The present study has identified a number of differences between parotid gland B cells of a patient with SS compared with B cells obtained from the peripheral blood of the same patient. This patient manifested increased titers of autoantibodies (anti-Ro and anti-La), hypergammaglobulinemia and enlargement of the parotid glands, and could therefore be considered to have active disease. The data provide evidence that B cells that infiltrate the salivary glands in SS were highly selected. The repertoire differences were especially noteworthy in the productive and, therefore, the expressed VL gene repertoire, and they supported the conclusion that the parotid gland B cells were highly selected. B cells from the parotid gland were a distinct population exhibiting significantly elevated mutational frequencies in both productive Vκ and Vλ gene rearrangements, and showing preferential expansion and somatic mutation of particular VL chain rearrangements (VκA27–Jκ5, VκA19–Jκ2 and Vλ1C–Jλ2/3) compared with peripheral blood B cells. Of interest, parotid gland VL gene rearrangements showed an increased number of silent mutations in the productive repertoire with no increase in the R/S ratio compared with the peripheral blood, suggesting that replacement mutations may have been negatively selected from this population. Mutations within RGYW/WRCY sequences appeared to be positively selected in VL gene rearrangements. Altogether, these results indicate that parotid gland B cells in SS represent a unique and highly selected B-cell population.
VL gene usage
Strong selective influences were detected in the parotid gland of the patient, with B cells that rearranged VκA27, VκA19 and Vλ2E as well as Vλ1C being preferentially expanded. Since these VL genes were not found to be over-represented in the nonproductive VL gene repertoire of the parotid gland, they appeared to result from positive selection. Furthermore, there was evidence of clonal expansion of VκA27–Jκ5 and VκA19–Jκ2 rearrangements in the patient's parotid gland only, as well as of Vλ1C–Jλ3 rearrangements in both the peripheral blood and parotid gland of this patient with SS.
One feature of the present patient was that the κ/λ ratio of B cells in the patient's peripheral blood was significantly lower than that found in normal subjects (0.7 versus 1.8) [10
]. When the patient's serum was enriched, the κ/λ ratio was found to be 1.77, suggesting that different influences may effect selection of memory B cells versus plasma cells. It is also possible that the reduced κ/λ ratio in the blood represents preferential migration of κ-expressing B cells (e.g. VκA27 and VκA19) from the blood into the parotid gland. Notably, the κ/λ ratio in the parotid gland was 1.8, consistent with this possibility.
Positive selection of particular VL
chain genes by foreign or autoantigens present in the parotid gland appears to shape the productive VL
chain repertoire in the inflamed tissue. A restriction of the VL
chain repertoire has been described following vaccination. As an example, antibodies against Haemophilus influenzae B
that develop as part of a Th2 response have been identified to be frequently encoded by VκA2, VκO8/O18, VκL11, VκA17, and VκA27 [15
]. Moreover, Vλ genes of the Vλ2 and Vλ7 families were found in the Hib-antibody encoding VL
gene repertoire [15
]. In addition, VκA27 and Vλ2C, Vλ2E, Vλ2A2 or Vλ10A were also shown to encode anti-Streptococcus pneumoniae
]. Interestingly, VκA27 and Vλ2E that were frequently found in the parotid gland of this patient, with VκA27 expanded clonally, have also been shown to encode anti-rabies virus antibodies [17
]. Microbial antigens, including bacterial and viral epitopes that could be involved in the pathogenesis of SS [2
], could thus also be involved in the selective processes shaping the VL
gene repertoires of B cells accumulating in the parotid gland of this SS patient.
Also, autoantigens might be involved in the accumulation of parotid gland B cells in this patient. In this regard, VκA27 was frequently used by RFs in patients with rheumatoid arthritis [17
]. RF is typically present in sera of patients with SS [18
] and was also detected in the saliva or in salivary gland biopsies [19
] of these patients. In this regard, Martin et al.
described two salivary gland lymphomas that developed in SS patients from RF-specific B cells [20
]. Moreover, VκA27 has been reported to be frequently employed by lymphomas developing in the gland of SS patients [21
]. Despite the presence of clonally expanded B cells expressing VκA27, the current patient did not develop lymphoma during a follow-up period of 3 years after the examination. This observation indicates that additional factors or further persistence of the chronic B-cell proliferation are essential for the development of lymphoma.
Histological studies suggest that inflamed ductal epithelial cells represent the focus of the inflammatory response in the salivary glands of patients with SS. There is clear evidence of an inflammatory environment with presentation of self-antigens characteristic of SS [22
] that may permit the production of autoantibodies. Systemic B-cell activation, characterized by hyperimmunglobulinemia and the production of autoantibodies, however, can precede disease manifestations in SS [22
]. This suggests the possibility that enhanced migration or homing of activated lymphocytes into the salivary glands from other sites of B-cell activation may play an important role in disease pathogenesis.
In this context, activated epithelial duct cells have been shown to secrete specific chemokines, such as SDF-1 (CXCL-12) and BCA-1 (CXCL-13), that are capable of attracting specific B lymphocytes into the glands [22
]. H & E staining of the parotid tissue revealed lymphoid follicles as well as diffuse plasma cell infiltration of the organ (Hansen et al
., manuscript submitted). This is inline with the assumption that plasma and memory B cells accumulate in the parotid gland but cannot clarify the origin of these cells. Whatever the primary aberration in the induction of the salivary inflammation, one abnormality relates to the generation of ectopic germinal center-like structures in the inflamed glands. Abnormal migration of B cells into the salivary glands could contribute to this process.
JL gene usage
In contrast to the skewed VL gene repertoire, no differences in the JL gene usage were observed when comparing peripheral blood B cells and parotid gland B cells of the patient. These data rather suggest that selective processes are dependent on the rearranged VL gene.
Mutational analysis supported the conclusion that a distinct B-cell subpopulation accumulated in the parotid gland. The mutational frequency and the percentage of mutated light chain genes were greater in the productive VL chain rearrangements of B cells from the parotid gland compared with those from peripheral blood, but they accumulated a large number of silent mutations. Interestingly, productively rearranged Vλ genes of the parotid gland exhibited a significantly greater mutational frequency than the Vκ gene rearrangements. Altogether, in the parotid gland and in the peripheral blood of the SS patient, nonproductive VL chain rearrangements showed a significantly lower mutational frequency than productive VL chain genes, suggesting that mutations were clearly selected.
A positive selection of mutations was previously identified in VL
gene rearrangements of normal subjects [10
], but not in that of a patient with systemic lupus erythematosus [24
]. In the parotid gland, expanded B cells expressing VκA27 and Vλ2E as well as clonally expanded VL
chains were mutated at a significantly higher frequency compared with the remainder of the repertoire. This finding suggests that B cells bearing particular receptors may have undergone antigen-triggered somatic hypermutation.
Several groups have previously described germinal center-like structures in the parotid gland [3
]. The parotid gland might therefore be able to act as a secondary lymphoid organ, facilitating somatic hypermutation and selection of antigen-specific B cells. Antigen-driven germinal center reactions might proceed within ectopic lymphoid follicles in the parotid gland, giving rise to highly mutated antigen-specific B cells. On the contrary, migration of highly mutated antigen-specific B cells from the patient's blood to the parotid gland could also contribute to the observed differences in the mutational frequencies.
The analysis of somatic mutations of the VL gene rearrangements of the B cells provided evidence of selection against replacement mutations. In addition, the marked increase of RGYW/WRCY mutations in the productive B-cell repertoire of the parotid indicates that positive selection of mutations in these highly targeted motifs occurred in the salivary gland. Selection thus appears to have diminished some mutations while increasing others. The analysis of the R/S ratio and the mutational 'hot spots' of productive VL chain rearrangements of peripheral blood and parotid gland B cells revealed no major abnormalities when compared with normal donors. This indicates intact mechanisms of selection against replacement mutations in the FRs that might cause structural constrains of the immunoglobulin molecule. The frequency of silent mutations was found to be increased in the productive light chain repertoire of B cells from the parotid gland, consistent with a reduced R/S ratio in the CDR and FR.
Overall, replacement mutations were selected against in the parotid gland. This is in line with the observations of Gellrich et al.
], Stott et al.
] and Miklos et al.
], who reported a decrease in the R/S ratio in VH
gene rearrangements of B cells obtained from the salivary glands of SS patients or in B cells from mucosa-associated lymphoid tissue lymphoma in SS patients. Furthermore, Stott et al.
described a decreased R/S ratio in the CDR of VH
gene rearrangements of B cells obtained by minor salivary gland biopsies from two patients with SS [6
Detailed analyses of the frequency of occurrence and mutations of the highly mutable motifs RGYW and WRCY revealed that mutations in nonproductive VLrearrangements of B cells from the parotid gland were less targeted towards RGYW on both DNA strands. These mutations of RGYW on both DNA strands were, however, selected positively in VL gene rearrangements of B cells from the parotid gland. Although no firm conclusion can be drawn, it might be possible that the clear pattern of these targeted mutations is basically generated in the parotid gland of the patient, with particular retention of selected VL rearrangements. Altogether, the influences of selection appeared to be overall intact in the present SS patient.