the study was approved by the ethical committee of the hospital district of southwestern finland .
fourteen patients with relapsing - remitting ms ( 10 women ) initiating natalizumab treatment ( tysabri , 300 mg iv every 4 weeks ; biogen idec , cambridge , ma ) at the neurology outpatient clinic of the division of clinical neurosciences at the turku university hospital , turku , finland ( december 2008 to december 2009 ) , were prospectively recruited into the study .
characteristics included the following : mean age 37.2 ( range 2146 ) years ; disease duration from ms diagnosis 1.9 ( 03.75 ) years ; expanded disability status scale score 3.7 ( 17.5 ) ; number of relapses 4.1 ( 110 , total ) and 1.9 ( 05 , year before ) ; and disease - modifying therapy year before , -interferon ( n = 6 ) and glatiramer acetate ( n = 1 ) .
the sample size was determined by the number of patients initiating natalizumab treatment and willing to participate during the recruitment period of 1 year . heparinized blood samples ( 10 ml ) were obtained shortly before the initiation of treatment and at 1 week and 1 , 3 , and 12 months of treatment .
two patients discontinued the treatment ( after 1 and 8 months [ infusion reaction and fear of progressive multifocal leukoencephalopathy , respectively ] ) , and 3 patients did not show up at 1 week .
one patient experienced a relapse 10 days after initiation of natalizumab treatment and received a course of methylprednisolone pulse therapy .
immunofluorescence staining was performed using whole blood ( ethylenediaminetetraacetic acid  anticoagulated peripheral blood , 100 l ) with the following antibodies : cd19-pe - cy7 , cd5-apc , cd10-apc , cd19-apc , cd20-apc - cy7 , cd45-apc - cy7 , cd45-percp - cy5.5 , cd4-apc , cd3-pe - cy7 ( bd biosciences , franklin lakes , nj ) ; cd49d , cd183-pe , cd196-pe , cd195-fitc , cd3-pe - cy7 , cd3-percp - cy5.5 ( bd pharmingen , san diego , ca ) .
samples were processed according to euroflow standard operating procedure for sample preparation and staining ( euroflow.org ) and analyzed using flow cytometry ( facscanto ; bd , san jose , ca ) .
rationale for the choice of target molecules was the following : cd19 and cd20 were included as b cell determinants .
cd183/cxcr3 and cd49d / vla-4 evaluation on b cells was the hypothesis - driven main objective of the study .
other chemokine receptors and cell surface molecules not immediately associated with b cell cns homing ( i.e. , cd196/ccr6 , cd195/ccr5 , cd5 ) were included as controls . from each tube ,
100,000 events were collected , and data were analyzed using facsdiva version 6.1.3 ( bd ) .
on the same day , the blood lymphocyte counts were obtained from differential white blood cell count analyzed at 0 , 1 , 3 , and 12 months using sysmex xe2100 ( sysmex , kobe , japan ) automated hematology analyzer according to standard clinical laboratory procedures , and absolute counts for b cell subpopulations were calculated from lymphocyte counts with assistance of flow cytometry results .
statistical analyses were performed separately for each antibody using sas system ( sas institute , cary , nc ) for windows 9.4 .
if residuals were normally distributed , time comparisons were done using mixed - model repeated - measures analysis of variance with heterogeneous compound symmetry covariance structure and followed by dunnett adjustment for pairwise comparisons .
otherwise , comparisons were made using signed - rank or paired t test with bonferroni adjustment multiple comparisons .
samples taken during natalizumab treatment were compared to the baseline sample . corrected 2-sided p values < 0.05 were considered statistically significant .
the study was approved by the ethical committee of the hospital district of southwestern finland .
fourteen patients with relapsing - remitting ms ( 10 women ) initiating natalizumab treatment ( tysabri , 300 mg iv every 4 weeks ; biogen idec , cambridge , ma ) at the neurology outpatient clinic of the division of clinical neurosciences at the turku university hospital , turku , finland ( december 2008 to december 2009 ) , were prospectively recruited into the study .
characteristics included the following : mean age 37.2 ( range 2146 ) years ; disease duration from ms diagnosis 1.9 ( 03.75 ) years ; expanded disability status scale score 3.7 ( 17.5 ) ; number of relapses 4.1 ( 110 , total ) and 1.9 ( 05 , year before ) ; and disease - modifying therapy year before , -interferon ( n = 6 ) and glatiramer acetate ( n = 1 ) .
the sample size was determined by the number of patients initiating natalizumab treatment and willing to participate during the recruitment period of 1 year . heparinized blood samples ( 10 ml ) were obtained shortly before the initiation of treatment and at 1 week and 1 , 3 , and 12 months of treatment .
two patients discontinued the treatment ( after 1 and 8 months [ infusion reaction and fear of progressive multifocal leukoencephalopathy , respectively ] ) , and 3 patients did not show up at 1 week .
one patient experienced a relapse 10 days after initiation of natalizumab treatment and received a course of methylprednisolone pulse therapy .
immunofluorescence staining was performed using whole blood ( ethylenediaminetetraacetic acid  anticoagulated peripheral blood , 100 l ) with the following antibodies : cd19-pe - cy7 , cd5-apc , cd10-apc , cd19-apc , cd20-apc - cy7 , cd45-apc - cy7 , cd45-percp - cy5.5 , cd4-apc , cd3-pe - cy7 ( bd biosciences , franklin lakes , nj ) ; cd49d , cd183-pe , cd196-pe , cd195-fitc , cd3-pe - cy7 , cd3-percp - cy5.5 ( bd pharmingen , san diego , ca ) .
samples were processed according to euroflow standard operating procedure for sample preparation and staining ( euroflow.org ) and analyzed using flow cytometry ( facscanto ; bd , san jose , ca ) .
rationale for the choice of target molecules was the following : cd19 and cd20 were included as b cell determinants .
cd183/cxcr3 and cd49d / vla-4 evaluation on b cells was the hypothesis - driven main objective of the study .
other chemokine receptors and cell surface molecules not immediately associated with b cell cns homing ( i.e. , cd196/ccr6 , cd195/ccr5 , cd5 ) were included as controls . from each tube ,
100,000 events were collected , and data were analyzed using facsdiva version 6.1.3 ( bd ) .
on the same day , the blood lymphocyte counts were obtained from differential white blood cell count analyzed at 0 , 1 , 3 , and 12 months using sysmex xe2100 ( sysmex , kobe , japan ) automated hematology analyzer according to standard clinical laboratory procedures , and absolute counts for b cell subpopulations were calculated from lymphocyte counts with assistance of flow cytometry results .
statistical analyses were performed separately for each antibody using sas system ( sas institute , cary , nc ) for windows 9.4 .
if residuals were normally distributed , time comparisons were done using mixed - model repeated - measures analysis of variance with heterogeneous compound symmetry covariance structure and followed by dunnett adjustment for pairwise comparisons .
otherwise , comparisons were made using signed - rank or paired t test with bonferroni adjustment multiple comparisons .
samples taken during natalizumab treatment were compared to the baseline sample . corrected 2-sided p values < 0.05 were considered statistically significant .
natalizumab treatment led to increased absolute numbers of circulating b cells , and b cell subpopulations ( table , figure ) .
the increase in circulating b cells was seen already after 1 month of treatment , and it persisted throughout the study period .
proportionately , the increase was greatest among cd10cd19cd20 pre  b cells ( median 8.3-fold ) and cxcr3cd19 b cells ( 4.7-fold ) .
natalizumab - induced persistent increase in the proportion of cd19cd20 b cells of all peripheral blood lymphocytes was observed already after 1 week of treatment ( p = 0.004 at 1 week , p = 0.0005 at 1 month , p = 0.002 at 3 months , p = 0.008 at 12 months ; table , figure ) .
natalizumab treatment increased the proportion of cd10cd19cd20 pre  b cells during the first 3 months ( p = 0.02 , p = 0.01 , p = 0.004 , and p = 0.2 , respectively ; table , figure ) .
the proportion of vla-4cd19 cells decreased already after 1 week of natalizumab treatment ( p < 0.0001 at all time points ; table , figure ) .
there was no change in the proportion of cd5cd19 native transitional b cells ( table , figure ) .
the proportion of cxcr3cd19 cells was increased after 1 month ( p = 0.014 at 1 and 3 months , p = 0.0058 at 12 months ; table , figure ) .
natalizumab - treatment did not alter the proportion of ccr6cd19 or ccr5cd19 cells ( table , figure ) .
natalizumab - induced increases in cell counts and alterations in proportions of circulating b cells in patients with multiple sclerosis ( a  h ) the absolute number of total lymphocytes and all investigated b cell populations and surface molecules , except ccr5 at 12 months , increases significantly and persistently in patients with multiple sclerosis during natalizumab treatment .
( i ) the proportion of cd19cd20 b cells of all peripheral blood lymphocytes increases in patients with multiple sclerosis already after 1 week of natalizumab treatment .
( j ) the proportion of cd10cd19cd20 pre  b cells increases already after 1 week of natalizumab treatment .
( k ) the proportion of cd5cd19cd20 b cells is not altered during natalizumab treatment .
( l ) the proportion of vla-4cd19 cells decreases already after 1 week of natalizumab treatment .
( m ) the proportion of ccr5cd19 cells is not altered during natalizumab treatment ( n ) the proportion of ccr6cd19 cells is not altered during natalizumab treatment .
( o ) the proportion of cxcr3cd19 cells increased after 1 month of natalizumab treatment .
( p ) the proportion of cxcr3cd19 cells increased in 11 of 13 patients from baseline to 3 months during natalizumab treatment .
median , lower and upper quartiles , and range ( a  h ) or mean  standard error ( i  o ) is shown except ( p ) , where lines represent intraindividual changes.*p < 0.05 , * * p < 0.01 , * * * p < 0.001 compared to baseline ( 0 months ) .
m = month ; vla-4 = very late activation antigen 4 ; w = week .
hence , blocking vla-4 using natalizumab most likely both enhances the release of b cells from lymphoid tissues and bone marrow , and prevents b cell entry from blood into tissue .
this results with increased numbers of circulating b cells as shown in this study and by others .
our result of the apparent partial decrease in b cell vla-4 expression following natalizumab treatment is in line with previous results .
in addition to adhesion molecules , chemokine receptors and chemokines contribute to recruitment of leukocytes into sites of inflammation .
not much is known , however , about the specific chemokine receptors mediating entry of b cells into the cns under inflammatory conditions .
we show here for the first time that the proportion of circulating b cells expressing the chemokine receptor cxcr3 is significantly and persistently increased during natalizumab treatment .
cxcr3 is expressed in a subset of circulating b cells and , of note , cxcr3 expression on b cells in the csf is higher in active ms patients than in controls .
it is thus possible that the increase in the proportion of cxcr3-expressing circulating b cells following natalizumab treatment is related to the reduced migration ability of these cells into the inflamed cns .
moreover , the observed reduction in the level of cxcr3-binding chemokines within the cns following natalizumab treatment might hamper the trafficking of cxcr3 b cells into cns , which further contributes to the increased proportion of these cells in the blood .
natalizumab treatment did not alter the proportions of ccr5 or ccr6 b cells , highlighting the fact that natalizumab has divergent effects on various b cell populations expressing various homing determinants .
in addition , natalizumab seems to influence differently the proportions of cxcr3-expressing circulating b and t cells , as cxcr3cd8 t cells were shown to decrease in circulation during natalizumab treatment .
our study demonstrates the early ( demonstrable already after 1 month ) and persistent ( up to 1 year ) increase in absolute numbers of circulating cd19cd20 b cells and cd10cd19cd20 pre  b cells during natalizumab treatment , which probably is caused by the rapid release of these cells from lymphoid tissues and bone marrow due to vla-4 blockage .
this phenomenon is so pronounced that it leads to an increase in absolute numbers of any b cell population studied ( table ) .
however , when proportions of various b cell subpopulations are calculated , the only increase is observed in cxcr3-expressing b cells . it has been recognized that vla-4 has an important role in mediating t lymphocyte entry into the cns , but adhesion molecules controlling the b cell entry have been much less understood .
our study suggests that some of the treatment effect of natalizumab might be attributable to prevented entry of disease - relevant b cells into the cns .
the beneficial effect on cns inflammation of b cell vla-4 expression elimination in mice supports this hypothesis .
however , the mechanistic demonstration of the role of cxcr3 in b cell entry into the inflamed cns remains to be seen .
maija saraste : analysis of the data , drafting and revising the manuscript for intellectual content .
tarja - leena penttil : design of the antibody panels for flow cytometry analysis , analysis of the data .
laura airas : design and conceptualization of the study , critical revision of manuscript for intellectual content .
maija saraste is financially supported by the university of turku doctoral programme of clinical investigation , the maud kuistila memory foundation , the finnish ms foundation , and the orion research foundation .
the sponsor had no influence on the analysis and interpretation of data , in the writing of the report , or in the decision to submit the paper .
m. saraste received travel funding from the turku university foundation , received research support from the university of turku doctoral programme of clinical investigation , the maud kuistila memory foundation , the finnish ms foundation , the orion research foundation .
l. airas served on the scientific advisory board for teva , roche , biogen , genzyme , novartis , received travel funding and/or speaker honoraria from teva , genzyme , biogen idec , sanofi - aventis , merck , novartis , received research support from merck , novartis , biogen idec .
 objective : to study the effects of natalizumab treatment on subgroups of circulating peripheral blood b cell populations.methods:we studied the proportions and absolute numbers of cd19+cd20 + , cd10 + , and cd5 + b cell populations , and determined very late activation antigen-4 and chemokine receptor cxcr3 , ccr5 , and ccr6 expression on b cells in the peripheral blood of 14 natalizumab - treated patients with relapsing - remitting multiple sclerosis . 
 five blood samples per patient were obtained longitudinally before and during the first year of treatment . 
 blood samples were analyzed by 6-color flow cytometry.results:proportions of b cells and cd10 + pre  b cells were significantly increased , and very late activation antigen-4 expression on the b cell surface was significantly decreased already after 1 week of natalizumab treatment . 
 natalizumab - induced sustained increase in the proportion and absolute number of cxcr3-expressing b cells was statistically significant after 1 month of treatment . 
 there were no changes in the proportions of ccr5- or ccr6-expressing b cells.conclusions:the rapid and persistent increase in circulating cxcr3-expressing b cells in response to natalizumab treatment possibly reflects the relevance of this chemokine receptor in controlling migration of b cells into the cns in humans in vivo . 
METHODS
Standard protocol approvals, registrations, and patient consents.
Patients.
Flow cytometry.
Statistical analyses.
RESULTS
DISCUSSION
AUTHOR CONTRIBUTIONS
STUDY FUNDING
DISCLOSURE