Applicability, safety, and biological activity of regulatory T cell therapy in liver transplantation

Abstract

Regulatory T cells (Tregs) are a lymphocyte subset with intrinsic immunosuppressive properties that can be expanded in large numbers ex vivo and have been shown to prevent allograft rejection and promote tolerance in animal models. To investigate the safety, applicability, and biological activity of autologous Treg adoptive transfer in humans, we conducted an open-label, dose-escalation, Phase I clinical trial in liver transplantation. Patients were enrolled while awaiting liver transplantation or 6-12 months posttransplant. Circulating Tregs were isolated from blood or leukapheresis, expanded under good manufacturing practices (GMP) conditions, and administered intravenously at either 0.5-1 million Tregs/kg or 3-4.5 million Tregs/kg. The primary endpoint was the rate of dose- limiting toxicities occurring within 4 weeks of infusion. The applicability of the clinical protocol was poor unless patient recruitment was deferred until 6-12 months posttransplant. Thus, only 3 of the 17 patients who consented while awaiting liver transplantation were dosed. In contrast, all six patients who consented 6-12 months posttransplant received the cell infusion. Treg transfer was safe, transiently increased the pool of circulating Tregs and reduced anti-donor T cell responses. Our study opens the door to employing Treg immunotherapy to facilitate the reduction or complete discontinuation of immunosuppression following liver transplantation.

Keywords: T cell biology; cellular transplantation (nonislet); immunosuppression/immune modulation; liver transplantation/hepatology; tolerance; translational research/science.

Figure 1

Identification, enrollment, and follow‐up of eligible subjects. A, Original trial design targeting patients awaiting liver transplantation. B, Amended trial design targeting patients 6‐12 months posttransplant

Figure 2

In‐depth phenotypic characterization of expanded and circulating Tregs employing CyTOF. A, Barplots displaying the results of flow cytometric experiments assessing the sequential changes in the proportion of Tregs (defined by either CD4⁺CD25highCD127⁻ or CD25highFOXP⁺ expression) among circulating CD4⁺ T cells in the three patients receiving 1 million Tregs/kg and the six patients receiving 4.5 million Tregs/kg. Asterisks denote P < .05. B, Representative dot plot showing the expression of CD25 and Foxp3 in expanded (left panel) and circulating (right panel) Tregs after gating for CD3⁺, CD4⁺, and CD8⁻ cells employing CyTOF. C, Dendogram derived from a hierarchical clustering analysis of the patterns of variation in the expression of the 29 phenotypic markers shown in D in the 24 samples analyzed using CyTOF. The horizontal axis corresponds to the samples; the vertical axis corresponds to the dissimilarity between clusters. D, Heatmap displaying the median expression of 29 markers employed to characterize expanded and circulating Tregs by CyTOF (gated as described in B) before and at different time points following cell infusion: Preinfusion, 1‐week postinfusion (Post 1W), 1‐month postinfusion (Post 1M), 3‐months postinfusion (Post 3M). Rows represent individual markers and columns represent patient samples. The color in each cell reflects the relative expression level of the corresponding marker in the corresponding sample. Asterisks denote P < .05 when comparing the expanded Tregs and the circulating preinfusion Tregs. E, Expression levels of CD38 in expanded and circulating Tregs at different time points following cell infusion [Color figure can be viewed at https://www.wileyonlinelibrary.com]

Figure 3

Sequential changes in circulating Treg subsets following infusion of 4.5 million Tregs/kg. A, SPADE algorithm clustering of circulating Tregs before cell infusion based on the viSNE analysis of the markers assessed by CyTOF and described in Figure 2B. Data show all viable single cells hierarchically clustered according to similar protein expression levels. The nodes 1, 9, and 10 identify the Treg subpopulations expressing the highest levels of the 10 parameters more differentially expressed in expanded Tregs as compared to preinfusion circulating Tregs (CD38, Ki67, OX40, CD25, CD69, GATA3, CCR4, CTLA4, PD1, and HLA‐DR). Bubble size and color intensity correspond to population density. B, Cumulative frequency of circulating Tregs clustered on nodes 1, 9, and 10 at different time points before and after cell infusion grouping all six patients together. C, Representative viSNE density plots (top panel) and SPADE analyses (bottom panel) corresponding to circulating Tregs assessed at different time points before and after infusion. For SPADE analyses, bubble size represents population density and color denotes the magnitude of expression of the three representative markers (KI67, CD38, and HLA‐DR). An increase in nodes 1, 9, and 10 is noticeable 1 week, but not 1 month or 3 months, after cell infusion [Color figure can be viewed at https://www.wileyonlinelibrary.com]

Figure 4

Liver tests and serum cytokine patterns in patients receiving Treg infusion. A, Sequential changes in liver tests and blood cell count of P04 following infusion of 4.5 million Treg/kg. B, Sequential changes in serum cytokine levels in all six patients receiving 4.5 million Tregs/kg [Color figure can be viewed at https://www.wileyonlinelibrary.com]

Figure 5

Sequential changes in donor and third‐party alloimmune responses. A, Representative dot plots corresponding to P07, displaying the expression of CD154 on memory CD8⁺ T cells collected before Treg infusion and cultured with surrogate donor or third‐party cells. B, Sequential allospecific (left panel) and CMV‐specific (right panel) memory CD8⁺ T cell responses in the six patients receiving 4.5 million Tregs/kg. C, Sequential allospecific memory CD8⁺ T cell responses in the three patients receiving 1 million Tregs/kg. For all experiments, dot plots display median and standard deviation of the proportion of CD45RO⁺ CD8⁺ T cells expressing CD154 in response to surrogate donor or third‐party cells, CMV pp65, or phorbol 12‐myristate 13‐acetate (PMA), as described [Color figure can be viewed at https://www.wileyonlinelibrary.com]