9/30/2016 The Reality of Immunosuppression Clinical Trials: Identifying the role of TREGs Triple Immunosuppression to Prevent Graft Rejection Sang-Mo Kang, MD Division of Transplantation Department of Surgery University of California, San Francisco UCSF Transplant Symposium 2016 Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside Immunosuppression Overview � Brief background of regulatory T cells � Non-specific inhibition of immune responses ∆ against transplanted organ � Role of Regulatory T cells (Treg) in transplantation tolerance ∆ against pathogens ∆ against cancer � Numerous immunosuppression related � Considerations for therapeutic use of Treg in transplantation metabolic complications � Long term outcomes have largely plateaued � Treg Manufacturing � The most pressing need in transplantation is the � Ongoing/planned clinical trials induction of tolerance Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside 1
9/30/2016 Foxp3+ Tregs are essential for immune homeostasis The Emergence of Tregs in Transplant Tolerance Identity of suppressor T cells in Transplantation transplant CD4+CD25+ tolerance Suppressor Immune Treg therapy T cells tolerance in GvHD CD25 CD4 1950 1960 1970 1980 1990 2000 2010 The mutation in the FOXP3 gene leads to massive immune dysregulation (autoimmune polyendocrinopathy; autoimmune CD4+CD25+ Tregs in autoimmunity diabetes; hypothyroidism; autoimmune hemolytic anemia; autoimmune thrombocytopenia lymphadenopathy Foxp3 Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside 6 Immune system control of autoimmunity depends on a professional regulatory T cell Elimination of Tregs Leads to Rapid Death General Immune Homeostasis IL -15 IL -6 TNF- α Natural Treg IL -1 β Activated T reg Macrophage IL -23 AutoAb IL -12 Ag IFN- γ MHC/pep TCR naive T eff Tcell Bcell/DC CD3 T GF- β IL -10 T reg Local Regulation Kim JM et. al Nature Immunology 2007 Adaptive Treg Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside 2
9/30/2016 Growing evidence that Tregs are potential Role of Treg in Immunity therapeutics in transplantation � Tregs have been shown to prevent and even reverse � Tregs are critical to maintaining homeostasis and preventing autoimmunity in animal models autoimmunity � Tregs have been shown to be effective in preventing graft � Treg infiltration into tumors appears to provide a “privileged” versus host disease in humans microenvironment � Treg have been shown to be critical to the development and � Donor-specific Treg therapy does not appear to inhibit responses to viral pathogens or vaccines in mouse models maintenance of allospecific graft tolerance in numerous animal models � Treg therapy for graft versus host disease does not appear to ∆ Spontaneous liver transplant tolerance inhibit anti-tumor responses in bone marrow transplant models � Can Treg therapy be applied to reduce or eliminate non- specific immunosuppression in humans? Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside Donor-reactive Tregs have limited capacity to prolong Polyclonal vs Antigen Reactive Tregs allogeneic graft survival in normal hosts � Polyclonal Tregs are “unselected”, easy to expand Tregs Tregs Tx Tx � “donor antigen reactive” Treg (DAR Treg) have been selected BALB/c → B6 BALB/c → B6 for reactivity to the donor Islet transplantation heart transplantation 5 to 30 million � Approximately 1 in 10 polyclonal Tregs will have donor 100 reactivity None (n=7) Graft survival (%) 4C Treg (n=5) 75 ∆ Therefore at least 10 times less potent on a cell per cell basis Donor specific DAR Treg 50 poly Treg 25 0 0 20 40 60 80 100 Days after transplantation Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside 3
9/30/2016 Depletion of donor-reactive Teff cells is critical to efficacy of Treg Why Don’t Treg work well in normal hosts? DST Cyclophosphamide DST Cyclophosphamide Tregs Tregs Islet Tx Islet Tx � Almost all examples of transferring transplantation tolerance with Treg has been in the setting of co-adoptive transfer into 100% lymphopenic hosts (very few lymphocytes), with a limited Graft Survival (%) 80% Depletion + donor-specific Tregs number of T effector cells (Teff) 60% 40% � This suggests that the balance of Treg to Teff is important 20% None Depletion + polyclonal Tregs Depletion � 5-10% of ALL T cells are reactive to a fully mismatched donor 0% 0 7 14 21 28 35 42 49 56 63 70 ∆ Compared to approximately 1 in 10 6 for a conventional antigen Days after iTx ∆ is there a role for depletion of Teff? Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside Treg Therapy Increases Treg Frequency in the Allografts General Principles of Treg Rx from Mouse Models � Donor-specific Tregs are more effective than polyclonal, Deletion + Treg Deletion alone 40 unselected Treg ∆ 5-10% of polyclonal Tregs are donor reactive 30 % Tregs � Depletion of the donor-specific T effector cells is required for 20 optimal efficacy of Treg therapy 10 0 CD4 Foxp3 Ly5.1 Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside 4
9/30/2016 How many Tregs are needed to block transplant rejection in humans ? � Early adoptive co-transfer studies in mice showed that a ratio of at least 1:3 Treg/Teff ratio is needed If a ~1:3 Treg/Teff ratio is necessary for efficacy, � Tolerogenic treatments, such as sirolimus and anti-CD40L, leads to early accumulation of 30% Tregs in grafts how many Tregs do you need to give? � Alloantigen-specific Treg protected grafts have 30% Tregs in the first two weeks after transplant � 30% Tregs in immunosuppressive tumor micro-environment Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside Numbers of CD4+ T cells and Tregs in humans How to get Treg to 30%? Lymphocyte Tissue %CD4 Total CD4 %Treg Total Treg % Total Treg s Approaches Endogenous Endogenous Type of Number to % Tregs CD4 Tregs therapeutic Tregs infuse 0.25 x 10 9 10 x 10 9 5 x 10 9 Blood 50% 5% 1.9% Infuse Tregs after 165.5 x10 9 13.1 x10 9 Polyclonally 52 x10 9 30% 190 x 10 9 95 x 10 9 7.6 x 10 9 Lymph nodes 50% 8% 57.8% ex vivo expansion expanded 70 x 10 9 14 x 10 9 0.7 x 10 9 Spleen 20% 5% 5.3% 16.5 x10 9 1.3 x10 9 0.2 x10 9 Lymphodepletion + Isolated, 9% non-expanded banked without 50 x 10 9 10 x 10 9 2.5 x 10 9 Bone marrow 20% 25% 19% Tregs expansion Thymus a 50 x 10 9 10% 5 x 10 9 0.45 x 10 9 3.4% 9% 16.5 x10 9 1.3 x10 9 1.4 x10 9 Lymphodepletion + Polyclonally 16% expanded Tregs expanded Lung 30 x 10 9 40% 12 x 10 9 7% 0.84 x 10 9 6.4% Lymphodepletion + 1.65 x10 9** .13 x10 9** Donor antigen 0.4 x10 9 32% Liver 10 x 10 9 25% 2.5 x 10 9 2% 0.05 x 10 9 0.38% expanded Tregs expanded 50 x 10 9 17 x 10 9 0.5 x 10 9 Intestines 30-50% 3% 3.8% (Donor Specific) Others b 10 x 10 9 50% 5 x 10 9 5% 0.25 x 10 9 1.9% 165.5 x10 9 8% 13.1 x10 9 ** delete 90% of all T cells, leaving 1% donor reactive T effector Total 460 x 10 9 100% cells Tang and Lee Curr Op Organ Transpl, Aug, 2012 Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside 5
9/30/2016 Donor-reactive Treg expansion Considerations for Treg Therapy in Human Transplantation 5 10 4 CD25 10 3 10 � Treg therapy with unexpanded Treg will not achieve 0 high enough levels of Treg 3 4 5 0 10 10 10 CD127 � Treg therapy with polyclonal Treg will also be difficult Antigen-specific • Also potential for non-specific suppression primary expansion with donor B cells 11 days � Expansion of donor-specific Treg along with lymphodepletion will be necessary for clinical Donor Polyclonal secondary B cell activation translation Harvest & expansion using anti- 10 days Release assays CD3/28 beads 10-12 hrs 5 days Irradiated GMP K562-hCD40L cells Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside Putnam et al Am J Transpl 2013 Phenotype of Expanded Tregs - Examples Large Scale Expansion of Donor-Reactive Tregs Un-gated Treg culture Gated on CD4+ cells 1024 100 % Treg by TSDR 80 200-1600 fold CD62L 256 Helios 60 CD3 CD4 Fold Expansion 40 64 CD19 Foxp3 CD27 20 CD8 0 g g v e e n r r o T T c 16 y r T d l r o d p 100 drTreg % Suppression PolyTreg 80 4 TSDR: 60 T reg -specific- 40 demethylated 1 20 0 2 4 6 8 10 12 14 16 region 0 1:5 1:25 1:125 Days in Culture medium Allo APC Treg:Tresponder ratio aCD3/28 beads Clinical donor-reactive Treg manufacturing approved by FDA Treg Therapy in Transplantation: Bench to Bedside Treg Therapy in Transplantation: Bench to Bedside 6
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