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. 2016 Dec;20(8):1072-1080.
doi: 10.1111/petr.12795. Epub 2016 Oct 26.

Mass cytometry reveals a distinct immunoprofile of operational tolerance in pediatric liver transplantation

Audrey H Lau  1   2 , Matthew J Vitalone  2 , Kelly Haas  1 , Todd Shawler  2 , Carlos O Esquivel  2 , William E Berquist  1 , Olivia M Martinez  2   3 , Ricardo O Castillo  1 , Sheri M Krams  2   3
Affiliations

Mass cytometry reveals a distinct immunoprofile of operational tolerance in pediatric liver transplantation

Audrey H Lau et al. Pediatr Transplant. 2016 Dec.

Abstract

Long-term IS in transplant patients has significant morbidity, poorer quality of life, and substantial economic costs. TOL, defined as graft acceptance without functional impairment in the absence of IS, has been achieved in some pediatric LT recipients. Using mass cytometry, peripheral blood immunotyping was performed to characterize differences between tolerant patients and patients who are stable on single-agent IS. Single-cell mass cytometry was performed using blood samples from a single-center pediatric LT population of operationally tolerant patients to comprehensively characterize the immune cell populations in the tolerant state compared with patients on chronic low-dose IS. Specific T-cell populations of interest were confirmed by flow cytometry. This high-dimensional phenotypic analysis revealed distinct immunoprofiles between transplant populations as well as a CD4+ TOT (CD4+ CD5+ CD25+ CD38-/lo CD45RA) that correlates with tolerance in pediatric LT recipients. In TOL patients, the TOT was significantly increased as compared to patients stable on low levels of IS. This TOT cell was confirmed by flow cytometry and is distinct from classic Treg cells. These results demonstrate the power of mass cytometry to discover significant immune cell signatures that have diagnostic potential.

Keywords: T cells; cytometry by time of flight; immune cells; peripheral blood mononuclear cells; post-transplant.

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Figures

Figure 1
Figure 1. Mass cytometry identifies a T cell subset of Operational Tolerance (TOT)
PBMC from pediatric LT recipients that were defined as operationally tolerant, (TOL, n=7); stable on immunosuppression (IS, n=8); and PBMC from healthy controls (HC, n=5) were analyzed for 22 parameters using specific antibodies for CyTOF. (a) Visual representation of unsupervised hierarchical clustering. Results are shown for live cells and with analysis of 22 parameters. Major immune cell populations are delineated based on canonical lineage markers (CD3+CD4+ for CD4+ T cells, CD3+CD8+ for CD8+ T cells, CD14+ cells for monocytes, CD11c+HLA-DR+ cells for dendritic cells, CD16+ cells for NK cells, and CD19+CD20+ for B cells). The color scale indicates the median intensity of expression of the relative marker, in this case CD4, compared to all other cells while node sizes are based on the frequency of cells. Specific nodes are labeled A, B1-4, C1-4 and D and represent populations with significant differences between groups. (b) The relative abundance ± SD, on a linear scale, of node A for the TOL, IS and HC groups is shown (left). Nodes B1-4, C1-4, and D are shown in Figure S2. Selected histograms for CD3, CD4, CD5, CD8, CD25, CD38, and CD45RA for node A are shown (right). For each marker, the node of interest (red) against the background expression of the same marker on all other live cells (blue) is shown. (c) The relative abundance ± SD on a linear scale for the TOL and IS groups is shown (left). Selected histograms for CD3, CD4, CD5, CD8, CD25, CD38, and CD45RA for this node are shown (right). (d) CD4+CD25+Foxp3+ Treg cells and CD3+CD4+CD25+CD5+CD38−/loCD45RA− TOT cells were manually gated from CyTOF data and frequency compared between TOL (n=7) and IS (n=8) groups and plotted as the subset frequency of total live CD3+ lymphocytes. Data are expressed as percent of total live CD3+ lymphocytes. Mean of each group is shown. *, p < 0.01. **, p = 0.001.
Figure 2
Figure 2. Differential immunoprofile of TOL from HC and IS
The frequency of twenty-two immune cell populations (Figure S1a) as assessed by CyTOF between patients who are operationally tolerant (TOL, n=7) or on low dose single agent immunosuppression (IS, n=8) compared to healthy controls (HC, n=5) with mean shown for each group. * p < 0.05; ** p ≤ 0.01; and *** p ≤ 0.001. (b) Principal component analysis (PCA) showing the first two principal components of 38 immune cell populations (Table S3) for TOL (n=7), IS (n=8), and HC (n=5) comparison (combined proportion of variance 57.2%). (c, d) TOT, as determined by flow cytometry, differentiate TOL patients from IS. Peripheral blood mononuclear cells from TOL (n=5) and IS (n=6) were analyzed by flow cytometry for (c) T regulatory cells (CD4+CD25+Foxp3+) and (d) TOT (CD3+CD4+CD25+CD5+CD38−/loCD45RA−). Patient demographics are in Table S1. The gating strategy is shown in Figures S1b and S3a. Data are expressed as percent of total live CD3+ lymphocytes. Mean of each group is shown. *, p < 0.05.
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