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Cited by 204Research on Leishmaniasis StudiesT-cell and B-cell ImmunologyImmune responses and vaccinations

The DOI matches its Crossref record; 41 of the 41 references it cites were found in a registry or an index.

Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection

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Glennie, Nelson D., Yeramilli, Venkata A., Beiting, Daniel P., Volk, Susan W., Weaver, Casey T., Scott, Phillip·2015·Journal of Experimental Medicine
DOI 10.1084/jem.20142101PMC PMC4548053
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Abstract

Leishmaniasis causes a significant disease burden worldwide. Although Leishmania-infected patients become refractory to reinfection after disease resolution, effective immune protection has not yet been achieved by human vaccines. Although circulating Leishmania-specific T cells are known to play a critical role in immunity, the role of memory T cells present in peripheral tissues has not been explored. Here, we identify a population of skin-resident Leishmania-specific memory CD4+ T cells. These cells produce IFN-γ and remain resident in the skin when transplanted by skin graft onto naive mice. They function to recruit circulating T cells to the skin in a CXCR3-dependent manner, resulting in better control of the parasites. Our findings are the first to demonstrate that CD4+ TRM cells form in response to a parasitic infection, and indicate that optimal protective immunity to Leishmania, and thus the success of a vaccine, may depend on generating both circulating and skin-resident memory T cells.

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Figure 1. L. major–specific CD4+ T cells are present in skin distant from the primary infection site. Skin was collected from either naive C57BL/6 mice (wild-type or IFN-γ–reporter mice) or mice that had been previously infected with L. major in the left ear and resolved their lesions (referred to as immune mice). Skin isolated from infected mice included the left ear (primary ear), contralateral ear (CL), and immune (Imm.) flank; skin from naive mice (N.) included the ear and the flank. (A–C) Cells from the skin were incubated with either uninfected BM-derived DCs or BMDCs that were infected with L. major (iDC) and, 12 h later, IFN-γ production in CD4+ T cells (gated on live, CD45, CD90.2, CD4 cells) was assessed by expression of Thy1.1 (A and B) or by IFN-γ intracellular staining (C). (A) Representative flow plots. (B and C) Each dot represents an individual mouse, and data are combined from at least three experiments. (D) FITC anti-CD45.2 antibody was injected i.v. into immune mice (15 wk after infection) and 3 min later blood, flank, primary ear (Prim.), and contralateral (CL) ear were collected and incubated with infected BMDCs as above, and FITC staining was assessed on CD4+ IFN-γ+ cells. Data shown are from one representative experiment of 2 (n = 5 mice). (E) Cells isolated from the flank or spleen of immune mice were incubated with L. major infected BMDCs as above, and each bar represents the mean (±SEM) percent of IFN-γ–producing CD4+ T cells detected by flow cytometry (gated on live, CD45, CD4 cells). Data shown are combined from 3 experiments (n = 10 mice per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 1. L. major–specific CD4+ T cells are present in skin distant from the primary infection site. Skin was collected from either naive C57BL/6 mice (wild-type or IFN-γ–reporter mice) or mice that had been previously infected with L. major in the left ear and resolved their lesions (referred to as immune mice). Skin isolated from infected mice included the left ear (primary ear), contralateral ear (CL), and immune (Imm.) flank; skin from naive mice (N.) included the ear and the flank. (A–C) Cells from the skin were incubated with either uninfected BM-derived DCs or BMDCs that were infected with L. major (iDC) and, 12 h later, IFN-γ production in CD4+ T cells (gated on live, CD45, CD90.2, CD4 cells) was assessed by expression of Thy1.1 (A and B) or by IFN-γ intracellular staining (C). (A) Representative flow plots. (B and C) Each dot represents an individual mouse, and data are combined from at least three experiments. (D) FITC anti-CD45.2 antibody was injected i.v. into immune mice (15 wk after infection) and 3 min later blood, flank, primary ear (Prim.), and contralateral (CL) ear were collected and incubated with infected BMDCs as above, and FITC staining was assessed on CD4+ IFN-γ+ cells. Data shown are from one representative experiment of 2 (n = 5 mice). (E) Cells isolated from the flank or spleen of immune mice were incubated with L. major infected BMDCs as above, and each bar represents the mean (±SEM) percent of IFN-γ–producing CD4+ T cells detected by flow cytometry (gated on live, CD45, CD4 cells). Data shown are combined from 3 experiments (n = 10 mice per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.CC BY-NC-SA · PubMed Central
Figure 2. L. major–specific CD4 T cells are resident in immune skin. (A) Flank skin was collected from naive or immune mice at various time points after L. major infection, and IFN-γ–producing CD4+ T cells detected as in Fig. 1. Data shown represent the mean (±SEM) percent IFN-γ–producing CD4+ T cells detected by flow cytometry (n = 3–4 mice per time point). (B) Flank skin from immune mice (13–14 wk after infection) was grafted onto naive mice, and at various times the grafts were harvested and IFN-γ–producing CD4+ T cells were detected as in Fig. 1. Quantification of L. major–specific cells in skin grafts before grafting (time 0) and 1, 2, or 4 wk later is shown. Data are combined from two separate experiments and each dot represents one mouse. *, P < 0.05.
Figure 2. L. major–specific CD4 T cells are resident in immune skin. (A) Flank skin was collected from naive or immune mice at various time points after L. major infection, and IFN-γ–producing CD4+ T cells detected as in Fig. 1. Data shown represent the mean (±SEM) percent IFN-γ–producing CD4+ T cells detected by flow cytometry (n = 3–4 mice per time point). (B) Flank skin from immune mice (13–14 wk after infection) was grafted onto naive mice, and at various times the grafts were harvested and IFN-γ–producing CD4+ T cells were detected as in Fig. 1. Quantification of L. major–specific cells in skin grafts before grafting (time 0) and 1, 2, or 4 wk later is shown. Data are combined from two separate experiments and each dot represents one mouse. *, P < 0.05.CC BY-NC-SA · PubMed Central
Figure 3. L. major–immune mice rapidly up-regulate IFN and chemokine signaling after challenge. Naive or immune mice (20 wk after infection) were infected in the flank with L. major, and the flank skin was harvested 12 h later for transcriptional profiling by microarray. (A) Heat map of 145 differentially expressed genes between 4 naive and 4 immune mice challenged in the flank (fold change ≥ 2, false discovery rate ≤ 0.05). (B and C) Gene set enrichment analysis (GSEA) plot for gene sets IFN signaling (B) and chemokines and chemokine receptors (C). Heat maps show the top up-regulated genes from the IFN-signaling gene set or chemokines and chemokine receptors gene set between naive and immune mice (red, high; blue, low). Normalized enrichment score (NES) with false discovery rate q-value as calculated by GSEA. (D) Cells were isolated from the flank skin of immune mice (20 wk after infection) were incubated with or without L. major parasites for 12 h and gene expression assessed by qRT-PCR. Data shown are the fold induction of IFN-regulated genes after exposure to L. major. Each dot represents an individual animal, and is from one representative experiment of 2. *, P < 0.05.
Figure 3. L. major–immune mice rapidly up-regulate IFN and chemokine signaling after challenge. Naive or immune mice (20 wk after infection) were infected in the flank with L. major, and the flank skin was harvested 12 h later for transcriptional profiling by microarray. (A) Heat map of 145 differentially expressed genes between 4 naive and 4 immune mice challenged in the flank (fold change ≥ 2, false discovery rate ≤ 0.05). (B and C) Gene set enrichment analysis (GSEA) plot for gene sets IFN signaling (B) and chemokines and chemokine receptors (C). Heat maps show the top up-regulated genes from the IFN-signaling gene set or chemokines and chemokine receptors gene set between naive and immune mice (red, high; blue, low). Normalized enrichment score (NES) with false discovery rate q-value as calculated by GSEA. (D) Cells were isolated from the flank skin of immune mice (20 wk after infection) were incubated with or without L. major parasites for 12 h and gene expression assessed by qRT-PCR. Data shown are the fold induction of IFN-regulated genes after exposure to L. major. Each dot represents an individual animal, and is from one representative experiment of 2. *, P < 0.05.CC BY-NC-SA · PubMed Central
Figure 4. L. major–specific TRM cells enhance T cell recruitment after challenge with L. major. Splenocytes from L. major (A–C) or LCMV (D) immune mice were CFSE labeled to track them after transfer. They were injected i.v. into either naive or L. major–immune mice (20 wk after infection) that were then challenged with L. major or PBS in the flank. Recruitment of cells to the challenge site was assessed 20 h later. (A) Representative flow plots of CFSE+ CD4+ T cells comparing the blood and the flank in naive or immune mice challenged with L. major. (B) Frequency and number of transferred L. major immune CD4+ and CD8+ T cells in the blood and flank skin of naive and immune mice 20 h after challenge with L. major. (C) Frequency and number of transferred L. major–immune CD4+ T cells 20 h after challenge with L. major or injection of PBS in naive or immune mice. (D) Frequency and number of transferred LCMV immune lymphocytes in the blood and flank skin of naive and immune mice 20 h after challenge with L. major. (E) L. major–immune mice were treated with anti-CD4 mAb, and the frequency of CD4+ T cells in the skin of untreated or anti-CD4–treated immune mice is shown. (F and G) Naive L. major–immune or L. major–immune mice treated with anti-CD4 mAb (F) or anti-CXCR3 mAb (G) received CFSE-labeled splenocytes from immune mice, and were then infected in the flank with L. major. After 20 h, the frequency and number of the transferred cells in the blood and flank was determined. (H and I) Flank skin from naive or immune mice was grafted onto the flank of naive mice. After 7 d, the recipient mice were injected i.v. with CFSE-labeled immune cells, each graft was infected with L. major, and, 20 h later, cells were isolated from the grafts. (H) Representative flow plots of CFSE+ CD4+ T cells comparing the blood and naive or immune skin grafts challenged with L. major. (I) Frequency and number of transferred L. major–immune CD4+ T cells in naive or immune skin grafts (cells were gated on live, CD45, CD90.2, CD4 cells). All data are from one representative experiment of two or more with three or more mice per experiment, and each bar represents the mean (±SEM) of individual mice (A–G) or combined data from two experiments where each dot represents a mouse (H and I). Immune mice used in all these experiments were 14 to 20 wk after infection. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 4. L. major–specific TRM cells enhance T cell recruitment after challenge with L. major. Splenocytes from L. major (A–C) or LCMV (D) immune mice were CFSE labeled to track them after transfer. They were injected i.v. into either naive or L. major–immune mice (20 wk after infection) that were then challenged with L. major or PBS in the flank. Recruitment of cells to the challenge site was assessed 20 h later. (A) Representative flow plots of CFSE+ CD4+ T cells comparing the blood and the flank in naive or immune mice challenged with L. major. (B) Frequency and number of transferred L. major immune CD4+ and CD8+ T cells in the blood and flank skin of naive and immune mice 20 h after challenge with L. major. (C) Frequency and number of transferred L. major–immune CD4+ T cells 20 h after challenge with L. major or injection of PBS in naive or immune mice. (D) Frequency and number of transferred LCMV immune lymphocytes in the blood and flank skin of naive and immune mice 20 h after challenge with L. major. (E) L. major–immune mice were treated with anti-CD4 mAb, and the frequency of CD4+ T cells in the skin of untreated or anti-CD4–treated immune mice is shown. (F and G) Naive L. major–immune or L. major–immune mice treated with anti-CD4 mAb (F) or anti-CXCR3 mAb (G) received CFSE-labeled splenocytes from immune mice, and were then infected in the flank with L. major. After 20 h, the frequency and number of the transferred cells in the blood and flank was determined. (H and I) Flank skin from naive or immune mice was grafted onto the flank of naive mice. After 7 d, the recipient mice were injected i.v. with CFSE-labeled immune cells, each graft was infected with L. major, and, 20 h later, cells were isolated from the grafts. (H) Representative flow plots of CFSE+ CD4+ T cells comparing the blood and naive or immune skin grafts challenged with L. major. (I) Frequency and number of transferred L. major–immune CD4+ T cells in naive or immune skin grafts (cells were gated on live, CD45, CD90.2, CD4 cells). All data are from one representative experiment of two or more with three or more mice per experiment, and each bar represents the mean (±SEM) of individual mice (A–G) or combined data from two experiments where each dot represents a mouse (H and I). Immune mice used in all these experiments were 14 to 20 wk after infection. *, P < 0.05; **, P < 0.01; ***, P < 0.001.CC BY-NC-SA · PubMed Central
Figure 5. L. major–specific TRM cells provide increased protection against secondary challenge. Flank skin from naive or immune mice was grafted onto the flank of naive mice. After 7 or 30 d, grafts from both the naive and immune mice were challenged with L. major and, 2 wk later, the grafts were isolated and parasite burdens were measured. Some mice received 20 × 106 splenocytes (i.v.) from L. major–immune mice at the time of challenge. (A) Naive or immune skin grafts in the presence or absence of adoptively transferred L. major–immune cells were challenged with L. major and the parasite burden was assessed at 2 wk. Data shown are the mean (±SEM) parasite burden. (B) Intact naive or immune mice, or naive and immune skin grafted onto naive mice were challenged with L. major. The mice with skin grafts also received L. major–immune cells as above, and the parasite burden was assessed at 2 wk. The data shown are the mean (±SEM) of the parasite burden. (C) Naive or immune skin was grafted onto naive mice, and 30 d later mice received L. major–immune cells as above and both grafts were challenged with L. major. The data shown are the mean (±SEM) of the parasite burden 2 wk after L. major challenge. Data from A and B are representative of 2 experiments (n = 5 mice per group), and C is combined data from 2 experiments (n = 6 mice). Immune mice used in all these experiments were 14 to 20 wk after infection. *, P < 0.05.
Figure 5. L. major–specific TRM cells provide increased protection against secondary challenge. Flank skin from naive or immune mice was grafted onto the flank of naive mice. After 7 or 30 d, grafts from both the naive and immune mice were challenged with L. major and, 2 wk later, the grafts were isolated and parasite burdens were measured. Some mice received 20 × 106 splenocytes (i.v.) from L. major–immune mice at the time of challenge. (A) Naive or immune skin grafts in the presence or absence of adoptively transferred L. major–immune cells were challenged with L. major and the parasite burden was assessed at 2 wk. Data shown are the mean (±SEM) parasite burden. (B) Intact naive or immune mice, or naive and immune skin grafted onto naive mice were challenged with L. major. The mice with skin grafts also received L. major–immune cells as above, and the parasite burden was assessed at 2 wk. The data shown are the mean (±SEM) of the parasite burden. (C) Naive or immune skin was grafted onto naive mice, and 30 d later mice received L. major–immune cells as above and both grafts were challenged with L. major. The data shown are the mean (±SEM) of the parasite burden 2 wk after L. major challenge. Data from A and B are representative of 2 experiments (n = 5 mice per group), and C is combined data from 2 experiments (n = 6 mice). Immune mice used in all these experiments were 14 to 20 wk after infection. *, P < 0.05.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Glennie, N. D., Yeramilli, V. A., Beiting, D. P., Volk, S. W., Weaver, C. T., & Scott, P.
Affiliations
  • University of Pennsylvania (US)
  • University of Alabama at Birmingham (US)
Corresponding author
Scott, Phillip
ORCID
Venue
Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2015-07-27
Volume
212
Issue
9
Pages
1405-1414
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.20142101
PMC
PMC4548053
OpenAlex
W2162538566
Cited by
204
References
41
Citations by year
2026: 10 · 2025: 10 · 2024: 12 · 2023: 14 · 2022: 14 · 2021: 29 · 2020: 21 · 2019: 21 · 2018: 26 · 2017: 22 · 2016: 20 · 2015: 5
FWCI
26.10
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
National Institute of Allergy and Infectious Diseases (R21AI110869, T32AI007532)
Topics
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Research on Leishmaniasis Studies
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Life Sciences › Immunology and Microbiology › Immunology › Immune responses and vaccinations
Keywords
Leishmania major, cutaneous leishmaniasis, skin-resident memory T cells, CD4+ T cells, interferon-gamma, CXCR3, skin grafting, protective immunity, leishmaniasis vaccine, parasite control
MeSH terms
Animals, Humans, Immunologic Memory, Interferon-gamma, Mice, Transgenic, Skin, CD4-Positive T-Lymphocytes, Leishmaniasis, Cutaneous, Leishmania major, Mice, Receptors, CXCR3

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