Mixed Chimerism in T Cells after Allogeneic SCT Allows Early Diagnosis of Graft Rejection and Successful Treatment with Immunosuppression Withdrawal and/or Donor Leukocyte Infusion.
Authors: Buño Ismael , Balsalobre Pascual , Simón Ainhoa , Manzano Cintia , Barroso Diana , Serrano David , Carrión Rafael , Gómez-Pineda Alfonso , Díez-Martín José L. 
Published: April 2020

Abstract
Background: Graft rejection constitutes a serious complication, infrequent after conventional (ablative) allogeneic SCT but with higher incidence after reduced intensity conditioning (RIC) or T-cell depleted (TCD) SCT.
Objective: To evaluate the usefulness of chimerism monitoring for the early diagnosis of graft rejection in different SCT settings, as well as for the follow up of the response to treatment with immunosuppression withdrawal (ISW) and/or donor leukocyte infusions (DLI).
Patients and Methods: The study includes 68 SCT (32 ablative, 19 RIC, 17 TCD -including 8 haploidentical-). Chimerism analysis was performed by FISH for the sex chromosomes or STR-PCR (sensitivity 1%). Samples obtained on days +30, +100, +180, +365 and once a year thereafter, included bone marrow (BM) and peripheral blood (PB). Moreover, chimerism was analyzed in PB and leukocyte lineages (T lymphocytes CD3+, B lymphocytes CD19+ and myeloid cells CD15+ isolated (purity &gt;95%) by immunomagnetic means, AutoMACS, Miltenyi Biotec), every 2 weeks, starting on day +15 (except in ablative), and until complete chimerism (CC) was achieved.
Results: After initial engraftment in all patients, graft rejection was diagnosed in 8 (2 (6%) ablative, 2 (10%) RIC, 4 (23%) TCD), either established (severe pancytopenia and BM aplasia) or incipient (progressive decrease in PB and BM cell counts) a median of 52.5 days (range 23–110) after SCT. All patients showed mixed chimerism (MC) in BM and PB with higher percentages of recipient cells (%R) in PB. In 7/7 patients studied, T cells showed persistent MC with high %R (&gt;50% in 6/7; &lt;50% and &gt;25% in 1/7). B cells showed MC in 5/6 patients studied, with lower %R (&lt;15% in 3/5, &gt;50% in 1/5). Only 2 patients showed MC, transient in one of them, in myeloid cells. 2 patients were not treated due to concurrent multiorgan failure and subsequently died. Reduction of IS in 5 patients obtained 1 response (normal PB and BM counts, and CC). The other 4 patients underwent ISW but no further response was obtained. One of them received a second SCT while the other 3 were treated with DLI, and all of them responded. The last patient (transplanted from a haploidentical family donor) who was not receiving IS, responded to treatment with DLI. Time from therapeutic intervention to response was variable with a median of 2 months (range 1–7). 4 patients developed GVHD&gt;I, which was the cause of death in one and was controlled in the other three. One patient died from sepsis in complete remission (CR) 9 months after the transplant. 4 patients are alive in CR a median of 50 months after SCT (range 16–72).
Conclusions: The observation of MC, mainly in T lymphocytes, together with a decrease in PB and BM cell counts, allowed early diagnosis and successful treatment (6/6 patients) of graft rejection.