Open access Short report J Immunother Cancer: first published as 10.1136/jitc-2021-002426 on 30 June 2021. Downloaded from IFN-γ- and IL-17- producing CD8+ T (Tc17-1) cells in combination with poly- ICLC and peptide vaccine exhibit antiglioma activity Takayuki Ohkuri,1,2 Akemi Kosaka,1,2 Maki Ikeura,2 Andres M Salazar,3 1,2,4,5 Hideho Okada To cite: Ohkuri T, Kosaka A, ABSTRACT led to Food and Drug Administration Ikeura M, et al. IFN-γ- and IL- Background While adoptive transfer of T- cells has been approvals for patients with B- cell malig- 17- producing CD8+ T (Tc17-1) a major medical breakthrough for patients with B cell nancies. However, the development of cells in combination with poly- malignancies, the development of safe and effective T- ICLC and peptide vaccine exhibit effective T- cell therapies for GBM remains cell- based immunotherapy for central nervous system antiglioma activity. Journal a challenge due to multiple barriers, (CNS) tumors, such as glioblastoma (GBM), still needs to for ImmunoTherapy of Cancer including restricted T- cell homing to GBM overcome multiple challenges, including effective homing 2021;9:e002426. doi:10.1136/ and the antigenic heterogeneity, as we jitc-2021-002426 and persistence of T- cells. Based on previous observations 2 that interleukin (IL)-17-producing T-cells can traffic to the reviewed recently. Especially, the central + nervous system (CNS) has anatomical ► Additional online CNS in autoimmune conditions, we evaluated CD8 T- cells supplemental material is that produce IL-17 and interferon-γ (IFN-γ) (Tc17-1) cells barriers (eg, blood–brain barrier) which 3 published online only. To view, in a preclinical GBM model. tightly regulate the entry of immune cells. please visit the journal online Methods We differentiated Pmel-1 CD8+ T- cells into In the experimental autoimmune enceph- (http:// dx. doi. org/ 10. 1136/ jitc- Tc17-1 cells and compared their phenotypic and functional alomyelitis (EAE) models, interleukin + 2021- 002426). characteristics with those of IFN-γ-producing CD8 T (IL)-17- producing Type17 helper T-cells (Tc1) and IL-17- producing CD8+ T (Tc17) cells. We also TO and AK contributed equally. can migrate to the CNS through their evaluated the therapeutic efficacy, persistence, and tumor- Accepted 16 May 2021 expression of CCR6, thereby mediating homing of Tc17-1 cells in comparison to Tc1 cells using a the neuroinflammation.4 5 Moreover, CD8+ mouse GL261 glioma model. In vitro, Tc17-1 cells demonstrated profiles of Type17 T- cells (Tc17 cells) accumulate in Results http://jitc.bmj.com/ the spinal cord through IL-17 production both Tc1 and Tc17 cells, including production of both IFN-γ 6 and IL-17, although Tc17-1 cells demonstrated lesser to exacerbate EAE. These findings led us © Author(s) (or their degrees of antigen- specific ytotoxicc activity compared to hypothesize that IL-17- and interfer- + employer(s)) 2021. Re- use with Tc1 cells. In mice- bearing intracranial GL261- Quad on-γ (IFN-γ)- producing CD8 T (Tc17-1) permitted under CC BY- NC. No commercial re- use. See rights tumor and treated with temozolomide, Tc1 cells, but not cells would exhibit enhanced homing to and permissions. Published by Tc17-1, showed a significant prolongation of survival. the CNS with potent antitumor activity, BMJ. However, when the T- cell transfer was combined with leading to improved clinical outcomes in on September 28, 2021 by guest. Protected copyright. 1Department of Neurological poly- ICLC and Pmel-1 peptide vaccine, both Tc1 and Tc17- adoptive T- cell therapy for GBM. Surgery, University of Pittsburgh 1 cells exhibited significantly prolonged survival associated School of Medicine, Pittsburgh, with upregulation of very late activation antigen−4 on PA, USA Tc17-1 cells in vivo. Glioma cells that recurred following 2Brain Tumor Program, the therapy lost the susceptibility to Pmel-1- derived MATERIALS AND METHODS University of Pittsburgh Medical cytotoxic T- cells, indicating that immuno- editing was a Mice Center (UPMC) Hillman Cancer mechanism of the acquired resistance. Pmel-1 T- cell receptor (TCR) transgenic and Center, Pittsburgh, PA, USA Conclusions Tc17-1 cells were equally effective as Tc1 3Oncovir, Inc, Washington, C57BL/6J mice were purchased from The cells when combined with poly- ICLC and peptide vaccine District of Columbia, USA Jackson Laboratory. 4Department of Neurological treatment. Surgery, University of California Cell lines San Francisco, San Francisco, California, USA BACKGROUND The GL261 cell line was provided by Dr. 5Parker Institute for Cancer Malignant gliomas, including glioblastoma Robert M. Prins (University of California, Immunotherapy, San Francisco, (GBM), represent a highly unmet clinical Los Angeles). GL261-Quad cells expressing CA, USA 1 need, and their prognosis remains poor. human gp10025–33 (hgp10025-33), chicken Correspondence to Adoptive T- cell transfer therapy, specif- OVA257–264 and OVA323–339, and mouse I- Eα52–68 7 Dr Hideho Okada; ically chimeric antigen receptor (CAR) were generated and provided by Dr. John R. hideho. okada@ ucsf. edu T- cell therapy, has proven effective and Ohlfest (University of Minnesota). Ohkuri T, et al. J Immunother Cancer 2021;9:e002426. doi:10.1136/jitc-2021-002426 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-002426 on 30 June 2021. Downloaded from Reagents of mice on Kaplan-Meier plots among the groups. Mean Detailed information on antibodies and relevant reagents values between the two groups were compared using is available in online supplemental table 1. Student’s t- test. T-cell differentiation + - Pmel-1 CD8 CD44 T- cells were sorted using CD8α RESULTS magnetic MicroBeads system (Miltenyi Biotec) and then Phenotypic and functional properties of Tc1, Tc17, and Tc17-1 purified by MoFlo (Beckman Coulter). These cells were cells in vitro stimulated by mitomycin- C- treated splenocytes loaded Using Pmel-1 mouse- derived CD8+CD44- T- cells, we first with 2 µg/mL hgp100 peptide in the presence of + 25-33 generated IL-17- and IFN-γ-producing CD8 T (Tc17-1) the following cytokines and antibodies from days 0 to cells and compared their characteristics with those of 3: Tc1 cells, 100 U/mL IL-2, 2 ng/mL IL-12 and 5 µg/ IFN-γ-producing CD8+ T (Tc1) and IL-17-producing mL anti- IL4 mAb; and Tc17 and Tc17-1 cells, 10 ng/mL CD8+ T (Tc17) cells. Tc1 and Tc17 cells predominantly TGF-β1, 10 ng/mL IL-6, 10 µg/mL anti- IFN-γ mAb and 5 produced IFN-γ or IL-17, respectively, while Tc17-1 cells µg/mL anti- IL4 mAb. After day 4, T- cells were cultured represent heterogeneous cell populations, with the with 100 U/mL IL-2 for Tc1, 10 ng/mL IL-23 for Tc17, majority of cells producing IL-17 and approximately 10% and 1 µg/mL IL-12 and 10 ng/mL IL-23 for Tc17-1 cell producing both IL-17 and IFN-γ (figure 1A).Consistent differentiation. with flow cytometry data, Tc17-1 cells expressed lower Flow cytometry levels of Ifng mRNA than Tc1 cells (figure 1B). However, Intracellular cytokine staining Cytofix/Cytoperm Kit was Tbx21 mRNA (encoding T- bet) levels were comparable purchased from BD. The data were analyzed using BD between Tc1 and Tc17-1 cells (figure 1B). On the other Accuri C6 flow cytometer and software. hand, mRNA levels of Il17a and Rorc (encoding RORγt) expressed in Tc17-1 cells were at least as high as those RNA isolation and quantification of gene expression in Tc17 cells (figure 1C). Tc17-1 cells expressed CCR6 at Total RNA was extracted and subjected to quantitative higher levels than Tc1 but slightly lower than Tc17, while real time- PCR analysis as described previously.8 Relative Tc17-1 and Tc17 cells expressed lower levels of CXCR3 expression of mRNAs compared with control samples was than Tc1 cells (figure 1D and online supplemental figure calculated by the ddCt method. 1), suggesting that the CCR6 and CXCR3 expression profiles in Tc17-1 cells were closer to those of Tc17 cells CTL analysis than to Tc1 cells. 51 In vitro cytotoxicity was conducted using a 6- hour Cr- re- To investigate the functional properties of Tc1, Tc17, 9 lease assay as described previously. In brief, Tc1, Tc17, and Tc17-1 cells, we stimulated them with GL261 glioma and Tc17-1 cells were incubated with GL261 cells loaded cells in the absence or presence of exogenous hgp10025-33 with or without hgp10025-33. In some experiments, Tc1 peptide (figure 1E,F). Tc1 cells demonstrated the highest http://jitc.bmj.com/ and Tc17-1 cells were sorted from cervical lymph nodes level of IFN-γ production and antigen- specific cytotoxicity (CLNs) by MoFlo. compared with those by Tc17-1 and Tc17. While Tc17-1 cells exhibited low but significant antigen- specific IFN- ELISA γ production and cytotoxicity, Tc17 showed no cytotoxicity The amount of IFN-γ and tumor necrosis factor (TNF)-α above background levels. On the other hand, Tc17-1 cells in the supernatant was measured by BD OptEIA ELISA produced the highest levels of TNF-α among the three- on September 28, 2021 by guest. Protected copyright. sets. cell types. Taken together, Tc17-1 cells showed transcrip- Therapeutic mouse models tional characters of both Tc1 and Tc17 cells, while their C57BL/6J mice were intracranially inoculated with cytokine and chemokine receptor expression profiles, as GL261- Quad (1×105) cells as described previously.8 Tumor- well as cytotoxic activity, appear to be closer to those of bearing mice received temozolomide (TMZ; 330 µg/ Tc17 cells in vitro. mouse) intraperitoneally on day 9, then intravenously (IV) infusion of Tc1 or Tc17-1 (5×106) cell on day 10 followed Tc17-1 cells did not show effective antitumor activity in by intramuscular injections of polyinosinic- polycytidylic glioma-bearing mice treated with TMZ acid stabilized with poly- L- lysine carboxymethylcellulose We next assessed the therapeutic efficacy of intravenously (poly- ICLC) (2.5 mg/kg) with the peptide vaccine (100 transferred Pmel-1- derived Tc17-1 in mice bearing GL261- Quad gliomas which express the Pmel-1 epitope hgp10025- µg/mouse) on day 11 after tumor inoculation.