of February 13, 2018.This information is current as

Tumor in a Murine Glioma ModelPrimed as a Function of Proximity to theand Quantitative Defects in CD8 T Cells Vaccine Injection Site Matters: Qualitative

Andres M. Salazar and Michael R. OlinJunzhe Xia, Christopher A. Pennell, Lauryn E. Swier, John R. Ohlfest, Brian M. Andersen, Adam J. Litterman,

http://www.jimmunol.org/content/190/2/613doi: 10.4049/jimmunol.1201557December 2012;

2013; 190:613-620; Prepublished online 17J Immunol 

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The Journal of Immunology

Vaccine Injection Site Matters: Qualitative and QuantitativeDefects in CD8 T Cells Primed as a Function of Proximity tothe Tumor in a Murine Glioma Model

John R. Ohlfest,* Brian M. Andersen,* Adam J. Litterman,* Junzhe Xia,*

Christopher A. Pennell,† Lauryn E. Swier,* Andres M. Salazar,‡ and Michael R. Olin*

Malignant gliomas are lethal brain tumors for which novel therapies are urgently needed. In animal models, vaccination with

tumor-associated Ags efficiently primes T cells to clear gliomas. In clinical trials, cancer vaccines have been less effective at priming

T cells and extending survival. Generalized immune suppression in the tumor draining lymph nodes has been documented in mul-

tiple cancers. However, a systematic analysis of how vaccination at various distances from the tumor (closest to farthest) has not been

reported. We investigated how the injection site chosen for vaccination dictates CD8 T cell priming and survival in an OVA-

transfected murine glioma model. Glioma-bearing mice were vaccinated with Poly:ICLC plus OVA protein in the neck, hind

leg, or foreleg for drainage into the cervical, inguinal, or axillary lymph nodes, respectively. OVA-specific CD8 T cell number, TCR

affinity, effector function, and infiltration into the brain decreased as the vaccination site approached the tumor. These effects were

dependent on the presence of the tumor, because injection site did not appreciably affect CD8 T cell priming in tumor-free mice.

Our data suggest the site of vaccination can greatly impact the effectiveness of cancer vaccines. Considering that previous and

ongoing clinical trials have used a variety of injection sites, vaccination site is potentially a critical aspect of study design that is

being overlooked. The Journal of Immunology, 2013, 190: 613–620.

Active tumor immunotherapy shows great promise in an-imal models but has yet to achieve widespread successin the clinic. Vaccines have been extensively tested in

clinical trials for the treatment of glioma. Glioma patients havebeen vaccinated in multiple sites including the scapula draininginto the axilla (1), the anterior upper thigh (2), the upper arm (3),and the cervical regions (4, 5). Data are insufficient to correlateresponse rates with vaccination site. Few basic studies have exam-ined priming after vaccination as it relates to anatomic location.The sentinel lymph nodes (draining lymph node [DLN] nearest

to the tumor) are in direct lymphatic drainage from the primarytumor (6, 7) and are the DLNs most prone to immune suppression(8–10). In breast cancer and melanoma patients, T cells isolatedfrom the sentinel lymph nodes have suppressed activation and pro-liferation in response to various mitogens compared with T cellsisolated from the more distal lymph nodes (11–14). Multiple mech-anisms contribute to this local suppression. Tumor-elaborated solublefactors, such as TGF-b and PGE2 (15–18), can act at the tumor

site or DLN to dampen T cell reactivity. In experimental systems,additional documented mechanisms of local immune suppressionat the DLN include regulatory T cell–mediated killing of tumorAg-presenting dendritic cells (DCs) (19) and TCR nitration bymyeloid-derived suppressor cells (20).With respect to brain tumors, the immunologically specialized

nature of the brain and its draining cervical lymph nodes mustalso be considered. Initial experiments revealed that vaccinationwith Ag into the brain can trigger higher Ab titers comparedwith vaccination in the periphery (21). In contrast, Th1-mediated,delayed-type hypersensitivity responses are absent or bluntedwhen the same Ag is delivered to the brain (reviewed in Ref. 22).These findings support a model whereby the cervical lymph nodeshave an intrinsic Th2 bias in steady-state conditions. Additionalexperiments showed that CD8 T cells undergo initial expansionafter intracerebral tumor cell challenge, but fail to differentiateinto CTLs (23). However, it was unclear whether this was due totumor-induced immune suppression, a lack of costimulation, or anintrinsic bias against CTL development in the cervical lymphnodes. Normal mouse cerebrospinal fluid can suppress CD8T cell activation in ex vivo assays, which is restored by a TGF-bblocking Ab (24), implicating brain-derived TGF-b as one solublemediator of CTL suppression in the cervical lymph nodes.Despite support for Th2 immune deviation in the brain DLNs,

there is evidence that CD8 T cell responses play a tumoricidal rolein human gliomas. Infiltration of CD8 T cells is a positive prog-nostic factor in glioma patients (25). Furthermore, immunologicalsynapses between CD8 T cells and glioma cells have been docu-mented in humans (26). Interestingly, glioblastoma patients re-ceiving autologous tumor lysate-pulsed DC vaccines had superiorsurvival when their gene expression was of mesenchymal ratherthan the proneural molecular signature; the mesenchymal signa-ture is inflammatory and was correlated with significantly moreinfiltrating CD8 T cells at the tumor site compared with proneuraltumors (27). Regardless of these spontaneous or vaccine-induced

*Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455;†Department of Laboratory Medicine and Pathology, University of Minnesota, Min-neapolis, MN 55455; and ‡Oncovir, Inc., Washington, DC 20008

Received for publication June 6, 2012. Accepted for publication November 8, 2012.

This work was supported by National Institutes of Health Grants T32 GM008244(Medical Scientist Training Program; to B.M.A.), T32 CA009138 (Cancer BiologyTraining Grant to B.M.A.), R01 CA154345 (to J.R.O.), and R01 CA160782 (to J.R.O.);a Torske Klubben Fellowship for Minnesota Residents (to B.M.A.); American CancerSociety Grant RSG-09-189-01-LIB (to J.R.O.); the Minnesota Medical Foundation(J.R.O.); the Hedberg Family Foundation (J.R.O.); the Children’s Cancer ResearchFund (J.R.O.); and the Randy Shaver Foundation (M.R.O.).

Address correspondence and reprint requests to Dr. Michael R. Olin, Departmentof Pediatrics, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail address:olin0012@umn.edu

Abbreviations used in this article: BIL, brain-infiltrating lymphocyte; DC, dendriticcell; DLN, draining lymph node; MFI, mean fluorescent intensity; NTF, normalizedtotal fluorescence.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

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T cell responses, global immune suppression has been widelyaccepted to occur in glioma patients. Many studies done to estab-lish this conclusion were conducted with leukocytes harvestedafter treatment with glucocorticoids or chemotherapy, cloudingthe contribution of the tumor versus the treatment on dampenedimmunity. More recent data suggest that treatment with gluco-corticoids and alkylating chemotherapy plays a significant role ininducing global immune suppression, because both drugs are as-sociated with rapid posttreatment lymphopenia, and elevation inregulatory T cell or myeloid-derived suppressor cell frequency(28, 29). The severity of lymphopenia following the standard ofcare (steroids, chemotherapy, and radiation) negatively correlateswith overall survival in glioblastoma patients (30).There is stronger experimental evidence for profound local im-

mune suppression at the tumor site in gliomas. Studies in spontaneousmurine models suggest that gliomas accrue immune-suppressivecell populations even at early, asymptomatic stages (19). As gli-omas develop, recruitment of myeloid-derived suppressor cells ismediated, in part, through a cyclooxygenase-2 and PGE2 axis (31),decreasing CTL recruitment to the tumor bed. Local immunesuppression is further enforced by hypoxia, leading to reducedeffector function of T cells via increased production of immuno-suppressive factors (32). Moreover, glioma cells express inhibitoryligands that can trigger T cell tolerance including HLA-E and B7-H1 (33, 34). However, little is known about how these mechanismsof suppression at the tumor site impact priming responses in thecervical lymph nodes. One purpose of this study was to shed in-sight on this poorly understood area.Based on evidence that much of the tumor-derived immune sup-

pression in other malignancies is anatomically graded rather thanglobal (8–10, 35, 36), we reasoned that vaccination distal to gliomascould be exploited to improve active immunotherapy. We focusedour investigation on the relation between vaccination sites and theensuing CD8 T cell response. Vaccination in close proximity to thetumor DLN (cervical or axillary regions) resulted in no survivalbenefit. In contrast, vaccinations in DLN further from the brain (inthe inguinal region) increased survival of glioma-bearing mice.Mice vaccinated in the cervical region had CD8 T cells with lowerTCR affinity and weakened effector functions compared withpriming at the inguinal region. Considering that previous clinicaltrials have used a variety of injection sites, these data reveala neglected, yet potentially critical variable for trial design. In-jection site is a crucial aspect of active tumor immunotherapy inthis murine model that demands investigation in glioma patients.

Materials and MethodsAnimal models and cell lines

Tumors were implanted into female C57BL/6 mice (6–8 wk old) that werepurchased from The Jackson Laboratory and maintained in a specificpathogen-free facility according to the guidelines of the University ofMinnesota Animal Care and Use Committee. The GL261 orthotopictransplant model was established in C57BL/6 (B6) or B6 Nur77GFP

transgenic mice (37) (kind gift from Dr. Kristin Hogquist, University ofMinnesota Center for Immunology) by inoculation with 15,000 GL261cells or OVA-transfected GL261 cells (GL261-OVA hereafter) in 1 ml PBS.Tumors were implanted stereotactically into the right striatum; coordinateswere 2.5 mm lateral, 0.5 mm anterior of bregma, and 3 mm deep from thecortical surface of the brain (38).

To generate GL261-OVA, we constructed a minigene encoding fourpeptides presented by H-2b class I or II molecules as a single coding se-quence. The nucleotide sequences were codon optimized for expressionin mice and were synthesized (GenScript, Piscataway, NJ). The peptideswere encoded in the following order: human gp10025–33 (presented by Db),chicken OVA257–264 (presented by Kb), chicken OVA323–339 (presented byI-Ab), and mouse I-Ea52–68 (presented by I-Ab). To facilitate Ag process-ing, each peptide was flanked on both sides by the six amino acid residuesthat surrounded the peptide in the corresponding native protein. This

construct was inserted into the multiple cloning site of the pIRES-DsRed-Express vector (Clontech, Mountain View, CA) linearized with BamHI andEcoRI. Parental GL261 cells were transfected with this vector and selectedusing 2 mg/ml G418. The selected cells were subcloned by limiting di-lution, and a clone with a high level of Ag expression (as assessed byDsRed Express fluorescence) was chosen and used for all subsequentexperiments. GL261-OVA and parental GL261 were cultured in DMEMcontaining 10% FBS, penicillin/streptomycin (100 U/ml), and 0.1 mg/mlNormocin (Invivogen). A total of 500 mg/ml G418 was added to maintainselection of GL261-OVA cells during routine culture. GL261 cells fortumor lysate vaccine were cultured in neural stem cell media consistingof DMEM/F12 (1:1) with L-glutamine, sodium bicarbonate, penicillin/streptomycin (100 U/ml), B27 and N2 supplements (Life Technologies),and 0.1-mg/ml Normocin (Invivogen). Cultures were maintained at 5% O2

and supplemented with 20 ng/ml EGF and FGF semiweekly (R&D Sys-tems, Minneapolis, MN).

Vaccine preparation and injection

Tumor lysates were prepared by dissociating cells with nonenzymaticdissociation solution (Sigma), washing twice with PBS, resuspending in500 ml PBS, and freezing initially by placing in280˚C overnight. Cells werefurther lysed by five cycles of freezing in liquid nitrogen and thawing ina 56˚C water bath. Cell debris was pelleted by centrifugation at 14,000relative centrifugal force, and the protein concentration of the supernatantwas determined using a Bradford assay. Pellets were resuspended, andlysates were stored at 280˚C until use. Each vaccine was prepared on theday of vaccination and consisted of 65 mg protein tumor lysate and 50 mgphosphorothioated type-B CpG ODN 685 (59-tcgtcgacgtcgttcgttctc-39; SBIBiotech, Tokyo, Japan) in a final volume of 100 ml, injected intradermallyat the indicated site. For survival studies, vaccines were administered weeklystarting 3 d posttumor implantation for a total of 6 doses. For OVA vacci-nations, 100 mg OVA (Fisher Bioreagents) and 10 mg Poly:ICLC (Oncovir)in 100 ml were injected intradermally in hind leg, foreleg, or back of theneck. For the GL261-OVA survival study, mice were vaccinated on days 14–17 postinoculation, then on days 21–24. For other CD8 T cell primingexperiments, the animals were intradermally vaccinated on 4 consecutivedays with 100 mg OVA and 10 mg Poly:ICLC, and once more on day 7.

TCR affinity measurements

Nur77GFP mouse splenocytes were collected and plated at a concentrationof 7.5 3 105 cells/well in a 96-well plate. Cells were stimulated witheither 1026, 1028, 10210, or 10212 moles of SIINFEKL peptide. Non-stimulated cells were used as a control. After 8-h incubation, cells wereharvested and stained with CD8 and the SIINFEKL/Kb dextramer. GFPexpression within the CD8+ SIINFEKL/H-2Kb dextramer+ gate was an-alyzed by flow cytometry.

Measurement of relative TCR affinity by a competitive tetramer fall-offassay was adapted from a previously described method (39). In brief, 2 3107 splenocytes from the vaccinated Nur77GFP mice were stained with theSIINFEKL/Kb dextramer and anti-CD8 Abs as described above. After twowashes, cells were incubated with or without 0.5 mg unlabeled competitoranti-SIINFEKL-H-2Kb Ab (clone eBio25-D1.16; eBioscience) at 4˚C. Atthe indicated times, 100-ml aliquots were removed and added to an equalvolume of 2% PFA in PBS and run on a Becton Dickinson Canto three-laser flow cytometer. Normalized total fluorescence minus backgroundstaining was calculated using the following formula: (normalized totalfluorescence)time i = (% positive 3 MFI)time i/(% positive 3 MFI)time 0,where MFI is mean fluorescent intensity (as in Ref. 39).

Flow cytometry

A Becton Dickinson Canto three-laser flow cytometer was used for dataacquisition. Anti-mouse CD8a-Pacific Blue (clone 53-6.7) was purchasedfrom eBioscience. A SIINFEKL/Kb dextramer-PE was used for detectionof SIINFEKL/Kb-binding CD8 T cells (Immunodex). For whole-bloodstaining, 50 ml blood was obtained via orbital bleed and placed in 100 mlof a 1:10 dilution of heparin and PBS. Five microliters of SIINFEKL/Kb

dextramer was added to blood and incubated at room temperature for10 min, and 1 ml (0.5 mg) Abs was added to cells and incubated for anadditional 20 min at room temperature. Blood was lysed by adding 1 ml1:10 dilution lysis buffer (BD Pharmingen), incubated for 10 min at roomtemperature, centrifuged twice and resuspended in 100 ml PBS, and ana-lyzed. For analysis of secondary lymphoid tissues, cells were isolated fromDLNs and were suspended at 53 105 cells in 100 ml PBS. Five microlitersSIINFEKL/Kb dextramer was added to cells and incubated at room tem-perature for 10 min; 1 ml (0.5 mg) CD8 Ab was added to cells and incu-

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bated for an additional 20 min at 4˚C; cells were then washed twice andanalyzed by flow cytometry.

Brain-infiltrating lymphocyte isolation

Brain-infiltrating lymphocytes (BILs) were prepared by perfusing four miceper treatment group with PBS. Brains were minced into a crude suspensionin cold RPMI 1640 complete media with 10% FBS. Suspensions were thenpassed through a 40-mm filter, washed once in cold PBS, and spun on thefollowing Percoll gradient: 3 ml 70% (v/v, in PBS) bottom, 3 ml 37%middle, and 4 ml 30% with resuspended pellet. BILs were centrifuged at800 relative centrifugal force at 4˚C for 20 min and collected from the cellinterface and washed in cold RPMI 1640 complete media with 10% FBS.

CTL analyses

These assays were conducted as previously described (40). In brief, DLNswere harvested, dissociated, and lymphocytes were incubated with CFSE-labeled GL261 or GL261-OVA cells for 4 h at E:T ratios of 0:1 and 25:1,and analyzed for cytotoxicity according to the manufacturer’s protocol(Immunochemistry). After incubation, the percentage of CFSE-labeledtarget cells that stained with 7-AAD was determined by flow cytometry.Data were reported as specific lysis by correcting for background 7-AADstaining using the following formula: % CFSE+ 7-AAD+ at 25:1 E:T ratio 2% CFSE+ 7-AAD+ at 0:1 E:T ratio.

IFN-g detection

Lymphocytes isolated from DLNs were plated at a concentration of 53 105

cells/well and pulsed with 2 mg SIINFEKL peptide for 12 h. After incu-bation, 50 ml culture supernatant was analyzed for IFN-g using a flowcytometric bead array according to the manufacturer’s protocol (BDBiosciences). Cells were then harvested, surface-stained with CD8 andSIINFEKL/Kb dextramer, then intracellularly stained for IFN-g accordingto the manufacturer’s protocol (BD Biosciences). To determine the numberof secreted molecules of IFN-g per Ag-specific CD8 T cell, IFN-g con-centration was converted to molarity and divided by the number of platedcells per well. In addition, lymphocytes were harvested from the lymphnodes, stained for CD8, then intracellularly stained for IFN-g. Flowcytometry was used to determine the fraction of viable cells that were Ag-specific CD8 T cells, and a hemocytometer was used to determine thenumber of viable cells plated.

Statistical analysis

Statistical comparisons were made by ANOVA, followed by post hoccomparisons using a two-tailed t test. Differences in animal survival wereevaluated by log-rank test. All tests were performed with Prism 4 software(GraphPad Software). The p values ,0.05 were considered significant.Column statistics were calculated to validate significance within the valuesof each treatment group.

ResultsVaccination in the hind leg enhances survival benefit

We previously demonstrated that vaccinating glioma-bearing micewith a poly-Ag tumor lysate/CpG vaccine enhanced survival (40).However, these previous studies relied on vaccination in two sitesin the same animal: the hind leg and the neck, which is in contrastwith most clinical trials that use single vaccination sites. To in-vestigate potential differences in efficacy of vaccination amongthree different sites, we vaccinated glioma-bearing mice in theback of the neck, fore leg, or hind leg with GL261 lysate mixedwith CpG. Mice primed in the hind leg exhibited significantlyincreased long-term survival compared with mice vaccinated inthe back of the neck, foreleg, or saline control, which had nosurvival benefit (Fig. 1A).Although this experiment was suggestive of differential T cell

priming, the use of a poly-Ag lysate vaccine did not permittracking of Ag-specific T cells. To test an Ag-specific response, wedeveloped a model whereby mice bearing intracranial GL261-OVAwere vaccinated with OVA plus Poly:ICLC in three different sites.In this experiment, there was an incremental decrease in survival asthe vaccination site approached the tumor. Mice vaccinated in theback of the neck showed no survival benefit compared with control

animals, whereas mice vaccinated in the hind leg demonstrated thegreatest survival benefit (Fig. 1B).

CD8 T cell priming is suppressed through an anatomicgradient

We measured priming of an endogenous CD8 T cell responseagainst the OVA Ag by flow cytometry using a SIINFEKL/H2-Kb

dextramer. Mice were inoculated with GL261-OVA and vacci-nated in the back of the neck, foreleg, or the hind leg for drainageinto the cervical, axillary, or inguinal lymph nodes, respectively.In all tissues analyzed, there was a stepwise decline in the fre-quency of dextramer-binding CD8 T cells as the vaccination siteapproached the tumor in the following order: hind leg. foreleg.neck (Fig. 2B). There were several notable differences in CD8 Tfrequency in each tissue. In the blood, dextramer-binding CD8T cells averaged from ∼11 to 28% of the CD8 T cell compartmentdepending on the site of priming. Mice primed in the rear leg hadroughly twice as many dextramer-staining cells compared withother sites. In the lymph nodes draining the respective vaccinationsite, CD8 T cell frequency was much lower than blood, averaging∼1.5–2.8% depending on the site of priming. The BILs followeda similar trend compared with the vaccine DLNs, but at muchhigher frequency, ranging from 18 to 60% of the CD8 T cellsduring dextramer binding. There was a 3-fold increase in thedextramer-binding CD8 T cell frequency in mice vaccinated in thehind leg compared with back of the neck. There was also a changein the absolute number of BILs that paralleled the trend of CD8T cell frequency, but this failed to reach statistical significancebecause of high animal-to-animal variability (Fig. 2B).

Presence of the tumor locally alters the affinity of the TCR

Subsequent experiments were conducted to determine whether thelower T cell priming was due to an intrinsic bias of the cervical

FIGURE 1. Vaccination in the hind leg has superior therapeutic benefit.

(A) Vaccination with GL261 tumor lysate and CpG was administered

weekly starting 3 d post-GL261 implantation. (B) OVA plus Poly:ICLC

vaccination was administered for 4 consecutive days starting 14 d post–

OVA-GL261 implantation, then on days 21–24 for a total of eight doses.

Mice were monitored for survival. Kaplan–Meier plot illustrating survival

(n = 10/group; *p , 0.01, log-rank test). Data are representative of two

independent experiments.

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lymph nodes themselves or from the presence of the tumor. Theseexperiments also addressed how the presence of the cognate Ag(OVA) being expressed in the tumor impacted priming. To answerthese questions, we inoculated mice with the GL261-OVA or theparental GL261cells; saline injection was performed in the “notumor” group of mice to control for brain injury. Mice werevaccinated with OVA plus Poly:ICLC in the back of the neck fordrainage into the cervical lymph nodes. Whole blood was col-lected and analyzed as described above. As shown in Fig. 3A, thepresence of the tumor reduced the dextramer-binding CD8 T cellfrequency by half regardless of whether the tumor expressed OVA.GL261 and GL261-OVA groups were not significantly different.Thus, the presence of the tumor in the brain is responsible for thesuppression in priming in the cervical lymph nodes. Furthermore,the change in priming is not dependent on expression of the Agused for vaccination in the tumor.We then focused on qualitative differences in CD8 T cells primed

in the rear leg or neck, exclusively in tumor-bearing mice, becausethis is most relevant to a clinical scenario. The MFI of dextramer-binding CD8 T cells was significantly lower when vaccination wasadministered in the neck compared with the rear leg (Fig. 3B),suggesting that TCR affinity might be altered. To test TCR affinity,we first used a novel Nur77GFP reporter mouse that was recentlydescribed (37). In this mouse model, GFP expression is a surro-gate for TCR signaling strength and is not affected by costimu-lation delivered by the APC (37). Glioma-bearing Nur77GFP micewere repeatedly vaccinated in the neck or rear leg. One day after

the last vaccine, mice were sacrificed and their splenocytes werestimulated with various concentrations of SIINFEKL peptide.Dextramer-binding CD8 T cells were gated on and analyzed forGFP expression. Importantly, there was no appreciable differencein GFP expression in the absence of exogenous SIINFEKL pep-tide, demonstrating that the baseline TCR signaling was compa-rable in the splenocytes harvested from each group (Fig. 3C).There was a striking decrease in TCR signaling in CD8 T cellsfrom the cervical group relative to the inguinal group when Agconcentration was limiting; .2-fold differences at 10212–1028 MSIINFEKL. To confirm that GFP expression truly reflected TCRaffinity, we also conducted a standard tetramer fall-off assay ona portion of splenocytes harvested from the same Nur77GFP mice.Fig. 3D shows that dextramer-binding CD8 T cells primed byvaccination draining to the cervical lymph nodes had a signifi-cantly faster rate of tetramer fall off compared with the inguinalgroup. Collectively, these data reveal a qualitative change in TCRaffinity as affected by the location of vaccination in relation to thetumor DLNs.

Effector function of CD8 T cells is changed as a consequenceof vaccination site

We investigated how vaccination site alters effector cytokineelaboration and cytotoxic function of CD8 T cells. Mice wereprimed at multiple sites as before, and the lymph nodes draining thevaccination sites were harvested and used as a source of effectorcells. Lymphocytes were stimulated for 12 h in vitro with the OVA-

FIGURE 2. T cell priming is suppressed as vaccination site approaches the tumor. Glioma-bearing mice were vaccinated for 4 consecutive days and

boosted on day 7. On day 8, mice were sacrificed and analyzed using flow cytometry. (A) Gating scheme used. (B) Whole blood, lymph nodes, and BILs

were analyzed for an endogenous CD8 T cell response. Error bars are representative of SD (n = 8 mice/group for blood and lymph nodes, n = 4 for BILs;

*p , 0.05, **p , 0.01, t test). Data are representative of two independent experiments.

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derived SIINFEKL peptide or unpulsed as a control. There wasa .10-fold increase in soluble IFN-g measured in responder cellsfrom the inguinal lymph node compared with axillary (Fig. 4A).Cells from the cervical lymph node completely failed to elaborateIFN-g in response to stimulation with SIINFEKL.To better characterize the responding cells, we used flow

cytometry to determine the frequency of dextramer-binding cells inthe CD8 gate secreting IFN-g, and the amount of IFN-g producedper cell, after peptide stimulation in vitro. The number of solubleIFN-g molecules elaborated per dextramer-binding CD8 T cellwas calculated (Fig. 4B), revealing large differences in func-tional capacity on a per cell basis as affected by vaccination site.Consistent with previous experiments, mice primed in the neckin the absence of a tumor had a significant restoration of effectorfunction as measured by IFN-g response to SIINFEKL stimulation.Intracellular flow cytometry confirmed that dextramer-binding CD8T cells were the source of IFN-g (Fig. 4C). To assess the in vivoeffector function of tumoricidal T cells, unstimulated lymphocytes

were isolated from the various lymph nodes and analyzed intra-cellularly for IFN-g production. Mice primed in the hind leg hada significant increase of IFN-g compared with the other treatmentgroups (Fig. 4D). This response correlated with previous experi-ments demonstrating that priming near the tumor environment hadsuppressed effector function.We next investigated CD8 T cell tumoricidal function in tumor-

bearing mice primed with OVA and Poly:ICLC vaccination.Lymphocytes isolated from lymph nodes draining the vaccinationsite were used as a source of CTLs. CTLs from mice vaccinated inthe hind leg had significantly greater tumoricidal function com-pared with mice vaccinated in the foreleg (Fig. 4E). Cells isolatedfrom the cervical lymph nodes had negligible killing activity,but tumoricidal function was restored in tumor-free mice. Killingwas mostly dependent on the expression of OVA because parentalGL261 targets had diminished specific lysis. However, cellsprimed by vaccination in the hind leg or foreleg had tumoricidalfunction above background against parental GL261, implying

FIGURE 3. Presence of the tumor alters

TCR affinity after vaccination. Wild-type mice

were vaccinated on days 3–6 and boosted on

day 10 after tumor inoculations. Saline injec-

tion was used to control for brain injury in

tumor-free mice. Mice were sacrificed on day

11. (A) Whole blood was analyzed using flow

cytometry for an endogenous CD8 T cell

response. (B) MFI of the CD8+SIINFEKL/

Kb+ Dextramer population was measured on

splenocytes from Nur77GFP mice and (C) an-

alyzed for GFP expression as a readout of

TCR signaling. (D) Tetramer fall-off assay

conducted on splenocytes used in (C) to fur-

ther document TCR affinity. Error bars are

representative of SD [(A) n = 12/group, (B–D)

n = 7/group; *p , 0.05, **p , 0.01, t test].

(A) Data are representative of two indepen-

dent experiments.

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spontaneous epitope spreading to endogenous GL261 Ags in theselymph nodes. Also consistent with this explanation is the findingthat CTLs from tumor-free mice that were vaccinated with OVAhad no tumoricidal function against parental GL261 targets (Fig.4E). In addition, no significant killing activity was measured withinsaline-vaccinated mice at any site, ruling out differences in base-line killing function in cervical, axillary, and inguinal lymph nodes(data not shown).

DiscussionTumor-induced immune suppression consists of several func-tionally distinct mechanisms, most of which have been reported tocorrupt not only the tumor microenvironment but also local sec-ondary lymphoid tissues in direct drainage of the tumor site (41,42). In this study, we examined for the first time, to our knowl-edge, the relation between vaccination site and treatment efficacyin a glioma model. From an efficacy standpoint measured bysurvival, the results were generalizable to different vaccine adju-vants (CpG or Poly:ICLC) and different Ag sources (lysate orsingle protein). Our data demonstrate a profound suppression ofCD8 T cell priming within the cervical lymph nodes. This sup-pression is tumor dependent, but is not dependent on expression ofthe vaccine Ag in the tumor. This suppressive response is locationrelated, decreasing in severity as the distance from the tumorincreases. Such suppression is likely to occur from secretedtumor-derived factors that reach secondary lymphoid organs ratherthan direct contact with tumor cells because GL261 does notmetastasize to extracranial sites. A clear limitation to our study isthe use of mice with recently established tumors to explore thesequestions. At best, this is a model of minimal residual disease in

a clinical setting. How this animal model relates to humans whohave coevolution of the immune system and tumor for months isan open question. Nonetheless, the results are informative andraise intriguing questions that can be explored in human subjects.Tumor vaccines break tolerance by reversing anergy and/or

cross-priming naive CD8 T cells. Crucial to both processes ispresentation of Ag on MHC class I and induction of costimulatorysignals by DCs (43). We sought to characterize the suppressivemechanisms present in our tumor model from site-based differ-ences in response to vaccination. We found no difference in ex-pression of CD80, CD86, or OX40L in DCs in the cervical lymphnodes of glioma-bearing and tumor-free mice (data not shown).Differential expression of other costimulatory molecules cannotbe ruled out, although a myriad of other suppressive mechanismsexists. Hyperactivation of immune checkpoint molecules such asCTLA-4 or PD-1 may occur from the local presence of tumor-derived factors. Such a mechanism has been associated withanergic or tolerant CD8 T cells whose tumoricidal function can berestored by Ab-mediated blockade (44). In addition, studies reportincreases in DC expression of PD-L1 in tumor DLNs (45). Also,local downregulation of MHC II expression has been shown tolimit CD4 help to tumor-specific CD8 T cells (46), but we did notdetect differences in expression of MHC class II by DCs in ourstudies (data not shown). Regulatory T cells are often increased intumor DLNs (47, 48). We have not tested regulatory T cells levelsin our model. Clearly, many additional studies are needed to morefully characterize the mechanisms of immune suppression in thecervical lymph nodes of glioma-bearing mice.TCR diversity exists in the CD8 T cell precursor clones capable

of responding to vaccination. Differential expansion of clones

FIGURE 4. Impaired effector function of CD8

T cells primed near the tumor. Tumor-bearing and

tumor-free mice were vaccinated on days 3–6 and

boosted on day 10 after tumor inoculation. Lympho-

cytes were isolated from DLNs on day 11, stimulated

with a SIINFEKL peptide, and (A) analyzed for IFN-g

secretion. (B) The molecules of IFN-g elaborated per

Ag-specific cell were determined by calculating the

number of dextramer-binding cells in the tissue cul-

ture in (A). (C) Intracellular flow cytometry was per-

formed on the cultures in (A). (D) Aggregate data from

(C). (E) Total intracellular IFN-g production, and (F)

CTL assays using lymphocytes isolated from the in-

dicated DLNs as effectors. Error bars are representa-

tive of SD [(A–D) n = 4/group, (E) n = 5, and (F) n = 8;

*p , 0.05, **p , 0.01, t test]. Data are representative

of two independent experiments.

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occurs in a hierarchy according to TCR affinity, enabling high-affinity clones to preferentially expand and dominate the re-sponse (39, 49, 50). We found relatively weaker TCR affinity ofCD8 T cells specific for the OVA Ag when vaccination drained tothe cervical lymph nodes of mice with gliomas. It is unlikely thatthymic emigrant populations could contribute to this effect, as1 wk elapsed from time of tumor implantation to vaccination.Rather, this difference may be because of other factors includingrelatively greater expansion of cells with weaker binding to cog-nate Ag, destabilized TCR coreceptor complex (51–53), or per-haps more likely is that cells with stronger TCR signaling areselectively deleted. Additional work is needed to address thesepossibilities. This study is the first, to our knowledge, to demon-strate a meaningful difference in TCR affinity as it relates tovaccination site in tumor-bearing hosts. We postulate that differ-ences in TCR affinity may underlie the inferior effector functionsof CD8 T cells from mice vaccinated near the tumor DLNs, be-cause only high levels of Ag triggered TCR signaling ex vivo inthese mice (Fig. 3C). Accordingly, because of impaired TCRsignaling, it is unlikely that levels of peptide–MHC I are suffi-ciently high in situ to trigger CD8 T cell tumoricidal function inanimals vaccinated near the tumor. Lower TCR affinity and pooreffector function would account for the inferior survival of ani-mals vaccinated nearer to the tumor DLNs.This study has potential implications for the design of transla-

tional research and clinical trials, where injection site is variableand often arbitrarily chosen. These data provide guidelines fora strategic choice of vaccination site in humans and raise severalquestions. For example, in metastatic melanoma, would betterimmune responses be achieved if vaccination sites were consis-tently altered to be farthest away from active tumors? What is theimmune competence of such distal lymph nodes related to thosethat have been studied to date, which are only located aroundthe tumor and resected as part of standard of care? Our results callfor testing if anatomically graded suppression occurs in humans,whose anatomic scale may follow different rules than that of mice.

AcknowledgmentsWe are indebted to Dan Sloper, Rob Shaver, Jovany Cortes, Joe Lalli, and

Nick Erickson for assisting in the harvest of tissues and with survival

studies.

DisclosuresThe authors have no financial conflicts of interest.

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