the university of california , san francisco ( ucsf ) institutional review board ( ucsf committee on human research ) approved acquisition and reporting of data obtained in this observational study . thirty - five patients with relapsing forms of ms from the ucsf ms center were included in this study between march 2013 and january 2015 . complete blood counts were collected at baseline and 3 months , 6 months , and 12 months ( 1.5 months ) after initiating treatment with dmf . obtained lymphocyte subsets on 25 of the patients before and during dmf treatment . at the time of analysis a total of 71.4% of patients were women ; 25.7% had been treated previously with glatiramer acetate , 28.6% with i m interferon -1a , 22.9% with natalizumab , and 2.9% with fingolimod . complete blood cell counts and lymphocyte subsets were examined by cross - sectional analysis . for complete blood cell counts : baseline ( n = 34 ) , month 3 ( n = 21 ) , month 6 ( n = 15 ) , month 12 ( n = 17 ) ; for lymphocyte subsets : baseline ( n = 21 ) , month 3 ( n = 13 ) , month 6 ( n = 13 ) , month 12 ( n = 14 ) . paired longitudinal analyses were conducted for cd4 and cd8 counts at baseline and month 12 ( n = 11 ) . statistical analyses were conducted using prism 6.0 ( graphpad software , la jolla , ca ) . statistical significance for cross - sectional analyses the university of california , san francisco ( ucsf ) institutional review board ( ucsf committee on human research ) approved acquisition and reporting of data obtained in this observational study . thirty - five patients with relapsing forms of ms from the ucsf ms center were included in this study between march 2013 and january 2015 . complete blood counts were collected at baseline and 3 months , 6 months , and 12 months ( 1.5 months ) after initiating treatment with dmf . obtained lymphocyte subsets on 25 of the patients before and during dmf treatment . at the time of analysis a total of 71.4% of patients were women ; 25.7% had been treated previously with glatiramer acetate , 28.6% with i m interferon -1a , 22.9% with natalizumab , and 2.9% with fingolimod . complete blood cell counts and lymphocyte subsets were examined by cross - sectional analysis . for complete blood cell counts : baseline ( n = 34 ) , month 3 ( n = 21 ) , month 6 ( n = 15 ) , month 12 ( n = 17 ) ; for lymphocyte subsets : baseline ( n = 21 ) , month 3 ( n = 13 ) , month 6 ( n = 13 ) , month 12 ( n = 14 ) . paired longitudinal analyses were conducted for cd4 and cd8 counts at baseline and month 12 ( n = 11 ) . statistical analyses were conducted using prism 6.0 ( graphpad software , la jolla , ca ) . statistical significance for cross - sectional analyses total leukocyte counts diminished over time , and this reduction was statistically significant ( p = 0.004 ) at month 12 of dmf therapy ( figure 1 , table ) . the absolute change in leukocytes primarily reflected a 50.1% decrease ( p < 0.0001 ) in lymphocytes . at month 12 , lymphocyte counts were below the lower limit of normal ( lln ) in 50% of patients . while we observed a reduction in eosinophils ( p = 0.006 ) , a previous report indicated that dmf use may be associated with transient eosinophilia after the first 4 weeks of treatment . of interest , one patient who had a normal baseline eosinophil level exhibited eosinophilia at month 6 of dmf treatment , but this was not sustained the level of neutrophils remained relatively stable , and although there were reductions in the numbers of monocytes and basophils , they were not significant . complete blood cell counts were obtained at baseline ( n = 34 ) and at month 3 ( n = 21 ) , month 6 ( n = 15 ) , and month 12 ( n = 17 ) of treatment . p values less than 0.05 were considered statistically significant . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . peripheral blood leukocyte and lymphocyte subset counts before and during dimethyl fumarate treatment lymphocyte subsets also decreased at month 12 of dmf treatment ( figure 2a ) . there was a 37.5% reduction of cd19 b cells ( p = 0.035 ) . while cd4 t cell counts diminished in a proportion ( 39.2% , p = 0.0006 ) similar to total cd3 t cell counts , cd8 t cell counts this relative reduction in cd8 t cells was reflected by a 35.5% increase in the cd4/cd8 ratio ( p = 0.007 ) . a paired analysis of cd4 and cd8 t cells at baseline and month 12 demonstrated a disproportionate reduction of cd8 t cells relative to cd4 t cells ( p = 0.007 ) ( figure 2b ) . at month 12 of dmf therapy , ( a ) lymphocyte subsets were obtained at baseline ( n = 21 ) and at month 3 ( n = 13 ) , month 6 ( n = 13 ) , and month 12 ( n = 14 ) of treatment . ( b ) paired longitudinal analysis ( n = 11 ) of cd4 counts , cd8 counts , and cd4/cd8 ratio between baseline and month 12 . percent reduction of cd4 and cd8 counts from baseline to month 12 was calculated for each individual . the mean reduction of cd8 t cells ( 55.5% ) was significantly greater than the reduction in cd4 t cells ( 43.4% ) ( p = 0.007 ) . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . dmf and other faes activate nuclear factor ( erythroid - derived 2)related factor 2 ( nrf2 ) , a pathway that promotes expression of products that protect against oxidative damage and that can inhibit proliferation of lymphocytes and hematopoietic stem cells . in the phase 3 ms clinical trials , dmf treatment was associated with a roughly 30% reduction in total lymphocytes . in ms clinical practice , lymphocyte counts decreased below 500 cells/l ( grade 3 lymphopenia ) in approximately 6% of patients . cd4 t cells participate in cellular immune responses and direct b - cell differentiation of antibody - secreting plasma cells . cd8 t cells , which are dependent on interleukin-2 production from cd4 t cells , participate in antiviral immunity . thus , it is important to evaluate how dmf influences each of these subsets . as part of our routine management for patients treated with dmf , we monitored lymphocyte subsets . although we examined a relatively small number of patients , the reduction in cd8 t cells was prominent and more significant than the decrease in b cells or cd4 t cells . of interest , in 1998 it was reported that fae ( combination of dmf and monoethyl fumarate ) treatment of psoriasis vulgaris was associated with a larger reduction in peripheral cd8 t cells than cd4 t cells . our report is the second study showing that an fae reduces cd8 t cells more prominently and the first study demonstrating that dmf monotherapy causes a more significant reduction in cd8 t cells than cd4 t cells . therefore , it seems that this effect is a general property of faes and should not be attributed to differences in formulation , including changes in divalent cations used in fae salt preparations . immune suppression that occurs in hiv , treatment of malignancies , or transplant rejection increases the risk of pml . the risk of pml with the use of faes , including dmf , may be quite low . currently , all pml cases associated with fae treatment have been associated with sustained lymphopenia . recently , one case of pml was reported in a patient with ms who was treated with dmf for 4.5 years and was lymphopenic for 3.5 years . in this patient , our results indicate that cd8 t lymphocytes , which are likely critical to defend against the jc virus , are reduced to a greater extent than cd4 t lymphocytes during dmf treatment . while the risk of pml with dmf treatment of ms is exceptionally low , it is plausible that a sustained reduction in cd8 t cells could be one factor predisposing to jc virus activation . second , we have not yet monitored sufficient numbers of patients beyond 12 months of dmf treatment . third , our evaluation of peripheral blood lymphocyte subsets in dmf treatment of ms may not reflect the influence of dmf on immune cells in other compartments , including the cns . nevertheless , our findings should provide incentive to conduct larger prospective investigations to examine lymphocyte subsets and immune function of cd8 and cd4 t cells in dmf treatment of ms . currently , the manufacturer recommends that lymphocyte counts be monitored every 612 months on dmf therapy and that interruption be considered when lymphocyte counts decrease below 500 cells/l and remain below that level for more than 6 months . we have observed that cd8 t lymphocyte counts fell below the lln in some patients at 12 months of dmf treatment , even though total lymphocyte counts were greater than 500 cells/l . monitoring t lymphocyte subsets during dmf treatment of ms may be beneficial . crabtree - hartman has consulted for teva , novartis , and biogen and has been on the speakers ' bureau for teva neuroscience and biogen . k. lehmann - horn received research support from the deutsche forschungsgemeinschaft ( dfg ; le 3079/1 - 1 ) and us national multiple sclerosis society ( fg 2067-a-1 ) . cree is an editor for annals of neurology ; has consulted for abbvie , biogen idec , emd serono , genzyme , medimmune , novartis , sanofi aventis , and teva neurosciences ; received research support from acorda , avanir , biogen idec , emd serono , hoffman la roche , medimmune , novartis , and teva neurosciences ; and was an expert consultant for biogen idec . zamvil received honoraria for serving on the data safety monitoring boards from bioms , teva pharmaceuticals , and eli lilly ; is the deputy editor for neurology : neuroimmunology & neuroinflammation ; has a patent pending for aquaporin-4 peptides and methods for using the same ; received speaker honoraria from biogen idec , teva neuroscience , and genzyme ; has consulted for biogen idec , teva neuroscience , emd serono , genzyme , and novartis ; is on the speakers ' bureau for advanced health media and biogen idec ; and received research support from nih ( ro1 ai073737 and ro1 ns063008 ) , nmss ( rg 4768 and rg 5180 ) , maisin foundation , and guthy jackson charitable foundation .
objective : to evaluate the influence of dimethyl fumarate ( dmf , tecfidera ) treatment of multiple sclerosis ( ms ) on leukocyte and lymphocyte subsets.methods:peripheral blood leukocyte and lymphocyte subsets , including cd3 + , cd4 + , and cd8 + t cells ; cd19 + b cells ; and cd56 + natural killer ( nk ) cells , were obtained at baseline and monitored at 3 months , 6 months , and 12 months after initiation of dmf treatment.results:total leukocyte and lymphocyte counts diminished after 6 months of dmf therapy . at 12 months , lymphocyte counts 
 had decreased by 50.1% ( p < 0.0001 ) and were below the lower limit of normal ( lln ) in one - half of patients . 
 cd3 + t lymphocyte counts fell by 44.2% ( p < 0.0001 ) . among subsets , 
 cd8 + t cell counts declined by 54.6% ( p < 0.0001 ) , whereas cd4 + t cell counts decreased by 39.2% ( p = 0.0006 ) . 
 this disproportionate reduction of cd8 + t cells relative to cd4 + t cells was significant ( p = 0.007 ) and was reflected by a 35.5% increase in the cd4/cd8 ratio ( p = 0.007 ) . 
 a majority of cd8 + t cell counts , but not cd4 + t cell counts , were below the lln even when total lymphocyte counts were greater than 500 cells/l . cd19 + b 
 cell counts were reduced by 37.5% ( p = 0.035 ) . 
 eosinophil levels decreased by 54.1% ( p = 0.006 ) , whereas levels of neutrophils , monocytes , basophils , and nk cells were not significantly altered.conclusion:subsets of peripheral blood leukocytes and lymphocytes are differentially affected by dmf treatment of ms . 
 reduction of cd8 + t cells is more pronounced than that of cd4 + t cells . 
 these findings may have implications for cell - mediated antiviral immunity during dmf treatment .