Faculty Bibliography

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.

Introduction: 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. Methods: NK cells were purified from the peripheral blood of melanoma patients. They were evaluated for the expression of activating and inhibitory receptors. Cytotoxicity was measured by Lamp-1 expression. IFN-gamma production was measured after 4h stimulation with rhIL-12. Proliferation was quantified by CFSE after 6 days in the presence of rhIL- 2. MICA and HMGB1 expression on patients' plasma/sera was measured by ELISA. Event-time distributions were estimated with the use of the Kaplan-Meier method. Two tailed t-test unpaired was used to compare samples from different stages. Results: NK cells gradually develop a phenotypic and functional profile consistent with progressive exhaustion, from stage I to stage IV characterized by: 1) up-regulation of inhibitory receptors (Tim-3, KIRB1 and KIRNKAT2); 2) down-regulation of activating receptors (NKG2D and NKp46); 3) loss of IFN-gamma production, proliferation and cytotoxicity. Interestingly, the expression of Tim-3 is higher, while the cytotoxicy and IFN-gamma production is reduced in patients with melanomas thicker than 1mm. Moreover, higher expression of Tim-3 and KIRB1, and a lower cytotoxic ability and T-bet expression is associated with local or distant metastases. Higher expression of MICA in the plasma was associated with worse prognosis, as was validated in an independent cohort (Figure 1). Conclusions: These data demonstrate that NK cells become progressively exhausted in the context of melanoma progression and that Tim-3 blockade possibly earlier in disease may have some benefit on innate immune function. Finally, our data suggest that soluble MICA is a potential prognostic marker which may contribute to the NK cell exhaustion through its interactions with NKG2D

T cells play a critical role in host defense against viruses, intra- and extracellular microbes, and tumors. Because foreign antigen is presented amongst a vast majority of self-antigens, T cells have evolved the unique ability to discriminate "self" from "non-self" with high sensitivity and selectivity, enabling the elimination of foreign pathogens while largely avoiding self-reactivity. However, tissue-specific autoimmunity and tolerance to or eradication of cancer does not fit neatly into the self/non-self paradigm because the T cell responses in these situations are not directed to an exogenous pathogen, but rather most often to non-mutated self-proteins. Therefore, an important question is how the immune system establishes suitable thresholds that allow positively selected T cells to interact with selfligands in the periphery without causing overt activation. One hypothesis to explain how a T cell distinguishes among different types of self-ligands is the kinetic proof-reading theory, which relates signaling efficacy to the lifetime of the TCR (T cell receptor)-pMHC (peptide-major histocompatibility complex) interaction. More recently, T cell maturation associated signaling feedback pathways have also been hypothesized to play a role in T cell discrimination of between self-ligands. We are taking a variety of biophysical and cellular imaging approaches to determine how specific thresholds for T cell recognition of self-antigens are set. Our recent results [1] indicate that antitumor activity and autoimmunity are coupled and have a similar kinetic threshold; reducing autoimmunity cannot be accomplished without sacrificing efficacy of tumor killing. Therefore, an "optimal TCR affinity range" that leads to optimal tumor regression and minimal autoimmunity is elusive and treatment strategies focusing on increasing TCR affinities to a supraphysiological level has most likely little therapeutic benefit. Therefore, other approaches are needed to improve the balance between anti-tumor responses and autoimmunity. Our strategy to overcome this issue includes novel methods for careful biophysical engineering of tumor-specific TCRs to carefully balance tumorreactivity and autoimmunity. Furthermore, our recent preliminary data show that TCR-proximal signaling differs significantly between effector memory and central memory T cells due to differential constitutive activity and localization of signaling molecules. Understanding how activation signaling contributes to differences in memory T cell subset sensitivity may provide insight into how T cells can be manipulated to achieve optimal anti-tumor sensitivity. This could lead to adjuvants that target and enhance antigenspecific T cell anti-tumor efficacy. Together may lead to development of cancer immunotherapy approaches with improved outcomes

Current surgical treatment of primary melanoma is uniform for all histosubtypes, although certain types of melanoma, such as acral lentiginous melanoma (ALM), have a worse prognosis. No study has explored the effectiveness of standard melanoma treatment guidelines for managing ALM compared with nonacral melanoma (NAM). Study subjects were identified from a prospectively enrolled database of patients with primary melanoma at New York University. Patients with ALM were matched to those with NAM (1:3) by gender and melanoma stage, including substage (ALM, 61; NAM, 183). All patients received standard-of-care treatment. Recurrence and survival outcomes in both cohorts were compared. ALM histologic subtype was an independent negative predictor of recurrence-free survival (hazard ratio [HR], 2.24; P=.001) and melanoma-specific survival (HR, 2.58; P=.001) compared with NAM. Recurrence was significantly more common in patients with ALM than in those with NAM (49% vs 30%; P=.007). For tumors less than 2 mm in thickness, a significantly higher recurrence rate was seen with ALM versus NAM (P=.048). No significant difference was seen in recurrence for tumors greater than 2 mm (P=.12). Notably, the rate of locoregional recurrence was nearly double for ALM compared with NAM (P=.001). The data presented herein reveal a high rate of locoregional failure in ALM compared with NAM when controlling for AJCC stage. These results raise the question of whether ALM may require more aggressive surgical treatment than nonacral cutaneous melanomas of equal thickness, particularly in tumors less than 2 mm thick. Larger multicenter trials are necessary for further conclusions.

BackgroundMatrix metalloproteinase-23 (MMP-23) can block the voltage-gated potassium channel Kv1.3, whose function is important for sustained Ca2+ signaling during T cell activation. MMP-23 may also alter T cell activity and phenotype through cleavage of proteins affecting cytokine and chemokine signaling. We therefore tested the hypothesis that MMP-23 can negatively regulate the anti-tumor T cell response in human melanoma.MethodsWe characterized MMP-23 expression in primary melanoma patients who received adjuvant immunotherapy. We examined the association of MMP-23 with the anti-tumor immune response - as assessed by the prevalence of tumor-infiltrating lymphocytes and Foxp3+ regulatory T cells. Further, we examined the association between MMP-23 expression and response to immunotherapy. Considering also an in trans mechanism, we examined the association of melanoma MMP-23 and melanoma Kv1.3 expression.ResultsOur data revealed an inverse association between primary melanoma MMP-23 expression and the anti-tumor T cell response, as demonstrated by decreased tumor-infiltrating lymphocytes (TIL) (P inverted question mark= inverted question mark0.05), in particular brisk TILs (P inverted question mark= inverted question mark0.04), and a trend towards an increased proportion of immunosuppressive Foxp3+ regulatory T cells (P inverted question mark= inverted question mark0.07). High melanoma MMP-23 expression is also associated with recurrence in patients treated with immune biologics (P inverted question mark= inverted question mark0.037) but not in those treated with vaccines (P inverted question mark= inverted question mark0.64). Further, high melanoma MMP-23 expression is associated with shorter periods of progression-free survival for patients receiving immune biologics (P inverted question mark= inverted question mark0.025). On the other hand, there is no relationship between melanoma MMP-23 and melanoma Kv1.3 expression (P inverted question mark= inverted question mark0.27).ConclusionsOur data support a role for MMP-23 as a potential immunosuppressive target in melanoma, as well as a possible biomarker for informing melanoma immunotherapies.