Knowing that immunotherapy that works in mice often fails in patients with solid tumors, and that mouse models mostly utilize tumor cell lines from advanced tumors, Singhal et al. decided to bypass murine studies entirely and instead analyzed the functions of macrophages and monocytes and their interactions with tumor-specific T cells within early-stage human lung tumors. The results, recently published in Science Translational Medicine, demonstrated that the microenvironment in early-stage cancers is quite different from the microenvironment in advanced tumors, suggesting that different immunotherapy strategies may be necessary for the treatment of various tumor stages.

To begin, the researchers phenotypically profiled immune cells from lung cancer tissue, adjacent lung parenchyma, and peripheral blood in 93 patients with stage 1 or 2 non-small cell lung cancer (NSCLC). They found that most CD45⁺CD11b⁺ myeloid cells within the tumor were either CD14⁺ monocyte/macrophage lineage cells (MMLCs) or CD66b⁺ granulocytic lineage cells. The team observed that the MMLCs did not preferentially accumulate in the tumors. Based on markers and morphology, Singhal et al. determined that CD14⁺HLA-DR^hi cells were tumor-associated macrophages (TAMs) and CD14⁺HLA-DR^int cells were monocytes. Many tumors were enriched in TAMs or monocytes, however, there was only a low frequency of CD14⁺HLA-DR^lo/- monocytic myeloid-derived suppressor cells (MDSCs) present in the early-stage tumors.

Next, Singhal et al. observed that the CD14⁺ cells within the tumor, and to a lesser extent, the adjacent lung tissue, had high expression of both T cell co-inhibitory and costimulatory molecules. Moreover, TAMs co-expressed both M1 (pro-inflammatory) and M2 (anti-inflammatory, pro-tumor) polarization markers in the early-stage tumors. Surprisingly, PD-L1 expression in TAMs was correlated with expression of T cell costimulatory molecules, and the tumors were enriched in PD-L1^hi TAMs.

The team then analyzed the interactions between tumor-associated MMLCs and T cells. In an antigen-nonspecific assay, tumor-derived CD14⁺ cells generally did not suppress autologous T cell proliferation or IFNγ production, and in some cases, they even exerted a stimulatory effect on the T cells. To study the effect of MMLCs on tumor-specific T cell responses, the researchers utilized an in vitro model system comprising human T cells transduced with Ly95 – a high-affinity transgenic TCR recognizing an HLA-A*0201-restricted NY-ESO-1 peptide – and A549 human lung adenocarcinoma cells modified to express the NY-ESO-1 peptide. Coculture of these two cell types led to Ly95 T cell activation and production of IFNγ and granzyme B. The addition of tumor-derived CD14⁺ cells to the coculture had minimal effect on Ly95 T cell response for most patients, although in some cases, either suppressive or stimulatory effects were observed. These effects were mostly antigen-specific and required direct cell-to-cell contact. The researchers sought to elucidate the source of the discrepancies between patients, but found no correlation between CD14⁺ cell-mediated suppressive effects and M2 macrophage marker expression or MDSC presence.

The researchers then examined the effect of tumor PD-L1 expression on T cell function by co-culturing Ly95 T cells with the target tumor cells that were modified to express PD-L1. Not surprisingly, tumor cell PD-L1 expression inhibited T cell activation, cytolytic activity, and IFNγ production, and these effects were mostly reversed by anti-PD-1. In contrast, PD-L1⁺CD14⁺ cells (with or without anti-PD-L1) generally did not affect T cell responses, although PD-L1^hi TAMs sometimes stimulated the T cells. To study this further, the team loaded TAMs with long peptides specific for the Ly95 T cells and confirmed that these TAMs directly stimulated cognate T cell responses despite high PD-L1 expression. Further experiments demonstrated that in the event of cognate tumor antigen cross-presentation, high PD-L1 expression on TAMs protected them from tumor-specific T cell killing. Thus, paradoxically, anti-PD-1 treatment may limit the immune stimulatory effect of TAMs, diminishing the antitumor effect in early-stage tumors.

Overall, Singhal et al. bring to light and begin to unravel the dynamic and complex roles that MMLCs may have in various stages of tumor development. CD14⁺ MMLCs in early-stage NSCLC tumors generally did not affect T cell response in vitro, and the accumulation of MMLCs, TAMs, and monocytes in the tumors did not significantly affect the overall survival of the patients. The absence or presence of PD-L1 on CD14⁺ MMLCs did not alter their effect on T cell response; however, PD-L1^hi TAMs sometimes stimulated tumor-specific T cells, whereas PD-L1⁺ tumor cells suppressed T cell effector function. The immune stimulatory effect of PD-L1^hi TAMs, coupled with the co-expression of M1 and M2 polarization markers on TAMs in early-stage lung tumors, underscores the importance of understanding the tumor microenvironment at different stages and has implications for immunotherapy design.

by Anna Scherer