Pratik Bhojnagarwala

Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

Intraoperative Near-Infrared Imaging Can Distinguish Cancer from Normal Tissue but Not Inflammation

Introduction Defining tumor from non-tumor tissue is one of the major challenges of cancer surgery. Surgeons depend on visual and tactile clues to select which tissues should be removed from a patient. Recently, we and others have hypothesized near-infrared (NIR) imaging can be used during surgery to differentiate tumors from normal tissue. Methods We enrolled 8 canines and 5 humans undergoing cancer surgery for NIR imaging. The patients were injected with indocyanine green (ICG), an FDA approved non-receptor specific NIR dye that accumulates in hyperpermeable tissues, 16–24 hours prior to surgery. During surgery, NIR imaging was used to discriminate the tumor from non-tumor tissue. Results NIR imaging identified all tumors with a mean signal-to-background ratio of 6.7. Optical images were useful during surgery in discriminating normal tissue from cancer. In 3 canine cases and 1 human case, the tissue surrounding the tumor was inflamed due to obstruction of the vascular supply due to mass effect. In these instances, NIR imaging could not distinguish tumor tissue from tissue that was congested, edematous and did not contain cancer. Conclusions This study shows that NIR imaging can identify tumors from normal tissues, provides excellent tissue contrast, and it facilitates the resection of tumors. However, in situations where there is significant peritumoral inflammation, NIR imaging with ICG is not helpful. This suggests that non-targeted NIR dyes that accumulate in hyperpermeable tissues will have significant limitations in the future, and receptor-specific NIR dyes may be necessary to overcome this problem.

An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells

Careful preparation of human tissues is the cornerstone of obtaining accurate data in immunologic studies. Despite the essential importance of tissue processing in tumor immunology and clinical medicine, current methods of tissue disaggregation have not been rigorously tested for data fidelity. Thus, we critically evaluated the current techniques available in the literature that are used to prepare human lung tumors for immunologic studies. We discovered that these approaches are successful at digesting cellular attachments and ECMs; however, these methods frequently alter the immune cell composition and/or expression of surface molecules. We thus developed a novel approach to prepare human lung tumors for immunologic studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This enzymatic digestion cocktail optimized cell yield and cell viability, retrieved all major tumor‐associated cell populations, and maintained the expression of cell‐surface markers for lineage definition and in vivo effector functions. To our knowledge, we present the first rigorously tested disaggregation method designed for human lung tumors.

The timing of TGF-β inhibition affects the generation of antigen-specific CD8+ T Cells

BackgroundTransforming growth factor (TGF)-β is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-β slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-β before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-β is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation.MethodsBALB/c mice were pretreated with a blocking soluble TGF-β receptor (sTGF-βR, TGF-β-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-βR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells.ResultsInhibition of TGF-β before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p < 0.05). This effect was due to the inhibition of CTLs, as it was not present in mice with severe combined immunodeficiency (SCID) or those depleted of CD8+ T cells. Furthermore, pretreatment with sTGF-βR inhibited tumor-specific CTL activity in a Winn Assay. Tumors grew to a much larger size when mixed with CD8+ T cells from mice pretreated with sTGF-βR than when mixed with CD8+ T cells from mice in the control group: 96 mm3 vs. 22.5 mm3, respectively (p < 0.05). In addition, fewer CD8+ T cells were generated in Ad.E7-immunized mice pretreated with sTGF-βR than in mice from the control group: 0.6% total CD8+ T cells vs. 1.9%, respectively (p < 0.05).ConclusionsThese studies provide the first in vivo evidence that TGF-β may be necessary for anti-tumor immune responses in certain cancers. This finding has important implications for our understanding of anti-tumor immune responses, the role of TGF-β in the immune system, and the future development of TGF-β inhibiting drugs.

Adenoviral-based immunotherapy provides local disease control in an orthotopic murine model of esophageal cancer.

Despite recent advances in the development of novel therapies, esophageal carcinoma remains an aggressive cancer associated with a poor prognosis. The lack of a high throughput, reproducible syngeneic animal model that replicates human disease is partly responsible for the paucity of novel therapeutic approaches. In this report, we present the first successful syngeneic, orthotopic model for esophageal cancer. This model was used to test an established adenoviral-based tumor vaccine. We utilized a murine esophageal cancer cell line established from the ED-L2-cyclin D1;p53–/– mouse that was transduced to express a viral tumor antigen, the Human Papilloma Virus (HPV) E7 protein. The tumor was established in its natural microenvironment at the gastroesophageal junction. Tumor growth was consistent and reproducible. An adenoviral vaccine to E7 (Ad.E7) induced an E7-specific population of functionally active CD8+ T cells that trafficked into the tumors and retained cytotoxicity. Ad.E7 vaccination reduced local tumor growth and prolonged overall survival. These findings suggest that orthotopic tumor growth is a reasonable preclinical model to validate novel therapies.

Human neutrophils can mimic myeloid-derived suppressor cells (PMN-MDSC) and suppress microbead or lectin-induced T cell proliferation through artefactual mechanisms

We report that human conventional CD15+ neutrophils can be isolated in the peripheral blood mononuclear cell (PBMC) layer during Ficoll gradient separation, and that they can impair T cell proliferation in vitro without concomitant neutrophil activation and killing. This effect was observed in a total of 92 patients with organ transplants, lung cancer or anxiety/depression, and in 18 healthy donors. Although such features are typically associated in the literature with the presence of certain myeloid-derived suppressor cell (PMN-MDSC) populations, we found that commercial centrifuge tubes that contained membranes or gels for PBMC isolation led to up to 70% PBMC contamination by CD15+ neutrophils, with subsequent suppressive effects in certain cellular assays. In particular, the suppressive activity of human MDSC should not be evaluated using lectin or microbead stimulation, whereas assays involving soluble or plate-bound antibodies or MLR are unaffected. We conclude that CD15+ neutrophil contamination, and associated effects on suppressor assays, can lead to significant artefacts in studies of human PMN-MDSC.

Abstract A02: The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer

To date there has been an increasing focus on the interactions between inflammatory myeloid cells and T cells in the tumor microenvironment because cytotoxic anti-tumoral T cells represent the chief effector mechanism of anti-tumoral immunity. Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in lung tumors; however, whether specialized neutrophil subpopulations capable of regulating T cell responses exist in human cancers is unknown. Our goal was to identify subsets of TANs and determine their specific roles in the regulation of T cell responses in patients with early stage lung cancer. To address this question, freshly isolated tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. An extensive phenotypic analysis of 55 early-stage human lung tumors revealed that TANs, defined as CD11b + Arg1 + MPO + CD66b + CD15 + cells, contained two major sub-populations. One subset of canonical TANs expressed classic neutrophil markers. A second subset of TANs displayed a combination of neutrophil markers plus markers (CD14 + HLA-DR + CCR7 + CD86 + ) normally expressed on antigen-presenting cells (APC). This subset of TANs was found in lung tumor tissue but not in adjacent uninvolved lung. We termed this unique neutrophil population “APC-like hybrid TANs”. The frequency of these hybrid TANs varied widely within lung cancers and ranged from 0.5-25% of all TANs. Interestingly, the frequency of this hybrid population declined as tumors enlarged, and they were almost completely absent in tumors greater than 5 cm in diameter. Mechanistically, we determined that low doses of IFN-γ and GM-CSF in the tumors were required for the development of APC-like hybrid neutrophils. The high proportion of hybrid TANs (>10% of all TANs) directly correlated with the presence of IFN-γ and GM-CSF in the autologous tumor tissue. Using bone marrow-derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC-like hybrid neutrophils originate from CD11b + CD15 + CD10 - CD16 -/low/int neutrophil progenitors in the presence of IFN-γ and GM-CSF or in tumor-conditioned media. By contrast, mature CD11b + CD15 + CD10 + CD16 hi neutrophils did not acquire the phenotype of hybrid TANs, when cultured under these conditions. In addition, we determined that IFN-γ and GM-CSF synergistically exerted APC promoting effects on immature neutrophils via the downregulation of the transcription factor, Ikaros. However, the development of APC-like hybrid neutrophils was profoundly inhibited under hypoxic conditions. Functionally, the APC-like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) produce APC cytokines such as TNF and IL-12 after stimulation, 2) stimulate antigen non-specific autologous T cell responses induced by plate-bound anti-CD3 antibodies 3) directly stimulate antigen-specific autologous memory T cell responses to virus-derived antigens, 4) augment NY-ESO-1 specific effector T cell responses by providing a co-stimulatory signals through the OX40L, 4-1BBL CD86, CD54 molecules, and 5) cross present tumor-associated antigen as IgG-immune complex. In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early-stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC-like hybrid neutrophils. These hybrid TANs had enhanced ability to trigger and support T cell responses in direct cell-cell interactions. This property of hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Shaun O9Brien, Edmund Moon, Tom Stephen, Abhishek Rao, Alfred Garfall, Wayne Hancock, Jose Conejo-Garcia, Charuhas Deshpande, Michael Feldman, Sunil Singhal, Steven Albelda. The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A02.

Surgical cytoreduction restores the antitumor efficacy of a Listeria monocytogenes vaccine in malignant pleural mesothelioma

Recent studies suggest that immunotherapy may offer a promising treatment strategy for early-stage malignant pleural mesothelioma (MPM), but advanced tumor burden may limit the efficacy of immunotherapy. Therefore, we hypothesized that surgical cytoreduction could restore the efficacy of vaccine-based immunotherapy for MPM. We developed a murine model of MPM through transduction of a mesothelioma cell line with mesothelin. We used this model to evaluate the efficacy of a Listeria monocytogenes vaccine expressing mesothelin. Tumor growth was significantly inhibited at four weeks in animals vaccinated two weeks prior to tumor cell inoculation as compared to those given an empty vector control (1371 ± 420 mm(3) versus 405 ± 139 mm(3); p < 0.01). Mice vaccinated one week prior to tumor challenge also displayed significant reduction in tumor volume (1227 ± 406 mm(3) versus 309 ± 173 mm(3); p < 0.01). The vaccine had no effect when administered concurrently with tumor challenge, or after tumors were established. Flow cytometry showed reduced mesothelin expression in large tumors, as well as tumor-associated immunosuppression due to increased myeloid derived suppressor cells (MDSCs). These factors may have limited vaccine efficacy for advanced disease. Surgical cytoreduction of established tumors restored the antitumor potency of the therapeutic vaccine, with significantly reduced tumor burden at post-operative day 18 (397 ± 103 mm(3) versus 1047 ± 258 mm(3); p < 0.01). We found that surgery reduced MDSCs to levels comparable to those in tumor-naïve mice. This study demonstrates that cytoreduction surgery restores the efficacy of cancer vaccines for MPM by reducing tumor-related immunosuppression that impairs immunotherapy.

Abstract A66: Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation

Tumor-recruited myeloid cells represent a significant portion of inflammatory cells within the tumor microenvironment and influence nearly all steps of tumor progression. Although tumor-associated neutrophils (TANs) are present in large numbers, the role of TANs in cancer progression remains unclear and has only been recently investigated in murine models. The goal of this study was to provide a phenotypic and functional characterization of TANs in freshly harvested surgically resected lung cancer samples. Tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. Importantly, fresh lung tumors from patients were processed within 20 minutes of removal from the patient. We developed an approach to prepare human lung tumors for immunological studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This approach retrieved all major tumor-associated cell populations with high cell yield and maintained the expression of cell surface markers and the functions of isolated cells. In multicolor tracings, TANs were defined as CD15hi/CD66b+/CD11b+/MPO+/Arg1+/IL-5Ra-/ cells. We found that TANs comprised 5—25% of cells isolated from all digested human lung tumors (n=42). Compared to blood neutrophils, TANs displayed an activated phenotype (CD62Llo/CD54hi) and a novel repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. They also produced substantial quantities of the pro-inflammatory factors MCP-1, IL-8, MIP-1a, and IL-6, as well as the anti-inflammatory IL-1R antagonist. TANs were not hypofunctional cells, since they were able to phagocytize bacteria and produce reactive oxygen species. Understanding the role of TANs in regulating T cell responses in cancer patients is particularly important because cytotoxic T lymphocytes are the chief effector cells mediating antigen-driven anti-tumor immunity. We found that TANs were able to stimulate antigen non-specific T cell proliferation and IFN-γ release. Crosstalk between TANs and activated T cells led to substantial up-regulation of CD54, CD86, OX40L, and 4-1BBL co-stimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive feedback loop. This stimulatory activity of TANs appeared to be related to tumor size, as TANs from the majority of the smaller tumors ( Thus, our data suggest that in patients with early-stage lung cancer, TANs do not significantly contribute to inhibition of T cell responses. Rather, the majority of neutrophils recruited into the tumor microenvironment undergo phenotypic and functional changes that result in the formation of cells that resemble the murine anti-tumor “N1 TANs” and could potentially augment and support T cells responses. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Tom Stephen, Anjana Ranganathan, Charuhas Deshpande, Tatiana Akimova, Anil Vachani, Leslie Litzky, Wayne Hancock, Jose Conejo-Garcia, Michael Feldman, Sunil Singhal, Steven Albelda. Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A66.

Abstract 1291: Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells

Aim: Advances in immunotherapy require a detailed understanding of host immunity. Data from murine models indicate solid tumors of various types can recruit Tregs and/or promote iTreg development, and thereby curtail antitumor responses. However, clinical data have been restricted to either evaluation of Treg numbers in tumors, or studies of PBMC Treg in patients with cancers. Surprisingly, no studies of Treg function have ever been reported using Tregs isolated from tumors themselves. Methods: Using our recently reported technique, we isolated Tregs within 20 mins of resection from 13 lung cancer patients, stages Ia-IIIa, and 2 control non-cancer patients (diaphragmatic hernia and lung granuloma). The mean age of the cancer patients (10 males, 3 females) was 65.4±2.2 years; 7 patients had adenocarcinoma, 3 had squamous cell carcinoma, and the remainder had large cell neuroendocrine carcinoma, colon adenocarcinoma and high-grade carcinoma. The mean size of their tumors was 4.0±0.8 cm, and 1 of the 13 patients developed metastases during the follow-up period. We compared the suppressive function of Tregs isolated from each patient9s freshly resected tumor, LN and blood, using cryopreserved healthy donor PBMC aliquots as responders, and area-under-curve (AUC) analysis. We controlled for comparable CD4+ and FOXP3+ purity in all isolated Treg samples, and cell viability, excluding from further analysis all data from non-comparable FOXP3+ subsets. Thus, we were able to avoid artifacts arising from different cell conditions (e.g. use of autologous responder cells) or from unequal FOXP3+ content in Treg isolates. Results: Tumor-infiltrating Tregs had significantly enhanced suppressive function when compared with Tregs isolated from other locations in the same patient. Thus, tumor Tregs were significantly more suppressive than lung LN Tregs (p = 0.0003), and significantly more suppressive than blood Tregs (p = 0.03). Tregs isolated from LN in close proximity to tumors had comparable suppressive function to Tregs isolated from distant LN (p>0.05), and blood Tregs had comparable suppressive function to that of LN Tregs (p>0.05). Additional analyses showed that the enhanced suppressive function of tumor Treg was not due to Treg-induced killing/apoptosis of responder cells, cytokine and/or growth factor deprivation, or soluble factors produced by tumor Treg cells. Conclusions: We demonstrate for the first time that tumor-infiltrating human lung cancer Treg have significantly increased suppressive function. This finding is important for the development of effective anti-tumor therapy. While we are continuing to study the basis for such differences, our data suggest that studies of peripheral blood Tregs in cancer patients may be inadequate and inefficient approaches for understanding the fundamental mechanisms of limited host antitumor immune responses. Citation Format: Tatiana Akimova, Evgeniy B. Eruslanov, Pratik S. Bhojnagarwala, Jon G. Quatromoni, Jacqueline Morgen, Sunil Singhal, Steven M. Albelda, Wayne W. Hancock. Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1291. doi:10.1158/1538-7445.AM2015-1291