CXCL13 Is an Indicator of Germinal Center Activity and Alloantibody Formation Following Transplantation

Abstract

Donor-specific antibodies (DSA) are a recognized cause of allograft injury, yet biomarkers that indicate their development posttransplant or guide management are not available. CXCL13 (chemokine [C-X-C motif] ligand 1) is a chemoattractant produced within secondary lymphoid organs necessary for germinal center (GC) and alloantibody formation. Perturbations in serum CXCL13 levels have been associated with humoral immune activity. Therefore, CXCL13 may correlate with the formation of HLA antibodies following transplantation.

Methods: A murine skin graft model was utilized to define the production and kinetics of CXCL13 in response to alloantigen. Human Tfh:B-cell in vitro cocultures were performed to evaluate CXCL13 production by human lymphocytes, and serum from healthy controls and human transplant recipients with and without de novo DSA was tested for CXCL13.

Results: CXCL13 was detectable in the blood of allografted mice and correlated with Tfh and GC B-cell responses. Greater CXCL13 expression was observed in the draining lymph nodes of allografted mice as compared with naïve or syngeneic graft recipients, and serum levels preceded the detection of DSA posttransplant. Similarly, productive human Tfh:B-cell interactions that led to plasmablast differentiation and IgG formation also exhibited CXCL13 expression. CXCL13 levels in human transplant recipients with de novo DSA were greater than in healthy controls and stable transplant patients and also correlated with the development of alloantibodies in a small cohort of serially monitored recipients.

Conclusions: CXCL13 indicates GC alloreactivity and alloantibody formation and correlated with DSA formation in kidney transplant recipients, thereby introducing CXCL13 as a potential biomarker for HLA antibodies.

FIGURE 1.

CXCL13 expressed in graft-DLNs correlates with GC alloreactivity and indicates DSA formation following transplantation. (A) B6 mice were transplanted with BALB/c (allogeneic) or b6 (syngeneic) skin grafts and draining lymph nodes collected for flow cytometric and PCR analysis. Mice were also serially bled, and serum collected for CXCL13 and antibody analysis. (B) Serum CXCL13 levels in naïve and syngeneic or allogeneic skin-grafted mice 10 d posttransplant. (C) IL-21 and CXCL13 mRNA expression in graft-DLNs 5 d after primary and secondary skin grafts. (D) IL-21 and CXCL13 mRNA expression in sorted murine Tfh (PD1^hiCXCR5⁺) cells compared with CD4⁺ non-Tfh (CD44^hiCXCR5^–) cells. (E) Serum CXCL13 levels over time relative to graft-DLN Tfh cell (green), GC B cell (blue), and DSA (orange) kinetics. Summary data represent mean (SE) and are representative of at least 2 independent experiments with a total of 5–10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. CXCL13, chemokine (C-X-C motif) ligand 1; DLN, draining lymph node; DSA, donor-specific antibody; GC, germinal center; Tfh, T follicular helper.

FIGURE 2.

Human CXCL13 correlates with GC-like Tfh:B-cell reactivity in vitro. (A) Lymph node–derived human lymphocytes were sorted into CXCR5^– T and CXCR5⁺ Tfh cells and cultured in vitro with enriched B cells for 5 d. Cocultured cells were collected for flow cytometric and PCR analysis, and culture supernatants were collected for antibody assessment. (B) Representative flow plots and summary data of plasmablast (MHCII⁺CD27^hiCD38⁺ B cells) formation. (C) Summary data of IgG antibody production as measured by ELISA (450 nm OD). (D) Summary data of coculture IL-21 and CXCL13 mRNA expression. (E) Representative flow plots and summary data of CXCL13 production by CD4⁺ T cells. Summary data represent mean (SE) and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. CXCL13, chemokine (C-X-C motif) ligand 1; Tfh, T follicular helper.

FIGURE 3.

CXCL13 levels correlate with the development of alloantibodies in kidney transplant recipients. (A) Serum CXCL13 levels in HC (n = 19) and kidney transplant recipients with (DSA+, n = 15) and without (ST, n=8) de novo DSA. DSA+ samples were collected from patients within 2 wk of developing de novo DSA. (B) Serum CXCL13 levels (red line) and cumulative HLA antibodies (black bars) over time in 3 kidney transplant recipients with de novo DSA formation. Serum CXCL13 levels in 3 separate healthy controls (C) and 3 separate ST recipients without DSA (D) over time. Summary data represent mean (SE). *P < 0.05, ***P < 0.001. CXCL13, chemokine (C-X-C motif) ligand 1; DSA, donor-specific antibody; HC, healthy controls; ST, stable transplant.