Theodore H. Welling

Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments

Th17 cells play an active role in autoimmune diseases. However, the nature of Th17 cells is poorly understood in cancer patients. We studied Th17 cells, the associated mechanisms, and clinical significance in 201 ovarian cancer patients. Tumor-infiltrating Th17 cells exhibit a polyfunctional effector T-cell phenotype, are positively associated with effector cells, and are negatively associated with tumor-infiltrating regulatory T cells. Tumor-associated macrophages promote Th17 cells through interleukin-1beta (IL-1beta), whereas tumor-infiltrating regulatory T cells inhibit Th17 cells through an adenosinergic pathway. Furthermore, through synergistic action between IL-17 and interferon-gamma, Th17 cells stimulate CXCL9 and CXCL10 production to recruit effector T cells to the tumor microenvironment. The levels of CXCL9 and CXCL10 are associated with tumor-infiltrating effector T cells. The levels of tumor-infiltrating Th17 cells and the levels of ascites IL-17 are reduced in more advanced diseases and positively predict patient outcome. Altogether, Th17 cells may contribute to protective human tumor immunity through inducing Th1-type chemokines and recruiting effector cells to the tumor microenvironment. Inhibition of Th17 cells represents a novel immune evasion mechanism. This study thus provides scientific and clinical rationale for developing novel immune-boosting strategies based on promoting the Th17 cell population in cancer patients.

Induction of IL-17+ T Cell Trafficking and Development by IFN-γ: Mechanism and Pathological Relevance in Psoriasis

Th1 and Th17 T cells are often colocalized in pathological environments, yet Th1-derived IFN-γ inhibits Th17 cell development in vitro. We explored the physiologic basis of this paradox in humans. In this study, we demonstrate increased the number of CD4+ and CD8+ IL-17+ T cells in skin lesions of psoriasis. Furthermore, we show that myeloid APCs potently support induction of IL-17+ T cells, and that this activity is greatly increased in psoriasis. We tested stimuli that might account for this activity. Th1 cells and IFN-γ are increased in psoriatic blood and lesional skin. We show that IFN-γ programs myeloid APCs to induce human IL-17+ T cells via IL-1 and IL-23. IFN-γ also stimulates APC production of CCL20, supporting migration of IL-17+ T cells, and synergizes with IL-17 in the production of human β-defensin 2, an antimicrobial and chemotactic protein highly overexpressed by psoriatic keratinocytes. This study reveals a novel mechanistic interaction between Th1 and IL-17+ T cells, challenges the view that Th1 cells suppress Th17 development through IFN-γ, and suggests that Th1 and IL-17+ T cells may collaboratively contribute to human autoimmune diseases.

Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial

BACKGROUND For patients with advanced hepatocellular carcinoma, sorafenib is the only approved drug worldwide, and outcomes remain poor. We aimed to assess the safety and efficacy of nivolumab, a programmed cell death protein-1 (PD-1) immune checkpoint inhibitor, in patients with advanced hepatocellular carcinoma with or without chronic viral hepatitis. METHODS We did a phase 1/2, open-label, non-comparative, dose escalation and expansion trial (CheckMate 040) of nivolumab in adults (≥18 years) with histologically confirmed advanced hepatocellular carcinoma with or without hepatitis C or B (HCV or HBV) infection. Previous sorafenib treatment was allowed. A dose-escalation phase was conducted at seven hospitals or academic centres in four countries or territories (USA, Spain, Hong Kong, and Singapore) and a dose-expansion phase was conducted at an additional 39 sites in 11 countries (Canada, UK, Germany, Italy, Japan, South Korea, Taiwan). At screening, eligible patients had Child-Pugh scores of 7 or less (Child-Pugh A or B7) for the dose-escalation phase and 6 or less (Child-Pugh A) for the dose-expansion phase, and an Eastern Cooperative Oncology Group performance status of 1 or less. Patients with HBV infection had to be receiving effective antiviral therapy (viral load <100 IU/mL); antiviral therapy was not required for patients with HCV infection. We excluded patients previously treated with an agent targeting T-cell costimulation or checkpoint pathways. Patients received intravenous nivolumab 0·1-10 mg/kg every 2 weeks in the dose-escalation phase (3+3 design). Nivolumab 3 mg/kg was given every 2 weeks in the dose-expansion phase to patients in four cohorts: sorafenib untreated or intolerant without viral hepatitis, sorafenib progressor without viral hepatitis, HCV infected, and HBV infected. Primary endpoints were safety and tolerability for the escalation phase and objective response rate (Response Evaluation Criteria In Solid Tumors version 1.1) for the expansion phase. This study is registered with ClinicalTrials.gov, number NCT01658878. FINDINGS Between Nov 26, 2012, and Aug 8, 2016, 262 eligible patients were treated (48 patients in the dose-escalation phase and 214 in the dose-expansion phase). 202 (77%) of 262 patients have completed treatment and follow-up is ongoing. During dose escalation, nivolumab showed a manageable safety profile, including acceptable tolerability. In this phase, 46 (96%) of 48 patients discontinued treatment, 42 (88%) due to disease progression. Incidence of treatment-related adverse events did not seem to be associated with dose and no maximum tolerated dose was reached. 12 (25%) of 48 patients had grade 3/4 treatment-related adverse events. Three (6%) patients had treatment-related serious adverse events (pemphigoid, adrenal insufficiency, liver disorder). 30 (63%) of 48 patients in the dose-escalation phase died (not determined to be related to nivolumab therapy). Nivolumab 3 mg/kg was chosen for dose expansion. The objective response rate was 20% (95% CI 15-26) in patients treated with nivolumab 3 mg/kg in the dose-expansion phase and 15% (95% CI 6-28) in the dose-escalation phase. INTERPRETATION Nivolumab had a manageable safety profile and no new signals were observed in patients with advanced hepatocellular carcinoma. Durable objective responses show the potential of nivolumab for treatment of advanced hepatocellular carcinoma. FUNDING Bristol-Myers Squibb.

Renal Artery Aneurysms: A 35-year Clinical Experience With 252 Aneurysms in 168 Patients

ObjectiveTo define the relevance of treating renal artery aneurysms (RAAs) surgically. Summary Background DataMost prior definitions of the clinical, pathologic, and management features of RAAs have evolved from anecdotal reports. Controversy surrounding this clinical entity continues. MethodsA retrospective review was undertaken of 168 patients (107 women, 61 men) with 252 RAAs encountered over 35 years at the University of Michigan Hospital. Aneurysms were solitary in 115 patients and multiple in 53 patients. Bilateral RAAs occurred in 32 patients. Associated diseases included hypertension (73%), renal artery fibrodysplasia (34%), systemic atherosclerosis (25%), and extrarenal aneurysms (6.5%). Most RAAs were saccular (79%) and noncalcified (63%). The main renal artery bifurcation was the most common site of aneurysms (60%). RAAs were often asymptomatic (55%), with a diagnosis made most often during arteriographic study for suspected renovascular hypertension (42%). ResultsSurgery was performed in 121 patients (average RAA size 1.5 cm), including 14 patients undergoing unilateral repair with contralateral RAA observation. The remaining 47 patients (average RAA size 1.3 cm) were not treated surgically. Operations included aneurysmectomy and angioplastic renal artery closure or segmental renal artery reimplantation, aneurysmectomy and renal artery bypass, and planned nephrectomy for unreconstructable renal arteries or advanced parenchymal disease. Eight patients underwent unplanned nephrectomy, being considered a technical failure of surgical therapy. Dialysis-dependent renal failure occurred in one patient. There were no perioperative deaths. Late follow-up (average 91 months) was available in 145 patients (86%). All but two arterial reconstructions remained clinically patent. Secondary renal artery procedures included percutaneous angioplasty, branch embolization, graft thrombectomy, and repeat bypass for late aneurysmal change of a vein conduit. Among 40 patients with clearly documented preoperative and postoperative blood pressure measurements, 60% had a significant decline in blood pressure after surgery while taking fewer antihypertensive medications. Late RAA rupture did not occur in the nonoperative patients, but no lessening of this group’s hypertension was noted. ConclusionSurgical therapy of RAAs in properly selected patients provides excellent long-term clinical outcomes and is often associated with decreased blood pressure.

c-Met is a marker of pancreatic cancer stem cells and therapeutic target.

BACKGROUND & AIMS Growth of many different tumor types requires a population of self-renewing cancer stem cells (CSCs). c-Met is a marker of normal mouse pancreatic stem and progenitor cells; we investigated whether it is also a marker of human pancreatic CSCs that might be developed as a therapeutic target. METHODS We studied growth of primary human pancreatic adenocarcinoma in NOD SCID mice. The self-renewal capability of pancreatic cancer cells that expressed high levels of c-Met (c-Met(high)) was assessed using in vitro sphere assays and compared with those that were c-Met negative or expressed low levels of c-Met. The tumorigenicity of c-Met(high) pancreatic cancer cells was evaluated in NOD SCID mice. RESULTS c-Met(high) cells readily formed spheres, whereas c-Met-negative cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with small hairpin RNAs significantly inhibited tumor sphere formation. c-Met(high) cells had increased tumorigenic potential in mice; those that expressed c-Met and CD44 (0.5%-5% of the pancreatic cancer cells) had the capability for self-renewal and the highest tumorigenic potential of all cell populations studied. In pancreatic tumors established in NOD SCID mice, c-Met inhibitors slowed tumor growth and reduced the population of CSCs when given alone or in combination with gemcitabine. Administration of XL184 for 2 weeks after cardiac injection of cancer cells prevented the development of metastases. CONCLUSIONS c-Met is a new marker for pancreatic CSCs. It is required for growth and metastasis of pancreatic tumors in mice and is a therapeutic target for pancreatic cancer.

T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment

Human tumors progress despite the presence of tumor associated antigen (TAA)-specific T cells. Many different molecular and cellular mechanisms contribute to the failure of T cells to eradicate the tumor. These include immune suppressive networks that impair ongoing T cell function and enable tumor escape. Recent studies have started to reveal the nature of effector T cells in the tumor microenvironment. In this article we discuss T cell anergy, exhaustion, senescence, and stemness, and review the phenotype of dysfunctional T cell subsets and the underlying molecular mechanisms in the tumor microenvironments. We suggest that targeting T cell dysfunctional mechanisms and introducing/promoting T cell stemness are important approaches to treat patients with cancer.

Kupffer Cell Suppression of CD8+ T Cells in Human Hepatocellular Carcinoma Is Mediated by B7-H1/Programmed Death-1 Interactions

B7-H1 is a recently identified B7 family member that, along with one of its receptors, programmed death-1 (PD-1), has been involved in multiple immunopathologic scenarios. However, the nature of B7-H1 and PD-1 in human hepatocellular carcinoma (HCC) remains poorly defined. We investigated the expression and functional relevance of this pathway in patients with HCC. We showed that B7-H1 expression on Kupffer cells (KC) was increased in tumor tissues compared with surrounding nontumor liver tissues in patients with HCC and this correlated with poorer survival. Coculture of HCC cells with monocytes showed that tumor-associated interleukin-10 contributed to the induction of B7-H1 in the HCC environment. We further observed that the levels of PD-1(+)CD8(+) T cells were higher in tumor tissues than in nontumor tissues. B7-H1(+) KCs and PD-1(+) T cells were colocalized in the HCC stroma. PD-1(+)CD8(+) T cells had decreased proliferative ability and effector function as shown by reduced granule and cytokine expression compared with PD-1(-) T cells. Importantly, blocking KC B7-H1 interaction with PD-1(+)CD8(+) cells using neutralizing antibodies recovered effector T-cell function. Our data indicate that the B7-H1/PD-1 axis contributes to immune suppression in human HCC, with blockade of this pathway carrying important therapeutic implications.

Tumor-associated macrophages produce interleukin 6 and signal via STAT3 to promote expansion of human hepatocellular carcinoma stem cells.

BACKGROUND & AIMS Cancer stem cells (CSCs) can contribute to hepatocellular carcinoma (HCC) progression and recurrence after therapy. The presence of tumor-associated macrophages (TAMs) in patients with HCC is associated with poor outcomes. It is not clear whether TAMs interact with CSCs during HCC development. We investigated whether TAMs affect the activities of CSCs in the microenvironment of human HCCs. METHODS HCCs were collected from 17 patients during surgical resection and single-cell suspensions were analyzed by flow cytometry. CD14(+) TAMs were isolated from the HCC cell suspensions and placed into co-culture with HepG2 or Hep3B cells, and CSC functions were measured. The interleukin 6 (IL6) receptor was blocked with a monoclonal antibody (tocilizumab), and signal transducer and activator of transcription 3 was knocked down with small hairpin RNAs in HepG2 cells. Xenograft tumors were grown in NOD-SCID/Il2Rg(null) mice from human primary HCC cells or HepG2 cells. RESULTS CD44(+) cells from human HCCs and cell lines formed more spheres in culture and more xenograft tumors in mice than CD44(-) cells, indicating that CD44(+) cells are CSCs. Incubation of the CD44(+) cells with TAMs promoted expansion of CD44(+) cells, and increased their sphere formation in culture and formation of xenograft tumors in mice. In human HCC samples, the numbers of TAMs correlated with the numbers of CD44(+) cells. Of all cytokines expressed by TAMs, IL6 was increased at the highest level in human HCC co-cultures, compared with TAMs not undergoing co-culture. IL6 was detected in the microenvironment of HCC samples and induced expansion of CD44(+) cells in culture. Levels of IL6 correlated with stages of human HCCs and detection of CSC markers. Incubation of HCC cell lines with tocilizumab or knockdown of signal transducer and activator of transcription 3 in HCC cells reduced the ability of TAMs to promote sphere formation by CD44+ cells in culture and growth of xenograft tumors in mice. CONCLUSIONS CD44(+) cells isolated from human HCC tissues and cell lines have CSC activities in vitro and form a larger number of xenograft tumors in mice than CD44(-) cells. TAMs produce IL6, which promotes expansion of these CSCs and tumorigenesis. Levels of IL6 in human HCC samples correlate with tumor stage and markers of CSCs. Blockade of IL6 signaling with tocilizumab, a drug approved by the Food and Drug Administration for treatment of rheumatoid arthritis, inhibits TAM-stimulated activity of CD44(+) cells. This drug might be used to treat patients with HCC.

FOXP3 Defines Regulatory T Cells in Human Tumor and Autoimmune Disease

Activated T cells may express FOXP3. It is thought that FOXP3 is not a specific marker to determine regulatory T cells (Treg) in humans. Here, we examined the functional phenotype and cytokine profile of the in vitro induced FOXP3(+) T cells, primary FOXP3(+) and FOXP3(-) T cells in patients with ulcerative colitis and tumors including colon carcinoma, melanoma, hepatic carcinoma, ovarian carcinoma, pancreatic cancer, and renal cell carcinoma. We observed similar levels of suppressive capacity of primary FOXP3(+) T cells in blood, tumors, and colitic tissues. Compared with primary FOXP3(-) T cells in the same microenvironment, these primary FOXP3(+) T cells expressed minimal levels of effector cytokines, negligible amount of cytotoxic molecule granzyme B, and levels of suppressive molecules interleukin-10 and PD-1. Although the in vitro activated T cells expressed FOXP3, these induced FOXP3(+) T cells expressed high levels of multiple effector cytokines and were not functionally suppressive. The data reinforce the fact that FOXP3 remains an accurate marker to define primary Tregs in patients with cancer and autoimmune disease. We suggest that the combination of FOXP3 and cytokine profile is useful for further functionally distinguishing primary Tregs from activated conventional T cells.

Portal Vein Thrombosis and Survival in Patients with Cirrhosis

The effects of occlusive portal vein thrombosis (PVT) on the survival of patients with cirrhosis are unknown. This was a retrospective cohort study at a single center. The main exposure variable was the presence of occlusive PVT. The primary outcome measure was time‐dependent mortality. A total of 3295 patients were analyzed, and 148 (4.5%) had PVT. Variables independently predictive of mortality from the time of liver transplant evaluation included age [hazard ratio (HR), 1.02; 95% confidence interval (CI), 1.01‐1.03], Model for End‐Stage Liver Disease (MELD) score (HR, 1.10; 95% CI, 1.08‐1.11), hepatitis C (HR, 1.44; 95% CI, 1.24‐1.68), and PVT (HR, 2.61; 95% CI, 1.97‐3.51). Variables independently associated with the risk of mortality from the time of liver transplant listing included age (HR, 1.02; 95% CI, 1.01‐1.03), transplantation (HR, 0.65; 95% CI, 0.50‐0.81), MELD (HR, 1.08; 95% CI, 1.06‐1.10), hepatitis C (HR, 1.50; 95% CI, 1.18‐1.90), and PVT (1.99; 95% CI, 1.25‐3.16). The presence of occlusive PVT at the time of liver transplantation was associated with an increased risk of death at 30 days (odds ratio, 7.39; 95% CI, 2.39‐22.83). In conclusion, patients with cirrhosis complicated by PVT have an increased risk of death. Liver Transpl 16:83–90, 2010.