Ipilimumab increases activated T cells and enhances humoral immunity in patients with advanced melanoma.

Ipilimumab, a fully human monoclonal antibody, which blocks cytotoxic T-lymphocyte antigen-4, has demonstrated an improvement in overall survival in 2 phase III trials of patients with advanced melanoma. To gain an understanding of its mechanism of action, the effects of ipilimumab on T-cell populations and on humoral immune responses were studied in patients with advanced melanoma from 2 phase II trials. Antibody levels against 5 tumor antigens were assessed at baseline and up to 12 weeks after ipilimumab treatment. Serologic reactivity to the cancer-testis antigen NY-ESO-1 increased by at least 5-fold at week 12 of treatment in 10% to 13% of patients. Increased antibody levels were also observed to the tumor antigens Melan-A, MAGE-A4, SSX2, and p53. Immunocompetence was evaluated with tetanus boosters administered before ipilimumab and pneumococcal and influenza vaccines given 5 days after ipilimumab treatment. At week 7, most patients who received ipilimumab and vaccine showed greater humoral responses relative to baseline titers. For peripheral T-cell populations, statistically significant increases in the percent of activated (HLA-DR) CD4 and CD8 T cells with concomitant decreases in naive CD4 and CD8 T cells were observed after ipilimumab treatment. These changes were evident by week 4 of treatment. Increases were also observed in central memory, effector memory, and activated ICOS CD4 T cells, but not in ICOS CD8 T cells or in FoxP3 CD4 regulatory T cells. These results suggest that ipilimumab can enhance immune responses mediated by different T-cell populations, and humoral immunity, in melanoma patients.

Recommendations for the validation of flow cytometric testing during drug development: II assays.

Flow cytometry-based assays serve as valuable tools for various aspects of the drug development process ranging from target discovery and characterization to evaluation of responses in a clinical setting. The integrity of the samples and the appropriate selection and characterization of the reagents used in these assays are in themselves challenging. These concerns taken together with the flow-based technology makes the validation of flow cytometry assays a challenging effort. Therefore, apart from summarizing the role of flow cytometry technology in various stages of drug development, this manuscript focuses on recommendations for the validation of methods applying flow cytometry. Information is also provided on the relevant validation parameters for different types of flow cytometry assays to guide the users of this platform. Together, the recommendations and the information on regulatory guidelines provided in this manuscript represent the consensus of all the authors and can assist the flow cytometry user in implementing the appropriate method validation strategies.

Development and validation of flow cytometry methods for pharmacodynamic clinical biomarkers.

Flow cytometry is a powerful analytical tool for the analysis of multiple biological parameters of individual cells or particles within heterogeneous cell populations. It has been widely used in biomedical research to perform immunophenotyping, cell counting and numerous cell function assessments, such as intracellular cytokine production, protein phosphorylation, cell proliferation and apoptosis. The implementation of standardized flow cytometry-based biomarker assays in clinical trials remains a challenge due to the limited stability of clinical specimens and the technical variations between instruments. To ensure data quality, it is crucial to develop robust assays for clinical applications. In this review, we summarize current practice in developing, validating and implementing flow cytometry assays to evaluate biomarkers in clinical research.

Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand.

GD3, a ganglioside expressed on melanoma, is the only tumour-associated glycolipid described to date that can induce a CD1d-restricted natural killer T (NKT)-cell response. We analysed the fine specificity of GD3-reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3-reactive NKT cells. One population was CD4+ CD8- and was specific for GD3; the other population was CD4- CD8- and cross-reacted with GM3 in a CD1d-restricted manner, but did not cross-react with GM2, GD2, or lactosylceramide. This indicated that the T-cell receptors reacting with GD3 recognize glucose-galactose linked to at least one N-acetyl-neuraminic acid but will not accommodate a terminal N-acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT-cell response. Coimmunization of GM3-loaded antigen-presenting cells (APCs) with GD3-loaded APCs suppressed the NKT-cell response to GD3 in a CD1d-restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2-4 days. In vitro, GM3-loaded APCs also suppressed the interleukin-4 response, but not the interferon-gamma response, of NKT cells to alpha-galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2-like NKT-cell responses.

Cross-presentation of disialoganglioside GD3 to natural killer T cells.

GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2- CD1-) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of approximately 1:2000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.