Apoptosis and antigen affinity limit effector cell differentiation of a single naïve B cell by Justin J. Taylor, Kathryn A. Pape, Holly R. Steach, and Marc K. Jenkins Science Volume 347(6223):784-787 February 13, 2015 Published by AAAS

Fig. 1 Assessing the polyclonal APC-specific B cell response Fig. 1 Assessing the polyclonal APC-specific B cell response.(A) Detection and (B) quantitation of APC-specific B cells from pooled spleen and lymph node samples enriched using anti-APC microbeads from naïve mice (n = 18) or mice immunized with APC in CFA (n = 45) 7 days earlier. Assessing the polyclonal APC-specific B cell response.(A) Detection and (B) quantitation of APC-specific B cells from pooled spleen and lymph node samples enriched using anti-APC microbeads from naïve mice (n = 18) or mice immunized with APC in CFA (n = 45) 7 days earlier. (C) Detection and (D) quantitation of APC-specific plasma cells (Ighigh), GC (Ig+ B220+ CD38- GL7+), naïve/memory (M) (Ig+ B220+ CD38+ GL7–), and AP B cells (Ig+ B220+ CD38+ GL7+). Memory B cells were quantitated by the increase in CD38+ GL7– cells over uninjected controls. (E) Detection and (F) quantitation of CFSEhigh CD45.1+ APC-specific B cells from CD45.2+ mice that received 2 × 107 donor CD45.1+ B cells before immunization with APC in CFA, or CFA alone (n = 10). Numbers on the flow cytometry plots in (A), (C), and (E) reflect the percent of cells within the gated population. These percentages and knowledge of the total number of B cells in the enriched fraction were used to calculate the number of cells shown in (B), (D), and (F). The bars represent the mean and P values determined using an unpaired two-tailed Student’s t test. Data points were combined from two to six experiments. Justin J. Taylor et al. Science 2015;347:784-787 Published by AAAS

Fig. 2 Assessing the response of an individual naïve APC-specific B cell.(A) Detection of APC-specific donor cells from CD45.2+ recipients that received 0.2 or 2 × 106 CFSE or Celltrace violet (CTV)–labeled CD45.1+ B cells 1 to 3 days before immunization with APC in CFA. Samples were analyzed 7 days after immunization, after simultaneous CD45.1 and APC-based cell enrichment. Assessing the response of an individual naïve APC-specific B cell.(A) Detection of APC-specific donor cells from CD45.2+ recipients that received 0.2 or 2 × 106 CFSE or Celltrace violet (CTV)–labeled CD45.1+ B cells 1 to 3 days before immunization with APC in CFA. Samples were analyzed 7 days after immunization, after simultaneous CD45.1 and APC-based cell enrichment. The fourth and fifth plots show CFSE profiles for gated populations a and b from the second and third plots. Numbers on the plots reflect the percent of cells within the gated population. (B) Frequency of immunized recipient mice (n = 6 for mice that received 2 × 106 cells or n = 384 for those that received 0.2 × 106) containing an APC-specific CFSE/CTVlow donor population above the limit of detection (LOD) of two cells. (C) Total number of cells in APC-specific clonal populations from 74 mice that received 0.2 × 106 cells and contained a population above the LOD. (D) Frequency of each subset within polyclonal or clonal APC-specific populations. Subsets are gated as shown in Fig. 1C, and each row depicts an individual clone (n = 74) or the entire APC-specific population from a mouse (n = 45). (E) Frequency of APC-specific clones generating one, two, three, or four subsets. (F) Total number of cells produced by each clone, separated into groups based on the number of subsets produced. A Mann-Whitney test was used to generate the P values. Bars in (C) and (F) represent medians. Data points were combined from 12 experiments. Justin J. Taylor et al. Science 2015;347:784-787 Published by AAAS

Fig. 3 Assessing proliferation and apoptosis of APC-specific clones Fig. 3 Assessing proliferation and apoptosis of APC-specific clones.(A) Frequency of cells in each CFSE/CTV division bin (Div) in wild-type (n = 74) or Bim-deficient (n = 34) APC-specific clonal populations. Assessing proliferation and apoptosis of APC-specific clones.(A) Frequency of cells in each CFSE/CTV division bin (Div) in wild-type (n = 74) or Bim-deficient (n = 34) APC-specific clonal populations. Clones are displayed in the same order as in Fig. 2D. (B) Total number of cells detected for each wild-type (open circle) or Bim-deficient (black triangle) clone compared to the frequency of cells completing at least seven divisions. (C and D) Number of cells detected for each clone displayed as a percentage of the minimum number predicted based on CFSE/CTV dilution analysis, with the clones grouped in (D) based on the number of subsets produced. (E) Total number of cells produced by APC-specific Bim-deficient clones. (F and G) Frequency of clones that produced (F) the indicated number or types of subsets or (G) any of the indicated subset. The bars in (C) to (E) represent medians. P values were determined in (C) and (D) using a Mann-Whitney test and in (F) and (G) using Fisher’s exact test. Data points were combined from 3 to 12 experiments. Justin J. Taylor et al. Science 2015;347:784-787 Published by AAAS

Fig. 4 Assessing the response of an individual BCR transgenic B cell Fig. 4 Assessing the response of an individual BCR transgenic B cell.(A) Representative detection of CD45.2+ IgMa CFSElow donor cells from recipients of a limited number (5 to 15 cells) of MD4 Rag1−/− B cells 1 to 3 days before immunization with HEL–OVA or DEL-OVA in CFA. Samples were analyzed 7 days after immunization after CD45.2-based cell enrichment. Assessing the response of an individual BCR transgenic B cell.(A) Representative detection of CD45.2+ IgMa CFSElow donor cells from recipients of a limited number (5 to 15 cells) of MD4 Rag1−/− B cells 1 to 3 days before immunization with HEL–OVA or DEL-OVA in CFA. Samples were analyzed 7 days after immunization after CD45.2-based cell enrichment. The second and third plots show gated populations a and b from the first and second plots. Numbers on the plots reflect the percent of cells within the gated population. (B) Total number of cells in HEL-stimulated MD4 (black circles, n = 31), MD4 DEL-stimulated (gray circles, n = 40), or AID-deficient APC-specific (white circles, n = 27) clonal populations generated by immunization. (C and D) Frequency of clones that produced (C) the indicated subset combinations or (D) or any of the indicated subset. (E) Amount of APC staining of memory cells in clonal APC-specific populations that produced memory cells and plasma cells or memory cells but not plasma cells. The bars in (B) and (E) represent medians. P values were determined in (B) and (E) using a Mann-Whitney test and in (C) and (D) using Fisher’s exact test. Data points were combined from 17 MD4 experiments, 3 AID-deficient experiments, and 12 wild-type experiments. Justin J. Taylor et al. Science 2015;347:784-787 Published by AAAS