Cytotoxic immune cells do not affect TDP-43 and p62 sarcoplasmic aggregation but influence TDP-43 localisation

Sporadic inclusion body myositis (sIBM) is an idiopathic inflammatory myopathy with invasion of CD8 T cells in muscle and aggregation of proteins in the sarcoplasm. TDP-43 and p62 are two proteins that aggregate in affected muscle, and have been suggested as specific markers for sIBM over other inflammatory myopathies. TDP-43 is also mislocalised from the nucleus to the sarcoplasm in sIBM. It is not clear if inflammation precedes protein aggregation in sIBM. This study investigated if exposure to cytotoxic inflammatory cells caused TDP-43 and p62 aggregation or TDP-43 mislocalisation in cultured myotubes. TALL-104 coculture was highly cytotoxic to myotubes after 24 h. Secretion of IFNγ and TNFα were higher in cocultures compared to monocultured TALL-104 cells, indicating activation. TALL-104 cells attached to and infiltrated myotubes. There was no effect of TALL-104 coculture on TDP-43 or p62 sarcoplasmic aggregate size or frequency. However, there was decreased localisation of TDP-43 to the nucleus with TALL-104 coculture compared to control. In an in vitro setting, cytotoxic immune cells did not cause TDP-43 or p62 sarcoplasmic aggregation, suggesting cellular cytotoxicity may not trigger aggregation of these proteins. However TALL-104 coculture influenced TDP-43 localisation, suggesting cytotoxic immune cells may contribute to TDP-43 localisation shifts which is observed in sIBM.


Immunofluorescent microscopy
Skeletal muscle-derived cells were seeded on Gibco Geltrex (A1569601)-coated 13 mm round coverslips in 24 well plates.The cells were allowed to proliferate for 48 h before differentiation for 5 days before coculture with TALL-104 cells for 24 h.Cells were washed and fixed with 4% formaldehyde solution (Fisher Scientific, UK 12777847) for 10 min at room temperature.Cells were permeabilised with 0.1% v/v triton X100 in PBS for 15 min before blocking with 5% goat serum in PBS for 30 min to 1 h.Primary antibodies TDP-43 1:150 (10782-2-AP) and p62 1:100 (66184-1-Ig, both from Proteintech, UK) diluted in blocking buffer were added by inverting the coverslip onto a 30 µL drop of antibody solution on Parafilm®, and incubating in a humidified chamber overnight at 4 °C.Coverslips were incubated in appropriate Alexa Fluor™-conjugated secondary antibodies (Invitrogen,UK Alexa Fluor™ goat anti-rabbit 488 A-11008 or goat anti mouse 546 A-11030) for 1 h at room temperature.Cells were washed and HCS CellMask™ deep red (H32721) was added at 1:5000 in PBS for 30 min to visualise all cell

TDP-43 localisation
To analyse TDP-43 localisation, images were captured using an Olympus IX81-ZDC inverted widefield fluorescent microscope at 10× magnification.Four images were analysed per condition per myogenic donor.DAPI and HCS CellMask™ staining was used to observe subcellular compartments of the nucleus and sarcoplasm respectively.Based on the observed staining, TDP-43 was classified as being within the nucleus, sarcoplasm, nucleus and sarcoplasm, or neither (not expressed).Localisation was quantified for both single nucleated and multinucleated cells.Localisation was expressed as a percentage of total number of cells observed with HCS CellMask™ for each localisation compartment.

TD-43 and p62 aggregation analysis
For p62 and TDP-43 aggregation analysis, cells were imaged using a Zeiss LSM 800 confocal microscope.Eight z-stacks each composed of two images approximately 0.9 µm apart from one experiment were captured.Images were analysed using NIH Image J (USA).Obtained images were split into individual colour channels and converted to 8-bit.p62 and HCS CellMask™ channels were manually thresholded.
p62 particle frequency and size was determined in the p62 channel using NIH Image J using the "analyse particles" function with pixel size over 3. p62 frequency was the total number of particles obtained per image with the "analyse particles" function and p62 size was the average particle size per image.In the thresholded cell mask channel, percentage area of image coverage was obtained.p62 particle frequencies were normalised to percentage area coverage per image using HCS CellMask™ staining, to account for differences in myotube size.
The same images for p62 analysis were used for TDP-43 sarcoplasmic aggregate analysis.Sarcoplasmic aggregates were identified manually as punctate areas of staining.Using Image J, freehand regions were drawn around aggregates to measure their area in µm 2 .TDP-43 aggregate frequencies were normalised to percentage area coverage per image of cell mask staining using the cell mask coverage.TDP-43 colocalisation with p62 was assessed for each identified TDP-43 aggregate using thresholded p62 and TDP-43 channels.Any area of overlap between TDP-43 and p62 objects over two pixels was considered colocalised.Percentage colocalisation is the percentage of TDP-43 aggregates that colocalised with p62 puncta for all aggregates observed for one donor under one condition.p62 puncta and TDP-43 aggregate analysis was conducted blinded.

ELISA
DuoSet® Enzyme-linked immunosorbent assays (ELISA) were purchased from R&D systems, UK.Human IFN gamma (DY285B), and human TNF alpha (DY210) were used.ELISA were performed following manufacturer's instructions.Nunc MaxiSorp™ immunoplates were coated with recommended dilutions of capture antibody, blocked with 0.2 µm filtered 1% w/v BSA (Sigma, UK, A7030) (reagent diluent) for 1 h, before incubation of samples and standards diluted in reagent diluent for 2 h.Recommended concentrations of detection antibody diluted in reagent diluent were added for 2 h in sealed plates.Streptavidin horse radish peroxidase (HRP) diluted in reagent diluent was incubated for 20 min before washing and addition of substrate solution (1:1 mix hydrogen peroxide and tetramethylbenzidine, R&D Systems, UK) for 20 min.Colour development was halted with 2N sulphuric acid.Optical density was determined using a Multiskan™ FC plate reader by subtracting readings at 540 nm from 450 nm to account for any imperfections in the plate.

Statistics
Statistical analysis and graph plotting was performed with GraphPad Prism 7. Normal distribution of data was tested with Shapiro Wilk test before choosing appropriate statistical tests.All statistical tests performed were two-tailed.Data are displayed as mean ± standard error of mean for normally distributed data or median ± interquartile range for non-normally distributed data.

TALL-104 coculture was cytotoxic to myotubes
To examine if direct coculture with TALL-104 cells was cytotoxic to myotubes, lactate dehydrogenase release was measured from cocultures at different E:T ratios after 4, 24, and 48 h.The E:T ratios used were 0.5:1, 1:1, 2.5:1, and 5:1.IL-2-containing medium was used as a control.IL-2 is a cytokine whose main roles are within the immune system 34 , therefore it was not expected that the inclusion of this cytokine in controls would have any detrimental effect on myogenic cells, and IL-2-containing medium showed low myotube cytotoxicity.These myogenic cultures show limited continuous differentiation after day 4 (supplementary Fig. S1), therefore IL-2 is not expected to interfere with differentiation.
In cocultures, large areas of floating debris surrounded by TALL-104 cells were observed.Myotubes that remained attached to the surface appeared thinner than those in the control condition (Fig. 1A).At 4 h, only the highest E:T ratio of 5:1 showed a significant increase in cytotoxicity compared to myotubes cultured with IL-2 control (Fig. 1B).By 24 h, all E:T ratios showed increased cytotoxicity of myotubes compared to no TALL-104 IL-2 control, which was also true for 48 h co-culture (Fig. 1C,D).At 24 h, the mean percentage cytotoxicity was 13.23 ± 1.73% in the control, 53.66 ± 3.4% for 0.5:1, 68.97 ± 3.04% 1:1, 75.02 ± 9.07% 2.5:1, and 93.91 ± 8.92% 5:1.When comparing cytotoxicity over time, all E:T ratios showed higher cytotoxicity at 24 h compared to 4 h, whereas only 5:1 ratio showed increased cytotoxicity from 24 to 48 h, suggesting most cytotoxic effects occur between 4 and 24 h (Fig. 1E).

TALL-104 cells attached to and invaded myotubes
TDP-43 and p62 immunofluorescent staining was conducted in myotube TALL-104 cocultures.In some immunofluorescent images of cocultures, small rounded cells with a high nuclear to cytoplasm ratio were observed which were likely TALL-104 cells.TALL-104 cells were observed attaching to myotubes (Fig. 3A).They were also seen in the sarcoplasm of myotubes, with a halo of darkness surrounding them, which may show localised myotube destruction (Fig. 3B).Furthermore, TDP-43 and p62 were visible in TALL-104 cells.TDP-43 was observed in the nucleus of TALL-104 cells, as well as strongly staining the edges of the cells.p62 was less pronounced but was also localised towards the periphery of the cell cytoplasm.

TALL-104 coculture did not affect p62 and TDP-43 aggregation, but TDP-43 aggregates were less likely with coculture
To examine if direct coculture with cytotoxic immune cells caused p62 aggregation in myotubes, immunofluorescent microscopy was used to quantify size and frequency of p62 puncta.24 h coculture with 1:1 E:T ratio of TALL-104 cells was used.Figure 4A shows p62 in myotubes.p62 was mostly located in the sarcoplasm with variable sized puncta and some areas of diffuse staining, occasional p62 puncta in the nucleus could also be observed.p62 puncta frequency relative to cell area was not affected by TALL-104 coculture compared to IL-2 control (Fig. 5A).There was also no difference in p62 puncta size between IL-2 control and TALL-104 coculture (Fig. 5B) with mean p62 puncta size of 0.267 ± 0.023 µm 2 in the IL-2 control group.

TDP-43 subcellular localisation was affected by TALL-104 coculture
The localisation of TDP-43 within myogenic cells was assessed when cultured with 1:1 E:T ratio of TALL-104 for 24 h compared to skeletal muscle derived cells treated with IL-2-containing media for the same length of time.There was a significant difference in the interaction between TDP-43 localisation and treatment condition, showing TDP-43 localisation was altered with TALL-104 coculture (two-way ANOVA) (Fig. 6).The only subcellular localisation with a difference between IL-2 control and TALL-104 coculture with Tukey's post-hoc testing was "nucleus only", showing fewer cells with TDP-43 only in the nucleus in TALL-104 coculture compared to IL-2 control.In immunofluorescent images of the TALL-104 coculture group there appeared more cells with weak TDP-43 sarcoplasmic (and nuclear) expression.There was also a significant difference in the localisation of TDP-43 between the subcellular compartments (two-way ANOVA p < 0.0001).The localisation with the largest percentage of TDP-43 regardless of treatment condition was nucleus and sarcoplasm with mean 83 ± 3.71% (Tukey's post-hoc test).

Discussion
This study aimed to investigate the effects of cell-mediated cytotoxicity on TDP-43 and p62 degenerative features in skeletal muscle.Other proteins are also implicated in sIBM, most notably amyloid-β [35][36][37] .However, the importance of amyloid-β in sIBM has been called into question 38 .The presence of amyloid β aggregates in sIBM myofibres may be low, with some studies showing as low as between 0 39 and 0.4% 40 of myofibres containing amyloid deposits.On the other hand, amyloidβ was found over-represented in rimmed vacuoles compared to other areas of the sarcoplasm 15 .It was decided not to focus on this protein here.Whilst TDP-43 and p62 are suggested as specific sIBM markers, aggregates of these proteins have been observed in other muscular diseases.TDP-43 aggregation is seen in oculopharyngeal muscular dystrophy, desminopathy, myotilinopathy, and hereditary IBM 41,42 , however, these diseases are not immune-mediated.With no distinctive TDP-43 aggregate pattern in sIBM compared to other diseases 42 , the combined presence of TDP-43 and CD8 + T cells may serve as selective markers of sIBM.p62 is identified in other inflammatory myopathies, particularly immune-mediated necrotising myositis (IMNM) but undetectable in normal muscle 43,44 .This study builds upon previous work examining cytotoxic immune cell interactions with skeletal muscle cells.Coculture of antigen-specific CD8 + T cells and transgenic antigen presenting myotubes was used to model polymyositis.These myotubes died with increasing lysis at increasing densities of CTLs and the CD8 T cells infiltrated into the myotubes 45 , similar to what was observed here.The effect of coculturing isolated sIBM CD8 + T cells with autologous myotubes has been tested, showing 1 out of 4 sIBM patient's CTLs showed cytotoxicity against autologous myotubes 46 .This suggests some sIBM patient CTLs may be primed against skeletal muscle antigens.
Previous in vitro models of sIBM have been created.IL-1β and IFNγ treatment of healthy human myotubes was used to investigate the effects of these cytokines on a range of degenerative characteristics altered in sIBM [47][48][49] .In rat myotubes, treatment with IL-1β triggered TDP-43 mislocalisation to the sarcoplasm, without triggering inclusion body-like protein aggregates 50 .These results show inflammation can precede non-inflammatory sIBM features in myotubes.However, sIBM-derived myotubes cultured under electrical stimulation showed TDP-43 and p62 aggregation 51 , highlighting a potential muscle-intrinsic mechanism of aggregation in sIBM.The effects of cell-mediated cytotoxic immunity on TDP-43 and p62 in myotubes has not previously been investigated.The work presented here contributes to understanding the interplay of inflammation on degenerative sIBM-like features.
Here, TALL-104 cells were used as a proxy for CD8 T cells due to their continued growth in culture and easy availability.TALL-104 is a lymphoblastic leukaemic cells line expressing CD8, CD3, and T cell receptor proteins as well as natural killer (NK) cell markers including CD56 and CD161 32,33 .Blocking NK cell receptors NKp46, NKG2D, and 2B4 prevented TALL-104-mediated lysis, therefore cytotoxicity is mediated through NK cell receptors, independent of MHC recognition 32 .Instead, they resemble a mixed lineage sharing features of NK and CD8 T cells [52][53][54][55][56] .It was expected TALL-104 coculture would not be cytotoxic as myotubes should lack epitopes that trigger cytotoxicity.Furthermore, TALL-104 spare normal cells whilst targeting tumourigenic cells [57][58][59][60] .NK cells can target autologous skeletal muscle cultures from healthy patients, whilst autologous myotubes were not targeted or killed by CD8 cytotoxic T cells 61 .This suggests culturing myogenic cells may reveal epitopes capable of stimulating NK cells that are usually hidden or absent in vivo.The lack of reactivity of TALL-104 cells to normal brain cells 57 indicates cytotoxicity against myotubes is unlikely to be due to HLA-mismatching or anti-self recognition.
The results presented here suggest exposure to cytotoxic immune cells does not precede TDP-43 and p62 sarcoplasmic aggregation.The decreased likelihood of images containing TDP-43 aggregates may reflect a change in homeostatic functions of TDP-43, such as RNA processing in myo-granules 62 where TDP-43 forms puncta with muscle-specific mRNAs.In cells undergoing apoptosis there is a rapid degradation of mRNA 63 , therefore TDP-43's homeostatic roles could cease with TALL-104 coculture.However, selection bias where only cells with a higher propensity to survive TALL-104 cytotoxicity may have influenced these results.Whilst a decrease in nuclear TDP-43 was observed with TALL-104 coculture, the localisation did not shift to the sarcoplasm only, showing a "mislocalisation" of TDP-43 was not recapitulated.However, this suggests cytotoxic inflammatory cell exposure can contribute to TDP-43 subcellular shifts, which could contribute to the overall phenotype of TDP-43 mislocalisation as seen in sIBM.
There are several limitations to this study.Firstly, TALL-104 cells mediate cytotoxicity through MHC-independent mechanisms, as well as being a neoplastic cell line.sIBM CD8 + T cells are highly differentiated, therefore the TALL-104 cell line is unlikely to share similar activation features to native sIBM CTLs.TALL-104 were identified based on size and high nuclear to cytoplasmic ratio, but co-staining with CD8 or other T cell markers would have been preferrable.The inflammatory state of the myotubes themselves was not tested, which could have been investigated by examining major histocompatibility complex I and other inflammatory markers on myotubes.To further characterise the cytotoxic reactivity of TALL-104 to myotubes, granzyme and perforin analysis could be conducted.Finally, in comparison to the slowly progressive nature of sIBM, the cytotoxicity of TALL-104 cells here was acute, limiting the relevance of these experiments.
Overall, the exploratory results in this study show exposure to cytotoxic immune cells did not trigger TDP-43 or p62 sarcoplasmic aggregation as seen in sIBM, suggesting in vitro aggregation of these proteins were not preceded by inflammatory cytotoxicity.Conversely, TALL-104 cells triggered a shift in TDP-43 subcellular localisation, showing cytotoxic cells may contribute to the sIBM-like characteristic of TDP-43 mislocalisation to the sarcoplasm.This study helps towards understanding whether inflammation or protein aggregation arises first in sIBM.

Figure 5 .
Figure 5. Coculturing TALL-104 cells with myotubes did not affect p62 or TDP-43 aggregate size or frequency.(A) There was no difference in p62 puncta frequency between IL-2 control and TALL-104 coculture at 1:1 effector to target ratio (p = 0.0796).(B) There was no difference in p62 puncta size between IL-2 control and TALL-104 coculture (p = 0.697).Student's T-test.(C) There was no difference between IL-2 control and TALL-104 co-culture for TDP-43 aggregate frequency relative to cell area (p = 0.728), (D) aggregate size (p = 0.548), (E) or colocalisation with p62 puncta (p = 0.369).(F) There was a higher frequency of images containing TDP-43 aggregates in the IL-2 control group compared to TALL-104 coculture (Fisher's exact test p = 0.0018, eight images (with two z-stacks) for each of 5 donors).(A-E) Student's T-test or Mann-Whitney U test.n = 5 myogenic donors.

Figure 6 .
Figure 6.TDP-43 subcellular localisation was affected by TALL-104 coculture.(A) Representative images of TDP-43 localisation with IL-2 control and TALL-104 coculture.Scale bar = 100 µm.(B) There was a difference in the distribution of TDP-43 between the subcellular compartments between the two treatment conditions (twoway ANOVA interaction factor p = 0.02).There was a decrease in nuclear only expression of TDP-43 with TALL-104 coculture compared to IL-2 control (p = 0.0401), but there was no difference for the nuclei and sarcoplasm (p = 0.241), sarcoplasm only (p = 0.974), or neither (p > 0.999) groups.Two-way ANOVA with Sidak's multiple comparisons.n = 5 myogenic donors.