PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors

Abstract

Vaccination with irradiated B16 melanoma cells expressing either GM-CSF (Gvax) or Flt3-ligand (Fvax) combined with antibody blockade of the negative T-cell costimulatory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) promotes rejection of preimplanted tumors. Despite CTLA-4 blockade, T-cell proliferation and cytokine production can be inhibited by the interaction of programmed death-1 (PD-1) with its ligands PD-L1 and PD-L2 or by the interaction of PD-L1 with B7-1. Here, we show that the combination of CTLA-4 and PD-1 blockade is more than twice as effective as either alone in promoting the rejection of B16 melanomas in conjunction with Fvax. Adding αPD-L1 to this regimen results in rejection of 65% of preimplanted tumors vs. 10% with CTLA-4 blockade alone. Combination PD-1 and CTLA-4 blockade increases effector T-cell (Teff) infiltration, resulting in highly advantageous Teff-to-regulatory T-cell ratios with the tumor. The fraction of tumor-infiltrating Teffs expressing CTLA-4 and PD-1 increases, reflecting the proliferation and accumulation of cells that would otherwise be anergized. Combination blockade also synergistically increases Teff-to-myeloid-derived suppressor cell ratios within B16 melanomas. IFN-γ production increases in both the tumor and vaccine draining lymph nodes, as does the frequency of IFN-γ/TNF-α double-producing CD8 + T cells within the tumor. These results suggest that combination blockade of the PD-1/ PD-L1-and CTLA-4-negative costimulatory pathways allows tumorspecific T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby shifting the tumor microenvironment from suppressive to inflammatory.he interaction between the T-cell receptor complex (TCR) and antigenic peptides bound in surface MHC molecules provides the specificity that defines adaptive T-cell immunity. In addition to TCR activation, costimulation via ligation of the coreceptor CD28 on T cells by B7 molecules on antigenpresenting cells (APCs) is required for optimal T-cell activation (1). Once mobilized, however, T cells begin to express other members of the CD28/B7 receptor family that attenuate the immune response through inhibition of proliferation and cytokine production (2). Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is rapidly up-regulated following T-cell activation and binds to B7 molecules with a higher affinity than does CD28. The receptor programmed death-1 (PD-1) is also expressed on T cells following activation, where, on binding to its ligands PD-L1 and PD-L2, it promotes T-cell anergy, apoptosis, and exhaustion. Recently, an additional coinhibitory ligand/receptor interaction has been described that involves binding of PD-L1 on T cells to B7-1 on APCs or vice versa (3). Although the existence of so many layers of T-cell inhibition may seem surprising, the severe and sometimes fatal autoimmunity that results when even one of these pathways is disrupted attests to their necessity.Malignant transformation was classically defined by the ability to avoid the norm...

Targeted agents and immunotherapies: optimizing outcomes in melanoma

“…As the clinical development of anti-PD-1 anti bodies proceeded, a growing body of research suggested an added benefit in tumour response when administered in combination with an anti-CTLA-4 antibody 91 . From a mechanistic perspective, this finding was consistent with the contrasting role of PD-1 in regulating the effector phase of the immune response, as opposed to the role of CTLA-4 in mediating the priming phase.…”

Targeting the PD1/PD-L1 axis in melanoma: Biological rationale, clinical challenges and opportunities

“…Curran et al reported, in mice xenograft, that the combination of CTLA-4 and PD-1 blockade determines the accumulation of CTLA-4/PD-1 double-positive T effector cells within B16 melanoma cell lines [77]. These data suggest that T cells that would otherwise be functionally and proliferatively repressed are instead able to continue expanding and carrying out effector functions.…”

Cocktails for cancer with a measure of immunotherapy

“…Although the targets of these drugs both do the same job, silencing T cells, they do so in different ways: CTLA 4 prevents the activation of T cells; PD 1 blocks the cells once they have infil trated the tumour and its environment. And treating mice with compounds that block both proteins yielded a more inflamed tumour as well 3 . "There was reason to think that if you block both, the T cells will be even more ready to kill the tumours, " says Michael Postow, an oncologist at Memorial Sloan Kettering.…”