![]() ![]() 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). (E) Bar plots of V gene usage frequency in each tissue. For both the DLNs and the tumor, the p-value <0.05 was considered statistically significant, ****p ≤ 0.0001. Statistical significance was determined by using an nonparametric, unpaired, two-sided, Mann-Whitney t-test. 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). (D) CDRH3 length distribution of intra-tumoral and DLN unqiue V H seqeunces. (C) Hill diversity (log 10) – used as a measure of clonal diversity – was calculated for all clones in each tissue. Error bars represent the standard error of the mean (SEM). The lower the value, the higher the polarization level of the B cell response. ![]() (B) Polarization curve the mean number of clones (log 10) that contribute to 80% of all V H sequences reads in each tissue is shown on the y-axis. As the number of clones increases, the polarization decreases. (A) Schematic representation of clonal polarization. BM, bone marrow DLN, draining lymph node.Ĭlonal analysis, CDRH3 length distribution, and V gene usage of B cells isolated from different tissues. R s, Spearman rank-order coefficient p-values are indicated. (F) Rarefaction curves for sequences from all tissues. (E) Correlation between library duplicates from all tissues. Each sequence is represented by a blue dot. X and Y axes are on a logarithmic scale and represent the transcript counts for each sequence. Transcript counts for each V H sequence were correlated between duplicates. The vertical dashed line indicates the percent of V H sequences reads in which unique V H sequence diversity reaches 99%. (C) Rarefaction curve for the shared V H sequences as identified in both technical duplicates. X-axis represents the percent of sampled V H sequences reads in each iteration out of the total number of V H sequences reads in the sample, and Y-axis represents diversity of unique V H sequences that were sampled as a percent out of the total number of unique V H sequences (B) Venn diagram of the duplicates, showing an example of the number of unique V H sequences in each duplicate and the number of shared unique V H sequences between duplicates. (A) Rarefaction curves for each duplicate originating from the same sample. The data thus suggests that antibody repertoire signatures can serve as indicators for identifying tumor-reactive B cells.ĪIRR-seq B cell BCR-Seq VDJ recombination antibody repertoire next generation sequencing triple negative breast cancer tumor infiltrating lymphocytes.Ĭopyright © 2021 Aizik, Dror, Taussig, Barzel, Carmi and Wine.ĭuplicate approach for removal of errors from BCR-Seq data. Tracing the distribution of TIL-B clones across various compartments indicated that they migrate to and from the TME. Importantly, TIL-Bs were highly mutated but non-class switched, suggesting that class-switch recombination may be inhibited in the TME. We found that TIL-Bs exhibit distinct antibody repertoire measures, including high clonal polarization and elevated somatic hypermutation rates, suggesting a local antigen-driven B-cell response. Here, we utilized B cell receptor high-throughput sequencing (BCR-Seq) to profile the antibody repertoire signature of tumor-infiltrating lymphocyte B cells (TIL-Bs) in comparison to B cells from three anatomic compartments in a mouse model of triple-negative breast cancer. The role of B cells in the tumor microenvironment (TME) has largely been under investigated, and data regarding the antibody repertoire encoded by B cells in the TME and the adjacent lymphoid organs are scarce. ![]()
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