Cerebrospinal fluid B cells from multiple sclerosis patients are subject to normal germinal center selection

Abstract

Previous findings from our laboratory demonstrated that some clonally expanded cerebrospinal fluid (CSF) B cells from MS patients exhibit diminished mutation targeting patterns in comparison to typical B cells selected in the context of germinal centers (GCs). In order to determine whether the overall CSF B cell repertoires adhered to mutation patterns typical of GC-selected B cells, we analyzed the immunoglobulin repertoires from CSF B cells of 8 MS patients for mutation characteristics typical of GC-derived B cells. Mutation targeting was preserved. Thus, clonal expansion of some CSF B cells may occur independently of GC, but the CSF B cell pool is governed by typical GC selection. Interestingly, the heavy chain CDR3's of CSF B cells from MS patients had a net acidic charge, similar to GC-derived B cells, but a tendency towards longer CDR3's, consistent with autoreactive B cells. How these findings may support current hypotheses regarding the origin of CSF B cells is discussed.

Figure 1. Frequency of B cell subsets in PB and CSF of OND and MS patients

Cells isolated from PB and CSF of MS patients (n=10) and OND patients (n=7) were analyzed by flow cytometry using anti-CD19 and anti-CD27 mAbs. Total CD19⁺ cells (defined as a percentage of total lymphocytes) were analyzed for expression of CD27 to determine naïve (CD27⁻) and memory (CD27⁺) B cell subsets. Average frequencies (±standard error) of each subset are as indicated below the x-axis.

Figure 2. Heavy and Kappa Variable and Junction Chain usage in HCPB, OND CSF, and MS CSF B cells

Antibody rearrangements were analyzed from single CSF B cells as described in Materials and Methods from MS one CIS and two OND patients as indicated. (A) Heavy(H) and Kappa(κ), Variable(V) and Junction(J) segment usage in HCPB and CSF CD19⁺ B cells are presented as a percentage of total sequences obtained from each patient sample. Center oval “n” indicates number of productive sequences obtained and analyzed. H chain V- and J-segment data is summarized on the left, κ chain V- and J-segment data on the right. (B) H and κ chain, V and J segment usage in CSF B cells from MS patients. Data is presented as indicated in Panel A.

Figure 2. Heavy and Kappa Variable and Junction Chain usage in HCPB, OND CSF, and MS CSF B cells

Antibody rearrangements were analyzed from single CSF B cells as described in Materials and Methods from MS one CIS and two OND patients as indicated. (A) Heavy(H) and Kappa(κ), Variable(V) and Junction(J) segment usage in HCPB and CSF CD19⁺ B cells are presented as a percentage of total sequences obtained from each patient sample. Center oval “n” indicates number of productive sequences obtained and analyzed. H chain V- and J-segment data is summarized on the left, κ chain V- and J-segment data on the right. (B) H and κ chain, V and J segment usage in CSF B cells from MS patients. Data is presented as indicated in Panel A.

Figure 3. Heavy Chain CDR3 amino acid length

CDR3 lengths from the heavy chains of CSF B cells from 8 MS, one CIS and two OND patients and HCPB were determined and separated into 4 groups of varying length. Average CDR3 lengths are listed above (+/− standard error of the mean, * indicates significance of p•.05, mann-whitney t-test).

Figure 4. Heavy Chain CDR3 total amino acid charge

Amino acid sequence of heavy chain CDR3s of the CSF B cells isolated from MS, CIS and OND patients were analyzed to determine total CDR3 charge at pH 7. Charges are grouped from each patient repertoire and listed as a proportion of total sequences that were analyzed from each patient. Center circle “n” indicates number of sequences included in the analysis.