Fig 2: Triple immunohistochemistry for expression of CD3, CD20, and proliferating cell nuclear antigen in spleen and lymph nodes. (A) Spleen or (B) lymph node sections from arthritic monkeys were triple-stained for CD3 (blue), CD20 (red), and PCNA (green) and analyzed by confocal microscopy. The three panels were merged. Colocalization of CD20 (red) with PCNA (green) resulted in a yellowish-green color. Stained sections were analyzed using a laser scanning confocal microscope (LSM 780). (C) CD20+PCNA+ intensity scores. The intensity of fluorescence was graded on a scale of 1 (weak) to 4 (strong fluorescence intensity); n = 4 per group. The means of groups at each time points were compared using student t-tests. *Significant (p < 0.05) differences from the control group are indicated. Normal: healthy cynomolgus monkey; RA: cynomolgus monkey with collagen-induced arthritis; control: control group; anti-CD154: anti-CD154 treatment group.

Fig 3: Abemaciclib treatment induces immune infiltration and T/B cell aggregation. (A) Representative immunohistochemical staining for CD45, CD8, CD19 and PD-L1 (400×), scale bars: 50 μm. (B) TIL was quantified by counting positive signals in four randomly selected fields (400×) in each tumor section using ImageJ, n=6 mice. Statistical comparisons were performed using an unpaired T test. (C) Representative immunofluorescence images of CD3 (green), CD19 (red) and nuclear staining (DAPI, blue) showing different types of cell aggregates (400×). Both CD3+ T cells and CD19+ B cells in the control group were scattered, while the numerous infiltrated T cells and B cells formed ectopic lymphoid-like structures with different shapes in the abemaciclib-treated group, scale bars: 50 μm. (D) Quantification of T cell/B cell aggregates in 3 randomly selected 100× fields in each tumor section.

Fig 4: T cell infiltration precedes loss in allograft viability in organ transplant recipients.(A) Photographs of transplanted cardiac ear pinna iso (top) and allografts (bottom) at days 6 (d6), d9, and d12 after transplantation. (B and C) Electrocardiogram (ECG) of transplanted heart measured from electrodes placed on edge of iso (B) and allograft (C). Scale bars: 2 seconds. (D) Bioluminescent images depicting graft viability at d6, d9, and d12 after transplantation in a WT BALB/c mouse. Left ear; BALB/c cardiac isograft expressing firefly luciferase (FL). Right ear; C57BL/6 cardiac allograft expressing FL. Scale bar represents total flux (photon /second); red, high; blue, low. Images are representative of 9 dual graft recipient mice from 2 independent experiments. (E) Violin plot of total flux measured from cardiac allografts (n = 9) and isografts (n = 9) at each respective time point indicated on x axis. Purple, allograft (Allo); Orange, isograft (Iso). Dashed line indicates mean. **P = 0.0028 calculated by Mann-Whitney U test. (F) Images of H&E staining of allograft (top) and isograft (bottom) on d9 after transplantation. Inset box 1, skin surface region; mononuclear cell infiltration was shown in both allo- and isograft surface. Inset box 2, graft parenchyma. Massive mononuclear cell infiltration was shown in allograft parenchyma. (G) Images of immunofluorescent staining for CD3 (Green) and DAPI (Blue). Left; low magnification, right; high magnification of inset region. CD3+ T cells infiltrating allograft parenchyma.

Fig 5: Phenotypic analysis of B cells, DCs, and TAMs from PBS- or abemaciclib-treated ID8 tumors by flow cytometry, n=6 mice. (A) Representative zebra plots of activation marker expression and cytokine levels in CD45+CD3-CD11c- B220+ B cells. (B) Histogram overlays show different costimulatory molecules expression on CD45+CD11c+MHC-II+ dendritic cells. (C) Representative histogram overlay of MHC-II shows M1-polarized TAMs and that of CD206 shows M2-polarized TAMs.