Fig 1: Pathological changes in anti-HMGCR antibody-positive necrotizing myopathy patients showing muscular Bcl-2-positive lymphocyte infiltration and lymphoid follicle-like structures. (A) Inflammatory cell infiltrates to the endomysium and perivascular areas. (B, C) CD4-positive/CD8-positive lymphocytes infiltrated to the perivascular area and endomysium. (D) CD20-positive lymphocytes were rarely observed in mild cases with anti-HMGCR antibody-positive myopathy. (E) Bcl-2-positive lymphocytes are observed in the perivascular area. (F) Bcl-2-positive lymphocytes infiltrate to endomysium. (G) CCR4-positive lymphocytes were scattered in both perimysium and endomysium. (H) Lymphocytic accumulations were scattered in severe cases with anti HMGCR antibody-positive myopathy. (I, J) Lymphocytes were positive for CD3 and CD20 in these accumulations. (K) Lymphocytes were positive for Bcl-2 in lymphocytic accumulations. (L) CCR4-positive lymphocytes were observed in both endomysium and lymphocytic accumulations. (M) a-SMA was negative. (N) Bcl-2 indexes in anti-HMGCR antibody-positive myopathy were significantly highest in each group. (O) CCR4 indexes were highest in anti-HMGCR antibody-positive myopathy cases (***p < 0.001). (A–G) Patient 12. (H–K, M) Patient 11. (L) Patient 18. Scale bar: 100 µm.
Fig 2: Pathological changes in skin of anti-HMGCR antibody-positive necrotizing myopathy patients also showed Bcl-2-positive lymphocyte infiltration and lymphocytic accumulations. (A) Skin biopsy specimens show superficial perivascular dermatitis. (B) CD3-positive lymphocytes are observed in epidermis and dermis. (C) CD20-positive lymphocytes are not observed. (D) Lymphocytes infiltrating skin are positive for Bcl-2. (E) In severe cases, lymphocytic accumulations are observed in dermis. (F, G) CD3-positive/CD20-positive lymphocytes infiltrate to cutis including these accumulations. (H) Bcl-2-positive lymphocytes were diffusely observed in skin tissues including these accumulations. (I) CCR4-positive lymphocytes were also scattered. (J) α-SMA was negative except for vessels. (K) Bcl-2 indexes of skin in anti-HMGCR antibody-positive myopathy were significantly highest in each group (***p < 0.001). (L) CCR4-positive lymphocytes were observed only in anti-HMGCR antibody-positive myopathy (***p < 0.001). (A–E) Patient 14. (F–J) Patient 10. Scale Bars: (A–E) 100 µm, (F–H, J) 500 µm, (I) 50 µm.
Fig 3: Bcl-2 and CCR4 immunopositivity in muscle of other IIMs. (A, B) Focal endomysial Bcl-2- and CD45-positive lymphocytes infiltrations forming hotspot were observed, especially in sIBM cases. (C) The muscle biopsy specimen of sIBM patient with HTLV-1 infection showed aberrant CCR4-positive lymphocytes. (D) Bcl-2-positive perivascular cuffings were scattered most frequently in cases with antimitochondria M2 antibody-positive myositis. (E) Superficial perivascular dermatitis in cases without anti-HMGCR antibody. (F) CD45-positive lymphocytes infiltrated mainly in perivascular areas. (G) Bcl-2-positive lymphocytes are scattered. (H) CCR4-positive cells were not observed. Scale bar: 100 µm.
Fig 4: Fratricide changes the Th-subset proportion with decreased Th2, Th17, and Treg cytokine production while sparing Th1 cytokine production. (A) Chemokine receptor expression profiles of CAR T-cell products. The Th1-like subset was defined as CD4(+), CXCR3(+), CCR4(–), CCR6(–), and CCR10(–). Th2-like subset was defined as CD4(+), CXCR3(–), CCR4(+), CCR6(–), and CCR10(–) by FCM. Phenotypes were analyzed by gating the CAR-positive population, with the exception of the UTD group. Dots and bars represent individual data from each donor and SEM (3 experiments from 3 donors). ***P < .001; ****P < .0001; ns, not significant (vs CD19-CAR T group) by 1-way ANOVA with Tukey’s post hoc test. (B) Profiles of cytokine production by CD19- and CCR4-CAR T cells. CAR T cells were stimulated with PMA/Iono. Cytokine levels in the supernatant 24 hours after stimulation were analyzed using the LUMINEX assay. Data are representative of 3 experiments from 3 donors. Bars represent the mean and standard deviation of technical duplicates. (C) Depletion of CCR4-positive subsets by adding CCR4-CAR T cells to CD19-CAR T cells. T cells were stimulated and transduced with either the CCR4 CAR or CD19 CAR. 10% CCR4-CAR T cells were mixed with CD19-CAR T cells 5 days after stimulation. CCR4 expression in CD19-CAR–positive cells was analyzed on day 10. The numbers denote the percentage of cells in each quadrant. Data are representative of 3 experiments from 3 donors. (D) Heat map of Th-related cytokine production by CD19-CAR T cells pretreated with CCR4-CAR T cells compared with untreated CD19-CAR T cells. CAR T cells were stimulated using the indicated stimulators. The cytokine levels in the supernatant at 24 hours after stimulation were analyzed using LUMINEX assay. The cytokine levels from untreated CD19-CAR T cells were set to 1 and the relative expression levels are indicated. Data are representative of 3 experiments from 3 donors.
Fig 5: CCR4-CAR T cells specifically lyse CCR4-positive T-cell lymphoma cell lines and produce cytokines upon stimulation, whereas some CCR4 CAR constructs display ligand-independent T-cell degranulation. (A) Lytic activity of CAR T cells against tumor cell lines. CAR T cells and tumor cells expressing luciferase were incubated at the indicated E:T ratios. Specific lysis after 16 hours of coculture was determined based on the luminescence. Bars represent the means and standard deviation from technical triplicates. Data are representative of 3 experiments from 3 donors. (B) Degranulation of CCR4-CAR T cells. CAR T cells were stimulated with media, PMA-Iono, or HH cells at a 1:1 R:S ratio with CD107a detecting antibody and incubated for 6 hours. The cells were analyzed by FCM. The upper panels are representative flow plots for CD107a expression, the lower panels are pooled data from 3 experiments, and the 3 donors indicate baseline degranulation of CCR4CART cells derived from h1567 or its derivatives. The numbers in each column indicate the percentage of CD107a-positive populations. Dots and bars represent individual data from each donor and the SEM, respectively. (C) Cytokine production by CCR4-CAR T cells. CAR T cells were stimulated with media, PMA-Iono, or HH cells at a 1:1 R:S ratio and incubated in the presence of monensin for 6 hours. The cells were analyzed for intracellular cytokines using FCM. The upper panels are representative flow plots for IL-2 and TNFa expression, and the lower panels are pooled data from 3 experiments with 3 donors. The numbers in each column indicate the percentage of IL-2 TNF-a positive populations. Dots and bars represent individual data from each donor and SEM, respectively. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; ns, not significant (vs CD19-CAR T group) using 1-way ANOVA with Tukey’s post hoc test.
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