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. 2014 Dec;66(12):3445-56.
doi: 10.1002/art.38872.

Antibody-secreting cell specificity in labial salivary glands reflects the clinical presentation and serology in patients with Sjögren's syndrome

Jacen S Maier-Moore  1 , Kristi A Koelsch, Kenneth Smith, Christopher J Lessard, Lida Radfar, David Lewis, Biji T Kurien, Nina Wolska, Umesh Deshmukh, Astrid Rasmussen, Kathy L Sivils, Judith A James, A Darise Farris, R Hal Scofield
Affiliations

Antibody-secreting cell specificity in labial salivary glands reflects the clinical presentation and serology in patients with Sjögren's syndrome

Jacen S Maier-Moore et al. Arthritis Rheumatol. 2014 Dec.

Abstract

Objective: The serologic hallmark of primary Sjögren's syndrome (SS) is the presence of IgG antibodies specific for Ro (SSA) and La (SSB). The molecular characteristics of gland-derived B cells at the site of primary SS inflammation have been described previously; however, parallels between glandular antibody-secreting cells (ASCs) and serologic antibody specificities have not been evaluated. We used recombinant monoclonal antibody (mAb) technology to study the specificities of salivary gland (SG)-derived ASCs, evaluate their molecular characteristics, and identify IgG antibody specificity.

Methods: Human antibodies were generated from glandular IgG ASCs. Heavy chain and light chain use and immunoglobulin subclass were analyzed by sequencing. Enzyme-linked immunosorbent assay, indirect immunofluorescence, enzyme immunoassay, and (35) S-labeled protein immunoprecipitation analysis were used to determine antibody specificity.

Results: Evaluation of single ASCs in SG biopsy specimens from a patient with primary SS and a patient with SS and overlapping systemic lupus erythematosus revealed significant concordance between serum autoantibody and glandular ASC specificities. Gland-derived ASC heavy chains and light chains were extensively somatically hypermutated, which is indicative of antigen-driven responses. Specifically, we produced the first fully human mAb derived from SGs.

Conclusion: In patients with SS, the SGs are a site for the production of antibodies that extend beyond the canonical Ro and/or La SS specificities. Glandular antibody production strongly reflected the serologic humoral response in the 2 patients whom we studied.

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Conflict of interest statement

Conflict of Interest: The authors have no conflicts of interest to report.

Figures

Figure 1
Figure 1. Histological evaluation of hematoxylin- and eosin-stained salivary gland biopsies from an SLE patient with secondary SS and a primary SS patient with Raynaud’s syndrome
Diffuse periductal and perivascular lymphocytic infiltration (Fig 1A, left), sclerosis and fatty replacement (Fig 1A, center) can be seen in the biopsy of the SS/SLE patient. A structure resembling an ectopic germinal center was also detected within the gland (Fig 1A, right, black arrows). Significant perivascular and periductal lymphocytic infiltration (Figs 1B, left and center) can be observed in the pSS patient, along with periductal foci within the gland (Fig 1B, right).
Figure 2
Figure 2. Labial salivary gland-derived human recombinant antibodies produced by an SS/SLE Patient have specificity to Ro, La and SmRNP
Recombinant human monoclonal antibody screening for affinity to Sm (■), SmRNP (▲), Ro (◆) and La (▼) by ELISA (Positive cutoff ≥ 0.5 OD405). High affinity binding of A2-G03 anti-Ro by ELISA (A, left panel; Kd=7×10−10M) and A3-E03 to SmRNP (A, right panel; Kd=9.0×10−11M). INNO-LIA™ analysis of A2-G03 anti-Ro antibody to both 52 kD and 60kD Ro antigens (B, center strip), and SmB by A2-G05 antibody (B, right strip). Anti-nuclear antibody staining for A2-G03 (C, top panel) and A3-E03 anti-SmRNP antibody (C, bottom panel). 35S protein immunoprecipitation (35S IP) and RNA co-immunoprecipitation (RNA IP) of HeLa cell lysate (D) for A2-G03 to 60 kD Ro (35S IP, 3rd from left) and proteins from the SmRNP complex by A3-E03 (35S IP, 4th from left, arrows A and B/B′). RNA IP analysis showed co-precipitation of hYRNA3, hYRNA4 and hYRNA5 by A2-G03 (RNA IP, 2nd from left) and of multiple U-Small nuclear RNP complexes, particularly U1RNP by A3-E03 (RNA IP, 3rd from left). A U3-Ro standard (RNA IP, left) serum and total nucleic acids (TNA) (RNA IP, right) were included as controls.
Figure 3
Figure 3. Labial salivary gland-derived human recombinant antibodies produced by a primary SS patient can be polyreactive, clonal and bind multiple antigens
Recombinant human monoclonal antibody screening for affinity to Sm (■), nuclear RNP (▲), Ro (◆) and La (▼) by ELISA (Positive cutoff ≥ 0.5 OD405). An antibody lacking binding was included as a negative control (A, Neg Control). Anti-nuclear antibody staining by HEp-2 indirect immunofluorescence analysis showing diffuse cytoplasmic/cytoplasmic fiber staining (such as B2-E04 and B3-C06) and, in some cases, cytoplasmic and low titer nuclear speckled staining (such as B4-G03, B4-A01 and B4-B03). Crithidia kinetoplast immunofluorescence testing for B4-C03 (B, lower right panel). 35S protein immunoprecipitation of HeLa cell lysate for B2-G05 and B4-E06 bound a single band consistent with 60 kD Ro (C). RNA IP showed co-precipitation of hYRNAs1-5 by B4-E06 compared to a U3-Ro standard control (D). The antibodies B2-E04 and B4-G01 showed co-precipitation of a band consistent with PL12 around 100kD (D, 2nd and 3rd from left) and verified by RNA IP for B4-G01 shown next to a PL12 control serum (D, right panel).
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