The immunohistology of the spleen from the SLE patient produced a picture similar to the cellular architecture of healthy spleens in mice [15
] and humans [16
], which is known to be interspersed with GCs. In the present study, however, a mantle surrounding the GC was not identified. Each mature GC is generally derived from one to three B-cell clones, which manage to survive a significant reduction in clonal diversity and then go on to endure V(D)J hypermutation. The GC is of most interest because it is the site of antigen driven V(D)J hypermutation and selection [15
] where antigen-specific B cells acquire point mutations in the V regions of transcriptionally active rearranged immunoglobulin genes. These mutations accumulate steadily during expansion of B-lymphocyte clones in the dark zone of the GC. This clonal evolution occurs independently in each GC, because little trafficking of B cells between GCs has been observed [17
The cellular components necessary for a GC response were present in both GC A and GC B (i.e. B cells, FDCs capable of presenting antigen and T cells). However, none of the GCs and B-cell clusters had a discernible mantle zone. No plasma cells were located within the GCs themselves but they were loosely distributed in the surrounding tissue within close proximity to the GCs (data not shown). The immunohistochemistry also demonstrates that both GCs are of approximately equal size, which validates comparison of the data produced from each.
A recent study in Science
] identified autoreactive B cells in MRL.Faslpr
mice proliferating in the T-cell zone of lymphoid tissues. This was thought to be due to their deficiency of the Fas receptor, because these cells would normally be deleted through the Fas receptor/Fas ligand-mediated pathway of apoptosis. A similar explanation may account for the GCs identified in this study not exhibiting a traditional mantle, and explain the lack of negative selection illustrated by the low number of nonfunctional genes.
Of the total number of rearranged VH
genes, 19% were found to contain stop codons and out-of-frame rearrangements, and were therefore deemed to be nonfunctional, the majority being found in cluster D. This correlates with a similar study conducted by Jacobi et al
], who found 13% of PBL VH
gene sequences amplified from an SLE patient to be nonfunctional, as compared with 53% of genes amplified from PBLs of a healthy individual. Those investigators observed a similar R/S ratio in the productive rearrangements to that seen in the nonproductive rearrangements. It was therefore suggested that there may be some abnormality in selection in SLE related to an intrinsic failure of B-cell apoptosis or enhanced B-cell activation by T cells, which overwhelms protective mechanisms that are effective in normal individuals.
The most abundantly expressed VH
gene family was VH
3, which is not surprising because it is the largest family and has been found to be the most dominant in the normal repertoire [20
]. What is perhaps more interesting is the large number of VH
5 sequences (16.6%) as compared with the 3.9% expected to be produced randomly in the normal human repertoire. VH
5–51 has also been isolated from breast tumours (Nzula et al
., unpublished data) and thymic GCs from a patient with myasthenia gravis [9
] previously in our laboratory, but not from GCs from the salivary glands of two patients with Sjögren's syndrome, using the same primers [21
]. It is therefore unlikely that the family VH
5 primers used here preferentially amplify VH
5 sequences have been consistently associated with IgE antibodies, and it has been suggested that these antibodies may be associated with an unidentified superantigen [22
]. Two studies analyzing anti-DNA antibodies in SLE both identified a heavy chain clone comparable to VH
]. Comparison of our V-D-J rearrangements with the two anti-DNA specific antibodies revealed very few somatic mutations in common, however. One hotspot highlighted at position 77 (in the tip of the FR3 loop) [25
] was found to be present in the anti-DNA specific antibodies as well as in most of the analyzed VH
5–51 sequences from this study, but this was not often the same amino acid substitution.
VH5–51 was also used by two human IgG monoclonal antibodies that bind phospholipid, derived from the PBL of a SLE patient. In this instance the J segment used was JH6b, which was not found in combination with VH5–51 in the present study. It may be significant that patient M produced anticardiolipin antibodies, but not anti-dsDNA, although there is no direct evidence that the cardiolipin antibodies used VH5–51 because no information about the specificity of these antibodies was obtained.
A complete absence of VH
1 family genes was observed in the present study of splenic GC, which contrasts with the theoretically expected value of 20–30%. This was also found to be the case in another study of SLE patients conducted by Hansen et al
]. They found that 13% of functional PBL V genes from healthy control individuals were from the VH
1 family, but only about 1% of the VH
genes expressed by PBLs of a SLE patient used this family. de Wildt et al
], on the other hand, found very little difference between expression of VH
1 in healthy control individuals and SLE patients. The PBL B-cell repertoire may not reflect the repertoire of splenic B cells.
Using high-fidelity Taq polymerase and the same primers as used here, the PCR error rate for VH
genes was less than one base per four VH
]. The average numbers of single base mutations in the VH
genes from GC A and GC B were 7.6 and 15.8, respectively, and for clusters C and D they were 15 and 14.9, respectively. This is significantly higher than the Taq polymerase error rate, demonstrating somatic hypermutation in vivo
. The difference in mutation rates between the two GCs may indicate that they are in different states of maturity or that GC B might have been founded by a memory B cell that was already mutated (e.g. the founder cell of clone B-b; Fig. ). Hypermutation within a GC is closely linked to antigen-induced B-cell proliferation; thus, from the data presented here, this appears to be the case in both GCs and the B-cell clusters. The second phase of this process, during an immune response against a xenoantigen, is selection of B cells that express high-affinity antigen receptors resulting from rare mutations, by competition for binding to antigen on the surface of FDCs. This process is generally believed to result in an increase in the ratio of replacement to silent mutations, especially within CDRs, and often in selection against replacement mutations in the FRs. The evidence presented here is supportive of antigen selection in GC B and clusters C and D, in which the R/S ratio is higher than random in the CDRs. Selection for replacement mutations in the FRs has previously been observed, for example in two anti-hen egg lysozyme antibodies (HyHEL-5 and HyHEL-10) [28
], in which both had contact residues in the FRs. It is also possible that the high R/S ratio in the FR region of the sequences in this study are the result of antigen selection, but this was not confirmed because of the absence of data on specificity.
A serine codon bias was also observed (not shown), with 70% of the serines in CDR1 and CDR2 represented by AGC or AGT, both of which are recognized targets of the hypermutation machinery (for review [30
]). Only AGC and AGT serine codons produced replacement mutations in the CDRs. Of these, the mutation from serine to asparagine was the most prevalent, which is in accordance with mutational analysis performed on patients with myasthaenia gravis [9
The clonal genealogies show that the groups of rearranged VH
genes included sequences that could be assigned to parental and daughter cells on the basis of shared mutations and junctional sequences. By far the most dominant sequences were VH
3–30*01 and VH
5–51*01 (approximately 16% each). In studies of PBLs in SLE, the most common VH
segment observed was VH
3–23 (12%) [27
], which we have seen in similar numbers (11%).