Fig 2: PKCθ releases IL-1β through activation of NFκB pathway in PAs. (A) Volcano plot showing the distribution of DEGs of osteogenic cytokines in PRKCQ high expression group versus PRKCQ low expression group in PAs (The data analyzed are described in Figure 2A; OGCs, osteogenic cytokines). (B,C) Immunohistochemistry for IL-1β in clinical specimens of GH-PAs (n = 20) and PRL-PAs (n = 20) (B) (×200; scale bar, 100 μm). Quantification, right (*** p < 0.001; ** p < 0.01). The relevance of expression levels of IL-1β and PKCθ (C). (D) ELISA to detect the secretion level of IL-1β levels of GH3 and ATT20 in treatment with sotrastaurin and PMA (n = 6 independent experiments; *** p < 0.001). (E) ELISA to detect the secretion level of IL-1β levels of GH3 and ATT20 cells in groups of vector and shPKCθ with PMA (n = 6 independent experiments; *** p < 0.001). (F) Schematic diagram of GH3 or ATT20 CM were administered to induce RAW264.7 differentiation towards osteoblasts. (G) TRAP staining of RAW264.7 administrated IL-1β recombinant protein and CM of GH3 cells in groups of vector and shPKCθ (×40; scale bar, 500 μm). Quantification, right (n = 6 independent experiments; *** p < 0.001). (H) HE staining exhibited bone destruction of xenograft tumor of GH3 while shPKC and administration of IL-1β recombinant protein (×200; scale bar, 100 μm). Quantification of bone erosion score, right (n = 6 mice per group; *** p < 0.001). (I) TRAP staining of RAW264.7 administered IL-1β mAb and CM of GH3 cells in treatment of PMA (×40; scale bar, 500 μm). Quantification, right (n = 6 independent experiments; *** p < 0.001). (J) HE staining exhibited bone destruction of xenograft tumor of GH3 while administered IL-1β mAb and PMA (×200; scale bar, 100 μm). Quantification of bone erosion score, right (n = 6 mice per group; *** p < 0.001). (K–N) WB detection of NF-κB pathway in GH3 (K,M) and ATT20 (L,N) while administered PMA combine with sotrastaurin (K,L) or shPKCθ (M,N). (O) STRING protein–protein interaction network analysis exhibiting proteins interacted with PKCθ. (P) CoIP reveals the ability of PKCθ to bind IKKα, P-IKKα/β, IKBα, and p-IKBα while treated with PMA. (Q) Schematic diagram exhibited PKC transcriptionally regulates IL-1β through NF-kB pathway.

Fig 3: Genomic PRKCQ amplification contributes to the constitutive activation of TCR signaling in malignant T cells.(A) Chromatin aberration detected by WGS of genomic DNA extracted from PBMCs before anti–PD-1 treatment compared with granulocyte genomic DNA in this patient. (B) The violin plot shows the expression level of PRKCQ in CD4+ tumor T cells, CD8+ T cells, and Tregs at the single-cell level in PBMCs before anti–PD-1 treatment. The data were analyzed using the Wilcoxon ranked-sum test with continuity correction. (C) IHC staining of PKCθ and p-PKCθ (T538) on skin tumors. The images in the left panel are representative of the results obtained using skin tumors from 5 patients with MF without PRKCQ amplification. The images in the right panel are results obtained using tumor sample obtained before the patient received anti–PD-1 treatment. Scale bar: 25 μm. (D) Western blot results showing PKCθ and p-PKCθ (T538) expression in the cell lysate of PBMCs after anti–PD-1 treatment from the patient. Cell lysates of PBMCs from 2 healthy donors served as the normal control (NC). (E–G) The violin plots show the expression levels of markers of T cell activation on CD4+ tumor T cells and CD8+ T cells before and after anti–PD-1 treatment at the single-cell level in PBMCs. The data were analyzed using the Wilcoxon ranked-sum test with continuity correction. (H and I) Proliferation of PBMCs that were obtained from the patient before (H) and after (I) anti–PD-1 therapy and treated with or without Compound 20 (5 μM), a PKCθ-specific inhibitor. Student’s t test was utilized. The results are shown as the mean ± SEM of data from 3 independent experiments for (H). Data from one experiment are shown for I.

Fig 4: Schematic model of mechanism underlying hyperprogression after anti–PD-1 treatment in the cutaneous T cell lymphoma patient.PD-1 acts as a tumor suppressor for malignant T cells with oncogenic TCR activation caused by PRKCQ amplification; PD-1 blockade treatment released oncogenic TCR signaling and led to the proliferation and activation of malignant T cells.

Fig 5: Vimentin deficiency in Tregs leads to accumulation and persistence of Helios+IL-18R+ Tregs in the lung after influenza infection(A) Flow cytometry contour plots and quantification of Helios+ Treg frequency among lung Tregs in WT and vimentin cKO mice after influenza infection.(B) Helios expression on lung Tregs of WT and vimentin cKO after influenza infection.(C) Helios+ Treg number in whole lung after influenza infection.(D and G) Heatmap of differentially expressed genes (p < 0.05).(E, F, H, I, and N) Normalized expression of Nt5e, Entpd1, Il18r1, Areg, and Prkcq in Helios+ and Helios− Tregs.(J) Frequency of Helios+IL-18R+, Helios+IL-18R−, and Helios−IL-18R+ cells among lung Tregs after influenza infection.(K and L) Frequency and absolute number of Helios+IL-18R+ Tregs in the whole lung tissue after influenza infection.(M) Relative mRNA expression of Il18r1, Areg, and Vim in WT and Vim-deficient splenic nTregs. Data are mean ± SD, n = 4–5 mice/group.p values were calculated by two-way ANOVA (A–C and K–M) and two-tailed unpaired Student’s t test (E, F, H, I, and N). *p < 0.05, ***p < 0.0005, and ****p < 0.0001.