Alexander W. MacFarlane

PD-1 Expression on Peripheral Blood Cells Increases with Stage in Renal Cell Carcinoma Patients and Is Rapidly Reduced after Surgical Tumor Resection

MacFarlane and colleagues show that tumor resection reverses PD-1 expression on peripheral blood (PB) immune cells and suggest that PD-1 blockade for renal cell carcinoma patients with PD-1 expression on PB cells would be most effective prior to surgery, especially in early-stage cancer. Programmed death-1 (PD-1) receptor is an inhibitory receptor on hematopoietic cells that can negatively regulate immune responses, particularly responses to tumors, which often upregulate PD-1 ligands. PD-1/PD-1 ligand blocking antibodies can reverse the inhibition and show significant therapeutic promise in treating renal cell carcinoma (RCC), lung cancer, and melanoma. While PD-1 expression on tumor-infiltrating lymphocytes has been associated with poor outcome in RCC, we sought to define immune cell biomarkers, including PD-1, on peripheral blood mononuclear cells (PBMC) that could predict disease progression of RCC patients before and after nephrectomy. We analyzed expression of numerous immune cell markers on fresh PBMCs from 90 RCC patients preoperatively and 25 age-matched healthy controls by 10-color flow cytometry. Postoperative blood samples were also analyzed from 23 members of the RCC patient cohort. The most striking phenotypic immune biomarker in RCC patients was a significant increase in PD-1 expression on certain PBMCs in a subset of patients. Increased PD-1 expression on CD14bright myelomonocytic cells, effector T cells, and natural killer (NK) cells correlated to disease stage, and expression was significantly reduced on all cell types soon after surgical resection of the primary tumor. The results indicate that PD-1 expression on fresh peripheral blood leukocytes may provide a useful indicator of RCC disease progression. Furthermore, measuring PD-1 levels in peripheral blood may assist in identifying patients likely to respond to PD-1 blocking antibodies, and these therapies may be most effective before and immediately after surgical resection of the primary tumor, when PD-1 expression is most prominent. Cancer Immunol Res; 2(4); 320–31. ©2013 AACR.

Ubiquitylation of an Internalized Killer Cell Ig-Like Receptor by Triad3A Disrupts Sustained NF-κB Signaling

Killer cell Ig-like receptor (KIR) with two Ig-like domains and a long cytoplasmic domain 4 (2DL4; CD158d) is a unique KIR expressed on human NK cells, which stimulates cytokine production, but mechanisms regulating its expression and function are poorly understood. By yeast two-hybrid screening, we identified the E3 ubiquitin ligase, Triad3A, as an interaction partner for the 2DL4 cytoplasmic domain. The protein interaction was confirmed in vivo, and Triad3A expression induced polyubiquitylation and degradation of 2DL4. Overexpression of Triad3A selectively abrogated the cytokine-producing function of 2DL4, whereas Triad3A short hairpin RNA reversed ubiquitylation and restored cytokine production. Expression of Triad3A in an NK cell line did not affect receptor surface expression, internalization, or early signaling, but significantly reduced receptor turnover and suppressed sustained NF-κB activation. 2DL4 endocytosis was found to be vital to stimulate cytokine production, and Triad3A expression diminished localization of internalized receptor in early endosomes. Our results reveal a critical role for endocytosed 2DL4 receptor to generate sustained NF-κB signaling and drive cytokine production. We conclude that Triad3A is a key negative regulator of sustained 2DL4-mediated NF-κB signaling from internalized 2DL4, which functions by promoting ubiquitylation and degradation of endocytosed receptor from early endosomes.

Measuring Intracellular Calcium Signaling in Murine NK Cells by Flow Cytometry

This chapter describes a method by which activating receptor-mediated calcium signaling can be measured in individual murine NK cells using a flow cytometer fitted with a UV laser. One major advantage of this method is that the calcium response of the minority NK cell population and even smaller NK cell subpopulations can be measured simultaneously from a mixture of freshly prepared total splenocytes without resorting to prior cell sorting or expansion in culture. Briefly, cells are harvested and stained to mark the populations of interest, then loaded with indo-1 AM dye and analyzed on the flow cytometer. After an appropriate baseline is established, the cells are treated with a biotinylated antibody to activating receptors, which are subsequently cross-linked by addition of streptavidin. The increase in intracellular calcium is quantified by measuring a shift in the indo-1 emission spectrum that takes place when the dye becomes bound to calcium.

Cutting edge: Role of NK cells and surfactant protein D in dendritic cell lymph node homing: Effects of ozone exposure

The roles of NK cells, surfactant protein D (SP-D), and IFN-γ, as well as the effect of ozone (O3) inhalation, were studied on recirculation of pulmonary dendritic cells (DC) to the mediastinal lymph nodes. O3 exposure and lack of SP-D reduced NK cell IFN-γ and lung tissue CCL21 mRNA expression and impaired DC homing to the mediastinal lymph nodes. Notably, addition of recombinant SP-D to naive mononuclear cells stimulated IFN-γ release in vitro. Because NKp46, a glycosylated membrane receptor, was necessary for dose-dependent SP-D binding to NK cells in vitro and DC migration in vivo, we speculate that SP-D may constitutively stimulate IFN-γ production by NK cells, possibly via NKp46. This mechanism could then initiate the IFN-γ/IL-12 feedback circuit, a key amplifier of DC lymph node homing. Inhibition of this process during an acute inflammatory response causes DC retention in the peripheral lung tissue and contributes to injury.

NK cell dysfunction in chronic lymphocytic leukemia is associated with loss of the mature cells expressing inhibitory killer cell Ig-like receptors

ABSTRACT A prospective analysis of natural killer (NK) cell phenotype and function was performed on fresh peripheral blood samples from untreated patients with B-cell chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). Compared to healthy controls, CD56dim NK cells in CLL patients displayed reduced expression of the NKG2D activating receptor and increased CD27 expression, which indicates declines in mature cells. In addition, NK cells from CLL patients showed reduced degranulation responses toward transformed B cells alone or with rituximab and were more sensitive to activation-induced cell death. We further noted a striking reduction in the frequency and viability of NK cells expressing the inhibitory killer cell Ig-like receptors (KIR)2DL1 and/or KIR3DL1, which progressed over time in most patients. Comparisons between a CLL patient and healthy monozygotic twin were consistent with our results in the larger cohorts. Functional and biomarker alterations were less pronounced on NK cells from SLL patients, which have lower tumor burden in peripheral blood than CLL, but significant reduction in degranulation under ADCC conditions and lower frequency and viability of KIR-expressing NK cells were still evident in SLL. We conclude that mature KIR-expressing NK cells respond to the high circulating B cell tumor burden in CLL, but undergo activation-induced apoptosis. Consequently, CLL patients may benefit from therapies that augment NK cell survival and function.

mTOR-Dependent and Independent Survival Signaling by PI3K in B Lymphocytes

Peripheral B lymphocyte survival requires the B cell receptor (BCR) and B cell activating factor (BAFF) binding to its receptor (BAFF-R). Deletion of the BCR, or its signal transducing chaperone Igβ, leads to rapid loss of mature B cells, indicating that signals initiated at the BCR are crucial for B cell survival. BAFF or BAFF-R deficiency also significantly reduces the numbers of mature B cells despite normal BCR expression. Together, these observations indicate that continued BCR and BAFF-R signaling are essential for the survival of mature resting B cells in the periphery. Here we demonstrate that tonic BCR signals up-regulate p100 (Nfkb2) as well as Mcl-1 protein expression at a post-transcriptional level via a PI3K-dependent pathway. p100 expression is mTOR-independent, whereas Mcl-1 expression is mTOR-dependent. BAFF treatment further elevated Mcl-1 levels by an mTOR-independent pathway, while consuming p100. Accordingly, Mcl-1 induction by BAFF is abrogated in Nfkb2-/- B cells. We propose that the cumulative effects of the BCR and BAFF-R signaling pathways increase Mcl-1 levels beyond the threshold required for B cell survival.

Erratum: Cutting edge: Role of NK cells and surfactant protein D in dendritic cell lymph node homing: Effects of ozone exposure (Journal of Immunology (2016) 196 (553-557))

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved. Copyright

Abstract 2537: Antibody dependent cytotoxicity is enhanced by Ari-4175 through NK cell activation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We previously reported that the prolyl peptidase inhibitor, Ari-4175, showed dramatic anti-tumor effects in KRAS mutant colorectal cancer xengrafts when given either alone or in combination with cetuximab. In vitro data suggest that the therapeutic effect of 4175 might partially be due to the augmentation of ADCC through elevating expression of CD16 on NK cells. However, our data indicates that 4175 does not have a direct effect on isolated NK cells. Therefore, we believe that the observed effect on NK cells and augmentation of ADCC is as a result of activation of another cell type possibly working through the production of a cytokine. We noted a significant expansion of neutrophils in 4175-treated mice that occurred prior to increased expression of CD69 and CD16 on NK cells. Therefore, we hypothesized that neutrophils might be stimulated by 4175, which results in subsequent activation of NK cells to enhance their ADCC response. Ari-4175 was administered orally to nude mice at 200 µg q.d. x5 days/week. Peripheral blood or spleens were assayed for immune parameters, ex vivo, at various time points. Expression of surface markers on myeloid and NK cells were monitored by flow cytometry. Natural cytotoxicity and ADCC were assayed using HCT116 cells and cetuximab in a flow cytometry-based assay. Neutrophil depletion experiments were accomplished by administration of an anti Ly-6G antibody (clone 1A8, cat#BE0075-1, BioXCell), specific for neutrophils, (1 mg per mouse, injected on the days 0, 2, and 4). An Isotype control antibody (rat IgG2a; cat#BE0089, BioXCell) was given at the same dose and by the sameschedule. We were able to effectively eliminate neutrophils and the previously reported cell population (CD45+ CD3- NKp46- CD11c- CD19- CD11b+ Ly6C+ Ly6G+) that appeared to be upregulated following treatment with 4175. Preliminarily, depletion of neutrophils and this cell subset did not have any impact on ADCC augmentation or activation of NK cells. ADCC was significantly greater with 4175 and cetuximab treatment compared to 4175 alone with or without neutrophil depletion. Additionally, results of xenograft studies with panitumomab, an anti EGFR antibody that does not induce ADCC, and 4175 seem to suggest that there is less activity with this combination than with 4175 and cetuximab which is capable of inducing ADCC.Our results indicate that the immunomodulatory effects of Ari-4175 on NK cells appear to be independent of neutrophils. However, in these experiments treatment with 4175 and neutrophil depletion were initiated at the same time. Additional experiments to better elucidate the mechanism of action of this agent are in progress. Citation Format: Alexander MacFarlane, Tetyana Bagnyukva, Jiping Zhang, Kerry Campbell, Barry Jones, William Bachovchin, Hossein Borghaei. Antibody dependent cytotoxicity is enhanced by Ari-4175 through NK cell activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2537. doi:10.1158/1538-7445.AM2014-2537

Abstract 494: Immunomodulatory effects of a small molecule dipeptidyl peptidase inhibitor, Ari-4175, are mediated through NK cell activation in a colorectal cancer model.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: A possible mechanism of the antitumor effect of many therapeutic monoclonal antibodies is antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells. We recently observed that an inhibitor of dipeptidyl peptidase (DPP)4-like serine proteases, Ari-4175, significantly slowed the growth of K-RAS mutated HCT-116 tumor xenografts in nude mice, either as a single agent or in combination with cetuximab. Ari-4175 is not directly cytotoxic in vitro, and since it was able to overcome, in vivo, the resistance to blockade of the epidermal growth factor receptor engendered by the KRAS mutation in the HCT-116 colorectal cancer (CRC) cell line, we tested the hypothesis that the mechanism of the antitumor activity involves the activation of NK cells, resulting in the enhancement of ADCC. Methods: Ari-4175 was administered orally to C57Bl/6 mice at 200 μg q.d. x5 days/week. Peripheral blood or spleens were assayed for immune parameters, ex vivo, at various time points. Expression of surface markers on myeloid and NK cells were monitored by flow cytometry. Natural cytotoxicity and ADCC were assayed using HCT116 cells and cetuximab using a flow cytometry-based assay. Cytokines were measured in mouse serum using Luminex assays. Results: Treatment of mice with Ari-4175 induced up-regulation of the activation marker CD69 on NK cells on day 2, followed by up-regulated expression of FcRIIIA (CD16, which mediates ADCC) on day 7. Coordinate with these changes in surface markers detected in peripheral blood samples, splenocytes from Ari-4175-treated mice exhibited significantly increased in vitro natural cytotoxicity responses toward the HCT116 colon cancer cell line. The treated mice exhibited increased serum levels of inflammatory cytokines, including IL-1, IL-6, MCP-1, GCSF, and IL-2. In addition to the impacts on NK cells, we also observed a significant expansion of a distinct CD45+CD14+Gr-1+MHC-II−CDllb+CD11cvariable myeloid cell population in both peripheral blood and spleens of mice after one week of treatment with Ari-4175. Conclusion: The prolyl peptidase inhibitor, Ari-4175, showed dramatic anti-tumor effect in KRAS mutant colorectal cancer xengrafts when given alone or in combination with cetuximab. In vitro data suggest that the therapeutic effect of 4175 might partially be due to the augmentation of ADCC through elevating expression of CD16 on NK cells. In addition, Ari-4175 appears, in vivo, to expand a unique myeloid cell population, which may be responsible for an inflammatory cytokine response and subsequent activation of NK cells. Our study provides a mechanistic rationale for testing Ari-4175 in a clinical trial and possible biomarker endpoints for evaluation in peripheral blood samples. Citation Format: Alexander W. MacFarlane, Jiping Zhang, Barry Jones, William Bachovchin, Kerry Campbell, Hossein Borghaei. Immunomodulatory effects of a small molecule dipeptidyl peptidase inhibitor, Ari-4175, are mediated through NK cell activation in a colorectal cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 494. doi:10.1158/1538-7445.AM2013-494

Abstract 5226: Potential anti-tumor effect of a small molecule dipeptidyl peptidase inhibitor, 4175, in colorectal cancer

Introduction: Cetuximab is an effective therapeutic agent in a number of malignancies. Current data indicates that about 30% of colorectal cancer patients bearing mutated K-ras do not benefit from this agent. A possible mechanism of the antitumor effect of cetuximab is mediated through antibody-dependent cell-mediated cytotoxicity (ADCC). The present study is aimed to investigate the potential activity of Ari-4175, a broad -spectrum inibitor of prolyl peptidases and a potent immune stimulator in the treatment of K-ras mutant colorectal cancer xenografts as a single agent or in combination with cetuximab. Methods: The effect of Ari-4175 alone or in combination with cetuximab was evaluated both in vitro and in vivo. In vitro, the proliferation of K-ras mutant colon cancer cell lines DLD-1 and HCT-116 was detected after three days of culture in the medium containing various concentrations of Ari-4175 or cetuximab. In vivo, nude mice bearing DLD-1 or HCT-116 xenograft tumors were randomly divided into four groups, control, Ari-4175 alone, cetuximab alone and Ari-4175 plus cetuximab. Ari-4175 was administered orally at 100 ug, q.d or b.i.d and cetuximab was injected i.p at 200 ug per week. Tumor measurements were conducted twice a week. Peripheral blood was collected from the mice before the treatment or after 7 to 14 days of the treatment. CD16 expression on NK cells was monitored by flow cytometry. Results: Ari-4175 (10 nM ∼ 200 uM) alone or in combination with cetuximab did not show significant cytotoxicity on either DLD-1 or HCT-116 in cell culture. However, the growth of both DLD-1 and HCT-116 tumors were significantly blocked by the application of Ari-4175 in a dose-dependent manner. The combination of Ari-4175 with cetuximab lead to a further decrease in tumor size although not statistically significant, probably due to lower number of animals. Cetuximab alone did not show any therapeutic effect on HCT-116 xenograft but did have moderate efficacy on DLD-1 tumors. Treatment with Ari-4175 also increased the level of CD16 on mouse NK cells. Conclusion: The prolyl peptidase inhibitor, Ari-4175, showed anti-tumor effect in K-ras mutant colorectal cancer xengrafts when given alone or in combination with cetuximab. The therapeutic effect of 4175 might partially be due to the augmentation of ADCC through elevating expression of CD16 (FcγRIIIA) on NK cells. Our study provides the basis of testing Ari-4175 in a clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5226. doi:1538-7445.AM2012-5226