BACKGROUND: Follicular lymphoma (FL) arises from a singe original B cell within the germinal center. These monoclonal B cells maintain an intimate relationship with host immune cells, especially T cells that both sustain and oppose their growth. These T cells express numerous co-stimulatory molecules such as OX40, GITR and ICOS as well as inhibitory receptors such as PD-1, CTLA-4 and TIGIT that act as checkpoints to prevent destructive immune responses and maintain peripheral self-tolerance.Tumor B cells can over-express ligands for these checkpoints causing the T cells to be non-responsive, exhausted and even apoptotic, thereby evading immune surveillance.
Presently, checkpoint antibodies that block the inhibitory receptors on the infiltrating T cells have been used in therapy of cancer with mixed results. Blocking the PD-1/PD-L1 interaction in FL has resulted in about a 40% overall response rate. (Brockelmann, et al. Haematologica; 2017.)Antibodies against other immune checkpoints are currently being developed and may be effective as single agents or in combination with antibodies that activate T cells or with other therapeutic agents. Indeed, the number of possible candidates and combinations exceeds the available patients for clinical trials. Therefore, methods are needed to first select targets expressed or co-expressed on tumor-immune infiltrating T cells.
High throughput single-cell RNA sequencing has made it possible to interrogate the transcriptome of tumor-infiltrating T cells, providing insight into hundreds of genes per cell. Because it is not focused or limited to surface proteins already targeted by existing antibodies, it allows for novel target discovery. However, the presence of mRNA does not guarantee translation of the protein or its trafficking to the surface where it can act as an antibody target. Also, detectable mRNA is not required for the protein to be available on the surface of the infiltrating T cells. The turnover of cell surface protein may be discordant with the mRNA encoding it and, in some cases, a small amount of mRNA may translate into large amounts of targetable protein.
The goal of this study was to compare, at the single-cell level, the presence of mRNA and its corresponding protein in tumor-infiltrating T cells in biopsy specimens of FL.
METHODS: Cryopreserved single-cell suspensions of samples from patients with FL were thawed and surface stained with antibodies against phenotypic markers as well as the co-stimulatory target OX40 and the inhibitory target PD-1. The cells were then fixed and permeabilized allowing probes for OX40 and PD-1 to label the corresponding mRNA using the PrimeFlow® RNA Assay Kit (Thermo Fisher Scientific). Cells were analyzed on an LSRII (BD Biosciences) and the data were analyzed using web-based software (Cytobank.org).
RESULTS: FL samples from three patients have been studied so far focusing on the question of co-expression of OX40 and PD-1 and their corresponding mRNA in various T cell subpopulations. In each case, detection of mRNA via the labeled probe correlates with a presence of surface protein but not always with the amount of surface expression. Comparing surface PD-1 high CD4 T cells to those that are negative for PD-1, the median fold change of PD-1 protein is 100 times greater whereas the mRNA is only 1.5. In some cases, surface protein of either OX40 or PD-1 was found but no RNA could be detected. This is most noticeable in specific subsets of T cells, such as CD4 positive T regulatory cells (TRegs) and exhausted CD8 T cells as seen in Figure 1 below.
CONCLUSION: In the search for novel targets or combinations that may act synergistically to activate the immune system, it is appealing to turn to a technique which can interrogate a large part of the genome in an unsupervised fashion such as single-cell RNA sequencing. Our data suggests that mRNA expression may positively predict the presence of the corresponding cell surface target, but that its absence is not necessarily a negative predictor.
No relevant conflicts of interest to declare.
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