Human HLA-A*0201 monocytes were obtained from two sources, purified either by counterflow centrifugal elutriation (Advanced Biotechnologies) or negative magnetic cell sorting (Biological Specialty Corporation). Similar results were obtained with both sources. The monocytes were aliquoted into vials and cryopreserved in 90% FBS, 10% DMSO. For each experiment, one or more vials were thawed and washed in C10 medium: RPMI 1640 with 10% FBS, 2 mM l-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1× nonessential amino acids, 50 μM 2-ME, and Primocin (InvivoGen). Unless otherwise indicated, medium components were from HyClone; low endotoxin products were chosen where available. A total of 4-5 × 106 monocytes were cultured per well of a 6-well plate in 2.5 ml C10 medium supplemented with 1000 U/ml each of IL-4 and GM-CSF (C10GF; cytokines from R & D Systems). After 2 and 4 days of culture, each well was topped up with 0.5 ml C10GF; after 6 days of culture, floating and loosely adherent immature monocyte-derived DCs were harvested by gentle resuspension.
Vials of a human CD8+ T cell line specifically recognizing the peptide NLVPMVATV in the context of HLA-A*0201 were purchased from Pro-Immune. Each vial was thawed and cultured overnight in RPMI 1640 with 10% FBS and 5 ng/ml IL-2 and used the next day.
Yeast surface display
Plasmids for yeast surface display were based on pCT-CON (21
) and were transformed into EBY100 (20
), a strain that expresses Aga1p under galactose induction, using the Frozen EZ Yeast Transformation II kit (Zymo Research). Yeast colonies were cultured to mid-log phase at 30°C in selective SD-CAA medium (2% dextrose, 0.67% yeast nitrogen base, 0.5% casamino acids, 0.1 M sodium phosphate, pH 6.0) and then induced in SG-CAA (SD-CAA with galactose replacing dextrose) for 48 h at 20°C. Single copies of some expression cassettes were integrated into the EBY100 yeast chromosome using the integrating shuttle vector pRS304 (22
). The resulting yeast strains were grown up in rich YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) and induced in YPG (1% yeast extract, 2% peptone, and 2% galactose) for 36 h at 20°C. Yeast medium nitrogen sources were obtained from BD Biosciences. Surface display levels were measured by flow cytometry with chicken α
(Invitrogen) or 9e10 mAb (Covance). The number of copies per yeast cell was estimated by comparison with Quantum Simply Cellular beads (Bangs Laboratories).
After 6 days of differentiation, immature monocyte-derived DCs were seeded in 96-well round-bottom plates at 1 or 2 × 105 cells in 200 μl C10GF per well. Appropriate numbers of yeast cells (measured by OD at 600 nm with 1 OD ≈ 107/ml) were rendered nonviable by UV-irradiation (2 × 1000 J/m2 in a Stratalinker; Stratagene), pelleted by centrifugation, and added to the DCs. For inhibition experiments, DCs were preincubated with Z-FL-COCHO (10 μM; Calbiochem), lactacystin (5 μM; Calbiochem), or chloroquine (25 μM) for 1 h before yeast samples were introduced. Twenty-four hours later, half the medium was replaced with a T cell suspension, with 0.7-1 × 105 T cells per well. Following 4 h of coculture, the contents of each well were transferred to tubes for labeling with Miltenyi’s IFN-γ secretion assay kit according to the recommended protocol. Briefly, cells were labeled with a bispecific Ab that captures secreted IFN-γ on the cell surface during a 45 min incubation period in medium at 37°C, and then labeled on ice for 30 min with α-CD8-FITC (BD Biosciences) and α-IFN-γ-PE (Miltenyi Biotech). In experiments involving FITC-conjugated yeast, α-CD8-Alexa Fluor 647 (BD Biosciences) was substituted. The percentage of CD8+ cells that were IFN-γ+ was determined by flow cytometry (Coulter Epics XL or BD FACSCalibur). The cut-off PE fluorescence was set for each experiment such that ~0.5% of T cells were IFN-γ+ in a negative control sample (no yeast or peptide). The positive control with 1 μM of the extended peptide ARNLVPMVATVQGQN (synthesized by GenScript) resulted in 45-70% IFN-γ+ T cells.
Yeast intracellular expression
Intracellular expression of the same fusion protein as is expressed by surface display was achieved by deleting the signal peptide of Aga2p, followed by transformation into BJ5464α (Yeast Genetic Stock Center). BJ5464α is isogenic to the parent strain of EBY100 and lacks the galactose-inducible Aga1p gene. The resulting colonies were grown up in SDCAA and induced in SG-CAA for 12 h at 30°C.
Slot blot comparison of Ag levels
A total of 6 OD.ml of each yeast culture was washed with PBS, resuspended in 300 μl 25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Soltec Ventures) in PBS, and incubated for on ice for 30 min. The proteins released into solution by the reducing agent were pooled with those from a second 30-min extraction with 25 mM TCEP. The yeast pellets were then washed with spheroplast buffer (50 mM Tris-HCl, pH 7.5, 1.4 M sorbitol, and 40 mM 2-ME), incubated with 2.4 U Zymolyase (Zymo Research) in 120 μl spheroplast buffer containing a protease inhibitor mixture (Roche) for 15 min at 37°C, and boiled in 2% SDS for 5 min. The protein extracts were blotted onto nitrocellulose membrane with a slot-blotting apparatus (Bio-Rad). The membrane was blocked with 5% milk powder, incubated with 9e10 ascites fluid (Covance) followed by goat α-mouse-HRP (Pierce), developed with SuperSignal West Dura substrate (Pierce), and imaged on a FluorS Imager (Bio-Rad).
Surface display Ag dose normalization
In experiments where different linkers were used to surface-display Ag, several cultures of each yeast sample were induced, and cultures with mean Ag levels within 10% of each other were selected to minimize the effect of variable Ag dose on cross-presentation. However, the variability in expression level across the panel of initial constructs (deleted linker, unchanged, and C1-5) was too high for this approach to be satisfactory. Therefore, each yeast sample was mixed with the appropriate amount of EBY100 yeast to normalize the Ag dose while maintaining the 20:1 ratio of yeast to DCs.
Measuring linker susceptibility to Cathepsin S
A total of 0.2 OD.ml of each yeast sample was washed and incubated with the indicated amounts of recombinant human Cathepsin S (CatS; Calbiochem) in 100 μl PBS at 37°C. The yeast samples were washed and labeled with 12CA5 mAb (α-HA; Roche) and chicken α-c-myc, followed by goat α-mouse-PE (Sigma-Aldrich) and goat α-chicken-Alexa Fluor 488 (Invitrogen). The mean c-myc fluorescence of the HA+ population was compared against that of yeast samples that had not been treated with CatS.
DCs (2 × 105/well) were seeded in 96-well round-bottom plates, with separate plates for each time point. After adding the yeast samples (5 × 105/well), the plates were immediately centrifuged briefly (200 × g, 1 min) to settle the yeast and were returned to the incubator. At each time point, a plate was placed on ice and 90% of the medium in each well was replaced with cold radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich). The well contents were moved to tubes, vortexed to promote cell lysis, and centrifuged to pellet the released yeast. The yeast was washed with RIPA buffer and PBS with 0.1% BSA before being labeled for HA and c-myc epitopes as described above.
Fluorescein-binding single-chain variable fragment (scFvs)
The fluorescein-binding scFvs used here were products of directed evolution for decreased dissociation rate using yeast surface display (23
). These scFvs were subcloned into pRS316-based plasmids with an improved α
mating factor pre-pro sequence (J. A. Rakestraw, A. Piatesi, and K. D. Wittrup, unpublished observations). Codons encoding the extended peptide ARNLVPMVATVQGQN were inserted between the C terminus and the c-myc
epitope. The resulting constructs were transformed into the protein disulfide isomerase-overexpressing yeast strain YVH10 (24
) together with a dummy plasmid bearing the trp
nutritional marker. Transformants were grown up in SD-CAA and induced in YPG containing 0.1 M sodium phosphate, pH 6.0 for 3 days at 20°C. The culture supernatants containing ~10 mg/L of scFv-Ag were adjusted to pH 7.4 and dialyzed against PBS.
UV-irradiated BJ5654α yeast cells were washed three times in 0.4 M sodium carbonate, pH 8.4, and resuspended in 10 μl/OD.ml of a freshly prepared 0.15 mg/μl solution of fluorescein-PEG-NHS (MW 5000; Nektar) in sodium carbonate buffer. The reaction was allowed to proceed for 30 min at room temperature, after which the yeast was washed six times with PBS containing 0.1% BSA. Fluorescein-conjugated yeast was loaded with Ag by incubation with scFv-Ag culture supernatants (1 ml/107 yeast) for 1 h on ice. Flow cytometry analysis (c-myc labeling) of the loaded yeast showed that the Ag levels mediated by 4M2.3, 4M3.12, and 4M4.5 were within ~5% of each other, but the level of 4M5.3-Ag was ~15% higher. Labeling fluorescein-conjugated yeast with 4M5.3 fusion protein for 30 min followed by 30 min, 37°C incubation in pH 5.4 PBS containing 0.1% BSA and 1 μM fluorescein-biotin resulted in a final Ag level comparable to that mediated by the other scFvs. This method of Ag level normalization was performed for the cross-presentation assay. In addition, to reduce Ag loss before phagocytosis, the plate was centrifuged (at 200 × g for 1 min) immediately after addition of the yeast to the DCs.
The mathematical model consisted of the following equations describing the amounts of yeast-bound Ag (Ab), free Ag within the phagosome (Af), and cytoplasmic Ag (Ac) relative to the initial amount:
For 0 < t
, where tpre
is the time before phagocytosis
, where tmat
is the time taken for a phagolysosome formation
≤ 24 h
These equations were solved in Matlab with the initial conditions Ab
= 1, Af
= 0. For a given set of parameter values, the final value of Ac
(at 24 h) was determined for a wide range of koff
values. Ten logarithmically spaced values spanning three orders of magnitude were tested for each of the following parameters: c1
(0.1 - 10), c2
(10 - 1000), c3
(10 - 1000), kesc
(0.01 - 1 min-1
), and kdeg
(0.01 - 1 min-1
). An optimal t1/2
was found to exist between 10 and 105
min for all 105
possible combinations of parameter values, which should span all reasonable biological values. The time parameters, tpre
, were fixed at 30 min and 20 min, respectively. The former value is an estimate but is not a critical value because it is always multiplied by c1
, which was varied widely. The latter value was deemed reasonable based on the postphagocytosis time course analysis we performed.