Dual Targeting of CD21 and TLR7: A New Strategy to Suppress Autoreactive B-Cell Activation in SLE

Systemic lupus erythematosus (SLE) is a highly heterogeneous chronic autoimmune disease characterized by breakdown of B-cell tolerance, massive production of autoantibodies against nuclear antigens, and subsequent systemic inflammation and organ damage. During autoantibody production, the extrafollicular (EF) B-cell differentiation pathway is the primary source of antibody-secreting cells (ASCs) and is closely associated with disease activity. Among these, a B-cell subset with low CD21 expression (CD21^lo) and high CD11c expression accumulates in aging, infection, and autoimmune diseases and has been identified as a key precursor of extrafollicular ASCs; however, the developmental trajectory and receptor regulatory mechanisms of this subset remain unclear.

Toll-like receptor 7 (TLR7), an innate immune receptor that recognizes single-stranded RNA, plays a crucial role in SLE pathogenesis by promoting B-cell tolerance escape and autoantibody production. In contrast, complement receptor 2 (CD21), a co-receptor of the B-cell receptor (BCR), normally enhances B-cell activation, yet it is paradoxically downregulated in lupus-associated B-cell subsets. This contradictory phenomenon suggests a more complex role for CD21 in autoimmunity. To address these questions, the research team established a TLR7-driven adoptive transfer lupus model to systematically investigate the regulatory role and molecular mechanisms of CD21 in the extrafollicular differentiation of autoreactive B cells.

The research team adoptively transferred splenic B cells from wild-type (WT) and TLR7-knockout (Tlr7-KO) mice at a 1:1 ratio into 564Igi transgenic recipient mice, which provide an autoimmune microenvironment rich in nuclear antigens and T-cell help. Seven days post-transfer, mature B cells, germinal center (GC) B cells, and non-GC B cells derived from Tlr7-KO donors were significantly reduced. Notably, ASCs and DN2-like cells (CD11c⁺CD21^-) decreased by 24-fold and 9.7-fold, respectively, confirming that both B-cell tolerance breakdown and germinal center development depend on TLR7 signaling. Blocking GC formation using Bcl6 conditional knockout mice did not affect ASC numbers. Combined with kinetic analysis showing WT donor B-cell expansion starting on day 4 and ASC accumulation in the splenic red pulp from day 5, the study clearly demonstrated that autoreactive B cells in this model primarily differentiate into ASCs via the extrafollicular pathway, driven by TLR7 signaling.

Figure 1. The 564Igi adoptive transfer model is TLR7-dependent and supports extrafollicular (EF) differentiation of autoreactive B cells

To dissect the relationship between B-cell proliferation and differentiation, donor B cells were labeled with CellTrace Violet (CTV). The results showed that CD21 expression gradually decreases during donor B-cell proliferation, with most ASCs requiring at least seven divisions. Phenotypic clustering divided donor B cells into marginal zone-like, follicular-like, and DN2-like subsets. The CD21^loCD23^- subset predominantly consisted of terminally divided cells, whereas in Tlr7-KO mice this subset contained significantly more resting cells and failed to complete terminal divisions. Late-division cells also upregulated the ASC-associated transcription factors BLIMP1 and IRF4, indicating a pre-programmed division-linked differentiation pathway for autoreactive ASCs. CD21 downregulation is tightly linked to TLR7 signaling; TLR7 deficiency impedes continuous cell-cycle progression and differentiation.

Figure 2. Naïve autoreactive B cells downregulate CD21 and establish TLR7 sensitivity during a fixed division-differentiation program

Using CD21-Cre×Rosa26^{Lox-Stop-Lox-tdTomato} fate-mapping mice, nearly all donor-derived CD21^loCD23^- cells in 564Igi recipients at day 7 were tdTomato⁺, confirming their origin from initially CD21⁺ B cells rather than a pre-existing CD21^lo pool. Serial adoptive transfer experiments showed that sorted CD21^lo cells preferentially differentiated into CD138⁺ ASCs in secondary recipients, whereas follicular B cells primarily formed GC B cells. In vitro stimulation with TLR7 agonist and cytokines further demonstrated that CD21^lo cells efficiently differentiate into plasmablast-like cells and produce large amounts of autoantibodies against cytoplasmic components, establishing CD21^lo cells as direct precursors of extrafollicular ASCs with strong autoantibody-secreting potential.

Figure 3. CD21^lo cells derive from CD21⁺ cells and are poised for ASC and autoantibody production

Purified naïve follicular B cells (CD21^midCD23⁺), marginal zone B cells, and transitional T1 B cells were separately transferred into 564Igi recipients. Only naïve follicular B cells robustly generated CD21^loCD23^- cells by day 5.5 post-transfer, with most cells undergoing ≥7 divisions expressing CD138. Terminally divided cells were markedly enriched for the CD21^loCD23^- subset, and a single mouse's starting follicular B cells contributed ~20% of total ASCs. Marginal zone B cells produced few ASCs while largely retaining their original phenotype, and T1 B cells failed to differentiate efficiently into ASCs. These results confirm that naïve follicular B cells are the dominant source of both CD21^lo precursors and early autoreactive ASCs, requiring passage through the CD21^lo intermediate state for extrafollicular differentiation.

Figure 4. CD21⁺ ASC precursor B cells originate from naïve follicular B cells (FoB)

To define the clonal evolution from naïve follicular B cells (FoB) to autoreactive cells, bulk heavy- and light-chain BCR sequencing was performed on donor subsets 6.5 days post-transfer. Hill diversity analysis showed the lowest receptor diversity and most pronounced clonal expansion in CD21^lo cells, while ASCs exhibited higher clonal dominance despite greater richness, indicating that CD21^lo cells are transitional effector precursors under strict clonal selection in ASCs. κ/λ light-chain ratio analysis and shared heavy-chain gene usage (e.g., Ighv1–82, Ighv1–80) further supported early selection in CD21^lo cells. Phylogenetic analysis revealed that the most expanded ASC clones belonged to the same evolutionary branch as the 564Igi autoreactive heavy chain, with transitional BCR sequences in CD21^lo cells bridging toward ASCs, confirming that naïve FoB progressively acquire autoreactivity through clonal selection and receptor editing, with CD21^lo cells representing a critical intermediate stage.

Figure 5. BCR repertoire sequencing reveals the developmental trajectory of follicular B cells toward autoreactivity

Using CD21-knockout and human CD21-transgenic mouse models, mCd21-KO donor B cells showed increased early division but failed to reach terminal divisions, indicating that CD21 loss does not affect initial activation but blocks proliferation progression. Blocking the C3d-binding site on hCD21⁺ B cells with a monoclonal antibody reduced terminal division, ASC generation, and red pulp ASC accumulation while preserving the CD21⁺ population and decreasing CD21^loCD23^- cells. In vitro and in vivo experiments confirmed that ligand (C3d/iC3b) binding triggers rapid CD21 internalization, which is completely prevented by antibody blockade or C3 deficiency. Thus, ligand engagement drives CD21 downregulation, and functional blockade inhibits this process and subsequent proliferation, ultimately reducing ASC formation.

Figure 6. CD21 downregulation during proliferation is suppressed by functional receptor blockade

Serial transfer of CD21-tdTomato reporter cells showed that some CD21^lo cells formed in primary recipients partially restored CD21 expression in secondary 564Igi hosts, demonstrating that the CD21^lo state is not irreversible but depends on continuous ligand presence in the microenvironment. Delayed in vitro blockade experiments revealed that CD21 downregulation stops immediately upon antibody addition and correlates with ligand exposure duration. In vivo delayed blockade starting day 3 post-transfer maintained CD21 levels at the day-3 state and produced bimodal CD21 expression in terminally divided cells. Notably, donor B cells at day 3 were mostly undivided, whereas by day 7 both blocked and unblocked groups reached terminal divisions. These results establish that CD21 downregulation precedes proliferation, represents an early activation event, and full downregulation serves as a critical fate checkpoint for autoreactive B-cell differentiation into ASCs; incomplete downregulation allows cells to revert and alter their differentiation trajectory.

Figure 7. CD21 downregulation requires sustained ligand binding and occurs before B-cell proliferation

Through integrated analyses of cell fate mapping, clonal evolution, and molecular regulation, this study comprehensively elucidates the mechanism of extrafollicular differentiation of autoreactive B cells. It clarifies the origin, differentiation potential, and regulatory mechanisms of CD21^lo cells and demonstrates the therapeutic value of targeting CD21 and TLR7—acting at the initiation and maintenance phases of differentiation, respectively. Dual targeting offers a more precise strategy to suppress autoreactive B-cell activation, providing a novel "dual-target" intervention approach for SLE and mechanistic insight into similar B-cell subsets in infection and aging.

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