Immune responses to coiled coil supramolecular biomaterials.

Self-assembly has been increasingly utilized in recent years to create peptide-based biomaterials for 3D cell culture, tissue engineering, and regenerative medicine, but the molecular determinants of these materials' immunogenicity have remained largely unexplored. In this study, a set of molecules that self-assembled through coiled coil oligomerization was designed and synthesized, and immune responses against them were investigated in mice. Experimental groups spanned a range of oligomerization behaviors and included a peptide from the coiled coil region of mouse fibrin that did not form supramolecular structures, an engineered version of this peptide that formed coiled coil bundles, and a peptide-PEG-peptide triblock bioconjugate that formed coiled coil multimers and supramolecular aggregates. In mice, the native peptide and engineered peptide did not produce any detectable antibody response, and none of the materials elicited detectable peptide-specific T cell responses, as evidenced by the absence of IL-2 and interferon-gamma in cultures of peptide-challenged splenocytes or draining lymph node cells. However, specific antibody responses were elevated in mice injected with the multimerizing peptide-PEG-peptide. Minimal changes in secondary structure were observed between the engineered peptide and the triblock peptide-PEG-peptide, making it possible that the triblock's multimerization was responsible for this antibody response.

Gamma interferon signaling in macrophage lineage cells regulates central nervous system inflammation and chemokine production.

Intracranial (i.c.) infection of mice with lymphocytic choriomeningitis virus (LCMV) results in anorexic weight loss, mediated by T cells and gamma interferon (IFN-gamma). Here, we assessed the role of CD4(+) T cells and IFN-gamma on immune cell recruitment and proinflammatory cytokine/chemokine production in the central nervous system (CNS) after i.c. LCMV infection. We found that T-cell-depleted mice had decreased recruitment of hematopoietic cells to the CNS and diminished levels of IFN-gamma, CCL2 (MCP-1), CCL3 (MIP-1alpha), and CCL5 (RANTES) in the cerebrospinal fluid (CSF). Mice deficient in IFN-gamma had decreased CSF levels of CCL3, CCL5, and CXCL10 (IP-10), and decreased activation of both resident CNS and infiltrating antigen-presenting cells (APCs). The effects of IFN-gamma signaling on macrophage lineage cells was assessed using transgenic mice, called "macrophages insensitive to interferon gamma" (MIIG) mice, that express a dominant-negative IFN-gamma receptor under the control of the CD68 promoter. MIIG mice had decreased levels of CCL2, CCL3, CCL5, and CXCL10 compared to controls despite having normal numbers of LCMV-specific CD4(+) T cells in the CNS. MIIG mice also had decreased recruitment of infiltrating macrophages and decreased activation of both resident CNS and infiltrating APCs. Finally, MIIG mice were significantly protected from LCMV-induced anorexia and weight loss. Thus, these data suggest that CD4(+) T-cell production of IFN-gamma promotes signaling in macrophage lineage cells, which control (i) the production of proinflammatory cytokines and chemokines, (ii) the recruitment of macrophages to the CNS, (iii) the activation of resident CNS and infiltrating APC populations, and (iv) anorexic weight loss.

Identification of molecular targets for immunotherapy of patients with head and neck squamous cell carcinoma.

To identify molecular targets for immunotherapy of head and neck squamous cell carcinoma (HNSCC) patients, we analyzed gene expression profile in matched tumor (HN) and normal fibroblast (FB) cell lines established from a HNSCC patient using microarray technique followed by real-time RT-PCR. Screening against a series of established normal and malignant cell lines followed by screening against a panel of normal human tissues led to the identification of 7 genes (AREG, CDH3, KLK10, NmU, SLPI, ANAX3 and MAL2), which were over-expressed at least 10-fold in tumors over any of the normal tissues. We determined the expression of mRNA encoding these genes against a panel of 15 HNSCC primary tumor samples. Relative expression of these genes was at least 20-fold. Expression of AREG, CDH3, KLK10, NmU and SLPI at the protein level was determined by immunohistochemistry in seven supraglottic laryngeal cancer specimens. All five proteins were expressed in these tumor samples with high intensity. We conclude that these molecules are potential targets for immunotherapy of HNSCC patients.

Recombinant vaccinia virus expressing IL-2 generates effective anti-tumor responses in an orthotopic murine model of head and neck carcinoma.

We evaluated the efficacy of a replication-competent, attenuated recombinant vaccinia virus (rvv) expressing IL-2 as a tumor vaccine in an immunocompetent murine model of head and neck squamous cell carcinoma. We implanted oral tumors by injection of tumor cells (SCC VII/SF) into the floor of the mouth of the syngeneic C3H/HeJ mice. Previous studies with this model suggested the presence of tumor-induced immune suppression. To circumvent the immune suppression we subcutaneously (s.c.) immunized the mice with irradiated, rvv-infected tumor cells prior to or along with intratumoral (i.t.) vaccination. A single s.c. vaccination invoked tumor-specific T cell proliferation and cytotoxicity. Mice treated by this protocol survived longer compared to those treated with i.t. vaccination alone. Tumor growth was significantly inhibited (P < 0.0002) and tumor regression was observed in all mice. The numbers of CD4(+) and CD8(+) lymphocytes as well as macrophages in the tumor beds and in tumor-draining lymph nodes were significantly increased in the mice treated by s.c. plus i.t. vaccination compared to s.c. or i.t. vaccination alone. These results suggested that s.c. vaccination along with i.t. vaccination increased antitumoral immunity and that CD4(+), CD8(+) lymphocytes as well as macrophages may play an important role in this antitumoral immunity.