Natural killer cell infiltration is discriminative for antibody-mediated rejection and predicts outcome after kidney transplantation
Authors: Saleh Yazdani, Jasper Callemeyn, Stéphane Gazut, Evelyne Lerut, Henriette de Loor, Max Wevers, Line Heylen, Carole Saison, Alice Koenig, Olivier Thaunat, Lieven Thorrez, Dirk Kuypers, Ben Sprangers, Laure-Hélène Noël, Leentje Van Lommel, Frans Schuit, Marie Essig, Wilfried Gwinner, Dany Anglicheau, Pierre Marquet, Maarten Naesens
Journal: Kidney International (2018)

Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell–mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation. Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell–mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation. The existing immunosuppressive armamentarium is insufficient in preventing kidney transplant recipients from developing humoral alloreactivity1Thaunat O. Koenig A. Leibler C. et al.Effect of immunosuppressive drugs on humoral allosensitization after kidney transplant.J Am Soc Nephrol. 2016; 27: 1890-1900Crossref PubMed Scopus (41) Google Scholar with the occurrence of donor-specific antibodies (DSAs) to human leukocyte antigen (HLA) and ABMR, which is hallmarked by microcirculation inflammation and complement split product (C4d) deposition.2Nankivell B.J. Alexander S.I. Rejection of the kidney allograft.N Engl J Med. 2010; 363: 1451-1462Crossref PubMed Scopus (433) Google Scholar, 3Loupy A. Hill G.S. Jordan S.C. The impact of donor-specific anti-HLA antibodies on late kidney allograft failure.Nat Rev Nephrol. 2012; 8: 348-357Crossref PubMed Scopus (272) Google Scholar In recent years, DSAs were demonstrated to be a crucial prognostic factor for graft outcome and ABMR is recognized as a prime reason for graft failure after kidney transplantation.4El-Zoghby Z.M. Stegall M.D. Lager D.J. et al.Identifying specific causes of kidney allograft loss.Am J Transplant. 2009; 9: 527-535Crossref PubMed Scopus (616) Google Scholar, 5Sellares J. de Freitas D.G. Mengel M. et al.Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence.Am J Transplant. 2012; 12: 388-399Crossref PubMed Scopus (1079) Google Scholar, 6Naesens M. Kuypers D.R. De Vusser K. et al.The histology of kidney transplant failure: a long-term follow-up study.Transplantation. 2014; 98: 427-435Crossref PubMed Scopus (110) Google Scholar The underlying mechanisms of ABMR have been intensively studied over the past decade to identify potential therapeutic targets. Given the central role of anti-HLA antibodies in the occurrence of ABMR, B-cell inhibition (e.g., by B cell–depleting rituximab treatment), plasma cell inactivation (by proteasome inhibition using bortezomib), or HLA antibody depletion by plasma exchange or the IgG-degrading enzyme derived from Streptococcus pyogenes has been tested in clinical studies.7Jordan S.C. Lorant T. Choi J. et al.IgG endopeptidase in highly sensitized patients undergoing transplantation.N Engl J Med. 2017; 377: 442-453Crossref PubMed Scopus (160) Google Scholar However, most of these therapies had only limited success in the prevention or treatment of ABMR.8Sandal S. Zand M.S. Rational clinical trial design for antibody mediated renal allograft injury.Front Biosci (Landmark Ed). 2015; 20: 743-762Crossref PubMed Scopus (6) Google Scholar Also, complement inhibition by eculizumab or other complement inhibitors is being tested in ABMR, but pilot data suggest that terminal complement inhibition is effective only in a subset of ABMR cases.9Jordan S.C. Choi J. Kahwaji J. et al.Complement inhibition for prevention and treatment of antibody-mediated rejection in renal allograft recipients.Transplant Proc. 2016; 48: 806-808Crossref PubMed Scopus (11) Google Scholar, 10Kulkarni S. Kirkiles-Smith N.C. Deng Y.H. et al.Eculizumab therapy for chronic antibody-mediated injury in kidney transplant recipients: a pilot randomized controlled trial.Am J Transplant. 2017; 17: 682-691Crossref PubMed Scopus (77) Google Scholar, 11Lefaucheur C. Viglietti D. Hidalgo L.G. et al.Complement-activating anti-HLA antibodies in kidney transplantation: allograft gene expression profiling and response to treatment.J Am Soc Nephrol. 2018; 29: 620-635Crossref PubMed Scopus (67) Google Scholar Therefore, novel and innovative preventive or therapeutic approaches for ABMR are needed to improve outcome after transplantation. Nevertheless, despite the partial elucidation of different processes that play a role in the pathophysiology of ABMR,12Halloran P.F. Reeve J.P. Pereira A.B. et al.Antibody-mediated rejection, T cell-mediated rejection, and the injury-repair response: new insights from the Genome Canada studies of kidney transplant biopsies.Kidney Int. 2014; 85: 258-264Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar it remains largely unclear which of these pathways actually determine ABMR activity and outcome after transplantation and which processes are the most promising targets for the prevention and treatment of ABMR. In this study we aimed to elucidate the pathways and the repertoire of graft-infiltrating immune cells that distinguish ABMR from TCMR and that determine graft outcome after kidney transplantation. Transcriptomic data from 95 samples of the Biomarkers of Renal Graft Injuries in Kidney Allograft Recipients (BIOMARGIN) case-control study, all pertaining to unique subjects, were used as the initial test cohort. ABMR was present in 15 of 95 cases (15.8%), TCMR in 11 of 95 cases (11.6%), and mixed rejection (ABMR + TCMR) in 6 of 95 cases (6.3%). The other 63 of 95 biopsies (66.3%) did not have rejection (no rejection). The demographic characteristics of this cohort are listed in Supplementary Tables S1 and S2. Patients with ABMR (including mixed cases) had significantly higher degrees of proteinuria (0.57 ± 0.64 g/l vs. 0.17 ± 0.42 g/l; P = 0.001) but similar estimated glomerular filtration rate (37.7 ± 18.2 ml/min per 1.73 m2 vs. 43.6 ± 19.4 ml/min per 1.73 m2; P = 0.15) than did patients without ABMR (TCMR or no rejection). C4d deposition was present in 45% of cases with ABMR. Implementing the described statistical pipeline, 783 probe sets were found with a multivariate ABMR score of >0.25, reflecting discriminative performance for ABMR across all comparisons in the 95 samples of the BIOMARGIN study. The 783 selected probe sets represented ∼1.5% of the transcripts. To further enrich the probe set list with ABMR-specific hits, 131 transcripts with a TCMR score of >0.20 were eliminated, which yielded 652 ABMR-specific transcripts, corresponding to 503 unique identified genes. In these 503 unique genes specific for ABMR, canonical pathway enrichment analysis of the identified transcripts using Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA) demonstrated significant enrichment of genes and pathways involved in innate and adaptive immunity, including graft versus host disease (P < 0.001), allograft rejection (P < 0.001), antigen presentation (P < 0.001), communication between the innate and adaptive immune systems (P < 0.001), crosstalk between dendritic and NK cells (P < 0.001), and NK cell signaling (P < 0.001) (Figure 1a; Supplementary Table S3). Because these pathways share many genes, all genes from 6 of the top 10 pathways were selected and plotted in an overlapping (Figure 1b) and an interaction network (Figure 1c) structure. All genes except TRGV9, which was omitted from the interaction network, were strongly connected to each other, either directly or indirectly. Notably, interferon-gamma and tumor necrosis factor were at the center of this network, each regulating a large set of molecules associated with ABMR (140 and 143, respectively; more than one-third of all genes in the ABMR gene set: 196 of 503 genes). We then predicted upstream regulators within our ABMR gene set by using Ingenuity Pathway Analysis. Most of the top factors were cytokines such as interferon-gamma, tumor necrosis factor, and interleukins (Supplementary Table S4), which is also consistent with the central place of these cytokines in the network structure shown in Figure 1c. Transcription regulators were also predicted to explain differential expression of the ABMR gene set including STAT1/3, IRF7, NKX2-3, and SMARCA4. Interestingly, STAT1, STAT4, IRF1, and IRF8 were also present in the ABMR gene set. In Ingenuity Pathway Analysis of the 503 genes associated with ABMR (Supplementary Table S5), we noted significant enrichment of leukocyte activation (129 of 503 genes) and immune cell proliferation (131 of 503 genes). This observation was confirmed with Enrichr (a comprehensive gene set enrichment analysis web server) using public gene expression data from the human gene atlas,13Chen E.Y. Tan C.M. Kou Y. et al.Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.BMC Bioinformatics. 2013; 14: 128Crossref PubMed Scopus (3021) Google Scholar, 14Kuleshov M.V. Jones M.R. Rouillard A.D. et al.Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.Nucleic Acids Res. 2016; 44: W90-W97Crossref PubMed Scopus (3951) Google Scholar which specifically showed enrichment of genes expressed in CD56+ NK cells (P < 0.001), CD14+ monocytes (P < 0.001), and CD33+ myeloid cells (P < 0.001). Considering that many of the differentially expressed transcripts were not specific for immune cell subsets, we next performed more detailed deconvolution analyses to elucidate the nature of the graft-infiltrating immune cell subsets. To estimate the differential relative leukocyte distribution among rejection types, the CIBERSORT algorithm15Newman A.M. Liu C.L. Green M.R. et al.Robust enumeration of cell subsets from tissue expression profiles.Nat Methods. 2015; 12: 453-457Crossref PubMed Scopus (5003) Google Scholar was applied to the transcriptomic data of the BIOMARGIN samples and an external validation cohort (GSE21374, a publicly available database). For this purpose, the LM22 gene signature matrix was used, which contains 547 genes that can distinguish between 22 leukocyte subtypes, corresponding to 11 major leukocyte types. In our cohort, a significantly higher relative percentage of NK cells in ABMR was demonstrated in comparison to TCMR and no rejection (Figure 2; Supplementary Figure S1A and Supplementary Table S6). Similarly, NK cell distribution was increased when considering mixed and "pure" ABMR cases, respectively. To obtain an estimation of absolute leukocyte cell subset infiltration, we adjusted the CIBERSORT deconvolution data (relative fractions of 22 leukocyte subtypes) for the expression of the pan-leukocyte marker CD45/PTPRC (Supplementary Figure S2). We then correlated absolute leukocyte infiltration of all 11 major leukocyte types in the BIOMARGIN data set with different transplant rejection phenotypes (ABMR or TCMR) relative to infiltration in samples without rejection. Among the leukocyte types, NK cells, both resting and activated subtypes, were the only cells that differentiated ABMR from TCMR (Figure 3a and b; Supplementary Figure S1B). Other leukocyte types such as monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells were highly enriched in ABMR, but did not discriminate ABMR from TCMR (Supplementary Figure S2B). Next, we studied whether NK cell infiltration is associated with the presence of DSAs and ABMR-specific histological lesions, such as microcirculation inflammation (defined as glomerulitis plus peritubular capillaritis) (Figure 3c). Of the 24 biopsies with microcirculation inflammation, 22 (91.7%) had NK cell infiltration above the median value (Figure 3d), with good discriminative performance (area under the receiver operating characteristic curve, 0.89; P < 0.001). There was no association between NK cell infiltration and transplant glomerulopathy. Tubulitis was also associated with estimated NK cell infiltration. All other lesions including interstitial inflammation and chronic histological lesions did not associate with NK cell infiltration. Of the 22 biopsies with both microcirculation inflammation score ≥2 and increased NK cell infiltration, 15 (68.2%) had detectable DSAs. All 15 biopsies were classified as ABMR by central pathology. The remaining 7 cases (31.8%) did not have circulating DSAs, and 3 of these did not have any detectable HLA antibodies. The cause of the histology and molecular profile of these biopsies with microcirculation inflammation and molecular signs of NK cell infiltration, in the absence of DSAs, remained unknown. C4d deposition in peritubular capillaries was demonstrated in only 8 of 22 biopsies (36.3%) with a high estimated NK cell infiltration and microcirculation inflammation. All patients with C4d deposition in peritubular capillaries had increased NK cell infiltration (Figure 3e). To confirm the deconvolution-inferred NK cell increase in ABMR, computer-assisted histological quantification was used. We compared the density of NK cells between a subset of samples with ABMR (n = 9), samples with TCMR (n = 11), and samples without rejection (n = 9) by using the consensus phenotypic marker NKp46/CD335 (Figure 4a).16Walzer T. Blery M. Chaix J. et al.Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46.Proc Natl Acad Sci U S A. 2007; 104: 3384-3389Crossref PubMed Scopus (362) Google Scholar No mixed rejection phenotypes were included. ABMR samples had significantly higher NK cell infiltration than did samples without rejection (Figure 4b). The number of infiltrating NK cells significantly associated with the presence of DSAs, C4d deposition in peritubular capillaries, and microcirculation inflammation. Also, samples diagnosed with TCMR had higher NK cell infiltration than did samples without rejection. Although the NK cell count was higher in ABMR than in TCMR, this trend did not reach statistical significance. Nevertheless, individual lesions of TCMR such as tubulitis or interstitial inflammation did not associate with increased NK cell infiltration, illustrating that the molecular signatures of NK cell infiltration correspond better with the detailed and semiquantitative histological presentation than with the dichotomous histological diagnoses according to Banff consensus (Figure 4c). To evaluate which immune cell type associates with graft outcome, we applied the CIBERSORT algorithm with the LM22 gene signature matrix to a publicly available database (GSE21374),16Walzer T. Blery M. Chaix J. et al.Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46.Proc Natl Acad Sci U S A. 2007; 104: 3384-3389Crossref PubMed Scopus (362) Google Scholar comprising microarray data of 282 indication biopsies from 105 patients. Rejection was reported in 77 of 282 biopsies (27.3%), although no further information on the specific phenotype was available in the data set. Importantly, the occurrence of graft failure in the first 3 years postbiopsy was reported. Similar to our study population, infiltration of all major leukocyte subtypes was increased in cases of rejection (Supplementary Table S6). However, activated NK cells were the leukocytes that best predicted graft failure at both 1 year and 2 years postbiopsy (area under the receiver operating characteristic curve, 0.74) (Figure 5a–c). Although intrarenal plasma cells were also predictive of graft failure, this association was lost when only biopsies with histologically proven rejection were considered but persisted for NK cells (data not shown). In multivariate Cox proportional hazards analysis, NK cell infiltration predicted graft failure better than (P < 0.001), and independent of, the diagnosis of rejection according to the Banff classification (P = 0.039). In this study, we demonstrate that ABMR after kidney transplantation associates with an intrarenal expression signature enriched with immune-related pathways in general and NK cell pathways in particular. The specific association of NK cell infiltration with the diagnosis of ABMR and with microcirculation inflammation, estimated using immune cell deconvolution of microarray expression data, further corroborates the importance of NK cells in ABMR. Although this is not the first article reporting the involvement of NK cells in ABMR, its importance lies in the demonstration that activated NK cells are the sole cell type that discriminates ABMR from TCMR and correlates with transplant outcome. We describe the specificity of NK cell infiltration for microcirculation inflammation, a hallmark of ABMR activity, whereas no association was found with other histological lesions such as interstitial inflammation and interstitial fibrosis. The lack of association with chronic lesions, including transplant glomerulopathy, may implicate a crucial role for NK cells in the early pathogenesis of active ABMR. An association of intrarenal NK cell transcripts with ABMR has been shown in a previous human study.17Hidalgo L.G. Sis B. Sellares J. et al.NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection.Am J Transplant. 2010; 10: 1812-1822Crossref PubMed Scopus (306) Google Scholar In contrast to our findings, this earlier study showed that a DSA-associated signature could not differentiate ABMR from TCMR while NK cell–selective transcripts associated with the presence of DSAs and microcirculation inflammation in late, but not in early, ABMR biopsies. This leads to questions about the specificity of these transcripts for ABMR.18Hidalgo L.G. Sellares J. Sis B. et al.Interpreting NK cell transcripts versus T cell transcripts in renal transplant biopsies.Am J Transplant. 2012; 12: 1180-1191Crossref PubMed Scopus (94) Google Scholar The more specific NK cell signatures for ABMR in our BIOMARGIN set are likely explained by the case-control study design and focus on well-defined single pathologist histological phenotyping, including only few mixed cases, whereas the previous analyses had a cross-sectional study design and less centralized phenotyping methods. In addition, in our study, the proportions of ABMR cases and TCMR cases occurring within the first transplant year compared with those occurring at later time points were similar, which probably minimized the influence of time-dependent lesions and transcripts that was seen in these previous studies.19Halloran P.F. Pereira A.B. Chang J. et al.Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM).Am J Transplant. 2013; 13: 2865-2874Crossref PubMed Scopus (124) Google Scholar A similar NK cell profile was recently reported in heart transplantation, where NK cell, endothelial cell/angiogenesis, monocyte, and interferon-gamma–inducible transcripts were the main molecular signatures of cardiac ABMR.20Loupy A. Duong Van Huyen J.P. Hidalgo L. et al.Gene expression profiling for the identification and classification of antibody-mediated heart rejection.Circulation. 2017; 135: 917-935Crossref PubMed Scopus (97) Google Scholar This corroborates the general validity of our findings and the potential canonical value of these ABMR signatures across different organ types. The importance of NK cells in ABMR in humans is supported by animal studies that provided compelling evidence that these cells are involved in cardiac and kidney ABMR.17Hidalgo L.G. Sis B. Sellares J. et al.NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection.Am J Transplant. 2010; 10: 1812-1822Crossref PubMed Scopus (306) Google Scholar, 18Hidalgo L.G. Sellares J. Sis B. et al.Interpreting NK cell transcripts versus T cell transcripts in renal transplant biopsies.Am J Transplant. 2012; 12: 1180-1191Crossref PubMed Scopus (94) Google Scholar, 21Hirohashi T. Chase C.M. Della Pelle P. et al.A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody.Am J Transplant. 2012; 12: 313-321Crossref PubMed Scopus (177) Google Scholar, 22Akiyoshi T. Hirohashi T. Alessandrini A. et al.Role of complement and NK cells in antibody mediated rejection.Hum Immunol. 2012; 73: 1226-1232Crossref PubMed Scopus (53) Google Scholar, 23Hadad U. Martinez O. Krams S.M. NK cells after transplantation: friend or foe.Immunol Res. 2014; 58: 259-267Crossref PubMed Scopus (24) Google Scholar, 24Zhang Z.X. Huang X. Jiang J. et al.Natural killer cells mediate long-term kidney allograft injury.Transplantation. 2015; 99: 916-924Crossref PubMed Scopus (33) Google Scholar, 25Resch T. Fabritius C. Ebner S. et al.The role of natural killer cells in humoral rejection.Transplantation. 2015; 99: 1335-1340Crossref PubMed Scopus (35) Google Scholar, 26Kohei N. Tanaka T. Tanabe K. et al.Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts.Kidney Int. 2016; 89: 1293-1306Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar Mechanistically, although a strong association with NK cell infiltration is demonstrated, it is unclear where they are involved in the pathogenesis of ABMR, that is, as a late downstream effector or as an early mediator directly triggered by DSAs. After transplantation, NK cells were shown to be primarily involved in complement-independent rejection mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC), in both animal models and human studies.21Hirohashi T. Chase C.M. Della Pelle P. et al.A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody.Am J Transplant. 2012; 12: 313-321Crossref PubMed Scopus (177) Google Scholar, 27Rajalingam R. The impact of HLA class I-specific killer cell immunoglobulin-like receptors on antibody-dependent natural killer cell-mediated cytotoxicity and organ allograft rejection.Front Immunol. 2016; 7: 585Crossref PubMed Scopus (29) Google Scholar Indeed, whole-genome and targeted expression analyses also suggested that ADCC plays a role in acute ABMR.28Venner J.M. Hidalgo L.G. Famulski K.S. et al.The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD16a Fc receptors.Am J Transplant. 2015; 15: 1336-1348Crossref PubMed Scopus (97) Google Scholar, 29Gupta A. Broin P.O. Bao Y. et al.Clinical and molecular significance of microvascular inflammation in transplant kidney biopsies.Kidney Int. 2016; 89: 217-225Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 30Dos Santos D.C. Campos E.F. Saraiva Camara N.O. et al.Compartment-specific expression of natural killer cell markers in renal transplantation: immune profile in acute rejection.Transpl Int. 2016; 29: 443-452Crossref PubMed Scopus (14) Google Scholar It has also been shown that increased ADCC reactivity can be used to predict the development of chronic allograft injury in kidney transplant recipients, even independent of DSA detection.31Legris T. Picard C. Todorova D. et al.Antibody-dependent NK cell activation is associated with late kidney allograft dysfunction and the complement-independent alloreactive potential of donor-specific antibodies.Front Immunol. 2016; 7: 288Crossref PubMed Scopus (24) Google Scholar Lately, Parkes et al.32Parkes M.D. Halloran P.F. Hidalgo L.G. Evidence for CD16a-mediated NK cell stimulation in antibody-mediated kidney transplant rejection.Transplantation. 2017; 101: e102-e111Crossref PubMed Scopus (58) Google Scholar studied CD16a-inducible transcripts in primary human NK cells in vitro and showed the involvement of some of these transcripts in NK cell activation in ABMR, including the central involvement of interferon-gamma, tumor necrosis factor, and colony-stimulating factor 2, which we also identified as key upstream regulators in our ABMR signature. The importance of NK cells in ABMR seems contradictory to the observed small relative fraction, as they constitute only a minority of infiltrating leukocytes. In addition, NK cells have an important role in producing cytokines and chemokines and interact with other immune and nonimmune cells. The action of very few infiltrating cells could thus be much broader than is perhaps suggested by their numbers. Although this might implicate a rather immunoregulatory role for NK cells, another explanation may be offered by considering the effector site. DSAs interact with the vascular endothelium and trigger multiple effector mechanisms, including ADCC, which are initiated in the dynamic endovascular compartment. Blood circulation may allow rapid clearance of a majority of NK cells, with only a minority infiltrating into the perivascular compartment. Further characterization of the infiltrating NK cell is necessary to investigate their origin (tissue-resident vs. infiltrating peripheral blood cell), reactivity (expression of CD16, killer-cell immunoglobulin-like receptor, etc.), and effector mechanism (regulatory vs. cytotoxic). Of all the cases with NK cell infiltration and microvascular inflammation, 62% did not have C4d deposition in peritubular capillaries, whereas all cases with C4d deposition had increased NK cell presence. Although C4d deposition can be a useful epiphenomenon for the diagnosis of ABMR, this suggests that complement activation and C4d deposition are not necessary for NK cell–mediated graft injury, which is supported by the variable results of studies on complement inhibitors for this disease.9Jordan S.C. Choi J. Kahwaji J. et al.Complement inhibition for prevention and treatment of antibody-mediated rejection in renal allograft recipients.Transplant Proc. 2016; 48: 806-808Crossref PubMed Scopus (11) Google Scholar, 10Kulkarni S. Kirkiles-Smith N.C. Deng Y.H. et al.Eculizumab therapy for chronic antibody-mediated injury in kidney transplant recipients: a pilot randomized controlled trial.Am J Transplant. 2017; 17: 682-691Crossref PubMed Scopus (77) Google Scholar, 11Lefaucheur C. Viglietti D. Hidalgo L.G. et al.Complement-activating anti-HLA antibodies in kidney transplantation: allograft gene expression profiling and response to treatment.J Am Soc Nephrol. 2018; 29: 620-635Crossref PubMed Scopus (67) Google Scholar Despite the association between NK cell infiltration, ADCC, and ABMR in one-third of cases with microvascular inflammation and increased NK cell infiltration, no circulating DSAs were detected. This finding remains ill-explained and could be related to intragraft adsorption of DSAs or presence of non-HLA DSAs, which therefore remain undetected.33Bachelet T. Couzi L. Lepreux S. et al.Kidney intragraft donor-specific antibodies as determinant of antibody-mediated lesions and poor graft outcome.Am J Transplant. 2013; 13: 2855-2864Crossref PubMed Scopus (62) Google Scholar However, antibody-independent NK cell activation, for example, through endothelial stress ligands or missing self, should be considered.20Loupy A. Duong Van Huyen J.P. Hidalgo L. et al.Gene expression profiling for the identification and classification of antibody-mediated heart rejection.Circulation. 2017; 135: 917-935Crossref PubMed Scopus (97) Google Scholar, 23Hadad U. Martinez O. Krams S.M. NK cells after transplantation: friend or foe.Immunol Res. 2014; 58: 259-267Crossref PubMed Scopus (24) Google Scholar, 24Zhang Z.X. Huang X. Jiang J. et