Mechanisms of Gene Therapy for Tolerance: B7 Signaling Is Required for Peptide-IgG Gene-Transferred Tolerance Induction

Abstract

LPS-activated B cells, transduced with IgG fusion proteins, are highly tolerogenic APCs. To analyze the mechanisms for this B cell-delivered gene therapy, we first followed the fate of CFSE-labeled B cell blasts. These cells primarily localized to the spleen, where a small population persisted for at least 1 mo after injection. By day 7 after injection, ∼95% of the transduced cells had divided at least once, presumably an effect of the in vitro LPS activation into the cycle, because resting cells did not divide. B cells from gld donors were not tolerogenic, initially suggesting a role for Fas ligand (FasL) in tolerance. Because transduced normal B cells expressed only low levels of FasL and did not kill Fas-expressing Jurkat or A20 B lymphoma cells in vitro, these data suggest that gld B cells are not tolerogenic due to unique characteristics of these B cells rather than the lack of functional FasL expression. The transduced B cell blasts displayed significant up-regulation of both B7 costimulatory molecules, and B7.2 up-regulation was maintained through day 7 in vivo. When B cells from B7 knockout donors were transduced to express Ig fusion proteins, they were not tolerogenic in two different mouse strains and Ag models. Moreover, anti-B7 Ab blocked tolerance induction in this model, a result consistent with a role for B7 in tolerance induction. We propose that tolerance may be induced in this model by B7-driven negative regulatory signaling, but tolerance is maintained by a lack of signal 2, because expression of B7 is eventually lost in vivo.

Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice

“…In addition, MZ B cells also express higher levels of B7.1 and B7.2 than FO B cells do, thus facilitating effective antigen presentation to CTLA-4 ϩ regulatory T cells. 23,24 Through fluorescence-activated cell sorter analysis, we found that the B7 costimulatory molecules were further up-regulated on IgG1 anti-CD20 treatment plus high-dose FVIII exposure. In contrast, control IgG plus FVIII treatment had no significant effect on this phenotype of MZ B cells, compared with nontreated naive mice (Figure 3).…”

Tolerance without Clonal Expansion: Self-Antigen-Expressing B Cells Program Self-Reactive T Cells for Future Deletion

“…On the other hand, we have performed experiments in which the B cells of B MOG mice were activated in vivo using LPS or anti-CD40, but naive T cells transferred to these mice were not sufficiently stimulated to proliferate. It has previously been shown that activated transferred B cells need to express B7 molecules to induce tolerance (10). It is possible that, in our system, tolerance can also be achieved by costimulatory molecules other than B7.…”

“…On the other hand, memory T cells may not need costimulation and could therefore be activated by B cells. A problem of this hypothesis is that also activated B cells, which normally do express B7 molecules, can induce tolerance of T cells (10). It is therefore not clear whether it is indeed the absence of costimulation what causes B cells to induce tolerance.…”

In Vivo Induction of Type 1-Like Regulatory T Cells Using Genetically Modified B Cells Confers Long-Term IL-10-Dependent Antigen-Specific Unresponsiveness

“…Though the number of B cells used to induce tolerance was relatively high, it is unlikely that this contributed to an specific effect, because control mice injected with an equal number of nontransduced or GFP-transduced B cells showed no induction of an immune response. Moreover, in earlier studies performed by others (27,28), a comparable number of B cells (15-20 ϫ 10 6 ) were transferred to confer Ag-specific immune response.…”