Immune Tolerance and Alloreactivity in Liver Transplant Recipients on Different Monotherapy Immunosuppressive Agents

Study Overview
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Brief Summary
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This study is being done with the purpose of trying to understand if and why transplant recipients may develop tolerance to their transplanted organ. Tolerance means being able to lower or take away immunosuppression (anti-rejection medications) without causing organ rejection.

Detailed Description
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Life-long immunosuppressive therapy is typically required in the majority of liver allograft recipients. In the early years of liver transplantation (LT), the majority of deaths occurred secondary to graft loss from acute or chronic rejection despite immunosuppression (IS). With the advent of more powerful and specific IS agents, e.g. calcineurin-inhibitors (CNIs) cyclosporine (CyA) and tacrolimus (TAC), graft rejection rates significantly declined and short and long term graft/patient survival dramatically improved. However, along with the advance in survival rates came the adverse effects of long term immunosuppression (IS), e.g. morbidity and mortality from cardiovascular events, renal insufficiency, infectious complications, recurrent viral hepatitis and malignancy. These events are exacerbated by pre-existing conditions and an aging transplant population. Immunosuppression tapering or withdrawal could lower the incidence of these complications and improve long term graft and patient survival. Therefore, the study proposed is a laboratory investigation (using blood samples collected from the subjects) comparing immune tolerance and alloreactivity profiles in LT recipients on monotherapy IS or converted to rapamycin monotherapy, to determine tolerogenic properties of the different IS agents. Knowledge of these properties would support the need for specific IS therapy to promote immune tolerance and consider IS withdrawal. Monotherapy patients will be identified by the organ transplant database and medical charts at Northwestern. Patients will be invited to participate in the study and asked to undergo venipunctures for our analysis. Patient demographics, laboratories and other clinical data will be recorded. Patients on CNI monotherapy are continuously being identified for conversion to rapamycin monotherapy during clinic visits or chart reviews at Northwestern. Patients are selected for conversion due to significant CNI side effects, e.g. chronic kidney disease (creatinine clearance < 50 in the absence of significant proteinuria > 1g, poorly controlled diabetes mellitus/hypertension/hyperlipidemia, peripheral neuropathy). In general, patients are converted from CNIs to rapamycin over 2-3 weeks once therapeutic rapamycin levels are achieved. Study procedures will be carried out by the investigators and associated personnel. Patients will be assigned a number in numerical order, to remove patient identifiers from the data analysis. A separate screening/enrollment log will be kept separate from the data. Baseline characteristics of the patients will be recorded: age, sex, liver disease, past medical history, history of acute rejection or other graft dysfunction, other post-LT complications, previous and current IS regimens. Monotherapy patients (10 from CyA, Tacrolimus, and MMF; 5 rapamycin) will be identified as above and asked to participate. Blood will be drawn at one time point for the following analysis: Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). HLA microchimerism & HLA G Ten patients who have been pre-selected for rapamycin conversion will have the above assays performed two weeks prior to conversion and 3-6 months following conversion. They will also have liver function and drug level tests.

Official Title
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Immune Tolerance and Alloreactivity in Liver Transplant Recipients on Different Monotherapy Immunosuppressive Agents

Conditions
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Liver Transplant Rejection, Immunosuppression

Intervention / Treatment
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* Procedure: Blood Draw - Rapamycin
* Procedure: Blood Draw from Control Subjects
* Procedure: Blood Draw - CyA
* Procedure: Blood Draw - Tacrolimus
* Procedure: Blood Draw - MMF
* Procedure: Blood Draw - Rapamycin


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age ≥18 years Orthotopic or Living-Related liver transplant (LT) recipient Monotherapy patients: > 6 months with stable graft function on current monotherapy (CNI, MMF, or rapamycin) Converting patients: CNI therapy converting to rapamycin or MMF monotherapy and > 6 months of stable graft function. >1 years post-LT without an acute rejection episode or chronic rejection Normal liver function tests (no recurrent HCV, chronic rejection, autoimmune hepatitis, etc.) No history of induction or lymphocyte depletion therapy Exclusion Criteria: Multi-visceral organ recipients Graft dysfunction of any etiology Inadequate follow-up or available outcomes Unable to understand, sign or ask questions regarding the informed consent process and protocol

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Control Group<br>Ten Healthy individuals will have blood drawn (4 green top tubes(40 ml = 8 tsp.)) at one time point at Northwestern for control purposes. Blood will be drawn to conduct the following tests: Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells), and HLA microchimerism & HLA G. | Procedure: Blood Draw from Control Subjects<br>* Ten healthy individuals will have blood drawn (40 ml = 8 teaspoons (tsps.)).Blood will be drawn at one time point for the following: Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). HLA microchimerism & HLA G<br>|
| Monotherapy Group<br>Monotherapy patients [cyclosporine (CyA) (10 patients), Tacrolimus (5 patients), mycophenolate mofetil (MMF) (10 patients), rapamycin (10 patients)]: Blood will be drawn at one time point (4 green top tubes (40 ml = 8 tsp.)) to conduct the following tests: Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells), and HLA microchimerism & HLA G. | Procedure: Blood Draw - CyA<br>* Blood drawn from 10 patients on cyclosporine (CyA) (40 ml = 8 tsp.)). Blood will be drawn at one time point for the following: Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). HLA microchimerism & HLA G<br>* Other names: cyclosporine;Procedure: Blood Draw - Tacrolimus<br>* Blood drawn from 5 patients on Tacrolimus (40 ml = 8 tsp.) at one time point for the following: Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). HLA microchimerism & HLA G<br>* Other names: Tacrolimus;Procedure: Blood Draw - MMF<br>* Blood will be drawn from 10 patients on mycophenolate mofetil (MMF) (40 ml or the equivalent of 8 teaspoons). Blood will be drawn at one time point for the following analysis: Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). HLA microchimerism & HLA G<br>* Other names: mycophenolate mofetil;Procedure: Blood Draw - Rapamycin<br>* Blood drawn from 10 patients on rapamycin (40 ml = 8 tsp.)) at one time point for the following: Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). HLA microchimerism & HLA G<br>* Other names: Rapamycin;|
| Conversion Group<br>Ten CNI monotherapy/dual therapy (CNI + MMF) patients pre-selected for conversion to rapamycin or wean to MMF monotherapy. Assays performed 2 weeks prior to conversion, 3-6 months following successful conversion. Liver function/drug levels monitored weekly during conversion until stable levels achieved. Patients converting from CNI monotherapy to rapamycin monotherapy (2-4 wks.): CNI discontinued when 2 therapeutic rapamycin levels (5-10) reached, graft function stable (clinical care protocol). MMF conversion: MMF dose slowly increased to 3 g/day (max.) while CNI therapy reduced by 1-2 mg/day (FK506) or 25-50 mg/day (CyA) monthly until CNI discontinued (1-6 months) (clinical care protocol). Monthly liver function/drug levels performed after successful conversion (standard of care). | Procedure: Blood Draw - Rapamycin<br>* Ten calcineurin-inhibitors (CNI) monotherapy or dual therapy (CNI+MMF) patients will have blood taken (40 ml=8 tsp.) 2 wks. prior to conversion, 3-6 months post successful conversion. 1) Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28-FOXP3+CD127low cells). 2) Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers shown to induce regulatory T cells (ILT3; ILT4), 3) Soluble HLA G, and 4) Liver function/drug levels. If problems develop during conversion (e.g. acute rejection, significant drug side effects) requiring discontinuation of rapamycin, MMF and/or reversion to CNI therapy, assays will not be performed. Monthly liver function/drug levels performed after successful conversion (standard of care).<br>* Other names: Sirolimus;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Track interval outcome measures for development of higher percentages of FOXP3+ T1 regulatory cells in stable liver transplant recipients on rapamycin or MMF monotherapy compared to CNI monotherapy. | Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). Liver function and drug levels. | Two weeks prior to conversion, Months 3 & 6 following conversion | 
| Track interval outcome measures for development of higher percentages of FOXP3+ T regulatory cells in liver transplant recipients after conversion from CNI to rapamycin comparing to MMF monotherapy. | Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). | Two weeks prior to conversion, Months 3 & 6 following conversion | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Track interval outcome measures for development of higher percentages of immunophenotypic markers associated with regulatory T cell production in stable liver transplant recipients on rapamycin or MMF monotherapy compared to CNI monotherapy. | Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). HLA microchimerism & HLA G (dendritic cell ratios, soluble HLA G) Liver function and drug levels. | Two weeks prior to conversion, Months 3 & 6 following conversion | 
| Track interval outcome measures for development of higher percentages of immunophenotypic markers associated with regulatory T cell production in liver transplant recipients after conversion from CNI to rapamycin or MMF monotherapy. | Dendritic cell assays: myeloid vs. lymphoid (CD11c; CD123); maturation and ability to process antigens (CD83; CD205); markers that have been shown to induce regulatory T cells (ILT3; ILT4). Regulatory/Suppressor Cells (CD4+CD25+FOXP3+CD127low; and CD8+ CD28- FOXP3+CD127low cells). HLA microchimerism & HLA G (dendritic cell ratios, soluble HLA G)(hypertension, hyperlipidemia, renal insufficiency, diabetes, neuropathy) | Two weeks prior to conversion, Months 3 & 6 following conversion | 
| Document improvement in adverse CNI side effects after conversion to rapamycin or MMF monotherapy, comparing designated time points. | CNI side effects - hypertension, hyperlipidemia, renal insufficiency, diabetes, neuropathy. Review liver function and drug levels at designated time points. | Two weeks prior to conversion, Months 3 & 6 following conversion | 
| Document the development of any adverse rapamycin or MMF side effects after conversion, comparing designated time points. | Document Rapamycin side effects - oral ulcers, edema, pancytopenia, gastrointestinal dysfunction; or MMF side effects - pancytopenia, gastrointestinal dysfunction. Review liver function and drug levels at designated time points. | Two weeks prior to conversion, Months 3 & 6 following conversion | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Immunosuppression