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CD16+ natural killer (NK-) cells play, together with donor-specific antibodies (DSA) and via antibody-dependent cellular cytotoxicity (ADCC), an important role in the pathogenesis of antibody-mediated rejection (ABMR) in lung-transplant recipients (LTRs). Cytotoxic CD16+NKG2C+ NK cells proliferate in response to human Cytomegalovirus (HCMV) infections via the presentation of HCMV-encoded and highly polymorphic UL40 peptides. In our study, we aimed to clarify whether infections with HCMV-strains carrying different UL40 peptide variants are associated with the shift of the NK cell repertoire and the development of ABMR in LTRs.
We included 30 DSA+ABMR+, 30 DSA+ABMR− and 90 DSA−ABMR− LTRs. In all patients, 1 episode of high-level HCMV-replication occurred. In all DSA+ABMR+ LTRs, HCMV-replication occurred prior to ABMR diagnosis. The association of HCMV UL40 variants with the expansion of CD16+ NK cell subsets and ABMR was assessed in NK cell proliferation and ADCC assays.
Our study revealed that the VMAPRTLIL and VMTPRTLVL UL40 variants were significantly overrepresented in DSA+ABMR+ LTRs. Both peptides were associated with a pronounced proliferation of cytotoxic and proinflammatory CD16+NKG2C+ NK cells. The stimulation with both peptides led to a shift of the NK cell repertoire towards CD16+NKG2C+ NK cells, which was associated with strong ADCC responses after stimulation with endothelial cells and plasma from DSA+ABMR+ LTRs.
Distinct UL40 peptide variants of the infecting HCMV-strain are associated with the development of ABMR after lung transplantation, due to a shift towards a highly cytotoxic CD16+NKG2C+ NK cell population. These peptides are thus potential prognostic markers for ABMR.
NK cells respond to non-self-cells by the direct lysis and the production and secretion of proinflammatory effector molecules, such as interferon-γ (IFNγ) and tumor necrosis factor α (TNFα). NK cells are important effector cells in the antibody-mediated rejection (ABMR), which presents a significant challenge for long-term graft survival in LTRs.
Especially mature and blood resident NK cells express high levels of the FcγR CD16a, which mediates the antibody-dependent activation of NK cells by a process termed antibody-dependent cellular cytotoxicity (ADCC).
Human Cytomegalovirus (HCMV) may cause severe and potentially life-threatening infections in LTRs. HCMV-replication in the lung-allograft was recently associated with release of proinflammatory chemokines and increased risk of graft-rejection.
In response to HCMV-replication, a specialized subset of NKG2C+, and to a lower extent also NKG2A+ NK cells expand and persist in immunocompetent individuals and immunocompromised solid organ recipients.
NKG2C and NKG2A, both together with CD94, bind to the nonclassical MHC class I molecule HLA-E, which is on the surface of HCMV-infected cells complexed and stabilized with the HCMV-encoded UL40 peptide.
After primary infection, HCMV establishes a life-long persistence, from which sporadic reactivations and re-infections occur. The consequential repeated stimulation of the immune system results in in a shift of the human NK cell repertoire, hallmarked by the accumulation of long-living NKG2C+ and NKG2A+ NK cells.
The overall long-term consequences of such an HCMV-specific imprint in the NK cell repertoire for graft survival after lung transplantation, and in particular for ABMR are so far, however, unknown. The main aim of the present study was thus to reveal, whether there is an association between the HCMV-UL40 variants and the risk for ABMR and to which extent the HCMV-UL40 mediated expansion of CD16+NKG2C+ and CD16+NKG2A+ NK cells increase the risk for ABMR in LTRs.
Material and methods
Patients and samples
In our study, 150 patients were included, 30 DSA positive (DSA+) patients with AMBR and 120 matched controls. The controls were matched to the ABMR group in regard of age at transplantation, gender, D/R serostatus, as well as time, compartment and duration of the episode of high-level HCMV replication using case-control matching (Supplementary Methods, Table 1).
All patients were either HCMV-seropositive (R+) or received the organ from an HCMV-seropositive donor (D+/R-). The patients received induction therapy with 30mg Alemtuzumab (Berlex), and the maintenance therapy regimen included tacrolimus, corticosteroids and mycophenolate mofetil. HCMV-Immunoglobulin (Cytotect, 100 units/kg) was administered once weekly for 4 weeks posttransplantation and all patients received antiviral (Val-)Ganciclovir prophylaxis, R+ patients for 3, D+/R- patients for 12 months. All patients were followed-up by quantitative HCMV-PCR, weekly for 2 months, monthly to bimonthly for 1 year after transplantation and in larger intervals thereafter. HCMV-DNA levels of >1000 copies/ml plasma were preemptively treated with (Val-)Ganciclovir.
The standard posttransplantation care was performed as recently described in detail.
Surveillance bronchoscopy with a transbronchial biopsy and bronchoalveolar lavage, spirometry with body plethysmography were performed at 2 weeks and routinely thereafter. Computed tomography (CT) scan was performed each year and additionally in cases of lung function deterioration.
LTRs were routinely tested for de novo DSA at 2 weeks and 1,2,3,6,9,12,18, and 24 months after transplantation, and in cases of lung function deterioration, using a bead array technique as recently described.
Reactions with an MFI >1,000 were considered positive.
NK cell assays
Details of the antibody-dependent ABMR assays, NK cell proliferation assays and antibody-dependent ADCC assays are presented in detail in the supplementary material & methods section. For the ABMR assays, NK cells were cocultured together with the patient's plasma and human umbilical vein endothelial cells (HUVEC), followed by flow-cytometry analysis. For the NK cell proliferation assays, CFSE-stained NK cells were co-cultured together with UL40 peptide pulsed antigen-presenting RMA-S/HLA-E/LFA-3 cells, followed by analysis of proliferating and non-proliferating NK cell subsets. For the ADCC assays, NK cells were stimulated with UL40 peptides pulsed RMA-S/HLA-E/LFA-3 cells and proliferating and non-proliferating NK cells were subsequently cocultured either with Rituximab-coated Raji cells or with patient's plasma and HUVEC cells. NK cell activation was subsequently analyzed by flow-cytometry or LDH release assays.
The frequencies of the UL40 variants between the study cohorts were compared using the χ2-test. Repeated measures 1-way analysis of variance (RM ANOVA, with the Geisser-Greenhouse correction) and Tukey-post test (RM ANOVA: p < 0.05) were used to compare the means of proliferating and activated NK cell subsets between the cohorts. The time between the HCMV-episode and ABMR was compared between the UL40 variants using the Mantel-Cox test. The study was approved by the institutional review board of the Medical University of Vienna (EK-No.1687/2018).
Characteristics of the study cohort
The study cohort consisted of 30 patients who developed de novo DSA and clinical ABMR (DSA+ABMR+) within 2 years-post transplantation. In all DSA+ABMR+ LTRs, 1 episode of high-level HCMV-replication (>1000 copies/mL) occurred either in blood (N = 20) or in the bronchoalveolar lavage (BAL,N = 10), in median 130 (88-231) days prior to ABMR diagnosis. The median duration of these viral episodes was 14 (10-31) days.
As matched controls 30 LTRs who developed de novo DSA but had no clinical, but sub-clinical ABMR (DSA+ABMR−), and 90 patients who had neither DSA nor ABMR (DSA−ABMR−) within 2 years posttransplantation were selected. In contrast to DSA+ABMR+ LTRs, DSA+ABMR− and DSA−ABMR− had no allograft dysfunction, as defined by a FEV1% ≤80% at baseline for ≥3 weeks, at the diagnosis of ABMR.
From each patient and control, samples were available at 2 time points in the follow-up: The first plasma or BAL sample was available during the peak of the high-level HCMV-replication. The second sample was plasma, which was available after the diagnosis of ABMR in DSA+ABMR+ LTRs, before the patients underwent plasmapheresis or extracorporeal immunoadsorption for the elimination for DSA, or at a matched time point for DSA+ABMR− or DSA−ABMR− LTRs, respectively.
NKG2C± NK cells are highly activated by DSA and HUVEC cells
First, we aimed to analyze the contribution of CD16+CD94+NKG2C+, CD16+CD94+NKG2A+ and CD16+CD94− NK cells in the development of ABMR. Therefore, we first isolated CD56+CD16+ NK cells from 24 healthy, HCMV-seropositive blood donors and compared the CD16a expression levels between the NK cell subsets. As shown in Figure 1A, CD16+CD94+NKG2C+ showed significant higher CD16a expression levels, compared to CD16+CD94+NKG2A+ and CD16+CD94− NK cells. No different CD16a expression levels were however found between CD16+CD94+NKG2A+ and CD16+CD94− NK cells.
We then tested the isolated CD16+ NK cells in a flow-cytometry based in vitro antibody-dependent ABMR assay, using HUVECs and plasma samples from DSA+ABMR+ patients after the first ABMR diagnosis or from DSA−ABMR−, DSA+ABMR− at a comparable time point (Figure. S1). As shown in Figure 1B-G, stimulation with plasma samples from DSA+ABMR− and DSA+ABMR+ LTRs lead to a significant activation of CD16+CD94+NKG2C+, CD16+CD94+NKG2A+ and CD16+CD94− NK cells, compared to DSA−ABMR− LTRs. This was reflected by significantly higher percentages and expression levels of the cytotoxicity marker CD107a (Figure. 1B-C) as well as the pro-inflammatory cytokines IFNγ (Figure. 1D-E) and TNFα (Figure. 1F-G).
We then also compared the antibody-dependent activation levels between CD16+CD94+NKG2C+, CD16+CD94+NKG2A+ and CD16+CD94− NK cells. When stimulated with plasma from DSA+ABMR− or DSA+ABMR+ LTRs, CD16+CD94+NKG2C+ NK cells showed generally a significantly higher activation level for all tested markers compared to CD16+CD94+NKG2A+ and CD16+CD94− NK cells (all: p < 0.001,ANOVA and Dunn's Test). In contrast, we observed no significant difference between CD16+CD94+NKG2A+ and CD16+CD94− NK cells (all:ns, ANOVA and Dunn's Test). In summary, this shows that especially CD16+CD94+NKG2C+ cells are highly activated in DSA+ LTRs and thus may be the main CD16+ NK cell subset, which contributes to tissue injury in DSA+ LTRs during ABMR.
HCMV-UL40 variants are associated with the development of ABMR
As the expansion of CD16+CD94+NKG2C+, and to a lower extent also CD16+CD94+NKG2A+ NK cells depends on HCMV-encoded UL40 peptides,
we next analyzed the UL40 variants of the infecting HCMV-strains of the DSA−ABMR−, DSA+ABMR− and DSA+ABMR+ LTRs by Sanger-sequencing. Overall, 5 different UL40 variants dominated in the study cohort: VMAPRTLIL (N = 48,32%), VMAPRTLLL (N = 38,25.3%), VMTPRTLIL (N = 26,17.3%), VMTPRTLVL (N = 12,8%) and VMAPRTLVL (N = 8,5.3%). Other UL40 variants were detected each only in 1 or 2 LTRs and were consequently summarized as “unusual” variants (N = 20,13.3%).
We then compared the distribution of the UL40 variants between DSA−ABMR−, DSA+ABMR− and DSA+ABMR+ LTRs and found a different pattern between DSA+ABMR+ LTRs and the controls (Figure 2, Figure S2). While DSA−ABMR− and DSA+ABMR− LTRs showed overall a similar UL40 variant diversity, in DSA+ABMR+ LTRs 2 variants, VMAPRTLIL (43.3%) and VMTPRTLVL (40%), dominated. Especially the VMTPRTLVL variant was solely found in patients with ABMR (PPV:1, NPV:0.4), while VMTPRTLIL as well as “unusual” variants occurred only in DSA−ABMR− and DSA+ABMR− LTRs (both: p < 0.0001, F-Test).
We then also compared the time between the occurrence of a high-viral load and ABMR between the UL40 variants. No significant differences were found (all: p > 0.05, Mantel-Cox test).
HCMV-UL40 variants are associated with the shape of the NK cell repertoire
We then tested to which extent the 5 major UL40 variants (VMAPRTLIL,VMAPRTLLL,VMTPRTLIL,VMTPRTLVL,VMAPRTLVL) were associated with a shift of the human CD16+ NK cell repertoire towards highly cytotoxic CD16+CD94+NKG2C+ NK cells. Therefore, we cocultured CD16+ NK cells with respective UL40-peptide pulsed antigen presenting cells or HCMV-infected HUVEC cells for 14 days. After 7 and 14 days, respectively, we assessed the proliferating and non-proliferating NK cell subsets by flow-cytometry (Figure S3). UL40 peptides (Figure 3) and HCMV-infected cells (Figure S4) elicited a significant proliferation of CD16+CD94+NKG2C+ and CD16+CD94+NKG2A+ NK cells. In contrast, the proliferation of CD16+CD94− NK cells dominated in response to antigen presenting cells without peptide or noninfected HUVEC (Figure 3, Figure S4).
We then compared the percentages of the proliferating CD16+CD94+NKG2C+, CD16+CD94+NKG2A+ and CD16+CD94− NK cell subsets between the different UL40 peptides. We found that especially the VMAPRTLIL and VMTPRTLVL peptides were associated with the proliferation of CD16+NKG2C+ NK cells after 7 and 14 days of coculture, respectively. Compared to the VMAPRTLLL,VMTPRTLIL or VMAPRTLVL, stimulation with the VMAPRTLIL or VMTPRTLVL UL40 peptides resulted in a significantly higher percentage of CD16+CD94+NKG2C+ after 14 days of co-culture (all:p < 0.01, ANOVA and Dunn's Test).
Proliferating NKG2C± NK cells are highly potent mediators of ADCC
To test whether the proliferating and nonproliferating CD16+CD94+NKG2C+, CD16+CD94+NKG2A+ and CD16+CD94− NK cells are active and capable to elicit ADCC after 14 days of expansion, we tested the NK Cells in an in vitro flow-cytometry based ADCC assay, using Rituximab-coated Raji cells and the cytotoxicity marker CD107a. As shown in Figure 4A-D, all proliferating and nonproliferating CD16+CD94+NKG2C+, CD16+CD94+NKG2A+ and CD16+CD94− NK cells were capable of ADCC, as reflected by the Rituximab concentration-dependent increase of CD107a in all tested CD16+ NK cell subsets.
We then also compared the percentage of activated NK cells as well as the expression levels of CD107a between the proliferating and non-proliferating CD16+CD94+NKG2C+, CD16+CD94+NKG2A+ and CD16+CD94− subsets and found, that CD16+CD94+NKG2C+ showed a significantly higher degree of activation, compared to CD16+CD94+NKG2A+ and CD16+CD94− NK cells. No significant differences were however found between CD16+CD94+NKG2A+ and CD16+CD94− NK cells (Figure 4A-D).
Next, we compared the activation level between proliferating and non-proliferating CD16+CD94+NKG2C+, CD16+CD94+NKG2A+ and CD16+CD94− NK cell subsets, respectively. All proliferating NK cell subsets showed generally a higher degree of activation, compared to non-proliferating cells (all:p < 0.01,ANOVA and Dunn's Test).
HCMV UL40-mediated shape of the NK cell repertoire is associated with ABMR
We then also tested whether the HCMV-UL40 mediated shift of the NK cell repertoire towards highly potent CD16+CD94+NKG2C+ NK cells result in an increased ADCC response. Therefore, we expanded NK cells with the UL40 peptides for 14 days and cocultured the NK cells then with HUVEC cells and the plasma samples of DSA−ABMR−, DSA+ABMR− and DSA+ABMR+ LTRs. As shown in Figure 4E only plasma from DSA+ABMR− and DSA+ABMR+ LTRs, together with expanded NK cells, lead to a significant lysis of HUVEC cells, as reflected by a higher LDH release. Importantly, no significant differences were found in the lysis of HUVEC cells between plasma from DSA+ABMR− and DSA+ABMR+ LTRs.
We finally compared the level of lysis of HUVECs induced by NK cells, which were stimulated with the different UL40 peptides. As shown in Figure 4F, CD16+ NK cells, stimulated with the VMAPRTLIL and VMTPRTLVL variant lead to a significantly higher antibody-mediated lysis of HUVEC cells in the presence of patient plasma, compared to NK cells, stimulated with the VMAPRTLLL,VMTPRTLIL or VMAPRTLVL variant (all:p < 0.01,ANOVA and Dunn's Test).
In the present study, we demonstrate that the specific UL40 variant of the infecting HCMV-strain and the resulting shaping of the human NK cell repertoire, especially towards CD16+NKG2C+ NK cells, play an important role in the antibody-mediated allograft injury during ABMR after lung transplantation.
So far, it remained an open question why of all DSA+ LTRs only some developed ABMR, while other DSA+ patients show no evidence of humoral rejection over time. A recently published study in kidney transplant recipient (KTRs) found that the presence of DSA alone is not a risk factor for rejection or graft failure.
In the present study, we revealed that all DSA+ patients, independent of whether they developed ABMR or not, show a comparable ADCC response against endothelial cells, when tested in the in vitro antibody-dependent ABMR assay. However, our study revealed that the UL40 peptide variant, present in the HCMV-strain infecting the patients, plays a significant role in the development of AMBR and was signifantly different between DSA+AMBR+ and DSA+AMBR− patients. It has previously been shown that the highly polymorphic UL40 peptide occurs in different variants in HCMV-strains infecting LTRs.
In the present patient population, we identified in overall 18 different UL40 variants in the infecting HCMV-strains, detected during an episode of high-level virus replication in blood or lung. Of these however, 2 HCMV-encoded UL40 peptides, VMAPRTLIL and VMTPRTLVL, were especially prevalent, and the peptide VMTPRTLVL was even detected exclusively in DSA+ patients, developing ABMR in the follow-up. Our data are of special interest, as predictive factors for ABMR in de novo DSA+ LTRs are still scarce.
In our patient population, the occurrence of the VMTPRTLVL variant, detected in an episode of virus replication was reliably associated with the subsequent occurrence of ABMR in LTRs.
As a potential mechanism behind the association between the distinct UL40 variants and the development of ABMR, we revealed that the VMAPRTLIL and VMTPRTLVL variants are associated with a strong shift towards highly potent and proinflammatory CD16+NKG2C+ NK cells. This shift, demonstrated by NK cell proliferation assays, further results in an overall increase in the NK cell-mediated ADCC response, as revealed in the DSA-dependent ABMR assays. Our data thus demonstrate that HCMV-infections with VMAPRTLIL- and VMTPRTLVL-encoding strains lead to an increase in the proliferation of CD16+NKG2C+ NK cells, which then may mediate ABMR via an overall enhanced ADCC response.
Different studies associated so far the occurrence of ABMR to the level of complement binding of the DSA in the single patients, as measured by C1q fixation or C3d-binding activity.
that highlights the role of FcγR-expressing effector cells, such as NK cells. The overall importance of NK cells for the development of ABMR was also recently demonstrated in KTRs, in whom the level of intra-graft NK cell infiltrates and transcripts strongly predicted ABMR and graft survival.
These studies are in line with our present data, and all together demonstrate an important role of the antibody-mediated activation of NK cells during humoral rejection.
We showed that especially the shift towards the CD16+NKG2C+ NK cell population was important for ABMR. In our study, we demonstrated that CD16+NKG2C+ NK cells did not only provide cytotoxic potential but also secreted high levels of IFNγ and TNFα. These findings are of special interest, as recently published transcriptomic studies in KTRs with de novo DSA demonstrated that ABMR and allograft loss is hallmarked by IFNγ-inducible transcripts, such as the pro-inflammatory CXCL10 and CXCL11,
It was consequently hypothesized that IFNγ may play a major role in the development and pathogenesis of ABMR. The effects of IFNγ include the upregulation of HLA and the activation of pro-inflammatory macrophages, both possibly facilitating the development of ABMR. TNFα is known as an important factor for the expression of adhesion molecules on endothelial cells, which facilitates the migration of lymphocytes into the allograft. It is thus reasonable that a strong CD16+NKG2C+ NK cell-mediated cytokine response plays an important role in the pathogenesis of ABMR, by inducing downstream donor-specific innate and adaptive immune responses.
In summary, the present study reveals that distinct variants of the HCMV UL40 peptide present in the infecting HCMV-strain are associated with the following development of ABMR in LTRs, and that this is caused by a shift of the human NK cell repertoire towards a highly cytotoxic CD16+NKG2C+ NK cell population. Further studies are needed to evaluate whether the identification of the HCMV UL40 peptide may serve as a potential prognostic marker for ABMR and to further clarify the association of different HCMV-strains with the risk of allograft rejection and ABMR.