Flow Cytometry Based Chimerism Analysis Can Predict Graft Rejection in Pediatric Patients Receiving HLA Mismatched Stem Cell Transplantation
Authors: Teltschik Heiko-Manuel , Kreil Hanna , Schlegel Patrick , Feuchtinger Tobias F. , Schumm Michael , Handgretinger Rupert , Lang Peter 
Published: October 2019

Abstract
Abstract 4156
Graft failure is a life-threatening complication after allogeneic stem cell transplantation and its probability increases with the degree of HLA disparity between donor and recipient. In HLA-mismatched transplantation, specific T cells against mismatched HLA class I alleles were shown to be associated with rejection. Moreover, HLA mismatches allow to monitor the chimerism status of several cell populations by flow cytometry with specific monoclonal antibodies against HLA alleles with high sensitivity even in very low cell numbers. We report our experience with this method in a cohort of 90 pediatric patients who received T and B cell depleted haploidentical stem cells after myeloablative conditioning regimens. Diagnoses were: leukemias n=43, solid tumors n=28, non malignant diseases n=19. Graft manipulation was carried out by magnetic microbeads and the clinimacsÒ device with 4 to 5 log depletion efficacy. Conditioning regimens were TBI, Bu or Mel based and comprised ATG or OKT3. Immunosuppression with mycophenolate mofetil (MMF) was given in most patients (if donor T cells in the graft exceeded 25.000/kg body weight) Flow cytometry with HLA antibodies for detection of chimerism status of T and NK cell subsets in peripheral blood was started at day 0 and was repeated at least weekly. 14 out of 90 patients (15%) rejected the graft between day 19 and 86 (median day 33). Two different rejection patterns were observed: early rejections (diagnosis of rejection, day 19–40, median day 28) with cytokine release syndrome including high fever, massive elevation of CRP, ferritin, LDH and D-Dimers or late rejections without signs of inflammation (diagnosis of rejection, day 53–86). In all patients, residual T cells of recipient origin were still detectable after the conditioning procedure. The portion of residual recipient T cells within the first week (day 0–7) was predictive: Non-rejectors had a median percentage of 46+-5% recipient T cells detectable (n=42 analyses) whereas rejectors showed a significant higher percentage (85+-7%, n=13 analyses, p<0.001). In more than 90% of the rejectors, the portion of detectable autologous recipient T cells exceeded 60%. Thus, the risk of rejection for patients with >60% detectable recipient T cells was 41% (12 out of 29 rejected), whereas patients with <60% recipient T cells had a risk of 4% (only 1 out of 27 rejected; p=0.001). In non-rejectors, recipient T cells where detectable at a very low level (median 0,7/μl) up to day 60 without proliferation tendency, whereas strongly increasing and significantly higher numbers were observed in rejectors (from 3/μl at day 0–3 up to 617/μl at day 15–18; p< 0,05 for all data pairs). Based on these data, donor lymphocyte infusions (DLI) and cessation of MMF were performed in 12 patients with increasing residual recipient T cells to prevent imminent rejection (1–4 infusions, median number: 1; starting dose: 25.000 CD3+cells/kg bw). Recipient T cells were reduced/eradicated in 7/12 patients (60%) after DLIs and no rejection occurred. In 5 patients, DLIs had no impact on increasing recipient T cells, resulting in graft rejection in 4 out of these 5 patients.

Conclusions:
Flow cytometry with specific monoclonal antibodies against HLA alleles allows to analyze the origin of lymphocyte subsets and their kinetics after HLA mismatched transplantation with high sensitivity even in very low cell numbers. The percentage of residual recipient T cells in the first week posttransplant was predictive, since patients with >60% detectable recipient T cells had a significantly higher risk to experience graft rejection. In all cases, rejection was associated with increasing recipient T cells. Intervention with DLI and cessation of immune suppression may contribute to prevent rejection.

Disclosures:
No relevant conflicts of interest to declare.