By utilizing a combination of different bioinformatic prediction models, including predictions for HLA affinity and cathepsin processing trained using publicly available datasets, we investigated the potential immunogenicity of BCR IGHV repertoires in patients with MS, compared to patients with other inflammatory neurological diseases (OIND). Key findings were high predicted affinities of idiotopes to disease-associated HLA-DRB1*15:01, and a generally high predicted affinity for
complementarity determining region (CDR) 3 derived idiotopes for HLA-DR and DQ in general. A similar variability was not found for HLA class I molecules. Additionally, we identified areas in the framework region (FW) with higher affinity and that these were associated with high probability of cleavage by cysteine cathepsins. Different IGHV families were predicted to differential degradation, due to structural differences. IGHV4 in particular was predicted vulnerable to cathepsin S. Average rarity of TCEM in the BCRs were found to be higher in MS patients than in OIND patients in some IGHV positions. By combining these outputs, we found that 42% of highly transcribed IGHV sequences in MS patients have at least one idiotope with high predicted HLA-DR affinity, high probability of cathepsin cleavage and contain rare TCEM.
Figure 7. Graphical summary – Paper I
“Proportion of immunoglobulin heavy chain variable (IGVH) fragments that were predicted to have the potential to engage in idiotope-driven T–B cell collaboration. (…)” Each mode of prediction is highlighted by
T-B collaboration figure. “The upper panels show the proportion of fragments at each complementarity determining region 3 (CDR3) relative position that fulfils each criterion. The lower panel shows the proportion
that fulfils all criteria. Nearly all fragments inhabiting all three features occur in the CDR3 region (yellow shading).” Modified figure and text from Paper I – Høglund et al. (2017) Front. Immunol. 8:1255 (93).
CC-BY 4.0 https://creativecommons.org/licenses/by/4.0/)
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7.2 Paper II
In this paper we sought to validate the cathepsin predictions made in paper I. This was done by first assessing the accuracy of cathepsin cleavage prediction models17 by using monomeric CNS proteins that also are known or potential substrates for cathepsins. Cathepsins S, L or B were mixed individually with substrates and sampled after several time points under similar conditions as the dataset used for training the models. We found that higher predicted probability of cleavage was clearly associated with higher occurrence of cleavages. Secondly, we assessed whether the predictions also were as accurate for tetrameric IgGs, and found accuracy was reduced. We hypothesized this reduction may be a methodological issue, as the nanoliquid chromatography mass spectrometry (nLCMS) used may not detect all cleavages in the larger IgGs. Finally, we described in detail the IgG degradation patterns by the three cathepsins, identifying a varying activity across different acidities, and confirmed that these cathepsins degrade variable regions differently, while simultaneously degrading the constant regions similarly across six different IgGs.
Figure 8. Graphical summary – Paper II
Monoclonal IgGs and recombinant CSF proteins were subject to in silico and in vitro processing to predict and detect cleavage activity by cysteine cathepsins. Predicted results were compared to actual mass-spectrometry
detected cleavages, and cleavages were mapped within the proteins. (Figure from Paper II - Høglund et al (2019), Int. J. Mol. Sci., 20(19), 4843. (174). CC-BY 4.0. (https://creativecommons.org/licenses/by/4.0/)
17 Same model as used in paper I, trained on publicly available datasets.
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7.3 Paper III
We utilized the predictive models described in papers I and II, including predicted HLA-DR affinities and cathepsin cleavage, to guide selection of potentially immunogenic idiotopes and negative controls from the CSF IGHV repertoire described in paper I. Nine of the patients had available
cryopreserved PBMC, sampled simultaneously as the lumbar puncture. The predicted idiotope peptides were synthesized and used to identify T cells specific for these in a flow cytometry-based activation assay, detecting expression of CD154 (CD40L) as the designated activation marker after stimulating the PBMC for 12 hours. We identified idiotope-specific memory T cells, frequently expressing C-C chemokine receptor (CCR) 6, in all patients assessed, in some patients we detected multiple robust responses. The idiotope peptides generating a response were derived from CDR3 related peptides, predicted to be released by cathepsins S or B expressed in B cells, and were associated with mutations that could influence affinity or cathepsin cleavage. The findings indicate that these MS patients all have idiotope-specific memory T cells, capable of entering the CNS.
Figure 9 Graphical summary – Paper III
“Flow cytometry based idiotope-specific T cell activation assay. A) IGHV amino acid sequences from nine MS patients were run through predictive models to identify likely antigenic idiotopes based on HLA class II affinity, cathepsin cleavage and frequency classification (FC) of T cell exposed motifs (TCEM). B) 500,000 PBMC were stimulated with synthetic idiotope peptides predicted to be stimulatory, tolerogenic or inert as well as positive and negative controls for 12 hours in presence of anti-CD40 antibodies. B cells or other professional APCs with
idiotope peptides bound to their HLA class II receptor may activate cognate CD4+ T cells. C) CD4+CD45RO+ memory T cells specifically activated by idiotope peptides were detected by surface expression of CD154, upregulated upon TCR stimulation. The example shows a detected memory T cell response to an idiotope
peptide.” Figure and text from Paper III - Høglund et al., manuscript (2020)
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7.4 Paper IV
We compared the B cell populations in blood and CSF of patients treated with either dimethyl fumarate (DMF) or other alternatives (glatiramer acetate, beta-interferons or no therapy) in a cross-sectional, explorative study. We found a reduced population of memory B cells in the blood during DMF therapy, thereby confirming other studies finding the same, and further found the reduction correlated with treatment duration. In CSF the absolute count of mononuclear cells was significantly lower in DMF treated than the others, and there was also a disproportionate decrease in plasmablasts.
The study thus supports a potential B cell depleting mechanism of DMF but does not answer whether the effect of the drug is mainly on circulating or intrathecal B cells. As the study was explorative with a limited number of included patients, further studies to confirm our findings are necessary.
Figure 10: Graphical summary – Paper IV
Changes detected with flow cytometry of CSF and blood B cells in DMF-treated MS patients compared to control group. Figure also illustrates differences in blood and CSF concentration of DMF metabolite monomethyl fumarate (MMF). A modified figure was first presented on the poster P1214 “Dimethyl fumarate
alters the composition of B cells in the blood and cerebrospinal fluid of patients with multiple sclerosis”, (Høglund et al.) presented at the ECTRIMS congress, Berlin, Oct 12th, 2018.
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