Antibodies that block programmed death 1 (PD-1) protein improve survival in patients with advanced non–small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, a condition in which little progress has been made during the past decade. In this pilot study, we administered two preoperative doses of PD-1 inhibitor nivolumab in adults with untreated, surgically resectable early (stage I, II, or IIIA) NSCLC. Nivolumab (at a dose of 3 mg per kilogram of body weight) was administered intravenously every 2 weeks, with surgery planned approximately 4 weeks after the first dose. The primary end points of the study were safety and feasibility. We also evaluated the tumor pathological response, expression of programmed death ligand 1 (PD-L1), mutational burden, and mutation-associated, neoantigen-specific T-cell responses. Neoadjuvant nivolumab had an acceptable side-effect profile and was not associated with delays in surgery. Of the 21 tumors that were removed, 20 were completely resected. A major pathological response occurred in 9 of 20 resected tumors (45%). Responses occurred in both PD-L1–positive and PD-L1–negative tumors. There was a significant correlation between the pathological response and the pretreatment tumor mutational burden. The number of T-cell clones that were found in both the tumor and peripheral blood increased systemically after PD-1 blockade in eight of nine patients who were evaluated. Mutation-associated, neoantigen-specific T-cell clones from a primary tumor with a complete response on pathological assessment rapidly expanded in peripheral blood at 2 to 4 weeks after treatment; some of these clones were not detected before the administration of nivolumab. Neoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced a major pathological response in 45% of resected tumors. The tumor mutational burden was predictive of the pathological response to PD-1 blockade. Treatment induced expansion of mutation-associated, neoantigen-specific T-cell clones in peripheral blood. (Funded by Cancer Research Institute–Stand Up 2 Cancer and others; ClinicalTrials.gov number, NCT02259621.)

We examined the immune microenvironment of primary colorectal cancer using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry, and functional analysis of tumor-infiltrating lymphocytes. A subset of colorectal cancer displayed high infiltration with activated CD8⁺ cytotoxic T lymphocyte (CTL) as well as activated Th1 cells characterized by IFNγ production and the Th1 transcription factor TBET. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly upregulated expression of multiple immune checkpoints, including five—PD-1, PD-L1, CTLA-4, LAG-3, and IDO—currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of colorectal cancer. Significance: The findings reported in this article are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair–defective subset of colorectal cancer selectively upregulates at least five checkpoint molecules that are targets of inhibitors currently being clinically tested. Cancer Discov; 5(1); 43–51. ©2014 AACR. See related commentary by Xiao and Freeman, p. 16 This article is highlighted in the In This Issue feature, p. 1

PD-1 blockade unleashes CD8 T cells¹, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens², and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay³in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIT^highTRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

T _reg Responses to Eos: CD4 ⁺ regulatory T cells (T _regs ) are critical for keeping our immune system in check: They prevent immune responses from getting out of hand and keep autoimmunity at bay. By activating the expression of some genes and turning off expression of others, the master regulatory transcription factor of T _regs , Foxp3, endows these cells with the appropriate gene expression program to mediate their suppressive effects. Pan et al. (p. 1142 , published online 20 August) now demonstrate that the transcription factor Eos is selectively required for Foxp3-mediated gene suppression in mice. Genes normally suppressed by Foxp3 in T _regs remained “on” when Eos expression was suppressed, whereas genes activated by Foxp3 were unaffected. T _reg function was also affected by Eos suppression. With half their genetic program disrupted, these cells resembled an intermediate between T _regs and conventional CD4 ⁺ T cells—unable to suppress immune responses properly and partially responsive to T cell–activating stimulation.