Sequence homology between HLA-bound cytomegalovirus and human peptides: A potential trigger for alloreactivity.

Human cytomegalovirus (hCMV) reactivation may often coincide with the development of graft-versus-host-disease (GVHD) in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n = 50; matched related donor (MRD), n = 27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (≥6) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC5

HCMV cross-reactivity potential of 77 SCT DRP from whole exome sequencing data using sliding window match analysis.

<p>HCMV cross-reactivity potential of 77 SCT DRP from whole exome sequencing data using sliding window match analysis.</p

GVHD tissue-specific immunogenic CMV peptide matches, CMV reactivation before GVHD patients.

<p>Patient-specific process of cross comparison along with the source genes, IC50 values to indicate inverse binding affinity and GVHD organ specific tissue involvement. *Note: Twelve patients with CMV reactivation/infection before GVHD onset exhibited previously identified immunogenic CMV peptide matches with gene expression specific to the tissues affected by GVHD (above); The filter of immunogenicity provides a connection to T cell reactivity shown <i>in vitro</i> to the listed CMV genes in a separate patient population [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178763#pone.0178763.ref036" target="_blank">36</a>]; Patient 79 with muscle/fascia GVHD showed no muscle-specific previously <i>known immunogenic</i> CMV peptide matches but still had three relevant CMV peptide matches expressed in the skeletal muscle (not shown); Tissues in parentheses were also affected by GVHD but without immunogenic matches/expression by patient; Patients 67 and 71 experienced <i>de novo</i> CMV infection; All 13 patients exhibited multiple CMV peptide matches with unknown immunogenicity.</p

Bioinformatic pipeline of the alloreactive peptide library comparison with the CMV proteome.

<p>A determination of the potential for Pro-GVHD DRP cross-reactivity upon CMV reactivation correlated with GVHD-tissue specific distribution of peptides & immunogenicity.</p

CMV- Human match analysis per DRP (IC50 <500 nM) by donor type according to continuous amino acid sequence homology (one standard deviation from the mean value).

<p>CMV- Human match analysis per DRP (IC50 <500 nM) by donor type according to continuous amino acid sequence homology (one standard deviation from the mean value).</p

Human-CMV short sequence homology in GVHD tissue specific peptide and gene distribution from GTEx analysis (n = 18).

<p>GVHD incidence denotes the specific organs affected in each patient; Peptides, lists the number of unique peptide-HLA complexes matched between human and CMV peptide library; the column, <i>Genes</i> lists the source genes for the aforementioned peptides; <i>GVHD tissue specific peptides</i> lists the number of peptides which bind HLA with an IC50 <500nM, and are expressed in tissues affected by GVHD; <i>GVHD tissue gene expression</i> denotes the number of genes expressed at an RPKM >10 corresponding to the GVHD tissue specific peptides. Note: *- All patients with an asterisk following their numeric representation experienced CMV reactivation prior to GVHD (except Patients 67 and 71, <i>de novo</i> CMV infected) and patients without an asterisk experienced GVHD prior to CMV reactivation. **- human peptides may have overlapping areas of homology yielding a higher number of matches. Immunogenic CMV genes in this context refer to the genes associated with HCMV ORF-specific CD8+ T memory cell responses measured in frequency among CMV seropositive patients previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178763#pone.0178763.ref036" target="_blank">36</a>].</p

Matrix depicting T cell clonal cross-reactivity between CMVp-HLA and mHA-HLA.

<p>*-indicates response of the alloreactive T cell clone to a viral pathogen peptide,bound to the same HLA as the mHA and vice versa. For example, TC₁ recognizes, mHA₁ HLA + CMVp₂HLA, TC_1CMV recognizes, mHA₁ HLA + CMVp₁ HLA, and so on.</p

Matrix depicting the T cell clonal interaction with mHA-HLA.

<p>Matrix depicting the T cell clonal interaction with mHA-HLA.</p

CMV+GVHD cross reactivity potential.

<p>Patient-specific peak CMV peptide matches intersecting peak alloreactive peptides (IC50<500 nM) as a cross-reactivity potential stratified by donor type contained within each DRP alloreactive peptide library.</p

Sequence homology between HLA-bound cytomegalovirus and human peptides: A potential trigger for alloreactivity

<div><p>Human cytomegalovirus (hCMV) reactivation may often coincide with the development of graft-versus-host-disease (GVHD) in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n = 50; matched related donor (MRD), n = 27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (≥6) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50<500nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, an average of 29,658 matched the MRD DRP alloreactive peptides and 52,910 matched MUD DRP peptides (p<0.001). <i>In silico</i> analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner. hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset. Analysis of patients with GVHD identified potential cross reactive peptide expression within affected organs. We propose that hCMV peptide sequence homology with human alloreactive peptides may contribute to the pathophysiology of GVHD.</p></div