Anne Gallois

Reversal of NK-Cell Exhaustion in Advanced Melanoma by Tim-3 Blockade

Silva and colleagues show that Tim-3 expression in NK cells from melanoma patients correlates with poor prognosis; blockade of Tim-3 induces NK-cell proliferative and cytolytic capacity and responsiveness to growth factors, indicating that Tim-3 is a therapeutic target for patients with advanced melanoma. The immunoregulatory protein T-cell immunoglobulin- and mucin-domain–containing molecule-3 (Tim-3) mediates T-cell exhaustion and contributes to the suppression of immune responses in both viral infections and tumors. Tim-3 blockade reverses the exhausted phenotype of CD4+ and CD8+ T cells in several chronic diseases, including melanoma. Interestingly, natural killer (NK) cells constitutively express Tim-3; however, the role of Tim-3 in modulating the function of these innate effector cells remains unclear, particularly in human diseases. In this study, we compared the function of Tim-3 in NK cells from healthy donors and patients with metastatic melanoma. NK cells from the latter were functionally impaired/exhausted, and Tim-3 blockade reversed this exhausted phenotype. Moreover, Tim-3 expression levels were correlated with the stage of the disease and poor prognostic factors. These data indicate that Tim-3 can function as an NK-cell exhaustion marker in advanced melanoma and support the development of Tim-3–targeted therapies to restore antitumor immunity. Cancer Immunol Res; 2(5); 410–22. ©2014 AACR.

TLR4 Engagement during TLR3-Induced Proinflammatory Signaling in Dendritic Cells Promotes IL-10–Mediated Suppression of Antitumor Immunity

Toll-like receptor (TLR) agonists are promising adjuvants for immune therapy of cancer, but their potential efficacy as single or combinatorial agents has yet to be fully evaluated. Here, we report that among all TLR agonists tested, dendritic cells (DC) stimulated with the TLR3 agonist polyI:C displayed the strongest activity in stimulating proinflammatory responses and the production of melanoma antigen-specific CD8(+) T cells. Simultaneous treatment with TLR7/8 agonists further improved these responses, but the inclusion of bacterial lipopolysaccharide (LPS), a TLR4 agonist, suppressed proinflammatory cytokine production. This inhibition was contingent upon rapid induction of the suppressive cytokine interleukin (IL)-10 by LPS, leading to dysregulated immune responses and it could be reversed by signal transducers and activators of transcription 3 knockdown, p38 blockade or antibodies to IL-10 and its receptor. Our findings show how certain TLR agonist combinations can enhance or limit DC responses associated with antitumor immunity, through their relative ability to induce IL-10 pathways that are immune suppressive.

Reversal of natural killer cell exhaustion by TIM-3 blockade.

Natural killer (NK) cells are innate immune cells that become progressively exhausted in advanced stage cancer, crippling their ability to execute antitumor functions. We previously characterized the nature of NK cell exhaustion in metastatic melanoma patients, reporting a correlation with high expression of TIM-3. Blockade of this immune checkpoint molecule reversed the exhausted phenotype and improved NK cell function.

Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors

In osteoclasts, Src controls podosome organization and bone degradation, which leads to an osteopetrotic phenotype in src–/– mice. Since this phenotype was even more severe in src–/– hck–/– mice, we examined the individual contribution of Hck in bone homeostasis. Compared to wt mice, hck–/– mice exhibited an osteopetrotic phenotype characterized by an increased density of trabecular bone and decreased bone degradation, although osteoclastogenesis was not impaired. Podosome organization and matrix degradation were found to be defective in hck–/– osteoclast precursors (preosteoclast) but were normal in mature hck–/– osteoclasts, probably through compensation by Src, which was specifically overexpressed in mature osteoclasts. As a consequence of podosome defects, the 3‐dimensional migration of hck–/– preosteoclasts was strongly affected in vitro. In vivo, this translated by altered bone homing of preosteoclasts in hck–/– mice: in metatarsals of 1‐wk‐old mice, when bone formation strongly depends on the recruitment of these cells, reduced numbers of osteoclasts and abnormal developing trabecular bone were observed. This phenotype was still detectable in adults. In summmary, Hck is one of the very few effectors of preosteoclast recruitment described to date and thereby plays a critical role in bone remodeling.—Vérollet, C., Gallois, A., Dacquin, R., Lastrucci, C., Pandruvada, S. M. N., Ortega, N., Poincloux, R., Behar, A., Cougoule, C., Lowell, C., Al Saati, T., Jurdic, P., Maridonneau‐Parini, I., Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors. FASEB J. 27, 3608–3618 (2013). www.fasebj.org

Dendritic Cell-Targeted Approaches to Modulate Immune Dysfunction in the Tumor Microenvironment

There has been enormous progress this past decade in the understanding of the biology of dendritic cells (DCs) along with increasing attention for the development of novel dendritic cell (DC)-based cancer therapies. However, the clinical impact of DC-based vaccines remains to be established. This limited success could be explained by suboptimal conditions for generating potent immunostimulatory DCs as well as immune suppression mediated by the tumor microenvironment (TME). Therefore, strategies that optimize the potency of DC vaccines along with newly described therapies that target the TME in order to overcome immune dysfunction may provide durable tumor-specific immunity. These novel interventions hold the most promise for successful cancer immunotherapies.

A needle in the 'cancer vaccine' haystack

Vaccines can prevent infections by several pathogens. Success, however, has been limited for other chronic diseases, reflecting a constraint for effectively manipulating the human immune system. The results from four studies describe a novel dendritic cell (DC) subset in humans that may be crucial for the design of vaccines against cancer and other chronic diseases.

Abstract 5410: Tim-3 expression and function in natural killer cells from metastatic melanoma patients

BACKGROUND: Tim-3 (T-cell immunoglobulin domain and mucin domain 3) is a type-I glycoprotein receptor with pivotal roles in immune regulation and tolerance induction. Tim-3 has opposing roles in innate and adaptive immunity. It synergizes with TLR signaling in DCs inducing the production of inflammatory cytokines, while it acts as a negative regulator of Th1/Tc1 cell function by triggering T cell apoptosis upon interaction with its ligand, galectin-9. Tim-3 is also responsible for the functional exhaustion of T cells observed in some chronic infectious diseases and tumors, such as metastatic melanoma (MM). Natural Killer (NK) cells, innate immune cells which eliminate tumors and infected cells through cytotoxicity and IFNγ production, constitutively express Tim-3. Moreover, it has been described that some tumor cells (including melanoma cells) express and secrete galectin-9. However, little is known about the role of Tim-3 on NK cells, particularly in the presence of galectin-9, in healthy or melanoma patients. METHODS: Purified NK cells from PBMCs of 20 melanoma donors (MD) and 40 healthy donors (HD) were characterized according to the expression of Tim-3 by flow cytometry. Lamp-1 expression on NK cell surface was used to measure NK cell cytotoxicity against the following target cells: i) K562 cells which don9t express Galectin-9 and ii) Gmel galectin-9+ and Gmel galectin-9- sorted melanoma cells. IFNγ production was assessed by intracellular staining after 4 hours culture in presence of IL-12. RESULTS: MD NK cells express higher levels of Tim-3 compared to HD NK cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5410. doi:1538-7445.AM2012-5410

Melanoma progression is associated with NK cell exhaustion

The immunoregulatory protein T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) contributes towards T cell exhaustion in several chronic diseases, including melanoma [1]. NK cells from the latter were shown to be functionally impaired/exhausted, as they failed to proliferate, produce cytokines or kill target cells. In addition they down regulated activating receptors (NKG2D and NKp46) and upregulated inhibitory receptors (KIRB1, KIRNKAT2 and Tim-3). Notably Tim-3 blockade reversed this exhausted phenotype, implicating this molecule as a major checkpoint inhibitor in advanced melanoma [2]. To further evaluate NK cell phenotype and function as a consequence of progressive melanoma, we monitored NK cells from a large cohort of patients with stage I-IV melanoma tested the association between NK cell phenotype and clinicopathological variables associated with melanoma prognosis. Expression of MICA (NKG2D ligand) and HMGB1 (Tim-3 ligand) in the plasma/sera of our main cohort was also monitored in an independent validation cohort.

Abstract 465: Characterization of NK cell exhaustion in melanoma at different stages.

BACKGROUND: The concept of CD8+ T cell exhaustion in the context of metastatic cancer has been reinforced by the recent success of immunotherapies targeting the exhaustion markers CTLA-4 and PD-1 in advanced melanoma. Exhausted T cells are characterized by: 1) an over expression of inhibitory receptors or exhaustion markers such as CTLA-4, PD-1 and Tim-3; 2) a loss of function (cytotoxicity, cytokine production and proliferation); 3) a down-regulation of cytokine receptors, rendering them refractory to cytokine stimulation; and 4) changes in transcription factors, including downregulation of T-bet expression. T cell exhaustion has been extensively studied in the context of chronic infectious diseases and different types of cancer, however little is known about exhaustion of NK cells in the same background. NK cells, innate immune cells that eliminate tumors through cytotoxicity and IFN-γ production, have a key role in immune surveillance of tumors, including melanoma. In this study, we characterize and compare the phenotype of NK cells from patients with melanoma of different stages (I, II, III, IV) and healthy donors. METHODS: We compared NK cells from 10 patients with melanoma at each stage of presentation (I, II, III, IV) and from 20 healthy donors as it relates to membrane expression of activating (NKG2D and NKp46) and inhibitory receptors (KIRB1 and KIRNKAT2), function (cytotoxicity, IFN-γ production and proliferation) and the intracellular expression of the transcription factor T-bet. NK cells’ cytotoxicity was measured by Lamp-1 expression using K562 cells as target cells. IFN-γ production was measured after 4h stimulation with rhIL-12. Proliferation was quantified by CFSE after 6 days in the presence of rhIL-2. RESULTS: As the stage of the melanoma advanced we observed a pattern of gradual increase of inhibitory receptors (with a higher difference between stage II and stage III) and a concomitant decrease of activating receptors (with a higher difference between stage III and stage IV). With regard to the three most important NK cell functions, IFN-γ production and proliferation were impaired beginning with stage I and advancing to stage IV, while cytotoxicity was reduced only at stage III and stage IV. Similarly, T-bet expression also began to decrease at stage III. CONCLUSIONS: These data suggest that NK cells from melanoma patients gradually develop a phenotypic and functional profile consistent with exhaustion, beginning with stage I and advancing to stage IV. The exhaustion phenotype begins with initial loss of IFN-γ production and proliferation ability, and finally cytotoxicity. Therefore, the concept that NK cells also become exhausted make them an interesting population to target therapeutically. Furthermore, as the exhaustion phenotype is acquired progressively through the stages, and not exclusive to stage IV, it may be possible to intervene early in the course of tumor progression. Citation Format: Ines Pires Da Silva, Anne Gallois, Iman Osman, Nina Bhardwaj. Characterization of NK cell exhaustion in melanoma at different stages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 465. doi:10.1158/1538-7445.AM2013-465

Abstract SY40-02: Interplay between microenvironment and cancers

Tumors are highly adept at evading the immune system through a multitude of mechanisms, including the secretion of factors that modulate both innate and adaptive immunity. Matrix metalloproteinase-2 (MMP-2) is a proteolytic enzyme that degrades the extracellular matrix and is over expressed by many tumors, including melanoma. We recently documented the presence of MMP-2-specific CD4(+) T cells in tumor-infiltrating lymphocytes (TILs) in several melanoma patients (Cancer Cell 19:333, 2011). Strikingly, MMP-2-specific CD4(+) T cells displayed an inflammatory T(H)2 profile, mainly TNF-α, IL-4, and IL-13 and expressing GATA-3. When exposed to MMP-2, immature human dendritic cells (DCs) primed naive CD4(+) T cells to differentiate into an inflammatory T(H)2 phenotype through OX40L expression and inhibition of IL-12p70 production. MMP-2 was subsequently found to degrade the type I IFN receptor, thereby preventing STAT1 phosphorylation, which is necessary for IL-12p35 production, and the subsequent formation of bioactive IL-12. More recently we have explored the mechanisms underlying the up regulation of OX40L and have identified signaling pathways and potential receptors that lead to this modulation. Active MMP-2, therefore, acts as an endogenous type 2 “conditioner” providing an explanation for the observed prevalence of detrimental type 2 responses in melanoma. Novel properties of MMP-2 on other components of the immune system, and on tumor progression, will be discussed along with DC-based strategies to alleviate immune suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY40-02. doi:1538-7445.AM2012-SY40-02