Impaired complement regulation drives chronic lung allograft dysfunction after lung transplantation

Abstract

A greater understanding of chronic lung allograft dysfunction (CLAD) pathobiology, the primary cause of mortality after lung transplantation, is needed to improve outcomes. The complement system links innate to adaptive immune responses and is activated early post-lung transplantation to form the C3 convertase, a critical enzyme that cleaves the central complement component C3. We hypothesized that LTx recipients with a genetic predisposition to enhanced complement activation have worse CLAD-free survival mediated through increased adaptive alloimmunity. We interrogated a known functional C3 polymorphism (C3R102G) that increases complement activation through impaired C3 convertase inactivation in two independent LTx recipient cohorts. C3R102G, identified in at least one out of three LTx recipients, was associated with worse CLAD-free survival, particularly in the subset of recipients who developed donor-specific antibodies (DSA). In a mouse orthotopic lung transplantation model, impaired recipient complement regulation resulted in more severe obstructive airway lesions when compared to wildtype controls, despite only moderate differences in graft-infiltrating effector T cells. Impaired complement regulation promoted the intragraft accumulation of memory B cells and antibody-secreting cells, resulting in increased DSA levels. In summary, genetic predisposition to complement activation is associated with B cell activation and worse CLAD-free survival.

Figure 1.. A functional C3 polymorphism confers increased risk of chronic lung allograft dysfunction (CLAD) or death in two independent cohorts.

CONSORT diagrams for (A) Washington University/Barnes-Jewish Hospital (BJH) and (B) University of California-San Francisco (UCSF) cohorts. (C and E) Kaplan Meier plot of survival from CLAD or death in the BJH (C) and UCSF (E) cohort stratified by rs2230199 genotypes. (D and F) Kaplan Meier plot based on the multivariate Cox Regression analysis of CLAD-free Survival in the BJH (D) and UCSF (F) cohorts by rs2230199 status. P value represents log-rank test. aHR: adjusted hazard ratio; CI: confidence intervals.

Figure 2.. Complement-mediated CLAD or death is dependent on donor-specific antibodies (DSA).

CLAD-free survival was worse in the recipients with donor-specific antibodies (DSA) and the C3 R102G polymorphism (CC/GC). The BJH (A) and UCSF (B) cohorts were stratified by DSA-negative and DSA-positive recipients. p value represents log-rank test. (C and D) Kaplan Meier plot based on the multivariate Cox Regression analysis of CLAD-free survival in the UCSF cohort separated by genotype, stratified by DSA status. aHR: adjusted hazard ratio, CI: confidence interval.

Figure 3:. Dysregulated complement activation promotes CLAD in a mouse lung transplantation model.

(A) Immunoblot of serum and bronchoalveolar lavage (BAL) C3-alpha and -beta fragments from resting Crry^+/+ and Crry^−/− mice (N=4/group). The data shown is a representative result of 2 experiments. (B) Mouse orthotopic left lung transplant model of CLAD. (C, upper panel) POD 16 immunofluorescent staining of club and ciliated cells using antibodies specific for club cell secretory protein (CCSP) and acetylated tubulin (Ac-Tubulin), respectively (N=5/group). (C, middle panel) Trichrome and (C, lower panel) Hematoxylin and Eosin (H&E) staining of POD16 tissue used for blinded A and B rejection scoring (bar graphs). The histology shown is representative of at least six transplants per group. (D) Gating strategy to identify CCSP⁺CD326⁺ club cells. Data displayed is a representative result of N=5/group. (E) Intragraft total and indicated subset CD4⁺ and CD8⁺ T cell numbers at POD 16. The bar graph and dot plots show mean ± standard deviation for (C) Mann-Whitney U test and (D, E) Welch’s t-test, where *p <0.05 and ***p <0.001.

Figure 4:. Enhanced intragraft B cell accumulation and activation in lung recipients with a defect in complement regulation.

(A) Levels of C3d bound to B cells two days after the induction of doxycycline-induced allograft club cell injury. Representative C3d B cell staining and mean fluorescence intensity plots (N=4/group) using flow cytometry (FACS). (B) FACS analysis of intragraft B cell abundance and ki67 staining (proliferation) with a plot of total B cell numbers on POD 16. FACS contour plots and histograms are representative results from N = 4/group. (C) % of BAL CD19⁺ lymphocytes in UCSF cohorts who carry the GG genotype and C3 R102G (CC&GC) polymorphisms. P=0.02 by an unpaired t-test. (D) Indicated IgM and IgG DSA levels on POD 16 (N=5/group). (E) Intragraft CD138⁺ IgM⁺ antibody-secreting cells abundance as shown by representative FACS dot blots (N=5/group) and bar graphs on POD16. IgM expression was measured by intracellular (i.c.) staining. (F). A representative gating strategy was used to identify intragraft CD80⁺CD73⁺CD273⁺ memory B cells with bar graphs showing the total intragraft numbers of these cells on POD16. Bar graphs and dot blots show means ± standard deviations where (A) 2-way ANOVA with Sidak’s multiple comparisons test, and (B, D, E) Welch’s t-test was conducted where ns is not significant, *p < 0.05, **p < 0.01, ***p < 0.001.