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induce disease regression more selectively while providing durable protection from relapse through the establishment of memory responses.
We have developed a personalized tumor vaccine in which patient-derived tumor cells are fused with autolo- gous dendritic cells (DC) such that a broad array of tumor- derived antigens, including neo-antigens, is presented in the context of DC-mediated co-stimulation, thereby effectively capturing tumor heterogeneity.4,5 In a phase I/II clinical trial, vaccination of AML patients who achieved chemotherapy- induced remission induced durable expansion of leukemia- specific T cells in the peripheral blood and bone marrow. Remarkably, despite the vaccinated patients having a medi- an age of 63 years, 71% of them remained in remission at a median of 5 years of follow-up.6 These results were in stark contrast to historical data suggesting a 3-year progression free survival of 10-15% in this age group.7
A potential challenge to therapeutic efficacy of active vac- cination is dysfunction of the T-cell repertoire, character- ized by upregulation of pathways that promote exhaustion and senescence, particularly in the microenvironment of advanced disease.8 A transformative advance in the field of immunotherapy was the finding that therapeutic blockade of the programmed death 1 (PD-1)/programmed death lig- and 1 (PD-L1)-negative co-stimulatory pathway resulted in dramatic disease response in a subset of solid tumors, such as melanoma, characterized by a high mutational burden and the presence of neo-antigens and an associated intrinsic T-cell response.9 In contrast, checkpoint blockade has shown minimal therapeutic efficacy in patients with hema- tologic malignancies such as AML,10 potentially because of the relatively low mutational burden and lack of a signifi- cant population of tumor-reactive lymphocytes within the tumor microenvironment.
We postulated that a combination of vaccine and check- point inhibitor therapy would demonstrate unique synergy in which vaccination would provide functionally compe- tent leukemia-specific T-cell populations while the intro- duction of checkpoint blockade would enhance the effec- tiveness and persistence of these cells. Because an exhaust- ed T-cell phenotype can be due to several immunoinhibito- ry signals working in concert,11 we hypothesize that simul- taneous checkpoint blockade may be advantageous.
In the present study in an immunocompetent murine model of aggressive leukemia, we interrogated the immunological response to and therapeutic efficacy of fusion cell vaccination in conjunction with blockade of neg- ative co-stimulatory pathways using antibodies targeting PD-1 and T-cell immunoglobulin and mucin domain-con- taining protein 3 (TIM3), critical mediators of the immune suppressive milieu of the bone marrow.12-14 We also targeted repulsive guidance molecule b (RGMb), a co-receptor for bone morphogenetic proteins which play a role in the maintenance of hematopoietic progenitors including sup- port for AML cells in the marrow niche15,16 and may also mediate immune tolerance via binding to PD-L2 on myeloid cells.17
We demonstrated that the combination of a DC/AML vaccine and checkpoint blockade was uniquely effective in preventing disease progression and inducing a memory response as manifested by protection from tumor re-chal- lenge. Vaccination followed by checkpoint blockade result- ed in upregulation of genes regulating activation and prolif- eration of memory and effector T cells as well as enhanced T-cell clonal diversity.
Vaccine overcomes checkpoint inhibitor limitation
Methods
Cell lines
The murine AML cell line TIB-49 was purchased from the American Type Culture Collection. For all experiments, cell lines were transduced with luciferase/Mcherry using a lentiviral vector (pCDH-EF-eFFLy-T2A-mCherry). Murine LSK cells were obtained from transgenic C57BL/6J mice expressing mIDH2 (IDH2R140Q) and subsequently transduced with Hoxa9-GFP and Meis1a-YFP oncogenes, as previously described.18 Further details are provided in the Online Supplementary Methods.
Vaccination with dendritic cell/acute myeloid leukemia fusions and/or treatment with checkpoint inhibitors
in vivo
All animal studies were approved by the institutional animal care and use committee of Beth Israel Deaconess Medical Center. Murine syngeneic DC/AML fusion cells were generated as previ- ously described.19 C57BL/6J mice were inoculated retro-orbitally with 5×104 luciferase/mCherry TIB-49 murine leukemia cells (lmTIB) or 20×104 mutant IDH2/ Hoxa9-GFP/Meis1a-YFP primary AML cells using tail vein injections. Cohorts of mice were assigned to treatment with 100×103 DC/AML fusion cells via subcutaneous injection 24 h after AML challenge; intraperitoneally with 200 mg each of rat anti-mouse PD-1 (29F.1A10) rat IgG2a,k (BioXCell, NH, USA); mouse anti-mouse TIM3 (T3A.1A10) mIgG1,k; rat anti- mouse RGMb (307.9D1) rat IgG2a,k or all three monoclonal anti- bodies starting 4 days after AML challenge and continued every 3 days for six doses; or the combination of DC/AML fusion vaccine and monoclonal antibody treatment. Further details are provided in the Online Supplementary Methods.
Assessment of leukemia-specific immunity
On day 14-17 following tumor challenge, peripheral blood or spleen-derived T cells were isolated from the treated animals and exposed to either syngeneic TIB-49 tumor lysate or primary mutant IDH2 tumor lysate for 3 days. Intracellular interferon- gamma (IFN-γ) expression was then evaluated by intracellular flow cytometric analysis as a measure of leukemia-specific recognition.
To assess antigen-specific anti-tumor immunity the splenocytes underwent flow cytometric analysis using H-2 Db pentamers. Cells were stained with anti-CD8APC-Cy7 and murine survivi- specific APC-conjugated pentamers ATFKNWPFL (ProImmune, Inc; Sarasota, FL, USA). Cytomegalovirus-specific PE-conjugated pentamers HGIRNASFI (ProImmune, Inc; Sarasota, FL, USA) were used as the control. The percentage of pentamer-positive CD8 T cells was assessed using multichannel flow cytometry.
Analysis of T-cell receptor diversity
Targeted T-cell receptor (TCR) diversity was interrogated using a SMARTer human α/b profiling kit (Takara, CA, USA). Initially total RNA was extracted and purified from mouse blood using an RNeasy mini kit (Qiagen, Germantown, MD).
The quality of the sequencing data was checked in order to remove low-quality reads and then the data were aligned against TCR sequences from the GenBank and IMGT databases20 using MiXCR software.21 Further details are provided in the Online Supplementary Methods.
Single-cell RNA sequencing
Single-cell RNA-sequencing was performed on peripheral blood mononuclear cells isolated from control or treated mice. The 10X Genomics Chromium Controller system21 was employed to cap- ture single cells in the context of uniquely barcoded primer beads together in tiny droplets enabling large-scale parallel single-cell
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