Thomas F. Gajewski

Innate and adaptive immune cells in the tumor microenvironment

Most tumor cells express antigens that can mediate recognition by host CD8+ T cells. Cancers that are detected clinically must have evaded antitumor immune responses to grow progressively. Recent work has suggested two broad categories of tumor escape based on cellular and molecular characteristics of the tumor microenvironment. One major subset shows a T cell–inflamed phenotype consisting of infiltrating T cells, a broad chemokine profile and a type I interferon signature indicative of innate immune activation. These tumors appear to resist immune attack through the dominant inhibitory effects of immune system–suppressive pathways. The other major phenotype lacks this T cell–inflamed phenotype and appears to resist immune attack through immune system exclusion or ignorance. These two major phenotypes of tumor microenvironment may require distinct immunotherapeutic interventions for maximal therapeutic effect.

Helper T Cell Differentiation Is Controlled by the Cell Cycle

Helper T (Th) cell differentiation is highly regulated by cytokines but initiated by mitogens. By examining gene expression in discrete generations of dividing cells, we have delineated the relationship between proliferation and differentiation. Initial expression of IL-2 is cell cycle-independent, whereas effector cytokine expression is cell cycle-dependent. IFNgamma expression increases in frequency with successive cell cycles, while IL-4 expression requires three cell divisions. Cell cycle progression and cytokine signaling act in concert to relieve epigenetic repression and can be supplanted by agents that hyperacetylate histones and demethylate DNA. Terminally differentiated cells exhibit stable epigenetic modification and cell cycle-independent gene expression. These data reveal a novel mechanism governing Th cell fate that initially integrates proliferative and differentiative signals and subsequently maintains stability of the differentiated state.

Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells.

CD8+ T cells induce immunosuppressive mechanisms in the tumor microenvironment. The Great Escape One of the long-standing questions in cancer biology is how tumors evade the patrolling immune response. Many potential explanations have been proposed, including that the tumor itself suppresses the immune response. Indeed, some of the most promising new immunotherapies work by blocking inhibitory molecules, which enhances immune activity against the tumor. Now, Spranger et al. show that immunosuppressive mechanisms in the tumor may involve negative feedback loops dependent on an infiltrating immune response. The authors noticed that even tumors that are infiltrated with CD8+ T cells are not rejected and that this correlated with expression of three types of immunosuppressives: indoleamine-2,3-dioxygenase (IDO), PD-L1/B7-H1, and regulatory T cells. Mechanistic studies in mice suggested that CD8+ T cells were required to be in the tumor microenvironment for the up-regulation of these immunosuppressives. Extending these studies to the clinic, patients with inflammatory tumor infiltrates will thus be more likely to benefit from immune checkpoint therapies. Tumor escape from immune-mediated destruction has been associated with immunosuppressive mechanisms that inhibit T cell activation. Although evidence for an active immune response, including infiltration with CD8+ T cells, can be found in a subset of patients, those tumors are nonetheless not immunologically rejected. In the current report, we show that it is the subset of T cell–inflamed tumors that showed high expression of three defined immunosuppressive mechanisms: indoleamine-2,3-dioxygenase (IDO), PD-L1/B7-H1, and FoxP3+ regulatory T cells (Tregs), suggesting that these inhibitory pathways might serve as negative feedback mechanisms that followed, rather than preceded, CD8+ T cell infiltration. Mechanistic studies in mice revealed that up-regulated expression of IDO and PD-L1, as well as recruitment of Tregs, in the tumor microenvironment depended on the presence of CD8+ T cells. The former was driven by interferon-γ and the latter by a production of CCR4-binding chemokines along with a component of induced proliferation. Our results argue that these major immunosuppressive pathways are intrinsically driven by the immune system rather than being orchestrated by cancer cells, and imply that cancer immunotherapy approaches targeting negative regulatory immune checkpoints might be preferentially beneficial for patients with a preexisting T cell–inflamed tumor microenvironment.

Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy.

Gut microbes affect immunotherapy The unleashing of antitumor T cell responses has ushered in a new era of cancer treatment. Although these therapies can cause dramatic tumor regressions in some patients, many patients inexplicably see no benefit. Mice have been used in two studies to investigate what might be happening. Specific members of the gut microbiota influence the efficacy of this type of immunotherapy (see the Perspective by Snyder et al.). Vétizou et al. found that optimal responses to anticytotoxic T lymphocyte antigen blockade required specific Bacteroides spp. Similarly, Sivan et al. discovered that Bifidobacterium spp. enhanced the efficacy of antiprogrammed cell death ligand 1 therapy. Science, this issue, p. 1079 and p. 1084; see also p. 1031 Gut microbes modulate the effectiveness of cancer immunotherapies in mice. [Also see Perspective by Snyder et al.] T cell infiltration of solid tumors is associated with favorable patient outcomes, yet the mechanisms underlying variable immune responses between individuals are not well understood. One possible modulator could be the intestinal microbiota. We compared melanoma growth in mice harboring distinct commensal microbiota and observed differences in spontaneous antitumor immunity, which were eliminated upon cohousing or after fecal transfer. Sequencing of the 16S ribosomal RNA identified Bifidobacterium as associated with the antitumor effects. Oral administration of Bifidobacterium alone improved tumor control to the same degree as programmed cell death protein 1 ligand 1 (PD-L1)–specific antibody therapy (checkpoint blockade), and combination treatment nearly abolished tumor outgrowth. Augmented dendritic cell function leading to enhanced CD8+ T cell priming and accumulation in the tumor microenvironment mediated the effect. Our data suggest that manipulating the microbiota may modulate cancer immunotherapy.

Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity

Melanoma treatment is being revolutionized by the development of effective immunotherapeutic approaches. These strategies include blockade of immune-inhibitory receptors on activated T cells; for example, using monoclonal antibodies against CTLA-4, PD-1, and PD-L1 (refs 3, 4, 5). However, only a subset of patients responds to these treatments, and data suggest that therapeutic benefit is preferentially achieved in patients with a pre-existing T-cell response against their tumour, as evidenced by a baseline CD8+ T-cell infiltration within the tumour microenvironment. Understanding the molecular mechanisms that underlie the presence or absence of a spontaneous anti-tumour T-cell response in subsets of cases, therefore, should enable the development of therapeutic solutions for patients lacking a T-cell infiltrate. Here we identify a melanoma-cell-intrinsic oncogenic pathway that contributes to a lack of T-cell infiltration in melanoma. Molecular analysis of human metastatic melanoma samples revealed a correlation between activation of the WNT/β-catenin signalling pathway and absence of a T-cell gene expression signature. Using autochthonous mouse melanoma models we identified the mechanism by which tumour-intrinsic active β-catenin signalling results in T-cell exclusion and resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody therapy. Specific oncogenic signals, therefore, can mediate cancer immune evasion and resistance to immunotherapies, pointing to new candidate targets for immune potentiation.

PD-L1/B7H-1 Inhibits the Effector Phase of Tumor Rejection by T Cell Receptor (TCR) Transgenic CD8+ T Cells

Although increased circulating tumor antigen-specific CD8+ T cells can be achieved by vaccination or adoptive transfer, tumor progression nonetheless often occurs through resistance to effector function. To develop a model for identifying mechanisms of resistance to antigen-specific CTLs, poorly immunogenic B16-F10 melanoma was transduced to express the Kb-binding peptide SIYRYYGL as a green fluorescent protein fusion protein that should be recognized by high-affinity 2C TCR transgenic T cells. Although B16.SIY cells expressed high levels of antigen and were induced to express Kb in response to IFN-γ, they were poorly recognized by primed 2C/RAG2−/− T cells. A screen for candidate inhibitory ligands revealed elevated PD-L1/B7H-1 on IFN-γ-treated B16-F10 cells and also on eight additional mouse tumors and seven human melanoma cell lines. Primed 2C/RAG2−/−/PD-1−/− T cells showed augmented cytokine production, proliferation, and cytolytic activity against tumor cells compared with wild-type 2C cells. This effect was reproduced with anti-PD-L1 antibody present during the effector phase but not during the priming culture. Adoptive transfer of 2C/RAG2−/−/PD-1−/− T cells in vivo caused tumor rejection under conditions in which wild-type 2C cells or CTLA-4-deficient 2C cells did not reject. Our results support interfering with PD-L1/PD-1 interactions to augment the effector function of tumor antigen-specific CD8+ T cells in the tumor microenvironment.

Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8α+ dendritic cells

The generation of antitumor CD8+ T cell responses requires type I interferon responsiveness in host antigen-presenting cells

Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment.

Despite the frequent detection of circulating tumor antigen-specific T cells, either spontaneously or following active immunization or adoptive transfer, immune-mediated cancer regression occurs only in the minority of patients. One theoretical rate-limiting step is whether effector T cells successfully migrate into metastatic tumor sites. Affymetrix gene expression profiling done on a series of metastatic melanoma biopsies revealed a major segregation of samples based on the presence or absence of T-cell-associated transcripts. The presence of lymphocytes correlated with the expression of defined chemokine genes. A subset of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10) was confirmed by protein array and/or quantitative reverse transcription-PCR to be preferentially expressed in tumors that contained T cells. Corresponding chemokine receptors were found to be up-regulated on human CD8(+) effector T cells, and transwell migration assays confirmed the ability of each of these chemokines to promote migration of CD8(+) effector cells in vitro. Screening by chemokine protein array identified a subset of melanoma cell lines that produced a similar broad array of chemokines. These melanoma cells more effectively recruited human CD8(+) effector T cells when implanted as xenografts in nonobese diabetic/severe combined immunodeficient mice in vivo. Chemokine blockade with specific antibodies inhibited migration of CD8(+) T cells. Our results suggest that lack of critical chemokines in a subset of melanoma metastases may limit the migration of activated T cells, which in turn could limit the effectiveness of antitumor immunity.

Blocked Ras Activation in Anergic CD4+ T Cells

T cell anergy is a state of functional unresponsiveness characterized by the inability to produce interleukin-2 (IL-2) upon T cell receptor stimulation. The mitogen-activated protein kinases ERK-1 and ERK-2 and the guanosine triphosphate-binding protein p21Ras were found to remain unactivated upon stimulation of anergic murine T helper cell 1 clones. The inability to activate the Ras pathway did not result from a defect in association among Shc, Grb-2, and murine Son of Sevenless, nor from a defect in their tyrosine phosphorylation. This block in Ras activation may lead to defective transactivation at activator protein 1 sites in anergic cells and may enable T cells to shut down IL-2 production selectively during anergy.

Efficacy and safety of ipilimumab monotherapy in patients with pretreated advanced melanoma: a multicenter single-arm phase II study

BACKGROUND This phase II study evaluated the safety and activity of ipilimumab, a fully human mAb that blocks cytotoxic T-lymphocyte antigen-4, in patients with advanced melanoma. PATIENTS AND METHODS Patients with previously treated, unresectable stage III/stage IV melanoma received 10 mg/kg ipilimumab every 3 weeks for four cycles (induction) followed by maintenance therapy every 3 months. The primary end point was best overall response rate (BORR) using modified World Health Organization (WHO) criteria. We also carried out an exploratory analysis of proposed immune-related response criteria (irRC). RESULTS BORR was 5.8% with a disease control rate (DCR) of 27% (N = 155). One- and 2-year survival rates (95% confidence interval) were 47.2% (39.5% to 55.1%) and 32.8% (25.4% to 40.5%), respectively, with a median overall survival of 10.2 months (7.6-16.3). Of 43 patients with disease progression by modified WHO criteria, 12 had disease control by irRC (8% of all treated patients), resulting in a total DCR of 35%. Adverse events (AEs) were largely immune related, occurring mainly in the skin and gastrointestinal tract, with 19% grade 3 and 3.2% grade 4. Immune-related AEs were manageable and generally reversible with corticosteroids. CONCLUSION Ipilimumab demonstrated clinical activity with encouraging long-term survival in a previously treated advanced melanoma population.