Delivery of Foreign Antigen Selectively to Membrane IgD on CLL and Normal B Cells Leads to Superior T-Cell Activation: A Possible Approach to Restore/Enhance T-Cell Immunity and T-Cell Help

TCR mediated T cell activation is less robust in patients with CLL than healthy controls (HC), contributing to an immune deficiency that impairs quality and longevity of life. In HCs, T cell activation begins with myeloid or B cells acquiring and processing antigen and then presenting antigenic peptide-MHCII complexes to T cells. Activated B cells are more effective antigen-presenters than resting cells. In CLL, the process is defective, particularly for immune synapse formation with T cells.

Most CLL cells and unswitched normal B cells express surface membrane (sm) IgM and IgD. Since both molecules bear the same antigen binding site, each initiates the antigen processing and presentation activity. However, documentation that both do this in the same way is lacking. We investigated if delivery of foreign antigen to CLL and normal B cells selectively by sm IgM or by smIgD influences antigen delivery, processing, and presentation, and subsequent T-cell activation.

We recombinantly modified two murine mAbs of comparable affinities for IgM or for IgD so they bear human IgG4 constant regions to obviate Fc receptor interaction (r-αIgM, r-αIgD), and so they carry (or not) the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (r-αIgM-RBD, r-αIgD-RBD). Using the CXCR4^DimCD5^Bright, proliferative fraction (PF) of CLL cells, which is enriched in antigen presentation molecules and capacities, we assessed the efficiency of internalization of smIg and of associated RBD, and the ability of preferentially targeted B cells to present RBD peptides to T cells; we used flow cytometry to measure induction of sm CD69, CD134, CD137, and intracellular (ic) IFNγ and IL-4. For confocal microscopy, we used pHrodo labelled r-αIgM (Deep Red) and r-αIgD (Green) to determine if IgM and IgD deposit antigen in distinct ic compartments.

Exposing CLL cells to the mAbs at 4^oC, showed although the PF has significantly more smIgM than smIgD (P=0.0141), preferential engagement of sm IgD or IgM at 37^oC led to significantly higher levels of ic IgD than IgM (P=0.0402). Similar data were found for normal B cells (P=0.0302).

Next, we tested if delivery of RBD selectively to smIgM or smIgD led to differences in T cell activation based on increases in activation antigen and Th1 and Th2 cytokine levels. Consistent with previous observations, incubation of resting T cells with PF cells led to increases in T cell numbers (Total: P=0.0370; CD4: P=0.0322; CD8: P=0.0420). So this value was used as the baseline for all comparisons. Exposure of T+PF to r-αIgM or r-αIgD did not significantly change the 5 readouts (not shown). Notably, engagement of smIgM on PF cells by r-αIgM-RBD never led to increases in T cell numbers, T cell activation antigen expression, or cytokine production over baseline. In contrast, engagement of smIgD with r-αIgD-RBD increased T cell numbers and in 3 T cell readouts over the same baseline (CD8⁺P=0.0445; CD4⁺CD134⁺P= 0.368; CD8⁺CD134⁺P=0.0385).

Additionally, significant differences were found after comparing r-αIgD vs r-αIgD-RBD for multiple readouts (CD3⁺P=0.0087; CD4⁺P=0.0161; CD8⁺P=0.0029; CD4⁺CD69⁺P=0.0013; CD4⁺CD134⁺P=0.0010; CD4⁺CD137⁺P=0.0172; CD4⁺IL-4⁺P=0.0334; CD8⁺CD69⁺P=0.0020; CD8⁺CD134⁺P=0.0008; CD8⁺CD137⁺P=0.0006; CD8⁺IFNg⁺P=0.0008; CD8⁺IL-4⁺P=0.0012). This was never the case for r-αIgM vs r-αIgM-RBD.

Finally, T cell activation was significantly greater when comparing r-αIgD-RBD to r-αIgM-RBD (CD3⁺P=0.0070; CD4⁺P=0.0045; CD8⁺P=0.0062; CD4⁺CD69⁺P=0.0012; CD4⁺CD134⁺P=0.0014; CD4⁺CD137⁺P=0.0278; CD8⁺CD69⁺P=0.0042; CD8⁺CD134⁺P=0.0004; CD8⁺CD137⁺P=0.0004; CD8⁺IFNg⁺P=0.0157; CD8⁺IL-4⁺P=0.0206).

Thus, antigen delivered by smIgD is more stimulatory to T cells than if delivered by smIgM, and these activating events are occurring on RBD-specific T cells. Similar results were observed in normal B cells.

To determine if the ic “handling” of antigens differed based on the route of entry, we incubated CLL cells with r-αIgM and r-αIgD, each labeled with a distinct fluorophore that emits at acid pH. This showed that r-αIgM and r-αIgD entered distinct compartments.

CLL B cells activated through IgD more effectively induce T-cell activation than when activated through IgM, suggesting the presence of a distinct B cell activation pathway in CLL and normal B cells. Using this immunostimulatory pathway might allow enhanced immune responses to foreign and potentially tumor antigens.

Allen:C4 Therapeutics: Current holder of stock options in a privately-held company; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Ghia:AstraZeneca: Consultancy; Janssen: Consultancy; BeiGene: Consultancy; Bristol Myers Squibb: Consultancy; Roche: Consultancy; Merck Sharp & Dohme Corp.: Consultancy; Loxo@Lilly: Consultancy; AbbVie: Consultancy. Chiorazzi:Abbvie: Consultancy.