Fig 2: Endogenous retrovirus activity promotes the accumulation of T cells in the skin following S. epidermidis colonization, related to Figure 2(A) Volcano plot of expressed loci from sorted Sca-1+CD49f+ keratinocytes from unassociated or S. epidermidis-associated at day 7 post-association. Underlined denotes locus containing an active reverse transcriptase. (B) Heatmap showing transcript levels of the indicated ERVs measured by qRT-PCR of mRNA isolated from ear pinnae from unassociated or daily S. epidermidis-associated mice at day 7 post association. Values were normalized to Gapdh expression in the same sample. (C) Schematic showing encoded regions of LINE-1 elements in the C57BL/6J (WT) mice genome. Regions highlighted in yellow represent the nucleotide regions encoded in the given locus. (D) Calculated iMFI of MLV SU expression detected by flow cytometry on the surface of CD49f+Sca-1+ primary keratinocytes isolated from neonatal mice stimulated with TLR agonists. (E-G) WT mice were treated daily with either vehicle control (S. epi + vehicle) or antiretroviral (S. epi + antiretroviral) beginning at 1 week before S. epidermidis association for a total of 3 weeks. Two weeks post association, T cell populations were evaluated by flow cytometry. (E and F) Absolute number of CD8+ T cells, CD4+ T cells, ?dTCRlow T cells, IL-17A+ (Tc17) or IFN-?+ (Tc1) CD8+ T cells, IL-17A+ (Th17) or IFN-?+ (Th1) CD4+ T cells, or IL-17A+ ?dTCRlow T cells (E) in the ear pinnae or (F) the spleen. (G) Absolute number of bead-enriched f-MIIINA:H2-M3 tetramer positive CD8ß+ T cells in the spleen. Data are represented as mean ± SEM. Each dot represents an individual mouse. (H) WT mice were treated daily with either vehicle control (S. epi + vehicle) or antiretroviral (S. epi + antiretroviral) beginning 1 week before daily S. epidermidis association for a total of 2 weeks. Seven days post-association, RNA was purified from sorted Sca-1+CD49f+ keratinocytes and sequenced. The expression of the S. epidermidis-induced ERV, Chr5 23.7M is highlighted in red. (I) Enumeration of S. epidermidis colony-forming units (CFU) at day 14 post association in WT mice treated daily with either vehicle control (S. epi + vehicle) or antiretroviral (S. epi + antiretroviral) beginning at 1 week before S. epidermidis association. (J) Growth curve (OD600) of S. epidermidis grown in media treated with vehicle, tenofovir and emtricitabine alone or with a combination of tenofovir and emtricitabine at different concentrations. *P ? 0.05, **P ? 0.01, ***P ? 0.001, ****P ? 0.0001 as calculated with one-way ANOVA with Holm-Šidák multiple comparison test (C, E-H). Data are representative of two (A-D, H-J) or three (E) and (F) independent experiments using 4-5 mice per group (A), (B), E-I). Graphs in (C) and (J) represent the average of a technical triplicate.

Fig 3: Dermal ?dT Cell Activation Requires Both IL-1R and IL-23R Signaling Pathways(A) Gene set enrichment analysis identifies transcriptional downregulation of the IL-12 production regulatory pathway and IL-1R pathway in psoriasis patients effectively treated with glucocorticoid. The top 10 leading edge genes in these two pathways are shown.(B) Whole skin cell suspensions from WT, IL-1R KO, and IL-23R KO mice were labeled with CFSE and stimulated with IL-23, IL-1ß, or IL-23 plus IL-1ß for 3 days. Cell proliferation and intracellular IL-17 were analyzed by flow cytometry. Flow plots gated on CD3+?dTCRintV?4 or CD3+?dTCRintV?6 cells are representative of at least two independent experiments with similar results. Each experiment includes at least three mice from WT, IL-1R KO, or IL-23R KO strains.(C) Summarized percentages of dermal ?dT17 cells are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t test).(D) Summarized dermal ?dT cell proliferation with different subsets of dermal ?dT cells (V?4 and V?6) is shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t test).

Fig 4: S. epidermidis induced T cells accumulation in the skin is independent of STING signaling in CD11c+ cells, related to Figure 4Frequency and absolute number of CD8+ T cells, CD4+ T cells, ?dTCRlow T cells, IL-17A+ (Tc17) or IFN-?+ (Tc1) CD8+ T cells, IL-17A+ (Th17) or IFN-?+ (Th1) CD4+ T cells and IL-17A+?dTCRlow cells in the ear pinnae of Cd11ccre-Stingflox/flox and Cd11ccre-Stingflox/flox mice two weeks post-association with S. epidermidis. Data are represented as mean ± SEM. Each dot represents an individual mouse.

Fig 5: cGAS-STING signaling promotes T cells accumulation in the skin in response to S. epidermidis colonization, related to Figure 4(A) Frequency of CD8+ and CD4+ (left) T cells, percentage change of the absolute numbers of CD8+, CD4+, ?dTCRlow T cells (middle) and absolute number of IFN-?+ CD8+ T cells (Tc1) (right) in the ear pinnae of naive (unassociated) WT, cGas–/– and Sting–/– mice. (B) Absolute number of CD8+, CD4+, ?dTCRlow T cells and/or MAIT cells and frequency of IL-17A+ or IFN-?+ CD8+ or CD4+ T cells and IL-17A+ ?dTCRlow or MAIT cells in the ear pinnae from WT, cGas–/– and Sting–/–mice associated with S. epidermidis (day 14) or left unassociated. (C) S. epidermidis CFU enumeration at 14 days post-association in WT and Sting–/– mice topically associated with S. epidermidis (S. epi) or left unassociated. (D) Expression of Chr5 23.7 locus determined by RNASeq in Sca-1+CD49f+ keratinocytes purified from unassociated or daily S. epi-associated Krt14cre-Stingflox/flox and Krt14cre+Stingflox/flox mice at day 7 post-association. ** FDR < 0.01, **** FDR < 0.001. (E) Absolute number of CD8+, CD4+, ?dTCRlow T cells and/or MAIT cells and frequency of IL-17A+ or IFN-?+ CD8+ or CD4+ T cells and IL-17A+ ?dTCRlow or MAIT cells in the ear pinnae from Krt14cre-Stingflox/flox and Krt14cre+Stingflox/flox mice associated with S. epidermidis (day 14) or left unassociated. (F) Proposed model for ERV control of T cell responses to the microbiota: 1- Discrete sensing of microbiota by keratinocytes (via, in part, TLR2-specific ligands) promotes the expression of defined ERVs; 2- Reverse transcription of ERVs leads to cytosolic accumulation of ERV-derived cDNAs that are sensed by the cGAS/STING pathway. Resulting activation of keratinocytes is associated with an antiviral program and type I IFN production; 3- Discrete keratinocyte “hot spots” could promote an environment favorable to the capture of microbiota-derived antigens by DCs and subsequent migration of these cells to the lymph node; 4- commensal-specific T cells migrate back to the skin where their accumulation and function could be promoted by ERV-activated keratinocytes. In the context of non-classical T cells such as MAIT cells, local responses to ERVs may be sufficient to control their local proliferation and accumulation in the skin. The result of this sequence of events is the accumulation of a network of commensal specific T cells able to broadly promote tissue physiology including tissue repair. All cells were stimulated with PMA and ionomycin. Each dot represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as calculated with two-way ANOVA with Holm-Šidák multiple comparison test. Data are represented as mean ± SEM. Data are representative of two independent experiments using 4-6 mice per group.