Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Abstract

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunoglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunoglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (V lambda 2E) and variable kappa chain genes (V kappa A27). Moreover, clonally related V(L) chain rearrangements were identified; namely, V kappa A27-J kappa 5 and V kappa A19-J kappa 2 in the parotid gland, and V lambda 1C-J lambda 3 in the parotid gland and the peripheral blood. V kappa and V lambda rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V(L) chain genes caused by selection and clonal expansion of B cells expressing particular V(L) genes. In addition, the data document an accumulation of B cells bearing mutated V(L) gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.

Figure 1

Distribution of individual Vλ genes in B cells from the peripheral blood and from the parotid gland of a patient with Sjögren's syndrome (SS) compared with those of normal healthy subjects (NHS). The Vλ gene usage of normal donors is published elsewhere [11]. Vλ genes are arranged starting with the genes located within the A-cluster of the Vλ locus (J-proximal). The significant differences in the frequency of occurrence of 3H (P < 0.05)/7A^§ (P < 0.05)/1G^* (P < 0.005)/10A° (P < 0.005) gene rearrangements comparing the nonproductive and productive Vλ gene repertoire suggest processes of positive and negative selection of these Vλ gene segments.

Figure 2

Vλ1c–Jλ3b rearrangements obtained from the peripheral blood (D10IVL1F9 and D10IIVL1E12) and from the parotid gland (PaIVL1E11 and PaIVL1G12) of the patient with Sjögren's syndrome.

Figure 3

Distribution of individual Vκ genes in B cells from the peripheral blood and from the parotid gland of a patient with Sjögren's syndrome(SS) compared with those of peripheral blood B cells from normal healthy subjects (NHS). ^# The significant difference in the frequency of occurrence of VκB2 comparing the nonproductive and productive Vκ gene repertoire suggests negative selection of this gene segment (P < 0.005). Vκ gene usage of normal donors has been published elsewhere [10]. Vκ genes are arranged in order from J-proximal to J-distal.

Figure 4

Frequency of replacement mutations (black) and silent mutations (white) of each codon of productively rearranged V_L genes of B cells isolated from peripheral blood and from the parotid gland of a patient with Sjögren's syndrome. The frequency of mutation of each codon is calculated as the percentage of sequences that contain mutations in particular codon positions. Mutational 'hot spots' of replacement mutations are shown. Nonproductive V_L genes were not analyzed because of the small number of mutations. CDR, complementary determining regions.