CTLA-4 blockade enhances polyfunctional NY-ESO-1 specific T cell responses in metastatic melanoma patients with clinical benefit

Blockade of inhibitory signals mediated by cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to enhance T cell responses and induce durable clinical responses in patients with metastatic melanoma. The functional impact of anti-CTLA-4 therapy on human immune responses is still unclear. To explore this, we analyzed immune-related adverse events and immune responses in metastatic melanoma patients treated with ipilimumab, a fully human anti-CTLA-4 monoclonal antibody. Fifteen patients were selected on the basis of availability of suitable specimens for immunologic monitoring, and eight of these showed evidence of clinical benefit. Five of the eight patients with evidence of clinical benefit had NY-ESO-1 antibody, whereas none of seven clinical non-responders was seropositive for NY-ESO-1. All five NY-ESO-1 seropositive patients had clearly detectable CD4+ and CD8+ T cells against NY-ESO-1 following treatment with ipilimumab. One NY-ESO-1 seronegative clinical responder also had a NY-ESO-1 CD4+ and CD8+ T cell response, possibly related to prior vaccination with NY-ESO-1. Among five clinical non-responders analyzed, only one had a NY-ESO-1 CD4+ T cell response and this patient did not have detectable anti-NY-ESO-1 antibody. Overall, NY-ESO-1-specific T cell responses increased in frequency and functionality during anti-CTLA-4 treatment, revealing a polyfunctional response pattern of IFN-γ, MIP-1β and TNF-α. We therefore suggest that CTLA-4 blockade enhanced NY-ESO-1 antigen-specific B cell and T cell immune responses in patients with durable objective clinical responses and stable disease. These data provide an immunologic rationale for the efficacy of anti-CTLA-4 therapy and call for immunotherapeutic designs that combine NY-ESO-1 vaccination with CTLA-4 blockade

Integrated NY-ESO-1 antibody and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated with ipilimumab

Ipilimumab, a monoclonal antibody against cytotoxic T lymphocyte antigen 4 (CTLA-4), has been shown to improve survival in patients with advanced metastatic melanoma. It also enhances immunity to NY-ESO-1, a cancer/testis antigen expressed in a subset of patients with melanoma. To characterize the association between immune response and clinical outcome, we first analyzed NY-ESO-1 serum antibody by ELISA in 144 ipilimumab-treated patients with melanoma and found 22 of 140 (16%) seropositive at baseline and 31 of 144 (22%) seropositive following treatment. These NY-ESO-1–seropositive patients had a greater likelihood of experiencing clinical benefit 24 wk after ipilimumab treatment than NY-ESO-1–seronegative patients (P = 0.02, relative risk = 1.8, two-tailed Fisher test). To understand why some patients with NY-ESO-1 antibody failed to experience clinical benefit, we analyzed NY-ESO-1–specific CD4+ and CD8+ T-cell responses by intracellular multicytokine staining in 20 NY-ESO-1–seropositive patients and found a surprising dissociation between NY-ESO-1 antibody and CD8 responses in some patients. NY-ESO-1–seropositive patients with associated CD8+ T cells experienced more frequent clinical benefit (10 of 13; 77%) than those with undetectable CD8+ T-cell response (one of seven; 14%; P = 0.02; relative risk = 5.4, two-tailed Fisher test), as well as a significant survival advantage (P = 0.01; hazard ratio = 0.2, time-dependent Cox model). Together, our data suggest that integrated NY-ESO-1 immune responses may have predictive value for ipilimumab treatment and argue for prospective studies in patients with established NY-ESO-1 immunity. The current findings provide a strong rationale for the clinical use of modulators of immunosuppression with concurrent approaches to favor tumor antigen-specific immune responses, such as vaccines or adoptive transfer, in patients with cancer

Analysis of the High-Affinity Iron Uptake System at the Chlamydomonas reinhardtii Plasma Membrane ▿

Multicopper ferroxidases play a vital role in iron metabolism in bacteria, fungi, algae, and mammals. Saccharomyces cerevisiae utilizes a channeling mechanism to couple the ferroxidase activity of Fet3p to Fe3+ transport into the cell by Ftr1p. In contrast, the mechanisms by which mammals couple the ferroxidase reaction to iron trafficking is unclear. The human ferroxidases ceruloplasmin and hephaestin are twice the size of Fet3p and interact with proteins that are not expressed in fungi. Chlamydomonas FOX1 is a homolog of the human ferroxidases but likely supports iron uptake in a manner similar to that of yeast, since Chlamydomonas reinhardtii expresses a ferric iron permease homolog, FTR1. The results presented support this hypothesis. We show that FOX1 is trafficked to the plasma membrane and is oriented with its multicopper oxidase/ferroxidase domain in the exocytoplasmic space. Our analysis of FTR1 indicates its topology is similar to that of S. cerevisiae Ftr1p, with a potential exocytoplasmic iron channeling motif and two potential iron permeation motifs in membrane-spanning regions. We demonstrate that high-affinity iron uptake is dependent on FOX1 and the copper status of the cell. Kinetic inhibition of high-affinity iron uptake by a ferric iron chelator does not reflect the strength of the chelator, supporting a ferric iron channeling mechanism for high-affinity iron uptake in Chlamydomonas. Last, recombinant FOX1 (rFOX1) has been isolated in a partially holo form that exhibits the UV-visible absorbance spectrum of a multicopper oxidase and the catalytic activity of a ferroxidase