Fig 2: The association between the CGS and immune infiltration in The Cancer Genome Atlas lung adenocarcinoma database. (A) Three independent bioinformatic methods (ImmuCellAI, Timer, and quanTIseq) were utilized to estimate the abundance of different immune cells in the high- and low-CGS groups. (B) Immunohistochemistry analyses of early-stage LUAD tissues from Wuhan Union hospital demonstrated that the abundance of tumor-infiltrating CD4+ T cells was higher in the low-CGS group. ns, P=0.05; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. The LUAD tissues were fixed in 4% paraformaldehyde and then embedded in paraffin. The paraffin blocks that contained LUAD tissues were cut into sections and processed for antigen activity restoration and endogenous peroxidase activity quenching. The anti-CD4 antibody (ab133616, Abcam, Cambridge, MA, USA) was used for immunostaining. The estimation of the infiltration of CD4+ T cells in the high- and low-CGS groups was performed by 2 independent pathologists. Briefly, the CD4 staining score was estimated by the intensity score [0 (no response), 1 (weak response), 2 (mild response), 3 (strong response)] and the proportional score [1 (0–25%), 2 (26–50%), 3 (51–75%), 4 (76–100%)]. The final CD4 score was calculated by multiplying the intensity score and the proportional score. A score of 0–4 indicated low infiltration of CD4+ T cells, and a score of 5–12 indicated high infiltration of CD4+ T cells. CGS, clock gene signature; IHC, immunohistochemical; LUAD, lung adenocarcinoma.

Fig 3: HER2 CAR T cells persist in the brain, spleen and lymph nodes of recipient mice. Representative flow cytometry of live CD45RO+ cells from spleen (A) and lymph node (C) showing CD4+ and CD8+ CAR T cell engraftment in NSG SU-DIPG36 PDX tumor-bearing mice (n = 5 per group) week 6 post CAR infusion (experimental endpoint). The mean percentage of live CD4+ and CD8+ CART cells is quantified from spleen (B) and lymph nodes (D) and shown is the mean % cells ± SEM for the individual 5 mice. n.s = not significant, *P = .025, ***P = .001 non parametric Student’s t-test. (E) Endpoint (6 weeks post CAR T cell infusion) H&E staining of representative mouse brains from a SU-DIPG36 tumor-bearing mouse treated with HER2-specific CAR T cells show infiltration of human CD4+ and CD8+ cells (black arrows) and some small numbers of GFP + tumor cells. Staining was performed with anti-CD4 (ab133616, Abcam, 1/500), anti-CD8 (MA5-14548, Invitrogen, 1/100) or anti-GFP (ab6556), Histofine Simple Stain MAX (Nichirei Biosciences Inc.) secondary antibodies conjugated to peroxidase were then used and signal was developed with diaminobenzidine followed by counterstaining with hematoxylin. Scale bar is 1 mm in top image and 0.5 mm in bottom panel.