Figure 1.
BLT humanized mice exhibit profound plasma hypogammaglobulinemia.
(A) BLT mouse plasma (n = 13) was collected and analyzed by ELISA for the presence of human IgM, IgA and IgG. Log transformed plasma Ig levels in BLT mice are plotted on a linear y-axis and shown superimposed over the mean (center horizontal line) and 95% range (shaded area) Ig values from adult humans. (B) At harvest, ELISPOT analyses for plasma cells were performed with mononuclear cells isolated from the indicated tissues of BLT mice (n = 4). Statistical comparisons were 1 way ANOVA with Bonferroni's multiple comparisons tests. Plot colors are matched to the colors in (A). * indicates a p value less than 0.05. ** indicates a p value less than 0.01. *** indicates a p value less than 0.001. (C) Sections from the spleens (left) and lymph nodes (right) of BLT mice analyzed for lymphoid architecture via H&E staining. BALB/c tissues were included as positive controls for the appearance of normal immune structures. Scale bars = 50 µm in all images. Boxes indicate the areas that are shown at higher magnification in the images below.
Figure 2.
BLT mouse peripheral blood harbors few unswitched and switched memory B cells relative to adult human peripheral blood.
(A–B) Representative flow cytometry analyses for CD27 and IgD expression on B cells in adult human PB and BLT mouse PB shows a paucity of unswitched/switched memory B cells in BLT mouse blood. (adult PB, n = 5; BLT PB, n = 18). (C–D) Flow cytometry plots and bar graph for CD38 and IgD expression on CD19+ cells reveal an absence of switched memory B cell populations in BLT mouse PB while these populations are present in adult human PB (adult PB, n = 5; BLT PB n = 16). * indicates a p value less than 0.05. ** indicates a p value less than 0.01. *** indicates a p value less than 0.001. Comprehensive statistical analyses are presented in Table 1.
Table 1.
Comparison of B cell populations between BLT mouse and adult human peripheral blood using CD27/IgD and CD38/IgD co-expression patterns.
Figure 3.
B cell ontogeny in the bone marrow of BLT mice follows canonical patterns.
(A–B) Flow cytometry and scatter plots reveal the relative paucity of mature B cells in the BM as defined according to CD10 and CD20 expression. (n = 15; 1 way ANOVA with Bonferroni's multiple comparisons tests). (C–D) Flow cytometry and scatter plots reveal that, as expected, immature B cells exhibited higher levels intracellular TdT expression, a protein that is active during B cell receptor rearrangement. (n = 15; 1 way ANOVA with Bonferroni's multiple comparisons tests). (E–F) Flow cytometry and scatter plots for CD10 expression show that the majority of BLT mouse BM CD19+ cells are Pre-B/Immature B cells. (n = 13, t-test). (G–H) More BLT mouse Pre-B/Immature B cells identified in (E–F) express the pre-B cell receptor surrogate light chain component lambda 5 than CD10neg mature B cells. (n = 4, t-test). (I–J) Conversely, more CD10neg mature B cells expressed intracellular IgM than did the Pre-B/Immature B cells present in BLT mouse BM (n = 13, t-test). ** indicates a p value less than 0.01. *** indicates a p value less than 0.001.
Figure 4.
Most B cells in BLT mouse tissues exhibit an immature phenotype.
(A–B) Flow cytometry plots and bar graph for CD27 and IgD expression on B cells reveal an absence of unswitched/switched memory B cells in BLT mouse BM, spleen, LN, liver and lungs (n = 5 for all tissues). (C–D) Flow cytometry plots and bar graph for CD38 and IgD expression on CD19+ cells reveal an absence of switched memory B cell populations in BLT mouse tissues (n = 5 for all tissues). * indicates a p value less than 0.05. ** indicates a p value less than 0.01. *** indicates a p value less than 0.001. Comprehensive statistical analyses are presented in Table 2.
Table 2.
B cell populations in BLT mice according to CD27/IgD and CD38/IgD co-expression patterns.
Figure 5.
Repeated immunizations with PC-KLH induced B cell differentiation in BLT mouse peripheral blood towards mature phenotypes and increased plasma antigen-specific IgM.
(A) Immunization schedule. (B–E) The B cell populations in the PB of BLT mice (n = 7) were characterized before, during and after repeated PC-KLH immunizations. Representative flow cytometry analyses for CD27 and IgD expression on PB B cells (B) and longitudinal analysis of B cell populations (C) are shown. Similarly, (D–E) depict representative flow cytometry and longitudinal data regarding CD38 and IgD expression of PB B cells. (F) The percentage of CD38++IgDneg B cells that also express CD27 was calculated pre- and post-immunization to determine the contribution of atypical switched memory B cells to the CD27negIgDneg population in (B). (G) Plasma levels of antigen-specific IgM and IgG were measured longitudinally throughout the course of repeated PC-KLH immunizations (n = 7).
Table 3.
Comparison of B cell populations between naïve and PC-KLH immunized BLT mice using CD27/IgD and CD38/IgD co-expression patterns.
Figure 6.
Repeated immunizations with PC-KLH induced B cell differentiation in BLT mouse tissues towards mature phenotypes.
(A–B) B cell populations in the BM, spleen, LN, liver and lungs were characterized in naïve and immunized BLT mice. CD27 and IgD (A) (naïve, n = 5; immunized, n = 7 for all tissues; t-tests) and CD38 and IgD (C) (naïve, n = 5; immunized, n = 7; t-tests) co-expression patterns revealed increased levels of CD19+ cells exhibiting mature phenotypes throughout the body following the repeated PC-KLH immunizations. (C) The percentage of CD38++IgDneg B cells that also express CD27 was calculated in naive BLT mice and PC-KLH immunized BLT mice to determine the contribution of atypical switched memory B cells to the CD27negIgDneg populations from each tissue in (A). (D) Sections from spleen (left) and lymph node (right) of PC-KLH immunized BLT mice analyzed for lymphoid architecture via H&E staining. Scale bars = 50 µm in all images. Boxes indicate the areas that are shown at higher magnification in the images below. * indicates a p value less than 0.05. ** indicates a p value less than 0.01. *** indicates a p value less than 0.001. Comprehensive statistical analyses are presented in Table 3.
Figure 7.
PC-KLH induced systemic production of PC-specific IgM expressing plasma cells in BLT mice.
ELISPOT analyses revealed the presence of PC-specific IgM producing plasma cells in the BM (n = 6), spleen (n = 6), LN (n = 6), liver (n = 6) and lungs (n = 3) of immunized BLT mice. PC-specific IgG, KLH-specific IgM and KLH-specific IgG producing plasma cells were rare in each of the tissues sampled. Statistical analyses were 1 way ANOVA with Bonferroni's multiple comparisons tests. *** indicates a p value less than 0.001.