Fig 2: Dot1L activity in B cells controls proliferation in response to in vitro T cell-dependent stimuli, class switch recombination in response to in vitro T-independent stimuli, and viability in response to both the stimuli ARepresentative flow cytometry plots showing gating strategy to identify and compare relative frequency of IgG1 switched cells from WT and KO mice after 4 days of activation with anti CD40 + IL-4.B, CNumber of cell divisions traced by CTV dilution of B cells after 4 days of stimulation with LPS (B) or LPS + IL-4 (C). Data represent three biological replicates for each genotype.D, EStatistical analysis of the percentage of IgG1 switched cells per generation of proliferating B cells after 4 days of stimulation with LPS (D) or LPS + IL-4 (E). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 3, KO; n = 3).F, GRepresentative flow cytometry plots showing gating strategy to identify and compare relative frequency of IgG1 switched cells from WT and KO mice after 4 days of activation with LPS (F) or LPS + IL-4 (G).HRepresentative flow cytometry plot from single cells showing gating strategy to identify live (Zombie NIR-) and dead cells (Zombie NIR+) after 4 days of stimulation with LPS + IL-4.I–KStatistical analysis of dead cells indicated as the percentage of total single cells after 4 days of stimulation with LPS (I), LPS + IL-4 (J) or anti-CD40 + IL-4 (K). Results represent the data from one experiment and numbers represent biological replicates for each group (I: (WT) n = 3, (KO) n = 3; J: (WT) n = 4, (KO) n = 4; K: (WT) n = 4, (KO) n = 4).LRepresentative histograms indicating the levels of cleaved caspase-3 in live (Zombie NIR-) after 4 days of stimulation with LPS + IL-4. Cleaved caspase-3 levels on dead (Zombie NIR+) population shown, as positive control.MIntracellular flow cytometry staining for H3K79me2 in activated B-cell after 4 days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control. Data represent three biological replicates for each treatment.NStatistical analysis of Median Fluorescence Intensity (MFI) of H3K79me after 4 days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control is shown. Results represent the data from one experiment, and numbers represent biological replicates for each treatment (n = 4).O, PRepresentative flow cytometry plots showing gating strategy to identify and compare relative frequency of switched cells after 4 days of stimulation of naive B cells with LPS (O) or LPS + IL-4 (P) either in the presence of DOT1L inhibitor, Pinometostat, or DMSO as a control.Q, RStatistical analysis of dead cells indicated as the percentage of total single cells after 4 days of stimulation with LPS (Q) or LPS + IL-4 (R) either in the presence of DOT1L inhibitor, Pinometostat, or DMSO as a control. Results represent the data from one experiment, and numbers represent biological replicates for each treatment (n = 4).SNumber of cell divisions traced by CTV dilution of B cells after 4 days of stimulation with LPS either in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Data represent four biological replicates for each treatment.TStatistical analysis of the percentage of IgG3 switched cells per generation of proliferating B cells after 4 days of stimulation with LPS either in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment, and numbers represent biological replicates for each treatment (n = 4). Data information: Statistical analyses were performed using Student’s two-tailed unpaired t-test. Statistical significance was determined by calculating P-value. P-value less than 0.05 was considered as significant. Bars and error bars indicate mean ± SD.

Fig 3: Class switch recombination and proliferative potential of Dot1L-deficient B cells in response to T cell-dependent and T cell-independent stimuli in vitro AStatistical analysis of IgG1 switching of Dot1L-proficient (WT) and Dot1L-deficient (KO) B cells after 4 days of activation with anti-CD40 + IL-4. Results represent the data from one experiment, and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4).BNumber of cell divisions traced by CTV dilution of B cells stimulated for 4 days with anti-CD40 + IL-4. Data represent three biological replicates for each genotype.CStatistical analysis of the percentage of IgG1 switched cells per generation of proliferating WT and KO B cells after 4 days of stimulation with anti-CD40 + IL-4. Results represent the data from one experiment, and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4).D, EStatistical analysis of switching of WT and KO naïve B cells activated for 4 days with LPS alone (IgG3 switching) (D) or with LPS + IL-4 (IgG1 switching) (E). Results represent the data pooled from at least four independent experiments, and numbers represent biological replicates for each group (D: (WT) n = 8, (KO) n = 9; E: (WT) n = 11, (KO) n = 13).FStatistical analysis of IgG1 switching of naïve B cells after 4 days of activation with anti-CD40 + IL-4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment, and numbers represent biological replicates for each treatment (n = 4).GNumber of cell divisions traced by CTV dilution of B cells stimulated for 4 days with anti-CD40 + IL-4 either in the presence of DOT1L inhibitor Pinometostat or DMSO as a control. Data represents three biological replicates for each treatment.HStatistical analysis of the percentage of IgG1 switched cells per generation of proliferating WT B cells after 4 days of stimulation with anti-CD40 + IL-4 either in the presence of DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment, and numbers represent biological replicates for each treatment (n = 3).IStatistical analysis of IgG3 switching of naïve B cells after 4 days of activation with LPS alone in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment, and numbers represent biological replicates for each treatment (n = 4). Data information: Statistical analyses were performed using the Student’s two-tailed unpaired t-test. Statistical significance was determined by calculating the P-value. A P-value of less than 0.05 was considered as significant. Bars and error bars indicate mean ± SD.

Fig 4: A conjugate CDP-RBD vaccine elicits faster antibody responses compared to its unconjugated counterpart(A) Timeline of mouse vaccinations. Mice were immunized with 100 µg of RBD or CDP-RBD on days 0 and 14. Sera were collected from all mice before vaccination (day 0) and at experimental days 13, 21, 28, and 35 (n = 10 for days 13 and 21, n = 5 for days 28 and 35).(B) Vaccine proteins were mixed with Montanide ISA 720 together with CpG 1826 oligonucleotide (MnC).(C) Anti-RBD total immunoglobulin (Ig) endpoint titers for both vaccine groups at experimental days 13, 21, 28 and 35 as assessed by ELISA. The number of responding mice is provided for each timepoint.(D) Anti-RBD total immunoglobulin (Ig) titers at day 21 (left) and day 35 (right).(E) Analysis of endpoint titers of RBD-specific IgG subclasses (IgG1, IgG2a, IgG2b, IgG3) at day 21. The number of responding mice is provided for each IgG subclass.(F) Surrogate virus neutralization assay. Circulating neutralizing antibodies in the sera of vaccinated mice inhibit the interaction between RBD and the ACE-2 receptor.(G and H) Surface Plasmon Resonance (SPR) biosensor assay. Binding of anti-RBD antibodies of mouse sera toward commercial RBD. Sera were diluted 1:100.(I) Total immunoglobulin (Ig) endpoint titers of CDP-RBD immunized mice at experimental day 21 against the wild type-RBM, Delta-RBM and Omicron-RBM were assessed by ELISA. Sera were diluted 1:100.Data are shown as geometric mean values ± geometric SD (C and E), as mean values ±SD (D, F, and H) or as Box-and-whisker plots (I). Statistical significance was determined by an unpaired, Mann-Whitney test for each time point (C and H) or IgG subclass (E) or by a two-way ANOVA followed by Sidak’s multiple-comparison test (F and I). (*p < 0.05, **p < 0.01).

Fig 5: Dot1L-deficient B cells show reduced serum Ig titers and aberrant plasma cells formation A–EStatistical analysis of resting serum titers of IgM (A), IgA (B), IgG1 (C), IgG2b (D), and IgG3 (E) from WT and KO mice. P-value for IgA (B) was calculated by Mann–Whitney test. Results represent the data from two independent experiments and numbers represent biological replicates for each group (WT; n = 8, KO; n = 8).FRepresentative flow cytometry plots showing gating strategy to identify and compare plasma cells (Sca-I+CD138+) between WT and KO cells after 4 days of stimulation with LPS + IL-4 (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from three independent experiments, and numbers represent biological replicates for each group (WT; n = 10, KO; n = 10).GRepresentative flow cytometry plots showing gating strategy to identify and compare activated B cells (CD138+ Blimp1+), Pre-PB cells (CD138- Blimp1+) and plasma blast (PB) cells between WT and KO cells after 4 days of stimulation with LPS + IL-4 (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment, and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4).HRepresentative flow cytometry plots showing the relative surface density for B220 (CD45R) of WT and KO cells after 4 days of stimulation with LPS + IL-4 (left panel) and statistical analysis of MFI of B220 for each group (right panel). Results represent the data from three independent experiments, and numbers represent biological replicates for each group (WT; n = 10, KO; n = 10).IRepresentative histograms showing the relative surface density for CD19 on total live WT and KO cells after 4 days of stimulation with LPS + IL-4 (left panel) and statistical analysis of MFI of CD19 for each group (right panel). Results represent the data from one experiment, and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4). Data information: Statistical analyses were performed using the Student’s two-tailed unpaired t-test. Statistical significance was determined by calculating the P-value. A P-value less than 0.05 was considered as significant. Bars and error bars indicate mean ± SD.