Significance of HLA-E and its two NKG2 receptors in development of complications after allogeneic transplantation of hematopoietic stem cells

Abstract

Transplantation of hematopoietic stem cells (HSCT) is a procedure commonly used in treatment of various haematological disorders which is associated with significantly improved survival rates. However, one of its drawbacks is the possibility of development of post-transplant complications, including acute and chronic graft-versus-host disease (GvHD) or CMV infection. Various studies suggested that NK cells and their receptors may affect the transplant outcome. In the present study, patients and donors were found to significantly differ in the distribution of the NKG2A rs7301582 genetic variants - recipients carried the C allele more often than their donors (0.975 vs 0.865, p<0.0001). Increased soluble HLA-E (sHLA-E) levels detected in recipients' serum 30 days after transplantation seemed to play a prognostic and protective role. It was observed that recipients with higher sHLA-E levels were less prone to chronic GvHD (11.65 vs 6.33 pg/mL, p=0.033) or more severe acute GvHD grades II-IV (11.07 vs 8.04 pg/mL, p=0.081). Our results also showed an unfavourable role of HLA-E donor-recipient genetic incompatibility in CMV infection development after transplantation (OR=5.92, p=0.014). Frequencies of NK cells (both CD56dim and CD56bright) expressing NKG2C were elevated in recipients who developed CMV, especially 30 and 90 days post-transplantation (p<0.03). Percentages of NKG2C+ NK cells lacking NKG2A expression were also increased in these patients. Moreover, recipients carrying a NKG2C deletion characterized with decreased frequency of NKG2C+ NK cells (p<0.05). Our study confirms the importance of NK cells in the development of post-transplant complications and highlights the effect of HLA-E and NKG2C genetic variants, sHLA-E serum concentration, as well as NKG2C surface expression on transplant outcome.

Keywords: HLA-E; HSCT; NK cell receptors; NK cells; NKG2A; NKG2C; sHLA-E; transplant outcome.

Figure 1

The gating strategy (A–G): grey - singlets; green - lymphocytes; blue - lymphocytes T CD3+; orange - NK cells. (A) - discrimination of doublets (FSC-A vs. FSC-H); (B) - discrimination of debris and lymphocytes gating; (FSC-A vs. SSc-A); (C) - lymphocyte subpopulations: lymphocytes T CD3+ (blue) and NK cells (orange) - (CD3 vs. SSc-A); (D) - NK cells (CD56 vs. CD16); (E) - NK cells NKG2C positive (NKG2C vs. SSc-A); (F) - NK cells NKG2A positive (NKG2A vs. SSc-A); (G) - NK cells double positive CD94+CD57+ (CD94 vs. CD57); (H) - NK cells double positive NKG2A+NKG2C+ (NKG2A vs. NKG2C); (I) - NK cells double positive CD314+NKG2A+ (CD314 vs. NKG2A).

Figure 2

Differences in NKG2A rs7301582 T allele frequencies between AML patients and donors.

Figure 3

Associations between HLA-E incompatibility and post-transplant complications development; CMV infection (A) and chronic GvHD (B) occurred more often among patients after HLA-E mismatched HSCT. Acute GvHD was developed irrespective of HLA-E compatibility (C).

Figure 4

Soluble HLA-E levels detected in recipients’ serum 30 days after transplantation were lower in recipients who developed chronic (A) or acute (B) GvHD in comparison to recipients without these complications.

Figure 5

Serum sHLA-E concentration in HSCT recipients at day 30 and 90 after transplantation. A significant time-dependent increase has been observed.

Figure 6

Recipients who developed severe acute GvHD grades II-IV carried the NKG2C del variant more frequently.

Figure 7

Changes in frequencies of T and NK cells (A) and NK cell subpopulations (B) during the observation period.

Figure 8

Differences in expression of NKG2C on NK cells in HSCT recipients with CMV infection at various time points after transplantation. Frequency of NKG2C+ cells was increased 60 (*p=0.005) and 90 (**p=0.007) days after HSCT in patients who developed CMV infection as compared to patients without this complication (A). Frequency of NKG2C+ NK CD56bright cells was higher among recipients with CMV infection (day +60 *p=0.006 and day +90 **p=0.009, (B). Similarly, frequency of NKG2C+ NK CD56dim cells was increased in recipients with CMV infection (day +60 *p=0.025 and day +90 **p=0.003, (C). Patients with CMV infection characterized with a significant increase in frequency of NK cells expressing NKG2C cells between days 30 and 90 after HSCT (*p=0.016, D). A significant difference between days 30 and 90 after HSCT was also observed for the frequencies of NKG2C+ NK CD56bright cells (*p=0.008, E) as well as NKG2C+ NK 56dim cells (*p=0.030, in patients with CMV infection F).

Figure 9

Differences in expression of NKG2A-/NKG2C+ NK cells in patients with CMV infection at various time points after transplantation. Increased frequency of NKG2A-/NKG2C+ NK cells in recipients with CMV infection 90 days after HSCT (p=0.003, A). Frequency of NKG2A-/NKG2C+ CD56bright NK cells was significantly increased in CMV patients 30 and 90 days after transplantation (p=0.002 and p=0.002, respectively, B). Higher frequency of NKG2A-/NKG2C+ population of CD56dim NK cells was observed in 90 days after HSCT (p=0.004, C).

Figure 10

Differences in frequencies of NKG2C+ NK cells in patients with CMV infection carrying various NKG2C genotypes. Decreased frequency of NK cells expressing NKG2C receptor in recipients carrying NKG2C deletion (A *p=0.048, B *p=0.042 for NK cells and NK CD56bright subpopulation, respectively). Significantly higher frequency of NKG2C+ NK CD56dim cells in recipients with NKG2A rs7301582 CC and NKG2C wt genotype at +90 day after HSCT (p=0.005, C).