Modulation of CD4+ T lymphocyte lineage outcomes with targeted, nanoparticle-mediated cytokine delivery.

Within the immune system there is an exquisite ability to discriminate between "self" and "non-self" that is orchestrated by antigen-specific T lymphocytes. Genomic plasticity enables differentiation of naive CD4+ T lymphocytes into either regulatory cells (Treg) that express the transcription factor Foxp3 and actively prevent autoimmune self-destruction or effector cells (Teff) that attack and destroy their cognate target. An example of such plasticity is our recent discovery that leukemia inhibitory factor (LIF) supports Treg maturation in contrast to IL-6, which drives development of the pathogenic Th17 effector phenotype. This has revealed a LIF/IL6 axis in T cell development which can be exploited for modulation using targeted cytokine delivery. Here we demonstrate that LIF-loaded nanoparticles (NPs) directed to CD4+ T cells (i) oppose IL6-driven Th17 development; (ii) prolong survival of vascularized heart grafts in mice; and (iii) expand FOXP3+ CD4+ T cell numbers in a non-human primate model in vitro. In contrast, IL-6 loaded nanoparticles directed to CD4+ T cells increase Th17 development. Notably, nanoparticle-mediated delivery was demonstrated to be critical: unloaded nanoparticles and soluble LIF or IL-6 controls failed to recapitulate the efficacy of cytokine-loaded nanoparticles in induction and/or expansion of Foxp3+ cells or Th17 cells. Thus, this targeted nanoparticle approach is able to harness endogenous immune-regulatory pathways, providing a powerful new method to modulating T cell developmental plasticity in immune-mediated disease indications.

A LIF/Nanog axis is revealed in T lymphocytes that lack MARCH-7, a RINGv E3 ligase that regulates the LIF-receptor.

Nanog is a stem cell transcription factor required for self-renewal and for maintaining pluripotency, and Nanog itself is regulated at least in part by leukaemia inhibitory factor (LIF)--a pluripotent cytokine of the IL6 family. MARCH-7 is an E-3 ligase linked to regulation of the LIF-receptor in T lymphocytes and T cells from mice that lack expression of MARCH-7 are hyper-responsive to activation signals and show a five-fold increase in LIF activity. Here we ask, does MARCH-7 influence the expression profile of Nanog during the synchronized entry of T cells into the cell cycle? We discovered that lack of MARCH-7 was permissive for Nanog expression at both transcript and protein levels during G1/S: moreover, addition of exogenous LIF to the MARCH-7 null cells caused a further 13-fold induction of Nanog; other measured transcripts including TGFß, p53 and STAT3 were relatively unchanged. Since lack of MARCH-7 altered responsiveness to activation signals we sought evidence for pre-existing regulatory miR's that might correlate with MARCH-7 gene dose using head-to-head comparisons between MARCH-7 null, heterozygous and wt spleen cells. 34 miRs were found including miR-346 that is known to target LIF transcripts and miR-346 is one of 16 miRs differentially expressed between hESCs and induced hiPSCs. Of the 34 miRs, 12 were known to be temporally regulated in embryonic nerve cells. In summary, in the absence of MARCH-7 a new signaling pathway is unmasked that involves Nanog expression in the T cell lineage. This is the first demonstration that T cells retain responsiveness to a LIF/Nanog axis and that this axis is linked to MARCH-7.