CD4+ binding proteins as immunosuppression factors

Robey, Frank

Abstract

It is well-known that patients suffering from head and neck squamous cell carcinoma (HNSCC) have prognostically significant deficiencies in natural cellular and humoral immunity. As a general observation, patients with stronger immune responses against the HNSCC live longer and have a better chance of survival than HNSCC patients with poorer immune responses. Although the immunosuppression remains localized to the tumor in the early stages of the disease, immunosuppression becomes systemic as the disease spreads. In the early stages of HNSCC, immune competence was evaluated and results indicated that only 20% of the newly diagnosed patients suffering from HNSCC had normal cellular immune functions. These findings show that even early in the disease, severe cellular immunosuppression is present, yet the cause of this is not clear.Since the percentage of patients who survive HNSCC is small compared to many others cancers, it is of great importance to develop an understanding of the immune suppression mechanism in HNSCC as immunotherapy may be the major therapeutic needed to defeat HNSCC. At a minimum, to defeat HNSCC by any means, it appears that HNSCC-induced immunosuppression must be contained and reversed.We have been building on our knowledge of immunosuppression caused by the envelope protein from HIV, gp120, as a template for studying HNSCC-induced immunosuppression. We, and others have learned that gp120 itself can bind to CD4 on the surface of T cells and, in doing so, stimulate a p56lck related signalling mechanism in the cell. The same was observed by us for the C4 peptides that are derived from gp120 and were shown to bind to CD4. Others have shown that gp120 can attenuate the ability of the T cells to make IL-2 in response to various stimuli. We have shown that synthetic helical peptides from the C4 domain of gp120 also can cause the attenuation of IL-2 production by T cells in culture.Detergent extracts of solid tumors of HNSCC contain factors that are capable of binding to recombinant soluble CD4 in a fashion that is very closely related to the way gp120 binds to CD4. The same was found to be true of proteins secreted by cell cultures containing HNSCC. By binding to CD4, these proteins inhibit the ability of gp120 to bind to CD4 and synthetic mimetics of the gp120 binding site for CD4. Studies using fluorescently labeled CD4 and HNSCC showed extensive labelling throughout the cells in culture and this indicates perhaps that natural proteins in the tumor, upon expression on the surface of the tumor or upon release from the tumor, can bind cell surface CD4 and induce CD4-mediated immunosuppression in T cells.We have treated Jurkat cells in culture with 4-day-old conditioned media from HNSCC and have found that the cells remain healthy and viable over a three day period at 37 oC. However, when the cells were stimulated with conconavalin A, there was a very clear reduction in the amount of IL-2 that was secreted into the culture media after two days when compared with the amount of IL-2 produced by cells that were not pre-treated with the HNSCC conditioned media. This result shows that immunosuppression caused by HNSCC conditioned media may closely parallel some of the immunosuppression caused by gp120 from HIV. The key difference however may be in the fact that HIV immunosuppression is systemic whereas HNSCC, at the early stages appears to be localized. The results show the importance of IL-2 in developing a full immune response against the tumors of the head and neck; without IL-2 it will be impossible for tumor infiltrating lymphocytes to expand.Gel filtration experiments were performed in an effort to isolate and identify the factors from HNSCC that bind CD4 and can induce the attenuation of IL-2 by stimulated T cells. The profiles of the eluants is very broad and indicates that the factors from the HNSCC are multiple, probably proteolytically fragmented from parent proteins and may contain various amounts of phosphorylated and glycosylated amino acids. There was some CD4-binding activity eluted from anion exchange resins and this paralleled similar properties that have been reported for the boine-derived protein, bone sialoprotein (BSP).Using immunohistochemistry and ELISAs we have found BSP in HNSCC in cultured cells and in the conditioned media. With purified recombinant samples of BSP we have been able to show some CD4 binding and to a small degree, attenuation of IL-2 production by Con A-stimulated Jurkats. This parallels the findings from other labs in which osteopontin have been found to be associated with cancer-derived immunosuppression. Preliminary results have shown us that osteopontin itself can bind CD4. Whether the HNSCC-derived bone proteins osteopontin and BSP are involved in immunosuppression is a primary topic of investgation at the present time