TLR2 is expressed on activated T cells as a costimulatory

Mousa Komai-Koma†, Louise Jones‡, Graham S. Ogg‡, Damo Xu†§, and Foo Y. Liew†§

†Division of Immunology, Infection, and Inflammation, University of Glasgow, Glasgow G11 6NT, United Kingdom; and ‡The Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, United Kingdom

Communicated by Salvador Moncada, University of London, London, United Kingdom, January 14, 2004 (received for review November 28, 2003) Toll is the founder of a group of pattern recognition receptors that Methods play a critical role in the innate immunity in . At least 10 Cells and Cultures. Cord blood was obtained from the Yorkhill distinct Toll-like receptors (TLRs), recognizing -associated Maternity Hospital, Glasgow, from informed consented mothers. molecular patterns, have now been identified in humans. Most Peripheral blood was obtained from healthy laboratory donors. investigations on TLRs have focused on cells of the innate system. We CD4ϩ T cells were purified by negative selection according to the report here that naı¨ve human T cells expressed high levels of cell- manufacturers’ instructions (Miltenyi Biotec, Auburn, CA) fol- surface TLR2 after activation by anti- receptor and lowed by double positive (CD3ϩ and CD4ϩ) selection by fluores- IFN-␣. Activated cells produced elevated levels of in re- cence-activated cell sorting (FACS). Peripheral blood CD4ϩ T cells sponse to the TLR2 ligand, bacterial lipopeptide. Furthermore, were further separated into CD45RAϩ and CD45ROϩ cells (12). ؉ ؉ CD4 CD45RO memory T cells from peripheral blood constitutively The cells were cultured in RPMI medium 1640 ϩ 10% FCS at 37°C expressed TLR2 and produced IFN-␥ in response to bacterial lipopep- ϫ 6 in 5% CO2 (2 10 cells per ml) in 24-well plates for 72 h in the tide, which also markedly enhanced the proliferation and IFN-␥ presence or absence of immobilized anti-CD3 antibody (1–5 ␮g͞ml, ؉ production by CD45RO T cells in the presence of IL-2 or IL-15. Thus, BD Bioscience), IFN-␣ (100–1,000 units͞ml, Insight Biotechnol- TLR2 serves as a costimulatory receptor for -specific T cell ogy, Wembley, U.K.), (LPS, 1–10 ␮g͞ml, development and participates in the maintenance of T cell memory. ␮ ͞ Sigma), (BLP, Pam3Cys-SK4, 1–10 g ml, This suggests that , via their pathogen-associated molec- EMC, Tuebingen, Germany), or a combination of these reagents as IMMUNOLOGY ular patterns, may contribute directly to the perpetuation and acti- indicated in the figures. For blocking experiments, cells were vation of long-term T cell memory in both antigen-dependent and activated with anti-CD3 antibody Ϯ IFN-␣ overnight followed by independent manner. adding anti-human TLR2 antibody or control IgG (both 10 ␮g͞ml, eBioscience, San Diego). BLP (2 ␮g͞ml) was added1hlater.In oll was originally found in Drosophila as a pattern recognition some experiments, cells were cultured with IL-2 (10–1,000 units͞ Treceptor associated with defense against fungal and bacterial ml, GlaxoSmithKline, Stevenage, U.K.) or IL-15 (20–200 ng͞ml, infection (1, 2). Toll was subsequently found in mammals and was Immunex) added at the beginning of the cultures. We have titrated named Toll-like receptor (TLR). At least 10 distinct TLRs have the concentrations of anti-CD3 antibody, IL-2, and IL-15 and found now been identified in humans (3–9). TLRs are activated by that there was no significant difference in the range of concentra- pathogen-associated molecular patterns with target selectivity. The tions used above. For routine analysis, cell proliferation ([3H]thy- pathogen-associated molecular patterns are integral structural midine incorporation) and production were determined at components of the pathogens and are thus expected to be essential 72 h of culture (48 h for IL-2). Cytokine concentrations were to the survival of the infectious organisms. Therefore, pathogen- determined by ELISA by using paired (BD Bioscience). associated molecular patterns are expected to be conserved among Results are expressed as mean Ϯ SEM (n ϭ 3–5; *, P Ͻ 0.05; **, a range of pathogens, including , bacteria, and . P Ͻ 0.01 by Student’s t test). It should be noted that, because of the TLRs act as primary sensors of microbial products and heterogeneity nature of human population, as expected there was activate signaling pathways that lead to the induction of immune considerable individual variation in response to T cell receptor and inflammatory (10). TLRs belong to a broader family (TCR) activation and BLP. All experiments were performed at of , which include receptors for the proinflammatory least three times from three individuals. Results presented are cytokines IL-1 and IL-18 and the orphan receptor T1͞ST2 (11). representative from a single blood donor. All members of this superfamily signal in a very similar manner because of the presence of a conserved Quantitative PCR. This was carried out as described (13). The sequence in the cytosolic domain called the Toll͞IL-1 receptor primers and probes used are as follows: for TLR2, GGT- domain, which activates common signaling pathways, most no- TCAAGCCCCTTTCTTCTTTA (forward), TGTGAGAT- tably those leading to the activation of the transcription factor GAGAAAAAAGAGATGTTTC (reverse), and CATTCT- NF-␬B and stress-activated protein kinases (11). TAAACTTACTGGGAAATCCTTACAAAACCCTAGG Most investigations on TLRs have focused on cells of the (probe); for TLR4, CAGAGTTTCCTGCAATGGATCA (for- innate system, because TLRs are closely associated with innate ward), TGCTTATCTGAAGGTGTTGCACAT (reverse), and response. Although innate immunity may constitute the primary CCATTCGTTCAACTTCCACCAAGAGCTG (probe); for functions of TLRs, there is no a priori reason why TLRs may not CD14, CCGCTGTGTAGGAAAGAA (forward), GCGCTC- have a direct function on adaptive immunity. We have investi- CATGGTCGATAA (reverse), and TTCCAGAGCCTGTC- gated the expression and functions of TLRs on T cells. We report CGGA (probe); and for MD2, TCCATATTTACTGAAGCTC here that TLR2 is expressed on the surface of activated and memory T cells. Furthermore, it functions as a costimulator Abbreviations: APC, antigen-presenting cell; BLP, bacteria lipoprotein; EBV, Epstein–Barr receptor molecule for T cell activation and helps to maintain T virus; FACS, fluorescence-activated cell sorter; LPS, lipopolysaccharide; TCR, T cell receptor; cell memory. These data provide a role for TLR2 and may help TLR, Toll-like receptor. to explain how memory T cells are sustained in an immune §To whom correspondence may be addressed. E-mail: [email protected] or competent host. The finding should have important implications [email protected]. in our understanding of the host response to infections. © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400171101 PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 ͉ 3029–3034 Downloaded by guest on September 29, 2021 (forward), ATTGCATTTTATCACAGTA (reverse), and TT- Results GGGTCTGCAACTCATCC (probe). Expression of TLR2 and TLR4 on Activated Human CD4؉ T Cells. To demonstrate the expression of TLRs on CD4ϩ T cells, cord blood Flow Cytometry. Purified ex vivo T cells or activated T cells were (containing mostly naı¨ve T cells) was collected and CD4ϩ T cells ␣ stained with directly conjugated antibodies: CD4(PE) and purified by negative selection followed by positive selection on a cell ␣ ␣ ␣ HLA-DR(FITC) from Sigma, CD3(ApC), CD45RA(PE), sorter (Ͼ99.9% pure). The cells were cultured with medium alone ␣ and CD45RO(PE) from BD Bioscience, and normal IgG or immobilized anti-CD3 antibody, and the expression of TLR2 and control (PE or ApC) from BD Bioscience. All samples were TLR4 was determined by quantitative PCR and flow cytometry. preincubated for 30 min with human IgG (Sigma) to block FcR. Naı¨ve CD4ϩ T cells expressed significant levels of mRNA and The cells were analyzed on a FACSCalibur flow cytometer intracellular proteins of TLR2, TLR4, and MD2 but not CD14. This (Becton Dickinson). All results are representative of at least was not affected by anti-CD3 ϩ IFN-␣ activation (Fig. 1a). In three independent experiments. The anti-TLR2 antibody (clone contrast, only activated T cells expressed appreciable levels of TL2.1) and anti-TLR4 antibody (clone HTA125) used here have cell-surface TLR2 or TLR4 (Fig. 1b). Kinetic studies show that been extensively characterized (14–17), and similar results were cell-surface TLR expression peaked between 12 and 72 h after obtained with a different source of antibody. activation and were still at a substantial level by 96 h (data not ϩ ϩ shown). The cell-surface expression of TLR2 and TLR4 on CD4 Immunofluorescence Microscopy. CD4 T cells were cytocentrifuged T cells were also visualized by immunofluorescence microscopy, ϫ at 400 g for 5 min (Cytospin-3, Shandon, Pittsburgh) and then which clearly showed TLRs expressed in a polarized manner on fixed with 1% paraformaldehyde (Sigma) for 10–15 min. Cells were CD3ϩ and CD4ϩ T cells (Fig. 1c). Similar results were obtained subsequently processed for analysis of CD3, CD4, TLR2, and TLR4 with cord blood CD8ϩ T cells (data not shown). Because the T cell ␣ expression as described above for flow cytometry (except CD3- preparation was Ͼ99.9% pure and the majority (but not all) of the PE, which was obtained from Sigma). Cells were analyzed with a cells stained positive for TLR2 or TLR4, it is unlikely that the Zeiss Axiovert microscope (Zeiss) by using an FITC and Texas red staining was due to APC, such as and dendritic cells. specific filter set (Glen Spectra, Middlesex, U.K.). Fluorescent The polarized staining pattern is intriguing. It is tempting to images were captured by using an ORCA cooled CCD camera speculate that TLRs tend to aggregate forming raft for (Hamamatsu Photonics, Hamamatsu City, Japan) and processed by effective function in a manner similar to TCR. We added IFN-␣ in OPENLAB software (Improvision, Coventry, U.K.). our culture medium as it enhanced the expression of the TLRs. The mechanism for this synergistic effect of TCR engagement and Tetramer and TLR Analysis of Epstein–Barr Virus (EBV)-Specific Cell IFN-␣ stimulation for TLR expression is currently under investi- Line. Complexes were synthesized as described (18). Briefly, puri- gation. We have elected to use plate-bound anti-CD3 antibody to fied HLA-A*0201 heavy chain and b2 microglobulin were synthe- activate the T cells in our culture system instead of the more sized by means of a prokaryotic expression system, with the heavy conventional soluble anti-CD3 plus irradiated APC, to avoid the chain modified by deletion of the transmembrane͞cytosolic domain potential contribution of APC, which carries TLRs and thus may and C-terminal addition of a sequence containing the BirA enzy- potentially affect indirectly the activation of T cells by means of matic biotinylation site. HLA heavy chain, b2 microglobulin, and cytokines. peptide (EBV BMLF1 protein 280–8 GLCTLVAML) (19) were refolded by dilution. The 45-kDa refolded product was isolated by Expression of TLR2 and TLR4 on Activated Antigen-Specific EBV؉ CD8؉ FPLC and then biotinylated by BirA (Avidity, Denver) in the T Cells. To demonstrate antigen specificity and relevance to ϩ presence of biotin, ATP, and Mg2 (Sigma). Streptavidin– infection of TLR expression, we generated CD8ϩ T cell lines phycoerythrin conjugate (Sigma) was added in a 1:4 molar ratio, from EBV-infected patients. The cell lines were analyzed by and the tetrameric product was concentrated to 1 mg͞ml. FACS by using MHC class I-EBV peptide tetramer and anti- TLR antibodies. Fig. 2 shows that 5.1% and 7.5% activated ؊ ؊ Culture of Cells from TLR2 / Mice. and cells EBV-specific CD8ϩ T cells from a donor were TLR2 and TLR4 Ϫ Ϫ ϩ were harvested from WT or TLR2 / mice (20). CD4 T cells positive, respectively. The percent of TLRϩ T cells varied among were purified by negative selection. The purity was 95%. The individuals ranging from 5% to 25%. cells (2 ϫ 106 per ml) were cultured in 24-well plates with CD3ϩ cell-depleted spleen cells (2 ϫ 105 per ml, without detectable BLP, but Not LPS, Acts as a Costimulatory Molecule for T Cell Activa- ϩ ϩ CD4 CD3 cells) from WT mice. Cell purification was carried tion. TLRs have been associated principally with Th1 cell response out with kits from Miltenyi Biotec and purity analyzed by FACS. (21). To determine the functional significance of TLR expression The cells were cultured for 3 days in the presence of immobilized on T cell differentiation, purified naı¨ve CD4ϩ T cells were cultured anti-CD3 antibody (1 ␮g͞ml, BD Bioscience). The activated with anti-CD3 antibody and IFN-␣ in the presence or absence of ϩ CD4 T cells were then collected and purified by positive BLP (Pam3Cys-SK4) or LPS, the ligands for TLR2 and TLR4, selection (purity Ͼ99.5%) and cultured with immobilized anti- respectively (8, 9). The proliferation of activated T cells was not CD3 antibody with or without BLP (2 ␮g͞ml) for 3 more days. affected by LPS but modestly enhanced by BLP. However, BLP Cell proliferation and IFN-␥ concentration in the culture super- markedly enhanced the production of IFN-␥, IL-2, and tumor natant were then determined as above. This series of experi- necrosis factor ␣ by activated CD4ϩ T cells (Fig. 3a). Similar results ments, which ensured equal potential WT antigen-presenting were also obtained when the cells were cultured with two other cell (APC) contamination in the activated WT and TLR2Ϫ/Ϫ TLR2 ligands, zymosan A, and peptidoglycan (data not shown). In CD4ϩ T cell populations, was designed to test whether BLP acted contrast, LPS had little or no effect on the cytokine production by directly on T cells or via APC. Experiments were also performed the T cells. We therefore focused on the function of TLR2. Fig. 3b by deliberately adding WT APC into the cultures of CD4ϩ T cells shows that the cytokine-enhancing effect of BLP was specifically from WT or TLR2Ϫ/Ϫ mice. Purified CD4ϩ T cells (1 ϫ 106 per blocked by an anti-TLR2 antibody, further demonstrating the ml) from WT or TLR2Ϫ/Ϫ mice were cultured for 3 days with functional specificity of BLP as TLR2 dependent. It should be immobilized anti-CD3 antibody alone or with 5% WT APC. Cell noted that because of heterogeneity of human population, as proliferation and IFN-␥ production were then determined as expected, there was considerable variation in the degree of en- above. hancement of cellular proliferation and cytokine production be-

3030 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400171101 Komai-Koma et al. Downloaded by guest on September 29, 2021 IMMUNOLOGY

Fig. 1. Naı¨ve CD4ϩ T cells activated with anti-CD3 antibody and IFN-␣ expressed cell-surface TLR2 and TLR4. (a)Naı¨ve CD4ϩ T cells purified from cord blood were activated with anti-CD3 antibody and IFN-␣ and analyzed for TLR2, TLR4, and MD2 mRNA at various time points by real-time PCR. (b) Cells activated with anti-CD3 antibody and IFN-␣, or culture medium alone (nonactivated), for 72 h were washed extensively and triple stained with ␣TLR4-FITC, ␣TLR2-FITC, ␣CD3-ApC, and ␣CD4-PE antibodies or control isotype-match IgG. Cells were analyzed by FACS. (c) Cells were double stained with ␣CD-PE and ␣TLR-FITC or ␣CD3-PE and ␣TLR-FITC and analyzed by fluorescent microscopy. Results are representative of three experiments.

tween individual cord blood donors. Nevertheless, the pattern of cell-depleted WT APC for 3 days. CD4ϩ T cells were then elevation by BLP was consistent. harvested, purified, and cultured with immobilized anti-CD3 for a further 3 days in the presence or absence of BLP. In such situations, BLP Acts Directly on T Cells and Not via APC. To further exclude the T cells from both TLR2Ϫ/Ϫ and WT mice should contain similar possibility that the observed effect of BLP on T cell activity may be level of contaminating WT APC. Whereas T cells from WT mice due to an indirect effect on contaminating APCs, we performed produced enhanced proliferation and IFN-␥ production in response similar experiments with T cells from TLR2 knockout mice. to BLP, T cells from the TLR2Ϫ/Ϫ mice failed to respond to BLP Purified (Ͼ95%) CD4ϩ T cells from TLR2Ϫ/Ϫ or WT mice were (Fig. 4a). We then cultured purified CD4ϩ T cells from TLR2Ϫ/Ϫ cultured in vitro with plate-bound anti-CD3 antibody and CD3ϩ T or WT mice with immobilized anti-CD3 and deliberately added

Komai-Koma et al. PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 ͉ 3031 Downloaded by guest on September 29, 2021 Fig. 2. BMLF1-specific CD8ϩ T cells were expanded in vitro and stained with the BMLF1͞HLA-A*0201 phycoerythrin-labeled tetramer (y axis) and FITC- labeled antibodies (x axis) directed to TLR4 (Top Right), TLR2 (Middle Right), and isotype control (Bottom Right). Of the CD8͞tetramer double-positive cells, 7.5% were positive for TLR4, and 5.1% were positive for TLR2.

WT APC in the presence or absence of BLP. Although T cells from WT mice produced increased proliferation and IFN-␥ production in the presence of BLP, T cells from TLR2Ϫ/Ϫ failed to do so even in the presence of up to 10% deliberately added CD3-depleted APC from WT mice. It should be noted that, unlike human T cells, murine T cells proliferated significantly in response to BLP. Fur- thermore, IFN-␣ did not influence murine T cell activation by anti-CD3 antibody (data not shown).

TLR2 Is Constitutively Expressed on Memory T Cells. We then inves- tigated the relative expression and function of TLR2 on memory and naı¨ve CD4ϩ T cells. CD4ϩ T cells were purified from peripheral blood of healthy donors and separated into CD45RAϩ (naı¨ve) and CD45ROϩ (memory) subsets. Cell-surface TLR2 expression was then analyzed by flow cytometry. As expected, naı¨ve CD45RAϩ cells did not express appreciable amounts of TLR2 in the absence of activation. In contrast, CD45ROϩ memory T cells expressed significant levels of TLR2 ex vivo (Fig. 5). The high level of TLR2 expression on CD45ROϩ cells could not be further enhanced by stimulation with anti-CD3 or anti-CD3 ϩ BLP. In contrast, TLR2 expression by the naı¨ve CD45RAϩ cells was significantly increased by the stimulation with anti-CD3, and this was further enhanced by the presence of BLP. The expression of TLR2 paralleled the activation status of the cells as evident by their coexpression of HLA-DR antigen, an activation marker.

␥ Fig. 3. The TLR2 ligand (BLP) but not the TLR4 ligand (LPS) acts as a BLP Enhances Proliferation and IFN- Production by Memory T Cells. costimulatory molecule for the activation of CD4ϩ T cells. (a) CD4ϩ T cells We then investigated the functional role of TLR2 expression in were purified from cord blood and cultured with immobilized anti-CD3 and ϩ ϩ activated naı¨ve CD4 CD45RA T cells and memory IFN-␣ in the presence of graded concentrations of LPS or BLP. T cell CD4ϩCD45ROϩ T cells in response to BLP in vitro. The cells were proliferation and cytokine production were determined at 72 h. Vertical cultured with plate-bound anti-CD3 antibody Ϯ BLP, and cellular bars represent SEM (n ϭ 4) of three experiments. *, P Ͻ 0.05; **, P Ͻ 0.01 (vs. culture with anti-CD3 ϩ IFN-␣ without BLP). (b) The costimulatory effect proliferation and cytokine production was determined. Naı¨ve and ϩ ϩ of BLP is blocked by anti-TLR2 antibody. Cord blood CD4 T cells were memory CD4 T cells did not show any detectable response when cultured with immobilized anti-CD3 antibody ϩ IFN-␣ with or without BLP cultured with BLP alone (Fig. 6). Both cell types produced high (2 ␮g͞ml). The enhancing effect of BLP was significantly blocked by the levels of proliferation when cultured with anti-CD3 antibody, which presence of an anti-TLR2 antibody (10 ␮g͞ml) but not by an isotype- ϩ was not affected by BLP. Naı¨ve CD4 T cells cultured with matched normal IgG2a (not shown). Results are representative of three anti-CD3 produced a low level of IFN-␥, which was modestly experiments. *, P Ͻ 0.05 vs. column without BLP.

3032 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400171101 Komai-Koma et al. Downloaded by guest on September 29, 2021 Fig. 4. APC from WT mice failed to modify the lack of effect of BLP on CD4ϩ T cells from TLR2Ϫ/Ϫ mice. (a) CD4ϩ T cells from WT or TLR2Ϫ/Ϫ mice were cultured for 3 days with CD3ϩ cell-depleted APC from WT mice (T:APC, 10:1) in the presence of immobilized anti-CD3 antibody. CD4ϩ T cells were then harvested, purified, and recultured with anti-CD3 antibody for a further 3 days. Cell prolif- ␥ ϩ IMMUNOLOGY eration and IFN- production were then analyzed. (b and c) CD4 T cells from WT Fig. 5. Expression of TLR2 on naı¨ve (CD45RAϩ, Left) and memory (CD45ROϩ, Ϫ/Ϫ or TLR2 mice were cultured for 3 days either alone (b) or with 5% APC from WT Right) peripheral blood CD4ϩ T cells. Flow cytometry was carried out on CD4ϩ ␥ mice (c) in the presence of anti-CD3 antibody. Cell proliferation and IFN- pro- T cells by using anti-CD45RO (PE), anti-CD45RA (PE), and anti-TLR2 (FITC) Ϯ ϭ duction were then determined. Data are mean SEM (n 3) from three antibodies. (a) Cells were stained for TLR2 ex vivo; mIgG denotes murine IgG Ͻ Ͻ experiments. *,P 0.05; **,P 0.01 (vs. figures of WT cells cultured without BLP). isotype control. (b) Anti-CD3 activation induced TLR2 expression on naı¨ve cells. This was further enhanced by the presence of BLP. The constitutively ex- ϩ ϩ pressed TLR2 by memory cells was not significantly increased further by enhanced by BLP. In contrast, memory CD4 CD45RO cells anti-CD3 and BLP stimulation. (c) TLR2 expression paralleled HLA-DR expres- activated with anti-CD3 produced significant amounts of IFN-␥, sion on these cells. Results are representative of three donors. which was markedly enhanced by BLP (Fig. 6a). IL-4 production was not detected (data not shown). We further compared the role ϩ self-amplification loop for TLR functions. (ii) TLR2 and TLR4 are of BLP on naı¨ve and memory CD4 T cells under a bystander ϩ activation condition with IL-2 and IL-15, two cytokines closely also expressed on subsets of activated EBV-specific CD8 T cells associated with memory cell expansion (12, 22–25). Naı¨ve derived from EBV-positive patients. This result extends our finding ϩ ϩ to clinically relevant T cells beyond the CD4ϩ subset. (iii) Although CD4 CD45RA cells did not produce appreciable levels of pro- ϩ liferation or cytokines when cultured with IL-2 or IL-15, and this activated CD4 T cells expressed similar levels of TLR4 and TLR2, was not affected by BLP. In contrast, CD4ϩCD45ROϩ memory T the cells did not respond to LPS, a recognized ligand for TLR4 (33). ␥ This is consistent with a large number of reports in the literature cells produced significant levels of proliferation and IFN- when ϩ ϩ cultured with IL-2 or IL-15, and these functions were markedly (34–36). LPS has recently been reported to activate CD4 CD25 enhanced by the presence of BLP (Fig. 6b). IL-4 or IL-10 was not T cells in mice (32). However, this finding remains to be confirmed. ϩ ϩ detected in the cultures (data not shown). In the CD4 CD25 T cell system, the potential involvement of cell types other than T cells could not be excluded. Using highly purified Discussion T cells, we were not able to activate murine CD4ϩCD25ϩ T cells to TLR messages have been reported to be present in T cells (26–32). respond consistently to LPS (data not shown). The reason for the We have now directly demonstrated the expression and function of failure of human T cells, in contrast to cells of the monocytic TLRs on the cell surface of antigen-specific T cells. The following lineage, to respond to LPS is currently unclear, but it may be due lines of evidence support the functional expression of TLR2 on T to the lack of coreceptors (such as CD14 shown in Fig. 1a)onthese cells: (i) the T cell population tested had no detectable CD14 T cells essential for LPS-induced activation. (iv) Activated or message (Fig. 1a); (ii) the majority of a 99.9% pure population of memory human T cells also failed to respond to PolyI:C (a ligand CD4ϩCD3ϩ cells stained positive for TLR2 (Fig. 1 b and c); (iii) for TLR3) or CpG (a ligand for TLR9) (data not shown), suggesting antigen-specific tetramer-positive cells also stained positive for that TLR2, whose ligands are perhaps the most conserved among TLR2 (Fig. 2); (iv) BLP alone did not induce a detectable level of all of the known TLRs, may be unique in its ability to function as cytokine or proliferation (Fig. 3a); and (v) in contrast to WT T cells, a costimulator receptor of T cells. (v) Induction of TLR expression anti-CD3-activated T cells from TLR2Ϫ/Ϫ mice did not respond to on T cells is TCR activation-dependent. Once induced, TLR2 BLP even in the presence of up to 5% of WT APC (Fig. 4). signaling further enhances the T cell functions. This TCR–TLR Several features reported here are noteworthy. (i) Surface ex- interaction pathway suggests that TLR2 may be essential for pression (and hence function) of TLR2 was evident only after TCR optimal antigen-specific T cell response. (vi) TLR expression is cell activation, and this was enhanced by the presence of the TLR2 activation-dependent and is enhanced by the presence of IFN-␣, ligand, BLP. Although the mechanism for the induction of TLR another innate cytokine. This demonstrates the networking role of migration from the cytoplasm to cell surface is currently unknown, TLR2 with other innate mediators in enhancing adaptive immune the effect of BLP in this process may represent an important response.

Komai-Koma et al. PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 ͉ 3033 Downloaded by guest on September 29, 2021 antigen-specific Th1 cell function, in addition to an indirect role through the activation of APC (9, 21). Our results therefore demonstrate the existence of a T cell activation pathway via TLR2 that recognizes the largest spectrum of pathogen-associated mo- lecular patterns and stress proteins among the TLRs (9, 31, 37). The constitutive and sustained expression of TLR2 on memory T cells may represent an important host device to mount an immediate strong response on encountering a reinfecting pathogen. Because BLP is highly conserved among bacterial strains, results reported here may help to explain the often observed ‘‘flare up’’ of T cell-mediated autoimmunity during microbial infection. Another important feature for the expression of TLR2 on memory T cells could be the contribution to the expansion and maintenance of memory T cells in the absence of specific antigen but in the presence of memory-sustaining cytokines, including IL-2 and IL-15 pro- duced by adaptive as well as innate immune cells. This mechanism could contribute to the perpetuation of long-term memory by infectious agents with shared TLR2 ligand but are otherwise unrelated to the antigen specificity of the memory T cells. It should also be noted that BLP alone did not activate naı¨ve or memory T cells. It does so only in the presence of TCR activation or a bystander effect of cytokines such as IL-2 or IL-15. This dual- signaling mechanism should avoid excessive T cell proliferation by BLP alone. Recently, it was reported that receptor triggering led to up-regulation of TLR9, which was constitutively expressed in memory B cells (38). Furthermore, human memory B cell prolif- erated and differentiated into plasma cells in response to CpG Fig. 6. BLP increases the proliferation and IFN-␥ production by memory CD4ϩ DNA (39). Our findings reported here suggest that there may be a T cells in response to anti-CD3 activation or IL-2 or IL-15 stimulation. (a) parallel between T and B cells in that TLR signaling helps to CD45RAϩ and CD45ROϩ T cells from peripheral blood were cultured with maintain long-term memory of these cells. This observation may anti-CD3 antibody. Proliferation and IFN-␥ production was determined. *, P Ͻ ϩ also help to resolve a current central controversy on the require- 0.05; **, P Ͻ 0.01 (vs. culture with immobilized anti-CD3 alone). (b) CD45RA ϩ ment for the continuous presence of specific pathogen for the or CD45RO cells were cultured with IL-2 or IL-15 in the presence or absence maintenance of long-term specific T cell memory (40–42). of BLP. *, P Ͻ 0.05; **, P Ͻ 0.01 (vs. cultures of respective cytokine). We thank Dr. James Brewer (University of Glasgow) for assistance with immunofluorescence microscopy. We also thank Dr. Sabine Totemeyer The present studies establish TLR2 as a costimulatory receptor Ϫ Ϫ (University of Cambridge) for providing lymphoid cells from the TLR2 / for pathogen-derived ligand, as opposed to conventional T cell mice originally provided by Dr. Shizou Akira (University of Osaka). This costimulation ligands expressed on autologous cells. TLR2 may work was supported by the Medical Research Council, the Wellcome Trust, play an important role in adaptive immunity by directly enhancing the Chief Scientist’s Office, Scotland, and the European Commission.

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