Faculty Bibliography

The TLR7/8 agonist, Resiquimod has been used as an immune adjuvant in cancer vaccines. We evaluated the safety and immunogenicity of the cancer testis antigen NY-ESO-1 given in combination with Montanide with or without Resiquimod in high risk melanoma patients. In Part I of the study, patients received 100ug full length NY-ESO-1 protein emulsified in 1.25mL Montanide (day 1) followed by topical application of 1000mg of 0.2% Resiquimod gel on days 1 and 3 (Cohort 1) versus days 1, 3, and 5 (Cohort 2) of a 21 day cycle. In Part II, patients were randomized to receive 100ug NY-ESO-1 protein plus Montanide (day 1) followed by topical application of placebo gel (Arm-A; N=8) or 1000mg of 0.2% Resiquimod gel (Arm-B; N=12) using the dosing regimen established in Part I. The vaccine regimens were generally well-tolerated. NY-ESO-1 specific humoral responses were induced or boosted in all patients, many with high titers. In Part II, 16 of 20 patients in both arms had NY-ESO-1-specific CD4+ T cell responses. CD8+ T cell responses were only seen in 3 of 12 patients in Arm B. Patients with TLR7 SNP rs179008 had a greater likelihood of developing NY-ESO-1-specific CD8+ responses. In conclusion, NY-ESO-1 protein in combination with Montanide with or without topical Resiquimod is safe and induces both antibody and CD4+ cell responses in the majority of patients; the small proportion of CD8+ cell responses suggests that the addition of topical Resiquimod to Montanide is not sufficient to induce consistent NY-ESO-1 specific CD8+ cell responses.

Eradication of human immunodeficiency virus-1 (HIV-1) from an infected individual requires a means of inducing production of virus from latently infected cells and stimulating an immune response against the infected cells. We report the development of lentiviral vectors that transduce dendritic cells (DCs) to both induce production of virus from latently infected cells and stimulate antigen-specific cytotoxic T lymphocytes (CTLs). The vectors package Vpx, a lentiviral accessory protein that counteracts the SAMHD1-mediated block to DC transduction, allowing for long-term expression of vector-encoded proteins. The vectors encode influenza or HIV-1-derived epitopes fused via a self-cleaving peptide to CD40L that releases the peptide into the endoplasmic reticulum for entry into the antigen presentation pathway. Expression of CD40L caused transduced DCs to mature and produce Th1-skewing cytokines. The DCs presented antigen to CD8 T cells, enhancing antigen-specific CTLs. Coculture of the transduced DCs with latently infected cells induced high-level virus production, an effect that was mediated by tumor necrosis factor alpha. The ability of a DC vaccine to reactivate latent HIV-1 and stimulate an adaptive immune response provide a means to reduce the size of the latent reservoir in patients. This strategy can also be applied to develop DC vaccines for other diseases.

We sought to define cellular immune mechanisms of synergy between tumor-antigen-targeted monoclonal antibodies and chemotherapy. Established B16 melanoma in mice was treated with cytotoxic doses of cyclophosphamide in combination with an antibody targeting tyrosinase-related protein 1 (alphaTRP1), a native melanoma differentiation antigen. We find that Fcgamma receptors are required for efficacy, showing that antitumor activity of combination therapy is immune mediated. Rag1(-/-) mice deficient in adaptive immunity are able to clear tumors, and thus innate immunity is sufficient for efficacy. Furthermore, previously treated wild-type mice are not significantly protected against tumor reinduction, as compared with mice inoculated with irradiated B16 alone, consistent with a primarily innate immune mechanism of action of chemo-immunotherapy. In contrast, mice deficient in both classical natural killer (NK) lymphocytes and nonclassical innate lymphocytes (ILC) due to deletion of the IL2 receptor common gamma chain IL2gammac(-/-)) are refractory to chemo-immunotherapy. Classical NK lymphocytes are not critical for treatment, as depletion of NK1.1(+) cells does not impair antitumor effect. Depletion of CD90(+)NK1.1(-) lymphocytes, however, both diminishes therapeutic benefit and decreases accumulation of macrophages within the tumor. Tumor clearance during combination chemo-immunotherapy with monoclonal antibodies against native antigen is mediated by the innate immune system. We highlight a novel potential role for CD90(+)NK1.1(-) ILCs in chemo-immunotherapy.

HIV-1 infection is characterized by myeloid dendritic cell (DC) dysfunction which blunts their responsiveness to vaccine adjuvants. We previously showed that non-viral factors in HIV-seropositive plasma are partially responsible for mediating this immune suppression. In this study we investigated recombinant Listeria monocytogenes (Lm) vectors, which naturally infect and potently activate DCs from seronegative donors, as a means to overcome DC dysfunction associated with HIV infection. Monocyte-derived DCs were cocultured with plasma from HIV-infected donors (HIV-moDCs) to induce a dysregulated state and infected with an attenuated, non-replicative vaccine strain of Lm expressing full length clade B consensus gag (KBMA Lm-gag). Lm infection stimulated cytokine secretion (IL-12p70, TNFalpha, IL-6) and Th-1 skewing of allogeneic naive CD4 T cells by HIV-moDCs, in contrast to the suppressive effects observed by HIV plasma on moDCs upon toll-like receptor ligand stimulation. Upon coculture of KBMA Lm-gag-infected moDCs from HIV-infected donors with autologous cells, expansion of polyfunctional, gag-specific CD8+ T cells was observed. Reactivation of latent proviruses by moDCs following Lm infection was also observed in models of HIV latency in a TNFalpha-dependent manner. These findings reveal the unique ability of Lm vectors to contend with dysregulation of HIV-moDCs , while simultaneously possessing the capacity to activate latent virus. Concurrent stimulation of innate and adaptive immunity and disruption of latency may be an approach to reduce the pool of latently infected cells during HIV infection. Further study of Lm vectors as part of therapeutic vaccination and eradication strategies may advance this evolving field.

Matrix metalloproteinase-2 (MMP-2) is involved in several physiological mechanisms, including wound healing and tumor progression. We show that MMP-2 directly stimulates dendritic cells (DCs) to both upregulate OX40L on the cell surface and secrete inflammatory cytokines. The mechanism underlying DC activation includes physical association with Toll-like receptor-2 (TLR2), leading to NF-kappaB activation, OX40L upregulation on DCs, and ensuing TH2 differentiation. Significantly, MMP-2 polarizes T cells toward type 2 responses in vivo, in a TLR2-dependent manner. MMP-2-dependent type 2 polarization may represent a key immune regulatory mechanism for protection against a broad array of disorders, such as inflammatory, infectious, and autoimmune diseases, which can be hijacked by tumors to evade immunity.

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.

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.