The Kinetics of Early T and B Cell Immune Recovery after Bone Marrow Transplantation in RAG-2-Deficient SCID Patients

The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT

BCR rearrangement analysis and clinical outcome.

<p>Two different IgH BCR gene rearrangements (FR2 and FR3) were PCR amplified followed by Gene Scan analysis in all studied patients 2–4 months post-bone marrow transplant (BMT) and in an age-matched healthy control (a). Parallel detection of KREC levels and the FR3 IgH BCR gene rearrangement were PCR amplified, followed by RQ-PCR analysis (KREC) or by Gene Scan analysis (BCR repertoire) in patients #7 (upper panel) and #8 (lower panel) from the time of BMT and up to one year post-transplant. For KREC, both signal joint and coding joint were examined (b).</p

Pre-BMT immune workup of the 10 RAG2-deficient SCID patients, including flow cytometry analysis, and cell-mediated and humoral-immune evaluations.

<p>Control levels are presented in bracket.</p><p>ND – not done, UD- undetectable, PHA – phytohemagglutinin, SI – stimulation index (patient/control), TCR - T cell receptor, TREC - TCR excision circles (copies per 0.5 mcg DNA), ALC - absolute lymphocyte count.</p

Early presence of KREC and transplant-related morbidity.

<p>KREC signal joint (sj) copies were detected in all studied patients (#1–#10) at either 2–4 months (a) or various time lengths (b) post-bone marrow transplant (BMT). KREC copies were determined by real-time quantitative (RQ) PCR and calculated by comapring the obtained cycle threshold (Ct) for each patient to the Ct of the same patient's KREC levels before undergoing BMT, using the ΛΛCt relative quantification analysis. The percent ratios between KREC-cj copies and KREC-sj copies were measured in patients with early presentation of KRECs (#7–#10) (c). Four age-matched healthy individuals were used as controls. Each sample was detected in triplicate in all experiments.</p

Characteristics of the BMT procedure and its related complications.

<p>MRD - match related donors, MUD - match unrelated donor, MMRD - mismatch related donor, Bu – Busulfan, Cy – cyclophosphamide, ATG – anti thymocytes globulin, Tre – Treosulfan, Flu – Fludarabine, Thi – Thiotepa, Mel – melphalan, PBSC –peripheral blood stem cells, BM – Bone marrow, UCB – Umbilical cord blood, CSA – Cyclosporin, MTX – methotrexate, MMF – mycophenolate mofetil.</p

Simultaneous recovery of the ordered TCR rearrangement events and its relations to clinical outcome.

<p>Four different TCR rearrangement events (TCRD-Dδ2-Dδ3, TCRD-Dδ2-Jδ1, TCRD-Vδ1-Jδ1, TCRA-REC), indicative of different thymic maturation stages, were analyzed by real-time quantitative (RQ)-PCR overtime post transplant in 5 patients. The fold of change in these rearrangements was calculated for each patient by comparing the obtained cycle threshold (Ct) value to the Ct of his/her level before undergoing BMT, using the ΛΛCt relative quantification analysis. Three healthy controls were analyzed. Their average values are presented as a dotted line for each rearrangement event.</p

Clinical outcome and cell -mediated and humoral-immune reconstitution after BMT in 10 RAG2-deficient SCID patients.

<p>ND-not done, UD-undetectable, PHA – phytohemagglutinin, SI – stimulation index (patient/control), ALC - absolute lymphocyte count.</p

The clinical features of the SCID patients and their specific RAG2 genetic defect.

<p>SCID – severe combined immunodeficiency.</p

List of primers used by real time PCR analysis to detect the amount of TRECs, signal joint KRECs, coding joint KRECs and the various TCR rearrangements.

<p>List of primers used by real time PCR analysis to detect the amount of TRECs, signal joint KRECs, coding joint KRECs and the various TCR rearrangements.</p

Early presence of TREC and transplant-related mortality.

<p>TREC copies were detected in all studied patients (#1–#10) at either 2–4 months (a) or various time lengths (b) post-bone marrow transplant (BMT). TREC copies were determined by real-time quantitative (RQ) PCR and calculated by comparing the obtained cycle threshold (Ct) value of a patient sample to the standard curve using an absolute quantification algorithm. Each sample was detected in triplicate.</p