TRIF-mediated TLR3 and TLR4 signaling is negatively regulated by ADAM15.

TLRs are a group of pattern-recognition receptors that play a crucial role in danger recognition and induction of the innate immune response against bacterial and viral infections. The TLR adaptor molecule, Toll/IL-1R domain-containing adaptor inducing IFN (TRIF), facilitates TLR3 and TLR4 signaling and concomitant activation of the transcription factors, NF-κB and IFN regulatory factor 3, leading to proinflammatory cytokine production. Whereas numerous studies have been undertaken toward understanding the role of TRIF in TLR signaling, little is known about the signaling components that regulate TRIF-dependent TLR signaling. To this end, TRIF-interacting partners were identified by immunoprecipitation of the TRIF signaling complex, followed by protein identification using liquid chromatography mass spectrometry. Following stimulation of cells with a TLR3 or TLR4 ligand, we identified a disintegrin and metalloprotease (ADAM)15 as a novel TRIF-interacting partner. Toward the functional characterization of the TRIF:ADAM15 interaction, we show that ADAM15 acts as a negative regulator of TRIF-mediated NF-κB and IFN-ß reporter gene activity. Also, suppression of ADAM15 expression enhanced polyriboinosinic polyribocytidylic acid and LPS-mediated proinflammatory cytokine production via TRIF. In addition, suppression of ADAM15 expression enhanced rhinovirus 16 and vesicular stomatitis virus-mediated proinflammatory cytokine production. Interestingly, ADAM15 mediated the proteolytic cleavage of TRIF. Thus, ADAM15 serves to curtail TRIF-dependent TLR3 and TLR4 signaling and, in doing so, protects the host from excessive production of proinflammatory cytokines and matrix metalloproteinases. In conclusion, to our knowledge, our study clearly shows for the first time that ADAM15 plays an unexpected role in TLR signaling, acting as an anti-inflammatory molecule through impairment of TRIF-mediated TLR signaling.

PD1 Blockade Enhances ICAM1-Directed CAR T Therapeutic Efficacy in Advanced Thyroid Cancer.

PURPOSE: Advanced thyroid cancers, including poorly differentiated and anaplastic thyroid cancer (ATC), are lethal malignancies with limited treatment options. The majority of patients with ATC have responded poorly to programmed death 1 (PD1) blockade in early clinical trials. There is a need to explore new treatment options. EXPERIMENTAL DESIGN: We examined the expression of PD-L1 (a ligand of PD1) and intercellular adhesion molecule 1 (ICAM1) in thyroid tumors and ATC cell lines, and investigated the PD1 expression level in peripheral T cells of patients with thyroid cancer. Next, we studied the tumor-targeting efficacy and T-cell dynamics of monotherapy and combination treatments of ICAM1-targeting chimeric antigen receptor (CAR) T cells and anti-PD1 antibody in a xenograft model of ATC. RESULTS: Advanced thyroid cancers were associated with increased expression of both ICAM1 and PD-L1 in tumors, and elevated PD1 expression in CD8+ T cells of circulating blood. The expression of ICAM1 and PD-L1 in ATC lines was regulated by the IFNγ-JAK2 signaling pathway. ICAM1-targeted CAR T cells, produced from either healthy donor or patient T cells, in combination with PD1 blockade demonstrated an improved ability to eradicate ICAM1-expressing target tumor cells compared with CAR T treatment alone. PD1 blockade facilitated clearance of PD-L1 high tumor colonies and curtailed excessive CAR T expansion, resulting in rapid tumor clearance and prolonged survival in a mouse model. CONCLUSIONS: Targeting two IFNγ-inducible, tumor-associated antigens-ICAM1 and PD-L1-in a complementary manner might be an effective treatment strategy to control advanced thyroid cancers in vivo.

Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity.

Adoptive transfer of high-affinity chimeric antigen receptor (CAR) T cells targeting hematological cancers has yielded impressive clinical results. However, safety concerns regarding target expression on healthy tissue and poor efficacy have hampered application to solid tumors. Here, a panel of affinity-variant CARs were constructed targeting overexpressed ICAM-1, a broad tumor biomarker, using its physiological ligand, LFA-1. Anti-tumor T cell potency in vitro was directly proportional to CAR affinity and ICAM-1 density. In a solid tumor mouse model allowing simultaneous monitoring of anti-tumor potency and systemic off-tumor toxicity, micromolar affinity CAR T cells demonstrated superior anti-tumor efficacy and safety compared to their nanomolar counterparts. Longitudinal T cell tracking by PET/CT and concurrent cytokine measurement revealed superior expansion and contraction kinetics of micromolar affinity CAR T cells. Therefore, we developed an ICAM-1 specific CAR with broad anti-tumor applicability that utilized a reduced affinity targeting strategy to significantly boost efficacy and safety.

CAR T Therapy Targeting ICAM-1 Eliminates Advanced Human Thyroid Tumors.

Purpose: Poorly differentiated thyroid cancer and anaplastic thyroid cancer (ATC) are rare yet lethal malignancies with limited treatment options. Many malignant tumors, including papillary thyroid cancer (PTC) and ATC, are associated with increased expression of ICAM-1, providing a rationale for utilizing ICAM-1-targeting agents for the treatment of aggressive cancer. We developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 to leverage adoptive T-cell therapy as a new treatment modality.Experimental Design: ICAM-1 CAR T cells were applied to multiple malignant and nonmalignant target cells to investigate specific target cell death and "off-tumor" toxicity in vitroIn vivo therapeutic efficacy of ICAM-1 CAR T cells was examined in ATC mouse models established from a cell line and patient-derived tumors that rapidly develop systemic metastases.Results: ICAM-1 CAR T cells demonstrated robust and specific killing of PTC and ATC cell lines in vitro Interestingly, although certain ATC cell lines showed heterogeneous levels of ICAM-1 expression, addition of cytotoxic CAR T cells induced increased ICAM-1 expression such that all cell lines became targetable. In mice with systemic ATC, a single administration of ICAM-1 CAR T cells mediated profound tumor killing that resulted in long-term remission and significantly improved survival. Patient-derived ATC cells overexpressed ICAM-1 and were largely eliminated by autologous ICAM-1 CAR T cells in vitro and in animal models.Conclusions: Our findings are the first demonstration of CAR T therapy against both a metastatic, thyroid cancer cell line and advanced ATC patient-derived tumors that exhibit dramatic therapeutic efficacy and survival benefit in animal studies. Clin Cancer Res; 23(24); 7569-83. ©2017 AACR.

Longitudinal PET imaging demonstrates biphasic CAR T cell responses in survivors.

Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.

The TIR-domain containing adaptor TRAM is required for TLR7 mediated RANTES production.

Toll-like receptor 7 (TLR7) plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV) and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM) plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM(-/-) murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-ß, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNß, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNß and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM(-/-) cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.

Comparative performance of the Uni-Gold™ HSV-2 Rapid: a point-of-care HSV-2 diagnostic test in unselected sera from a reference laboratory.

BACKGROUND: HSV-2 diagnosis is typically by viral culture, viral DNA amplification of lesion material or by serology in cases of subclinical presentation. These methods can be time consuming and expensive. The Uni-Gold™ HSV-2 Rapid is a fast, point-of-care diagnostic test that can be performed outside a full service laboratory. OBJECTIVE: To evaluate the ability of the Uni-Gold™ HSV-2 Rapid to correctly diagnose the presence or absence of anti-HSV-2 antibodies in patient serum samples in comparison to the University of Washington HSV Western blot (UWWB). STUDY DESIGN: Sera from 100 adult patients in the USA were tested for HSV-2 specific antibodies by Uni-Gold™ HSV-2 Rapid and results were compared to those of the UWWB to determine the test's sensitivity and specificity. RESULTS: Of 18 patients seropositive for HSV-2 by UWWB, 17 were correctly identified as such by the Uni-Gold™ HSV-2 Rapid. Of 76 patients who were seronegative for HSV-2 by UWWB, 75 were correctly identified by the rapid test. Six sera had indeterminate results by UWWB. Sensitivity for the Uni-Gold™ HSV-2 Rapid was 94% and specificity was 99%. CONCLUSION: The Uni-Gold™ HSV-2 Rapid had high sensitivity and specificity in a small sample of unselected, adults seeking care in the Seattle, USA area. An accurate, near-person test allows immediate counseling directed toward symptom recognition, treatment, and practices that can limit the risk of HSV-2 transmission.