Cd3e pe

Fresh isolated 2 × 10⁶ cells/mouse of splenocytes or MLNLs for each group were analyzed by flow cytometry. Separated cells were dyed with rabbit anti-mouse CD16/CD32, CD3e-PE, CD19-PE-Cy7, and CD11c-APC (BD Biosciences, Shanghai, China), as well as goat anti-mouse CCR10-Axlexa Flour 488 (Abcam, Cambridge, UK), at 4 °C for 30 min. The stained cells were washed and detected on a BD FACSAria III platform. The absolute number of CD3e⁺, CD19⁺, or CD11c⁺ cells (1 × 10⁵/mouse) from each immunization group and control group (pcDNA3.1) was analyzed by flow cytometry. CCR10 expression on CD3e⁺, CD19⁺, or CD11c⁺ cells was analyzed using FlowJo V10 software (BD Biosciences, Shanghai, China).

Flow-cytometry antibodies included CD3e-PE, PD1-BV605, CD45-BV786 (BD-Biosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-APC, CD45.1-PE (eBioscience), CD45.2-Alexa700, CD-8α-Percp-Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas-red (Life Technologies, Carlsbad, CA, USA). Blocking PD-1 antibody (clone RMP1-14, BioXCell, Branford, CT, USA) was administered systemically by intraperitoneal injections of 250 μg [41 (link)]. Blocking anti-CD40L (clone MR1, BioXCell) was administered systemically by intraperitoneal injections of 250 μg [19 (link)].

Mice of generation x + 1 with signs of emerging hind limb paralysis or elevated CD19⁺ blast levels in the peripheral blood were anesthetized with ketamine/xylazine, and the animal was exsanguinated via the retrobulbar venous plexus. After cervical dislocation for confirmation of death, spleen and femoral bones were dissected and collected in cold PBS. Bone marrow and spleen cells were isolated as described before (Richter et al. 2020 (link)). For flow cytometric characterization of the blast population using FACSVerse (Beckton Dickinson) and FACSuite software, the following antibodies were used: CD3e-FITC, CD11b-FITC, CD8a-PE (all Miltenyi Biotec), CD34-FITC, CD45R-FITC, CD3e-PE, Sca-1-PE, CD4-APC, c-kit-APC, CD45-PerCP-Cy5.5 (all BD), CD19-PE, IgM-APC (all Biolegend). Doubling times of leukemic cell populations were calculated from peripheral blood blast frequencies using the following formula with t₁ and t₂ indicating the age of the animal in days at the respective time points of sample analysis: $$\mathrm{Doubling}\mathrm{time}=\frac{\left(t_2,-,t_1\right)\times \log (2)}{\log (\mathrm{blast}\mathrm{frequency}{(t}_2))-\log (\mathrm{blast}\mathrm{frequency}(t_1))}$$
Cytospins were prepared from spleen and bone marrow cell suspensions and stained as described before (Richter et al. 2019 (link)). For monitoring of CD19⁺ blast frequencies, blood (< 50 µl) was sampled from the tail vein, and CD19⁺, c-kit⁺ and Sca-1⁺ cell populations were quantified as described above.