T-Bet+ IgM Memory Cells Generate Multi-lineage Effector B Cells

Abstract

Immunoglobulin M (IgM) memory cells undergo differentiation in germinal centers following antigen challenge, but the full effector cell potential of these cells is unknown. We monitored the differentiation of enhanced yellow fluorescent protein (eYFP)-labeled CD11c⁺ and CD11c^neg T-bet⁺ IgM memory cells after their transfer into naive recipient mice. Following challenge infection, many memory cells differentiated into IgM-producing plasmablasts. Other donor B cells entered germinal centers, downregulated CD11c, underwent class switch recombination, and became switched memory cells. Yet other donor cells were maintained as IgM memory cells, and these IgM memory cells retained their multi-lineage potential following serial transfer. These findings were corroborated at the molecular level using immune repertoire analyses. Thus, IgM memory cells can differentiate into all effector B cell lineages and undergo self-renewal, properties that are characteristic of stem cells. We propose that these memory cells exist to provide long-term multi-functional immunity and act primarily to maintain the production of protective antibodies.

Figure 1.. Characterization of Aicda-Expressing IgM+ Memory Cells In Vivo

E.-muris-infected (AID-creERT2 × ROSA26-eYFP) F1 mice were administered tamoxifen on days 7 and 10 post-infection, and splenocytes were analyzed on day 70 post-infection. (A) eYFP+ GL7neg CD138neg IgM+ memory cells (R1), CD19hi B cells (R2), CD19+ follicular B cells (R3), and eYFP+ GL7neg CD138neg IgMneg switched memory cells (R4) were identified. Data from a representative experiment are shown in the plots at the top; the plots at the bottom are aggregate data indicating the frequency of each ofthe populations. *p < 0.05, ***p < 0.001, and ****p < 0.0001. (B) The B cells identified in the regions defined in (A) were monitored for their expression of a panel of markers previously characterized on IgM memory B cells (Yates et al., 2013). Cells in R1 are shown in blue and R2 in red; R3 cells are indicated with a black line (open histograms). (C) The expression of the indicated markers was analyzed on eYFP+ GL7neg CD138neg IgM+ memory cells (R4; orange histogram) and eYFP+ GL7neg CD138neg IgMneg memory cells (R1; blue histogram); overlapping cells appear as green. (D) The expression of CD11b was analyzed in eYFP+ GL7neg CD138neg CD11c+ (purple histogram) and CD11cneg IgM+ memory cells (green histogram). The data in (A)-(D) are representative of two experiments that used 4 mice per group. (A) Statistical significance was determined using a repeated- measures one-way ANOVA with Tukey’s multiple comparison test for the left (p < 0.0001; F = 0.678; df =11) and middle panels (p < 0.0001; F = 0.0002; df = 11) or a two-tailed paired t test for the data in the right panel (p < 0.0001;t = 59;df = 3). In (C) and (D), **p < 0.01, ***p < 0.001, and ****p < 0.001.(E) A Venn diagram is shown that illustrates the relationships between the various populations that were characterized. CD11c+ and CD11cneg cells and cells expression Aicda are indicated by the colors. IgM and swIg memory cellsare indicated by cross-hatching. See text for details.

Figure 2.. IgM Memory Cells Differentiated Only after Challenge Infection

(A) Purification ofIgM memoryBcells. eYFP+GL7neg CD138neg IgGneg splenic B cellswere purified by flow cytometry; representative dot plots show expression of the indicated cell surface markers among the eYFP+ cells prior to (open histograms) and after (red histograms) purification. (B) Purified IgM memory cells were transferred into naive mice (top panels), mice infected for50 days (middle panels), or naive micethat were challenged at the time of cell transfer; each of the groups of recipient mice were analyzed 12 days post-cell transfer. eYFP+ donor cells (gated in plots on left) were analyzed for expression ofGL7 and CD138 (middle plots). Expression of IgM was analyzed in GL7neg CD138neg memory cells (plots on right; red histograms); analyses ofthe GL7neg CD138+ASCs(bottom right plots; open histogram) and GL7+ CD138neg GC cells(blue histogram) are shown only for the challenged mice. The percentage of eYFP+ cells in each ofthe populations in the recipient mice is quantified in the plot on right. Statistical significance was determined using an ordinary one-way ANOVA (p < 0.0001; F = 948.2; df = 35) and a Holm-Sidak’s multiple comparison test. (C) Spleen cell numbers in the mice analyzed in (B) are shown. Statistical significance was determined using an ordinary one-way ANOVA (p < 0.0001; F = 28.7; df = 12) and with Tukey’s multiple comparison test. The data in (A)-(C) are representative of one experiment that used 4 or 5 mice per group. (D) Recipient mice were administered BrdU from day 14to21 following transferofT-cell-depleted splenocytes from infected (AID-Cre-ERT2 × Rosa26 eYFP) F1 mice; the recipient mice were infected immediately following cell transfer. Spleen IgM+, GL7neg and CD138neg, CD19+ B cells were analyzed on day 21 for BrdU incorporation. Aggregate data are shown in the panel on the right. Statistical significance was determined using a two-tailed paired t test (p = 0.0019; t = 7.289; df = 4). In (C) and (D), **p < 0.01, ***p < 0.001, and ****p < 0.001.

Figure 3.. IgM Memory B Cells Generated All Effector and Memory Subsets following Challenge Infection

(A) Purified eYFP+ IgM memory cells were transferred into naive mice, the recipient mice were infected, and splenic B cells were analyzed 31 days post-transfer. Representative analyses of differentiated eYFP+ donor-cell-derived B cells are shown; aggregate data are shown in the plots on the right. See text for details. Statistical significance was determined using a repeated-measure (RM) one-way ANOVA with Tukey’s multiple comparison test in upper (p < 0.0001; F = 196; df = 26) and lower (p < 0.0001; F = 125.8; df = 26) panels. (B) CXCR3 expression among eYFP+ CD38lo GL7+ GC B cells (shaded histogram) and eYFP+CD38hi GL7neg CD138neg memory cells (open histogram) on day 31 post-transfer. The MFI of each of the populations is shown in the plot on the right. Statistical significance was determined using a two-tailed Wilcoxon test (p = 0.125). (C) IgM and CD138 expression on eYFP+ donor-derived cells in the BM of recipient mice on day 31 post-transfer. Statistical significance was determined using an RM one-way ANOVA with Tukey’s multiple comparison test (p < 0.0001; F = 72.29; df = 23). The data in (A)-(C) are representative of 3 experiments that used 2–4 mice per group. In (A) and (C), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 4.. IgM Memory B Cell Differentiation Occurred Independently of CD11c

(A) Purification of CD11c⁺ and CD11c^neg IgM memory cells. eYFP⁺ GL7^neg CD138^neg IgG^neg memory cells were separated on the basis of CD11c expression. The purified CD11c⁺ (dashed histogram) and CD11c^neg (shaded histogram) B cells are shown. (B) Equal numbers CD11c⁺ or CD11c^neg eYFP⁺ IgM memory cells were transferred into naive mice, and the recipient mice were infected. Differentiation of eYFP⁺ cells in the spleen and BM of recipient mice was analyzed on day 30 post-transfer, as in Figure 3. The percentage of eYFP⁺ cells in each population and the percentage of CD11c⁺ among the memory cells are quantified in the plots at the bottom. Statistical significance was determined by doing a comparison between recipient mice for each population, using an ordinary one-way ANOVA (p < 0.0001; F = 26.79; df = 51) with Sidak’s multiple comparison test (left panel), two tailed unpaired t test (middle panel; p = 0.413; t = 0.8531; df = 10.3), and a Kruskal-Wallis test (p < 0.0001) with Dunn’s multiple comparison test (right panel). (C) CD11c expression among eYFP⁺ CD38^lo GL7⁺ GC B cells and eYFP⁺ CD38^hi GL7^neg CD138^neg CD11c⁺ memory cells in mice that received CD11c⁺ IgM memory cells 30 days post-transfer. MFI values are quantified in the plot below. Statistical significance was determined using a two-tailed paired t test (p < 0.0001; t = 46.5; df = 3). (D) Steady-state interconversion of IgM memory cells. CD11c⁺ or CD11c^neg eYFP⁺ IgM memory cells were transferred into recipient mice that had been infected for 47 days, and differentiation of donor cells in the spleen of the recipient mice was analyzed 21 days later. Statistical significance was determined using a two-tailed unpaired t test in the middle panel (p = 0.0031; t = 4.294; df = 7.335). The data are representative of 2 experiments (A-C), each containing 3 or 4 mice per group, or one experiment with 5 mice per group (D). In (C) and (D), **p < 0.01, ****p < 0.0001.

Figure 5.. IgM Memory B Cells Retained Their Multi-lineage Potential after Serial Transfer

(A) eYFP⁺ IgM memory cellswere purified from micethat had been recipientsofeYFP⁺ IgM memory cells 31 days prior; the donor cells were transferred into naive mice, which were subsequently infected. Splenocytes from the recipient mice were analyzed 30 days post-secondary transfer. Representative dot plots illustrating the gating forthe differentiation ofeYFP⁺ cells are shown (left); aggregate data are shown in the plots on the right. Statistical significance was determined using an RM one-way ANOVA with Tukey’s multiple comparison test for the upper panel (p < 0.0001; F = 57.8; df = 23) and lower panel (p < 0.0001; F = 269.1; df = 23). (B) Representative dot plots of IgM and CD138 expression on eYFP⁺ donor cells in the BM of recipient mice day 30 post-secondary transfer. Statistical significance was determined using an RM one-way ANOVA (p = 0.0001; F = 37.16; df = 27) with Tukey’s multiple comparison test. The data are representative of 3 experiments that used 2 or 3 mice per group. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 6.. IgH Chain Repertoire Analyses of Differentiated IgM Memory Cells

IgSeq analysis was performed on B cell populations generated from transferred IgM memory B cells. (A) Frequency of IgH V-gene usage in the donor IgM memory population and each IgM memory cell-derived B cell population. V-regions are indicated on the ordinate. (B) A Venn diagram is shown that illustrates the number of clonotypes shared among B cell populations in a representative mouse. Regions of overlap represent the number of clonotypes shared between the overlapping populations. (C) Clonal enrichment. The plot on the right shows the fold clonal enrichment (i.e., the clonal frequency divided by the expected frequency; assuming equal representation) of clones unique to each population and clones identified in all effector populations. For frequencies less than one, the negative reciprocal was plotted. (D) Percent of clonotypes shared among B cell populations. The blue bar segments represent the frequency of clones within each population that were also found in one other population; the red bars represent clones found in two other populations, and the green bars represents clones found in three other populations. The plot on the right shows the percentage of clones shared between at least one other population within an individual mouse. Each mouse was compared to a different recipient mouse from a different time point (control). Statistical significance was determined using a RM one-way ANOVA (p = 0.0008; F = 16.92; df = 29) with Dunnett’s multiple comparisons test. (E) A Circos plot representing the clonal overlap in pairwise comparisons of the different B cell populations is shown. (F) A lineage tree illustrating common clones identified in all of the isolated B cell populations. Black circles represent a germline clone, and white circles represent inferred clones. Daughter clones are shown in color, based on the effector population from which they were identified. The number of mutations from germline is indicated by the number shown in each circle. (G) The number of replacement and silent mutations, from germline, in the V-region of the indicated populations is shown. Statistical significance was determined by an ordinary one-way ANOVA (p < 0.0001; F = 1244; df = 145,175) with Tukey’s multiple comparisons test. ****p < 0.0001. (H) The percentage of clones with the indicated number of replacement and silent mutations in the V-region of the IgM memory donor population (left) and the IgM memory recipient population on day 27 post-challenge (right) is shown. Outliers have been removed for clarity. All data of the recipient mice are from 2 experiments with 3 mice per group. The donor population is representative of two experiments with three pooled mice per experiment.