PMC5221476_01
PMC5221476_02
Disease course. Shown is a summary of the patient's disease course with relapses and disability progression represented by the blue line. CSF findings in 1997 and 2013 are shown at the top of the graph; thoracic spinal cord lesion count represented by the black dots; rituximab treatment cycles are indicated by the red arrows. Weekly IFN-ss1a was given from 1999 to 2004; three-times weekly IFN-ss1a was given from 2004 to 2006, followed by treatment with rituximab. Secondary progression without relapses became apparent starting 2009.
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In 2013, brain MRI scans showed multiple T2/supratentorial fluid attenuated inversion recovery (FLAIR) hyperintense lesions with a predominantly periventricular distribution pattern typical of MS (Supplementary Figure 1A). Prior to starting rituximab in 2006, an increasing lesion count was seen and a nearly stable lesion count thereafter (Fig. 2A), with one new subpial cerebellar lesion first noted in 2013 (Supplementary Figure 1B and C). Figure 1D and E), plus development of several new thoracic cord lesions (Supplementary Figure 1D, F and 2C, D). Furthermore, brain and cervical cord atrophy rates indicated increased tissue volume loss compared to rates in normal aging, despite treatment with rituximab (Fig 2B, E and F).2, 3 We searched for cerebral meningeal infiltrates in this patient, but no definite enhancement could be detected on post-gadolinium T2-weighted FLAIR images (not shown).
Longitudinal MRI examinations reveal increase in lesion load and lesion volume and decrease in parenchymal volume. Shown are brain lesion load (# of T2 lesions, (A), and percentage brain volume change (% relative to baseline, (B); cervical, thoracic spinal cord T2 lesion load (# of T2 lesions, C), volume (mm3, D) and cervical cord area longitudinally (mm2, T2W contrast, E) and cross-sectionally for total cord area, white matter area and gray matter area, each at C2-3 level (PSIR, mm2, (F). Plots (open circles; closed circles; triangles) in the graphs represent the respective time point of detection of the illustrated findings by MRI. The gray vertical dashed line indicates the start of rituximab therapy. The horizontal dashed line in panel E indicates the atrophy rate for normal aging. The dashed lines in panel F indicate lower level of normal. In general, CSP lesion load remains stable, while CSP lesion volume and TSP lesion load and volume increase over time. A temporary increase of TSP lesion volume in 2000 was due to a relapse with transverse myelitis at level T1/2 (see Fig. S1D), followed by clinical remission and a reduced lesion volume. Prior to starting rituximab treatment, a doubling of brain lesions occurred between 1997 and 2006. Only a slight increase in brain lesion number was observed after starting rituximab. MRI, magnetic resonance imaging; T2W, T2-weighted; PSIR, phase-sensitive inversion recovery MRI sequence; CSP, cervical spinal cord; TSP, thoracic spinal cord.
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Prior to starting rituximab in 2006, an increasing lesion count was seen and a nearly stable lesion count thereafter (Fig. 2A), with one new subpial cerebellar lesion first noted in 2013 (Supplementary Figure 1B and C). Figure 1D and E), plus development of several new thoracic cord lesions (Supplementary Figure 1D, F and 2C, D). Furthermore, brain and cervical cord atrophy rates indicated increased tissue volume loss compared to rates in normal aging, despite treatment with rituximab (Fig 2B, E and F).2, 3 We searched for cerebral meningeal infiltrates in this patient, but no definite enhancement could be detected on post-gadolinium T2-weighted FLAIR images (not shown).
Clonally related B-cell receptors (BCR) in PB (2013, 2014) and CSF (2013) indicate temporal persistence of B cells in blood and CSF. In MS patients not treated with anti-CD20 therapies, hundreds of different clusters of clonally related B-cell receptor/immunoglobulin heavy chain variable region (Ig-VH) transcripts are normally found in peripheral blood. In this long-term B cell depleted patient, only very few BCR IgM- or IgG-heavy VH sequences were found in PB or CSF; no IgM/G-VH could be amplified from the November 2011 PB sample. In this figure, each dot (node) represents a group (cluster) of clonally related Ig-VH transcripts (clusters 1-41); germline immunoglobulin IGHV/IGHJ usage and most common CDR3 per cluster of related B cells are shown in the table. Blue nodes indicate IgM-VH or IgG-VH sequences belonging to B cells that were present in CSF in November 2013 and/or also present in PB in November 2013 or October 2014; red nodes indicate BCRs found in PB at either time point. Blue lines in the graph or shades in the table indicate related B cells found in CSF and PB at both time points; red lines in the graph or shades in the table indicate related B cells in PB only, present at either/both time points; time (T/gray circle) markers in table indicate B cells present across both time points. Overall, Ig-VH obtained from peripheral blood or CSF by polymerase chain reaction suggests persistence of recirculating memory B cells, or long-lived plasma cells, unaffected by rituximab despite long-term anti-CD20 treatment. CSF, cerebrospinal fluid; PB, peripheral blood; IgG, immunoglobulin G; IgM, immunoglobulin M; IGHV, immunoglobulin heavy chain variable; IGHJ, immunoglobulin heavy chain joining; CDR3, complementarity determining region three.
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Indeed, by applying immune repertoire sequencing, we found evidence for a small number of intrathecal B cells with clonally related counterparts in the periphery that persisted over time (Fig 3).