Supplementary Figure Legends
Figure S1.Flow cytometry analysis of antigen-specific reagents.
A. Representative flow cytometry gating strategy for colostrum B cell phenotype analysis. A broad lymphocyte gate was drawn on a forward versus side scatter plot (top far left plot), followed by exclusion of clumps and doublets using side scatter and forward scatter gating (top center plots). Total live cells were gated as Aqua Vital Dye–, shown in the top far right plot. Total B cells were gated as CD19+ and CD3–/CD14–/CD16–/CD235a– (blue box, bottom far left plot, 14.4%). The population of total B cells was then analyzed to determine the proportion of IgD– cells, IgD–/CD38Hi cells (red box, bottom left center plot, 1.8%), IgD–/CD27+ cells (blue box, bottom right center plot, 83%), and HIV-1 Env-specific cells (blue box, bottom far right plot, 3.9%).
B. Compensation matrix adjustment. To assess whether the observed diagonal was due to undercompensation of the sample, we adjusted the compensation matrix to assess the change in the diagonal of antigen-specific B cells. The original matrix is shown on the left and is the same as the bottom right panel of S1A. Increasing amounts of compensation applied to the sample are shown. In the upper row, AF647 signal is subtracted from the BV421 signal; in the bottom row, BV421 signal is subtracted from the AF647 signal; in the middle row, both are done simultaneously. As expected, the position of the diagonal changes slightly with increasing applied compensation, but even with high levels of applied compensation (50%), the diagonal is not eliminated.
C. Phenotyping of uninfected PBMC. A sample of PBMC from an HIV-1 uninfected woman was phenotyped using the same panel as in S1A. Using a strategy analogous to the one above, lymphocytes, singlets, viable cells, B cells, and memory B cells are gated on, followed by analysis of the antigen-specific B cell population. No antigen-specific B cells were detected. No antigen-specific B cells were found in the colostrum B cells from the same participant (data not shown).
Figure S2. Env specificities of antibodies isolated from blood and colostrum B cells. HIV-1 Env-specific antibody populations from blood (left) and colostrum (right) are illustrated in donut charts for each of the eight subjects from which colostrum and blood Env-specific B cells were isolated. Env specificity is indicated by color according to the legend on the right. CH404 is the only transmitting subject from whom multiple HIV-1 Env-specific colostrum antibodies were isolated.
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