Human PD-1 agonist treatment alleviates neutrophilic asthma by reprogramming T cells

Abstract

Background: Neutrophilic asthma is associated with disease severity and corticosteroid insensitivity. Novel therapies are required to manage this life-threatening asthma phenotype. Programmed cell death protein-1 (PD-1) is a key homeostatic modulator of the immune response for T-cell effector functions.

Objective: We sought to investigate the role of PD-1 in the regulation of acute neutrophilic inflammation in a murine model of airway hyperreactivity (AHR).

Methods: House dust mite was used to induce and compare neutrophilic AHR in wild-type and PD-1 knockout mice. Then, the therapeutic potential of a human PD-1 agonist was tested in a humanized mouse model in which the PD-1 extracellular domain is entirely humanized. Single-cell RNA sequencing and flow cytometry were mainly used to investigate molecular and cellular mechanisms.

Results: PD-1 was highly induced on pulmonary T cells in our inflammatory model. PD-1 deficiency was associated with an increased neutrophilic AHR and high recruitment of inflammatory cells to the lungs. Consistently, PD-1 agonist treatment dampened AHR, decreased neutrophil recruitment, and modulated cytokine production in a humanized PD-1 mouse model. Mechanistically, we demonstrated at the transcriptional and protein levels that the inhibitory effect of PD-1 agonist is associated with the reprogramming of pulmonary effector T cells that showed decreased number and activation.

Conclusions: PD-1 agonist treatment is efficient in dampening neutrophilic AHR and lung inflammation in a preclinical humanized mouse model.

Keywords: Neutrophilic asthma; PD-1 agonist; T cells; humanized mice; lung inflammation.

Figure 1:. Neutrophilic inflammation is associated with PD-1 induction in lung immune cells

(A) BALB/cByJ mice (WT) mice were sensitized via subcutaneous (s.c.) tail base injection of HDM (200 μg) mixed in CFA (1:1 v/v). After 13 days, mice were intranasally (i.n.) challenged with HDM (100 μg). On day 14, lung resistance was measured before euthanasia, BAL collection and lung processing. (B) Variations in lung resistance measured in restrained ventilated mice. (C) Total number of CD45⁺ cells, (D) CD3⁺ T lymphocytes and (E) B lymphocytes. (F) Representative flow cytometry plots of neutrophils and (G) the quantification of lung neutrophils and eosinophils presented as the percentage among CD45⁺ cells. (H) Composition of PD-1⁺ cells in the lung suspensions. (I) Representative flow cytometry plots of PD-1 induction on CD3⁺ CD45⁺ cells and the corresponding quantification presented as the percentage of PD-1⁺ T cells in the lungs. (J) Comparison between the percentage of CD4⁺ and CD8⁺ T cells expressing PD-1 in the lungs of HDM-treated mice within PD-1⁺ T cells. (K) Composition of PD-1⁺ CD4⁺ T cells according to FOXP3 and CD62L markers. Data are representative of at least 2 independent experiments and are presented as means ± SEM (two-tailed Student’s t-test; n=5).

Figure 2:. PD-1 deficiency increases neutrophilic AHR and lung inflammation

WT and PD-1 KO mice were sensitized via s.c. tail base injection of HDM (200 μg) mixed in CFA (1:1 v/v). After 13 days, mice were i.n. challenged with HDM (100 μg). Control mice received PBS only. On day 14, lung functions were measured before euthanasia and BAL collection. (A) Lung resistance and (B) dynamic compliance measured in tracheostomized mechanically ventilated mice exposed to increasing concentrations of methacholine. (C) Absolute count of CD45⁺ cells, (D) neutrophils, (E) T cells, (F) B cells, (G) eosinophils, and (H) alveolar macrophages quantified in the BAL using the count precision beads in flow cytometry analysis. (I) Levels of TNF-α, (J) IL-17A, (K) IFN-γ, (L) IL-6, (M) MCP-1, (N) CXCL-10, (O) G-CSF, and (P) IL-4 quantified in the BAL. (Q) Hematoxylin and eosin (H&E) staining of lung sections (scale bar = 20 μm). Data are representative of at least 2 independent experiments and are presented as means ± SEM (two-tailed Student’s t-test; n=5).

Figure 3:. PD-1 axis is comparably induced in humanized PD-1 mice and WT mice following HDM challenge

(A) Representation of extracellular, transmembrane, and intracellular domains of PD-1 in hPD-1 mouse. (B)hPD-1 mice were sensitized via s.c. tail base injection of HDM (200 μg) mixed in CFA (1:1 v/v). After 13 days, mice were i.n. challenged with HDM (100 μg). On day 14, lung and spleen processing followed euthanasia. (C) Representative flow cytometry plots of human PD-1 induction on CD45⁺ cells from hPD-1 mice. (D) PD-1⁺ CD45⁺ cell percentage in the lungs of WT and hPD-1 mice following HDM challenge. (E) Representative flow cytometry plots of human PD-1 induction on CD3⁺ cells and (F) the corresponding quantification presented as the percentage of PD-1⁺ T cells in the lungs of hPD-1 mice. (G) Percentage of PD-1⁺ T cells in the spleens of hPD-1 mice. (H) Representative flow cytometry plots of PD-L1 and PD-L2 induction on CD45⁺ cells and (I) the corresponding quantification presented as the percentage of PD-L1⁺ PD-L2⁻ cells and (J) PD-L1⁺ PD-L2⁺ cells in the lungs of hPD-1 mice. Data are representative of at least 2 independent experiments and are presented as means ± SEM (two-tailed Student’s t-test; n=4).

Figure 4:. PD-1 agonist downregulates AHR and controls neutrophilic lung inflammation

(A) Humanized PD-1 mice were sensitized via s.c. tail base injection of HDM (200 μg) mixed in CFA (1:1 v/v). After 13 days, mice were i.n. challenged with HDM (100 μg) and received 500 μg of PD-1 agonist via the intraperitoneal route or the corresponding isotype, while the dose was reduced to 250 μg via the intravenous route on day 14. Naïve mice were not sensitized, challenged, or treated. On day 15, lung function was measured. BAL and lungs were collected after euthanasia. (B) Lung resistance measured in tracheostomized ventilated mice. (C) Absolute count of CD45⁺ cells, (D) neutrophils, (E) T cells, (F) alveolar macrophages, and (G) eosinophils quantified in the BAL. (H) Levels of IFN-γ, (I) IL-6, (J) TNF-α, (K) IL-17A, (L) MCP-1,(M) CXCL-10, and (N) G-CSF quantified in the BAL. (O) Representative images of H&E and AB-PAS-stained histology sections (scale bar = 50 μm) with the corresponding quantifications of (P) recruited immune cells and (Q) airway epithelial thickness. Data are representative of at least 2 independent experiments and are presented as means ± SEM (two-tailed Student’s t-test; n=4–5). Mouse image provided with permission from Servier Medical Art.

Figure 5:. scRNAseq analysis reveals the impact of PD-1 agonist treatment on the lung immune landscape

Single-cell RNA-seq was performed on sorted CD45⁺ cell suspensions pooled from 3 lungs per group (isotype- versus PD-1 agonist- treated mice). Samples were analyzed using Partek Flow genomic analysis software. (A) tSNE plot (2D graph) revealing the effect of PD-1 agonist on main immune cell populations annotated using SingleR. (B) Bubble heatmap showing the expression of relevant genes implicated in cytokine pathways. (C) tSNE plot (3D graph) of CD45⁺ cells colored by Pdcd1 expression. The intensity of the blue color, as well as the dot size, are dependent on the level of Pdcd1 expression. Green circles represent a cluster of Pdcd1⁺ high cells. (D) Dot plot showing the intensity of Pdcd1 expression in total CD45⁺ lung cells.

Figure 6:. PD-1 agonist treatment downregulates the effector phenotype of lung T cells

Following the induction of neutrophilic lung inflammation, humanized PD-1 mice received two injections of the PD-1 agonist or the corresponding isotype, as previously described. On day 15, lungs were washed and collected after euthanasia. (A) Absolute count of CD45⁺ cells, (B) neutrophils, (C) CD3⁺ T cells. (D) Volcano plot showing significantly affected pathways in lung immune cells from PD-1 agonist-treated mice as compared to isotype-treated mice (MSigDB_Hallmark_2020 gene set). (E) tSNE plot representation based on the expression of Cd44 and Sell genes in lung immune cells. The size of dots is dependent on the level of Cd3e expression. (F) Expression of genes related to the T effector phenotype expressed as fold change (PD-1 agonist vs isotype group). (G) Representative flow cytometry plots showing the gating strategy for the identification of CD4⁺ T cells subsets according to CD44 and CD62L expression. (H) Percentages of effector memory CD4⁺ T cells, (I) central memory CD4⁺ T cells, (J) naïve CD4⁺ T cells, and (K) Teff:Treg ratio in the lungs. Data are representative of at least 2 independent experiments and are presented as means ± SEM (two-tailed Student’s t-test; n=4).