Kelch-like Protein 11 Antibodies in Seminoma-Associated Paraneoplastic Encephalitis

Abstract

A 37-year-old man with a history of seminoma presented with vertigo, ataxia, and diplopia. An autoantibody specific for kelch-like protein 11 (KLHL11) was identified with the use of programmable phage display. Immunoassays were used to identify KLHL11 IgG in 12 other men with similar neurologic features and testicular disease. Immunostaining of the patient's IgG on mouse brain tissue showed sparse but distinctive points of staining in multiple brain regions, with enrichment in perivascular and perimeningeal tissues. The onset of the neurologic syndrome preceded the diagnosis of seminoma in 9 of the 13 patients. An age-adjusted estimate of the prevalence of autoimmune KLHL11 encephalitis in Olmsted County, Minnesota, was 2.79 cases per 100,000 men. (Funded by the Rochester Epidemiology Project and others.).

Figure 1.. Discovery and Orthogonal Validation of Anti-KLHL11 Antibodies in Serum Samples Obtained from Men with Paraneoplastic Rhombencephalitis.

Panel A shows immunoprecipitation from a human phage display (10¹⁰ plaque-forming units [PFU] per milliliter) in cerebrospinal fluid (CSF) or serum samples (asterisks) obtained from the patients and healthy controls. Ma2 protein antigen (PNMA2) was identified in patients with encephalitis and autoantibodies to Ma2, and kelch-like protein 11 (KLHL11) antigen was identified in patients with novel, anti-Ma2–negative, seminoma-associated paraneoplastic encephalitis. Panels B through E show orthogonal validation of KLHL11 autoantibodies. Panel B shows detection of immunoprecipitated KLHL11 by Western blotting with an anti-FLAG antibody. KLHL11–MYC–FLAG was successfully immuno-precipitated by the serum or CSF IgG obtained from patients with autoimmune KLHL11 encephalitis (Patients 3, 5, 6, 7, 9, 10, and 11) and by a commercial antibody to KLHL11 (+), but not by the IgG of patients with anti-Ma2–associated encephalitis (Patients A and B) or protein A/G–negative controls (−). M denotes the protein marker. Panel C shows colocalization of the immunofluorescence signal from the IgG of Patient 11 (left) and anti-FLAG antibody (middle) in a cell-based assay with KLHL11–MYC–FLAG overexpression. The scale bar corresponds to 10 μm. Panels D and E show colocalization of the immunofluorescence signal from the KLHL1 IgG of Patient 11 (Panel D) and Patient 3 (Panel E) with a commercial antibody to KLHL11 on mouse brain tissue. The insets in Panels D and E show the degree of red and green channel overlap, or “yellow” signal at a higher magnification. (Areas represented in the insets are shown with dashed lines.) The scale bars correspond to 50 μm. Validation experiments in Panels B and C were completed with the use of the HEK293T cell overexpression system. For microscopy in Panels C through E, cell nuclei were identified with the use of DAPI (4′,6-diamidino-2-phenylindole) stain.