Ab253297

The protocol of IHC of formalin-fixed paraffin-embedded sections was performed as previously described (19 (link)). Anti-PLIN2 (ab181452, Abcam, Waltham, MA, USA) was used at a dilution ratio of 1:300, and the serial sections were incubated with primary antibodies such as anti-CD4 (ZSGB-BIO, ZM-0418), anti-CD8 (ZSGB-BIO, ZA-0508), anti-CD19 (ZSGB-BIO, ZM-0038), anti-CD56 (ZSGB-BIO, ZM-0057), anti-CD68 (ZSGB-BIO, ZM-0464), and anti-FOXP3 (ab253297, Abcam). We used PBS to replace the primary antibody as negative control. The IHC staining results of PLIN2 was assessed by ImageJ software (20 (link), 21 (link)). Digital photos were acquired from the tumor center and invasive front, and the average values were calculated for further analysis (22 (link)).

Hepa1-6 cells, derived from the BW7756 tumor in a C57L mouse, were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were cultured in high-glucose Dulbecco’s modified eagle medium (DMEM) (BasalMedia Technologies Co., Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (Yeasen Biotechnology Co., Shanghai, China) and 1% penicillin–streptomycin (Yeasen Biotechnology Co., Shanghai, China) at 37 °C under a 5% CO₂ atmosphere.
Lenvatinib was obtained from Eisai (Ibaraki, Japan). Anti-mouse PD-1 Ab (anti-PD-1 Ab; clone RMP1- 14) and mouse isotype control IgG (control IgG; clone 2A3) were purchased from Bio X Cell (West Lebanon, NH, USA).
Primary Abs used for immunohistochemical staining included: anti-CD4 Ab (#25229, CST), anti-CD8α Ab (#98941,CST), anti-Forkhead box protein p3 (Foxp3) Ab (ab253297, Abcam), anti-F4/80 Ab (#70076, CST), anti-CTLA4 Ab (A00020, Boster), anti-T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) Ab (A01962, Boster), anti-TIM3 Ab (ab241332, Abcam) and, anti-V domain-containing Ig suppressor of T-cell activation (VISTA) Ab (#54979, CST).

IHC was performed as previously described [28 (link)], and serial sections were incubated with primary antibodies such as anti‐CD161 (67537‐1‐Ig, Proteintech), anti‐LLT1 (ab197341, Abcam), anti‐CD4 (ZM‐0418, ZSGB‐BIO), anti‐CD8 (ZA‐0508, ZSGB‐BIO), anti‐CD19 (ZM‐0038, ZSGB‐BIO), anti‐CD68 (ZM‐0464, ZSGB‐BIO), and anti‐Foxp3 (ab253297, Abcam).
The IHC staining results of CD161 and LLT1 were independently and double‐blindly evaluated by two senior pathologists (Liang Ding and Qingang Hu) who were blinded to the patients' data, and the average values were calculated for further analysis. IHC staining was scored according to the percentage of positive cells and staining intensity. The percentage of stained cells was defined as 0 = 0–5%; 1 = 6–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 75–100%. The staining intensity was defined as follows: 0 = negative staining; 1 = weak staining; 2 = moderate staining; and 3 = strong staining. The IHC score was calculated by multiplying the grade of the staining intensity by that of the staining percentage. High and low expression of LLT1 were defined as the median of IHC scores. The readout score of CD161 lymphocytes was subdivided into values for the tumor center and invasive margin, and high and low expression of CD161 lymphocytes were defined according to the median of the sum of these two values.