FCGR3A and FCGR2A Genotypes Differentially Impact Allograft Rejection and Patients' Survival After Lung Transplant

Abstract

Fc gamma receptors (FcγRs) play a major role in the regulation of humoral immune responses. Single-nucleotide polymorphisms (SNPs) of FCGR2A and FCGR3A can impact the expression level, IgG affinity and function of the CD32 and CD16 FcγRs in response to their engagement by the Fc fragment of IgG. The CD16 isoform encoded by FCGR3A [158V/V] controls the intensity of antibody-dependent cytotoxic alloimmune responses of natural killer cells (NK) and has been identified as a susceptibility marker predisposing patients to cardiac allograft vasculopathy after heart transplant. This study aimed to investigate whether FCGR2A and FCGR3A polymorphisms can also be associated with the clinical outcome of lung transplant recipients (LTRs). The SNPs of FCGR2A ([131R/H], rs1801274) and FCGR3A ([158V/F], rs396991) were identified in 158 LTRs and 184 Controls (CTL). The corresponding distribution of genotypic and allelic combinations was analyzed for potential links with the development of circulating donor-specific anti-HLA alloantibodies (DSA) detected at months 1 and 3 after lung transplant (LTx), the occurrence of acute rejection (AR) and chronic lung allograft dysfunction (CLAD), and the overall survival of LTRs. The FCGR3A [158V/V] genotype was identified as an independent susceptibility factor associated with higher rates of AR during the first trimester after LTx (HR 4.8, p < 0.0001, 95% CI 2.37-9.61), but it could not be associated with the level of CD16- mediated NK cell activation in response to the LTR's DSA, whatever the MFI intensity and C1q binding profiles of the DSA evaluated. The FCGR2A [131R/R] genotype was associated with lower CLAD-free survival of LTRs, independently of the presence of DSA at 3 months (HR 1.8, p = 0.024, 95% CI 1.08-3.03). Our data indicate that FCGR SNPs differentially affect the clinical outcome of LTRs and may be of use to stratify patients at higher risk of experiencing graft rejection. Furthermore, these data suggest that in the LTx setting, specific mechanisms of humoral alloreactivity, which cannot be solely explained by the complement and CD16-mediated pathogenic effects of DSA, may be involved in the development of acute and chronic lung allograft rejection.

Keywords: Fc-gamma receptors; HLA antibodies; allograft rejection; chronic lung allograft dysfunction; lung transplantation; natural killer cells.

Figure 1

Distribution of FCGR3A and FCGR2A genotypes in LTRs and CTLs. The proportion of the FCGR2A (left Panel, A) and FCGR3A (right panel, B) genotypes resulting from the SNP allelic combination were analyzed in a cohort of 158 LTRs and 184 CTLs recruited in southern France.

Figure 2

Study flow chart analysis of the detection by single antigen flow bead Luminex Assay of de novo DSA and C1q binding activity of DSA at M1 and at M3 post-LTx.

Figure 3

Analysis of the influence of Fc-gamma Receptor polymorphisms on Rejection Free survival. (A) Impact of FCGR3A genotype on freedom from acute lung rejection. Kaplan–Meir Survival analysis links the FCGR3A [158V/V] (solid line, n = 20) to lower acute-rejection-free survival after 3 months post-LTx in LTRs, when compared with the FCGR3A [158V/F] and FCGR3A [158F/F] group (dashed line, n = 81). LTRs with the FCGR3A [158V/V] show an increased rate of acute rejection events occurring during the first 3 months post-LTx. (B) Impact of FCGR2A genotype on CLAD free survival. Kaplan–Meir Survival analysis links the FCGR2A [131R/R] (solid line, n = 39) to a lower CLAD-free survival rate in LTRs over time or study follow-up (years, x axis), when compared with the FCGR2A [131H/H] and FCGR2A [131R/H] group (dashed line, n = 119).

Figure 4

Representative illustration of the NK-Cellular Humoral test (NK-CHAT) used to evaluate serum- and DSA- induced NK cell activation toward B cell lines expressing cognate HLA class I or HLA-DQ7 donor-specific allo-antigens. (A) PBMC effectors obtained from FCGR3A [158 V/V] CTL were exposed to B-EBV cell lines previously coated with DSA-negative serum. This allowed for evaluation of the baseline expression level of CD16 expression (CD16MFI) within the CD3-CD56+CD16+ NK cell compartment when PBMC were exposed to B cell targets. As a positive control indexing the level of IgG-induced CD16 down regulation (CD16DRI), the same B-EBV cell lines were coated in the presence of 10γ/ml Rituximab (anti CD20 IgG) prior to exposure to the effector PBMC. This allowed for calculation of the CD16 down regulation index (CD16DRI: baseline CD16MFI NK cells exposed to B cells coated with CTL DSA-negative serum/CD16 MFI of NK cells exposed to the same B targets pre-coated in presence of Rituximab). In this context, CD16DRI in response to Rituximab = 19, i.e., 90/4.8. (B) The CD16 DRI of NK cells was evaluated in response to the serum collected at M1 in one LTR (LTR 1, Table 5) with detectable levels of DSA recognizing the HLA-A3 and -B7 expressed on the B cell targets. Non-immunized serum (DSA-negative) collected from LTR 1 at time of LTx (D0) was used as the reference baseline CD16 MFI value to calculate the CD16DRI. Despite a cumulative MFI of DSA >17,000, DSA detected at M1 in LTR1 failed to induce CD16-dependent NK cell alloreactivity (CD16DRI = 1). (C) The CD16 DRI of NK cells was evaluated in response to the serum collected at time of ABMR diagnosis in one kidney transplant recipients (KTR 1, Table 5) with detectable levels of DSA recognizing the HLA-DQ7 (MFI: 13,000) expressed on the B cell targets. The CD16DRI of KTR1 (CD16DRI: 45) was greater than that observed in response to the Rituximab (CD16DRI:19, A). (D) The CD16 DRI of NK cells was evaluated in response to the serum collected at time of ABMR diagnosis in a second KTR (KTR 2, Table 5) with detectable levels of DSA recognizing the HLA-DQ7 (MFI: 9,000) expressed on the B cell targets. The CD16DRI of KTR1 (CD16DRI: 30) was greater than that observed in response to the Rituximab (CD16DRI:19, A). (E) The CD16 DRI of NK cells was evaluated in response to the serum collected at M3 (time of acute rejection) in a second LTR (LTR 17, Table 5) with detectable levels of DSA recognizing the HLA- DQ7 (MFI: 13,400) expressed on the B cell targets. Non-immunized serum (DSA-negative) collected from LTR 17 at D0 was used as the reference baseline CD16 MFI value to calculate the CD16DRI (CD16DRI = 1). The MFI DSA of LTR 17 (MFI: 13,400) was similar to the MFI DSA of ABMR KTR 1 serum as illustrated in (C). (F) The CD16 DRI of NK cells was evaluated in response to the serum collected at M3 (time of acute rejection) in a third LTR (LTR 20, Table 5) with detectable levels of DSA recognizing the HLA- DQ7 (MFI DSA: 9,000) expressed on the B cell targets. The DSA-negative serum of LTR 20 collected at D0 was used as a baseline CD16 MFI value to calculate the CD16DRI (CD16DRI = 1.9). The MFI DSA of LTR 20 (MFI: 9,000) was similar to the MFI DSA of ABMR KTR 2 serum evaluated in (D).

Figure 5

Kaplan-Meir survival analysis of lung allograft survival stratified according to FCGR2A [131R/H] genotypes. The FCGR2A [131R/R] homozygous genotype (solid black line, n = 39) is associated with lower survival rates in LTRs when compared to FCGR2A [131H/R] (gray line, n = 68) or the FCGR2A [131H/H] (dashed line, n = 51).