patients with ms under daclizumab therapy mount normal ... · activation-induced cell death6,7 and...

Yen Chih Lin PhDPaige Winokur BSAndrew Blake BSTianxia Wu PhDJody Manischewitz MSLisa R King BSElena Romm MSHana Golding PhDBibiana Bielekova MD

Correspondence toDr Bielekovabibibielekovanihgov

Supplemental dataat Neurologyorgnn

Patients with MS under daclizumabtherapy mount normal immune responsesto influenza vaccination

ABSTRACT

Objective The purpose of this study was to assess the potential immunosuppressive role of da-clizumab a humanized monoclonal antibody against the a chain of the interleukin 2 receptorin vivo by comparing immune responses to the 2013 seasonal influenza vaccination betweenpatients with multiple sclerosis (MS) on long-term daclizumab therapy and controls

Methods Previously defined subpopulations of adaptive immune cells known to correlate with theimmune response to the influenza vaccination were evaluated by 12-color flow cytometry in 23daclizumab-treated patients with MS and 14 MS or healthy controls before (D0) and 1 day (D1)and 7 days (D7) after administration of the 2013 Afluria vaccine Neutralizing antibody titers andCD41 CD81 T cell B cell and natural killer cell proliferation to 3 strains of virus contained in theAfluria vaccine were assessed at D0 D7 and 180 days postvaccination

Results Daclizumab-treated patients and controls demonstrated comparable statistically signif-icant expansions of previously defined subpopulations of activated CD81 T cells and B cells thatcharacterize the development of effective immune responses to the influenza vaccine whileproliferation of T cells to influenza and control antigens was diminished in the daclizumab cohortAll participants fulfilled FDA criteria for seroconversion or seroprotection in antibody assays

Conclusion Despite the mild immunosuppressive effects of daclizumab in vivo demonstrated byan increased incidence of infectious complications in clinical trials patients with MS under dacli-zumab therapy mount normal antibody responses to influenza vaccinations Neurol Neuroimmunol

Neuroinflamm 20163e196 doi 101212NXI0000000000000196

GLOSSARYAb 5 antibody Ag 5 antigen CMV 5 cytomegalovirus DAC-HYP 5 daclizumab high-yield process DC 5 dendritic cellEBV 5 Epstein-Barr virus Flu-HA 5 influenza hemagglutinin Ig 5 immunoglobulin IL-2R 5 interleukin 2 receptor ILC 5innate lymphoid cell MS 5 multiple sclerosis NK 5 natural killer PBMC 5 peripheral blood mononuclear cell RRMS 5relapsing-remitting multiple sclerosis

Daclizumab high-yield process (DAC-HYP [Biogen Idec Boston MA and AbbVie IncNorth Chicago IL]) a humanized monoclonal antibody (Ab) against CD25 the a chainof the high-affinity interleukin 2 receptor (IL-2R) with proven clinical efficacy in multiplesclerosis (MS)12 was conceptually developed as a selective blocker of activated T cells becauseT cells upon activation upregulate CD25 and consume IL-23 Although in vitro studies usingnonphysiologically high concentrations of daclizumab supported a direct inhibitory role ofdaclizumab on T cells polyclonally activated T cells isolated from patients under daclizumabtherapy had unhindered proliferation and cytokine production45 Conversely via inhibition ofactivation-induced cell death67 and FoxP31 regulatory T cells89 daclizumab augments sur-vival of activated T cells in vivo Consistent with these observations both CD25-deficientmice and humans experience lymphoproliferation10ndash12 However CD25-deficient humans are

From the Neuroimmunological Diseases Unit Neuroimmunology Branch (YCL PW AB ER BB) and Clinical Neuroscience Program(TW) National Institute of Neurological Diseases and Stroke NIH Bethesda MD FDA (JM LRK HG) CBER and NIH Center forHuman Immunology (BB) NIH Bethesda MD

Funding information and disclosures are provided at the end of the article Go to Neurologyorgnn for full disclosure forms The Article ProcessingCharge was paid by NINDS

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CCBY-NC-ND) which permits downloading and sharing the work provided it is properly cited The work cannot be changed in any way or usedcommercially

Neurologyorgnn copy 2016 American Academy of Neurology 1

ordf 2016 American Academy of Neurology Unauthorized reproduction of this article is prohibited

also immunocompromised and daclizumabtreatment causes a slight increase in infectiouscomplications in phase II13ndash15 and phase IIItrials12

Trying to explain this apparent paradox wediscovered that daclizumab limits activationpriming of antigen (Ag)-specific CD41 andCD81 T cells indirectly by limiting dendriticcell (DC)-mediated trans-presentation of IL-25 This early IL-2 signal delivered at the timewhen naive T cells do not yet express high-affinity IL-2R is crucial for their differentia-tion to T cell effectors Daclizumab also hasunanticipated effects on innate lymphoid cells(ILCs) promoting differentiation of ILC pre-cursors away from proinflammatory lymphoidtissue inducer (subtype of ILC3) cells andtoward CD56bright natural killer (NK) cells716

These immunoregulatory NK cells can killactivated autologous T cells1617 thus provid-ing overlapping functions with regulatory Tcells

These multiple and unique mechanisms ofaction underlie efficacy of daclizumab inrelapsing-remitting MS (RRMS)1213ndash15 The

question remains how potently immunosup-pressive daclizumab therapy really is willdescribed effect on innate immunity preventactivation of CD41 CD81 T cells and B cellsplasma cells to common infectious pathogensTherefore the purpose of this study was toassess the potential immunosuppressive roleof daclizumab in vivo by comparing immuneresponses from patients with MS on long-termdaclizumab therapy and controls to the sea-sonal influenza vaccination

METHODS Standard protocol approvals registrationsand patient consents The study was approved by the NIH

institutional review board and all patients provided written con-

sent The study was performed under investigational new drug

application (IND 107973 IND sponsor BielekovaNational

Institute of Neurological Disorders and Stroke [NINDS]) as part

of NINDS clinical trial 10-N-0125 ldquoInvestigating mechanism of

action of DAC-HYP in the treatment of high-inflammatory

multiple sclerosis (MS)rdquo (ClinicalTrialsgov identifier

NCT01143441)

Participants Participant demographics and diagnosis are

presented in table 1 Twenty-three patients with RRMS

received DAC-HYP 150 mg subcutaneously every 4 weeks for

a minimum of 36 months 609 (1423) of these patients were

treated with a previous formulation of daclizumab (Zenapax

Hoffmann-La Roche Basel Switzerland) for up to 6 years

Table 1 Optimized combination of commercially available fluorochrome-conjugated antibodies to reliably quantify T cell and monocyte B celland RORgt1 immune cells

Conjugation T cell and monocyte B cell Intracellular staining

FITC Anti-human CD69 antibody (BD FN50) Anti-human CD40 antibody (Invitrogen HB14) Anti-human CD56 antibody (BD MEM188)

PE Anti-human CD40 antibody (BioLegend HD14) Anti-human CD21 antibody (Beckman CoulterA32524)

Anti-human RORgd antibody (RampD600380)

PerCP-Cy55 Anti-human HLA-DR antibody (BD Tu36) Anti-human CD20 antibody (BD 2H7) Anti-human CD123 antibody (eBioscience7G3)

PE-Cy7 Anti-human CD86 antibody (BioLegend IT22) Anti-human CD86 antibody (BioLegend IT22) Anti-human CD11c antibody (eBioscience39)

V450 Anti-human CD4 antibody (BD RPA-T4) Anti-human CD80 antibody (BD L3074) Anti-human CD45 antibody (BD HI30)

Brilliant Violet510

Anti-human CCR7CD197 antibody (BioLegendG043H7)

Anti-human IgM antibody (Biolegend MHM-88)

AmCyan Anti-human CD8 antibody (BD SK1)

Qdot 605 Anti-human CD8 antibody (Invitrogen 3B5) Anti-human CD27 antibody (Invitrogen CLB-271) Anti-human CD19 antibody (eBioscienceHIB19)

Qdot 655 Anti-human CD14 antibody (Invitrogen TuK4) Anti-human CD19 antibody (Invitrogen SJ25-C1) Anti-human CD3 antibody (eBioscienceOKT3)

Qdot 705 Anti-human CD4 antibody (InvitrogenS35)

Qdot 800 Anti-human CD45 antibody (Invitrogen H130) Anti-human CD45 antibody (Invitrogen H130)

APC Anti-human CD25 antibody (BioLegend M-A251) Anti-human IgD antibody (BioLegend HIT2)

Alexa Fluor 700 Anti-human CD38 antibody (BioLegend HIT2) Anti-human CD38 antibody (MACS Miltenyi130-094-553)

Anti-human CD14 antibody (BioLegendHCD14)

APC-Cy7 Anti-human CD3 antibody (BioLegend SK7) Anti-human CD10 antibody (BioLegend HI10a) Anti-human HLA-DR antibody (eBioscienceLN3)

Abbreviations BD 5 Becton Dickinson FITC 5 fluorescein isothiocyanate IgM 5 immunoglobulin M RampD 5 RampD SystemsPresented as name of antibody (company clone)

2 Neurology Neuroimmunology amp Neuroinflammation


before enrollment in the 10-N-0125 protocol DAC-HYP has

the identical amino sequence of Zenapax but because of

expression in different cell types its glycosylation changes

affect its binding to Fc receptors18 Controls (10 patients with

RRMS and 4 controls with no evidence of CNS inflammation

see table e-1 at Neurologyorgnn for details) were prospectively

recruited from the natural history protocol 09-N-0032 (table e-

1) Diagnosis of RRMS was based on the 2010 revisions to the

McDonald diagnostic criteria19 The local patients (DAC-HYP

n 5 17 controls n 5 14) had sample collection and analysis

performed at all time points (day 0 [D0] D1 D7 and D180)

whereas patients requiring long-distance travel to NIH had

analysis only at D0 D1 and D180 (DAC-HYP n 5 6

controls none)

Sample preparation and peripheral blood mononuclearcell isolation All samples were assigned an alpha-numeric code

Personnel generating data were blinded to the participantsrsquo

diagnoses or treatment assignment The data obtained from MS

and non-MS controls were overlapping and therefore we did not

sub-divide this control group based on diagnostic conclusions

Peripheral blood mononuclear cells (PBMCs) were iso-

lated from blood immediately after collection in LeucoSep

tubes before the vaccine (D0) 1 day later (D1) and 7 days

later (D7) according to manufacturerrsquos instruction Briefly

LeucoSep tubes were prepared by spinning 15 mL of lym-

phocyte separation medium (Lonza) at room temperature for

30 seconds at 1000g Twenty-five milliliters of freshly col-

lected blood was added into tubes which were then spun for

Figure 1 Flu immunizationndashinduced changes in the proportions and absolute numbers of T cells and theirrelevant subpopulations

CD41 and CD81 T cells and their subsets that were previously shown to change in response to influenza vaccines (HLA-DR1

effector cells and CD691-activated CD8 T cells) were assessed by multicolor flow cytometry before (D0) and 1 day (D1) and7 days (D7) postimmunization with Afluria 2013 vaccine The 2 left panels in each row represent absolute numbers ofidentified cell populations in the daclizumab-treated patients and controls while the 2 right panels represent proportions ofthe same cell populations in the identical cohorts The vertical box plots show the median and 25ndash75 range while thewhiskers reflect minimum and maximum values for each diagnostic group The scatter dot plots correspond to individualpatientrsquos data p 005 0001 p 005 p 0001 Abs 5 antibodies DAC 5 daclizumab

Neurology Neuroimmunology amp Neuroinflammation 3


10 minutes under the same conditions Monocytes were

removed from the tube wall via scraping and the supernatants

were transferred to new 50-mL centrifugation tubes and

washed twice The cells were counted by hemocytometer

and aliquotted between flow cytometry and Ag-specificity

assays

Autologous plasma was preserved for quenching the Trace-

CellViolet staining reaction (see below) Serum was collected at

each time point into BD Vacutainer SST tubes (Becton Dickin-

son Franklin Lakes NJ) spun aliquotted and frozen (280degC)

until analysis of neutralizing Abs while remaining serum was

used fresh for T cell Ag specificity assays

Figure 2 Flu immunizationndashinduced changes in the proportions and absolute numbers of B cells and theirrelevant subpopulations

CD191 B cells and their subsets (naive CD861 B cells transitional IgD1CD271CD381 B cells and 2 closely relatedpopulations of memory B cells IgD2CD271CD381 and IgD2CD271CD801) were assessed by multicolor flow cytometrybefore (D0) and 1 day (D1) and 7 days (D7) postimmunization with Afluria 2013 vaccine The 2 left panels in each rowrepresent absolute numbers of identified cell populations in the daclizumab-treated patients and controls while the 2 rightpanels represent proportions of the same cell populations in the identical cohorts The vertical box plots show the medianand 25ndash75 range while the whiskers reflect minimum and maximum values for each diagnostic group The scatter dotplots correspond to individual patientrsquos data p 005 0001 p 005 p 0001 Abs 5 antibodies DAC 5

daclizumab IgD 5 immunoglobulin D



Ex vivo flow cytometry immunophenotyping For ex vivoimmunophenotyping multicolor staining combinations were de-

signed (table 1) to include T and B cell subpopulations that were

found to be responsive to influenza vaccination in the previous

study20 The 2 3 106 blood cells (after lysing red blood cells) or

PBMCs were stained according to a previously published proto-

col21 that includes blockade of Fc receptors by 2 IV immuno-

globulin (Ig) For intracellular staining of ROR-gt a minimum of

106 blood cells were stained for surface markers Then cells were

fixed by CytofixCytoperm buffer and washed by PermWash

buffer (all BD Biosciences) before staining with intracellular Ab

Cells were immediately acquired on a BD LSR II equipped

with a High Throughput Sampler and analyzed with FACSDiva

61 software (all BD Biosciences) Gating was based on isotype

controls (see figure e-1 for gating strategy)

T cell Ag specificity assay PBMCs were stained with 5 mM

CellTraceViolet (Invitrogen Carlsbad CA) according to the

manufacturerrsquos protocol with the modification that saved autol-

ogous plasma was used for quenching of the staining reaction

After washing CellTraceViolet-stained PBMCs were counted

and resuspended in autologous serum and seeded at 1 3 106

cells100 mLwell into U-bottom 96-well plates Ags (influenza

hemagglutinin [Flu-HA Afluria vaccine] brain homogenate

isolated from brain of a patient with MS obtained by rapid

autopsy and prepared as described22 and 2 control viral

complex antigens commercially available lysates of Epstein-Barr

virus [EBV] and human cytomegalovirus [CMV Advanced

Biotechnology]22) all used at 1 mgmL final concentration were

added to appropriate wells in 100 mL of X-vivo medium and

incubated at 37degC 5 CO2 On day 6 of incubation wells

that depleted nutrients and acidified medium were split into 2

new wells (in autologous serum to X-vivo medium at 11 ratio)

On day 9 cell pellets were collected and proliferation of different

cell populations was assessed by flow cytometry after surface

staining with the following antibodies CD19 (Mouse IgG1k

Clone HIB19 BD Biosciences) CD56 (Mouse IgG1k Clone

MY31 BD Biosciences) CD8 (Mouse IgG1k Clone RPA-T8

BD Biosciences) CD3 (Mouse IgG2ak Clone OKT3

eBiosciences) and CD4 (Mouse IgG2b Clone OKT4

eBiosciences) Before acquisition by flow cytometer 10 mL

SPHERO AccuCount Ultra Rainbow Fluorescent Particles was

added to each well to proportionally enumerate cells between

different Ag conditions

Neutralizing Ab titers Viral-neutralizing activity was analyzedby a microneutralization assay in MDCK cells using minor mod-

ifications of previously published methods of the pandemic influ-

enza reference laboratories of the Centers for Disease Control and

Prevention23 Specifically Ab-neutralization titers were measured

against the 3 influenza strains in the inactivated influenza virus

(TIV) vaccine used for immunization of study participants Sera

were tested at an initial dilution of 120 and those that were

negative (120) were assigned an arbitrary titer of 10 All sera

were tested in triplicate using the geometric mean value for final

analysis

Statistical analysis For each T cell or B cell expression abso-

lute numbers or proportions the time effect (D0 D1 D7 or

the difference between 3 time points) was examined for daclizu-

mab and control group separately using 1-way repeated-

measures analysis of variance (figures 1ndash3) with a compound

symmetry structure among the time levels For each cell

proliferation the effect of condition (with 5 levels control

BM CMV EBV and Flu) was examined for daclizumab and

control group separately using 1-way analysis of variance

Figure 3 Flu immunizationndashinduced changes in the proportions and absolute numbers of RORgt-expressingCD41 and CD81 T cells

Expression of RORgt the master regulator of Th17 lineage was assessed in CD41 and CD81 T cells by intracellular stainingbefore (D0) and 1 day (D1) and 7 days (D7) postimmunization with Afluria 2013 vaccine The 2 left panels in each rowrepresent absolute numbers of identified cell populations in the daclizumab-treated patients and controls while the 2 rightpanels represent proportions of the same cell populations in the identical cohorts The vertical box plots show the medianand 25ndash75 range while the whiskers reflect minimum and maximum values for each diagnostic group The scatter dotplots correspond to individual patientrsquos data p 005 0001 p 005 p 0001 Note the differences in scalesbetween the daclizumab and control cohorts Abs 5 antibodies DAC 5 daclizumab



(figure 4) For the B cell titers of 3 viruses in the Afluria vaccine

the time effect was evaluated for daclizumab and control group

separately using a mixed model including time and virus effect

with participants as random effect and a compound symmetry

structure among the time levels within the virus The

interaction between time and virus and age as covariate were

examined and dropped from the model

Pairwise multiple comparisons between the 3 time levels or 5

condition levels using Tukey method were applied to the response

variables with significant time (or condition) effect As most of

the response variables did not have normal distribution Box-

Cox procedure was used to choose the power (or lambda) for data

transformation Shapiro-Wilk test was applied to the residuals for

normality test Statistical analyses were performed using SAS ver-

sion 92 (SAS Institute Cary NC)

RESULTS Ex vivo immunophenotyping We observedthat seasonal Flu-HA immunization (Afluria 2013vaccine) induced analogous statistically significantexpansion of specific T cell (figure 1) and B cell(figure 2) subpopulations in the current study aswere previously described in relationship to 2009seasonal and pandemic H1N1 Flu vaccines20

Consistent with preexisting immunity observed inthe majority of participants in both studied cohortsthe activationexpansion of the cells of adaptiveimmunity occurred early 1 day postvaccination

On the T cell side we observed significantincrease in the absolute numbers and proportions ofrecently activated HLA-DR1 CD41 T cells (ID 10in the original publication20 of Flu-induced immunechanges on which the current study was designed seefigure e-1 for gating strategy) and HLA-DR1 CD81

T cells (ID42) In the daclizumab cohort there was atrend for proportional expansion of these cells whichdid not reach significance after adjustment for multi-ple comparisons In contrast CD691 CD81 T cells(ID39) were significantly expanded in both cohorts

On the B cell side Flu-HA induced proportionalincrease in B cells which was entirely driven by bothabsolute and proportional increase in memory B cellsboth IgD2CD271CD381 (ID96) and IgD2CD271CD801 (ID97) which were expanded tosimilar and highly statistically significant levels inboth cohorts Transitional B cells (IgD1CD272CD381 ID103) were expanded proportionally buttheir absolute numbers varied between different par-ticipants while naive-activated B cells (IgD1CD272CD861 ID110) were not influenced byFlu vaccination on a group level consistent withthe preexisting immune response in the majority oftested participants

Figure 4 Flu immunizationndashinduced changes in cellular proliferation toexogenously added complex antigens

Freshly isolated peripheral blood mononuclear cells from blood samples collected before(D0) and 7 days (D7) after vaccination with Afluria 2013 vaccine were stained with CellTra-ceViolet vital fluorescent dye before addition of complex antigens (brain homogenate [BH]cytomegalovirus [CMV] Epstein-Barr virus [EBV] and Afluria 2013 vaccine [Flu]) in thepresence of autologous serum (and therefore with in vivondashachievable concentrations ofDAC-HYP in treated patients) Proliferation of CD41 and CD81 T cells B cells and NK cellswasmeasured 9 days later by quantifying proportion of cells that diluted CellTraceViolet andis expressed as stimulation index (ratio of proliferation measures in wells with exogenouslyadded Ag as compared to control wells where no exogenous Ag was added) Results aredepicted for daclizumab-treated patients (left column) and controls (right column) Thevertical box plots show the median and 25ndash75 range while the whiskersreflect minimum and maximum values for each diagnostic group The scatter dot plots

correspond to individual patientrsquos data p 0050001 p 005 p 0001 Ag 5 antigen DAC 5

daclizumab DAC-HYP 5 daclizumab high-yield processNK 5 natural killer



Intracellular staining for RORgt Because IL-2signaling was shown to antagonize Th17differentiation24 we asked whether Flu-HAndashdriveninduction of the master regulator of Th17 T cellsRORgt is altered by daclizumab therapy Toanswer this we used intracellular staining forRORgt (table 1 last column) We reasoned that ifIL-2 signaling inhibits Th17 differentiation theninhibiting IL-2 signaling during evolving immuneresponse by daclizumab would enhance Th17differentiation which could be detected byincreased RORgt expression in T cells Contrary tothis assumption while we observed increased absolutenumbers and proportions of RORgt expressingCD41 and CD81 T cells in both cohorts thechanges in CD41 T cells reached statisticalsignificance only in the controls Furthermoreabsolute numbers as well as proportions ofRORgt1 CD41 and CD81 T cells were higher(not significant) in controls than in daclizumab-treated patients (figure 3 please note differences inscales)

Cellular Ag specificity Both cohorts had preexistingimmunity to Flu-HA as evidenced by highstimulation indices (ratio of numbers ofproliferating cells in the condition with Ag dividedby background proliferation observed in the wellswhere no exogenous Ag was added) observedalready at D0 before administration of the vaccine(figure 4) In fact the difference in proliferation ofT cells or B cells to Flu-HA Ag between D0 and D7did not reach statistical significance in either cohortWe also observed robust proliferation to CMV andEBV in the control group but not in daclizumab-treated patients Neither cohort demonstratedsignificant proliferation to complex autoantigenbrain homogenate

Humoral immunity to Afluria vaccine Neutralizing Abtiters to the 3 viral variants present in 2013 Afluriavaccine (ie 2 A strains ACA2009 and AVicto-ria3612011 one B strain BMass022012) wereassessed before vaccination (D0) and 7 (D7) and180 days (D180) postvaccination Consistent withour data obtained in T cell specificity assays weobserved that all studied participants had preexistingimmunity (ie Ab titers $140) to at least one ofthe viral variants (figure 5A) In the control group 9of 42 (214) possible patientvariant combinationsdid not fulfill criteria for seroprotection at D0 Nineof 9 (100) patientvariant pairs from the controlgroup that did not have preexisting immunity sero-converted (ie achieved $4-fold increase in Abtiters) at D7 postimmunization The average foldincrease in Ab titers in the newly seroconvertedgroup was 356 In daclizumab-treated patients 8 of

51 (157) patientvariant combinations (note thatbecause of long-distance travel we had D7 follow-updata only on 17 daclizumab-treated patients) did notfulfill criteria for seroprotection at D0 Seven of 8(875) patientvariant pairs that did not havepreexisting immunity seroconverted at D7postimmunization The average fold increase in Abtiters in this group was 987 The seroconversion ratefor both groups was highly significant (figure 5B)

DISCUSSION DAC-HYP is emerging therapy forRRMS1213 As our armamentarium of therapeuticagents for this disease expands their efficacy is care-fully weighed against their side effects Because noneof the current or emerging pharmaceutical agents arecurative patients with RRMS are expected to receivetherapy for years or decades and the effects of suchlong-term treatment on immune responses againstinfectious agents or vaccinations need to beconsidered Clinical trials of daclizumab(Zenapax)1314 as well as of its new formulationDAC-HYP1215 recorded a mild but reproducibleincrease in infectious complications althoughopportunistic infections have not been reportedThese in vivo observations are consistent within vitro mechanisms of action which includeinhibition of trans-presentation of IL-2 byautologous DCs to primed T cells leading todecreased generation of Ag-specific CD41 andCD81 T effectors522 Activated T cell responses arefurther affected during daclizumab therapy bycytotoxicity of expanded immunoregulatoryCD56bright NK cells1617 However in contrast tomice CD25 is not necessary for development ofhuman Ab-secreting memory B cells or plasma cellsand consequently no inhibitory effects ofdaclizumab on serum IgM IgA IgE and IgG levels(IgG isotypes) have been observed13 This is incontrast to intrathecal Ab production (measured asIgG index) which declined upon daclizumabtreatment of MS7 suggesting that daclizumabtargets pathogenic immune responses mediatingautoimmunity more potently than it does immuneresponses in general Nevertheless the extent towhich daclizumab therapy interferes withdevelopment of protective immunity has not beendirectly investigated

Although current analysis of cellular and humoralimmune responses to seasonal influenza vaccinationwas performed within the context of an open-labelclinical trial all samples were processed and analyzedin blinded fashion The major drawback of the cur-rent study is the limited number of participants Itis practically impossible to apply complex functionalstudies performed on freshly isolated T cells to alarge number of participants Our previous studies



demonstrated that reproducible results for within-participant variability can be obtained with comparablesample sizes (ie 10ndash20 participants)9161725 Further-more our diverse assays were complementary and theresults are consistent across different assays

We observed that all studied participants fromboth cohorts had preexisting cellular and humoralimmunity to at least one of the 3 viral variants presentin the 2013 Afluria vaccine This explains the rapidactivation of adaptive immune responses measured

by immunophenotyping of blood samples Using thisAg-nonspecific assay we observed greater activationof CD41 and CD81 T cells in controls as comparedto daclizumab-treated patients with MS Correspond-ingly in Ag-specific assays we also observed greaterCD41 and CD81 T cell reactivity to multiple testedAg in the control cohort as compared to daclizumab-treated patients Using autologous serum assured thatT cell activation occurred in the presence of physio-logic concentrations of daclizumab thus mimickingthe in vivo situation as much as possible Overall theobtained results are consistent with previouslydescribed inhibition of DC-driven activation of Ag-specific T cells5 explaining the decreased prolifera-tion of all CD41 and CD81 T cells irrespective ofthe Ag studied We do not believe that our data sug-gest that daclizumab-treated patients with MS havelower preexisting immunity to EBV or CMV becausewe have investigated this issue previously using anassay that bypasses effect of daclizumab on DCsand observed comparable levels of CD41 andCD81 T cell reactivity to EBV and CMV in theblood of daclizumab-treated and untreated patientswith MS22

The study design also allowed us to investigate theintriguing possibility that IL-2 signaling blocks differ-entiation of T cells to Th17 effectors24 Because da-clizumab blocks high-affinity IL-2 signaling inhuman T cells9 if the above-mentioned mechanismoccurred in humans one could expect increased gen-eration of Th17 effectors in patients on daclizumabtherapy To avoid nonphysiologic changes introducedby a long in vitro culture we focused on the T cellexpression of the master regulator of Th17 lineageRORgt Although we observed transient upregula-tion of RORgt intracellular expression in CD41

and CD81 T cells which coincided with their acti-vation (as measured by expression of activationmarkers HLA-DR CD69 and CD38) at D1 post-immunization this increase was actually more robust(and highly statistically significant) in controls ascompared to the daclizumab-treated cohort This sug-gests that high-affinity IL-2 signaling does not havean inhibitory role on Th17 differentiation in humansa result supported by a recent study of participantswith different genetic variants of IL2RA26 Alterna-tively the decrease in RORgt induction could haveresulted from the aforementioned inhibitory effect ofdaclizumab on DC-mediated T cell activation

In contrast to the observed inhibitory role on Tcells the expansion of memory B cells (immunophe-notyping results) and increase in neutralizing Ab titerswere not affected by daclizumab therapy All but onedaclizumab-treated participant who did not have pre-existing protective immunity was able to achieveseroprotection at D7 postimmunization and the

Figure 5 Flu immunizationndashinduced changes in neutralizing Ab titers against 3viral variants present in Afluria 2013 vaccine

(A) The change in neutralizing Ab titers to all 3 viruses present in the Afluria vaccine betweenD0 (before vaccination) and 7 days (D7) and 180 days (D180) postvaccination graphed sep-arately for control group (left panels) and DAC-HYPndashtreated patients (right panels) (B) Sub-group of control (left panel) and daclizumab-treated participants (right panel) who did nothave preexisting Ab immunity at D0 to at least one of the viral variants are plotted sepa-rately to demonstrate that all participants increased neutralizing Ab titers 7 days postvac-cination (D7) The vertical box plots show the median and 25ndash75 range while thewhiskers reflect minimum and maximum values for each diagnostic group The scatter dotplots correspond to individual patientrsquos data p 005 0001 p 005 p 0001Ab 5 antibody DAC-HYP 5 daclizumab high-yield process



seroconversion titers were actually higher in daclizu-mab as compared to control cohorts These resultsindicate that humoral immunity is largely unaffectedby daclizumab therapy

The current study provides strong support to previ-ously described inhibitory mechanisms of daclizumabon T cell immunity while demonstrating that thisinhibitory effect is not complete and allows generationof protective immunity against environmental Ag suchas seasonal influenza immunization However becauseof preexisting immunity in the majority of participantsthe question of whether daclizumab therapy inhibitsdevelopment of T cell responses to neo-antigens remainsopen and may need to be investigated in future studies

AUTHOR CONTRIBUTIONSDr Lin study concept and design acquisition of data analysis and inter-

pretation critical revision of the manuscript for important intellectual con-

tent Mrs Winokur acquisition of data Mr Blake acquisition of data Dr

Wu Statistical analysis Mrs Manischewitz acquisition of data Mrs King

acquisition of data Mrs Romm acquisition of data Dr Golding study

concept and design Dr Bielekova study concept and design critical revi-

sion of the manuscript for important intellectual content study supervision

ACKNOWLEDGMENTThe authors thank Anne Mayfield for scheduling patients and Jenifer

Dwyer Jamie Cherup and the clinical team for sample collection and

expert patient care

STUDY FUNDINGThe study was supported by the intramural research program of the

National Institute of Neurological Disorders and Stroke (NINDS) and col-

laborative agreement (CRADA) between NINDS and AbbVieBiogen Idec

DISCLOSUREYC Lin P Winokur A Blake T Wu and J Manischewitz report no

disclosures LR King is employed by the FDA E Romm and H Gold-

ing report no disclosures B Bielekova is coinventor on several NIH pat-

ents related to daclizumab and as such has received patent royalty

payments from the NIH and received research support from the NIH

BiogenAbbVie and Santhera Pharmaceuticals Go to Neurologyorgnn

for full disclosure forms

Received July 31 2015 Accepted in final form November 25 2015

REFERENCES1 Gold R Giovannoni G Selmaj K et al Daclizumab high-

yield process in relapsing-remitting multiple sclerosis

(SELECT) a randomised double-blind placebo-controlled

trial Lancet 20133812167ndash2175

2 Giovannoni G Gold R Selmaj K et al Daclizumab high-


(SELECTION) a multicentre randomised double-blind

extension trial Lancet Neurol 201413472ndash481

3 Waldmann TA Goldman CK Bongiovanni KF et al

Therapy of patients with human T-cell lymphotrophic

virus I-induced adult T-cell leukemia with anti-Tac a

monoclonal antibody to the receptor for interleukin-2

Blood 1988721805ndash1816

4 Caudy AA Reddy ST Chatila T Atkinson JP

Verbsky JW CD25 deficiency causes an immune dysre-

gulation polyendocrinopathy enteropathy X-linked-like

syndrome and defective IL-10 expression from CD4 lym-

phocytes J Allergy Clin Immunol 2007119482ndash487

5 Wuest SC Edwan JH Martin JF et al A role for

interleukin-2 trans-presentation in dendritic cell-

mediated T cell activation in humans as revealed by da-

clizumab therapy Nat Med 201117604ndash609

6 Lenardo MJ Interleukin-2 programs mouse T lympho-

cytes for apoptosis Nature 1991353858ndash861

7 Perry JS Han S Xu Q et al Inhibition of LTi cell devel-

opment by CD25 blockade is associated with decreased

intrathecal inflammation in multiple sclerosis Sci Transl

Med 20124145ra106

8 Oh U Blevins G Griffith C et al Regulatory T cells are

reduced during anti-CD25 antibody treatment of multiple

sclerosis Arch Neurol 200966471ndash479

9 Martin JF Perry JS Jakhete NR Wang X Bielekova B

An IL-2 paradox blocking CD25 on T cells induces IL-2-

driven activation of CD56(bright) NK cells J Immunol

20101851311ndash1320

10 Wakabayashi K Lian ZX Moritoki Y et al IL-2 receptor

alpha(22) mice and the development of primary biliary

cirrhosis Hepatology 2006441240ndash1249

11 Roifman CM Human IL-2 receptor alpha chain defi-

ciency Pediatr Res 2000486ndash11

12 Sharfe N Dadi HK Shahar M Roifman CM Human

immune disorder arising from mutation of the alpha chain

of the interleukin-2 receptor Proc Natl Acad Sci USA

1997943168ndash3171

13 Bielekova B Richert N Howard T et al Humanized anti-

CD25 (daclizumab) inhibits disease activity in multiple

sclerosis patients failing to respond to interferon-beta Proc

Natl Acad Sci USA 20041018705ndash8708

14 Bielekova B Howard T Packer AN et al Effect of anti-

CD25 antibody daclizumab in the inhibition of inflam-

mation and stabilization of disease progression in multiple


15 Wynn D Kaufman M Montalban X et al Daclizumab in

active relapsing multiple sclerosis (CHOICE study) a

phase 2 randomised double-blind placebo-controlled

add-on trial with interferon beta Lancet Neurol 20109

381ndash390

16 Bielekova B Catalfamo M Reichert-Scrivner S et al Reg-

ulatory CD56bright natural killer cells mediate immuno-

modulatory effects of IL-2R-alpha-targeted therapy

(daclizumab) in multiple sclerosis Proc Natl Acad Sci

USA 20061035941ndash5946

17 Jiang W Chai NR Maric D Bielekova B Unexpected

role for granzyme K in CD56bright NK cell-mediated

immunoregulation of multiple sclerosis J Immunol

2011187781ndash790

18 Bielekova B Daclizumab therapy for multiple sclerosis

Neurotherapeutics 20131055ndash67

19 Polman CH Reingold SC Banwell B et al Diagnostic

criteria for multiple sclerosis 2010 revisions to the McDo-

nald criteria Ann Neurol 201169292ndash302

20 Tsang JS Schwartzberg PL Kotliarov Y et al Global anal-

yses of human immune variation reveal baseline predictors

of postvaccination responses Cell 2014157499ndash513

21 Han S Lin YC Wu T et al Comprehensive immunophe-

notyping of cerebrospinal fluid cells in patients with neuro-

immunological diseases J Immunol 20141922551ndash2563

22 Wuest SC Mexhitaj I Chai NR et al A complex role of

herpes viruses in the disease process of multiple sclerosis

PLoS One 20149e105434

23 Rowe T Abernathy RA Hu-Primmer J et al Detection of

antibody to avian influenza A (H5N1) virus in human



serum by using a combination of serologic assays J Clin

Microbiol 199937937ndash943

24 Laurence A Tato CM Davidson TS et al Interleukin-2

signaling via STAT5 constrains T helper 17 cell genera-

tion Immunity 200726371ndash381

25 Bielekova B Goodwin B Richert N et al Encephalito-

genic potential of the myelin basic protein peptide (amino

acids 83-99) in multiple sclerosis results of a phase II

clinical trial with an altered peptide ligand Nat Med

200061167ndash1175

26 Hartmann FJ Khademi M Aram J et al Multiple

sclerosis-associated IL2RA polymorphism controls GM-

CSF production in human TH cells Nat Commun

201455056



DOI 101212NXI000000000000019620163 Neurol Neuroimmunol Neuroinflamm

Yen Chih Lin Paige Winokur Andrew Blake et al influenza vaccination

Patients with MS under daclizumab therapy mount normal immune responses to

This information is current as of January 27 2016

ServicesUpdated Information amp

httpnnneurologyorgcontent31e196fullhtmlincluding high resolution figures can be found at

Supplementary Material httpnnneurologyorgcontentsuppl2016012731e196DC1

Supplementary material can be found at

References httpnnneurologyorgcontent31e196fullhtmlref-list-1

This article cites 26 articles 9 of which you can access for free at

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectioncerebrospinal_fluidCerebrospinal Fluidfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

2016 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

also immunocompromised and daclizumabtreatment causes a slight increase in infectiouscomplications in phase II13ndash15 and phase IIItrials12

Trying to explain this apparent paradox wediscovered that daclizumab limits activationpriming of antigen (Ag)-specific CD41 andCD81 T cells indirectly by limiting dendriticcell (DC)-mediated trans-presentation of IL-25 This early IL-2 signal delivered at the timewhen naive T cells do not yet express high-affinity IL-2R is crucial for their differentia-tion to T cell effectors Daclizumab also hasunanticipated effects on innate lymphoid cells(ILCs) promoting differentiation of ILC pre-cursors away from proinflammatory lymphoidtissue inducer (subtype of ILC3) cells andtoward CD56bright natural killer (NK) cells716

These immunoregulatory NK cells can killactivated autologous T cells1617 thus provid-ing overlapping functions with regulatory Tcells

These multiple and unique mechanisms ofaction underlie efficacy of daclizumab inrelapsing-remitting MS (RRMS)1213ndash15 The

question remains how potently immunosup-pressive daclizumab therapy really is willdescribed effect on innate immunity preventactivation of CD41 CD81 T cells and B cellsplasma cells to common infectious pathogensTherefore the purpose of this study was toassess the potential immunosuppressive roleof daclizumab in vivo by comparing immuneresponses from patients with MS on long-termdaclizumab therapy and controls to the sea-sonal influenza vaccination

METHODS Standard protocol approvals registrationsand patient consents The study was approved by the NIH

institutional review board and all patients provided written con-

sent The study was performed under investigational new drug

application (IND 107973 IND sponsor BielekovaNational

Institute of Neurological Disorders and Stroke [NINDS]) as part

of NINDS clinical trial 10-N-0125 ldquoInvestigating mechanism of

action of DAC-HYP in the treatment of high-inflammatory

multiple sclerosis (MS)rdquo (ClinicalTrialsgov identifier

NCT01143441)

Participants Participant demographics and diagnosis are

presented in table 1 Twenty-three patients with RRMS

received DAC-HYP 150 mg subcutaneously every 4 weeks for

a minimum of 36 months 609 (1423) of these patients were

treated with a previous formulation of daclizumab (Zenapax

Hoffmann-La Roche Basel Switzerland) for up to 6 years

Table 1 Optimized combination of commercially available fluorochrome-conjugated antibodies to reliably quantify T cell and monocyte B celland RORgt1 immune cells

Conjugation T cell and monocyte B cell Intracellular staining

FITC Anti-human CD69 antibody (BD FN50) Anti-human CD40 antibody (Invitrogen HB14) Anti-human CD56 antibody (BD MEM188)

PE Anti-human CD40 antibody (BioLegend HD14) Anti-human CD21 antibody (Beckman CoulterA32524)

Anti-human RORgd antibody (RampD600380)

PerCP-Cy55 Anti-human HLA-DR antibody (BD Tu36) Anti-human CD20 antibody (BD 2H7) Anti-human CD123 antibody (eBioscience7G3)

PE-Cy7 Anti-human CD86 antibody (BioLegend IT22) Anti-human CD86 antibody (BioLegend IT22) Anti-human CD11c antibody (eBioscience39)

V450 Anti-human CD4 antibody (BD RPA-T4) Anti-human CD80 antibody (BD L3074) Anti-human CD45 antibody (BD HI30)

Brilliant Violet510

Anti-human CCR7CD197 antibody (BioLegendG043H7)

Anti-human IgM antibody (Biolegend MHM-88)

AmCyan Anti-human CD8 antibody (BD SK1)

Qdot 605 Anti-human CD8 antibody (Invitrogen 3B5) Anti-human CD27 antibody (Invitrogen CLB-271) Anti-human CD19 antibody (eBioscienceHIB19)

Qdot 655 Anti-human CD14 antibody (Invitrogen TuK4) Anti-human CD19 antibody (Invitrogen SJ25-C1) Anti-human CD3 antibody (eBioscienceOKT3)

Qdot 705 Anti-human CD4 antibody (InvitrogenS35)

Qdot 800 Anti-human CD45 antibody (Invitrogen H130) Anti-human CD45 antibody (Invitrogen H130)

APC Anti-human CD25 antibody (BioLegend M-A251) Anti-human IgD antibody (BioLegend HIT2)

Alexa Fluor 700 Anti-human CD38 antibody (BioLegend HIT2) Anti-human CD38 antibody (MACS Miltenyi130-094-553)

Anti-human CD14 antibody (BioLegendHCD14)

APC-Cy7 Anti-human CD3 antibody (BioLegend SK7) Anti-human CD10 antibody (BioLegend HI10a) Anti-human HLA-DR antibody (eBioscienceLN3)

Abbreviations BD 5 Becton Dickinson FITC 5 fluorescein isothiocyanate IgM 5 immunoglobulin M RampD 5 RampD SystemsPresented as name of antibody (company clone)
















controls none)
























assays































































analysis





































































expert patient care







































Med 20124145ra106







20101851311ndash1320









1997943168ndash3171













381ndash390





USA 20061035941ndash5946




2011187781ndash790














PLoS One 20149e105434














200061167ndash1175




201455056


















Reprints

















controls none)
























assays































































analysis





































































expert patient care







































Med 20124145ra106







20101851311ndash1320









1997943168ndash3171













381ndash390





USA 20061035941ndash5946




2011187781ndash790














PLoS One 20149e105434














200061167ndash1175




201455056


















Reprints









assays































































analysis





































































expert patient care







































Med 20124145ra106







20101851311ndash1320









1997943168ndash3171













381ndash390





USA 20061035941ndash5946




2011187781ndash790














PLoS One 20149e105434














200061167ndash1175




201455056


















Reprints























































analysis





































































expert patient care







































Med 20124145ra106







20101851311ndash1320









1997943168ndash3171













381ndash390





USA 20061035941ndash5946




2011187781ndash790














PLoS One 20149e105434














200061167ndash1175




201455056


















Reprints



























































expert patient care







































Med 20124145ra106







20101851311ndash1320









1997943168ndash3171













381ndash390





USA 20061035941ndash5946




2011187781ndash790














PLoS One 20149e105434














200061167ndash1175




201455056


















Reprints













200061167ndash1175




201455056


















Reprints



















Reprints




patients with ms under daclizumab therapy mount normal ... · activation-induced cell death6,7 and...

Documents