TLR2 Or TLR5 Stimulation Subject to Immunomodulation by Direct T Cells Are + Activated Human Neonatal

The Journal of Immunology

Activated Human Neonatal CD8؉ T Cells Are Subject to Immunomodulation by Direct TLR2 or TLR5 Stimulation1

Mark McCarron2*† and Denis J. Reen*†

In conditions of optimal priming, the neonate possess competency to mount quantitatively adult-like responses. Vaccine formu- lations containing sufficiently potent adjuvants may overcome the neonates’ natural tendency for immunosuppression and provoke a similarly robust immune response. TLR expression on T cells represents the possibility of directly enhancing T cell immunity. We examined the ex vivo responsiveness of highly purified human cord blood-derived CD8؉ T cells to direct TLR ligation by a repertoire of TLR agonists. In concert with TCR stimulation, only Pam3Cys (palmitoyl-3-Cys-Ser-(Lys)4) and flagellin monomers significantly enhanced proliferation, CD25؉ expression, IL-2, IFN-␥, TNF-␣, and intracellular granzyme B expression. TLR2 and TLR5 mRNA was detected in the CD8؉ T cells. Blocking studies confirmed that the increase in IFN-␥ production was by the direct triggering of surface TLR2 or TLR5. The simultaneous exposure of CD8؉ T cells to both TLR agonists had an additive effect on IFN-␥ production. These data suggest that a combination of the two TLR ligands would be a potent T cell adjuvant. This may represent a new approach to TLR agonist-based adjuvant design for future human neonatal vaccination strategies requiring a .CD8؉ component. The Journal of Immunology, 2009, 182: 55–62

mmaturity of the neonatal immune system compared with overcome. Consequently, it is thought that adjuvant choice for ne- adults, including possession of a T cell repertoire that is ex- onates is likely to play a key role in creating the necessary priming I clusively naive, is thought to contribute to the increased sus- conditions to elicit a protective adult-like immune response (6). At ceptibility to infection and the high rate of infection-related deaths present, alum salts continue to monopolize human vaccine formu- during the neonatal period (1–3). lations, yet they are relatively poor adjuvants at inducing cellular Vaccination represents an extremely efficient and cost-effective immune responses (11). public health intervention (4), particularly when administered dur- TLR ligands, predominantly through their activation of innate ing the neonatal period, as access to medical care is greater at this cells, represent attractive candidates as future vaccine adjuvants time point than later in life in many geographical areas (5). Vac- (12). To date, 10 members of the TLR family have been identified cines that are effective in the high-risk period of the first 3 mo of (TLRs 1–10) in humans and a number of ligands for each, with the life and that can confer protection from “birth to death” remain an exception of TLR10, have also been characterized (13). The broad increasingly urgent unmet need. The slow induction of vaccine- array of pathogen-derived ligands enables the innate immune sys- mediated infant protection correlates with the gradual postnatal tem to survey the body and detect most pathogen types. Broadly maturation of the infant immune system (6). speaking, TLR2 (with TLR1 or TLR6) is triggered by lipopeptides 3 However, in certain circumstances neonates can overcome their (“mimicked” by palmitoyl-3-Cys-Ser-(Lys)4 (Pam3Cys) or fibro- evolutionary conserved tendency for immunosuppression and blast-stimulating lipopeptide-1 (FSL-1)); TLR3 by dsRNA (mim- mount adult-like CD8ϩ T cell responses (7). The induction of po- icked by poly(I:C)); TLR4 by LPS; TLR5 by flagellin monomers; tent CD8ϩ T cell responses is likely to be critical in the design of TLR7 by a guanosine anolog, loxoribine (7-allyl-7,8-dihydro-8- modern prophylactic vaccines against viruses and intracellular oxo-guanosine); TLR8 by ssRNA; and TLR9 by unmethylated bacteria (8). The most convincing evidence of adult-like CD8ϩ T CpG dinucleotides within particular sequence contexts (CpG cell responses in human neonates are the “proof of principle” ex- motifs). periments provided by nature whereby functionally mature cyto- The activation of the innate immune response via TLRs repre- lytic CD8ϩ T memory lymphocytes have been detected in the cord sents the traditional interface, as foreseen by Janeway (14), be- blood of newborns with congenital infections (9, 10). This indi- tween pathogens and the immune system resulting in an adaptive cates that, under appropriate inflammatory conditions, the well- immune response that is propagated independently of direct con- documented deficiencies in the neonatal immune system can be tact with the intact pathogen or its TLR ligands. However, the direct interaction of TLR ligands with TLR-expressing adaptive T cells is an emerging concept (15), and these cells may represent *Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ire- novel TLR responsive targets in future vaccine adjuvants. In a land; and †University College Dublin Conway Institute of Biomolecular and Biomed- number of recent well-controlled studies, the presence of various ical Research, University College Dublin, Dublin, Ireland TLRs on human CD4ϩ T cells (16, 17), CD4ϩCD25ϩ T regulatory Received for publication February 28, 2008. Accepted for publication October 7, ␥␦ 2008. cells (18–20), and T cells (21) have been described. Few studies have addressed the expression of TLRs on human The costs of publication of this article were defrayed in part by the payment of page ϩ charges. This article must therefore be hereby marked advertisement in accordance or murine CD8 T cells. The immunomodulatory action of TLR2 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the Children’s Medical and Research Foundation (RP350) (to M.M.). 3 Abbreviations used in this paper: Pam3Cys, palmitoyl-3-Cys-Ser-(Lys)4; FSL-1, 2 Address correspondence and reprint requests to Dr. Mark McCarron, Children’s fibroblast-stimulating lipopeptide-1. Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin 12, Ireland. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org 56 TLR EXPRESSION ON NEONATAL CD8ϩ T CELLS

ϩ on murine effector (22) and naive (23) CD8 T cells has been sciences). The suspension was incubated at room temperature for 10 min, demonstrated. In contrast, a recent human study failed to detect followed by a wash in PBS and analysis on an LSR II flow cytometer, and the data were analyzed using BD FACSDiva software (BD Biosciences). TLR2 mRNA and demonstrated only functional TLR3 expression ϩ ϩ To determine the frequency of activated CD8 T cells responding to the on effector and effector memory CD8 T cells (24). TLR ligands, cells were stained intracellularly with CFSE (Invitrogen). In light of these conflicting findings, it was of interest to exam- Briefly, the cells were suspended at 10 ϫ 106/ml in serum-free RPMI 1640 ine TLR expression and function on human CD8ϩ T cells. In par- at a final CFSE concentration of 0.5 ␮M. The tube was incubated at 37°C ticular, the use of cord blood-derived CD8ϩ T cells in an APC- for 5 min and unincorporated extracellular CFSE was quenched by the addition of two volumes of heat-inactivated filtered FCS followed by fur- independent model allowed a full elucidation of the TLR repertoire ϩ ther 15 min of incubation. Cells were then washed twice in PBS, counted, on human neonatal naive CD8 T cells. resuspended, activated for the examination of proliferation as described above, and examined on the LSR II. To enumerate the percentage of gran- Materials and Methods zyme B-producing cells, intracellular staining was performed using anti- Cell purification human granzyme B-PE (Caltag Laboratories) and the Cytofix/Cytoperm kit (BD Biosciences) in accordance with the manufacturers’ recommenda- Venous blood from the umbilical cords of healthy babies delivered tions. To accurately define positive events, appropriate isotype controls either via natural delivery or elective caesarian section was collected were used for all flow cytometric analysis. into endotoxin-free 50-ml polypropylene tubes (Corning) and antico- agulated with EDTA (10 mM; Sigma-Aldrich). In all cases informed ELISA consent was sought and obtained as directed by the local ethical insti- Cell-free supernatants that had been appropriately diluted were analyzed tute. PBMCs were obtained by density centrifugation over Lymphoprep according to the manufacturer’s instructions for IFN-␥, TNF-␣, or IL-2 (Axis-Shield). A two-step purification procedure was employed to en- ϩ content using ELISA kits where the specific monoclonal capture Ab had sure the isolation of highly purified CD8 T cells that were free from been precoated onto microtiter wells (Invitrogen). The wells were spectro- Ag-presenting cell contamination. ϩ photometrically analyzed at 450 nm using a VersaMax microplate reader First, CD8 T cells were negatively selected from fresh PBMCs using (Molecular Devices). The concentration of each unknown sample was de- a negative selection Ab cocktail (human CD8ϩ T cell enrichment cocktail, ϩ ϩ ϩ termined automatically from the standard curve and analyzed using the Stemcell Technologies) containing mAb to CD4 , CD14 , CD16 , four-parameter algorithm on SoftMax Pro software (Molecular Devices). CD19ϩ, CD56ϩ, and glycophorin A. The Ab-labeled cells were cross- linked to magnetic nanoparticles (StemSep magnetic colloid, Stemcell RNA isolation and real-time PCR Technologies) and bound to a magnetic column allowing the elution of CD8ϩ T cells. Total RNA was extracted using TRIzol reagent (Invitrogen) in RNase-free The partially purified CD8ϩ T cells were then maintained overnight in polypropylene tubes (Ambion) as per the manufacturers’ instructions. The 6-well plates (Corning) in complete medium that consisted of RPMI 1640 homogenates were stored at Ϫ70°C until they were processed. The RNA (Cambrex BioScience) supplemented with L-glutamine, penicillin, and was then solubilzed in diethyl pyrocarbonate-treated water and the residual streptomycin (Sigma-Aldrich) and 10% heat-inactivated filtered FBS (FBS DNA was degraded by incubation with DNase I as instructed (Invitrogen). Gold; PAA Laboratories). The following day, double-positive FACS on a The RNA was reverse transcribed using a SuperScript first strand synthesis BD FACSAria was performed on the CD8ϩ T cells based on their surface system (Invitrogen) using the random primers according to the manufac- expression of CD3ϩ and CD8ϩ (BD Biosciences). Reanalysis of the sorted turer’s instructions. Real-time quantitative PCR was performed on an ABI T cells revealed a routine purity of Ͼ99.5% CD3ϩCD8ϩ T cells. For use Prism 7900HT sequence detection system (Applied Biosystems) using pre- as a positive PCR control, monocytes were isolated by positive selection designed TaqMan gene expression assays specific for human TLR2 using CD14ϩ microbeads that magnetically retained CD14ϩ expressing (Hs01872448_s1) and TLR5 (Hs01920773_s1). Specific gene expression cells in an LS column (Miltenyi Biotec). was normalized to 18S ribosomal RNA. Relative expression of TLR2 and TLR5 mRNA levels in cells activated for 24 h compared with resting cells Cell stimulation and proliferation was calculated using the 2Ϫ⌬⌬Ct method of analysis (Applied Biosystems). Duplicate samples were analyzed in each experiment. Highly purified human CD8ϩ T cells were washed in endotoxin-free Dul- becco’s PBS (Invitrogen), resuspended at 5 ϫ 105/ml in complete cell TLR studies medium, and activated ex vivo in an APC-independent system using anti- CD3/CD28-coated beads (Dynabeads; Invitrogen) as surrogate APCs. For For blocking experiments, cells were activated by anti-CD3/CD28 beads the study of effector functions, cells were activated in the presence of low (cell-bead ratio of 8:1) in the presence of IL-12 (5 U/0.4 ng) for 24 h dose (5 U/0.4 ng) exogenous IL-12 (R&D Systems). IL-12 acts as a third followed by the addition of either anti-human TLR2 Ab (InvivoGen), anti- signal that is essential for the differentiation of highly purified, activated human TLR5 Ab (InvivoGen), or control IgG at the indicated graded con- naive murine CD8ϩ T cells (25) and human neonatal naive CD8ϩ T cells centrations. The activated cells were incubated with the blocking Abs for (M. McCarron and D. J. Reen, manuscript in preparation) activated in the 1.5 h. Pam3Cys or flagellin was then added and supernatants were collected absence of APCs. T cells are unresponsive to IL-12 in the absence of cell after a further 48-h incubation in the presence of the agonists and stored at activation. Various cell-bead ratios were used as indicated in the figure Ϫ70°C until examined. ϩ 5 legends. With the exception of kinetic and blocking studies, TLR ligands For the cooperative studies, CD8 T cells (5 ϫ 10 /ml) were activated were added at the indicated concentrations at the initiation of cultures. The as before and either left untreated or treated with the TLR agonists alone or in combination. Proliferation was examined as described above and following TLR stimuli were used: TLR2 agonist: Pam3Cys (EMC Micro- collections), TLR4 agonist: ultra-pure LPS (Escherichia coli-0111: B4 supernatants were obtained and stored at Ϫ70°C. strain; InvivoGen), and the TLR5 agonist: flagellin (Salmonella thyphi- murium; InvivoGen). PHA (Sigma-Aldrich) responsiveness was examined Statistical analysis by adding PHA to a final concentration of 5 ␮g/ml. Results are expressed as means with SD. Statistical significance was de- 4 For proliferation studies, cells were plated in triplicate at 5 ϫ 10 per termined using a Student’s paired t test. Only p-values Ͻ0.05 were con- 3 well in 96-well round-bottom plates (Corning) and 1 ␮Ci of [ H]thymidine sidered statistically significant. Sample sizes are indicated in the figure (specific activity 25 Ci/mmol; Amersham Biosciences) was added to each legends. Student’s paired t test was performed with Minitab statistical soft- well for the last 18 h of a 72-h culture. Cells were harvested with a Tomtec ware version 13.32. Harvester 96 onto 90 ϫ 120-mm glass fiber filtermats (Wallac), and [3H]thymidine uptake was determined in a 1450 MicroBeta liquid scintil- Results lation counter (Wallac). In all other assays, cells were incubated in endo- ϩ toxin-free 5 ml (12 ϫ 75-mm)-polystyrene round-bottom tubes (BD Bio- Highly purified human neonatal CD8 T cells, isolated sciences) at 2 ϫ 105 cells per tube in complete medium. This was done to independently of contaminating TLR-expressing cells, are promote cell and anti-CD3/CD28 bead contact. activated by artificial surrogate APCs Flow cytometry Human umbilical cord blood contains a relatively homogeneous ϩ ϩ ϩ population of resting naive neonatal CD45RA CD8 T cells (26). To determine cell activation status, the purified CD8 T cells were either ϩ ϩ left resting or stimulated by anti-CD3/CD28 beads for the indicated time, CD3 CD8 T cells were purified by a combination of column and resuspended in 100 ␮l PBS, and stained with anti-CD25ϩ-PE (BD Bio- FACSAria cell sorting resulting in a routine final purity of Ͼ99.5% The Journal of Immunology 57

FIGURE 1. FACS-purified human neonatal CD8ϩ T cells are not responsive to PHA and proliferate in response to anti-CD3/CD28 beads. A, Cord blood CD8ϩ T cells were purified on StemSep columns followed by FACS purification and analyzed by flow cytometry. The results are repre- sentative of data routinely obtained following every cell sorting. The number (99.7%) represents the percentage of cells positively expressing both FIGURE 2. Activated human CD8ϩ T cells proliferate in response to ϩ ϩ ϫ 4 ϩ CD3 and CD8 on their cell surface. B, Cells (5 10 /well) were seeded flagellin and Pam3Cys but not LPS. FACS-purified CD8 T cells were following column purification (Column) or FACS purification (FACS) into activated by anti-CD3/CD28 beads at a cell-bead ratio of 8:1. Graded con- ␮ a 96-well plate in triplicate and stimulated with PHA (5 g/ml). Prolifer- centrations (0.1–10,000 ng/ml) of (A) Pam3Cys, (B) flagellin, or (C) ultra- ation was examined by incubation in the presence of [3H]thymidine (1 pure LPS were added to the activated cells for the full 72-h incubation, and ␮Ci) for the last 18 h of a 72-h culture. Results represent the mean with SD proliferation compared with cells that had been activated by anti-CD3/ of three independent samples. C, FACS-purified cells were incubated with CD28 alone (3/28) was examined. The results show the mean and SD from .p Ͻ 0.05 ,ء .various ratios (2:1 to 64:1) of CD8ϩ T cells to anti-CD3/CD28 beads and three, four, and three independent experiments, respectively proliferation was examined following 72 h of incubation. The results represent the mean with SD of three independent samples.

this concentration was used in subsequent experiments. The TLR4 (Fig. 1A). The use of a two-step purification methodology ensured ligand ultrapure LPS did not alter the proliferation pattern of the that the CD8ϩ T cell population was unlikely to contain contam- anti-CD3/CD28-activated T cells over the range of concentrations inating TLR-expressing cells such as B cells, NK cells, and mono- tested (Fig. 2C). Similarly, activated T cells were found to be cytes. Additionally, the FACS-purified CD8ϩ T cells failed to pro- unresponsive to agonists to TLR3, TLR7, TLR8, and TLR9 over a liferate to PHA, a polyclonal T cell activator that requires the range of concentrations (data not shown). There was no cell divi- presence of accessory cells (27), thereby confirming the absence of sion or detectable cytokine secretion in human neonatal CD8ϩ T TLR-expressing accessory cells (Fig. 1B). This lack of responsive- cells when TLR agonists were added in the absence of anti-CD3/ ness contrasts with the proliferation of column-purified CD8ϩ T CD28 activation of the cells. cells before FACS cell sorting (Fig. 1B). Flagellin and Pam3Cys independently regulate activated human CD3/CD28 Dynabeads, uniform polystyrene beads of approxi- ϩ mate leukocyte cell size containing bound Abs to mimic TCR trig- neonatal CD8 T cell division and activation gering and costimulation, were used as a physiological relevant Following activation, CD8ϩ T cells undergo cellular division be- system of T cell stimulation (28). A range of cell-bead ratios were fore differentiation and acquisition of effector functions (29). We ϩ examined to establish optimal ratios for neonatal CD8 T cell examined the effect of TLR2 and TLR5 agonists on the cell divi- activation (Fig. 1C). A cell-bead ratio of 8:1 was found to be the sion of activated CD8ϩ T cells using CFSE.

lowest ratio to produce a consistently potent proliferative response. The addition of ﬂagellin and Pam3Cys to anti-CD3/CD28- ϩ ϩ activated CD8 T cells signiﬁcantly decreased the percentage of Upon activation, human neonatal CD8 T cells become undivided cells from 62.8% (63.2 Ϯ 3.4%) to 23.9% (23.1 Ϯ responsive to TLR2 or TLR5 stimulation 1.7%) and 42.4% (43.8 Ϯ 2.6%), respectively (Fig. 3A). Flagellin Ϯ The addition of the TLR2/TLR1 agonist Pam3Cys to anti-CD3/ proved a more potent costimulator, driving 16% (16.7 3.2%) of CD28-activated T cells increased cell proliferation, with maximum cells into a third division compared with 3.9% (4.3 Ϯ 1.2%) of ␮ Ϯ proliferation being obtained at 1 g/ml (Fig. 2A). The TLR2/TLR6 cells activated in the presence of Pam3Cys and 1.5% (1.6 0.4%)

agonist FSL-1 provoked a similar proliferation pattern to Pam3Cys by cells activated by anti-CD3/CD28 alone. (data not shown). More strikingly, the TLR5 agonist ﬂagellin in- The expression of IL-2R and the production of IL-2 for auto- duced a dose-dependent augmentation of CD8ϩ T cell prolifera- crine consumption simultaneously promote clonal expansion and tion (Fig. 2B). This was most potently observed at 0.1 ␮g/ml and survival of activated T cells (30). We examined the expression of 58 TLR EXPRESSION ON NEONATAL CD8ϩ T CELLS

FIGURE 4. Flagellin and Pam3Cys enhance the effector functions of activated human CD8ϩ T cells. FACS purified CD8ϩ T cells were acti- FIGURE 3. Flagellin and Pam Cys enhance CD8ϩ clonal expansion ac- 3 vated by anti-CD3/CD28 beads at three ratios (8:1, 12:1, 16:1) in the pres- companied by an increase in the percentage of CD25ϩ cells and signifi- ence of exogenous IL-12 with either flagellin (3/28 ϩ Flagellin) or cantly enhanced IL-2 production. A, Cells were stained with CFSE and Pam Cys (3/28 ϩ Pam Cys) and supernatants were examined following a incubated in 12-well plates in the presence of anti-CD3/CD28 beads alone 3 3 72-h incubation for either (A) IFN-␥ production or (B) TNF-␣ production. (3/28; cell-bead ratio of 8:1) or in the presence of flagellin (0.1 ␮g; 3/28 ϩ These results represent the mean with SD of six and four independent Flagellin) or Pam Cys (1 ␮g; 3/28 ϩ Pam Cys). Following 72 h of incu- p Ͻ 0.05. C, The percentage of cells positive for ,ء .samples, respectively 3 3 bation, the cells were examined on an LSR II flow cytometer. A represen- ϩ intracellular granzyme B in purified human CD8 T cells that were acti- tative sample of three samples is demonstrated. B, The percentage of ac- vated by anti-CD3/CD28 beads (cell-bead ratio of 8:1) in the presence or tivated CD8ϩ T cells with surface expression of CD25 was examined absence of flagellin or Pam Cys was examined as outlined in Materials and following 24, 48, or 72 h of incubation with either anti-CD3/CD28 stim- 3 Methods after 24, 48, and 72 h of incubation. The results show the mean ulation alone (cell-bead ratio of 8:1) and with flagellin or Pam Cys added. .p Ͻ 0.05 ,ء .and SD from three independent samples 3 ,ء .The results represent the mean with SD of three independent samples p Ͻ 0.05. C, Cells were activated at a cell-bead ratio of 4:1 in the presence of exogenous IL-12 and examined by ELISA for IL-2 production following 24, 48, or 72 h of incubation in the presence or absence of flagellin or Flagellin monomers and Pam3Cys independently enhance the ϩ Pam3Cys. These results show the mean and SD from four independent effector functions of activated human neonatal CD8 T cells p Ͻ 0.05. ϩ ,ء .samples Following cell differentiation, CD8 T cells express a range of effector molecules that mediate defense against pathogens or tu- mors (31). ␥ the IL-2R ␣-chain (CD25), a central component of the IL-2 re- Both flagellin and Pam3Cys enhanced IFN- (Fig. 4A) and sponsive receptor and prototypical cell activation marker, on TNF-␣ (Fig. 4B) production at various cell-bead ratios examined. CD8ϩ T cells activated in the presence of the two TLR ligands. The effect of TLR2 and TLR5 ligation on the cytolytic capacity of ϩ Both flagellin and Pam3Cys significantly enhanced the percentage activated CD8 T cells was studied by examining the percentage ϩ of activated CD8ϩ T cells with CD25 surface expression at 48 and of CD8 T cells expressing intracellular granzyme B at 24, 48, and

72 h following activation (Fig. 3B). 72 h in the presence of the ligands. Both flagellin and Pam3Cys Additionally, CD8ϩ T cells activated in the presence of either sustained significantly higher levels of granzyme B in cells acti- TLR agonists produced IL-2 that was 4-fold greater in the presence vated for 72 h compared with cells activated by anti-CD3/CD28 of flagellin and 3-fold greater in the presence of Pam3Cys than that alone (Fig. 4C). ϩ produced by CD8 T cells activated in the absence of the TLR ϩ agonists following 72 h of incubation (Fig. 3C). Human neonatal CD8 T cells express TLR2 and TLR5 mRNA, ␥ Collectively, these results indicate that the TLR agonists are and TLR ligand-mediated IFN- production occurs in a capable of enhancing clonal expansion by enhancing CD25 ex- TLR2- or TLR5-specific manner pression on activated CD8ϩ T cells as well as significantly in- We next examined highly purified human neonatal CD8ϩ T cells creasing secretion of IL-2. for both TLR2 and TLR5 mRNA expression before and after The Journal of Immunology 59

FIGURE 6. Dual stimulation of human neonatal CD8ϩ T cells with

ﬂagellin and Pam3Cys enhances clonal expansion and has an additive effect of IFN-␥ production. A, CD8ϩ T cells were activated (cell-bead ratio of

8:1) and exposed to flagellin, Pam3Cys, or a combination of both agonists ϩ ϩ (3/28 Flagellin Pam3Cys) and proliferation was examined following a 72-h incubation. This result represents the mean and SD of four inde- p Ͻ 0.05. B, CD8ϩ T cells, activated (cell-bead ratio ,ء .pendent samples of 8:1) in the presence of exogenous IL-12, were exposed to either flagel- ␥ ϩ lin, Pam3Cys, or both agonists for the duration of culture (72 h) and IFN- FIGURE 5. Human neonatal CD8 T cells express TLR2 and TLR5, production was examined by ELISA. This result shows the mean and SD ␥ .p Ͻ 0.05 ,ء .and TLR-mediated enhancement of IFN- production is specifically ab- of three independent samples lated by specific blocking of surface TLR expression. A, Resting CD8ϩ T cells were either collected into TRIzol following purification or were activated (cell-bead ratio of 8:1) ex vivo for 24 h by anti-CD3/CD28 beads TLR5 blocking Abs and then exposed to the respective TLR ago- and then collected into TRIzol and stored at Ϫ70°C until processing. RNA nists for a further 48 h. isolation, reverse transcription, and quantitative RT-PCR were conducted Anti-TLR5 specifically blocked flagellin-mediated IFN-␥ pro- for both TLR2 and TLR5. TLR expression was normalized with the 18S duction in a dose-dependent manner (Fig. 5B). A similar ablation rRNA level, and TLR levels are expressed as relative levels compared with of Pam Cys-mediated IFN-␥ production was achieved by anti- ϩ 3 transcript levels in resting CD8 T cells (media) that were assigned a value TLR2, although a higher concentration of anti-TLR2 was neces- .p Ͻ 0.05 ,ء .of 1. Data are means and SD from three independent samples ϩ sary (Fig. 5C). This likely reflects a less specific inhibition by the B and C, CD8 T cells were activated (cell-bead ratio of 8:1) in the pres- anti-TLR2 Ab because of the heterodimeric nature of the TLR2 ence of exogenous IL-12 for 24 h. Graded concentrations of blocking Abs receptor compared with the homodimeric TLR5 receptor. specific for (B) TLR5 (0.0125–12.5 ␮g) and (C) TLR2 (0.125–25 ␮g) were Collectively, these results demonstrate that human neonatal added for 1.5 h and flagellin or Pam3Cys was added to the cells for a further ϩ 48 h. Supernatants were collected following the total 72 h incubation and CD8 T cells are specifically responsive to flagellin via TLR5 and frozen at Ϫ70°C until IFN-␥ levels were examined. The TLR-mediated Pam3Cys via TLR2 and that these ligands specifically alter the ϩ IFN-␥ enhancement was not blocked by an isotype-matched control, and effector functions of CD8 T cells. the specificity of the blocking Abs was confirmed as IFN-␥ production was unaltered when the blocking Ab and the unmatched TLR ligand (anti- Flagellin and Pam3Cys mutually enhance human neonatal ϩ TLR2 and flagellin or anti-TLR5 and Pam3Cys) were incubated together CD8 T cell activation (data not shown). The results show the mean and SD from three and four A recent area of intense research in APC biology is the examina- .p Ͻ 0.05 ,ء .independent samples, respectively tion of the combinatorial activation of TLRs to uncover the additive, synergistic, or antagonistic effects on various cellular functions in the hope of tailoring adjuvant mixtures to elicit stronger anti-CD3/CD28 activation. TLR2 and TLR5 mRNA was detected and more durable immune responses (32). To date, an examination in resting CD8ϩ T cells using quantitative RT-PCR (Fig. 5A). of this on TLR-expressing T cells has not been undertaken. Con- Crellin et al. previously demonstrated that TLR5 mRNA levels in sequently, we examined the effect of dual TLR stimulation on ϩ ϩ human CD4 T cells decrease following cell activation (18). In activated CD8 T cells with Pam3Cys and flagellin. agreement, both TLR2 and TLR5 mRNA levels were significantly The exposure of activated CD8ϩ T cells to both agonists sig- reduced by an average of 50% and 58%, respectively, following nificantly enhanced human CD8ϩ T cell clonal expansion com- cell activation for 24 h compared with resting CD8ϩ T cells. pared with stimulation with each agonist alone (Fig. 6A). More To examine whether the described enhancement of IFN-␥ (Fig. strikingly, there was an additive effect on IFN-␥ production when 4A) was mediated by the triggering of surface TLRs on the CD8ϩ both TLR agonists were present (Fig. 6B). T cells, we examined IFN-␥ production from activated CD8ϩ T Thus, a considerable enhancement of a key effector molecule in cells that had been pretreated with specific anti-TLR2 and anti- human neonatal CD8ϩ T cells was achieved by dual stimulation 60 TLR EXPRESSION ON NEONATAL CD8ϩ T CELLS

with flagellin and Pam3Cys compared with each agonist alone. The ulation are a mouse and rat study. In the mouse study, TLR5 complementary use of these agonists as adjuvants in CD8ϩ T cell was not detected and, in particular, anti-CD3ϩ-stimulated vaccination strategies may prove advantageous. mouse naive CD8ϩ T cells were functionally unresponsive to flagellin stimulation (23). In the rat model, TLR5 mRNA was Discussion detected in the CD4ϩ and T regulatory cells, but the anti-CD3ϩ- This study further demonstrates that TLRs can act downstream of activated cells were unresponsive to flagellin stimulation (37). their traditional role in the inflammatory response, providing an- Collectively, these studies are conflicting on whether murine other link in the previously bifurcated fields of innate and adaptive CD4ϩ T cells possess TLR5 mRNA. Moreover, the two func- immunity. tional studies on anti-CD3ϩ-activated murine T cells imply ϩ Our results show that activated neonatal naive CD8 T cells are that, if present, TLR5 is nonfunctional. functionally responsive to direct stimulation by TLR2 or TLR5 This raises the intriguing possibility that human naive CD8ϩ T ϩ agonists. Both resting and activated neonatal naive CD8 T cells cells uniquely express functional TLR5, which, as described in this were unresponsive to ligands directed against the remaining TLRs. study, can act as an extremely potent costimulatory receptor. It is Flagellin and Pam3Cys functioned directly to enhance cellular ac- already known that human but not murine dendritic cells express tivation, clonal expansion, and cell effector function beyond that functional TLR5 (38). Perhaps this differential expression should which was achieved by normal cellular activation. In general, not be surprising given the vastly different microbial challenges flagellin tended to be a more potent agonist than Pam3Cys. Sig- that the human species must encounter compared with the murine nificantly, the combined stimulation of neonatal T cells with both species and the large number of immunological differences be- TLR ligands resulted in increased clonal expansion and an additive tween the species, which have already been described and re- effect on IFN-␥ secretion. These results suggest a significant co- ϩ viewed by Mestas and Hughes (39). stimulatory role for TLR2 and TLR5 in human CD8 T cell There is growing evidence that TLR signaling on both murine function. ϩ and human T cells is physiologically important. In two murine To date, the investigation of TLR expression on CD4 T cells pathological models, direct TLR costimulation of murine T cells and T regulatory cells has provided the impetus for research in this was implicated in a MyD88-dependent colitis model (40) and a area (15). Consequently, there is a dearth of studies examining TLR9-dependent experimental autoimmune myocarditis model TLR expression on either murine or human CD8ϩ T cells. (41). In a murine model of Candida infection (20) and a tumor Two studies have emerged with regard to TLR2 expression and model (19), the direct action of TLR2 and TLR8 ligands, respec- function on murine CD8ϩ T cells. Sobek et al. found that a murine tively, on T regulatory cells temporarily reversed T regulatory cell CD8ϩ cytolytic T effector cell line was functionally responsive to suppression and reduced Candida infection load and tumor size. TLR2 stimulation (22). Cottalorda et al. extended this observation ϩ The direct action of TLR9-triggered murine CD4 T cells in en- to purified murine naive CD8ϩ T cells demonstrating that, follow- hancing humoral immunity by causing increased Ig titers has also ing TCR sensitization, Pam Cys enhanced cellular activation and 3 been described (42). An indirect association with human disease effector functions (23). Taken together, these studies demonstrated ϩ has also been posited through the demonstration of alterations in that activated naive and effector murine CD8 T cells are respon- the expression of various TLRs on T cell subsets between healthy sive to TLR2 stimulation, and this in turn enhances their activation and ill humans during filarial (43), hepatitis C (44), and HIV (45) and increases IFN-␥ production. However, studies on TLR2 ex- ϩ infections. pression in human CD8 T cells are less clear. Komai-Koma et al. first suggested that, similar to human naive CD4ϩ T cells, cord The emerging consensus in the literature appears to be that T blood CD8ϩ T cells expressed TLR2 mRNA and protein (16). cells require activation as a prerequisite to TLR responsiveness (15). This activation-dependent checkpoint has been extended to However, these data were not demonstrated. A more recent study ϩ found that freshly purified human CD8ϩ T cells express mRNA for human CD8 T cells in our study. A requirement for prior TCR TLR3 but do not express mRNA for TLR2 (24). activation would presumably prevent nonspecific activation of T Our observations conclusively demonstrate that human CD8ϩ T cells by exogenous or endogenous ligands. However, this may not cells express TLR2 mRNA and, following TCR stimulation, are accurately reflect the full effect of TLR ligands on T cells, as a functionally responsive to the most commonly used TLR2 agonist, TCR-independent effect on T cells has been reported. Zanin- Zhorov et al. demonstrated that the endogenous chaperone heat Pam3Cys. We did not find naive cells to be responsive to a TLR3 ϩ agonist. However, Tabiasco et al. found that following sorter pu- shock protein 60 alone can activate total adult human CD3 T rification of CD8ϩ T cell subsets, TLR3 expression was restricted cells via TLR2 stimulation, resulting in inhibition of cellular che- to effector and effector memory T cell subsets and, in agreement motaxis and increased cellular adhesion to fibronectin (46). This with our study, was not present on the human naive CD8ϩ T cells effect has since been replicated in a murine study (47). Recently, (24). This may indicate differential regulation between T cell sub- the ability of Pam3Cys to induce homotypic aggregation in human ϩ sets depending on their differentiation state, which is commonly total CD4 T cells independently of TCR sensitization has been ϩ the case in the APC compartment (33). It remains unclear why observed (45). A similar effect on neonatal naive CD8 T cells Tabiasco et al. failed to detect TLR2 mRNA in naive CD8ϩ T was observed during the course of our study (data not shown). cells. Collectively, these results may indicate that T cells, or at least Functional expression of TLR5 on human CD4ϩ and T regula- subsets within the T cell compartment, are sensitive to TLR ligand tory cells has been described (18). The human Jurkat cell line is stimulation and modulation of certain functions in the absence of also responsive to flagellin (34). However, data on TLR5 expres- TCR stimulation. Furthermore, the possibility that human neonatal ϩ sion on T cells is sparser in other nonhuman species. In two murine CD8 T cells possess mRNA or surface expression of additional studies, there are conflicting views on whether naive CD4ϩ T cells TLR receptors that are not responsive to their respective ligands possess TLR5 mRNA (35, 36). Additionally, neither study at- based on traditional T cell activation parameters has not been pre- tempted to examine the responsiveness of the cells to flagellin cluded by this study. The ability of these receptors to modulate stimulation. To our knowledge, the only studies to examine the chemotaxis or the costimulatory effect of the TLR2 and TLR5 functional responsiveness of nonhuman T cells to flagellin stim- agonists should be addressed in future studies. The Journal of Immunology 61

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