Fig 1.
Antigen-specific memory CD8+ T cells rapidly traffic into VacV-infected skin and provide protective immunity.
(A) Naïve P14 CD8+ T cells were transferred into B6 mice and infected with LCMV. At 75 days post LCMV-infection, mice were challenged with VacV-GP33 on the left ear skin and trafficking of memory P14 CD8+ T cells into the skin was quantified. (B) Proliferation of memory CD8+ T cells from (A) was analyzed in the indicated tissue by BrdU incorporation. (C) Quantification of (B). (D) Expression of CD127 and KLRG1 on memory P14 CD8+ T cells that trafficked in to the skin as shown in (A). (E) Quantification of (D). (F) Naïve and LCMV-immune mice were infected with 1 x 106 PFU of VacV-GP33 on the left ear skin. On the indicated day post-infection, viral load was quantified in the infected skin. LOD (limit of detection) is indicated by the dashed line.
Fig 2.
CD62L-dependent lymph node trafficking is required for naïve CD8+ T cells to become activated following VacV skin infection, but not systemic LCMV infection.
(A) Equal numbers of WT (Thy1.1/1.2) and CD62L-/- (Thy1.1/1.1) naïve P14 CD8+ T cells were transferred into B6 mice and infected with either VacV-GP33 by skin scarification or LCMV by i.p injection. Frequencies of the transferred P14 CD8+ T cells was evaluated on the peak day of expansion (day 10 for VacV-GP33, day 7 for LCMV). (B) Quantification of (A). (C) WT or CD62L-/- naïve P14 CD8+ T cells were transferred into separate naïve B6 mice and infected with VacV-GP33 on the left ear skin. Expansion of the transferred T cells was then quantified. (D) Same as (C) except mice were infected with LCMV. (E, F) Quantification of circulating memory WT and CD62L-/- P14 CD8+ T cells on day 40 post-infection.
Fig 3.
Memory CD8+ T cells traffic into the skin independent of CD62L, then express CD69 following VacV infection.
(A) Expression of CD62L on WT and CD62L-/- memory P14 CD8+ T cells on day 60 post-LCMV infection. (B) WT and CD62L-/- memory P14 CD8+ T cells were identified in the spleen and cervical lymph node by flow cytometry. (C,D) Quantification of total numbers of memory P14 CD8+ T cells shown in (B). (E) WT or CD62L-/- memory P14 CD8+ T cells from (A) were stimulated with IL-15 and the capacity to bind to P- and E-selectin was quantified. (F) LCMV-immune mice containing WT or CD62L-/- memory P14 CD8+ T cells were infected with VacV-GP33 on the left ear skin and trafficking of the memory cells into the skin was quantified on days 3 and 7 post-infection. (G) Same as (F) except the expansion of the memory P14 CD8+ T cells was analyzed in the peripheral blood. *P<0.01 (H) Quantification of total numbers of WT and CD62L-/- memory P14 CD8+ T cells in the skin on day 40 post VacV-GP33 skin infection. (I) Expression of CD69 and CD103 on naïve or memory P14 CD8+ T cells in the skin on day 40 post VacV-GP33 skin infection. (J) Quantification of (I).
Fig 4.
CD69+ memory CD8+ T cells that remain in the skin are protected from antibody-mediated depletion and produce IFNγ following topical peptide challenge.
(A) LCMV immune mice were infected with VacV-GP33. On day 40 post VacV-GP33 infection, mice received control IgG or anti Thy1.1 antibody as described in Materials and Methods. Seven days later, memory Thy1.1+ P14 CD8+ T cells from the skin were analyzed for expression of CD69 and CD103. (B) Quantification of frequencies of skin P14 CD8+ T cells from (A). (C,D) Quantification of total numbers of (C) CD69+ and (D) CD69- memory P14 CD8+ T cells from (A). (E) Naïve YFP-IFNγ P14 CD8+ T cells were transferred into B6 mice and infected with LCMV. On day 60 post-infection, mice were then infected with VacV-GP33 on the left ear skin. On day 40 after the VacV-GP33 infection, the ear skin was challenged with either GP33-41 peptide or control peptide (NP396-404). Expression of IFNγ (YFP) was analyzed 6 hours post-peptide challenge. (F,G) Quantification of (F) CD69 expression and (G) YFP expression from (E). (H) Same mice as (E) except ear skin swelling was quantified at 6 hours post-peptide challenge.
Fig 5.
Circulating memory CD8+ T cells become CD69+ TRM following local antigen recognition in the skin.
(A) LCMV-immune mice were infected with VacV-GP33 on the left ear skin and VacV-OVA on the right. On day 15 post-infection, total number of memory P14 CD8+ T cells in the skin were quantified. (B) CD69 expression on memory P14 CD8+ T cells from (A). (C) Quantification of (B). (D) On day 40 post-infection, the number of CD69+ memory P14 CD8+ T cells was quantified in the left and right ear skin.
Fig 6.
TCM CD8+ T cells become CD69+ TRM following VacV infection.
(A) 1 x 105 TCM (CD62L+) or TEM (CD62L-) memory P14 CD8+ T cells were sorted by FACS and transferred into B6 mice, which were then infected on the left ear skin with VacV-GP33. (B) On day 7 post-infection, trafficking of memory CD8+ T cells into the skin was analyzed. (C) The total number of memory P14 CD8+ T cells in the skin from (B) was quantified over time. (D, E) Expression of (D) CD69 and (E) CD103 was analyzed in the skin on day 14 post-infection. (F) Total number of memory P14 CD8+ T cells in the skin on day 40 post-infection. (G) Expression of CD69 and CD103 on P14 CD8+ T cells in the skin on day 40 post VacV-GP33 skin infection that originated from either naïve or TCM populations.
Fig 7.
Naïve CD8+ T cells form tissue-residents following VacV skin infection better than circulating TCM CD8+ T cells.
(A) CD62L+ naïve or TCM P14 CD8+ T cells were sorted by FACS. (B) Equal numbers of naïve (Thy1.1/1.2) and TCM (Thy1.1/1.1) CD8+ T cells from (A) were transferred into naïve B6 mice (Thy1.2/1.2) and infected with VacV-GP33 on the left ear skin. (C) Expression of CD103 on day 15 post-infection. (D) Quantification of (C). (E) Expression of CD69 on day 15 post-infection. (F) Log2 ratio of naïve to TCM P14 CD8+ T cells in the spleen and VacV infected skin. (G) On day 40 post-infection, expression of CD69 and CD103 was quantified on P14 CD8+ T cells in the skin that originated from either naïve or TCM CD8+ T cells.
Fig 8.
Previously naïve CD8+ T cells express more CD103 than memory CD8+ T cells when stimulated with TGF-β following VacV skin infection.
(A) Naïve (Thy1.1/1.2) and either purified TCM or TEM (Thy1.1/1.1) P14 CD8+ T cells were co-transferred into naïve B6 mice that were then infected on the left ear skin with VacV-GP33. On day 10 post-infection, splenocytes were stimulated with 1 ng/ml TGF-β1 for 48 hours and expression of CD103 was analyzed. (B) Same experimental design as (A) except CD103 expression was quantified following stimulation with the indicated concentration of TGF-β1.
Fig 9.
Circulating memory CD8+ T cells require perforin, but not CD62L, to provide protective immunity against VacV skin infection.
(A) WT or Prf1-/- B6 mice were infected with 2 x 107 CFU of attenuated LM-OVA. On day 40 post-infection, LM-OVA-immune animals or naïve controls were infected with 1 x 106 PFU VacV-OVA on the left ear skin and the frequency of H2-Kb OVA257-264 specific CD8+ T cells were quantified in the blood. (B) Quantification of (A). (C) Viral titers in the ear skin of mice from (A) on day 7 post-infection. (D) WT and Lsel-/- mice were infected with attenuated LM-OVA (2 x 107 CFU). On day 40 post-infection, naïve controls or LM-OVA immune mice were infected with VacV-OVA on the left ear skin. On day 7 post-VacV-OVA skin infection, H2-Kb-OVA257-264 specific CD8+ T cells were analyzed in the blood. (E) Quantification of (D). (F) Viral load was quantified from the ear skin.