Victoria Dean

Abstract CD8 + T cells are critical mediators of antitumor immunity but differentiate into a dysfunctional state, known as T cell exhaustion, after persistent T cell receptor stimulation in the tumor microenvironment (TME). Exhausted T (T ex ) cells are characterized by upregulation of coinhibitory molecules and reduced polyfunctionality. T cells in the TME experience an immunosuppressive metabolic environment via reduced levels of nutrients and oxygen and a buildup of lactic acid. Here we show that terminally T ex cells uniquely upregulate Slc16a11 , which encodes monocarboxylate transporter 11 (MCT11). Conditional deletion of MCT11 in T cells reduced lactic acid uptake by T ex cells and improved their effector function. Targeting MCT11 with an antibody reduced lactate uptake specifically in T ex cells, which, when used therapeutically in tumor-bearing mice, resulted in reduced tumor growth. These data support a model in which T ex cells upregulate MCT11, rendering them sensitive to lactic acid present at high levels in the TME.

Neonatal pig bone marrow stromal cells (PBMSC) were tested in vivo and in vitro to establish their use as a large-animal model for the study of skeletogenesis. When implanted in diffusion chambers in athymic mice for 6-8 weeks, both freshly isolated pig bone marrow and passage 2 PBMSC formed partially mineralized cartilage, bone-like material, and fibrous tissue. The cartilage showed metachromatic, perilacunar staining with toluidine blue and safronin O, alcian blue staining for chondroitin and keratan sulfate, and intense immunostaining for type II collagen. Osteocalcin was immunolocalized to the mineralized regions, consistent with the formation of bone. Alkaline phosphatase was primarily observed in cell layers at boundaries between tissue types. Unstimulated monolayer cultures of PBMSC produced type I but not type II collagen, responded to dexamethasone (10(-8) M) with a 1.7-fold increase in alkaline phosphatase activity, and were stimulated to divide by basic fibroblast growth factor (1.5-fold; EC50 1 ng/ml). Transforming growth factor beta (TGF-beta) blocked both dexamethasone-induced alkaline phosphatase expression (EC50, 1 ng/ml of TGF-beta) and the mitogenic effects of bFGF (EC50 0.06 ng/ml of TGF-beta). When incubated for 10-14 days in medium containing dexamethasone, beta-glycerophosphate and ascorbate PBMSC formed mineralized nodules. Calcification occurred in the middle of the aggregates and was associated with intensely alkaline phosphatase positive cells and a dense type I collagen-rich matrix. PBMSC also displayed colony-forming unit-fibroblastic activity, with approximately 1 in 80 of the plated cells formed colonies > 128 cells over 14-21 days. PBMSC therefore mimic the known activities of stromal cells from other species, including the human, suggesting that they are a valid model for skeletal research.

While checkpoint blockade immunotherapies have widespread success, they rely on a responsive immune infiltrate; as such, treatments enhancing immune infiltration and preventing immunosuppression are of critical need. We previously generated αPD-1 resistant variants of the murine HNSCC model MEER. While entirely αPD-1 resistant, these tumors regress after single dose of oncolytic vaccinia virus (VV). We then generated a VV-resistant MEER line to dissect the immunologic features of sensitive and resistant tumors. While treatment of both tumor types induced immune infiltration and IFNγ, we found a defining feature of resistance was elevation of immunosuppressive cytokines like TGFβ, which blunted IFNγ signaling, especially in regulatory T cells. We engineered VV to express a genetically encoded TGFβRII inhibitor. Inhibitor-expressing VV produced regressions in resistant tumor models and showed impressive synergy with checkpoint blockade. Importantly, tumor-specific, viral delivery of TGFβ inhibition had no toxicities associated with systemic TGFβ/TGFβR inhibition. Our data suggest that aside from stimulating immune infiltration, oncolytic viruses are attractive means to deliver agents to limit immunosuppression in cancer.

The distribution of several native extracellular matrix proteins (type I, III, and IV collagens and fibronectin) using immunofluorescent localization is described for in two different malignant gliomas (BT4A and BT4An). In addition, antibodies against denatured forms of type I and III collagens were used to localize areas of active degradation within the tumors. We have shown that both tumors express the native connective tissue components studied, although the distribution of these components within and between the tumors was different. In addition, native type I and III collagens and fibronectin were overexpressed in the tumors compared to the normal brain. Morphometry on immunostained type IV collagen sections showed an increase in vascular elements in both tumors compared to normal brain tissue. The BT4A tumor, which by light microscopy showed a degradative mode of invasion, expressed denatured type I and III collagens at the tumor-brain border zone, suggesting that this tumor has collagenolytic activity. The present article suggests that the distribution and changes in extracellular matrix protein synthesis and degradation may play an important role in the progressive growth of brain tumors in vivo.