Skin-resident CD4⁺ T cells protect against <i>Leishmania major</i> by recruiting and activating inflammatory monocytes

<div><p>Tissue-resident memory T cells are required for establishing protective immunity against a variety of different pathogens, although the mechanisms mediating protection by CD4⁺ resident memory T cells are still being defined. In this study we addressed this issue with a population of protective skin-resident, IFNγ-producing CD4⁺ memory T cells generated following <i>Leishmania major</i> infection. We previously found that resident memory T cells recruit circulating effector T cells to enhance immunity. Here we show that resident memory CD4⁺ T cells mediate the delayed-hypersensitivity response observed in immune mice and provide protection without circulating T cells. This protection occurs rapidly after challenge, and requires the recruitment and activation of inflammatory monocytes, which limit parasites by production of both reactive oxygen species and nitric oxide. Overall, these data highlight a novel role for tissue-resident memory cells in recruiting and activating inflammatory monocytes, and underscore the central role that skin-resident T cells play in immunity to cutaneous leishmaniasis.</p></div

Early protection is not enhanced by circulating memory T cells.

<p>A) Naive, immune, and immune mice treated with FTY-720 or αCXCR3 were challenged intradermally in the ear with 2x10⁶ dsRed <i>L</i>. <i>major</i>. B) DTH response was monitored over the course of 3 days. C, D) Parasite burden (C) and the number of Ly6C⁺ monocytes in the infection site (D) was determined at 72 hrs. Data shown are from one experiment representative of two (n = 3 mice per group). E) Naive, immune, naive parabiotic, and immune parabiotic mice were challenged intradermally in the ear with 2x10⁶ dsRed <i>L</i>. <i>major</i>. F) DTH response was monitored over the course of 3 days. G, H) Parasite burden (G) and the number of Ly6C⁺ monocytes in the infection site (H) was determined at 72 hrs. Data shown are from one experiment representative of two (n = 3 mice per group). P < 0.05 = *; P < 0.01 = **; P < 0.001 = ***.</p

Circulating memory T cells are not required to control low dose <i>L</i>. <i>major</i> infection.

<p>A) Naive and immune mice were challenged in the ear with 10³ parasites and DTH was monitored over 72 hrs. B) Proportions of total CD45⁺ population in naive and immune ears 72 hrs after low dose challenge. C) Representative histogram showing frequency of MHCII cells in naive (black line) and immune (gray line) mice 72 hrs after low dose challenge. Data shown are from one experiment of two (n = 3 mice per group). D) Naive and immune flank skin were grafted side-by-side onto naive WT or RAG^-/- mice, then each graft was challenged with 10³ dsRed <i>L</i>. <i>major</i>. E) Parasite burden at 4 weeks was compared between intact naive and immune mice as well as naive and immune grafts on WT recipients. Data shown are from one experiment representative of two (n = 5 or 9 mice per group). F) Parasite burden at 4 weeks was compared between naive and immune grafts on WT or RAG^-/- recipients. Data shown are combined from two experiments (n = 5 mice per group). P < 0.05 = *; P < 0.01 = **; P < 0.001 = ***.</p

<i>L</i>. <i>major</i> immune mice are protected within 72 hrs of challenge in a CD4⁺ T_RM dependent manner.

<p>A) Naive and immune mice were challenged intradermally in the ear with 2x10⁶ dsRed <i>L</i>. <i>major</i> and DTH response was monitored over the course of 3 days. (B-D) Representative parasite burden in naive and immune mice 72 hrs after challenge as measured by limiting dilution (B), quantitative PCR (C), or flow cytometry (D). Representative data shown are from one experiment representative of eight (n = 3–4 mice per group). E) Naive, immune, and immune mice depleted of CD4⁺ or CD8⁺ T cells were challenged intradermally in the ear with 2x10⁶ dsRed <i>L</i>. <i>major</i>. F) DTH response was monitored over the course of 3 days. G) Parasite burden was determined at 72 hrs. Data shown are from one experiment representative of two (n = 3 mice per group). H) Naive and immune flank skin were grafted side-by-side onto naive recipients, then each graft was challenged intradermally with 2x10⁶ dsRed <i>L</i>. <i>major</i>. I) Parasite burden was determined at 72 hrs. Data shown are from one experiment representative of four (n = 5 mice per group). P < 0.05 = *; P < 0.01 = **; P < 0.001 = ***.</p

Rapid protection in <i>L</i>. <i>major</i> immune mice is associated with recruitment of inflammatory monocytes.

<p>A) Gating strategy for distinguishing myeloid cell populations. B) Numbers of T cells and myeloid cell populations in ear skin 72 hrs after challenge. C) Proportions of total CD45⁺ population in naive and immune ears 72 hrs after challenge. Data shown are combined from six experiments (n = 18 mice per group). D) Representative histogram or contour plots showing frequency of MHCII⁺, DHR⁺, and iNOS⁺ monocytes in naive (black line) and immune (gray line) mice 72 hrs after challenge. Data shown are from one experiment of two (n = 3 mice per group). E) The proportion of infected CD11b⁺ dsRed⁺ cells comprised of each cell type 72 hrs after challenge. Data shown are combined from six experiments (n = 18 mice per group) F) Representative plot of dsRed expression in CD11b⁺ cells from uninfected (gray fill), naive (black line), or immune (gray line) mice. G) Quantification of dsRed MFI in Ly6C⁺ cells from naive or immune skin 72 hrs after challenge. Data shown are combined from two experiments (n = 8 mice per group). H) Number of parasites per infected cell counted from 50 cells per cytospin slide. Data shown are combined from two experiments (n = 5–6 mice per group). P < 0.05 = *; P < 0.01 = **; P < 0.001 = ***.</p

Engagement of NKG2D on Bystander Memory CD8 T Cells Promotes Increased Immunopathology following <i>Leishmania major</i> Infection

<div><p>One of the hallmarks of adaptive immunity is the development of a long-term pathogen specific memory response. While persistent memory T cells certainly impact the immune response during a secondary challenge, their role in unrelated infections is less clear. To address this issue, we utilized lymphocytic choriomeningitis virus (LCMV) and <i>Listeria monocytogenes</i> immune mice to investigate whether bystander memory T cells influence <i>Leishmania major</i> infection. Despite similar parasite burdens, LCMV and <i>Listeria</i> immune mice exhibited a significant increase in leishmanial lesion size compared to mice infected with <i>L. major</i> alone. This increased lesion size was due to a severe inflammatory response, consisting not only of monocytes and neutrophils, but also significantly more CD8 T cells. Many of the CD8 T cells were LCMV specific and expressed gzmB and NKG2D, but unexpectedly expressed very little IFN-γ. Moreover, if CD8 T cells were depleted in LCMV immune mice prior to challenge with <i>L. major</i>, the increase in lesion size was lost. Strikingly, treating with NKG2D blocking antibodies abrogated the increased immunopathology observed in LCMV immune mice, showing that NKG2D engagement on LCMV specific memory CD8 T cells was required for the observed phenotype. These results indicate that bystander memory CD8 T cells can participate in an unrelated immune response and induce immunopathology through an NKG2D dependent mechanism without providing increased protection.</p></div

LCMV memory T cells migrate to leishmanial lesions and upregulate gzmB expression.

<p>B6 mice received CD45.1+ P14 cells (P14 chimeras) and were infected with LCMV for 5 days (effector time point) or 30 days (memory time point). At the indicated time post LCMV infection, splenocytes were harvested from naïve P14 mice or P14 chimeras and the numbers of P14 CD8 T cells were quantified. Equal numbers (5×10⁵) of P14 CD8 T cells were transferred into congenically marked B6 mice that had been infected with <i>L. major</i> for 2 weeks (A). After 36 hours, spleens (B), infected ears (C), and contralateral uninfected ears (D) were harvested from the recipients and P14 frequency was analyzed by flow cytometry (E). P14 CD8 T cells present in the spleen (F) or infected ear (G) were incubated with BFA alone for 5 hours and analyzed for production of gzmB and IFN-γ. The relative mean fluorescence intensity for each was calculated (H). Data are representative of two independent experiments (n = 3–5 per group).</p

CD8 T cells infiltrating the leishmanial lesions express gzmB but low levels of IFN-γ.

<p>B6 mice were infected with LCMV Armstrong or left uninfected. After 30 days, mice were infected with <i>L. major</i>. After 4 weeks, infected ears (A) and peripheral blood (C) were taken for analysis by flow cytometry. Cells from the infected ears were incubated with BFA, monensin and CD107a antibody for 5 hours and then stained for additional cell surface markers and intracellular proteins. Representative dot plots (A) and total cell numbers (B) are shown. Peripheral blood was taken and white blood cells were isolated and stained for cell surface markers and intracellular proteins. Representative dot plots are shown (C). Whole ear tissue was homogenized and supernatants were analyzed for gzmB and IFN-γ by ELISA (D). Flow data are representative of five independent experiments (n = 4–5 mice per group). Ear supernatant data are representative of two independent experiments (n = 4 mice per group). Percentages are shown as mean ± SEM.</p

LCMV specific CD8 T cells are present in the leishmanial lesion and express gzmB but low levels of IFN-γ.

<p>B6 mice were infected with LCMV Armstrong or left uninfected. After 30 days, mice were infected with <i>L. major</i>. After 4 weeks, <i>L. major</i> infected skin was taken and incubated with a pool of 20 LCMV derived peptides and BFA for 6 hours before staining for surface and intracellular proteins. Representative plots (A) and pooled data (B) are shown. Data are representative to 2 independent experiments (n = 5 per group). P14 T cells were transferred into B6 mice and the next day a group was infected with LCMV. After 30 days, GFP+ OTI T cells were transferred into all groups and mice were infected with <i>L. major</i>-OVA (C). After 4 weeks, <i>L. major</i> infected skin was harvested and incubated with BFA for 5 hours. The samples were analyzed for the presence of P14 cells or OTI cells (D) and numbers of each were calculated (E). The OTI or P14 cells from the same infected ear of an LCMV immune <i>L. major</i> infected mouse were also incubated with BFA, monensin, and CD107a antibody then stained for additional surface and intracellular proteins (F). Data are representative of four independent experiments (n = 4–5 per group). Percentages are shown as mean ± SEM.</p

Exacerbated immunopathology is lost following depletion of CD8 T cells in LCMV immune mice prior to <i>L. major</i> infection.

<p>B6 mice were infected with LCMV or left uninfected for 45 days. Mice in each group were then treated with CD8-depleting antibody every 3 days for 15 days or left untreated. Blood was taken from animals in each group prior to <i>L. major</i> infection to assess CD8 depletion efficiency (A) and at 4 weeks post <i>L. major</i> infection to monitor reconstitution of the CD8 T cell compartment (B). All mice were with metacyclic <i>L. major</i> and ear thickness was measured weekly (C). Pictures were taken 4 weeks post <i>L. major</i> infection (D). Infected skin was taken at 4 weeks post infection and parasite burden was assessed using a limiting dilution assay (F). Infected skin was analyzed 4 weeks after <i>L. major</i> infection and the number of CD8 T cells present in each group was calculated (E). Data are representative of two independent experiments (n = 10 per group).</p