Immune surveillance is a critical component of the antitumor response in vivo, yet the specific components of the immune system involved in this regulatory response remain unclear. In this study, we demonstrate that autoantibodies can mitigate tumor growth in vitro and in vivo. We generated two cancer cell lines, embryonal carcinoma and glioblastoma cell lines, from monkey-induced pluripotent stem cells (iPSC) carrying a homozygous haplotype of major histocompatibility complex (MHC, Mafa in Macaca fascicularis). To establish a monkey cancer model, we transplanted these cells into monkeys carrying the matched Mafa haplotype in one of the chromosomes. Neither Mafa-homozygous cancer cell line grew in monkeys carrying the matched Mafa haplotype heterozygously. We detected in the plasma of these monkeys an IgG autoantibody against GRP94, a heat shock protein. Injection of the plasma prevented growth of the tumor cells in immunodeficient mice, whereas plasma IgG depleted of GRP94 IgG exhibited reduced killing activity against cancer cells in vitro. These results indicate that humoral immunity, including autoantibodies against GRP94, plays a role in cancer immune surveillance. Cancer Res; 77(21); 6001–10. ©2017 AACR.

The establishment of an experimental monkey model of cancer is important to develop new treatments for cancer. Cynomolgus macaques are genetically closer to humans than are mice, and the structures of biologically relevant molecules in monkeys and the organization of the hematopoietic system in macaques are similar to those in humans. Indeed, more than half of the antibodies against human molecules react to molecules of cynomolgus monkeys (1, 2). Furthermore, recent genomic studies have revealed that several monkey cytochrome P450s, which work in drug metabolism, are apparently orthologous to human P450s (3). Therefore, a cancer model in cynomolgus macaques may be more useful than a cancer model in mice for preclinical experiments (4, 5). However, spontaneous neoplasms and malignant tumors in cynomolgus monkeys are uncommon (6). In addition, no cancer model in cynomolgus monkeys has been established by transplantation of tumor cell lines as in mouse cancer models because there is no inbred line in cynomolgus monkeys. To solve this problem, we established major histocompatibility complex (MHC) homozygous tumor cell lines of cynomolgus macaques for transplantation to MHC-matched heterozygous monkeys.

We established induced pluripotent stem cells (iPSC) from fibroblasts of a cynomolgus macaque carrying homozygous MHC genes by introduction of the four Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc; refs. 7, 8) and colonies of cynomolgus macaques carrying the identical MHC haplotype heterozygously (7, 9). From an immunological aspect, cells derived from MHC homozygous iPSCs are more acceptable than MHC-mismatched cells by hosts carrying identical MHC genes. This strategy enables the establishment of various types of tumor models transplantable to cynomolgus macaques as various somatic cells have been developed from iPSCs including neural cells, hematopoietic cells, and pancreatic cells (10–14).

We established cancer cell lines from iPSCs of a cynomolgus macaque carrying a homozygous MHC haplotype; however, MHC-matched hosts rejected these cell lines after transplantation. We found antibodies reacting to the cancer cells in plasma of the macaques that rejected transplanted cells. The plasma also reacted to other immortalized cells transduced with oncogenes that were not transplanted. The IgG in plasma recognized glucose-regulated protein (GRP) 94, a kind of heat shock proteins that is considered as a target of tumor therapy. GRP94 is a common antigen of oncogene-transduced cells that we made in the present study. Cytotoxic T cells are thought to be the main effector of tumor rejection, while B-cell responses including anticancer antibodies in the cancer-immune surveillance have not attracted attention (15–19). In the present study, we found that not only T cells and natural killer (NK) cells but also B cells producing autoantibodies against GRP94 play a role in cancer immune surveillance in the nonhuman primate model.

Culture of iPSCs

iPSCs were established from skin fibroblasts of a 3-year-old female MHC homozygous cynomolgus macaque (9) by transducing human OCT3/4, SOX2, KLF4, and c-MYC genes by a lentivirus according to a previously reported method (7, 8).

iPSCs were cultured with mouse embryonic fibroblasts (MEF; ReproCELL #RCHEFC003) as feeder cells in Primate ES Medium (ReproCELL #RCHEMD001) supplemented with 4 ng/mL of recombinant human basic fibroblast growth factor (bFGF; WAKO #068-05384) on a dish coated with 0.1% gelatin (Sigma-Aldrich #G1890) at 37°C in a 5% CO2 atmosphere.

Embryoid body formation and differentiation of iPSCs

For the generation of CD34+ CD45+ hematopoietic stem and/or progenitor cells, mesodermal embryoid bodies made in a low attachment plate, AggrewellT400 (STEMCELL Technologies #27945), added to which 100 ng/mL of bone morphogenetic protein 4 (BMP4; PeproTech #120-05) were cultured with a mouse mesenchymal stem cell line, C3H 10T1/2, as feeder cells in the presence of vascular endothelial growth factor (VEGF, 50 ng/mL; PeproTech #100-20) and stem cell factor (SCF, 100 ng/mL; PeproTech #250-03) for 14 to 18 days (10, 11).

NPCs were induced from iPSCs as previously reported (12, 13). Briefly, embryoid bodies were cultured on plates coated with Matrigel in 20% fetal bovine serum (FBS)-containing Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Sigma-Aldrich #D6421) supplemented with bFGF (20 ng/mL, WAKO), SB431542 (5 μmol/L, Sigma-Aldrich #4317) and dorsomorphin (5 μmol/L, Sigma-Aldrich #P5499).

Retroviral vectors

Retroviral vector plasmids except for the constitutive active form of RAS (HRASV12) and human papilloma virus type-16 (HPV-16) E6/E7 genes were previously reported (Supplementary Table S1; refs. 20–22). pBABE puro-RASV12 retroviral vector was purchased from Cell Biolabs (#RTV-101). pZip-NeoSV(X)1 E6/E7-encoded human HPV-16 E6/E7 proteins were kindly provided by Dr. Tohru Kiyono (University Tokyo, Tokyo, Japan). We established individual retroviruses using amphotropic retrovirus-packaging cells, PA317 cells, as described previously (23).

Animals and tumor transplantation

MHC homozygous and MHC heterozygous cynomolgus macaques were identified in the Filipino macaque population, which carried a particular set of Mafa haplotype alleles called HT1 (9). All protocols for animal experiments were approved by the Shiga University of Medical Science Animal Experiment Committee (Permit numbers: 2012-7-1HHH, 2012-7-4, 2013-2-10, and 2014-1-5H). The animal experiments were carried out in strict accordance with the Guidelines for the Husbandry and Management of Laboratory Animals of the Research Center for Animal Life Science at Shiga University of Medical Science, the guidelines of an Institutional Animal Care and Use Committee and Standards Relating to the Care and Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. Regular veterinary care and monitoring, balanced nutrition, and environmental enrichment were provided by the Research Center for Animal Life Science at the Shiga University of Medical Science.

Under ketamine/xylazine anesthesia, 2 × 107 PTY and 2 × 107 NPCPR cells were injected into the subcutaneous tissue and the brain, respectively. A brain stereotaxis apparatus was used for injection into the brain (24). The MRI equipment (Magnex Scientific) in the Molecular Neuroscience Research Center of Shiga University of Medical Science was used for taking T1- and T2-enhanced images of the brain after NPCPR injection under anesthesia.

NOD/Shi-scid, IL-2Rγnull mice (NOG mice) were purchased from the Central Institute for Experimental Animals (CIEA, Kawasaki, Kanagawa, Japan). Tumor cell lines, 5 × 106 cells, suspended in 50% Matrigel (BD Biosciences #354230) were injected subcutaneously into the necks of 6- to 8-week-old NOG mice. NPCPR was injected intracranially into NOG mice with a head-fixation system.

Tumor tissues were collected about 90 days after transplantation. Half of the grafts were fixed in 10% formalin for histologic analysis, and the other half were minced with scissors under an aseptic condition and cultured in the appropriate medium with antibiotics to remove the cells derived from NOG mice.

To assess the in vivo efficacy of plasma taken from monkeys rejecting tumors, we injected 2 × 107 PTY cells together with 300 μL of normal monkey plasma, plasma taken from monkeys rejecting tumors or PBS into NOG mice (day 0). We used plasma without inactivating complements because NOG mice did not have complement-dependent cytotoxic activity (25). We injected the monkey plasma for 6 days after tumor cell injection and measured the tumor diameter every day. Tumor volume was calculated by the formula ab2/2 (a, width; b, length).

Histologic analysis and immunohistochemistry

Formalin-fixed and paraffin-embedded graft tissues were sectioned and stained with hematoxylin and eosin using a standard protocol. For immunohistochemistry, the deparaffinized sections were incubated with diluted primary antibodies overnight at 4°C after inactivation of endogenous peroxidase with methanol and H2O2. Then the sections were incubated with the secondary antibody conjugated with horseradish peroxidase (HRP; NICHIREI Bioscience #424152) for 1 hour at room temperature. A peroxidase substrate, 3,3′-diaminobenzidine tetrahydrochloride (DAB, NICHIREI Bioscience #415172), was used for color development. The primary antibodies were as follows: anti-AFP (clone ZSA06, NICHIREI Bioscience, #422221), anti-OCT3/4 (clone: C10, SANTA CRUZ BIOTECHNOLOGY, INC, #sc-5279), anti-PLAP (clone: 8A9, DAKO, #M7191), anti-S-100 (rabbit polyclonal antibody, DAKO, #Z0311), anti-GFAP (clone: 6F2, DAKO, #Z0334), and Ki67 (clone: MIB1, DAKO, #N1633).

Flow cytometry

Single-cell suspensions of tumor cells and other cells were immunostained with a purified mouse anti-CD30 antibody (Clone: HRS4, Immunotech #A87939), purified rat anti-GRP94 (Clone: 9G10, Abcam #ab2791), purified mouse anti-GRP78 (Clone: C38, eBioscience #14-9768), and purified mouse anti-GRP75 (Clone: JG1, Abcam #ab2799) followed by staining with fluorescein isothiocyanate (FITC)-conjugated goat polyclonal anti-mouse IgG (Nordic Immunological Laboratories #GAM/Fab/FITC), cyanine 3 (CY3)-conjugated goat anti-mouse IgG (Abcam #ab97035), and Texas Red (TR)-conjugated goat polyclonal anti-rat IgG (Abcam #ab6843) as secondary antibodies. Dead cells were labeled with 2 μg/mL propidium iodide (Sigma-Aldrich #P4170). In all of the flow cytometry experiments, isotype-matched antibodies corresponding to each antibody were used as controls. The samples were analyzed by a FACS Calibur instrument (Becton, Dickinson and Company) in the Central Research Laboratory of Shiga University of Medical Science.

Detection of antibodies in plasma of the monkeys after transplantation

After heat-inactivation, plasma was diluted 10 times with PBS. Fifty microliters of the diluted plasma was added to the cell suspension and the cells were incubated for 30 minutes on ice. After washing with PBS twice, the cells were incubated with 2 μL of FITC-labeled goat polyclonal anti-monkey IgG for another 30 minutes on ice. The cells were analyzed by flow cytometry after washing with PBS twice.

In vitro killing assay by complement-dependent cytotoxity

Fifty microliters of each of the inactivated plasma samples diluted at various concentrations or 100 μL of IgG in plasma of monkeys rejecting tumors or 5 μL of monoclonal rat anti-GRP94 (Clone: 9G10, final concentration of 0.05 mg/mL, Abcam) was added to 2 × 105 PTY cells suspended in 100 μL of PBS and incubated on ice for 30 minutes. After washing twice, PTY cells were treated with 500 μL of guinea pig complement (Gibco #19195-015) or normal monkey serum, which were diluted 5 times in a complete medium for 45 minutes at 37°C. Dead cells were labeled with 2 μg/mL propidium iodide (Sigma-Aldrich), and live cells and dead cells were counted using flow cytometry.

ELISPOT assay

Heparinized peripheral blood was collected from the cancer-rejecting monkeys before and after transplantation. After lysing red blood cells, 5 × 105 cells per well were cultured in an ELISPOT plate with PTY cell lysate, which was made from 5 × 104 PTY cells by a freeze and thaw method. The experiments were usually performed in triplicate wells for each condition, but duplicate culture was used when the number of peripheral blood cells was not sufficient. After culture for 3 days, the number of interferon (IFN)-γ spots was counted by the analyzer ImmunoSpot (Cellular Technology Limited). Stimulation indices (SI) were calculated by the following formula: number of spots in culture of the blood cells plus tumor lysate/number of spots in culture of the blood cells only.

Purification of IgG fraction in plasma of cancer-rejecting monkeys

The IgG fraction in plasma of cancer-rejecting monkeys was purified using protein G-Sepharose (GE Healthcare UK Ltd. #28903134). Briefly, monkey plasma diluted in PBS at one hundred times was passed through protein G-Sepharose after treatment with a binding buffer (20 mmol/L sodium phosphate, pH 7.0). Trapped IgG fractions were recovered by an elution buffer (0.1 M glycine-HCl, pH 2.7), and eluted IgG fractions were immediately neutralized by 400 μL of 1 M Tris-HCl, pH 9.0. The eluted buffer was exchanged with PBS by dialysis in cellulose membrane tubes (EIDIA Co. Ltd. #UC27-32-100).

SDS–PAGE and Western blot analyses

A plasma membrane protein extraction kit (101Bio, LCC. #P503L) was used to purify plasma membrane proteins from PTY cells according to the manufacturer's instructions. Recombinant human GRP94 protein (ProSpec-Tany TechnoGene Ltd. #HSP091), recombinant GRP78 protein (StressMarq Bioscience Inc. #SPR-107), and PTY plasma membrane proteins precipitated by plasma IgG of the cancer-rejecting monkeys were loaded into 10% SDS slab gels at a concentration of 0.1 mg/mL in SDS-PAGE sample buffer (60 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 1% 2-mercaptoethanol).

After separation by 6% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins were electrotransferred to Immobilon-P Transfer Membranes (Millipore #IPVH00010) by a wet transfer apparatus. The membranes were blocked in Tris-buffered saline containing 5% nonfat milk and 0.01% Tween-20 at room temperature for 1.5 hours and then incubated with the plasma IgG of the cancer-rejecting monkeys, mouse monoclonal anti-GRP78 antibody (clone C38), or mouse monoclonal anti-GRP94 antibody (clone 2H3, Abcam #ab63469) overnight at 4°C. After washing, the membranes were incubated at room temperature for 1 hour with HRP-conjugated goat polyclonal anti-monkey IgG (Nordic Immunological Laboratories #GANon/IgG(FC)/PO) at 1:1,000 dilution in RIA buffer supplemented with 5% milk powder. HRP activity on the membranes was developed with Western Blotting Luminal Reagent (Millipore #WBLURO500) according to the manufacturer's instructions.

Detection of an antibody specific for GRP94 and GRP78 by ELISA

Recombinant GRP94 protein (0.5 μg/mL) or recombinant GRP78 protein (0.5 μg/mL) was seeded in 96-well flat-bottom assay plates (Coaster #3368). After blocking with 3% BSA in PBS, diluted monkey plasma or the IgG fraction in the plasma of monkeys rejecting tumors was added. After 2-hour incubation at room temperature, the plates were washed 6 times with PBS containing 0.05% Tween 20. HRP-conjugated goat polyclonal anti-monkey IgG was incubated for 2 hours at room temperature. After washing 6 times with PBS containing 0.05% Tween 20, HRP activity was assessed using 3, 3′, 5, 5′-tetramethyl benzidine substrate (100 μL). The reaction was stopped by the addition of 1 M hydrogen chloride (100 μL). Optical density (OD) was measured at 450 nm and 620 nm. Results are shown after subtraction of OD at 620 nm from OD at 450 nm. The experiments were performed in triplicate wells for each condition.

Absorption of an antibody specific for GRP94 from the IgG fraction in plasma of monkeys rejecting tumors

Fifty microliters of recombinant GRP94 protein (10 mg/mL) was seeded in 96-well flat-bottom assay plates (Coaster #3368). PBS was used for a control instead of recombinant GRP94 protein. After blocking with 3% BSA in PBS, 0.1 mg/mL plasma IgG of the cancer-rejecting monkeys was added. After overnight incubation at 4°C, the seeded plasma IgG was collected. Absorption of IgG specific for GRP94 from plasma IgG of the cancer-rejecting monkeys was verified by ELISA before using for an in vitro killing assay.

Embryonal carcinoma cell line PTY and glioblastoma cell line NPCPR are produced from iPSCs of an MHC homozygous cynomolgus macaque

Human somatic cells require more than 4 changes in oncogenes for tumorigenic potential, that is, (i) activation of telomerase, (ii) inactivation of the pRB pathway, (iii) inactivation of the p53 pathway, and (iv) overexpression of RAS, PI3CA, and cyclin D1, which disrupt cell-cycle regulation and lead to constitutive proliferation of cells (26–29). Therefore, we transduced four human oncogenes, TERT, p53CT (dominant-negative form of the p53 gene and C-terminal region of wild-type p53), CDK4, and HRASV12 (constitutive active form of HRAS) simultaneously with a retrovirus into iPS cells in a hematopoietic differentiation culture and further transduced two oncogenes, c-MYC and mutant PI3CA (Supplementary Fig. S1A and S1B), for which gene expression in the cells was confirmed by RT-PCR (Supplementary Fig. S1C and Supplementary Table S2). Cells with a polygonal shape and island-like structure, named “PTY” cells, formed larger colonies with a larger number in the soft-agar colony forming assay than did cells without PI3CA mutation or c-MYC introduction (Fig. 1A; Supplementary Fig. S1B and S1D).

Figure 1.

PTY cells and NPCPR cells that are derived from Mafa-homozygous iPSCs show an embryonal carcinoma phenotype and a glioblastoma-like phenotype, respectively. A, Stereoscopic image of cultured PTY cells. B, Tumor volumes of PTY cells in four NOG mice injected subcutaneously. C–F, Histologic analysis of a representative PTY tumor taken from NOG mouse #2. C, Hematoxylin and eosin staining. D–F, Immunohistochemical staining of AFP (D), OCT3/4 (E), and PLAP (F). G, Flow cytometric analysis of PTY cells stained with anti-CD30 antibody. H and I, Hematoxylin and eosin staining of an NPCPR tumor. H, NPCPR tumor invasion in the cortex of the brain. Arrows encircle the tumor. I, Necrosis and microvascular proliferation. J–L, Immunohistochemical staining for S-100 (J), GFAP (K), and Ki-67 (L).

Figure 1.

PTY cells and NPCPR cells that are derived from Mafa-homozygous iPSCs show an embryonal carcinoma phenotype and a glioblastoma-like phenotype, respectively. A, Stereoscopic image of cultured PTY cells. B, Tumor volumes of PTY cells in four NOG mice injected subcutaneously. C–F, Histologic analysis of a representative PTY tumor taken from NOG mouse #2. C, Hematoxylin and eosin staining. D–F, Immunohistochemical staining of AFP (D), OCT3/4 (E), and PLAP (F). G, Flow cytometric analysis of PTY cells stained with anti-CD30 antibody. H and I, Hematoxylin and eosin staining of an NPCPR tumor. H, NPCPR tumor invasion in the cortex of the brain. Arrows encircle the tumor. I, Necrosis and microvascular proliferation. J–L, Immunohistochemical staining for S-100 (J), GFAP (K), and Ki-67 (L).

Close modal

PTY cells formed tumors in immunocompromised NOG mice (Fig. 1B) after subcutaneous injection. The tumor formed from PTY cells in NOG mice was composed of cuboidal epithelioid cells with pleomorphic and vesicular nuclei and with large nucleoli, and the cells were arranged in solid, tubular, and papillary patterns with aggressive invasion (Fig. 1C). The PTY tumor cells were positive for α-fetoprotein (AFP), OCT3/4, and placental alkaline phosphatase (PLAP) in immunohistochemical staining (Fig. 1D–F) and weakly positive for CD30 in flow cytometry analysis (Fig. 1G). These findings are consistent with human embryonal carcinoma (30, 31).

Next, we established a glioblastoma cell line derived from monkey iPSCs via neural progenitor cells (NPC). NPCs were first produced and then p53CT, CDK4, hTERT, and c-MYC genes were transduced for immortalization, and further transduction of mutant PI3CA and HRASV12 genes was performed to generate a glioblastoma cell line (12, 13, 25–29, 32, 33). As a result, NPCs with mutant PI3CA and HRASV12 increased in number more vigorously in vitro and formed larger colonies in soft agar than did NPCs with mutant PI3CA alone (Supplementary Fig. S1E). The gene expression in the oncogene-transduced cells was confirmed by RT-PCR (Supplementary Fig. S1C and Supplementary Table S2).

We performed a tumorigenesis assay of the cells derived from induced NPCs in vivo (Fig. 1H–L). In NOG mice in which cells had been injected into the parietal lobe of the brain, round-shaped tumor cells with large nuclei and a small cytoplasm invaded the surface of the cortex with necrosis and microvascular proliferation (Fig. 1H and I). Thus, the cell line named “NPCPR” showed a phenotype of glioblastoma, and the cells were positive for S-100, GFAP, and Ki67 (Fig. 1J–L; ref. 34).

The induced cancer cell lines PTY and NPCPR are immunologically rejected by MHC-matched hosts

To establish a monkey cancer model, we injected PTY and NPCPR cells into MHC-matched monkeys. PTY cells were injected subcutaneously into five MHC-matched monkeys, but all of them rejected the cells within 4 to 5 weeks after injection regardless of immunosuppression (Fig. 2A and B, data not shown). NPCPR cells were injected into the frontal lobe of the brain in an MHC-matched monkey (Fig. 2C). MRI study revealed that NPCPR cells were rejected about 4 weeks after injection despite the fact that the brain is considered to be an immune-privileged site (Fig. 2D).

Figure 2.

PTY and NPCPR cells are immunologically rejected in an MHC-matched monkey. A and B, PTY cells were injected into five MHC-matched monkeys with or without immunosuppression. Representative data for one of the five monkeys without immunosuppression are shown. A, A shoulder of the monkey 1 week after subcutaneous injection of PTY cells. B, No subcutaneous tumor was detected 4 weeks after injection. C and D, NPCPR cells were injected into the left frontal lobe of an MHC-matched monkey. C, A T2-enhanced image of MRI immediately after injection of NPCPR cells (red circle). D, A T2-enhanced image of MRI 4 weeks after injection (red circle). E–G, MHC-matched fibroblasts (E), PTY cells (F), and NPCPR cells (G) were stained with plasma of a monkey rejecting the tumor. IgG antibodies bound to the tumor surface were detected with FITC-conjugated anti-monkey IgG and a flow cytometer. Fibroblasts were taken from the same monkey from which iPSCs were derived. Plasma of three other monkeys showed similar results. H, Complement-dependent cytotoxic assay of PTY cells using plasma of the monkey rejecting PTY cells. PTY cells were treated with plasma serially diluted as indicated in the x-axis and complements to detect dead cells labeled with propidium iodide. *, significant differences (P < 0.05) between with and without complement. I,In vivo inhibition of tumor growth by monkey plasma. PTY cells were injected into NOG mice on day 0. After injection, PBS, normal monkey plasma or tumor-rejecting monkey plasma was injected from day 0 to day 6 (arrows). Tumor diameters were measured every day to calculate tumor volume. Significant differences were detected by Student t test (N = 3; *, P < 0.05). J, Production of IFNγ by peripheral blood cells against PTY antigen. The numbers #1–#5 indicate individual monkeys. Monkey #5 was transplanted with PTY cells twice with an 8-month interval.

Figure 2.

PTY and NPCPR cells are immunologically rejected in an MHC-matched monkey. A and B, PTY cells were injected into five MHC-matched monkeys with or without immunosuppression. Representative data for one of the five monkeys without immunosuppression are shown. A, A shoulder of the monkey 1 week after subcutaneous injection of PTY cells. B, No subcutaneous tumor was detected 4 weeks after injection. C and D, NPCPR cells were injected into the left frontal lobe of an MHC-matched monkey. C, A T2-enhanced image of MRI immediately after injection of NPCPR cells (red circle). D, A T2-enhanced image of MRI 4 weeks after injection (red circle). E–G, MHC-matched fibroblasts (E), PTY cells (F), and NPCPR cells (G) were stained with plasma of a monkey rejecting the tumor. IgG antibodies bound to the tumor surface were detected with FITC-conjugated anti-monkey IgG and a flow cytometer. Fibroblasts were taken from the same monkey from which iPSCs were derived. Plasma of three other monkeys showed similar results. H, Complement-dependent cytotoxic assay of PTY cells using plasma of the monkey rejecting PTY cells. PTY cells were treated with plasma serially diluted as indicated in the x-axis and complements to detect dead cells labeled with propidium iodide. *, significant differences (P < 0.05) between with and without complement. I,In vivo inhibition of tumor growth by monkey plasma. PTY cells were injected into NOG mice on day 0. After injection, PBS, normal monkey plasma or tumor-rejecting monkey plasma was injected from day 0 to day 6 (arrows). Tumor diameters were measured every day to calculate tumor volume. Significant differences were detected by Student t test (N = 3; *, P < 0.05). J, Production of IFNγ by peripheral blood cells against PTY antigen. The numbers #1–#5 indicate individual monkeys. Monkey #5 was transplanted with PTY cells twice with an 8-month interval.

Close modal

We measured levels of antibodies against the tumor in the monkey plasma to try to determine a cause of immunologic rejection. PTY cells were stained with plasma collected 4 weeks after tumor injection, while MHC-matched fibroblasts were not stained (Fig. 2E and F). NPCPR cells were also stained with the monkey plasma 2 and 4 weeks after injection of NPCPR cells, although the cells were not stained with plasma before injection (Fig. 2G). These results indicated that the monkeys rejecting the tumor cells had antibodies against the corresponding tumors and that immunologic responses against MHC-matched transplanted cells were induced in the monkeys.

To assess the contribution of the antibodies in plasma to the rejection, the complement-dependent cytotoxity (CDC) of the plasma was examined. Dilution-dependent killing activities of the plasma were detected in vitro (Fig. 2H) as well as in vivo (Fig. 2I). The antibodies in plasma were thought to be one of the causes of rejection in the monkey model as well as IFNγ-producing T-lymphocytes specific against PTY (Fig. 2J).

Oncogene-transduced cells react with IgG antibodies in plasma taken from monkeys rejecting tumors

To assess the antigens recognized by antibodies in plasma taken from monkeys rejecting tumors, we firstly confirmed cross-reactivity of the plasma against other cancer cells (Fig. 3). The plasma of the monkey injected with PTY cells also reacted with the cytoplasm and plasma membrane of NPCPR cells as well as MHC-matched fibroblasts immortalized by transduction of the oncogenes p53CT, CDK4, and hTERT, whereas fibroblasts without transduction of any oncogene were not recognized by the plasma (Fig. 3A and B).

Figure 3.

Cross-reaction of plasma with various cancer cells and immortalized cells. A, PTY cells, NPCPR cells, and immortalized MHC-matched fibroblasts were stained with plasma of a monkey rejecting PTY cells. B, PTY cells, non-immortalized fibroblasts, and fibroblasts immortalized by introduction of three oncogenes (p53CT, CDK4, and hTERT) on slide glasses were stained with plasma of a monkey rejecting PTY cells. C, MHC-matched fibroblasts in which the indicated genes had been introduced were stained with plasma of the monkey rejecting the tumor. a, MHC-matched fibroblasts; b–i, MHC-matched fibroblasts transduced with p53CT (b), CDK4 (c), hTERT (d), p53CT and CDK4 (e), CDK4 and hTERT (f), p53CT and hTERT (g), p53CT, CDK4 and hTERT (h), or hTERT and human papilloma virus E6/E7 (i).

Figure 3.

Cross-reaction of plasma with various cancer cells and immortalized cells. A, PTY cells, NPCPR cells, and immortalized MHC-matched fibroblasts were stained with plasma of a monkey rejecting PTY cells. B, PTY cells, non-immortalized fibroblasts, and fibroblasts immortalized by introduction of three oncogenes (p53CT, CDK4, and hTERT) on slide glasses were stained with plasma of a monkey rejecting PTY cells. C, MHC-matched fibroblasts in which the indicated genes had been introduced were stained with plasma of the monkey rejecting the tumor. a, MHC-matched fibroblasts; b–i, MHC-matched fibroblasts transduced with p53CT (b), CDK4 (c), hTERT (d), p53CT and CDK4 (e), CDK4 and hTERT (f), p53CT and hTERT (g), p53CT, CDK4 and hTERT (h), or hTERT and human papilloma virus E6/E7 (i).

Close modal

MHC-matched fibroblasts without introduction of an oncogene and fibroblasts transduced with one oncogene, p53CT, CDK4, or hTERT, did not react with plasma taken from monkeys rejecting tumors (Fig. 3C, a–d). On the other hand, fibroblasts co-transduced with p53CT + CDK4, CDK4 + hTERT, p53CT + hTERT, or p53CT + CDK4 + hTERT and fibroblasts immortalized by introduction of hTERT + HPV-16 E6/E7 genes instead of p53CT + CDK4 genes reacted to the plasma (Fig. 3C, e–i). Thus, the antibodies in plasma taken from monkeys rejecting tumors recognized common antigens expressed on the cells transduced with two or more oncogenes. Therefore, introduction of at least two genes among the genes used in the present study changed the antigenicity of MHC-matched cells.

GRP94 and GRP78 are expressed on the surfaces of the cancer cell lines and oncogene-transduced cells.

To identify the common antigens expressed on the surfaces of both tumorigenic cells and nontumorigenic cells and recognized by the antibodies in plasma taken from monkeys rejecting tumors, we performed SDS-PAGE after immunoprecipitation of the cell lysates of PTY cells and fibroblasts immortalized by p53CT, CDK4, and hTERT by plasma taken from monkeys rejecting tumors and a normal monkey. Several common bands between 75 and 110 kDa were found between PTY cells and immortalized fibroblasts (Supplementary Fig. S2). LC-MS/MS analysis predicted that digested peptides were derived from HSP90β and GRP78 in common with PTY cells and immortalized fibroblasts (Supplementary Table S3). GRP94, an HSP90β paralogue, and GRP78 are intracellular heat shock proteins stabilizing unfolding proteins in somatic cells, and they have been reported to be expressed on the surface of cancer cells (35, 36). Therefore, we focused on these heat shock proteins as common antigens recognized by plasma taken from monkeys rejecting tumors.

To confirm the LC-MS/MS results, we examined the expression of GRP94 and GRP78 on the plasma membrane of oncogene-transduced cells. Plasma membrane proteins of the PTY cell line were precipitated with IgG fractions of two plasma samples taken from monkeys rejecting tumors, and GRP94 was detected with an anti-GRP94 antibody (Fig. 4A). GRP78 was not detected in the plasma membrane protein fraction of PTY cells with an anti-GRP78 antibody (Fig. 4B).

Figure 4.

Plasma IgG of a monkey rejecting PTY cells contains the specific IgG for GRP94. Membrane-bound molecules were collected from lysates of PTY cells, and then the molecules bound to plasma IgG of the cancer-rejecting monkeys were precipitated. The precipitated molecules were separated and transferred to membranes that were blotted with anti-GRP94 (A) and anti-GRP78 (B) antibodies. The total membrane-bound proteins of PTY tumor cells were used as a positive control for staining. #1 pre IgG IP protein, PTY membrane protein precipitated by plasma IgG before tumor injection in monkey #1. #1 post IgG IP protein, PTY membrane protein precipitated by plasma IgG after tumor rejection in monkey #1. #2 pre IgG IP protein, PTY membrane protein precipitated by plasma IgG before tumor injection in monkey #2. #2 post IgG IP protein, PTY membrane protein precipitated by plasma IgG before tumor rejection in monkey #2. C and D, Plasma IgG of monkey #1 (right) and monkey #2 (left) against recombinant GRP94 (C) and GRP78 (D) was examined using ELISA. Pre, plasma IgG before tumor injection; Post, plasma IgG 4 weeks after tumor rejection. Averages of triplicate wells are shown. Significant differences between pre- and postrejection were calculated by Student t test (*, P < 0.05). The IgG fraction in the plasma of tumor-rejecting monkey #1 of Fig. 4 was absorbed to remove IgG specific for GRP94 by recombinant GRP94 protein. E, ELISA of GRP94 using the IgG fraction after removing GRP94-specific IgG. The x-axis shows the concentration of GRP94 used for the absorption of IgG specific for GRP94. The y-axis shows absorbance. F, Complement-dependent cytotoxity of the IgG fraction after removing GRP94-specific IgG. PTY cells were treated with each of the removed IgG fractions. A rat monoclonal antibody specific for GRP94 was used as a positive control, and PBS was used instead of plasma IgG as a negative control. The y-axis shows the percentage of detected dead cells labeled with propidium iodide (PI; *, P < 0.05).

Figure 4.

Plasma IgG of a monkey rejecting PTY cells contains the specific IgG for GRP94. Membrane-bound molecules were collected from lysates of PTY cells, and then the molecules bound to plasma IgG of the cancer-rejecting monkeys were precipitated. The precipitated molecules were separated and transferred to membranes that were blotted with anti-GRP94 (A) and anti-GRP78 (B) antibodies. The total membrane-bound proteins of PTY tumor cells were used as a positive control for staining. #1 pre IgG IP protein, PTY membrane protein precipitated by plasma IgG before tumor injection in monkey #1. #1 post IgG IP protein, PTY membrane protein precipitated by plasma IgG after tumor rejection in monkey #1. #2 pre IgG IP protein, PTY membrane protein precipitated by plasma IgG before tumor injection in monkey #2. #2 post IgG IP protein, PTY membrane protein precipitated by plasma IgG before tumor rejection in monkey #2. C and D, Plasma IgG of monkey #1 (right) and monkey #2 (left) against recombinant GRP94 (C) and GRP78 (D) was examined using ELISA. Pre, plasma IgG before tumor injection; Post, plasma IgG 4 weeks after tumor rejection. Averages of triplicate wells are shown. Significant differences between pre- and postrejection were calculated by Student t test (*, P < 0.05). The IgG fraction in the plasma of tumor-rejecting monkey #1 of Fig. 4 was absorbed to remove IgG specific for GRP94 by recombinant GRP94 protein. E, ELISA of GRP94 using the IgG fraction after removing GRP94-specific IgG. The x-axis shows the concentration of GRP94 used for the absorption of IgG specific for GRP94. The y-axis shows absorbance. F, Complement-dependent cytotoxity of the IgG fraction after removing GRP94-specific IgG. PTY cells were treated with each of the removed IgG fractions. A rat monoclonal antibody specific for GRP94 was used as a positive control, and PBS was used instead of plasma IgG as a negative control. The y-axis shows the percentage of detected dead cells labeled with propidium iodide (PI; *, P < 0.05).

Close modal

The expression of GRP94 and GRP78 on the oncogene-transduced cells was examined using flow cytometry analysis and immunofluorescence staining of the GRPs in various cells (Supplementary Fig. S3). GRP94 and GRP78 were detected on the surfaces of cells with oncogenes hTERT, p53CT, and CDK4, regardless of whether they were tumorigenic or not (Supplementary Fig. S3A-S3H). Monkey IgG in plasma taken from monkeys rejecting tumors co-existed on the surface of the cells (Supplementary Fig. S3I–S3P), but GRP78 mainly co-existed with monkey IgG in the cytoplasm (Supplementary Fig. S3M and S3N). These results were consistent with the results of an immune precipitation assay (Fig. 4B).

Plasma IgG against GRP94 in tumor rejection

We examined the IgG specific for GRP94 and GRP78 in normal monkeys. Western blot analysis (Supplementary Fig. S4A and S4B) and ELISA (Supplementary Fig. S4C and S4D) using recombinant proteins of GRP94 and GRP78 revealed the presence of IgG against GRP94 and GRP78 in not only plasma of monkeys rejecting tumors but also in plasma of normal monkeys as an autoantibody. We compared IgG titers with GRP94 and GRP78 in pretransplantation plasma and posttransplantation plasma by ELISA with recombinant GRP94 and GRP78 (Fig. 4C and D). Levels of the plasma IgG against GRP94 after transplantation in monkeys #1 and #2 were significantly higher than those before transplantation. Cytotoxicity by the IgG specific for GRP94 was examined with IgG after absorption of the antibody bound to GRP94 protein. CDC activity was significantly decreased after absorption of the IgG specific for GRP94 (Fig. 4E and F). On the other hand, titers of the IgG against GRP78 in the plasma after transplantation were higher than those before transplantation, but the titers were very low (Fig. 4D). This result and low expression level of GRP78 on the cell surface (Fig. 4B; Supplementary Fig. S3E and S3F) indicated that anti-GRP94 antibodies participated more than anti-GRP78 antibodies in the rejection of cancer.

In the present study, we established two cancer cell lines, embryonal carcinoma and glioblastoma cell lines, from Mafa-homozygous iPS cells. Contrary to our expectation, the Mafa-homozygous cancer cell lines did not grow in Mafa-heterozygous matched monkeys. In the monkeys rejecting tumors, an antibody against tumor cells was detected. Plasma IgG of the monkeys rejecting tumors contained autoantibodies against the heat shock protein GRP94, which was commonly expressed on the surfaces of PTY, NPCPR, and other immortalized cells. The autoantibodies against GRP94 participated in the tumor rejection and the immune surveillance during oncogenesis in the MHC-matched host.

PTY and NPCPR cells expressed GRP94, which was detected in the mouse embryonal carcinoma cell line F9 and in human glioblastoma cells (37, 38). GRP94, which is known as an ER chaperone protein, enhances ER activity to synthesize a large amount of proteins for rapid proliferation of cancer cells. GRP94 is essential for the processing of proteins that have been implicated in oncogenesis, such as insulin-like growth factor 1 (IGF1) and integrins. These proteins stabilized by GRP94 changed cell growth, differentiation and migration in the situation of hypoxia and glucose starvation of the tumor microenvironment (39). As GRP94 has a high capacity to bind Ca2+, GRP94 prevents Ca2+ flux from the ER to the cytosol under an ER stress condition and inhibits caspase activation to protect cancer cells from apoptosis (40). Furthermore, it was shown that the expression of GRP94 was gradually upregulated during carcinogenesis in colorectal tumors (41), being consistent with our results showing that GRP94 was expressed in not only tumorigenic cells but also nontumorigenic cells transduced with at least two oncogenes (Fig. 3C). The exact mechanisms of the translocation of GRP94 from the ER to the cell surface during carcinogenesis are still unclear. GRP94 overexpression by gene transduction without sequences for the plasma membrane anchoring domain did not lead to the expression of GRP94 on the cell surface (data not shown). Thus, unknown anchor molecules other than CD91, TLRs, MHC-class I, MHC class II, and HER2, which are already known as ligands or client proteins expressed on the cell surface, may be needed for GRP94 to stay on the surface of oncogenic cells (36).

Many studies have shown that T cells and NK cells play an important role in cancer immunosurveillance (15–19). On the other hand, there are several reports about humoral immunity in immunosurveillance (42). Autoantibodies in cutaneous malignant melanoma patients were a better prognostic factor during interferon α-2b therapy (43). As autoantibodies have been studied for their potential use as cancer biomarkers, higher levels of anti-cyclin B1 and anti-mutant p53 antibodies in healthy individuals than in cancer patients are related to cancer-free status (44–46). However, as cyclin B1 and mutant p53 are internal proteins and are not expressed on the cell surface, anti-cyclin B1 and anti-p53 antibodies are not thought to be direct effector molecules in antitumor immunity. Our results showed that the plasma including autoantibodies against GRP94 prevented tumor growth in NOG mice. After removing GRP94 from plasma IgG, CDC activity was significantly reduced. The results suggest that humoral immunity including autoantibodies against GRP94 works in immune surveillance by not only CDC but also by antigen-dependent cell-mediated cytotoxicity (47).

Anticancer immune therapy against both GRP94 and GRP78 has already been attempted in patients (48, 49), though the expression of GRP78 on PTY and NPCPR cells was not apparent. Our data support the possible efficacy of immunotherapy with an antibody and raise the following question: Why are clinical cancers not rejected despite GRP94 and GRP78 expression on cancer cells? It is possible that a tumor in vivo expresses GRP at a lower level than does a tumor in vitro due to immunosurveillance during oncogenesis and/or immune tolerance against GRP94 and GRP78. Spontaneous cancer cells in patients might already be immune-edited to be less antigenic.

In conclusion, we found that humoral immunity prevented the growth of cancer cells and that the transformed cells were killed during oncogenesis not only by T cells and NK cells but also by immunoglobulins including anti-GRP94 autoantibodies produced by plasma cells.

No potential conflicts of interest were disclosed.

Conception and design: H. Ishigaki, M. Nakayama, Y. Itoh, K. Ogasawara

Development of methodology: H. Ishigaki, H. Inoue, T. Akagi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Ishigaki, T. Maeda, T. Sasamura, H. Ishida, T. Inubushi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Ishigaki, T. Maeda, Y. Itoh

Writing, review, and/or revision of the manuscript: H. Ishigaki, Y. Itoh, K. Ogasawara

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Maeda, H. Inoue, J. Okahara, T. Shiina, M. Nakayama

Study supervision: K. Ogasawara

We thank Ms. Yachiyo Mitsuishi for support in the culture of cells, Ms. Chiaki Masuda for performing RT-PCR of neural progenitor cells, Dr. Eiji Yamada for giving suggestions regarding the histologic diagnosis of tumors, Mr. Akira Yokoe for making sections and performing staining, and Dr. Hideaki Tsuchiya, Dr. Shinichiro Nakamura, Mr. Takahiro Nakagawa, and Mr. Ikuo Kawamoto for animal care. We also thank Dr. Shin Kaneko for instructions to develop hematopoietic cells from iPSCs and kindly providing C3H10T1/2 cells.

H. Ishigaki was awarded Grants-in-Aid for Young Scientists (B) Grant number 23790442 and the Presidential Research Support 2010 and 2016 in Shiga University of Medical Science.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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