Figure 3.

Clonal analysis, CDRH3 length distribution, and V gene usage of B cells isolated from different tissues. (A) Schematic representation of clonal polarization. Each color represents a single clone. As the number of clones increases, the polarization decreases. (B) Polarization curve; the mean number of clones (log₁₀) that contribute to 80% of all V_H sequences reads in each tissue is shown on the y-axis. The lower the value, the higher the polarization level of the B cell response. Error bars represent the standard error of the mean (SEM). (C) Hill diversity (log₁₀) – used as a measure of clonal diversity – was calculated for all clones in each tissue. Error bars represent standard deviation. (D) CDRH3 length distribution of intra-tumoral and DLN unqiue V_H seqeunces. The median CDRH3 length of clones from the tumor or the DLNs of treated mice (orange/purple bars) was compared to that of naïve clones (red bars). Statistical significance was determined by using an nonparametric, unpaired, two-sided, Mann-Whitney t-test. For both the DLNs and the tumor, the p-value <0.05 was considered statistically significant, ****p ≤ 0.0001. (E) Bar plots of V gene usage frequency in each tissue. All V genes were sorted according to the frequency of V genes observed in the naïve B cells subset (IgM⁺ B cells from the bone narrow of naïve mice). Number of V genes used in each tissue is detailed on the bar plot. Naïve - naïve mice, Treated - tumor-bearing mice.