Fig. 2

PLX3397 pre-treatment (days 0–30 post-transplant) does not prevent the onset nor attenuate clinical symptoms and histopathology of cGVHD. A Experimental timeline for irradiation, adoptive cell transfer and PLX3397 treatment. Lethally irradiated B6D2F1 mice were transplanted with 5 × 10⁶ BM cells and 0.5 × 10⁶ mature splenic T cells from either B6D2F1 (syngeneic controls) or C57Bl/6 (allogeneic) donor mice to induce cGVHD. From post-transplant days 0–30, mice were also fed either control chow or PLX3397-incorporated chow at 150 ppm or 300 ppm, before being all switched back to standard chow for post-transplant days 30–70. Mice were scored for pathology weekly until post-transplant day 70 (n = 5–6 mice/group). B Temporal profile of clinical scores over the duration of the experiment, i.e., until day 70 post-transplant. Note that cGVHD mice consistently scored higher than syngeneic controls. PLX3397 pre-treatment did not reduce clinical scores, either during the treatment period (post-transplant days 0–30) or during the 40 days of cGVHD progression (post-transplant days 30–70). Magenta asterisks are ‘cGVHD/control chow’ versus ‘Syngeneic/control chow’. C Clinical scores in syngeneic and cGVHD mice on day 28 post-transplant, control chow or PLX3397 treatment at 150 ppm or 300 ppm. Note that PLX3397 pre-treatment (i.e., prior to cGVHD development) had no impact on clinical symptoms. Asterisks indicate post-hoc results. D Clinical scores in syngeneic and cGVHD mice on day 70 post-transplant, with 30 days of control or PLX3397 pre-treatment (at 150 ppm or 300 ppm) followed by 40 days of control chow. Note that PLX3397 pre-treatment had no impact on the severity of the clinical symptoms of cGVHD. Asterisks indicate post-hoc results. E, F Quantification of the IBA1^pos immunoreactive area (E) and IBA1^pos mean fluorescence intensity (MFI) values (F) in skin tissue from syngeneic and cGVHD mice, with and without PLX3397 pre-treatment. Asterisks indicate post-hoc results. G (Left) Representative confocal images of IBA1^pos macrophages (green) in the skin. Cell nuclei are labelled with DAPI (blue). (Right) Representative confocal images of CD68^pos macrophages (red) in the skin from syngeneic and cGVHD mice, with and without PLX3397 pre-treatment. Cell nuclei are labelled with DAPI [blue (H, I) quantification of the CD68^pos immunoreactive area (H) and CD68 MFI values (I)] in skin tissue from syngeneic and cGVHD mice, with and without PLX3397 pre-treatment. Asterisks indicate post-hoc results. J, K Histopathology scores (J) and representative images of skin tissue stained with haematoxylin and eosin (K) from syngeneic and cGVHD mice, with and without PLX3397 pre-treatment. Arrowheads point at cGVHD pathology in the skin as per Table 2. L Mansson’s trichrome stained skin samples from syngeneic and cGVDH treated with control or PLX3397 chow. Scale bar = 100 µm. M Fibrosis score for skin samples from syngeneic and cGVHD mice, with and without PLX3397 pre-treatment. Asterisks indicate post-hoc results. N Dermal thickness quantified using Masson’s trichrome stained skin samples. Asterisks indicate post-hoc results. O Quantification of CD3^pos T cells in the epidermal layer of skin tissue from syngeneic and cGVHD mice, with and without PLX3397 pre-treatment. Asterisks indicate post-hoc results. P Representative confocal images of CD3^pos T cells in the skin from syngeneic and cGVHD mice, with and without PLX3397 pre-treatment. Statistics: repeated measure two-way ANOVA with Bonferroni’s multiple comparisons (B), two-way ANOVA (C–H, J, M–O) followed by Bonferroni post hoc comparison. Dots in box plots represent individual mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar = 100 µm (L, P), 50 µm (G, J)