CD16+ natural killer cells in bronchoalveolar lavage are associated with antibody-mediated rejection and chronic lung allograft dysfunction

Abstract

Acute and chronic rejections limit the long-term survival after lung transplant. Pulmonary antibody-mediated rejection (AMR) is an incompletely understood driver of long-term outcomes characterized by donor-specific antibodies (DSAs), innate immune infiltration, and evidence of complement activation. Natural killer (NK) cells may recognize DSAs via the CD16 receptor, but this complement-independent mechanism of injury has not been explored in pulmonary AMR. CD16⁺ NK cells were quantified in 508 prospectively collected bronchoalveolar lavage fluid samples from 195 lung transplant recipients. Associations between CD16⁺ NK cells and human leukocyte antigen mismatches, DSAs, and AMR grade were assessed by linear models adjusted for participant characteristics and repeat measures. Cox proportional hazards models were used to assess CD16⁺ NK cell association with chronic lung allograft dysfunction and survival. Bronchoalveolar lavage fluid CD16⁺ NK cell frequency was associated with increasing human leukocyte antigens mismatches and increased AMR grade. Although NK frequencies were similar between DSA+ and DSA- recipients, CD16⁺ NK cell frequencies were greater in recipients with AMR and those with concomitant allograft dysfunction. CD16⁺ NK cells were associated with long-term graft dysfunction after AMR and decreased chronic lung allograft dysfunction-free survival. These data support the role of CD16⁺ NK cells in pulmonary AMR.

Keywords: acute lung injury; biomarker; inflammation; innate immunity.

Figure 1.

Inclusion and exclusion criteria for study participants.

Figure 2.. CD16+ NK cells are mature and proliferative.

(A) NK cell action is determined by the integration of signals from a range of activating and inhibitory receptors. CD16 is a potent activating signal after binding the Fc component of antibodies. NKG2C recognizes CMV-infected cells through HLA-E. Killer cell immunoglobulin-like receptors (KIR) bind HLA and can send activating or inhibiting signals. NKG2A ligation of HLA-E sends an inhibitory signal. (B) Flow cytometry phenotypes for CD16 negative and CD16 positive NK cells in 506 bronchoscopy samples were analyzed. Individual data points are shown bound by boxes at 25^th and 75^th percentiles and medians depicted with bisecting lines. Compared to CD16- NK cells, CD16+ NK cells are more proliferative (ki67, p < 0.0001), had increased KIR expression (p < 0.0001), had decreased NKG2A (p < 0.0001), and had increased NKG2C (p < 0.0001). P values were generated with generalized estimating equation models adjusted for age and cytomegalovirus serostatus.

Figure 3.. The percentage of CD16-positive NK cells are increased with increasing donor and recipient HLA mismatches but not donor-specific antibodies.

NK cells were measured in BAL and quantified by absolute numbers and as percentage positive for CD16. (A) With increasing donor and recipient HLA mismatches, the percentages of CD16 on CD3-CD56+ NK cells were increased. (B) Percentage of NK cells expressing CD16 on BAL NK cells at the time of positive donor-specific antibodies (n = 84) or negative donor-specific antibodies (n = 484). (C) Percentage of NK cells expressing CD16 during transient or persistent DSA. (D) The absolute numbers of CD16+ NK cells among recipients with and without DSA. (E) The absolute numbers of CD16+ NK cells among recipients by DSA persistence. Individual data points are shown bound by boxes at 25^th and 75^th percentiles and medians depicted with bisecting lines. P values were generated with generalized estimating equation models adjusted for age, cytomegalovirus serostatus, time after transplant. Pairwise differences were adjusted for multiple comparisons using Benjamini-Hochberg corrections.

Figure 4.. CD16+ NK cells are increased during antibody-mediated rejection.

Antibody-mediated rejection for participants was graded during serial bronchoscopies. (A) The percentage of NK cells expressing CD16 within bronchoalveolar lavage was increased across AMR grades and subtypes. (B) The absolute numbers of bronchoalveolar lavage CD16+ NK cells were increased in participants with definite AMR only. Individual data points are shown bound by boxes at 25^th and 75^th percentiles and medians depicted with bisecting lines. Analyses employed cumulative linked mixed models of AMR as an ordinal variable adjusted for repeat observations among study participants, age, cytomegalovirus serostatus, and time after transplant. Pairwise differences were adjusted for multiple comparisons using Benjamini-Hochberg corrections. P values: * < 0.05; *** < 0.001, **** < 0.0001.

Figure 5.. Bronchoalveolar lavage CD16+ NK cells are increased by AMR recurrence and graft dysfunction.

(A) The percentage of NK cells expressing CD16 according to frequency of AMR detections (1 episode [bronchoscopy n = 78], 2 episodes [bronchoscopy n = 60], 3 episodes [bronchoscopy n = 41] compared to no AMR. (B) The percentage of NK cells expressing CD16 in AMR stratified by participants with and without allograft dysfunction. (C) The absolute counts of NK cells expressing CD16 in AMR stratified by participants with and without allograft dysfunction. Individual data points are shown bound by boxes at 25^th and 75^th percentiles and medians depicted with bisecting lines. Associations were established with logistic regression models adjusted for repeat measures, age, and cytomegalovirus serostatus and time after transplant. P values: * < 0.05; ** < 0.01.

Figure 6.. Percentage of BAL CD16+ NK cells predict CLAD-free survival.

Extended Kaplan-Meier plots are shown in each panel, left censored at 6 months. (A) CLAD-free survival according to AMR grade. (B) Time to CLAD or death stratified by median NK cell CD16 percentage among participants with AMR. (C) Time to CLAD or death stratified by median NK cell CD16 percentage across all participants. P-values represent results from log rank tests.

Figure 7.. Pulmonary AMR occurs through complement and Fc-dependent mechanisms.

(A) After antibody binds to the allograft, complement can be activated leading to C4d deposition and cell death via the complement cascade (B) Through the CD16, CD32, and CD64 Fc receptors, effector cells can recognize bound antibodies in the process of antibody-dependent cellular cytotoxicity (ADCC). (C) Neutrophils are common AMR pathologic features and may become activated through Fc receptors. (D) Of the Fc receptors, CD16 has high affinity and is an activating-only receptor. The finding of CD16+ NK cells in the bronchoalveolar lavage of AMR participants supports this alternate mechanism of lung allograft injury.