Induction of hyporesponsiveness to intact foreign protein via retroviral-mediated gene expression: the IgG scaffold is important for induction and maintenance of immune hyporesponsiveness

Abstract

IgG molecules can be highly tolerogenic carriers for associated antigens. Previously, we reported that recipients of bone marrow or lipopolysaccharide-stimulated B-cell blasts, both of which were retrovirally gene-transferred with an immunodominant peptide in-frame with the variable region of a murine IgG heavy chain, were rendered profoundly unresponsive to that epitope. To further investigate whether tolerance to larger molecules can be achieved via this approach and whether the IgG scaffold is important for induction and maintenance of immunological tolerance, we engineered two retroviral constructs encoding the cI lambda repressor (MBAE-1-102 and MBAE-1-102-IgG) for gene transfer. Our results show that recipients of bone marrow or peripheral B cells, transduced with the MBAE-1-102-IgG recombinant, are hyporesponsive to p1-102. In addition, the self-IgG scaffold enhanced the induction and maintenance of such an immune hyporesponsiveness. Thus, our studies demonstrate that in vivo-expressed IgG heavy chain fusion protein can be processed and presented on the appropriate MHC class II, resulting in hyporesponsiveness to that antigen and offering an additional therapeutic approach to autoimmune diseases.

Figure 1

T-cell responses to the dominant epitopes of p1–102 in CB6 F₁ recipients of 1–102-IgG-transduced LPS B-cell blasts. CB6 F₁ mice were injected with mock-transduced or F12.7 gene-transduced LPS B-cell blasts. Mice then were immunized with p1–102 and HEL. Two weeks later, lymph nodes were restimulated with dilutions of synthetic peptides, representing H-2^d (p12–26, Left) or H-2^b (p73–88, Right) immunodominant epitopes and pulsed with [³H]thymidine. The incorporated [³H]thymidine was detected by using a direct beta counter. One set of representative experiments is shown, with 3–5 mice per group. All mice respond equally to HEL. Stimulation index refers to (cpm/backgroup cpm). Background cpm vary from 150 to 300 in this set of experiments. Data are presented as mean ± SE.

Figure 2

Antibody responses to p1–102 and epitopes in CB6 F₁ recipients of 1–102-IgG-transduced B-cell blasts. CB6 F₁ mice were pretreated as described in Fig. 1. Mice were primed and boosted with p1–102 and HEL. Antibody IgG immune response was measured by ELISA. One set of representative experiments is shown, with 3–4 mice per group. All mice respond equally to HEL.

Figure 3

Cytokine responses to p1–102 in CB6 F₁ recipients of 1–102-IgG-transduced B-cell blasts. CB6 F₁ mice were pretreated and immunized as described in Fig. 1. One set of representative experiments is shown, with 3–5 mice per group. Data are presented as mean ± SE.

Figure 4

Humoral hyporesponsiveness to p1–102 in CB6 F₁ mice receiving 1–102-IgG-transduced BM. CB6 F₁ mice were irradiated with 400 rad and injected with mock-transduced or F12.7 gene-transduced BM cells. Mice were primed and boosted with p1–102 and HEL. Left Y ordinate represents the IgG titer against p1–102, and the right Y ordinate represents the titers against epitopes. Antibody IgG titers against p1–102 and p73–88 in recipient mice of F12.7 transduced BM (filled columns) are significantly lower than those in mock controls (empty columns) (P < 0.05). Note that antibody against the minor epitope (p55–69) also is reduced in the recipient mice of F12.7-transduced BM.

Figure 5

Efficacy in tolerance induction and maintenance between 1–102-IgG and 1–102 gene transfer. (A) LPS blast recipients. (B) BM recipients. (Upper) Primary immune response. (Lower) Secondary immune response.

Figure 6

Difference in memory T-cell responses between 1–102-IgG and 1–102 gene transfer. CB6 F₁ mice were pretreated, immunized, and boosted as described in Fig. 4. T-cell proliferation and cytokine production of splenic T cells was measured 1.5 months after immunization. One set of representative experiments is shown, with five mice per group. Data are presented as mean ± SE.

Figure 7

Comparable levels of p1–102 mRNA expression between 1–102-IgG and 1–102. Spleen samples from the same recipients as in Fig. 6 were harvested (approximately 4 months after gene transfer), and the p1–102-specific mRNAs were measured by reverse transcription–PCR followed by Southern blotting using p1–102 as a probe.