several persistent trends. Consistent with the more rapid rate of early cell division noted in Figures and, CD8 cells expanded more rapidly than CD4 cells in response to either stimulus. Focusing on CD4 cells, this subset expanded more rapidly in response to beads than anti-CD3 (Figure ) and this difference was statistically significant at days 7, 14, and 21. There was a trend to more rapid expansion of CD8 cells in response to anti-CD3, particularly at days 7 and 14, but these differences did not achieve statistical significance (Figure ). Consistent with these reciprocal trends in CD4 and CD8 expansion, cultures stimulated with anti-CD3 accumulated a significantly higher proportion of CD8 cells at all three time points than matched bead-treated cultures (Table ).
CD45RA expression on cultured T cells is a typical characteristic of terminally differentiated effector T cells, but unlike conventional effectors , the CD45RA+ CD8 T cells noted after anti-CD3 treatment were consistently CD27+ (Figure ) and CD57- (data not shown). Effector T cells typically produce intracellular cytokines within 4 hours after stimulation in vitro , but the CD27+ anti-CD3-expanded CD8 cells (in contrast with the CD27- cells in the same preparation) produced little intracellular IFNγ or TNFα in response to PMA/A23187 stimulation (Figure ). T cell response to restimulationOn occasion, the number of cells generated by one cycle of T cell expansion may be insufficient for the desired purpose, and further expansion would be desirable. To compare impact of restimulation, cells previously expanded using anti-CD3/CD28 beads were CFSE-labeled 6 to 63 days later, and restimulated with fresh anti-CD3/CD28 beads, anti-CD3 plus irradiated autologous MNCs, or, as a control, maintained in medium plus IL2 alone. Like fresh cells, restimulated cells showed an increased rate of division 40-50 hours post stimulation (Figure and ). Unlike fresh cells (Figure.