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Purpose: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody-based immunotherapy that includes the activation of natural killer (NK) cells.Experimental Design: HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibodydependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcgR3A singlenucleotide polymorphism (SNP).Results: VTX-2337 selectively activates TLR8 with an EC 50 of about 100 nmol/L and stimulates production of TNFa and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNg production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNg production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcgR3A genotypes (V

Standard in vitro methods for assessing T-cell activation have typically measured either the proliferative responses of peripheral blood mononuclear cell (PBMC) cultures to various provocative stimuli employing tritiated thymidine incorporation or the secretion of specific cytokines from activated cells. These bulk assay methods suffer the drawback of being lengthy assays, and, in addition, they do not provide information about functional responses of individual lymphocyte subsets. This report describes a three-color flow cytometric method for the rapid analysis (4 hours) of individual T-cell subsets in whole blood responding to various provocative stimuli, including pokeweed mitogen, the comitogenic monoclonal antibodies (mAbs) CD2/CD2R, the superantigen staphylococcal enterotoxin B (SEB), and the speciac antigen Candlda albicans. After 4 hours, CD69 expression in response to CD2/ CDZR paralleled thymidine incorporation measured after 72 hours. Variations in the proportions of CD4+ and CD4-T cells expressing CD69 were observed with Merent stimuli. These observations demonstrate the potential of multiparameter flow cytometry for the investigation of functional responses of individual T-cell subsets to a variety of stimuli in whole blood. o 1995 wiley-Liss, Inc.

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Background: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, “cross-priming” can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor’s blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual’s APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8+ T cells, and a lower proportion of IFN-γ-producing CD4+ and/or CD8+ T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8+ T cells in breast cancer patients was almost completely CD28+CD45RA− (memory phenotype). CMV-responsive CD8+ T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27−CD28−CD45RA+) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.