Tolerance Associated Gene Expression following Allogeneic Hematopoietic Cell Transplantation

<div><p>Biologic markers of immune tolerance may facilitate tailoring of immune suppression duration after allogeneic hematopoietic cell transplantation (HCT). In a cross-sectional study, peripheral blood samples were obtained from tolerant (n = 15, median 38.5 months post-HCT) and non-tolerant (n = 17, median 39.5 post-HCT) HCT recipients and healthy control subjects (n = 10) for analysis of immune cell subsets and differential gene expression. There were no significant differences in immune subsets across groups. We identified 281 probe sets unique to the tolerant (TOL) group and 122 for non-tolerant (non-TOL). These were enriched for process networks including NK cell cytotoxicity, antigen presentation, lymphocyte proliferation, and cell cycle and apoptosis. Differential gene expression was enriched for CD56, CD66, and CD14 human lineage-specific gene expression. Differential expression of 20 probe sets between groups was sufficient to develop a classifier with > 90% accuracy, correctly classifying 14/15 TOL cases and 15/17 non-TOL cases. These data suggest that differential gene expression can be utilized to accurately classify tolerant patients following HCT. Prospective investigation of immune tolerance biologic markers is warranted.</p></div

Receiver operating characteristic (ROC) plot for diagnostic accuracy of gene classifier.

<p>*ROC plot for diagnostic accuracy presents true positive rate vs. false positive rate (or sensitivity x 1-specificity) for gene expression-based phenotypic classifier of TOL and non-TOL patient groups. AUC 0.97 (95% CI 0.82–0.97).</p

Direction and magnitude of change in TOL group vs. non-TOL and control: Genes with decreased expression in TOL group.

<p>*TLR4—toll-like receptor 4; TLR8—toll-like receptor 8; PELI2—pellino homolog 2; IRAK3—interleukin-1 receptor-associated kinase 3; LILRA2—leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 2; LILRA5—leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 5; PDLIM5—PDZ and LIM domain 5; ARRB1—arrestin, beta 1; PAK1—p21 protein (Cdc42/Rac)-activated kinase 1; SOCS2—suppressor of cytokine signaling 2; RHOA—ras homolog gene family, member A; IL-13RA—interleukin 13 receptor, alpha 1; TNFSF13B—tumor necrosis factor (ligand) superfamily, member 13b (BAFF); TNFSF12—tumor necrosis factor (ligand) superfamily, member 12 (APRIL); GSN—gelsolin; SMAD1—SMAD family member 1; VNN1—vanin 1; PPT1—palmitoyl-protein thioesterase 1; SOD2—superoxide dismutase 2, mitochondrial; DAPK1—death-associated protein kinase 1; EVI5—ecotropic viral integration site 5; CCNY—cyclin Y.</p

Comparison of patient, transplantation, and GVHD variables across tolerant and non-tolerant groups.

<p>*categorical data compared with Fisher’s exact test or Chi-square, continuous data utilized wilcoxon rank sum test</p><p>* AA—aplastic anemia; ALL—acute lymphoblastic leukemia; AML—acute myelogenous leukemia; CML—chronic myelogenous leukemia; FL—follicular lymphoma; HD—Hodgkin lymphoma; IMF—idiopathic myelofibrosis; MCL—mantle cell lymphoma; MDS—myelodysplastic syndrome; MM—multiple myeloma; MPD—myeloproliferative neoplasm; PBSC—peripheral blood stem cells; BM—bone marrow harvested stem cells; MMUD—mismatched unrelated donor; MRD—matched sibling donor; MUD—matched unrelated donor; HLA—human leukocyte antigen; CMV—cytomegalovirus; neg—negative; pos—positive; Bu—busulfan; Cy—cyclophosphamide; Flu—fludarabine; ATG—anti-thymocyte globulin; R—rituximab; BCNU—carmustine; VP16—etoposide; TBI—total body irradiation; pento—pentostatin; CSA—cyclosporine; TAC—tacrolimus; MMF—mycophenolate mofetil; MTX—methotrexate; aGVHD—acute graft vs. host disease; pred—prednisone; rapa—rapamycin (sirolimus); ECP—extra-corporeal photopheresis; cGVHD—chronic graft vs. host disease</p><p>Comparison of patient, transplantation, and GVHD variables across tolerant and non-tolerant groups.</p

Cell lineage enrichment analysis conducted using Initial 2 group SAM comparison (TOL vs. non-TOL).

<p>*Data are presented for cell-lineage specific gene sets for which significant enrichment was demonstrated. Data for CD4, CD8, and CD19 not presented (p = NS).</p><p>*TOL—tolerant; non-TOL—not tolerant; ES—enrichment score; NES—normalized enrichment score; p value—significance level; FDR—false discovery rate.</p><p>Cell lineage enrichment analysis conducted using Initial 2 group SAM comparison (TOL vs. non-TOL).</p

Comparison of immune cell subsets among TOL and non-TOL patients.

<p>*Numbers indicate proportion of examined PBMC with the identified phenotype.</p><p>*NK—natural killer cell; DC—dendritic cell; IEL—intra-epithelial lymphocyte; Treg—regulatory T cell; NKT—NKT cells; TOL—tolerant patients; non-TOL—non-tolerant patients</p><p>Comparison of immune cell subsets among TOL and non-TOL patients.</p

Direction and magnitude of change in non-TOL group vs. TOL and control: Genes with increased expression in non-TOL group.

<p>*CTSS—cathepsin S; FCER1G—Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide; FCGR1B—Fc fragment of IgG, high affinity Ib, receptor (CD64); CD93—CD93 molecule; CR1—complement component (3b/4b) receptor 1; TLR1—toll-like receptor 1; VSIG4—V-set and immunoglobulin domain containing 4; DUSP6—dual specificity phosphatase 6; MNDA—myeloid cell nuclear differentiation antigen; GAPT—GRB2-binding adaptor protein, transmembrane; FKBP1A—FK506 binding protein 1A, 12kDa; TNFSF13B—tumor necrosis factor (ligand) superfamily, member 13b (BAFF); CDKN2B—cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4); HHEX—hematopoietically expressed homeobox; RHOB—ras homolog gene family, member B; CARD16—caspase recruitment domain family, member 16; SOD2—superoxide dismutase 2, mitochondrial.</p

Confirmation of differential gene expression using NanoString: Genes with decreased expression in TOL group.

<p>Confirmation of differential gene expression using NanoString: Genes with decreased expression in TOL group.</p

Enriched cellular process networks shared between current experimental data and published tolerance-associated gene expression data in solid organ transplantation.

<p>*Cellular process networks are ranked in descending order based on p value for magnitude of enrichment to annotated networks using MetaCore by GeneGo software (for each process network, solid organ = published solid organ transplant data,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117001#pone.0117001.ref002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117001#pone.0117001.ref005" target="_blank">5</a>] and HCT = HCT experimental data.</p><p>Enriched cellular process networks shared between current experimental data and published tolerance-associated gene expression data in solid organ transplantation.</p

Direction and magnitude of change in TOL group vs. non-TOL and control: Genes with increased expression in TOL group.

<p>*TNIK—TRAF2 and NCK interacting kinase; FCRL3—Fc receptor-like 3; NKG7—natural killer cell group 7 sequence; GZMH—granzyme H (cathepsin G-like 2, protein h-CCPX); CD8A—CD8a molecule; LAG3—lymphocyte-activation gene 3; TOX—thymocyte selection-associated high mobility group box; TBX21—T-box 21 (T-bet); EOMES—eomesodermin; TGFBR3—transforming growth factor, beta receptor III; IFNγ—interferon, gamma; IL-28RA—interleukin 28 receptor, alpha (interferon, lambda receptor); CCL5—chemokine (C-C motif) ligand 5; PLEKHF1—pleckstrin homology domain containing, family F (with FYVE domain) member 1; GZMH—granzyme H (cathepsin G-like 2, protein h-CCPX); CDK17—cyclin-dependent kinase 17; CEP78—centrosomal protein 78kDa.</p