Functional Consequences of Memory Inflation after Solid Organ Transplantation

Abstract

CMV is a major infectious complication following solid organ transplantation. Reactivation of CMV leads to memory inflation, a process in which CD8 T cells expand over time. Memory inflation is associated with specific changes in T cell function, including increased oligoclonality, decreased cytokine production, and terminal differentiation. To address whether memory inflation during the first year after transplantation in human subjects alters T cell differentiation and function, we employed single-cell-matched TCRαβ and targeted gene expression sequencing. Expanded T cell clones exhibited a terminally differentiated, immunosenescent, and polyfunctional phenotype whereas rare clones were less differentiated. Clonal expansion occurring between pre- and 3 mo posttransplant was accompanied by enhancement of polyfunctionality. In contrast, polyfunctionality and differentiation state were largely maintained between 3 and 12 mo posttransplant. Highly expanded clones had a higher degree of polyfunctionality than rare clones. Thus, CMV-responsive CD8 T cells differentiated during the pre- to posttransplant period then maintained their differentiation state and functional capacity despite posttransplant clonal expansion.

Figure 1:. CMV IE-1 responsive CD8 T cells have a heterogeneous but highly polyfunctional phenotype:

A) Genes quantified by sequencing and markers measured by index sorting. UMAP dimensionality reduction used to display gene expression data for all genes across all patients and time points. DBSCAN clustering used to identify clusters within B) all or D) CD8 T cells. Gray represents cells that did not fall into any clusters. Heatmap of gene expression of each DBSCAN cluster for C) all T cells or E) CD8 T cells, with unsupervised clustering applied. Values shown are z-score transformed (across clusters) cluster mean expression level for corresponding gene/marker F) Table describing differentiation state of each cluster of CD8 T cells. * diff.=differentiation n=6 subjects.

Figure 2:. Expanded clones have greater polyfunctionality than rare clones:

Batch corrected gene expression data analyzed for degree of functionality, or combination of functions IFNγ, CD107a, GZMB, PRF1, and TNF expressed. (A and C) Frequency of indicated cells expressing each combination of the five functions at each time point for A) all clones together or C) clones representing <2% (top) or ≥2% (bottom) of the population. Gray represents one and red represents five functions. Combinations indicated in key below the pie charts. Values represent median percentage of the sample with each combination of function across subjects. (B and D) Pairwise frequency of shared functions across all subjects at each time point for B) all clones together or D) clones representing <2% (top) or ≥2% (bottom) of the population. Columns are numerator, and rows are denominator. E.g. for row CD107a and column GZMB, the coloration of the heatmap represents the % of GZMB⁺ cells that also express CD107a. Values represent median value with each combination of function across subjects. Statistical analysis in B and D completed using two-way ANOVA with Tukey correction for multiple comparisons without assuming sphericity. * represents p<0.05. n=6 subjects.

Figure 3:. The phenotype of clonally expanded cells changes post-transplant:

Clones from each subject overlaid onto UMAP projection of CD8 T cells. Each clone ≥2% is depicted with different colored dots as detailed on the right with associated TCRß CDR3 amino acid sequences. Clones <2% are represented in gray.

Figure 4:. Clones with increasing size increase polyfunctionality only from pre- to post-transplant:

CD8 T cells from all subjects overlaid onto UMAP projection color coded by A) clones ≥2% (black) or <2% (gray) of the sample or B) induction therapy rATG (pink) or no rATG (purple). (C – D) Polyfunctionality index (PI) calculated as described.(20) Clone size (CS) determined at each time point as the frequency of the clone out of total sorted CD8 T cells. CS depicted on each heatmap represents the value at the final time point. Fold change (FC) was the log2 fold change calculated between the CS values at the two time points represented in the heatmap. Heatmaps clustered based on magnitude FC. For all clones representing C) ≥2% at either month 0 or month three and present at the other time point, or D) ≥2% at either month three or month twelve and present at the other time point, heatmap depicts PI at each time point, CS at month twelve, and FC in clone size from the earlier to the later month. Statistical analysis computed with (C–D) Wilcoxon matched-pairs signed rank test. n=6 subjects.

Figure 5:. Change in clone size associated with distinct gene expression from pre- to post-transplant:

Heatmaps depict effect size (Hedges’ g) of change in gene expression within clones between indicated time points. Far left columns represent change in clone size. Unsupervised clustering performed based on gene expression. Heatmaps display data for A) comparison of pre- and three months post-transplant and C) comparison of three and 12 months post-transplant. n=6 subjects. (B and D) Clones defined as increasing, decreasing, or maintained in size in Figures 5A and 5B were grouped, and distribution of expression of IFNγ, CD107a, GZMB, PRF1, TNF computed for each tertile. Kolmgorov-Smirnov test computed for pairwise comparison between B) pre- and three months post-transplant and D) three and 12 months post-transplant. False discovery rate used to determine significance given use of multiple comparisons. Threshold of significance was α=0.05. Arrows indicate statistical difference associated with a change in that direction. – indicates no statistical difference. n=6 subjects.