Immunotransplant Preferentially Expands Teffector Cells over Tregulatory Cells and Cures Large Lymphoma Tumors

doi:10.1182/blood-2008-05-155457Prepublished online September 23, 2008;

Joshua D Brody, Matthew J Goldstein, Debra K Czerwinski and Ronald Levy cells and cures large lymphoma tumorsImmunotransplant preferentially expands Teffector cells over Tregulatory

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Immunotransplant Preferentially Expands Teffector Cells over Tregulatory Cells and Cures Large Lymphoma Tumors

Joshua D. Brody, Matthew J. Goldstein, Debra K. Czerwinski, Ronald Levy Division of Oncology, Department of Medicine, Stanford University Medical Center, Stanford, California

Blood First Edition Paper, prepublished online September 23, 2008; DOI 10.1182/blood-2008-05-155457

Copyright © 2008 American Society of Hematology




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Abstract Ex vivo expanded tumor-infiltrating lymphocytes infused into lymphodepleted recipients has clear anti-tumor efficacy. More practical sources of such anti-tumor lymphocytes would broaden the application of this approach.

Previously, we described an in situ vaccination combining chemotherapy with intra-tumoral injection of CpG-enriched oligonucleotides, which induced T-cell immunity against established lymphoma. An ongoing clinical trial of this maneuver has demonstrated clinical responses in lymphoma patients.

Here, we use this vaccine maneuver to generate immune cells for transfer into irradiated, syngeneic recipients. Transferred tumor-specific Teffector cells preferentially expanded, increasing the Teffector:Treg ratio in these ‘immunotransplant’ recipients, and curing large and metastatic tumors. Donor T cells were necessary for tumor protection and CD8 T cell immune responses were enhanced by post-transplant booster vaccination.

Hematopoietic stem cell transplant is a standard therapy for lymphoma. Therefore, in situ tumor vaccination followed by immunotransplant of harvested tumor-specific T cells could be directly tested in clinical trials to treat otherwise resistant malignancies.




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Introduction Previously, we have described a practical way of achieving therapeutic anti-tumor immunity by administering cytotoxic therapy to release tumor antigens combined with intra-tumoral injection of an immunostimulatory CG-enriched oligodeoxynucleotide (CpG). This vaccination maneuver induces tumor-specific CD8 T-cell immunity and causes tumor remission both in a pre-clinical model of murine B-cell lymphoma1 and in patients with B-cell lymphomas. To further improve this therapeutic maneuver, we have considered possible obstacles to the development of anti-tumor immunity such as regulatory cells and myeloid suppressor cells. Myeloablative therapy with hematopoietic stem cell transplantation—a standard therapy for hematologic malignancies—could address these problems by eliminating regulatory immune cells2,3 and making ‘space’ for the expansion of specific anti-tumor T cells. Rosenberg and co-workers have demonstrated a remarkable anti-tumor effect of transferring cultured tumor infiltrating T cells (TILs) into lymphodepleted melanoma patients4. However, it is unknown whether transferred peripheral blood mononuclear cells (PBMCs) from a vaccinated host could be as powerful. One concern is that transferred PBMCs would contain Tregs, which may have persistent, suppressive effects on transferred tumor-specific Teffectors. The cytokines chiefly responsible for homeostatic T-cell proliferation—IL-7, IL-15, and IL-21—have been shown to preferentially affect Teffectors over Tregs

5,6. We hypothesized that adoptive transfer of vaccine-primed, anti-cancer T cells into syngeneic hematopoietic stem cell transplant recipients— ‘immunotransplant’—would cause relatively greater proliferation of tumor-specific Teffectors and skewing against transferred Tregs, resulting in a potent anti-tumor state. We have devised such an ‘immunotransplant’ maneuver and demonstrated its preferential proliferative effect on transferred, tumor-specific Teffectors as well as its impressive power to treat large tumors. We anticipate that these results could be directly translated from the pre-clinical model to a clinical trial in patients with lymphoma. Methods Reagents CpG 1826 5'-TCCATGACGTTCCTGACGTT was provided by Coley Pharmaceutical Group (Ottawa, Ontario, Canada). Cyclophosphamide (CTX) and Neomycin were purchased from Sigma-Aldrich. Luciferin was purchased from Xenogen (Alameda, CA). Cell lines and animal models A20, a BALB/c B cell lymphoma line expressing MHC class I and class II H-2d molecules, was obtained from American Type Culture Collection. Tumor cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (HyClone Laboratories), 100 U/ml penicillin, 100 µg/ml streptomycin (both from Invitrogen Life Technologies), and 50 µM 2-ME (Sigma-Aldrich). Cells were grown in suspension




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culture at 37°C in 5% CO2. 8- to 10-wk-old female BALB/c mice were purchased from Harlan Sprague Dawley Laboratories. All experiments were conducted in accord with NIH guidelines and with approval of the Stanford University Institutional Animal Care and Use Committee. Flow cytometry The following monoclonal antibodies were used in this study: PE anti-mouse IFNγ, FITC anti-mouse CD8, PE anti-mouse CD8β, PerCP anti-mouse CD3, PerCP anti-mouse CD4, PerCP anti-mouse CD8, and APC anti-mouse CD44 (BD Pharmingen). Cells were stained in phosphate buffered saline (PBS), 1% BSA, and 0.01% sodium azide and analyzed by flow cytometry on a BD FACSCalibur System. Donor splenocytes were labeled with CFSE (Invitrogen) in a 5μM solution in PBS. CpG/CTX Vaccination A20 lymphoma cells (107 in 100 µl of PBS) were implanted s.c. on the lower back in 8- to 10-wk-old BALB/c mice. Treatments began when tumors reached a size of ~100mm2, typically at day 14 after tumor inoculation. The chemotherapy agent cyclophosphamide (CTX) was then administrated i.p. at a dose of 100 mg/kg on each of two consecutive days. CpG was then injected intra-tumorally at 100 µg/dose in 100 µl PBS on each of the 5 days following chemotherapy. Using this maneuver, a CD8 T cell response caused most mice to be cured1. Such mice were used as splenocyte donors 7 days after the completion of vaccination. Immunotransplant Eight to 10-week old, naïve, recipient mice were irradiated with 900cGy TBI in a Philips x-ray unit (250 kV, 15 mA). Irradiated recipients were injected intravenously with 5 x 106 bone marrow (BM) cells in 0.5 mL RPMI 1640 medium in the tail vein admixed with splenocytes. Unless otherwise specified, splenocyte dose is 1 spleen per 1 recipient. In all experiments, bone marrow was taken from the same source as donor spleens to more closely parallel the clinical application of this approach. Beginning 2 days prior to ablative therapy and thereafter, drinking water was supplemented with neomycin 1mg/ml for gut decontamination. In the tumor-protective setting, recipient mice were implanted s.c. on day 3 after transplant with 107 A20 cells. In the tumor-therapeutic setting mice were challenged with 107 A20 cells 14 days prior to transplant/transfer, and selected at the time of treatment such that all cohorts harbored tumors of approximately 100mm2. The growth of tumor was monitored by calipers three times per week. In some experiments, donor splenocytes were labeled with CFSE. In some experiments a vaccine ‘boost’ was administered, consisting of 1x106 A20 cells cultured in the presence of CpG1826 at final concentration of 3μg/mL for 72 hours and irradiated to 50Gy immediately prior to injection. These boosts were administered either i.v. or s.c. Immunotransplants were performed using donor splenocyte cell subsets by positively isolating using Dynabeads® FlowComp™ Mouse CD4 or CD8 kits or depleting using Dynabeads® Mouse pan T (Thy 1.2) kits (Invitrogen) as per protocols. Of note, FlowComp™ Mouse CD8 positive selection is based on anti-CD8α mAb binding such that




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selected cells can be phenotypically assessed using anti-CD8β mAb without risk of selecting/detecting mAb interference. Detection of tumor reactive T cells Blood was collected from tail vein, anticoagulated with EDTA 2mM in PBS, then diluted 1:1 with Dextran T500 (Pharmacosmos, Denmark) 2% in PBS and incubated at 37°C for 45 minutes to precipitate red cells. Leukocyte-containing supernatant was removed, centrifuged and remaining red cells were lysed with ACK buffer (Quality Biological, Gaithersburg, MD). PBMCs were then co-cultured with 5 x 105 irradiated A20 cells for 24 hours with 0.5µg of anti-mouse CD28mAb (BD Pharmingen) and in the presence of monensin (Golgistop – BD Biosciences) for the last 5 hours at 37°C and 5% CO2. Tumor-specificity of the response was assessed by parallel experiments co-culturing PBMCs with 5 x 105 irradiated CT26 cells—a balb/C colon cancer cell line (Supplemental Fig. 1). Cells were then washed and stained with anti-CD8 FITC and anti-CD4 allophycocyanin (BD Biosciences). Intracellular IFNγ expression was assessed using BD Cytofix/Cytoperm Plus kit (catalog no. 554715) per instructions, and BD anti-IFNγ PE-conjugated Ab. Cells were analyzed on a FACScalibur flow cytometer. Bioluminescence The A20-luficerase cell line was provided by R. Negrin7. Mice were anaesthetized with isofluorane and an aqueous solution of luciferin (150 mg/kg intraperitoneally) (Xenogen, Alameda, CA) was injected 5 minutes prior to imaging. Animals were placed into the light-tight chamber of the CCD camera system (IVIS-200, Xenogen). Photons emitted from luciferase-expressing cells within the animal body were quantified using the software program ‘Living Image’ (Xenogen) as an overlay on Igor (Wavemetrics, Seattle, WA). Results Tregs increase in CpG/CTX vaccinated mice, but immunotransplant increases the Teffector:Treg ratio Our prior work described an intra-tumoral CpG plus systemic cyclophosphamide (CTX) vaccine maneuver (CpG/CTX). Briefly, mice with subcutaneous A20 tumors received systemic CTX followed by five daily intra-tumoral injections of CpG-1826 resulting in tumor regression mediated by CD8 T cells. As described in other tumor vaccine systems8, there was an absolute increase in the number of Treg cells post-vaccination despite the development of tumor-specific immunity (Fig. 1B). Therefore, we asked whether the transfer of splenocytes into lymphodepleted recipients could skew the T cell population against Tregs. CFSE labeled splenocytes were transferred into untreated (‘full’) or lethally irradiated (‘empty’) recipients. Recipient T cells were assessed at day 15 for CFSE dilution. ‘Empty’ recipients showed marked Teffector cell proliferation—3.6 fold more so than foxP3(+) Tregs (Fig. 1C). Minimal proliferation occurred in ‘full’ recipients. An immunotransplant maneuver enhances tumor-specific T-cell responses and protects against tumor challenge




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The effect of immunotransplant on tumor-specific T cells was assessed. CpG/CTX vaccinated donors and immunotransplant recipients were tumor-challenged 3 days after transplant using 100-fold above the A20 minimal lethal dose to provide a stringent test of the potency of the immune response. Fifteen days after transplant PBMCs were assayed for IFNγ response to A20 tumor cells. Vaccinated donor mice demonstrated a significant tumor-specific T-cell response (Fig. 2B). Transfer of splenocytes from these vaccinated donor mice into ‘full’ recipients resulted in no detectable tumor-specific T cells (Fig. 2B, 3rd panel), despite the measurable persistence of the transferred population over the same time course (Fig. 2D, left panels, CFSE (+) population). In contrast, ‘empty’ recipients demonstrated a robust, tumor-specific T-cell response, an order of magnitude greater than either ‘full’ recipients or vaccinated donors. Challenged tumors grew rapidly in untreated mice and in ‘full’ recipients of splenocyte transfer, (Fig. 2C) whereas vaccinated donors and ‘empty’ immunotransplant recipients were completely protected. Interestingly, there was brief tumor growth in the ‘empty’ recipients peaking at day 14 followed by complete regression (Fig. 2C, closed squares), indicating anti-tumor immunity increasing over time. Measurement of CFSE intensity of transferred T cells demonstrated their proliferation over time in ‘empty’ but not ‘full’ recipients (Fig. 2D, upper panels). Tumor-specific T cells proliferated to an even greater degree than other T cells (mean CFSE = 5.1 vs. 12.1). Because homeostatic proliferation can induce a memory phenotype in transferred T cells9,10, we compared CD44 expression in T cells repopulating ‘full’ versus ‘empty’ recipients (CD44hi = 1.7% vs. 22.5%). Donor and ‘empty’ recipient mice were also re-tested for tumor-specific immunity 4 weeks from the time of immunotransplant. Intact tumor-specific memory was observed in donor mice and to an even higher degree in ‘empty’ recipients (3% vs. 5% of memory CD8 cells) (Fig. 2E). Immunotransplant protects against systemic tumor burden We next tested the efficacy of immunotransplantation on systemic tumor. Recipient mice were lethally irradiated and rescued with bone marrow cells and splenocytes from donors that received either no vaccine (Fig. 3A) or CpG/CTX vaccination (Fig. 3B). One day after transplant, mice were challenged i.v. with high dose (1x106) A20-LUC (50x minimal lethal dose7) and followed by bioluminescence. All mice had initial tumor growth. Mice receiving unvaccinated-donor splenocytes rapidly progressed while mice receiving vaccinated-donor splenocytes had rapid remission of disease. Similar results were demonstrated using intraperitoneal tumor challenge (data not shown). Immunotransplanted mice were separately challenged with wild type A20 cells and followed for survival (Fig. 3C). Recipient mice receiving bone marrow and splenocytes from unvaccinated donors succumbed by day 25 while recipients of vaccinated-donor splenocytes all survived. Immunotransplant induces T cell mediated tumor protection




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Because immunotransplant of splenocytes from CpG/CTX vaccinated mice resulted in a ~10 fold increase in CD8 T cell tumor-specific immune response (Fig, 2B) but no apparent increase in tumor-specific immune response of other cell types (data not shown), we tested the relative importance of different cell subsets in mediating tumor protection. On day 8 post CpG/CTX vaccination, donor splenocyte populations are comprised of ~11% CD8 T cells, 43% CD4 T cells, and 46% non-T cells (Fig. 4B). Using monoclonal-antibody-conjugated ferromagnetic beads, purified splenocyte subsets were obtained and transferred into irradiated recipient mice at total cell numbers normalized to that population’s proportion in donor splenocytes (i.e. ~105 x106 complete splenocytes, 49x106 non-T cells, 45x106 CD4 T cells, or 12x106 CD8 T cells were transferred). Recipients were challenged with s.c. tumor on post-transplant day 3 and followed for tumor-growth and survival. These data demonstrated that T cells were necessary to transfer complete tumor protection and suggested that CD8 cells were more potent in this regard (Fig. 4C). Complete donor vaccination is required to transfer anti-tumor immune responses and tumor protection and can be enhanced by booster vaccination We next tested the requirements for vaccination of immune donors. Donor mice received either no treatment, tumor implantation alone, tumor implantation followed by systemic CTX, or the complete CpG/CTX vaccination maneuver (Fig. 5A). At the time of splenocyte harvest, tumor-bearing and CTX-treated donors both had measurable tumors. CpG/CTX vaccinated donors had little or no measurable disease. Recipient mice that received splenocyte transfer from unvaccinated, A20-bearing, or CTX-treated donors did not demonstrate significant tumor-specific CD8 T-cell responses, nor were they protected from tumor challenge (Fig. 5B,C). Mice receiving splenocytes from A20-bearing donors were also not protected from the smaller i.v. tumor-challenge of A20 cells contaminating the splenocyte population. In contrast, recipients receiving splenocytes from fully vaccinated donors demonstrated significant tumor-specific CD8 T-cell responses and 100% tumor protection (Fig. 5B,C 4th panel). We hypothesized that if transferred CD8 T cells mediate immunotransplant anti-tumor immunity, then delivering ‘signals 1 and 2’ (cognate antigen in the context of costimulatory molecules) along with the homeostatic proliferative signal would enhance this immune response. A20 tumor cells were cultured in vitro with CpG (to increase costimulatory molecule expression1), irradiated, and given with immunotransplant either as an i.v. (Fig. 5A) or s.c. (data not shown) boost. There was a marked, 3-4 fold increase in the proportion of anti-tumor CD8 T cells with both routes of immunization which was highly tumor-specific (Fig. 5B and Supplemental Fig. 1) . We also observed a suggestion of more potent tumor protection, as the initial tumor growth usually observed in challenged immunotransplant recipients no longer occurred (Fig. 5C). Additionally, late recurrences (beyond day 75) observed in a minority of challenged immunotransplanted mice did not occur in ‘boosted’ cohorts. (All cohorts were followed for 90 days and late recurrences were seen in 0-40% of recipients in repeated experiments). Immunotransplant-induced tumor immunity increases with time and with serial transplants




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To investigate the mechanism of tumor protection in immunotransplant and to determine the requirement for ‘emptiness’, we varied the conditioning-radiation dose. Recipients received 900, 600, or 300 cGy TBI followed by immunotransplant and then received high dose A20 tumor challenge (Fig. 6B). As previously, mice receiving full dose TBI demonstrated transient tumor growth peaking by day 20, but regressing by day 25. Mice receiving lower TBI doses showed variable protection, though there appeared to be a threshold dose. A two-fold reduction (600 to 300cGy) reduced tumor protection from 100% to 30%. In addition, tumor immunity in these recipients appeared to decrease with time, such that several mice demonstrated remission followed by rapid tumor recurrence (Fig. 6C, third column). To determine the potency of CpG/CTX donor splenocytes we transferred them at varying doses into lethally irradiated recipients: one donor spleen → one recipient versus 1/4 or 1/8 dose (Fig. 6C). As previously, recipient mice receiving the complete dose of splenocytes were protected from tumor, with some recipients exhibiting transient tumor growth peaking at day 10 and regressing by day 15. Recipients of lower splenocyte doses were variably protected. Also, peak tumor immunity appeared to increase with time. In lower splenocyte-dose cohorts initial immunity was insufficient to stop rapid tumor growth but by day 20, tumor immunity was powerful enough to eradicate tumors as large as 200mm2. Though the ‘empty signal’ increases the anti-tumor effect of transferred T cells, it is a transient effect of ablative therapy and diminishes with hematopoietic reconstitution. Because T-cell transfer into the ‘empty’ mouse induces both proliferation as well as qualitative changes (e.g. increased proportion of tumor-specific and memory T cells), we asked whether these changes were reversible (i.e. dependent upon continued ‘emptiness’ of the recipient) or irreversible (i.e. persistent after secondary transfer to a ‘full’ recipient). To address this question we performed serial immunotransplants using cured recipients as donors into either ‘empty’ or ‘full’ secondary recipients. These secondary recipients received high dose tumor challenge on day 3 post-transplant. The secondary transfer of immunotransplant-cured donor splenocytes into ‘full’ recipients demonstrated significant protection. We observed delayed tumor growth compared to tumor-challenged control mice and cure in 50% of recipients (Fig. 6E). This differed markedly from the transfer of CpG/CTX vaccine-primed splenocytes into ‘full’ recipients, which yielded no tumor protection (Fig. 2B). Additionally, serial immunotransplant into ‘empty’ secondary recipients protected 100% of these recipients from subsequent tumor challenge without the transient tumor growth seen in standard immunotransplant recipients (Figs. 2B, 5C, 6B,C) indicating an increased anti-tumor effect. Immunotransplant induces a tumor-specific immune response and cures mice with large tumors Because of the striking regression of large tumors in the above prophylactic experiments, we asked whether immunotransplant could be effective in the therapeutic setting. On day 14 after s.c. implantation of 1x107 A20 cells, tumors are ~100mm2 , radiation resistant, and uniformly lethal in <20 days. These tumor-bearing mice were used as immunotransplant recipients to test the therapeutic effect of this maneuver.




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Mice bearing 100mm2 tumors received either no irradiation or 900cGy TBI and received splenocytes and bone marrow from CpG/CTX vaccinated donors, as previously. The un-irradiated recipients also received a ‘boost’ of irradiated, CpG-treated A20 cells to optimize their chance of tumor protection. On day 15 post-transplant, PBMCs were assayed for tumor-specific IFNγ producing cells. In these large tumor-bearing recipients immunotransplant led to the induction of tumor-specific IFNγ-producing CD8 T cells and to rapid cure (within 10 days) of 100% of mice (Fig. 7B,C right panel). In contrast, splenocyte transfer from unvaccinated donors neither induced tumor-specific CD8 T cells nor caused any reduction in tumor size (Fig. 7B,C mid panel). These results were identical to mice receiving BMT alone without splenocyte transfer (data not shown). Similarly, in un-irradiated recipient mice, transfer of bone marrow plus vaccinated-donor splenocytes plus CpG-A20 ‘boost’ was ineffective (Fig. 7B,C left panel). The 100% cure rate of mice bearing 100mm2 tumors prompted us to treat mice with larger tumors. Mice with tumors as large as 400mm2 received immunotransplant from either untreated or CpG/CTX-vaccinated donors. In only the latter group we observed multiple instances of remarkable tumor regressions (Fig. 7D). To determine if there was an influx of effector cells to the tumor site—paralleling the development of tumor-specific T cells in the peripheral blood—we excised tumors on day 8 post-transplant from tumor-bearing recipients treated with 0 or 900 cGy TBI and receiving immunotransplant from either unvaccinated or CpG/CTX-vaccinated donors. A significant proportion of tumor infiltrating CD3 cells was seen only in recipients receiving the complete immunotransplant maneuver (Fig. 7E).

Discussion The remarkable therapeutic effect of transferring ex vivo primed TIL into lymphodepleted melanoma patients11 compels the further development of this approach. It has been suggested that more aggressive lymphodepletion (i.e. lethal myeloablation with stem cell rescue) would make this approach more powerful12 and that discovering a way to replace TILs with vaccine-primed T cells would facilitate broader clinical application. Though such an attempt by the same group was not encouraging13, our findings suggest that some vaccine strategies are more effective in inducing transferable immunity. It is possible that more effective vaccinations, such as the CpG/CTX used here, will be the key to making immunotransplantation effective without the need for ex-vivo expansion of T cells. Here, we demonstrate that an in situ vaccine maneuver, while generating tumor-specific T-cell response also generates an increased number of Tregs (Fig. 1B)8. Using vaccine-primed donors as a source of T cells for immunotransplant prompts the question of whether this increase in Tregs will hinder the transferred immunity. Supporting recent results by Mirmonsef et al.14 in a transgenic T-cell system, herein, we show that immunotransplant increases the Teffector:Treg in transferred splenocytes (Fig. 1D) and causes preferential proliferation of tumor-specific CD8 T cells (Fig. 2D).




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The tumor-specific, IFNγ-producing, CD8 T-cell response occurs primarily amongst memory CD44hi T cells, a subset also known to be proportionally increased by homeostatic proliferation9,10. The preferential proliferation of tumor-specific T cells may be related to this memory phenotype or other qualities of these cells that make them more responsive to ‘homeostatic cytokines’. Alternately, the tumor-specific T-cell preferential expansion could be the additive or synergistic effect of the ‘empty’ environment combined with encountering their cognate antigen (in tumor-bearing mice). The lack of tumor-specific T-cell proliferation in the presence of cognate antigen but absence of the homeostatic-proliferative signal (i.e. in the ‘full’ recipients—Fig. 2D, 1st panel) suggests synergy between the two signals. Along with these changes in the transferred T cells, we have shown the consequent increased T-cell memory (Fig. 2E) and the induction of tumor protection against even high-dose subcutaneous or systemic tumor challenge (Fig. 2C, 3). This protection is entirely dependent on both the ‘emptiness’ of recipients as well as the potent vaccination of donors. In contrast to studies showing an inherent immune response to murine and human tumors15-17, we have shown that donors with lesser ‘vaccinations’, such as tumor alone or tumor treated with chemotherapy, do not transfer anti-tumor immunity against high-dose tumor challenge (Fig. 4). Of note, Borrello et al. used i.v. tumor-challenged donors and transferred tumor-purged splenocytes into lymphodepleted recipients14,18, whereas our experiments use no in vitro tumor purge. Though their system differs from ours in the degree of tumor protection, both demonstrate the enhanced anti-tumor immunity of T cells transferred into the ‘empty’ recipient. The additional improvement of our immunotransplant maneuver by a vaccine ‘boost’ gives an indication of its mechanism and ultimate clinical application. The proliferative and qualitative changes that transferred T cells undergo in the ‘empty’ recipient are primarily due to increased availability of cytokines such as IL-7 and IL-15 which function independently of TCR stimulation and co-stimulation. Culturing A20 cells with CpG in vitro increases the expression of CD4019, CD80, and CD861. We therefore reasoned that a post-transplant boost with CpG-stimulated A20 should optimally drive proliferation of tumor-specific T cells by simultaneously sending homeostatic proliferative signals along with signals 1 and 2. Our data show that both i.v. and s.c. routes of vaccine boost enhance tumor-specific immunity (Fig. 4 and data not shown) and avoid late recurrences that sometimes occur in the absence of the boost. The clinical benefit of boosting argues for its inclusion in clinical translation of immunotransplant. This is consistent with a clinical trial of immunotransplant of pneumonia vaccine-primed T cells demonstrating that such a boosting approach was necessary for effective T-cell expansion20. Tumor protection in our immunotransplant maneuver demonstrates a strict dependence on the degree of irradiation (Fig. 5B). This is consistent with our previous findings of the strict dependence on ‘emptiness’ for homeostatic proliferation of transferred T cells (data not shown). We hypothesize that it is the ‘emptiness’ of lethally irradiated recipients that induces the changes seen in transferred tumor-specific T-cells as opposed to other effects of recipient irradiation such as microbial translocation12, radiation-induced ‘cytokine storm’21,22, or other radiation-induced immune effects23. We conjecture that those latter types of direct irradiation effects would be greatest shortly after treatment, whereas the homeostatic proliferation induced by emptiness is a time-dependent process, taking weeks




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to maximally increase T cell numbers24. Using lower-dose splenocyte transfer, we demonstrate that immunity does increase over time, allowing for the late elimination of tumors as large as 200mm2 – despite the 300 fold greater cell number compared to the time of tumor challenge (Fig. 5C). Several studies have shown pre-clinical efficacy of immunotherapies against established, but minimal, disease burden18,25-27. Therefore, it is not surprising that translating such approaches into successful clinical trials has been difficult. Our initial results were encouraging enough to study the therapeutic setting of large, established tumors, which differ in several ways from standard-dose tumor challenge. First, in the A20 model, tumors of less than 0.25 mm2 are radio-sensitive and cured by lethal dose TBI (data not shown), whereas tumors of 100mm2 are radio-resistant (Fig. 6C, middle panel). Perhaps the greatest obstacle is that mechanisms of immune evasion change with increasing tumor size. Specifically, Elpek et al. demonstrated in the A20 tumor model that an immunotherapy effective against lower tumor burdens had no effect on larger tumors28. Therefore, the cure of 100% of immunotransplant recipients (Fig. 6C) is in accord with our initial hypothesis: immunotransplant eliminates multiple components of tumor immune-evasion strategies. These results allowed us to test even larger tumors that are more applicable to the clinical situation (Fig. 6D). The superior anti-tumor potency of immunotransplant in curing large tumors could feasibly predict its successful translation into clinical trials. As the in situ vaccine maneuver combining cytotoxic therapy and intra-tumoral CpG has already translated well from the pre-clinical model to objective clinical responses, we are optimistic that immunotransplant will also translate to an improved clinical effect. Authorship Contribution: JDB - designed the research, performed research, analyzed data, wrote the paper MJG - designed the research, performed research, analyzed data, wrote the paper DKC - designed the research, performed research, analyzed data RL - designed the research, analyzed data, wrote the paper. Conflict-of-interest-disclosure: All authors declare no competing financial interests. Correspondence to: Ronald Levy, Division of Oncology, Department of Medicine, Stanford University Medical Center, Center for Clinical Sciences Research Room 1105, 269 Campus Drive, Stanford, CA 94305-5151. Phone: 650-7256452; Fax: 650-7361454; e-mail: [email protected]. Acknowledgements We thank Dr. Shoshana Levy and Eric Berlin for helpful discussion in preparation of this manuscript. This work was supported by grants from the National Institute of Health (CA-34233, CA-33399, CA-49605), and by a Career Development Award from the Lymphoma Research Foundation (J.D.B.). R.L. is an American Cancer Society Clinical Research Professor.




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19. Rieger R, Kipps TJ. CpG oligodeoxynucleotides enhance the capacity of adenovirus-mediated CD154 gene transfer to generate effective B-cell lymphoma vaccines. Cancer Res. 2003;63:4128-4135. 20. Rapoport AP, Stadtmauer EA, Aqui N, et al. Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer. Nat Med. 2005;11:1230-1237. 21. Baron F, Beguin Y. Preemptive cellular immunotherapy after T-cell-depleted allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2002;8:351-359. 22. Ferrara JL. Cytokine dysregulation as a mechanism of graft versus host disease. Curr Opin Immunol. 1993;5:794-799. 23. Hallahan D, Kuchibhotla J, Wyble C. Cell adhesion molecules mediate radiation-induced leukocyte adhesion to the vascular endothelium. Cancer Res. 1996;56:5150-5155. 24. Singh NJ, Schwartz RH. The lymphopenic mouse in immunology: from patron to pariah. Immunity. 2006;25:851-855. 25. Chung Y, Qin H, Kang CY, Kim S, Kwak LW, Dong C. An NKT-mediated autologous vaccine generates CD4 T-cell dependent potent antilymphoma immunity. Blood. 2007;110:2013-2019. 26. Adam C, Mysliwietz J, Mocikat R. Specific targeting of whole lymphoma cells to dendritic cells ex vivo provides a potent antitumor vaccine. J Transl Med. 2007;5:16. 27. Meziane el K, Bhattacharyya T, Armstrong AC, et al. Use of adenoviruses encoding CD40L or IL-2 against B cell lymphoma. Int J Cancer. 2004;111:910-920. 28. Elpek KG, Lacelle C, Singh NP, Yolcu ES, Shirwan H. CD4+CD25+ T regulatory cells dominate multiple immune evasion mechanisms in early but not late phases of tumor development in a B cell lymphoma model. J Immunol. 2007;178:6840-6848.




Figure 1: Treg increase in CpG/CTX vaccinated mice and preferential Teffector proliferation during immunotransplant. (A) Mice received CpG/CTX vaccination as described. (B) On day 15 post-vaccination, donor mice splenocytes were assessed for T-cell subsets by flow cytometry. (C) Splenocytes were taken from wild type donor mice and labeled with CFSE 5μg/ml and injected along with unlabeled bone marrow cells into recipients that received either no (0 cGy) or lethal (900cGy) total body irradiation (TBI). (D) On day 15 post-transplant, splenocytes were taken from 3 recipient mice and separately measured by flow cytometry for CFSE, surface CD4, and intra-cellular foxP3. Data shown are gated on live, CFSE (+), CD4(+) cells. Data shown are representative of the 3 individual recipients.

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Figure 2: Immunotransplant enhances tumor-specific T-cell responses and protects against high dose tumor challenge. (A) Mice received either no vaccine, vaccination with CpG/CTX, or syngeneic bone marrow and CFSE labeled splenocytes from vaccinated donors after either no irradiation (‘full’ recipients) or 900cGy TBI (‘empty’ recipients). On post-transplant day 3, mice (10 per cohort) were challenged with 1x107 A20 cells s.c. (B,D) On day 15 post-transplant, mice were bled and PBMCs tested for tumor-specific IFNγ production. (B) Graphs are gated for CD3(+) lymphocytes and statistics are IFNγ(+)cells as a percentage of all CD44hi cells. (C) Tumor growth curves are composites for each cohort with error bars signifying ±1 S.D. (D) Upper right panel, mean fluorescent intensity (MFI) of IFNγ producing cells related to CFSE dilution. The MFI of IFNγ(+) cells - 5.1 - is lower than that of IFNγ(-), - 12.1. Lower panels, ‘empty’ mice contain more (22.5%) CD3(+)CD44hi cells [defined as in (A)]. than their ‘full’ mice counterparts (1.7%) , (E) On day 45 post-transplant, 9 mice were re-bled and PBMC separately assayed as in (B,D) except that gated are CD8(+) cells, indicated are the proportion of IFNγ(+)cells as a percentage of CD44hi cells.

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Figure 3: Immunotransplant protects against systemic tumor burden. Donor mice received (A) no treatment or (B)CpG/CTX vaccination as described. On day 0 (7 days after the completion of the CpG/CTX vaccine), recipient mice received 9Gy TBI followed by transplant of 5x106 BM cells and splenocytes from donors. Mice were challenged on day 1 post-transplant with 106 A20-LUC cells i.v. and followed clinically and per their bioluminescence. The same representative mice are shown over time. (C) Cohorts of recipient mice (n=10) receiving the same treatments and tumor challenge with 106 wild type A20 cells i.v. were followed for clinical signs of illness and survival.

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Figure 4: Immunotransplant of CD8 T cells is both necessary and sufficient for tumor protection. (A) Donor mice received CpG/CTX vaccination and splenocyte cell subsets were purified from CpG/CTX vaccinated donors using mAb-conjugated ferromagnetic beads to either positively select or deplete specific populations. (B) Resulting populations were gated on live lymphocytes and purity was assessed by flow cytometry. (C) CpG/CTX donor splenocyte subsets were used in immunotransplant as above. Recipient mice received high dose tumor challenge day 3 post-transplant and were followed for bi-dimensional tumor size. Proportions of tumor-free mice are indicated. One mouse in the “CD4+CD8” group died within a week post-transplant, but still had palpable tumor.

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Figure 5: Donor requirements to transfer anti-tumor immunity. (A) Donor mice received no treatment, A20 tumor challenge, A20 tumor challenge followed by CTX, or A20 tumor challenge followed by CTX and intra-tumoral CpG. On day 0 (7 days after the completion of the CpG/CTX vaccine), recipient mice received 9Gy TBI followed by 5x106 BM cells and splenocytes from donors. One group received a simultaneous boost of 106 A20 cells, which were cultured with CpG-1826 at 3 μg/ml for 72 hours, then irradiated (50Gy). On day 3 post-transplant, all cohort of mice received high dose tumor challenge. (B) On day 15, post-transplant/transfer mice were bled and assayed by flow cytometry for tumor-specific CD8 T-cell responses as described. Indicated are the percentage of IFNγ producing CD8(+) live lymphocytes (n=3 per cohort). (C) Cohorts of mice were followed for bi-dimensional tumor size and proportions of tumor-free mice are indicated. One mouse in the “no treatment donor” cohort (first column) showed minimal subcutaneous growth, but manifested systemic disease (with hind-limb paralysis) and was sacrificed on day 30.

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Figure 6: Immunotransplant-induced tumor immunity increases with time and with serial transplants. (A) Donor mice received CpG/CTX vaccination and recipient mice received TBI. (B) Donor splenocytes and bone marrow were transferred to recipients that received either 900cGy, 600cGy, or 300cGy as depicted. (C) Recipient mice received 900cGy TBI followed by 5x106 BM cells and splenocytes from donors at donor:recipient ratios of 1:1, 1:4, or 1:8, as depicted. (B,C) Recipient mice received high dose tumor challenge day 3 post-transplant and were followed for bi-dimensional tumor size. Proportions of tumor-free mice are indicated. (D) Serial Immunotransplant: Cured immunotransplant recipients from experiments conducted as in (A) were subsequently used as donors in a serial immunotransplant. Secondary recipients received either no irradiation or 900cGy TBI as depicted, followed by 5x106 BM cells and splenocytes from cured immunotransplant mice. On day 3 post-(secondary) transplant, recipients were challenged with 1 x 107 A20 cells s.c. and followed for tumor-growth.

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Figure 7: Cure of large tumors by Immunotransplant. (A-E) Donor mice received no treatment or CpG/CTX vaccination as depicted in (A). Recipient mice were challenged with 1x107 A20 cells s.c. 14 days earlier, then received either no irradiation or 900cGy TBI followed by 5x106 BM cells and splenocytes from donors. Un-irradiated mice also received an i.v. boost of A20 cells stimulated with CpG followed by irradiation (‘CpG-A20’ cells) at the time of transplant. (B) On day 15 post-transplant mice were bled and IFNγ producing tumor-specific CD8 T-cells were assayed by flow cytometry, as described (n=3). (C) Cohorts of mice (n=10) were followed for bi-dimensional tumor size and proportions of tumor-free mice are indicated. Three mice from the “no vaccine donor” cohort died within 3 days post-transplant, still with palpable tumor. (D) 400mm2 tumor-bearing mice were used as immunotransplant recipients as in (A-C) from donors receiving no treatment or CpG/CTX vaccination, shown are photographs of the same mice over time. (E) 100mm2 tumor-bearing mice were used as immunotransplant recipients and were sacrificed on day 8 post-transplant. Excised tumors were stained for CD3 and visualized per standard immunoperoxidase protocol. 20X micrographs shown are representative of 10 fields examined.




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Immunotransplant Preferentially Expands Teffector Cells over Tregulatory Cells and Cures Large Lymphoma Tumors