disruption of cd8þ treg activity results in expansion of t … · 2014. 2. 19. · class ib...

Research Article

Disruption of CD8þ Treg Activity Results in Expansion ofT Follicular Helper Cells and Enhanced Antitumor Immunity

Diana A. Alvarez Arias1,3, Hye-Jung Kim1,3, Penghui Zhou1,3, Tobias A.W. Holderried1,3,5,Xuan Wang1, Glenn Dranoff1,2,4, and Harvey Cantor1,3

AbstractTumor growth is associated with the inhibition of host antitumor immune responses that can impose serious

obstacles to cancer immunotherapy. To define the potential contribution of Qa-1–restricted CD8 regulatory Tcells (Treg) to the development of tumor immunity, we studied B6.Qa-1 D227Kmice that harbor a pointmutationin theMHC class Ib molecule Qa-1 that impairs CD8 Treg suppressive activity. Here, we report that the growth ofB16 melanoma is substantially delayed in these Qa-1–mutant mice after therapeutic immunization with B16melanoma cells engineered to express granulocyte macrophage colony-stimulating factor compared with Qa-1B6-WT controls. Reduced tumor growth is associated with enhanced expansion of follicular T helper cells,germinal center B cells, and high titers of antitumor autoantibodies, which provoke robust antitumor immuneresponses in concert with tumor-specific cytolytic T cells. Analysis of tumor-infiltrating T cells revealed that theQa-1 DK mutation was associated with an increase in the ratio of CD8þ T effectors compared with CD8 Tregs.These data suggest that the CD8þ T effector–Treg ratio may provide a useful prognostic index for cancerdevelopment and raise the possibility that depletion or inactivation of CD8Tregs represents a potentially effectivestrategy to enhance antitumor immunity. Cancer Immunol Res; 2(3); 207–16. �2013 AACR.

IntroductionRecent advances in cancer immunology have generated

considerable interest in approaches that interrupt inhibitorypathways that can constrain antitumor immunity (1, 2). Forexample, definition and analysis of CTLA-4 expression byregulatory and other T cells led to the generation of aCTLA-4 monoclonal antibody (ipilimumab) that substantiallyextends survival in patients with metastatic melanoma (3, 4)and shows promise for achieving durable remissions of othertypes of cancer. Although ipilimumab has been approved as astandard-of-care therapy for advancedmelanoma since 2011, arelatively small fraction (approximately 1/5) of patients treatedwith this drug achieved long-term clinical responses, suggest-ing that targeting additional inhibitory pathways mightincrease the efficacy of this general therapeutic strategy.

Most current approaches directed at regulatory T cells(Treg) have targeted cells within the CD4 lineage. However,there is increasing evidence that CD8 T cells also include aregulatory cell lineage that inhibits T-cell responses (5). Theseregulatory CD8 T cells (CD8 Treg) eliminate activated Tfollicular helper (TFH) cells through recognition of the MHCclass Ib molecule, Qa-1 (HLA-E in human) expressed at theirsurface. This regulatory subset represents about 5% of CD8 Tcells and expresses a triad of surface receptors, CD44, CD122,and Ly49, that can be used to purify them (6, 7). Qa-1 knock-inmice (B6.Qa-1 D227K; B6-DK) have been generated to express apoint mutation (position 227, D ! K) that disrupts bindingof Qa-1 to the T-cell receptor (TCR)–CD8 complex of CD8Tregs (8). These mutant mice develop increased germinalcenters, high titers of autoantibodies, and a lethal systemiclupus erythematosus (SLE)-like autoimmune disease (6),highlighting the contribution of this Qa-1–restricted inhib-itory interaction to regulation of the immune response andself-tolerance.

These considerations led us to examine the potential con-tribution of Qa-1–restricted inhibitory pathways to antitumorimmunity. Analysis of mice that had been inoculated withgranulocyte macrophage colony-stimulating factor (GM-CSF)–expressing B16 melanoma cells revealed robust upregu-lation of Qa-1 on lymphocytes and tumor infiltration of CD8Tregs. We reasoned that increased targeting of Qa-1þ TFH cellsby CD8 Tregsmight inhibit tumor immunity, and disruption ofthis inhibitory interaction might enhance the protectiveimmune response. We therefore analyzed the response ofB6-DK mice challenged with B16 melanoma after vaccinationwith irradiated GM-CSF–transduced tumor cells. We find that

Authors' Affiliations: Departments of 1Cancer Immunology and AIDS and2Medical Oncology, Dana-Farber Cancer Institute; 3Department of Micro-biology and Immunobiology, Division of Immunology and 4Department ofMedicine, Harvard Medical School, Boston, Massachusetts; and 5Depart-ment of Gastroenterology, Hepatology, and InfectiousDiseases, Universityof D€usseldorf, D€usseldorf, Germany

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

D.A. Alvarez Arias and H.-J. Kim contributed equally to this work.

CorrespondingAuthor:HarveyCantor, Dana-Farber Cancer Institute, 450Brookline Avenue, Boston, MA 02215. Phone: 617-632-3348; Fax: 617-632-4630; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0121

�2013 American Association for Cancer Research.

CancerImmunology

Research

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genetic disruption of CD8 Treg activity results in enhancedantitumor immunity that is associated with a robust antibodyresponse to tumor-associated antigens (TAA) that cooperateswith CD8 effector T cells (Teff) to constrain tumor growth.

Materials and MethodsMice

C57BL/6J (B6; Jackson Laboratory), B6.Rag2�/� (Taconic),and B6-DK mice [N11; 99% congenic according to single-nucleotide polymorphism (SNP) analysis], OT-I [C57BL/6-Tg(TcraTcrb)1100 Mjb/J], and OT-II [B6.Cg-Tg(TcraTcrb)425Cbn/J] mice were housed in pathogen-free conditions. Allexperiments were performed in compliance with federal lawsand institutional guidelines, as approved by the InstitutionalAnimal Care and Use Committee (IACUC) of the Dana-FaberCancer Institute (Boston, MA).

Cell lines and tissue cultureB16F10 (AmericanTypeCulture Collection) andGVAX (B16-

GM-CSF)were cultured in complete Dulbecco'sModified EagleMedium (DMEM) with 10% fetal calf serum (FCS). B16-OVAwas cultured in complete DMEM with 10% FCS supplementedwith 250mg/mLG418 (Invitrogen). All lines weremaintained at37�C and 5% CO2. All cell lines used were checked for myco-plasma. B16-OVA was authenticated by PCR for OVA expres-sion. No additional authentication was performed.

Reagents and flow cytometrySingle-cell suspensions were stained with target antibodies

for 30 minutes in the dark at 4�C in ice-cold fluorescence-activated cell sorting (FACS) buffer (2%FCS, 0.1%NaN3 in PBS),washed, and analyzed using a FACSCalibur or LSR Fortessa(BD Biosciences) and FlowJo software (TriStar). Tumors weredigested with collagenase/dispase for 2 hours at 37�C withagitation and fractionated on Nyco-Prep 1.007 separationmedium (PROGEN) with centrifugation at 2,000 rpm at roomtemperature. Lymphoid fraction was collected, washed exten-sively with ice-cold FACS buffer, and analyzed. Tumor infil-trates were defined as CD45þ cells. Anti-B220, anti-CD44, anti-CD45, anti-CD122, anti-Ly49 C/I/F/H, anti-NK1.1, anti-CD11b,anti-CD25, anti-CXCR5, anti-ICOS, IgG1 isotype, anti–Qa-1b,anti-CD69, anti-Fas, and anti-IgM (BDBioscience) or anti-CD4,anti-CD8, anti-CD3, anti-CD200, anti-BTLA4, anti–PD-1, anti-FoxP3 (eBioscience), and CD1d tetramer (NIH Tetramer CoreFacility at Emory University) were used for cell analysis.

Tumor challenge and treatmentSix- to 12-week-old female B6-WT or B6-DK mice were

challenged subcutaneously with 2–5 � 105 B16 (or B16-OVA)tumor cells and immunized with irradiated 1� 105 GVAX (B16retrovirally transduced with GM-CSF) subcutaneously on theopposite flank on day 0. Mice were treated by immunizationwith 5� 105 irradiated (150 Gy) B16 (or B16-OVA) and 5� 105

GVAX s.c. on days 7 and 14 after challenge on alternating sides.Tumors were measured 2 to 3 times per week using calipersand tumor volume was presented as (x � y � z)/2 mm3. Miceexhibiting signs of distress orwith tumors reaching 2 cmon thelongest axis were sacrificed according to IACUC guidelines.

Survival plot was created using Prism 6.0 (GraphPad Software,Inc.). In some experiments, mice were vaccinated with irradi-ated 5 � 105 B16-OVA and 5 � 105 GVAX s.c. 1 week beforechallenge with 1 � 106 B16-OVA and tumor incidence wasrecorded. In serum transfer experiments, Rag2�/� hosts werechallenged with 1 � 106 B16-OVA and treated with 400 mLserum intraperitoneally on days 0, 8, and 15 after challenge.Mice also received 2 � 106 na€�ve OT-I Tg CD8 T cells intra-venously on day 2 and 1 mg IL15–IL15Ra complex intraperi-toneally on days 2, 7, and 14 after challenge. Serum wasprepared from either B6-WT or B6-DK mice vaccinated ondays 0, 7, 10, and 17 with irradiated 1 � 106 B16-OVA and 5 �105 GVAX. Serum was collected beginning on day 13 after thefirst immunization, and equal volumes from each collectiondate were pooled before treatment. Serum was prepared asdescribed (9), total IgG was obtained by precipitation withammonium sulfate (Sigma), diluted in PBS, and desalted on50,000 MW Amicon filters (EMD Millipore).

ELISADetection of NP-specific antibodies by ELISAwas performed

as described (6). Serum harvested 14 days after immunizationwith NP19-KLH in complete Freund's adjuvant (CFA) andreimmunization with NP19-KLH in IFA was used as a standard.A 1:1,000 dilution of this immune serum was defined as 100 U/mL. OVA-specific antibodies were detected on ELISA platescoated with 10 mg/mL OVA (Pierce) overnight at 4�C. TotalIgG1 was detected by incubating plates with biotinylated anti-mouse IgG1 conjugated to streptavidin peroxidase. Anti-OVAIgG1 standard was from Abcam. Anti–double-stranded DNA(dsDNA) and anti-nuclear antigen (ANA) antibodies in mousesera were determined by ELISA according to the manufac-turer's protocol (Alpha Diagnostic International). Anti-macro-phagemigration inhibitory factor (MIF) and anti-angiopoietin-2 (Ang-2) IgG antibodies were measured as described (10).Recombinant mouse MIF and Ang-2 were from R&D Systems.IFN-g in mouse sera was measured using an OptEIA ELISA Kit(BD Biosciences); interleukin (IL)-21 was measured using anELISA Duo Set (R&D Systems).

ImmunohistochemistrySections from frozen spleens and tumors, 7 mm and 10 mm,

respectively, were fixed in cold acetone, air-dried, blocked with3% bovine serum albumin in PBS and 1 µg/mL Fc block (BDBiosciences) for 30 minutes at room temperature, and stainedwith antibodies overnight in blocking buffer in the dark at 4�Cusing a moist chamber. Images were acquired on a Nikoninverted wide-field fluorescence microscope with 100� mag-nification. Germinal centers in spleen were defined by labelingwith phycoerythrin (PE)-conjugated anti-B220 antibodies (BD,RA3-6B2) and fluorescein isothiocyanate (FITC)–conjugatedanti–GL-7 (BD, clone GL7) antibodies. IgG deposition in tumorwas detected with FITC-conjugated anti-mouse IgG antibody(Sigma), complement membrane-activated complex (MAC)with biotinylated C5b-9 antibody (Bioss), macrophages withF4/80 antibody (eBioscience), desmin with anti-desmin Ab(Abcam), and fibroblast with anti-ERTR7 Ab (Santa CruzBiotechnology). Data were quantified using ImageJ software

Alvarez Arias et al.

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and presented as fluorescent area (pixels2) per field of view(FOV) or usingNIS Elements software (Nikon Instruments Inc.)and presented as fluorescent area (pixels2) per region ofinterest (ROI). Vasculature was visualized using PE-conjugatedanti-CD31 antibody (eBioscience) and vessel diameter wasmeasured on the longest axis by NIS Elements software.

Adoptive transfer experimentsNa€�ve CD4þCD25� cells were purified from spleens of OT-II

Tg WT or B6-DK mice using a CD4 Cell Enrichment Setand biotinylated anti-CD25 antibody (BD Biosciences). CD4cells were in vitro polarized to TFH cells with 50 ng/mLIL-21, 20 ng/mL IL-6, 10 mg/mL anti–IL-4, 10 mg/mL anti–IFN-g , 20 mg/mL anti–TGF-b, and cultured for 5 days in thepresence of 1 mg/mLOT-II peptide and antigen-presenting cells.Na€�veB cellswere isolated fromthe spleens ofB6-WTmiceusinga B Lymphocyte Enrichment Set (BD Biosciences). B-cell andCD4cell puritywas>95%, according toFACSanalysis. CD8Tregswere isolated from the spleens of B6-WT mice immunizedintraperitoneally with 100 mg of keyhole limpet hemocyanin(KLH) in CFA, and reimmunized 7 days later with 100 mg of KLHin incomplete Freund's adjuvant (IFA). CD8 Tregs were firstenriched with a CD8 Cell Enrichment Set (BD Bioscience)followed by sorting for CD3þCD8þCD44þCD122þLy49þ T cells.Rag2�/� mice were adoptively transferred with 1.5 � 106 OT-IITFH cells, 3.5 � 106 na€�ve B cells, and 1 � 105 CD8 Tregs.Immediately after transfer, mice were immunized intraperito-neally with 100 mg of NP13-OVA in CFA, and after 10 days, micewere reimmunized intraperitoneally with 50 mg of NP13-OVA inIFA and challenged with 1 � 106 B16-OVA s.c. 4 days later.Tumor size was monitored as mentioned above.

Statistical analysesData are presented as mean� SEM. Statistical analyses were

performedusingPrism6.0 software, theWilcoxon–Mann–Whit-ney rank sum test for comparison of two groups or conditions,unless otherwise noted. P < 0.05 was considered to be statisti-cally significant (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

ResultsGenetic disruption of CD8Treg activity is associatedwithreduced melanoma growth and enhanced TFH cellresponsesWe utilized the B16 melanoma model to investigate the

contribution of CD8Tregs to antitumor immunity (11). Tumor-bearing B6 mice were vaccinated with irradiated B16 melano-ma cells engineered to express GM-CSF (GVAX) to induce animmune response to the tumor (12). Following GVAX, wenoted substantial upregulation of Qa-1 expression by spleno-cytes and by tumor-infiltrating lymphocytes (TIL), but not bytumor cells (Fig. 1A). As B16 tumor cells do not expressdetectable Qa-1, host cells in the spleen and tumor infiltratesrepresent potential targets of Qa-1–restricted CD8 Tregs.Upregulation of Qa-1 expression by immune cells was associ-ated with the infiltration of B16 tumors by cells expressingmarkers of the CD8 Treg phenotype (Fig. 1B). Increasedproportions of CD8 Tregs within tumor-infiltrating CD8 Tcells correlated with rapid tumor growth (�day 20; Fig. 1B,

right). We directly tested the contribution of Qa-1–restrictedCD8 Tregs to tumor rejection using Qa-1 knock-in mice (B6-DK) that harbor defective Qa-1–restricted CD8 Treg activitysecondary to a Qa-1 point mutation that disrupts TCR recog-nition of Qa-1–peptide ligands (8). We inoculated B6-WT andB6-DK mice with B16 tumor cells and immunized them withirradiated GM-CSF–transduced B16 cells on day 0, 7, and 14.B6-DK mice showed significantly extended survival andreduced tumor growth compared with B6-WT mice (Fig. 1C).

Further analysis of B6-DK mice revealed increased numbersof TFH cells (CD4þICOSþCD200þ) compared with B6-WTmice(Fig. 1D), consistent with previous findings that immunizationof B6-DK mice with foreign antigens results in increasedexpansion of TFH cells and high titers of autoantibodies (6).Moreover, tumor-infiltrating CD4 T cells in Qa-1–mutant micedisplayed a more activated phenotype, as judged by levels ofICOS expression (Fig. 1D; ref. 3). No significant difference wasnoted in the numbers of CD4 Tregs or NK cells (Fig. 1E).Interestingly, increased intratumoral expansion of effector CD8T cells and reduced numbers of CD8 Tregs were detected in B6-DKmice comparedwith B6-WT controls (Fig. 1E). This resultedin a substantial increase in the intratumoral CD8þ Teff–Tregratio that was associated with the Qa-1 DK mutation.

Vaccination of B6-DKmice results in enhanced antibodyresponses to TAA

In view of the increased numbers of TFH cells (e.g., Fig. 1D)and germinal center B cells (see below) in tumor-bearingB6-DK mice, we asked whether these Qa-1–mutant micedeveloped antibody responses to surrogate TAA. We immu-nized B6-WT and B6-DK mice with irradiated GM-CSF–trans-duced B16 tumor cells that expressed an ovalbumin transgene(B16-OVA; ref. 13). Two to four weeks later, we detected hightiters of OVA-specific antibody in the sera from B6-DK, but notB6-WT mice (Fig. 2A). Increased anti-TAA antibody formationwas associated with enhanced germinal center formation inB6-DK mice (Fig. 2B). Vaccination of B6-DK mice with irradi-ated B16-OVA and GVAX 7 days before inoculation with B16-OVA tumor cells also induced high titers of anti-OVA anti-bodies. This antibody response was maintained during thecourse of B16 tumor growth and correlated with increasedtumor protection (Supplementary Fig. S1), suggesting thatantibodies induced by vaccination might have durable anti-tumor activity. Moreover, higher titers of anti-OVA antibodiesin B6-DKmicewere also detectedwhenmicewerefirst injectedwith B16-OVA tumor cells 7 days before immunization withirradiated B16-OVA and GVAX, supporting the idea that theenhanced response to TAA in B6-DK mice might have atherapeutic value (Fig. 2C).

To evaluate the contribution of TFH cells to antitumorantibody responses in Qa-1–mutantmice, we adoptively trans-ferred in vitro polarized TFH cells from B6-WT OT-II or B6-DKOT-II TCR Tgmice into Rag2�/� hosts along with na€�ve B6-WTB cells and CD8 Tregs. Following inoculation with B16-OVAcells and challenge with NP-OVA, recipients of TFH cells fromB6-DK mice produced substantially higher titers of both totalanti-NP (anti-NP23 IgG1) and high affinity (anti-NP4 IgG1)antibodies and anti-OVAantibodies than recipients of TFH cells

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Figure 1. GVAX immunization combinedwith the disruption ofCD8 Treg activity inB6-DKmice significantly potentiates antitumor immunity. A, B6-WTmicewerechallenged with 2 � 105 B16 cells s.c. and immunized with irradiated 1 � 106 GVAX (gray box) or not (black box) on days 5, 8, and 11. Qa-1 expression wasdetermined on gated subsets of splenocytes and TILs 20 to 24 hours after the last immunization. Data are representative of two independent experimentsand presented as the ratio of mean fluorescence intensity (MFI) of specific Qa-1 staining and MFI of background (bg) staining with streptavidin only.B, CD8 Tregs efficiently infiltrate into tumors. Mice were challenged with 5� 105 B16 cells. The lymphoid fraction of collagenase/dispase digested tumors wascollectedbygradientcentrifugationon10, 20,and30daysafter challenge.TheproportionsofCD8Treg (CD122þLy49þ)weredeterminedbyFACSaspercentageof CD8þCD3þ cells. A representative FACS plot from day 20 after challenge is shown on the left. Tumor growth is shown on the right. C, B6-WT (&) or B6-DK(&) mice were challenged with 2 � 105 B16 and immunized on the same day with irradiated 1 � 105 GVAX followed by immunization with irradiated5 � 105 B16 and 5 � 105 GVAX s.c. on days 7 and 14; n ¼ 15 mice per group. Data are presented as cumulative survival (left) or tumor growth (right) of B16-challenged mice from two independent experiments. D, B6-WT (&) or B6-DK (&) mice were treated as in B. Percentage (left) or absolute number(right) ofCD4TFH cells (CD200

þICOSþPD-1hi gated fromCD3þCD4þ cells) fromeither spleens or tumors (Tm) of treated animalswasdetermined 3 days after thelast immunization. ICOS expression by tumor-infiltrating CD3þCD4þ lymphocytes was determined by FACS 3 days after the last immunization. Data arerepresentative of at least three independent experiments. E, cellular analysis of tumor-bearing B6-WT (&) or B6-DK (&) mice immunized with GVAX. Theproportion and absolute number of CD8, Ly49þ CD8, NK (NK1.1þCD3�), or CD4 Treg (CD25þFoxP3þ gated from CD3þCD4þ) lymphocytes from the spleens,tumor draining lymph nodes (dLN), or tumors (Tm) were determined by FACS 3 days after the last immunization. Data are representative of at least twoindependent experiments. P values (�) are defined in Materials and Methods.






from B6-WT donors, and this response was associated withreduced tumor growth (Fig. 3A and B). We again observedenhanced expansion of TFH cells (but not CD8 Tregs) inrecipients of B6-DK TFH cells (Fig. 3C). The expansion ofTFH cells was accompanied by increased levels of IL-21, butnot IFN-g in the sera of B6-DK mice (Fig. 3D), indicating thatthe expanded T cells were producing a canonical TFH cytokine.Next, we analyzed the repertoire of antibodies present in the

serum of GVAX-vaccinated B6-WT and B6-DKmice. We foundincreased titers of autoantibodies in the sera of B6-DK mice,including anti-dsDNA antibodies (Fig. 4A). The same trendwasobserved after treatment of tumor-bearing mice with GVAX

(Fig. 4B). In addition, we noted several types of autoantibodiesthat have been reported to have antiangiogenic properties(10, 14, 15) in a portion of the B6-DKmice, but not B6-WTmice(Fig. 4C). These antibodies were induced upon GVAX vacci-nation andwere not detectable in na€�ve B6-WT or B6-DKmice.To determine whether the tumor vasculature was affected bythe therapeutic antibodies in B6-DK mice, we performedhistology on tumor sections and immunostaining with CD31antibodies. Although the number of vessels was similar, thetumor vessels appeared smaller in tumors of B6-DK than in B6-WT controls (Fig. 4D), suggesting that increased vasculaturepathologywas associatedwith increased antibody responses ofB6-DK mice, which may contribute to delayed tumor growth.

Most analyses of protective tumor immunity have focusedon cell-mediated immunity, including the host cytotoxicresponse. Our analysis of enhanced antitumor immunity inanimals with defective CD8 Treg activity suggested a role forautoantibody responses, including antibodies to TAA. Todetermine whether antibodies produced after GVAX immuni-zation of B6-DK mice contributed to antitumor immunity, Igwas prepared from the serum of B6-WT or B6-DK mice afterGVAX immunization. Rag2�/�mice were inoculated with B16-OVA and treated with 400 mL of serum-derived Ig on days 0, 8,and 15. Mice also received 2� 106 na€�ve OT-I cells on day 2 and1 mg IL15/IL15Ra complex on days 2, 7, and 14 after tumorinoculation as a source of activated CD8 T cells. We found that60% of the mice that received antibodies from B6-DK miceremained tumor free, whereas all mice that received serum Igfrom WT controls developed tumors (Fig. 5A). This antitumorresponse did not depend on the contribution of anti-OVAantibody alone because the provision of high titers of anti-OVA Ig (500mg anti-OVA IgG1) toRag2�/�micewithout T cellshad no detectable effect on tumor growth (Supplementary Fig.S2). Further analysis of B16 tumors revealed increased IgGdeposition in the tumors of B6-DK mice after treatment withGVAX (Fig. 5B), which colocalized with the C5b-C9 comple-ment MAC (Fig. 5B) and was associated with macrophageinfiltrates (Fig. 5C); Ig did not colocalize with other tumorstromal components, including CD31þ and ERTR-7þ cells (Fig.5D). We also used a polyclonal antibody to the desmin stromalprotein that cross-reacts with single- and double-strandedDNA (16) for additional histologic analysis (SupplementaryFig. S3). Immunofluorescence analysis using anti-desmin anti-body showed colocalization with IgG deposits in addition tothe presence of filamentous structures normally detected bythis antibody (Fig. 5D). Areas containing colocalized anti-desmin antibody accounted for a substantial fraction of theintratumoral IgG foci (�30%–50%), suggesting that a signifi-cant proportion of intratumoral Ig may represent anti-DNAantibodies derived from sera of tumor-bearing B6-DK mice.

DiscussionWe report here that antitumor immunity against melanoma

is enhancedby the genetic disruption ofCD8Treg activity that isassociatedwith the expansion ofTFH cells and germinal centerBcells, the generation of anti-TAA antibodies, and delayed tumorgrowth. These findings concerning the antitumor response areconsistent with previous findings that targeting of TFH cells

Figure 2. GVAX immunization induces robust antibody response to TAA inB6-DK mice. A, B6-WT (&) or B6-DK (&) mice were vaccinated withirradiated 1 � 106 B16-OVA and 5 � 105 GVAX on days 7 and 14. Serawere collected 14, 21, and 28 days after vaccination and analyzed foranti-OVA IgG1 antibodies by ELISA, n ¼ 3 mice per group. Data arerepresentative of two independent experiments. B, histology of spleensfrom A on day 28. Green, GL7; red, B220. Germinal center (GC) area,pixel2 per FOV and GC numbers per FOVwere quantifiedwith ImageJ. C,mice were challenged with 5 � 105 B16-OVA and immunized withirradiated 1� 105 GVAX followed by immunization with irradiated 1� 106

B16-OVA and 5� 105 GVAX s.c. on days 7 and 14; n¼ 5mice per group.Sera were collected on day 25 after challenge and analyzed for anti-OVAIgG1 antibodies by ELISA. Data are representative of two independentexperiments.






by CD8 Treg constrains the expansion of TFH cells and thedevelopment of high-affinity antibody and autoantibodyresponses. Excessive production of IL-21 by activated TFH

cells in B6-DK mice may also stimulate a CD8 T-cellresponse to exert antitumor activity (17).

The therapeutic activity of the antibody response mountedby B6-DK mice was confirmed from studies of tumor-bearinghosts infused with IgG from immunized tumor-bearing B6-DK, but not from B6-WT mice. Transfer of IgG from theformer, but not the latter donors resulted in significantprotection from lethal tumor growth in adoptive hosts (Fig.5A). The antibody response in B6-DK mice included anti-dsDNA, anti-MIF, and anti–Ang-2 specificities after GVAXtreatment and tumor challenge (Fig. 4). Increased IgG depo-sition in tumors of B6-DK mice was associated with tumortissue destruction and often colocalized with MACs (C5b-C9)that have the potential to lyse tumor cells. Whether anti-bodies generated in this setting directly target B16 melanomaor act through the activation of tumor-infiltrating immunecells, such as macrophages, is not clear. NK-mediated cytol-ysis of B16 melanoma does not appear to be involved in thisprocess because NK cells did not colocalize with intratumoralIgG and we did not observe antibody-dependent cell-medi-ated cytotoxicity against B16 cells after incubation with B6-DK–derived antibody.

Considerable attention has been given to the association ofinflammatory and autoimmune responses and the develop-ment of cancers (18–22). However, the influence of chronicautoimmunity on the natural history of solid tumors is not wellunderstood. For example, although patients with SLE havedecreased risk of breast cancer andmelanoma (23), the under-lying mechanisms and biologic significance of these observa-tions are unclear. It may be relevant that longitudinal analyses

of sera from long-term surviving melanoma patients aftervaccination with GM-CSF–secreting autologous melanomacells have revealed that the generation of autoantibodies wasassociated with increased tumor destruction (15).

These findings raise the intriguing possibility that interrup-tion of CD8 Treg activity may act in part through enhancedantibody responses to "classical" autoimmune target antigens,including dsDNA. While the full repertoire of antibodies gen-erated in B6-DKmice upon GVAX treatment and their relativecontribution to antitumor immunity has not been determined,the production of high levels of anti-dsDNA antibody in tumor-bearing B6-DKmice after GVAX treatmentwas associatedwithprotective antitumor immunity. Although the development ofanti-dsDNA antibody is considered a pathogenic sign in thecontext of SLE, the appearance of these autoantibodies inpatients with cancer has been associated with improvedclinical outcome (24). Anti-dsDNAantibodiesmay affect tumorgrowth through direct binding and induction of apoptosis (25,26). Cell-penetrating anti-DNA antibodies, for example, inhibitDNA single- and double-stranded DNA repair in tumor cellsand sensitize them to DNA-damaging therapy (27). Anti-dsDNA antibody complexed to dsDNA can also stimulate Bcells and dendritic cells in a TLR9-dependent manner toproduce increased complement-fixing antibodies and IFN-a,respectively, which can have inhibitory effects on malignancy(28, 29). These considerations support the notion that gener-ation of a broad-based range of autoantibodies, includingdsDNA, in the absence of CD8 Treg activity may contributeto the enhanced anti-melanoma responses noted here as wellas to the response against a wide range of tumors.

The ability of antibodies specific for intracellular rather thancell surface molecules to mediate antitumor immunity is notwell understood. The pathogenic effects of antibodies to the

Figure 3. Disrupted CD8 Treg activity in B6-DK mice is associated with decreased tumor formation and increased antibody response to TAA. Rag2�/� micewere adoptively transferred with OT-II WT or B6-DK polarized in vitro to TFH cells, naïve B cells, and CD8 Tregs. Mice were immunized with NP-OVAin CFA, boosted on day 10 with NP-OVA in IFA, and challenged with 5 � 105 B16-OVA 4 days later. A, tumor volume was recorded every 2 to 3 days.B, sera were collected on day 15 or 16 after challenge and analyzed for anti-OVA IgG1 or anti-NP IgG1 antibodies by ELISA. C, absolute numbers of TFH,GC B cells (B220þFasþIgM�), or CD8 Tregs (CD3þCD8þ CD122þLy49þ) from B6-WT (&) or B6-DK (&) mice were determined in the spleens,tumor draining lymphnodes (dLN), or tumors (Tm). D, IL-21and IFN-g levelswerequantifiedbyELISA in serumcollected fromB6-WT (&) orB6-DK (&)mice onday 15 or 16 after challenge. Data are representative of at least three independent experiments; n ¼ 5 mice per group.






cytoplasmic enzyme glucose-6-phosphate isomerase (GPI) intheK/BXNarthritismousemodelmay be instructive.Here, anti-GPI antibodies initiate local inflammatory changes leading toincreased extracellular GPI and enhanced vascular permeabilityto serum autoantibodies and inflammatory cells (30, 31). In thepresent study, antibody-mediated antitumor effects dependedon the cooperative action of tumor-specificCD8T cells (Fig. 5A).Induction of inflammatory foci in the tumor environment mayenhance the release of intracellular antigens (e.g., dsDNA),resulting in increased permeability of the vasculature andmobilization of antigen-specific CD8þ cytolytic T cells into thetumor sites. This coordinated humoral and cellular responsemay be essential for optimal antitumor activity. Indeed, acombined antibody and CD8þ antitumor immune response tothe intracellular melanoma antigen NY-ESO-1 has been corre-lated with clinical benefit in patients with advanced melanomatreated with anti-CTLA-4 antibody (ipilimumab; ref. 32).Increased ICOSþ CD4 cells within the tumor and enhancedIL-21 production in the serum of B6-DK mice are consistentwith previousfindings thatTFH cells are preferentially expandedin B6-DK mice. Additional experiments, for example, withICOS�/� mice, are needed to document the contribution ofelevated ICOSþ CD4 cells to antitumor responses and delineatethe potential link between ICOSþ CD4 cells, IL-21 production,and enhanced antitumor responses. Recent analysis of spatio-temporal dynamics of the tumor–immune interaction duringtumor progression revealed that CXCL13 and IL-21 constitutethe pivotal antitumor players, and increased numbers of TFHandB cells are strongly correlatedwith a positive prognosis (33).Promotion of effector T cells by IL-21 in a mouse melanomamodel, and the efficacy of IL-21 treatment of patients withmetastatic melanoma suggest a multifaceted role for IL-21, inaddition to its potential contribution to antibody-mediatedantitumor responses noted here (34, 35).

Here we find that an increase in the ratio of CD8 Teff to CD8Treg (and no change in CD4 Treg) within tumors of B6-DKmice compared with those in B6-WT mice is associated withenhanced antitumor activity after tumor vaccination. Ourobservations are congruent with previous findings thatincreased ratios of CD8 T cells to FoxP3þCD4þ Treg areassociated with favorable prognosis in the context of humanand mouse cancers (36–38). Blockade of CD8 Treg-dependentinhibition may tip the balance within the tumor microenvi-ronment in favor of the Teff compartment, resulting inenhanced antitumor immunity.

We also detected anti-MIF and anti–Ang-2 autoantibodiesin some GVAX-treated tumor-bearing B6-DK mice, but not inB6-WT controls (Fig. 4C). These antibodies may exert antitu-mor effects through the inhibition of the Tie-2 pathway in

Figure 4. High autoantibody titers induced after GVAX immunization in theabsence of CD8 Treg activity. A,micewere vaccinatedwith irradiated 5�105 B16-OVA and 5 � 105 GVAX on day 7 and 14. Sera were collected 4weeks later and analyzed for anti-dsDNA IgG or anti-ANA IgG antibodiesbyELISA. B,micewere challengedwith 5�105B16-OVAand immunizedwith irradiated1�105GVAX followedby immunizationwith irradiated 5�105B16-OVA and 5� 105GVAX s.c. on day 7 and 14. Serawere analyzedas above. C, mice (1–5) were challenged and treated as in B. Sera wereanalyzed for anti-MIF or anti–Ang-2 IgG antibodies by ELISA.

Data are representative of at least two independent experiments. D,remodeling of the tumor microenvironment with therapeutic antibodiesresults in decreased tumor vasculature. Histology of B16-OVA tumorsthat were collected on day 20 from either B6-WT or B6-DK mice. Micewere immunized with irradiated 5 � 105 B16-OVA and 5 � 105 GVAX ondays 7 and 14 after challenge. Vessel numbers were counted manuallyper slide and vessel diameter was measured on the longest axis by NISElements software. Data are representative of three independentexperiments.






macrophages and MMP-9 production (10). Interestingly,increases in both anti-MIF and anti–Ang-2 antibodies werealso noted in response to treatment with GVAX and anti-CTLA-4 antibody therapy and correlated with increased tumorvasculopathy (10). Whether antibody activity present in IgG-enriched sera from B6-DK mice directly contributes to theantitumor response requires further experiments, including

evaluation of the effects of absorption of purified IgG by tumorlysate. These considerations suggest that the disruption ofnormal immunoregulatory T cells may favor increased auto-antibody responses that can target molecular elements thatcontribute to tumor growth and spread.

Here we identify a new immune phenotype of Qa-1–mutantmice. Genetic disruption of the inhibitory interaction between

Figure 5. Antitumor antibodies produced inB6-DKmice enhance tumor rejection and remodel the tumormicroenvironment. A, serumwasprepared fromeitherB6-WT (&) or B6-DK (&) mice serially vaccinated with irradiated 1� 106 B16-OVA and 5� 105 GVAX.Rag2�/� hosts were challenged with 1� 106 B16-OVAand treated with concentrated serum intraperitoneally on days 0, 8, and 15 after challenge. Mice also received naïve OT-I Tg CD8 T cells on day 2 and1 mg IL15/IL15Ra complex on days 2, 7, and 14 after challenge (mice, n¼ 4). B, histology of B16-OVA tumors that were collected on day 20 from either B6-WT(&) or B6-DK (&) mice. Mice were either untreated or immunized with irradiated 5 � 105 B16-OVA and 5 � 105 GVAX on day 7 and 14 after challenge.Green, anti-mouse IgG; red, complement MAC; blue, 40,6-diamidino-2-phenylindole (DAPI). Data were quantified using ImageJ and are presented asfluorescent area, pixels2 per FOV (right). Data are representative of at least two independent experiments. C, histology of B16-OVA tumors that werecollected on day 20 from either B6-WT (&) or B6-DK (&) mice. Mice were untreated or immunized with irradiated 5 � 105 B16-OVA and 5 � 105 GVAXon day 7 and 14 after challenge.Green, anti-mouse IgG; red, F4/80. Data belowwere quantified usingNIS Elements software and are presented as fluorescentarea, pixels2 per ROI or using ImageJ and presented as fluorescent area, pixels2 per FOV (right). Data are representative of at least two independentexperiments. P values were calculated to compare F4/80þ fluorescent area within IgGþ and IgG� regions. D, tissue sections obtained from above wereanalyzed for IgG deposition (green) in relation to stromal components by staining with anti-CD31 (red), anti-ERTR7 (red), and anti-desmin (red). Nuclei werestained with DAPI (blue). Data shown are representative of the IgG deposit area within tumors from B6-DK mice containing 5 to 10 distinct foci.






CD8Treg and Qa-1þTFH cells results in the induction of robustautoimmune antitumor responses driven by enhanced TFH cellhelper activity and the upregulation of therapeutic antibodyproduction by B cells. Our data indicate that the CD8þ Teff–Treg ratio in tumor lymphocyte infiltrates may represent auseful prognostic index for cancer development. These dataalso suggest that specific depletion of CD8 Treg represents anovel and potentially effective strategy for cancer treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

TheEditor-in-Chief ofCancer Immunology Research is an author of this article.In keeping with the AACR's editorial policy, the paper was peer reviewed and anAACR journal editor not affiliated with Cancer Immunology Research renderedthe decision concerning acceptability.

Authors' ContributionsConception and design: D.A. Alvarez Arias, H.-J. Kim, G. Dranoff, H. CantorDevelopment of methodology: D.A. Alvarez Arias, H.-J. Kim, H. CantorAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.A. Alvarez Arias, H.-J. Kim, P. Zhou, T.A.W.Holderried, X. Wang, H. Cantor

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.A. Alvarez Arias, H.-J. Kim, G. Dranoff, H. CantorWriting, review, and/or revision of themanuscript:D.A. Alvarez Arias, H.-J.Kim, G. Dranoff, H. CantorAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D.A. Alvarez Arias, T.A.W. HolderriedStudy supervision: H.-J. Kim, H. Cantor

AcknowledgmentsThe authors thank Dr. Lisa Cameron (DFCI Microscopy Core) for help with

image preparation and analysis, A. Angel for manuscript and figure preparation,and V. Garcia for technical assistance.

Grant SupportThis work was supported in part by NIH research grant AI 037562 and a gift

from the LeRoy Schecter Research Foundation (to H. Cantor) and NRSA Fellow-ships (T32AI07386; to D. Alvarez Arias) and (T32CA070083; to H-J. Kim). T.A.W.Holderried is supported by the University of D€usseldorf (D€usseldorf, Germany).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 13, 2013; revised November 12, 2013; accepted December 9,2013; published OnlineFirst December 31, 2013.

References1. Kantoff PW, Schuetz TJ, Blumenstein BA, Glode M, Bilhartz DL,

Wyand M, et al. Overall survival analysis of a phase II randomizedcontrolled trial of a Poxviral-based PSA-targeted immunotherapy inmetastatic castration-resistant prostate cancer. J Clin Oncol 2010;28:1099–105.

2. Vanneman M, Dranoff G. Combining immunotherapy and targetedtherapies in cancer treatment. Nat Rev Cancer 2012;12:237–51.

3. Sharma P, Wagner K, Wolchok JD, Allison JP. Novel cancer immu-notherapy agents with survival benefit: recent successes and nextsteps. Nat Rev Cancer 2011;11:805–12.

4. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, HaanenJB, et al. Improved survival with ipilimumab in patients with metastaticmelanoma. N Engl J Med 2010;363:711–23.

5. Kim HJ, Cantor H. Regulation of self tolerance by Qa-1-restrictedCD8þ regulatory T cells. Semin Immunol 2011;23:446–52.

6. Kim HJ, Verbinnen B, Tang X, Lu L, Cantor H. Inhibition of follicular Thelper cells by CD8þ Treg is essential for self tolerance. Nature 2010;467:328–32.

7. KimHJ,WangX, Radfar S, Sproule TJ, RoopenianDC,Cantor H.CD8þ

T regulatory cells express the Ly49 class I MHC receptor and aredefective in autoimmune-proneB6-Yaamice. ProcNatl AcadSci USA2011;108:2010–5.

8. Lu L, KimHJ,WerneckMB, Cantor H. Regulation of CD8þ regulatory Tcells: interruption of the NKG2A-Qa-1 interaction allows robust sup-pressive activity and resolution of autoimmune disease. Proc NatlAcad Sci U S A 2008;105:19420–5.

9. Reilly RT, Machiels JP, Emens LA, Ercolini AM, Okoye FI, Lei RY, et al.The collaboration of both humoral and cellular HER-2/neu-targetedimmune responses is required for the complete eradication of HER-2/neu-expressing tumors. Cancer Res 2001;61:880–3.

10. Schoenfeld J, Jinushi M, Nakazaki Y, Wiener D, Park J, Soiffer R, et al.Active immunotherapy induces antibody responses that target tumorangiogenesis. Cancer Res 2010;70:10150–60.

11. Klein C, Bueler H, Mulligan RC. Comparative analysis of geneticallymodified dendritic cells and tumor cells as therapeutic cancer vac-cines. J Exp Med 2000;191:1699–708.

12. Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, et al.Vacciniation with irradiated tumor cells engineered to secrete murinegranulocyte-macrophage colony-stimulating factor stimulates potent,specific, and long-lasting anti-tumor immunity. Proc Natl Acad SciU S A 1993;90:3539–43.

13. Bellone M, Cantarella D, Castiglioni P, Crosti MC, Ronchetti A,Moro M, et al. Relevance of the tumor antigen in the validation ofthree vaccination strategies for melanoma. J Immunol 2000;165:2651–6.

14. Leow CC, Coffman K, Inigo I, Breen S, Czapiga M, Soukharev S, et al.MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibitsangiogenesis and tumor growth in human tumor xenograft models. IntJ Oncol 2012;40:1321–30.

15. Sittler T, Zhou J, Park J, Yuen NK, Sarantopoulos S, Mollick J, et al.Concerted potent humoral immune responses to autoantigens areassociated with tumor destruction and favorable clinical outcomeswithout autoimmunity. Clin Cancer Res 2008;14:3896–905.

16. Kamei H. Nuclear antigens, as DNA, of DSB389 MAb and some otheranti-desmin monoclonal antibodies. Cell Biol Int 1996;20:769–75.

17. Zeng R, Spolski R, Finkelstein SE, Oh S, Kovanen PE, Hinrichs CS,et al. Synergy of IL-21 and IL-15 in regulating CD8þ T cell expansionand function. J Exp Med 2005;201:139–48.

18. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, andcancer. Cell 2010;140:883–99.

19. Coussens LM, Zitvogel L, Palucka AK. Neutralizing tumor-promotingchronic inflammation: a magic bullet? Science 2013;339:286–91.

20. Bernatsky S, Kale M, Ramsey-Goldman R, Gordon C, Clarke AE.Systemic lupus and malignancies. Curr Opin Rheumatol 2012;24:177–81.

21. Ekstrom K, Hjalgrim H, Brandt L, Baechlund E, Klareskog L, EkbomA, et al. Risk of malignant lymphomas in patients with rheumatoidarthritis and in their first-degree relatives. Arthritis Rheum 2003;48:963–70.

22. Ngalamika O, Zhang Y, Yin H, Zhao M, Gershwin ME, Lu Q. Epige-netics, autoimmunity and hematologic malignancies: a comprehen-sive review. J Autoimmun 2012;39:451–65.

23. Bernatsky S, Easton DF, Dunning A, Michailidou K, Ramsey-GoldmanR, Gordon C, et al. Decreased breast cancer risk in systemic lupuserythematosus: the search for a genetic basis continues. Lupus 2012;21:896–9.

24. Syrigos KN, Charalambopoulos A, Pliarchopoulou K, Varsamidakis N,Machairas A, Mandrekas D. The prognostic significance of autoanti-bodies against dsDNA in patients with colorectal adenocarcinoma.Anticancer Res 2000;20:4351–3.

25. Torchilin VP, Iakoubov LZ, Estrov Z. Antinuclear autoantibodies aspotential antineoplastic agents. Trends Immunol 2001;22:424–7.






26. Cao Q, Xu W, Wen Z, Xu L, Li K, Chu Y, et al. An anti-double-strandedDNAmonoclonal antibody induced by tumor cell-derived DNA inhibitsthe growth of tumor in vitro and in vivo via triggering apoptosis. DNACell Biol 2008;27:91–100.

27. Hansen JE, Chan G, Liu Y, Hegan DC, Dalal S, Dray E, et al. Targetingcancer with a lupus autoantibody. Sci Transl Med 2012;4:157ra42.

28. Tarhini AA, Gogas H, Kirkwood JM. IFN-alpha in the treatment ofmelanoma. J Immunol 2012;189:3789–93.

29. Kis-Toth K, Szanto A, Thai TH, Tsokos GC. Cytosolic DNA-activatedhuman dendritic cells are potent activators of the adaptive immuneresponse. J Immunol 2011;187:1222–34.

30. Matsumoto I, Maccioni M, Lee DM, Maurice M, Simmons B, BrennerM, et al. How antibodies to a ubiquitous cytoplasmic enzyme mayprovoke joint-specific autoimmune disease. Nat Immunol 2002;3:360–5.

31. Binstadt BA, Patel PR, Alencar H, Nigrovic PA, Lee DM, Mahmood U,et al. Particularities of the vasculature can promote the organ spec-ificity of autoimmune attack. Nat Immunol 2006;7:284–92.

32. Yuan J, Adamow M, Ginsberg BA, Rasalan TS, Ritter E, Gallardo HF,et al. Integrated NY-ESO-1 antibody and CD8þ T-cell responsescorrelate with clinical benefit in advanced melanoma patients treatedwith ipilimumab. Proc Natl Acad Sci U S A 2011;108:16723–8.

33. BindeaG,Mlecnik B, Tosolini M, Kirilovsky A,WaldnerM,Obenauf AC,et al. Spatiotemporal dynamics of intratumoral immune cells reveal theimmune landscape in human cancer. Immunity 2013;39:782–95.

34. Kim-Schulze S, Kim HS, Fan Q, Kim DW, Kaufman HL. Local IL-21promotes the therapeutic activity of effector T cells by decreasingregulatory T cells within the tumor microenvironment. Mol Ther 2009;17:380–8.

35. Petrella TM, Tozer R, Belanger K, Savage KJ, Wong R, Smylie M, et al.Interleukin-21 has activity in patients with metastatic melanoma: aphase II study. J Clin Oncol 2012;30:3396–401.

36. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, et al.Intraepithelial CD8þ tumor-infiltrating lymphocytes and a highCD8þ/regulatory T cell ratio are associated with favorable progno-sis in ovarian cancer. Proc Natl Acad Sci U S A 2005;102:18538–43.

37. Quezada SA, Peggs KS, Curran MA, Allison JP. CTLA4 blockadeand GM-CSF combination immunotherapy alters the intratumorbalance of effector and regulatory T cells. J Clin Invest 2006;116:1935–45.

38. Hodi FS, Butler M, Oble DA, Seiden MV, Haluska FG, Kruse A, et al.Immunologic and clinical effects of antibody blockade of cytotoxicT lymphocyte-associated antigen 4 in previously vaccinated can-cer patients. Proc Natl Acad Sci U S A 2008;105:3005–10.






2014;2:207-216. Published OnlineFirst December 31, 2013.Cancer Immunol Res Diana A. Alvarez Arias, Hye-Jung Kim, Penghui Zhou, et al. Follicular Helper Cells and Enhanced Antitumor Immunity

Treg Activity Results in Expansion of T+Disruption of CD8

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