Ole Audun Werner Haabeth

Inflammation driven by tumour-specific Th1 cells protects against B-cell cancer

The immune system can both promote and suppress cancer. Chronic inflammation and proinflammatory cytokines such as interleukin (IL)-1 and IL-6 are considered to be tumour promoting. In contrast, the exact nature of protective antitumour immunity remains obscure. Here, we quantify locally secreted cytokines during primary immune responses against myeloma and B-cell lymphoma in mice. Strikingly, successful cancer immunosurveillance mediated by tumour-specific CD4+ T cells is consistently associated with elevated local levels of both proinflammatory (IL-1α, IL-1β and IL-6) and T helper 1 (Th1)-associated cytokines (interferon-γ (IFN-γ), IL-2 and IL-12). Cancer eradication is achieved by a collaboration between tumour-specific Th1 cells and tumour-infiltrating, antigen-presenting macrophages. Th1 cells induce secretion of IL-1β and IL-6 by macrophages. Th1-derived IFN-γ is shown to render macrophages directly cytotoxic to cancer cells, and to induce macrophages to secrete the angiostatic chemokines CXCL9/MIG and CXCL10/IP-10. Thus, inflammation, when driven by tumour-specific Th1 cells, may prevent rather than promote cancer.

How Do CD4+ T Cells Detect and Eliminate Tumor Cells That Either Lack or Express MHC Class II Molecules?

CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR) transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor-specific antigen by host antigen-presenting cells (APCs) appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315), where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR-transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-γ stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed.

A model for cancer-suppressive inflammation

In oncology, inflammation is generally regarded as a cancer-promoting process only. Here, we argue that this view may represent a misleading oversimplification. We present evidence from our own work and from the literature documenting cancer-suppressive aspects of inflammation. We propose that specific types of inflammation, in particular inflammation driven by tumor-specific Th1 cells, may repress rather than promote cancer. Th1 cells collaborate with tumor-infiltrating M1 macrophages to efficiently recognize and eliminate malignant cells. In a Th1 environment, pro-inflammatory cytokines such as interleukin (IL)-1α, IL-1β, IL-6 and tumor-necrosis factor α (TNFα) enhance anti-cancer immunity. Inducing Th1-type inflammation may significantly improve immunotherapeutic strategies against cancer.

Interleukin-1 is required for cancer eradication mediated by tumor-specific Th1 cells

The role of inflammation in cancer is controversial as both tumor-promoting and tumor-suppressive aspects of inflammation have been reported. In particular, it has been shown that pro-inflammatory cytokines, like interleukin-1α (IL-1α), IL-1β, IL-6, and tumor necrosis factor α (TNFα), may either promote or suppress cancer. However, the cellular and molecular basis underlying these opposing outcomes remains enigmatic. Using mouse models for myeloma and lymphoma, we have recently reported that inflammation driven by tumor-specific T helper 1 (Th1) cells conferred protection against B-cell cancer and that interferon-γ (IFN-γ) was essential for this process. Here, we have investigated the contribution of several inflammatory mediators. Myeloma eradication by Th1 cells was not affected by inhibition of TNF-α, TNF-related weak inducer of apoptosis (TWEAK), or TNF-related apoptosis-inducing ligand (TRAIL). In contrast, cancer elimination by tumor-specific Th1 cells was severely impaired by the in vivo neutralization of both IL-1α and IL-1β (collectively named IL-1) with IL-1 receptor antagonist (IL-1Ra). The antitumor functions of tumor-specific Th1 cells and tumor-infiltrating macrophages were both affected by IL-1 neutralization. Secretion of the Th1-derived cytokines IL-2 and IFN-γ at the incipient tumor site was severely reduced by IL-1 blockade. Moreover, IL-1 was shown to synergize with IFN-γ for induction of tumoricidal activity in tumor-infiltrating macrophages. This synergy between IL-1 and IFN-γ may explain how inflammation, when driven by tumor-specific Th1 cells, represses rather than promotes cancer. Collectively, the data reveal a central role of inflammation, and more specifically of the canonical pro-inflammatory cytokine IL-1, in enhancing Th1-mediated immunity against cancer.

Indirect CD4 + T cell-mediated elimination of MHC II NEG tumor cells is spatially restricted and fails to prevent escape of antigen-negative cells

Tumor‐specific Th1 cells can activate tumor‐infiltrating macrophages that eliminate MHC class II negative (MHC IINEG) tumor cells. Activated M1‐like macrophages lack antigen (Ag) receptors, and are presumably unable to discriminate and thus kill both Ag‐positive (AgPOS) and Ag‐negative (AgNEG) tumor cells (bystander killing). The lack of specificity of macrophage‐mediated cytotoxicity might be of clinical importance as it could provide a means of avoiding tumor escape. Here, we have tested this idea using mixed populations of AgPOS and AgNEG tumor cells in a TCR‐transgenic model in which CD4+ T cells recognize a secreted tumor‐specific antigen. Surprisingly, while AgPOS tumor cells were recognized and rejected, AgNEG cells grew unimpeded and formed tumors. We further demonstrated that macrophage‐mediated cytotoxicity was spatially restricted to areas dominated by AgPOS tumor cells, sparing AgNEG tumor cells in the vicinity. As a consequence, macrophage tumoricidal activity did not confer bystander killing in vivo. The present results offer novel insight into the mechanisms of indirect Th1‐mediated elimination of MHC IINEG tumor cells.

Molecular profiling of tumor-specific TH1 cells activated in vivo

The central role of tumor-specific TH1 cells in anticancer immune responses is becoming increasingly appreciated. However, little is known about how these cells are generated in vivo. Here, we used flow cytometry and gene expression microarrays to characterize the primary activation and TH1 differentiation of naïve tumor-specific CD4+ T cells in a mouse model of cancer immunosurveillance. We took advantage of T-cell receptor-transgenic mice in which CD4+ T cells recognize a tumor-specific antigen secreted by MHC class II-negative MOPC315 myeloma cells. Cancer cells were injected subcutaneously and T-cell activation was analyzed in draining lymph nodes and at the incipient tumor site 8 d later. Upon activation and migration to incipient tumor sites, tumor-specific CD4+ T cells exhibited the upregulation of 29 cell-surface molecules (CD2, CD5, CD11a, CD18, CD25, CD28, CD44, CD45, CD49d, CD51, CD54, CD69, CD71, CD83, CD86, CD90, CD95, CD102, CD122, CD153, CD166, CD200, CD249, CD254, CD274, CD279, Ly6C, MHC class I and CCR7) and the downregulation of five (CD27, CD31, CD45RB, CD62L and CD126). Activated CD4+ T cells produced interferon γ, a cytokine consistent with a TH1-polarized response, tumor necrosis factor α as well as interleukin (IL)-2, IL-3 and IL-10. The activation of naïve tumor-specific CD4+ T cells in draining lymph nodes resulted in the upregulation of 609 genes and the downregulation of 284 genes. The bioinformatic analysis of differentially expressed genes identified functional pathways related to tumor-specific TH1 cell activation. This study may represent a useful resource to guide the development of TH1-based immunotherapies against cancer.

Adoptive Transfer of Tumor-Specific Th2 Cells Eradicates Tumors by Triggering an In Situ Inflammatory Immune Response

Adoptive cell therapy (ACT) trials to date have focused on transfer of autologous tumor-specific cytotoxic CD8+ T cells; however, the potential of CD4+ T helper (Th) cells for ACT is gaining interest. While encouraging results have been reported with IFNγ-producing Th1 cells, tumor-specific Th2 cells have been largely neglected for ACT due to their reported tumor-promoting properties. In this study, we tested the efficacy of idiotype-specific Th2 cells for the treatment of mice with MHC class II-negative myeloma. Th2 ACT efficiently eradicated subcutaneous myeloma in an antigen-specific fashion. Transferred Th2 cells persisted in vivo and conferred long-lasting immunity. Cancer eradication mediated by tumor-specific Th2 cells did not require B cells, natural killer T cells, CD8+ T cells, or IFNγ. Th2 ACT was also curative against B-cell lymphoma. Upon transfer, Th2 cells induced a type II inflammation at the tumor site with massive infiltration of M2-type macrophages producing arginase. In vivo blockade of arginase strongly inhibited Th2 ACT, consistent with a key role of arginase and M2 macrophages in myeloma elimination by Th2 cells. These results illustrate that cancer eradication may be achieved by induction of a tumor-specific Th2 inflammatory immune response at the tumor site. Thus, ACT with tumor-specific Th2 cells may represent a highly efficient immunotherapy protocol against cancer. Cancer Res; 76(23); 6864-76. ©2016 AACR.

Idiotype-specific CD4(+) T cells eradicate disseminated myeloma.

T-cell immunotherapy is emerging as a very promising immunotherapeutic treatment strategy, and is the subject of numerous ongoing clinical trials in the management of various malignant diseases. Little is known about the therapeutic potential of adoptive T-cell therapy (ACT) in diseases affecting the bone marrow. The unique growth pattern of myeloma cells, with early dissemination and a predilection for the bone marrow compartment, raises important issues about the accessibility of such cells to T-cell immunotherapy. Similar patterns of tumor cell distribution are observed in other hematological malignancies, as well as in the advanced stages of other malignant diseases, where bone marrow metastases are a common occurrence. We therefore wanted to explore the applicability of T-cell therapy in the setting of multiple myeloma.

Tumor-specific CD4 + T cells eradicate myeloma cells genetically deficient in MHC class II display

CD4+ T cells have been shown to reject tumor cells with no detectable expression of major histocompatibility complex class II (MHC II). However, under certain circumstances, induction of ectopic MHC II expression on tumor cells has been reported. To confirm that CD4+ T cell-mediated anti-tumor immunity can be successful in the complete absence of antigen display on the tumor cells themselves, we eliminated MHC II on tumor cells using CRISPR/Cas9. Our results demonstrate that ablation of the relevant MHC II (I-Ed) in multiple myeloma cells (MOPC315) does not hinder rejection by tumor-specific CD4+ T cells. These findings provide conclusive evidence that CD4+ T cells specific for tumor antigens can eliminate malignant cells in the absence of endogenous MHC class II expression on the tumor cells. This occurs through antigen uptake and indirect presentation on tumor-infiltrating macrophages.

Limitations of bystander killing in Th1/M1 immune responses against a secreted tumor antigen.

T-cell recognition of tumor antigens presented on tumor-infiltrating macrophages (TAMs) induces a tumoricidal M1-like phenotype. Resultant indirect immune responses could eliminate not only antigen secreting (AgPOS), but also antigen negative (AgNEG) tumor cells via bystander killing. Such broad-spectrum response could eliminate antigenically heterogeneous tumors. Using an in vivo model of CD4+ T-cell mediated immunity against MHC II negative myeloma cells, bystander killing of AgNEG cells was ineffective due to strict spatial constraints of Th1-induced TAM cytotoxicity.