Assessment of T-Cell Reconstitution After Two Step Haploidentical Stem Cell Transplants by Measurement of T-Cell Receptor Excision Circles (TREC)

Hematopoietic stem cell transplantation (HSCT) can associated with T cell immunodeficiency which can contribute to a high incidence of infections in patients.  In the thymus, hematopoietic progenitor cells undergo rapid proliferation and differentiation to become mature T cells. Factors which can affect the function of the thymus include: age, high dose chemotherapy and radiotherapy, GVHD, relapse and the occurrence of some opportunistic infections.  T cell rearrangement continues even in the elderly, and because the TRECs are non-replicating episomal DNA and their number can be decreased only by cell death, dilution through cell division or both, the measurement of the number of TRECs can be a measure of immunity.  The purpose of this study was to measure T cell reconstitution after hematopoietic stem cell transplantation in adult patients with hematological malignancies by measuring TREC at various time points after transplant.

            To measure thymic output after HSCT, TREC levels were measured in sixteen patients ages 23-68 (median age 53).  Two patients had CLL, one had follicular lymphoma and the rest had acute leukemia (ten AML and three ALL).  Ten patients received myeloablative transplants and six received reduced intensity transplants.  All patients achieved full donor chimerism at the time of analysis and all survived a minimum of 6 months after transplant (all but one survived at least 500 days post-transplant). For the myeloablative transplants, patients received 12 Gy of TBI followed by a donor lymphocyte infusion of 2 x 108 CD3 cells/kg followed two days later by cyclophosphamide at 60mg/kg/day for two days followed one day  later by a CD34 selected stem cell product.  For the non-myeloablative transplants, patients received fludarabine 30mg/m2 for four doses and cytarabine 2gm/m2 for four doses followed by 2 Gy of TBI then as described for the myeloablative transplants.  All patients received tacrolimus and mycophenolate mofetil for GVHD prophylaxis.  The CD34 dose ranged from 1.40 - 5.89 x 106 cells/kg (median dose 3.34 x 106 cells/kg).

            Peripheral blood samples were purified using the AUTOMACS magnetic cell sorter (Miltenyi Biotec).  To determine the purity of the CD4+ and CD8+ cells, two color cell staining using fluorochrome-conjugated antibodies against CD3+, CD4+, CD8+ and CD56+ and flow cytometry was performed. TRECs were quantified by real-time polymerase chain reaction (PCR) analysis using the 5’ nuclease (Taqman) assay. A standard curve was plotted using samples with known amounts of TREC, and TREC values for each sample was calculated by the ABI7700 software. Samples were run and analyzed in triplicate. 

Our analysis of the data showed that in general there were more TREC’s per 50,000 CD8 cells then CD4 cells.  Recipients with donors that had higher TRECs had more TRECs after transplant and that with one exception the number of TRECs in the recipient did not exceed that of the donor.