converting lymphoma cells into potent antigen-presenting cells … · 2017. 7. 3. · cd20-ifna or...

Research Article

Converting Lymphoma Cells into PotentAntigen-Presenting Cells for Interferon-InducedTumor RegressionJing Liao1,2, Yan Luan3, Zhenhua Ren1,2, Xiaojuan Liu1,2, Diyuan Xue1,2, Hairong Xu1,Zhichen Sun1,2, Kaiting Yang1,2, Hua Peng1, and Yang-Xin Fu4

Abstract

Anti-hCD20 is a therapeutic mAb that is clinically used to treatB-cell lymphoma. Some lymphomas are resistant to anti-hCD20;others relapse after treatment with anti-hCD20. Using a syngeneicimmunocompetent mouse model, we observed that targetinglymphoma with interferon-a (IFNa) abolished resistance ofB-cell lymphoma to anti-CD20 while limiting interferon (IFN)-associated systemic toxicity in the host. Control of tumors by afusion of anti-CD20 and IFNa (anti–CD20-IFNa) depended onexisting tumor-infiltrating CD8þ T cells. Although lymphomas

were resistant to IFN-directed killing, IFN-exposed tumor cellsbecame the dominant antigen-presenting cells (APC) for thereactivation of tumor-infiltrating CD8þ T cells that then con-trolled those lymphomas. Anti–CD20-IFNa also abolishedcheckpoint blockade resistance in advanced B-cell lymphoma.Our findings indicate that anti–CD20-IFNa eradicates B-celllymphoma by employing tumor cells as APCs to reactivatetumor-infiltrating CD8þ T cells and synergizing with anti–PD-L1treatment. Cancer Immunol Res; 5(7); 560–70. �2017 AACR.

IntroductionLymphoma is one of the most common cancers (1). Non-

Hodgkin lymphomas (NHL) account for 80% to 90% of alllymphomas. Approximately 85% of all NHLs are of B-cell origin(B-NHL; ref. 2). The antibody (Ab) to hCD20, rituximab(Rituxan), which is effective against many B-NHLs (3), functionsby induction of tumor apoptosis (4), Ab-dependent cellularcytotoxicity (ADCC) and phagocytosis (ADCP), Ab stimulationof complement-dependent cytotoxicity (CDC), complement-enhanced ADCC (CR3-ADCC), a vaccine effect, and a T cell–dependent immune response (5, 6). Although rituximab is stan-dard therapy for many B-NHLs, patients have an overall responserate of only 40% to 50% in relapsed or refractory low-grade B-NHLs and amedian time to progression (MTTP) of approximately9 months (7). New therapeutic options are needed to overcomeanti-CD20 resistance and effectively treat B-cell lymphoma.

Interferon-a (IFNa), a protein of the type I interferon family, isused to treat NHLs and other cancers (8). IFNa functions in cancer

immunotherapy via direct antiproliferation or proapoptosis oftumor cells and by upregulating CD20 expression on tumor cells,enhancing the effect of ADCC or ADCP, and bridging the innateandadaptive immune responses (9).Useof IFNa as amaintenancetherapy for follicular lymphoma (FL) improves progression-freesurvival (PFS). However, systemic administration of IFNa cancause severe toxicities, such as neutropenia, fatigue, liver injury,pulmonary embolism, and psychiatric disorders (10, 11). Theshort half-life of IFNa in the blood and the wide distribution ofthe type I interferon receptor (IFNAR)on all nucleated cellsmake itdifficult for IFNa to reach target tissues (12, 13).

Human IFNa2b fused to amAb to hCD20 (veltuzumab) had apotent antitumor effect. Treatment with the fusion protein pro-duced a survival rate of 100%, whereas survival was 20% witheither agent alone, or 40% with rituximab therapy in a humanlymphoma xenograftmodel (14). Targeted delivery of IFNa to thetumor microenvironment by hCD20 mAb induces tumor cellapoptosis in vitro and could lead to tumor control in vivo (15).Treatment with anti–hCD20-hIFNa can reverse the rituximabresistance of B-NHL in vitro by inhibiting cell proliferation andinducing cell death (16).However, not all lymphomas respond toanti-CD20 or IFN with apoptosis. Here, we used a syngeneicimmunocompetent mouse model and observed that targetingB-cell lymphoma with anti–CD20-IFNa abolished anti-CD20resistance in B-cell lymphoma while limiting IFN-associatedtoxicity. Tumor regression occurred via a mechanism dependenton preexisting tumor-infiltrating CD8þ T cells. Understanding theimmune mechanism of IFN-mediated tumor control will lead tomore effective treatment combinations.

Materials and MethodsMice

Wild-type (WT) BALB/c and BALB/c nudemice were purchasedfromVital River Laboratories. CL-4micewere purchased fromThe

1Key Laboratory of Infection and Immunity, Institute of Biophysics, ChineseAcademy of Sciences, Beijing, China. 2University of Chinese Academy ofSciences, Beijing, China. 3DingFu Biotarget Co. Ltd., Suzhou, Jiangsu, China.4Department of Pathology, University of Texas Southwestern Medical Center,Dallas, Texas.

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

Corresponding Authors: Yang-Xin Fu, Department of Pathology, Univer-sity of Texas Southwestern Medical Center, 6000 Harry Hines Blvd,Dallas, TX 75235. Phone: 773-307-5478; Fax: 773-834-8940; E-mail:[email protected]; and Hua Peng, Key Laboratory of Infec-tion and Immunity, Institute of Biophysics, Chinese Academy of Sciences,15 Datun Road, Beijing 100101, China. Phone: 8610-6488-1152; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-16-0221

�2017 American Association for Cancer Research.

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Jackson Laboratory. CD11c-DTR (depletion of CD11c-expressingcells via the diphtheria toxin receptor)micewere bred and housedat the Institute of Biophysics, CAS. mMT�/� mice were kindlyprovided byDr. Zhihai Qin of the Institute of Biophysics, CAS. Allof the mice were maintained under specific pathogen-free con-ditions and were used between 6 and 12 weeks of age in accor-dance with the experimental animal guidelines set by the Insti-tutional Animal Care and Use Committee of the Institute ofBiophysics, CAS (SYXK2014-44).

Cell lines and reagentsA20 is a murine B-cell lymphoma cell line with a BALB/c back-

ground and was purchased from ATCC in 2013. A20-HA wasselected as a single clone with 2 mg/mL puromycin (InvivoGen)after being transduced by lentivirus expressing hemagglutinationantigen (HA) in 2014. BL3750 is a B-cell lymphoma cell line witha C57BL/6 background and was provided by Dr. Holbrook Kohrt(Stanford University Medical Center, Stanford, CA) in 2015. L929is a murine fibroblast cell line with a C3H/An background, whichwas provided by Dr. Zhihai Qin (Institute of Biophysics, CAS,Beijing, China) in 2013. All cell lines were maintained accordingto the method used by the ATCC and were tested and found to befree of mycoplasma contamination. The cell lines were authen-ticated by flow cytometry and morphology. Anti–PD-L1 blockingmAb (10F.9G2) was purchased from BioXCell. The FcgRII/IIIblocking Ab (clone 2.4G2), CD8-depleting Ab (clone 2.43),and CD4-depleting Ab (clone GK1.5) were produced in house.Diphtheria toxin (DT) was purchased from CALBIOCHEM(Darmstadt) and prepared according to the manufacturer's in-structions. Clophosome, which was used to deplete macrophagecells, was purchased from FormuMax (Sunnyvale, CA).

Production of the anti–CD20-IFNa fusion proteinThe variable region sequence of the mAb to mouse CD20

(18B12, Biogen) was synthesized by Invitrogen and cloned intothe pEE12.4 expression plasmid (Lonza as a single-chain variablefragment (ScFv) with a human IgG1 Fc in the C-terminal region.Murine IFNa4a was inserted into the N-terminal region of ScFvwith a (SG4)4 linker to make the anti–CD20-IFNa fusion protein.The plasmid was transiently transfected into FreeStyle 293-F cells,and the fusion protein in the supernatant was purified using aprotein A-Sepharose column (GE Healthcare). The nontargetingcontrol protein IFNa-IgG1 was obtained in a similar mannerexcept that the variable region sequence of the Ab was from anti-HBsAg H25B10 hybridoma cells. Please refer to the supplemen-tary material for all protein gene sequences

Tumor growth and treatmentsA total of 2 � 106 to 3 � 106 A20 tumor cells were subcuta-

neously (s.c.) transplanted into the flanks of the mice. Tumorvolumes were measured along three orthogonal axes (a, b, and c)and calculated as tumor volume¼ abc/2. Tumors were grown for9 to 14 days to reach a size of 100mm3 and then treatedwith anti–CD20-IFNa or control hIgG intratumorally or intravenously. Forthe CD8-depletion experiment, 200 mg of anti-CD8 Ab (clone2.43) was injected intraperitoneally one day before anti–CD20-IFNa treatment. For the CD4-depletion experiment, 200 mg ofCD4 mAb (clone GK1.5) was injected intraperitoneally one daybefore anti–CD20-IFNa treatment. To block lymphocyte traffick-ing, 25mg of FTY720was injected intraperitoneally one day beforeanti–CD20-IFNa treatment. A total of 10 mg of FTY720 was

administered every day to maintain the blockade. For the PD-L1blockade experiment, 50 mg of anti-mouse PD-L1 Ab (clone10F.9G2) was injected intratumorally simultaneously withanti–CD20-IFNa treatment.

Binding assayA20 cells (2 � 105) were stained with indicated proteins and

single-cell suspensions of A20 cells (2� 105)were incubatedwithanti-CD16/32 (anti-FcgIII/II receptor, clone 2.4G2) for 20 min-utes. Anti-CD20, anti–CD20-IFNa, and hIgGwere serially dilutedand added to the cells to achievefinal dose of 0–500ng. Cellswereincubated on ice for 20 minutes, washed twice, and then incu-bated with anti-human IgG Fcg-PE for 20 minutes on ice. Afterwashing twice, samples were run on a FACSCalibur (BD). Datawere analyzed using FlowJo software (Tree Star, Inc.).

Flow cytometric sorting and analysisSingle-cell suspensions were incubated with anti-CD16/32

(anti-FcgIII/II receptor, clone 2.4G2) for 20 minutes and thenstained with conjugated Abs. All fluorescent-labeling mAbs werepurchased fromBioLegendor eBioscience. Sampleswere analyzedonaFACSCalibur (BD)or FACSFortessaflowcytometer (BD), andcells were sorted on a FACSAria III Cell Sorter (BD). Data wereanalyzed using FlowJo software (Tree Star, Inc.).

Apoptosis assayA total of 5 � 104 A20 cells and BL3750 cells were incubated

with mitomycin C (1 mg/mL) or 2,000 pmol/L hIgG, IFNa-IgG,anti-CD20 and anti–CD20-IFNa in 96 round-well plates at 37�Cfor 48 hours. Cells were stained with Annexin V-Alexa Fluor 647and propidium iodide (PI) to distinguish populations of earlyapoptotic (Annexin VþPI�), late apoptotic (Annexin VþPIþ) andnecrotic (Annexin V�PIþ) cells. The percentage of apoptotic cellswas calculated for each sample as the sum of early apoptotic andlate apoptotic cells.

Measurement of IFNg-secreting tumor-infiltrating CD8þ T cellsby ELISPOT assay

A20-bearingmicewere intratumorally treatedwith3mgof anti–CD20-IFNa and control Abs on day 16. Two days later, tumortissues were digested, and CD8þ T cells were purified by FACSsorting. Approximately 2 � 104 CD8þ T cells and 5 � 104 WTBALB/c mice splenocytes were mixed together with 0.1 mg/mLmitomycin C-treated A20 cells for 44 hours. IFNg production wasdetermined with an IFNg ELISPOT assay kit according to themanufacturer's manual (BD). The visualized cytokine spots werequantified using the ImmunoSpot Analyzer (CTL).

Generation of bone marrow chimerasWTBALB/cmicewere irradiatedwith a single dose of 1,000 rad.

Irradiated mice were adoptively transferred intravenously with 5� 106 CD11c-DTR Tg of donor bone marrow cells the next day.Mice were maintained on sulfamethoxazole and trimethoprim(Bactrim) antibiotics diluted in drinking water for 4 weeks afterreconstitution.Micewere injectedwith tumor cells approximately12 weeks after reconstitution.

APC presentation assayFor the ex vivo APC cross-presentation assay, A20-HA tumor-

bearing BALB/c nude mice were treated intratumorally with 3 mg

Targeting Lymphoma with Antibody-Interferon

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of anti–CD20-IFNa or anti-CD20 Ab on day 12. Two days later,the mice were sacrificed, and the tumor cells were digestedwith 1 mg/mL collagenase IV (Sigma-Aldrich) and 20 U/mLDNase I (Sigma-Aldrich) at 37�C for 40 minutes. Tumor cells(CD45þB220þ), DCs (CD45þB220�CD11cþF4/80�), andmacrophages (CD45þB220�CD11c�F4/80þ) were sorted using aFACSAria III Cell Sorter (BD). A total of 2� 104 purifiedCL4 T cellswere mixed with an approximate number of APCs in ratios of 4:1,8:1, and 16:1. Two days later, the supernatants were collected, andIFNg wasmeasuredbyELISA. For the in vitro tumor cell presentationassay, 2.5� 105 A20-HA cells and 5� 104 purifiedCL4 T cells wereincubated with PMA/ionomycin (50 ng/mL PMA and 1 mg/mLionomycin,obtained fromSigma-Aldrich)or50pmol/Lanti-CD20and anti–CD20-IFNa in 96 round-well plates at 37�C. Eighteenhours later, brefeldin A was added to the supernatants at a finalconcentrationof5 mg/mL. Six hours later, the cellswerefirst stainedwith APC-conjugated anti-mouse CD8a Ab (53-6.7) as a surfaceCD8 marker before fixation/permeabilization and intracellularlystained for IFNg (XMG1.2). All of the reagents and Abs werepurchased from eBioscience, and surface and intracellular stainingwas performed according to the manufacturer's protocols.

Statistical analysesData were analyzed using Prism 6.0 software (GraphPad) and

presented as the mean � SEM. The P values were assessed usingtwo-tailed unpaired Student t test or two-way analysis of variance

with the following thresholds for statistical significance: �, P <0.05; ��, P < 0.01; and ��, P < 0.001.

ResultsDelivery of IFNa to the tumor inhibits B-cell lymphoma growth

Both anti-CD20 (3, 5) and IFNa (9) are therapeutically ben-eficial in the treatment of NHLs. We asked whether combinedanti-CD20 and IFNa treatment would synergistically reduce theCD20þ A20 B-cell lymphoma burden in a syngeneic immuno-competent mouse model. BALB/c mice were inoculated s.c. withA20 cells. Nine days later, 100 mg of anti-CD20 was administeredintravenously to depletemost normal B cells (Supplementary Fig.S1). On days 10–13, mice were daily given 10 mg of IFNa-IgG1,anti-CD20, or control PBS via intravenous injection. Systemicadministration of anti-CD20 or IFNa-IgG1 could not control thetumor growth (Fig. 1A). This result indicates that this model is anappropriate for mimicking clinical anti-CD20 treatment resis-tance. Considering that all nucleated cells express IFNAR (13),and that less than 0.01% of s.c.-injected IFNa would accumulatein the target tissue (12), targeting IFNa to the tumor environmentcould enhance tumor control. To test this hypothesis, we admin-istered 3 mg of IFNa-IgG1, anti-CD20, or control hIgG intratu-morally on days 12 and 14. The intratumoral delivery of this doseof IFNa-IgG1 eliminated the tumor (Fig. 1B). Thus, administra-tion of IFNa directly to the tumor controlled tumor growth more

Figure 1.

Targeting delivery of IFNa to the tumor microenvironment using anti-CD20 efficiently enhances the antitumor effect. A, WT BALB/c mice (n ¼ 5/group)were injected subcutaneously (s.c.) with A20 cells (3 � 106). Then, 100 mg of anti-CD20 was administered intravenously (i.v.) on day 9. A total of 10 mgof IFNa-IgG1, anti-CD20, or control PBS was administered intravenously on days 10, 11, 12, and 13. B, WT BALB/c mice (n ¼ 5/group) were injected s.c.with A20 cells (3 � 106) and treated intratumorally (i.t.) with 3 mg of IFNa-IgG1, anti-CD20, or control hIgG on days 12 and 14. C, Structure of theanti–CD20-IFNa fusion protein. D, WT BALB/c mice (n ¼ 5/group) were injected s.c. with A20 cells (3 � 106); then, 100 mg of anti-CD20 was administeredintravenously (i.v.) on day 9. The average tumor size was 68 mm3. A total of 10 mg of IFNa-IgG1, anti-CD20, anti–CD20-IFNa, or control PBS was administeredintravenously on days 10, 11, 12, and 13. Data represent mean � SEM of two (B, D) or three (A) independent experiments. �� , P < 0.01; �� , P < 0.001.

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effectively thandid systemic administration. This suggests that thetarget for IFNa-induced tumor control is likely inside the tumormicroenvironment.

We proposed that targeting the delivery of IFNa to lymphomaby linking IFNa to anti-CD20 Ab could enhance antitumoractivity. Anti–CD20-IFNa fusion proteins were constructed (Fig.1C) and purified (Supplementary Fig. S2A) as described in theMaterials and Methods section. Binding of the fusion proteins tomurine CD20 (mCD20)was assessed by flow cytometry using theA20 cell line. The anti–CD20-IFNa fusion protein and anti-CD20both bound mCD20-expressing murine lymphoma cells withsimilar avidity (Supplementary Fig. S2B). We analyzed IFNabioactivity of the anti–CD20-IFNa fusion protein using a VSV-GFP (Vesicular stomatitis virus expressing green fluorescent pro-tein) infection assay. Anti–CD20-IFNa and IFNa-IgG equallyinhibited VSV-GFP infection (Supplementary Fig. S2C). We con-cluded that our anti–CD20-IFNa fusion protein maintained thebinding activity of anti-CD20 and the bioactivity of IFNa.

We asked whether delivery of IFNa to the tumor site throughanti-CD20 Abs could overcome anti-CD20 and IFNa resistance.In tumor-bearing mice intravenously treated with the fused anti–CD20-IFNa (Fig. 1D), growth of A20 lymphoma was inhibited;neither anti-CD20 Ab nor IFNa systemic treatment alone had anytherapeutic effect. After fusion protein treatment, 40% of tumorswere eradicated. Thus, targeted delivery of IFNa by anti-CD20 tothe tumormicroenvironment is more effective than anti-CD20 orIFNa-hIgG1 treatment alone.

Tumor regression mediated by anti–CD20-IFNa depends onadaptive immunity

Anti–CD20-IFNa administered intravenously can effectivelycontrol B-cell lymphoma (Fig. 1D).We administered anti–CD20-IFNa intratumorally or intravenously, to determine whetherperipheral lymphoid tissue, or the tumor, was the primary loca-tion where anti–CD20-IFNa functions. The antitumor effect wasbetter after intratumoral, rather than the intravenous, adminis-tration of anti–CD20-IFNa (Fig. 2A), similar to the effectobserved with IFNa-IgG1 in both A20 and BL3750 tumormodels(Supplementary Fig. S3A and S3B). This indicates that the tumortissue is the main site for the effect of anti–CD20-IFNa. To studyhow Ab-IFN works inside tumor tissues and rule out effects fromperipheral tissues, we used intratumoral injection. IFNa caninduce tumor cell apoptosis and cause tumor regression (15).We tested the sensitivity of A20 cells to IFN-mediated apoptosisusing an in vitro culture system. In contrast to BL3750 cells, whichare B-cell lymphoma cells with a C57BL/6 background and aresensitive to IFNa-induced apoptosis, IFNa did not induce apo-ptosis of A20 cells (Fig. 2B and Supplementary Fig. S4). This raisesthe possibility that IFN-mediated tumor regression may dependnot on direct IFN-mediated killing but instead on hostimmune-mediated tumor clearance. To test this hypothesis,we s.c. inoculated A20 cells into syngeneic BALB/c nude mice(T-cell deficiency). Three doses of intratumoral anti–CD20-IFNa had no effect on tumor growth in nude mice (Fig. 2C);this result indicates that T cells are essential for the therapeuticeffect of anti–CD20-IFNa.

Tumor-infiltrating CD8þ T cells play a key role in eliminatingtumors

To determine which subsets of T cells are involved in tumorregression mediated by anti–CD20-IFNa, WT BALB/c mice bear-

ing established A20 tumors were intratumorally treated withfusion protein with CD8þ or CD4þ T cells ablated by intraper-itoneal administration of anti-CD8 or anti-CD4 mAbs. CD8þ T-cell depletion abolished the therapeutic effect of anti–CD20-IFNaadministered either intravenously (Supplementary Fig. S5) or

Figure 2.

The antitumor effects of anti–CD20-IFNa depend on T cells. A, WT BALB/cmice (n ¼ 6/group) were injected subcutaneously (s.c.) with A20 cells(3 � 106) and treated intratumorally (i.t.) with 3 mg of anti–CD20-IFNa orcontrol hIgG on days 11 and 14. B, BL3750 (n ¼ 5/group) or A20 cells(n ¼ 8/group) were incubated with 2,000 pmol/L of the respectivetreatment for 48 hours. The percentage of apoptotic cells was quantified foreach sample as the sum of early apoptotic and late apoptotic cells. C, BALB/cnude mice (n ¼ 5/group) were injected subcutaneously (s.c.) with A20 cells(2 � 106) and treated intratumorally with 3 mg of anti-CD20-IFNa orcontrol hIgG on days 11, 13, and 15. Data represent mean � SEM of two(B, C) or three (A) independent experiments. �� , P < 0.001.






intratumorally (Fig. 3A). Depletion of CD4þ T cells did not affecttreatment efficacy (Supplementary Fig. S6). Thus,CD8þT cells butnot CD4þ T cells are essential for mediated tumor regressionmediated by anti-CD20-IFNa.

As intratumoral injection of the IFN-fusion protein resulted in amore potent and rapid antitumor effect than did systemic deliveryof IFNa, IFNa might induce antitumor immunity inside tumortissues through reactivating exhausted T cells. We investigatedwhether the preexisting TILs in the A20 model were sufficient forthe responsiveness of this model to anti–CD20-IFNa treatment.FTY720, a sphingosine 1-phosphate (S1P) receptor agonist, canpotently inhibit the egression of na€�ve and effector lymphocytesfrom the LN into the circulation and peripheral tissues (17) andthus limit migration of primed T cells into tumor tissues. One dayafter FTY720 treatment initiation, there was a nearly 90% reduc-tion in peripheral T cells. The number of circulating T cells wasfurther decreased after long-term administration of FTY720 (Sup-plementary Fig. S7A and S7B). Antitumor effects of the fusionprotein remained potent in the presence of FTY720 (Fig. 3B).Furthermore, we depleted tumor-infiltrating CD8þ T cells by

intratumoral administration of low doses (20 mg) of anti-CD8mAb. In the absence of tumor-infiltrating CD8þ T cells afterFTY720 treatment, the therapeutic effect of anti–CD20-IFNa wasabrogated (Fig. 3B). To explore whether anti–CD20-IFNa treat-ment could enhance the tumor-specific T-cell response in thetumor microenvironment, we compared CD8þ T cell IFNg pro-duction after anti-CD20-IFNa or control Ab treatment. Miceharboring established A20 tumors were treated intratumorallywith anti–CD20-IFNaor control Ab, andCD8þT cellswere sortedfrom the tumor and restimulated with mitomycin-C–treated A20tumor cells. IFNgþ cells were measured by an enzyme-linkedimmunospot (ELISPOT) assay 2 days after coculture. More IFNgspot-forming cells were observed in the IFNa-treated group thanin the anti-CD20 treatment group (Fig. 3C). In addition, toexplore the activity of CD8þ T cells after anti–CD20-IFNa treat-ment, mice harboring established A20 tumors were treated intra-tumorally with anti–CD20-IFNa or control hIgG on days 14, 16,and 18. CD8þ T cells were sorted from the tumor tissue on day 24and transferred to tumor-bearing nude mice. The CD8þ T cellsfrom the anti-CD20-IFNa–treated immunocompetent mice

Figure 3.

The therapeutic effect of anti–CD20-IFNa is dependent on tumor-infiltrating CD8þ T cells. A, WT BALB/c mice (n ¼ 5/group) were injected subcutaneously (s.c.)with A20 cells (3 � 106) and treated intratumorally (i.t.) with 3 mg of anti–CD20-IFNa or control hIgG on days 14 and 18. CD8þ T-cell–depleting Ab (clone2.43; 200 mg/mouse) was administered intraperitoneally (i.p.) twice a week starting on day 13. B, WT BALB/c mice (n ¼ 5/group) were injected s.c. with 3 � 106

A20 cells on the right flank and treated intratumorally with 3 mg of anti–CD20-IFNa or control hIgG on days 10 and 12. One day before administration ofthe fusion proteins, tumor-bearingmice received 25mgof FTY720 i.p., and the dosewas reduced to 10mgpermouse every 2 days starting onday9. A total of 20mgofCD8-depleting Ab was administered intratumorally on days 10, 12, and 14. C, WT BALB/c mice (n ¼ 5/group) were injected s.c. with A20 cells (3 � 106) onthe right flank. Two days after treatment with 3 mg of IFNa-IgG1, anti-CD20, anti–CD20-IFNa, or control hIgG1 on day 16, tumor-infiltrating CD8þ T cells were sorted,and an IFNg ELISPOT assay was performed with mitomycin C-treated A20 cells and na€�ve splenocytes from WT mice. D, WT BALB/c mice (n ¼ 15/group)were injected s.c. with A20 cells (3 � 106) on the right flank and treated intratumorally with 3 mg of anti–CD20-IFNa or control hIgG on days 14, 16, and 18.Tumor-infiltrating CD8þ T cells were sorted on day 24. BALB/c nude mice (n ¼ 5/group) were injected s.c. with A20 cells (2 � 106), and the cells weretransferred with previously sorted CD3þCD8þ T cells (5 � 105) on day 1. Data represent mean � SEM of two independent experiments. � , P < 0.05;�� , P < 0.01; �� , P < 0.001.

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effectively inhibited tumor growth in immune-deficientmice (Fig.3D). Thus, tumor-infiltrating CD8þ T cells play a key role in theeradication of B-cell lymphoma.

Conventional APCs are not essential for tumor controlThe IFNAR on DCs is essential for anti-EGFR-IFNb–mediated

tumor regression, The DC is a major APC for cross-presentation(18). To test whether DCs are the dominant APCs activatingtumor-infiltrating CD8þ T cells, we used CD11c-DTR–reconsti-tuted BALB/c mice bearing A20 tumors. Anti–CD20-IFNa erad-icated the tumor even in the absence ofDCs (Fig. 4A). Anti–CD20-IFNa therapy generated protection against rechallenge (Fig. 4B),suggesting development of a protective memory response. Theantitumor vaccine effect of anti-human CD20mAb is engaged bymacrophages, which selectively transfer antigens to DCs to pro-mote long-term immunity against cancer (19). Therefore, we usedClophosome, a drug that can deplete macrophages, to determinewhethermacrophages play a key role in controlling tumor growth.However, the antitumor effect of anti–CD20-IFNa was observedin the absence of macrophages (Fig. 4C), along with the estab-lishment of a protective memory response (Fig. 4D). To exclude

the compensatory effects between DCs and macrophages, wedepleted both of these cells simultaneously. No differences wereobserved for tumor growth in the mice deficient in both DCs andmacrophages (Fig. 4E). Given that B cells may function as APCs,we used mMT�/� mice (20), which are deficient in mature B cells,to study whether B cells are required for tumor regression. Hostnormal B cells also did not function as APCs (Fig. 4F). Thus,traditional APCs are not essential for controlling tumor growth inA20 B-cell lymphoma.

Tumor cells as APCs activate the tumor-infiltrating CTLresponse

The antitumor effect started early on day 3 post-treatment(Figs. 2A and 3C), in an intratumor CTL-dependent manner,suggesting that A20 B-cell lymphoma cells could act as APCs.We confirmed that CD86 and MHC class I expression levels ontumor cells increased in the presence of IFNa (Fig. 5A and B).Next, we evaluated whether these activated tumor cells couldbe APCs and stimulate antigen-specific CD8þ T cells. CL4 Tcells, whose TCR is specific for the hemagglutinin antigen(HA) CD8 epitope, were incubated with A20 or A20-HA

Figure 4.

Conventional APCs are not required forCD8þ T-cell–dependent tumor control.A, A20-bearing CD11c-DTR–reconstituted BALB/c mice (n ¼ 5/group) were injected subcutaneously(s.c.) with A20 cells (1� 107) and treatedintratumorally (i.t.) with 3 mg of anti–CD20-IFNa or control hIgG on days 18and21.DTwas administered every 2daysstarting on day 17. B, Approximately 45days after the tumor rejection fromA, themice were rechallenged with A20 cells(3 � 107). Na€�ve WT BALB/c mice wereused as the control. C,WT BALB/c mice(n¼8/group)were injected s.c.withA20cells (3� 106) and treated intratumorallywith 3 mg of anti–CD20-IFNa or controlhIgG on day 10. The macrophage-depleting reagent (Clophosome, 200mg/mouse) or control liposome wasadministered intraperitoneally (i.p.) ondays 9 and 12. D, Approximately 45 daysafter the tumor rejection in C, the micewere rechallenged with A20 cells (1.5 �107). Na€�ve WT BALB/c mice were usedas the control. E, A20-bearing CD11c-DTR–reconstituted BALB/c mice (n ¼5/group) were injected s.c. with 1 � 107

A20 cells and treated intratumorallywith3 mg of anti–CD20-IFNa or control hIgGon days 10 and 13. DT was administeredintraperitoneally on days 9, 11, and 13.Clophosome (200 mg/mouse) or controlliposome was administeredintraperitoneally on days 9 and 13. F,mMT�/� BALB/c mice (n ¼ 6/group)were injected s.c. withA20cells (3� 106)on the right flank and treatedintratumorally with 3 mg of anti–CD20-IFNa or control hIgG on days 10 and 12.Data represent mean � SEM of twoindependent experiments. �� , P < 0.001.






(HA-expressing) cells in the presence of PMA/ionomycin, anti-CD20, and anti–CD20-IFNa. Anti–CD20-IFNa alone couldnot activate CL4 T cells in the absence of its antigens. How-

ever, anti–CD20-IFNa induced CL4 T cells to produce IFNgonly in the presence of A20-HA cells (Fig. 5C). These dataindicate that anti–CD20-IFNa can stimulate self-presentation

Figure 5.

The direct antigen-presenting ability of tumor cells is dramatically enhanced after anti–CD20-IFNa treatment. A and B, A20 cells (5 � 104) per well wereincubated with 20 pmol/L indicated antibodies in 96 round-well plates (n¼ 6/group). 48 hours later, tumor cells were collected and the expression of CD86 (A) orMHC class I (B) was analyzed by flow cytometry. C, Splenocytes and inguinal LN cells were collected from CL4 Tg mice and Thy1.1þTCRVb8þCD8þ T cellswere sorted. Sorted T cells were cocultured with A20-HA cells in the presence of PMA/ionomycin (50 ng/mL PMA and 1 mg/mL ionomycin) or 50 pmol/L anti-CD20and anti–CD20-IFNa in 96 round-well plates for 24 hours (n ¼ 8/group). Six hours before flow cytometry detection, brefeldin A (5 mg/mL) was added. Flowcytometry patterns and gate frequencies in percentages (left), and statistical results (right). D, BALB/c nude mice (n ¼ 5/group) were injected subcutaneously(s.c.) with 2 � 106 A20-HA cells and treated intratumorally (i.t.) with 3 mg of anti-CD20 or anti–CD20-IFNa on day 12. Two days later, macrophages(CD45þB220�CD11c�F4/80þ), DCs (CD45þB220�CD11cþF4/80�), and tumor cells (CD45þB220þ) were sorted from tumor tissues. A total of purified CL4 T cells(2 � 104) were mixed with an approximate number of APCs in ratios of 4:1, 8:1, and 16:1. Two days later, the supernatants were collected, and IFNg wasmeasured by ELISA. Data represent mean � SEM of two independent experiments. �� , P < 0.001.

Liao et al.





by tumor cells, which then activate antigen-specific tumor-infiltrating CTLs.

To verify whether tumor cells could be the dominant APCs,we sorted out various APCs from the Ab-treated tumor tissuesand co-incubated them with CL4 T cells in the presence of 20pmol/L Abs for 48 hours. Compared with the group treatedwith anti-CD20, the antigen-presenting ability of APCs wasimproved after anti–CD20-IFNa treatment (Fig. 5D). Tumorcells, which make up more than 90% of the CD45þ populationin the tumor microenvironment, displayed the most powerfulantigen-presenting ability among three types of APCs on a percell basis (Fig. 5D). Thus, tumor cells, but not conventionalAPCs, are the dominant APCs responsible for the anti-CD20-IFNa–mediated tumor control.

Anti–PD-L1 therapy could enhance anti–CD20-IFNa treatmentPersistent antigenic stimulation leads to CD8þ T-cell exhaus-

tion (21). Advanced tumors might develop adaptive resistanceand exhaust TIL over time. Classic Hodgkin lymphoma (cHL)frequently exhibits genetic alterations that lead to overexpressionof the programmeddeath-1 (PD-1) ligands; such tumorsmight bevulnerable to PD-1 blockade. Indeed, anti–PD-L1 Ab is associatedwith a favorable safety profile, but complete response is still rare(22, 23). Type I and II IFNs can induce PD-L1 upregulation in Bcells (24). Consistent with this report, our in vitro (Fig. 6A) andin vivo (Fig. 6B) results demonstrated that PD-L1 levels wereincreased in tumor cells after IFNa treatment. This increaseexplains the difficulty in controlling advanced cancers using thefusion protein and raises the possibility that PD-L1 may be anadaptive resistance mechanism that allows lymphoma to escape

immune destruction and resist IFN therapy.We proposed that thecombination therapy of PD-L1 checkpoint blockade and IFNamight overcome the resistance of large tumors to a single treat-ment. To test the efficacy of this treatment strategy, WT BALB/cmice bearing established A20 tumors that were not responsive tosingle treatment were treated with the fusion protein and anti–PD-L1 Abs simultaneously. PD-L1 blockade further enhanced theantitumor efficacy of anti–CD20-IFNa for the advanced largetumors, and approximately 40% of the tumors were eradicated(Fig. 6C). Mice with complete tumor regression after combina-tional treatment resisted the A20 tumor rechallenge with 5 timesthe lethal dose; this result suggests long-term memory protectionfrom relapse (Fig. 6D). Together, these data indicate that PD-L1checkpoint blockade can enhance the therapeutic effect of anti–CD20-IFNa.

DiscussionSome patients with B-cell lymphoma respond to treatment

with either anti-CD20 or IFN; others do not respond or developresistance. Toxicity with systemic delivery limits clinical use ofIFN. Human IFNa2b fused to anti-hCD20 Ab (veltuzumab)exhibited more potent antitumor efficiency than either agentalone or in combination in human lymphoma xenografts (14).Linking type I IFN to tumor antigen-associated Abs increased theantitumor effect of type I IFN in a xenograftmodel by direct killingof IFNa-sensitive 38C13-hCD20 tumor cells (15). Treatmentwithanti–hCD20-hIFNa reversed rituximab resistance of B-NHLin vitro, resulting in inhibition of cell proliferation and inductionof cell death (16). Becausemany lymphomas are resistant to anti–

Figure 6.

Anti–PD-L1 can enhance the therapeutic effect of anti–CD20-IFNa to control advanced B-cell lymphoma. A, A20 cells (5 � 104) per well were incubated with20 pmol/L of indicated antibodies in 96 round-well plates (n ¼ 6/group). 48 hours later, tumor cells were collected and the expression of PD-L1 wasanalyzed by flow cytometry. B,WTBALB/cmice (n¼ 5/group) were injected subcutaneously (s.c.) with A20 cells (3� 106). A total of 3 mg of IFNa-IgG1, anti-CD20,anti-CD20-IFNa, or control hIgG was administered intratumorally (i.t.) on day 16. The average tumor size was 193 mm3. Two days later, tumor cells werecollected and PD-L1 expression was analyzed by flow cytometry. C, WT BALB/c mice (n ¼ 13/group) were injected s.c. with A20 cells (3 � 106) and treatedintratumorally with 3 mg of anti–CD20-IFNa or control hIgG on days 16, 18, and 20. Fifty micrograms of anti–PD-L1 (10F.9G2) or Rat IgG (rIgG) was administeredintratumorally on the same time. D, Approximately 45 days after the tumor rejection from C, the mice were rechallenged with A20 cells (1.5 � 107). Na€�veWT BALB/c mice were used as the control. Data represent mean � SEM of two independent experiments. �� , P < 0.01; �� , P < 0.001.






CD20- or IFN-mediated apoptosis, we wondered whether target-ing tumor cells with IFNa could mobilize the adaptive immunesystem for tumor control. Using a syngeneic tumor model ofIFNa-resistant A20 B-cell lymphoma, we observed that tumorregression mediated by anti–CD20-IFNa depends on adaptiveimmunity. Anti–CD20-IFNa enhanced the antigen-presentingfunction of lymphoma cells to reactivate tumor-infiltrating CD8þ

T cells, which play a key role in eliminating the tumor. However,anti–CD20-IFNa also induced adaptive resistance after severaldoses of administration. Therefore, we used a combination ofimmune checkpoint blockade and anti–CD20-IFNa to overcometreatment-induced adaptive resistance. Indeed, anti–CD20-IFNaenhanced anti–PD-L1 treatment to enhance the antitumor effectfor advanced B-cell lymphoma (Fig. 7).

Addition of rituximab or IFNa treatment to chemotherapycould increase patients' response rates and effectively extendprogression-free survival (25–27). Response to IFNa and rituxi-mab/IFNa combination treatment is similar, and longer progres-sion-free survival was observed in patients with extended IFNatreatment (20.9 months vs. 48.7 months; ref. 28). Many B-celllymphoma patients have no effective response to the current

clinical treatment. Similar to many human lymphomas, A20tumors are resistant to chemotherapy and to anti–CD20 Ab- orIFNa-induced tumor apoptosis and have poor responses to bothanti-CD20 therapy and chemotherapy. These characteristics indi-cate that the A20 model may mimic lymphoma that is resistant toclinical treatment. Administration of IFNa intratumorally reducedtumor burden; systemic administration of anti–CD20-IFNa wasless effective. Thus, a proper IFNa concentration in the tumor iscritical for tumor control. Therefore, we constructed an anti–CD20-IFNa fusion protein in order to achieve target delivery of IFNa intothe tumor microenvironment. Some lymphomas may be moresensitive to direct killing by either anti-CD20 Ab or IFNa, whereasothers depend more on adaptive immune responses.

IFN is a potent cytokine that activates antigen-presenting cellsand increases cross-priming in the draining LN, which can thenexpand tumor-specific T cells. However, preexisting tumor-infiltrat-ing CD8þ T cells played a key role in eliminating B-cell lympho-ma by using FTY720, an inhibitor of T-cell trafficking from thedraining LN to tumor tissues. Injection of FTY720 before andduring treatment did not change the therapeutic effect of anti–CD20-IFNa. This result raises the possibility that targeting the

Figure 7.

Proposed model for anti–CD20-IFNa–mediated tumor regression. During the process of tumor establishment, infiltrating activated T cells become exhausted inthe tumormicroenvironment. Anti–CD20-IFNa fusion protein could deliver targeted IFNa to the tumor and dramatically enhance the antigen-presenting ability of tumorcells. In this situation, tumor cells could rapidly activate tumor-infiltrating CD8þ T cells, leading to a reduction of the tumor burden. Although both type I IFN andtype II IFN could upregulate PD-L1 expression on tumor cells, anti–CD20-IFNa could also enhance anti–PD-L1 treatment to mediate the antitumor response.

Liao et al.





tumormicroenvironment with IFNamight be sufficient to activatelocal APCs for TILs. DCs are the chief APCs that prime the CTLresponse (29, 30). The antitumor vaccine effect of anti–human-CD20 Ab is engaged by macrophages via selective transfer ofantigens to DCs; this result further promotes long-term immunityagainst cancer (19). Neither DCs nor macrophages were essentialfor tumor regression mediated by anti–CD20-IFNa or establish-ment of immune protection in our syngeneic B-cell lymphoma. Inaddition, anti–CD20-IFNa retained its antitumor function in theabsence of host normal B cells. CD8þ T cells directly interact withantigen-expressing B lymphoma (31–33). A20 lymphoma B cellscould be the APCs that affect the tumor-infiltrating CTL response.Unlike anti-CD20–treated tumor cells, the antigen-specific CD8þ Tcells were activated by tumor cells treated with anti–CD20-IFNaand were the largest cell population in the tumor microenviron-ment. Tumor-infiltrating CD8þ T cells in the previously treatment-resistant tumor microenvironment might be activated when theAPC function of lymphoma cells is significantly enhanced bytreatment with the targeted Ab anti–CD20-IFNa. Our studydemonstrates that tumor cells could be antigen-presenting cellsbut does not rule out that host cells can also contribute to antigenpresentation.

During tumor progression, a single treatment loses its thera-peutic efficacy because of intrinsic resistance and acquiredimmune tolerance (5, 34, 35). Many combination therapies havebeen proposed to treat advanced cancers, including costimulatoryreceptor agonists (anti-CD137; ref. 36) or blockade of the anti-phagocytic ("don't eat me") signal (anti-CD47; ref. 37) in com-bination with CD20mAb. Immune checkpoint therapy has led tobreakthroughs in cancer therapy (38). The ligands for PD-1, PD-L1 (B7-H1), and PD-L2 (B7-DC) could also be upregulated inresponse to inflammation (39). Many B-cell lymphomas remainunresponsive or become unresponsive after initial favorableresponses to anti–PD-L1 or IFN treatment. As a combinationtherapy, PD-L1 blockade could enhance the antitumor efficacy ofanti–CD20-IFNa and reduce relapse rates for advanced largetumors that are resistant to either anti-CD20 Ab or the anti–CD20-IFNa fusion protein. Thus, the promising future of anti–

CD20-IFNa therapy might be in combination with other immu-notherapies, including anti–PD-L1, for the treatment of cancer.

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

Authors' ContributionsConception and design: J. Liao, Y.-X. FuDevelopment of methodology: J. Liao, Z. RenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Liao, X. Liu, H. XuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Liao, Y.-X. FuWriting, review, and/or revision of the manuscript: J. Liao, H. Peng, Y.-X. FuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Luan, D. Xue, H. Xu, Z. Sun, K. Yang, H. PengStudy supervision: H. Peng, Y.-X. Fu

AcknowledgmentsThe authors thank Dr. Mingzhao. Zhu (Institute of Biophysics, CAS) for

helpful suggestions and comments on the project.We also thankDaryl HarmonandCasey Timmerman for editing.mMT�/�micewere kindly provided byZ.Qin(the Institute of Biophysics, CAS). The authors also thankH. Su,Q. Li, and S.Wei(Institute of Biophysics, CAS) for expert technical assistance.

Grant SupportThis work was supported by National Natural Science Foundation of

China grant (No. 81172814) to H. Peng (No 881202328) to Y. Luan, Keydeployment project from Chinese Academy of Sciences (No. KFZD-SW-205)to H. Peng, Ministry of Science and Technology of China grant (No.2016YFC1303400 and No. 2011DFA31250) to Y.-X. Fu, and National Scienceand Technology Major Project of China grant (No. 2012ZX10001006) to H.Peng. This research was in part supported by the U.S. National Institutes ofHealth grants CA141975 to Y.-X. Fu.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 30, 2016; revisedDecember 9, 2016; acceptedMay 15, 2017;published OnlineFirst May 22, 2017.

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Liao et al.




2017;5:560-570. Published OnlineFirst May 22, 2017.Cancer Immunol Res Jing Liao, Yan Luan, Zhenhua Ren, et al. for Interferon-Induced Tumor RegressionConverting Lymphoma Cells into Potent Antigen-Presenting Cells

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