CD8+ tissue-resident memory T cells induce oral lichen planus erosion via cytokine network

  1. Maofeng Qing
  2. Dan Yang
  3. Qianhui Shang
  4. Jiakuan Peng
  5. Jiaxin Deng
  6. Jiang Lu
  7. Jing Li
  8. HongXia Dan
  9. Yu Zhou
  10. Hao Xu
  11. Qianming Chen

  • Curated by eLife

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    eLife assessment

    Overall, this is an important study that characterizes human oral lichen planus via single-cell analysis. Although the work is descriptive, it can represent an important resource for future studies and highlights potentially relevant biology. However, the claims are a bit overstated and some of the analyses that lead to interpretations remain incomplete.

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Abstract

CD8 + tissue-resident memory T (CD8 + Trm) cells play key roles in many immune-inflammation-related diseases. However, their characteristics in the pathological process of oral lichen planus (OLP) remains unclear. Therefore, we investigated the function of CD8 + Trm cells in the process of OLP. By using single-cell RNA sequencing profiling and spatial transcriptomics, we revealed that CD8 + Trm cells were predominantly located in the lamina propria adjacent to the basement membrane and were significantly increased in patients with erosive oral lichen planus (EOLP) compared to those with non-erosive oral lichen planus (NEOLP). Furthermore, these cells displayed enhanced cytokine production, including IFN-γ (Interferon-gamma, a pro-inflammatory signaling molecule), TNF-α (Tumor Necrosis Factor-alpha, a cytokine regulating inflammation), and IL-17 (Interleukin-17, a cytokine involved in immune response modulation), in patients with EOLP. And our clinical cohort of 1-year follow-up was also supported the above results in RNA level and protein level. In conclusion, our study provided a novel molecular mechanism for triggering OLP erosion by CD8 + Trm cells to secrete multiple cytokines, and new insight into the pathological development of OLP.

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  1. Version published to 10.7554/elife.83981 on eLife
  2. eLife

    Author Response

    Reviewer 2 (Public Review):

    1. My major criticism of the study is that the authors argue for CD8+ Trm activity as a key mechanism for OLP pathogenesis but have presented mostly descriptive datasets. The data strongly argue for CD8+ Trm cells as a defining feature of erosive OLP, but there is no data to support their involvement in disease pathogenesis. The authors note the lack of a mouse model for OLP which represents a significant technical barrier to interrogating the role of CD8+ Trm cells in OLP pathogenesis.

    Thank you for bringing this to our attention, and please accept our apologies for any confusion caused by our previous article. The pathogenesis of OLP is responsible for the immune disease caused by multiple factors, but there is no corresponding animal model at present, which has obvious limitations on the research. Therefore, we focus on the research on the reasons for the change of the clinical state of the disease. Our study found that CD8+ TRM cells play an important role in the changes observed in the local presentation of OLP, specifically erosions. However, it is important to note that they are not the primary driver of the disease. In addition, we use cohort studies combined with transcriptome data to increase the strength of evidence for causal effects. We have revised and emphasized this point in the updated text.

    The modified description in introduction is as follows:

    Notably, EOLP has a significantly higher risk of malignant transformation than non-erosive oral lichen planus (NEOLP) (Danielsson et al., 2013). To reduce the psychological and economic burden of OLP patients, improve their quality of life, and decrease the risk of cancer, it is crucial to maintain the disease in a relatively stable non-erosive stage for as long as possible. However, clinical experience suggests that OLP often exhibits a prolonged and recurrent disease course, with alternating periods of non-erosive and erosive lesions. Despite this, the underlying causes and mechanisms of lesion type switching remain unclear (Husein-ElAhmed and Steinhoff, 2022). (Page 4, lines 13-21)

    1. Another criticism is the lack of strong findings in the analysis of CD8+ Trm cells isolated from non-erosive and erosive OLP tissues. The authors note increases in CD8+ Trm cell recovery, however, they only observe minor changes in CD8+ Trm activity upon restimulation. Analyzing the activation status or proliferative capacity of CD8+ Trm cells from non-erosive and erosive OLP could be informative and more robust measures of functional changes.

    We appreciate your suggestion to test the activation status and proliferation of sorted CD8+ Trm cells to further investigate the differences between the two groups. However, due to the limited amount of tissue available for our study, it was so hard to obtain sufficient numbers of CD8+ Trm cells for these experiments. Additionally, there is a lack of established methods for in vitro culture of CD8+ Trm cells, which further limited our options for functional studies.

    To investigate the function of CD8+ Trm cells in the two tissue groups, we instead measured inflammatory factors in the supernatant of CD8+ Trm cells after in vitro stimulation. This allowed us to indirectly assess the activity of CD8+ Trm cells in non-erosive and erosive OLP. We used ELISA assay to measure the levels of several inflammatory cytokines, which are known to be produced by activated T cells, including CD8+ Trm cells.

    We acknowledge that this method has limitations and is an indirect measure of CD8+ Trm cell function. However, we believe that our approach provides useful information on the potential role of CD8+ Trm cells in oral lichen planus and represents a valuable contribution to the field.

    1. A minor criticism is the formatting of the data presented in Figure 4. The authors should clearly label each marker used in the flow cytometry experiments as well as clearly labeling y-axes for graphs 4H and 4I.

    Thank you for your valuable comments, I have modified the flow cytometry diagram accordingly and labeled each step of the gating strategy, also modified the other two diagrams. And 4H and 4I figure numbers changed to 4G and 4H.

  3. eLife

    eLife assessment

    Overall, this is an important study that characterizes human oral lichen planus via single-cell analysis. Although the work is descriptive, it can represent an important resource for future studies and highlights potentially relevant biology. However, the claims are a bit overstated and some of the analyses that lead to interpretations remain incomplete.

  4. eLife

    Reviewer #1 (Public Review):

    Qing et al. hypothesize that CD8+ tissue-resident memory T (Trm) cells contribute to the pathogenesis of oral lichen planus. They compare oral mucosal lesions from patients with non-erosive oral lichen planus (NEOLP; n=3) and erosive oral lichen planus (EOLP; n=1) using single-cell RNA-sequencing and spatial transcriptomics and report that CD8+ Trm is enriched and more functionally active in EOLP. Their principal findings are 1) increased proportion of CD8+ Trm in EOLP (vs NEOLP), 2) CD8 Trm in OLP lesions produce TNF, IFNg, and IL-17, and 3) CD8 Trm exist in the healthy epithelium and in lamina propria adjacent to the damaged epithelium in NEOLP/EOLP. The strength of evidence for findings reported in the manuscript is weak.

    Strengths:
    The pathogenic CD8+ T cell response in lichen planus is a relatively unexplored topic and oral lichen planus is a debilitating disease, thus advancements in its understanding are impactful. The authors' approach is innovative; the manuscript's spatial transcriptomics data are completely novel. The logistical regression analyses that tie CD8+ T cell transcriptional signatures to clinical outcomes are compelling.

    Weaknesses:
    The authors' data do not firmly support their conclusions. The methods section and figures/figure legends lack important details and labels which makes it difficult to interpret the data. For instance, it is unclear to me how the authors have defined CD8+ tissue-resident memory T (Trm) cells. Human CD8 Trm expresses specific surface markers (CD69, CD103, and CD49a) and a core transcriptional signature (Kumar et al Cell Rep 2017; Cheuk et al Immunity 2017; Fonseca et al Nat Immunol 2022) that are not described herein. In Figures 1-2, the authors do not describe how they annotated their scRNA-seq data, what samples they are including/comparing, criteria used to identify relevant gene expression changes, and they report T cell phenotypes that are inconsistent with published reports and make me question the validity of their T cell/NK cell cluster annotation. Double positive (CD4+CD8+) T cells are not thought to exist outside of the thymus. Human CD8+ Trm has been described to express Itga1 (CD49a), Itgae (CD103), and granzymes yet the authors' CD8 Trm cluster (Fig 2B) exhibits little to no expression of these genes. Also, the authors report il23a expression by CD8 Trm when T cells are not a recognized source of IL-23.

    Impact
    In my opinion, the main comparison made (NEOLP vs EOLP) is not meaningful. The authors' main conclusion is that there is more CD8+ Trm-mediated inflammation occurring in erosive OLP compared to non-erosive OLP. This is in line with what one would predict, as erosive OLP is a more severe form of the disease. Thus, I don't believe this manuscript significantly advances the understanding of lichen planus immunopathogenesis. The utility of exploring the pathogenesis of human disease is it may identify new targets of intervention and lead to better treatments. The methods used within the manuscript (scRNA-seq, spatial transcriptomics) have the potential to yield significant insights into OLP however in their present form, the authors' analyses do not support the premise that CD8 Trm is the pathogenic cell type in OLP. Thus I do not feel that the authors achieved their central aim.

  5. eLife

    Reviewer #2 (Public Review):

    Qing et al conducted high-resolution single-cell RNA sequencing and spatial transcriptomic profiling to characterize the immunological state of oral mucosa tissue from non-erosive OLP and erosive OLP patients. They find that tissue from erosive OLP patients possessed greater numbers and displayed enhanced activation of CD8+ tissue-resident memory T (CD8+ Trm) cells when compared to non-erosive OLP patients. The authors also designed a cohort study that demonstrated that tissues from patients with recent bouts of erosion displayed a more activated immunological state assessed by transcriptional profiling. Finally, the authors conducted immunological assays to demonstrate greater recovery and higher activation of CD8+ Trm cells from erosive OLP patients.

    The sequencing data presented in the study are of high quality and demonstrate key immunological differences between patients with non-erosive OLP and erosive OLP. The authors focused on T cells due to their strong correlation with OLP pathogenesis, but they also observe significant changes to B cell and mast cell levels in erosive OLP compared to non-erosive OLP. Further commentary on the contribution(s) of B cells and mast cells to OLP pathogenesis would be helpful to fully capture the importance of the sequencing dataset.

    My major criticism of the study is that the authors argue for CD8+ Trm activity as a key mechanism for OLP pathogenesis but have presented mostly descriptive datasets. The data strongly argue for CD8+ Trm cells as a defining feature of erosive OLP, but there is no data to support their involvement in disease pathogenesis. The authors note the lack of a mouse model for OLP which represents a significant technical barrier to interrogating the role of CD8+ Trm cells in OLP pathogenesis.

    Another criticism is the lack of strong findings in the analysis of CD8+ Trm cells isolated from non-erosive and erosive OLP tissues. The authors note increases in CD8+ Trm cell recovery, however, they only observe minor changes in CD8+ Trm activity upon restimulation. Analyzing the activation status or proliferative capacity of CD8+ Trm cells from non-erosive and erosive OLP could be informative and more robust measures of functional changes.

    A minor criticism is the formatting of the data presented in Figure 4. The authors should clearly label each marker used in the flow cytometry experiments as well as clearly labeling y-axes for graphs 4H and 4I.

  6. Version published to 10.1101/2022.10.18.22281149v1 on medRxiv