Zm 0464

The formalin‐fixed, paraffin‐embedded glioma tissue was stained according to our previous procedure,²⁶ which included 25 glioma samples. Written informed consent was obtained for all patients. Briefly, brain tumor sections were incubated with the PD‐L1 (1:100, ab213524, Abcam, USA), IBA1 (1:100, ab213524, Abcam, USA), TMEM119 (1:100, ab213524, Abcam, USA), CD68 (ZM‐0464, ZSGB‐BIO, China), CSF1R (1:200, 25,949‐1‐AP, Proteintech, USA), and TGFB1 (1:300, 21,898‐1‐AP, Proteintech, USA) antibody overnight at room temperature, respectively. Then, the stained sections were scored by two independent pathologists. The staining intensity was 0–3 points: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The extent of staining reflected the percentage of positive cells: 0 (<5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (>75%). Staining index was defined as the product of staining intensity and staining extent. Also, PD‐L1 protein expression in The Cancer Proteome Atlas (TCPA, http://tcpaportal.org) was analyzed.

MIF staining was conducted at Nanjing Freethinking Biotechnology Co. Ltd. (Nanjing, PR China). Formalin‐fixed paraffin‐embedded sections were prepared as previously described [28 (link)]. The slides were first deparaffinized in xylene, rehydrated, and washed before boiling in Tris–EDTA buffer (50×, pH 9, Beyotime, Shanghai, PR China). After blocking the endogenous peroxidases by incubation in an antibody block for 10 min, the slide was detected in each round, including primary antibody incubation, secondary antibody incubation, and tyramide signal amplification visualization, followed by labeling the next antibody after epitope retrieval and protein blocking as described above. Sections were serially incubated with primary antibodies such as anti‐CD161 (67537‐1‐Ig, Proteintech, Wuhan, PR China), anti‐LLT1 (ab197341, Abcam, Cambridge, UK), anti‐CD4 (48274, Cell Signaling Technology, Beverly, MA, USA), anti‐CD8 (85336, Cell Signaling Technology), anti‐pan‐CK (ZM‐0464, ZSGB‐BIO, Wuxi, PR China), and anti‐Foxp3 (ab215206, Abcam, Shanghai, PR China). Finally, the slides were stained with 4′,6‐diamidino‐2‐phenylindole (Selleckchem, Shanghai, PR China) for nuclei and mounted with anti‐quenching sealing tablets.

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.