Effects of activating NK cell receptor expression and NK cell reconstitution on the outcomes of unrelated donor hematopoietic cell transplantation for hematologic malignancies

Abstract

Inhibitory NK cell receptors are recognized as important determinants of NK cell activity in hematopoietic cell transplantation (HCT). The role of activating receptors and their acquisition after HCT is less certain. Therefore, we comprehensively evaluated both inhibitory and activating receptors in 59 patients receiving unrelated donor HCT. NK cell numbers normalized quickly relative to B and T cells; however, the expression of both inhibitory and activating isoforms of killer immunoglobulin-like receptors (KIRs) was delayed. Most NK cells expressed an immature phenotype during the first 6 months post-HCT; however, we found high expression of activating NKp46 and NKp44 natural cytotoxicity receptors (NCRs), and cytotoxicity was preserved. Early reconstituting NK cells from unmanipulated grafts showed lower cytotoxicity than those from T-cell-depleted grafts. Differences in NK cell reconstitution had significant effects on clinical outcomes. Patients whose NK cells reconstituted earlier had better survival and lower relapse rates. The best survival group was recipients who possessed HLA-C2 but their donor lacked the cognate-activating KIR2DS1. Collectively, our data underscore the clinical relevance of reconstituting NK cells and their activating KIRs and NCRs. In addition to NK cell quantification and genotyping, comprehensive assessment of NK cell functions and phenotypes, including activating receptors, is essential.

Figure 1. Reconstitution of lymphocyte subsets

Multicolor flow cytometry analyses were used for enumeration of (A) CD3⁻CD56⁺ NK cells, (B) CD3⁺ T cells, (C) CD3⁺CD4⁺ T cells, (D) CD3⁺CD8⁺ T cells, and (E) CD19⁺ B cells per microliter of blood. The solid lines in each panel represent the upper and lower limits of normal for each lymphocyte subset. The normal range was determined by testing 57 healthy children older than 12 months at our institution.

Figure 2. Comparison of NK-cell and T-cell reconstitution

(A) Time-to-reconstitution to normal range for NK cells and T cells. (B) T:NK cell ratio. The solid line represent the median T:NK cell ration (5.4) in normal children. The normal range and normal ratio were determined using blood samples from 57 healthy children as described in the legend of Figure 1.

Figure 3. Surface expression of KIR and CD94/NKG2A

Mean percentages of NK cells expressing different KIR genes and CD94/NKG2A as determined by multicolor flow cytometry.

Figure 4. Amount of KIR gene transcripts

(A-F) The mean number of copies of each KIR gene transcript per NK cell in the donor and in comparison to those in the patient at various time-points after HCT. (G) Relative abundance of KIR gene transcripts over time. (H) Time-to-normalization of inhibitory versus activating KIR genes.

Figure 5. Natural cytotoxicity and NCR expression

(A) Mean cytotoxicity against K562 cells. (B) Mean percentages of NK cells that were NKp46^bright, or (C) that were NKp44⁺, as determined by multicolor flow cytometry.

Figure 6. Comparison of natural cytotoxicity and NK cell numbers between patients who had received a T cell depleted graft versus those with a T cell replete graft

(A) In the first 4 months post-HCT, patients who received a T cell depleted graft showed a greater degree of natural cytotoxicity than patients who received T cell replete grafts. This difference was statistically significant (p<0.0001). There was no difference between the two graft types after 6 months. There was no difference in the quantitative reconstitution of NK cells in T-cell depleted grafts (-○-) versus unmanipulated grafts (-●-) (p>0.24 on all time points).

Figure 7. Overall survival

Patients who were positive for HLA-C group 2 and received a donor graft which was negative for KIR2DS1 had a significantly better survival (p=0.02).