Mixed Chimerism I: Renal Allograft Tolerance Through Mixed Chimerism

Studies from our laboratories over the past 15 years have demonstrated in several preclinical animal models that long-term tolerance to organ allografts can be achieved through the induction of mixed lymphohematopoietic chimerism. The present proposal is designed to extend these studies to a clinical protocol for renal transplantation in patients with end-stage renal failure.

Basis/Rationale: We have previously demonstrated in monkeys that a non-myeloablative conditioning regimen achieved transient mixed lymphohematopoietic chimerism and long-term renal allograft survival in full MHC mismatches in >70% of recipients. Our recent clinical studies have demonstrated efficacy in achieving similar mixed chimerism using a modified preparative regimen for the treatment of refractory lymphomas. In a related study, a combination of this regimen with renal transplantation from HLA-identical siblings has resulted in the successful treatment of three patients suffering from myeloma and renal failure.

Clinical Protocol Summary: We propose initially to treat 10 adult patients in end-stage renal failure with an available living related donor, mismatched for one HLA haplotype and not showing evidence of presensitization. Recipients will receive Cyclophosphamide, 60 mg/kg on days –5 and –4, MEDI-507, 0.1 mg/kg on day –2 and 0.6 mg/kg on days –1, 0 and +1, and 7 Gy thymic irradiation on day -1. On day 0, they will receive donor bone marrow (>2xl0^8 nucleated cells/kg) and a donor kidney transplant. Post-transplant, patients will receive a standard dose of cyclosporin for 60 days, followed by tapering doses over the next month.

Significance: Induction of transplantation tolerance has the potential to provide the following benefits to the renal transplant recipient:

1. Freedom long-term from chronic immunosuppressive medications and their complications
2. Freedom from the effects of chronic rejection
3. Lack of sensitization to other antigens present in the transplanted organ
4. Return of normal immune status and resistance to opportunistic infections

Mechanistic Studies: As part of the trial, the following studies will be conducted:

• Assessment of chimerism by flow cytometry: Flow cytometric analysis of blood after the donor cell infusion will be performed for the assessment of chimerism. Testing of percent chimerism will be done for multiple lineages.
• Immune Reconstitution Assays: T cell recovery, including naïve and memory-type CD4 and CD8 cells, as well as B cell and NK cell recovery, will be monitored. Flow cytometry will be used to monitor these PBMC subsets (CD2, CD3, CD4, CD8, CDIR, CD25, CD30, CD33, CD45, CD45RA, CD45RO, CD19, CD56, CD62L, HLA-DR).
• Tolerance Mechanisms Assays: CML and MLR are performed for in vitro analysis of tolerance to donor and host, and of anti-third party alloresponses. To assess tolerance by suppression of cell-mediated immunity, sequential recipient anti-donor MLR and CML assays will be performed. In addition co-culture MLR and CML analyses will be performed, using recipient PBL, frozen at the time of the transplant as naïve responder cells. We will then determine whether or not re-exposure of fresh host responders to donor antigens leads to suppression of the frozen naïve recipient responders in co-culture.
• PCR, Gene Chip: Samples will be collected to perform real-time PCR by the ITN core PCR laboratory. The PCR analyses will focus on several genes implicated in acute rejection, tolerance or tolerance breakdown. These genes include, but are not limited to, cyclo-oxygenase, perforin, IL-10, IL-7 and IL-15. Quantitative PCR will be performed to assess relative expression of such genes in participants at various time points. These genes will be used to track such parameters as disease onset, progression and response to therapeutics.
  The samples collected to isolate RNA for PCR analysis will also be used to supply RNA for gene chip assays. These samples will be analyzed using the 95 Affymetrix gene chip set or a custom array containing a subset of genes known to be important in immune cell function or expressed in peripheral blood.
• Vb Expression: Samples of RNA will be returned from the ITN repository to our laboratory for assays of V-beta expression.

When sufficient T cell numbers have recovered, ELISPOT and intracellular cytokine staining assays will be performed to measure cytokine production in response to donor, host and third party antigens. In addition, TREC analyses of sorted CD4+ and CD8+ populations will be used as a measure of thymic recovery.