Ag-specific Ab-secreting B cells generated during an immune response are thought to arise from distinct differentiation pathways and comprise a heterogeneous population with specific effector properties. It is the combination of these effector cells that is responsible for providing effective short-term and long-term immunity. Here we investigate the generation of plasma cells during the T cell–dependent immune response to NP in Aiolos-deficient mice and provide new insight into the regulatory requirements responsible for the generation of different plasma cell populations. The long-lived high affinity plasma cells that reside in the BM are absent in the Aiolos-deficient mice after both primary and secondary antigenic challenge. This indicates that a defect is manifested not only during the initial generation of this type of plasma cell from the GC, but also in their subsequent production from memory B cells. However, the short-lived low and high affinity plasma cell populations found in the spleen remain unaffected.
Early in the response, naive B cells that have encountered Ag can directly differentiate into low affinity plasma cells ( , population A). These plasma cells are found in the spleen and BM and constitute the first line of defense against pathogens. They have not undergone somatic hypermutation or isotype switching and disappear quickly after antigenic encounter. Aiolos deficiency does not interfere with this early and short-lived plasma cell response.
Later in the response, a high affinity plasma cell population accumulates in the BM (, population C′) that is long-lived and responsible for the long-term systemic production of high affinity Abs required for protective immunity. Lack of Aiolos selectively ablates this BM plasma cell population. Aio−/− mice show no significant number of plasma cells in the BM for several months after immunization, a period during which their number in the BM of WT mice has peaked. These findings indicate that there is no delay in the formation of AFCs. Thus, Aiolos is required for the generation of either a GC-derived high affinity AFC (, population C′) or a GC-derived AFC precursor that homes to the BM (, population C). It is possible that Aiolos is necessary for the differentiation, homing, or survival of this population.
The presence of long-lived plasma cells in the BM is dependent on the GC reaction. Disruption of the GC by anti-CD40L Abs leads to a reduction of high affinity plasma cells in the BM while sparing the extrafollicular and low affinity subsets (22
). Because formation of the high affinity BM plasma cells is impaired in Aio−/−
mice, we examined whether there was a defect in the GC reaction. Previous experiments have shown the existence of GC in the spleens of aging unimmunized Aio−/−
). Upon immunization, young Aio−/−
mice show a similar increase in the number of GCs (not depicted). Thus, there is no intrinsic defect in the signals that drive B cell differentiation into a GC reaction in Aio−/−
GCs, however, appear to sustain B cell clones with decreased BCR affinity for Ag, as an increased number of noncanonical V(D)J rearrangements was recovered 13 d after primary immunization. Even within B cell clones with canonical sequences, changes reflective of antigenic selection such as high R/S ratios in CDR, W→L mutation at position 33, and usage of the YYGS/N motif in the CDR3, were decreased in Aio−/−
GC B cells. Analysis of the 3′ flanking region of the VH
gene of GC B cells in Peyer's patches, which is not subject to selection, showed no defect in somatic hypermutation. Thus, there seems to be a relaxed negative selection in B cells from Aio−/−
mice, allowing survival of low affinity GC B cells that would otherwise be lost during the primary response. This could be explained by the fact that Aio−/−
B cells have decreased BCR signaling thresholds, allowing survival of cells in Aio−/−
mice that would have otherwise died by neglect in the GC.
Although there is a partial impairment in the selection of high affinity B cell clones in the GCs of Aio−/−
mice, an altered level of signaling through the BCR is not likely to account for the severe reduction of plasma cells in the BM. There is recent evidence that very low affinity cells can undergo a GC reaction and mount AFC responses, and that low affinity plasma cell clones can survive long-term in the BM (23
). In addition, FcγRIIB-deficient mice, whose B cells also display a hyperresponsive phenotype, have normal to increased humoral responses (24
). As high affinity B cell clones are seen in the GCs of Aio−/−
mice, one would expect some contribution from these cells to the BM plasma cell compartment, which is not the case. Furthermore, high affinity memory B cells ( A) are generated in normal numbers, indicating that B cells exiting the GC have undergone antigenic selection. Upon restimulation, these memory B cells (, population D) are responsible for the rapid but transient appearance of short-lived high affinity plasma cells in the spleen (, population E), resulting in a small temporal increase in high affinity anti-NP Ab titers. However, the BM plasma cell population remains severely impaired (, population F), suggesting that their deficit lies beyond the GC. Molecular analysis of sorted GC B cells supports this hypothesis because the expression of genes involved in clonal selection, survival, and plasma cell differentiation within the GC was normal in Aio−/−
mice (). It is possible that the development of naive precursors of high affinity BM plasma cells are specifically affected by lack of Aiolos. Potential differences in the Aio−/−
naive anti-NP repertoire relative to WT may also be responsible for the BM AFC deficit.
To determine whether the lack of BM plasma cells was due to a defect in homing or migration, AFC activity was assayed at other immunological sites. In mice deficient in the chemokine receptor CXCR4, homing of plasma cells or their precursors to the BM is impaired and plasma cells are found in elevated numbers in the blood and spleen of these mice for 2 wk after immunization (25
). In Aio−/−
mice, no increase in the number of NP-specific AFC activities was found in the blood or the lymph nodes. Examination of populations enriched for GC B and plasma cells as well as in vitro LPS-differentiated plasma cells revealed no defect in the expression of the chemokine receptors CXCR4, CXCR5, and CCR7 in Aio−/−
cells. Expression of the adhesion molecules CD44, VLA-4, and LFA-1, which are up-regulated in plasma cells and involved in their stromal interaction (26
), was also examined but no significant difference was found between Aio−/−
and WT levels. Thus, it is unlikely that misexpression of these receptor and adhesion molecules causes altered homing or retention of plasma cells in the Aio−/−
mice. It is possible that misexpression of other chemokine receptors with a potential role in plasma cell homing (27
) is deregulated. Nonetheless, the low titer of high affinity NP-specific Abs in the serum of Aio−/−
mice detected immediately to a few weeks after immunization correlates with the severe reduction of high affinity plasma cells and rules out an alternative site for their retention and short-term survival.
It is possible that BM plasma cells are generated in Aio−/−
mice but are unable to survive due to a stromal defect. BM stroma was recently shown to be required for selection and survival of long-lived BM plasma cells (26
). In addition, stromal cells secrete IL-6, a growth factor required for plasma cell survival within the BM (26
). Reconstitution studies showed that Aio−/−
B cells were not able to differentiate into plasma cells even in the presence of WT BM stroma ( C). These experiments rule out a stromal defect as the underlying cause of the AFC deficiency. Mixed chimera experiments have also confirmed that the plasma cell defect is intrinsic to the B cell compartment and not caused by insufficient help from Aio−/−
T cells. These studies also rule out the possibility that plasma cell interactions with other BM B cells, which are altered in Aio−/−
mice (i.e., pre-B/immature B; reference 12
), were responsible for their BM AFC deficit.
Previous studies have shown that Ikaros and Aiolos are essential components of chromatin remodeling complexes within the nucleus that modulate the structure of chromatin to effect gene expression and control lymphocyte differentiation and proliferation (30
). Gene expression studies on populations enriched for plasma cells and their precursors failed to reveal a major deficit in any of the previously described plasma cell differentiation factors. However, the low frequency of NP-specific plasma cells (~1% of CD138+
plasma cells in WT) could limit our ability to visualize changes in their gene expression profile within a heterogeneous plasma cell–like population. Therefore, we can only conclude that Aiolos does not affect expression of genes previously shown to be required for differentiation of all plasma cells. Another possibility is that Aiolos controls unidentified genes required for the differentiation or imminent survival of the long-lived high affinity plasma cell in the BM.
It has been recently shown that short- and long-lived plasma cells are derived from phenotypically distinct plasma cell precursor populations (33
). This investigation provides further evidence that T cell–dependent generation of long-lived high affinity plasma cells occurs independently of other plasma cell subsets. It provides support for the hypothesis that these are a separate entity of Ab-secreting cells with a differentiation program that is distinct from that of short-lived high affinity as well as low affinity plasma cells. Future studies will aim to determine whether Aiolos is involved in generating and/or determining the fate of the plasma cell precursor populations. In contrast to the transcription factors Blimp-1
, and IRF4
, whose activities affect all plasma cells, Aiolos
is the first transcription factor described to be specifically required for the differentiation and/or survival of the long-lived high affinity subset.