Antigen reactivity profile of the leukemic clonotypic B cell receptor Immunoglobulins

Chronic lymphocytic leukemia (CLL) and Splenic marginal zone lymphoma (SMZL) are types of cancer that start from white blood cells, called B lymphocytes or B cells.

A key player in providing external activatory stimuli to leukemic cells is the B cell receptor (BcR), a transmembrane receptor protein unique to each B cell consisting of a membrane –bound immunoglobulin (IG) molecule associated with the Iga and Igb co-receptors. The BcR IG binds to antigens (i.e. molecules capable of inducing an immune response) and thereby initiates an intracellular signaling cascade resulting in cell activation. Of note, inhibition of BcR IG-mediated signaling has emerged as a highly efficacious treatment mode for these tumors, highlighting their dependence on extrinsic triggering. Nevertheless, open issues abound regarding the precise role of the BcR IG in lymphomagenesis, particularly regarding the range of interactions with antigens and their functional impact on clonal behaviour and patient outcome.

IGs are Υ-shaped molecules that consist of four polypeptide chains, two identical heavy chains and two identical light chains, connected by disulfide bonds. IGs are able to recognize a great number of antigens in order to protect the organism from various threats. This ability rest to the vast IG diversity that results from the combined effect of specific mechanisms that function during B cell development. Amongst them, the random assembly of one each of multiple distinct genes (IGHV, IGHD, IGHJ for the IG heavy chain and IGKV and IGKJ or IGLV and IGLJ genes for the kappa and lambda light chain respectively) for the synthesis of an IG molecule leads to a huge variety of combinations. Another mechanism taking place in later phases of B cell ontogeny, subsequent to antigen encounter is somatic hypermutation (SHM) that leads to the introduction of point mutations into the rearranged IG genes. Some of the altered IGs generated by SHM possess enhanced ability to recognize and bind the specific antigen and, thus, are preferentially stimulated by the antigen to survive and proliferate. Overall, SHM of IG genes is a fundamental process for the creation of a diverse and effective B-cell repertoire.

Within IGpath project we investigated the antigen binding profile of the clonotypic BcR IG of CLL and SMZL cases in order to gain insight into the type of antigenic stimuli that could lead to BcR triggering and immune signaling and may be involved in disease ontogeny.

Chronic lymphocytic leukemia

We focused in particular on a specific subset of CLL cases that is defined by the expression of IGHV4–34 BcR IGs that have been targeted by SHM (CLL stereotyped subset #201). The human IGHV4-34 gene is known to encode autoreactive antibodies. Furthermore, increased serum concentration of IGHV4-34 antibodies is observed following infections with herpesviruses such as Epstein–Barr virus (EBV), cytomegalovirus (CMV) and Mycoplasma pneumoniae. Subset #201 is also noteworthy owing to the high frequency of novel N-glycosylation motifs created by SHM within the VH FR3. N-linked glycosylation is a widespread post-translational modification of proteins, including IGs and, importantly, it can modulate IG affinity towards antigen.

That said, until recently nothing was known about the antigen reactivity of subset #201 BcR IG and whether/how it could be affected through SHM-induced changes of N-linked glycosylation. We investigated this issue taking advantage from the recombinant DNA technology: we produced the clonotypic BcR IG of subset #201 cases as soluble monoclonal antibodies (mAbs) and studied thoroughly their antigen reactivity profile. In particularly we generated mAbs in either the authentic SHM state (i.e. mAbs that are identical with the BcR IGs expressed on the surface of leukemic B cells) or after reverting specific SHMs that altered N-glycosylation sites using molecular biology methodology (i.e. site-directed mutagenesis).

In contrast to other CLL mAbs that have been previously shown to be polyreactive (i.e. antibodies that bind with low affinity to a variety of structurally unrelated antigens), we found that subset #201 mAbs did not bind to a series of self-and foreign antigens but bind selectively to viable human lymphoblastoid and epithelial cell line cells.  Importantly, this profile was critically affected by several mutations introduced to the mAbs by the SHM process. Altogether, these results emphasize the importance of SHM in shaping the distinct (auto)antigenic recognition profile of CLL mAbs.

Splenic marginal zone lymphoma

We investigated the antigen binding profile of the clonotypic BcR immunoglobulin (IG) of SMZL cases in order to gain insight into the type of antigenic stimuli that could lead to BcR triggering and immune signaling. The clonotypic BcR IG of SMZL patients were produced as soluble mAbs and were tested for the recognition of a variety of antigens. We found that SMZL BcR IGs recognize a wide range of autoantigens (native DNA, nuclear Hep-2 cell extract, actin, myosin, thyroglobulin, β-amyloid, carbonic anhydrase, F(ab’)2) and the non-self hapten trinitrophenyl, indicating that SMZL B cell clones may arise from polyreactive B cells, likely resident in the normal splenic marginal zone.