Foreign antigen delivered to CLL B cells via membrane IgD leads to a distinct antigen processing pathway that allows antigen-specific T cell activation that is superior to membrane IgM delivery Free

Effective T-cell-dependent immune responses are initiated by the uptake of antigen by antigen presenting cells (APCs) that then cleave the antigen into fragments and present these, in association with major histocompatibility molecules, to a T cell with antigen receptors of appropriate specificity. In the normal setting, the most relevant APCs are dendritic cells, macrophages, and B lymphocytes, with the former capturing antigens non-specifically and the latter specifically via surface membrane immunoglobulin (smIg) with appropriate antigen specificity. Notably, in patients with CLL, the outcome of the antigen-presentation process, and hence the activation of an effective immune response, is disrupted due to defective immune synapse formation between the APC and the antigen-specific T cell. Although this process has been studied in detail in CLL, the mechanisms of antigen capture and delivery to endosomes that precede actual antigen presentation have not been as precisely defined. We have studied the latter, focusing on the consequences of antigen captured and delivered by smIgM versus smIgD.

We engineered two murine monoclonal antibodies (mAbs) that bind human IgM or IgD with comparable affinities to express the human IgG4 constant region to which the SARS-CoV-2 receptor-binding domain (RBD) was (or not) bound: r-αIgM, r-αIgM-RBD, r-αIgD, r-αIgD-RBD. These mAbs were used to target the FACS sorted, proliferative fraction (PF; CD19⁺CXCR4^DimCD5^Bright) of CLL cells that is enriched in antigen-processing machinery. PF cells were exposed to antigen for 1h before incubating with autologous T cells for 48h. Antigen-specific T-cell activation was determined by upregulation of CD69, CD134, CD137 and production of IFNγ, IL-4. Confocal microscopy was used to track antigen internalization and trafficking.

Efficiency of internalization of smIgM and smIgD was analyzed after 1h exposure to the mAbs in 10 CLL and 10 healthy donor (HD) samples. Despite detecting greater expression, defined by MFI, of smIgM than smIgD on PF cells at 4^oC (P=0.0141), there were greater amounts of intracellular IgD than IgM (P=0.0402) at 37^oC; this was also found with B cells from HD(P=0.0302).

Antigen delivery via r-αIgD-RBD, but not r-αIgM-RBD, significantly enhanced T-cell activation of 16 patients (8 U-CLL and 8 M-CLL), especially among CD8⁺ T cells (CD8⁺ P=0.0445; CD8⁺CD134⁺0.0385). Compared to their respective controls, r-αIgD-RBD induced higher expression of CD4⁺CD69⁺ (P=0.0012), CD4⁺CD134⁺(P=0.0014), CD8⁺CD137⁺ (P=0.0004), and CD8⁺IL-4⁺ (P=0.0012). In contrast, B cells from 19 HD responded to both isotypes, though r-αIgD-RBD consistently triggered stronger T-cell activation than r-αIgM-RBD. Thus, CLL cells show an IgD-specific advantage and HD B cells also favor IgD, but less exclusively.

We next examined whether baseline or post-stimulation HLA-DR expression on CLL cells correlated with their capacity to present antigen to T cells. Using samples from 10 M-CLL and 10 U-CLL patients, at starting point, U-CLL samples exhibited the highest overall HLA-DR expression. Next, we measured HLA-DR levels following a 1h incubation with smIg-binding mAbs. HLA-DR was significantly upregulated in PF cells, with an increase observed after r-IgD-RBD stimulation compared to control, r-αIgD, and r-αIgM-RBD (P=0.0061;P= 0.0007; P=0.0019 ). A similar trend was observed for HLA-ABC expression.

To assess whether BCR isotype influences intracellular antigen handling, we performed confocal microscopy using fluorophore-labeled r-αIgM and r-αIgD. Notably, the two isotypes trafficked to distinct intracellular compartments, suggesting that IgM and IgD follow divergent processing pathways that could influence the distinct capacities to activate T cells.

We believe that these data indicate, for the first time, that smIgD bound antigen is more efficiently taken up into distinct endosomal compartments and presented in a way that leads to superior activation of antigen-specific T cells than IgM. This is the case for people with CLL and healthy individuals. Exploiting this IgD-driven pathway may allow development and expansion of antigen-specific T cells that could support subsequent B and T cell effector responses against pathogens and tumor antigens, particularly in an immunocompromised setting such as CLL.