Stable Tregs and Related Materials and Methods

A method to make stable iTreg cells, comprising introducing an inducer composition to an inducible first cell culture,

• wherein the inducer composition comprises anti-CD3 mAb/anti-CD28 mAb and at least one composition selected from the group consisting of:
• anti-IL-2;
• antiCD25 mAb;
• or Janus tyrosine kinase (Jak3) inhibitor, and
• wherein the first cell culture comprises CD4+Foxp3/GFP− cells in a medium with or without syngeneic antigen presenting cells.

A two-phase method of obtaining a stable regulatory T-cell, comprising

• a first induction phase and a second expansion phase,
• wherein the first induction phase comprises contacting a naive CD4 T cell in a first medium with a stimulatory signal and an appropriate amount of:
• one or more factors comprising:
• i) a combination of anti-IL-2 mAb, or anti-IL-2 receptor mAb, or Jak3 inhibitor;
• ii) an anti-T-cell receptor-β (TCRβ) mAb;
• or, iii) an anti-CD11a MAb;
• wherein the stimulatory signal is a combination of an anti-CD3 antibody and an anti-CD28 antibody, and
• wherein the second expansion phase comprises replacing the first medium from the first induction phase with a fresh culture medium containing an appropriate amount of IL-2 and without addition of the factors used in the second induction phase for a sufficient time to generate at least one stable regulator T-cell wherein the generated stable regulatory T-cell expresses forkhead 3 (Foxp3) transcription factor, a GITR marker, a CTLA4 marker, a CD25 marker, and suppresses at least 80% proliferation of an effector T cell in an in vitro suppressor assay at a 1:1 ratio.