New markers for murine memory B cells that define mutated and unmutated subsets

The study of murine memory B cells has been limited by small cell numbers and the lack of a definitive marker. We have addressed some of these difficulties with hapten-specific transgenic (Tg) mouse models that yield relatively large numbers of antigen-specific memory B cells upon immunization. Using these models, along with a 5-bromo-2′-deoxyuridine (BrdU) pulse-label strategy, we compared memory cells to their naive precursors in a comprehensive flow cytometric survey, thus revealing several new murine memory B cell markers. Most interestingly, memory cells were phenotypically heterogeneous. Particularly surprising was the finding of an unmutated memory B cell subset identified by the expression of CD80 and CD35. We confirmed these findings in an analogous V region knock-in mouse and/or in non-Tg mice. There also was anatomic heterogeneity, with BrdU+ memory cells residing not just in the marginal zone, as had been thought, but also in splenic follicles. These studies impact the current understanding of murine memory B cells by identifying new phenotypes and by challenging assumptions about the location and V region mutation status of memory cells. The apparent heterogeneity in the memory compartment implies either different origins and/or different functions, which we discuss

CD73 expression is dynamically regulated in the germinal center and bone marrow plasma cells are diminished in its absence.

CD73 catalyzes the conversion of extracellular nucleosides to adenosine, modulating inflammatory and T cell responses. Elevated expression of CD73 marks subpopulations of murine memory B cells (MBC), but its role in memory development or function is unknown. Here, we demonstrate that CD73 is progressively upregulated on germinal center (GC) B cells following immunization, is expressed at even higher levels among T follicular helper cells, but is absent among plasma cells (PC) and plasmablasts (PB). We analyzed the T-dependent B cell response in CD73 knockout mice (CD73KO). During the early response, CD73KO and wild type (WT) mice formed GCs, MBCs and splenic PBs and PCs similarly, and MBCs functioned similarly in the early secondary response. Late in the primary response, however, bone marrow (BM) PCs were markedly decreased in CD73KO animals. Tracking this phenotype, we found that CD73 expression was required on BM-derived cells for optimal BM PC responses. However, deletion of CD73 from either B or T lymphocytes alone did not recapitulate the phenotype. This suggests that CD73 expression is sufficient on either cell type, consistent with its function as an ectoenzyme. Together, these findings suggest that CD73-dependent adenosine signaling is prominent in the mature GC and required for establishment of the long-lived PC compartment, thus identifying a novel role for CD73 in humoral immunity

The BM PC compartment is diminished in the absence of BM-derived CD73.

<p>(<b>A</b>) Schematic of experimental design. Chimeric animals were established from adoptive transfer of WT or CD73KO donor BM into irradiated WT or CD73KO hosts. Donor and hosts were allotypically distinct (CD45.1 and CD45.2) in all chimeric combinations except the CD73KO donor/CD73KO host controls. 6-weeks post BM transfer, chimeric animals were immunized i.p. with NP-CGG in alum, and 11-weeks later, BM PCs were enumerated by ELISpot. Extent of chimerism was evaluated by flow cytometry analysis of splenocytes and is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092009#pone.0092009.s004" target="_blank">Figure S4</a> and in the text; 1–3 million events were collected per sample. (<b>B</b>) Evaluation of chimeric mice 11-weeks post-immunization. Frequency of IgG₁ NP-specific PCs per million BM cells, determined by ELISpot analysis. Each point represents an individual mouse. Data shown are pooled from 6 (WT into WT), 5 (KO into WT) and 1 (WT into KO and KO into KO) individual experiments. Mean values are depicted by heavy horizontal lines. * indicates Student's t-test p values of <0.05.</p

BM-resident PCs are diminished late in the primary response in the absence of CD73.

<p>On the indicated days post i.p. immunization with NP-CGG in alum, CD73KO (empty circles, dashed lines) or WT (filled squares, solid lines) spleen and BM were harvested for ELISpot analysis of NP-binding IgG₁⁺ PB/PCs. (<b>A</b>) Splenic Elispots. (<b>B</b>) BM Elispots. In A and B, upper and lower panels show frequencies of NP16- and NP2-binding Elispots, respectively. Day 28 and day 11 represent 4 and 2 independent experiments, respectively, each with 4–10 individual mice. Each other point is averaged from 4–10 individual mice in a single experiment. Error bars depict standard deviations. The symbols *, ** and **** represent t test values of <0.05, <0.01 and <0.0001, respectively. BM NP₁₆ paired t test for day 55–85 was 0.03. A day 72 BM NP₂ value was excluded due to technical variation. (<b>C</b>) Complied BM NP₁₆ day 70, 72, 77 and 85 ELISpots. Student's <i>t</i> test gave p<0.0001.</p

CD73 expression is modulated among responding antigen-specific B lineage cells in response to immunization with T-dependent antigen.

<p>Splenocytes and BM cells from B6 WT and control CD73KO mice were stained and analyzed by flow cytometry on the indicated days post i.p. immunization with NP-CGG in alum. Representative FACS histograms of CD73 expression are shown. 3 million events were collected per sample. (<b>A</b>) CD73 expression on splenic GC B cells from WT (solid line) or CD73 KO (shaded gray), identified as NIP⁺ kappa^lo CD38^low CD95^hi CD19⁺. For comparison, staining of WT kappa⁺ CD38^hi CD95^lo CD19⁺ cells, which are predominately naïve, is shown (dashed line). (<b>Upper right</b>) Percentage of CD73⁺ GC B cells as a function of time; each point represents an individual mouse. On days 72–77, GC B cells were gated as NIP⁺ kappa^lo CD95^hi CD19⁺ (without CD38 gating). (<b>B</b>) CD73 staining of PB/PC populations from the spleen and marrow of WT (solid line) and CD73KO (shaded gray) mice. Antigen specific splenic and BM PB/PC populations were identified as intracellular NIP₅BSA^hi surface B220⁻, and were a mixture of CD138^hi and CD138^lo cells. Similar results were seen when gated on IgG₁^hi.</p

Absence of CD73 does not significantly affect MBCs frequency, quality or function.

<p>(<b>A</b>) WT (filled squares, solid lines) and CD73KO (empty circles, dashed lines) mice were immunized i.p. with NP-CGG precipitated in alum, and at indicated times post immunization, the splenic MBC compartment was analyzed by FACS. Shown is fraction of live lymphocytes that were IgG₁-class-switched memory phenotype antigen specific B cells (IgG₁⁺ CD38⁺ NIP-binding κ⁻ CD19⁺). Day 28 represents 4 independent experiments and day 11 represents 2 independent experiments. 1.3–3 million events were collected per sample. Each point represents pooled data from 3–14 individual mice. Error bars represent standard deviations. (<b>B</b>) Comparison of mutational load within Vλ₁ sequences from IgG₁⁺ CD38⁺ NIP-binding κ⁻ MBCs purified by FACS from CD73KO (empty circles) and WT (filled squares) spleens. Each point represents an individual Vλ₁ sequence. Vλ₁ mutation rates did not significantly differ between groups by Student's <i>t</i> test. (<b>C</b>) Schematic of experimental design to analyze the secondary response mounted by MBCs formed in the absence of CD73. B6 WT or CD73KO mice were immunized i.p. with NP-CGG in alum. 8 or 12 weeks later, splenocytes were harvested and depleted of T cells. Resulting purified B cells containing 4.5×10³ IgG₁⁺ CD38⁺ NP-specific memory cells were transferred i.v. into CD45.1 congenic recipients. Recipients were i.p. immunized with NP-CGG in alum 16 hours post transfer. Splenocytes were analyzed 4 days later by FACS and ELISpot. (<b>D</b>) Numbers of memory phenotype IgG₁⁺ CD38⁺ NIP-binding splenic B cells after MBC adoptive transfer and secondary immunization. There were no significant differences between groups when analyzed by the Student's <i>t</i> test. For comparison, immunized recipient strain non-transferred “no cell control” (NCC) mice are shown (solid circle). (<b>E</b>) Frequencies of NP-binding IgG₁⁺ splenic PCs detected by ELISpot analysis post adoptive transfer and secondary immunization. There were no significant differences between groups (Student's <i>t</i> test). Symbols and shading as in D.</p

GCs are modestly altered in the absence of CD73.

<p>B6 WT (filled squares, solid lines) and CD73KO (empty circles, dashed lines) mice were immunized i.p. with NP-CGG precipitated in alum or alum alone. At the indicated times post immunization, splenocytes were analyzed by flow cytometry. Each point represents pooled data from 3–22 (NP-CGG) or 1–5 (alum alone) individual mice. Day 28 represents 4 independent experiments and day 11 represents 2 independent experiments. 1.3–2 million events were collected per sample. Error bars represent standard deviation of samples. (<b>Upper panel</b>) Frequencies of NP-specific GC phenotype B cells (CD95^hi CD38⁻ NIP-binding kappa^lo CD19⁺) cells among total live lymphocytes. Paired <i>t</i> test over d8-28: p = 0.0679. (<b>Middle panel</b>) Frequencies of GC phenotype (CD95^hi CD38⁻) cells among total NIP-binding kappa^lo CD19⁺ B cells. Paired <i>t</i> test over d22-85: p = 0.0409. (<b>Lower panel</b>) Fraction of IgG₁ class switched cells among total antigen specific GC B cells. Paired <i>t</i> test over days 15–85: p = 0.010.</p