Inhibitory Receptors Beyond T Cell Exhaustion

Abstract

Inhibitory receptors (iRs) are frequently associated with "T cell exhaustion". However, the expression of iRs is also dependent on T cell differentiation and activation. Therapeutic blockade of various iRs, also referred to as "checkpoint blockade", is showing -unprecedented results in the treatment of cancer patients. Consequently, the clinical potential in this field is broad, calling for increased research efforts and rapid refinements in the understanding of iR function. In this review, we provide an overview on the significance of iR expression for the interpretation of T cell functionality. We summarize how iRs have been strongly associated with "T cell exhaustion" and illustrate the parallel evidence on the importance of T cell differentiation and activation for the expression of iRs. The differentiation subsets of CD8 T cells (naïve, effector, and memory cells) show broad and inherent differences in iR expression, while activation leads to strong upregulation of iRs. Therefore, changes in iR expression during an immune response are often concomitant with T cell differentiation and activation. Sustained expression of iRs in chronic infection and in the tumor microenvironment likely reflects a specialized T cell differentiation. In these situations of prolonged antigen exposure and chronic inflammation, T cells are "downtuned" in order to limit tissue damage. Furthermore, we review the novel "checkpoint blockade" treatments and the potential of iRs as biomarkers. Finally, we provide recommendations for the immune monitoring of patients to interpret iR expression data combined with parameters of activation and differentiation of T cells.

Keywords: T cell exhaustion; activation; checkpoint blockade; differentiation; immune monitoring; immunotherapy; inhibitory receptors.

Figure 1

CD8 T cell subsets are inherently different in iR expression and effector functions. The various differentiation subsets that are found in circulation in healthy donors are depicted with the iRs that they predominantly express. The relative levels of each iR are indicated below each subset, in parallel to the capacity for cytokine secretion and CD107a translocation. The graphs are based on data from a 6-h intracellular cytokine production assay using PBMC from healthy donors (75). Also shown is iR expression in activated cells (“activated blast”), together with activation markers, based on a 3-day stimulation assay on CD8 T cells from healthy donors (75). The size of the receptor depicted on cells reflects the relative expression of iRs.

Figure 2

Discriminating differentiation subsets is the minimal analysis to assess the link between iR expression and functionality of CD8 T cells. The significance of iR expression on CD8 T cell function can be assessed by analyzing cytokine production by iR-positive versus iR-negative cells. The relative capacity for cytokine production in the various subsets is depicted by the numbers of orange intracellular dots. As an example, the expression of two iRs are shown: BTLA and PD1. The percentage below each subset name indicates the relative frequency of that subset within total CD8 T cells. The percentages next to each receptor indicate the fraction of cells within a given subset that express the receptor (the size of the receptor depicted on cells also reflects relative expression of iRs). These percentages are estimated based on average values in PBMC of healthy donors (75). When iR-positive cells are compared to iR-negative cells within the total CD8 T cells (red arrow), artifactual conclusions can be driven due to the inherent differences in iR expression and effector function of the various subsets. For example, BTLA+ cells in total CD8 T may show less cytokine secretion than their BTLA− counterparts, but this is due to the enrichment of Naïve cells in BTLA+ cells, which do not produce cytokines as compared to the enrichment of differentiated cells in BTLA−. With each subset, BTLA+ and BTLA− are comparable cytokine producers. Therefore, it is not “cytokine production capacity” (=artifactual conclusion) but “differentiation status” what accounts for differences in comparing BTLA+ and BTLA− cells. As a minimal requirement, analysis of individual subsets is necessary to adequately address the link between iR expression and effector function.

Figure 3

Levels of iR may peak at the effector phase, and may further modulate differently during acute versus chronic immune responses. Naïve cells express mainly BTLA and low levels of TIM3. Effector cells express a wider variety of iRs. The levels of certain iRs such as PD1, CTLA-4, LAG3, and TIM3 may peak at the effector phase. Thereafter, iR expression differs in chronically stimulated cells (“exhausted cells”) where iRs are relatively maintained, as opposed to memory cells after clearance of an acute infection where iRs are down-modulated.