*Purpose: The role of alloreactive memory B (mB) cells in the pathogenesis of antibody-mediated rejection (ABMR) is increasingly recognized. Yet, a comprehensive characterization of mB cell heterogeneity during ongoing donor-specific antibody (DSA) responses is lacking.

*Methods: Using 22-color flow cytometry and in vitro assays, we investigated the immune phenotype and function of blood subsets of mB cells in patients who did not develop DSA (DSA-, N=48) and in patients who developed DSA (DSA+, N=48) post kidney transplantation and in healthy controls (HC, N=17).

*Results: We have identified three distinct subsets of blood CD20⁺CD38^lo mB cells: resting memory (RM, CD27⁺CD21⁺Tbet^–), activated memory (AM, CD27⁺CD21^–Tbet^int) and tissue-like memory (TLM, CD27^–CD21^–Tbet^hi) B cells. AM and TLM were significantly expanded in blood of DSA+ patients (P=0.001 for both), while RM mB cells were the predominant subset in DSA- patients and in HC. AM, unlike RM or TLM mB cells, strongly correlated with the plasmablast response (r=0.57, P=0.003), and circulating DSA levels (r=0.73, P<0.001). In DSA+ patients who developed ABMR (N=20), AM but not RM or TLM mB cells were selectively expanded and peaked at the time of biopsy-proven rejection. Phenotypically, AM mB cells upregulated multiple activation markers (Ki67, CD86 and CD95 ; P<0.001 for all) and readily expressed plasma cell markers (Blimp and IRF4 ; P<0.001 for both). In contrast, TLM mB cells expressed increased inhibitory receptors (FcγRIIb, CD72 and FcRL5 ; P<0.001 for all) and strongly downregulated CD40 (P=0.002), suggesting their hypo-responsiveness to T-cell help. Consistently, when co-cultured with T follicular helper cells (T_FH), TLM poorly differentiated into plasmablast and did not produce IgG, while AM mB cells highly differentiated into plasmablast and produced DSA in vitro.

*Conclusions: We identified functional heterogeneity in CD21^– mB cells during ABMR. AM were enriched for alloantigen-specific cells and prone to plasmablast differentiation upon T_FH-cell help, while TLM mB cells were T_FH-cell independent and prone to regulation/exhaustion. Timely detection and selective targeting of deleterious AM mB-cell response represent valuable strategies to prevent ABMR.

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