all patients gave written informed consent for the provision of blood for the purpose of this research . the protocol under which these samples were obtained was approved by the ethics committee of the university of tbingen , germany . patient characteristics white blood cell counts were determined by routine testing at the central laboratory of our hospital . flow cytometric analysis was performed with fresh whole blood specimens after staining with fluorochrome - labeled antibodies . the following antibodies were used : anti - human cd3-apc ( 1/100 ) clone ucht1 , anti - human cd4-fitc ( 1/100 ) clone sk3 , anti - human cd8-percp ( 1/100 ) clone sk1 , anti - human cd19-fitc ( 1/100 ) clone hib19 , anti - human cd27-pe ( 1/50 ) clone l128 , anti - human cd38-apc ( 1/50 ) clone hit2 , anti - human cd56-pe ( 1/50 ) clone b159 , anti - human cd197(ccr7)-pe ( 1/50 ) clone 3d12 , all from bd biosciences , and anti - human cd45ra - apc ( 1/50 ) clone hi100 from biolegend . as a reference absolute numbers of cd4 and cd8 t cells were calculated by multiplying lymphocyte numbers obtained from the white blood cell count with the frequency of cd4 and cd8 cells in flow cytometric analysis . jcv copy numbers were determined by quantitative pcr at the institute for virology , heinrich heine university , dsseldorf , germany . statistical analysis was performed by unpaired t test using spss 22 ( ibm corp . , all patients gave written informed consent for the provision of blood for the purpose of this research . the protocol under which these samples were obtained was approved by the ethics committee of the university of tbingen , germany . white blood cell counts were determined by routine testing at the central laboratory of our hospital . flow cytometric analysis was performed with fresh whole blood specimens after staining with fluorochrome - labeled antibodies . the following antibodies were used : anti - human cd3-apc ( 1/100 ) clone ucht1 , anti - human cd4-fitc ( 1/100 ) clone sk3 , anti - human cd8-percp ( 1/100 ) clone sk1 , anti - human cd19-fitc ( 1/100 ) clone hib19 , anti - human cd27-pe ( 1/50 ) clone l128 , anti - human cd38-apc ( 1/50 ) clone hit2 , anti - human cd56-pe ( 1/50 ) clone b159 , anti - human cd197(ccr7)-pe ( 1/50 ) clone 3d12 , all from bd biosciences , and anti - human cd45ra - apc ( 1/50 ) clone hi100 from biolegend . as a reference absolute numbers of cd4 and cd8 t cells were calculated by multiplying lymphocyte numbers obtained from the white blood cell count with the frequency of cd4 and cd8 cells in flow cytometric analysis . jcv copy numbers were determined by quantitative pcr at the institute for virology , heinrich heine university , dsseldorf , germany . statistical analysis was performed by unpaired t test using spss 22 ( ibm corp . , armonk , ny ) . three female and 2 male patients between 39 and 56 years of age were recruited at our institution at the initial presentation of pml between 2012 and 2015 ( table ) . two patients had hiv - associated pml , 2 patients were on treatment with fumaric acid ( fumaderm ) for psoriasis for more than 6 months , and one patient with relapsing - remitting multiple sclerosis was on natalizumab for more than 24 months . pml outcome was related to the jcv copy numbers within the csf at the time of pml diagnosis . in one patient with negative jcv - pcr , an immune reconstitution inflammatory syndrome ( iris ) developed in both patients with hiv - pml following initiation of combined antiretroviral therapy and in the natalizumab - treated patient with multiple sclerosis following plasma exchange . iris was characterized by the development of new neurologic symptoms and new contrast - enhancing lesions on cerebral mri . two patients with less than 500 jcv copies per ml csf had only moderate residual symptoms and 2 patients who displayed > 10,000 copies of the jcv dna per ml of csf died of pml within 44 and 61 days , respectively . all patients had reduced lymphocyte numbers at the initial diagnosis of pml ( table ) . at the initial diagnosis of pml , peripheral blood samples were obtained from all patients and cells were directly analyzed by flow cytometry after staining with the indicated fluorochrome - labeled antibodies ( figure 1 ) . as compared with healthy controls , the frequency of central memory cd4 t cells was highly significantly reduced ( figure 2 ; p < 0.00001 ) , as was the frequency of naive cd4 t cells ( p = 0.04 ) . the proportion of effector memory cd4 t cells was increased ( p = 0.01 ) . in contrast , the frequencies of naive , central memory and effector memory cd8 t cells ( figure 2 ) , cd19 b lymphocytes , cd19cd38 plasma cells , cd19cd27 memory b cells , and cd56 natural killer cells ( figure 3 ) were not significantly different from healthy controls . expression of ccr7 and cd45ra was determined on live cd4 and cd8 cells , and expression of cd38 and cd27 was determined on live cd19 cells as indicated . reduced frequency of central memory cd4 t cells ( cd4tcm ) ( p < 0.00001 ) , naive cd4 t cells ( p = 0.04 ) , and effector memory cd4 t cells ( cd4tem ) ( p = 0.01 ) in patients with pml . healthy controls ( hc ) are depicted in the first column of each dotplot , patients with pml in the second . frequency of cd19 b lymphocytes , cd19cd38 plasma cells , cd19cd27 memory b cells , and cd56 nk cells does not significantly differ from healthy controls ( hc ) . healthy controls are depicted in the first column of each dotplot , patients with pml in the second . nk = natural killer ; pml = progressive multifocal leukoencephalopathy . longitudinal analysis of peripheral immune cells was performed during the course of pml in patients 1 and 2 . patient 1 displayed a high jcv viral load within the csf ( 27,300 copies / ml csf ) and died 61 days after initial pml diagnosis . he displayed highly reduced frequencies of naive cd4 t cells and central memory cd4 t cells and an increased proportion of effector memory cd4 t cells throughout the progressive course of the disease ( figure 4 ) . in patient 2 , the reduced frequencies of naive cd4 t cells and central memory cd4 t cells returned almost to normal levels after initiation of combined antiretroviral therapy and during subsequent resolution of pml . patient 1 neither clinically nor radiologically showed signs of iris whereas patient 2 already showed radiologic signs ( edema , gadolinium enhancement ) when diagnosed with pml . frequency of naive , central memory ( tcm ) , and effector memory ( tem ) cd4 t cells over the course of pml in patient 1 ( open circles ) and patient 2 ( closed triangles ) . gray areas indicate normal values ( mean values  2 sds of healthy controls ) . three female and 2 male patients between 39 and 56 years of age were recruited at our institution at the initial presentation of pml between 2012 and 2015 ( table ) . two patients had hiv - associated pml , 2 patients were on treatment with fumaric acid ( fumaderm ) for psoriasis for more than 6 months , and one patient with relapsing - remitting multiple sclerosis was on natalizumab for more than 24 months . pml outcome was related to the jcv copy numbers within the csf at the time of pml diagnosis . in one patient with negative jcv - pcr , an immune reconstitution inflammatory syndrome ( iris ) developed in both patients with hiv - pml following initiation of combined antiretroviral therapy and in the natalizumab - treated patient with multiple sclerosis following plasma exchange . iris was characterized by the development of new neurologic symptoms and new contrast - enhancing lesions on cerebral mri . two patients with less than 500 jcv copies per ml csf had only moderate residual symptoms and 2 patients who displayed > 10,000 copies of the jcv dna per ml of csf died of pml within 44 and 61 days , respectively . all patients had reduced lymphocyte numbers at the initial diagnosis of pml ( table ) . at the initial diagnosis of pml , peripheral blood samples were obtained from all patients and cells were directly analyzed by flow cytometry after staining with the indicated fluorochrome - labeled antibodies ( figure 1 ) . as compared with healthy controls , the frequency of central memory cd4 t cells was highly significantly reduced ( figure 2 ; p < 0.00001 ) , as was the frequency of naive cd4 t cells ( p = 0.04 ) . the proportion of effector memory cd4 t cells was increased ( p = 0.01 ) . in contrast , the frequencies of naive , central memory and effector memory cd8 t cells ( figure 2 ) , cd19 b lymphocytes , cd19cd38 plasma cells , cd19cd27 memory b cells , and cd56 natural killer cells ( figure 3 ) were not significantly different from healthy controls . expression of ccr7 and cd45ra was determined on live cd4 and cd8 cells , and expression of cd38 and cd27 was determined on live cd19 cells as indicated . nk = natural killer cells ; nkt = natural killer t cells . reduced frequency of central memory cd4 t cells ( cd4tcm ) ( p < 0.00001 ) , naive cd4 t cells ( p = 0.04 ) , and effector memory cd4 t cells ( cd4tem ) ( p = 0.01 ) in patients with pml . healthy controls ( hc ) are depicted in the first column of each dotplot , patients with pml in the second . frequency of cd19 b lymphocytes , cd19cd38 plasma cells , cd19cd27 memory b cells , and cd56 nk cells does not significantly differ from healthy controls ( hc ) . healthy controls are depicted in the first column of each dotplot , patients with pml in the second . longitudinal analysis of peripheral immune cells was performed during the course of pml in patients 1 and 2 . patient 1 displayed a high jcv viral load within the csf ( 27,300 copies / ml csf ) and died 61 days after initial pml diagnosis . he displayed highly reduced frequencies of naive cd4 t cells and central memory cd4 t cells and an increased proportion of effector memory cd4 t cells throughout the progressive course of the disease ( figure 4 ) . in patient 2 , the reduced frequencies of naive cd4 t cells and central memory cd4 t cells returned almost to normal levels after initiation of combined antiretroviral therapy and during subsequent resolution of pml . patient 1 neither clinically nor radiologically showed signs of iris whereas patient 2 already showed radiologic signs ( edema , gadolinium enhancement ) when diagnosed with pml . frequency of naive , central memory ( tcm ) , and effector memory ( tem ) cd4 t cells over the course of pml in patient 1 ( open circles ) and patient 2 ( closed triangles ) . gray areas indicate normal values ( mean values  2 sds of healthy controls ) . herein , we provide data indicating that alterations in the composition of peripheral cd4 helper t cell subpopulations are associated with development of pml . cd4 t cells have a pivotal role in the defense against viral infections as has been shown in numerous human diseases including hiv , hepatitis c virus , and jcv . the development of pml is known to be associated with low total numbers of cd4 t cells in patients with late - stage hiv infections and patients with idiopathic cd4 t cell lymphopenia . our patients with pml did not display any significant alterations in the frequencies of naive , central memory and effector memory cd8 t cells , b lymphocyte populations , and natural killer cells . in contrast , the proportions of cd4 central memory and naive t cells were highly significantly decreased and the proportion of effector memory cd4 t cells was increased . central memory t cells express the chemokine receptor crr7 and cd62l and recirculate through secondary lymphoid organs . effector memory cd4 t cells do not express ccr7 and cd62l , molecules required for entry into lymph nodes , but express chemokine receptors such as ccr5 , which direct their migration to the sites of inflammation . they are preferentially located within nonlymphoid tissues or remain within the blood and rapidly exhibit effector functions after a new antigen encounter . the fact that the proportion of cd4 effector memory t cells was high in the peripheral blood of patients with pml thus indicates that a large proportion of peripheral cd4 t cells is actively engaged in viral defense . this is consistent with the previously observed prominent infiltration of cd4 t cells into inflammatory cns lesions in pml and further supports the central role of cd4 rather than cd8 t cells in immune defense against jcv . before the diagnosis of pml , 2 patients had been on immunomodulatory treatment with fumaric acid and one patient with natalizumab . these treatments are known to induce alterations in peripheral immune cells and may thus have influenced our results . dimethyl fumarate has been shown to both reduce the frequency of lymphocytes in peripheral blood and to disproportionally reduce the frequency of cd8 t cells , while natalizumab increases peripheral lymphocyte counts and the proportion of peripheral b lymphocytes . the fact that the reduction of naive and central memory cd4 t cells was partially restored in patient 2 after resolution of pml and not in patient 1 who displayed a fatal course of pml may further indicate that the frequencies of these cells negatively correlate to pml disease activity . collectively , these observations are in line with previous reports that found reduced frequencies of cd62l - expressing cd4 t cells in natalizumab - treated patients with relapsing - remitting multiple sclerosis who developed pml . in our cohort , the frequency of central memory cd4 t cells was reduced in patients with hiv - associated pml as well as in patients who developed pml under immunomodulatory treatment . this further supports the notion that the reduced frequency of central memory cd4 t cells represents a state of the immune system with reduced immunocompetence against jcv . furthermore , these data indicate that additional in - depth analysis of the role of cd4 helper t cell populations during the course of pml could provide important insights into the immune conditions that predispose to this disease . reconstitution of cd4 t cells in longitudinal analysis might be a predictor for clinical outcome . obviously , this investigation is limited by the small number of patients , by the different conditions and medications under which pml developed , and the different courses of disease ( i.e. , with and without development of iris ) . if confirmed , the observed alterations in cd4 t cell subpopulations could provide new insights into the pathogenesis of pml and may ultimately represent a biomarker for pml disease activity or to detect patients at risk of pml development . this would be urgently needed as jcv infections of the nervous system including pml , jcv meningitis , and cerebellar granule cell neuronopathy become more clinically prominent . study concept and design : f.b . acquisition of data : e.d . analysis and interpretation : e.d . e. dubois reports no disclosures . c. ruschil is on the scientific advisory board for novartis pharma gmbh . f. bischof served on the scientific advisory board for genzyme , novartis , and roche , received speaker honoraria and travel funding from biogen idec , genzyme , and novartis , and received research support from novartis .
objectives : to assess alterations in the composition of peripheral immune cells in acute progressive multifocal leukoencephalopathy ( pml).methods : fresh blood samples from 5 patients with acute pml and 10 healthy controls were analyzed by flow cytometry for naive , central memory and effector memory cd4 and cd8 t cells , b lymphocytes , plasma cells , memory b cells , plasma blasts , and natural killer ( nk ) cells . 
 the frequency of central memory cd4 t cells was determined longitudinally during the course of pml in 2 patients.results:the frequencies of naive , central memory and effector memory cd8 t cells , b cells , plasma cells , and nk cells were not altered in patients with pml . 
 in contrast , the frequencies of naive cd4 t cells ( p = 0.04 ) and central memory cd4 t cells ( p < 
 0.00001 ) were reduced and the frequencies of effector memory cd4 t cells were increased ( p = 0.01 ) . 
 longitudinal analysis showed that this pattern was preserved in a patient with fatal pml outcome and restored in one patient who recovered from pml.conclusions:these data indicate that pml is associated with reduced frequencies of peripheral central memory helper t cells but not with alterations in the frequencies of cytotoxic t cell populations , b lymphocytes , plasma cells , or nk cells .