of March 31, 2018.This information is current as T Cells+Fraction of CD8

LargerImmunogenicity and Cross-Priming a Stability Is Associated with IncreasedDeterminant To Improve Peptide/MHC Modification of a Tumor Antigen

and Todd D. SchellAlan M. Watson, Lawrence M. Mylin, Megan M. Thompson

ol.1102221http://www.jimmunol.org/content/early/2012/11/18/jimmun

published online 21 November 2012J Immunol 

MaterialSupplementary

1.DC1http://www.jimmunol.org/content/suppl/2012/11/19/jimmunol.110222

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Modification of a Tumor Antigen Determinant To ImprovePeptide/MHC Stability Is Associated with IncreasedImmunogenicity and Cross-Priming a Larger Fraction ofCD8+ T Cells

Alan M. Watson,*,1 Lawrence M. Mylin,† Megan M. Thompson,†,2 and Todd D. Schell*

Altered peptide ligands (APLs) with enhanced binding to MHC class I can increase the CD8+ T cell response to native Ags,

including tumor Ags. In this study, we investigate the influence of peptide–MHC (pMHC) stability on recruitment of tumor

Ag-specific CD8+ T cells through cross-priming. Among the four known H-2b–restricted CD8+ T cell determinants within SV40

large tumor Ag (TAg), the site V determinant (489QGINNLDNL497) forms relatively low-stability pMHC and is characteristically

immunorecessive. Absence of detectable site V–specific CD8+ T cells following immunization with wild-type TAg is due in part to

inefficient cross-priming. We mutated nonanchor residues within the TAg site V determinant that increased pMHC stability but

preserved recognition by both TCR–transgenic and polyclonal endogenous T cells. Using a novel approach to quantify the fraction

of naive T cells triggered through cross-priming in vivo, we show that immunization with TAg variants expressing higher-stability

determinants increased the fraction of site V–specific T cells cross-primed and effectively overcame the immunorecessive phe-

notype. In addition, using MHC class I tetramer–based enrichment, we demonstrate for the first time, to our knowledge,

that endogenous site V–specific T cells are primed following wild-type TAg immunization despite their low initial frequency, but that

the magnitude of T cell accumulation is enhanced following immunization with a site V variant TAg. Our results demonstrate that site

V APLs cross-prime a higher fraction of available T cells, providing a potential mechanism for high-stability APLs to enhance

immunogenicity and accumulation of T cells specific for the native determinant. The Journal of Immunology, 2012, 189: 000–000.

The T cell response to viral and tumor Ags is typicallyskewed toward a subset of immunodominant determi-nants. Such determinants have been the main focus of

research for vaccine development and immunotherapy. However,T cells targeting subdominant determinants, which induce fewerresponder T cells, can also play a role in the control of tumors (1, 2)and virus infections (3–5). Limited immunological tolerance to-ward subdominant determinants found in self-tumor–associatedAgs in mice (6–8) and humans (9) makes them attractive targetsfor CD8+ T cell–based immunotherapy approaches. Understanding

the mechanisms contributing to the subdominant phenotype may

facilitate inclusion of T cells targeting a broader repertoire ofdeterminants for vaccination and immunotherapy.Expression of the SV40 large TAg (TAg) oncoprotein in primary

mouse fibroblasts leads to cell immortalization and transformation

in vitro and also serves as a tumor rejection Ag when these cellsare injected into immunocompetent mice (10). The CD8+ T cell

response to SV40 TAg in C57BL/6 mice is characterized by areproducible immunodominance hierarchy directed toward four

determinants, designated sites I, II/III, IV, and V (11). T cellsspecific for sites I, II/III, and IV are detected following immuni-

zation with wild-type TAg (wt-TAg), whereas the endogenous site

V–specific T cell response is detected only following immuniza-tion with TAg variants lacking sites I, II/III, and IV or following

immunization with a site V minigene-based vaccine (12, 13). Wepreviously found that injection of site V–specific TCR–transgenic

T cells (TCR-V) into C57BL/6 mice was not sufficient to over-come the weak response to site V in the context of wt-TAg im-

munization, indicating that precursor frequency is not the sole

limiting factor (14). Rather, we found that site V is weakly cross-presented in vivo compared with the more dominant determinants

from TAg. Based on previous data showing a kinetic difference inthe stability of site V/H-2Db complexes relative to peptide–MHC

(pMHC) formed with the other known TAg determinants (13), weproposed that unstable pMHC interactions may contribute to

limited cross-presentation and the immunorecessive phenotype

of site V.Amino acid substitutions within weakly immunogenic T cell

determinants can lead to improved immunogenicity. Such al-

tered peptide ligands (APLs) have been shown to induce highermagnitude CD8+ T cell responses, particularly in the setting of

self-tolerance, and can increase CD8+ T cell–mediated antitumor

*Department of Microbiology and Immunology, The Pennsylvania State UniversityCollege of Medicine, Hershey, PA 17033; and †Department of Biological Sciences,Messiah College, Mechanicsburg, PA 17055

1Current address: Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA.

2Current address: Department of Molecular, Cellular, and Biomedical Sciences, Uni-versity of New Hampshire, Durham, NH.

Received for publication August 1, 2011. Accepted for publication October 15, 2012.

This work was supported by Grant RO1 CA025000 from the National CancerInstitute/National Institutes of Health. A.M.W. was supported by Predoctoral Train-ing Grant T32 CA60395 from the National Cancer Institute/National Institutes ofHealth.

Address correspondence and reprint requests to Dr. Todd D. Schell, Department ofMicrobiology and Immunology, The Pennsylvania State University College of Med-icine, Hershey, PA 17033. E-mail address: tschell@hmc.psu.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: APL, altered peptide ligand; B6, C57BL/6; DC,dendritic cell; Flu, influenza; FSC-A, forward scatter; MFI, mean fluorescence in-tensity; MHC-I, MHC class I; P, position; pAPC, professional APC; pMHC, peptide–MHC; RT, room temperature; rVV, recombinant vaccinia virus; SJL, B6.SJL-Ptprca

Pepcb/BoyJ; SSC-A, side scatter; TAg, SV40 large TAg; TCR-I, site I–specific TCR–transgenic T cell; TCR-V, site V–specific TCR–transgenic T cell; UD, undivided; wt,wild-type.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102221

Published November 21, 2012, doi:10.4049/jimmunol.1102221 by guest on M

arch 31, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

immunity (15–18). APLs with engineered substitutions at TCRcontact residues can directly enhance T cell activation by in-creasing TCR signal transduction (17). A second class of APLseffects residues that improve peptide affinity for MHC class I(MHC-I) and/or increase stability of pMHC. The basis for im-proved immunogenicity of this latter class of APLs may resultfrom structural changes in the pMHC complex that improvesTCR–pMHC affinity (19). Alternatively, changes in peptide af-finity for MHC-I may promote higher levels of Ag presentationthat could lead to more durable T cell–APC interactions and moreefficient T cell activation (18, 20). Although evidence suggeststhat a minimal affinity for MHC-I is necessary for a peptide to beimmunogenic (21, 22), multiple studies additionally show a strongcorrelation between pMHC stability (t1/2) and immunogenicity(23–25). In fact, a recent extensive study by Harndahl and col-leagues (26) provides strong support that pMHC stability canaccurately predict immunogenicity. Thus, APLs containing aminoacid substitutions that increase pMHC stability are likely to bemore immunogenic, although no consensus has been reached re-garding the basis for this effect.Cell-associated Ags, such as tumor Ags, can induce CD8+ T cell

responses by accessing the cross-presentation pathway that isactive in professional APCs (pAPCs) (27, 28). Thus, pMHC sta-bility could potentially impact the efficiency of cross-priming byinfluencing the level and/or duration of pMHC cross-presentation.We evaluated the extent to which site V APLs promote increasedimmunogenicity following expression of variant TAgs in C57BL/6(B6) fibroblasts. Using newly defined APLs and a unique approachto monitor the initial recruitment of naive TCR–transgenic T cells,we find that APLs with enhanced pMHC stability increase thefraction of naive TCR–transgenic T cells recruited by cross-primingand overcome the immunorecessive nature of site V by theendogenous T cells. In addition, using MHC tetramer enrich-ment, we show that wt-TAg immunization induces limitedaccumulation of endogenous site V–specific T cells and thatthis response is dramatically enhanced by immunization withAPL-expressing TAg.

Materials and MethodsMice

B6, B6.129S2-Tap1tm1Arp (tap12/2), and B6.PL-Thy1a/CyJ (Thy1.1) micewere purchased from The Jackson Laboratory and bred on site. TCR-V (14)mice and site I–specific TCR–transgenic (TCR-I) (29) mice were describedpreviously. TCR-V mice were bred onto the B6.SJL-PtprcaPepcb/BoyJ(SJL; Taconic Farms) background. All mice were maintained under spe-cific pathogen-free conditions in the Penn State Hershey animal facility.All studies were performed in accordance with an approved Penn StateHershey Institutional Animal Care and Use Committee protocol.

Synthetic peptides

Peptides were synthesized at the Penn State Hershey MacromolecularCore Facility using Fmoc chemistry. Peptides were dissolved in DMSO,then diluted to the appropriate concentration in RPMI 1640 medium withGlutaMAX (Invitrogen), and stored at 280˚C. Peptides correspond tothe SV40 TAg site V (QGINNLDNL; Pep-V), site I (SAINNYAQKL;Pep-I), and a control H-2Db–binding peptide corresponding to influenzavirus nucleoprotein 366–374 (ASNENMETM; Pep-Flu). The followingsite V variant peptides were used: Q489A (AGINNLDNL), G490A(QAINNLDNL), I491F (QGFNNLDNL), N492A (QGIANLDNL), L494A(QGINNADNL), D495A (QGINNLANL), and N496A (QGINNLDAL).

Site-directed mutagenesis

TAg constructs expressing the Q489A mutation were produced viaQuikchange II XL (Stratagene) site-directed mutagenesis using oli-gonucleotides 59-TTGCCT TCA GGT GCT GGA ATT AAT AAC CTGGAC-39 (sense) and 59-GTC CAG GTT ATT AAT TCC AGC ACCTGA AGG CAA-39 (antisense). G490A substitutions were introducedinto SV40 TAg by the Altered Sites II mutagenesis procedure (11)(Promega) alone or in combination with MHC anchor residue alaninesubstitutions inactivating sites I, II/III, and IV. The mutagenic oligo-nucleotide encoding G490A was 59-TTG CCT TCA GGT CAG GCT ATTAAT AAC CTG GAC-39. Determinants I, II/III, and IV were inactivatedby alanine substitutions using the mutagenic oligonucleotides N210A,59-GTG TCT GCT ATT AAT GCT TAT GCT CAA AAA TTG-39;N227A, 59-ATT TGT AAA GGG GTT GCTAAG GAATAT TTG ATG-39; and F408A, 59-TCA GTG GTG TAT GAC GCT TTA AAATGC ATGGTG-39.

Cells

Cell lines are summarized in Table I and referred to in the text using thecommon name. Q489A, TAP Q489A, and TAP G490A cell lines wereproduced by immortalization of primary mouse kidney cells from B6 orTAP12/2 mice following transfection with TAg plasmids by the calciumphosphate precipitation method (30). G490A and G490A V-only cellswere derived using the Fugene 6 method of transfection (Roche). TAg-transformed cell lines were maintained in DMEM supplemented with 5%or 10% FCS and 100 U penicillin/ml, 100 mg streptomycin/ml, 100 mgkanamycin/ml, 2 mM L-glutamine, 10 mM HEPES buffer, and 0.075%(w/v) NaHCO3. Primary site V–specific T cell cultures were initiated byincubation of 1 3 107 RBC-depleted spleen cells from previously im-munized B6 mice with 5 3 105 g-irradiated (100 Gy) K-3,1,4 cells/wellof 12-well plates. Thereafter, T cell cultures were passed every 7 d intofresh wells containing stimulator cells with 5 U/ml recombinant humanIL-2 (Amgen). TCR-V and TCR-I T cell lines were initiated in vitro withRBC-depleted spleen cells from naive TCR-V and TCR-I mice. At thetime of primary culture, 100 nM synthetic site V or site I peptide wasused for stimulation. TCR-V and TCR-I cultures were maintained asdescribed above by restimulation with irradiated wt-TAg cells. The siteI–specific clone K-11 (33), site II/III–specific CTL clone K-19 (33), andsite IV-specific CTL line SV2168T (34) were maintained as describedpreviously. T cells were used for assays on days 4 to 5 of the growthcycle. Lymphocytes and TAP2-deficient RMA/s cells (35) were main-tained in complete RPMI 1640 medium with GlutaMAX supplementedwith 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin, 25 ng/mlsodium pyruvate, 50 mM 2-ME, and 10 mM HEPES.

Table I. Cell lines used in this study

Common Name Background Transforming Agent Determinants Expressed Formal Name Reference No.

wt-TAg B6 SV40 I, II/III, IV, V B6/WT-19 (30)V-only B6 pSLM-361-11 V B6/T 116A1 Cl-C (12)Q489A B6 pAMW 4-8 I, II/III, IV, Q489A Q489A WT Bulk 4-8 Current studyG490A B6 pMS02-7 I, II/III, IV, G490A G490A WT C1.1 Current studyG490A V-only B6 p413S06 C3-13 G490A G490A V-only D2.2 Current studyNull B6 pLMTS364-1 None B6/122B1 (12)TAP wt TAP12/2 pPVU0 I, II/III, IV, V TAP WT-DNA (14, 31)TAPV-only TAP12/2 pSLM-361-11 V TAP(361-11)c (14)TAP Q489A TAP12/2 pAMW 4-4 I, II/III, IV, Q489A Tap2/2 Q489A WT 4-4 B1 Current studyTAP G490A TAP12/2 pMS02-7 I, II/III, IV, G490A Tap2/2 G490A WT A1 Current studyK-3,1,4 B6 V B6/K-3,1,4 (32)K-1,4,5 B6 None B6/K-1,4,5 (32)

2 APLs ENHANCE CROSS-PRIMING OF CD8+ T CELLS

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Measurement of relative affinity and H-2Db complex stability

Assays to estimate relative affinity have been previously described (36, 37).RMA/s cells (38) were grown overnight at 26˚C, 5% CO2, and then 5 3105 cells were plated per well of a 96-well round-bottom plate and theindicated concentrations of peptide added. Cultures were incubated at 37˚C,5% CO2, for 4 h and then stained with 28-14-8s anti-Db mAb for 30 min at4˚C. Bound primary Ab was detected by addition of goat anti-mouse IgG-FITC (MP Biomedicals) for 30 min at 4˚C. Samples were collected on anFACSCanto instrument (BD Biosciences) and the geometric mean of FITCstaining determined using FlowJo Software (Tree Star). To calculate thepercent maximum mean fluorescence intensity (MFI), the following for-mula was used: (MFIpeptide 2 MFIno peptide)/(MFIHi 2 MFIno peptide) 3100, where MFIHi is the peptide concentration in which the highest MFIwas achieved. Data were plotted using Prism software (GraphPad), and therelative affinity (Kd) of peptide binding to H-2Db was estimated usingnonlinear regression analysis fitted to one-site saturation binding kinetics.The relative decay rate of synthetic peptide–stabilized H-2Db complexeson RMA/s cells was determined as previously described (13).

Cytotoxicity assay

Cytotoxicity assays were performed as previously described (39). Briefly,T-25 flasks of target fibroblasts were labeled overnight with 200 mCi so-dium 51chromate. Target cells were treated with trypsin in versene, and 104

cells were seeded into 96-well V-bottom plates. Effector cells were platedwith targets in triplicate at the indicated E:T ratio in a total volume of200 ml. The plates were incubated for 4 h at 37˚C and 5% CO2 and thencentrifuged to pellet cells and debris. A total of 100 ml supernatant wasremoved from each well. Percent specific lysis was determined as de-scribed previously (39). Effector cell lines specific for sites I, II/III, IV, andV were as follows: TCR-I cells, clone K-19, line SV2168T, and TCR-Vcells, respectively.

Immunizations

Cell lines for immunization were grown under uniform conditions in asingle batch for each experiment and frozen at a concentration of 2 3 107

cells/ml at280˚C in DMEM supplemented with 10% FBS and 5% DMSO.On the day of immunization, cells were quickly thawed at 37˚C, washedtwice with PBS, and resuspended at a concentration of 108 cells/ml in PBS.Mice were immunized i.p. with 5 3 107 cells (0.5 ml). Peptide immuni-zations were performed by s.c. injection at the base of the tail with 100 mgPep-V, Pep-Q489A, or Pep-I and 160 mg hepatitis B virus core helperpeptide 128–140 in 100 ml IFA as previously described (40). Immunizationwith recombinant vaccinia viruses (rVV)-ES-V and rVV-ES-I (13) wascarried out by i.v. injection of 107 PFU in 0.2 ml PBS into the tail vein.

Fine specificity measurements and intracellular cytokinestaining

Fine specificity of responding T cells was determined following primaryimmunization with 5 3 107 of the indicated cells followed by a boosterimmunization 21 d later with either B6/V-only cells or B6/G490A V-onlycells as indicated. After 8 d, splenocytes were restimulated ex vivo with1 mM site V peptide or each of the site V single amino acid substitutionanalogs and the percent of CD8+ cells producing IFN-g determined byintracellular cytokine staining and flow cytometry. The percentage of wtsite V response was calculated according to the formula: (percent responseto analog/percent response to site V) 3 100.

To detect the intracellular production of IFN-g, 1 to 23 106 spleen cellsor cultured T cells were stimulated for 5 to 6 h with synthetic peptides ina solution of 1 mg/ml brefeldin A as previously described (12). For assaysusing TAg-transformed cells as stimulators, T cells lines were mixed 1:1with stimulator cells (3 3 105 cells each) for 1 h followed by addition of 1mg/ml brefeldin A and incubation at 37˚C, 5% CO2, for a further 5 h.Intracellular IFN-g was detected using the Cytofix/Cytoperm kit (BDPharmingen) according to the manufacturer’s instructions.

In vivo proliferation assays

Single-cell suspensions of lymphocytes from TCR-V/SJL-transgenic miceand/or Thy1.1+ mice were labeled with 5 mM CFSE (Molecular Probes) aspreviously described (14). Mice received 1 3 106 (unless otherwise noted)CD8+/Tet-V+ cells by i.v. injection and the following day were immunized.Three to 7 d following immunization, spleens were harvested, and TCR-Vcells were visualized by FACS. For Thy1.1-spike experiments, totallymphocytes from TCR-V/SJL and Thy1.1+ mice were mixed prior totransfer. The number of undivided (UD) TCR-V cells (CFSE-Hi TCR-V)and UD-Thy1.1 cells (CFSE-Hi CD8+thy1.1+) was determined, and a ratio

was calculated according to the equation: UD–TCR-V/UD-Thy1.1 to de-fine the fraction of TCR-V cells that had not undergone initial cell division.

Flow cytometry and Abs

Ex vivo staining of lymphocytes was performed on single-cell suspensionsfrom spleens created by pushing the particulate tissues through a stainless-steel 60-mesh screen (Bellco Glass) as previously described (12). Allstaining was performed in FACS buffer (PBS supplemented with 2% FBSand 0.1% sodium azide) and completed at room temperature (RT). Cellswere incubated in a 1:100 dilution of rat anti-mouse CD16/CD32 (Fcblock) for 15 min at RT and then washed one time with FACS buffer.Fluorophore-conjugated Abs (1:150 dilution) and MHC-tetramer (1:200dilution) were added to the cells and incubated for 15 min at RT. Cellswere washed three times with FACS buffer, fixed in 2% paraformaldehydein PBS, and analyzed using FACScan II, FACSCalibur, FACSCanto, orLSR II flow cytometer (BD Biosciences). Unless otherwise noted,100,000–300,000 live events were recorded based on forward scatter (FSC-A)/side scatter (SSC-A) gating, and data were analyzed using FlowJo soft-ware (Tree Star). MHC-I tetramers specific for H-2Db/TAg site V (Tet-V)or H-2Db/TAg site I (Tet-I) were produced and characterized as de-scribed previously (12). The following Abs were purchased from BDBiosciences or eBioscience: CD8a (clone 53-6-7), CD45.1 (clone A20),Thy1.1 (clone H1S51), Thy1.2 (53-2.1), IFN-g (clone XMG1.2), and DUMP(CD19 [MB19-1], MHC class II [M5/114.15.2], F4/80 [BM8], CD11b [M1/70], and CD4 [GK1.5]).

Tetramer-based enrichment of site I– and site V–specific CD8+

T cells from mice

At least 10 d prior to the experiment, positive control mice were immunizedwith wt-TAg or Q489A cells, and at least 1 d prior to the experiment, micereceived 105 naive TCR-V cells i.v. Immunization plus adoptive transfer ofTCR-V cells in positive control mice yielded detectable populations of bothsite I– and site V–specific T cells to facilitate identification of tetramer-positive cells during gating. A variation of previously published protocols(41–43) was used for tetramer enrichment. Briefly, on the day of enrich-ment, the spleen and superficial cervical, brachial, inguinal, lumbar, andmesenteric lymph nodes were harvested. Organs were mechanically dis-rupted, and the resulting suspension was incubated in RPMI 1640 sup-plemented with 150 U/ml collagenase (17018-029; Life Technologies) and100 U/ml rDNAse I (04 536 282 001; Roche) at 37˚C for 30 min with rocking.Tissues were passed through a 60-mesh screen, RBCs were lysed using Tris-ammonium-chloride, and the cells were washed once with FACS buffer. Cellswere resuspended in 0.75 ml FACS buffer and costained with Tet-V–PE andTet-I–allophycocyanin for 30 min at RT in FACS buffer containing Fc block.

MHC tetramer–stained cells were washed twice with 5 ml MACS buffer(PBS supplemented with 0.5% BSA and 2 mM EDTA), and stained with0.1 ml each anti-PE and anti-APC MicroBeads (Miltenyi Biotec) in a totalvolume of 1 ml for 15 min at 4˚C. Cells were washed with 10 ml MACSbuffer and passed through a cell strainer (70-mesh; BD Biosciences orFischer Brand) before centrifugation. Cells were resuspended in 3 mlMACS buffer and passed over an LS column (Miltenyi Biotec) at 4˚C.Columns were washed three times with 3 ml MACS buffer, and cellsbound to the column were eluted twice with 5 ml MACS buffer. Elutedcells were washed with MACS buffer.

Eluted cells were stained in a 96-well plate with the following Abs:unlabeled anti-CD16/CD32, FITC DUMP (Abs to CD19, MHC class II, F4/80, CD11b, and CD4), anti–CD8-PerCP Cy5.5, anti–Thy1.2-PE-C7, Tet-V–PE, and Tet-I–allophycocyanin for 15 min at RT. Cells were washedthree times with FACS buffer and resuspended in 2% paraformaldehyde.All samples were run on a BD FACSCanto II or LSRII (BD Biosciences).The entire eluted cell fraction was acquired. First, live cells were deter-mined by FSC-A/SSC-A gating followed by gating on FITC-negative/CD8+ cells and then Thy1.2+ cells. Dot plots were displayed as Tet-I+

versus Tet-V+ cells. Tetramer-positive populations from positive controlmice were used as a guide to set up the tetramer+ gates.

Statistics

All data are displayed as mean6 SD. The p values were evaluated using anunpaired Student t test unless otherwise noted.

ResultsIdentification of site V analogs with increased pMHC stability

Immunogenicity requires that a peptide determinant have a mini-mum affinity for the presenting MHC molecule (21, 22). In ad-

The Journal of Immunology 3

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

dition, several studies suggest that the stability of pMHC com-plexes is predictive of the immunogenicity of CD8+ T celldeterminants (23–26) and has been directly linked to the estab-lishment of immunodominance hierarchies for CD4+ T cells (44).We sought to identify site V APLs that could form pMHC withincreased stability. Despite the formation of relatively unstablepMHC complexes, the wt site V determinant, encompassing TAgresidues 489–497 (QGINNLDNL), harbors the canonical Db an-chor residues at position (P) P5-N and P9-L (Fig. 1A). Thus, tostabilize pMHC interactions, we considered the role of nonanchorresidues to enhance pMHC binding without disrupting TCR rec-ognition (36, 45, 46). P3-I and P8-N were previously shown to becritical for recognition by site V–specific CTL (47) and, asdemonstrated in other systems, P3, P4, P6, P7, and P8 can pro-trude from the MHC binding cleft and/or serve as TCR contactresidues (48, 49). Thus, we identified positions P1 and P2 forsubstitution with alanine residues. The resulting analogs are des-ignated Q489A and G490A, respectively. We evaluated both therelative binding affinity of the Q489A and G490A peptides forH-2Db (Fig. 1B) as well as the stability of H-2Db moleculesformed with each peptide (Fig. 1C). To determine the relativeaffinity of peptide binding to H-2Db, TAP2 mutant RMA/s cellswere incubated overnight at 26˚C and then aliquots of cells mixed

with increasing concentrations of each peptide. After 4 h at 37˚C,cells were stained for H-2Db surface expression and the relativeaffinity determined (Fig. 1B). The results indicate that peptideQ489A has an increased relative affinity for Db (4-fold), whereasthat of G490A is similar to wt site V. Thus, only the Q489A analogshowed an increased relative affinity, which was similar to thedominant site I peptide.The stability of peptide-loaded Db complexes was estimated by

measuring the decay of pMHC from RMA/s cells following re-moval of excess peptide. Complexes formed with the Q489A,G490A, and site V peptides had t1/2 of ∼4, 2.75, and 2 h, re-spectively (Fig. 1C). The site I peptide was the most stable, witha t1/2 .4 h. These results indicate that both Q489A and G490Aformed more stable pMHC than the wt site V peptide, althoughonly Q489A peptide showed a measurable change in relativeaffinity for H-2Db.We next evaluated cross-recognition of Q489A and G490A by

site V–specific T cells. T cell lines derived from TCR-V (Fig. 1D,1E) or polyclonal site V–specific T cells isolated from immunizedmice (Fig. 1F, 1G) were stimulated with decreasing concentrationsof site V, Q489A, G490A, or control Flu (NP366–374) peptide. Atthe highest concentration, site V, Q489A, and G490A peptidesstimulated a similar percentage of cells to produce IFN-g for both

FIGURE 1. Site V substitution mutants Q489A and G490A enhance pMHC stability and conserve recognition by site V–specific T cells. (A) Sequence of

synthetic peptides representing the Q489A and G490A mutants. (B) Relative affinity of synthetic peptides bound to H-2Db was determined by incubation of

RMA/s cells overnight at 26˚C followed by addition of the indicated concentrations of each peptide to aliquots of RMA/s cells and incubation at 37˚C for

4 h. Cells were stained for H-2Db expression and the data expressed as the percent maximum MFI of Db expression for each peptide. Relative affinity (Kd)

was determined as described in the Materials and Methods using nonlinear regression analysis. (C) RMA/s cells were incubated with the indicated peptides

overnight at 29˚C, excess peptide was removed by washing, and the cells were incubated at 37˚C for the indicated times. Cells were held on ice prior to

staining for H-2Db. Data are presented as percent of maximum arbitrary fluorescence units (AFU) for each peptide. Cultures of TCR-V T cells (D, E) or

polyclonal endogenous site V–specific T cells (F, G) were stimulated with 1027 M (D, F) or 1027–10212 M (E, G) peptide in the presence of brefeldin A, and

intracellular IFN-g was assessed by flow cytometry. Values represent the percent of CD8+ T cells producing IFN-g (D, F) or the percent of maximum CD8+

IFN-g+ cells for each peptide (E, G). All results are representative of at least two independent experiments in which similar results were obtained.

4 APLs ENHANCE CROSS-PRIMING OF CD8+ T CELLS

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

TCR–transgenic and polyclonal T cells (Fig. 1D, 1F). The wt siteV and Q489A peptides demonstrated similar functional avidity forclonal TCR-V (Fig. 1E) and polyclonal (Fig. 1G) T cell lines at allpeptide dilutions, indicating no discernable difference in T cellrecognition. Functional avidity for G490A was reduced between5- and 10-fold for TCR-V and polyclonal T cells, respectively.Overall, these results demonstrate that compared with wt site V,Q489A binds to Db with a higher relative affinity, forms morestable pMHC, and is recognized by site V–specific T cells witha similar efficiency. The G490A analog forms pMHC with mod-erately increased stability compared with wt site V, but hasa similar relative affinity for Db and provides a less efficient targetfor site V–specific T cells in vitro, indicating that the functionalavidity of site V–specific T cells for the G490A variant is reduced.Thus, two site V APLs were identified with increased pMHCstability, but which varied in the other parameters measured.

Q489A and G490A TAg variants are processed and presentedfor T cell recognition

We produced full-length TAg variants containing the Q489A orG490A substitutions in the context of both wt and site V-only TAg.Primary mouse B6 and tap1 knockout primary kidney cells weretransfected with plasmid DNA, and immortalized cell lines werederived for each of the constructs except Q489A V-only TAg(Table I), which was unable to immortalize primary kidney cells.Relative expression of TAg in each cell line was verified byimmunoprecipitation/Western blot and did not vary by .39%(A.M. Watson and T.D. Schell, unpublished observations). In-corporation of the particular site V variant in each cell line wasverified by sequencing of PCR-amplified genomic DNA (A.M.Watson and T.D. Schell, unpublished observations). We confirmedthat the TAg determinants were processed and presented from theQ489A and G490ATAg variants using in vitro recognition by siteI and site V–specific CTL lines. T cells produced IFN-g in re-sponse to B6-derived, but not tap1 knockout–derived, cell lines(Fig. 2), demonstrating direct presentation of the expected deter-minants by the B6-derived cell lines. Furthermore, CTL killing ofQ489A or G490A B6-derived cell lines was similar to that of wt-TAg-expressing cells, and the TAg variants did not interfere withtarget cell killing by site I–, II/III–, and IV–specific T cells(Supplemental Fig. 1). Thus, Q489A and G490A are processedand presented in a TAP-dependent manner, and the B6-derivedcells present the expected array of TAg determinants.

Immunization with Q489A or G490A induces endogenous siteV–specific T cell responses

Endogenous site V–specific T cell responses have not previouslybeen detected following immunization with TAg-expressing cellsthat encode both site V and dominant TAg determinants. To testthe immunogenicity of the Q489A and G490A TAgs, mice wereimmunized with wt-TAg, V-only, Q489A, G490A, or G490A V-only B6-derived cell lines (Fig. 3A, left panels). Discernable tet-ramer V+CD8+ T cells were observed for one of three mice andtwo of three mice immunized with Q489A and G490A, respec-tively. Two of three mice had detectable site V–specific CD8+

T cell responses following immunization with V-only cells. Al-though previous studies had used pooled lymphocytes from V-only–immunized mice (12), we consistently found using individualmice that an average of one in three mice could mount a detect-able ex vivo response to site V (data not shown). In contrast,immunization with G490A V-only cells produced strong ex vivoresponses in all mice ranging from 1.18–2.75% of CD8+ T cells(Fig. 3A, left panels), indicating that the combination of theG490A analog and the absence of the dominant determinants wasoptimal for the induction of site V cross-reactive CD8+ T cells. Nosignificant accumulation of site V–specific T cells was detected inwt-TAg–immunized or unimmunized mice. We observed no dis-cernable relationship between the magnitude of the site V–specificT cell response and the site I– or IV–specific T cell response inindividual mice (Fig. 3B, 3C, respectively), indicating that the siteV variants did not compromise the response to the dominantdeterminants and likewise the detection of site V–specific T cellswas not facilitated by reduced responses to the dominant deter-minants.To discern whether low numbers of site V–specific T cells were

primed in some immunized mice, splenocytes were restimulatedin vitro with K-3,1,4 cells, which express a TAg variant lackingsites I, II/III, and IV. In general, we found that mice with detectableex vivo Tet-V+ cells demonstrated marked expansion of T cells inculture (Fig. 3A, right panels), with the exception of mouse 1immunized with V-only TAg. Additionally, mouse 2 from theQ489A-immunized group showed accumulation of a prominentpopulation of Tet-V+ cells despite no detection ex vivo. Consistentwith previous studies, site V–specific T cells did not expand fromwt-TAg–immunized mice or from unimmunized mice followingin vitro culture, indicating that the expansion of site V–specificT cells was not due to in vitro priming. These data demonstrate thatQ489A and G490A are immunogenic when coexpressed with thedominant TAg determinants, overcoming the immunorecessivephenotype of site V. Even so, the site V–specific response remainedquantitatively subdominant to both sites I and IV.

Site V–reactive T cells primed by Q489A and G490A havea similar phenotype to those primed by wt site V

The increased immunogenicity of Q489A and G490A could beexplained by recruitment of additional T cell clones. To investigatewhether an overall change in the site V–reactive population occursfollowing immunization with Q489A and G490A, we evaluatedboth the fine specificity of the responding site V–reactive T cellsand the TCR b-chain usage by these T cells. Because only lowlevels of T cells can be detected ex vivo after primary immuni-zation with Q489A and G490A cells (Fig. 3), we used a prime/boost approach in which mice were first immunized with Q489Aor G490A cells followed by booster immunizations with V-onlycells to expand any site V cross-reactive T cells. Some mice re-ceived prime/boost with either V-only cells or G490AV-only cellsfor comparison. Ex vivo analysis of responding cells was per-formed at day 8 postboost, prior to the time when CD8+ T cell

FIGURE 2. Direct presentation of Q489A and G490A determinants by

B6-derived cells. B6-derived and tap1 knockout–derived cell lines

expressing wt-TAg or TAg variants expressing the Q489A or G490A

substitutions were tested for their ability to stimulate IFN-g production by

either site I–specific CTL clone K-11 or bulk polyclonal site V–specific

CTL. K-1,4,5 cells lack expression of the four known H-2b–restricted

determinants. Cells were stained for intracellular IFN-g production and

analyzed by flow cytometry.

The Journal of Immunology 5

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

responses to primary site V–only immunization can be detected.CD8+ T cell responses were tested against a panel of site V variantpeptides containing single amino acid substitutions at all positionsexcept for N493 and L497, which serve as the primary anchorresidues and for which substitutions interrupt peptide binding.CD8+ T cells from each of the four groups of mice showed similarfine specificities (Fig. 4), with substitutions at Q489 and G490having the least impact, residue L494 having the greatest impact,and the remaining four residues having intermediate impacts onT cell recognition. For the G490A and G490AV-only–immunizedgroups, there was a trend toward greater impact for substitutions atD495 and N496, but these changes were not statistically differentfrom the V-only–immunized group. These results indicate that thefine specificity of site V–reactive T cells is maintained followingpriming with the Q489A and G490A variants.We also broadly determined the TCR b-chain usage of site V–

reactive T cells derived from mice primed with wt site Vor site Vvariants. We first compared the profiles of site V–reactive CD8+

T cells that accumulate following a prime and boost with either V-only cells or G490A V-only cells, because these immunizationsinduce higher levels of T cells facilitating direct ex vivo analysis.All mice showed dominant accumulation of Vb7+ T cells, withsome mice in both groups also using Vb2 (Supplemental Fig. 2A).One mouse in the G490AV-only group had a minor population ofVb17+ cells, which was not observed in the V-only–immunizedmice. We also evaluated TCRb expression by site V–reactiveCD8+ T cells expanded in vitro from mice initially primed with V-only or Q489A cells (Supplemental Fig. 2B). In vitro expansion

was necessary due to the limited accumulation of cells followingimmunization with Q489A. This analysis also revealed predomi-nant accumulation of Vb7+ T cells in the V-only and Q489A

FIGURE 3. Q489A and G490A prime endoge-

nous site V–specific T cell responses in the pres-

ence of the dominant TAg determinants. (A–C) B6

mice were immunized with the indicated B6-de-

rived cell lines. Ten days following immunization,

spleens were harvested, and cells were stained with

anti-CD8 and site V MHC tetramer. The percentage

of endogenous site V–specific CD8+ T cells was

assessed ex vivo or following two rounds of in vitro

restimulation with K-3,1,4 cells. (A) Values indicate

the percent of CD8+ T cells that are site V MHC

tetramer positive. For comparison purposes, the

data for site I (B) and site IV (C) are plotted along

with the CD8+Tet-V+ T cell response to site V. Each

data point represents an individual mouse, and the

open and closed paired symbols represent data from

the same animal. Results are representative of at

least two independent experiments with three mice

per group.

FIGURE 4. Fine specificity of CD8+ T cells primed with Q489A and

G490A is similar to those primed against wt site V. Groups of three

C57BL/6 mice were immunized i.p. with the indicated B6-derived cell

lines and rested for 3 wk. Mice received booster vaccinations with either

V-only cells or G490AV-only cells. On day 8, splenocyte responses to site

V wt and variant peptides containing single amino acid substitutions were

tested ex vivo using intracellular staining for IFN-g production by CD8+

cells. Data are presented as the percentage of response to the wt site V

peptide. All mice were tested individually, and the error bars represent SD.

No statistically significant differences were observed among the immuni-

zation groups. The data are representative of two independent experiments

in which similar results were obtained.

6 APLs ENHANCE CROSS-PRIMING OF CD8+ T CELLS

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

groups. Taken together, the fine specificity and TCR b-chainanalyses suggest that the responsive repertoire of T cells is similarfollowing priming with wt site V compared with the site V var-iants. Although we cannot exclude CDR3 differences among theresponding clones, the results indicate that site V–reactive TCRsuse a restricted set of TCR b-chain families.

Q489A and G490A variants enhance cross-priming of TCR-VT cells

Previous evaluation of mechanisms regulating the immunogenicityof site V indicated that the wt determinant is inefficiently cross-presented, resulting in proliferation of only a fraction of adop-tively transferred naive TCR-V T cells (14). To determine whetherthe site V variants would promote more efficient triggering of TCR-V

T cells through cross-priming, we developed an assay to quantifythe fraction of cells remaining undivided following immunization.In this approach, naive CFSE-labeled TCR-V cells are cotransferredwith CFSE-labeled spleen cells from naive Thy1.1+ mice prior toimmunization (Fig. 5A). Because all mice received the same ratio ofTCR-V to CD8+Thy1.1+ cells, and the Thy1.1+ cells did not dividefollowing immunization (Fig. 5A, bottom right panel), the CFSE-hiCD8+Thy1.1+ population provides an internal reference to evaluatechanges in the undivided TCR-V population. In this way, the fre-quency of cells that initially proliferate can be quantified indepen-dent of the expansion and survival of the divided cells. Data arerepresented as the number of undivided CFSE-hi TCR-V cells di-vided by the number of CFSE-hi Thy1.1+CD8+ cells such that areduced ratio indicates more naive cells are triggered.

FIGURE 5. A greater fraction of naive TCR-V cells undergoes proliferation in response to Q489A or G490A. (A–D) Total CFSE-labeled splenocytes

from TCR-V mice and Thy1.1-congenic mice were mixed together (Thy1.1-spike), and aliquots containing 13 106 TCR-V T cells were transferred into B6

mice. The following day, mice were immunized with the indicated cell lines (A–C) or with the indicated peptide or rVVexpressing endoplasmic reticulum–

targeted minigene products (D). Three days following immunization, splenocytes were stained with Tet-V, anti-CD8, and anti-Thy1.1. The ratio of CFSE-hi

UD TCR-V/CFSE-hi CD8+Thy1.1+ cells was calculated as in the example shown in (A). (B) Results show the fraction of undivided TCR-V cells in each

immunization group. The control group consists of unimmunized mice or mice immunized with null cells. (C) The percent of naive T cells that divided was

calculated by the equation 100 3 (1 2 [I/U]), where I is the mean ratio from a group of immunized mice, and U is the mean ratio from the group of

unimmunized mice. (B and C) Results from two independent experiments were normalized and combined. The p values were determined by unpaired t tests.

(D) Data are presented as in (B) and the percentages shown as in (C). For mice immunized with peptide, U is the mean ratio from peptide I–immunized

mice. For mice immunized with rVV-ES-V, U is the mean ratio from rVV-ES-I–immunized mice. The data are representative of two independent

experiments with groups of three mice.

The Journal of Immunology 7

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

To restrict the T cell response to cross-presented Ag, we im-munized mice with TAP12/2 cell lines TAP-wt, TAP-Q489A, andTAP-G490A. As expected (14), immunization with TAP-wt cellstriggered a small fraction of TCR-V cells to divide (Fig. 5B).Immunization with TAP-Q489A or TAP-G490A cells resulted insignificantly more TCR-V cells dividing compared with TAP-WTimmunization. In summary, 33.4% of TCR-V cells divided fol-lowing immunization with TAP-wt, whereas 69.9 and 69.4% ofTCR-V cells divided following immunization with TAP-Q489Aand TAP-G490A, respectively (Fig. 5C). These results demon-strate that Q489A and G490A cross-prime a higher fraction ofTCR-V cells than the wt-TAg.In contrast to these results, we previously found that almost all

TCR-I cells divided in response to cross-presented Ag followingimmunization with wt-TAg cells (14), raising the possibility thata subset of TCR-V cells may be incapable of responding to the Agin vivo. To examine this question, we asked whether bypassingcross-presentation would initiate priming of a higher fraction ofthe TCR-V donor cells. Mice were immunized with syntheticpeptides (40) or rVV expressing an endoplasmic reticulum–tar-geted minigene (e.g., rVV-ES-V) (13). These approaches bypassthe need for Ag processing and cross-presentation of Ag fromdonor cells. The results revealed that almost all of the TCR-V cellsunderwent division following immunization with peptide V andpeptide Q489A or rVV-ES-V but not with control site I immuni-zations (Fig. 5D). These results rule out the possibility of anunresponsive subpopulation of TCR-V T cells and support theconclusion that cross-priming constitutes a major limitation in theresponse to TAg site V. Taken together, these data reveal thatenhanced cross-presentation of site V is associated with increasedpMHC stability, but that methods of immunization that bypasscross-presentation are most efficient in activation of TCR-V cellsin vivo.

TCR-V precursor number does not influence the efficiency ofcross-priming

Incomplete cross-priming of TCR-V cells following immunizationwith TAg-transformed cells could result from transfer of largenumbers of TCR–transgenic T cells because T cells of the samespecificity can compete for available pMHC during priming (50).Thus, we transferred decreasing numbers (106–104) of TCR-Vcells plus Thy1.1+ cells into mice prior to immunization withTAP-wt cells. Three days following immunization, CD8+ T cellswere enriched from splenocytes, and the fraction of undividedTCR-V T cells was measured. The results indicate that a similarfraction of naive TCR-V cells divided regardless of the number oftransferred TCR-V cells (Fig. 6A). This result is not due to alteredT cell seeding efficiencies because the percentage of CD8+

Thy1.1+ cells detected at each dilution was reduced proportion-ately (Fig. 6B). These data indicate that the fraction of naive TCR-V cells that is cross-primed remains the same when the number ofprecursors is between 106 and 104 per mouse and suggest thatcompetition among responding TCR-V T cells does not contributetoward the incomplete response. Extrapolating these results sug-gests that as naive T cells approach more physiologic numbers[103 and below (41–43)], incomplete cross-priming of availablesite V–specific T cells remains a limiting factor.

Endogenous site V–specific T cells are primed followingimmunization with wt-TAg

Although an endogenous site V–specific T cell response has notbeen detected following wt-TAg immunization, our results usingtransferred TCR-V–transgenic cells suggest that limited primingmight occur. Lack of detection could be explained by a combina-

tion of inefficient cross-priming and low precursor number. Wedetermined the number of site V–specific precursor T cellsavailable in naive mice using MHC-tetramer–based magneticenrichment (41–43). Site I–specific T cells were coenriched fromeach mouse so that relative levels of site I– and site V–specificT cells could be compared in individual mice. Cells isolated fromspleens and lymph nodes of individual mice were stained andgated as described in the Materials and Methods and depictedin Fig. 7A. The values reported represent the number of cellsdirectly detected per mouse following analysis of the entire cellpopulation. On average, 6.4 site V–specific precursor T cells and19.2 site I–specific T cells were detected per naive mouse (Fig.7C). Thus, naive mice contained ∼3:1 Tet-I/Tet-V–specific pre-cursor T cells, indicating that naive site V–specific T cells areinitially present at lower numbers than site I–specific T cells.The ability to detect low numbers of endogenous site V–specific

T cells provided an opportunity to readdress whether endogenoussite V–specific T cells are primed following wt-TAg immunizationand to evaluate the early kinetics of response to the site V analogs.Mice were immunized with wt-TAg cells or Q489A cells andisolated at day 7 postimmunization. We found that endogenoussite V–specific T cells accumulated in mice following wt-TAgimmunization (Fig. 7B). Our cumulative results demonstratea 22-fold expansion (141.4 cells/mouse; Fig. 7C) of endogenoussite V–specific T cells compared with naive mice. By comparison,site I–specific T cells expanded 184-fold (3524 cells per mouse)following immunization with wt-TAg, suggesting that site I–spe-cific T cells accumulate at a higher rate over the 7-d period.Immunization with Q489A cells resulted in endogenous site V–specific T cells expanding to 2868 cells/mouse, a 450-fold ex-pansion of the precursor population, representing an ∼20 fold(2868/141.4) greater expansion of site V–specific T cells relative

FIGURE 6. The number of precursor TCR-V cells does not alter the

fraction of naive T cells that are cross-primed. (A and B) B6 mice received

the indicated number of CFSE-labeled TCR-V cells plus Thy1.1-spike as

in Fig. 5 so that the initial ratio of TCR-V cells to CD8+Thy1.1+ cells

remained the same in all groups. Three days following immunization with

TAP-wt cells, spleens were harvested and cells enriched using CD8 magnetic

beads. The ratio of UD TCR-V/CD8+Thy1.1+ cells (A) or Thy1.1+ cells as a

percentage of total CD8+ cells (B) is plotted. The line in (B) is the linear

regression of the data (r2 = 0.8844).

8 APLs ENHANCE CROSS-PRIMING OF CD8+ T CELLS

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

to wt-TAg immunization. We observed that Q489A immunizationalso promoted accumulation of higher numbers of site I–specificT cells (22,159 cells per mouse), suggesting that the Q489A cellsmay be inherently more immunogenic than wt-TAg cells. Becausesite I– and site V–specific T cells were coisolated from individualmice, we were able to directly compare the ratio of site I– and siteV–specific CD8+ T cells in naive mice versus that obtained frommice immunized with wt-TAg or Q489A cells. As shown in Fig.7D, the ratio of site I/site V–specific T cells was significantlydecreased in Q489A-immunized mice compared with mice im-munized with wt-TAg cells, indicating that the Q489A substitutionresults in enhanced in vivo expansion of site V–specific T cellsrelative to site I–specific T cells. These results suggest that higheraccumulation of site V–specific T cells following Q489A cellimmunization is not solely due to enhanced immunogenicity ofthe cell line, but rather that Q489A is more immunogenic. Takentogether, our results demonstrate that endogenous site V–specificT cells are present at relatively low levels in naive mice but ex-pand in vivo following immunization with wt-TAg cells. T cellaccumulation was increased following immunization with Q489Acells at early time points (Fig. 7) and continued to increase at latertime points to allow direct ex vivo detection (Fig. 3).

DiscussionIdentification of APLs that form stable pMHC-I complexes pro-vides an effective way to enhance determinant-specific immuni-zation for cancer or infectious diseases (18, 20, 51). Although themechanisms promoting increased immunogenicity of APLs mayvary, previous studies indicate that agonist peptides can modify

the molecular contacts between the TCR and pMHC, resulting inmore effective triggering through the TCR (18, 52). Alternatively,quantitative increases in pMHC on APCs may enhance primingof naive CD8+ T cells by allowing prolonged T cell–APC inter-actions (53). The resulting augmented accumulation of T cells ispotentially explained by more complete recruitment of the avail-able naive T cells or through greater expansion and increasedsurvival of primed T cells. In the current study, we provide evi-dence that APLs with increased pMHC stability both enhanceendogenous T cell accumulation and cross-prime a higher fractionof available naive TCR–transgenic CD8+ T cells specific for animmunorecessive determinant. This mechanism may contribute tothe overall increase in T cell numbers following immunization,thereby overcoming the immunorecessive phenotype.A potential consequence of the increased pMHC stability of the

site V variants is higher or more durable pMHC presentation onAPCs. High-stability peptides are typically presented in greaternumbers than low-stability peptides (25) due to preferentialloading of high-stability peptides by the MHC-I Ag presentationmachinery (54). The consequences of increased pMHC presenta-tion for T cell activation have been revealed by intravital mi-croscopy studies showing that the number of pMHC presented onpAPCs during priming in the lymph nodes determines the rate atwhich naive CD8+ T cells are activated. Specifically, the numberof pMHCs presented by dendritic cells (DCs) inversely correlatedwith the speed of T cell transition through the three phases ofT cell–APC interaction leading to T cell activation (53). ThoseT cells unable to form prolonged contacts with DCs due to en-countering subthreshold Ag levels were unable to transition to the

FIGURE 7. Endogenous site V–specific T cells proliferate following immunization with wt-TAg cells. (A–C) Total splenocytes and lymphocytes were

stained with Tet-I–APC and Tet-V–PE and isolated by magnetic enrichment. (A) The flow cytometric gating scheme was based on FSC-A versus SSC-A

followed by cells positive for CD8 and negative for the DUMP stain and finally CD8+Thy1.2+ cells (A, top panel). Representative dot plots from positive

and negative control mice and naive mice are displayed (A, bottom panel). Positive control mice received immunization with wt-TAg cells and transfer of

TCR-V cells prior to the day of the experiment. The negative controls were naive mice that received no tetramer staining prior to magnetic enrichment. (B)

Representative plots of TAg-specific T cells obtained from mice 7 d following immunization with wt-TAg or Q489A cells. (C) Data compiled from all

experiments. Values indicate the number of cells in each gate (A, B) or the mean value of all Tet-V+ or Tet-I+ cells in all experiments (C). The data are

representative of two (B) or three (A) experiments comprising at least three mice per group. (D) The ratio of Tet I+ cells to Tet V+ cells from (C) is plotted

for individual mice. Bars indicate mean values. The p values were calculated using the Mann–Whitney U test.

The Journal of Immunology 9

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

latter stages of T cell activation. Relevant to the current study, thenumber of pMHCs presented by DCs in vivo was dependent onpMHC stability. Thus, site V analogs that form stable pMHC maytrigger a higher proportion of site V–reactive CD8+ T cells due tosustained cross-presentation of increased pMHC complexes.Differences in pMHC stability can potentially influence both

direct presentation by the TAg-transformed cells and cross-presentation by APCs. However, the impact on cross-presentationmay be more dramatic because Ag capture by DCs is downregu-lated following initial activation and licensing (55, 56), suggestingthat DCs take up a fixed amount of Ag for cross-presentation. Thisscenario differs from the constant supply of directly presenteddeterminants generated in cells that synthesize the Ag de novo.Shortly following acquisition of Ag, DCs may present a broadrange of determinants that later becomes biased toward high-stability pMHC due to rapid loss of low-stability pMHC. In-deed, Yu and colleagues (51) found that pMHC t1/2 on DCs wasdirectly related to the increased immunogenicity of a self/tumorAg determinant. Although the short t1/2 of site V–pMHC com-plexes on the cell surface (between 2 and 3 h) may be sufficientfor immunogenicity, it may limit the proportion of T cells thatcan achieve sustained signaling through the TCR required forT cell activation (57, 58). This may explain why a subpopula-tion of naive TCR-V T cells never divides in response to cross-presented TAg.Changes in pMHC stability or epitope production may influence

the number of pMHC complexes available for T cell priming;however, increased immunogenicity of the site V variants mayadditionally be explained by a direct change in TCR–pMHCcontacts, leading to more efficient triggering through the TCR. Forsome APLs, amino acid substitutions that increase pMHC stabilityand immunogenicity do not influence the overall structure of thepMHC complex, indicating that increased immunogenicity wasprimarily due to formation of more stable pMHC (18, 20). Thismechanism may best explain the increased immunogenicity of theQ489A variant, which showed the most dramatic increase inpMHC stability and relative affinity. Alternatively, some APLswith increased pMHC stability alter TCR contacts, resulting inincreased affinity of the TCR for the pMHC complex (19). Sucha mechanism may explain the increased immunogenicity of theG490A variant that showed a modest increase in pMHC stabilitywithout a net increase in the relative affinity for MHC. However,we note that the functional avidity of site V–specific polyclonalT cells and TCR–transgenic T cells was not increased for eitherthe Q489A or G490A variant peptides in vitro, suggesting that thevariant pMHC do not inherently increase the efficiency of TCRsignaling among site V–specific T cells. Further discernment ofthe contribution of pMHC stability and pMHC conformationtoward improved immunogenicity of the site V variants wouldrequire structural analysis of the pMHC and pMHC–TCR com-plex by x-ray crystallography (18, 19).Increased immunogenicity of the site V variants for the en-

dogenous site V–specific CD8+ T cells could be explained byactivation of new clonotypes (59, 60). We found only minordifferences in TCR-V b-chain family usage among site V–reac-tive CD8+ T cells induced by wt and Q489A or G490A TAgs,suggesting that the repertoire is generally restricted to the dom-inant TCR-V b-chains. However, our results leave open thepossibility of increased recruitment of T cells using TCRs withunique CDR3 regions within the dominant TCR-Vb families. Ourdata demonstrating that the site V variants prime a higher fractionof naive TCR–transgenic T cells are consistent with the idea thatadditional site V T cells in the naive polyclonal repertoire couldbe recruited.

We previously considered that T cell competition for pAPCscontributes to the immunorecessive phenotype of TAg site V (14).Mice coimmunized with wt-TAg and V-only TAg expressed inseparate cells developed T cells specific for both site V and theimmunodominant determinants. In contrast, site V–specific T cellswere undetectable following immunization with cells coexpress-ing wt-TAg and V-only Tag. These results suggested that T cellsspecific for the immunodominant determinants limit priming oraccumulation of site V–specific T cells as has been observed inseveral different systems (61–63). A recent study by Galea andcoworkers (64) provides evidence that T cells specific for thedominant TAg site I or site IV determinants compete with T cellsspecific for site V at the early stages of T cell priming. In addition,they found that modulation of the site IV determinant to reducethe t1/2 of pMHC complexes alleviated immunodomination oversite V when the determinants were coexpressed in tandem ina DNA vaccine. Our observation that enhanced site V/MHC sta-bility is associated with relieving the immunorecessive phenotypemay be explained in part by decreased competition from CD8+

T cells responding to the immunodominant TAg determinants,resulting in more efficient priming or expansion of site V–specificT cells. However, optimal site V–specific responses were bestobtained when the G490A variant was expressed in the absence ofthe dominant determinants, suggesting a continued role for T cellcompetition.Although the site V variants were able to produce a detectable

endogenous T cell response, site V was not raised to immuno-dominant status. Our data suggest that if the site V– and site I–specific naive T cell precursor numbers were equal, site V wouldremain subdominant due to the limited ability to cross-prime theavailable precursors. Thus, for site V to move up the immuno-dominance hierarchy, both an increase in the number of precursorsand the proportion of precursors that are triggered would be re-quired. This scenario differs from systems in which subdominantCD8+ T cells outnumber their immunodominant counterparts,such that an increase in cross-priming alone might be sufficient toincrease immunodominance. For example, La Gruta and col-leagues (65) found that the frequency of subdominant Flu deter-minant–specific CD8+ T cell precursors is significantly higher thanthat of their immunodominant counterparts. These authors foundincomplete recruitment of the available CD8+ T cell clonotypesspecific for the subdominant Flu Db-PB1-F262–70 determinant,which has an estimated t1/2 of ∼3 h (66). In contrast, CD8+ T cellsspecific for two dominant Db-Flu determinants with t1/2 estimatedto be ∼4 (NP366–374) and 16 h (PA224–233) (66) underwent moreefficient recruitment (65). We hypothesize that naive T cell re-cruitment may be nearly complete when pMHC stability is abovea yet undetermined threshold and variably incomplete whenpMHC stability falls below that threshold.We also show for the first time, to our knowledge, that site

V–specific CD8+ T cells are primed following immunization withwt-TAg–expressing cells. The inability to detect site V–specificT cells in previous studies is likely explained by the limitedaccumulation of these T cells following immunization, whichremain below the limit of detection using standard flow cytometricanalysis. Only through MHC tetramer enrichment was this re-sponse detected. Considering that T cells were isolated from ∼13108 total cells, the frequency of site V–specific T cells detected at7 d postimmunization with wt-TAg cells was 1 in 1.4 3 106. Incontrast, the frequency of site V–specific T cells detected fol-lowing immunization with Q489A cells was ∼5-fold higher at 1 in2.9 3 105. These site V–specific T cells continue to expand overthe next several days following immunization with the site Vvariants, whereas the response in wt-TAg–immunized mice never

10 APLs ENHANCE CROSS-PRIMING OF CD8+ T CELLS

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

reaches detectable levels (Fig. 3A). We speculate that site V–specific T cells triggered by wt-TAg may undergo abortive pro-liferation, whereas the site V variants induce a more characteristicextended T cell expansion in vivo.In summary, our results suggest that more complete recruitment

of the available naive T cell population can be achieved throughapproaches that increase pMHC stability or bypass cross-presentation. This mechanism may explain the enhanced immu-nogenicity of some APLs derived from similar subdominant orcryptic determinants and provide a strategy to expand the repertoireof determinants available for control of cancer and infectiousdiseases.

AcknowledgmentsWe thank Jeremy Haley and Melanie Epler for excellent technical assis-

tance and Dr. Satvir S. Tevethia for insightful suggestions. We also thank

Sandip Savaliya for maintenance of mouse colonies and the preparation

of genomic DNA for sequencing by the Penn State Hershey Molecular Ge-

netics Core and Nate Scheaffer and Dr. David Stanford from the Penn State

Hershey Flow Cytometry Core for technical expertise and assistance.

DisclosuresThe authors have no financial conflicts of interest.

References1. Ryan, C. M., and T. D. Schell. 2006. Accumulation of CD8+ T cells in

advanced-stage tumors and delay of disease progression following secondaryimmunization against an immunorecessive epitope. J. Immunol. 177: 255–267.

2. Feltkamp, M. C., G. R. Vreugdenhil, M. P. Vierboom, E. Ras, S. H. van der Burg,J. ter Schegget, C. J. Melief, and W. M. Kast. 1995. Cytotoxic T lymphocytesraised against a subdominant epitope offered as a synthetic peptide eradicatehuman papillomavirus type 16-induced tumors. Eur. J. Immunol. 25: 2638–2642.

3. Frahm, N., P. Kiepiela, S. Adams, C. H. Linde, H. S. Hewitt, K. Sango,M. E. Feeney, M. M. Addo, M. Lichterfeld, M. P. Lahaie, et al. 2006. Control ofhuman immunodeficiency virus replication by cytotoxic T lymphocytes targetingsubdominant epitopes. Nat. Immunol. 7: 173–178.

4. van der Most, R. G., A. Sette, C. Oseroff, J. Alexander, K. Murali-Krishna,L. L. Lau, S. Southwood, J. Sidney, R. W. Chesnut, M. Matloubian, andR. Ahmed. 1996. Analysis of cytotoxic T cell responses to dominant and sub-dominant epitopes during acute and chronic lymphocytic choriomeningitis virusinfection. J. Immunol. 157: 5543–5554.

5. Remakus, S., D. Rubio, X. Ma, A. Sette, and L. J. Sigal. 2012. Memory CD8+T cells specific for a single immunodominant or subdominant determinant in-duced by peptide-dendritic cell immunization protect from an acute lethal viraldisease. J. Virol. 86: 9748–9759.

6. Grossmann, M. E., T. Davila, and T. Celis. 2001. Avoiding tolerance againstprostatic antigens with subdominant peptide epitopes. J. Immunother. 24: 237–241.

7. Schell, T. D. 2004. In vivo expansion of the residual tumor antigen-specific CD8+T lymphocytes that survive negative selection in simian virus 40 T-antigen-transgenic mice. J. Virol. 78: 1751–1762.

8. Uram, J. N., C. M. Black, E. Flynn, L. Huang, T. D. Armstrong, and E. M. Jaffee.2011. Nondominant CD8 T cells are active players in the vaccine-induced an-titumor immune response. J. Immunol. 186: 3847–3857.

9. Friedman, R. S., A. G. Spies, and M. Kalos. 2004. Identification of naturallyprocessed CD8 T cell epitopes from prostein, a prostate tissue-specific vaccinecandidate. Eur. J. Immunol. 34: 1091–1101.

10. Tevethia, S. S., and T. D. Schell. 2002. The immune response to SV40, JCV, andBKV. In Human Polyomaviruses. K. Khalili, and G. L. Stoner, eds. John Wiley &Sons, Inc., New York, p. 585–610.

11. Mylin, L. M., R. H. Bonneau, J. D. Lippolis, and S. S. Tevethia. 1995. Hierarchyamong multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simianvirus 40 T antigen. J. Virol. 69: 6665–6677.

12. Mylin, L. M., T. D. Schell, D. Roberts, M. Epler, A. Boesteanu, E. J. Collins,J. A. Frelinger, S. Joyce, and S. S. Tevethia. 2000. Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large Tantigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformedcells, or vaccinia virus recombinants expressing full-length T antigen or epitopeminigenes. J. Virol. 74: 6922–6934.

13. Fu, T.-M., L. M. Mylin, T. D. Schell, I. Bacik, G. Russ, J. W. Yewdell,J. R. Bennink, and S. S. Tevethia. 1998. An endoplasmic reticulum-targetingsignal sequence enhances the immunogenicity of an immunorecessive simianvirus 40 large T antigen cytotoxic T-lymphocyte epitope. J. Virol. 72: 1469–1481.

14. Otahal, P., S. C. Hutchinson, L. M. Mylin, M. J. Tevethia, S. S. Tevethia, andT. D. Schell. 2005. Inefficient cross-presentation limits the CD8+ T cell responseto a subdominant tumor antigen epitope. J. Immunol. 175: 700–712.

15. Overwijk, W. W., A. Tsung, K. R. Irvine, M. R. Parkhurst, T. J. Goletz, K. Tsung,M. W. Carroll, C. Liu, B. Moss, S. A. Rosenberg, and N. P. Restifo. 1998. gp100/pmel 17 is a murine tumor rejection antigen: induction of “self”-reactive,tumoricidal T cells using high-affinity, altered peptide ligand. J. Exp. Med. 188:277–286.

16. Dyall, R., W. B. Bowne, L. W. Weber, J. LeMaoult, P. Szabo, Y. Moroi,G. Piskun, J. J. Lewis, A. N. Houghton, and J. Nikolic-Zugic. 1998. Heterocliticimmunization induces tumor immunity. J. Exp. Med. 188: 1553–1561.

17. Slansky, J. E., F. M. Rattis, L. F. Boyd, T. Fahmy, E. M. Jaffee, J. P. Schneck,D. H. Margulies, and D. M. Pardoll. 2000. Enhanced antigen-specific antitumorimmunity with altered peptide ligands that stabilize the MHC-peptide-TCRcomplex. Immunity 13: 529–538.

18. van Stipdonk, M. J. B., D. Badia-Martinez, M. Sluijter, R. Offringa, T. van Hall,and A. Achour. 2009. Design of agonistic altered peptides for the robust in-duction of CTL directed towards H-2Db in complex with the melanoma-associated epitope gp100. Cancer Res. 69: 7784–7792.

19. Chen, J.-L., G. Stewart-Jones, G. Bossi, N. M. Lissin, L. Wooldridge, E. M. L. Choi,G. Held, P. R. Dunbar, R. M. Esnouf, M. Sami, et al. 2005. Structural andkinetic basis for heightened immunogenicity of T cell vaccines. J. Exp. Med.201: 1243–1255.

20. Borbulevych, O. Y., T. K. Baxter, Z. Yu, N. P. Restifo, and B. M. Baker. 2005.Increased immunogenicity of an anchor-modified tumor-associated antigen isdue to the enhanced stability of the peptide/MHC complex: implications forvaccine design. J. Immunol. 174: 4812–4820.

21. Sette, A., A. Vitiello, B. Reherman, P. Fowler, R. Nayersina, W. M. Kast,C. J. Melief, C. Oseroff, L. Yuan, J. Ruppert, et al. 1994. The relationship be-tween class I binding affinity and immunogenicity of potential cytotoxic T cellepitopes. J. Immunol. 153: 5586–5592.

22. Assarsson, E., J. Sidney, C. Oseroff, V. Pasquetto, H.-H. Bui, N. Frahm,C. Brander, B. Peters, H. Grey, and A. Sette. 2007. A quantitative analysis of thevariables affecting the repertoire of T cell specificities recognized after vacciniavirus infection. J. Immunol. 178: 7890–7901.

23. van der Burg, S. H., M. J. Visseren, R. M. Brandt, W. M. Kast, and C. J. Melief.1996. Immunogenicity of peptides bound to MHC class I molecules depends onthe MHC-peptide complex stability. J. Immunol. 156: 3308–3314.

24. Lipford, G. B., S. Bauer, H. Wagner, and K. Heeg. 1995. In vivo CTL inductionwith point-substituted ovalbumin peptides: immunogenicity correlates withpeptide-induced MHC class I stability. Vaccine 13: 313–320.

25. Levitsky, V., Q. J. Zhang, J. Levitskaya, and M. G. Masucci. 1996. The life spanof major histocompatibility complex-peptide complexes influences the efficiencyof presentation and immunogenicity of two class I-restricted cytotoxicT lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4. J. Exp. Med.183: 915–926.

26. Harndahl, M., M. Rasmussen, G. Roder, I. Dalgaard Pedersen, M. Sørensen,M. Nielsen, and S. Buus. 2012. Peptide-MHC class I stability is a better pre-dictor than peptide affinity of CTL immunogenicity. Eur. J. Immunol. 42: 1405–1416.

27. Huang, A. Y., P. Golumbek, M. Ahmadzadeh, E. Jaffee, D. Pardoll, andH. Levitsky. 1994. Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science 264: 961–965.

28. Wolkers, M. C., G. Stoetter, F. A. Vyth-Dreese, and T. N. M. Schumacher. 2001.Redundancy of direct priming and cross-priming in tumor-specific CD8+ T cellresponses. J. Immunol. 167: 3577–3584.

29. Staveley-O’Carroll, K., T. D. Schell, M. Jimenez, L. M. Mylin, M. J. Tevethia,S. P. Schoenberger, and S. S. Tevethia. 2003. In vivo ligation of CD40 enhancespriming against the endogenous tumor antigen and promotes CD8+ T cell ef-fector function in SV40 T antigen transgenic mice. J. Immunol. 171: 697–707.

30. Tevethia, M. J. 1984. Immortalization of primary mouse embryo fibroblasts withSV40 virions, viral DNA, and a subgenomic DNA fragment in a quantitativeassay. Virology 137: 414–421.

31. Cavender, J. F., A. Conn, M. Epler, H. Lacko, and M. J. Tevethia. 1995. Simianvirus 40 large T antigen contains two independent activities that cooperate witha ras oncogene to transform rat embryo fibroblasts. J. Virol. 69: 923–934.

32. Tanaka, Y., and S. S. Tevethia. 1990. Loss of immunorecessive cytotoxic Tlymphocyte determinant V on SV40 T antigen following cocultivation with site-specific cytotoxic T lymphocyte clone Y-5. Intervirol. 31: 197–202.

33. Campbell, A. E., F. L. Foley, and S. S. Tevethia. 1983. Demonstration of multipleantigenic sites of the SV40 transplantation rejection antigen by using cytotoxicT lymphocyte clones. J. Immunol. 130: 490–492.

34. Schell, T. D., and S. S. Tevethia. 2001. Control of advanced choroid plexustumors in SV40 T antigen transgenic mice following priming of donor CD8(+)T lymphocytes by the endogenous tumor antigen. J. Immunol. 167: 6947–6956.

35. Ljunggren, H. G., and K. Karre. 1985. Host resistance directed selectivelyagainst H-2-deficient lymphoma variants. Analysis of the mechanism. J. Exp.Med. 162: 1745–1759.

36. Hudrisier, D., H. Mazarguil, F. Laval, M. B. Oldstone, and J. E. Gairin. 1996.Binding of viral antigens to major histocompatibility complex class I H-2Dbmolecules is controlled by dominant negative elements at peptide non-anchorresidues. Implications for peptide selection and presentation. J. Biol. Chem. 271:17829–17836.

37. Yadav, R., Y. Yoshimura, A. Boesteanu, G. J. Christianson, W. U. Ajayi,R. Shashidharamurthy, A. K. Stanic, D. C. Roopenian, and S. Joyce. 2003. TheH4b minor histocompatibility antigen is caused by a combination of geneticallydetermined and posttranslational modifications. J. Immunol. 170: 5133–5142.

38. Karre, K., H. G. Ljunggren, G. Piontek, and R. Kiessling. 1986. Selective re-jection of H-2-deficient lymphoma variants suggests alternative immune defencestrategy. Nature 319: 675–678.

The Journal of Immunology 11

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

39. Deckhut, A. M., and S. S. Tevethia. 1992. Effect of point mutations in the nativesimian virus 40 tumor antigen, and in synthetic peptides corresponding to the H-2Db-restricted epitopes, on antigen presentation and recognition by cytotoxicT lymphocyte clones. J. Immunol. 148: 3012–3020.

40. Schell, T. D., J. D. Lippolis, and S. S. Tevethia. 2001. Cytotoxic T lymphocytesfrom HLA-A2.1 transgenic mice define a potential human epitope from simianvirus 40 large T antigen. Cancer Res. 61: 873–879.

41. Kotturi, M. F., I. Scott, T. Wolfe, B. Peters, J. Sidney, H. Cheroutre, M. G. vonHerrath, M. J. Buchmeier, H. Grey, and A. Sette. 2008. Naive precursor fre-quencies and MHC binding rather than the degree of epitope diversity shapeCD8+ T cell immunodominance. J. Immunol. 181: 2124–2133.

42. Obar, J. J., K. M. Khanna, and L. Lefrancois. 2008. Endogenous naive CD8+T cell precursor frequency regulates primary and memory responses to infection.Immunity 28: 859–869.

43. Moon, J. J., H. H. Chu, M. Pepper, S. J. McSorley, S. C. Jameson, R. M. Kedl, andM. K. Jenkins. 2007. Naive CD4(+) T cell frequency varies for different epitopesand predicts repertoire diversity and response magnitude. Immunity 27: 203–213.

44. Lazarski, C. A., F. A. Chaves, S. A. Jenks, S. Wu, K. A. Richards, J. M. Weaver,and A. J. Sant. 2005. The kinetic stability of MHC class II:peptide complexes isa key parameter that dictates immunodominance. Immunity 23: 29–40.

45. Sigal, L. J., and D. E. Wylie. 1996. Role of non-anchor residues of Db-restrictedpeptides in class I binding and TCR triggering. Mol. Immunol. 33: 1323–1333.

46. Sigal, L. J., P. Goebel, and D. E. Wylie. 1995. Db-binding peptides from in-fluenza virus: effect of non-anchor residues on stability and immunodominance.Mol. Immunol. 32: 623–632.

47. Mylin, L. M., T. D. Schell, M. Epler, C. Kusuma, D. Assis, C. Matsko, A. Smith,A. Allebach, and S. S. Tevethia. 2007. Diversity of escape variant mutations inSimian virus 40 large tumor antigen (SV40 Tag) epitopes selected by cytotoxicT lymphocyte (CTL) clones. Virology 364: 155–168.

48. Velloso, L. M., J. Michaelsson, H.-G. Ljunggren, G. Schneider, and A. Achour.2004. Determination of structural principles underlying three different modes oflymphocytic choriomeningitis virus escape from CTL recognition. J. Immunol.172: 5504–5511.

49. Meijers, R., C.-C. Lai, Y. Yang, J.-H. Liu, W. Zhong, J.-H. Wang, andE. L. Reinherz. 2005. Crystal structures of murine MHC Class I H-2 D(b) andK(b) molecules in complex with CTL epitopes from influenza A virus: impli-cations for TCR repertoire selection and immunodominance. J. Mol. Biol. 345:1099–1110.

50. Kedl, R. M., W. A. Rees, D. A. Hildeman, B. Schaefer, T. Mitchell, J. Kappler,and P. Marrack. 2000. T cells compete for access to antigen-bearing antigen-presenting cells. J. Exp. Med. 192: 1105–1113.

51. Yu, Z., M. R. Theoret, C. E. Touloukian, D. R. Surman, S. C. Garman,L. Feigenbaum, T. K. Baxter, B. M. Baker, and N. P. Restifo. 2004. Poor im-munogenicity of a self/tumor antigen derives from peptide-MHC-I instabilityand is independent of tolerance. J. Clin. Invest. 114: 551–559.

52. Hemmer, B., I. Stefanova, M. Vergelli, R. N. Germain, and R. Martin. 1998.Relationships among TCR ligand potency, thresholds for effector functionelicitation, and the quality of early signaling events in human T cells. J.Immunol. 160: 5807–5814.

53. Henrickson, S. E., T. R. Mempel, I. B. Mazo, B. Liu, M. N. Artyomov, H. Zheng,A. Peixoto, M. P. Flynn, B. Senman, T. Junt, et al. 2008. T cell sensing of antigendose governs interactive behavior with dendritic cells and sets a threshold forT cell activation. Nat. Immunol. 9: 282–291.

54. Howarth, M., A. Williams, A. B. Tolstrup, and T. Elliott. 2004. Tapasin enhancesMHC class I peptide presentation according to peptide half-life. Proc. Natl.Acad. Sci. USA 101: 11737–11742.

55. West, M. A., R. P. A. Wallin, S. P. Matthews, H. G. Svensson, R. Zaru,H.-G. Ljunggren, A. R. Prescott, and C. Watts. 2004. Enhanced dendriticcell antigen capture via toll-like receptor-induced actin remodeling. Science305: 1153–1157.

56. Sallusto, F., M. Cella, C. Danieli, and A. Lanzavecchia. 1995. Dendritic cells usemacropinocytosis and the mannose receptor to concentrate macromolecules inthe major histocompatibility complex class II compartment: downregulation bycytokines and bacterial products. J. Exp. Med. 182: 389–400.

57. Huppa, J. B., M. Gleimer, C. Sumen, and M. M. Davis. 2003. Continuous T cellreceptor signaling required for synapse maintenance and full effector potential.Nat. Immunol. 4: 749–755.

58. Mempel, T. R., S. E. Henrickson, and U. H. Von Andrian. 2004. T-cell primingby dendritic cells in lymph nodes occurs in three distinct phases. Nature 427:154–159.

59. Stuge, T. B., S. P. Holmes, S. Saharan, A. Tuettenberg, M. Roederer, J. S. Weber,and P. P. Lee. 2004. Diversity and recognition efficiency of T cell responses tocancer. PLoS Med. 1: e28.

60. Baumgartner, C. K., A. Ferrante, M. Nagaoka, J. Gorski, and L. P. Malherbe.2010. Peptide-MHC class II complex stability governs CD4 T cell clonal se-lection. J. Immunol. 184: 573–581.

61. Willis, R. A., J. W. Kappler, and P. C. Marrack. 2006. CD8 T cell competition fordendritic cells in vivo is an early event in activation. Proc. Natl. Acad. Sci. USA103: 12063–12068.

62. Grufman, P., E. Z. Wolpert, J. K. Sandberg, and K. Karre. 1999. T cell com-petition for the antigen-presenting cell as a model for immunodominance in thecytotoxic T lymphocyte response against minor histocompatibility antigens. Eur.J. Immunol. 29: 2197–2204.

63. Weaver, J. M., F. A. Chaves, and A. J. Sant. 2009. Abortive activation of CD4T cell responses during competitive priming in vivo. Proc. Natl. Acad. Sci. USA106: 8647–8652.

64. Galea, I., J. Stasakova, M. S. Dunscombe, C. H. Ottensmeier, T. Elliott, andS. M. Thirdborough. 2012. CD8+ T-cell cross-competition is governed bypeptide-MHC class I stability. Eur. J. Immunol. 42: 256–263.

65. La Gruta, N. L., W. T. Rothwell, T. Cukalac, N. G. Swan, S. A. Valkenburg,K. Kedzierska, P. G. Thomas, P. C. Doherty, and S. J. Turner. 2010. Primary CTLresponse magnitude in mice is determined by the extent of naive T cell re-cruitment and subsequent clonal expansion. J. Clin. Invest. 120: 1885–1894.

66. Chen, W., K. Pang, K.-A. Masterman, G. Kennedy, S. Basta, N. Dimopoulos,F. Hornung, M. Smyth, J. R. Bennink, and J. W. Yewdell. 2004. Reversal in theimmunodominance hierarchy in secondary CD8+ T cell responses to influenza Avirus: roles for cross-presentation and lysis-independent immunodomination. J.Immunol. 173: 5021–5027.

12 APLs ENHANCE CROSS-PRIMING OF CD8+ T CELLS

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from