Novel Immune Targets in Melanoma—Response

We thank Nocentini and colleagues for their interest in our recent work in which we reported on the prevalence and distribution of the expression of novel checkpoint targets in melanoma, as published in Clinical Cancer Research (1). In their letter, the authors state that anti-GITR clone 621, an antibody used in our CyTOF analysis, but not immunohistochemical experiments, may not be as sensitive for detecting GITR as other clones. To address possible differences in the sensitivity of different anti-GITR antibodies, we performed a direct comparison of GITR staining by anti-GITR clone 621 and anti-GITR clone DT5D3 (the clone recommended by Nocentini and colleagues) using flow cytometry and compared the levels of GITR detected on immune cells in three human melanoma tumor dissociates.We found that the average levels of GITR detected on immune cell subsets was comparable between the two clones, albeit with 2% average increase in GITR being detected on CD4+ T cells by clone DT5D3, and elevated levels of GITR seen on myeloid cells with clone 621 (Fig. 1A). We note, however, that there were differences in the level of GITR detected by the two clones within the same tumor sample in some cases (Fig. 1B). Nonetheless, the results provided from anti-GITR clone DT5D3 suggest that <1% of CD8 T cells, approximately 2%–3% of CD4 T cells, and approximately 1%–2% of total T cells in the tumor microenvironment express GITR (Fig. 1A), reaffirming our previous conclusion that GITR is minimally expressed on T cells in the tumor microenvironment. While we previously reported an average 2% expression of GITR on B cells and monocytes with anti-GITR clone 621, results utilizing clone DT5D3 show that GITR expression on these cells was <1%.In one of the three melanoma tumor dissociates, we found sufficient numbers of FoxP3+ CD25hi T-regulatory cells to compare levels of GITR detected by anti-GITR clone DT5D3 and anti-GITR clone 621. In this sample, the anti-GITR clone DT5D3 did detect a significantly higher proportion of GITR+ regulatory T cells than with the anti-GITR clone 621, and with better resolution of GITR+ cells (Fig. 1C). Indeed, anti-GITR clone DT5D3 resolved GITR-positive cells better than anti-GITR clone 621 on all immune cell subsets examined (Fig. 1D).In conclusion, while differences in GITR expression levels were observed between anti-GITR clone DT5D3 and anti-GITR clone 621, the overall results from both antibodies were comparable, with the notable exception of GITR staining on regulatory T cells. The results provided by anti-GITR clone DT5D3 reaffirm our conclusions on low level GITR expression in the tumor microenvironment in melanoma, with the exception that a higher proportion of regulatory T cells are likely to be positive for GITR, previously undetected by anti-GITR clone 621.See the original Letter to the Editor, p. 5422G. V. Long is a consultant/advisory board member for Aduro, Amgen, Array Biopharma, Bristol-Myers Squibb, Merck Sharpe & Dohme, Novartis, Oncosec, Pierre Fabre, and Roche. R.A. Scolyer is a consultant/advisory board member for Merck Sharp & Dohme, Novartis, Myriad, and NeraCare. No potential conflicts of interest were disclosed by the other authors.This work was supported by Melanoma Institute Australia, the New South Wales Department of Health, NSW Health Pathology, the National Health and Medical Research Council of Australia (NHMRC) and Cancer Institute NSW. J.S. Wilmott, R.A. Scolyer, and G. Long are supported by NHMRC Fellowships. U. Palendira and A. Ferguson are supported by the Cancer Council NSW (RG18-08). G. Long is supported by the Melanoma Foundation of the University of Sydney through the University of Sydney Medical Foundation. J. Edwards is supported by the Research Training Program and Sydney Catalyst scholarships at The University of Sydney.