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. 2020 Jun 2;54(1):87-97.
doi: 10.1002/jmd2.12128. eCollection 2020 Jul.

Quantifying lymphocyte vacuolization serves as a measure of CLN3 disease severity

Willemijn F E Kuper  1 , Marlies Oostendorp  2 , Brigitte T A van den Broek  3   4 , Karin van Veghel  4 , Lourens J P Nonkes  2 , Edward E S Nieuwenhuis  5 , Sabine A Fuchs  1 , Tineke Veenendaal  6 , Judith Klumperman  6 , Albert Huisman  2 , Stefan Nierkens  4 , Peter M van Hasselt  1   3
Affiliations

Quantifying lymphocyte vacuolization serves as a measure of CLN3 disease severity

Willemijn F E Kuper et al. JIMD Rep. .

Abstract

Background: The CLN3 disease spectrum ranges from a childhood-onset neurodegenerative disorder to a retina-only disease. Given the lack of metabolic disease severity markers, it may be difficult to provide adequate counseling, particularly when novel genetic variants are identified. In this study, we assessed whether lymphocyte vacuolization, a well-known yet poorly explored characteristic of CLN3 disease, could serve as a measure of disease severity.

Methods: Peripheral blood obtained from healthy controls and CLN3 disease patients was used to assess lymphocyte vacuolization by (a) calculating the degree of vacuolization using light microscopy and (b) quantifying expression of lysosomal-associated membrane protein 1 (LAMP-1), using flow cytometry in lymphocyte subsets as well as a qualitative analysis using electron microscopy and ImageStream analysis.

Results: Quantifying lymphocyte vacuolization allowed to differentiate between CLN3 disease phenotypes (P = .0001). On immunofluorescence, classical CLN3 disease lymphocytes exhibited abundant vacuole-shaped LAMP-1 expression, suggesting the use of LAMP-1 as a proxy for lymphocyte vacuolization. Using flow cytometry in lymphocyte subsets, quantifying intracellular LAMP-1 expression additionally allowed to differentiate between infection and storage and to differentiate between CLN3 phenotypes even more in-depth revealing that intracellular LAMP-1 expression was most pronounced in T-cells of classical-protracted CLN3 disease while it was most pronounced in B-cells of "retina-only" CLN3 disease.

Conclusion: Lymphocyte vacuolization serves as a proxy for CLN3 disease severity. Quantifying vacuolization may help interpretation of novel genetic variants and provide an individualized readout for upcoming therapies.

Keywords: CLN3 disease; ImageStream; flow cytometry; lymphocyte vacuolization; lysosomal membrane‐associated protein‐1 (LAMP‐1); neuronal ceroid lipofuscinosis (NCL).

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

FIGURE 1
FIGURE 1
A, Quantifying lymphocyte vacuolization in healthy controls compared to (classical) CLN3 disease. Quantifying the degree of lymphocyte vacuolization—in this study performed along two axes: the percentage of vacuolated lymphocytes (log 10) and the number of vacuoles per affected lymphocyte (linear)—accurately distinguishes (classical) CLN3 disease patients from healthy controls. Among the healthy controls are some individuals who are heterozygous for the common 1 kb deletion in CLN3 in whom the degree of vacuolization appears slightly higher. B, Microscopical assessment of lymphocyte vacuolization in CLN3 disease. Left: light microscopy image of a vacuolated lymphocyte in classical CLN3 disease. Right: ImmunoFluorescence on 300 nm thick sections of a lymphocytes of a classical CLN3 disease patient (not the same cell as on the left panel) displaying abundant LAMP‐1 expression (in green) appearing at the membranes of the vacuoles surrounding the nucleus (in blue). C, Quantifying lymphocyte vacuolization in different CLN3 disease severity phenotypes. The percentage of vacuolated lymphocytes with a single positive LAMP‐1 expression measured on flow cytometry. Connected lines show repeated measurements per patient. The linear mixed model estimates stable percentages of singe positive vacuolated lymphocytes per CLN3 disease severity type over time (P = .26) which is represented by the black lines. LAMP‐1 = lysosomal‐associated membrane protein 1
FIGURE 2
FIGURE 2
Lymphocyte vacuolization and LAMP‐1 expression correlate with CLN3 disease severity. The percentage of vacuolated lymphocytes (left upper panel), percentage of LAMP‐1 single positive CD4 (right upper panel), CD8 (left lower panel), and CD20 (right lower panel) lymphocytes split per phenotypic subtype shown in bar plots. In the percentage of vacuolated lymphocytes, and single positive CD4 and CD8 lymphocytes a clear correlation with disease severity is seen. In the CD20 subset, a particularly high degree of LAMP‐1 expression is seen in the mildest CLN3 disease phenotype: CLN3‐associated retinal degeneration. LAMP‐1 = lysosomal‐associated membrane protein 1
FIGURE 3
FIGURE 3
Flow cytometry analyses of LAMP‐1 expression in CLN3 disease in different lymphocyte subsets. The percentage of lymphocytes with a single positive LAMP‐1 expression measured on flow cytometry. A, CD4; B, CD8; and C, CD20. Connected lines show repeated measurements per patient. The linear mixed model estimates stable percentages of singe positive lymphocytes per CLN3 disease severity type over time which is represented by the black lines. LAMP‐1 = lysosomal‐associated membrane protein 1
FIGURE 4
FIGURE 4
ImageStream analysis of LAMP‐1 expression and distribution CLN3 disease lymphocytes. ImageStream analysis of classical CLN3 disease lymphocytes compared to healthy control lymphocytes confirmed increased expression of LAMP‐1 in CLN3 disease lymphocytes distributed throughout a major area of the cell. A, Examples of LAMP‐1 expression in CD4 positive lymphocytes. The mean LAMP‐1 expression/cell was calculated for all lymphocytes per condition (healthy vs CLN3). Shown are representative examples of mean LAMP‐1 expression/cell at the 10th up to 90th percentile of the distributions. B, Median LAMP‐1 intensity expressed as % of cell above/equal to a specified intensity threshold value (x‐axis), CLN3 disease vs healthy control. Shaded ranges: dark band represents interquartile range (25th‐75th percentile), light band 2.5th to 97.5th percentile range. LAMP‐1 = lysosomal‐associated membrane protein 1
FIGURE 5
FIGURE 5
Immunoelectron microcopy imaging of LAMP‐1 expression and distribution in CLN3 disease lymphocytes. Immunoelectron microscopy of ultrathin cryosections labeled with LAMP‐1—10 nm protein A gold particles. Compared to a healthy control, lymphocytes from a classical CLN3 disease patient show an increased LAMP‐1 expression at the membranes of vacuolated lysosomes, but also around nonvacuolated lysosomes and late endosomes. LE = late endosome; Lys = Lysosome; M = mitochondrion; N = nucleus
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