Our data reveal several novel aspects of human peripheral B cell development. We show that human transitional B cells can be subdivided into multiple populations based on CD24/CD38 expression, a gradation of immature and mature surface marker expression, dye extrusion (reflecting the expression and functionality of the ABCB1 transporter), and functional responses. Notably, we identify a late transitional B cell population intermediate between T2 and mature naïve stages that is a normal B cell developmental intermediate, the dominant peripheral transitional B cell population in healthy adults, and substantially increased in the peripheral compartment of patients reconstituting after B cell depletion therapy. Overall, our results indicate that human transitional cells exist along a phenotypic and functional continuum between immature and mature, both confirming and substantially extending the prior characterization of human transitional B cells (7
). Similar to the mouse, we suggest that immune competence is gradually acquired as immature B cells transit through the transitional cell stage toward mature naïve (1
). Thus, mature surface markers are gradually acquired, the ABCB1 transporter is expressed, and proliferation and survival begin to be favored over apoptosis in response to BCR stimulation.
There have been a number of recent reports defining human transitional B cells (7
). These studies have demonstrated a population of CD24hiCD38hi B cells with distinct phenotype and function compared to mature naïve B cells. However, discrete developmental subsets have not been well characterized. Reduced proliferation, survival, differentiation, and chemotaxis of CD24hiCD38hi human transitional B cells have been demonstrated in vitro, but notably there are several discrepancies in the literature regarding the nature of the signals that mediate the survival and proliferation of distinct transitional B cell subsets. For example, one publication has proposed that human T1 cells have marked CD40L enhanced proliferation but little response to BAFF stimulation (26
). Other studies have demonstrated more of a gradation of response, with mature subsets simply having more proliferation (8
) (or entry into cell cycle) in response to CD40L/anti-Ig than their transitional cell counterparts. Similarly, Sims et al.
found BAFF to be a relatively inefficient inducer of cell cycle entry in human B cells, but still with a trend toward greater induction in mature than transitional B cell populations (7
). Our data are in accord with the view of mature human B cells having greater proliferative responses to a range of stimuli compared to transitional B cells. Thus, based on CFSE dilution, proliferation of the T1 subset is clearly induced by anti-Ig + either CpG or BAFF, although to a lesser degree than in the T2 and mature naïve subsets. It is of additional note in our study that CpG was the most highly effective stimulus for the proliferation and survival of human transitional cells, in accord with a recent publication (27
), and raising the question of how autoreactive transitional cells escape such stimulation in vivo
. We also demonstrate for the first time that human transitional B cells (T1/T2) have reduced calcium signaling compared to mature naïve B cells.
Of note, even within the murine literature there is some disagreement as to the precise delineation of transitional B cell subsets. Thus, Loder et al.
first proposed the partition of transitional B cells into two subpopulations based on CD21, CD23, and IgD: T1 and T2, with similar phenotypic properties to that described here with the exception of IgD expression on human T1 cells (3
). A notable parallel between this classification scheme and our characterization of human transitional B cells is the increasing expression of CD21 on maturation from T1 to T2. However, Allman et al.
have described an alternate classification scheme using the developmental marker AA4 and variable expression of IgM and CD23 to delineate three transitional populations T1-T3. Similar to this analysis, we find higher expression of CD23 on human transitional B cells upon maturation from T1 to T2 and decreasing IgM expression. The T2 populations defined by these different murine classification schemes are likely distinct, with the CD21high/CD23+ T2 population of Loder et al. recently postulated to be a marginal zone precursor (10
). This is interesting as other data indicates that B cells transferred into lymphopenic murine hosts, a situation possibly recapitulated after B cell depletion therapy, undergo rapid activation and differentiation into cells phenotypically indistinguishable from purported MZ precursors and MZ B cells (10
). Our detailed characterization of human spleen defines for the first time an analogous MZ precursor population, although notably MZ B cells are not expanded in the peripheral blood following BCD or in the context of the peripheral blood lymphopenia associated with SLE (29
As with the human T3 population characterized for the first time here, murine T3 cells would be included within the mature naïve compartment by conventional gating strategies. Their presence in human peripheral blood is revealed here by the inability to efficiently extrude R123/mitotracker, a higher CD24 and IgM expression, and a lower CD23 expression compared to conventional mature naïve B cells, functional immaturity based on increased susceptibility to apoptosis, and a dramatic expansion during immunologic reconstitution after B cell depletion therapy. The delineation of a T3 population in humans is an important departure from recent studies of human transitional B cells, where it has been suggested that human B cells may comprise only a single transitional stage or contiguous T1 and T2 stages. Regardless, it is clear that further identification of markers differentially expressed by subsets of human transitional B cells would greatly facilitate their resolvability. An additional layer of complexity has recently been proposed by Cambier and colleagues. Their results demonstrate that non-transgenic mature naïve B cells can develop into anergic cells with a T3 phenotype in response to chronic antigenic stimulation (12
). Along these lines is important to bear in mind that activated naïve B cells may also lose expression of the ABCB1 transporter and become unable to extrude rhodamine, although this generally parallels the acquisition of CD27 expression (19
). Hence, one possible alternative explanation for our results would be to postulate that peripheral blood B cells with a T3 phenotype represent mature anergic cells that have developed in response to repeated antigenic stimulation presumably by self antigens. These interpretations are not mutually exclusive as it is possible that T3 cells could indeed contain a fraction of mature or immature anergic B cells. However, we believe that the dominance of a T3 phenotype within cord blood as well as the ability of these cells to differentiate to mature naïve in vitro makes it unlikely that the majority of these cells would represent anergized mature cells rather than transitional cells.
The discrimination of multiple human transitional B cells is meaningful in that it provides important insight into the developmental process followed by human B cells. Thus, it should be noted that in mice immature T1 cells home to the spleen which has been purported be the main or only environment for transitional cells differentiation (9
). On the other hand, Lindsley et al. have recently described a later murine transitional B cell subset that develops in the BM in parallel with peripheral splenic T2 development (30
). The detection of abundant T2 and later transitional B cells in human peripheral blood post-rituximab indicates either substantially increased differentiation in and release from the bone marrow or greatly increased generation in and recirculation from the spleen. Based on the fact that in normal subjects the bone marrow contains mainly the T1 subset of transitional cells and the spleen is enriched for later T3 subsets and contains activated T1/T2 cells, we would suggest increased BM generation of T1 cells and differentiation in the spleen with subsequent recirculation in the peripheral blood. Extra-splenic maturation of B cells may also take place given the expansion of T2 and T3 subsets in cord blood as well. Of note, the finding of late transitional B cells in human spleen and cord blood has been documented here for the first time.
The factors that govern the survival and selection of human transitional cells are important to further delineate, particularly given that such processes shape the mature B cell repertoire, deviations from normal regulation may precipitate autoimmunity, and the potential restoration of proper regulation will determine the long-term outcome of B cell depletion therapy. There has been consensus in the mouse literature that T1 B cells are a primary target of negative selection (4
). However, the factors governing selection in the later transitional stages (T2 and T3) in the periphery are more controversial (24
). A general principle has existed that mature B cells are activated upon BCR stimulation, whereas the same signals on developing B cells lead to unresponsiveness or cell death. Critically, however, exactly where in the transitional cell spectrum this change in responsiveness occurs is unclear. Although it has been suggested that murine T2 B cells, when compared to T1 B cells, acquire
responsiveness to T-cell help signals (32
), other data supports a more gradual acquisition of immune competence within the transitional B cell compartment (1
). T2 and T3 subsets may also undergo negative selection vs. positive selection depending on the combination of antigen-receptor mediated and non-antigen-receptor mediated signals, the provision of T cell help, and cytokine stimulation (including BAFF) (24
). Later transitional B cells may be particularly responsive to BAFF (24
) and thus it is interesting to speculate whether the enhanced BAFF characteristic of a B cell depleted state (33
) may contribute to the prominent expansion of T3 cells during rituximab induced B cell depletion and reconstitution. Further definition of the signals that mediate survival of human transitional B cells will elucidate our understanding of normal tolerance mechanisms and biological events underlying autoimmunity. Moreover, an improved understanding of the progressive maturation stage of human B cells will greatly enhance our ability to elucidate the precise checkpoints and mechanisms involved in the enforcement of B cell tolerance.