Fig 2: Reactivity of HZ6 and HZ8 TCR engineered Jurkat T cells to NY-ESO-1157-165 WT-SCT-K562 or alanine-substituted pMHC-SCTs (SCT-K562 mutants) target cells. (A) Schematic of the functional evaluation assay of the TCRs in Jurkat T cells. TCR engineered Jurkat T cells co-transduced with human CD3-CD8 construct were co-cultured with K562 target cells that were transduced with NY-ESO-1157-165/HLA-A2 single-chain trimer (SCT-K562 cells). The reactivity of TCR engineered Jurkat T cells was evaluated by detection of the IL-2 secretion after co-culturing of effector cells and target cells. (B) Flow cytometry histogram comparison of NY-ESO-1157-165/HLA-A2 expression on the surface of K562 cells transduced with NY-ESO-1157-165 WT-SCT (WT-SCT-K562) and K562 cells without transduction. (C) Flow cytometry analysis of TCR expression on Jurkat T cells transduced with TCR and CD3-CD8 before and after14 days in vitro culture in the presence of 2 µg/ml puromycin. (D) ELISA measuring secretion of IL-2 from untransduced, HZ6-CD8, and HZ8-CD8 Jurkat effector cells following 48 h co-incubation with WT-SCT-K562 target cells or K562 cells as control. (E) Alanine scanning approach for HZ6 and HZ8 TCR in Jurkat T cells. HZ6-CD8-Jurkat cells or HZ8-CD8-Jurkat cells were co-cultured with WT-SCT-K562 or alanine-substituted pMHC-SCTs K562 (SCT-K562 mutants) cells. IL-2 concentrations in the supernatant were measured by ELISA. For (D,E), the data is a representative of 3 independent experiments with two technical replicates. Means ± SD for a representative experiment are shown.

Fig 3: Spontaneous and cell line derived murine UPS tumours are macrophage rich and lymphocyte poor.(A) Using FACS analyses, macrophages are the dominant intra-tumoural immune population (14%), whereas lymphocyte numbers are low (<2%). (B) In a separate analysis comparing spontaneous UPS, cell lined derived (transplanted) UPS and the inflamed M3-9-M ERMS model, CD4 and CD8 lymphocyres are low (1–4%) and significantly less than the infamed M3-9-M-Female model (6–9%). The cell line UPS model is more inflamed and contains more lymphocytes than the spontaneous model, althogh these differences did not reach stastistical significance. (C-D) NanoString® immune profiling of UPS tumours also demonstrates high macrophage scores, and negative lymphocyte, NK cell, dendritic cell and adaptive immune pathway scores compared to an inflamed sarcoma model.

Fig 4: CD8+ MILs have a higher proliferative potential than splenic CD8+ T cells and give rise to phenotypically comparable progeny cells.(A) Thy1.1+ TCR transgenic P14 CD8+ T cells (103) were adoptively transferred into Thy1.2/1.2 C57BL/6 recipients, which were subsequently infected with LCMV clone 13. Splenocytes and MILs were purified at 8 dpi and adoptively transferred into infection-matched recipients, which were euthanized 7 days later. (B to D) Percentages of total donor P14 CD8+ T cells in the spleen (Sp) and hindlimb muscles (Mus), and P14 CD8+ T cells expressing transcription factors (B), inhibitory receptors (C), or effector cytokines (D) are shown. Data are representative (FACS plots) of two independent experiments or cumulative (bar graphs); n = 6 mice. The bars represent the mean, and the error bars represent the SD.

Fig 5: L-XRT hampers tumor growth, activates T cells, increases NK cells and M1 macrophages, and downregulates the inhibitory cytokine TGF-ß. (A), The one-tumor mouse model was established by injecting 344SQ-P cells (0.5 × 106) into the right hind legs of 129Sv/Ev mice (n=5/group), after which L-XRT (1 Gy × 2 fractions) was delivered when tumors reached around 7–8 mm in diameter. Anti-PD1 (200 µg/inj.) and anti-CTLA-4 (50 µg/inj.) were administered intraperitoneally on days 5, 8, 12, and 16 post 344SQ-P injection. Mice were euthanized when tumor reached 14 mm, and survival was plotted by the Kaplan-Meier method. Experiment was repeated twice, and similar patterns were detected. (B and C) dLNs from the L-XRT dose groups were harvested at 3 days after radiation and analyzed with flow cytometry. Both CD4 (No XRT vs 1 Gy × 2, p=0.0318) and CD8 cells (No XRT vs 1 Gy × 2, p=0.0001) were activated after two 1 Gy fractions, as depicted by the activation marker CD44. (D and E), spleens (spl), dLNs, and TILs were harvested at 48 hours after two 1 Gy fractions for phenotyping by flow cytometry. (D) Cells were gated on the CD45+ population and then on CD49b+ to identify NK cells (TILs no XRT vs TILs 1 Gy × 2, p=0.0328). (E) Cells were gated on CD45+ and then on Gr1intermediate and CD11b+ to identify macrophages, and further gated on F4/80+ CD38hi to identify M1 macrophages (No XRT vs 1 Gy × 2, p=0.0463). (F) NanoString molecular counts (nCounter immunology panel) showed significant reduction in local TGF-ß expression 24 hours after two 1 Gy fractions of L-XRT (p=0.0007). Data for each group was represented as mean±SD and Student’s t-tests were used to compare groups. *p=0.05 was considered to indicate statistical significance. dLNs, draining lymph nodes; L-XRT, low-dose radiation; PD1, programmed cell death protein 1; TGF-ß, tumor growth factor beta; TILs, tumor-infiltrating leukocytes.