Short-course rapamycin treatment enables engraftment of immunogenic gene-engineered bone marrow under low-dose irradiation to permit long-term immunological tolerance

Abstract

BackgroundApplication of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a ‘niche’ so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained.MethodsBM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells.ResultsImmune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM.ConclusionsRapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0508-3) contains supplementary material, which is available to authorized users.

Transfer of antigen-encoding bone marrow under immune-preserving conditions deletes mature antigen-specific B cells in recipients and inhibits antigen-specific antibody production

“…To facilitate the testing of this hypothesis, we exploited a mild conditioning regime that enables engraftment of engineered BM but which largely preserves an existing immune repertoire (Coleman, Bridge et al 2013, AL-Kouba, Wilkinson et al 2017, Bhatt, Rudraraju et al 2017).…”

“…Low-dose irradiation serves as a mild conditioning regime that permits significant engraftment of transferred BM whilst substantially preserving recipient immunity (AL- Kouba, Wilkinson et al 2017, Bhatt, Rudraraju et al 2017. Transfer of BM encoding for antigen expression following low-dose irradiation is an effective means of ablating antigen-specific T-cell responsiveness (Coleman, Bridge et al 2013).…”

“…For non-myeloablative (300cGy) procedures, mice received a single dose of irradiation. Some mice were administered rapamycin (0.6mg/kg) daily for 3 weeks post-transplant (Bhatt, Rudraraju et al 2017).…”

“…Here we address this and ask can 'de novo peripheral tolerance' be induced in a pre-established B-cell compartment? We use recent advances in BM/HSCmediated gene therapy that use mild 'conditionng' regimes to enable the effects on established lymphocyte populations and their repertoires to be examined (Coleman, Bridge et al 2013, Coleman, Jessup et al 2016, Bhatt, Rudraraju et al 2017. Many studies of B-cell tolerance employ systems where the B-cell receptor (BCR) is genetically manipulated either to increase clonal frequency..…”

Defining B cell tolerance checkpoints and their modulation by immunotherapy

“…Antigen can also be genetically targeted for expression by tolerogenic cells post- In a therapeutic setting, where established pathogenic memory and effector populations against the encoded antigen exist, widespread expression of the antigen is not ideal because the host immune system mediates rejection of donor cells when antigen is expressed in the BM compartment [539]. Additionally, widespread expression often encourages the silencing of gene promoters, which turns off antigen expression, and therefore prevents tolerance [547].…”