*Purpose: Donor-specific anti-HLA antibodies (DSA) represent an important biomarker for acute rejection and are a major cause of long-term graft failure after kidney transplantation (KTx). The cellular mechanisms that lead to the development of DSA are not entirely understood. Here we analyzed the phenotypic polarization, activation and function status of CXCR5⁺CD45RO⁺CD4⁺ circulating (c)T follicular helper cell (T_FH), which are known to be critical for antibody production by B cells.

*Methods: We have consented 11 healthy controls (HC) and 47 Thymoglobulin-induced KTx patients participating in an ongoing observational cross-sectional study. Twenty-four patients were identified as DSA⁺ (MFI above 1000), including 13 asymptomatic DSA patients without proven antibody-mediated rejection (ABMR) and 11 undergoing biopsy proven ABMR. In addition, 23 DSA negative patients comprised of 9 patients with acute cellular rejection (TCMR) only, and 14 stable, rejection free patients. Whole blood was obtained and analyzed by flow cytometry to identify cT_FH subsets polarization (Th1, Th2, Th17) and activation (ICOS/PD1) status. For functional assays, FACS-sorted cT_FH were incubated with memory (m) B cells ± Staphylococcus aureus Enterotoxin B (SEB) for 6 days in vitro co-cultures and analyzed for their ability to trigger plasmablast formation and IgG production.

*Results: All DSA⁺ KTx patients display increased levels (%) of Th1/Th17-cT_FH cells with activated phenotypes as compared to HC (p= 0.007) or stable patients (p=0.05). Interestingly, only ABMR patients presented also significant higher levels of activated (CD62L^lowICOS^hiPD1^hi) Th2-cT_FH subsets (most functionally potent) compared to all other groups (0.0004 to HC, p=0.003 to stable, p=0.01 to TCMR, and p=0.03 to asymptomatic DSA⁺ patients). Principal component analysis (PCA) incorporating all 47 patients and repeat count values from 9 phenotypic markers, independently confirmed that ABMR KTx recipients cluster distinctly from asymptomatic DSA⁺, stable and TCMR KTx recipients. In addition, while cT_FH from all KTx patients are functional and support in vitro plasmablast formation and IgG production, cT_FH from ABMR patients triggered significant higher pathogenic IgG1/IgG3 production from mB cells compared to asymptomatic DSA⁺ patients.

*Conclusions: Our data underscore the value of cT_FH cell monitoring, in addition to the currently available DSA measurements, for a comprehensive mechanistic characterization of DSA responses, and support immunotherapeutic strategies to target T_FH cells for DSA⁺ KTx patients at risk of ABMR.

« Back to 2019 American Transplant Congress