From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Blood First Edition Paper, prepublished online September 18, 2014; DOI 10.1182/blood-2014-04-568956 GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 An autologous leukemia cell vaccine prevents murine acute 2 leukemia relapse after cytarabine treatment 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 John D. Gibbins1,2, Lindsay R. Ancelet1, Robert Weinkove1,3,4, Benjamin. J Compton5, Gavin F. Painter5, Troels R. Petersen1 and Ian F. Hermans1,2 1 Malaghan Institute of Medical Research, PO Box 7060, Wellington 6242, New Zealand 2 School of Biological Sciences, Victoria University of Wellington, PO Box 600 Wellington 6140, New Zealand 3 Capital and Coast District Health Board, Wellington Hospital, Wellington 6021, New Zealand 4 Department of Pathology and Molecular Medicine, University of Otago, Wellington, PO Box 7343, Wellington 6242, New Zealand 5 Ferrier Research Institute, Victoria University of Wellington, PO Box 33-436, Petone 5046, New Zealand Correspondence: Ian F. Hermans, Malaghan Institute of Medical Research, PO Box 7060, Wellington 6242, New Zealand. Phone: +6444996914; Fax: +6444996915; e-mail: [email protected] Short title: Chemotherapy enables potent leukemia immunotherapy Text word count: 4510 Abstract word count: 196 Number of figures: 7 Number of references: 75 Scientific category: Immunobiology Submitted: April 9, 2014 30 31 32 Key points 1 Copyright © 2014 American Society of Hematology From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 A cellular vaccine incorporating the glycolipid α-galactosylceramide prevents 2 relapse of acute leukemia following cytarabine chemotherapy 3 2 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 Abstract 2 Acute leukemias with adverse prognostic features carry a high relapse rate 3 without allogeneic stem cell transplantation (allo-SCT). Allo-SCT has a high 4 morbidity and is precluded for many patients due to advanced age or 5 comorbidities. Post-remission therapies with reduced toxicities are urgently 6 needed. The murine acute leukemia model C1498 was used to study the 7 efficacy of an intravenously administered vaccine consisting of irradiated 8 leukemia cells loaded with the natural killer T (NKT) cell agonist α- 9 galactosylceramide (α-GalCer). Prophylactically, the vaccine was highly 10 effective at preventing leukemia development through the downstream 11 activities of activated NKT cells, which was dependent on splenic langerin+ 12 CD8α+ dendritic cells and lead to stimulation of anti-leukemia CD4+ and CD8+ 13 T cells. However, hosts with established leukemia received no protective 14 benefit from the vaccine, despite inducing NKT cell activation. Established 15 leukemia was associated with increases in regulatory T cells and myeloid- 16 derived suppressor cells, and the leukemic cells themselves were highly 17 suppressive in vitro. Although this suppressive environment impaired both 18 effector arms of the immune response, CD4+ T cell responses were more 19 severely affected. When cytarabine chemotherapy was administered prior to 20 vaccination, all animals in remission post-therapy were protected against 21 rechallenge with viable leukemia cells. 22 3 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 Introduction 2 Induction chemotherapies for acute leukemias typically induce morphologic 3 remission, but without allogeneic stem cell transplantation (allo-SCT), most 4 patients with high-risk genetic features subsequently relapse.1-5 Allo-SCT has 5 a high morbidity and mortality, is costly, and is often precluded by age, co- 6 morbidities or lack of a suitable donor.6-8 There is an unmet need for effective 7 post-remission therapies that do not carry the toxicities and cost of allo-SCT.9 8 9 Relapse of acute leukemia is mediated by a population of blasts that fall 10 below the threshold used to define morphologic remission, but may be 11 detected using sensitive flow cytometric or molecular assays.10-14 In addition 12 to over expressing certain self-antigens,15 leukemic blasts harbor numerous 13 mutations,16 resulting in expression of tumor-specific antigens capable of 14 eliciting autologous CD4+ and CD8+ T cell responses.17 This can potentially be 15 exploited by post-remission immunotherapy.18,19 16 17 The use of irradiated whole leukemia cells in vaccines for post-remission 18 immunotherapy is technically feasible,20 and has the potential to elicit immune 19 responses against multiple leukemia-specific antigens without needing to first 20 define leukemia-specific T cell epitopes or patient tissue type. However, 21 administration of a vaccine without a suitable adjuvant is unlikely to elicit an 22 effective immune response and may lead to tolerance.21,22 The glycolipid α- 23 galactosylceramide (α-GalCer) has recently been shown to be a useful 24 adjuvant for whole tumor cell vaccination by eliciting stimulatory interactions 25 between dendritic cells (DCs) and natural killer T (NKT) cells.23-26 When DCs 4 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 acquire cellular material from irradiated tumor cells that have been treated 2 with α-GalCer, the protein content is presented as peptides via MHC 3 molecules to CD4+ and CD8+ T cells, while the α-GalCer is presented via the 4 MHC-like molecule CD1d to NKT cells. Interactions between DCs and NKT 5 cells promote CD40 signaling, leading to DC activation, which increases their 6 capacity to stimulate peptide-specific T cells.23,24 Significantly, vaccines 7 comprised of irradiated tumor cells pulsed with α-GalCer have been shown to 8 be effective in murine models of hematopoietic malignancies, including acute 9 myeloid leukemia (AML) and acute lymphoid leukemia (ALL),23,27,28 and in 10 other malignancies.25,27,29 11 12 Cancer-associated immunosuppression can present a significant barrier to 13 effective vaccine-based immunotherapy.30 AML generates an 14 immunosuppressive environment,31,32 characterised by impaired DC function33 15 and increased levels of regulatory T cells (Tregs).35-37 It follows that 16 immunotherapy may be most effectively used during morphologic remission 17 after induction chemotherapy. 18 19 Here we investigated the efficacy of a vaccine comprised of irradiated 20 leukemia cells pulsed with α-GalCer in a murine acute leukemia model. While 21 vaccination was capable of eliciting a leukemia-specific T cell response in 22 mice with established disease, the activity was impaired by leukemia- 23 associated immunosuppression. However, when the vaccine was 24 administered to mice in remission after cytarabine chemotherapy, it protected 25 against rechallenge with an increased dose of viable leukemic blasts. These 5 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 findings have implications for the design of clinical trials testing 2 immunotherapies for acute leukemias. 3 6 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY 1 CHEMOTHERAPY ENABLES POTENT LEUKEMIA Materials and Methods 2 3 Animal ethics 4 Inbred C57BL/6 mice were purchased from Jackson Laboratories, Bar Harbor, 5 Maine. Also used were: lang-EGFPDTR and lang-EGFP mice, which express 6 the human diphtheria toxin (DT) receptor and/or enhanced green fluorescent 7 protein (EGFP) under the langerin promoter;38 and FoxP3-GFP mice, that 8 have EGFP inserted into the first coding exon of the Foxp3 gene.39 All animals 9 were bred and housed at the Malaghan Institute of Medical Research 10 Biomedical Research Unit, Wellington, New Zealand. Experiments were 11 approved by the Animal Ethics Committee, Victoria University, Wellington, 12 New Zealand; reference 2012R28M. 13 14 Media and reagents 15 The acute leukemia line C1498,40 (ATCC, Manassas, VA, USA), was cultured 16 in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 5% fetal 17 bovine serum (FBS) (SAFC Bioscience, Auckland, New Zealand), 100 U/mL 18 penicillin/100 g/mL streptomycin, 50 M 2-mercaptoethanol (all from Invitrogen, 19 Auckland, New Zealand). α-GalCer was manufactured by synthesizing a 20 protected phytosphingosine derivative from phytosphingosine (TCI, P1765) as 21 previously described. 41.42 22 23 Leukemia challenge treatment with whole tumor vaccines 24 For leukemia challenge experiments, mice were administered 1 x 105 C1498 25 cells intravenously via the lateral tail vein, unless otherwise stated. To 7 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 generate vaccines, C1498 cells were cultured for 24 hours in IMDM with 200 2 ng/ml of α-GalCer, washed with phosphate-buffered saline (PBS), and γ- 3 irradiated to 150 Gy. Vaccines comprised of of 7.5 x 105 cells were 4 administered intravenously. Mice were monitored for onset of leukemia- 5 associated symptoms, such as weight loss or overt behavioral symptoms 6 (hunching, reduced activity or reduced grooming) and were euthanized 7 following symptom development. Although weight loss was monitored for all 8 mice in symptom-free survival experiments, the onset of symptoms often 9 preceded weight loss in this model. Experiments were conducted with 5-6 10 animals per treatment group. CD4+ or CD8+ T cells were depleted by injection 11 of anti-CD4 antibodies (GK1.5; 125 μg per mouse), or anti-CD8 antibodies 12 (2.43; 250 μg per mouse), respectively, administered five, twelve and 13 nineteen days following vaccination; depletion methods were sufficient to 14 maintain >95% depletion for GK1.5 and >90% for 2.43 over the course of the 15 experiment (Supplementary Figure 1). Anti-CD25 (clone PC61) was used to 16 deplete Tregs, resulting in >95% reduction of CD4+ CD25+ cells 17 (Supplementary Figure 2). Langerin+ DCs were depleted from Lang- 18 EGFPDTR mice by intraperitoneal administration of 350 ng of DT two days 19 before vaccine, resulting in >95% reduction of langerin+ CD8α+ DCs for three 20 days.43,44 In some experiments three doses of 3 mg of cytarbine (Pfizer, 21 Auckland, New Zealand) was administered ten hours apart the day following 22 leukemia challenge. 23 24 Histology 8 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 Femurs were placed in 4% formalin (Sigma-Aldrich, MO, U.S.A), decalcified 2 with 10% formic acid and processed. Paraffin embedded sections were 3 stained with haematoxylin-and-eosin (made in house) and blood smears were 4 stained with Romanowsky stain variant (Siemens Healthcare, Erlangen, 5 Germany). Slides were examined with an Olympus BX51 microscope 6 (Precision Microscopy Equipment Wellington, New Zealand) and captured 7 with an Olympus DP70 (Precision Microscopy Equipment, Wellington, New 8 Zealand) using Cell^F software (Olympus). 9 10 Cytokine production assay 11 Supernatant cytokine levels were measured by cytokine bead array (Biorad 12 Laboratories, Inc, Auckland, New Zealand) following culture with one-million 13 splenocytes and ten-thousand bone marrow-derived DCs (BM-DC) loaded 14 with C1498 lysate for 4 hours. BM-DCs were prepared from syngeneic bone 15 marrow cultured in IL-4 and GM-CSF for six days, followed by 18 hours with 16 100 ng/ml of LPS; lysate from C1498 was added at a ratio of one DC to the 17 equivalent of six tumor cells for the last four hours. 18 19 T cell suppression assay 20 Lymph node preparations from naïve C57BL/6 were stained with 21 carboxyfluorescein succinimidyl ester (CFSE), and cultured for 72 hours with 22 2 µg/mL anti-CD3 (clone 2C11) and 2 µg/mL anti-CD28 (clone 37.51) (both 23 prepared in house) in the presence of purified splenic CD11b+ cells or C1498 24 cells. CFSE dilution on T cells was analyzed by flow cytometry. 25 9 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 Flow cytometry 2 Nonspecific FcR binding was blocked with anti-CD16/32 clone 2.4G2 3 (prepared in house). Dead cells were excluded by staining with propidium 4 iodide (BD Pharmingen, California, USA), 4, 6-diamidino-2-phenylindole 5 (DAPI) (Invitrogen) or LIVE/DEAD® Fixable Blue (Invitrogen). For intracellular 6 staining, cells were restimulated for 20 hours with anti-CD3 and anti-CD28 7 antibodies and 1 µg/ml of monensin was added for the last four hours of 8 incubation. Flow cytometry was performed using a FACSCalibur or LSRII (BD 9 Biosciences) and analyzed using FlowJo software (TreeStar Inc.). Doublet 10 and dead cell exclusion was performed. The antibodies used were: anti-CD3- 11 FITC (145-2C11), CD3-PE-Cy7 (145-2C11) anti-CD4-A488 (GK1.5), anti- 12 CD8-A700 (53-6.7), anti-CD11b biotin (M170), anti-CD86 PE (GL-1) anti- 13 CD44-PerCP-Cy5.5 (IM7), anti-FoxP3-PE (FJK-16s) and anti-IFN-γ-PE-Cy7 14 (XMG1-2), all from eBioscience, Auckland, New Zealand; anti-CD11c-PE-Cy7 15 (N418), anti-CD4-APC (GK1.5), anti-CD8-FITC (53-6.7) anti-CD11c-APC 16 (N418), all from Biolegend, San Diego, USA; anti-CD8-Pacific blue (53-6.7) 17 anti-CD40-biotinylated (3/23), streptavidin-PE-Cy7, all from BD Bioscience. 18 Invariant NKT cells were detected using α-GalCer-loaded CD1d tetramers 19 (NIH Tetramer Core Facility, Atlanta, USA). 20 21 Statistical analyses 22 Bars and error bars depict the mean and standard deviation of the mean. For 23 comparisons of one variable, the Mann–Whitney test was used for unpaired 24 data, the Wilcoxon-matched pairs test for paired data, and a one-way analysis 25 of variance (ANOVA) with a Bonferroni post-test for experiments comparing 10 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 more than two groups. The log-rank test was used to determine significance 2 between Kaplan Meier survival curves. Analysis was performed with Prism 5.0 3 software (GraphPad Software, Inc.); P values of <0.05 were considered 4 significant. 11 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY 1 CHEMOTHERAPY ENABLES POTENT LEUKEMIA Results 2 3 A vaccine comprised of irradiated α-GalCer-pulsed leukemia cells 4 protects against acute leukemia through the activities of CD4+ and CD8+ 5 T cells and langerin+ CD8α+ DCs 6 Mice intravenously challenged with the cell line C1498 developed leukemia, 7 characterized by replacement of normal bone marrow hematopoiesis and 8 leukocytosis with circulating blasts (Figure 1A-C). However, mice vaccinated 9 with irradiated leukemia cells pulsed with the glycolipid adjuvant α-GalCer 10 (leukemia/α-GalCer) seven days before C1498 challenge did not develop 11 leukocytosis (Figure 1C) and no blasts were seen in the peripheral blood 12 smear or by histologic examination of bone marrow (Figure 1A-C). Animals 13 vaccinated with the leukemia/α-GalCer vaccine were protected from leukemia 14 development and remained symptom-free for the duration of the experiment 15 (Figure 1D). Vaccination with irradiated leukemia cells without α-GalCer, or 16 with α-GalCer alone, did not protect hosts from leukemia development (Figure 17 1D). 18 19 To determine the effector cells responsible for vaccine-induced protection 20 against leukemia development, mice were vaccinated and depleted of CD4+ 21 or CD8+ cells two days before C1498 challenge to ensure that the depletion 22 would not interfere with cells potentially important for immune priming. 23 Vaccine-induced protection was reduced following depletion of either CD4+ or 24 CD8+ cells, suggesting that both CD4+ and CD8+ T cells mediated the 25 protection afforded by the vaccine (Figure 1E). 12 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 2 In the spleen, CD8α+ DCs are thought to be responsible for phagocytosing 3 circulating apoptotic cells,21 and a subpopulation of these cells in the marginal 4 zone that express CD103 and langerin have been shown to induce robust 5 cytotoxic T cell responses through efficient CD8+ T cell cross-priming.44-46 To 6 establish whether langerin+ DCs were involved in the protection afforded by 7 the leukemia/α-GalCer vaccine, lang-EGFPDTR hosts were depleted of 8 langerin-expressing cells by DT treatment before vaccination as previously 9 described.44 This led to complete abrogation of the protective effect of 10 leukemia/α-GalCer (Figure 1F), indicating that langerin+ DCs are essential for 11 vaccine efficacy. 12 13 Vaccination is ineffective in the presence of established leukemia 14 despite retaining capacity to stimulate NKT cells and DCs 15 Having shown that prophylactic leukemia/α-GalCer vaccination can elicit an 16 anti-tumor effect, we next determined whether the vaccine could prolong 17 survival in mice with established leukemia. Mice inoculated with C1498 one 18 week before vaccination with leukemia/α-GalCer received no therapeutic 19 benefit, and the onset of symptoms was comparable to non-vaccinated 20 controls (Figure 2A). 21 22 To explore why therapeutic vaccination of mice with established leukemia was 23 ineffective, we investigated the cascade of immune activation involved in α- 24 GalCer-adjuvanted vaccination. Since the adjuvant effect of α-GalCer involves 25 reciprocal interaction and activation of NKT cells and DCs,47-50 we first 13 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 assessed the level of NKT cell activation by analyzing NKT cell number and 2 function in animals vaccinated seven or fourteen days after C1498 challenge. 3 A substantial increase in the number of splenic NKT cells was observed in all 4 vaccinated animals compared with unvaccinated controls, although there was 5 a trend towards reduced accumulation of NKT cells in leukemia-bearing 6 animals (Figure 2B,C). A notable feature of activated NKT cells is the ability to 7 rapidly produce high levels of the cytokines IL-4 and IFN-γ, which can be 8 detected in serum.47 Both cytokines were detected in host serum after 9 vaccination and levels were similar in mice with and without established 10 leukemia (Figure 2D-E). Together these data suggest that the capacity of the 11 vaccine to stimulate NKT cells in established leukemia is largely intact. 12 13 We next determined whether the presence of established leukemia impaired 14 the ability of the vaccine to activate DCs. Lang-EGFP hosts were used to 15 identify langerin+ CD8α+ DCs by flow cytometry. A decrease in the proportion 16 of the langerin+ CD8α+ subset of DCs was found in the spleens of all 17 vaccinated mice regardless of presence of established leukemia, which is 18 consistent with previous reports showing these DCs are depleted in response 19 to NKT cell stimulation (Figure 2F and G).21,45,51 The remaining langerin+ 20 CD8α+ DCs upregulated CD40 and CD86 after vaccination, irrespective of the 21 presence of established leukemia (Figure 2H and I), although the expression 22 of CD86 on langerin+ CD8α+ DCs was reduced in mice with established 23 leukemia. The expression of CD40 and CD86 was similarly upregulated in 24 CD8α+ DCs (Figure 2J and K). 25 14 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 The interaction between DCs and NKT cells involves CD40/CD40 ligand 2 interaction that contributes to significant production of IL-12, primarily from 3 langerin+ CD8α+ DCs.44 Serum IL-12 was analyzed five hours after vaccine 4 administration in the presence or absence of established leukemia, and 5 similar levels were observed (Figure 2L). Together, these results indicate that 6 the inefficacy of the vaccine was not attributed to a failure to activate NKT 7 cells or DCs. 8 9 Vaccine-induced activation of CD4+ T cells is suppressed in the 10 presence of established leukemia 11 To establish whether leukemia suppressed effector T cells, the vaccine- 12 induced T cell response was examined. Splenocytes from animals vaccinated 13 in the presence or absence of established leukemia were cultured with bone 14 marrow-derived DCs loaded with C1498 lysate and levels of IFN-γ in the 15 supernatant were quantified. Splenocytes from vaccinated animals had 16 elevated supernatant IFN-γ following restimulation, suggesting a leukemia 17 antigen-specific immune response had been induced, although overall levels 18 from leukemia-bearing animals were not significantly different from animals 19 without leukemia (Figure 3A). Flow cytometric analysis of splenic CD8+ T cells 20 seven days after vaccination showed that upregulation of the activation 21 marker CD44 and intracellular IFN-γ production were not significantly impaired 22 by the presence of established leukemia (Figure 3C-D). However, established 23 leukemia prevented vaccine-induced CD44 and IFN-γ expression on CD4+ T 24 cells (Figure 3E-F). Therefore, in the established leukemia setting, the 25 induction of leukemia-specific CD4+ T cells was impaired. 15 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 2 Established leukemia is associated with numerous suppressive 3 mechanisms 4 Increased numbers of Tregs are found in patients with acute leukemias, such 5 as AML and ALL.52-57 To determine the involvement of regulatory cells in 6 C1498, the percentage of CD4+ FoxP3+ Tregs was determined by flow 7 cytometry. Mice with established leukemia had a significantly increased 8 percentage of CD4+ FoxP3+ cells in both the liver and spleen (Figure 4A-B). 9 To determine whether alleviation of Treg-related immunosuppression could 10 enhance the therapeutic effect of the leukemia/α-GalCer vaccine, an anti- 11 CD25 antibody was used to deplete Tregs from leukemia-bearing mice before 12 vaccination.58 Mice depleted of Tregs prior to vaccination had increased 13 survival, although all animals ultimately succumbed to leukemia outgrowth 14 (Figure 4C). This was dependent on the vaccine because Treg depletion 15 alone did not delay symptom onset. These results indicate that Tregs are 16 partially responsible for the inefficacy of therapeutic leukemia/α-GalCer 17 vaccination. 18 19 Since myeloid-derived suppressor cells (MDSC) can also potentially 20 contribute to immunosuppression in patients with acute leukemias,52-56 the 21 proportion of MDSCs was assessed in the spleens of leukemic animals. A 22 significantly increased percentage of CD11b+ Ly6G+ cells was observed in 23 animals with established leukemia (Figure 4D-E). These cells could be 24 distinguished from leukemic blasts by CD11b, which is not expressed in 25 C1498 (J.D.G, unpublished data, January 14, 2013). When splenic CD11b+ 16 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 cells were isolated 20 days after leukemia challenge and cultured with CFSE- 2 labeled lymphocytes, the proliferation of CD4+ T cells was significantly 3 reduced relative to cultures containing CD11b+ cells from control mice, 4 suggesting greater suppressive activity on a per cell basis (Figure 4F). 5 Although a trend towards reduced CD8+ T cell proliferation was also observed, 6 this failed to reach significance in all experiments (Figure 4G) (J.D.G, 7 unpublished data, January 14, 2013). 8 9 To analyze whether the C1498 line itself had immunosuppressive capabilities, 10 C1498 cells were cultured with CFSE-labeled lymphocytes and T cell 11 proliferation was compared to cultures containing naïve CD11b+ splenocytes. 12 Proliferation of CD4+ and CD8+ T cells was severely impaired by co-culture 13 with C1498 cells, although suppression of CD4+ T cell proliferation was more 14 pronounced (Figure 4H-I). Overall, this data indicates that there may be 15 several immunosuppressive activities at play in the context of established 16 leukemia. 17 18 Cytarabine pretreatment does not suppress vaccine-induced responses 19 While induction chemotherapy can drastically reduce the tumor cell burden in 20 leukemic patients, potentially providing an opportunity for immunotherapeutic 21 intervention, it can also induce lymphopenia and an expansion of Tregs.59 To 22 determine whether cytarabine chemotherapy could be used successfully in 23 combination with immunotherapy, the effect of cytarabine treatment on T cells 24 was analyzed in mice with acute leukemia. While the percentage of T cells 25 was reduced in the spleens of mice with untreated leukemia, mice treated with 17 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 cytarabine after leukemia challenge had similar proportions of CD4+ and CD8+ 2 T cells compared to naïve controls (Figure 5A-B). Moreover, while expression 3 of the activation marker CD44 on CD8+ and CD4+ T cells was reduced in 4 animals with leukemia, in cytarabine-treated animals expression was similar 5 to that seen in naïve healthy controls (Figure 5C-D), surprisingly suggesting 6 that rather than suppressing endogenous immune responses, cytarabine may 7 be instead restoring the T cell compartment. 8 9 We next wanted to determine the effect of cytarabine pretreatment on the 10 vaccine-induced response. C1498-challenged mice were treated with 11 cytarabine and 20 days later administered the leukemia/α-GalCer vaccine, or 12 left unvaccinated, and the proportions of Tregs and MDSCs was assessed 13 one week following vaccination. Although we observed a reduction in the 14 percentages of Tregs in both the spleen and liver of vaccinated animals 15 compared to untreated leukemic mice, the addition of cytararbine to the 16 treatment regime had no effect (Figure 6A-B). Similarly, there were no 17 changes in the proportions of MDSCs (Figure 6C-D). The ratio of effector 18 CD8+ T cells (CD44hi) to Tregs was increased in both the spleen and liver of 19 vaccinated mice relative to untreated animals and was also unaffected by 20 cytarabine pretreatment. We observed a similar trend in the ratio of effector 21 CD4+ T cells (CD44hi) to Tregs, again establishing that the addition of 22 cytarabine did not impact the vaccine-induced response in leukemic mice 23 (Figure E-H). 24 18 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 The different treatments were associated with significant changes in 2 proportions of T cells in the bone marrow, the major site of leukemia cell 3 accumulation. Vaccinated animals had an increased proportion of CD3+ T 4 cells compared to unvaccinated mice that was unaffected by cytarbine (Figure 5 6I). Interestingly, leukemia challenge was associated with an increase in the 6 percentage of CD8+ T cells expressing the T cell exhaustion marker 7 programmed-death 1 (PD-1) in the bone marrow, which was unchanged 8 following cytarabine treatment alone. However, treatment with cytabarine 9 followed by vaccination resulted in reduced percentages of PD-1-expressing 10 CD8+ T cells compared to untreated C1498-challenged animals, similar to that 11 observed in vaccinated, non-leukemic mice (Figure 6J). Similarly, the lowest 12 percentage of PD-1 expressing CD4+ T cells was observed in mice that 13 received the combination therapy (6K). These results demonstrate that 14 cytarabine pretreatment does not suppress vaccine-induced immune 15 responses and suggests rather, that the therapies may be successfully 16 combined. Importantly, chemotherapy to induce minimal residual disease for 17 acute leukemia may provide a window for immunotherapy. 18 19 20 Vaccination following cytarabine treatment protects against leukemia 21 rechallenge 22 Since cytarbine pretreatment did not negatively impact vaccine-induced 23 immune responses, we next assessed the efficacy of the leukemia/α-GalCer 24 vaccine as a post-remission therapy (Figure 7A). Mice were challenged with 25 C1498 and then treated with cytarabine. After 20 days, one group was 19 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 vaccinated with the leukemia/α-GalCer vaccine. Cytarabine treatment 2 prolonged survival in both groups after C1498 challenge (Figure 7B) as 3 previously reported,61,62 although all animals eventually relapsed 4 (Supplementary Figure 3). Surviving cytarabine treated animals, as well as 5 additional naïve controls, were challenged with an elevated dose of viable 6 C1498 cells to determine whether they had developed protective immunity to 7 acute leukemia. Strikingly, animals that received vaccination after cytarabine 8 chemotherapy had superior protection from C1498 rechallenge, whereas all 9 animals that received chemotherapy alone developed symptoms associated 10 with leukemia progression within 20 days (Figure 7B). Therefore, vaccination 11 provided durable protection against leukemia when administered during 12 remission following cytarabine chemotherapy, suggesting that an α-GalCer- 13 pulsed irradiated leukemia cell vaccine may be a promising post-remission 14 immunotherapy for acute leukemia. 15 16 17 18 19 Discussion 20 There is an unmet need for effective post-remission therapies for acute 21 leukemia that have reduced toxicity and cost compared to allo-SCT. Using the 22 aggressive acute leukemia cell line C1498,63 we show that a simple vaccine 23 comprised of whole irradiated leukemia cells pulsed with the glycolipid 24 adjuvant α-GalCer protected against leukemia development in vivo. Despite 20 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 displaying immunologic activity, the vaccine did not delay disease progression 2 in mice with established disease, at least in part due to leukemia-related 3 immunosuppressive activities. However, in animals that were in remission 4 following cytarabine chemotherapy, the vaccine protected against leukemia 5 rechallenge. 6 7 The glycolipid α-GalCer acts as an adjuvant via a third-party mechanism by 8 binding to CD1d on DCs and recruiting NKT cells to create a stimulatory 9 environment that leads to enhanced peptide-specific responses by 10 conventional CD4+ and CD8+ effector T cells.23,47,64 While CD1d is weakly 11 expressed on C1498 and has been identified in other acute leukemic cell lines, 12 including AML-ETO9a 23, EL4, and WEHI-3B (L.R.A, unpublished data, June 13 14, 2014), we have previously demonstrated that a CD1d-negative α-GalCer- 14 pulsed glioma vaccine can provide protection against glioma challenge. 25 We 15 have also shown that CD1d-deficient DCs can transfer α-GalCer to host 16 resident CD1d-expressing antigen-presenting cells in vivo to induce potent 17 iNKT cell activation, which likely reflects transfer of α-GalCer embedded 18 within membranes of the injected cells.46 It is therefore not necessary for NKT 19 cells to interact directly via CD1d on the cells of the vaccine to provide 20 adjuvant activity. Rather we favor the hypothesis that host DCs acquire α- 21 GalCer from the leukemia cells of the vaccine; these DCs then become 22 licensed by presenting the α-GalCer via CD1d to NKT cells, in turn leading to 23 enhanced induction of peptide-specific CD4+ and CD8+ effector T cells. 24 Consistent with this mechanism, the activity of the leukemia/α-GalCer vaccine 21 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 required langerin-expressing DCs, and involved CD4+ and CD8+ T cells for full 2 efficacy. 3 4 Therapeutic administration of the leukemia/α-GalCer vaccine was unable to 5 prolong survival in mice with established leukemia, despite stimulating NKT 6 cells, and leading to activation of DCs. We identified several mechanisms by 7 which C1498 could suppress immune responses, including increased 8 proportions of Tregs and MDSCs, and a direct suppressive effect of the 9 leukemia cells on T cell proliferation. It is notable that acute leukemias have 10 been reported to produce arginase65 and indolamine 2,3-dioxygenase66 which 11 may be responsible for this direct effect. Our results suggest that leukemia- 12 specific CD4+ T cells play a significant role in the efficacy of the leukemia/α- 13 GalCer vaccine and depletion experiments indicated that CD4+ T cells were of 14 similar importance as CD8+ T cells for vaccine efficacy. In leukemia-bearing 15 mice, IFN-γ production by CD4+, but not CD8+ T cells was impaired. 16 Interestingly, MDSCs from leukemic mice induced a more pronounced 17 suppression of CD4+ T cells compared to those harvested from non-leukemic 18 animals, although we were unable to show a similar reproducible effect on 19 CD8+ T cells. Also, the potent suppression of T cell proliferation by the 20 leukemia cells themselves appeared to be greater on CD4+ T cells than on 21 CD8+ T cells. It is possible therefore that the leukemic environment induces 22 broad CD4+ T cell dysfunction, perhaps including the inability to provide T cell 23 help to CD8+ T cells. In this context, it is notable that vaccine efficacy in the 24 prophylactic setting was dependent on langerin+ CD8α+ DCs, which have 25 been shown to have a potent capacity for stimulating cytotoxic T cells; the 22 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 vaccine may ultimately depend on CD4+ T cell help to mobilize effective 2 cytotoxic T cells through these DCs. However, given that some anti-tumor 3 activity was seen in the absence of CD8+ T cells, other CD4+ T cell functions 4 must be involved, perhaps as effectors in their own right,67 or through 5 interactions with other MHC class II-expressing cells. 6 7 Other groups have also shown that α-GalCer pulsed acute leukemia cells can 8 be used as a prophylactic vaccine. Using the AML cell line AML-ETO9a, 9 Mattarollo et al23 showed that irradiated α-GalCer-pulsed AML cells prevented 10 AML development in the prophylactic setting but only delayed progression 11 when administered therapeutically, and Shimizu et al29 demonstrated effective 12 prophylactic vaccination using the myelomonocytic WEHI3B model. Our 13 experiments extend these findings by elucidating immunosuppressive 14 activities in established leukemia, and indicating that protection against acute 15 leukemia can also be invoked during remission after cytarabine chemotherapy. 16 As this chemotherapeutic agent is in routine clinical use for leukemia induction 17 and consolidation,68,69 these results are of particular relevance clinically.27,29 18 Our results support a previous in vivo study with C1498, in which mice treated 19 with a GM-CSF-secreting irradiated cell vaccine five or seven days after 20 cytarabine treatment were protected against leukemia development, despite 21 transient development of severe neutophenia and lymphopenia following 22 chemotherapy. 60 23 24 We have demonstrated successful combination of immunotherapy following 25 chemotherapy pretreatment of leukemic mice, and have shown that 23 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 cytarabine alone did not induce changes to the immune environment that 2 could be detected by day 20. Given the suppressive effects of C1498, we 3 expected that cytarabine pretreatment would have alleviated some of the 4 leukemia-induced suppression and consequently, more robust immune 5 responses would be detected in mice that received the combined treatment. 6 In contrast to this, we observed little improvement in vaccine-mediated 7 responses following cytarabine. We expect that although the leukemic burden 8 was certainly reduced in cytarabine treated mice, the number of circulating 9 tumor cells may have been substantially increased by time of vaccination, 10 which was delayed until nearly three weeks following chemotherapy. 11 Evidence for this is reported in a study by Lin et al., where aggressive C1498 12 progression was demonstrated by in vivo imaging, and a ten-thousand fold 13 increase in whole body photon counts of leukemic cells was observed within 2 14 weeks of tumor challenge 60. Comparable with our findings, relapse also 15 occurred in mice treated with the same dose of cytarabine we used in this 16 study, despite using half the dose of viable C1498 cells for leukemic challenge. 17 It may be possible that earlier administration of the vaccine following 18 cytarabine treatment could generate more robust immune responses in this 19 group. Since we also found little evidence to support a role for cytarabine in 20 modulating proportions of Tregs and MDSCs, the efficacy against rechallenge 21 we observe when cytarabine is combined with vaccination may simply reflect 22 the capacity of this drug to drastically reduce the burden of leukemia cells, 23 and the pre-existing immunity afforded by the vaccine before rechallenge. 24 24 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY CHEMOTHERAPY ENABLES POTENT LEUKEMIA 1 In comparison to vaccination with defined antigens, an α-GalCer-loaded 2 autologous whole cell vaccine for acute leukemia has the advantages of 3 stimulating both innate and adaptive immunity, and covering a broad range of 4 leukemia-associated antigens without being limited by HLA phenotype.71,72 5 Since leukemic blasts are readily obtained from the blood or bone marrow of 6 patients at diagnosis,73 and α-GalCer has been used safely in early-phase 7 clinical trials,75-77 vaccination with α-GalCer-pulsed irradiated leukemia cells 8 may represent a feasible post-remission immunotherapy. We are aware that 9 other approaches to adjuvanting autologous tumor cell vaccines have been 10 described for hematological malignancies, including toll-like receptor (TLR) 11 ligands.78 While we have not directly compared α-GalCer and TLR ligands in 12 this model, the two approaches are not mutually exclusive and it is possible 13 that these adjuvants may synergize to provide enhanced therapy 71,72. 14 15 In summary, an α-GalCer-adjuvanted whole leukemia cell vaccine that is 16 effective at preventing leukemia development in naïve animals is ineffective in 17 the presence of established leukemia, due to suppressive activities of Tregs, 18 MDSCs and the leukemia cells themselves. However, in the setting of 19 remission after cytarabine treatment, the vaccination leads to durable 20 protection against subsequent leukemia rechallenge. We suggest that post- 21 induction immunotherapy with an autologous irradiated leukemia cell vaccine 22 adjuvanted with α-GalCer may prove a useful strategy for prevention of high- 23 risk acute leukemias. 24 25 25 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 CHEMOTHERAPY ENABLES POTENT LEUKEMIA Acknowledgements This research is supported by the Marsden Fund Council from Government funding, administered by the Royal Society of New Zealand, and by the New Zealand Ministry of Science and Innovation (C08X0808). We acknowledge the NIH Tetramer Core Facility (contract HHSN272201300006C) for provision of CD1d tetramers and thank Chingwen Tang and Taryn Osmond for their technical assistance. Contribution: J.D.G., L.R.A., R.W., T.R.P. and I.F.H. designed the research; J.D.G., L.R.A., R.W., and I.F.H. analyzed and interpreted the data and wrote the manuscript; J.D.G. and L.R.A. conducted the experiments; G.F.P. and B.J.C. synthesized α-GalCer. Conflict-of-interest disclosure: The authors declare no competing financial interests. 26 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 CHEMOTHERAPY ENABLES POTENT LEUKEMIA References 1. Magenau J, Couriel DR. Hematopoietic stem cell transplantation for acute myeloid leukemia: to whom, when, and how. Curr Oncol Rep 2013; 15(5): 436-44. 2. Terwijn M, van Putten WLJ, Kelder A, et al. High prognostic impact of flow cytometric minimal residual disease detection in acute myeloid leukemia: data from the HOVON/SAKK AML 42A study. J Clin Oncol 2013; 31(31): 3889-97. 3. 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Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines. Plos One 2011; 6(3): e17657. 47. Hermans IF, Silk JD, Gileadi U, et al. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J Immunol 2003; 171(10): 5140-7. 48. Silk JD, Hermans IF, Gileadi U, et al. Utilizing the adjuvant properties of CD1d-dependent NK T cells in T cell-mediated immunotherapy. J Clin Invest 2004; 114(12): 1800-11. 49. Fujii S-I, Liu K, Smith C, Bonito AJ, Steinman RM. The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation. The Journal of experimental medicine 2004; 199(12): 1607-18. 50. Liu K, Idoyaga J, Charalambous A, et al. Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells. 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GIBBINS et al IMMUNOTHERAPY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 CHEMOTHERAPY ENABLES POTENT LEUKEMIA Figure 1) An α-GalCer-pulsed whole leukemia cell vaccine protects against acute leukemia and is dependent on CD4+ and CD8+ T cells and langerin+ CD8α+ DCs. A vaccine comprising α-GalCer-pulsed irradiated C1498 cells was administered intravenously seven days before C1498 challenge. (A) Bone marrow histology, (B) peripheral blood smear and (C) peripheral white blood cell counts were performed at symptom onset in unvaccinated animals, 40 days after C1498 challenge in vaccinated animals, and in naïve control mice. (D) Kaplan Meier plot showing symptom-free survival of vaccinated and unvaccinated mice after leukemia administration on day zero. Symbols represent treatments: unvaccinated ( ), vaccinated with α-GalCer-pulsed irradiated leukemia cells ( ), vaccinated with unpulsed irradiated leukemia cells ( ), vaccinated with free α-GalCer ( ). Statistical analysis compares unvaccinated and vaccinated with α-GalCer-pulsed irradiated leukemia cells groups. (E) Kaplan Meier plots showing symptom-free survival of mice vaccinated with leukemia/α-GalCer and challenged with C1498 at day zero. Symbols represent treatments: unvaccinated ( ), vaccinated with α-GalCer-pulsed irradiated leukemia cells ( ), depletion of CD4+ cells ( ) or depletion of CD8+ cells (). Statistical analyses compares the depletion groups to mice vaccinated with αGalCer-pulsed irradiated leukemia cells. (F) Lang-EGFPDTR mice were prophylactically vaccinated and one group was administered DT. Symbols represent treatment groups: unvaccinated ( ), prophylactic α-GalCer vaccination ( ), prophylactic vaccination and DT treatment (). Symptom-free survival was analyzed and is graphed. *P<0.05 (one-way ANOVA with a Bonferroni post test). Figure A-C represents a single experiment, Figure D represents three experiments and Figures E-F represent two experiments; five mice per group were used for each experiment. **P<.01 (Mantel-Cox log-rank test). *P<.05, ***P<.001 (Mantel-Cox log-rank test). Figure 2) Leukemia/α-GalCer vaccination is ineffective in the presence of established acute leukemia despite NKT cell and DC activation. Mice were challenged with C1498 cells i.v. one week before vaccination with α-GalCer-pulsed or unpulsed irradiated leukemia cells. (A) Kaplan Meier graph showing survival of mice. Symbols represent treatment groups: unvaccinated ( ), therapeutic α-GalCer vaccination ( ), treatment with unpulsed irradiated leukemia cells ( ). This graph represents three experiments, each with five mice per group. (B-E) Mice were inoculated with C1498 cells seven or fourteen days before vaccination with irradiated α-GalCer-pulsed leukemia cells. (B) Representative flow cytometry plots showing 33 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 CHEMOTHERAPY ENABLES POTENT LEUKEMIA identification of splenic NKT cells (CD3+ α-GalCer–loaded CD1d tetramer+). (C) Frequency of splenic NKT cells following vaccination in mice with and without established acute leukemia. (D) Serum IL-4 levels and (E) serum IFN-γ levels two hours after vaccination (F-G) The splenic langerin+ CD8α+ DC population in LangEGFP mice was analyzed 24 hours following vaccination. (F) Representative flow cytometry plots showing identification of splenic langerin+ CD8α+ DCs (CD11c+ GFP+). (G) Frequency of splenic langerin+ CD8α+ DCs. (H-I) The expression of CD40 and CD86 on langerin+ CD8α+ DCs respectively. (J-K) The CD8α+ DC population in C57BL/6 mice was analyzed 24 hours following vaccination. The expression of CD40 and CD86 on CD8α+ DCs, respectively. (L) Serum IL-12p70 was quantified five hours following vaccination. These results are indicative of two independent experiments, each with five mice per group. **P<.01, ***P<.001 (one-way ANOVA with a Bonferroni post test). Figure 3) Established leukemia disrupts leukemia/α-GalCer vaccine-mediated CD4+ T cell function. Mice were challenged with C1498 i.v. seven days before vaccination and responses were analyzed one week later. (A) Splenocytes were cultured for 24 hours with () or without () DCs loaded with C1498 lysate. Supernatant IFN-γ was quantified. (B-F) The splenic CD4+ and CD8+ T cell populations were analyzed by flow cytometry. (B) Representative flow cytometry plots showing identification of CD4+ and CD8+ T cells from mouse spleens. IFN-γ+ cells were determined by comparison to an isotype control antibody (lower left). (C, E) MFI of CD44 expressed on CD8+ and CD4+ T cells respectively. (D, F) The proportion of CD8+ and CD4+ cells producing IFN-γ respectively. Panel A represents three experiments. Statistical analysis compares experiments performed in the presence of DCs only (one-way ANOVA, Bonferroni post test) and panels B-F represent two experiments, each with five mice per group. *P<.05, **P<.01, ***P<.001 (one-way ANOVA with a Bonferroni post test). Figure 4) Elevated immune suppression in hosts with established acute leukemia. (A-B) Mice were challenged with C1498 i.v. and the immune response in the liver and spleen was analyzed 20 days later. (A) Flow cytometric identification of Tregs. (B) Percentage of Tregs in the spleens and livers of naïve and leukemiachallenged mice. (C) Symptom-free survival of mice challenged C1498 and vaccinated seven days later. One group was depleted of Tregs the day after 34 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 CHEMOTHERAPY ENABLES POTENT LEUKEMIA leukemia challenge. Symbols represent treatment groups: unvaccinated ( ), therapeutic α-GalCer vaccination ( ), therapeutic α-GalCer vaccination plus PC61 ( ), irradiated C1498 cells plus PC61 (). Statistical analysis compares therapeutic α-GalCer vaccination plus PC61 to therapeutic α-GalCer vaccination. (D-G) mice were challenged C1498 i.v. and euthanized 20 days later. (D) Flow cytometric identification of splenic CD11b+ Ly6G+ MDSCs. (E) The proportion of MDSCs within live splenocytes. (F,G) Splenic CD11b+ cells were isolated from naïve or C1498 challenged mice and cultured with CFSE labeled lymphocytes and anti-CD3 and antiCD28. A representative histogram of CFSE dilution of CD4+ cells (F) and CD8+ cells (G) incubated with CD11b cells from naïve mice (black line) or C1498 challenged mice (dotted line); unstimulated (shaded) and graph of reduced CFSE. (H-I) CFSE labeled lymph node cells stimulated with anti-CD3 and anti-CD28 were cultured with naïve splenocytes or C1498 cells for 72 hours. A representative histogram of CFSE dilution of CD4+ (H) and CD8+ (I) T cells culted with naïve splenocytes (black line) or C1498 cells (dotted line); unstimulated (shaded). The percent divided of CD4+ cells (H) or CD8+ cells (I). This figure represents three experiments, each with five mice per group. Panels B, and E-H *P<.05, **P<.01 (t-test with Mann Whitney). Panel C *P<.05 (log-rank Mantel-Cox test). Figure 5) Chemotherapy restores the T cell compartment in leukemic mice. Mice were challenged with C1498 cells and 24 hours three 3 mg doses of cytarabine were administered i.p. ten hours apart. (A-D) The T cell populations in the spleen were analyzed and identified as CD3+ expressing either CD8 or CD4. The proportion of live cells expressing CD3 and CD8 (A) or CD3 and CD4 (B). The MFI of CD44 CD8+ cells (C) and CD4+ cells (D). A-D represents three experiments, with five mice per group *P<.05, **P<.01 (one-way ANOVA with a Bonferroni post test). Figure 6) Cytarabine pretreatment does not suppress vaccine-induced immune responses. Mice were challenged with C1498 cells and 24 hours three 3 mg doses of cytarabine were administered i.p. ten hours apart. On day 23 one group of chemotherapy treated mice was vaccinated with leukemia/α-GalCer and responses were analyzed one week later. The percentage of CD4+ FoxP3+ cells of CD3+ in the spleen (A) and liver (B). The percentage of CD11b+ Ly6G+ of CD3- in the spleen (C) and liver (D). The ratio of CD44hi CD8+ effector T cells to CD4+ FoxP3+ Tregs and ratio of CD44hi CD4+ effector T cells to CD4+ FoxP3+ Tregs in the spleen (E, G) and liver (F,H), respectively. (I) The percentage of CD3+ T cells in the bone marrow. The 35 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. GIBBINS et al IMMUNOTHERAPY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 CHEMOTHERAPY ENABLES POTENT LEUKEMIA percentage of PD-1+ CD8+ and CD4+ T cells in the bone marrow (J, K), respectively. Graphs represent one experiment, with five-six mice per group *P<.05, **P<.01, ***P<.001 (one-way ANOVA with a Bonferroni post test). Figure 7) Vaccination following chemotherapy protects against leukemic rechallenge. (A-B) Mice were challenged with C1498 cells and 24 hours three 3 mg doses of cytarabine were administered i.p. ten hours apart. On day 23 one group of chemotherapy treated mice were administered the leukemia/α-GalCer vaccine and symptom free survival was monitored. (A,C) Surviving mice were rechallenged on day 45 with a five-fold increased dose of 5 x 105 viable C1498 cells and symptomfree survival was again followed. Symbols represent treatment groups: leukemia only (), n = 10, cytarabine treatment ( ) n =19, cytarabine plus α-GalCer vaccination () n = 20. B-C represent two experiments *P<.05, **P<.01 (one-way ANOVA with a Bonferroni post test). P<.0001 (Log-rank Mantel-Cox Test). 15 36 Figure 1 A Naive C1498 alone Vaccine + C1498 25 μ μM Naive C1498 alone Vaccine + C1498 E * 60 * 40 20 0 C1498 Vaccine - + - 80 60 40 * 20 * 50 Time (days) ** 80 60 40 20 0 0 10 100 20 30 40 50 40 50 Time (days) F 0 100 + + 100 0 Symptom Free Survival (%) D 80 Symptom Free Survival (%) C White blood cells per litre of blood (x109) 50 μ μM Symptom Free Survival (%) B 100 *** 80 60 40 20 0 0 10 20 30 Time (days) Figure 2 80 85.2 CD1d tet FSC-A 20 40 60 80 1000 500 1500 200 + I *** + + + *** 4000 L *** 2000 500 1000 0 C1498 14 days + Vaccine + + + K J 3000 *** *** ** 2000 0 C1498 7 days C1498 14 days + Vaccine + + + 4000 1000 + + + + + 0 C1498 7 days C1498 14 days + Vaccine 4000 ** *** ** 3000 2000 0 C1498 7 days C1498 14 days + Vaccine 0 6000 2000 5000 1000 C1498 14 days + Vaccine - + + + 15 + + + + + + + ** 10 5 C1498 14 days + Vaccine + + 3000 1000 0.354 Langerin FSC-A C1498 7 days C1498 14 days + Vaccine + + + + 58.3 IL-12 pg/ml CD40 MFI on Lang+ CD11c+ Cells C1498 7 days C1498 14 days + Vaccine 400 + + + + 600 0 0 + G SSC-A 1500 0 F *** 800 IFN-y (pg/ml) ** E *** CD86 MFI on Lang+ CD11c+ Cells 2000 ** 5 C1498 7 days C1498 14 days + Vaccine 100 MFI of CD86 on CD8+ CD11c+ Cells IL-4 pg/ml 2500 10 CD11c 0 D CD40 MFI on CD8+ CD11c+ Cells ** Percent of CD11c+ Cells Expressing Langerin 20 CD3 2.53 40 0 *** 20 From www.bloodjournal.org by guest on February 4, 2015. For15personal use only. 60 Time (days) H Percent of CD3+ cells that are tetramer positive C B 100 SSC-A Symptom Free Survival (%) A + + + Figure 3 C 600 400 400 200 200 0 C1498 Vaccine E * 800 600 *** **2015. For personal use only. From www.bloodjournal.org by guest on February 4, ** MFI of CD44 on CD3+ CD4+ cells IFN-y (pg/ml) 800 MFI of CD44 on CD3+ CD8+ cells A + B C1498 Vaccine + + *** 600 400 200 0 0 + - *** + - + C1498 Vaccine + + + - + + + D CD8 CD3 SSC-A 87.9 FSC-H FSC-A CD4 23.3 CD8 * 30 20 10 C1498 Vaccine IFN-γ IFN-γ 0.332 CD8 51 F 40 0 + - + + + Percent of CD4+ cells that are IFN- + 39 Percent of CD8+ cells that are IFN- + 33.6 C1498 Vaccine 4 *** ** 3 2 1 0 + - + + + ** 50 C ** Symptom Free Survival (%) B Symptom Free Survival (%) A Percent of CD4+ cells that are FoxP3+ Figure 4 100 FoxP3 20 10 0 Naive C1498 Naive SSC-A CD11b E FSC-A 100 Percent divided of CD4+ cells % of Max 60 40 20 *** 20 0 0 10 Spleen H ** 20 30 Time (days) 1.0 0.5 60 40 20 Naive 0 101 AML 40 30 20 102 103 104 G 103 104 105 60 40 0 0 CFSE C1498 Naive C1498 100 Percent divided of CD8+ cells 100 80 % of Max Naive 60 40 20 0 0 2 10 3 10 CFSE 4 10 10 5 95 90 85 20 0 0 20 0 10 1 10 2 10 3 CFSE 10 4 10 5 40 ** 40 20 Naive C1498 * 100 80 20 40 60 0 105 100 10 ** 60 80 CFSE I 80 Time (days) 0 0.0 0 0 102 40 100 100 100 80 1.5 * 50 80 40 % of Max F Ly6G 2.0 60 % of Max D Percent of live cells that are CD11b+ Ly6G+ Liver C1498 80 Percent divided of CD4+ cells FSC-A 30 Percent divided of CD8+ cells CD4 SSC-A 40 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. 80 60 40 20 0 Naive C1498 60 ** ** 10 5 0 C1498 Cytarabine - + - ** 2000 MFI of CD44 on CD3+ CD8+ cells C + + ** *** 15 10 5 0 AML Cytarabine D * 1500 - + - ** 3000 + + * 2000 1000 1000 500 0 AML Cytarabine Percent of live cells that are CD3+ CD4+ cells B From www.bloodjournal.org by guest 20 on February 4, 2015. For personal use only. 15 MFI of CD44 on CD3+ CD4+ cells A Percent of live cells that are CD3+ CD8+ cells Figure 5 0 - + - + + AML Cytarabine - + - + + Figure 6 4 2 0 + - + + - + + 2 N.S. 1 0 C1498 Vaccine Cytarabine + + + Percent of CD3+ T cells N.S. 6 C1498 Vaccine Cytarabine + - + + + + - + + + N.S. 2 1 0 + - + + + + - J 20 N.S. 15 10 5 0 + - + + - + + + + + 10 5 - + - + + + - + + - H N.S. *** 15 10 Ratio of C1498 Vaccine Cytarabine 5 0 - + - + + + - + + - + + + K ** N.S. 20 10 2 0 + - - + + + - + + - + + + *** 30 20 10 - + - + + + - + + - + + + ** * 60 N.S. 40 20 0 - + - + + + - + + - *** **** 200 + + + N.S. 150 100 50 C1498 Vaccine Cytarabine N.S. N.S. * 40 0 C1498 Vaccine Cytarabine + + + *** 30 0 CD4+ eff/Tregs N.S. 15 C1498 Vaccine Cytarabine G *** F Ratio of CD8+ eff/Tregs 20 Ratio of CD4+ eff/Tregs Ratio of CD8+ eff/Tregs E 4 C1498 Vaccine Cytarabine * ** ** * 0 C1498 Vaccine Cytarabine + + + N.S. ** *** Percent of CD3- cells that are CD11b+ Ly6G+ 3 C1498 Vaccine Cytarabine * 6 C1498 Vaccine Cytarabine D Percent of CD3- cells that are CD11b+ Ly6G+ C I use only. B by guest on February 4, 2015. For personal ** From www.bloodjournal.org * * * 3 8 Percent PD-1+ of CD8+ T cells 8 * N.S. Percent PD-1+ of CD4+ T cells Percent of CD3+ that are FoxP3+ CD4+ T cells A Liver Spleen Percent of CD3+ that are CD4+ FoxP3+ cells N.S. 0 - + - + + + - + + - + + + C1498 Vaccine Cytarabine - + - + + + - + + - + + + Figure 7 A Cytarabine Vaccination From www.bloodjournal.org byC1498 guestrechallenge on February 4, 2015. For personal use only. Monitor Symptom-free Survival C **** 100 80 60 40 20 0 0 10 20 30 Time (days) 40 50 Symptom Free Survival (%) B Symptom Free Survival (%) C1498 challenge 100 80 **** 60 40 20 0 0 20 40 Time (days) 60 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Prepublished online September 18, 2014; doi:10.1182/blood-2014-04-568956 An autologous leukemia cell vaccine prevents murine acute leukemia relapse after cytarabine treatment John D. Gibbins, Lindsay R. Ancelet, Robert Weinkove, Benjamin J. Compton, Gavin F. Painter, Troels R. Petersen and Ian F. 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