1. How to prove a hypothesis:
• Develop better tools over louder voices
2. Regulation of developmental and cellular biology:
• There is a vast checks and balances network for Treg
3. Clinical translation of Treg.
• local expertise (in vitro expanded Treg)
• Developing therapeutics and curing disease
• New jobs/careers to contemplate

The rise of the Ts empire (1970-1985): Infectious tolerance (Gershon), day 3 vs. day 7 Tx (Nishizuka and Sakakura).

Dark Ages (1985 to 1995): Adoptive transfer of Lyt-1 cells suppresses disease (Sakaguchi)

Renaissance Age (~1995 to 2003): First molecular marker for T suppressor cells (Sakaguchi, 1995)

Modern Age (2003-present): Identification of a transcriptional repressor that programs Treg and its mutation is responsible for Scurfy mice/IPEX humans (Ziegler, Rudensky 2003)

• Caveats
• pre-MHC restriction and BCR/TCR
• Limited monoclonal antibodies
• Few clonal T cell lines
• in vivo readout for suppression was Ab secret.
• Interpretation of data
• Ts secreted soluble factors
• I-J+ locus in MHC
• Ts had anti-Id specificity
• Lack of proper molecular tools made hard to prove models correct - nearly impossible to prove wrong

The rise of molecular immunology and fall of Ts empire

• Few stable CD8+ with antigen specific suppressive acitvity were isolated
• Biochem. nature of soluble suppressor factors was not elucidated (or reported erroneously)
• T-T suppressor hybridomas did not transcribe TCR genes
• Ts were not MHC restricted, but CD8+CTL are class I restricted

• Induction of Autoimmune Diseases in male nu/nu mice by the transfer of spleen Lyt subsets from male nu/+ mice
• Lyt-1:CD5
• Lyt-2:CD8

• Like anti-Lyt1, anti-CD25 depleted cells induce AI (Sakaguchi)

• T cell expansion regulated during thymic development and in the periphery
• Self reactive T cells
• Most deleted in thymus (AIRE)
• Peripheral tolerance
• "Crash" keeps total T cell #s down and prevents bystander activation
• AICD (activation induced cell death)
• Peripheral induction of Treg for chronic infect.

• Treg develop in thymus
• TCR has intermediate affinity for self antigen
• Upregulate Foxp3 (transcription factor req for Treg development)
• Low affinity for self - short time 15 days
• Int affinity for self - longer time 30 days

• Mice lacking Treg = autoimmunity
• Foxp3 knockout phenotype is like scurfy mouse
• Overexpression of Foxp3 can cure autoimmune disease
• Too many Treg (via transgene or adoptive transfer) increases risk of certain infections and tumor persistance

Adoptive transfer of Foxp3+ cells can cure autoimmune disease in mice

• Suppression of the Scurfy phenotype
• Reversal of diabetes with expanded Tregs (total or antigen specificity)
• The first hint at the clinical relevance of Treg came from experiments using adoptive transfer to cure IPEX/Scurfy phenotype and T1 diabetes.

Murine studies have shown that regulatory activity can be induced in non-Treg if they are stimulated under appropriate conditions

• Naive T cell
• TGFbeta or IL-10 or Retinoic Acid or AhR ligands changes naive t cell into activated converted Treg like cell

• Inhibitory cytokines
• Cytolysis
• Metabolic disruption
• Through gap junctions (cAMP)
• Death due to cytokine deprivation
• Targeting DCs
• Inhibition of DC maturation and function

• However the same process that drives the anti-leukemic effect can also induce GVHD
• GVHD can cause extensive destruction of host tissues and even death

• Leukemia relapse
• Umbilical Cord Blood HSCT has even lower rates of relapes that Matched related donor HSCT

• Decreasing non-relapse mortality after HSCT is critical
• 20-35% of all HSCT recipients die from TRM
• In addition because of high TRM some patients are not even offered HSCT
• TO improve the safety of BMT goal is to reduce or eliminate GVHD

• BALB/c ----> bone marrow (+/-) splenocytes ----> C57BL/6
• L-Selecting (hi) but not the L-selectin (lo) CD4+25+ T reg cells are potent inhibitors of GVHD and BM graft rejection

• Potential advantages of induced Treg
• Increased initial cell # (Treg 3% non Treg 97%)
• Ease of isolation (isolated with anti-CD4 beads)
• Degree of expansion (>1000 fold)
• Treg can be from graft donor

• Are as potent as nTreg in vivo at suppressing xenogeneic GVHD

• Hippen (Human Immunotherapy Lab) - Basic research, Drug discovery, Proof of principle, Tox. Profile, Scale up, Phase 1-3
• MCT:GMP Lab (toxicity and Phase 1-3)
• Clinical Physicians (Phase 1-3)

• Trials race rashly ahead for regulatory immune cells

• Phenotype is stable
• Frozen cells do not persist at all.... not viable?
• Ways to go before we reach Xeno levels
• Include explanations: Lymphopenia

• Identifying that nTreg were safe and effective in this model allowed us to initiate the world’s first clinical trial with in vitro expanded nTreg
• Which was headed up by Dr. Claudio Brunstein from the Dept. of Med. Heme/onc.
• Monitor Treg# and phenotype