One group of representative experiments is shown, with five mice per group. hyporesponsiveness. Thus, Leucyl-phenylalanine our studies demonstrate that (13) exhibited the specific tolerogenic properties in adult BALB/c mice of a chimeric molecule consisting of residues 12C26 of the cI repressor protein (p1C102) fused to the N terminus of a murine IgG H chain. In addition, animals receiving retrovirally encoded 12C26-IgG were shown to be profoundly unresponsive to the 12C26 peptide at both the humoral and cellular levels (14). In cases where immunodominant epitopes have not yet been mapped, it would be desirable to fuse the Leucyl-phenylalanine entire protein to the IgG scaffold for tolerance induction. However, it is unclear whether a chimeric molecule consisting of full-length protein would be efficiently processed and presented and in turn tolerize as effectively as selected epitopes. Furthermore, it is unclear whether the IgG scaffold is essential for induction and maintenance of tolerance. To address these questions, we established two MBAE retroviral constructs encoding either p1C102 Rabbit Polyclonal to DAPK3 or p1C102-IgG heavy chain. The constructs were used to transduce B cells or bone marrow (BM) cells, which were adoptively transferred to syngeneic mice to test for tolerance. When challenged with p1C102, mice receiving either LPS B-cell blasts or BM transduced with the 1C102-IgG-encoding gene failed to respond as effectively as the mock controls to the major epitopes of p1C102 recognized by mice of these haplotypes. Moreover, compared with 1C102 alone, the 1C102-IgG fusion protein Leucyl-phenylalanine was more effective in the induction of hyporesponsiveness. The former induced only a transient form of hyporesponsiveness, suggesting that this IgG scaffold is usually Leucyl-phenylalanine important in the maintenance of tolerance. These results show that retrovirally mediated transfer of a gene encoding full-length protein fused to IgG is an effective approach for the purpose of inducing hyporesponsiveness to multiple epitopes. MATERIALS AND METHODS Mice. CB6 F1 mice were purchased from The Jackson Laboratory at 6C8 weeks of age and housed in pathogen-free micro-isolater cages in our animal facility. Retroviral Constructs Encoding cI p1C102 or p1C102-IgG and Virus Producer Cell Lines. A 320-bp DNA fragment encoding p1C102 was amplified by PCR (30 cycles: 94C, 15 sec; 55C, 15 sec; 72C, 1.5 min) from pRB104 (a kind gift from Richard M. Breyer, Vanderbilt Medical Center, Nashville, TN). The 5 primer, GCG GTC GAC ATG AGC ACA AAA AAG AAA CC, contained a M15 (pREP4) (QIAgen Expressionist Kit). The p1C102 protein was prepared and purified by using a Ni-nitrilotriacetic acid column according to the manufacturers instructions. Eluted p1C102 fractions were dialyzed against PBS (pH 7. 2), filter-sterilized, and antigenically verified by ELISA and Western blot analysis (data not shown). Western blot analysis was performed by using the mAb B3.11, specific for the 12C26 epitope of p1C102 (13, 14). The major antigenic peptides of p1C102 in H-2d, H-2b, and H-2k mice, residues 12C26 (LEDARRLKAIYEKKK), residues 73C88 (VEEFSPSIAREIYEMY), and residues 55C69 (NALNAYNAALLAKIL), respectively (6, 18) were synthesized in the Molecular Biology Core of the Holland Laboratory by using a solid-phase method and were purified to 95% homogeneity by HPLC. Retroviral-Mediated Gene Transfer to BM and LPS-Stimulated B-Cell Blasts. Retroviral-mediated gene transfer into BM and bacterial LPS (055:B5, Sigma)-stimulated splenic B cells has been described (14, 15, 19). Briefly, cells were cultured (3 106/ml, 5 ml cultures) for 48 hr with irradiated (2,000 rad) F5.19/F12.7 or mock control packaging cell lines in the presence of 6 g/ml of polybrene and either 50 g/ml of LPS for B-cell blasts or 200 units/ml of IL-3, IL-6, and IL-7 (Genzyme) for BM. For adoptive transfer of BM, adult CB6 F1 mice exposed to 400-rad irradiation were injected i.v. with 1C2 106 gene-transferred or mock-transduced BM cells. For adoptive transfer of LPS B-cell blasts, nonirradiated CB6 F1 mice were injected with at least 1 107 transduced LPS blasts. Immunologic Protocols. Ten days after receiving transduced LPS B-cell blasts or 6C8 weeks in.