Rationale: Changes in peripheral blood cell populations have been observed but not detailed at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: To provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from IPF patients and controls were profiled using 10x Chromium 5 prime single-cell RNA sequencing (scRNA-seq). Flow cytometry was used for validation. Protein concentrations of Regulatory T-cells (Tregs) and Monocytes chemoattractants were measured in plasma and lung homogenates from patients and controls. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched controls yielded 149,564 cells that segregated into 23 subpopulations, corresponding to all expected peripheral blood cell populations. Classical monocytes were increased in progressive and stable IPF compared to controls (32.1%, 25.2%, 17.9%, respectively, p<0.05). Total lymphocytes were decreased in IPF vs controls, and in progressive vs stable IPF (52.6% vs 62.6%, p=0.035). Tregs were increased in progressive IPF (1.8% vs 1.1%, p=0.007), and were associated with decreased survival (P=0.009 in Kaplan-Meier analysis). Flow cytometry analysis confirmed this finding in an independent cohort of IPF patients. Tregs were also increased in two cohorts of lung scRNA-seq. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF, reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

NK is a scientific founder at Thyron, served as a consultant to Biogen Idec, Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Theravance, LifeMax, Three Lake Partners, Optikira, Astra Zeneca, RohBar, Veracyte, Augmanity, CSL Behring, Galapagos, Fibrogen, and Thyron over the last 3 years, reports Equity in Pliant and Thyron, and grants from Veracyte, Boehringer Ingelheim, BMS and non-financial support from MiRagen and Astra Zeneca. AU reports receiving research funding from Boehringer Ingelheim, and personal consulting fees or honoraria from Boehringer Ingelheim, Kamada, RemedyCell, Augmanity Nano, Splisense, Veracyte, and 1E Therapeutics in the last 36 months. JCS reports receiving honoraria and travel support from Boehringer Ingelheim. All other authors report no conflict of interest.

This study was funded by NIH R01HL127349, R001HL141852, U01HL145567, R21HL161723, P01HL11450, and an unrestricted gift from Three Lake Partners (NK), Pulmonary Fibrosis Foundation Scholars Award (AU), 1K08HL151970-01 (CR), R01HL153604 and R03HL154275 (JLG), Ubben Center for Pulmonary Fibrosis Research Fund (JHM), R01HL152677 and R01HL16163984 (ELH).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The study was performed on deidentified, cryopreserved PBMC samples of IPF patients and matched controls, obtained with informed consent on protocol approved by the Institutional Review Board at the Yale School of Medicine (#1307012431).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Results can be further explored through our user-friendly data-mining website (http://ildimmunecellatlas.com), and raw sequencing data will be available in GEO upon peer-reviewed publication.

http://ildimmunecellatlas.com