Macrophage-hybridomas: generation, structure, and function

We report the generation of macrophage-hybridomas, obtained by somatic cell fusion between macrophage-enriched C3H.eB spleen cell population, and a drug-resistant MPC-11 myeloma cell line, designated as 4T00.1L1 clone. Screening for hybridomas possessing macrophage properties was carried out by assaying the presence of two macrophage-specific enzymes: lysozyme and nonspecific esterase. Two hybridomas, E2-7 and E2-10, were selected for further studies. We found that clones of E2-7 (E2-7.7) did not express Fc receptors but possessed cell-surface Ia molecules. In contrast, clones of E2-10 (E2-10.20) possessed Fc receptors but were devoid of Ia molecules. E2-7.7 did, however, express Fc receptors after mitomycin treatment, whereas E2-10.20 eliminated the expression of Fc receptors after treatment with mitomycin C. Opsonized erythrocytes were phagocytized by E2-10.20 cells, but not by E2-7.7. Phagocytosis was thus correlated with the possession of Fc receptors. Testing the response of KLH-primed lymph node cells to KLH-pulsed hybridoma cells, we found that E2-7.7 cells caused antigen-specific lymphoproliferative response, whereas E2-10.20 did not. Thus, antigens could be presented by E2-7.7 but not by E2-10.20 cells. The response was shown to be mediated by T but not by B lymphocytes. The difference in antigen-presenting capacity could not be attributed to differences in antigen uptake by the different hybridomas, because the two hybridomas manifested the same level of pinocytosis. Both hybridomas produced IL1. The differences in the properties of the two hybridomas may indicate that the normal partners represent two distinct subpopulations of macrophages. The segregation of functional properties among the hybridoma clones may lead to a clarification of the dependence of distinct functions on defined molecular structures.     

Cryoimmunotherapy with local co-administration of ex vivo generated dendritic cells and CpG-ODN immune adjuvant,elicits a specific antitumor immunity

Cryoablation is a low-invasive surgical procedure for management of malignant tumors. Tissue destruction is obtained by repeated deep freezing and thawing and results in coagulative necrosis and in apoptosis. This procedure induces the release of tumor-associated antigens and proinflammatory factors into the microenvironment. Local administration of immature dendritic cells (DCs) potentiates the immune response induced by cryoablation. To further augment the induction of long-lasting antitumor immunity, we investigated the clinical value of combining cryoimmunotherapy consisting of cryoablation and inoculation of immature DCs with administration of the immune adjuvant, CpG oligodeoxynucleotides. Injection of the murine Lewis lung carcinoma, D122-luc-5.5 that expresses the luciferase gene, results in spontaneous metastases, which can be easily monitored in vivo. The local tumor was treated by the combined treatment. The clinical outcome was assessed by monitoring tumor growth, metastasis in distant organs, overall survival, and protection from tumor recurrence. The nature of the induced T cell responses was analyzed. Combined cryoimmunotherapy results in reduced tumor growth, low metastasis and significantly prolonged survival. Moreover, this treatment induces antitumor memory that protected mice from rechallenge. The underlying suggested mechanisms are the generation of tumor-specific type 1 T cell responses, subsequent induction of cytotoxic T lymphocytes, and generation of systemic memory. Our data highlight the combined cryoimmunotherapy as a novel antitumor vaccine with promising preclinical results. Adjustment of this technique into practice will provide the therapeutic benefits of both, ablation of the primary tumor and induction of robust antitumor and antimetastatic immunity.     

Tumor-Associated Antigen Peptides as Anti-Metastatic Vaccines

Summary Cytotoxic T-lymphocytes (CTLs) kill abnormal cells. CTLs recognize major histocompatibility complex class I molecules in complex with peptides derived from relevant antigens. The identification of tumor associated antigen peptides enabled the design of anti-tumor and anti-metastatic vaccines in a murine lung carcinoma.     

Peptide fragments from the tuftsin containing domain of immunoglobulin G synthesis and biological activity

Peptides corresponding to sequences of the Fc-portion of immunoglobulin G (IgG) surrounding and containing the tuftsin molecule were synthesized. The compounds were assayed for their ability to compete with [3H-Arg4]tuftsin in binding to mouse peritoneal macrophages and to stimulate the cell's capacity to phagocytize. Despite the sensitivity that tuftsin has demonstrated to various chemical modifications and structural alterations which usually cause reduction or total loss of biological activity, IgG-related analogs possess potent tuftsin-like activity. The activity is not caused by enzymatic breakdown and release of tuftsin. The fact that the elongated tuftsin analogs can specifically be attached to and activate macrophages may indicate a possible connection between Fc and tuftsin's receptors.     

Glyco-tuftsin derivatives modulate interleukin-1 and tumor necrosis factor production

Six Thr1 (O-glyco)-derivatives of the "phagocytosis stimulating peptide" tuftsin, H-Thr-Lys-Pro-Arg-OH and the N-glycosylated undecapeptide H-Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asn(beta-D-GlcNAc)-Ser-Thr-OH, which correspond to the "tuftsin-region" at the Fc-domain of immunoglobulin G (amino acid residues 289-299), were evaluated in comparison with tuftsin and rigin, H-Gly-Gln-Pro-Arg-OH, for their capacity to evoke the release of interleukin-1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dose-dependent manner, the release of the two cytokines from both cell types.     

Membrane-anchored beta 2-microglobulin stabilizes a highly receptive state of MHC class I molecules

The magnitude of response elicited by CTL-inducing vaccines correlates with the density of MHC class I (MHC-I)-peptide complexes formed on the APC membrane. The MHC-I L chain, beta2-microglobulin (beta2m), governs complex stability. We reasoned that genetically converting beta2m into an integral membrane protein should exert a marked stabilizing effect on the resulting MHC-I molecules and enhance vaccine efficacy. In the present study, we show that expression of membranal human beta2m (hbeta2m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 10(4)- to 10(6)-fold lower than parental RMA-S, as detected by complex-specific Abs and by T cell activation. Moreover, saturation of the transfectants' MHC-I by exogenous peptide occurs within 1 min, as compared with approximately 1 h required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3- to 5-fold higher. Expression of native hbeta2m only results in marginal effect on the binding profile. Soluble beta2m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hbeta2m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/beta2m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. In vivo, peptide-loaded transfectants are considerably superior to parental cells in suppressing tumor growth. Our findings support the role of an allosteric mechanism in determining ternary MHC-I complex stability and propose membranal beta2m as a novel scaffold for CTL induction.     

Induction of antitumor immunity by CTL epitopes genetically linked to membrane-anchored beta2-microglobulin

Level and persistence of antigenic peptides presented by APCs on MHC class I (MHC-I) molecules influence the magnitude and quality of the ensuing CTL response. We recently demonstrated the unique immunological properties conferred on APCs by expressing beta2-microglobulin (beta2m) as an integral membrane protein. In this study, we explored membrane-anchored beta2m as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. We expressed in mouse RMA-S cells two H-2Kb binding peptides from MO5, OVA257-264, and TRP-2181-188, each genetically fused with the N terminus of membranal beta2m via a short linker. Specific Ab staining and T cell hybridoma activation confirmed that OVA257-264 was properly situated in the MHC-I binding groove. In vivo, transfectants expressing both peptides elicited stronger CTLs and conferred better protection against MO5 than peptide-saturated RMA-S cells. Cells expressing OVA257-264/beta2m were significantly superior to OVA257-264-charged cells in their ability to inhibit the growth of pre-established MO5 tumors. Our results highlight the immunotherapeutic potential of membranal beta2m as a universal scaffold for optimizing Ag presentation by MHC-I molecules.     

Polytuftsin: a potential precursor for slow release of the phagocytosis stimulating peptide tuftsin

1. Polytuftsin (Thr-Lys-Pro-Arg)n, was synthesized through polycondensation of an amino-free and carboxyl-activated derivative of tuftsin, H2N-Thr-Lys(Z)-Pro-Arg(Tos)-OSu, following suitable deprotection and fractionation steps. 2. Digestion of polytuftsin by trypsin, as well as by normal human serum, at 37 degrees C, yielded free tuftsin. 3. Polytuftsin affected the decreased formation of lung-metastasis, in B16 melanoma treated mice and prolonged the survival of animals more efficiently than tuftsin. 4. Tuftsin was found to be totally degraded by serum enzymes within approximately 60 min at 37 degrees C.