neonatal cd8+ t cells are slow to develop into lytic effectors after hsv infection in vivo

Neonatal CD8+ T cells are slow to develop into lyticeffectors after HSV infection in vivo

Marian A. Fernandez1, Ingrid A. C. Evans1,2, Eddy H. Hassan1,2,Francis R. Carbone3 and Cheryl A. Jones1,2

1 Centre for Perinatal Infection Research, The Children's Hospital at Westmead,Westmead, Australia

2 Discipline of Paediatrics and Child Health, University of Sydney, Sydney, Australia3 Department of Microbiology and Immunology, University of Melbourne, Melbourne,Australia

HSV is an important neonatal pathogen. We defined the kinetics of the primary CTLresponse to HSV-2 in vivo in neonatal mice. Using a replication-defective HSV-2 virus,we demonstrate that neonates mount a primary HSV-specific CTL effector response inthe draining LN, with delayed onset and shortened peak activity, in contrast to the rapid,strong response observed in adult mice. The shortened peak neonatal CTL response isindependent of HSV dose and is associated with retarded CD8+ T cell expansion,reduced expansion of HSV-specific tetramer-positive CD8+ T cells and a reduced CD8+

T cell IFN-c response. Paradoxically, neonatal CD8+ T cells display enhanced non-specific early activation that is not sustained. Neonatal HSV-specific TCR-transgenicCD8+ T cells showed reduced proliferation in vivo when transferred into HSV-infectedneonatal mice compared to adult T cell controls. Our data suggest that early events inCD8+ T cell priming underlie the attenuated newborn CTL response to HSV.

Introduction

The newborn have increased susceptibility to herpes-viruses like HSV which is an important cause of birthdefects, preterm births and perinatal death [1, 2]. Aftermucocutaneous HSV infection, CD8+ T cells areresponsible for late clearance of virus from the skinand the peripheral nervous system [3, 4] and forcontrolling reactivation from neuronal latency [5–7].CTL are important mediators of antiviral immunity, buttheir role in the newborn defence against HSV has notbeen fully evaluated. Neonatal T cells were previously

thought to be functionally deficient, but have morerecently been shown to be capable of adult-likeresponses under conditions of optimal priming[8–11]. Strong memory CD8+ T cell responses havebeen reported in the newborn to alloantigens [12], toviruses including HIV [13, 14], inactivated and replica-tion-competent murine retroviruses [10, 15, 16], andafter subclinical congenital CMV infection [17], toprotozoa [18] and to DNA vaccines or attenuated viralvectors [19–21]. In contrast, human infants have beenshown to have reduced memory CD4+ and CD8+ T cellresponses to HSV [22, 23]. Thus, not all herpesviruseselicit protective CTL responses in the newborn, andstudy of the earliest events of CD8+ Tcell priming to HSVmay define the conditions needed to induce a protectiveCTL response from the neonatal period.

After viral infection, DC activate naive T cells bypresenting Ag to MHC receptors covalently linked to theTCR, and provide costimulatory signals through CD80/86 to CD28 on the T cell surface. TCR stimulationinduces entry of activated T cells into the cell cycle and

Immunity to infection

Correspondence: Assoc. Prof. Cheryl Anne Jones, Centre forPerinatal Infection Research, The Children's Hospital atWestmead, Locked Bag 4001, Westmead, NSW 2145, AustraliaFax: +61-2-98453389e-mail: [email protected]

Received 1/12/06Revised 29/8/07

Accepted 8/11/07

[DOI 10.1002/eji.200636945]

Key words:CTL � Herpes simplex

virus � Kinetics� Neonate

Abbreviations: Cas : Cas-Br-E murine leukaemia virus � gB :glycoprotein B � p.i. : post infection

Marian A. Fernandez et al. Eur. J. Immunol. 2008. 38: 102–113102

f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

rapid expansion of Ag-specific effectors, followed by acontraction phase, which leaves a pool of memoryeffectors [24]. The size of T cell expansion is thought todetermine the size of the memory pool phase [24]. Theexpansion of CD8+ T cells and development of cytotoxiceffector activity to viruses and other pathogens has beenwell characterised in adults [24, 25]. In neonatal mice,the primary kinetics of CD8+ Tcell expansion and IFN-cproduction have been defined in the spleen afterinfection with Cas-Br-E murine leukaemia virus (Cas)[10, 15, 26] and polyoma virus [27]. Low-dose neonatalinfection with Cas induces a smaller expansion of Ag-specific CD8+ Tcells and a slower peak CD8+ Tcell IFN-cresponse compared to adults. However, a greaterproportion of neonatal CD8+ T cells express IFN-c atthe time of the peak response [15, 26], and CTLcytotoxicity develops to adult-like levels with similarkinetics [10, 26]. Similarly, polyoma virus infection oftumour-resistant and tumour-susceptible strains ofnewbornmice induces vigorous expansion of Ag-specificCTL, and a strong but slightly delayed CD8+ Tcell IFN-cresponse [27]. However, ex vivo Ag-specific cytotoxicactivity is significantly reduced in tumour-susceptibleneonates compared to adults and tumour-resistantneonates [27]. Thus, the kinetics of Ag-specific CTLresponses after viral infection in the newborn period canbe quite diverse depending on the pathogen, andneonatal CD8+ T cell expansion, IFN-c productionand cytotoxicity are not always coordinated.

In contrast to Cas and polyoma virus, the lytic cycle ofHSV is rapid, with production of infectious progeny inthe skin of adult mice occurring by 20 h post attachmentand entry [28], and clearance of infectious virus by7–10 days post infection (p.i.) [29]. CD8+ T celleffectors expand vigorously after HSV infection, despitea very low HSV-specific CTL precursor frequency in thedraining LN compared to many other viruses [30]. Theonset and duration of cytotoxic effector function afterHSV infection is also more rapid than after Cas orpolyoma virus infection, with armed CTL effectorsevident in the draining LN of adult mice as early as day 2p.i. [31]. The kinetics of the neonatal CTL effectorresponse to HSV has not been fully elucidated. We havepreviously shown that neonatal mice have a reducedprimary CTL effector response to HSV in the draining LNat the time of the peak adult response, day 5 p.i. [32].However, as this represented only one time point, herewe sought to rigorously define the kinetics of CD8+ Tcellexpansion, activation and effector function (cytotoxicity,IFN-c production) after primary HSV-2 infection inneonatal mice. We employed highly sensitive in vivoT cell methodologies that had been previously used todefine the primary CTL response to HSV-1 in adult mice[31]. HSV infection primarily occurs in the muco-epithelium. Therefore, we analysed the extent and

magnitude of the CD8+ T cell response in the drainingLN, the site of T cell activation. Infection was bysubcutaneous inoculation because it mimics the com-monest route of newborn infection with this virus [1, 2].

Results

Neonatal mice mount a peak primary HSV-specific CD8+ T cell response of slow onset andshort duration

We have demonstrated that neonatal mice mount areduced primary CTL response to HSV at the time of thepeak adult response (day 5 p.i.) using a conventional exvivo assay of CD8+ Tcell cytotoxicity [32]. To determinewhether this reflected an age-dependant difference inthe kinetics of the primary CD8+ Tcell effector responseto HSV infection, we used a highly sensitive in vivo assayof HSV-specific CTL cytotoxicity [31]. Neonatal andadult mice were injected with WT HSV-2 or PBS s.c. inthe footpads, and the CFSE intensity of adoptivelytransferred HSV peptide-primed CTL targets (CFSEhigh)and unprimed controls (CFSElow) was determined in thedraining popliteal LN from days 1 to 3 p.i. We observedthat neonatal mice had reduced CD8+ HSV-2-specificCTL activity compared to adults postWTHSV-2 infectionup to the onset of lethal encephalitis in neonatal mice(days 1, 2 p.i., p <0.05, and day 3 p.i., p <0.001, two-tailed Student's t-test) (Fig. 1A). To examine theneonatal CTL response beyond 3 days p.i., we used areplication-defective HSV-2 strain, dl5-29 (UL5

– UL29–),

which is non-virulent in neonatal mice [33] and whichinduces a CTL response in adults equivalent to WT virus[32, 34]. We measured the HSV-specific CTL response invivo from days 1 to 15 post HSV infection and observedthat the neonatal response was significantly reduced inthe draining LN compared to adults up to day 5 p.i. andagain at day 7 p.i. (Fig. 1B; p <0.001, days 3–5 and 7p.i., two-tailed Student's t-test), and in the spleens up today 7 p.i. and again at day 12 p.i. (Fig. 1C; p <0.01days 5–7 p.i., p <0.05 days 9 and 12 p.i., two-tailedStudent's t-test). The duration of peak HSV-specificCD8+ cytotoxicity (i.e. >65% specific lysis) was alsoshorter in neonates compared to adults at these sites(3 days versus 7 days in LN, 3 days versus 6 days inspleens, of neonates versus adults, respectively). Thus,neonatal mice mount a primary CD8+ cytotoxic T cellresponse to acute HSV infection in the draining LN andspleen with slow onset and shortened duration of peakactivity compared to adults.

To test if the delayed neonatal CD8+ T cell responseto the immunodominant determinant reflected theresponse to non-dominant epitopes, we measured thein vivo CTL responses to the major subdominant CTL

Eur. J. Immunol. 2008. 38: 102–113 Immunity to infection 103


epitope to HSV ribonucleotide reductase (HSV-2RR1829–836) [35] at day 5 post HSV infection, i.e. thetime of the peak adult response. We observed that theneonatal CD8+ cytotoxic Tcell response to RR1829–836 inthe draining LN remained significantly reduced com-pared to the response in adult mice (8 � 1.3% versus18 � 2.7%, HSV-infected neonates compared to adults,respectively, at day 5 p.i. p <0.05, two-tailed Student'st-test; not shown). Thus, the limited neonatal CTLresponse to HSV-2 is not associated with enhancedresponses to subdominant epitopes.

The age-dependant difference in the primary CTLresponse to HSV is not dependant on virus dose

In the previous experiments, we tested the HSV-specificCTL response to the same dose of virus. We nextcompared the CTL response at one time point (day 4p.i.) in the two age groups to a range of doses of HSV-2virus, dl5-29 (2 � 100–2 � 105 PFU/mouse). Neonataland adult mice were observed to mount a reduced HSV-specific CTL response at high doses in both draining LNand spleen at day 4 p.i. (Fig. 2A, B). The adult responsewas stronger at all doses tested in the draining LN, butsignificantly so only at the highest doses (p <0.001, two-

Figure 1. Neonatal mice mount a short, delayed primary HSV-2-specific CD8+ CTL response in vivo. Adult or neonatal mice (n = 5/strain/age/time point) were inoculated s.c. with (A) WT HSV-2, or (B, C) the replication-defective HSV-2 mutant (dl5-29). HSV-specific cytotoxicity in vivo was measured in the draining popliteal LN (A, B) or spleens (C) by FACS at the times shown afteradoptive transfer of CFSE-labelled gB498–505 peptide-pulsed syngeneic adult female splenocytes and unpulsed controls. Shown aremean percentages of HSV gB-specific lysis � SEM in HSV-infected adult and neonatal mice until day 15 p.i. or for WT HSV-2 (A),until the onset of lethal encephalitis (day 3 p.i.). Data are from one of two independent experiments with similar results. Non-specific lysis was not observed in mice mock-infected with PBS or to targets primed with irrelevant peptide (OVA, not shown).{p <0.05, *p <0.01, **p <0.001, two-tailed Student's t-test, adults versus neonates.

Figure 2. The age-dependant difference in the primary CTL response to HSV is not dependant on virus dose. Neonatal and adultmice (n = 5/age/dose) were injected s.c. with doses of the replication-defective HSV-2 virus (dl5-29) as shown. At day 4 p.i., equalconcentrations of syngeneic CFSE-labelled gB498–505 peptide-pulsed and unpulsed controls were adoptively transferred into themice. HSV-specific lysis wasmeasured in the draining LN (A) and spleen (B) by FACS. Shown aremean values� SEM of one of twoexperiments with similar results. (C) Data from (A) represented as HSV-specific lysis in draining LN by HSV dose adjusted fordraining LN APC number. Non-specific lysis was not observed in mice mock-infected with PBS. *p <0.001, two-tailed Student'st-test, adults versus neonates.



tailed Student's t-test, adults versus neonates,2 � 103 PFU/mouse and 2 � 105 PFU/mouse). How-ever, as 1-wk-old mice have approximately a log-fold lessnumber of CD11c+ DC in the draining LN compared toadults (Fernandez and Jones, unpublished results),using the data in Fig. 2A, we next compared the CTLafter normalising the virus dose for the DC number.Even after correcting dose for APC number, the age-dependent differences in HSV-specific CD8+ lysisremained evident (Fig. 2C). Thus, the observed age-related difference in the primary CTL response to HSVwas not solely due to excess Ag relative to the number ofAPC in the draining LN compared to adults.

Neonatal mice demonstrate slower expansion ofCD8+ T cells in the draining LN after HSV-2infection

Neonates have lower numbers of leukocytes, includingCD8+ Tcells in the draining LN, compared to adult mice[8]. Therefore, the delayed CTL response to primaryHSV infection could be due to the increased timerequired for a neonate to expand or recruit sufficientHSV-specific CD8+ T cell effectors into the LN. Toexplore this concept further, we analysed the kinetics ofthe total number (Fig. 3A) and fold increase (Fig. 3B) ofCD8+ T cells in the draining LN of neonatal mice andadult controls from days 1 to 15 p.i. with a single dose ofHSV replication-defective strain, dl5-29 (2 � 105 PFU/mouse). We found that neonatal mice took the sametime as adults to reach their peak total CD8+ T cellnumber in the draining LN at day 6 p.i. and then showedthe same rate of decline to day 15 p.i. (Fig. 3A).However, the rate of CD8+ T cell expansion wassignificantly less in neonates at early times (days 2–3)post HSV infection compared to adults (p <0.05,

Wilcoxon Rank Sum test; Fig 3B). Thus, the magnitudeand the early rate of expansion of neonatal CD8+ T cellsin the draining LN after HSV infection is significantlyreduced compared to adults.

As measurement of total CD8+ T cell numbers doesnot allow us to distinguish between Ag-specific expan-sion and non-specific proliferation or recruitment, wenext compared the frequency of HSV-specific CD8+

T cells in the draining LN of neonatal and adult mice attimes up to day 15 p.i. using a tetramer directed to theimmunodominant determinant from the HSVglycoprotein B (gB) (gB498–505) [36, 37] that has beenpreviously used to define the kinetics of the HSV-specificCD8+ Tcell response to HSV-1 in adult mice [31, 37, 38].HSV gB-specific T cells were first observed in thedraining LN of adults and neonates after HSV-2 infectionat day 4 p.i., with peak frequency detected on day 5 p.i.(Fig. 4A, B). The frequency of tetramer-positive CD8+

Tcells in the draining LN at the time of the peak response(day 5 p.i.) was significantly less in HSV-infectedneonates than the value observed in infected adults(2.2 � 0.2% versus 6.2 � 1.0%, adults versus neonates,p <0.01, Wilcoxon Rank Sum test). Thus, neonatal miceshow reduced expansion of HSV-specific CD8+ T celleffectors in the draining LN in response to primary HSVinfection.

Neonatal HSV-specific TCR-transgenic CD8+

T cells display adult-like ability to proliferatewhen primed ex vivo, but not in vivo in neonatalrecipients

One possible explanation for the age-related differencesin CD8+ T cell expansion after primary HSV infection isthat neonatal T cells have an inherently reduced abilityto proliferate upon TCR engagement with HSV-primed

Figure 3. HSV induces a greater increase in draining LN cell number in adult mice compared to neonates at early times p.i.Neonatalmice and adult controls (n = 5/time point/age) were inoculated s.c. with the replication-defective HSV-2mutant (dl5-29),ormock-infectedwith an equivalent volume of PBS. At the times shown, total cell numbers in the draining LNwere determined byTrypan blue exclusion. CD8+ T cell numbers (A) and the fold increase (number of CD8+ T cells on each day p.i. divided by thenumber of CD8+ T cells in mock-infected age-matched controls) (B) were calculated from FACS data obtained after staining withanti-CD8+ antibody. Shown are themean values from one of two separate experiments � SEM. *p <0.05, Wilcoxon Rank Sum test,neonate versus adult fold increase in CD8+ T cells.



DC. To test this notion, we measured the ability ofneonatal and adult HSV gB-specific T cells to proliferatein response to DC from mock-infected adult syngeneicfemale mice that had been primed in vitro with HSV gBpeptide (gB498–505). We used peptide-primed adult DCto remove the influence of any age-dependant differ-ences in APC function. HSV-specific CD8+ T cells wereobtained from neonatal and adult gBT-I.1 transgenicmice which express a TCR that recognises the HSVgB498–505 determinant presented by MHC class I [39],and labelled with CFSE. CD8+ T cell proliferation wasmeasured by flow cytometry after 60 h of co-culturewith primed DC and expressed as a percentage of cells

that proliferated. Non-specific CD8+ T cell proliferationwas measured by analysing the response to adult DCprimed with an irrelevant MHC class I-restricted Ag(OVA), or to DC that were left unprimed. Over 80%(84 � 2.9%) of neonatal CD8+ Tcells and 66 � 0.7% ofadult TCR-transgenic CD8+ T cells were observed toproliferate in response to the HSV peptide-primed adultDC (Fig. 5A). A smaller percentage of both adult andneonatal T cells were observed to proliferate non-specifically in response to DC primed with OVA or tounprimed DC in multiple experiments (Fig. 5A). Adult-like CD8+ T cell proliferation was also observed whenneonatal HSV TCR-transgenic T cells were cultured for

Figure 5. Neonatal HSV CD8+ T cells exhibit adult-like ability to proliferate ex vivo but not in vivo. (A) CFSE-labelled HSV TCR-transgenic T cells fromneonatal and adultmicewere incubatedwith naive female adult DC that had been primed in vitrowithHSVgB498–505 peptide, peptide (OVA257–264), or with medium alone (Mock). CD8+ T cell proliferation was determined by flow cytometryafter 60 h. (B) CFSE-labelled TCR-transgenic T cells were transferred into naive female neonates (adult or neonatal T cells) orfemale adults (adult T cells only) prior toWTHSV-2 infection andCD8+ T cell proliferationwasmeasured in the draining LNby flowcytometry at 48 h p.i. (C) Female neonatal CFSE-labelled TCR-transgenic T cells were transferred into naive female neonatal oradult mice prior to infection, and proliferation was analysed at 48 h p.i. (A–C) Shown are the mean percentages of HSV-specificCD8+ T cell proliferation � SEM. n = LN from 3–4 (A) or 4–6 mice (B, C)/age group/type of recipient. *p <0.01, two-tailed Student'st-test.

Figure 4. Expansion of HSV-specific Kb-gB498–505 tetramer-positive CD8+ T cells is reduced in neonatal mice. Neonatal and adultmice were inoculated s.c. with the replication-defective HSV-2 mutant (dl5-29), or mock-infected with an equivalent volume ofPBS. At the times shown, cells in the draining LN were stained for CD8 and Kb-gB tetramer and analysed by flow cytometry. Cellswere gated on CD8+ T cells. (A) Mean frequencies of CD8+ Kb-gB498–505

+ cells � SEM. Mean levels of background staining in mock-infected controls are indicated by the dotted line. Data from two independent experiments with similar results (n = 6–8 mice/agegroup/time point). (B) Representative FACS dot plot of HSV-specific (tetramer-positive) CD8+ T cells from draining LN of HSV- ormock-infected neonatal and adult mice at day 5 p.i. *p <0.05, **p <0.01 HSV-infected adults versus neonates, Wilcoxon Rank Sumtest.



shorter periods of time (24 and 48 h, not shown). CD8+

T cells cultured in the absence of DC did not proliferate(not shown). Thus, neonatal TCR-transgenic CD8+

T cells proliferate as strongly as adult CD8+ T cells inresponse to Ag-primed adult DC in vitro.

To test if the adult-like ability of neonatal transgenicT cells to proliferate in vitro to HSV reflected thesituation in vivo, CFSE-labelled adult or neonatal HSVTCR-transgenic T cells were adoptively transferred intonaive neonatal C57BL/6 mice 24 h prior to WT HSV-2infection. In the same experiment, adult TCR-transgenicT cells were also injected into adult recipients 24 h priorto infection as a positive control. HSV-specific CTLproliferation in vivo was measured by loss of CFSEintensity of CD8+Va2+ cells by flow cytometry at 48 hp.i. We observed that neonatal HSV TCR-transgenicT cells showed reduced proliferation to HSV infection invivo in neonatal recipients compared to adult HSV-specific CD8+ T cells (24 � 2.3% versus 64 � 1.6%,p <0.01, neonatal versus adult T cells, respectively, two-tailed Student's t-test; Fig. 5B). We next measured theproliferation of neonatal HSV TCR-transgenic CD8+

Tcells in naive adult or neonatal recipients at 48 h p.i. asabove. We observed that neonatal TCR-transgenic CD8+

Tcells after HSV infection in vivo proliferated to the sameextent, irrespective of the age of the recipient mouse(38 � 1.1% versus 30 � 1.0% in adult and neonatalrecipients, respectively, two-tailed Student's t-test,

p >0.05). Thus, after HSV infection, neonatal transgenicT cells do not proliferate as well as adult transgenicT cells in neonatal mice. Furthermore, neonatal T cellsdo not show enhanced proliferation in vivo whentransferred into adult mice but can be induced toproliferate like adult T cells in vitro when primed withadult DC.

Neonatal CD8+ T cells show reduced activation atlate but not early times after acute HSV infection

Activation of CD8+ T cells through TCR engagement iscritical to their development as cytotoxic effectors [24].We therefore tested if the reduced HSV-specificcytotoxicity was associated with reduced CD8+ T cellactivation. For this, we compared the expression of theearly T cell activation marker, CD69, the IL-2 receptora-chain, CD25, and the LN homing receptor CD62L(which is down-regulated upon activation) on CD8+

Tcells in the draining LN of neonates and adults at timesup to 7 days p.i. with the HSV-2 virus, dl5-29. Weobserved that a significantly greater proportion of CD8+

T cells from the draining LN of infected neonatesexpressed CD69 shortly after infection compared toadults (6, 12 and 24 h p.i., p <0.01, two-tailed Student'st-test, adults versus neonates) (Fig. 6A). However,neonatal CTL activation beyond 24 h p.i. with HSV(as measured by CD25 expression) was reduced

Figure 6. Neonatal CD8+ T cells display early increased activation compared to adults after HSV infection that is not sustained.Neonatalmice and adult controls (n = 5/age/time point) were inoculated s.c. with the replication-defective HSV-2mutant (dl5-29),or mock-infected with an equivalent volume of PBS. Draining LN cells were stained at the times shown for surface expression ofCD8, andCD69 (A), CD25 (B), CD62L (C) and, on day 5 only, CD25 andKb-gB498–505 (D), and CD62L andKb-gB498–505 (H), then analysedby FACS. Cells were gated on CD8+. The total number of CD8 T cells was determined by Trypan blue exclusion, and the number ofCD69+ (E), CD25+ (F), and CD62Llow (G) cells was calculated from the flow cytometry data. Shown are themean values� SEM fromone of two separate experiments with similar results. *p <0.01, infected adults versus neonates, Student's two-tailed t-test.



compared to infected adults (p <0.01, respectively,days 4 and 5 p.i, adults versus neonates, Student's t-test)(Fig. 6B). Similarly, there was a significant proportion ofCD62Llow CD8+ T cells in neonates at days 3 and 5 p.i.(Fig. 6C; p <0.01, adults versus neonates, two-tailedStudent's t-test). Significantly reduced CD25 expressionin neonates was also evident at day 5 p.i. on tetramer-positive HSV-specific CD8+ T cells (p <0.01, adultsversus neonates, Student's t-test) (Fig. 6D). Fig. 6 alsoshows the total number of CD8+ T cells that expressCD69 (Fig. 6E), CD25 (Fig. 6F) or CD62Llow (Fig. 6G) inage-matched mock- or HSV-infected neonatal or adultmice at the indicated times p.i. As shown, despite theincreased proportion of neonatal CD8+ T cells thatexpress CD69 shortly after infection, the total number ofCD8+ CD69+ T cells in neonatal LN remains smallcompared to adults. Within the small population oftetramer-positive CD8+ T cells in the neonate at thistime, a greater proportion were CD62Llow compared toadults (67% versus 42%, p <0.01, neonates versusadults, two-tailed Student's t-test) (Fig. 6H). Thus, thedelayed primary CD8+ cytotoxic effector response toHSV-2 in neonatal mice is associated with reducedactivation of the total CD8+ T cell population at late butnot early time points after infection, and with reducedIL-2R expression by HSV-specific T cells at later times.

Neonatal CD8+ T cells show reduced IFN-csecretion in response to acute HSV infection

Concurrent with the initiation of proliferation, CD8+

Tcells are activated by TCR-dependant signals to expressIFN-c and TNF-a which recruit and/or activate othereffector cells, and restrict intracellular viral replication[24]. We have previously shown that neonatal CD8+

T cells taken from the draining LN at day 5 p.i. produce

less IFN-c compared to adult controls after 3 days ofculture ex vivo with Ag [32]. To test if the kinetics ofneonatal CD8+ T cell cytokine production is similarlydelayed in vivo in response to HSV, we measured thefrequency of CD8+ T cells from the draining LN of HSV-infected neonatal and adult mice that express IFN-c byintracellular cytokine staining on days 3, 5 ,7 and 9 p.i.with HSV-2 virus, dl5-29. Significantly fewer neonataltotal CD8+ T cells at days 3 and 5 p.i. expressed IFN-ccompared to adult Tcells (Fig. 7A; p <0.01, adults versusneonates, Wilcoxon Rank Sum test). The frequency ofneonatal HSV-gB-specific tetramer-positive CD8+ Tcellswas also significantly reduced compared to adults atday 5 p.i. (Fig. 7B; p <0.01, adults versus neonates,Wilcoxon Rank Sum test). IFN-c expression hadreturned to baseline levels in both adults and neonates

Figure 7. The CD8+ T cell IFN-c response in the draining LN ofneonatal mice is reduced after HSV infection compared toadults. Neonatal and adult mice (n = 5/age/time point) wereinoculated s.c. with the replication-defective HSV-2 mutant(dl5-29), or mock-infected with an equivalent volume of PBS.Draining LN cells were restimulated overnight with HSVpeptide gB498–505, then stained for IFN-c and CD8 (A) � Kb-gB498–505 tetramer (B) at days 3, 5, 7 p.i. Cells were analysed byFACS and gated on CD8+. Shown are mean values of CD8+

T cells that expressed IFN-c from one of two separateexperiments. *p <0.01, infected adults versus neonates, Wil-coxon Rank Sum Test.

Table 1. Profile of IFN-c expression by total and tetramer-positive CD8+ T cells in draining LN of neonatal and adult mice on day 5post HSV infectiona)

CD8+ T cells (%)b,c)

IFN-c (total) IFN-c+ tetramer+ IFN-c+ tetramer– Tetramer+ (total)

Neonates

Mock 0.14 � 0.13 0.05 � 0.03 0.09 � 0.04 0.12 � 0.05

HSV dl5-29d) 0.61 � 0.27e) 0.17 � 0.02e) 0.39 � 0.07 1.11 � 0.35

Adults

Mock 0.48 � 0.36 0.09 � 0.03 0.39 � 0.26 0.73 � 0.11

HSV dl5-29d) 3.61 � 0.44e) 1.03 � 0.15e) 2.58 � 0.40f) 3.85 � 0.80e)

a) n = 6 mice/per treatment group/ageb) Shown are mean percentages � SEMc) Determined by flow cytometry after staining for CD8a, IFN-c, and MHC class I-restricted Kb-gB tetramerd) HSV dl5-29 (2 � 105 PFU/mouse) given via s.c. injection into footpade) HSV-infected neonates versus adults, p <0.01, Wilcoxon Rank Sum testf) HSV-infected neonates versus adults, p <0.05, Wilcoxon Rank Sum test



by day 7 p.i. As shown in Table 1, IFN-c+ tetramer+

CD8+ Tcells represented approximately one third of theIFN-c+ CD8+ T cell population in both neonates andadults. Thus, consistent with the cytotoxic effectorresponse, production of IFN-c by neonatal CD8+ T cellsis significantly reduced after HSV infection compared toadult controls.

Discussion

Immunological immaturity persists from foetal life intothe newborn period and can result in a poorly protectiveresponse to intracellular pathogens, such as viruses,making neonates highly susceptible to severe disease[8]. CD8+ T cells are important effectors of life-longantiviral immunity. Here, we define for the first time thekinetics of the newborn primary CD8+ Tcell response toHSV, an important neonatal viral pathogen. We havemade the key observations that the primary CTLresponse to HSV in neonatal mice is associated withreduced CD8+ T cell expansion and a CD8+ T cellcytotoxic effector response of slow onset and shortduration compared to adult controls, yet paradoxicallywith rapid early activation of neonatal CD8+ T cells thatis not sustained. We have used a replication-defectiveHSV mutant to define the late times p.i., as WT HSV-2 israpidly lethal to the newborn even in low doses [34].This strain induces T cell responses equal to WT virus inadult mice [32]. Although our system removes the effectof virus spread on the induction of the CTL, it does allowus to define the earliest events in switching on CTLeffectors in newborn mice.

The induction of the primary CTL response to virusesincluding HSV type 1 has been well characterised inadult mice [25, 31, 37, 40]. Here we show that after HSVtype 2 infection there is rapid activation and arming ofAg-specific CD8+ cytotoxic effectors in the draining LNand spleens of adult mice, with kinetics similar to thereported response to HSV-1 [31, 38]. In adult mice, CTLactivity against HSV-2 was first detected in the LN fromday 3 p.i., with peak CTL responses evident from day 3to 9 p.i. In contrast, there was a slow rise to the peakHSV-specific CTL response in neonates from day 3 today 6 p.i. in both the LN and spleen. This was followedby a rapid loss of CD8+ T cell cytotoxicity after 3 days,rather than the strong, sustained response that occurredin adults. Rapid expansion of dominant T cell popula-tions has been reported to suppress T cell responses tosubdominant determinants [41]. Thus, the limitedneonatal response to gB498–505, the immunodominantCTL determinant to HSV, could have been associatedwith an enhanced response to subdominant determi-nants. Here, we show that this was not the case, as theneonatal CTL response to the subdominant epitope to

HSV-2, RR1829–836 [35, 36], was similarly reduced.Numerous studies have defined the memory CD8+ Tcellresponse to viruses in the newborn [10–16, 18],including a disabled infectious single-cycle HSV-1 strain[42], but few studies [10, 15, 26, 27], like ours, haveexamined the kinetics of the primary CTL response inthe neonatal period after viral infection. Fadel et al.reported the primary CTL cytotoxic responses in new-born mice to i.p. infection with low doses of Cas. Incontrast to our study, neonatal CTL responses occurredwith similar kinetics to adults [15, 26]. Possibleexplanations for the different observations includecontrasting life cycles of the virus, different routes ofinoculation (i.p. versus s.c.), which would employdifferent subtypes of APC to activate the CTL, anddifferent timing of analyses. Another difference was thatthe age-associated difference in the neonatal CD8+ Tcellresponse to HSV remained evident even when virus dosewas adjusted for APC number, whereas the impairedneonatal CTL response to Cas was dose dependant. Thissuggests that the differences we have observed are notsolely related to the functional immaturity of theneonatal immune system, but are also potentiallycompounded by specific immunoevasive properties ofHSV.

Another study of the kinetics and magnitude of theprimary CTL response in neonates was by Moser andcoworkers [27], who reported robust Ag-specific CTLresponses to polyoma virus infection in tumour-resistantnewborn mice after a short delay, but functional defectsin the CTL response of tumour-susceptible neonates.Like our study, analyses were performed after s.c.infection, but only splenic CD8+ T cell responses werereported. In contrast to our study, the reduced neonatalAg-specific cytotoxicity occurred despite a massiveexpansion of Ag-specific CD8+ T cells and strongCD8+ T cell IFN-c and perforin responses (also seenafter Cas infection). Furthermore, the impairment wasreversed after Ag restimulation of neonatal CD8+ T celleffectors in vitro. Previous work has demonstratedreduced ex vivo CTL responses to HSV with Agrestimulation in neonates at the time of the peakresponse [32]. Taken together, this suggests that CTLfrom tumour-susceptible neonates are armed afterpolyoma virus infection, even though they fail to elicita cytotoxic response, whereas those from HSV-infectedneonates are not.

After contact with a foreign Ag, Ag-specific T cellsproliferate and differentiate via signalling through theTCR. The magnitude of clonal CD8+ T cell expansion isdriven by the duration of antigenic stimulation and localIL-2 production, with brief stimuli resulting in lowendogenous IL-2 production and activated CD8+ T cellapoptosis [43]. The peak of CD8+ Tcell activity in adultsappears to depend on Ag dose, whereas the kinetics of



CD8+ T cell homeostasis remains similar irrespective ofdose [24]. Despite a lower lymphocyte number in the LNand spleen of neonates, many studies have reportedvigorous expansion/recruitment of CD8+ Tcell effectorsafter neonatal infection [15, 26, 27]. Here, we show thatthe expansion of total and Ag-specific (Kb-gB498–505

tetramer-positive) CD8+ Tcell in the draining LN occurswith different kinetics in newborn mice compared toadult controls. Unlike the rapid burst of proliferationseen in adults, there is limited expansion of neonatalCD8+ T cells after HSV infection until day 4 p.i., evenwhen T cell number is taken into account. Moser andcoworkers similarly reported a low percentage oftetramer-positive CD8+ T cells after polyoma virusinfection of newborn mice compared to adults with thesame infection [27]. Like us, they observed that a greaterproportion of total neonatal CD8+ T cells expressedIFN-c, compared to the proportion of Ag-specifictetramer+ CD8+ T cells that expressed IFN-c. In theirstudy, neonatal CD8+ IFN-c+ cells showed decreasedtetramer staining, purportedly as a result of ligand-induced TCR down-regulation in response to antigenicrestimulation in vitro. In contrast, we observed that thereduced proportion of tetramer-positive IFN-c+ CD8+

Tcells was not associated with reduced tetramer bindingin neonates or adults.

Labelled neonatal HSV TCR-transgenic CD8+ T cellsshowed reduced proliferation in vivo compared to adultTcell controls in neonatal recipients uponHSV infection.Vigorous proliferation of neonatal HSV-specific TCR-transgenic CD8+ T cells was observed, however, after invitro co-culture with peptide-primed adult DC. Thissuggests that slow expansion of CD8+ Tcells in neonatalmice after HSV infection in vivo is likely due to acombination of inherent properties of neonatal T cells,the competency of the APC, and to the environment inwhich they are stimulated. Adult transgenic T cellsproliferated well in either adults or neonates, whereasneonatal HSV TCR-transgenic T cells showed reducedproliferation even when transferred into adult mice. Thecytokine milieu in the neonatal LN may therefore besuboptimal for neonatal T cell activation, but sufficientfor adult Tcells. The IL-2 family of cytokines is importantfor CD8+ T cell viability after activation [44]. Thus, itwill be important to determine if the slow expansion ofneonatal CD8+ T cells is associated with reducedproduction of IL-2 and IL-2 family cytokines in thedraining LN in vivo. DC maturation is also critical to thedevelopment of a protective effector T cell response.Mature DC express high levels of costimulatorymolecules and produce cytokines that promote T cellsurvival and/or pro-inflammatory cytokine production[45]. In contrast, sub-optimally matured DC produceimmunosuppressive cytokines such as IL-10, andputatively inhibit Tcell effector development and induce

activated T cell apoptosis by as yet undefined mechan-isms [45]. Studies of neonatal APC and cytokineresponses in vivo after HSV infection are beingperformed to test these theories.

Adult-like memory CD8+ T cell responses have beenreported to a number of viruses and vaccines afterneonatal priming [13, 17, 18, 21]. Some studies haveeven reported enhanced CD8+ T cell memory responsesin later life after neonatal priming with viral pathogens[15, 26]. Thus, neonatal T cells have the capacity tomount protective CD8+ T cell responses under certainconditions. In contrast, PBMC from HSV-infected new-born infants show reduced proliferation and IFN-cresponses to HSV restimulation in vitro compared toinfected older children and adults [23], and newbornmice immunised with large doses of an HSV-1 DISCvaccine showed reduced memory CD8+ T cell cytotoxicresponses at 4 wk of age without rechallenge [42]. Itwill thus be important to determine whether there isreduced expansion and function of memory CD8+ T cellresponses after neonatal HSV infection. This is currentlyunder evaluation. Given that the adult-like CTLresponses have been reported in neonates to otherviruses but not to HSV, the age-dependant differenceswe have observed may be specific to the immunobiologyof HSV in the newborn system and not simply due to afunctional immaturity of T cells.

Stimulation of T cells correlates with the expressionof T cell activation markers [46]. We have previouslyshown that neonatal CD8+ T cells from the draining LNdisplay reduced expression of the IL-2 receptor, CD25, atthe time of the peak adult CTL response [32]. Here, weextend this observation and demonstrate that CD8+

T cells from the draining LN of neonatal mice expressCD25 with the same kinetics as adult mice after primaryHSV infection, but to a lesser extent from day 3 p.i. Incontrast to the reduced activation at later times, stainingfor CD69 expression revealed that the neonatal CD8+

T cells were activated more rapidly and to a greaterextent than adult CD8+ Tcells in the draining LN shortlyafter HSV infection. This data is consistent with thefindings of Adkins and coworkers [47], who demon-strated that neonatal T cells show rapid up-regulation ofearly activation markers in response to polyclonalactivation with anti-CD3 antibody or mitogens in avariety of mouse strains. The early T cell activation wasassociated with more rapid entry of neonatal CD8+

T cells into the cell cycle compared to adult T cells.However, Adkins also reported enhanced CD25 expres-sion on neonatal CD8+ T cells in response to anti-CD3antibody and mitogens. In contrast, we show thatneonatal CD8+ T cell CD25 and CD62Llow expression isreduced in neonates after HSV infection beyond day 3p.i. and associated with reduced CTL effector function.We are unable to separate non-specific activation from



HSV-specific CD8+ T cell activation at early times p.i.due to the limited sensitivity of CD8+ tetramer staining.However, as the CD8+ T cell IFN-c expression was alsodelayed and of reduced magnitude in HSV-infectedneonates compared to adults, it is likely that themajorityof CD8+ Tcells that express CD69 in the neonate at theseearly times are non-specifically activated. At the presenttime, the fate of these activated T cells remainsunknown. Thus, it will be important to define thekinetics of the contraction phase of the newborn Ag-specific T cell response to HSV and the molecularmechanisms that contribute to this process.

In summary, we have made the novel finding that theprimary CD8+ Tcell response to HSV in neonatal mice isassociated with reduced CD8+ T cell expansion andshort, attenuated peak effector activity compared toadult controls, yet paradoxically with enhanced earlyneonatal CD8+ T cell activation that is not sustained. Asthis is the time frame when innate cellular effectorsinteract with each other to instruct and regulate naiveT cells to mount a protective adaptive response againstHSV, future work will determine if differences in thecoordination or function of the neonatal innate responseaccount for the shortened peak neonatal adaptiveresponse. Further dissection of the earliest events ofCD8+ T cell priming shortly after HSV infection mayinstruct the requirements for inducing protectiveantiviral adaptive responses in the first weeks of life.This information will facilitate the development ofpostnatal immunotherapeutic interventions againstsevere perinatal viral pathogens including HSV thatcould also be applied in childhood and in later life.

Materials and methods

Viruses and cells

WT HSV-2 strain 186syn+-1 [48] and the replication-defectiveHSV-2 186syn+-1 mutant, dl5-29, defective for the UL5 geneand the UL29 gene [33], were propagated and stored aspreviously described [34]. The HSV-2 virus, dl5-29, and thecomplementary cell line, V5-29, were kindly provided by DavidKnipe, Harvard Medical School, Boston, MA.

Mice strains and inoculations

All experiments were conducted in accordance with TheChildren's Hospital at Westmead and Westmead HospitalAnimal Ethics guidelines and with the approval of the Office ofGene Technology and Recombination, Australia. AdultC57BL/6 mice from the Animal Resource Centre (Perth,Australia) were acclimatised for 1 wk prior to use. gBT-I.1TCR-transgenic mice which express a TCR that recognises theHSV-1 MHC class I-restricted gB498–505 determinant (SSIE-FARL) in complex with H-2Kb [39] were bred at WestmeadAnimal Facilities. Newborn mice were infected at 1 wk of age

to mimic the immune response of a human newborn at term[12]. Mice were inoculated either with 1 � 105 PFU/mouse ofWT HSV-2 virus or doses as indicated of the replication-defective HSV-2 strain, dl5-29, or mock-infected with anequivalent volume of low-endotoxin PBS (Invitrogen Corpora-tion, Carlsbad, CA) via s.c. inoculation (10 lL) into each hindfootpad. Adoptive transfer of splenocytes was by i.v. injection(100 lL) into the tail vein of adults or substernally into thesuperior vena cava of neonates.

In vivo HSV-specific CTL assay

Single-cell splenocyte preparations from naive female adultC57BL/6 mice were prepared as in vivo CTL target cells andcontrols, after removal of erythrocytes by osmotic lysis [31]. Inbrief, half the cells were pulsed with gB498–505 peptide(SSIEFARL) (1 lM) or with HSV-2 RR1829–836 peptide(RTFDFGML) (1 lM) where indicated (both from Auspep,Parkville, Australia) for 45 min at 37�C, then labelled with ahigh concentration (2.5 lM) of CFSE (CFSEhigh cells), and halfwere left unpulsed and labelled with a lower concentration(0.25 lM) of CFSE (CFSElow cells). Cells from each populationwere mixed together in an equal proportion and injected i.v.(1 � 107 cells/mouse) into HSV-infected adult or neonatalmice or mock-infected controls at times p.i. as indicated. At 4 hpost transfer, the draining LN and spleens were collected, madeinto single-cell suspensions, and then analysed by flowcytometry. The HSV gB-specific lysis was calculated bycomparing the percentages of high and low CFSE intensitiesaccording to the following formula: ratio = (percentageCFSElow/percentage CFSEhigh). Percentage specific lysis = [1 –(ratio unprimed/ratio primed) � 100].

Flow cytometry and tetramer staining

All antibodies were obtained from BD Biosciences (San Diego,CA) unless otherwise stated. Draining popliteal LN cells wereblocked in PBS containing 1% Fc block (2.4G2), 1% humanfoetal bovine serum (HFBS), 1% normal rat serum (NRS)(Animal Resource Centre, Perth, Australia) and 1% normalgoat serum (NGS) (Silenus, Hawthorn, Australia) for 30 minto 1 h at 4�C, and then stainedwith anti-CD8a-PerCP (53–6.7),anti-CD3-PE (145–2C11), and purified anti-CD25-biotin (7D4)or purified anti-CD69 antibody (HI.2F3) followed by strepta-vidin-APC at 4�C for 20 min for FACS analysis. The Kb-gB498–505 tetramer (conjugated to PE) was provided byA. Brooks, Dept. Microbiology & Immunology, University ofMelbourne. Tetramer staining of LN cells was for 20 min at37�C. Data were acquired on a BD FACSCalibur flow cytometerand analysed using CellQuest software (BD Biosciences, SanJose, CA).

In vitro HSV-specific CD8+ T cell proliferation assay

HSV-specific CD3+ CD8+ T cells were obtained from thespleens of TCR-transgenic (gBT-I.1) adult and neonatal miceusing a FACS Diva cell sorter (BD Bioscience), then labelledwith 0.5 lM CFSE (37�C for 10 min). Purified (�97%)CD11c+ DC were obtained from the LN of naive female adultmice by Nycodenz density gradient purification, immunomag-



netic antibody depletion to remove cells not of DC lineageusing anti-CD3 complex (17A2), anti-Thy-1/CD90 (G7), anti-Gr-1+Ly-6G (RB6–8C5), and anti-erythrocyte Ly-76 (TER-119)antibodies, then sorting using a FACS Diva cell sorter (BDBiosciences) after staining with anti-CD11c antibody (HL3).Purified DC were primed with gB498–505 peptide (SSIEFARL)(1 lM) (Auspep) for 45 min at 37�C, OVA257–264 peptide(SIINFEKL) (1 lM) (Auspep) or left unprimed, then co-cultured with the CFSE-labelled gBT-I.1 adult and/or neonatalT cells in 96-well V-bottom tissue culture plates (5 � 104 DCwith 5 � 104 CD8+ CTL/well) in 200 lL DMEMwith 10% FCS(Life Technologies), 2 mM L-glutamine and 50 lM 2-ME(Sigma-Aldrich, St. Louis, MO) for approximately 60 h. Cellswere then stained for Va2+-TCR (B20.1), which is expressed bythe majority of gBT-I.1 TCR-transgenic T cells, and analysed byflow cytometry [38]. Proliferation was determined by the lossof CFSE intensity of CFSE+ CD8+ Va2+-TCR+ cells [31].

In vivo CD8+ T cell proliferation assay

Spleens were removed from either naive female adult gBT-I.1mice or neonatal mice, made into single-cell suspensions,depleted of CD11c+ DC and purified by immunomagneticbeads using MidiMACS with LS separation columns (BDBiosciences). Splenocytes were labelled with CFSE (2.5 lM) at37�C for 10 min, and injected i.v. (4 � 106 CD8+ T cells/mouse) into adults or neonates. At 24 h post adoptive transfer,mice were inoculated s.c. in the footpad with WT HSV-2(1 � 105 PFU/mouse). At the indicated times p.i., mice wereeuthanised and the draining popliteal LN stained for CD8+

Va2+ cells and analysed by FACS. Proliferation of HSV gB-specific CD8+ cells was determined by assessing the reductionin CFSE-associated fluorescence intensities of CD8+ Va2+

T cells [31].

Intracellular cytokine staining

The method was as previously published [34]. In brief,draining popliteal LN cells from individual HSV-infected miceor mock-infected controls were cultured for 12–16 h in 96-wellplates (2 � 105 cells/200 lL, in triplicate) with gB498–505

peptide (1 lg/mL) at 37�C/5% CO2 in RPMI 1640 (InvitrogenCorporation) containing 10% FCS, 2 mM L-glutamine,2 � 10–2 mM 2-ME (Sigma) and 2% penicillin/streptomycin.We have previously optimised this duration of stimulation forour intracellular IFN-c assays. Brefeldin A (“GolgiPlug”) wasadded to a final concentration of 1 lg/mL for the final 4 h ofstimulation. Cells were then washed, fixed and permeablised(Cytofix/Cytoperm; BD Biosciences) for 20 min on ice, washedand blocked in Perm Wash (BD Biosciences), containing theblocking cocktail described above, stained for 20 min at 4�Cwith anti-IFN-c-FITC (XMG1.2) and anti-CD8a-PerCP anti-body, then analysed by FACS. For tetramer staining, cells werestained with Kb-gB498–505 tetramer for 20 min at 37�C andwashed prior to intracellular cytokine staining.

Statistics

All statistical analyses were performed using the two-tailedStudent's t-test or, for non-continuous data, a Wilcoxon Rank

Sum test. A p value of <0.05 was considered significant.Ack-nowledgements: This work was supported in part by agrant from the National Health and Medical ResearchCouncil of Australia (Grant No. 253684 to C.A.J.). Wethank Anthony Cunningham, Steve Alexander, and LisaSedger for their valuable discussions, Kate Herc, MarySartor and Sanda Lum for their technical assistance.

Conflict of interest: The authors declare no financial orcommercial conflict of interest.

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neonatal cd8+ t cells are slow to develop into lytic effectors after hsv infection in vivo

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