A new staining protocol for detection of murine antibody-secreting plasma cell subsets by flow cytometry

We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice. In mice, CD138, B220, and Blimp1 (B lymphocyte-induced maturation protein-1), a plasma cell (PC) signature transcription factor, are frequently used to quantify mature antibody-secreting PCs. Here, we show in a Blimp1:GFP (green fluorescent protein) reporter mouse that the CD138hi/B220low population in bone marrow (BM) contains a significant fraction of Blimp1-negative cells. Therefore, detection of B220 and CD138 alone is not sufficient to identify PCs unambiguously. To circumvent this problem, we developed a new 4-color PC staining protocol using antibodies against CD138, B220, transmembrane activator and CAML interactor (TACI) and CD19. This procedure detects a CD138hiTACIhi fraction with more than 98% Blimp1:GFP-positive cells. Our staining protocol also distinguishes proliferating plasmablasts (P1, CD138hi/TACIhi/B220int/CD19int) from two nonproliferating PC subsets, P2 (CD138hi/TACIhi/B220low/CD19int) and P3 (CD138hi/TACIhi/B220low/CD19low), without the need of a Blimp1:GFP reporter allele. In the BM, cytoplasmic IgG-positive plasma cells (which lack surface Ig) are detected in the CD19low P3 fraction, suggesting that the P3 population contains long-lived mature PCs. Hence, our 4-color staining technique identifies two nonproliferating mature CD138hi/TACIhi/B220low PC populations that differ in CD19 expression and is, therefore, superior to the commonly used CD138/B220 staining protocol. Plasma cells (PC) are the antibody-secreting workhorses of the adaptive humoral immune response. Mature nondiving PCs originate from an antigen-activated B cell through various stages of antibody-secreting cells. When antigen-activated B cells receive cognate T-cell help, they proliferate and undergo a germinal center (GC) reaction, during which they change their Ig heavy chain constant region and hypermutate their variable gene segments 1. Thereafter, GC B cells selected for high affine variable regions differentiate into memory B cells and early dividing precursor PCs, so-called plasmablasts (PBs) 2, 3. Newly generated PBs migrate to survival niches in the BM and may develop into nondividing mature PCs that can survive for years in mice 4, 5 and decades in humans 6, 7. The general belief is that signals provided during the GC reaction are required to enable B cells to become long-lived PCs. However, there is accumulating evidence that long-lived PCs also develop in the absence of T-cell help and GC formation 8. Terminal PC differentiation is controlled by a set of transcription factors, e.g. downregulation of Pax5 and upregulation of Blimp1 marks the differentiation of activated B cells into mature plasma cells 2, 9, 10. A reporter mouse line that express the GFP-encoding gene under the control of the endogenous Blimp1 promoter allowed to track PC development and revealed that early dividing CD138hi/B220intermediate (int) PB have a low expression of Blimp1, whereas nondividing mature CD138hi/B220low PCs in secondary lymphatic organs and survival niches show high levels of Blimp1 11. Therefore, the detection of high (hi) amounts of CD138 and downregulation of B220 developed by Smith and colleagues is frequently used to define and quantitate early and late antibody-secreting PCs by flow cytometry even in the absence of a Blimp1 reporter allele 12. However, when we stained cells from spleen and BM of SRBC-immunized Blimp1:GFP reporter mice for CD138 and B220, we found that the CD138hi/B220low fraction in the BM contained up to 21% GFP-negative cells (red gate in Fig. 1A, femur and tibia). Therefore, the so-called CD138hi/B220low “plasma cell” population in BM is heterogeneous and contains Blimp1-negative cells, which should be considered when “plasma cells” are isolated solely by high CD138 and low B220 expression. To solve this problem, we established a new 4-color staining protocol by using antibodies against CD138, B220, TACI, and the B lineage marker CD19. The survival receptor TACI is upregulated in activated B cells and PCs 13, 14. CD19 is controlled by Pax5, a transcription factor that is downregulated during PC differentiation 15. Therefore, surface abundance of CD19 gradually decreases during the differentiation of activated B cells into PCs, and PC populations with intermediate and low surface expression of CD19 have been described in humans ([16], and references therein). Our PC staining protocol detected a distinct CD138hi/TACIhi population in spleen and BM from SRBC-immunized Blimp1:GFP reporter mice (Fig. 1B, blue rectangle, and Supporting Information Fig. 1) and nonimmunized wild-type mice (Supporting Information Fig. 2A). The same population was found in spleen and BM from femur/tibia and spine of SRBC-immunized wild-type mice (Fig. 2A and Supporting Information Fig. 3, 4A), and the staining was compatible with intracellular staining of Ig (Supporting Information Fig. 4A, B), or Ki-67 (Fig. 2B and Supporting Information Fig. 5). The CD138hi/TACIhi population contained more than 98% Blimp1-positive cells in both the spleen and the BM (Fig. 1B). ELISpot of sorted CD138hi/TACIhi BM cells confirmed the presence of IgM-, IgG-, and IgA-secreting cells (Supporting Information Fig. 2). When we analyzed the CD138hi/TACIhi population for the presence of B220 and CD19, we detected four distinct subsets in both the spleen and the BM of femur/tibia (Figs. 1B, 2A and Supporting Information Fig. 3A–C, 4B, 5) of Blimp1:GFP reporter and wild-type mice. Almost all cells in the P1, P2, and P3 subsets in the reporter mouse expressed GFP and are, therefore, either PBs or mature PCs. The minor B220hi/CD19hi fraction in the CD138hi/TACIhi population consists very likely of early activated B cells because this population contained a large proportion of Ki-67-positive cells (data not shown) and a very low Blimp1:GFP fluorescence (Fig. 1B). Moreover, back-gating of B220hi/CD19hi cells into the CD138hi/TACIhi gate revealed only an intermediate TACI fluorescence (Supporting Information Fig. 6). A large fraction of the P1 population represents dividing PBs because this population showed an intermediate B220 fluorescence and included more than 60% Ki-67-positive cells (Fig. 2B). In contrast, P2 and P3 cells in spleen and BM expressed very low amounts of B220. While approximately 30% of P2 cells in the spleen were Ki-67-positive, the clear majority of P2 in the BM and P3 in both organs was Ki-67-negative. Therefore, most of the P2 and the P3 cells in the BM are mature nondividing PCs. Of all three subpopulations, BMP3 cells had the lowest abundance of surface CD19 (Fig. 2A, left histograms and diagram), and only a few Ki-67-positive cells could be detected (Fig. 2B). Therefore, P3 cells in the BM are mature resting PCs. BMP2 and P3 cells also differ in their distribution of IgH chain isotypes (Supporting Information Fig. 4B). For example, in the BM over 90% of the P2 cells expressed surface IgA (Supporting Information green gates in Fig. 4B), whereas IgG-producing cells (which lack surface expression, Supporting Information red gates in Fig. 4A) were enriched in the BMP3 fraction in wild-type mice immunized with SRBCs (red gate in Supporting Information Fig. 4B). Therefore, pregating on CD19-positive cells in flow cytometry-based PC isolations will result in the loss of the mature PC subset P3. It is still unclear whether P3 develop from P2, or if both mature PC subsets in the BM originate from different precursors. Based on the fluorescence intensities of CD138, TACI, B220, and CD19, we have established a new PC staining protocol that identifies three Blimp1-positive subsets in spleen and BM (P1–P3). To our knowledge, this is the first report that uses these four markers in one combined staining. As IgG-positive BM cells are detected in the P3 fraction, this CD19low PC subpopulation contains very likely long-lived PCs. In support, long-lived human PCs have recently been identified in the CD19low/negative PC fraction in the intestine by using the carbon-14 technique 7 or by detecting highly mutated antibodies 16, 17. Our PC staining protocol does not only distinguish early dividing PBs from late mature PCs, it also allows identifying and isolating pure PC populations without the need of a Blimp1:GFP reporter allele. We thank U. Appelt and M. Mroz for cell sorting. This work was supported, in part, by the Deutsche Forschungsgemeinschaft through research grant TRR130 (project P09) and the doctoral training program GRK1660 to H.-M.J. The authors declare no commercial or financial conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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