Antigen discovery unveils resident memory and migratory cell roles in antifungal resistance

Abstract

Priming at the site of natural infection typically elicits a protective T cell response against subsequent pathogen encounter. Here, we report the identification of a novel fungal antigen that we harnessed for mucosal vaccination and tetramer generation to test whether we can elicit protective, antigen-specific tissue-resident memory (Trm) CD4⁺ T cells in the lung parenchyma. In contrast to expectations, CD69⁺, CXCR3⁺, CD103^- Trm cells failed to protect against a lethal pulmonary fungal infection. Surprisingly, systemic vaccination induced a population of tetramer⁺ CD4⁺ T cells enriched within the pulmonary vasculature, and expressing CXCR3 and CX3CR1, that migrated to the lung tissue upon challenge and efficiently protected mice against infection. Mucosal vaccine priming of Trm may not reliably protect against mucosal pathogens.

Fig. 1:. Bl-Eng2 antigenicity, T cell epitope identification and tetramer validation.

(A) Lung CFU in SC vaccinated and control mice day 11 post-infection. Data are representative of 3 independent experiments (n=5–10 mice/ group). *p < 0.05 vs. IFA control mice. Number indicates n-fold difference vs. IFA control group. (B) Synthetic peptides (9 amino acid [aa] core plus 2 flanking aa on either side) used for ex vivo stimulation of Bl-Eng2 primed T cells harvested from splenocytes of SC vaccinated mice. (C) IFN-γ production in cell culture supernatants measured by ELISA. *p < 0.05 vs. all other groups. (D) At day 4 post-infection, CD4⁺ (top row) and CD8⁺ (bottom row) T cells from the lung were labeled with tetramer. Numbers indicate the percentage of tetramer⁺ cells of parent gate.

Fig. 2:. Induction and protection by Bl-Eng2-specific T cells after vaccination at the respiratory mucosa or skin.

(A) Mice received Bl-Eng2 in GCP either SC or IN three times, two weeks apart. CD4⁺ tetramer⁺ T cells were enumerated in the lung before (B) and after (C) challenge (day 4 post-infection). Anti-CD45 mAb was injected i.v. 5 minutes before euthanizing mice to stain lung vascular cells; plots in the second row show total tetramer⁺ cells distributed in the parenchyma and lung vasculature. Numbers in plots indicate either mean±SEM or percent of parent gate. Contour plots show combined data from five independent experiments (n=25–30 mice/group). (D) Cells from the spleen of unchallenged mice were pulled down with Bl-Eng2 tetramer and enumerated. Numbers in plots indicate mean±SEM (n=4 mice/group). *p < 0.05 for SC vs. IN vaccine groups. (E) Resistance to infection as determined by lung CFU. Mouse serum albumin (MSA) on GCPs served as a control (n=10 mice/group). CFU graph shows geometric mean with standard deviation. *p < 0.05 vs. control mice.

Fig. 3:. Polarization of antigen specific cells in the lung and skin.

Mice received Bl-Eng2 in GCP either SC or IN three times, two weeks apart. (A) Two weeks following the last boost, mice were challenged and lungs were harvested at day 4 post infection and stained for transcription factors T-bet, ROR-γt, and GATA3. Histograms show expression of each transcription factor within the designated tetramer⁺ cell populations. Adjacent bar graphs show corresponding MFI values for individual mice. (B) Dot plots display cytokine producing or foxp3⁺ lung tetramer⁺ cells at day 4 post infection; cells in plots for IFN-γ, IL-17, and IL-5 & IL-13 were stimulated ex vivo with Bl-Eng2 peptide for 5 hrs; foxp3 staining was performed before stimulation on a separate lung aliquot. Numbers in plot represent percent of parent gate. (C) Absolute numbers of cytokine producing T helper and regulatory T (foxp3⁺) cells. Data are from one representative experiment of two performed (n=5 mice/group). *p < 0.05 for SC vs. IN vaccine groups. (D) Lung CFU of SC vaccinated mice treated with cytokine neutralizing antibody. Mice vaccinated SC with Bl-Eng2 in GCP were given 100 μg i.v. of rat IgG or neutralizing cytokine antibody on the day of challenge and every other day thereafter until harvest at day 7 post-infection. Data shown are a combination of two independent experiments (n=10 mice/group). *p < 0.05 for rat IgG vs. indicated treatment groups.

Fig. 4:. Phenotypic analysis of Bl-Eng 2 primed T cells.

Mice were vaccinated with Bl-Eng2 in GCP either SC or IN. Two weeks after the last boost, lungs were harvested (A) prior to challenge and (B) at day 4 post-infection. Total lung tetramer⁺ T cells were separated into parenchymal and vasculature cells based on i.v. anti-CD45 mAb staining and were analyzed for the phenotypic markers CD69, CXCR3, CX3CR1, CD103 and KLRG1. Numbers in the dot plots and graphs below indicate percent of the parent gate, either tetramer⁺CD45 i.v.⁻ (parenchyma) or tetramer⁺CD45 i.v.⁺ (vasculature). Data are a combination of 3 independent experiments (n=15 mice/group). *p < 0.05 for SC vs. IN vaccine groups. (C+D) Heatmap tSNE plots for lung cells before (C) and after (D) challenge from one representative experiment of three performed (n=5 mice/group). In FlowJo, total tetramer⁺ cells from individual mice were concatenated into a single FSC file for SC and IN groups before and after challenge. tSNE parameters (iterations: 1,000; perplexity: 30) were computed for this concatenated file including the markers CD45 i.v., CD69, CXCR3, CD103, CX3CR1, and KLRG1. Heatmap tSNEs show individual vaccine and challenge groups plotted based on computed tSNE parameters 1 & 2; these parameters allow visualization of the six listed markers in two dimensional space by clustering cells with similar expression profiles together. The scale bar reflects the relative expression of the labeled marker; red and yellow indicate high expression compared to blue for low expression.

Fig. 5:. Migration of Bl-Eng2-specific T cells into the lungs after challenge is delayed and reduced in IN vaccinated mice.

(A) Mice were vaccinated with Bl-Eng2 in GCP either SC or IN three times, two weeks apart. Two weeks after the last boost, mice were challenged with B. dermatitidis yeast. 24 h before the harvest, and at serial time points post-infection, an initial anti-CD45 mAb (BV412) was given to mice by i.v. injection. On the day of harvest five minutes before euthanizing mice, a second anti-CD45 mAb conjugated to a different fluorochrome (Alexa 488) was also given i.v. Tetramer⁺ cells that stained for the second and initial mAb were termed vascular; those that stained for the initial but not the second mAb were termed migratory; and those that did not stain for either mAb were termed tissue. (B) Dot plots show the frequencies of tetramer⁺ cells that stained for the second vs. initial anti-CD45 mAb. The line graph shows total number of tetramer⁺ T cells over time. (C) Graphs show frequencies of tetramer⁺ cells found in corresponding compartments on serial days post infection. (D) Lung CFU at serial days post infection. (E) Numbers of total (left), vascular (middle) and migratory (right) tetramer⁺ cells positive for CXCR3⁺ or CX3CR1⁺. Data are from one representative experiment of two independent ones performed (n=5 mice/group). *p < 0.05 vs. corresponding IN vaccine group.

Fig. 6:. Redundant roles of CXCR3 and CX3CR1 in SC vaccinated mice.

C57BL6 (WT), CXCR3^−/−, and CX3CR1^−/− mice were SC vaccinated with Bl-Eng2 in GCP or MSA in GCP three times, two weeks apart. Two weeks after the last boost mice were challenged and lungs were harvested at day 4 post infection (A) Dots plots show tetramer⁺ T cells, numbers on plot indicate percent of parent gate. Corresponding bar graphs display absolute number of tetramer⁺ cells for WT and KO strains. Anti-CD45 mAb was injected i.v. 5 minutes before euthanizing mice to stain lung vascular cells; plots in the second row show total tetramer⁺ cells distributed in the parenchyma and lung vasculature. (B) Cytokine production by tetramer⁺ cells after stimulation ex vivo with Bl-Eng2 peptide. Dot plots show frequency among total tetramer⁺ cells and bar graphs below display absolute numbers of cytokine-producing tetramer⁺ cells. (C) Lung CFU at day 4 post-infection (n=5 mice/group). *p < 0.05 for naïve vs. vaccinated groups.

Fig. 7:. Proliferation of tetramer⁺ T cells in IN and SC vaccinated mice after challenge.

Mice were vaccinated with Bl-Eng2 in GCPs either SC or IN. Two weeks after the last boost, mice were harvested prior to challenge or at day 4 post-infection. 2 mg of BrdU was given by i.p. injection to all mice for three consecutive days before harvest. (A) Dot plots and graphs show the frequencies of tetramer⁺ (A) and cytokine⁺ (B) cells that are BrdU⁺ prior to challenge. (C+D) Dot plots and graphs illustrate analogous data at day 4 post-infection. Data are representative of two independent experiments (n=5 mice/group). *p < 0.05 for IN vs. SC vaccine groups.

Fig. 8:. Migratory T cells are necessary and sufficient to mediate vaccine protection.

Mice were vaccinated SC with CFA+Bl-Eng2 (A) Prior to challenge, CD4⁺ T cells were depleted in the lung (by i.t. delivery of 20 μg anti-CD4 mAb) or systemically (by i.v. delivery of 100 μg anti-CD4 mAb). Lung CFU were determined at day 6 post-infection. Data shown are a combination of three independent experiments (n=25–30 mice/group). *p < 0.05 vs. indicated groups. (B) Mice were vaccinated SC with Bl-Eng2 in GCP or CFA+Bl-Eng2. Two days before challenge and daily thereafter, mice were given 25 μg of FTY720 inhibitor i.p.. Lung CFU were determined at day 6 post-infection. Data are a combination of two independent experiments (n=20 mice/group). *p < 0.05 vs. non-treated control group. (C) Schematic of adoptive transfer of CD4⁺ T cells from the lung (Trm) or spleen (migratory) of SC vaccinated wild-type mice into TCR-α knockout mice. (D) C57BL6 wild type mice were vaccinated SC twice with CFA+Bl-Eng2. Two weeks after the boost, migratory CD4⁺ T cells were isolated by positive selection using anti-CD4 magnetic beads and lung resident T cells by purified by Percoll gradient. Cells were adoptively transferred into naïve TCR-α knockout mice. As a negative control, CD4⁺ T cells from the spleen of naïve wild type mice were transferred. At day 11 post-infection, lung CFU were determined. Data are a combination of two independent experiments (n=8–20 mice/group). *p < 0.05 vs. no transfer control group.