Background T cell mediated immunity is reported to play a pathogenic role in cardiac allograft vasculopathy (CAV) in heart transplant (HTx) patients. However, peripheral blood CD8+ T cells have not been previously characterized in CAV. This study aimed to identify potentially pathogenic circulating CD8+ T cell populations in high grade CAV patients using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq).

Methods Peripheral blood mononuclear cells (PBMC) collected from International Society for Heart and Lung Transplant (ISHLT) grade 2 or 3 CAV (high grade CAV; n=6) and normal HTx (n=12) patients were analyzed using CITE-seq and VDJ-seq. Key findings were validated by flow cytometry in an independent patient cohort of age-matched CAV (n=11) patients, normal HTx (n=12) patients and healthy donor subjects (n=11).

Results Among the seven peripheral CD8+ T cell clusters, high grade CAV patients demonstrated a significantly higher proportion of the CD38+HLA-DR+ CD8+effector memory T (Tem) cell cluster compared to normal HTx patients (median 6.2% vs 2.9%, p=0.01). CD38+HLA-DR+CD8+ Tem cells showed clonal expansion, activated interferon-γ (IFNG) signaling and enhanced cytotoxicity with granzyme B (GZMB) and perforin (PRF) overexpression. Significantly higher proportion of the proinflammatory and cytotoxic CD38+HLA-DR+ CD8+Tem cell cluster in high grade CAV compared to normal HTx patients was validated by flow cytometry. There was significantly increased clonal expansion of peripheral CD8+ T cells in high grade CAV compared to normal HTx patients (median Shannon index = 4.4 vs 6.1, p=0.03). CITE-seq identified LAIR2 as a potential biomarker for identifying high grade CAV patients as increased expression was found in CD38+HLA-DR+CD8+ Tem cells. Plasma LAIR2 was significantly elevated in the high grade CAV (n=20) compared to normal HTx patients (n=20; 16.0 pg/mL vs 70.3 pg/mL, p=0.02).

Conclusions We discovered and validated circulating CD38+HLA-DR+CD8+ Tem cells to be significantly increased in high grade CAV compared to normal HTx patients. The proinflammatory and cytotoxic phenotype of this CD8+ T cell cluster suggest its potential pathogenic role in human CAV.

What is new?

This is the first study to identify clonal expansion of circulating CD38+HLA-DR+effector memory CD8+ T cells in human cardiac allograft vasculopathy.

CD38+HLA-DR+ effector memory CD8+ T cells possess both proinflammatory and cytotoxic characteristics, suggesting their potential pathogenic role in human cardiac allograft vasculopathy.

LAIR2 is a potential signature gene of CD38+HLA-DR+effector memory CD8+ T cells.

What are the clinical implications?

Circulating CD38+HLA-DR+ effector memory CD8+ T cells and plasma LAIR2 protein are potential early biomarkers of cardiac allograft vasculopathy.

Evaluation of CD38+HLA-DR+ effector memory CD8+ T cells in longitudinal studies may reveal how this T cell cluster contributes to the development of human cardiac allograft vasculopathy.

Inhibiting the expansion of CD38+HLA-DR+effector memory CD8+ T cells and/or the LAIR2 pathway may become important therapeutic targets for prevention and treatment of human cardiac allograft vasculopathy.

The authors have declared no competing interest.

This study is supported by the National Center for Advancing Translational Sciences, NIH KL2TR001444 (PJK) and R35 HL145241(KL).

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:

This study was approved by the University of California, San Diego Health (UC San Diego Health) Office of IRB administration (No. 160808), and all participants provided written informed consent.

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