Myelin protein zero is naturally processed in the B cells of monoclonal gammopathy of undetermined significance of immunoglobulin M isotype: aberrant triggering of a patient's T cells

Abstract

Background: Monoclonal gammopathy of undetermined significance of immunoglobulin M isotype is a condition with clonally expanded B cells, recently suggested to have an infectious origin. This monoclonal gammopathy is frequently associated with polyneuropathy and antibodies against myelin protein zero, whereas the role of the T cells remains largely unknown. We analyzed protein zero-specific B cells, as antigen-presenting cells, and their capacity to activate T helper cells.

Design and methods: We used a well-characterized monoclonal gammopathy of undetermined significance-derived B-cell line, TJ2, expressing anti-protein zero immunoglobulin M. The ability of TJ2 cells to bind, endocytose, process, and present protein zero was investigated by receptor-clustering and immunofluorescence. The activation of protein zero-specific autologous T cells was studied by measuring interleukin-2 and interferon-gamma with flow cytometry, immunobeads, and enzyme-linked immunospot assays.

Results: Surface-receptor clustering and endocytosis of receptor-ligand (immunoglobulin M/protein zero) complexes were pronounced after exposure to protein zero. Naturally processed or synthetic protein zero peptide (194-208)-pulsed TJ2 cells significantly induced interleukin-2 secretion from autologous T cells compared to control antigen-pulsed cells (P<0.001). The numbers of interferon-gamma-producing T helper cells, including CD4(+)/CD8(+) cells, were also significantly increased (P=0.0152). Affinity-isolated naturally processed myelin peptides were potent interferon-gamma stimulators for autologous peripheral blood mononuclear cells, but not for control peripheral blood mononuclear cells.

Conclusions: We show for the first time that myelin protein zero is naturally processed in B cells from monoclonal gammopathy of undetermined significance of immunoglobulin M isotype, acting as aberrant antigen-presenting cells in activation of a patient's T helper cells. Our findings cast new light on the important role of autoreactive protein zero-specific B cells in the induction of the pathogenic T-cell responses found in nerve lesions of patients with monoclonal gammopathy of undetermined significance with peripheral neuropathy.

Figure 1.

Proposed model and experimental design of the current study. Myelin protein zero (P0) is a glycoprotein present in the membrane of Schwann cells in the peripheral nervous system. The crystal structure of the extracellular domain shown is from rat and is reprinted with kind permission from Dr Petri Kursula (Oulu, Finland). We hypothesize that P0 is exposed to the CD5⁺ P0 specific MGUS B cells, perhaps due to a nerve injury. (1) P0 binds to and induces clustering of BCR on the myelin-reactive MGUS B cells. (2) The BCR-antigen complex is endocytosed, processed and P0 peptides are loaded onto MHCII molecules. (3) P0 peptides are presented to and activate T cells. (4) The activated T cell starts producing IFNγ and IL2. The numbers shown in the Figure also correspond to the different experiments presented in the Results section. (1) Receptor clustering of surface IgM and co-localization of P0. (2) Co-localization of P0 and late endosomal/lysosomal marker LAMP-2. (3) Receptor clustering of MHCII and co-localization of P0. (4) Activation of autologous T cells by P0 incubated MGUS B cells as measured by intracellular IFNγ and secreted IL2. The ability of naturally processed P0 peptides to activate autologous T cells with the secretion of IFNγ was also investigated.

Figure 2.

Myelin/P0 binding to surface IgM, presence of P0 in the late endosome/lysosome compartment and presentation of myelin/P0 peptides in MHCII on the P0 specific MGUS B cell line TJ2. (A) Immunofluorescence images of myelin (green) co-clustered with surface IgM (red) on TJ2 cells after 5 h and 24 h. Yellow regions in the overlay images indicate co-localization. (B) Myelin binding to surface IgM was seen on a majority of TJ2 cells at all investigated time points (2.5 h, 5 h, 8 h, 16 h and 24 h). (C) Similar results were seen for P0 and surface IgM. (D) Images show uptake of P0 (green) in TJ2 cells after 0 h, 20 min and 24 h. Endosomal marker, LAMP-2, is shown in red. Yellow regions in the overlay images indicate co-localization, also shown by arrows. (E) Kinetic analysis of BCR-mediated P0 uptake in TJ2 cells. Percent P0-positive and LAMP-2- positive cells are shown after 0 h, 20 min, 40 min, 60 min, 4 h, and 24 h. Results from two independent experiments are shown (white and gray bars). For each time point, at least 300 LAMP-2 positive TJ2 cells were analyzed for the presence of P0. (F) The simultaneous uptake of P0 (green) and dextran (red) in the lysosomal compartment of TJ2 cells was investigated after 24 h. Yellow regions in the overlay images indicate co-localization. (G) Immunofluorescence images of myelin (green) co-clustered with HLAII (red) after 5 h and 24 h respectively. Yellow regions in the overlay images indicate co-localization. (H) Percentage of TJ2 cells exhibiting co-clustering of myelin with HLAII is shown for 2.5 h, 5 h, 8 h, 16 h, and 24 h. (I) Receptor clustering of MHCII and P0. (J) Negative control showing clustering of myelin, but no co-localization with control protein CD46.

Figure 3.

Naturally processed myelin peptides induce IFNγ secretion from PN-MGUS patient’s PBMC, but not from control PBMC. Affinity purified and FPLC separated myelin peptide fractions # 1 to 50 were analyzed by ELISPOT for the ability to stimulate IFNγ secretion in (A) autologous PBMC from a PN-MGUS patient and (B) control PBMC from a healthy donor. The spontaneous background was subtracted and mean values from triplicate samples are shown (left axis). Peptide fraction # 24–28 induced an increased secretion of IFNγ in autologous PBMC, but not in control PBMC, and fraction # 28 generated the highest IFNγ response. Peptide fraction # 24–28 coincides with the OD₂₈₀ absorbance peak from the FPLC separation (line) shown on the right axis.

Figure 4.

The PN-MGUS derived B-cell line TJ2 is capable of P0-specific T-cell activation. (A) Experimental design of the T-cell activation assay. CD19⁺ B cells, CD56⁺ NK cells and IFNγ-secreting, presumably EBV reactive cells, were eliminated from the PN-MGUS patient’s PBMC by magnetic separation. PBMC were subsequently co-cultured with antigen-pulsed TJ2 cells for 16 h. Intracellular IFNγ and secreted IL2 were measured after 6 h and 7 days, respectively. (B) Significantly increased levels of secreted IL2 in the 7-day cell cultures were seen after stimulation with P0 pulsed TJ2 cells (P<0.001), compared to control antigen, KLH-pulsed TJ2 cells. The results are mean values (±SD) of six separate experimental cultures. More than 30,000 cells from each culture were analyzed in FACS. (C, left panel) An increased level of intracellular IFNγ was seen in T lymphocytes after incubation with P0-primed TJ2 cells as compared to KLH (control antigen)-primed TJ2 cells. This increase was mainly seen in the CD4⁺ T lymphocyte population although a slight increase was also noted in the CD8⁺ T lymphocyte population. (C, right panel) T lymphoblasts also exhibited an increased accumulation of intracellular IFNγ after P0-primed TJ2 cell stimulation as compared to KLH (control antigen)-primed TJ2 cells. The IFNγ increase was seen in the CD4⁺ T blasts and in CD4⁺/CD8⁺ T blasts, but could not be detected in CD8⁺ T blasts. The P0 versus. KLH populations in C (both panels) were statistically analyzed using Fisher’s exact test and showed a significantly different response (P=0.0152).

Figure 5.

Crystal structure of the P0 extracellular domain from rat according to Shapiro et al. and Kursula P. Glycosylation site, Asn93, is shown as spheres and the HLA-DRB1*0701 predicted binding peptide amino acids 107–115 in black.