Transitional Type 1 and 2 B Lymphocyte Subsets are Differentially Responsive to Antigen Receptor Signaling

Mature B-lymphocytes develop sequentially from transitional type 1 (T1) and type 2 (T2) precursors in the spleen. To elucidate the mechanisms that regulate the developmental fate of these distinct B cell subsets, we investigated their biochemical and biological responses following stimulation through the B-cell antigen receptor (BCR). As compared with the T1 subset, T2 cells are more responsive to BCR engagement, as evidenced by their robust induction of activation markers, expression of the prosurvival protein Bcl-x(L), and enhanced proliferation. BCR stimulation of T2 cells leads to the appearance of B cells with mature phenotypic characteristics, whereas T1 cells die. All of these T2 responses are dependent on the BCR signal transducer Bruton's tyrosine kinase, which is dispensable for the T1 to T2 transition. Furthermore, the serine/threonine kinases ERK, p38 MAPK, and Akt are predominantly activated in T2 compared with T1 B cells following BCR cross-linking. We conclude that T1 and T2 B cells respond differentially to BCR engagement via the induction of stage-specific signaling pathways. In turn, these signaling pathways probably govern the development and selection processes that are critical for the formation of the mature B cell compartment.


Summary
Mature B-lymphocytes develop sequentially from transitional type 1 (T1) 1 and type 2 (T2) precursors in the spleen. To elucidate the mechanisms that regulate the developmental fate of these distinct B cell subsets, we investigated their biochemical and biological responses following stimulation through the B-cell antigen receptor (BCR). As compared with the T1 subset, T2 cells are more responsive to BCR engagement as evidenced by their robust induction of activation markers, expression of the pro-survival protein Bcl-x L , and enhanced proliferation.
BCR stimulation of T2 cells leads to the appearance of B cells with mature phenotypic characteristics, whereas T1 cells die. All of these T2 responses are dependent on the BCR signal transducer Bruton's Tyrosine Kinase (BTK), which is dispensable for the T1 to T2 transition.
Furthermore, the serine/threonine kinases extracellular signal-regulated kinase (ERK), p38 MAPK and Akt are predominantly activated in T2 compared to T1 B cells following BCR crosslinking. We conclude that T1 and T2 B cells respond differentially to BCR engagement via the induction of stage-specific signaling pathways. In turn, these signaling pathways likely govern the development and selection processes that are critical for the formation of the mature B cell compartment.

INTRODUCTION
B lymphocytes are generated throughout the life of most mammals. This process occurs in the fetal liver before birth and in the bone marrow (BM) thereafter. Production of functional B lymphocytes requires the normal progression of precursor B cells through both antigenindependent and antigen-dependent stages of development (1)(2)(3). The antigen-independent phase of B-cell differentiation that occurs in the fetal liver and the BM culminates in the expression of an assembled IgM molecule, which is displayed on the surface of immature B cells (3). Of the 10-20 million immature B cells (IgM + ) daily produced from the BM, only 10% reach the periphery; of these, only 10-30% join the long-lived B cell pool (IgM lo IgD hi ) (4)(5)(6)(7). Although the precise nature of this peripheral B cell loss is unclear, these observations suggest that immature B cells either compete with mature B cells for survival or they require selection signals to enter the long-lived mature B cell pool (6,8,9). Analysis of the peripheral B cell repertoire strongly suggests that the progression of immature B cells to the mature B cell stage is the result of positive selection (10)(11)(12)(13)(14)(15). In this context, BCR-derived signals are indispensable for both the formation of the B cell repertoire and the long-term survival of mature B lymphocytes (6,16).
Despite a critical requirement for the BCR during splenic B cell development, a specific role(s) for BCR signaling in this process remain(s) poorly understood (8,17,18 (2,4,5,8,17,20,21). These observations suggest that the BCR may deliver distinct signals at discrete stages of peripheral B cell development. Whether distinct BCR responses are developmentally regulated or stage-specific (T1, T2, and M) BCR signaling responses that determine the biological outcome are not known.
Affected animals display a 50% reduction in the number of M splenic B cells (37)(38)(39). The M B cell deficiency in these mice is likely due to a failure of T2 B cell transition into M B cells. A similar M B cell deficiency has been observed in mice with gene-targeted deletions in several other components of the BCR signalosome including Syk, Vav1, Vav2, B cell linker protein (BLNK), Phosphatidylinositol 3-kinase (PI3K) and PLC-γ2 (27,(40)(41)(42)(43)(44)(45)

Mice
Four to 8 week old C57BL/6 mice were used for all the experiments. The generation of btk-deficient mice (null mutant; btk -/-) has been described previously (34). These mice have a mixed genetic background of 129/SvxC57BL/6. For wild type controls, 129/SvxC57BL/6 or C57BL/6 mice (Jackson Laboratories, Maine) were used. All mice used as a source of cells were treated humanely in accordance with federal and state government guidelines and their use was approved by Vanderbilt's and UMMS's institutional animal committees.

Flow Cytometric Analysis
For flow cytometric analyses, splenocytes were harvested from WT and btk -/mice and depleted of red blood cells (RBCs). 1x10 6  for PE-30F1 and Ebioscience for anti-IgD). After washing in stain media, cells were resuspended in 1 mg/ml propidium iodide to exclude dead cells.
For the detection of intracellular Bcl-x L protein levels, splenocytes were incubated for 16 hours in the presence or absence of anti-IgM. 1x10 6 cells/sample were stained for surface expression of CD21 and HSA. Cells were then fixed in 4% paraformaldehyde and permeabilized in 0.3% saponin. Intracellular staining for Bcl-x L was achieved by incubating the permeabilized cells with FITC-conjugated anti-Bcl-x L antibodies in the presence of 15% mouse serum.
Samples were stained in parallel with a FITC-conjugated IgG 3 antibody as a background control.
All flow cytometric samples were assayed on a FACSCalibur TM or FACScan flow cytometer (Becton Dickinson) and the data was analyzed using CELLQuest TM (Becton Dickinson) or FlowJo (TreeStar Inc) software.

In Vitro Differentiation Assay
Splenic B cell subsets were isolated via a two-step process. First, B cells were purified from the spleens of WT and btk -/mice by a process of negative selection using an auto-MACS automated cell sorter (Milentyi Biotechnology). Briefly, pooled splenocytes were depleted of RBCs, incubated with anti-CD43 antibodies coupled to magnetic beads to deplete CD43-bearing 8 described in previous reports (19,46). Anti-IgM was specifically not used to avoid pre-activation through the BCR. Cells were sorted on a FACStar TM

Western Blotting
For western blot analysis of Bcl-x L , purified T1, T2 and, total B cells (as described in Fig.   2.) were used. Cells were washed and resupended in complete RPMI (supplemented with 10% FCS, 50 mM 2-ME and 100 µg/ml penicillin and 100 µg/ml streptomycin) at a concentration of 1x10 6 cells/ml and cultured for 16 hours with or without 10 µg/ml F(ab') 2 goat anti-mouse IgM antibodies (Jackson ImmunoResearch). For western blot analysis of MAP Kinases, cells were 9 resuspended in PBS at a concentration of 5x10 6 cells/ml and incubated with or without 20 µg/ml F(ab') 2 for indicated times as described above. After stimulation, whole cell extracts were prepared and resolved by 4-20% gradient denaturing SDS-PAGE, and blotted on to Immobilon (Millipore) membranes. The membranes were probed with rabbit anti-Bcl-x L (Pharmingen) or anti-phospho p38 (Thr180/Tyr182), anti-phospho-ERK1/2 (Thr202/Tyr204) anti-phospho-Akt

An Intact BCR Signaling Pathway is Required for the Development of Transitional Type 2 B cells into Mature B cells
In prior studies, we and others have demonstrated that the M B cell population is reduced in btk -/mice and that the majority of the remaining B cells display an immature phenotype (IgM bright IgD low and IgM bright IgD bright ) (19,33,34,47). BTK is an integral component of the BCR

Enhanced Expression of Activation Markers by Transitional 2 B cells
The ability of T2 B cells to develop into M B cells prompted us to investigate their activation responses following BCR stimulation. For these studies, splenocytes were cultured with or without anti-IgM antibodies as indicated. FACS analyses revealed that T2 B cells displayed heightened expression of the activation markers CD25, CD5, CD95, and CD86 compared to either T1 or M B cells prior to BCR stimulation. Following activation, the expression levels of these cell surface antigens increased on both M and T2 B cells, however T2 B cells displayed a greater increase than did M B cells. Interestingly, when input T2 cells were analyzed 72 hrs after anti-IgM treatment, the cells that retain T2 phenotype expressed more CD25 than the cells with a M phenotype ( Figure 4A). In contrast, the expression of these activation markers on untreated T1 cells was low or undetectable and did not significantly increase in response to treatment with anti-IgM ( Figure 4B). BCR signaling-defective btk -/-B 13 cells were defective for the up-regulation of these cell surface antigens upon anti-IgM stimulation (data not shown). Together, these findings suggest that the activation response of T2 B cells to BCR engagement relative to T1 B cells may be the result from distinct signaling program of the T2 B cell subset. Alternatively, the robust BCR responses of the T2 B cells may result from a developmental switch during T1 to T2 transition that enhances BCR-dependent responses of T2 B cells.

T2 B cells Proliferate More Efficiently than T1 B cells in Response to BCR Stimulation
Previous studies have shown that unlike mature B cells, immature splenic B cells (IgM bright HSA bright ) do not proliferate but instead undergo apoptosis (4,5,8,20,21,48).  Figure 3A). Furthermore, the corresponding btk -/-B cell subsets failed to proliferate in response to BCR stimulation at all time points tested ( Figure 5C and D). The proliferation of btk -/-B cells was comparable to WT when treated with PMA and ionomycin ( Figure 5E). Taken together, the observed lack of proliferation of T1 B cells in response to BCR stimulation is in agreement with previous reports (4,5,20,48). Importantly, the

BCR Activation Leads to Heightened Expression of Bcl-X L in T2 B Cells
Although many T2 B cells die in response to BCR engagement ( Figure 3A), a significant To confirm this interpretation, we initiated further studies to evaluate the upregulation of Bcl-x L protein levels by Western blotting. Cellular extracts were prepared from FACS-sorted and BCR-stimulated T1, T2, and M B cell populations (as in Figure 2). Consistent with the higher expression levels of Bcl-x L observed by FACS analysis (Figure 6A-C), the absolute levels of Bcl-x L protein markedly increased in T2 B cells stimulated with anti-IgM antibodies ( Figure   6D, compare lanes 5 and 6). In contrast, BCR stimulation did not significantly change the levels of Bcl-x L protein in T1 cells ( Figure 6D, compare lanes 1 and 2). These results indicate that the increased expression of Bcl-x L following engagement of the BCR is most pronounced for the T2 B cell subset. These observations are consistent with a role for Bcl-x L in promoting B cell survival to a sufficient extent such that B cells are able to initiate additional biological outcomes necessary for maturation and activation. Thus, the inability of T1 B cells to induce Bcl-x L to high levels may contribute in part to their enhanced apoptosis in response to BCR crosslinking (5,20,48). In contrast, induction of Bcl-x L by T2 cells may play a critical role in mediating their

DISCUSSION
In these studies, we have compared the effects of BCR signaling within peripheral B cell populations at discrete stages of development. Our results clearly establish that the two populations that comprise the immature splenic B cell compartment (T1 and T2) react distinctly to BCR stimulation. Specifically, T1 cells die in response to BCR signals, whereas T2 cells are stimulated to express activation markers and the pro-survival gene Bcl-x L , and to proliferate.  (46,57). This proposal is in agreement with the observed B cell developmental block at the T1 stage in BAFF -/mice (57), suggesting that the T1 to T2 B cell transition and subsequent development may require BAFF. Further studies of the intrinsic B cell signaling program and signals delivered by extracellular stimuli will be required to elucidate the molecular processes involved in the development of T1 into T2 B cells.
We demonstrate that in contrast to T1 B cells, T2 B cells respond to BCR stimulation by increasing the expression of Bcl-x L , proliferating robustly, and ultimately displaying a phenotype similar to M B cells. Consistent with this idea, we noted that enlarged IgD + cells in the spleen tend to have very high levels of IgD (as did in vitro-activated T2 cells) and are preferentially found in the T2 subset (Fig. 3C). It seems possible that these cells may be recent recipients of positive selection signals in vivo. Therefore, T1 and T2 B cell stages may represent two distinct stages during peripheral B cell development: The T1 stage may provide an opportunity to eliminate self-reactive B cells while the T2 stage may serve as the target for positive selection. The T2 B cell subpopulation proliferate in response to BCR stimulation (Fig.5).
Although the significance of this proliferation remains unclear, an increase in the T2 B cell population may be necessary to accommodate the cell loss that occurs during the recruitment of immature B cells into the pool of mature B cells (4)(5)(6)8,21). This is supported by the observation that a significant fraction of T2 B cells exposed to anti-IgM in vitro undergo apoptosis while only some cells mature to display a M B cell phenotype (Fig. 3). These results suggest that a Results in figure 7 show that BCR stimulation activates serine/threonine signaling pathways in T2 at a much higher level than in T1 B cells (Figure.7). The ERK1/2, p38 and Akt signaling pathways appear to regulate cell growth and differentiation in response to cellular stimulation (29,30,(59)(60)(61). Our findings suggest that activation of ERK1/2, p38 and Akt in T2 B cells may promote B cell survival and proliferation and may contribute to the molecular signals by guest on March 24, 2020 http://www.jbc.org/ Downloaded from underlying the T2 to M transition. In this regard, increased ERK1/2 phosphorylation has been shown to play a role at the checkpoint between pro-to pre-B cell transition (62). However, the mechanism of how the BCR is coupled to the downstream pathways in T1 versus T2 cells remains unclear. One possible reason for the observed signaling differences between T1 and T2 B cells may arise from differences in the components that comprise the BCR signalosome within these B cell subpopulations. Alternatively, robust signals generated at the T2 stage may simply be the consequence of the efficiency with which the BCR signaling components are recruited to the sphingolipid-rich membrane microdomains known as lipid rafts. These structures have been suggested to serve as platforms for initiating downstream BCR signaling cascades (63). Indeed, the BCR is poorly co-localized with lipid rafts in immature B cells compared to M B cells (64).
Taken together, these findings support a model in which T1 and T2 B cells may assemble distinct BCR signaling complexes and/or sequester similar complexes with differential efficiency into lipid rafts following BCR cross-linking. Clearly, more investigation is required to elucidate the genetic and biochemical reprogramming that must occur to induce the radically different biological outcomes observed in T1 and T2 B cells in response to activation through the same receptor. Regardless of the specific nature of such a mechanism, based on the results presented here, we propose that the T2 subset contains B cells that display a state of "heightened alertness" to more efficiently execute both mitogenic and differentiation signals mediated by the BCR as