Impact of HPV status on immune responses in head and neck squamous cell carcinoma.

The main objective of our study was to understand the impact of immune cell composition and the tumor-reactivity of tumor infiltrating lymphocytes (TIL) in HPV-positive (HPV+) and HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC). TIL cultures were established from primary HNSCC tumors, the T cell subsets were phenotypically characterized using flow cytometry, and Interferon (IFN)-γ ELISA assay was used to determine TIL function. NanoString Immune Profiler was used to determine an immune signature by HPV-status, and multiplex immunohistochemistry (MIHC) was used to quantify immune cell distributions and their spatial relationships. Results showed that HPV+ and HPV- HNSCC had similar capacity to expand IFN-γ reactive TIL populations, and these TIL populations had similar characteristics. NanoString analysis revealed increased differential expression of genes related to B cell functions in HPV+ HNSCC, which were significant at a Benjamini-Yekutieli adjusted p-value of < 0.001. MIHC also displayed increased CD8+ T cell and CD19/CD20+ B cell densities in the tumor region of HPV+ HNSCC as opposed to HPV- HNSCC (p < 0.01). Increases in a combined metric of tumor B cell content and stromal plasma cell content was associated with increased progression-free survival in HPV- HNSCC patients treated with immune checkpoint inhibitor therapy (p = 0.03). In summary, TIL populations expanded from HPV+ and HPV- HNSCC displayed similar IFN-γ reactivity. However, we identified a strong B-cell signature present within HPV+ HNSCC, and higher B and plasma cell content associated with improved PFS in HPV- HNSCC patients treated with immune checkpoint inhibitors.

Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis.

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.

Integrin alpha8beta1 mediates adhesion to LAP-TGFbeta1.

The development of fibrosis is a common response to a variety of injuries and results in the net accumulation of matrix proteins and impairment of normal organ function. We previously reported that the integrin alpha8beta1 is expressed by alveolar interstitial cells in normal lung and is upregulated during the development of fibrosis. TGFbeta1 is an important mediator of the inflammatory response in pulmonary fibrosis. TGFbeta1 is secreted as a latent protein that is non-covalently associated with latency-associated peptide (LAP) and requires activation to exert its effects. LAP-TGFbeta1 and LAP-TGFbeta3 contain the tripeptide sequence, arginine-glycine-aspartic acid (RGD), a known integrin recognition motif. The integrin alpha8beta1 binds to several ligands such as fibronectin and vitronectin through the RGD sequence. Recent reports demonstrate that the integrins alphavbeta1, alphavbeta6 and alphavbeta8 adhere to LAP-TGFbeta1 through the RGD site. Therefore, we asked whether LAP-TGFbeta1 might be a ligand for alpha8beta1 and whether this may be important in the development of fibrosis. We found that cell lines transfected with alpha8 subunit were able to spread on and adhere to recombinant LAP-TGFbeta1 significantly better than mock transfected cell lines. alpha8-transfected cells were also able to adhere to LAP-TGFbeta3 significantly better than mock transfected cells. Adhesion to LAP-TGFbeta1 was enhanced by activation of alpha8beta1 by Mn(2+), or 8A2, an integrin beta1 activating antibody. Furthermore, cell adhesion was abolished when we used a recombinant LAP-TGFbeta1 protein in which the RGD site was mutated to RGE. alpha8beta1 binding to LAP-TGFbeta1 increased cell proliferation and phosphorylation of FAK and ERK, but did not activate of TGFbeta1. These data strongly suggest that LAP-TGFbeta1 is a ligand of alpha8beta1 and interaction of alpha8beta1 with LAP-TGFbeta1 may influence cell behavior.