Expression profiling of TCR-engineered T cells demonstrates overexpression of multiple inhibitory receptors in persisting lymphocytes

Abstract

Despite significant progress in the development of adoptive cell-transfer therapies (ACTs) using gene-engineered T cells, little is known about the fate of cells following infusion. To address that, we performed a comparative analysis of gene expression between T-cell receptor-engineered lymphocytes persisting in the circulation 1 month after administration and the product that was infused. We observed that 156 genes related to immune function were differentially expressed, including underexpression of stimulators of lymphocyte function and overexpression of inhibitory genes in postinfusion cells. Of genes overexpressed postinfusion, the product of programmed cell death 1 (PDCD1), coinhibitory receptor PD-1, was expressed at a higher percentage in postinfusion lymphocytes than in the infusion product. This was associated with a higher sensitivity to inhibition of cytokine production by interaction with its ligand PD-L1. Coinhibitory receptor CD160 was also overexpressed in persisting cells, and its expression was associated with decreased reactivity, which surprisingly was found to be ligand-independent. These results contribute to a deeper understanding of the properties of transgenic lymphocytes used to treat human malignancies and may provide a rationale for the development of combination therapies as a method to improve ACT.

Trial registration: ClinicalTrials.gov NCT00509288 NCT00923195 NCT01273181.

Figures

Figure 1

Isolation of TCR-engineered lymphocytes from peripheral blood. (A) Timeline representing the outline of TCR-transfer clinical protocols and the experimental design for the isolation of cells used in gene-expression analysis. PBMCs are isolated ∼24 days before infusion by apheresis and subsequently stimulated with OKT3 in presence of IL-2 (Stim1). Retroviral transductions take place 23 and 22 days before infusion and a REP is started 2 weeks before administration of the cells. Culture media are supplemented with IL-2 during the ex vivo culture. The resulting cellular product is administered on day 0, and IL-2 is given for the first few days (1-3 days) to support engraftment. Approximately 1 month after infusion PBMCs are isolated by apheresis. Samples included in this study were prepared as follows: preinfusion samples are CD3+ cells isolated by negative selection, using magnetic bead sorting, from PBMCs collected at ∼day −24. Infusion samples are aliquots of the final cellular product, isolated from the infusion bag. Postinfusion samples are CD3+ mTCRb+ cells isolated from PBMC samples 1 month after infusion by FACS sorting. (B-C) FACS sorting of engineered T cells from PBMCs at 1 month postinfusion. (B) Presort staining of CD3 and mTCRb. (C) Postsort analysis showing enrichment of T cells expressing murine TCRs. Gated on lymphoid, single, PI− cells.

Figure 2

Gene-expression analysis. (A) PCA of 8 sets of preinfusion (blue), infusion (green), and postinfusion (red) samples analyzed by NanoString. (B) Venn diagram showing the number of differentially expressed genes identified in each of the 3 comparisons, among a code set of 511 targets. (C) Volcano plot for the 511 genes, comparing matched postinfusion and infusion engineered T cells. Horizontal line at y = 1.31 represents the threshold of statistical significance (P = .05) and vertical lines at x = +/−2 represent the threshold of FC set as cutoff values for the definition of differentially expressed genes.

Figure 3

Differentially expressed genes. (A) RNA expression levels for 18 genes that were differentially expressed in all three comparisons. Each line represents an individual patient. (B) Expression patterns of genes differentially expressed between postinfusion and preinfusion samples, which did not return to levels similar to preinfusion after engraftment.

Figure 4

PD-1 and CD160 are overexpressed in persisting transgenic lymphocytes. (A) Percentages of PD-1 expression in infusion and postinfusion CD8 T cells. (B) Reduced IFNγ production in CD8 T cells cocultured in vitro with relevant targets engineered to expressed PD-L1. (C) LAG-3, 2B4, and CD160 exhaustion markers surface expression in CD3+CD8+mTCRb+ infusion and postinfusion lymphocytes. Cells wre gated on lymphoid, PI−, CD3+, CD8+, mTCRb+ cells. (D) HVEM surface expression in melanoma tumor cells present in tumor digests of 4 melanoma patients. (E) Histograms showing surface staining of CD160 and costimulatory receptor LIGHT in persisting engineered lymphocytes of 5 patients. In all samples analyzed, LIGHT expression was lower than that of CD160.

Figure 5

CD160 is a marker of T cells with reduced IFNγ-secretion capacity. (A) Representative example of IFNγ intracellular staining in 1-month postinfusion MAGE A3 TCR+ CD8 lymphocytes (gated separately based on CD160 expression) cocultured against MAGE A3+ 624 and H1299-A2, or HVEM-transduced derivatives. (B) Summary of results from 10 infusion samples and 8 postinfusion samples cocultured with 624 or 624-HVEM. (C) Intracellular staining of IFNγ in infusion and postinfusion lymphocytes of patient 9 stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomicyn during 4 hours or nonstimulated controls. (D) Summary of 6 patients.