BackgroundThe skin contains many tissue-resident immune cells increasingly acknowledged as having key roles in maintaining tissue integrity and excluding infection. In lymphoid stress surveillance, lymphocytes are activated rapidly and in synchrony by stress ligands expressed by tissue cells after cellular stress and engage activating receptors such as NKG2D. Although much of this process has been worked out in model systems, whether this capability exists in human skin is not entirely clear. Therefore, we have characterised the human skin-resident lymphocyte compartment, and measured functional responsiveness of cells to see whether certain T cells have the capacity to respond rapidly to tissue stress. MethodsWe used normal adult human skin discarded after cutaneous surgery. Protocols were established to isolate and characterise lymphocytes by tissue disaggregation or three-dimensional explant culture. Skin-resident lymphocytes were characterised by multicolour flow cytometry, including cell-surface receptor expression and cytokine production after in-vitro stimulation. FindingsAfter ex-vivo isolation, distinct skin-resident lymphocyte subsets were identified, including the unconventional lymphocytes, γδ T cells and natural killer (NK) cells. Subsets were consistent across individuals; expressed as a mean (SD) percentage of leucocytes isolated, there were 46·1% CD4 T cells (15·2), 20·2% CD8 T cells (14·8), 1·6% γδ T cells (1·0), and 6·2% NK cells (5·0) (n=7–10). Explant culture greatly increased lymphocyte yield, enabling detailed functional characterisation. In addition, culture conditions greatly amplified the γδ T-cell compartment (1·6–4·0%, p=0·01, Student's t test). Skin-resident lymphocytes displayed skin-homing surface markers and a memory phenotype. Lymphocytes differed in expression of co-stimulatory receptors, with CD8 T cells, γδ T cells, and NK cells expressing the activating receptor NKG2D. Upon in-vitro activation, skin-resident lymphocytes readily produced cytokines and displayed distinct cytokine-producing profiles, with the response dependent on the strength of T-cell receptor-mediated activation. Skin-derived NKG2D+ lymphocytes had clear potential to produce both tumour necrosis factor α and interferon γ. InterpretationSkin-resident lymphocytes could be isolated from healthy human skin. Consistent with published data, cultured skin-resident αβ T cells maintained a tissue-resident memory (TRM) phenotype. In addition, that skin γδ T cell and NK cells also had a TRM-type phenotype was a novel finding. Distinct NKG2D+ lymphocytes were identified, which showed variable expression of additional activatory or inhibitory co-receptors, and were functionally competent with distinct cytokine-producing subsets. This system provides a platform to further characterise human skin-resident lymphocytes and their potential to be directly activated by surrounding tissue dysregulation. FundingUK Medical Research Council.