Targeted multi-omic analysis of human skin tissue identifies alterations of conventional and unconventional T cells associated with burn injury
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eLife assessment
This manuscript provides an important advance in our understanding of burn-associated T-cell responses. The evidence is convincing and the techniques are using the latest single-cell RNA-seq approaches in a rigorous manner. The studies are done directly on human skin so are highly clinically relevant.
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Abstract
Burn injuries are a leading cause of unintentional injury, associated with a dysfunctional immune response and an increased risk of infections. Despite this, little is known about the role of T cells in human burn injury. In this study, we compared the activation and function of conventional T cells and unconventional T cell subsets in skin tissue from acute burn (within 7 days from initial injury), late phase burn (beyond 7 days from initial injury), and non-burn patients. We compared T cell functionality by a combination of flow cytometry and a multi-omic single-cell approach with targeted transcriptomics and protein expression. We found a significantly lower proportion of CD8+ T cells in burn skin compared to non-burn skin, with CD4+ T cells making up the bulk of the T cell population. Both conventional and unconventional burn tissue T cells show significantly higher IFN-γ and TNF-α levels after stimulation than non-burn skin T cells. In sorted T cells, clustering showed that burn tissue had significantly higher expression of homing receptors CCR7, S1PR1, and SELL compared to non-burn skin. In unconventional T cells, including mucosal-associated invariant T (MAIT) and γδ T cells, we see significantly higher expression of cytotoxic molecules GZMB, PRF1, and GZMK. Multi-omics analysis of conventional T cells suggests a shift from tissue-resident T cells in non-burn tissue to a circulating T cell phenotype in burn tissue. In conclusion, by examining skin tissue from burn patients, our results suggest that T cells in burn tissue have a pro-inflammatory rather than a homeostatic tissue-resident phenotype, and that unconventional T cells have a higher cytotoxic capacity. Our findings have the potential to inform the development of novel treatment strategies for burns.
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eLife assessment
This manuscript provides an important advance in our understanding of burn-associated T-cell responses. The evidence is convincing and the techniques are using the latest single-cell RNA-seq approaches in a rigorous manner. The studies are done directly on human skin so are highly clinically relevant.
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Reviewer #1 (Public Review):
Most previous studies about burn injuries only considered systemic inflammation with analyses of blood specimens from patients. The current study is unique in the fact that it utilizes skin samples. The authors used single-cell analyses by flow cytometry and RNA-seq to characterize in detail the different T-cell populations. The differences are striking. Burned skins have higher degrees of CD69-negative T cells, which indicates that these are recruited from probably blood circulation. They are also substantially more responsive to stimulation by producing higher amounts of immunologic molecules, such as IFNG and TNFA. The results are compelling because they indicate that following burn injury, T cells infiltrate the lesions to potentially protect the damaged tissue from secondary infections.
However, there …
Reviewer #1 (Public Review):
Most previous studies about burn injuries only considered systemic inflammation with analyses of blood specimens from patients. The current study is unique in the fact that it utilizes skin samples. The authors used single-cell analyses by flow cytometry and RNA-seq to characterize in detail the different T-cell populations. The differences are striking. Burned skins have higher degrees of CD69-negative T cells, which indicates that these are recruited from probably blood circulation. They are also substantially more responsive to stimulation by producing higher amounts of immunologic molecules, such as IFNG and TNFA. The results are compelling because they indicate that following burn injury, T cells infiltrate the lesions to potentially protect the damaged tissue from secondary infections.
However, there is an important aspect missing. What does induce T-cell infiltration into the burned skins? A potential explanation is that resident myeloid cells directly or indirectly promote chemokine-mediated recruitment of T cells.
Another important consideration is the impact on other leukocyte populations. While the study is well focused on T cells, the immune system consists of a complex network of cells and molecules that interact with each other. The study does not address myeloid cells and innate lymphoid cells, which could also play important roles and display altered functions in burned injuries.
Nevertheless, the study provides important information about the "activation" statuses of several T cell populations following burned injuries and could help guide the development of better treatments.
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Reviewer #2 (Public Review):
In this study, Labuz and collaborators characterize the impact of burn injury on the T cell populations of the human skin. The authors use multiparametric flow cytometry and single-cell transcriptomics to analyze the numbers and the transcriptional profile of conventional and unconventional T cell populations in samples collected from patients with acute burn injury, late burn injury, and without burn injury. Their results show that burn injury disturbs the balance of T cell subtypes by increasing the percentage of CD4 T cells and decreasing the percentage of CD8 T cells. Both CD4 and CD8 T cells in the burn tissue presented lower expression of CD69 and higher expression of CD38, IFN-gamma, and TNF-alpha. The percentage of gamma delta T cells and MAIT cells positive for TNF-alpha and IFN-gamma also increased …
Reviewer #2 (Public Review):
In this study, Labuz and collaborators characterize the impact of burn injury on the T cell populations of the human skin. The authors use multiparametric flow cytometry and single-cell transcriptomics to analyze the numbers and the transcriptional profile of conventional and unconventional T cell populations in samples collected from patients with acute burn injury, late burn injury, and without burn injury. Their results show that burn injury disturbs the balance of T cell subtypes by increasing the percentage of CD4 T cells and decreasing the percentage of CD8 T cells. Both CD4 and CD8 T cells in the burn tissue presented lower expression of CD69 and higher expression of CD38, IFN-gamma, and TNF-alpha. The percentage of gamma delta T cells and MAIT cells positive for TNF-alpha and IFN-gamma also increased in the burn tissue. The authors then use single-cell RNA sequencing to gain further insights into how burn injury impacts the overall functions of skin T cells. This unbiased transcriptional profiling confirmed their previous observations that both conventional and unconventional T cells in the skin are replaced by new clusters that express lower levels of "tissue-resident" signature genes such as CD69 and higher levels of homing markers such as SELL and S1PR1. CD8 T cells of the burn skin samples show a clear reduction in the expression of cytotoxic molecules such as GZMK, GZMH, and GNLY, and this contrasts with the upregulation of cytotoxic molecules that are observed in the populations of unconventional T cell populations.
This is a relatively simple and descriptive work that will likely be an important resource for future studies investigating the role of T cell responses in skin wound healing and the maintenance of skin barrier function against pathogens following burn injury. A broader and more unbiased analysis of scRNA-seq data is necessary to better understand the biological processes and cellular responses that are being affected by the transcriptional changes observed in each T cell population as well as the possible implications of their findings.
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