Natural Killer (NK) Cell Reconstitution After Haploidentical Unmanipulated Bone Marrow Transplantation with Reduced Intensity Conditioning

Natural killer (NK) cells are innate immune effectors that directly lyse virally infected or malignant cells. There are 2 different subsets of NK cells with distinct phenotypic and functional characteristics: the CD56^dim subset, which composes 90% of peripheral blood NK cells and has a cytotoxic function, and the CD56^bright subset, which cooperates with dendritic cells and T cells in lymph nodes to secrete interferon and promote adaptive immune responses. NK cells are the first donor-derived lymphocyte subset to reconstitute after hematopoietic stem cell transplantation, reaching normal levels after 1 month. Nearly all phenotyping studies of NK subsets after haploidentical hematopoietic stem cell transplantation (HHSCT) reveal a rapid reconstitution of NK cells towards the CD56^bright subset. In addition, Y.-J. Chang et al found the highest 2-year survival in patients with a high number of CD56^bright NK cells after unmanipulated HHSCT.

We analyzed reconstitution of the NK compartment between days 90 and 180 after unmanipulated bone marrow HHSCT with reduced intensity conditioning (RIC).

Six adults received unmanipulated bone marrow HHSCT after RIC (fludarabine 30 mg/m² [day –6 to –2], cyclophosphamide 14.5 mg/kg [day –6 and –5], and busulfan i.v. 3.2mg/kg [day –3]) at our institution between July 2007 and July 2010. Prophylaxis for acute graft-versus-host disease (GvHD) consisted of cyclophosphamide 50mg/kg (days +3 and +4) and cyclosporine A and mycophenolate mofetil from day +5 onwards. We monitored the reconstitution kinetics of circulating NK cells (CD56+, CD3–), and the CD56^bright and CD56^dim subsets by multiparametric flow cytometry (FC 500 Beckman® Coulter) at day +90 and day +180 after transplantation. Patient characteristics and clinical outcomes are shown in Table 1. 6 patients who underwent allogeneic HLA-identical sibling HSCT with RIC during the same period were used as controls.

Table 1.

Patient characteristics and clinical outcome

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SCT, stem cell transplantation; GvHD, graft-versus-host disease; AML, acute myeloid leukemia; CR complete remission; aGvHD, acute GvHD; g-i III-IV, gastrointestinal grade III-IV; cut I-II, cutaneous grade I-II; MM, multiple myeloma; VGPR, very good partial response; cGvHD, chronic GvHD; L, lung; HD, Hodgkin disease; PR, partial response; MDS myelodysplastic syndrome.

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After HHSCT, NK cells reached normal levels in all patients but one at day +90, with a median number of NK cells of 111/mm³ (range, 25–195/mm³). At day +180 the median number of NK cells was 92/mm³ (range, 4–272/mm³).

When we analyzed the absolute number of CD56^bright and CD56^dim subsets at day +90, we observed 2 patterns: Two patients showed skewed NK cell reconstitution towards CD56^bright (Patient no. 3: 54 CD56^bright/mm³; 11 CD56^dim/mm³. Patient no. 4: 70 CD56^bright/mm³; 17 CD56^dim/mm³). Three patients reconstituted with a CD56^dim/CD56^bright ratio towards the CD56^dim cell subset, similar to that of healthy adults (Patient no. 1: 17 CD56^bright/mm³; 178 CD56^dim/mm³. Patient no. 5: 9 CD56^brigh/mm³; 135 CD56^dim/mm³. Patient no. 6: 20 CD56^bright/mm³; 116 CD56^dim/mm³). One patient did not achieve adequate NK cell reconstitution (Patient no. 2: 15 CD56^bright/mm³; 10 CD56^dim/mm³). In contrast, in the control group, an increase in the CD56^bright NK cell subset was not observed in any of the patients at any point.

It is worth noting that 2 of the 3 patients with better clinical outcome (no GvHD, no relapse), namely patients no. 3 and no. 4 were the ones with skewed NK cell reconstitution towards the CD56^bright NK cell subset. The other patient with a better clinical outcome (patient no. 6) had a normal CD56^dim/CD56^bright ratio at day +90. However, he showed an early CD56^bright reconstitution (363 CD56^bright/mm³; 34 CD56^dim/mm³) in an additional determination on day +30. NK cell subsets reconstitution kinetics is shown in Figure 1.

Figure 1.

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NK cell subsets reconstitution kinetics after HHSCT.

Figure 1.

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NK cell subsets reconstitution kinetics after HHSCT.

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In our experience, NK cell reconstitution is adequate after RIC unmanipulated bone marrow HHSCT.

Some patients recovered with a high proportion of CD56^bright NK cells, as previously reported in other studies on HHSCT.

Although limited by the sample size, our results are consistent with the previously observed survival advantage of patients with high early levels of CD56^bright NK cells after unmanipulated haploidentical transplantation.

No relevant conflicts of interest to declare.