Fig 2: Human lung ADCs lacking CHUK/IKKa are highly correlated with elevated TNF/TNFRSF1B-associated FOXP3 Treg-cell and macrophage scores. (A, Left) The array containing 90 human lung ADCs (BCS04017a; US Biomax, Inc.) was divided into two groups of low CD68 (L-CD68)- and high CD68 (H-CD68)-positive cells, analyzed by immunofluorescence (IF) staining. (Right) The correlation between macrophages (CD68) and Treg-cell (FOXP3) numbers was further analyzed. n, case numbers; ****P < 0.0001; Student’s t test. (B) Correlation of IKKa expression and macrophage numbers in the human lung ADC array (BCS04017a) was analyzed by IHC staining. The distribution of IKKa-stained intensities from strong (+++) to negative (-) staining was divided into two groups of L(low)-CD68 and H(high)-CD68. Data represent mean ± SEM. Data were analyzed by ?2 test. n, ADC numbers; ***P < 0.001. (C) Comparison of CD68 and CD163 expression patterns in human lung ADC and its adjacent tissue in human tissue arrays (HLung030PG02; US Biomax, Inc.), analyzed by IHC staining. Dark brown, positive cells; numbers on the Top, positions in the tissue array. (Scale bar, 50 µm.) (D) Correlation of CHUK expression levels with macrophage (Left) and Treg-cell (Right) scores in human lung ADCs determined by analyzing the data (14). Using quartile analysis, CHUK expression was divided into high (H-CHUK) and low (L-CHUK) expression levels. CHUK, IKKa; n, ADC numbers per group; P value at the Top of panels, Student’s t test. (E) Correlation of IKBKB expression levels with macrophage (Top) or Treg-cell (Bottom) scores in human lung ADCs determined by analyzing the data (14). Using quartiles and median analyses, IKBKB expression was divided into high IKBKB (H-IKBKB) and low IKBKB (L-IKBKB) expression levels. n, ADC numbers; P value at the Top of panels, Student’s t test. (F) Coexpression of the genes encoding molecules for macrophage and Treg-cell development in human lung ADCs determined by analyzing the data (6). P value at the Top of panels, two-tailed t test; n, ADC numbers; red lines, repression lines; r, Pearson correlation coefficient; -4 (-2 or -1), outliers that were removed from 230. (G) Correlation of H-CHUK or L-CHUK expression levels (using median and quartile analysis to divide CHUK expression groups) with the expression of CSF1, CSF1R, FOXP3, TNFRSF1B, TNF, or CCL22 genes in human lung ADCs (TCGA, PanCancer Atlas, cBioPortal). n, ADC numbers. P value at the Top of panels, Student’s t test.

Fig 3: A TNF/TNFRSF1B/c-Rel signaling cascade promotes Foxp3 Treg-cell induction. (A) Flow cytometric analyses of % Treg cells in CD4 T cells from the coculture of WT CD4+CD25- T cells with macrophages (macro) that were isolated from Kras-CL or KrasIKKaL lung ADCs (n = 3/group). Data represent mean ± SEM (three repeats). *P < 0.05; Student’s t test. (B) Flow cytometric analyses of % Treg cells in CD4 T cells from the coculture of WT CD4+CD25- T cells and peritoneal macrophages (PM) in a dose-dependent manner (ratio, 2:1 and 1:1) (Right). n = 4. Data represent mean ± SEM (three repeats). ***P < 0.001; ****P < 0.0001; Student’s t test (Left). (C) ROS (dichlorofluorescein [DCF]) levels in WT and Nox2KO PM (Left). Flow cytometric analyses of % Treg cells in CD4 T from the coculture of WT or Nox2KO PM with WT CD4 T cells (n = 3/group, Middle) and from the coculture of WT PM and WT CD4 T cells treated with NAC (n = 4), apocynin (n = 4), and vehicle control (n = 3) (Right). Data represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test. (D) Lung ADC burden (% area, Left) in WT mice receiving KrasIKKaL cells, treated with NAC (n = 5) and vehicle control (n = 4). ROS (DCF) levels in ADC-associated macrophage (Middle), and cytosolic and nuclear c-Rel levels in ADC-associated CD4 T cells detected by Western blotting, from NAC- and vehicle (Cont)-treated WT mice receiving KrasIKKaL cells (Right). LaminB1, a nuclear protein loading control; a-tubulin, a cytosolic protein loading control. Data represent mean ± SEM (three repeats); **P < 0.01; ***P < 0.001; Student’s t test. (E) Flow cytometric analyses of % Treg cells in CD4 T cells from the coculture of WT PM and WT CD4+CD25- T cells supplemented with TNF, H2O2, or a vehicle control (n = 3/group, Left), and c-Rel intensity (median fluorescence intensity, MFI) in these CD4 cells and CD25+Foxp3+ cells (Right). ns, not significant; **P < 0.01; ***P < 0.001. Data represent mean ± SEM (three repeats); Student’s t test. (F) Flow cytometric analyses of % Treg-cell induction in CD4 T cells from the coculture of WT PM and WT CD4 T cells treated with PTXF (50 µg/mL) and control. *P < 0.05. Data represent mean ± SEM (three repeats); Student’s t test. (G) Lung ADC burden (% area, Left) in WT mice receiving KrasIKKaL cells, treated with PTXF (50 mg/kg) or a vehicle control (n = 4/g/roup). IHC analyses of % Treg cells (Middle) in ADCs and ROS (DCF) levels in ADC-associated macrophages (Right) from WT mice receiving KrasIKKaL cells, treated with PTXF or a vehicle control (n = 4/group). Data represent mean ± SEM (three repeats); *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test. (H) Western blotting analyses of cytosolic and nuclear c-Rel levels in CD4 T cells isolated from KrasIKKaL lung ADCs in WT mice treated with PTXF or a vehicle control (Cont). Each example was obtained from multiple mice. LaminB1, a nuclear protein loading control; a-tubulin, a cytosolic protein loading control. (I) KrasIKKaL ADC burden (% area, Left) in WT and Tnf-/- (KO) mice (n = 6/group). Data represent mean ± SD. ****P < 0.0001; Student’s t test. Images of WT and Tnf KO lungs are representative (Right). Dark pink stained spots in hematoxylin and eosin slides represent tumors in the lungs. (J) IHC analyses of % Foxp3 Treg-cell (Left) and % F4/80 macrophage (Right) numbers in KrasIKKaL ADCs from WT and Tnf KO mice (n = 4/group; three sections per mouse). Data represent mean ± SD. ****P < 0.0001; Student’s t test. (K) TNFR2/TNFRSF1B expression in CD4 T cells isolated from lung ADCs of WT and Tnf KO mice, analyzed by RT-PCR. Data represent mean ± SEM (three repeats). ***P < 0.001; Student’s t test. (L–M) Flow cytometric analyses of % Treg cells in CD4 T cells (L) and c-Rel levels (MFI, M) in Treg cells from the coculture of WT PM with WT or Tnfrsf1b-/- (KO) CD4+CD25- T cells, treated with or without TNF. Data represent mean ± SEM (three repeats). *P < 0.05; **P < 0.01; ***P < 0.001; Student’s test. (N) ROS (DCF) levels in WT and Tnf KO peritoneal macrophages, analyzed by RT-PCR. Data represent mean ± SEM (three repeats). ****P < 0.0001; Student’s t test. (O) TNF expression in WT peritoneal macrophages treated with H2O2 or vehicle control. Data represent mean ± SEM (three repeats). *P < 0.05; Student’s t test. (P) Flow cytometric analyses of % Treg cells in CD4 T cells from the coculture of WT PM with WT or Tnfrsf1b-/- CD4 T cells as well as from the coculture of Tnf KO PM with WT or Tnfrsf1b-/- CD4 T cells, analyzed by flow cytometry. Data represent mean ± SEM (three repeats). ns, not significant; *P < 0.05; ***P < 0.001; Student’s test. (Q) Lung weight (Left) and KrasIKKaL ADC burden (Right) in irradiated CD45.1 mice receiving CD45.2 WT or Tnf-/- (KO) BM (n = 4). Data represent mean ± SD. *P < 0.05; Student’s test. (R) Flow cytometric analyses of ADC-associated CD4+Foxp3+ cells in CD45+ cells (Left) and c-Rel levels (MFI) in Foxp3 cells (Right) in CD45.1 mice receiving CD45.2 WT or Tnf KO BM cells (n = 3). Data represent mean ± SEM (three repeats). *P < 0.05; **P < 0.01; Student’s test. (S) Lung appearances of Tnfrsf1b-/-;Ikka?LU;KrasG12D and Ikka?LU;KrasG12D mice. (T) Lung ADC burden (% area, Left) of Tnfrsf1b-/-;Ikka?LU;KrasG12D and Ikka?LU;KrasG12D mice (n = 4/group). Flow cytometric analyses of Foxp3+ Treg-cell numbers (Middle, n = 3) and CD4 T cell c-Rel levels (Right, n = 3) in lung ADCs of Tnfrsf1b-/-;Ikka?LU;KrasG12D and Ikka?LU;KrasG12D mice. Data represent mean ± SEM (three experiments). *P < 0.05; **P < 0.01; Student’s test. (U) Survival rates for lung ADC patients expressing CHUK hemizygous deletions and TNFRSF1B (TNFR2) gain versus patients with ADC expressing CHUK and TNFRSF1B double diploid. The RNA-sequence data were obtained from cBioPortal, TCGA, ref. 6. Logrank test for P value analyses.

Fig 4: IKKa reduction up-regulates transcription of cytokine genes in human and mouse lung ADC cells. (A) Expression of CCL2, CSF1, TNF, ADAM8, and IL-23A in Kras-CL and KrasIKKaL ADC cells, analyzed by RT-PCR. Data represent mean ± SEM (three repeats). ***P < 0.001; **P < 0.01; Student’s t test. (B) Correlation of CHUK (IKKa) expression levels (using quartiles’ analysis to divide IKKa expression groups) with the expression levels of IL-23A or ADAM8 genes in human lung ADCs by analyzing the TCGA data (PanCancer Atlas, cBioPortal). H-CHUK, high CHUK; L-CHUK, low CHUK; P value at the Top of panels, Student’s t test; n, ADC numbers. (C–D) Expression of CHUK (IKKa), IL-23A (IL-23a), CCL22, CSF1, and TNF in human A549 (C) and mouse Kras-CL (D) lung ADC cells treated with Si-control (Si-Cont) or Si-IKKa RNA, analyzed by RT-PCR. Data represent mean ± SEM (three repeats). *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test. (E–F) ChIP analyses for binding of IKKa to the promoter regions of TNF (Tnf), CSF1 (Csf1), CCL22 (Ccl22), and IL-23A (Il-23a) genes by using an anti-IKKa antibody for immunoprecipitation, followed by PCR with primers for these genes in A549 cells (E) and in Kras-CL cells (F) treated with Si-control (Si-Cont) or Si-IKKa RNA. Data represent mean ± SEM (three repeats). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test. (G) ChIP-seq analyses for IKKa enrichment on the TNF gene in A549 cells. The enrichment peaks are marked by red lines. bp, nucleotide base pair; red arrow, 6,066 bp; Ab, antibody for immunoprecipitation. TNF gene: thickest lines, exons; second-thickest lines, untranslated sequences; thin lines, introns; red line, consensus sequences containing eight nucleotides. (H) A working model showing that ADC-IKKa reduction up-regulates the expression of TNF, CSF1, CCL-22, and IL-23A and macrophage recruitment, which generate a TNF/TNFRSF1B/c-Rel pathway for CD4 T cells to stimulate Treg-cell induction, accelerating KrasG12D ADC development. Arrows, promotion; lines, inhibition; M?, monocyte-derived macrophage.