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Adoptive ImmunotherapyCells Mediate Tumor Regression in Cancer In Vivo Sensitized and In Vitro Activated B

Li and Alfred E. ChangQiao Li, Seagal Teitz-Tennenbaum, Elizabeth J. Donald, Mu

http://www.jimmunol.org/content/183/5/3195doi: 10.4049/jimmunol.0803773August 2009;

2009; 183:3195-3203; Prepublished online 10J Immunol 

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In Vivo Sensitized and In Vitro Activated B Cells MediateTumor Regression in Cancer Adoptive Immunotherapy1

Qiao Li,2 Seagal Teitz-Tennenbaum, Elizabeth J. Donald, Mu Li,and Alfred E. Chang2

Adoptive cellular immunotherapy utilizing tumor-reactive T cells has proven to be a promising strategy for cancer treatment.However, we hypothesize that successful treatment strategies will have to appropriately stimulate not only cellular immunity, butalso humoral immunity. We previously reported that B cells in tumor-draining lymph nodes (TDLNs) may function as APCs. Inthis study, we identified TDLN B cells as effector cells in an adoptive immunotherapy model. In vivo primed and in vitro activatedTDLN B cells alone mediated effective (p < 0.05) tumor regression after adoptive transfer into two histologically distinct murinepulmonary metastatic tumor models. Prior lymphodepletion of the host with either chemotherapy or whole-body irradiationaugmented the therapeutic efficacy of the adoptively transferred TDLN B cells in the treatment of s.c. tumors as well as metastaticpulmonary tumors. Furthermore, B cell plus T cell transfers resulted in substantially more efficient antitumor responses than Bcells or T cells alone (p < 0.05). Activated TDLN B cells conferred strong humoral responses to tumor. This was evident by theproduction of IgM, IgG, and IgG2b, which bound specifically to tumor cells and led to specific tumor cell lysis in the presence ofcomplement. Collectively, these data indicate that in vivo primed and in vitro activated B cells can be employed as effector cellsfor cancer therapy. The synergistic antitumor efficacy of cotransferred activated B effector cells and T effector cells represents anovel approach for cancer adoptive immunotherapy. The Journal of Immunology, 2009, 183: 3195–3203.

T cell-based therapy of advanced cancers has been one ofthe few cellular strategies that have resulted in clinicallysignificant and durable responses. Dudley et al. have re-

ported that the adoptive transfer of tumor-infiltrating lymphocytescan result in regression of large burdens of metastatic tumor inmelanoma patients (1). Our laboratory has been involved in inves-tigating tumor-primed lymph nodes as a source of lymphoid cellsthat could be secondarily sensitized by in vitro methods to generateeffector T cells for adoptive immunotherapy (2–6). In patients withadvanced renal cell cancers, we have documented the ability toobtain durable clinical responses using vaccine-primed lymphnode cells in adoptive immunotherapy (7). However, clinical re-sponses have been confined to a subset of patients. Novel ap-proaches are needed to improve the efficacy of adoptive T celltherapies.

Cellular and humoral responses represent two critical arms ofimmunity. We hypothesize that any successful cancer treatmentstrategy will, in the final analysis, have to appropriately stimulateboth the humoral as well as the cellular immune responses. Un-fortunately, the current knowledge with respect to the effectiveinduction of anticancer humoral responses lags well behind that ofthe induction of cellular responses. To date, the predominant in-vestigative focus of adoptive immunotherapy for cancer has been

to understand the mechanisms involved in the induction, activa-tion, proliferation, and trafficking of effector T cells.

We have previously shown that approximately 60% of tumor-draining lymph node (TDLNs)3 cells are CD3� T cells. In vitroactivation of TDLN cells with anti-CD3/anti-CD28 mAbs resultsin the generation of therapeutic effector T cells (�90% CD3�

cells) (8–10). Nevertheless, the immune function of B cells, whichcomprise �30% of TDLN cells, and their potential antitumor re-activity have not been well characterized. To test our hypothesisthat TDLN B cells may function as APCs, we have reported thatthe simultaneous in vitro targeting of CD3 on T cells and CD40 onB cells or dendritic cells resulted in the generation of more potenteffector cells when adoptively transferred into tumor-bearing mice(5). These results established a role for engaging CD40 on TDLNB cells or dendritic cells as APCs in the generation of effector Tcells. Depletion of B cells and/or dendritic cells from the TDLNcells before in vitro activation abrogated the anti-CD3 and anti-CD40 augmented antitumor immunity (5).

More recently, we examined the immune-modulating effects ofIL-21 when administrated concomitantly with T cell transfer forcancer therapy (6). IL-21 administration promoted both T and Bcell antitumor reactivity. The humoral response associated withIL-21 administration was manifested by increased levels of tumor-specific IgG2b in the sera of animals. Use of B cell-deficient miceprovided direct evidence that host B cells contributed to T cell plusIL-21-elicited antitumor immunity because the IL-21-augmentedtherapeutic efficacy of the transferred T cells in the wild-type hostwas significantly reduced in the B�/� host.

In this present report, we have identified that B cells purifiedfrom TDLNs can function as effector cells in the adoptive cellulartherapy of established cancers. Furthermore, the combined use of

Division of Surgical Oncology, University of Michigan, Ann Arbor, MI 48109

Received for publication November 11, 2008. Accepted for publication July 1, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grant CA82529and by the Gillson Longenbaugh Foundation.2 Address correspondence and reprint requests to Dr. Alfred E. Chang and Dr. QiaoLi, University of Michigan Comprehensive Cancer Center, 1500 E. Medical CenterDrive, 3303 CC, Ann Arbor, MI 48109. E-mail addresses: aechang@umich.edu andqiaoli@umich.edu

3 Abbreviations used in this paper: TDLN, tumor-draining lymph node; CM, completemedium; TBI, total body irradiation.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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B effector cells and T effector cells in adoptive cellular transferresults in substantially more effective antitumor responses.

Materials and MethodsMice

Female C57BL/6 (B6) mice from The Jackson Laboratory were maintainedin a pathogen-free environment and used at age 8 wk or older. Principlesof laboratory animal care (National Institutes of Health publication 85-23,revised 1985) were followed. The University of Michigan Laboratory ofAnimal Medicine approved all the animal protocols.

Murine tumor cells

MCA 205 is a weakly immunogenic 3-methylcholanthrene-induced fibro-sarcoma that is syngeneic to B6 mice. The D5 melanoma tumor line is aclone of the B16-BL6 tumor line that is poorly immunogenic and synge-neic to B6 mice. D5G6 is a GM-CSF-secreting D5 melanoma tumor linegenerated in our laboratory (11). MCA 205 tumors were maintained in vivoby s.c. transplantation in B6 mice and used within the eighth transplantgeneration. Tumor cells were isolated from solid tumors and resuspendedin PBS for administration into mice or in complete medium (CM) for invitro assays as previously described (9).

TDLNs

To induce TDLNs, 1 � 106 MCA 205 or D5G6 tumor cells in 0.1 ml ofPBS were injected s.c. into the lower flanks of normal syngeneic mice. Thedraining inguinal lymph nodes were collected 9 days later and processedusing mechanical dissociation, then filtered through nylon mesh andwashed in HBSS. Multiple inguinal TDLNs were pooled from groups ofmice for lymphoid cell suspension preparation. CD3� T cells and CD19�

B cells were purified from TDLN cells by using Ab-coupled Microbeadsand a MACS separator (Miltenyi Biotec).

Cell activation and expansion

T cells and/or B cells were activated with immobilized anti-CD3 plus anti-CD28 mAbs in CM containing human recombinant IL-2 and/or LPS(Sigma-Aldrich) plus anti-CD40. Anti-CD3 and anti-CD28 (BD Bio-sciences) were immobilized on 24-well or 6-well tissue culture plates(Costar). Immobilization was achieved by placing 1 ml of Ab solution inPBS in the wells of the plates and incubating overnight at 4°C or at roomtemperature for 5 h. The coated plates were washed with PBS and thenused for TDLN cell activation at a concentration of 2 � 106 cells/ml. CMwas used for T cell activation in the presence of recombinant IL-2 (60IU/ml) (Novartis). These cells were activated/expanded at 37°C with 5%CO2 for 4 days. Anti-CD40 (FGK45) mAb ascites (1/300 dilution) wereused in a soluble form. The anti-CD40 ascites were produced by usingFGK45 hybridoma cells (American Type Culture Collection). The use ofanti-CD40 ascites at 1/300 dilution was determined by previous titratingtests to be optimal for B cell expansion in combination with LPS (5 �g/ml).We determined the IgG content in anti-CD40 ascites using ELISA andfound that the 1/300 dilution is equivalent to an IgG concentration of 0.2–0.5 �g/ml. Activated and expanded TDLN T and/or B cells were used foradoptive immunotherapy, phenotype, and immune function analyses.

Ab production assessment

Supernatants at the end of cell activation were collected and analyzed forIgM, IgG, and IgG2b production using ELISA (BD Pharmingen). Addi-tionally, we also tested IgG and IgM production in response to tumor Agstimulation. After activation and expansion, 1 � 106 TDLN T and B cellswere cocultured with 2.5 � 105 irradiated (6000 cGy) tumor stimulatorcells in 24-well tissue culture plates at 37°C with 5% CO2 for 24 h. Thesupernatant was then collected and analyzed for the production of IgG andIgM in response to tumor by ELISA.

Adoptive T and/or B cell therapy of pulmonary metastatic tumorand s.c. tumor

B6 mice were inoculated i.v. by tail vein with 2 � 105 MCA 205 or 1 �105 D5 tumor cells to establish pulmonary metastases. Three or 5 days aftertumor inoculation, the tumor-bearing mice were treated with tail vein in-jection of activated T cells and/or B cells purified from MCA 205 TDLNor D5G6 TDLN cells. In separate experiments, tumor-bearing mice weretreated with unfractionated TDLN cells activated with anti-CD3/anti-CD28/IL-2 or anti-CD3/anti-CD28/IL-2 plus LPS/anti-CD40. Commenc-ing on the day of the cell transfer, i.p. injections of IL-2 (40,000 IU) wereadministered in 0.5 ml of PBS and continued twice daily for eight doses.

Approximately 14 days after T cell transfer, all mice were sacrificed, andlungs were harvested for enumeration of pulmonary metastatic nodules.Subcutaneous tumors were treated in combination with whole body irra-diation as described below.

Treatment of s.c. tumors with purified B cells in combinationwith total body irradiation (TBI) and the induction oflymphodelpletion

Established (5-day) s.c. MCA 205 tumor-bearing mice were treatedwith TBI (5 Gy) followed by adoptive transfer of purified and activatedMCA 205 TDLN B cells. Tumor size was then monitored. In otherexperiments, we utilized a clinically relevant protocol for the inductionof nonmyeloablative lymphodepletion and executed B cell therapy ofcancer under lymphopenic conditions. In these experiments, we usedthe D5 melanoma tumor, which is resistant to cyclophosphamide andfludarabine chemotherapy. In this model, D5 tumor cells were inocu-lated on day 0, chemotherapy (1 mg of cyclophosphamide and 2 mg offludarabine) was administered i.p. on days 3 and 4, and activated B cellswere adoptively transferred i.v. on day 5.

Flow cytometry analysis

Cell surface expression of CD3, CD19, and B220 was analyzed by immu-nofluorescence assay and flow cytometry using a FACSCalibur flow cy-tometer (BD Biosciences). Fluorescence profiles were generated by ana-lyzing 10,000 cells and displayed as logarithmically increased fluorescenceintensity vs cell numbers. All FITC- or PE-conjugated Abs were from BDPharmingen. To block binding to the Fc receptors, mouse BD Fc Block(purified anti-mouse CD16/CD32; BD Pharmingen) was used to incubatethe cells before the addition of FITC- or PE-conjugated Abs. Binding ofIgG2b produced by activated B cells to tumor cells was detected usingFITC-anti-mouse IgG2b or matched isotype control (both from BD Bio-sciences) following the incubation of tumor cells with B cell culture su-pernatants. FACS data analysis was performed using the CellQuest soft-ware (BD Biosciences).

Ab and complement-mediated cytotoxicity

Tumor cell lysis mediated by Abs produced during TDLN activation wasassessed by incubating tumor cells with culture supernatants in test tubeson ice for 1 h followed by cell culture in the presence of rabbit complement(Calbiochem) in a 37°C water bath for another hour. Viable cells were thencounted under a microscope after trypan blue staining to calculate celllysis. Alternatively, cytotoxicity was analyzed in 96-well flat-bottom tissueculture plates and was determined using the Quick Cell Proliferation Assaykit (BioVision) according to the manufacturer’s instructions. OD valueswere measured via a multiwell spectrophotometer to evaluate tumor celllysis.

Statistical analysis

The significance of differences in numbers of metastatic nodules, the sizeof s.c. tumors, the concentration of Ig, and cell lysis was determined usingone-way ANOVA (Newman-Keuls post hoc test) or unpaired t test. Valuesof p �0.05 were considered statistically significant between the experimentgroups.

ResultsTumor-primed and ex vivo activated B cells produce IgG andIgM in response to tumor

To date, molecular targets chosen for Ab activation to generateantitumor effector cells have been confined on T cells. We reportedpreviously (5) that while TDLN cells are composed mainly of Tcells, which are �55% of the whole TDLN cell population, the restof the TDLN cells are CD3� cells. In this study, we further char-acterized these CD3� cells in the TDLNs. Flow cytometry analysis(Fig. 1) indicated that in addition to the 50–55% of CD3� TDLNT cells (Fig. 1A), the remainder of the �45% TDLN cells are Bcells. This was revealed by their positive staining of CD19 (Fig.1B) or B220 (Fig. 1C). PE-anti-CD19 and FITC-anti-B220 doublestaining revealed that these TDLN B cells express both CD19 andB220 (Fig. 1D).

To test our hypothesis that in vivo sensitized TDLN B cells mayhave the potential to mediate antitumor responses, we purified

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these B cells from the TDLN cells. Utilizing anti-CD19-coupledmagnetic beads, we have consistently obtained an enriched popu-lation of B cells from TDLNs (�95% CD19�, data not show).

Purified TDLN B cells were put into 24-well culture plates at 2 �106 cells/ml, 2 ml/well in CM, and activated with LPS or anti-CD40, respectively, for 4 days. Activated B cells were harvestedfor phenotype and function analyses. In vitro studies with varyingconcentrations of LPS or anti-CD40 revealed that LPS activationincreased IgG and IgM production by increasing B cell prolifera-tion (data not shown). Anti-CD40 increased Ig production by stim-ulating B cell differentiation (indicated by CD38 modulation, datanot shown). These activated B cells when cultured with irradiatedtumor cells resulted in significantly greater Ig production com-pared with the absence of tumor cells (B cells alone) (Fig. 2A).These experiments suggested that tumor-primed and LPS- or anti-CD40-activated TDLN B cells produce Ab in response to tumor.Furthermore, the combination of LPS and anti-CD40 resulted insignificantly ( p � 0.05) enhanced Ig production compared withLPS or anti-CD40 activation alone (Fig. 2B). We therefore usedthis LPS and anti-CD40 combination as TDLN B cell stimuli inthis study.

In vivo primed and in vitro activated TDLN B cells mediatesignificant tumor regression after adoptive transfer

To understand the immunological significance of Ab productionby in vivo sensitized and in vitro activated B cells derived fromTDLNs, we performed adoptive therapy experiments using wholeTDLN cells after simultaneous activation of T cells with anti-CD3/

FIGURE 1. CD19� B cells in the TDLNs. TDLN cell suspension wasprepared from freshly harvested TDLNs and stained with FITC-anti-CD3(A), PE-anti-CD19 (B), or FITC-anti-B220 (C), respectively, or double-stained with PE-anti-CD19 and FITC-anti-B220 (D). Stained cells werethen analyzed by flow cytometry using a FACScan flow microfluorometeras described in Materials and Methods. Data are representative of fiveexperiments performed.

FIGURE 2. Ig production of TDLNB cells after activation. A, Purified andactivated TDLN B cells produce IgGand IgM in response to tumor. B cellspurified from MCA 205 TDLNs wereactivated with LPS or anti-CD40, re-spectively. Activated TDLN B cells(1 � 106) were then restimulated withirradiated MCA 205 tumor cells for24 h. The culture supernatant with tu-mor restimulation (B cell � tumor) orwithout tumor restimulation (B cellalone) were collected and analyzedfor the secretion of IgG and IgM byELISA. B, LPS plus anti-CD40 acti-vation significantly enhanced IgMproduction of TDLN B cells com-pared with LPS or anti-CD40 activa-tion alone. Purified B cells fromTDLNs were activated with LPSand/or anti-CD40. Cultured superna-tants at the end of cell activation werethen collected and analyzed for Igproduction using ELISA.

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anti-CD28 plus activation of B cells with LPS/anti-CD40. Utiliz-ing the MCA 205 tumor model, we compared the antitumor reac-tivity of these activated TDLN cells with TDLN cells activatedwith anti-CD3/anti-CD28 alone in the adoptive immunotherapy of3-day established pulmonary metastases. As shown in Fig. 3A,unfractionated TDLN cells simultaneously activated with anti-CD3/anti-CD28 plus LPS/anti-CD40 to stimulate T cells and Bcells (group 2) resulted in significantly enhanced tumor regressionin adoptive transfer compared with an equivalent number of cellsactivated with anti-CD3/anti-CD28 only (T cells alone, group 1).By FACS, TDLNs activated with anti-CD3/CD28 alone had�95% T cells compared with �70%:30% T:B cells with anti-CD3/CD28 plus LPS/anti-CD40 activation. These experimentsclearly indicated that antitumor efficacy of in vitro activated T cellsplus B cells was more potent than using T cells alone.

We proceeded to investigate whether purified TDLN B cells alonewere capable of mediating tumor regression in adoptive transfer mod-

els. We enriched B cells by immunomagnetic beads and secondarilyactivated them with LPS and anti-CD40. These activated B cells wereadoptively transferred into mice with 3-day established lung metas-tases. The results are illustrated in Fig. 3B. Activated B cells alonemediated significant ( p � 0.01) tumor regression compared with un-treated control animals. We also evaluated the antitumor activity ofthe mixture of purified T and B cells from TDLNs that were activatedtogether with anti-CD3/anti-CD28 plus LPS/anti-CD40. When puri-fied T and B cells from TDLNs were mixed at a T:B ratio of 1:2 andactivated, a 65%:35% T:B cell mixture was obtained. These cellsmediated significantly greater reduction of tumor metastases (group 3)upon adoptive transfer compared with equal numbers of purified Tcells (group 2) or B cells (group 4) that were activated by anti-CD3/anti-CD28 or LPS/anti-CD40, respectively (Fig. 3B). Collectively,these data indicate that enhanced antitumor efficacy of cell therapycan be achieved by cotransferring T cells plus B cells after in vivopriming and in vitro activation. In vivo primed and in vitro activatedB cells may represent a novel effector cell type for cancerimmunotherapy.

Radiation and chemotherapy significantly enhance thetherapeutic efficacy of adoptively transferred B cells

Subcutaneous tumor models are generally more resistant to cellu-lar therapy than are pulmonary metastatic tumor models. We pro-ceeded by examining whether adoptively transferred effector Bcells could mediate effective antitumor effects in a s.c. tumormodel. In Fig. 4A, established (5-day) s.c. MCA 205 tumor-bear-ing mice were treated with TBI (5 Gy) followed by the adoptivetransfer of purified and activated MCA 205 TDLN B cells. WhileTBI alone slightly suppressed tumor growth, B cell transfer aloneshowed no therapeutic efficacy in the s.c. tumor model. However,B cell transfer following TBI synergistically and significantly in-hibited tumor growth.

In a similar way as described for the treatment of s.c. MCA 205tumors in Fig. 4A, we treated s.c. D5 tumors with TBI (5 Gy)and/or B cells (5 � 106). The results are summarized in Fig. 4B.Compared with the control (No treatment), TBI alone showed notherapeutic efficacy, while B cell transfer inhibited the s.c. D5tumor growth to a modest extent. Importantly, B cell transfer fol-lowing TBI resulted in significant inhibition of tumor growth ( p �0.03 compared with any other group).

TBI of 5 Gy induces lymphodepletion (12). The results revealedin Fig. 4, A and B, suggest that adoptively transferred TDLN Bcells suppress tumor growth more effectively in the lymphopenichost. To further evaluate the function of adoptively transferredeffector B cells in the lymphopenic host, we utilized a clinicallyrelevant protocol for the induction of nonmyeloablative lym-phodepletion and executed B cell therapy of cancer under theselymphopenic conditions. In this experiment, we used the D5 mel-anoma tumor because it is resistant to cyclophosphamide and flu-darabine chemotherapy. TDLN cells were induced by inoculatingB6 mice with a GM-CSF-secreting D5 tumor line in the flank (11).D5G6 TDLN B cells were enriched for subsequent activation withLPS and anti-CD40. These cells were transferred into chemother-apy-conditioned lymphopenic hosts for the treatment of estab-lished 5-day D5 melanoma lung metastases. Chemotherapy medi-ated a profound, but transient, reduction in total white blood cellcounts in the peripheral blood (data not shown). As demonstratedin Fig. 4C, chemotherapy alone had no significant therapeutic ef-fect on D5 tumor growth. In vivo sensitized and in vitro activatedB cells mediated D5 tumor regression ( p � 0.05) in a dose-de-pendent manner. These results verified the therapeutic efficacy ofTDLN B cells in a second tumor model tested in this study. More

FIGURE 3. Activated TDLN B cells mediated effective tumor regres-sion. A, Simultaneously activated T and B cells in unfractionated TDLNcells mediated significantly more efficient tumor regression than did T cellsalone. B6 mice were inoculated i.v. by tail vein with MCA 205 tumor cellsto establish pulmonary metastases. Three days after tumor inoculation, thetumor-bearing mice were treated with tail vein injection of various doses ofunfractionated MCA 205 TDLN cells activated with anti-CD3/anti-CD28/IL-2 or anti-CD3/anti-CD28/IL-2 plus LPS/anti-CD40, respectively. Ap-proximately 14 days after cell transfer, all mice were randomized and sac-rificed, and lungs were harvested for enumeration of pulmonary metastaticnodules. There were five mice in each group, and all of the animals formedtumor except for G2 when 3 � 106 cells were infused. Data are represen-tative of four experiments performed. B, Purified and activated TDLN Bcells alone mediated tumor regression, and the therapeutic efficacy can besignificantly augmented by purified B cells plus T cells. Three-day pulmo-nary metastases were established as in A, but treated with purified andactivated B cells alone, T cells alone, or T cells plus B cells. Lungs wereharvested for enumerations of pulmonary metastatic nodules as in A. Therewere five mice in each group, and all of the animals formed tumor exceptfor group 3 in both experiments. Data are representative of five experi-ments performed.

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importantly, for both doses of B cells infused, there was signifi-cantly ( p � 0.001) greater therapeutic effect in the lymphode-pleted hosts. These experiments indicate that induction of lym-phopenia before adoptive transfer of TDLN B cells couldsignificantly augment the therapeutic efficacy of these cells.

Therapeutic efficacy of TDLN B cells is associated with the Abproduction and tumor-specific cytotoxicity

To define the possible mechanisms underlying the antitumor ef-fects of TDLN B cells, we compared the Ab production and ex-amined the presence of cytotoxic Ab subclasses (e.g., IgG2b) afterculture of the purified B cells used for adoptive immunotherapy.

At the end of cell activation and expansion with anti-CD3/anti-CD28 and/or LPS/anti-CD40, cell culture supernatants were col-

lected for Ig detection (Fig. 5). Significantly increased IgM, IgG,and IgG2b production ( p � 0.001) in groups 3 and 4 correlatedwith the significantly increased therapeutic efficacy observed usingthese cells (T�B at 65–70%:30–35%) in adoptive immunotherapyof established pulmonary metastases (Fig. 3).

In Fig. 4, A and C, we tested the therapeutic efficacy of purifiedB cells to treat established (5-day) s.c. tumors in combination withTBI (Fig. 4A) or to treat established D5 pulmonary metastatic can-cer in a lymphodepleted host (Fig. 4C). Ab production was alsoassessed in the culture supernatant of these purified B cells usedfor adoptive transfer in these experiments. Table I shows that thesepurified TDLN B cells consistently produce IgM, IgG, and IgG2bafter activation with LPS/anti-CD40 before infusion.

IgG2b was reported to mediate hyperacute rejection in rat car-diac xenografts in a complement-dependent manner (13). Wetested the binding of IgG2b to tumor cells by flow cytometry in thefollowing experiments. Fig. 6 shows that MCA 205 TDLN B cell-produced IgG2b, as shown in Table I, binds to MCA 205 tumorcells (10.5% vs 0.6% control) in a tumor-specific fashion and didnot bind to D5 or Pan-02 tumor cells.

We then tested Ab- and complement-mediated cytotoxicity. Asindicated in Fig. 7A, Ab from supernatants where B cell activationswere performed (groups 3 and 4) demonstrated significantly moreefficient tumor cell killing compared with supernatants derivedfrom cell cultures where only T cell activations were performed(groups 1 and 2). This enhanced tumor cell killing correlated withthe significantly increased Ab production (Fig. 5) and therapeuticefficacy observed using these cells (T�B at 65–70%:30–35%; Fig.3) in adoptive immunotherapy of established pulmonary metastaticcancers. Alternatively, Ab-mediated cytotoxicity was analyzed bya spectrophotometric assay. In this assay, a special substrate4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene dis-ulfonate (WST-1) was added to tumor cell cultures at the end of Ab (cellculture supernatants 1–4) and complement incubation. The assay is basedon the cleavage of WST-1 to formazan dye by cellular mitochondrialdehydrogenases. Since cleavage of WST-1 to formazan dye occurs onlyin viable cells, the amount of dye produced, measured in OD values,directly correlates with the number of viable cells present in the culture.As shown in Fig. 7B, supernatants from cultures where B cells wereactivated (groups 3 and 4) demonstrated significantly increased tumor cellkilling compared with cultures where only T cells were activated (groups1 and 2). This was indicated by the significantly (p � 0.05) reduced ODvalues in these groups. These experiments verified the Ab plus comple-ment-mediated tumor cell lysis data measured by visual viable cell count-ing under the microscope (Fig. 7A).

The specificity of tumor cell lysis by TDLN B cell-produced Aband complement was also examined. As indicated in Fig. 8, D5-TDLN B cell culture supernatants (groups 6 and 8) plus comple-ment mediated significant ( p � 0.001) D5 tumor cell killing, whileMCA 205-TDLN B cell culture supernatant (group 7) showed noD5 cell lysis in the presence of complement compared with the useof complement alone ( p � 0.05). As indicated previously, IgM,IgG, and IgG2b production by these activated TDLN B cells isshown in Table I. In comparison, MCA 205-TDLN B cell culturesupernatant plus complement (group 7) mediated significant ( p �0.001) MCA 205 tumor cell killing, while D5-TDLN B cell culturesupernatants (groups 6 and 8) did not lyse MCA 205 cells in thepresence of complement and were similar to the use of comple-ment alone ( p � 0.05). Use of Pan-02 tumor cells further con-firmed that this tumor killing was immunologically specific.

Alternatively, Ab-mediated tumor-specific cytotoxicity was an-alyzed in the WST-1 assay described above. As shown in Fig. 9,D5-TDLN B cell culture supernatant plus complement (group 6)significantly killed D5 tumor ( p � 0.05), but not MCA 205 tumor

FIGURE 4. TBI or chemotherapy significantly augmented the therapeu-tic efficacy of adoptively transferred B cells. A, TBI significantly aug-mented the therapeutic efficacy of adoptively transferred B cells in the s.c.MCA 205 tumor model. Established (5-day) s.c. MCA 205 tumor-bearingmice were treated with TBI (5 Gy) followed by adoptive transfer of puri-fied and activated MCA 205 TDLN B cells. Animals treated with TBI orB cells alone served as controls. Tumor size was then monitored. Therewere six mice in each group, and all of the animals formed tumors. Data arerepresentative of two independently conducted experiments. B, TBI sig-nificantly augmented the therapeutic efficacy of adoptively transferred Bcells in the s.c. D5 tumor model. Established s.c. D5 tumor-bearing micewere treated with TBI and/or adoptive transfer of purified and activatedD5G6 TDLN B cells as in A. There were six mice in each group, and allof the animals formed tumor. Data are representative of two experimentsperformed. C, The poorly immunogenic tumor, D5 melanoma, was used inthis experiment. Five-day pulmonary metastases were established as in Fig.3. In relevance to inoculation of D5 tumor cells on day 0, chemotherapy (1mg of cyclophosphamide and 2 mg fludarabine) was administered i.p. ondays 3 and 4 to induce lymphodelpletion. Various doses of purified andactivated D5G6 TDLN B cells were given i.v. on day 5. Lungs were har-vested for enumerations of pulmonary metastatic nodules as in Fig. 3.There were six mice in each group, and all of the animals formed tumor.

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( p � 0.05). In comparison, MCA 205-TDLN B cell culture su-pernatant plus complement (group 5) significantly killed MCA 205tumor ( p � 0.001), but not D5 tumor ( p � 0.05). Additionally, theuse of Pan-02 tumor cells confirmed that this tumor killing wasindeed immunologically specific.

DiscussionThe experimental results we described in this study using twohistologically distinct weakly immunogenic and nonimmuno-genic tumors provide direct evidence that in vivo primed and invitro activated B cells isolated from TDLNs can serve as acellular reagent to mediate regression of established pulmonarymetastases and s.c. tumors upon adoptive transfer. Mechanisti-cally, we observed significantly enhanced tumor-associated Ab re-sponses. Cytotoxic Ab subclass IgG2b produced by these in vivosensitized and in vitro activated B cells bound specifically to tumorcells, and mediated specific tumor cell lysis in the presence ofcomplement. The application of activated B cells in this settingrepresents a novel approach that may significantly augment theefficacy of current T cell therapies. The only other report that weare aware of where activated B cells mediated tumor regressionwas by Harada et al. (14). In that report, normal B cells were boundto CD3 and mediated tumor regression presumably by binding toT cells. The mechanism by which activated normal B cells medi-ated tumor responses was not examined in that study.

Several recent studies have shown that T cells and B cells col-laborate to elicit immune responses. Down-regulated B cell func-tion decreased T cell immunity in two autoimmune disease models(15, 16). B cells are required for the generation of protective ef-fector and memory CD4 cells in response to infection (17). Both Tcells and B cells are required to mediate significant immune re-sponses in prion diseases (18). Recent studies suggested that Bcells can function as APCs to T cells in multiple sclerosis (19) andin hemolytic diseases (20). New experimental evidence has alsobeen documented that Ab plays an important role in renal trans-

plantation through circulating IgG-mediated hydrolysis of coagu-lation factors (21).

The role played by B cells in the host immune response to can-cer is complex and controversial. Depending on their state of ac-tivation, B cells have had divergent roles on T cell differentiationand effector function. In tumor models, resting B cells have beenreported to suppress T cell-mediated antitumor immunity (22–25).The mechanisms of the suppression by resting B cells on the an-titumor immune response is poorly understood (23, 25). On theother hand, activation of B cells with Ag or Ig cross-linking, butnot LPS activation, has been reported to up-regulate the chemokineCCL4 and attract CD4�CD25� regulatory T cells (26). As op-posed to resting B cells, several reports have indicated the efficacyof activated B cells in cellular immunotherapy of malignancies.Some of these reports have been focused on how activated B cellscan be used as effective APCs for T cell sensitization (27–31).Anti-CD40 activated human B cells were found to mediate tumorcell killing via a TRAIL/Apo-2L-dependent mechanism (32). CD4T cell therapy of cancer has not worked effectively in B�/� mice(33), and T cells by themselves were not enough in suppressingtumor growth in cancer patients (34). Kawakami et al. reported

FIGURE 5. Significantly increasedIgM, IgG, and IgG2b production in theculture of T plus B cells. Supernatant col-lected at the end of T and/or B cell acti-vation with anti-CD3/anti-CD28/IL-2and/or LPS/anti-CD40 were tested forIgM, IgG, and IgG2b using ELISA. Dataare representative of four independentlyperformed experiments.

FIGURE 6. TDLN B cell-produced IgG2b binds specifically to tumorcells. D5, MCA205, and Pan-02 tumor cells were incubated with the cul-ture supernatant (Sup) of MCA 205 TDLN B cells. Bound IgG2b onto thetumor cells was then detected by secondary Ab FITC-anti-mouse IgG2b.FITC-coupled secondary Ab isotype-matching control was also used.Numbers within each immunofluorescence dot plot indicate the percentageof viable cells stained positive with the FITC-coupled Abs. Data are rep-resentative of three independently performed experiments.

Table I. Secretion of Abs by activated B cells enriched from TDLNs(ng/ml, mean � SEM)

D5G6 TDLN B Cells MCA205 TDLN B Cells

Expt. 1 Expt. 2 Expt. 1 Expt. 2

IgM 186 � 50 158 � 7 122 � 14 410 � 153IgG 931 � 382 375 � 12 685 � 263 838 � 333IgG2b 86 � 29 47 � 4 73 � 26 62 � 18

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that the administration of a DNA-based vaccine resulted in anti-tumor effects of established murine tumors that were mediated byboth T and B cells (35). In this study, we have identified that Bcells from tumor-primed lymph nodes possess antitumor reactivityand can be secondarily activated to mediate tumor regression inadoptive transfer models. Other than Ab production and their spe-cific binding and cytotoxicity toward tumor cells as we describedherein, other mechanisms by which the transferred TDLN B cellsmediated tumor destruction are largely unknown. Evaluations ofthe interactions of tumor-primed T and B cells during in vitro

activation and in vivo after adoptive transfer warrant furtherinvestigation.

In our previously reported adoptive immunotherapy studies uti-lizing T cells, we have described the role played by IFN-� inantitumor responses both in animal studies (5, 6, 9, 10, 36) and inclinical trials (7). In this study, IFN-� production by activated Tcells, B cells, and T cells plus B cells derived from the TDLNs wasalso evaluated. While purified and activated T cells did not pro-duce Ab (Fig. 5), they produced large amounts of IFN-� (data notshow). In contrast, whereas purified and activated B cells produced

FIGURE 7. Ab and complement-mediated tumor cell lysis. A, IgG2b-enriched cell culture supernatant induces tumor cell cytotoxicity. Viable MCA 205tumor cells (0.5 � 106) were put in 4-ml test tubes in 450 �l of CM plus 50 �l of cell culture supernatant from four cell groups as indicated. Cells wereincubated on ice for 1 h. After centrifugation, culture supernatants were discarded and 450 �l of fresh CM plus 50 �l of rabbit complement was added toeach tube followed by incubation in a 37°C water bath for another 1 h. Control groups included MCA 205 cells similarly cultured without cell culturesupernatants or complement (MCA 205 only), or with complement alone (MCA 205 � C). Viable cells were then counted under the microscope after trypanblue staining. Percentage of viable cells was calculated by dividing the remaining viable cells by 0.5 � 106. Data are representative of two independentexperiments conducted. B, Ab and complement-mediated cytotoxicity was analyzed in 96-well culture plates and determined using the Quick CellProliferation Assay kit. This assay was performed according to the manufacturer’s instructions. MCA 205 tumor cells were incubated with or withoutAb-enriched cell culture supernatants and complement as in A. OD values were then measured via a multiwell spectrophotometer to evaluate cell lysis. Dataare representative of three independent experiments conducted.

FIGURE 8. Tumor-specific cytotoxicity mediated by Abs and complement. Viable D5, MCA 205, or Pan-2 tumor cells (0.5 � 106) were incubated with culturesupernatants of D5G6 TDLN B cells from two separate experiments (Sup#1 and Sup#3) or with culture supernatant of MCA 205 TDLN B cells (Sup #2) in 4-mltest tubes as indicated. After that, rabbit complement was added for incubation for 1 h. Control groups included tumor cells cultured with complete medium(Control), complement only (C), or Abs without complement. Viable cells were then counted under the microscope after trypan blue staining. Percentage of viablecells was calculated by dividing the remaining viable cells by 0.5 � 106.

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large amounts of Abs, they did not produce IFN-� (data notshown). Of note, activated T cells plus B cells released both Ab(Fig. 5) and IFN-� (data not shown) at high levels. These datademonstrated an association between T cell-mediated therapeuticefficacy with IFN-� production, as we reported before, and a cor-relation between B cell-mediated therapeutic efficacy with the ob-served Ab production. These data are supportive of our hypothesisthat the enhanced therapeutic efficacy mediated by T cells com-bined with B cells in this study is due to concurrent cellular andhumoral antitumor responses.

We found that radiation and chemotherapy significantly en-hanced the therapeutic efficacy of adoptively transferred B cells inthe s.c. and pulmonary metastatic tumor models, respectively. TBIleads to lymphodepletion (12). Our subsequent use of a clinicallyrelevant chemotherapy protocol (cyclophosphamide and fludara-bine) (1) for the induction of nonmyeloablative lymphodepletionfurther verified that B cell therapy of cancer could be significantlyenhanced in the lymphopenic host. A clinical trial of adoptivetransfer of autologous T cells in patients with advanced melanomapretreated with this nonmyeloablative but lymphodepleting che-motherapy regimen has been reported with promising clinical re-sponses (1). The intent of fludarabine and cyclophosphamide treat-ment is to induce lymphopenia and potentially eliminate cellularcytokine “sinks” for homeostatic cytokines, such as IL-7, IL-15,and IL-21, which can activate and expand tumor-reactive T cells aswell as result in the depletion of regulatory T cells (37–39). Mech-anisms involved in the enhanced tumor reactivity of adoptivelytransferred B cells in the lymphodepleted host have not been elu-cidated to date. These studies may provide more experiment evi-dence to support the use of B effector cells as a novel cellularreagent for cancer immunotherapy.

In a previous study (40), we tested the target recognition effi-ciency of TDLN-isolated T cells without in vitro activation. Wefound that freshly harvested, nonactivated TDLN T cells had verylimited immunity (e.g., IFN-� production) in response to tumorcell stimulation. In this study, we also performed experiments byculturing the TDLN-isolated B cells without LPS/anti-CD40 acti-vation. These B cells did not grow and were almost all dead by theend of 4 days in culture. B cells isolated form the spleens obtainedfrom the tumor-bearing mice as well as normal mice did not groweither without LPS/anti-CD40 activation (data not shown). Thevery limited B cell number (due to the lack of cell expansion) didnot allow us to perform adoptive transfer experiments to comparetheir antitumor efficacy with activated B cells. Also, significant celldeath in the absence of B cell activation did not allow us to collectculture supernatants for IgG and/or IgM detection or for binding

assays. These series of experiments underscore the essentialrequriment of in vitro activation for in vivo sensitized B cells tomediate tumor regression in cancer adoptive immunotherapy. Theuse of LPS plus anti-CD40 as TDLN B cell stimuli in this reportmay represent a combined stimulation of innate immunity (byLPS) and adaptive immunity (by anti-CD40 to mimic the CD40Lsignaling from Th cells). Evaluation of additional B cell activationpathways, such as the engagement of the BCR by using availabletumor-specific Ag or by using anti-Ig (e.g., anti-IgM or anti-IgD)in the forms of F(ab�)2 may lead to the better understanding ofrequirements and the optimization of methods for pre-effector Bcell secondary activation in vitro. Furthermore, successful T celltherapy required the infiltration of infused T cells within the tumormass (41). Studies on the infiltration and accumulation of infusedB cells in the s.c. tumors and the assessment of proliferation andsurvival of the infused B cells would increase our understanding ofthe events behind the observed therapeutic efficacy of adoptivelytransferred TDLN B cells.

In summary, we have identified that tumor-primed B cells thatare secondarily activated in vitro can mediate regression of estab-lished tumors upon adoptive transfer. This represents a novel ef-fector cell population that can enhance the therapeutic efficacy ofadoptive T cell therapies. Further investigations are required todetermine the mechanisms by which activated B cells mediate invivo tumor regression and how they interact with effector T cells.We have demonstrated that it is quite feasible to generate both Tand B effector cells together by in vivo sensitization and in vitromethods that can have direct clinical applications.

DisclosuresThe authors have no financial conflicts of interest.

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