1. Increased survival is a selective feature of human circulating antigen-induced plasma cells synthesizing high-affinity antibodies
by Inés González-García, Beatriz Rodríguez-Bayona, Francisco Mora-López, Antonio Campos-Caro, and José A. Brieva
Blood
Volume 111(2):
January 15, 2008
©2008 by American Society of Hematology

2. Detection of tetC− and 2 different subsets of tetC+ PCs in the peripheral blood of volunteers, 6 days after tet immunization.
Detection of tetC− and 2 different subsets of tetC+ PCs in the peripheral blood of volunteers, 6 days after tet immunization. (A) PCs were identified as CD19+ CD38HIGH cells in a CD19/CD38 dot plot obtained by FACS (left panel). The presence of tetCINT and tetCHIGH cell subsets was established by labeling permeabilized CD19 CD38HIGH cells with increasing quantities of FITC-tetC. Right panel shows a representative experiment. FITC-tetC concentrations of 500 ng/mL or less only distinguished 2 cell subsets (tetC− and tetC+; top left), while the use of higher concentrations revealed the presence of 3 different cell subsets (tetCNEG, tetCINT, and tetCHIGH; bottom left). (B) Representative control experiments showing the expression histograms of FITC-tetC staining (2 μg/mL) when the cells were coincubated with increasing quantities of unlabeled tet (left panels) or with 5% BSA (middle right), and when the cells were not permeabilized (lower right). Pale line denotes background control. (C) Temporal kinetics of tetCINT and tetCHIGH cells. The percentages of circulating tetCINT and tetCHIGH CD19+ CD38HIGH cells were obtained from day 3 to day 8 after booster. Results represent the mean plus or minus SEM (n = 7).
Inés González-García et al. Blood 2008;111:
©2008 by American Society of Hematology

3. Comparative flow cytometric phenotypic analysis of blood tetCNEG, tetCINT, and tetCHIGH PCs obtained 6 days after tet immunization.
Comparative flow cytometric phenotypic analysis of blood tetCNEG, tetCINT, and tetCHIGH PCs obtained 6 days after tet immunization. A representative experiment is presented in the top row (top series of dot plots) showing the expression of isotypic control, CD27, CD38, CD138, and HLA-DR, respectively, by tetCNEG, tetCINT, and tetCHIGHCD19 CD38HIGH cells. The bottom row summarizes the results observed in several donors. Values are expressed as the MFI of expression for each molecule in positive PCs. Results represent the mean plus or minus SEM (n = 6). *Statistically significant differences.
Inés González-García et al. Blood 2008;111:
©2008 by American Society of Hematology

4. Isolation of tetCNEG, tetCINT, and tetCHIGH PCs and comparison of the quantity of cytoplasmic Ig. (A) The 3 PC subsets were purified from peripheral blood by FAC sorting.
Isolation of tetCNEG, tetCINT, and tetCHIGH PCs and comparison of the quantity of cytoplasmic Ig. (A) The 3 PC subsets were purified from peripheral blood by FAC sorting. A May-Grunwald-Giemsa staining is shown in the top row; immunofluorescence confocal microscopy for FITC-tetC and IgG are shown in the middle and bottom rows, respectively (original amplification 40×/0.75 NA). (B) Top row: representative experiments showing the flow cytometry dot plots analysis of the intracellular expression of Igγ, Igκ, and Igλ light chains by tetCNEG, tetCINT, and tetCHIGH PCs. Bottom row: Bar histograms summarize the results obtained in different donors. Values are expressed as the MFI change (MFI of positive PCs minus MFI of negative PCs) for IgG and Igκ light-chain expression, and as MFI of positive PCs for Igλ light chain. Results represent the mean plus or minus SEM (n = 6). (C) Quantification of the IgG contained in purified tetCINT and tetCHIGH PCs. Whole-cell lysates were prepared in Triton X-100, and the lysed cells were centrifuged to remove the cell debris. The quantity of IgG released was assessed by ELISA. Results are expressed as picograms of IgG per cell obtained in 2 different experiments. (D) Comparison of the quantity of IGγ1 mRNA expressed by blood isolated tetCINT and tetCHIGH PCs. Values were normalized using as endogenous calibrators those obtained for tetCNEG PCs in each experiment, which were taken as 1. Results represent the mean plus or minus SEM (n = 6).
Inés González-García et al. Blood 2008;111:
©2008 by American Society of Hematology

5. Anti-tetC Ab contained in tetCHIGH and tetCINT PC subsets differed in their affinity for tetC.
Anti-tetC Ab contained in tetCHIGH and tetCINT PC subsets differed in their affinity for tetC. A flow cytometric assay was used to assess the specific tetC binding of the Ab synthesized by these PC subsets in which MFI of FITC-tetC staining is plotted against FITC-tetC concentration. A representative experiment of the saturation curves of tetCHIGH PC (top panel) and tetCINT PC (bottom panel) for FITC-tetC is shown. Insets show the corresponding Scatchard plots. Bmax and relative KD data are also included.
Inés González-García et al. Blood 2008;111:
©2008 by American Society of Hematology

6. Affinity maturation of IgG synthesized by tetCHIGH, tetCINT, and tetCNEG PCs. A total of 86 different IGVH3 gene sequences were obtained from isolated PC subsets of 4 different individuals, and the numbers of R and S mutations present in the CDR1 and CDR2, ...
Affinity maturation of IgG synthesized by tetCHIGH, tetCINT, and tetCNEG PCs. A total of 86 different IGVH3 gene sequences were obtained from isolated PC subsets of 4 different individuals, and the numbers of R and S mutations present in the CDR1 and CDR2, as well as the corresponding R/R + S ratio, were calculated. The theoretical R/R + S ratio value calculated for random mutations45 is shown as a dotted line. Results represent the mean plus or minus SEM of the different sequences obtained (n = 33, 32, and 16 for isolated tetCHIGH, tetCINT, and tetCNEG PCs, respectively). *Statistically significant differences.
Inés González-García et al. Blood 2008;111:
©2008 by American Society of Hematology

7. Functional capacities of tetCHIGH, tetCINT, and tetCNEG PC subsets.
Functional capacities of tetCHIGH, tetCINT, and tetCNEG PC subsets. (A-C) Left panels show a representative histogram of APO2.7 expression (A) and activated caspase-3 expression after a 24-hour culture period (B), and BrdU uptake after an 18-hour incubation period (C), by the 3 blood PC subsets. The corresponding bar histograms on the right summarize the results obtained from several donors. Values are expressed as the MFI of the expression of positive PCs (A,B) and as the percentage of positive PCs (C). Results represent the mean plus or minus SEM (n = 5 for panels A-C). (D) Recovery of tetCNEG, tetCINT, and tetCHIGH PCs in blood cell cultures. The percentage of tetCNEG, tetCINT, and tetCHIGH PCs recovered initially (0 hours), and after 24 and 48 hours of culture, were determined by flow cytometry. Results represent the mean plus or minus SEM (n = 6). (E) CXCL12-induced chemotaxis of tetCNEG, tetCINT, and tetCHIGH PCs. CXCL12 (1 μg/mL) was added to the bottom chamber of a transwell culture system. Values are expressed as the percentage of migrated PCs. Results represent the mean plus or minus SEM (n = 6). Asterisks in panels A through C indicate statistically significant differences among PC subsets, and in panels D and E indicate statistically significant differences within every PC subset among data from different times (D) or from different experimental conditions (E).
Inés González-García et al. Blood 2008;111:
©2008 by American Society of Hematology