Programmed cell death-1 (PD-1) at the heart of heterologous prime-boost vaccines and regulation of CD8+ T cell immunity

Abstract

Developing new vaccination strategies and optimizing current vaccines through heterologous prime-boost carries the promise of integrating the benefits of different yet synergistic vectors. It has been widely thought that the increased immunity afforded by heterologous prime-boost vaccination is mainly due to the minimization of immune responses to the carrier vectors, which allows a progressive build up of immunity against defined epitopes and the subsequent induction of broader immune responses against pathogens. Focusing on CD8+ T cells, we put forward a different yet complementary hypothesis based primarily on the systematic analysis of DNA vaccines as priming agents. This hypothesis relies on the finding that during the initiation of immune response, acquisition of co-inhibitory receptors such as programmed cell death-1 (PD-1) is determined by the pattern of antigen exposure in conjunction with Toll-like receptor (TLR)-dependent stimulation, critically affecting the magnitude and profile of secondary immunity. This hypothesis, based upon the acquisition and co-regulation of pivotal inhibitory receptors by CD8+ T cells, offers a rationale for gene-based immunization as an effective priming strategy and, in addition, outlines a new dimension to immune homeostasis during immune reaction to pathogens. Finally, this model implies that new and optimized immunization approaches for cancer and certain viral infections must induce highly efficacious T cells, refractory to a broad range of immune-inhibiting mechanisms, rather than solely or primarily focusing on the generation of large pools of vaccine-specific lymphocytes.

Figure 1

Representative studies to date, evaluating DNA priming - heterologous boosting.

Figure 2

Differential co-expression of inhibitory receptors by CD8⁺ T cells depending on priming. In brief, epitope-specific T cells from immunized mice were highly purified and analyzed without additional stimulation. Gene expression patterns were defined using hierarchical clustering; CD8⁺ T cells from naïve mice were used as a reference control. The bottom half of the figure summarizes the results pertaining to expression of inhibitory receptors such as PD-1, as average fold change of gene expression relative to control. There was coordinated up-regulation of gene expression corresponding to membrane receptors with inhibitory activity (yellow shaded section: Lag3, CTLA-4 and PD-1) in CD8⁺ T cells primed by peptide without adjuvant, but not DNA vaccine (summary of results in ref. [42]).

Figure 3

The responsiveness of CD8⁺ T cells is "imprinted" during the priming phase through PD-1 acquisition. The upper panel depicts the general methodology: mice were immunized by various regimens and specific T cells were restimulated ex vivo with HLA-A*0201-binding human Melan A 26-35 native peptide (EAAGIGILTV), in the presence of PD-1 blocking antibodies or control immunoglobulin. Ex vivo T cell proliferation was measured using a standard CFSE staining assay. The bottom panel depicts a summary of the results comparing the essential groups: T cells from Melan A plasmid versus Melan A 26-35 analogue peptide (ELAGIGILTV) immunized mice. While the epitope-specific T cells from DNA vaccinated mice had low PD-1 expression and high proliferative potential persistently, the T cells from peptide immunized mice had high PD-1 expression and low proliferative potential; however, their proliferation could be easily restored through blocking PD-1/PD-1L interaction, speaking to the critical role of PD-1 in determining the fate of CD8⁺ T cells post-priming (summary of results in refs. [42] and [48]).

Figure 4

The mechanism of prime-boosting in relation to PD-1-expression and central memory T cells. The flowchart in Figure 4A depicts schematically a proposed mechanism explaining the effectiveness of DNA priming - heterologous boosting in achieving superior immunity in immune competent organisms. Alternating DNA priming with heterologous boosting (viral vectors, recombinant proteins, peptides, cells, or cell lysates), achieves alternating production of 'central-memory' low PD-1 cells and highly differentiated effector T cells, respectively. Figure 4B is a temporal perspective on the synergy and differential output of priming and boosting vectors/regimens, respectively. It offers an explanation to why the exact prime-boost sequence is important based on the differential capability of vectors or regimens to elicit T cells with different properties such as susceptibility to negative regulatory mechanisms.

Figure 5

Schematic representation of the kinetics of various subsets of T cells within secondary lymphoid organs. This is a complementary perspective to that in Figure 4B, providing a rationale to why a specific sequence of priming and boosting is important to generating an elevated immune response.

Figure 6

Another dimension to the immune regulation of CD8⁺ T cells based on PD-1 expression. The lack of PD-1 up-regulation during priming may define a separate differentiation lineage. A current model (left side) depicts activation and differentiation of T cells, in relation to PD-1 expression, as a sequential upregulation and downregulation of PD-1, respectively. In this model, activated T cells unavoidably go through a stage in which they are sensitive to PD-1/PD-1L dependent negative regulatory mechanisms. Conversely, in the model depicted on the right side, the acquisition of PD-1 during T cell priming could be limited - depending on the priming regimen - thus yielding T cells that are not as susceptible to negative regulatory mechanisms associated with continuous or repeated antigen exposure. Thus, based on this model - and supported by recent evidence (42, 48) - immediate boosting would yield substantially higher immunity as opposed to immune 'exhaustion'. This enables the development of shortened immunization regimens utilizing a heterologous prime-boost strategy.

Figure 7

Co-regulation of PD-1 acquisition and functional avidity of T cells during immune priming. A and B show schematically the key parameters controlling two complementary features of T cells resulting from immune priming: PD-1 expression (A) and the functional avidity (B). Effective priming warrants optimal, balanced exposure to TCR-dependent and independent stimuli ("green zone") resulting in T cells with a desired effector profile upon boosting. Please note the inverse relationship between functional avidity and the amount of antigen. The table (bottom) depicts the major, synergistic features of priming and boosting vectors/regimens, as a pre-requisite to designing superior vaccination strategies. The model is based on published research (eg. refs [40,42,48,59,60]).