**7. Clues suggesting an antigen‐driven maturation of the intrathecal clonal population of B‐cells**

The IgG1 subclass is significantly elevated in most MS CSF although some authors recovered rare higher CSF IgG2 or IgG3 indexes [110, 154]. The Ig repertoire uses 51 functional germline genes and the normal repertoire of naïve peripheral blood CD19+ cells closely approximates the frequency of the VH family gene segments within the germline. On the other hand, over‐ representation of a single VH family is common in MS plaques and CSF but not in blood [155]. The VH4 family is observed in more than 35–95% of CD19+ or CD138+ cells. However, the VH1 family, which represents 20% of the functional germline, is nearly absent from CSF in MS [156, 157]. As observed for IgG, the VH4 germline predominates in CSF IgM [158]. This bias precedes the onset of OCB [155] and is typical of MS whereas different biases are observed in other CNS disorders [155]. The clonal expansion of a single ancestor gene was robustly demonstrated by the analysis of clonal diversification from an ancestor gene accumulating substitutions [156]. In CIS patients, the presence of a VH2‐VH4 CSF bias either in CD19 or in CD138 cells, which is observed in 7/10 patients, was highly correlated with a clinical conversion to MS in the following 6 months. By contrast, none of the CIS patients without the repertoire bias had developed MS at 2 years of follow‐up.

Somatic hypermutation (SHM) drives the maturation of the variable regions of the Ig genes under the control of activation‐induced cytidine deaminase (AID), with a preference to the hotspot regions (RGYW/WRCY motifs) of the CDR. The antigen‐driven affinity maturation process positively selects replacement mutations occurring in the CDR, which are in contact with the antigen, whereas silent mutations predominate in the framework regions. Therefore, the R/S ratio, which is lower than 2.9 before antigen‐driven maturation, is expected to be higher in CDR and the same or lower in the framework regions. In MS, the R/S ratio in the CDR of Ig genes was unchanged in blood B‐cells but elevated in CSF B‐cells [149, 158]. This ratio is elevated for both IgG‐ and IgM‐producing CSF B‐cells and occurs to the same extent in CSF, whereas the blood IgM ratio is about half of the IgG ratio [158]. The extent of SHM is in the expected range in CSF naïve B‐cells, whereas its level increases in switched memory B‐cells and plasma cells [159].

It is widely admitted that AID expression is a stage‐specific hallmark of germinal center B‐cells undergoing SHM and gene conversion. However, an aberrant expression of AID was dem‐ onstrated in CSF B‐cells producing IgG and IgM [158].

A signature score of hot/cold spots of higher/lower frequency replacement was established with six key positions, mostly situated in the CDR [160]. The signature score was 4.5 in the CSF of non‐MS patients, 2.0 in the blood of MS patients, and 10.9 ± 2.0 (7.6–11.9) in the CSF of MS patients (cutoff predictive of clinically defined MS ≥6.8). This technique was applied to B‐ cells extracted from the autopsied brains of four MS patients. The scores ranged from 10 to 14.5, so B‐cells from plaques and CSF share the bias [161]. Unfortunately, these bench results cannot be translated into common practice. Various clones displaying the same amino acid sequences but encoded by different nucleotides reinforce the hypothesis of antigen‐driven affinity maturation [158].

is more sensitive (Se 95%, Sp 91%) than the IgG index and OCB [153]. Since elevated CSF FLC is predictive of impairment [116], it has even been proposed as a therapeutic target [47].

**7. Clues suggesting an antigen‐driven maturation of the intrathecal clonal**

The IgG1 subclass is significantly elevated in most MS CSF although some authors recovered rare higher CSF IgG2 or IgG3 indexes [110, 154]. The Ig repertoire uses 51 functional germline genes and the normal repertoire of naïve peripheral blood CD19+ cells closely approximates the frequency of the VH family gene segments within the germline. On the other hand, over‐ representation of a single VH family is common in MS plaques and CSF but not in blood [155]. The VH4 family is observed in more than 35–95% of CD19+ or CD138+ cells. However, the VH1 family, which represents 20% of the functional germline, is nearly absent from CSF in MS [156, 157]. As observed for IgG, the VH4 germline predominates in CSF IgM [158]. This bias precedes the onset of OCB [155] and is typical of MS whereas different biases are observed in other CNS disorders [155]. The clonal expansion of a single ancestor gene was robustly demonstrated by the analysis of clonal diversification from an ancestor gene accumulating substitutions [156]. In CIS patients, the presence of a VH2‐VH4 CSF bias either in CD19 or in CD138 cells, which is observed in 7/10 patients, was highly correlated with a clinical conversion to MS in the following 6 months. By contrast, none of the CIS patients without the repertoire

Somatic hypermutation (SHM) drives the maturation of the variable regions of the Ig genes under the control of activation‐induced cytidine deaminase (AID), with a preference to the hotspot regions (RGYW/WRCY motifs) of the CDR. The antigen‐driven affinity maturation process positively selects replacement mutations occurring in the CDR, which are in contact with the antigen, whereas silent mutations predominate in the framework regions. Therefore, the R/S ratio, which is lower than 2.9 before antigen‐driven maturation, is expected to be higher in CDR and the same or lower in the framework regions. In MS, the R/S ratio in the CDR of Ig genes was unchanged in blood B‐cells but elevated in CSF B‐cells [149, 158]. This ratio is elevated for both IgG‐ and IgM‐producing CSF B‐cells and occurs to the same extent in CSF, whereas the blood IgM ratio is about half of the IgG ratio [158]. The extent of SHM is in the expected range in CSF naïve B‐cells, whereas its level increases in switched memory B‐cells

It is widely admitted that AID expression is a stage‐specific hallmark of germinal center B‐cells undergoing SHM and gene conversion. However, an aberrant expression of AID was dem‐

A signature score of hot/cold spots of higher/lower frequency replacement was established with six key positions, mostly situated in the CDR [160]. The signature score was 4.5 in the CSF of non‐MS patients, 2.0 in the blood of MS patients, and 10.9 ± 2.0 (7.6–11.9) in the CSF of MS patients (cutoff predictive of clinically defined MS ≥6.8). This technique was applied to B‐ cells extracted from the autopsied brains of four MS patients. The scores ranged from 10 to

**population of B‐cells**

64 Trending Topics in Multiple Sclerosis

and plasma cells [159].

bias had developed MS at 2 years of follow‐up.

onstrated in CSF B‐cells producing IgG and IgM [158].

In summary, B‐cells in CSF demonstrate the cardinal features of an antigen‐driven humoral immune response: clonal expansion and somatically hypermutated VH family sequences [155]. This suggests that these lineage cells were expanded by antigen and have undergone a germinal center reaction [162]. Moreover, there is evidence that the maturation process takes place at least partly in the CNS compartment [163].
