Loss of TDP-43 function and rimmed vacuoles persist after T cell depletion in a xenograft model of sporadic inclusion body myositis

Abstract

Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell–mediated autoimmunity. IBM muscle biopsies display nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative diseases, where nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43–mediated splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of subjects with IBM. Of 119 muscle biopsies tested, RT-PCR–mediated detection of cryptic exon inclusion was able to diagnose IBM with 84% sensitivity and 99% specificity. To determine the role of T cells in pathogenesis, we generated a xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from subjects with IBM displayed robust regeneration of human myofibers and recapitulated both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts showed invasion by human, oligoclonal CD8⁺ T cells and exhibited MHC-I up-regulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, associated with cryptic exon inclusion. Reduction of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppressed MHC-I up-regulation. However, rimmed vacuoles and loss of TDP-43 function persisted. These data suggest that T cell depletion does not alter muscle degenerative pathology in IBM.

Fig. 1.. Cryptic exon detection is a sensitive and specific assay for TDP-43 pathology in IBM biopsies.

(A) TDP-43 Western blot in myoblasts treated with TDP43 siRNA [knockdown (KD)] compared to control siRNA (C). (B) Visualization of cryptic exons (green arrows) in myoblast cells with TDP-43 knockdown (MyoKD) compared to control (MyoC) for TDP-43 target genes ACOT11, SLC39A8, PFKP, and RHEBL1. ACOT11, acyl-CoA thioesterase 11; SLC39A8, solute carrier family 39 member 8; PFKP, phosphofructokinase, platelet; RHEBL1, RHEB (ras homolog, mechanistic target of rapamycin kinase binding) like 1. (C) Immunohistochemical TDP-43 staining of muscle sections showing accumulation of TDP-43 in the cytoplasm (#) or nuclear clearing (arrows). Scale bar, 50 μm. (D) Visualization of cryptic exons (green arrow) in control myoblasts (MyoC), myoblasts with TDP-43 knockdown (MyoKD), or subject muscle biopsies [control (C) and IBM]; numbers indicate cases (table S2). (E) Representative gel showing cryptic exon expression from TDP-43 target genes GPSM2, ACSF2, HDGFRP2, and ZFP91 in skeletal muscle biopsies from IBM and control biopsies (DM, dermatomyositis; NA, neurogenic atrophy; C, normal muscle or mild nonspecific features).

Fig. 2.. IBM xenografts regenerate robustly in NRG mice.

(A) Representative images of 4-month control and IBM xenografts stained with hematoxylin and eosin (H&E), human spectrin (magenta), human lamin A/C (yellow), embryonic myosin heavy chain (eMHC) (yellow), and 4′,6-diamidino-2-phenylindole (DAPI) (cyan). Scale bar, 100 μm. (B to D) The number of fibers per xenograft area (B), the fraction of the xenograft covered by myofibers (C), and the percentage of eMHC⁺ regenerating fibers are similar between control and IBM xenografts (D). (E) The median cross-sectional area (CSA) of myofibers within the xenografts (*P < 0.05, Mann-Whitney test). For all graphs, each point denotes one subject (control, n = 10; IBM, n = 12).

Fig. 3.. Rimmed Vacuoles and TDP-43 pathology are observed in IBM xenografts.

(A) Representative images from GT stains of 4-month control and IBM xenografts. Examples of rimmed vacuoles (arrow) are exhibited by the IBM xenograft. Scale bar, 50 μm. (B) Quantification of the percentage of fibers with rimmed vacuoles (RV) in each group; each point denotes one subject (control, n = 10; IBM, n = 11). The control sample highlighted in green indicates the case 27 subject diagnosed with a vacuolar myopathy and was excluded from statistical analysis. Mann-Whitney test was used to determine significance (**P < 0.01). (C) TDP-43 aggregates and examples of nuclear clearing (arrows) in 4-month xenografts. Scale bar, 25 μm. (D) Cryptic exon expression from GPSM2 and ACSF2 in 3- and 4- month IBM xenografts and controls. (E) Cryptic exon expression from TDP-43 target genes GPSM2 and ACSF2 detected in IBM xenografts ranging from 3 to 8 months (B, subject biopsy).

Fig. 4.. IBM xenografts recapitulate pathological features of human disease.

(A) Representative images from MHC-I, CD3, and dual COX/SDH (COX-negative, SDH dark fibers stain blue) stains of 4-month control and IBM xenografts. (B) Serial sections of a 4-month IBM xenograft stained with H&E and anti-CD3 showing an example of T cell invasion of a non-necrotic fiber. (C) Quantification of the number of CD3⁺ T cells per xenograft area in 4-month collections. Each point denotes one subject (control, n = 10; IBM, n = 12). (D) Quantification of the percentage of COX-deficient fibers in each group; each point denotes one subject (control, n = 6; IBM, n = 11). Mann-Whitney test was used to determine significance (*P < 0.05). Scale bars, 50 μm.

Fig. 5.. T cells in IBM xenografts are phenotypically similar to subjects with IBM.

(A) Representative CD57 and KLRG1 stains of control and IBM xenografts. Scale bar, 100 μm. (B) Representative flow cytometry plots of PBMCs from a subject with IBM (top) and a 4-month xenograft (bottom) generated from the same subject. Treg, regulatory T cells; Tconv, conventional T cells. Quantification of (C) CD4/CD8 ratio and (D) percent of CD8⁺ T cells that are CD28⁻, CD57⁺, and KLRG1⁺ to evaluate T cell phenotypes in PBMCs and xenografts.

Fig. 6.. T cells in IBM xenografts show clonality and persistence.

TCR sequencing was performed to determine clonality (A) and the number of unique clonotype (richness) (B) in both control and IBM biopsies and xenografts (control biopsy, n = 1; control xenograft, n = 7; IBM biopsy, n = 8; IBM xenograft, n = 38). Mann-Whitney test was used to determine significance (*P < 0.05; **P < 0.01). (C) Heatmap displaying the Morisita-Horn index between biopsies and xenografts. (D) The proportion of TCR clones was compared between biopsies from subjects with IBM and corresponding xenografts at multiple time points as shown for two IBM xenograft cases. Each color represents a unique clonotype.

Fig. 7.. OKT3 treatment ablates T cells from IBM xenografts but does not affect myofiber regeneration.

(A) Representative H&E, CD3, lamin A/C (yellow), spectrin (magenta), eMHC (yellow), and DAPI stains of 4-month untreated and OKT3-treated IBM xenografts. Scale bar, 100 μm. Quantification of the number of CD3⁺ T cells over the xenograft area (B), the number of fibers over the xenograft area (C), fiber fraction (D), median fiber CSA (E), and percentage of eMHC⁺ fibers (F) are shown. For all graphs, each point denotes one xenograft (2-month untreated, n = 4; 2-month OKT3, n = 4; 4-month untreated, n = 12; and 4-month OKT3, n = 13). Xenografts were obtained from four subjects with IBM (cases 23, 26, 36, and 42). Mann-Whitney U test was used to test for significance (*P < 0.05; ****P < 0.0001).

Fig. 8.. OKT3 depletion of T cells reduces inflammation but not rimmed vacuoles or p62 pathology.

(A) Representative images from MHC-I, KLRG1, GT histological stains, and fluorescent costaining of p62 (yellow), spectrin (magenta), and DAPI in untreated and OKT3-treated 4-month IBM xenografts. Rimmed vacuoles and p62 aggregates are indicated by arrows. Scale bar, 100 μm. (B) Quantification of the percentage of MHC-I–positive fibers in untreated and OKT3-treated xenografts at 4 months (untreated, n = 5; OKT3, n = 5). Mann-Whitney test was used to determine significance (*P < 0.05). (C) Quantification of the number of KLRG1⁺ cells per xenograft cross section in untreated and OKT3-treated xenografts at both 2-month (untreated, n = 4; OKT3, n = 4) and 4-month (untreated, n = 11; OKT3, n = 12) time points. Mann-Whitney test was used to determine significance (***P < 0.001). (D) Quantification of the percentage of myofibers containing rimmed vacuoles (RV) in untreated and OKT3-treated xenografts at 4 months (untreated, n = 5; OKT3, n = 7). Mann-Whitney test was used to determine significance (xenograft samples from case 36 were excluded from this analysis as the subject biopsy did not display RVs). (E) Quantification of the percentage of fibers containing p62 aggregates in 4-month untreated (n = 5) and OKT3 (n = 5) treated xenografts. Mann-Whitney test was used to determine significance. (F) Expression from the ACSF2 cryptic exon compared to an ACSF2 conserved exon in 2-, 4-, and 8-month untreated and OKT3-treated IBM xenografts. The box diagrams show the primer design strategy for each PCR reaction. Top: One primer (black arrow) is designed to the cryptic exon (red), and the other is designed to a conserved exon (green) to detect cryptic exon (CE) expression. Bottom: Both primers are designed to a conserved exon to determine conserved exon expression as a control for total ACSF2 message.