Reconstitution Of NK Cells After Reduced Intensity Conditioned Unrelated Cord Blood Transplantation In Patients With Acute Myeloid Leukemia: Analysis Of a Prospective Phase II Multicentric Trial On Behalf Of Societe Française De Greffe De Moelle Osseuse Et Therapie Cellulaire (SFGM-TC) and Eurocord
Authors: Nguyen Stéphanie , Souchet Laetitia , Brahmi Abla , Vigouroux Stephane , Chevallier Patrice , Furst Sabine , Legrand Faezeh , Bay Jacques-Olivier , Socié Gérard , Ceballos Patrice , Huynh Anne , Cornillon Jérôme , Sy Françoise , Yakoubagha Ibrahim , Michel Gérard , Maillard Natacha , Margueritte Genevieve , Maury Sebastien , Uzunov Madalina , Bulabois Claude Eric , Michallet Mauricette , Clement Laurence , Dauriac Charles , Bilger Karin , Tabassome Simon , Beziat Vivien , Rocha Vanderson , Rio Bernard , Vieillard Vincent 
Published: October 2019

Background
Unrelated cord blood transplantation (UCBT) after reduced intensity conditioning regimen (RIC) has extended the use of cord blood in elderly patients and those with co-morbidities without an HLA identical donor, although relapse post transplant remains a concern in high risk AML patients. HLA incompatibilities between donor and recipient might enhance Natural Killer (NK) cell alloreactivity after allogeneic hematopoietic stem cell transplantation (HSCT). We previously observed that the quality of NK cell reconstitution was impaired after haploidentical HSCT, impacting on graft versus leukemia (GvL) effect, but was preserved after UCBT in a small cohort of patients.

Methods
To evaluate RIC-UCBT in patients with acute myeloid leukemia (AML), a prospective phase II multicentric trial was conducted in France, whose primary objective was to show a reduction in non-relapse mortality (NRM) from 40% (based on registry data) to 20%. Seventy-nine patients were enrolled for a de novo or secondary AML in complete remission (CR). The conditioning regimen consisted of cyclophosphamide (50mg/kg) + fludarabine (200mg/m2) + total body irradiation (2Gy), CsA +MMF as GVHD prophylaxis and GCSF from day +1. Patients were enrolled in 23 centers from October 2007 to September 2009. Engraftment rate was 87 % at day+60. At 2 years, overall survival, incidence of relapse and LFS were respectively 44%, 46% and 35%.
Peripheral blood samples were collected following UCBT in order to realize an extensive phenotypic and functional study of NK cells. Studies were started at 1 month (M1) post UCBT with available samples for 62 out of the 69 included patients, and were compared to 20 healthy donors and 15 cord blood (CB).

Results
Total CD3+ T-cells were 117 /mm3 at M1 (range 0-934), and 465 /mm3 at M3 (range 0-2917). CD19+ B-cells were 36/mm3, (range 0-524) and 342/mm3 (range 0-2990) at M1 and M3 respectively. NK cell recovery was prompt, representing 47% of the total lymphocyte population at M1 (186 CD3-CD56+ NK cells/mm3), 30% at M3 (239/mm3; range 2-767) and decreasing to normal rate at M6 (20% of lymphocytes).
At M1 post-UCBT, NK cells exhibited high rate of CD56bright, NKG2A, and KIR2DL4 associated with a decreased expression of CD8 and CD161, compared to CB and healthy donors. These immature characteristics were transient and return to normal value from M3 or M6 post-UCBT. Interestingly, we also observed a significant increased expression of the activation markers CD69, and HLA-DR during the whole period of the study, compared to CB and healthy donors, which probably reflects a persistent proliferation state of the NK cells. On the other hand, NK cells post-UCBT were indistinguishable from CB and healthy donors control samples for other receptor tested such as NKp30, NKp46, NKp80, and NKG2D. Notably, Expression of KIR2DL1 was decreased at M1 and M3 but reached similar values to controls at M6, whereas, KIR3DL1 was increased during the whole study.
To determine the significance of these phenotypic features, we assessed polyfunctional ability of NK cells following UCBT by a combined analysis of the degranulation (CD107a), and the production of IFN-γ and TNF-α. This study reveals that NK at M1 post-graft exhibited a transient higher ability to produce IFN-γ than healthy donors (p<0.0001), which reaches normal values by 6 months after UCBT, in correlation with the evolution of the immunoregulatory NKG2A+/CD56brightNK cells subset post transplant. Production of TNF-α was reduced in CB and at M1 as compared to healthy donors (p<0.0001) but quickly restored starting from M3. Degranulation’s ability was slightly impaired at M1 and M3, as compared to CB and healthy donors (p=0.002), but restored at M6.

Conclusion
This study shows that after RIC-UCBT, NK cells display some phenotypic features of activation associated with a prompt and complete restoration of their ability for polyfunctional activities. Further analyzes are needed to assess the impact of such observation on NK cell mediated GvL effect in this prospective trial of RIC-UCBT for AML patients in CR.

Disclosures:
No relevant conflicts of interest to declare.