patients with a diagnosis of rrms who had been prescribed bg-12 were enrolled in this prospective cohort study .
informed consent was obtained from all patients and controls prior to participation in the protocol , which was approved by the university of michigan institutional review board .
patients were between 18 and 65 years of age , had not received another nonsteroidal disease - modifying therapy within the last 30 days , and had not had iv immunoglobulin therapy or plasmapheresis in the last 6 months .
four of 5 patients who had received oral or injected steroid treatments discontinued use more than 1 month before taking bg-12 , and the other patient began a 6-week taper at the time of starting bg-12 .
table 1 lists previous nonsteroidal medications taken by each patient and the time of discontinuation .
patients were put on a dose escalation schedule and all reached 240 mg bg-12 twice daily within 15 weeks of treatment initiation .
a total of 13 patients with rrms were followed longitudinally from pretreatment to 46 months of treatment with bg-12 .
a baseline peripheral blood sample was drawn within 2 months prior to initiation of bg-12 , and after 46 months ( 20.5  3.8 weeks ) of reaching the standard dose of bg-12 .
sex and age of the patients was 8/13 ( 61.5% ) female , average age 46.0  8.7 years ( median 46.5 ; range 3158 ) .
the 5 men were 40.4  9.8 years old ( median 35 ; range 3357 ) .
eight of these patients had a follow - up sample taken after 12 months of treatment ( see table 1 ) .
peripheral blood mononuclear cells ( pbmc ) were also obtained from 6 healthy controls : 4/6 ( 66.7% ) female , average age 48.0  9.1 years ( median 47.0 ; range 3860 ) , and 2 men aged 31 and 49 years .
blood samples ( 2025 ml ) from all participants were collected in sodium citrate cell preparation tubes and processed immediately .
total b - cell numbers were calculated by multiplying percentages from flow cytometry and cbc lymphocyte counts .
patient demographics and clinical information isolated pbmc from patients and controls were stained with monoclonal antibodies reactive against the cell surface markers cd19 , cd27 , and cd43 purchased from bd biosciences ( east rutherford , nj ) and cd24 and cd38 from biolegend ( san diego , ca ) .
multicolor flow cytometry was performed for all samples on a beckman coulter ( sharon hill , pa ) lsr flow cytometer using standardized voltage and compensation settings .
data analysis was performed using tree star ( ashland , or ) flowjo version x.0.6 software .
purified b cells were isolated from pbmc by magnetic bead separation using anti - human cd19 magnetic - activated cell sorting beads and the protocol provided by miltenyi biotec ( bergisch gladbach , germany ) .
labeled cells were positively selected over ls columns ( miltenyi biotec ) and were routinely > 98% pure .
b cells were cultured in high glucose dulbecco modified eagle medium supplemented with 10% heat - inactivated fetal calf serum , penicillin , streptomycin , insulin ( 5 g / ml ) , and transferrin ( 50 g / ml ) with irradiated human cd40 ligand - transduced fibroblasts for 6 days .
cell - free supernatants were removed and analyzed for the presence of human il-10 using opteia kit ( bd biosciences ) following the manufacturer 's protocol .
exact p values were calculated using the appropriate statistical tests for each dataset as indicated in the figure legends .
two - way analysis of variance with dunn multiple comparison tests were used to establish statistical significance for datasets containing 3 or more groups .
comparisons of control and patient samples were performed using mann - whitney tests . paired comparisons of time point data for individual patients
patients with a diagnosis of rrms who had been prescribed bg-12 were enrolled in this prospective cohort study .
informed consent was obtained from all patients and controls prior to participation in the protocol , which was approved by the university of michigan institutional review board .
patients were between 18 and 65 years of age , had not received another nonsteroidal disease - modifying therapy within the last 30 days , and had not had iv immunoglobulin therapy or plasmapheresis in the last 6 months .
four of 5 patients who had received oral or injected steroid treatments discontinued use more than 1 month before taking bg-12 , and the other patient began a 6-week taper at the time of starting bg-12 .
table 1 lists previous nonsteroidal medications taken by each patient and the time of discontinuation . patients were put on a dose escalation schedule and all reached 240 mg bg-12 twice daily within 15 weeks of treatment initiation .
a total of 13 patients with rrms were followed longitudinally from pretreatment to 46 months of treatment with bg-12 .
a baseline peripheral blood sample was drawn within 2 months prior to initiation of bg-12 , and after 46 months ( 20.5  3.8 weeks ) of reaching the standard dose of bg-12 .
sex and age of the patients was 8/13 ( 61.5% ) female , average age 46.0  8.7 years ( median 46.5 ; range 3158 ) .
the 5 men were 40.4  9.8 years old ( median 35 ; range 3357 ) .
eight of these patients had a follow - up sample taken after 12 months of treatment ( see table 1 ) .
peripheral blood mononuclear cells ( pbmc ) were also obtained from 6 healthy controls : 4/6 ( 66.7% ) female , average age 48.0  9.1 years ( median 47.0 ; range 3860 ) , and 2 men aged 31 and 49 years .
blood samples ( 2025 ml ) from all participants were collected in sodium citrate cell preparation tubes and processed immediately .
total b - cell numbers were calculated by multiplying percentages from flow cytometry and cbc lymphocyte counts .
isolated pbmc from patients and controls were stained with monoclonal antibodies reactive against the cell surface markers cd19 , cd27 , and cd43 purchased from bd biosciences ( east rutherford , nj ) and cd24 and cd38 from biolegend ( san diego , ca ) .
multicolor flow cytometry was performed for all samples on a beckman coulter ( sharon hill , pa ) lsr flow cytometer using standardized voltage and compensation settings .
data analysis was performed using tree star ( ashland , or ) flowjo version x.0.6 software .
purified b cells were isolated from pbmc by magnetic bead separation using anti - human cd19 magnetic - activated cell sorting beads and the protocol provided by miltenyi biotec ( bergisch gladbach , germany ) .
labeled cells were positively selected over ls columns ( miltenyi biotec ) and were routinely > 98% pure .
b cells were cultured in high glucose dulbecco modified eagle medium supplemented with 10% heat - inactivated fetal calf serum , penicillin , streptomycin , insulin ( 5 g / ml ) , and transferrin ( 50 g / ml ) with irradiated human cd40 ligand - transduced fibroblasts for 6 days .
cell - free supernatants were removed and analyzed for the presence of human il-10 using opteia kit ( bd biosciences ) following the manufacturer 's protocol .
exact p values were calculated using the appropriate statistical tests for each dataset as indicated in the figure legends .
two - way analysis of variance with dunn multiple comparison tests were used to establish statistical significance for datasets containing 3 or more groups .
comparisons of control and patient samples were performed using mann - whitney tests . paired comparisons of time point data for individual patients
treatment of rrms with the food and drug administration  approved oral drug , bg-12 ( tecfidera ) , is known to reduce total pbmc counts compared to pretreatment levels .
decreases involve both the t- and the b - lymphocyte populations , yet detailed analysis within these major lymphocyte lineages has not been reported .
b cells can be subdivided into groups with distinct functions and states of activation that are distinguishable based on surface marker expression patterns . in this prospective cohort study ,
b - cell subsets were analyzed from 13 patients with rrms ( table 1 ) before and after 46 months of treatment with 240 mg twice daily oral bg-12 .
table 1 outlines the demographics and other clinical data for each patient including previous treatments and cbc lymphocyte counts .
patients 3 , 4 , 10 , and 11 had taken oral or injected corticosteroids that were discontinued prior to starting bg-12 therapy .
patient 1 had a flare that was treated with oral corticosteroids just prior to entering the study , and was tapered off of steroids immediately after bg-12 treatment was initiated .
most ( 11 of 12 ) of the patients had a marked reduction in peripheral blood lymphocytes ( pbl ) after 46 months treatment with bg-12 compared to pretreatment levels ( table 1 ) .
levels of pbl did not decrease between the 4 and 6 months and > 1 year time points for most patients . to determine the effects of bg-12 on b cells ,
six healthy individuals who had similar demographics for age , sex , and ethnicity were used as controls .
figure 1a shows the gating used select cd19 + b cells from a representative patient ( rrms 8) at baseline and 4 months after starting bg-12 .
the majority of these patients with rrms had an elevated percentage of cd19 + b cells prior to bg-12 treatment compared to controls ( figure 1b ) .
a significant decrease in the total number of b cells was observed at 46 months of treatment compared to pretreatment levels ( figure 1c and table 2 ) .
the total number of circulating cd19 + b cells in the patients with rrms did not significantly decrease between 4 and 6 months of treatment and more than 1 year of treatment ( table 2 ) .
peripheral blood mononuclear cells ( pbmc ) isolated from patients with relapsing - remitting multiple sclerosis ( rrms ) before and after bg-12 treatment and healthy controls were stained for b - cell markers .
( a ) representative forward scatter , side scatter , and anti - cd19 staining from a single patient ( rrms-8 ) shows standardized gating for lymphocytes , single cells , and total cd19 + b cells that was used for all of the samples .
( b ) comparison of % cd19 + b cells for healthy controls and patients with rrms before treatment .
each dot represents an individual sample and lines are the median interquartile range for each group .
the exact p value shown below the title was calculated using mann - whitney test .
( c ) total b - cell numbers / ml of blood for the patients with rrms were calculated before and after 46 months of treatment by multiplying the % cd19 + cells by the lymphocyte counts reported by the clinical pathology laboratory ( table 1 ) .
exact p value for paired patient data was determined using wilcoxon matched - pairs signed - rank test .
( d ) representative histograms from patient rrms-8 show gating used to define cd27 + memory b cells .
( e ) percentages of cd27 + memory b cells in patients with rrms before treatment compared with controls .
each dot represents data from one individual and lines are the median and interquartile range for each group .
( f ) longitudinal % cd27 + memory b - cell data for individual patients with rrms ( n = 8) before , after 46 months , and after more than 1 year of treatment with bg-12 .
total cell numbers of peripheral blood b - cell subsets cd27 has been commonly used to distinguish activated / memory b cells from other b - cell subsets or activation states .
cd19+cd27 + cells were analyzed before and after treatment with bg-12 ( figure 1 , d 
patients with rrms in this study had lower percentages of circulating cd27 + memory b cells prior to bg-12 treatment than the controls ( figure 1e ) , suggesting that the cd27 b cells , which include a mixture of immature , transitional , and naive b - cell subsets , were overrepresented in the patients with rrms before treatment .
as shown in figure 1f , there was some variability in the relative levels of memory vs naive b cells in the patients with rrms before treatment , and bg-12 had differential effects on the ratios .
treatment for 46 months led to decreases in the absolute number of circulating cd27 + b cells in all but one patient .
the exception was patient rrms-10 , who went on to have fewer memory b cells after 1 year of treatment than at baseline ( data not shown ) .
long - term treatment with bg-12 did not lead to further significant reductions in the total number of cd27 + b cells in most patients ( table 2 ) or a consistent change in the ratio of cd27 + to cd27 b cells ( figure 2c ) . taken together
, these results suggest that bg-12 treatment did not have a selective effect on either the cd27 + memory b cells or the cd27 immature , transitional , or naive b cells , but that there was a generalized decrease of both b - lymphocyte populations in patients with rrms . peripheral blood mononuclear cells ( pbmc )
isolated from healthy controls and patients with relapsing - remitting multiple sclerosis ( rrms ) before and after bg-12 treatment were stained and gated for cd19 + b cells as shown in figure 1 and then analyzed for coexpression of cd24 and cd38 or cd27 and cd43 .
( a ) representative staining from patient rrms-8 shows the gating used to select the cd24cd38 ( t2-mzp ) b - cell subset .
( b ) representative staining from patient rrms-8 shows gating used to analyze cd43 + cd27 + b-1 b cells before and after 4 months of bg-12 treatment .
( c ) comparison of t2-mzp b cells in controls and patients with rrms before treatment .
each dot represents an individual sample and lines are the median and interquartile range for each group .
( d ) percentages of b-1 b cells in controls and patients with rrms before treatment .
each dot represents an individual and lines are the median and interquartile range for each group .
( e ) percentages of t2-mzp b cells in individual patients with rrms before bg-12 therapy and after 46 months and > 12 months of treatment .
( f ) longitudinal changes in percentage of b-1 b cells for individual patients with rrms at baseline , 46 months , and > 12 months bg-12 treatment .
the cd27 and cd27 + b - cell populations can be further subdivided into functionally distinct subsets .
notably , a subset of regulatory b cells that produce il-10 has been found within the cd27 and cd27 + b - cell subsets .
cd27 b cells that are cd24cd38 ( figure 2a ) have been previously classified as transitional 2 marginal zone precursor ( t2-mzp ) b cells that produce il-10 .
circulating t2-mzp b cells were reduced in percentage in these patients with rrms prior to treatment in comparison to controls ( figure 2b ) . unlike the total cd27 b - cell subset
, increases in t2-mzp b cells were detected in 10 of 13 patients at 46 months treatment , and were higher than baseline in all 8 patients who were followed for more than 1 year ( figure 2c ) . despite reductions in total b cells , the number of circulating t2-mzp b cells tended to be higher after 46 months of bg-12 treatment and continued to increase over the longer term , resulting in significantly higher total t2-mzp b - cell numbers in the circulation of patients with rrms after 1 year of bg-12 treatment ( table 2 ) .
the cd27+cd43 + b - cell subset ( figure 2d ) has been identified as an innate - like b-1 b - cell subtype that , similar to its counterpart in mice , has the capacity to produce il-10 and to regulate immune responses .
prior to treatment , b-1 b cells were found in the circulation at very low levels in all but one of the 13 patients with rrms in our study in comparison to healthy controls ( figure 2e ) . despite the inclusion of patient rrms-3 , whose b-1 cells were within the normal range , the patients averaged a more than fourfold lower percentage of b-1 b cells than controls .
the b-1 b cells responded somewhat differently to bg-12 treatment compared to t2-mzp b cells ( figure 2f ) .
after 46 months of treatment with bg-12 , the percentage of circulating b-1 b cells was not significantly changed ( figure 2f ) , and there was variability in the response from patient to patient .
however , after 1 year of treatment , 6 of 8 patients with rrms had a substantially higher percentage of b-1 b cells than what was present prior to bg-12 treatment . with the exception of patient rrms-3 , who had 48,800 b-1 b cells / ml of blood at baseline compared to the 4,200  3,700 b-1 b cells / ml average for the other 7 patients
, there was a significant increase in the total number of circulating b-1 b cells after long - term treatment with bg-12 ( table 2 ) .
not all t2-mzp or b-1 b cells produce il-10 after stimulation , and patients with systemic lupus erythematosus had t2-mzp b cells that were defective in il-10 production . to determine whether b cells from the patients with rrms were functional , cd19 + b cells were purified using magnetic bead positive selection , and then cultured with a cd40 ligand - transduced cell line .
this assay was dependent on having enough purified b cells , which was not achieved for all patients and time points .
figure 3a shows il-10 data for the 3 patients with rrms in this study who were analyzed at both the pretreatment and 46 months of bg-12 treatment time points .
the percentages of t2-mzp and b-1 b cells in the sample prior to culture are shown in figure 3b .
the results indicated that levels of il-10 produced by these 3 patients were comparable to the combined levels of t2-mzp and b-1 b cells before or after treatment , but not well - correlated with the individual subsets .
a total of 9 patients with rrms were analyzed for cd40 ligand - induced il-10 production at the 46 months treatment time point ( figure 3c ) . while 7 of the 9 patients with rrms released levels of il-10 that correlated strongly with the percentage of t2-mzp plus b-1 b cells found in their blood , 2 patients with rrms appeared to have a reduction in the expected levels of b - cell il-10 production in this assay .
cd19 + b cells were positively selected by magnetic bead separation before and after 46 months of bg-12 treatment .
purified b cells were stimulated with irradiated hcd40l - fb for 6 days prior to measurement of il-10 in culture supernatants .
cultures containing hcd40l - fb alone or b cells with cd40l fibroblasts did not produce il-10 ( data not shown ) .
student t test calculated * p values for the data before and after treatment were p = 0.0018 , p < 0.0001 , and p = 0.2084 for patients rrms-1 , rrms-2 , and rrms-10 , respectively .
( b ) stacked bar graph of percentage of circulating t2-mzp b cells ( open bars ) and b-1 b cells ( hashed bars ) within the total cd19 + b - cell population before culture .
( c ) comparison of b - cell il-10 production vs combined levels of t2-mzp and b-1 b cells after 46 months treatment for 9 patients .
two patients did not produce il-10 despite high levels of regulatory b cells ( marked with x ) .
linear regression for the other 7 patients ( diamonds ) showed a high degree of correlation : y = 50.3x + 181 , r = 0.79 , f = 18.7 , p = 0.0075 .
 data point at 11.6% t2-mzp + b-1 b cells represents 2 patients , rrms-1 and rrms-2 , who produced il-10 = 1,010  45 and 1,008  99 pg / million cells , respectively .
treatment of rrms with the food and drug administration  approved oral drug , bg-12 ( tecfidera ) , is known to reduce total pbmc counts compared to pretreatment levels .
decreases involve both the t- and the b - lymphocyte populations , yet detailed analysis within these major lymphocyte lineages has not been reported .
b cells can be subdivided into groups with distinct functions and states of activation that are distinguishable based on surface marker expression patterns . in this prospective cohort study ,
b - cell subsets were analyzed from 13 patients with rrms ( table 1 ) before and after 46 months of treatment with 240 mg twice daily oral bg-12 .
table 1 outlines the demographics and other clinical data for each patient including previous treatments and cbc lymphocyte counts .
patients 3 , 4 , 10 , and 11 had taken oral or injected corticosteroids that were discontinued prior to starting bg-12 therapy .
patient 1 had a flare that was treated with oral corticosteroids just prior to entering the study , and was tapered off of steroids immediately after bg-12 treatment was initiated .
most ( 11 of 12 ) of the patients had a marked reduction in peripheral blood lymphocytes ( pbl ) after 46 months treatment with bg-12 compared to pretreatment levels ( table 1 ) .
levels of pbl did not decrease between the 4 and 6 months and > 1 year time points for most patients . to determine the effects of bg-12 on b cells ,
six healthy individuals who had similar demographics for age , sex , and ethnicity were used as controls .
figure 1a shows the gating used select cd19 + b cells from a representative patient ( rrms 8) at baseline and 4 months after starting bg-12 .
the majority of these patients with rrms had an elevated percentage of cd19 + b cells prior to bg-12 treatment compared to controls ( figure 1b ) .
a significant decrease in the total number of b cells was observed at 46 months of treatment compared to pretreatment levels ( figure 1c and table 2 ) .
the total number of circulating cd19 + b cells in the patients with rrms did not significantly decrease between 4 and 6 months of treatment and more than 1 year of treatment ( table 2 ) .
peripheral blood mononuclear cells ( pbmc ) isolated from patients with relapsing - remitting multiple sclerosis ( rrms ) before and after bg-12 treatment and healthy controls were stained for b - cell markers .
( a ) representative forward scatter , side scatter , and anti - cd19 staining from a single patient ( rrms-8 ) shows standardized gating for lymphocytes , single cells , and total cd19 + b cells that was used for all of the samples .
( b ) comparison of % cd19 + b cells for healthy controls and patients with rrms before treatment .
each dot represents an individual sample and lines are the median interquartile range for each group .
the exact p value shown below the title was calculated using mann - whitney test .
( c ) total b - cell numbers / ml of blood for the patients with rrms were calculated before and after 46 months of treatment by multiplying the % cd19 + cells by the lymphocyte counts reported by the clinical pathology laboratory ( table 1 ) .
exact p value for paired patient data was determined using wilcoxon matched - pairs signed - rank test .
( d ) representative histograms from patient rrms-8 show gating used to define cd27 + memory b cells .
( e ) percentages of cd27 + memory b cells in patients with rrms before treatment compared with controls .
each dot represents data from one individual and lines are the median and interquartile range for each group .
( f ) longitudinal % cd27 + memory b - cell data for individual patients with rrms ( n = 8) before , after 46 months , and after more than 1 year of treatment with bg-12 .
cd27 has been commonly used to distinguish activated / memory b cells from other b - cell subsets or activation states .
cd19+cd27 + cells were analyzed before and after treatment with bg-12 ( figure 1 , d 
patients with rrms in this study had lower percentages of circulating cd27 + memory b cells prior to bg-12 treatment than the controls ( figure 1e ) , suggesting that the cd27 b cells , which include a mixture of immature , transitional , and naive b - cell subsets , were overrepresented in the patients with rrms before treatment .
as shown in figure 1f , there was some variability in the relative levels of memory vs naive b cells in the patients with rrms before treatment , and bg-12 had differential effects on the ratios .
treatment for 46 months led to decreases in the absolute number of circulating cd27 + b cells in all but one patient .
the exception was patient rrms-10 , who went on to have fewer memory b cells after 1 year of treatment than at baseline ( data not shown ) .
long - term treatment with bg-12 did not lead to further significant reductions in the total number of cd27 + b cells in most patients ( table 2 ) or a consistent change in the ratio of cd27 + to cd27 b cells ( figure 2c ) .
taken together , these results suggest that bg-12 treatment did not have a selective effect on either the cd27 + memory b cells or the cd27 immature , transitional , or naive b cells , but that there was a generalized decrease of both b - lymphocyte populations in patients with rrms . peripheral blood mononuclear cells ( pbmc )
isolated from healthy controls and patients with relapsing - remitting multiple sclerosis ( rrms ) before and after bg-12 treatment were stained and gated for cd19 + b cells as shown in figure 1 and then analyzed for coexpression of cd24 and cd38 or cd27 and cd43 .
( a ) representative staining from patient rrms-8 shows the gating used to select the cd24cd38 ( t2-mzp )
( b ) representative staining from patient rrms-8 shows gating used to analyze cd43 + cd27 + b-1 b cells before and after 4 months of bg-12 treatment .
( c ) comparison of t2-mzp b cells in controls and patients with rrms before treatment .
each dot represents an individual sample and lines are the median and interquartile range for each group .
( d ) percentages of b-1 b cells in controls and patients with rrms before treatment .
each dot represents an individual and lines are the median and interquartile range for each group .
( e ) percentages of t2-mzp b cells in individual patients with rrms before bg-12 therapy and after 46 months and > 12 months of treatment .
( f ) longitudinal changes in percentage of b-1 b cells for individual patients with rrms at baseline , 46 months , and > 12 months bg-12 treatment .
the cd27 and cd27 + b - cell populations can be further subdivided into functionally distinct subsets .
notably , a subset of regulatory b cells that produce il-10 has been found within the cd27 and cd27 + b - cell subsets .
cd27 b cells that are cd24cd38 ( figure 2a ) have been previously classified as transitional 2 marginal zone precursor ( t2-mzp ) b cells that produce il-10 .
circulating t2-mzp b cells were reduced in percentage in these patients with rrms prior to treatment in comparison to controls ( figure 2b ) . unlike the total cd27 b - cell subset ,
increases in t2-mzp b cells were detected in 10 of 13 patients at 46 months treatment , and were higher than baseline in all 8 patients who were followed for more than 1 year ( figure 2c ) . despite reductions in total b cells , the number of circulating t2-mzp b cells tended to be higher after 46 months of bg-12 treatment and continued to increase over the longer term , resulting in significantly higher total t2-mzp b - cell numbers in the circulation of patients with rrms after 1 year of bg-12 treatment ( table 2 ) .
the cd27+cd43 + b - cell subset ( figure 2d ) has been identified as an innate - like b-1 b - cell subtype that , similar to its counterpart in mice , has the capacity to produce il-10 and to regulate immune responses .
prior to treatment , b-1 b cells were found in the circulation at very low levels in all but one of the 13 patients with rrms in our study in comparison to healthy controls ( figure 2e ) . despite the inclusion of patient rrms-3 , whose b-1 cells were within the normal range , the patients averaged a more than fourfold lower percentage of b-1 b cells than controls .
the b-1 b cells responded somewhat differently to bg-12 treatment compared to t2-mzp b cells ( figure 2f ) .
after 46 months of treatment with bg-12 , the percentage of circulating b-1 b cells was not significantly changed ( figure 2f ) , and there was variability in the response from patient to patient .
however , after 1 year of treatment , 6 of 8 patients with rrms had a substantially higher percentage of b-1 b cells than what was present prior to bg-12 treatment . with the exception of patient rrms-3 , who had 48,800 b-1 b cells / ml of blood at baseline compared to the 4,200  3,700 b-1 b cells / ml average for the other 7 patients ,
there was a significant increase in the total number of circulating b-1 b cells after long - term treatment with bg-12 ( table 2 ) .
not all t2-mzp or b-1 b cells produce il-10 after stimulation , and patients with systemic lupus erythematosus had t2-mzp b cells that were defective in il-10 production . to determine whether b cells from the patients with rrms were functional , cd19 + b cells were purified using magnetic bead positive selection , and then cultured with a cd40 ligand - transduced cell line .
this assay was dependent on having enough purified b cells , which was not achieved for all patients and time points .
figure 3a shows il-10 data for the 3 patients with rrms in this study who were analyzed at both the pretreatment and 46 months of bg-12 treatment time points .
the percentages of t2-mzp and b-1 b cells in the sample prior to culture are shown in figure 3b .
the results indicated that levels of il-10 produced by these 3 patients were comparable to the combined levels of t2-mzp and b-1 b cells before or after treatment , but not well - correlated with the individual subsets .
a total of 9 patients with rrms were analyzed for cd40 ligand - induced il-10 production at the 46 months treatment time point ( figure 3c ) . while 7 of the 9 patients with rrms released levels of il-10 that correlated strongly with the percentage of t2-mzp plus b-1 b cells found in their blood , 2 patients with rrms appeared to have a reduction in the expected levels of b - cell il-10 production in this assay .
cd19 + b cells were positively selected by magnetic bead separation before and after 46 months of bg-12 treatment .
purified b cells were stimulated with irradiated hcd40l - fb for 6 days prior to measurement of il-10 in culture supernatants .
cultures containing hcd40l - fb alone or b cells with cd40l fibroblasts did not produce il-10 ( data not shown ) .
student t test calculated * p values for the data before and after treatment were p = 0.0018 , p < 0.0001 , and p = 0.2084 for patients
( b ) stacked bar graph of percentage of circulating t2-mzp b cells ( open bars ) and b-1 b cells ( hashed bars ) within the total cd19 + b - cell population before culture .
( c ) comparison of b - cell il-10 production vs combined levels of t2-mzp and b-1 b cells after 46 months treatment for 9 patients .
two patients did not produce il-10 despite high levels of regulatory b cells ( marked with x ) .
linear regression for the other 7 patients ( diamonds ) showed a high degree of correlation : y = 50.3x + 181 , r = 0.79 , f = 18.7 , p = 0.0075 .
data point at 11.6% t2-mzp + b-1 b cells represents 2 patients , rrms-1 and rrms-2 , who produced il-10 = 1,010  45 and 1,008  99 pg / million cells , respectively .
our data confirm that treatment of patients with rrms with bg-12 leads to reductions in circulating lymphocytes , including b cells , as previously reported .
treatment with bg-12 was found to variably lower the total number of cd27 + memory b cells , as well as the cd27 immature , transitional , and naive b - cell subsets .
in contrast , cd24cd38 t2-mzp b cells increased in percentage after 46 months of treatment and remained higher in number than baseline in 8/8 ( 100% ) patients after 1 year of treatment .
long - term treatment also led to increased numbers of circulating cd43+cd27 + b-1 cells .
both t2-mzp and b-1 b cells have been independently shown to produce the immune regulatory cytokine , il-10 , and the production of il-10 by b cells in this study correlated with the combined presence of both of these cell subsets in 7/9 ( 78% ) patients , while 2 patients failed to produce il-10 despite relatively high levels of t2-mzp and b-1 cells .
thus , bg-12 treatment of patients with rrms appeared to favor b cells that had immune regulatory potential .
the mechanisms underlying these differential effects of bg-12 on circulating b - lymphocyte subsets have not yet been determined .
some possibilities for the reductions in the majority of b cells include direct induction of apoptosis or indirect killing of b cells by other cells .
another possibility is that bg-12 may have initially affected th cell viability or activation resulting in losses or changes to t - cell help that altered b - cell viability .
shifts in cytokine production from th1 toward th2 patterns have been noted in human t cells from patients treated with dmf .
these changes in t - cell cytokine output may have contributed to the increase in regulatory b - cell subsets , since the th2 cytokine il-5 has been shown to preferentially stimulate growth of immune regulatory mouse b-1 b cells that expressed fas ligand and il-10 .
further work is necessary to understand the cells and processes involved and their relative importance to the therapeutic effects of bg-12 .
direct effects of bg-12 or dmf on signal transduction in human b cells have not been previously reported .
bg-12 is known to activate the nuclear factor ( erythroid - derived 2)like 2 ( nrf2 ) pathway in neuronal cells . unlike the proapoptotic effects of dmf on t cells , our group recently demonstrated that activation of the nrf2 pathway by dmf can inhibit neuronal cell apoptosis as a possible mechanism of neuroprotection .
it will be of interest to determine whether bg-12 activates different signaling pathways and has a pro vs antiapoptotic effect on different subsets of human b cells .
one product of nrf2 activation that may play a direct role in the immune response is heme oxygenase1 ( ho-1 ) , which is known to stimulate the functions of treg cells .
in contrast , very little is known about the role of nrf2 and ho-1 in regulatory b - cell activation and function .
it remains to be determined whether bg-12 has direct effects on proliferation or protection from apoptosis of t2-mzp or b-1 b cells .
other possibilities for the observed increases in these cell populations include indirect effects through activation of other cell populations or homeostatic proliferation response resulting from the loss of other lymphocytes . in humans ,
cd24cd38 t2-mzp and cd27+cd43 + b-1 b cells have been designated as regulatory b - cell subsets based on their ability to produce il-10 . it should be noted that within these populations , il-10 production only occurs in a fraction of the total subset and is dependent on the method of stimulation .
both the t2-mzp and b-1 b - cell subsets were increased in total cell number and percentage following long - term treatment in the majority of the patients we studied .
the t2-mzp b cells responded earlier and more consistently than the b-1 b cells , suggesting that different mechanisms were involved in their proliferation .
increases in t2-mzp b cells have been noted following treatment of patients with rrms with other medications , suggesting that these cells may be highly important in the regulation of multiple sclerosis ( ms ) pathogenesis .
the response among b-1 b cells in bg-12-treated patients with rrms was more complicated and individualized , with the increase in b-1 b cells only detected after 1 year of treatment , and only in 6 of the 8 patients analyzed .
this may have been due in part to the fact that the majority of these innate b cells are resident in the lining of the gut and lungs , and the delayed increase in their numbers in the blood may reflect earlier activation or proliferation in the mucosa .
most of the patients had significantly lower levels of b-1 b cells prior to treatment , in agreement with findings of other investigators .
however , the previous authors concluded that treatment of patients with rrms with glatiramer acetate , interferon- , or natalizumab had no effect on cd43+cd27 + b-1 b cells .
this was in sharp contrast to the effects of glatiramer acetate treatment in mouse eae , which resulted in expansion and increased regulatory function of cd5 + b-1a cells .
it should be noted that cd5 is not a specific marker for b-1 cells in humans , complicating the interpretation of previous reports linking human cd5 + b cells to ms pathogenesis .
we did not assess il-10 production by intracellular staining , and therefore , were not able to determine whether the freshly isolated b cells were expressing il-10 in vivo .
the levels of il-10 produced by 7 out of 9 of the patients with rrms correlated with the combined percentages of t2-mzp and b-1 b cells in the sample , but not with either of the subsets independently . the data for these 7 patients contrasted with previous reports associating autoimmunity with poorly functioning regulatory b cells , and suggest that b cells in these patients with rrms would be capable of contributing to immune regulation .
notably , b cells from 2 of the patients with rrms in our study did not produce significant amounts of il-10 despite having relatively high levels of t2-mzp and b-1 b cells . it will be interesting to track clinical responses in these patients long term , and to perform similar analyses on a larger population to determine whether the beneficial effects of bg-12 correlate with the activation and competence of regulatory b cells .
it should be noted that this study was done with a limited number of patients , and that continued work should be done with larger groups and longer follow - up to confirm these findings .
study of more complex variables , such as the effects of prior treatments as well as duration and severity of disease at treatment initiation , will require much larger groups and may be important in determining which patients are most likely to benefit from bg-12 therapy . an important aspect of our study is the variability of the responses of b cells to bg-12 treatment and how this may relate to clinical responses .
none of these 13 patients has relapsed and there have been no new mri lesions during this study ( data not shown ) .
the patients have tolerated bg-12 treatment well with only a few reports of minor side effects that have subsided over time . with several cases of pml
having been reported , there is understandable concern about long - term effects of bg-12 and a need to screen for jc virus .
these issues highlight the need for more long - term studies with large cohorts of patients , which may lead to a better understanding of variables that relate not only to favorable outcomes , but also to severe negative side effects such as pml
. it will be interesting to determine whether the short - term effects of bg-12 on t2-mzp or b-1 b cells will predict the long - term clinical efficacy of bg-12 treatment .
lundy received nonprofit honorarium from university of connecticut , is on the editorial board for the open rheumatology journal , and received research support from merck , chugai , nih , university of michigan medical school , and edward t. and ellen k. dryer foundation .
y. mao - draayer served on the scientific advisory board for , received travel funding , and/or speaker honoraria from , and consulted for acorda , bayer , biogen idec , emd serono , genzyme , novartis , questor , chugai , and teva , and received research support from nih 
 niaid and ninds .
 objective : to test the hypothesis that dimethyl fumarate ( tecfidera , bg-12 ) affects b - cell subsets in patients with relapsing - remitting multiple sclerosis ( rrms).methods : peripheral blood b cells were compared for surface marker expression in patients with rrms prior to initiation of treatment , after 46 months , and at more than 1 year of treatment with bg-12 . 
 production of interleukin ( il)10 by rrms patient b cells was also analyzed.results:total numbers of peripheral blood b lymphocytes declined after 46 months of bg-12 treatment , due to losses in both the cd27 + memory b cells and cd27neg b - cell subsets . some interpatient variability was observed . 
 in contrast , circulating cd24highcd38high ( t2-mzp ) b cells increased in percentage in the majority of patients with rrms after 46 months and were present in higher numbers in all of the patients after 12 months of treatment . 
 the cd43+cd27 + b-1 b cells also increased at the later time point in most patients but were unchanged at 46 months compared to pretreatment levels . 
 purified b cells from 7 of the 9 patients with rrms tested after 46 months of treatment were able to produce il-10 following cd40 ligand stimulation , and the amount corresponded with the combined levels of t2-mzp and b-1 b cells in the sample . 
 none of the patients with rrms in this study have had a relapse while taking bg-12.conclusions:these data suggest that bg-12 differentially affects b - cell subsets in patients with rrms , resulting in increased numbers of circulating b lymphocytes with regulatory capacity . 
METHODS
Standard protocol approvals, registrations, and patient consents.
Demographics and blood samples.
Flow cytometry.
Human B-cell culture and IL-10 analysis.
Data collection and statistical analysis.
RESULTS
BG-12 treatment reduced circulating memory and naive B cells.
Decreased circulating memory and naive B cells in BG-12-treated patients with RRMS.
Increased CD24
DISCUSSION
STUDY FUNDING
DISCLOSURE