A GMCSF-Neuroantigen Tolerogenic Vaccine Elicits Systemic Lymphocytosis of CD4+ CD25high FOXP3+ Regulatory T Cells in Myelin-Specific TCR Transgenic Mice Contingent Upon Low-Efficiency T Cell Antigen Receptor Recognition

Abstract

Previous studies showed that single-chain fusion proteins comprised of GM-CSF and major encephalitogenic peptides of myelin, when injected subcutaneously in saline, were potent tolerogenic vaccines that suppressed experimental autoimmune encephalomyelitis (EAE) in rats and mice. These tolerogenic vaccines exhibited dominant suppressive activity in inflammatory environments even when emulsified in Complete Freund's Adjuvant (CFA). The current study provides evidence that the mechanism of tolerance was dependent upon vaccine-induced regulatory CD25+ T cells (Tregs), because treatment of mice with the Treg-depleting anti-CD25 mAb PC61 reversed tolerance. To assess tolerogenic mechanisms, we focused on 2D2-FIG mice, which have a transgenic T cell repertoire that recognizes myelin oligodendrocyte glycoprotein peptide MOG35-55 as a low-affinity ligand and the neurofilament medium peptide NFM13-37 as a high-affinity ligand. Notably, a single subcutaneous vaccination of GMCSF-MOG in saline elicited a major population of FOXP3+ Tregs that appeared within 3 days, was sustained over several weeks, expressed canonical Treg markers, and was present systemically at high frequencies in the blood, spleen, and lymph nodes. Subcutaneous and intravenous injections of GMCSF-MOG were equally effective for induction of FOXP3+ Tregs. Repeated booster vaccinations with GMCSF-MOG elicited FOXP3 expression in over 40% of all circulating T cells. Covalent linkage of GM-CSF with MOG35-55 was required for Treg induction whereas vaccination with GM-CSF and MOG35-55 as separate molecules lacked Treg-inductive activity. GMCSF-MOG elicited high levels of Tregs even when administered in immunogenic adjuvants such as CFA or Alum. Conversely, incorporation of GM-CSF and MOG35-55 as separate molecules in CFA did not support Treg induction. The ability of the vaccine to induce Tregs was dependent upon the efficiency of T cell antigen recognition, because vaccination of 2D2-FIG or OTII-FIG mice with the high-affinity ligands GMCSF-NFM or GMCSF-OVA (Ovalbumin323-339), respectively, did not elicit Tregs. Comparison of 2D2-FIG and 2D2-FIG-Rag1−/− strains revealed that GMCSF-MOG may predominantly drive Treg expansion because the kinetics of vaccine-induced Treg emergence was a function of pre-existing Treg levels. In conclusion, these findings indicate that the antigenic domain of the GMCSF-NAg tolerogenic vaccine is critical in setting the balance between regulatory and conventional T cell responses in both quiescent and inflammatory environments.

Approaches to inducing antigen‐specific immune tolerance in allergy and autoimmunity: Focus on antigen‐presenting cells and extracellular vesicles

“…According to the above considerations and studies on Treg cell activation by APCs, 55 one can speculate that surface expression of specific cytokines or growth factors on EVs can not only drive cellular trafficking and targeting of the latter, but also greatly support their tolerogenic function (Figure 1). In addition, our research findings showed that orally administered EVs may also efficiently suppress an ongoing immune response, as discussed below.…”

“…As a consequence, induced Treg cells ameliorated EAE. GM‐CSF binding to the corresponding receptors on myeloid APCs was reported to trigger selective cellular targeting and further promote tolerogenic presentation of MOG peptides to induce the generation of Treg lymphocytes instead of effector T cells 55 …”

“…On the other hand, equipping EVs with antigenic determinants complexed with MHC should direct them towards T cells‐expressing TCRs of corresponding specificities. Beyond that, covalent binding of cytokines or growth factors, like GM‐CSF, 55 and immune checkpoint molecules, like cytotoxic T‐lymphocyte‐associated protein‐4 (CTLA‐4), or their ligands, programmed death‐1 ligand‐1 (PD‐L1) especially, 95 to EV membranes may both increase the capacity of EV binding to desired cells via selective receptor ligation and greatly support the tolerogenic effect of EVs and their cargo. In such instances, the predicted antigen‐specificity of EV‐induced immune tolerance would be conferred by reprogramming, inhibiting and/or deleting the cells specifically targeted for mediating the immune response against a particular antigen, that is, APCs and antigen‐specific T cells.…”

Low-Zone IL-2 Signaling: Fusion Proteins Containing Linked CD25 and IL-2 Domains Sustain Tolerogenic Vaccination in vivo and Promote Dominance of FOXP3+ Tregs in vitro

“…The purified protein was activated by 10 min of exposure to 70 • C, and each prep was verified for bioactivity by induction of FOXP3 in MOG-stimulated 2D2-FIG splenocyte (SPL) cultures. Generation, expression, purification, and bioassay of GMCSF-MOG was performed as described in previous studies (16,19,20). This FP was comprised of the murine GM-CSF cytokine as the N-terminal domain, the peptide sequence comprising MOG35-55, and an eight-histidine C-terminus.…”

“…optimized by incorporating self-antigens that are recognized with intermediate TCR recognition efficiencies (14)(15)(16)(17)(18)(19)(20)(21). Conversely, vaccines incorporating antigens that are recognized with high TCR recognition efficiencies are optimal for induction of immunogenic Tcon responses (21)(22)(23).…”