Journal of Immunological Methods 220 Ž1998. 179–188
A yeast surface display system for the discovery of ligands that
trigger cell activation
Bryan K. Cho a , Michele C. Kieke a , Eric T. Boder b, K. Dane Wittrup b,
David M. Kranz a,)
Department of Biochemistry, UniÕersity of Illinois, 600 S. Mathews, Urbana, IL 61801, USA
Department of Chemical Engineering, UniÕersity of Illinois, 600 S. Mathews, Urbana, IL 61801, USA
Received 1 August 1998; accepted 19 August 1998
Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell
surface. For example, T cells are typically activated when the T cell receptor ŽTCR. and its associated costimulatory
molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast
expressing high cell-surface levels of a TCR ligand Ža recombinant antibody to the TCR Vb domain. were shown to act as
‘pseudo’ antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by
downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant
yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may
be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors
and thereby trigger specific cellular responses. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Yeast display; scFv; T cell activation; Antigen presentation; Biological screening
1. Introduction Fv antibody domains ŽscFv. 2 that bind to tumor-as-
sociate antigens ŽWinter et al., 1994.. It would be
Display systems have typically been used to dis- advantageous to develop a system that not only
cover novel molecules that bind to a specific ligand. identifies polypeptides that bind to ligands, but that
For instance, phage display has been used in lieu of also elicit defined cellular responses including apop-
B cell hybridoma technology to generate single-chain tosis, gene expression, cytokinesis, secretion, or en-
try into the cell cycle. Many of these responses are
the culmination of multiple amplification events that
follow ligation of cell surface receptors. By exploit-
Abbreviations: scFv, single-chain Fv; TCR, T cell receptor; ing these amplification processes, it is possible, in
RAG, recombination activating gene; AGA-HA, Aga2-hemag- principle, to greatly enhance the sensitivity with
glutinin; 7AAD, 7-aminoactinomycin D; FITC, 5-aminofluorescein
isothiocyanate; MHC, major histocompatibility complex
which displayed polypeptides are selected.
Corresponding author. Tel.: q1-217-244-2821; Fax: q1-217- To activate many cellular responses, high valency
244-5858; E-mail: email@example.com interactions between receptors on one cell with com-
0022-1759r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 1 5 8 - 6
180 B.K. Cho et al.r Journal of Immunological Methods 220 (1998) 179–188
plementary molecules on another cell are important cells even in the presence of a great excess of
Žreviewed in Metzger, 1992; Singer, 1992.. For ex- unmodified yeast cells, suggesting that this system
ample, stimulation of proliferative activity of a rest- may be suitable for screening ‘libraries’ for bioactive
ing T cell requires multiple interactions at the inter- peptides or proteins ŽPetsko, 1996..
face of the T cell and antigen presenting cell
ŽJorgensen et al., 1992; Allison and Krummel, 1995..
These interactions not only involve different proteins 2. Materials and methods
on the T cell surface and their respective polypeptide
ligands on the antigen presenting cell, but they typi-
cally require multivalent clustering of the T cell
receptor ŽValitutti et al., 1995; Scharenberg and
Kinet, 1996; Reich et al., 1997.. Because of this To produce the 2C TCRrrecombination activat-
circumstance, phage display systems which express ing gene ŽRAG.-1yry strain ŽH-2 b . of mice, 2C
few molecules per virion have a limited capacity to TCR transgenic mice were bred with RAG-1 knock-
induce many cellular responses. Although a recent out mice and the F1 generation was backcrossed to
report describes a modification of phage display to RAG-1yry mice ŽManning et al., 1997.. 2C
detect receptor activation through dimerized phage TCRrRAGyry mice contain ) 95% CD8q1B2q
particles ŽZaccolo et al., 1997., dimerization is not peripheral T cells and lack B cells and T cells of any
always sufficient. other specificity. Mice were maintained in barrier
In order to design a system that would allow for cages at the University of Illinois animal care facil-
rapid screening of polypeptides that trigger cell acti- ity. In the studies presented here, spleens from 2C
vation, we have explored the use of yeast cell surface TCRrRAGyry mice were isolated, red blood cells
display. In contrast to other display systems, yeast were lysed, and splenocytes were used without fur-
offers the advantage of protein folding pathways and ther enrichment.
codon usage that are closely related to mammalian
cells, thereby eliminating library biases that might be 2.2. Generation of scFÕ-yeast
encountered in phage or bacterial display. Yeast also
expresses as many as 10 5 fusion proteins per cell at The scFv-KJ16 gene ŽCho et al., 1995., including
the yeast surface ŽBoder and Wittrup, 1997., poten- a 3X c-myc epitope tag, was cloned as a downstream
tially allowing multivalent interactions to occur be- fusion to the yeast Aga2 gene in the yeast surface
tween the yeast and target cell. This system was first display vector pCT202 ŽBoder and Wittrup, 1997..
used to select for higher affinity antibody molecules This construct was transfected into the S. cereÕisiae
ŽscFv. against the hapten fluorescein ŽBoder and strain EBY100 Ž trp1 leu2 D1 his3D 200 pep4::HIS2
Wittrup, 1997.. We have also selected for higher prbD1.6R can1 GAL ŽBoder and Wittrup, 1997.. and
affinity scFv that recognize protein antigens such as these yeast were termed scFv-yeast. Yeast that only
the TCR from a library of scFv generated by random express the Aga2 gene linked to a hemagglutinin
mutagenesis ŽKieke et al., 1998.. In this report, a epitope tag Žtermed AGA-HA yeast. were prepared
model system is used to demonstrate that yeast are by transfecting EBY100 cells with pCT202, which
capable of inducing biological activity by virtue of does not contain a scFv gene. Individual colonies of
multivalently presented polypeptides that, as the transfected yeast were grown overnight in Trpy
monomers, do not trigger cell activation. Specifi- media containing glucose and harvested in log phase
cally, we express a single-chain Fv domain of an ŽOD600 s 0.5–1.5., then grown an additional ; 10
anti-Vb8 antibody ŽKJ16. on the surface of yeast, to 15 h in Trpy media containing galactose in order
and show that these yeast Žtermed scFv-yeast. stimu- to induce expression of the fusion protein gene,
late naive T cells to express ‘activation’ markers which was cloned behind a GAL1 promoter. Yeast
ŽCD69 and the IL-2 receptor ŽCD25.. and to down- were harvested by centrifugation and washed with
regulate TCR molecules on the responding T cell. sterile PBS Ž10 mM NaPO4 , 150 mM NaCl, pH 7.3.
We also demonstrate that scFv-yeast can activate T before use.
B.K. Cho et al.r Journal of Immunological Methods 220 (1998) 179–188 181
2.3. Flow cytometric analysis of scFÕ-KJ16 expres- vested, washed in PBS that contained 0.1% BSA and
sion 0.05% azide and stained with biotinylated clonotypic
antibody, 1B2 ŽKranz et al., 1984.. After washing,
Approximately 10 6 yeast cells Žone ml at one bound 1B2 and activation markers were detected
OD600 ; 10 7 cells. displaying either scFv-KJ16 or with a mixture of streptavidin: PE and FITC
Aga2-hemagglutinin ŽAGA-HA. were incubated with labeled-anti-CD69 or FITC labeled-anti-CD25 ŽIL-
a mouse anti-c-myc IgG antibody Ž1r100 dilution of 2R. antibody. The yeast: T cell mixture was ana-
9E10 ascites. and scTCR–biotin conjugate at ; 1.5 lyzed by flow cytometry, gating on 1B2-positive T
mM ŽSchodin et al., 1996. for 30 min on ice. The cells that were viable, based on exclusion of the vital
scTCR contained a Vb8 domain that is recognized dye 7-aminoactinomycin D Ži.e. PEq 7-AADy .. For
by the KJ16 antibody. Cells were washed, pelleted experiments monitoring the levels of surface TCR,
and resuspended in media containing fluorescein- biotinylated 1B2 antibody was detected with strepta-
labeled ŽFITC. goat anti-mouse IgG and streptavidin: vidin: PE and the yeast: T cell mixture was analyzed
phycoerythrin ŽPE. as secondary reagents. Yeast were by flow cytometry gating on T cells by light scatter
analyzed on a Coulter Epics XL flow cytometer at and cells that were 7AADy.
the Flow Cytometry Center of the University of
Illinois Biotechnology Center. From the fluorescence
intensities observed using anti-HA and anti-c-myc 3. Results and discussion
antibodies for scFv-KJ16-yeast in comparison with
other scFv-yeast systems where more quantitative 3.1. Induction and characterization of scFÕ-KJ16 on
assays were used ŽBoder and Wittrup, 1997; Kieke et the surface of yeast
al., 1998; and unpublished results., we estimated that
approximately 50,000 scFv-KJ16 molecules are ex- To explore the use of yeast cell surface display in
pressed per yeast cell. T cell activation, a scFv was expressed as a fusion
protein in which the scFv was linked to the C-
2.4. Preparation of KJ16 coupled latex microspheres terminus of Aga2p, an a mating type agglutinin
protein that binds to the corresponding a agglutinin
KJ16 IgG, purified as described by Cho et al. on the surface of a cells during yeast mating ŽBoder
Ž1995 ., was covalently coupled to 5 m m and Wittrup, 1997.. Since the agglutinins’ native
carboxylate-modified latex microspheres ŽBangs function involves external protein–protein recogni-
Laboratories, Indiana. using a two-step coupling pro- tion, the Aga2p molecules are positioned at the cell
cedure involving pre-activation with 1-Ž3-dimethyl- surface to allow for normal cell to cell interactions
aminopropyl.-3-ethylcarbodiimide, as recommended ŽLu et al., 1995.. The scFv derived from the anti-Vb8
by the manufacturer. KJ16 IgG was coupled in 0.1 antibody KJ16 binds to the TCR of a cytotoxic T
M borate buffer, pH 8.5 at a concentration of 1 lymphocyte ŽCTL. clone called 2C ŽRoehm et al.,
mgrml at room temperature overnight. Ethanolamine 1985; Cho et al., 1995.. The KJ16 scFv gene was
Ž5 mM. was used to mask uncoupled sites and cloned and linked to the 3X end of the Aga2 gene. A
microspheres were washed with sterile PBS prior to c-myc epitope tag was included at the C-terminus of
use in culture. As a control, an irrelevant monoclonal the Aga2rKJ16 scFv fusion, to allow surface levels
IgG Ž800E6. was coupled following the same condi- of scFv to be quantitated Žwith anti-c-myc antibody.
tions. independent of antigen binding ability. Aga2–scFv
fusion expression is induced by growth in galactose.
2.5. ActiÕation assays The scFv-yeast cells were examined by flow cy-
tometry to determine if the protein was expressed on
Splenocytes Ž10 5 . from 2C TCRrRAG-1y_y mice the cell surface and if so, whether they bound to
were incubated at 378C, 5% CO 2 with various num- TCR molecules. To detect the yeast surface protein,
bers of yeast cells that bear either scFv-KJ16 or a mouse antibody to the c-myc epitope was used
AGA-HA. After 20 h in culture, cells were har- together with a FITC-labeled secondary anti-mouse
182 B.K. Cho et al.r Journal of Immunological Methods 220 (1998) 179–188
IgG. To detect active scFv, the scFv-yeast cells were bated overnight with naive Vb8q T cells from 2C
incubated with biotinylated-soluble TCR containing TCRrRAG-1yry transgenic mice ŽManning et al.,
a Vb8 domain ŽSchodin et al., 1996., followed by 1997. ŽFig. 2A.. The fraction of T cells that express
phycoerythrin–streptavidin. Results were compared upregulated CD69 is dependent on the ratio of
with a negative control yeast strain that expressed KJ16rmicrospheres to T cells, whereas control mi-
only Aga2p ŽAGA-HA, Fig. 1. and yeast that ex- crospheres that contain an irrelevant IgG fail to
press an scFv domain from an antibody of unrelated activate cells even at ratios that are 100 times those
specificity Ždata not shown.. The scFv-yeast express- of the specific microspheres ŽFig. 2A..
ing a functional antibody domain on their surface Previous studies of scFv displayed on yeast have
bound both the anti-c-myc antibody and soluble shown that as many as 10 5 fusion protein molecules
Vb8q TCR. Binding of the soluble TCR was com- are expressed per cell ŽBoder and Wittrup, 1997.,
pletely inhibited by excess intact KJ16 antibody resulting in an antigen density considerably higher
Ždata not shown.. than the density of peptiderMHC complexes re-
quired for stimulating cytolytic activity ŽSykulev et
3.2. ActiÕation of 2C TCR r RAG1-1yry splenocytes al., 1996.. Furthermore, the average diameter of
with scFÕ-KJ16 yeast yeast Ž4–5 mm. is the optimal cell size for stimulat-
ing CTL responses ŽMescher, 1992.. To determine if
Soluble anti-Vb8 antibody ŽKJ16. or the scFv surface expressed scFv could activate naive T cells,
derived from this antibody are unable to induce T scFv-yeast or control yeast that only express the
cell activation ŽRojo and Janeway, 1988, and data Aga2 protein were incubated at various cell numbers
not shown.. However, like other anti-TCR antibodies with naive Vb8q T cells from the 2C TCRrRAG-
Že.g. Deeths and Mescher, 1997., KJ16 IgG pre- 1yry transgenic mice. After co-incubation of yeast
sented in multivalent form on five micron latex and resting T cells for 20 h, T cells were analyzed
microspheres can induce signals that result in T cell for the induction of the activation markers CD69 and
activation and upregulation of CD69, when incu- CD25 by flow cytometry. The clonotypic anti-TCR
Fig. 1. Yeast that display the Aga2rscFv-KJ16rc-myc fusion on their surface bind biotinylated-Vb8 scTCR. Yeast transfected with
Aga2rscFv-KJ16 or Aga2-hemagglutinin ŽAGA-HA. were incubated with anti-c-myc antibody Ž1r100 dilution of 9E10 ascites. and Vb8q
scTCR–biotin Ž; 1.5 mM. for 30 min on ice. Cells were washed, pelleted and resuspended in media containing FITC-labeled goat
anti-mouse IgG and streptavidin: PE as secondary reagents. Yeast were analyzed on a Coulter Epics XL flow cytometer at the Flow
Cytometry Center of the University of Illinois Biotechnology Center.
B.K. Cho et al.r Journal of Immunological Methods 220 (1998) 179–188 183
Fig. 2. Induction of T cell activation markers on naive splenocytes in the presence of KJ16rmicrospheres and scFv-yeast. Splenocytes Ž10 5 .
from 2C TCRrRAG-1y_y mice were cultured with ŽA. antibody-coupled microspheres or ŽB, C. yeast cells that bear either KJ16 scFv or
Aga2-hemagglutinin ŽAGA-HA. for 20 h at 378C, 5% CO 2 . Cells were harvested, washed, and stained with biotinylated clonotypic
antibody, 1B2. After washing, bound 1B2 and activation markers were detected with a mixture of streptavidin: PE and FITC
labeled-anti-CD69 ŽA,B. or FITC labeled-anti-CD25 ŽIL-2R. antibody ŽC.. The yeast: T cell mixture was analyzed by flow cytometry,
gating on 1B2-positive T cells that were viable, based on exclusion of the vital dye 7-aminoactinomycin D Ži.e. PEq 7-AADy .. For CD25
upregulation ŽC., results using a yeast to T cell ratio of 1:1 are shown. The percent of cells that were judged to be positive for CD69 Ži.e.
within the area of the cursor bar. are indicated in each panel.
184 B.K. Cho et al.r Journal of Immunological Methods 220 (1998) 179–188
antibody 1B2 was used in double staining to analyze able to completely inhibit the induction of the activa-
only 2C Vb8q T cells ŽKranz et al., 1984.. Both tion markers ŽFig. 3..
CD69 ŽFig. 2B. and CD25 ŽFig. 2C. were upregu- Another process that occurs during TCR-induced
lated on T cells exposed to the scFv-yeast. The activation of T cells is the down-regulation of sur-
amount of upregulation correlated with the number face TCR after serial triggering with ligands
of yeast used as activators and could be detected at Žpeptide–MHC complexes. of the appropriate affin-
yeast to T cell ratios as low as 0.1 to 1 ŽFig. 2B., ity ŽValitutti et al., 1995.. The clonotypic anti-TCR
similar to antibody-coupled microspheres ŽFig. 2A.. antibody, 1B2, and the anti-Vb8 antibody, KJ16,
This ratio is considerably lower than the 1:1 ratio bind to different and non-overlapping epitopes on the
typically used when transfected insect cells have TCR of 2C T cells ŽSchodin et al., 1996.. Therefore,
served as ‘antigen presenting cells’ ŽCai et al., 1996; the 1B2 antibody could be used to evaluate changes
Sun et al., 1996., even though the binding affinity of in the levels of TCR molecules on 2C T cells that
the KJ16 antibody Ž K D ; 120 nM; Cho et al., 1995. interacted with the scFv-yeast. As shown in Fig. 4,
is nearly the same as the peptiderMHC ligand, yeast that express cell surface scFv-KJ16 induced the
QL9rLd Ž K D ; 100 nM; Sykulev et al., 1994. used
in the latter studies.
The lack of T cell activation in the presence of the
control yeast ŽFig. 2B. indicated that yeast, like
insect cells ŽCai et al., 1996; Sun et al., 1996.,
provide a relatively inert surface from which
molecules important for eliciting a specific T cell
response can be characterized. However, unlike in-
sect cells, yeast are compatible with the optimal
temperature for mammalian cell culture Ž378C.. This
feature is particularly important for studies with im-
mune cells, as incubation of insect cells at 378C
leads to cell lysis and release of DNA that can result
in activation and thus may complicate the analyses
ŽCai et al., 1996; Sun et al., 1996.. For instance,
previous studies showed that DNA derived from
bacteria, yeast, and insect cells can induce spleen
cell activation, as determined by proliferation assays
ŽSun et al., 1996; Pisetsky, 1996.. Similarly, bacte-
rial display systems that have been developed for
combinatorial library screening ŽGeorgiou et al.,
1997. are also likely to trigger nonspecific inflamma-
tory responses. In addition, the lipopolysaccharide
layer could introduce a steric barrier that would
inhibit cell to cell contacts.
In the present study, three lines of evidence
demonstrate that the T cell activation induced by the
scFv-yeast does not involve DNA-mediated stimula-
tion. First, control yeast that do not bear the appro- Fig. 3. KJ16 Fab fragments inhibit T cell activation by scFv-yeast.
priate scFv do not induce T cell activation markers Splenocytes Ž10 5 . from 2C TCRrRAG-1y_y mice were incu-
ŽFig. 2B.. Second, the T cell population used here is bated at a 1:1 ratio with scFv-yeast in the presence or absence of
10 mgrml KJ16 Fab fragments or in the presence of a three-fold
from a TCRrRAGyry transgenic mouse that contain excess of AGA-HA yeast Žtop panel.. After 20 h, the cell mixtures
no B cells which are required in the DNA-induced were harvested and analyzed for CD69 induction as described in
response. Finally, soluble KJ16 Fab fragments are Fig. 2.
B.K. Cho et al.r Journal of Immunological Methods 220 (1998) 179–188 185
3.3. ActiÕation of 2C TCR r RAG1-1yry splenocytes
in the presence of irreleÕant yeast
The ability to use the yeast display system to
identify scFv or novel polypeptide ligands that act as
agonists or antagonists depends ultimately on the
ability to screen yeast libraries for a yeast cell that
bears a specifically reactive polypeptide among many
cells that bear different Žirrelevant. polypeptides. To
find out if yeast meet this requirement, T cells were
incubated with scFv-ŽKJ16. yeast in the presence or
absence of various excesses of yeast expressing only
the Aga2 polypeptide. After 20 h of incubation,
CD69 levels were measured by flow cytometry, gat-
ing on 1B2-positive Ž2C. T cells. All mixtures, in-
cluding the highest ratio of nonspecific yeast to
scFv-yeast tested Že.g. 30:1. induced levels of CD69
that were comparable to those induced by scFv-yeast
alone ŽFig. 5.. Similar results were obtained when
CD25 upregulation was monitored Ždata not shown.
indicating that specific cell activation could occur
even in the presence of a great excess of irrelevant
yeast. Therefore, by using high-throughput screening
assays in 96-well plate formats it should be possible
to screen libraries in the range of 10 5 to 10 6 inde-
pendent clones for yeast that express candidate poly-
peptides capable of inducing such cellular responses.
Over the past several years, many screening pro-
cedures have been developed to identify new ligands
that bind to a given receptor. However, procedures
that are based solely upon binding are likely to lack
Fig. 4. Downregulation of the TCR on T cells induced by scFv-
yeast. scFv-yeast were incubated with 10 5 splenocytes from 2C the sensitivity of other procedures that exploit the
TCRrRAG-1y_y mice at several different ratios of yeast to T great amplification that results from transducing sur-
cells. After 20 h the cells were harvested and incubated with face receptor binding into cellular responses. For
biotinylated-1B2 IgG followed by streptavidin: PE. TCR expres- instance, phage display typically selects for ligands
sion was monitored by flow cytometry gating on T cells by light
that bind to receptors in the mM to nM range. In
scatter and cells that were 7AADy. The population at the lower
PE levels Žlocated outside the region of the cursor bar. are yeast contrast, cellular responses to appropriate ligation of
cells that were not entirely excluded by the light scatter criteria. surface receptors by natural ligands can often be
Mean fluorescence units Žmfu. of the region within the cursor bar detected at pM concentrations or lower. However,
are indicated. the triggering of such receptors often requires dimer-
ization or oligomerization to induce activity Žre-
viewed by Heldin, 1995.. Many display systems lack
loss of TCR molecules from the 2C T cell surface, the ligand density required for activating cellular
whereas control yeast without surface scFv were responses, and therefore cannot take advantage of the
unable to induce this downregulation. These results signal amplification inherent in these processes. Ac-
are consistent with recent findings that TCR down- cordingly, previous reports that have screened for
regulation can occur in the absence of costimulation agonist molecules that induced receptor dimerization
ŽViola and Lanzavecchia, 1996.. have had to rely on multiple steps of which the first
186 B.K. Cho et al.r Journal of Immunological Methods 220 (1998) 179–188
is direct binding ŽWrighton et al., 1997; Xie et al., of interest, and triggers cellular responses. These
1997.. Because binding assays lack the sensitivity of findings suggest that the yeast Aga2-fusion system
cellular response assays, novel ligands may not be has the potential for generating and screening pep-
detected. tide or cDNA libraries for bioactive molecules. For
In this report, we describe a yeast display system example, thymus-specific cDNA libraries might be
that expresses a high density of cell surface fusion screened for novel cell surface ligands that are in-
proteins, crosslinks target receptors present on cells volved in particular T cell developmental functions
ŽCibotti et al., 1997. or covalently linked peptide–
MHC complexes ŽKozono et al., 1994. might be
screened as T cell agonists. It is also reasonable to
suggest that in cases where ligation of multiple
different receptors are required, that it will be possi-
ble to co-express different ligands using multiple
selectable markers on the appropriate Aga2-yeast
vectors. Although our results emphasize the utility of
this system as it applies to T cells, a similar approach
may be used to generate agonist or antagonist
molecules towards a wide variety of other cell sur-
face receptors that likewise induce cellular responses
upon crosslinking ŽHeldin, 1995..
It is important to note that yeast display is signifi-
cantly different from presentation of proteins on the
surface of inert latex or silica microspheres. For
instance, yeast display avoids the need to clone,
express, refold in vitro, andror purify the gene
productŽs. to be tested. Indeed, the inherent attrac-
tion for this system is its potential for screening the
biological activity of polypeptides derived from, as
of yet, unidentified genes or even from uncharacter-
ized open-reading frames. For these purposes, cur-
rent techniques provide yeast transformation effi-
ciencies that are on the order of bacterial transforma-
tions with library sizes of up to 10 7 independent
colonies ŽGietz et al., 1995 and unpublished data..
Furthermore, since yeast have a well-defined genetic
system with many selectable markers, they have the
Fig. 5. Yeast that bear scFv-KJ16 activate T cells in the presence
of excess irrelevant yeast. Yeast that express only Aga2 ŽAGA-HA.
were mixed with a constant number of scFv-ŽKJ16. yeast at
several different ratios and the mixture was incubated with 10 5
splenocytes from 2C TCRrRAG-1y_y mice. After 20 h in
culture, the levels of CD69 induction on T cells were determined
by flow cytometry, as described in Fig. 2. Similar results have
been obtained when T cells were analyzed for CD25. The control
Žtop panel. indicates the level of CD69 expression on T cells
when incubated only with irrelevant yeast, at the highest yeast to
T cell ratio used.
B.K. Cho et al.r Journal of Immunological Methods 220 (1998) 179–188 187
potential for displaying several polypeptides simulta- Jorgensen, J.L., Reay, P.A., Ehrich, E.W., Davis, M.M., 1992.
neously on the same yeast cell allowing for the study Molecular components of T-cell recognition. Annu. Rev. Im-
munol. 10, 835.
or even selection of co-receptor molecules. Kieke, M.C., Cho, B.K., Boder, E.T., Kranz, D.M., Wittrup, K.D.,
1998. Isolation of anti-T cell receptor scFv mutants by yeast
surface display. Protein Eng. 10, 1303.
Acknowledgements Kozono, H., White, J., Clements, J., Marrack, P., Kappler, J.,
1994. Production of soluble MHC class II proteins with cova-
lently bound single peptides. Nature 369, 151.
We thank Drs. Kappler and Marrack for originally Kranz, D.M., Tonegawa, S., Eisen, H.N., 1984. Attachment of an
providing the KJ16 hybridoma line and Gary Durack anti-receptor antibody to non-target cells renders them suscep-
and the University of Illinois Flow Cytometry Facil- tible to lysis by a clone of cytotoxic T lymphocytes. Proc.
Natl. Acad. Sci. USA 81, 7922.
ity for assistance with flow cytometry. We also thank
Lu, C.F., Montijn, R.C., Brown, J.L., Klis, F., Kurjan, J., Bussey,
Dr. Herman Eisen for helpful comments on the H., Lipke, P.N., 1995. Glycosyl phosphatidylinositol-depen-
manuscript. This work was partially supported by dent cross-linking of alpha-agglutinin and beta 1,6-glucan in
grants from the Whitaker Biomedical Engineering the Saccharomyces cereÕisiae cell wall. J. Cell Biol. 128, 330.
Foundation Žto KDW. and the NIH ŽR01AI35990 to Manning, T.C., Rund, L.A., Gruber, M.M., Fallarino, F., Gajew-
ski, T.F., Kranz, D.M., 1997. Antigen recognition and allo-
DMK.. E. Boder was supported by an NSF Graduate
genic tumor rejection in CD8qTCR transgenicrRAGyry
Fellowship. M. Kieke was supported as a trainee on mice. J. Immunol. 159, 4665.
NIH T32 GM07283. Mescher, M.F., 1992. Surface contact requirements for activation
of cytoxic T lymphocytes. J. Immunol. 149, 2402.
Metzger, H., 1992. Transmembrane signaling: the joy of aggrega-
tion. J. Immunol. 149, 1477.
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