T(CMV) cells exhibit a unique epigenetic signature

The differentiation of naive murine CD8⁺ T cells into the different types of memory and effector T cells is associated with specific changes at the transcriptional and epigenetic level [23,24,29,38,39]. Studies on human CD8⁺ T cell memory formation similarly identified changes in DNA methylation patterns in the cytotoxicity-related genes IFNG, GZMB, and PRF1 [40,41]. However, the global changes in DNA methylation patterns during the pathogen-induced formation of antigen-specific memory CD8⁺ T cells are only incompletely understood. Therefore, we isolated T(CMV) cells from 5 different CMV-seropositive donors to unravel their specific DNA methylation patterns. T(CMV) cells were identified as IFN-γ-secreting cells after restimulation with an overlapping peptide pool of the CMV-encoded pp65 protein (phosphoprotein of 65 kDa), which is the main component of the tegument layer of viral particles and an immunodominant target of T cell responses to CMV [42] (S1A Fig). An initial flow cytometric characterisation revealed that the vast majority of these IFN-γ⁺ T(CMV) cells showed a CD45RA⁺CD62L⁻ T_EMRA cell phenotype (S1B Fig), which is in line with recently published data [43,44]. For whole-genome bisulfite sequencing (WGBS), T(CMV) cells were isolated by flow cytometry-based cell sorting as CD3⁺CD4⁻CD8⁺IFN-γ⁺ cells, while memory CD8⁺ T cells (T_mem; CD3⁺CD4⁻CD8⁺CCR7⁻CD28^highCD27⁺CD45RA⁻; n = 5) and naive CD8⁺ T cells (T_N; CD3⁺CD4⁻CD8⁺CCR7⁺CD28^intKLRG1⁻CX3CR1⁻CD45RA^high; n = 4) from the same donors served as controls (S2 Fig). For each sorted sample, genomic DNA was isolated and the genome-wide DNA methylation profile was determined by WGBS. Global analysis of the DNA methylation data revealed a strong progressive loss of DNA methylation from T_N to both T_mem and T(CMV) cells (Fig 1A), which is congruent with previously published data on human CD4⁺ memory T cells [45]. Next, we identified DMRs in pairwise comparisons of all T cell subsets. We found the highest numbers of DMRs between T_mem vs. T_N cells (11,357) and T(CMV) vs. T_N cells (13,011), while a rather low number of only 104 DMRs were identified in the comparison of T(CMV) vs. T_mem cells (S1 Table). Accordingly, a principal component analysis (PCA) based on genome-wide DNA methylation levels revealed a close sample group relationship between T(CMV) and T_mem cells, whereas T_N cells were placed separately along the main principal component 1 (PC1) (Fig 1B). This finding was confirmed by hierarchical clustering of individual samples, which further demonstrated that the majority of DMRs showed a consistent methylation status in T(CMV) and T_mem cells, while being distinct to the largely methylated state of most DMRs in T_N cells (Fig 1C). Yet, it is important to note that all T(CMV) and T_mem cell samples clustered group-wise, indicating distinct methylation signatures between these cell types. The majority of DMRs across all pair-wise population comparisons were located in gene bodies and intergenic regions, while a smaller fraction was mapped to promoters (Fig 1D). In line with previous findings [33,46], we observed that many genes involved in the CD8⁺ T cell effector program contain demethylated regions in both T(CMV) and T_mem cells, but are methylated in T_N cells, including genes coding for molecules regulating T cell-mediated cytotoxicity (GZMA, GZMB, GZMK, IFNG), actin cytoskeletal organization (ACTA2, ACTB, ACTN4, DOCK2), integrin-dependent cell adhesion (ITGB1, ITGB1BP1, ITGA5), chemokine-mediated signalling (CCL1, CCL4, CCL5, CCR4, CCR5), TGFβ-mediated signalling (SMAD2, SMAD3, SMAD7), and WNT/β-catenin signalling (APC2, CTR9, CYLD, WNT11, WNT7B) (S1 Table). Taken together, the WGBS study demonstrated substantial changes of DNA methylation patterns during the differentiation of T_N into T(CMV) cells.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Genome-wide methylation profiling of T(CMV) cells.

CD8⁺ T cell subsets including T_N (n = 4), T_mem (n = 5), and T(CMV) cells (n = 5) were sorted using flow cytometry. From each sample, genomic DNA was isolated and converted by bisulfite treatment, followed by WGBS. (A) Circos plot showing DNA methylation levels for T_N, T_mem, and T(CMV) cells across the whole genome (in 10 kb tiling windows, aggregated all donors). CpG methylation levels are represented as histogram tracks across the genome based on the sample. High levels of methylation are indicated by dark blue, while low levels of methylation are indicated by light yellow. Brown-teal colour-coding between the bar tracks indicates differences in methylation levels relative to T_N cells. (B) Principal Component Analysis (PCA) per cell type and donor samples based on 50,000 highly variable 1 kb tiling regions. The percent of explained variance for each component is denoted in the axis labels. (C) Hierarchical clustering of 15,598 non-overlapping DMRs identified in pair-wise comparisons of methylomes from T_N, T_mem, and T(CMV) cells. The dendrogram on top corresponds to hierarchical clustering of the samples. The colour-code illustrates the mean methylation levels of the DMRs as indicated (yellow: methylation level = 0%, blue: methylation level = 100%). (D) Pie charts indicating the position of the pairwise DMRs identified in indicated group-wise comparisons relative to annotated genes. Numbers in parentheses show the number of DMRs in intergenic, promoter, or gene body regions according to their genomic position.

https://doi.org/10.1371/journal.ppat.1012581.g001

Next, we focused our analysis on the 104 DMRs that were identified in the comparison of T(CMV) vs. T_mem cells. A small fraction (25 DMRs) was also detected in the comparison of T_mem vs. T_N cells, and showed a more pronounced demethylation in T(CMV) cells (S1 Table). However, the majority (79 DMRs) was only detected in the comparison of T(CMV) vs. T_mem cells, and thus determine the unique epigenetic signature of T(CMV) cells. It is important to note that in a few cases more than one DMR was found in proximity to a given gene, resulting in 71 unique genes showing differential DNA methylation patterns between T(CMV) and T_mem cells (S1 Table). Some of the top demethylated DMRs in T(CMV) cells, identified in the pairwise comparison with T_mem cells, were associated to genes with known roles in differentiation and cytotoxic function of CD8⁺ T cells, including KLRD1, TBX21, ZEB2, and S1PR5 [47–50] (Fig 2). In addition, a number of DMRs being selectively demethylated in T(CMV) cells were associated to genes for which a role in CD8⁺ T cells has not been described yet, namely FGR, LINC01871, TMEM14C, MAD1L1, LMF1, and TBKBP1. TBK-binding protein 1 (TBKBP1) is an adaptor protein that can interact with TBK1 and promote its activation [51,52]. TBK1 is a central player during the type I IFN and pro-inflammatory cytokine innate immune response by mediating phosphorylation and thereby activation of the transcription factors interferon regulatory factor (IRF) 3 and 7 and also NF-κB [53–59]. The transcription signature of CD8⁺ T_EMRA cells can be regulated by several innate immune-related genes, including natural killer (NK) cell activation mediators and multiple NK receptors [33]. These findings prompted us to study the role of TBKBP1 in CD8⁺ T cells in more detail.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Methylation profiles of gene loci associated with top DMRs from epigenetic signature of T(CMV) cells.

Out of the 71 genes showing unique differential DNA methylation patterns between T(CMV) and T_mem cells the top 12 DMR-associated gene loci were selected. For each gene, CpG motifs (barcodes), DMRs (light grey boxes), and exons of the surrounding gene body (dark grey boxes, transcriptional start site indicated by arrows) are displayed. Coloured lines illustrate methylation values ranging from 0–100% of T_N (black), T_mem (cyan), and T(CMV) cells (red) in a linear manner. Mean values from all donors per CD8⁺ T cell subset are depicted.

https://doi.org/10.1371/journal.ppat.1012581.g002

Demethylation of the TBKBP1 DMR is associated with higher TBKBP1 gene expression in effector memory T cell subsets but not with alternative splicing

To characterise the methylation status of the newly identified TBKBP1 DMR within major human CD8⁺ T cell subpopulations, we isolated genomic DNA from sorted CD45RA⁺CCR7⁺ T_N, CD45RA⁺CCR7⁻ T_EMRA, CD45RA⁻CCR7⁻ T_EM, CD45RA⁻CCR7⁺ T_CM, and CD45RA⁺CCR7⁺CD28⁺CD62L⁺CD95⁺ T_SCM cells isolated from peripheral blood of CMV-seropositive donors and subjected them to pyrosequencing analysis. Analysis of the TBKBP1 DMR showed a pronounced demethylation pattern in T_EMRA cells and a partial demethylation in T_EM cells, while T_CM, T_SCM and particularly T_N cells were largely methylated at these sites (Fig 3A). Since it has been previously reported that CpG methylation levels around the promoter or first intron/exon region of a specific gene inversely correlate with gene expression [60–62], we next asked if the strong demethylation of the TBKBP1 DMR in T_EMRA cells is accompanied by a high TBKBP1 expression. Indeed, analysis of TBKBP1 gene expression revealed a high expression in T_EMRA cells, an intermediate expression in T_EM cells, a low expression in T_CM and T_SCM cells, while TBKBP1 expression was below the detection level in T_N cells (Fig 3B). Hence, we could observe a clear inverse correlation between DNA methylation rates and TBKBP1 gene expression levels (r = -0.7575; p-value = <0.00010) (Fig 3C). Interestingly, we identified a nearly identical TBKBP1 DMR methylation (S3A Fig) and mRNA expression pattern (S3B Fig) in sorted CD8⁺ T cell subsets isolated from human CMV-seronegative donors, also leading to an inverse correlation of the two data sets (r = -0.8965, p-value = <0.00010) (S3C Fig). In conclusion, our results indicate a DMR-mediated transcriptional control of the TBKBP1 gene locus, which seems to be independent of the CMV status.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Demethylation of TBKBP1 DMR correlates with increased TBKBP1 expression in T_EMRA and T_EM CD8⁺ T cell subsets and is stably maintained upon in vitro culture.

Indicated CD8⁺ T cell subsets were isolated from CMV-seropositive healthy donors and genomic DNA as well as RNA were isolated from sorted samples. Bisulfite-converted genomic DNA was subjected to pyrosequencing using primers targeting the TBKBP1 DMR and RNA was transcribed into cDNA to determine TBKBP1 expression levels by qRT-PCR. (A) Methylation profiles of the TBKBP1 DMR in indicated CD8⁺ T cell subsets. (Left) The methylation values from 1 representative donor were translated into a colour-code according to the scale ranging from yellow (0% methylation) via white (50% methylation) to blue (100% methylation), the position of the CpG motifs is depicted, and each rectangle represents the methylation value of a single CpG motif. (Right) The scatter plot shows the mean methylation level of all 5 CpG motifs of the TBKBP1 DMR in indicated CD8⁺ T cell subsets from 5 independent donors. Each dot represents data from one donor and mean values±SD are depicted. (B) The bar plot shows the relative TBKBP1 expression normalized to the housekeeping gene RPS9 in indicated CD8⁺ T cell subsets. Mean values±SD are depicted (n = 5). (C) Scatterplot and linear regression analysis show correlation of mean methylation of TBKBP1 DMR with mean TBKBP1 expression in indicated CD8⁺ T cell subsets from 5 donors (r, correlation coefficient; p, p-value).

https://doi.org/10.1371/journal.ppat.1012581.g003

Besides altering the accessibility of DNA-binding transcription factors and thus the rate of transcription, changes in DNA methylation can also affect splicing, leading to the generation of alternative transcripts [63]. Since the TBKBP1 DMR is located close to an exon/intron junction (S4A Fig), differences in DNA methylation at the TBKBP1 DMR might result in distinct alternative splicing of the TBKBP1 transcripts in CD8⁺ T cell subsets. Specific RT-PCRs were designed to discriminate between the different TBKBP1 transcripts reported in the Ensembl genome browser (S4A Fig). However, the statistical analysis of the TBKBP1-A/TBKBP1-B transcript ratios of sorted T_CM, T_EM and T_EMRA cells revealed no significant difference (S4B Fig), in line with a recently published study that did not detect TBKBP1 protein variants in total CD4⁺ and CD8⁺ T cells as well as NKT cells [64]. Thus, the DNA methylation changes at the TBKBP1 DMR does not result in distinct alternative splicing of the TBKBP1 transcripts.

Stable TBKBP1 DMR methylation pattern during long-term in vitro culture of CD8⁺ T cell subsets

During homeostatic proliferation, naive CD8⁺ T cells have been reported to undergo phenotypic alterations [65,66]. This observation raised the question whether the methylated and demethylated states of the TBKBP1 DMR in T_N and T_EMRA cells, respectively, remain stable during proliferation. To answer this question, we isolated CD8⁺ T_N and T_EMRA cells from peripheral blood of CMV-seropositive healthy donors and cultured them for up to 30 days in the presence of anti-CD3 and anti-CD28 antibodies plus IL-2. Every 5 days, an aliquot was taken for phenotypic characterisation by flow cytometry and pyrosequencing. Upon culture of T_N cells, we observed a transient downregulation of the homing receptor CCR7 reaching normal levels by day 20, and an acquisition of stem-like properties with the upregulation of CD62L and CD28 on day 15 (S5 Fig), as recently reported [41]. In contrast, T_EMRA cells more stably maintained their CD45RA⁺CCR7⁻ phenotype throughout the culture. Pyrosequencing analyses revealed that the TBKBP1 DMR remained fully methylated in cultured T_N cells and fully demethylated in cultured T_EMRA cells during the entire cultivation period (Fig 4). Together, these findings suggest that the epigenetic status of the TBKBP1 gene is rather stable and preserved in both cultured T_N and T_EMRA cells even after multiple rounds of T cell receptor (TCR)-triggered cell division.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Long-term in vitro culture of CD8⁺ T cell subsets does not alter the TBKBP1 DMR methylation patterns.

CD8⁺ T_N and T_EMRA cells were isolated from CMV-seropositive healthy donors and cultured in vitro with plate-bound anti-CD3/CD28 antibodies in the presence of exogenous human IL-2 for up to 30 days. Every 5 days, samples from cultured T_N (top) and T_EMRA cells (bottom) were taken to analyse the TBKBP1 DMR methylation status as described in Fig 3. The heatmaps are from a representative donor (left) and scatter plots summarize the mean methylation levels of all 5 CpG motifs of the TBKBP1 DMR from 5 independent donors. Each dot represents data from one donor and mean values±SD are depicted.

https://doi.org/10.1371/journal.ppat.1012581.g004

Elevated TBKBP1 expression levels correlate with increased TBK1 phosphorylation in CD8⁺ T cells

Although it is well known that the kinase TBK1 can be activated by various signals, including TCR signalling [53,67], the role of its adaptor protein TBKBP1 in CD8⁺ T cells remains unidentified. To characterize the status of the TBK1 signalling pathway in human CD8⁺ T cell subsets, we first analysed the total protein levels of TBKBP1 and TBK1 as well as phosphorylated TBK1 in T_N, T_CM, T_EM, and T_EMRA cells ex vivo isolated from CMV-seropositive healthy donors by immunoblotting. In accordance with the TBKBP1 mRNA expression data (Fig 3B), T_N cells hardly showed any TBKBP1 expression, while it was clearly detectable in T_CM, T_EM, and T_EMRA cells (Fig 5A). Similarly, TBK1 was only weakly expressed in T_N cells (Fig 5A), and T_CM cells showed slightly reduced TBK1 protein levels when compared to T_EM and T_EMRA cells, albeit not reaching statistical significance (Fig 5B). However, T_EMRA cells contained significantly more phosphorylated TBK1 when compared to T_CM cells (Fig 5C), indicating a higher basal activity of the TBK1 signalling pathway in terminally differentiated CD8⁺ T cells.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. T_EM and T_EMRA cells display an increased phosphorylation of TBK1 when compared to T_CM cells.

To characterize the status of the TBK1 pathway in CD8⁺ T cell subsets, T_N, T_CM, T_EM and T_EMRA cells were isolated from CMV-seropositive healthy donors, and lysates were generated and utilised for immunoblotting. (A) Representative immunoblot analysis of indicated CD8⁺ T cell subsets from one out of five donors showing the expression of TBKBP1, phosphorylated TBK1 (pTBK1) and TBK1. The analysis of GAPDH expression served as loading control. (B) The bar plots show GAPDH- or α-tubulin-normalized band intensities for TBK1 quantified from sorted T_CM, T_EM and T_EMRA cells of 5 donors. Symbols indicate samples from the same donor. (C) The box-and-whiskers plots show the ratio of GAPDH- or α-tubulin-normalized band intensities of phosphorylated TBK1 and TBK1 from sorted T_CM, T_EM and T_EMRA cells of 5 donors. For statistical analyses, a paired two-tailed student’s t test was conducted with *, p ≤ 0.05.

https://doi.org/10.1371/journal.ppat.1012581.g005

In a second step, we sought to get further insights into the role of TBKBP1 for the phosphorylation of TBK1. To this end, we retrovirally overexpressed TBKBP1 in CD8⁺ T cells and subsequently monitored TBK1 phosphorylation after short-term restimulation with anti-CD3 and anti-CD28 antibodies (S6A Fig). Successfully transduced CD8⁺ T cells were sorted by flow cytometry (S6B Fig) and TBKBP1-transdcued cells showed high TBKBP1 mRNA expression levels when compared to EV-transduced controls as expected (S6C Fig). In addition, immunoblotting analysis confirmed high TBKBP1 protein expression in CD8⁺ T cells retrovirally transduced with the TBKBP1 overexpression construct, and no differences in total TBKBP1 expression were observed between unstimulated and short-term restimulated CD8⁺ T cells (Fig 6A). Similarly, neither TBKBP1 overexpression nor the short-term restimulation influenced total TBK1 expression in CD8⁺ T cells (Fig 6B). Notably, while EV-transduced control cells did not show any increase in TBK1 phosphorylation when short-term restimulated cells were compared to unstimulated controls, the phosphorylation of TBK1 was significantly enhanced in TBKBP1-overexpressing CD8⁺ T cells after short-term restimulation (Fig 6C and 6D). Since a recent report had demonstrated that TBKBP1 can support the recruitment of TBK1 to protein kinase C-theta (PKCθ) in murine lung epithelial cells, thereby promoting TBK1 phosphorylation and activation [52], we here also investigated the role of PKCθ for the TBK1 signalling pathway in CD8⁺ T cells. First, we assessed the impact of PKCθ on the phosphorylation of TBK1 by adding a highly selective PKCθ inhibitor (HY-112681) to EV-transduced or TBKBP1-overexpressing CD8⁺ T cells during short-term restimulation. As already mentioned above, immunoblotting analysis confirmed high TBKBP1 protein expression in TBKBP1-overexpressing CD8⁺ T cells, and the TBKBP1 overexpression did not influence total TBK1 expression (S7 Fig). Notably, upon addition of the PKCθ inhibitor we did not observe any effect on the phosphorylation of TBK1, neither in TBKBP1-overexpressing nor in EV-transduced control cells (S7 Fig), suggesting that PKCθ is not promoting the phosphorylation of TBK1 in CD8⁺ T cells under the tested stimulation conditions. However, the PKCθ inhibitor had an impact on the functional properties of TBKBP1-overexpressing CD8⁺ T cells and caused reduced IFN-γ expression levels after a 4-hour restimulation period when compared to DMSO-treated controls (Fig 6E). Interestingly, this inhibitory effect was only observed in TBKBP1-overexpressing CD8⁺ T cells and not in EV-transduced controls. Taken together, our findings suggest that TBKBP1 expression can promote TBK1 phosphorylation in activated CD8⁺ T cells in a PKCθ-independent manner, likely resulting in the activation of further downstream signalling pathways.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. TBKBP1 overexpression in restimulated CD8⁺ T cells results in increased TBK1 phosphorylation.

PBMCs obtained from CMV-seronegative healthy donors were stimulated with plate-bound anti-human CD3 and anti-human CD28 for 48 hours and subsequently retrovirally transduced with TBKBP1-expressing vectors or empty vector (EV) controls. Successfully transduced CD8⁺mCherry⁺ T cells were sorted by flow cytometry, serum-starved overnight, and subsequently stimulated with soluble anti-human CD3, anti-human CD28, and cross-linking antibodies, while unstimulated cells served as additional controls. Samples from both unstimulated (-) and stimulated (+) EV-transduced or TBKBP1-overexpressing CD8⁺ T cells were subjected to immunoblotting to determine the expression of (A) TBKBP1, (B) TBK1, and (C) phosphorylated TBK1 (pTBK1). The analysis of GAPDH expression served as loading control. (D) Band intensities for pTBK1 and TBK1 were quantified and the fold change of TBK1 phosphorylation upon stimulation of EV-transduced or TBKBP1-overexpressing CD8⁺ T cells was determined by dividing the ratio of pTBK1/TBK1 band intensities of stimulated cells to the ratio of pTBK1/TBK1 band intensities of unstimulated samples. Data are combined from 4 independent donors analysed in 2 independent experiments. (E) EV-transduced or TBKBP1-overexpressing CD8⁺ T cells were stimulated in the presence of a PKCθ inhibitor (+) or treated with DMSO as control (-). Subsequently, CD3⁺CD8⁺ cells were analysed for intracellular IFN-γ expression by flow cytometry. The geometric mean fluorescent intensity (gMFI) of IFN-γ expression among CD3⁺CD8⁺ cells is depicted. Data are combined from 4 independent donors analysed in 2 independent experiments. (D, E) For statistical analyses, a paired two-tailed student’s t test was conducted with *, p ≤ 0.05.

https://doi.org/10.1371/journal.ppat.1012581.g006

Ectopic expression of TBKBP1 augments the virus-reducing capacity of CD8⁺ T cells

After having demonstrated that TBKBP1 overexpression has an effect on TBK1 phosphorylation, we next sought to investigate its functional role in CD8⁺ T cells during an immune reaction. To this end, we utilized ARMATA, an assay for rapid measurement of antiviral T cell activity [68]. In this assay, fibroblasts (MRC-5 cells) were infected with recombinant CMV expressing the fluorescent reporter mNeonGreen [69] and co-cultured with CMV-specific CD8⁺ T cells, which had been generated by retroviral overexpression of the human leukocyte antigen (HLA)-A*02:01-restricted high-avidity TCR recognising the CMVpp65-derived peptide NLVPMVATV (mTCR 5–2) [70]. Additionally, to assess the functional role of TBKBP1, cells were retrovirally transduced with the TBKBP1 overexpression construct, while EV-transduced cells served as control. Successfully transduced mTCR 5–2⁺mCherry⁺ CD8⁺ T cells were sorted by flow cytometry (S8 Fig) and added to the CMV-infected MRC-5 cells at different effector:target (E:T) ratios. The ARMATA measures the viral expression of mNeonGreen by temporal live cell imaging in 1-hour intervals and capture of representative microscopic images. As shown in Fig 7A, we observed a strong antiviral and cytopathic effect of mTCR 5–2⁺ CD8⁺ T cells, with an overall reduced number of viable mNeonGreen-expressing MRC-5 cells when compared to controls lacking T cells. Hourly measurements of the reporter signal revealed a continuous increase of the reporter signal in cultures without T cells, reaching a plateau around 12 hours with an only mild increase afterwards (Fig 7B, green curve). Upon addition of EV-transduced mTCR 5–2⁺ control CD8⁺ T cells, a marked decrease of the reporter signal was observed, starting at 12 hours and continuing until the end of the assay at 72 hours (Fig 7B, blue curve). This signal reduction was even more pronounced when TBKBP1-overexpressing mTCR 5–2⁺ CD8⁺ T cells were added, showing a significantly stronger neutralization of MRC-5-infected cells irrespective of the E:T ratio (Fig 7B, red curve). The improved virus reduction by TBKBP1-overexpressing CD8⁺ T cells was associated with significantly elevated levels of IFN-γ and Granzyme A, and a trend towards increased levels of Granzyme B, Perforin, MIP-1α (CCL3), and MIP-1β (CCL4) in supernatants of co-cultures with TBKBP1-overexpressing CD8⁺ T cells when compared to EV-transduced controls (Figs 7C and S9). Collectively, these data demonstrate that ectopic expression of TBKBP1 in human CD8⁺ T cells results in the upregulation of pro-inflammatory cytokines and chemokines, facilitating a significantly enhanced virus neutralization capacity.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. TBKBP1 overexpression enhances virus-reducing capacity of CD8⁺ T cells.

PBMCs obtained from CMV-seronegative healthy donors were stimulated with plate-bound anti-human CD3 and anti-human CD28 for 48 hours and subsequently retrovirally transduced with both the high-avidity TCR 5–2 containing murine constant regions (mTCR) and directed against the CMV HLA-A*02-peptide NLVPMVATV and also TBKBP1-overexpressing vectors or empty vector (EV) controls. Successfully transduced mTCR 5–2⁺mCherry⁺ CD8⁺ T cells were sorted by flow cytometry and added at indicated effector:target (E:T) ratios to MRC 5 cells infected with recombinant CMV expressing the reporter protein mNeonGreen. ARMATA measures the expression of mNeonGreen using live cell imaging. (A) Representative microscopic images 48 hours after infection showing the expression of mNeonGreen (fluorescence image) and the location of the cells in corresponding brightfield images in CMV-infected MRC-5 cells in the absence of added T cells (w/o T cells), in the presence of EV-transduced mTCR 5–2⁺ CD8⁺ T cells (T cells + TCR 5–2 +EV) or in the presence of TBKBP1-overexpressing mTCR 5–2⁺ CD8⁺ T cells (T cells + TCR 5–2 +TBKBP1). T cells were added at a E:T ratio of 1:1. (B) The ARMATA was quantified by hourly measurements of the reporter signal in cultures in the absence of added T cells (w/o T cells, green curve), in the presence of EV-transduced mTCR 5–2⁺ CD8⁺ T cells (T cells + TCR 5–2 +EV, blue curve) or in the presence of TBKBP1-overexpressing mTCR 5–2⁺ CD8⁺ T cells (T cells + TCR 5–2 +TBKBP1, red curve). (Left) The graph depicts mean values±SD from 2 technical replicates of 1 representative out of 3 independent experiments. (Right) The line plots summarize data from 3 independent experiments by showing the average area under the curve (AUC) ±95% CI for EV-transduced or TBKBP1-overexpressing mTCR 5–2⁺ CD8⁺ T cells cultured with CMV-infected MRC-5 at indicated E:T ratios. (C) 36 hours after infection, culture supernatants were harvested and cytokine profiles were determined from cultures of CMV-infected MRC-5 cells in the absence of added T cells (w/o T cells), in the presence of EV-transduced mTCR 5–2⁺ CD8⁺ T cells (T cells + EV, E:T = 1:1) or in the presence of TBKBP1-overexpressing mTCR 5–2⁺ CD8⁺ T cells (T cells + TBKBP1, E:T = 1:1). Data from 3 independent experiments with 2 technical replicates each are shown. Symbols indicate samples from the same donor. For statistical analyses, a paired two-tailed student’s t test (leaving out “w/o T cell” group) was conducted with *, p ≤ 0.05 and **, p ≤ 0.01.

https://doi.org/10.1371/journal.ppat.1012581.g007

Ethics statement

All human samples were obtained from the Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School (MHH). Written informed consent was obtained from all donors (Ethics Committee MHH, approval numbers: 3639–2017, 9001_BO-K, 9255_BO_K_2020).

Human donors

All human samples were obtained from male healthy donors. For the phenotypic characterisation and isolation of T(CMV) cells, CMV-seropositive HLA-typed donors showing >0.5% CMV phosphoprotein 65 (pp65)-specific T cells upon antigen-specific restimulation [88,89] were selected. The PKCθ inhibition experiments and the ARMATA were performed using cells from male CMV-seronegative, HLA-A*02 negative donors. In vitro cultivation experiments were conducted using cells from male CMV-seropositive donors regardless of their HLA type. All remaining experiments were performed with samples from both CMV-seronegative and -seropositive donors regardless of their HLA type. CMV-serological testing and HLA-typing were conducted at the Institute of Transfusion Medicine and Transplant Engineering, MHH.

Human sampling and cell isolation

For the phenotypic characterisation and whole genome bisulfite sequencing (WGBS) of T(CMV) cells, PBMCs were isolated from residual blood samples from disposable kits used during routine platelet apheresis at the Institute of Transfusion Medicine and Transplant Engineering, MHH, via discontinuous gradient centrifugation and immediately processed. For all remaining experiments, PBMCs were isolated from Leukocyte Reduction System (LRS) cones using Ficoll-based density gradient centrifugation (Lymphoprep; STEMCELL Technologies) and SepMate tubes (STEMCELL technologies). PBMCs were cryopreserved in FCS containing 10% DMSO. One day prior to the experiment, cryopreserved PBMCs were thawed, rinsed with excess TexMACS medium (Miltenyi Biotec) to remove DMSO, and rested overnight with TexMACS medium supplemented with 100 IU/ml of recombinant human IL-2 (Miltenyi Biotec).

Antibodies for flow cytometry

All monoclonal antibodies used for immunophenotyping and cell sorting were purchased from BioLegend and BD Biosciences. Characterisation of T(CMV) cells was performed using anti-CD3 BV510, anti-CD8 FITC, anti-IFN-γ PE, anti-CD45RA PerCP/Cy5.5, and anti-CD62L AF647. For WGBS, T(CMV) cells and control T cell subsets were isolated using anti-CD3 AF488, anti-CD4 APC-Cy7, anti-CD8 APC, anti-CD27 BV510, anti-CD28 PE-Dazzle, anti-CD45RA PerCP/Cy5.5, anti-KLRG1 PE/Cy7, anti-CCR7 BV421, and anti-CX3CR1 BV650. For pyrosequencing, quantitative real-time-PCR (qRT-PCR), and in vitro cultivation experiments, CD8⁺ T cell subsets were isolated using anti-CD3 AF488, anti-CD8 APC, anti-CD28 PE Texas Red, anti-CD45RA PerCP/Cy5.5, anti-CD62L PE-Cy7, anti-CD95 PE, and anti-CCR7 BV421. For the isolation of retrovirally-transduced CMV-specific CD8⁺ T cells, anti-CD8 APC and anti-mouse TCR APC-Cy7 along with LIVE/DEAD Fixable Blue Dead Cell dye were used (ThermoFischer Scientific).

Flow cytometry

PBMCs were prepared for flow cytometry assay as described previously [90]. Briefly, for surface staining, cells were resuspended in staining buffer (PBS and 0.5% BSA), and single-cell suspensions were labelled with antibodies for 30 minutes at 4°C, while chemokine receptor staining was carried out at 37°C for 30 minutes. For intracellular staining of IFN-γ, the Foxp3 staining kit (eBioscience) was used according to the manufacturer’s protocol. RatIgG (Dianova, final concentration of 40 μg/ml) was added to block unspecific binding. Following staining, cells were washed and resuspended in staining buffer. Samples were acquired on a FACSCanto10c, LSR-II flow cytometer (BD Biosciences) or LSRFortessa flow cytometer (BD Biosciences). Data were analysed using BD FACSDiva software version 8.0.1 and FlowJo software version 10 (both from BD Biosciences). Cell sorting of CD8⁺ T cell subsets and transduced cells was performed on a BD FACS ARIA II SORP (BD Biosciences).

Phenotypic characterisation of T(CMV) cells

For the phenotypic characterisation of T(CMV) cells, CD8⁺ T cells were enriched from PBMCs using the untouched CD8⁺ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Next, the enriched CD8⁺ T cells were stimulated with the CMVpp65 overlapping peptide pool PepTivator CMVpp65 (Miltenyi Biotec) at a concentration of 1 μg/ml of each peptide for 4 hours. Subsequently, IFN-γ-secreting cells were detected using IFN-γ Secretion Assay/Detection Kit (PE) (Miltenyi Biotec) according to the manufacturer’s instructions and subsequently phenotypically characterised by flow cytometry.

Isolation of CD8⁺ T cell subsets for WGBS, pyrosequencing, qRT-PCR, and in vitro cultivation experiments

For the isolation of CD8⁺ T cell subsets for WGBS, PBMCs were first enriched for CD8⁺ T cells and subsequently stimulated with CMVpp65 overlapping peptide pool as described above. After the detection of IFN-γ-secreting cells, cells were stained and T(CMV) cells were isolated by flow cytometry-based cell sorting as CD3⁺CD4⁻CD8⁺IFN-γ⁺ cells, while memory CD8⁺ T cells (T_mem; CD3⁺CD4⁻CD8⁺CCR7⁻CD28^highCD27⁺CD45RA⁻) and naive CD8⁺ T cells (T_N; CD3⁺CD4⁻CD8⁺CCR7⁺CD28^intKLRG1⁻CX3CR1⁻CD45RA^high) were sorted as controls.

For pyrosequencing, qRT-PCR and in vitro cultivation experiments, human CD8⁺ T cell subsets were isolated from cryopreserved PBMCs. First, CD8⁺ T cells were enriched using human anti-CD8 MicroBeads and the automated magnetic activated cell sorting (autoMACS) system (both Miltenyi Biotec) according to the manufacturer’s instructions. Subsequently, the CD8⁺ T cells were stained and sorted into T_N (CD45RA⁺CCR7⁺), T_SCM (CD45RA⁺CCR7⁺CD28⁺CD62L⁺CD95⁺), T_CM (CD45RA⁻CCR7⁺), T_EM (CD45RA⁻CCR7⁻), and T_EMRA (CD45RA⁺CCR7⁻) CD8⁺CD3⁺ T cell subsets by flow cytometry.

Whole-genome bisulfite sequencing (WGBS)

For WGBS, sorted T_N, T_mem, and T(CMV) cells from the blood of various donors were used to prepare genomic DNA with the DNeasy Blood & Tissue Kit (Qiagen). Approximately 50 ng genomic double-stranded DNA per donor was converted with sodium bisulfite using the EZ DNA Methylation-Lightning Kit (Zymo Research) and fragmented by sonication (Covaris S220, 10% duty cycle, 175W peak incident power, intensity 5, 200 cycles per burst, 120 seconds). The fragmented DNA served as input for the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) and resulted in libraries that were sequenced on an Illumina NovaSeq 6000 with depths between 216 and 298 million paired end reads (2 x 100 bp).

Sequencing data were processed using the nf-core/methylseq pipeline (version 2.2.0), using default parameters, genome assembly GRCh38 and—accel = true [91] (software freely accessible at http://doi.org/10.5281/zenodo.1343417). Briefly, the pipeline employs FASTQC (version 0.11.9), trimgalore (version 0.6.7) and bismark (version 0.24.0) [92] for read-level quality control, adapter trimming, bisulfite-aware alignment, and cytosine-level DNA methylation quantification.

Methylation calls were further processed using RnBeads (version 2.17.0) [93] using a minimum per CpG coverage of 2 and removing CpGs that were covered in less than 50% of the samples, that overlapped annotated SNPs, or that were located on sex chromosomes. Genome-wide methylation values were computed by coverage weighted aggregation of CpG-level methylation values across samples in groups for 1 kb and 10 kb tiling windows. Dimensionality reduction using principal component analysis (PCA) was performed on those 50,000 1 kb tiling windows that exhibited the highest variance in aggregate methylation levels across the dataset.

Pairwise DMRs between the groups were identified using Bsmooth/BSseq (version 1.30.0) [94]. Briefly, we applied the Bsmooth() command with default parameters on unfiltered CpG methylation calls. Subsequently, only CpGs with a coverage of at least 5 in two or more samples per group were retained. Based on these filtered CpGs, we computed t-statistics and DMRs using BSmooth.tstat (…, estimate.var = "same", local.correct = TRUE) and dmrFinder (…, qcutoff = c(0.01,0.99), maxGap = 200). Gene relations to nearest genes were annotated using GENCODE (version 44). Finally, DMRs containing at least 3 CpGs were selected using an absolute mean methylation level difference of 25%.

Sequencing data and methylation levels reported in this paper were uploaded to GEO under accession number GSE245832. Identified DMRs are listed in S1 Table.

In vitro cultivation of CD8⁺ T cell subsets

For in vitro cultivation, sorted T_N and T_EMRA cells were stimulated with plate-bound anti-human CD3 (OKT3; 1 μg/ml; BioLegend) and anti-human CD28 (CD28.2; 0.5 μg/ml; BioLegend) antibodies and cultured in TexMACS medium supplemented with 100 IU/ml human IL-2 at 37°C with 5% CO₂. Every 5 days during the 30-day culture period, cells were harvested and washed, and an aliquot was taken for pyrosequencing and phenotypic characterisation by flow cytometry. The remaining cells were re-stimulated with plate-bound anti-human CD3 and anti-human CD28 (both 0.5 μg/ml) antibodies and cultured in fresh TexMACS medium supplemented with 100 IU/ml human IL-2.

Pyrosequencing of the TBKBP1 DMR

Genomic DNA from ex vivo isolated CD8⁺ T cell subsets or in vitro cultured cells was extracted using the DNeasy Blood & Tissue Kits (Qiagen) and subsequently bisulfite-converted by using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturers’ recommendations. TBKBP1 DMR was pyrosequenced as described previously [95]. For amplification of the TBKBP1 DMR and subsequent pyrosequencing, we used the following primers:

‘forward’ 5’-TATTTAAGTTTGGGTGATAGAGTAAGAT-3’

‘reverse’ 5’-Bio-CCCAACCCTCAAAAATATAATATCT-3’

‘sequencing 1’ 5’-GGTGGTGTATGTTTGTAAT-3’

‘sequencing 2’ 5’-GGTTGAGGTGAGTTAAGAT-3’

Quantitative RT-PCR of TBKBP1

Total RNA was purified from isolated CD8⁺ T cell subsets using the RNeasy Mini Kit (Qiagen), spectrometrically quantified (DeNovix), and transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). TBKBP1-A primers (‘forward’ 5’-TTGCCCTCATCACTGCTTAC-3’; ‘reverse’ 5’-GGGTACTTGATCTCGTAGACTTTG-3’), TBKBP1-B primers (‘forward’ 5’-CAGGATCTGGCCTCCAAC-3’; ‘reverse’ 5’-CCCTGTAGGGAACTCAACTC-3’) or codon-optimized TBKBP1 primers (‘forward’ 5’-TCGCTCTCATCACTGCCTAC-3’; ‘reverse’ 5’-GGGTACTTGATTTCATAGACTTTA-3’) and SYBR green master mix (Roche) as well as cDNA were used in a qRT-PCR on a LightCycler 480 II (Roche) or QuantStudio 3 (Thermo Fisher Scientific). The data were analysed with LightCycler 96 SW 1.1 (Roche) or Design & Analysis Software v2.7.0 (Thermo Fisher Scientific). All procedures were performed according to the manufacturers’ recommendations.

Retroviral transduction

For retroviral overexpression of TBKBP1, an expression cassette consisting of codon-optimized TBKBP1 cDNA, porcine teschovirus-1 2A element (P2A), and mCherry cDNA was inserted into the pMP71 plasmid by gene synthesis (TWIST Bioscience). pMP71 containing only mCherry cDNA served as empty vector (EV) control. For the ARMATA, cells were not only retrovirally transduced with the TBKBP1 overexpression construct (or the EV control), but also with a pMP71-based plasmid coding for mTCR 5–2, a previously reported CMV-specific, HLA-A*02:01-restricted high-avidity TCR recognising the CMVpp65-derived peptide NLVPMVATV and containing a murine constant region (mTCR) for the isolation of successfully transduced cells [70].

Virus transduction of human PBMCs was performed as previously described [96]. In brief, the virus-packaging RD114 cell line was seeded in a 6-well plate at a density of 1.5 x 10⁶ cells/well 1 day prior to transfection. The next day, pMP71 vectors were transfected using calcium phosphate precipitation. Thereto, complete DMEM medium (Gibco) supplemented with 10% FBS (Gibco), 1% sodium pyruvate (Biochrom), and 1% HEPES (Sigma-Aldrich) was replaced 1 hour before transfection with complete medium containing 25 μM chloroquine diphosphate salt (Sigma-Aldrich). The plasmid-containing buffer was prepared by adding 18 μg of vector DNA and 15 μl of 2.5 M CaCl₂ (Applichem) to a final volume of 150 μl with water, which was then mixed with transfection buffer containing 280 mM sodium chloride, 42 mM HEPES, 3.5 mM disodium hydrogen phosphate, and 10 mM potassium chloride at 1:1 ratio by vortexing and subsequently incubated for 30 minutes at room temperature. The mixture was slowly added to RD114 cells (150 μl/well) in a dropwise manner and incubated for 15 hours at 37°C with 5% CO₂, followed by medium replacement with 3 ml complete DMEM medium. The supernatant containing retroviral particles was harvested 3 days later. For transduction, PBMCs were stimulated for 48 hours with plate-bound anti-human CD3 and anti-human CD28 (both 1 μg/ml). Additionally, retronectin-treated plates were prepared by coating with 12.5 μg/ml retronectin (TaKaRa) in PBS (Gibco) at 4°C overnight in a non-treated 24-well plate. Unbound retronectin was aspirated and plates were blocked with 2% BSA (Sigma-Aldrich) in PBS for 20 minutes at 37°C followed by washing twice with PBS. Supernatant containing retroviral particles was added to retronectin-coated plates followed by centrifugation at 2,000xg for 2 hours at 32°C. Following centrifugation, virus supernatant was aspirated and preactivated PBMCs (0.5–0.8 x 10⁶/well) in TexMACS medium supplemented with 100 IU/ml IL-2 and 6 ng/ml polybrene (Sigma-Aldrich) were added to each well. After spinoculating the cells onto virus-coated plates (1,000xg for 10 minutes at 32°C), the cells were incubated at 37°C with 5% CO₂ for 24 hours. After 24 hours, transduced human PBMCs were washed and resuspended in TexMACS medium supplemented with IL-2. Flow cytometry analysis was performed 5 days after spinoculation to determine transfection efficiency. Successfully transduced CD8⁺mCherry⁺ T cells (TBKBP1 overexpression or EV control) or CD8⁺mTCR⁺mCherry⁺ T cells (simultaneous overexpression of mTCR 5–2 and TBKBP1 or EV control) were sorted using flow cytometry and used for subsequent experiments.

Cell stimulation and immunoblotting

For sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent immunoblotting experiments, TBKBP1-overexpressing CD8⁺ T cells and control cells transduced with EV as well as ex vivo isolated CD8⁺ T_N, T_CM, T_EM and T_EMRA cells were generated and sorted as described above. Next, cells were serum-starved overnight and subsequently incubated with soluble 2 μg/ml anti-CD3 and anti-CD28 mAbs for 40 minutes on ice. Antibody-coated cells were then cross-linked with 4 μg/ml AffiniPure goat-anti-mouse IgG+IgM (H+L; Jackson ImmunoResearch) and incubated for 15 minutes at 37°C. Unstimulated controls were kept on ice. To stop the stimulation, excess ice-cold PBS was added and the cells were centrifuged at 4°C. Pellets were snap-frozen and stored at -80°C.

For preparing cell lysates from TBKBP1-overexpressing CD8⁺ T cells and control cells transduced with EV for SDS-PAGE and immunoblotting, cell pellets were resuspended in 40 μl of 2x SDS loading buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.02% bromphenol blue, 10% β-mercaptoethanol), heated at 95°C for 5 minutes, and cell debris was pelleted with 14,000xg at room temperature (RT) for 5 minutes. Per lane, 20 μl lysate was analysed on 10% polyacrylamide gels containing 0.1% SDS in Tris-glycine running buffer with 0.1% SDS at room temperature.

For preparing cell lysates from ex vivo isolated CD8⁺ T cell subsets for SDS-PAGE and immunoblotting, approximately 1 x 10⁶ cells were resuspended in 20 μl lysis buffer (20 mM Tris-base, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with protease and phosphatase inhibitors (both from Roche). Cells were lysed for 1h at 4°C and cell debris was precipitated at 14,000xg at 4°C for 5 minutes. Protein concentration was assessed colorimetrically via the bicinchoninic acid (BCA) protein assay (Takara), adjusted protein amounts were mixed with 4x SDS loading buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.04% bromphenol blue, 10% β-mercaptoethanol), lysates were heated at 95°C for 10 minutes, and loaded on 7.5% polyacrylamide gels containing 0.1% SDS in Tris-glycine running buffer with 0.1% SDS at room temperature.

Proteins were blotted on polyvinylidene difluoride membranes (Amersham Hybond P 0.2 μm) in a wet blot system in Tris-glycine transfer buffer containing 0.05% SDS and 20% methanol for 1 hour at 350 mA at 4°C. Membranes were subsequently blocked in 5% BSA in TBS with 0.1% Tween 20 for 1 hour at room temperature and incubated with anti-phospho-TBK1/NAK (Ser 172; Cell Signaling Technology), anti-TBK1/NAK (D1B4; Cell Signaling Technology) anti-SINTBAD (TBKBP1; D1A5; Cell Signaling Technology), anti-GAPDH (14C10; Cell Signaling Technology) or anti-α-Tubulin (Sigma-Aldrich) primary antibodies at 4°C overnight. Membranes were washed and incubation with HRP-coupled anti-rabbit or anti-mouse secondary antibodies (both from Dianova) was carried out for 1 hour at room temperature. Membranes were washed and developed with the Pierce ECL or SuperSignal substrate (Thermo Fisher Scientific) and a ChemoStar ECL Imager (INTAS). Quantitative analysis was performed with ImageJ software (version 1.52g, National Institute of Health). When necessary, membranes were stripped with Restore PLUS Western Blot Stripping Buffer (ThermoFisher Scientific) for 15 minutes at room temperature, before being washed, blocked, incubated with primary and secondary antibodies, imaged, and analysed as described above.

PKCθ inhibition

PKCθ inhibition experiments were carried out with EV-transduced or TBKBP1-overexpressing CD8⁺ T cells generated as described above. To assess the impact of PKCθ inhibition on TBK1 phosphorylation, sorted CD8⁺mCherry⁺ T cells were serum-starved overnight and subsequently preincubated for 1 hour with the PKCθ inhibitor (10 μM HY-112681, Hoelzel Biotech). Next, cells were restimulated for 15 minutes and further processed for SDS-PAGE and immunoblotting as described above. To assess the impact of PKCθ inhibition on the functional properties of CD8⁺ T cells, sorted CD8⁺mCherry⁺ T cells were preincubated for 1 hour with the PKCθ inhibitor as described above. DMSO-treated cells were used as control. Next, cells were stimulated for 2 hours using T cell TransACT (Miltenyi Biotech) as recommended by the manufacturer. Afterwards, Brefeldin A (10 μg/ml, Sigma-Aldrich) was added and cells were cultivated for additional 2 hours. At the end of the culture, cells were harvested and stained for flow cytometry as described above.

Assay for rapid measurement of antiviral T cell activity

For ARMATA [68], CD8⁺ T cells simultaneously overexpressing mTCR 5–2 and TBKBP1 (or the EV control) were generated and sorted as described above. One day prior to infection, MRC-5 cells were seeded in 96-well plates at a density of 20,000 cells/well and cultured in Eagle’s minimum essential medium with 10% FCS, 1 mM sodium pyruvate, and 1 mM glutamine. The following day, the MRC-5 cells were infected with a reporter CMV (TB40/BAC4 HCMV^3F) that was generated on the background of the clinical isolate TB40-E [69]. Briefly, after aspiration of the medium, the reporter CMV was added to the MRC-5 cells at a MOI of 0.1 and centrifuged at 700xg for 10 minutes at room temperature to enhance the infection and reduce infection heterogeneity. After centrifugation, the medium was immediately aspirated and the cells were washed with PBS. After washing, sorted mTCR⁺mCherry⁺ CD8⁺ T cells were added to the infected MRC-5 cells in a final volume of 200 μl medium at effector:target (E:T) ratios of 0.5:1 and 1:1. Coculture medium was composed of CTS OpTmizer T-cell expansion basal medium (ThermoFischer Scientific) supplemented with CTS Immune Cell SR (ThermoFischer Scientific) and 300 IU/ml IL-2. To analyse the virus reduction capacity of the T cells, the mNeonGreen signals from infected MRC-5 cells were monitored for 1 week using the Incucyte Live-Cell analysis system (Sartorius). Additionally, the confluence of cells was determined by the phase detector and images were analysed by the Incucyte Analysis Software. Data were exported to Prism 9.4.0 for graphical representation.

Cytokine multiplex assay

For measuring cytokines, 50 μl culture supernatants were harvested after 36 hours of the co-culture from the ARMATA. Supernatants were centrifuged at 500xg for 15 minutes at 4°C and kept at -80°C. For cytokine measurement, 50 μl thawed supernatant was diluted 1:1 with supplied assay buffer. Cytokines were measured using the MILLIPLEX Human CD8⁺ T Cell Magnetic Bead Panel Premixed 17 Plex-Immunology Multiplex Assay (Millipore) according to the manufacturer’s recommendations and analysis was done using the Bio-Plex Manager 6.1 software based on the standard curve.

Statistical analysis

Prism 9.4.0 software was utilized for statistical analysis. The normal distribution of data points was checked using the Shapiro-Wilk normality test. A paired two-tailed parametric student’s t test was conducted on samples that passed the normality test, and a non-parametric Wilcoxon matched-pairs signed rank test was performed on samples that failed in normality test. All data are presented as mean or mean ± SD, and p-values ≤ 0.05 deemed significant (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns; not significant).