Isolation of human monoclonal autoantibodies derived from pancreatic lymph node and peripheral blood B cells of islet autoantibody-positive patients.
OBJECTIVE
Autoantibodies against pancreatic islets and infections by enteroviruses are associated with type 1 diabetes, but the specificity of immune responses within the type 1 diabetic pancreas is poorly characterised. We investigated whether pancreatic lymph nodes could provide a source of antigen-specific B cells for analysis of immune responses within the (pre)diabetic pancreas.


METHODS
Human IgG antibodies were cloned from single B lymphocytes sorted from pancreatic lymph node cells of three organ donors positive for islet autoantibodies, and from the peripheral blood of a patient with type 1 diabetes. Antibodies to insulinoma-associated antigen 2 (IA-2), GAD65, zinc transporter 8 (ZnT8) and Coxsackie B virus proteins were assayed by immunoprecipitation and by immunofluorescence on pancreatic sections.


RESULTS
Human IgG antibodies (863) were successfully cloned and produced from 4,092 single B cells from lymph nodes and peripheral blood. Reactivity to the protein tyrosine phosphatase domain of the IA-2 autoantigen was detected in two cloned antibodies: one derived from a pancreatic lymph node and one from peripheral blood. Epitopes for these two antibodies were similar to each other and to those for circulating antibodies in type 1 diabetes. The remaining 861 antibodies were negative for reactivity to IA-2, GAD65 or ZnT8 by both assays tested. Reactivity to a Coxsackie viral protein 2 was detected in one antibody derived from a peripheral blood B cell, but not from lymph nodes.


CONCLUSIONS
We show evidence for the infrequent presence of autoantigen-specific IgG+ B lymphocytes in the pancreatic-draining lymph nodes of islet autoantibody-positive individuals.
Mara
M
Catani
Denise
D
Walther
Michael R
MR
Christie
Kerry A
KA
McLaughlin
Ezio
E
Bonifacio
Anne
A
Eugster
D000328:Adult; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001323:Autoantibodies / Q000302:isolation & purification*; D001324:Autoantigens / Q000276:immunology; D001402:B-Lymphocytes / Q000737:chemistry* / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000276:immunology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D007515:Islets of Langerhans / Q000276:immunology*; D008198:Lymph Nodes / Q000737:chemistry* / Q000276:immunology; D008297:Male; D008875:Middle Aged
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D007074:Immunoglobulin G