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. 2014 Apr 8;111(14):5307-12.
doi: 10.1073/pnas.1322292111. Epub 2014 Mar 24.

Persistence of skin-resident memory T cells within an epidermal niche

Ali Zaid  1 , Laura K Mackay, Azad Rahimpour, Asolina Braun, Marc Veldhoen, Francis R Carbone, Jonathan H Manton, William R Heath, Scott N Mueller
Affiliations

Persistence of skin-resident memory T cells within an epidermal niche

Ali Zaid et al. Proc Natl Acad Sci U S A. .

Abstract

Barrier tissues such as the skin contain various populations of immune cells that contribute to protection from infections. These include recently identified tissue-resident memory T cells (TRM). In the skin, these memory CD8(+) T cells reside in the epidermis after being recruited to this site by infection or inflammation. In this study, we demonstrate prolonged persistence of epidermal TRM preferentially at the site of prior infection despite sustained migration. Computational simulation of TRM migration within the skin over long periods revealed that the slow rate of random migration effectively constrains these memory cells within the region of skin in which they form. Notably, formation of TRM involved a concomitant local reduction in dendritic epidermal γδ T-cell numbers in the epidermis, indicating that these populations persist in mutual exclusion and may compete for local survival signals. Accordingly, we show that expression of the aryl hydrocarbon receptor, a transcription factor important for dendritic epidermal γδ T-cell maintenance in skin, also contributes to the persistence of skin TRM. Together, these data suggest that skin tissue-resident memory T cells persist within a tightly regulated epidermal T-cell niche.

Keywords: Brownian motion; Langerhans cells; intravital imaging HSV-1 infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Migration of skin TRM at the site of prior infection. (A) gBT-I.GFP CD8+ T cells (green) imaged by two-photon microscopy in the skin 32 d after HSV infection. Examples of different cell morphologies are magnified in i–iii. SHG (blue) delineates the collagen-rich dermis. Images correspond to Movie S1. (B) TRM are located within the basal epidermis (Ep), adjacent to the SHG+ dermis (D). (C) TRM contact the basement membrane at the dermis–epidermal border. Tissue sections of skin containing gBT-I.DsRed CD8+ T cells were costained with anti-laminin-γ2 antibodies. (D) Mean velocity and (E) displacement of TRM migrating within the epidermis at the indicated times after HSV infection. ***P < 0.0001; ns, not significant. (F) Epidermal location defines the dendritic morphology of skin TRM. Shown is a representative example of an epidermal and a dermal gBT-I T-cell 10 d after HSV infection. Sphericity measurements of dermal and epidermal gBT-I T cells 10 d (acute) and 32 d (memory) after HSV infection are plotted. (G) Time-lapse imaging of gBT-I TRM migration in the skin. (Left) The first frame of the movie; (Right) superimposed images taken at 3-min intervals over a 7.5-h period. Images correspond to Movie S4. (H) The average 2D surface area of individual TRM was calculated from 10 individual images per cell collected over a 60-min period. The total surface area covered per cell was calculated from superimposed images taken each minute for 1 h. The fold difference between the average surface area at time 0 and 60 is shown.
Fig. 2.
Fig. 2.
Persistence of skin TRM at the site of prior infection. (A) gBT-I CD8+ T cells were quantitated at the lesion scar and two defined distances either side of the lesion by two-photon microscopy of skin 36 and 280 d after HSV infection. Data were averaged from four to six images per region from six mice. (B) Mice were analyzed 20, 120, or 405 d after HSV infection. Strips of flank skin 0.5 cm wide by 2 cm long at the site of infection (lesion) and on either side were harvested for enumeration of CD103+ gBT-I CD8+ T cells. Error bars represent SEM, n = 4–8 mice pooled from two independent experiments. (C) Computational modeling of TRM migration. Movement of populations of cells distributed normally across the skin was simulated for 100 and 365 d based on an average cell velocity (V) of 1.12 μm/min (Left) or 3.36 μm/min (Right). The simulated region consisted of a 5-mm strip centered at 0. The frequency of cells within the simulated region was 50% at the beginning of the simulation (day 0) and the number of cells remaining within this region after the indicated time periods is shown.
Fig. 3.
Fig. 3.
TRM interact with LC in the epidermis. (A–C) Skin TRM interact frequently with LCs. (A) Representative image of gBT-I CD8+ T cells (red) migrating among LCs in Lg-GFP mice in the epidermis of HSV-immune mice. (B) Interaction between a TRM and LC in the skin at memory. Contact is represented by colocalization between the green and red channels (white). (C) Quantitation of the frequency of contacts between TRM and LCs. Images were acquired at 1-min intervals for 45–60 min. (D) No correlation between TRM and LC numbers in the skin after infection. Linear regression line and Pearson’s correlation coefficient (r) are shown. (E–G) Ablation of LCs for 2 wk influences TRM migration but not persistence. (E) Numbers of gBT-I CD8+ T cells in the skin and spleen and LCs in the skin of Lg-DTR mice following 2 wk of treatment with DT. n = 6–8 mice per group. (F) Example of TRM in Lg-DTR mice following 2 wk of treatment with DT. (G) Mean velocity of gBT-I CD8+ T cells in Lg-GFP or Lg-DTR mice after DT treatment. Error bars represent SEM, n = 4–7 mice per group, from two to three independent experiments.
Fig. 4.
Fig. 4.
TRM displace DETCs at the site of infection. (A) Two-photon microscopy of TRM and DETCs in the skin 30 d after HSV infection. A representative maximum intensity projection at the border of the lesion is shown. The sphericity and mean velocity of gBT-I.DsRed TRM and CXCR6GFP/+ DETC is shown. n = 6 mice. (B) Quantitation of the frequency of contacts between TRM and DETCs. Images were acquired at 1-min intervals for 45–60 min. Data are compiled from 158 TRM imaged from four mice. (C) TRM migrating in the epidermis can travel beneath DETCs. A maximum intensity projection across x, y, and z dimensions is shown. (D and E) TRM displace DETCs in the skin after infection. (D) Image of TRM and DETCs in CXCR6GFP/+ mice 30 d after HSV infection. (E) Quantitation of the density of cells within the indicated regions (B, border; D, distal region; and L, lesion) from the image in D. (F) Inverse correlation between TRM and DETC numbers in the skin after infection. Linear regression line and Pearson’s correlation coefficient (r) are shown. n = 7 mice.
Fig. 5.
Fig. 5.
TRM persist within an epidermal niche. (A) TRM form after intradermal injection and displace DETCs. Two representative examples of skin 30 d after injection of gBT-I effector T cells, showing a site with low numbers of TRM (region 1) and a second with high numbers (region 2). (B) Enumeration of TRM and DETCs in the images in A. (C) Enumeration of TRM and DETCs in the skin of naïve mice (N) and 30 d after intradermal injection of gBT-I T cells (ID). ns, not significant. (D) Negative correlation between TRM and DETC numbers in the skin after intradermal injection. Linear regression line and Pearson’s correlation coefficient (r) are shown. n = 6 mice. (E) Expression of AhR in memory T-cell subsets 30 d after HSV infection quantitated by RT-PCR. n = 3–4. (F) Ratio of WT to AhR−/− CD8+ T cells in the skin or spleen at the indicated times after i.v. transfer into mice treated with DNFB on the flank. Error bars represent SEM. Data pooled from two to three experiments with three to five mice per group.
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