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Leukemia Research 33 (2009) 525–538

Allogeneic CD3/CD28 cross-linked Th1 memory cells provide potentadjuvant effects for active immunotherapy of leukemia/lymphoma

M. Har-Noy a,d,∗, M. Zeira b, L. Weiss a, E. Fingerut d, R. Or a, S. Slavin c

a Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah-Hebrew University Medical Center, Jerusalem, 91120, Israelb Gene Therapy Institute, Hadassah-Hebrew University Medical Center, Jerusalem, 91120, Israel

c International Center for Cell Therapy & Cancer Immunotherapy (CTCI), Tel Aviv Medical Center, Tel Aviv, 64239, Israeld Immunovative Therapies Ltd., POB 974, Shoham, 60850, Israel

Received 21 July 2008; received in revised form 17 August 2008; accepted 21 August 2008Available online 1 October 2008

Abstract

The breaking of peripheral T-cell tolerance toward self-antigens expressed by tumor cells and the subsequent establishment of an effectivetumor protective immune response remains a major challenge for cancer immunotherapy. We report that both protective and therapeuticanti-tumor immune responses can be achieved in a mouse leukemia/lymphoma tumor model through the strong adjuvant effects providedby allogeneic CD3/CD28 cross-linked Th1 memory cells. The adjuvant effect of these cells is mediated by their ability to produce avariety of ‘danger signals’ which serve to deviate native non-protective Th2 anti-leukemia immune responses to effective Th1 immuneresponses.© 2008 Elsevier Ltd. All rights reserved.

Keywords: Active immunotherapy; Cancer vaccine; Adjuvant; Cryoimmunotherapy; Immunomodulation; Th1/Th2 immunity; B-cell leukemia/lymphoma

1. Introduction

Combination chemotherapy and monoclonal antibody(mAb)-based treatment strategies have improved survivalof patients afflicted with hematological malignancies. How-ever, patients that relapse after chemotherapy or autologousstem cell transplantation have a poor prognosis and very fewoptions for effective salvage treatment [1,2]. Therefore, itis an important therapeutic goal to develop treatment strate-gies that can prevent recurrence of disease after remissioninduction chemotherapy. Active immunotherapy concepts arebeing investigated in this setting [3,4].

The resident immune response to hematological malig-nancies usually is polarized to Th2 [5–9]. Clinical trials ofactive immunotherapy in hematological malignancies have

∗ Corresponding author at: Hadassah-Hebrew University Medical Center,Department of Bone Marrow Transplant and Cancer Immunotherapy, POB12000, Jerusalem, 91120, Israel. Tel.: +972 54 232 7077;fax: +972 3 970 2090.

E-mail address: [email protected] (M. Har-Noy).

been shown to promote this dominant Th2 humoral response,however, this has not translated into significant clinical effi-cacy [10–12].

Cancer eradication and maintenance of remission requiresTh1 immune activation [13]. Therefore, one of the goalsof active immunotherapy is to develop methods which arecapable of deviating a resident Th2 response to a Th1response. However, in some patients which develop a poten-tially effective Th1 immune response against a tumor [14–16]or are therapeutically immunized to develop a Th1 immuneresponse [17], the tumors still continue to grow unaffected[18].

This lack of efficacy in Th1 immune patients and theineffectiveness of native immune responses against tumorshas been attributed to the ability of tumors to employvarious strategies for evasion from immune attack [19].These immunoavoidance mechanisms employed by tumorsrender the immune system tolerant and permit tumors togrow unimpeded by immune surveillance even after specificupregulation of anti-tumor effector mechanisms by activeimmunotherapy. Therefore, active immunotherapy strategies

0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.leukres.2008.08.017


526 M. Har-Noy et al. / Leukemia Research 33 (2009) 525–538

require in addition to an immunomodulatory mechanismof action, a strategy to overcome tumor immunoavoidancemechanisms.

Establishment of self-tolerance to a tumor is thought tobe related to existing natural immune mechanisms whichare normally employed to prevent autoimmune disease. Thatthis normally beneficial effect may be responsible for tumorimmune evasion is supported by the observation that many ofthe tolerance mechanisms that prevent autoimmunity are thesame as employed by tumors to prevent immune destruction[20,21]. Therefore, immune mechanisms of autoimmunitymay hold clues to development of strategies for breakingtumor immune tolerance mechanisms.

The “danger hypothesis” proposes that the immune sys-tem does not primarily discriminate self from non-self, butinstead is mainly adapted to recognize and respond to anti-gens depending on the context in which the antigens arepresented to the immune system. Therefore, manipulating thecontext of tumor antigen presentation to the immune systemmay be a fruitful strategy for breaking immune tolerance totumors. The danger hypothesis proposes that antigens of anyorigin that are presented in the context of ‘danger signals’ willevoke an immune response [22]. This model is based on theidea that the crucial signals that alert to ‘danger’ for the ini-tiation of immune responses are endogenous, not exogenous[23].

In support of this hypothesis, induction of autoimmunityhas been shown to occur when self-antigens are presentedin the context of inflammation [24–26], demonstrating thatinflammation can serve as a ‘danger signal’. In addition,killing of normal melanocytes in the context of danger sig-nals (Hsp70 and CD40L) has been shown to break toleranceto the normal self-antigens shared with melanoma tumor cellsresulting in potent anti-tumor immune effects [27].

These data suggest that the use of an adjuvant that pro-vides danger signals may serve to increase the efficacy ofactive immunotherapy protocols. The use of adjuvants haslong been a strategy for influencing the immune response toantigens in a vaccine composition. Aluminum salts, and squa-lene oil in water emulsion (MF59) are the most widely usedadjuvants in human vaccines [28,29]. However, these adju-vants predominantly promote Th2 responses to antigens, andwhile effective at elevating serum antibody titers do not elicitsignificant cellular immune responses [30].

We have previously hypothesized [31] and then shown inthe BCL1 animal model of B-cell leukemia/lymphoma [32]that adoptive transfer of completely allogeneic CD3/CD28cross-linked Th1 memory cells can convert a resident Th2immune response to BCL1 to a Th1 response. This immunemodulatory effect correlated with significant prolongation ofsurvival and durable tumor remissions in up to 37.5% oflethally injected animals.

The allogeneic CD3/CD28 cross-linked Th1 memory cellsused in this previous study expressed numerous ‘danger sig-nals’, including alloantigens, production of large amountsof type 1 cytokines and expression of CD40L. This led us

to hypothesize that these same allogeneic CD3/CD28 cross-linked Th1 memory cells could serve as a potent adjuvant forstimulating Th1 anti-leukemia immunity when used togetherwith a source of BCL1 tumor antigen in active immunother-apy protocols.

In support of this hypothesis, we now report that activeimmunotherapy with BCL1 vaccine compositions contain-ing allogeneic CD3/CD28 cross-linked Th1 memory cells asan adjuvant were able to deviate the natural non-protectiveTh2 response to BCL1 to a Th1 response. This immunomod-ulatory effect was curative when the source of BCL1 antigenin the vaccine composition contained endogenous cell com-ponents. Translation of these results to the clinic may providea novel active immunotherapy for patients with hemato-logical malignancies in the setting of minimal residualdisease.

2. Materials and methods

2.1. Mice

Five- to six-week-old female Balb/c (H-2d/d) and male C57BL/6(H-2b/b) mice were purchased from the Hebrew University-Hadassah Medical School Animal Facility, Jerusalem, Israel. Allmice were kept under specific pathogen-free (SPF) conditions andgiven acidified water and food ad libitum. The study was approvedby the Animal Ethical Committee of the Hebrew University MedicalSchool (Project #MD-97.28-4). All mice were 6–8 weeks old whenplaced on experiment.

2.2. BCL1 tumor model

BCL1 is a spontaneous B-cell leukemia/lymphoma of Balb/corigin. The BCL1 tumor line is maintained in vivo by serial passagesin Balb/c recipients. In these experiments, animals were infusedintravenously through the tail vein with 2000 BCL1 cells on day 0which is lethal in 100% of the mice. In some experiments, 1 × 104

BCL1 were implanted subcutaneously in the flank of Balb/c mice.BCL1 forms a rapidly growing solid tumor in this setting resemblinglymphoma/plasmacytoma.

2.3. Preparation of allogeneic Th1 memory cells

Allogeneic Th1 memory cells were prepared as described previ-ously [32]. Briefly, CD4+ cells were isolated from male C57BL/6spleens and cultured for 6 days with anti-CD3 and anti-CD28-coated paramagnetic beads (CD3/CD28 T-cell Expander beads,Dynal/Invitrogen) at an initial bead:CD4 cell ratio of 3:1 and20 IU/ml recombinant mouse (rm)IL-2, 20 ng/ml rmIL-7, 10 ng/mlrmIL-12 (Peprotech, New Jersey) and 10 �g/ml antimurine IL-4 mAb (Becton Dickenson) in RPMI 1640 media containing10% FBS, penicillin–streptomycin–glutamine, nonessential aminoacids (NEAA) (Biological Industries, Israel) and 3.3 mM N-acetyl-cysteine (NAC; Sigma) (complete media). After 6 days in culture,the CD4 cells were harvested and debeaded by physical disruptionand passage over a magnet. These cells were either used fresh orstored in liquid nitrogen for future use. Prior to use, the cells were


M. Har-Noy et al. / Leukemia Research 33 (2009) 525–538 527

activated by incubation with anti-CD3/anti-CD28-coated nanobeadsfor 4 h in complete media. The cells are administered with the beadsattached.

2.4. Vaccinations

Mice were vaccinated with vaccine compositions suspended in0.1 ml of HBSS or complete media. Inoculations were made eitherin alternating foot pads or in the skin layer of the shaved flank.

2.5. BCL1 tumor antigen preparations

BCL1 lysates and irradiated BCL1 cells were used as sources oftumor antigens. Batches of BCL1 lysate were derived from 1 × 107

BCL1 cells suspended in 2 ml of HBSS and lysed by 3 freeze (in−80 ◦C freezer)–thaw (37 ◦C water bath) cycles. Total cell dis-ruption was microscopically validated using trypan blue staining.The lysate was mixed well to assure a homogenous solution andaliquoted into separate 0.1 ml doses. These doses were stored at−80 ◦C prior to use. Fresh BCL1 cells were irradiated at 20 Gy andused within an hour of treatment.

2.6. Monoclonal antibodies

The following monoclonal antibodies were used for surfacephenotyping: anti-mCD4-PerCP-Cy5 (IgG2a); isotype control ratIgG2a-PerCP-Cy5.5; anti-mCD62L-APC (IgG2a); isotype controlrat IgG2a-APC; anti-mCD45RB-PE (IgG2a); isotype control ratIgG2a-PE; anti-mCD8a-FITC (IgG2a); isotype control rat IgG2a-FITC; anti-mCD44-FITC (IgG2b); isotype control rat IgG2b-FITC;anti-mCD154(CD40L)-PE (IgG); isotype control rat IgG-PE;anti-mCD25-APC (IgG1); isotype control rat IgG1-APC; anti-mCD3e-PerCP-Cy5.5 (IgG); isotype control Armenian hamsterIgG-PerCP-Cy5.5 all from eBioscience Inc. (San Diego, USA).

2.7. ELISPOT assay

Single cell suspensions of spleen cells from immunized micewere prepared. The cells were aliquoted so that 2 × 106 viable cellswere plated in 2 ml of complete media in wells of a 24 well plate.The splenocytes in each well were pulsed with test antigens. Thetest antigens were prepared as freeze/thaw lysates suspended in com-plete media. Each well was pulsed with lysate from 1 × 107 cells:either BCL1, allogeneic Th1 cells or splenocytes from untreatedsyngeneic mice. The pulsed wells were cultured for 24 h at 37 ◦C ina humidified CO2 incubator. After 24 h, the non-adherent cells wereharvested, washed and counted. These cells were then plated in trip-licate at 100,000 viable cells per well on pre-coated anti-IFN-� andanti-IL-4 plates (eBioScience, San Diego, CA) and incubated foran additional 20 h in complete media supplemented with 20 IU/mlrmIL-2 (Peprotech). Fresh splenocytes from syngeneic mice acti-vated with PHA were used as a positive control for each plate (datanot shown). The plates were developed in accordance with the man-ufacturer’s instructions and read on an automated image analysissystem.

2.8. Cryoimmunotherapy tumor models

Two tumor models were used for the cryoimmunotherapy pro-tocols, a bilateral solid tumor model and solid tumor with systemic

disease model. The bilateral solid tumor model consisted of micegiven subcutaneous injections of 1 × 104 BCL1 tumor cells bilater-ally in the shaved flanks on day 0. For the solid tumor with systemicdisease model, mice received a single subcutaneous inoculation of1 × 104 BCL1 on day 0 on the left flank and also an intravenousinfusion of 2000 BCL1.

2.9. Cryoablation

Anesthetized mice (ketamine-HCL, 100 mg/kg, i.p.) underwentcryoablation treatment by applying mild pressure for 10 s withfrozen tweezers (which were kept in liquid nitrogen) applied to thetumor. Tumors were 16–25 mm2 when treated. The complete tumormass was ablated. To ensure complete thawing of the treated areabefore vaccination, intratumoral treatments were administered after1 h.

2.10. Statistics

Two-way ANOVA was used to determine significant differ-ences in cytokine levels, ELISPOT response frequencies and tumorvolume changes. A p value of <0.05 was considered signifi-cant. Logrank and hazard ratio analysis was used to compareKaplan–Meier survival curves (Graphpad Software; San Diego,CA). Animals that survived >60 days were censored from the anal-ysis.

3. Results

3.1. BCL1 immunogenicity

We characterized the native immune response to BCL1vaccination in Balb/c mice to serve as a baseline in whichto analyze the biological effects of adding an adjuvant. Wehypothesized that since BCL1 has been continuously pas-saged in vivo for many years that the current BCL1 clonewould prove to be non-immunogenic when administered tosyngeneic Balb/c mice without an adjuvant due to “immu-noediting” [33].

We tested the immunogenicity of two vaccine prepara-tions of BCL1, either irradiated whole BCL1 (irrad BCL1)or a freeze/thawed lysate of BCL1 (f/t BCL1). The “dangerhypothesis” [22,23] predicts that the f/t BCL1 lysate prepa-ration would be more immunogenic than the irrad BCL1preparation.

To test the immunogenicity of f/t BCL1 and irrad BCL1preparations they were each administered intradermally (i.d.)to Balb/c mice in 0.1 ml of HBSS once a week for 4 weeks.Inoculation of HBSS alone was used as a control. Duringthe 5th week after the first vaccination, the mice were sacri-ficed and spleens were removed and single cell suspensionswere prepared. The splenocytes were pulsed with f/t BCL1or with f/t lysate from syngeneic Balb/c splenocytes preparedin the same manner as the f/t BCL1 lysate which served asa control against reactivity against self-antigens. After 24 h,the non-adherent T-cells were harvested and aliquoted so that



Fig. 1. (A and B) Immune response to BCL1 vaccination with or withoutadjuvant. Balb/c mice (n = 3) were vaccinated four times intradermally atweekly intervals with either freeze/thawed BCL1 tumor lysate (f/t BCL1) orirradiated BCL1 (irrad BCL1) without adjuvant (1A) or mixed with 1 × 103

CD3/CD28 cross-linked allogeneic Th1 cells (allo Th1) as adjuvant (1B).On the 5th week following the first inoculation, animals were sacrificed andtheir spleens harvested, single cell suspension cultures of splenocytes werepulsed with f/t BCL1 or f/t splenocytes from a naïve Balb/c mouse as control.After 24 h, the non-adherent T-cells were removed and plated in triplicates at1 × 105 cells per well of anti-IFN-� and anti-IL-4 coated ELISPOT plates.After a culture of 20h, IFN-� and IL-4 spots were developed and countedby computer-assisted video image analysis. Each bar represents the meanspot number of triplicates ±S.E. out of 105 T-cells. Asterick (*) indicatessignificant difference (p < 0.05) and n.s. indicates not significant (p > 0.05)compared to control and between bracketed values (ANOVA 2-tailed test).

1 × 105 T-cells from each treatment were plated in triplicatein wells of anti-IFN-� and anti-IL-4 coated ELISPOT platesand cultured on these plates for an additional 20 h in cRPMIsupplemented with 20 IU mIL-2/ml.

Surprisingly, vaccination with both irrad BCL1 andf/t BCL1 without adjuvant were able to elicit significanttumor-specific immune responses (see Fig. 1A). The meanfrequency of tumor-specific T-cells (IL-4 + IFN-� spots) was1/64 after vaccination with irrad BCL1, which was sig-nificantly greater (3.5-fold) than the mean frequency of1/227 after f/t BCL1 vaccination. Both vaccination protocolsresulted in significantly greater frequencies of respondingT-cells compared to the control mean frequency of 1/3333(p < 0.001).

Both vaccine preparations caused tumor-specific T-cellresponses biased to Th2 (IL-4). T-cell IL-4 mean frequencyresponse to irrad BCL1 vaccination was 1/93 and the IFN-� response was significantly lower (p < 0.001) at 1/208. Themean frequency of IL-4 responders in f/t BCL1 vaccinatedmice was 1/322 which was significantly higher (p < 0.01) thanthe IFN-� mean response frequency of 1/769. The mean fre-quency of IFN-� responders in irrad BCL1 vaccinated micecompared to f/t BCL1 vaccinated mice were not significantlydifferent.

None of the mice vaccinated with irrad BCL1 or f/t BCL1were able to survive a lethal challenge of 2000 BCL1 cellsadministered intravenously through the tail vein (see Fig. 4),indicating the significant immune responses to both BCL1vaccination protocols were not protective.

3.2. Characterization of CD3/CD28 cross-linked Th1memory cells

Prior to testing the adjuvant activity of C57BL/6-derivedCD3/CD28 cross-linked Th1 memory cells, we first charac-terized these cells for the surface phenotype (Fig. 2A–C),cytokine production profile (Fig. 3A) and immune responseafter 4 weekly i.d. injections in allogeneic Balb/c mice(Fig. 3B).

In order to characterize the differentiation of cells overthe 6 day culture, we analyzed the surface expression ofCD62L, CD45RB, CD44, CD25 and CD40L by FACS of day0 CD4+ source cells and compared the staining patterns to thesame cells harvested after 6 days in culture before and afteractivation by CD3/CD28 cross-linking. Results are shown inFig. 2A.

The day 0 positively selected CD4+ cells from splenocytesstained CD62Lhi (naïve). This CD62Lhi population had sub-populations with different CD45RB and CD44 expressionresulting in a bimodal presentation upon FACS analysis, witha majority of cells with a CD45RBlo and CD44lo phenotype.A shift in phenotype occurred over the 6 day culture pro-cess, with the CD4+ cells changing from a predominatelyCD45RBlo, CD44lo phenotype to a CD45RBhi, CD44hi phe-notype, while remaining CD62Lhi. While the CD62L stainingremained high, the intensity was higher on day 6 comparedto day 0.

After activation by CD3/CD28 cross-linking, the day 6cells remained CD45RBhi, CD44hi but converted from aCD62Lhi phenotype to a CD62Llo phenotype. Therefore, theday 6 CD3/CD28 cross-linked cells expressed a phenotypeof CD4+, CD62Llo, CD45RBhi, CD44hi.

Phenotypically, mouse memory cells are normallyCD62Llo, CD45RBlo, CD44hi [34]. The CD62Llo,CD45RBhi, CD44hi phenotype of cells produced byour culture method express the same phenotype asmemory/effector cells that have been previously asso-ciated with autoimmune disease and allograft rejection[35–37].

Activation by CD3/CD28 cross-linking of day 6 cellsfor 4 h caused a significant increase in the number of cellsexpressing CD40L from 9.27 to 67.65% (Fig. 2B) which cor-related with an increase in cells expressing CD25 from 63.09to 91.52% (Fig. 2C).

We tested these CD3/CD28-activated CD4+, CD62Llo,CD45RBhi, CD44hi, CD40L+, CD25+ memory/effector cells(n = 6 batches) for cytokine production after activation byCD3/CD28 cross-linking of fresh day 6 harvested cells orday 6 harvested cells that had been frozen in liquid nitro-gen, thawed and activated. Activated day 0 CD4+ cells and



Fig. 2. (A–C) Characterization of CD3/CD28 cross-linked Th1 cells. C57BL/6-derived positively selected CD4+ T-cells were placed in culture on day 0in cRPMI supplemented with rmIL-12, rmIL-7, rmIL-2 and a neutralizing anti-IL-4 mAb and activated with CD3/CD28-conjugated T-cell Expanded beads(Dynal/Invirogen) at a 3:1 bead:cell ratio. The cells were split daily from day 3–6 and supplemented with additional T-cell Expander beads, rmIL-7, rmIL-2 andanti-IL-4 mAb. On day 6, the cells were harvested and activated with CD3/CD28-conjugated nanobeads. The phenotypic shift in CD45RB, CD62L and CD44from day 0 to day 6 is shown in Panel A. The black filled area represents the isotype control. The black line is the phenotype of day 0 CD4+ cells. The gray filledarea is the phenotype of day 6 cells prior to CD3/CD28 nanobead activation and the gray line represents the phenotype after CD3/CD28 nanobead activation.Panel B shows the phenotypic changes in the CD40L effector molecule expression in CD4+ cells placed in culture on day 0 compared to day 6 harvestedcells before and after CD3/CD28 nanobead activation. Only the activated cells expressed significant amounts of this effector molecule. Panel C represents thephenotypic change in CD25 expression in day 0 CD4+ cells compared to day 6 cells before and after CD3/CD28 nanobead activation. The phenotype of theactivated day 6 cells was CD4+, CD62Llo, CD45RBhi, CD44hi, CD40L+, CD25+.

non-activated fresh harvested day 6 cells were included forcomparison (see Fig. 3).

Fresh activated CD4+ memory/effector cells expressedsubstantial amounts of IFN-� (4210 ± 169.7 pg/ml/6 h)and negligible IL-4 (52.33 ± 6.8 pg/ml/6 h) and thuswe refer to these cells as Th1 memory cells. Whenthese Th1 memory cells are frozen in liquid nitro-gen prior to activation and later thawed and activated,

they maintain the Th1 phenotype but express approxi-mately 29% less IFN-� and IL-4 than the fresh cells(2985 ± 173.5 pg/ml/6 h of IFN-� and 37.2 ± 6.95 pg/ml/6 hof IL-4). Non-activated Th1 memory cells produced neg-ligible amounts of cytokines (13 ± 6.7 pg/ml/6 h IFN-�;8.8 ± 3.6 pg/ml/6 h IL-4). The source CD4+ cells isolatedby positive selection from a single cell suspension ofnormal C57BL/6 splenocytes produced cytokines upon acti-



Fig. 3. (A and B) Cytokine production (A) and immunogenicity (B) ofCD3/CD28 cross-linked Th1 memory cells. IFN-� and IL-4 cytokine pro-duction was analyzed in supernatants from 6 h cell cultures by ELISA. CD4cells positively selected from C57BL/6 cultured for 6 days in the presence ofIL-12 (day 1–3 only), IL-7 and IL-2 expanded 60–100-fold and differentiatedto CD45RBhi, CD44hi effector/memory cells. These cells were harvested onday 6, washed and reactivated with CD3/CD28 nanobeads, cultured for 6 hand the supernatants collected for analysis by ELISA (Panel A: fresh acti-vated Th1). These results were compared to supernatants from 6 h culturesof the same day 6 harvested cells that were first frozen in liquid nitrogenand then later thawed and activated with CD3/CD28 nanobeads (Panel A:thawed activated Th1). For comparison, supernatants from a sample of posi-tively selected CD4 cells (the same that were placed in culture on day 0) wereactivated with CD3/CD28 T-cell Expander beads at a 3:1 bead:cell ratio andcultured for 6 h (Panel A: CD4 naïve). Supernatants from day 6 harvestedcells were also cultured for 6 h without CD3/CD28 activation (Panel A: non-activated Th1 fresh).The immunogenicity of these cell compositions was tested by ELISPOT(Panel B). 1 × 104 fresh activated Th1 cells, fresh non-activated Th1 cellsor positively selected CD4 cells (control) derived from C57BL/6 mice wereinoculated intradermally in allogeneic Balb/c mice once a week for 4 weeks.During the 5th week, the mice (n = 3) were sacrificed, spleens removed andsingle cell splenocytes cultures established. The cultures were pulsed withfreeze/thawed lysates of CD57BL/6 splenocytes and cultured for 24 h. Thenon-adherent T-cell fraction was then harvested, washed, and 1 × 105 cellswere transferred to anti-IFN-� or anti-IL-4 coated ELISPOT plates in trip-licate and cultured another 20 h in the presence of 20 IU rmIL-2. Each barrepresents the mean spot number of triplicates ±S.E.

vation with CD3/CD28-conjugated microbeads with a Th2bias (254 ± 50.2 pg/ml/6 h IL-4; 53.5 ± 11.4 pg/ml/6 h IFN-�).

These data demonstrate that our culture conditions causeCD4+ naïve cells with a Th2 bias to differentiate to stronglypolarized Th1 memory/effector cells which express CD40L

upon CD3/CD28 cross-linking and express an unusualCD62Llo, CD45RBhi, CD44hi memory phenotype.

3.3. Immune response to allogeneic CD3/CD28cross-linked Th1 memory cells

To characterize the potential of allogeneic CD3/CD28cross-linked Th1 memory cells to provide adjuvant activ-ity for promotion of Type 1 immunity, we conducted astudy to determine if the C57BL/6-derived Th1 memorycells were able to elicit Type 1 immunity to their ownalloantigens in allogeneic Balb/c hosts. Mice were adminis-tered 1 × 104 CD3/CD28 cross-linked Th1 memory cells or1 × 104 Th1 memory cells without CD3/CD28 cross-linkingi.d. in 0.1 ml of cRPMI, inoculation of cRPMI alone was usedas a control. Mice (n = 6) were inoculated once weekly for 4weeks.

During the 5th week after the first vaccination, the micewere sacrificed and spleens were removed and single cell sus-pensions were prepared for ELISPOT analysis as describedpreviously. Results are shown in Fig. 3B.

Vaccination with allogeneic CD4 cells (day 0) aloneresulted in a base line allogeneic immune response meanfrequency (IFN-� + IL-4) of 1/348 T-cells. Allogeneic Th1cells (day 6) without CD3/CD28 cross-linking resulted in aresponse mean frequency of 1/268 T-cells, which was notsignificantly different than the CD4 cells alone. However,CD3/CD28 cross-linking of the allogeneic Th1 cells resultedin a mean frequency of 1/34 alloantigen-specific T-cells,which was significantly greater than the response frequencyof allogeneic CD4 cells and non-activated allogeneic Th1cells (p < 0.0001).

The immune response to allogeneic CD4 cells and non-activated allogeneic Th1 memory cells resulted in skewedTh2 immunity, while the CD3/CD28 cross-linked allogeneicTh1 memory cells elicited a strongly polarized Th1 response.CD4 cell vaccination resulted in a mean frequency of 1/181IL-4 producing T-cells, which was significantly greater(p < 0.0001) than the mean frequency of 1/4167 IFN-� pro-ducing T-cells. Non-activated allogeneic Th1 memory cellselicited a mean frequency of 1/147 IL-4 responding T-cells,which was significantly greater (p < 0.0001) than the meanfrequency of 1/1587 IFN-� responding T-cells. The syn-geneic splenocyte control did not elicit a detectable adaptiveimmune response (results not shown).

3.4. Protective vaccination and challenge

In order to determine if allogeneic Th1 cells could serve asan adjuvant to protect mice from a lethal challenge of BCL1tumor, we prepared vaccine mixtures of either freeze/thawedBCL1 lysate (f/t BCL1) or irradiated BCL1 cells (irrad BCL1)used alone (control) or mixed with 1 × 104 allogeneic Th1cells. Allogeneic Th1 cells alone without a source of tumorantigen were used as a control. Mice (n = 8 in each group)received four i.d. inoculations of the vaccine preparations



Fig. 4. Protective vaccination and tumor challenge. Kaplan–Meier survivalcurves of Balb/c mice vaccinated against BCL1 tumor (n = 8 for each group).All mice were infused i.v. with 2000 cells of BCL1 on day 0. Prior to tumorchallenge, mice were inoculated i.d. on day 22, day 15, day 8 and day1 with either media alone (control), 1 × 104 allogeneic CD3/CD28 cross-linked Th1 cells (Th1 alone), 1 × 104 allogeneic CD3/CD28 cross-linkedTh1 cells mixed with irradiated BCL1 (irrad BCL1 + Th1) or 1 × 104 allo-geneic CD3/CD28 cross-linked Th1 cells mixed with freeze/thawed BCL1tumor lysate (f/t BCL1 + Th1). The median survival of the control mice was21 days. Vaccination with irrad BCL1 alone (median survival = 20.5 days) orf/t BCL1 alone (median survival = 20.0 days) did not significantly affect sur-vival compared to control. The Th1 alone pretreatment significantly extendedsurvival to a mean of 24 days (hazard ratio = 3.14). Vaccination with irradBCL1 with Th1 as an adjuvant did not have a significant effect on survival(mean survival = 22 days). Mixing f/t BCL1 with Th1 resulted in 50% of themice surviving lethal tumor challenge and resulted in a median survival of46 days (hazard ratio = 6.08).

at weekly intervals on days 22, 15, 8 and 1. Media aloneinjections were used as a control. On day 0, all vaccinatedmice received a lethal i.v. challenge of 2000 BCL1 cells.

Mice were followed for survival (see Fig. 4). The mediansurvival of the media alone control mice was 21 days. Vac-cination with either irrad BCL1, f/t BCL1 or allogeneic Th1alone did not result in protection from tumor challenge. Inter-estingly, the allogeneic Th1 alone vaccination, while notprotective, significantly extended survival of challenged miceto a mean of 24 days (hazard ratio = 3.14). Vaccination with acomposition of irrad BCL1 + Th1 cells did not affect survival(median survival = 22 days) and did not provide protection.Vaccination with f/t BCL1 + Th1 cells resulted in a mediansurvival of 46 days (hazard ratio = 6.08) with 50% of the micesurviving lethal tumor challenge.

3.5. Therapeutic vaccine protocol

The previous experiment demonstrated the protectiveeffects of vaccination with f/t BCL1 and allogeneicCD3/CD28 cross-linked Th1 memory cells used as an adju-vant in animals that were vaccinated when they were tumorfree and then challenged with a lethal dose of tumor. In orderto determine if this protective effect could also provide a ther-apeutic effect, we investigated vaccination protocols on micewith pre-existing tumors.

The previous protective vaccination protocol included 4weekly i.d. inoculations (28 days) followed by tumor chal-lenge on day 35. This vaccination schedule could not betranslated to the therapeutic setting in our model, as mice

Fig. 5. Therapeutic vaccination. Kaplan–Meier survival curves of Balb/cmice (n = 6 per group) infused with 2000 BCL1 i.v. on day 0. Mice weregiven id injections of either 1 × 104 allogeneic CD3/CD28 cross-linked Th1memory cells alone (Th1) or f/t lysate of 1 × 106 BCL1 mixed with Th1(f/t BCL1 + Th1) i.d. on days 1, 8 and 15. Control mice survived a meanof 19.5 days. Mice vaccinated with Th1 alone survived significantly longerthan control with a mean of 26 days (logrank: p = 0.001; hazard ratio: 3.981).Mice vaccinated with f/t BCL1 + Th1 also survived significantly longer thancontrol mice with a mean survival of 34 days (logrank: p = 0.001; hazardratio: 3.981), which was not significantly different than the Th1 alone group.

succumb to disease 19–22 days after lethal BCL1 infusion.Therefore, mice (n = 6 each group) received a lethal doseof 2000 BCL1 cells intravenously on day 0 and therapeuticvaccinations of either 1 × 104 allogeneic CD3/CD28 cross-linked Th1 memory cells alone (Th1) or f/t lysate of 1 × 106

BCL1 mixed with allogeneic Th1 (f/t BCL1 + Th1) i.d. ondays 1, 8 and 15. Media alone inoculations on the sameschedule served as control. Results are shown in Fig. 5.

Control mice survived a mean of 19.5 days. Interestingly,mice vaccinated with Th1 alone without any source of tumorantigen survived significantly longer than control mice witha mean of 26 days (logrank: p = 0.001; hazard ratio: 3.981).Mice vaccinated with f/t BCL1 + Th1 also survived signifi-cantly longer than control mice with a mean survival of 34days (logrank: p = 0.001; hazard ratio: 3.981), but this was notsignificantly different than the allogeneic Th1 alone group.No mice were cured with either of these therapeutic vaccineprotocols.

3.6. Prime and therapeutic booster vaccination

Since no mice were cured in the therapeutic vaccine pro-tocols, we hypothesized that the 19–22 day lifespan of miceinfused with BCL1 tumors was not sufficient time to developan adoptive immune response that could overwhelm therapidly growing tumor. Therefore, we decided to test whethermice that were primed with allogeneic Th1-containing vac-cinations prior to tumor challenge would respond better totherapeutic vaccination. In these experiments, mice wereprimed by i.d. inoculations on day 22, day 15, day 8 andday 1 with either media alone (control), 1 × 104 allogeneicCD3/CD28 cross-linked Th1 cells (Th1) or 1 × 104 Th1mixed with freeze/thawed BCL1 tumor lysate (Th1 + f/tBCL1 prime). All mice were infused with 2000 BCL1 onday 0. On day 7, some mice were administered i.d. thera-peutic booster inoculations of either 1 × 104 Th1 alone or



Fig. 6. Prime with therapeutic booster. Kaplan–Meier survival curves ofBalb/c mice vaccinated against BCL1 tumor (n = 8 for each group). All micewere infused i.v. with 2000 cells of BCL1 on day 0. Prior to tumor challenge,mice were primed by inoculation i.d. on day 22, day 15, day 8 and day1 with either media alone (control), 1 × 104 allogeneic CD3/CD28 cross-linked Th1 cells (Th1) or 1 × 104 Th1 mixed with freeze/thawed BCL1tumor lysate (Th1 + f/t BCL1 prime). On day 7, some mice were admin-istered therapeutic booster inoculations i.d. with either Th1 alone or Th1mixed with f/t BCL1. Control mice survived a median of 20 days. Vacci-nation with Th1 alone resulted in significant survival extension to a medianof 23 days (logrank: p = 0.011; hazard ratio: 2.478). Vaccination with Th1alone followed by a Th1 booster resulted in significant survival advantage(median survival = 25 days) compared to control (logrank: p < 0.0001; haz-ard ratio = 3.915) and significant survival compared to Th1 alone (logrank:p = 0.03; hazard ratio = 2.337). Mice primed by vaccination with f/t BCL1with Th1 as adjuvant (Th1 + f/t BCL1 prime) resulted in median survival of57.5 days with 50% of mice surviving lethal challenge. Mice primed withTh1 + f/t BCL1 and administered a Th1 booster had 75% survival lethalchallenge. Mice primed with Th1 cells alone and administered a therapeuticbooster injection with f/t BCL1 + Th1 had a significant survival advantagecompared to control with median survival of 46 days (logrank: p < 0.0001;hazard ratio = 5.633) and 37.5% of mice surviving lethal challenge.

1 × 104 Th1 mixed with f/t BCL1. Results are shown inFig. 6.

Control mice survived a median of 20 days. Priming withTh1 alone again resulted in significant survival extensionto a median of 23 days (logrank: p = 0.011; hazard ratio:2.478). Priming with Th1 alone followed by a Th1 therapeu-tic booster resulted in significant survival advantage (mediansurvival = 25 days) compared to control (logrank: p < 0.0001;hazard ratio = 3.915) and significant survival compared toTh1 prime alone (logrank: p = 0.03; hazard ratio = 2.337).Mice primed by vaccination with f/t BCL1 with Th1 as adju-vant (Th1 + f/t BCL1 prime) had median survival of 57.5days with 50% of mice surviving lethal challenge, the sameresult obtained in our previous experiment (see Fig. 4). Miceprimed with Th1 + f/t BCL1 and administered a Th1 thera-peutic booster had 75% survival after a lethal challenge. Miceprimed with Th1 cells alone and administered a therapeuticbooster injection with f/t BCL1 + Th1 had a significant sur-vival advantage compared to control with median survival of46 days (logrank: p < 0.0001; hazard ratio = 5.633) and 37.5%of mice surviving lethal challenge.

The shape of the Kaplan–Meier survival curve for the Th1prime/Th1 + f/t BCL1 booster group was different when com-pared to other groups that also resulted in mice cured afterlethal BCL1 injection. The 50% of mice that did not sur-vive after Th1 + f/t BCL1 prime and the 25% that did notsurvive lethal challenge from the Th1 + f/t BCL1 prime/Th1

booster group showed immediate signs of leukemia (palpablesplenomegaly and significant weight gain) and succumbedto the disease very early at a mean of 24 days. By contrast,the Th1 prime + f/t BCL1 treatment group contained a subsetof mice (62.5%) also with obvious leukemia that survivedsignificantly longer than control mice and a separate subset(37.5%) that were apparently cured and never showed signsof leukemia.

3.7. Cryoimmunotherapy

In order to try to further improve the efficacy of thera-peutic vaccination, we hypothesized that in-situ tumor deathby necrosis would provide a more potent adaptive immuneresponse than our freeze/thawed lysate preparations. Necrot-ically killed cells are known to activate endogenous signalsof distress responsible for the recruitment and maturation ofdendritic cells (DC), stimuli that would not be generated byhealthy or apoptotically dying cells [38] and may be missingfrom our lysate preparations. Further, exposure of immatureDC to these stimuli provides maturation signals, critical forthe initiation of local and systemic Th1 immunity [39,40].

In order to cause in-situ death by necrosis, we decided touse a cryoablation protocol. Cryoablation surgery is a tech-nique that can be translated to the clinic and has been shown tobe a well-aimed and controlled procedure capable of induc-ing tissular necrosis [41]. The biologic changes that occurduring and after cryoablation have been studied in vitro andin vivo [42,43]. Cryoablation has been known to elicit an anti-genic stimulus capable of generating a specific immunologicresponse against autologous antigens of the frozen tissue [44].

To test this hypothesis, we established two tumor modelswhere subcutaneous tumors were available for cryoablation.In the solid tumor model, Balb/c mice were administeredsubcutaneous inoculations of 1 × 104 BCL1 tumor cellsbilaterally on the shaved flanks on day 0. In the systemictumor model, Balb/c mice were administered inoculations of1 × 104 BCL1 tumor cells on the left flank and 2000 BCL1intravenously through the tail vein on day 0. The left tumorof these animals (n = 8 per group) were then treated on day14 after the solid tumors had grown to an area >16 mm2 withcryoablation alone or cryoablation with intratumoral 1 × 103

allogeneic CD3/CD28 cross-linked Th1 memory cells (Th1),intratumoral Th1 cells alone or intratumoral complete mediaalone served as controls. The results are shown in Fig. 7Aand B.

In the systemic tumor model (Fig. 7A), the mean sur-vival of control mice was 21 days. Survival of mice treatedwith cryoablation alone and Th1 alone was not differentthan control. However, the combination of cryoablation withintratumoral Th1 treatment resulted in significantly extendedsurvival to a mean of 28.5 days (logrank: p = 0.0059; hazardratio: 3.194).

In the solid tumor model (Fig. 7B), the mean survival ofcontrol mice was 28 days. None of the treatments tested inthis model provided a significant survival advantage.



Fig. 7. (A–D) Solid tumor and systemic tumor response to cryoimmunotherapy. In the solid tumor model, Balb/c mice were administered subcutaneousinoculations of 1 × 104 BCL1 tumor cells bilaterally on the shaved flanks on day 0. In the systemic tumor model, Balb/c mice were administered inoculationsof 1 × 104 BCL1 tumor cells on the left flank and 2000 BCL1 intravenously through the tail vein on day 0. The Kaplan–Meier survival curves of systemictumor model (A) and solid tumor model (B) mice (n = 8 each group) treated on day 14 with cryoablation of all observable tumor of the left tumor mass eitheralone (cryo alone) or with intratumoral allogeneic CD3/CD28 cross-linked Th1 memory cells (Th1) (cryo + Th1) or with intratumoral Th1 cells alone withoutcryoablation (Th1 alone). Control mice survived a median of 21 days in the systemic model and 28.5 days in the solid tumor model. The cryo + Th1 treatmentresulted in significant survival advantage (logrank: p = 0.0059; hazard ratio = 3.194) in the systemic model. The survival advantage in the solid tumor modelwas not significant (n.s.). The same experiment was repeated with the addition of a 1 × 105 intravenous infusion of Th1 on day 7 for all mice. Part (C) showsthe tumor growth curves of the contralateral untreated tumor masses for mice treated with this protocol. Forty percent of the mice treated with cryo + Th1 werecured of disease. The growth of contralateral tumor was significantly suppressed (p < 0.01) in the other 60% of mice that eventually succumbed to disease. Inthe systemic tumor model (D), Kaplan–Meier survival curves are shown. Mice treated with intratumoral Th1 cells alone survived a mean of 28 days, whichwas significantly longer than control mice which survived a mean of 19 days (logrank: p < 0.0001; hazard ratio = 4.291). Forty percent of mice treated withcryo + Th1 survived >90 days.

Because the cryoablation protocol did not result in anymice cured as result of the treatment, we modified the treat-ment to include a 1 × 105 intravenous infusion of allogeneicTh1 on day 7 for all mice. We had previously shown thatintravenous infusion of 1 × 105 allogeneic Th1 cells on day7 caused a significant survival advantage in mice lethallyinjected with BCL1 on day 0 [32]. We hypothesized that thistreatment would provide more time for a potentially curativeadaptive immune response to develop and thus affect the sur-vival of mice undergoing cryoimmunotherapy. Further, wehypothesized that this treatment would prime for alloanti-gen immunity, shown in our previous experiment (Fig. 6) toprovide a survival advantage.

The results of these studies are shown in Fig. 7C (solidtumor model) and Fig. 7D (systemic tumor model). In thesolid tumor model, the area of tumor was determined bymeasurement of the longest width and length of the tumorwith calipers. After complete ablation of the left tumor bycryoablation, only the contralateral untreated tumor masswas measured. The tumor growth curves of the contralateraluntreated tumor masses are shown for control mice treatedwith cryoablation alone and mice treated with combinationof cryoablation and intratumoral allogeneic Th1 (cryo + Th1).

In support of our hypothesis, 40% of mice treated with thecombination therapy survived without evidence of tumor. Thecontralateral tumor area is displayed in Fig. 7C to separatelyshow the response of the 40% mice that survived and the 60%that eventually succumbed to disease. The growth of the con-tralateral tumors was significantly suppressed (p < 0.01) inthe 60% of mice that eventually succumbed to disease. Inthe systemic tumor model (Fig. 7D), mice treated with intra-tumoral allogeneic Th1 cells alone survived a mean of 28days, which was significantly longer than control mice whichsurvived a mean of 19 days (logrank: p < 0.0001; hazardratio = 4.291). 40% of mice treated with cryo + Th1 survived>90 days.

4. Discussion

The goal of therapeutic active immunotherapy is to elicita potent immune response that will cause the eradication ofthe tumor as well as generate a long-term memory responseto maintain complete remission. Although animals can beimmunized against the growth of some tumors, most ofthe attempts to use immunotherapy to cause the regres-



sion of existing animal and human tumors have not beensuccessful. We had previously observed that intravenousinfusion of allogeneic CD3/CD28 cross-linked Th1 mem-ory cells was capable of enhancing both circulating IFN-�and tumor-specific IFN-� production, while suppressingsystemic and tumor-specific IL-4 and systemic IL-10 inmice lethally inoculated with BCL1 compared to untreatedBCL1 inoculated mice [32]. Based upon this observation, wehypothesized that this immunomodulatory mechanism wouldprovide an adjuvant effect which would create systemic anti-leukemia immunity and break tolerance to self-antigens ofthe tumor. Our results indicate that the ‘danger signals’ andimmunomodulatory effects of these allogeneic Th1 cells arecapable of eliciting both protective and therapeutic anti-leukemia effects in the BCL1 tumor model.

In the absence of adjuvants, injection of purified pro-teins can induce tolerance [45]. Therefore, we expected thatirradiated and freeze/thawed BCL1 vaccinations, withoutany adjuvant, would not elicit an immune response in syn-geneic mice. Contrary to expectation, BCL1 vaccination withboth these vaccine compositions elicited high frequencies oftumor-specific T-cells, demonstrating that the current BCL1clone is in fact immunogenic.

However, the immune responses elicited by irradiatedand freeze/thawed BCL1 vaccinations without adjuvant werenot protective, as all mice vaccinated with these composi-tions were unable to survive a live tumor challenge. Bothvaccine compositions elicited Th2-biased adaptive immuneresponses. These results suggest that the selective pressureof in vivo passage of BCL1 resulted in a clone that evadesBalb/c immune elimination by provoking a non-protectiveTh2 immune response, rather than immunoediting and selec-tion of a non-immunogenic clone. This result correlates withprevious reports that a Th2 immune response against BCL1is associated with a fatal outcome, while a Th1 immuneresponse has been shown to be protective [46]. Similarly,a shift toward Th2 immunity has been reported to correlatewith disease progression in human B-CLL [47]. Our findingssupport previous studies which demonstrate that protectiveimmunity depends not only on the presence or absence of animmune response (immunogenicity), but also on the initiationof the proper type of immune response [48].

The finding of superior immunogenicity of irradiatedBCL1 (irrad BCL1) compared to the BCL1 lysate (f/t BCL1)was contrary to the ‘danger hypothesis’ prediction that apop-totic cells will be less immunogenic than lysed cells [49].The case of B-cell malignancies may be an exception to thisproposed concept of immunogenicity, as malignant B-cellsare a unique tumor type in that they express a tumor-associated immunoglobulin (Ig) that can serve as a target forhost-mediated adaptive immunity [50]. Expression of tumorassociated Ig may make B-cell tumors uniquely immuno-genic as compared to other tumors. In concurrence with ourresults, a previous study showed that vaccination with themembrane bound form of Ig was more immunogenic thanthe soluble form [51], which is consistent with our finding

that intact irrad BCL1 is more immunogenic than the f/tBCL1 lysate. However, immunization with BCL1 IgM hasbeen shown to have no significant therapeutic benefit [52], aswas the case in our study using irrad BCL1.

The type of cytokine profile present in the microenviron-ment of the vaccination sites at the time of DC maturationcan control the type of immune response that is generated.After uptake of antigen, DC mature to produce either IL-10or IL-12 and emigrate to the skin draining regional lymphnodes and present processed antigens to T-cells [53]. DC thatmatured in the presence of IL-12 promote the development ofa Th1 type response [54], while DC matured in the presenceof IL-10 [55,56], or in the absence of IL-12 [57], promoteTh2 type responses.

The strongly polarized Th2 immune response elicited byirrad BCL1 vaccination may be due to BCL1 production ofIL-10 in the microenvironment, since BCL1 and apoptoticcells in general are known to produce IL-10 [32,58]. However,vaccination with f/t BCL1 lysate, non-activated allogeneicTh1 cells and allogeneic CD4 naïve cells, all which did notproduce cytokines, also elicited polarized Th2 responses,albeit at lower frequencies than the irrad BCL1 preparation.This result is consistent with the genetic predisposition ofBalb/c mice to process and respond to antigens with a Th2-bias [59]. We also found that CD4 cells isolated from naïveBalb/c mice produced significant amounts of IL-4 when acti-vated. Since the host response to alloantigens results in T-cellactivation, the alloantigens in the vaccination compositionscontaining f/t BCL1, non-activated allogeneic Th1 cells andallogeneic CD4 naïve cells would be expected to result inIL-4 production into the microenvironment as a consequenceof host T-cell activation. DC exposure to IL-4 is known tosteer the immune response to Th2 [60], which is a possibleexplanation for our results.

By contrast, we showed that vaccination with allogeneicCD3/CD28-activated allogeneic Th1 cells elicited a stronglypolarized Th1 response. These activated Th1 cells wereshown to produce large amounts of IFN-� and expressCD40L, both of which are known to be capable of promot-ing Th1 immune responses. CD40L interaction with CD40expressed on DC has been shown to cause differentiation ofTh1 cells in part through the upregulation of IL-12 produc-tion [61]. IFN-� has been reported to play an indirect role inTh1 cell differentiation by upregulation of IL-12 productionby activated macrophages [62].

When combining irrad BCL1 or f/t BCL1 vaccine prepa-rations with these allogeneic Th1 cells, the anti-leukemiaimmune response to these tumor antigen preparations wasdeviated from Th2 domination to Th1, demonstrating thepotent immunomodulatory adjuvant effect of these cells.However, only the vaccine that contained f/t BCL1 cre-ated an immune response that could protect against lethaltumor challenge. While both tested vaccine preparationselicited dominate Th1 anti-leukemia responses, the quanti-tative and qualitative aspects of the responses were different.The ineffective irrad BCL1-containing vaccine elicited a



mixed Th1/Th2 response with Th1 dominance, while the f/tBCL1 preparation elicited a strongly polarized Th1 responsewithout a significant Th2 response and elicited a titer ofanti-leukemia specific Th1 cells approximately four timesgreater than that elicited by the irrad BCL1 preparation.These immunological differences may explain the differentialresults of these vaccination compositions.

The success of the f/t BCL1 preparation in elicitingprotective immunity to BCL1 may be due to endogenousheat shock proteins (hsp) that are likely contained withinour freeze/thawed lysates. Hsp are intracellular moleculeswhich act as chaperones for peptides [63,64], includingtumor-associated antigens [65]. Complexes of heat shockproteins and peptides can be isolated from freeze/thawedlysates [66]. Vaccination of mice with a cocktail of chap-erone proteins has been shown to provide protection froman aggressive BCR-ABL leukemia clone by steering immu-nity to Th1 [67,68]. Chaperone proteins provide an adjuvanteffect for Th1 immunity greater than using individual peptidevaccination alone [67] and can down-regulate the suppres-sor function of Treg cells [69] and cause DC maturation[70].

In order to elicit a curative immune response in animalswith existing disease, we attempted to first prime the animalsprior to tumor challenge with allogeneic Th1 cells alone orwith f/t BCL1 and then provide a therapeutic booster aftertumor challenge. This protocol resulted in high cure rates. Thef/t BCL1 + Th1 priming protocol protects 50% of mice from alethal BCL1 challenge. Providing a therapeutic booster withf/t BCL1 + Th1 to this protocol increases the cure rate to 75%.The need for priming prior to tumor inoculation for cura-tive immunity to develop suggests that protective anti-BCL1immunity takes time to develop and that the aggressivenessof the BCL1 clone that routinely causes death in mice in19–22 days does not allow sufficient time in which to buildprotective immunity in the therapeutic setting.

In the clinical setting, conventional approaches to mosthematological malignant disorders can achieve clinically evi-dent tumor responses in a substantial number of patients.However, persistence of minimal residual disease is thoughtto underlie the lack of survival benefit often reported inthese patients. Therefore, the adjuvant setting may be moreamenable for translation of our active immunotherapy proto-col, as patients with minimally residual disease presumablywould have life expectancies long enough for an activeimmunotherapy vaccination protocol to have time to developprotective immune responses.

Interestingly, priming animals by vaccination with allo-geneic Th1 cells alone without a source of tumor antigenand then providing a therapeutic booster with f/t BCL1 + Th1resulted in a 37.5% cure rate, showing that priming for tumor-specific immunity was not necessary for f/tBCL1 + Th1therapeutic efficacy. In addition, we found that primingwith allogeneic Th1 cells alone, without a therapeuticbooster, provided a significant survival advantage. Thissurvival advantage could be significantly enhanced by allo-

geneic Th1 alone booster vaccination after lethal tumorchallenge.

An immunological phenomenon called “heterologousimmunity” has been described where the immune sys-tem becomes biased by a high frequency of memory cellsspecific for a given pathogenic antigen, and where the activa-tion of these cells during an unrelated pathogen infectionsignificantly enhances clearance of an unrelated infection[71,72]. Our results seem to describe a possibly similarphenomenon whereby the vaccination with allogeneic Th1cells creates a Th1 footprint that when reactivated in thepresence of a tumor results in a ‘heterologous’ anti-tumoreffect.

It is interesting to hypothesize how this possible het-erologous anti-tumor immunity might be translated to theclinic. Since enhancement of circulating Th2 cells occursin elderly people [73,74] which is correlated with increasedsusceptibility to cancer [75], alloantigen vaccination mightbe an approach to correct this imbalance and improve tumorsurveillance. Should these patients later be diagnosed withcancer, therapeutic allogeneic booster vaccinations with orwithout autologous tumor antigen might be a strategy tocontrol the tumor growth.

Another approach to therapeutic vaccination we evaluatedwas a cryoimmunotherapy protocol that combined an intra-tumoral allogeneic Th1 treatment with tumor cryoablation.Cryoimmunotherapy has been shown previously to result insignificant survival advantage in a mouse model of metastaticlung cancer, which involved intratumoral injections of syn-geneic immature dendritic cells following cryoablation ofsolid tumors [76]. The anti-tumor effects of cryoimmunother-apy on remote solid tumors has been shown to be enhancedwhen intratumoral immature dendritic cells were combinedwith Bacillus-Calmette-Guerin (BCG) cell wall skeletonstimulation [77].

Our in-situ cryoimmunotherapy vaccine protocol resultedin significant survival advantage for mice with systemic dis-ease, but not for mice with bilateral solid lymphomas. Itis not clear as to why the immune response that occurs inour cryoimmunotherapy protocol would be effective againstsystemic disease but not against solid tumors. This resultsuggests a possible effector cell extravasation and/or traf-ficking problem that prevented effector cells from mediatingan effect on the contralateral solid tumor, but which had aneffect on systemic disease in the secondary lymphoid organsand circulation.

The differential control of trafficking of effector T-cellsto secondary lymphoid organs and to peripheral tissue iscontrolled by CD62L and CD44 expression. CD62L is wellknown as a lymph node homing receptor [78], while CD44 isinvolved in extravasation and trafficking of activated lympho-cytes to inflammatory sites [79]. In addition, subcutaneouslyinoculated tumors may not provide the same natural vascu-lature infrastructure necessary for effector cell extravasationand infiltration. In order to traffic to cutaneous tumor sites,T-cells have been shown to require expression of the skin



homing receptor, CLA [80]. However, a previous studydid demonstrate that subcutaneous tumor vaccination canresult in effector cells capable of homing to subcutaneouslyimplanted tumors [81].

It is also possible that the contralateral lymphoma maynot have expressed the necessary inflammatory signals andchemokines to recruit effector cells, or that effector cells arerecruited to the solid tumor but local suppressive cytokines,such as IL-10, or infiltrating Treg or other regulatory cellsinhibit their effector function.

When we added an intravenous infusion of allogeneic Th1cells to the cryoimmunotherapy protocol, curative immuneresponses were observed in 40% of mice in the solidtumor model. The mechanism by which the addition ofthis treatment to the cryoimmunotherapy protocol convertsan ineffective response to a curative response remains tobe elucidated. Our allogeneic Th1 cells were shown toexpress a CD62Llo, CD45RBhi, CD44hi phenotype. Thisis the phenotype of cells that are capable of extravasa-tion to the contralateral tumor bed. It is possible that theallogeneic Th1 cells traffic to the contralateral tumor andexert a graft-vs-leukemia effect. Cells with a CD62Llo,CD45RBhi, CD44hi phenotype have previously been shownto be associated with autoimmune disease and allograft rejec-tion [35–37].

Addition of an intravenous infusion of allogeneic Th1cells to the cryoimmunotherapy systemic tumor model alsoresulted in a 40% cure rate. This might also be related to agraft-vs-leukemia effect. In addition, CD40L expression onour allogeneic Th1 cells are capable of enhancing the hostproduction of IFN-� and enhancing NK activity [61] whichcould contribute to the enhanced efficacy of this protocol.Translation of this protocol to the clinic is possible in patientsthat present with plasmacytomas or lymphomas that might beaccessible for percutaneous cryoablation.

In conclusion, our CD3/CD28 cross-linked Th1 memorycells are capable of providing multiple important ‘danger sig-nals’, including expression of alloantigen, CD40L and IFN-�.These features, as well as the demonstrated immunomodu-latory effects, make these cells candidates for use as a noveladjuvant in active immunotherapy protocols. Translation ofthese results to the clinic may provide a new strategy forbreaking immune tolerance to tumors and improving theefficacy of active immunotherapy.

Conflict of interest statement

None.

Acknowledgements

This work was funded in part by a grant from the IsraelOffice of the Chief Scientist to M. H.-N. and support fromImmunovative Therapies Ltd. M. H.-N. is the founding sci-entist of Immunovative Therapies.

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Author's personal copy - Immunovative · 2015. 11. 25. · Author's personal copy Available online at Leukemia Research 33 (2009) 525 538 Allogeneic CD3/CD28 cross-linked Th1 memory