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Purpose: Activation of Toll-like receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcgamma receptor (FcgammaR) function, as they are critical mediators of antibody therapy., Experimental Design: The effect of the TLR7/8 agonist R-848 on cytokine production and antibody-dependent cellular cytotoxicity by human peripheral blood monocytes was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848 and Western blots were done to measure protein levels of FcgammaR. Murine bone marrow-derived macrophages from WT and knockout mice were examined to determine the downstream pathway involved with regulating FcgammaR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model., Results: Overnight incubation with R-848 increased FcgammaR-mediated cytokine production and antibody-dependent cellular cytotoxicity in human peripheral blood monocytes. Expression of FcgammaRI, FcgammaRIIa, and the common gamma-subunit was increased. Surprisingly, expression of the inhibitory FcgammaRIIb was almost completely abolished. In bone marrow-derived macrophage, this required TLR7 and MyD88, as R-848 did not increase expression of the gamma-subunit in TLR7(-/-) nor MyD88(-/-) cells. In a mouse solid tumor model, R-848 treatment superadditively enhanced the effects of antitumor antibody., Conclusions: These results show an as-yet-undiscovered regulatory and functional link between the TLR7/8 and FcgammaR pathways. This suggests that TLR7/8 agonists may be especially beneficial during antibody therapy., (Copyright 2010 AACR.)

Background: Regulation of gene expression by signal transducer and activator of transcription 1 (STAT1) within host tissues mediates the antitumor effects of interferon alfa (IFN α). We used a novel flow cytometric assay to examine phosphorylation-mediated activation of STAT1 within immune effector cell subsets following in vitro or in vivo IFN α treatments. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 17) or melanoma patients (n = 19) were treated in vitro with interferon alfa-2b (IFN α-2b) or phosphate-buffered saline (PBS) and subjected to multiparametric flow cytometry to measure the levels of phosphorylated STAT1 (P-STAT1) within immune cell subsets. We similarly analyzed PBMCs isolated from melanoma patients before and 1 hour after immunotherapy with IFN α-2b. All statistical tests were two-sided. Results: P-STAT1 levels in all major immune cell subsets increased within 15 minutes of in vitro IFN α-2b treatment of PBMCs; the increase was most pronounced in T lymphocytes and monocytes. Relatively low doses of IFN α-2b (i.e., 10²-10³ IU/mL) induced maximal STAT1 activation in vitro. Compared with melanoma patients, healthy donors had higher basal levels of P-STAT1 (specific fluorescence [Fsp]; i.e., FspPBS, the level of P-STAT1 in PBS-treated cells) in total PBMCs, natural killer (NK) cells, and T cells (mean FspPBS in total PBMCs: 5.5 in healthy donors versus 1.6 in patients, difference = 3.9, 95% confidence interval [CI] = 1.4 to 6.5, P = .004; mean FspPBS in NK cells: 4.6 in healthy donors versus 0.9 in patients, difference = 3.7, 95% CI = 1.7 to 5.7, P = .001; mean FspPBS in T cells: 6.8 in healthy donors versus 0.9 in patients, difference = 5.9, 95% CI = 2.5 to 9.3, P = .002). P-STAT1 was detected in the NK and T cells of two patients who received IFN α-2b immunotherapy (20 MU/m² [MU = million units], administered by intravenous injection). P-STAT1 levels in the PBMCs of a patient treated sequentially with 5 MU/m² and 10 MU/m² IFN α-2b (administered by subcutaneous injection) also increased in response to treatments with IFN α-2b but did not increase further with the increased dosage of IFN α-2b. Conclusion: This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN α immunotherapy. [ABSTRACT FROM AUTHOR]

This proof-of-concept study investigates the immune effects in metastatic breast cancer (MBC) patients after “vaccination” with activated T cells (ATC) armed with anti-CD3 x anti-HER2 bispecific antibody (HER2 BATs) followed by immune consolidation with immune ATC “boost” after high dose chemotherapy (HDC) and autologous stem cell transplant (SCT). Approximately 2 weeks after completion of vaccination portion of the study, immune T cells were obtained by leukopheresis, activated and expanded ex vivo and re-infused after HDC and SCT to test the hypothesis that transfer of immune unarmed ATC would accelerate reconstitution of anti-tumor activity after SCT. Eight metastatic breast cancer (MBC) patients received 8 infusions of HER2 BATs, low dose IL-2, and GM-CSF in the first part of the protocol to induce adaptive cellular and humoral responses. In the “boost” portion of the protocol, 6 of 8 patients received multiple infusions of unarmed ATC post SCT. There were no dose-limiting toxicities or delays in engraftment. Four of 6 patients tested for the immune correlative studies exhibited increases in anti-breast cancer (BrCa) cytotoxicity, antigen specific IFN-γ Elispots, anti-BrCa antibodies and increased IL-12 and Th1 serum cytokine levels after HER2 BATs infusions. Anti-BrCa tumor responses were seen as early as 2 weeks after SCT and persisted up to 2 years post-SCT. One out of 6 patients’ rapidly progressed and showed poor immune responses and high Th2 cytokine levels. There was a significant correlation (p < 0.002) between time to progression (TTP) and anti-BrCa cytotoxicity by immune T cells. This is the first study to show that adoptive transfer of immune T cells after SCT accelerates reconstitution of anti-BrCa specific immunity and correlates with delay TTP. [ABSTRACT FROM AUTHOR]