Regulatory Expression of Herpesvirus Entry Mediator Suppresses the Function of B and T Lymphocyte Attenuator-Positive Effector T Cells This information is current as of October 1, 2021. Ran Tao, Liqing Wang, Kenneth M. Murphy, Christopher C. Fraser and Wayne W. Hancock J Immunol 2008; 180:6649-6655; ; doi: 10.4049/jimmunol.180.10.6649

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulatory T Cell Expression of Herpesvirus Entry Mediator Suppresses the Function of B and T Lymphocyte Attenuator-Positive Effector T Cells1

Ran Tao,* Liqing Wang,* Kenneth M. Murphy,† Christopher C. Fraser,‡ and Wayne W. Hancock2*

The binding of herpesvirus entry mediator (HVEM) to B and T lymphocyte attenuator (BTLA) is known to activate an inhibitory signaling cascade in effector T (Teff) cells, but we now report that the HVEM-BTLA pathway is also important to the suppressive function of regulatory T cells (Tregs). Although naive T cells up-regulated BTLA upon TCR activation, Treg expression of BTLA remained low, regardless of TCR activation. Moreover, BTLA؊/؊ CD4؉CD25؉ Tregs had normal suppressive activity, whereas BTLA؊/؊ Teff cells were more resistant than wild-type Teff cells to suppression by Tregs, suggesting BTLA expression by Teff cells was required for their suppression by Tregs. In contrast to BTLA, HVEM expression was comparable in naive Tregs vs Teff cells, Downloaded from but after stimulation HVEM expression was quickly down-regulated by Teff cells, whereas HVEM was further up-regulated by Tregs. HVEM؊/؊ Tregs had decreased suppressive activity as compared with wild-type Tregs, indicating that Treg expression of HVEM was required for optimal suppression. Consistent with this, T cells from Scurfy mice (FoxP3 mutant) lacked HVEM expression, and adoptively transferred wild-type but not HVEM؊/؊ Tregs were able to control alloresponses in vivo by normal Teff cells. Our data demonstrate that Tregs can exert their effects via up-regulation of the negative costimulatory HVEM, which upon binding to BTLA expressed by Teff cells helps mediate the suppressive functions of Tregs in vitro and in vivo. The http://www.jimmunol.org/ Journal of Immunology, 2008, 180: 6649–6655.

he molecular interactions responsible for the suppressive success has been attained using Tregs to achieve long-term allo- activity of CD4ϩCD25ϩ regulatory T cells (Tregs)3 graft survival in normal, immunocompetent hosts (1–3). Therefore, T remain unclear, but Tregs are known to control immune a better understanding of how Tregs work is essential if progress responses to self-Ags, thereby preventing autoimmune disease, toward clinical therapy is to be attained. The detailed mechanisms and to regulate responses to nonself molecules in adaptive im- by which Tregs control immune responses in vivo are unknown. In

munity (1). Tregs have received much attention by transplant vitro studies often show a requirement for Treg/T effector (Teff) by guest on October 1, 2021 investigators because allograft tolerance is not routinely cell contact, although functionally active surface molecules on achieved clinically and transplant recipients require lifelong im- Tregs such as CTLA4 (CD152), -induced TNFR munosuppression and endure varying degrees of associated drug family-related (GITR, TNFRSF18), and the programmed toxicities. The identification and characterization of Tregs led to death receptor PD-1 (CD279), as well as soluble mediators includ- the expectation that these cells would be useful for therapy in ing IL-10 and TGF-␤ can also contribute to Treg suppressive func- transplantation. However, although clinical and experimental stud- tions, depending upon assay conditions and likely other factors (1). ies indicate that manipulating the balance between Tregs and re- Optimal T cell activation requires the TCR engagement of a sponder T cells can dampen host responses posttransplant, little cognate MHC-peptide complex and a costimulatory signal. The binding of CD28 to B7-1/B7-2 remains the best-characterized co-

*Department of Pathology and Laboratory Medicine, Joseph Stokes Jr. Research In- stimulatory pathway, but the persistence of T cell responses in Ϫ Ϫ stitute and Biesecker Pediatric Center, The Children’s Hospital of Philadelphia CD28 / mice led to the discovery of several CD28/B7 homologs, † and University of Pennsylvania, Philadelphia, PA 19104; Department of Pathology including the components of the ICOS/B7RP-1 and PD-1/PD-/ and Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110; and ‡Inflammation, Millennium Pharmaceuticals, PD-L2 pathways (4). The most recently recognized CD28 ho- Cambridge, MA 02139 molog, B and T lymphocyte attenuator (BTLA, CD272) (5), is Received for publication March 9, 2008. Accepted for publication March 9, 2008. unusual in that it interacts with the TNFR superfamily member, The costs of publication of this article were defrayed in part by the payment of page herpesvirus entry mediator (HVEM, TNFRSF14) (6). HVEM can charges. This article must therefore be hereby marked advertisement in accordance promote T cell activation by propagating positive signals from the with 18 U.S.C. Section 1734 solely to indicate this fact. TNF superfamily member ligand, LIGHT (TNFSF14, CD258), a 1 This work was supported in part by Grant R01-AI54720 from the National Institutes of Health (to W.W.H.). lymphotoxin-related inducible ligand that competes for glycopro- 2 Address correspondence and reprint requests to Dr. Wayne W. Hancock, Depart- tein D binding to HVEM on T cells (7). ment of Pathology and Laboratory Medicine, 916B Abramson Research Center, The A 2.8-Å crystal structure of the BTLA-HVEM complex shows Children’s Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318. E-mail address: [email protected] that BTLA binds the N-terminal cysteine-rich domain of HVEM and uses a unique binding surface compared with other CD28-like 3 Abbreviations used in this paper: Treg, ; Teff, effector T; GITR, glucocorticoid-induced TNFR family-related receptor; BTLA, B and T lymphocyte receptors (8). The BTLA binding site on HVEM overlaps with the attenuator; HVEM, herpesvirus entry mediator; IRES, internal ribosomal entry site; binding site for the HSV type 1 envelope D, but is WT, wild type. distinct from the binding site for LIGHT. HVEM has three cys- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 teine-rich domains. Competitive binding analysis and mutagenesis www.jimmunol.org 6650 SURFACE MOLECULES MEDIATING Treg SUPPRESSION reveals a unique BTLA binding site centered on a critical lysine and an internal ribosomal entry site (IRES) cassette downstream of the residue in cysteine-rich domain-1, the most membrane-distal do- cloning region and upstream of a cell surface-expressed nonsignaling nerve main, as opposed to LIGHT, which interacts largely through cys- growth factor receptor (15). Plasmid mHVEM-FL-GFP-RV was made from two PCR products, with primers 5Ј-BglII mHVEM and mHVEM/GFP teine-rich domain-3 (8–10). using mHVEM-FL-IRES-GFP-RV as template, and primers mHVEM-GFP The importance of the HVEM-BTLA pathway in control of Teff and 3Ј-GFP plus Sal using mHVEM-FL-IRES-GFP-RV as template; PCR cell functions was shown in several animal models. For example, products were annealed, amplified with primers 5Ј-BglII mHVEM and 3Ј- compared with controls, HVEMϪ/Ϫ mice had increased suscepti- GFP plus Sal, digested with BglII and SalI, and ligated into IRES-GFP-RV that was digested with BglII and SalI. The Phoenix ecotropic packaging bility to Con A mitogen-induced, T cell-dependent autoimmune Ϫ/Ϫ cell line was used for cotransfection with MinR1 Foxp3 and mHVEM hepatitis (11), and BTLA mice had greater acute allergic air- retroviral constructs in the presence of Lipofectamine 2000. Retroviral way inflammation (12), worse myelin oligodendrocyte glycopro- supernatants were added to CD4ϩCD25Ϫ T cells activated overnight tein peptide-induced experimental autoimmune encephalitis (4), with PMA, ionomycin, and IL-2; transduction efficiency was 90–95% and were unable to accept partially MHC-mismatched cardiac al- as determined by flow cytometry, and cells were centrifuged over Ficoll before use. lografts (13). In the current study, we propose a new model in which Tregs exert their suppressive effect via up-regulation of the Cardiac transplantation negative costimulatory ligand HVEM that, upon ligation with Intraabdominal vascularized mouse cardiac transplantation across a full BTLA expressed on the Teff cell side, enhances the suppressive MHC-mismatch was performed using BALB donors and C57BL/6 recip- function of Tregs and helps control immune response in vitro and ients, with anastomoses of donor ascending aorta and pulmonary artery in vivo. end-to-side to recipient infrarenal aorta and inferior vena cava, respectively (13). Grafts were assessed daily by abdominal palpation; rejection was Materials and Methods defined as cessation of cardiac contraction and confirmed by histology. For Downloaded from Ϫ/Ϫ adoptive transfer studies of Treg function, BALB/c were grafted into HVEM and other mice RAG2Ϫ/Ϫ recipients (B6 background), followed by i.v. transfer of 0.5 ϫ 6 ϩ Ϫ 6 Ϫ/Ϫ ϩ ϩ A targeting vector was constructed using a 7.8-kb genomic fragment con- 10 CD4 CD25 T cells plus 0.25 ϫ 10 WT or HVEM CD4 CD25 taining exons 3–8 of the HVEM gene, and a 0.8-kb sequence around exon Tregs; each cell population was isolated using magnetic beads for in vitro 3, including ATG, was replaced by pMC1 neo (14). The targeting vector assays. was linearized, electroporated into ES cells from 129 mice, HVEMϩ/Ϫ ES Statistics

cell clones were screened by Southern blot analysis, and chimeric mice http://www.jimmunol.org/ (B6/129) were derived by blastocyst injection. HVEMϩ/Ϫ mice were Ϫ Ϫ Allograft survival was used to generate Kaplan-Meier survival curves, and crossed to produce HVEM / mice and backcrossed at least eight gener- Ϫ Ϫ comparison between groups was performed by log- analysis. ations on a C57BL/6 background before study. BTLA / mice on the C57BL/6 background were previously described (5). We purchased wild- b d type (WT) C57BL/6 (H-2 ), BALB/c (H-2 ), C57BL/6/DBA2 F1 Results (B6D2F1), congenic Thy1.1, Scurfy and RAG2Ϫ/Ϫ C57BL/6 mice (The BTLA and HVEM mRNA expression by resting and activated Jackson Laboratory). Studies were performed with a protocol approved by T cells the Institutional Animal Care and Use Committee of Children’s Hospital of Philadelphia. We used quantitative real-time PCR to examine the expression of BTLA, HVEM, and LIGHT by purified CD4ϩCD25Ϫ (non-Tregs)

Flow cytometry by guest on October 1, 2021 vs CD4ϩCD25ϩ (Tregs) cells. CD4ϩCD25Ϫ T cells and naturally We purchased CFSE (Molecular Probes), PE-conjugated rat anti-mouse occurring CD4ϩCD25ϩ Tregs were isolated using magnetic beads Foxp3 mAb (eBioscience) and additional mAbs for flow cytometry (BD Pharmingen). Biotinylated BTLA tetramer was made as reported (6) and and stimulated in vitro with PMA/ionomycin plus IL-2. Cell acti- conjugated with streptavidin-PE or streptavidin-FITC at a molar ratio of vation led to BTLA up-regulation by non-Tregs but not by Tregs 1:4 before each staining. For Foxp3 studies, cells were first labeled with (Fig. 1a). In contrast, cell activation induced higher expression of cell surface markers, fixed, permeabilized, and stained with PE-conjugated HVEM ( p Ͻ 0.05) by Tregs than non-Tregs (Fig. 1b). Expression rat anti-mouse Foxp3 mAb (13). of a second HVEM receptor, LIGHT, was transiently up-regulated Quantitative PCR by4hofstimulation of CD4 T cells in vitro but returned to base- RNA extracted using RNeasy kits (Qiagen) was reverse transcribed with line levels thereafter (Fig. 1c). In addition, T cells from Scurfy random hexamers (ABI PRISM 5700; Applied Biosystems). Primer and mice, which have a truncated form of Foxp3 (16), lacked HVEM probe sequences for target were used for quantitative PCR amplifi- expression even after activation, suggesting the involvement of cation of total cDNA (ABI PRISM 5700 and TaqMan Predeveloped Assay Foxp3 in regulation of HVEM expression (Fig. 1d). Hence, upon Reagents; Applied Biosystems). Relative quantitation of target cDNA was determined by arbitrarily setting the control value to 1, and changes in the activation, activated Teff cells express increased BTLA mRNA but cDNA content of a sample were expressed as fold increases above the set down-regulate HVEM mRNA, whereas Tregs up-regulate HVEM control value. Differences in cDNA input were corrected by normalizing but not BTLA mRNA. signals obtained with specific primers to ribosomal RNA, and nonspecific amplification excluded by performing quantitative PCR in the absence of BTLA and HVEM expression by resting and activated target cDNA. T cells In vitro cellular assays Flow cytometry showed BTLA expression by 10–15% of naive CD4 T cells were negatively selected using magnetic beads (Miltenyi Bio- CD4ϩ and CD8ϩ T cells and Ͼ90% of B cells (Fig. 2a). Adoptive tec) and fractionated into CD25ϩ and CD25Ϫ populations (Ͼ95% purity by transfer of CFSE-labeled T cells into F1 mice led to alloactivation- flow cytometry). We used soluble CD3 mAb plus gamma-irradiated syn- induced up-regulation of BTLA protein by almost all CD4ϩ and geneic APC, CD3 and CD28 mAbs, or PMA/ionomycin plus IL-2 to ac- ϩ ϩ Ϫ tivate T cells in vitro. Treg function was assayed using MACS-purified CD8 T cells by 72 h (Fig. 2b). Similarly, most CD4 Foxp3 T CFSE-labeled CD4ϩCD25Ϫ T cells as effector cells and CD4ϩCD25ϩ cells cells highly expressed BTLA protein after in vitro TCR ligation or or retrovirally transduced Foxp3ϩ CD4 T cells; CD3 mAb-induced ϩ Ϫ TCR plus CD28 costimulation, but the extent of BTLA expression CD4 CD25 cell proliferation was assessed by flow cytometric anal- by CD4ϩFoxp3ϩ T cells remained low (Fig. 2c). Hence, Teff cells, ysis of CFSE dilution after 72 h (13). but not Tregs, up-regulated BTLA upon activation. Flow cytomet- Retroviral transduction of primary T cells ric analysis of HVEM expression using a PE-labeled BTLA tet- Murine Foxp3 was cloned into a murine stem cell virus-based bicistronic ramer showed HVEM was mainly expressed by resting T cells ϩ retroviral vector, MinR1, containing a 5Ј and 3Ј long terminal repeat site, (Fig. 2d); although some CD11b monocytes expressed HVEM, The Journal of Immunology 6651

FIGURE 1. BTLA, HVEM, and LIGHT mRNA expression by CD4ϩ CD25Ϫ vs CD4ϩCD25ϩ T cells. CD4ϩCD25Ϫ or CD4ϩCD25ϩ T cells from WT C57BL/6 (a–c)or Scurfy mice (d) were purified by MACS, with Ͼ95% purity by flow cytometry. Cells were stimulated with PMA (6 ng/ml), ionomycin (2 ␮M), and IL-2 (10 U/ml) and harvested as indicated for analysis of BTLA, HVEM, and LIGHT mRNA expression by quantitative PCR, as indicated. Relative quantitation of target cDNA was determined by setting the con- trol value to 1, and changes in the cDNA content of samples were ex- pressed as fold increases above the set control value. Data from triplicates were expressed as mean Ϯ SD and Downloaded from are representative of three experi- p Ͻ ,ء .ments with similar results p Ͻ ,ءءء p Ͻ 0.01; and ,ءء ;0.05 0.001 for CD4ϩCD25ϩ vs corre- sponding CD4ϩCD25Ϫ data. http://www.jimmunol.org/

there was no expression by B cells or dendritic cells. HVEM ex- HVEM on DC used in the Treg assays did not affect Treg sup- pression by CD4ϩ or CD8ϩ T cells was down-regulated by in vitro pression when assayed using WT Tregs, nor did they affect out- activation (Fig. 2e). Activation of Foxp3ϩ Tregs led to progres- comes using HVEMϪ/Ϫ Tregs (data not shown). Collectively, our sively increased surface expression of HVEM upon activation, in data suggest that HVEM is not only required for controlling T-T contrast to an almost complete lack of HVEM expression by interactions but is important to Treg function. Foxp3Ϫ cells in the same wells (Fig. 2f). These matching quanti- In concurrent studies of the role of BTLA expression on Teff by guest on October 1, 2021 tative PCR and flow cytometry data led us to propose that ligation cells vs Tregs, we found that BTLAϪ/Ϫ Teff cells proliferated as of BTLA on Teff cells, by HVEM on activated Tregs, contributed well as WT Teff cells in the absence of Tregs (third panel, Fig. 4b), to the suppressive function of Tregs. To test this proposition, we consistent with the lack of HVEM expression by Teff cells after generated HVEMϪ/Ϫ mice for in vitro and in vivo studies. 48 h of in vitro stimulation (Fig. 2b). However, as an increasing number of Tregs was added, the lack of Teff cell expression of Ϫ/Ϫ Ϫ/Ϫ Differing mechanisms by which HVEM and BTLA mice BTLA led to Teff cells being significantly more resistant to Treg have impaired Treg suppression suppression by Tregs than WT Teff cells (third panel, Fig. 4b). We used homologous recombination to disrupt exon 3 of the mu- Conversely, and consistent with the low expression of BTLA on Ϫ Ϫ rine HVEM gene (Fig. 3). Our studies of homozygous HVEMϪ/Ϫ Tregs in either naive or activated states, BTLA / Tregs showed mice backcrossed for over eight generations to the C57BL/6 strain normal suppressive activity compared with WT Tregs (fourth showed the mice were normal in appearance, growth and fertility, panel, Fig. 4b), indicating BTLA deficiency does not affect Treg had a normal number of T cells, B cells, monocytes, NK cells, and suppressive function. granulocytes and normal lymphoid architecture (data not shown). Moreover, analysis of 4- to 6-wk-old WT, BTLAϪ/Ϫ and HVEMϪ/Ϫ mice showed a comparable number of CD4ϩCD25ϩ Overexpression of HVEM enhances Treg suppressive function Foxp3ϩ Tregs in lymph nodes and (Fig. 4a), as well as in We used retroviral transduction, to test whether overexpression of and bone marrow samples (data not shown). HVEM would modify the function of Tregs. We have shown that CFSE-labeled CD4ϩCD25Ϫ T cells from WT or HVEMϪ/Ϫ transduction of murine CD4ϩCD25Ϫ cells with murine Foxp3 can mice were stimulated in vitro with CD3 mAb and syngeneic APC, efficiently convert Teff cells to Tregs (15), and we now transduced increasing numbers of Tregs from WT mice were added, and ef- Foxp3 plus HVEM, or Foxp3 alone, into CD4ϩCD25Ϫ T cells. fector T cell proliferation assessed by CFSE-based division pro- Efficiency of Foxp3 and HVEM transduction, monitored using hu- files. Regardless of the presence or absence of Tregs, the prolif- man nerve growth factor receptor and GFP reporters, respectively, eration of HVEMϪ/Ϫ Teff cells was significantly increased was Ͼ90% in each case (data not shown). Functions of compared with proliferation of cells in WT controls ( first panel, CD4ϩFoxp3ϩ Tregs, with or without associated HVEM overex- Fig. 4b), suggesting that HVEM is critical to down-regulation of pression, were assessed by the in vitro suppression assay. As the T-T interactions. However, when comparing the suppressive func- retroviral vector used to transduce HVEM contained a GFP re- tion of WT and HVEMϪ/Ϫ Tregs, HVEMϪ/Ϫ Tregs were signif- porter, we used CFSE-labeled Thy1.1 congenic CD4ϩCD25Ϫ T icantly less suppressive than WT Tregs (second panel, Fig. 4b), cells as Teff cells, and the suppressive effect was determined by the indicating that the increased expression of HVEM on Tregs after total number of Thy1.1ϩ T cells recovered after 72 h of incubation, activation is of functional significance. The presence or absence of and by their corresponding CFSE profiles. Forced expression of 6652 SURFACE MOLECULES MEDIATING Treg SUPPRESSION

FIGURE 2. Cell surface expres- sion of BTLA and HVEM by resting and activated T cells. a–c, BTLA ex- pression using hamster anti-mouse BTLA mAb (6A6) and are represen- tative of three experiments with similar results. a, WT or BTLAϪ/Ϫ splenocytes were dual stained with anti-BTLA mAb or hamster IgG- and FITC-conjugated anti-hamster IgG, plus PE-conjugated mAb to CD4, CD8, or CD19. b, CFSE-labeled C57BL/6 cells were adoptively trans- ferred to B6D2F1 mice for 72 h, fol-

lowed by gating on BTLA-positive Downloaded from cells lacking KdDd; donor CD4 and CD8 T cell expression of BTLA after each cell division is shown by dot plot (top) and mean fluorescence intensity (bottom). c, C57BL/6 splenocytes stimulated in vitro with CD3 or CD3/

CD28 mAbs were harvested at 24, 48, http://www.jimmunol.org/ and 72 h, stained with BTLA and T cell markers, fixed, permeabilized, and stained for Foxp3. d–f, HVEM expression using BTLA tetramer conjugated with streptavidin-PE or streptavidin-FITC, and are representa- tive of three experiments with similar results. d, HVEM expression by the indicated populations of C57BL/6 by guest on October 1, 2021 splenocytes. e, Serial HVEM expres- sion by CD4 and CD8 T cells acti- vated for up to 96 h with CD3 or CD3/ CD28 mAbs. f, Up-regulation of surface expression of HVEM by CD4ϩFoxp3ϩ Tregs activated in vitro using CD3 mAb or CD3/CD28 mAbs for the periods shown. Cells stained with BTLA tetramer conjugated with streptavidin-FITC, or stained with streptavidin-FITC alone, were fixed, permeabilized, and stained using PE- conjugated anti-Foxp3 mAb. The per- centage of positive cells is indicated.

HVEM by CD4ϩFoxp3ϩ T cells significantly enhanced their sup- impact of HVEM or BTLA deficiency on Treg function in vivo. pressive function, resulted in a decreased total number of Thy1.1ϩ We used a model in which fully MHC-mismatched murine cardiac Teff cells at 72 h, as a result of fewer Teff cells entering the cell allografts survive permanently in recipients receiving 2 wk of ther- cycle (Fig. 5a). These data show that HVEM is an important ef- apy with the histone deacetylase inhibitor, trichostatin A, plus fector molecule for Treg function. rapamycin, used at a subtherapeutic dosage (17). We had found that trichostatin A enhanced the function of Tregs in vivo, and that Ϫ/Ϫ Ϫ/Ϫ BTLA and HVEM mice are resistant to induction of a small dose of rapamycin was necessary to limit residual effector allograft tolerance T cell proliferation, in the early posttransplantation period, which Given our data on the importance of the HVEM-BTLA pathway in was not controlled by Tregs alone (18). As previously, trichostatin mediating the suppressive effects of Tregs in vitro, we tested the A plus rapamycin therapy achieved permanent allograft survival The Journal of Immunology 6653

FIGURE 3. Generation of HVEMϪ/Ϫ mice. a, Genomic organization of the mu- rine HVEM and the mutation induced by our targeting event; exons are shown as numbered boxes. An HVEM gene-targeting construct was generated to replace exon 3 of HVEM, containing the initiating methionine (ATG), with the neomycin resistance gene (NEO). b, Genomic DNA was extracted from mouse tail tissue, digested with EcoRI and analyzed by Southern blot using 3Ј probe, generating a 20-kb product for the WT allele (ϩ/ϩ) and a 5.0-kb product for the HVEM null allele (ϩ/Ϫ). c, Quantitative PCR analysis of RNA extracted from murine splenocytes stimulated with CD3 mAb or CD3 plus CD28 mAbs for 16 h. Downloaded from using the stringent BALB/c3C57BL/6 cardiac transplant model, lografts (Fig. 5b). However, we now found that long-term allograft whereas depletion of central and peripheral CD4ϩCD25ϩ Tregs by survival could not be achieved in recipients deficient in BTLA or pretransplant thymectomy and CD25 mAb caused rejection of al- HVEM (Fig. 5b), consistent with a role for the HVEM-BTLA http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Contrasting sites at which HVEM and BTLA contribute to Treg suppression. a, Comparable numbers of CD4ϩFoxp3ϩ Tregs in lymph nodes and of WT, BTLAϪ/Ϫ, and HVEMϪ/Ϫ mice. b, Impaired suppressive function of HVEMϪ/Ϫ CD4ϩCD25ϩ Tregs. CD4ϩCD25Ϫ and CD4ϩCD25ϩ T cells from WT or HVEMϪ/Ϫ mice were purified by MACS and used as Teff cells and Tregs, respectively; syngeneic B6 splenocytes depleted of Thy1.2ϩ cells were used as APC. A total of 5 ϫ 104 CFSE labeled CD4ϩCD25Ϫ T cells were stimulated in 96-well plates with CD3 mAb (0.5 ␮g/ml) and gamma-irradiated APC (5 ϫ 104/well), and an increasing number of CD4ϩCD25ϩ Tregs were added. Data shown are mean Ϯ SD of Teff proliferation after ,p Ͻ 0.001 for mutant vs WT cells. BTLAϪ/Ϫ Teff are more resistant than WT Teff to suppression by Tregs ,ءءء p Ͻ 0.005; and ,ءء ;p Ͻ 0.01 ,ء .h 72 as shown (third panel). All data are representative of three separate experiments. 6654 SURFACE MOLECULES MEDIATING Treg SUPPRESSION Downloaded from

FIGURE 5. HVEM expression promotes Treg-mediated suppression in vitro and in vivo. a, The suppressive function of CD4ϩFoxp3ϩ T cells with or ϩ Ϫ without overexpression of HVEM was tested by in vitro suppression assay. CD4 CD25 T cells activated with PMA/ionomycin and IL-2 were transduced http://www.jimmunol.org/ with retroviral vectors containing mouse Foxp3 and HVEM or control human nerve growth factor receptor and GFP vectors, cultured for 72 h with CD3 mAb-activated CFSE-positive Thy1.1ϩ Teff cells, and absolute number (left) and dividing cell number (right) were determined by flow cytometry. Data p Ͻ 0.001). b, trichostatin A (TsA) plus rapamycin (RPM) induces ,ءءء p Ͻ 0.005; and ,ءء ;p Ͻ 0.01 ,ء .are representative of three separate experiments Treg-dependent permanent allograft survival in WT but not HVEMϪ/Ϫ or BTLAϪ/Ϫ recipients. Fully MHC-mismatched cardiac allograft recipients (4–8 transplants/group) received 14 days of therapy with trichostatin A dissolved in DMSO (1 mg/kg/day, i.p.) plus a subtherapeutic dose of rapamycin (0.01 mg/kg/day, i.p.). Trichostatin A plus rapamycin led to permanent graft survival in all WT recipients, and tolerance was Treg-dependent as shown by allograft rejection in recipients undergoing pretransplant thymectomy plus Treg depletion using CD25 mAb. p Ͻ 0.001 compared with tolerant recipients). However, trichostatin A plus rapamycin therapy failed to induce permanent allograft survival in HVEMϪ/Ϫ or BTLAϪ/Ϫ recipients. p Ͻ 0.001 compared with WT recipients. c, RAG2Ϫ/Ϫ B6 mice were transplanted with BALB/c cardiac allografts and, on the same day, underwent adoptive transfer of 0.5 ϫ 106 WT B6 Teff cells plus 0.25 ϫ 106 WT or HVEMϪ/Ϫ B6 Tregs (3 allografts/group). WT Tregs induced long-term cardiac allograft survival, whereas by guest on October 1, 2021 recipients of HVEMϪ/Ϫ Tregs developed acute rejection of their allografts by 3 wk posttransplant (p Ͻ 0.01).

pathway in the optimal function of Treg in vivo. Consistent with is expressed at only low levels by Tregs, whereas HVEM is mainly this, adoptive transfer of WT but not HVEMϪ/Ϫ B6 Tregs to expressed by Tregs after T cell activation, resulting in negligible com- RAG2Ϫ/Ϫ recipients of BALB/c allografts was able to suppress petition for binding to HVEM between Tregs and Teff cells. This allograft rejection induced by cotransfer of WT Teff cells (Fig. 5c). differential pattern of expression for receptor and ligand on resting vs activated T cells provides an additional and hitherto unexpected Discussion means for fine tuning and regulation of T cell responses via the The current data show that TCR activation leads to BTLA up- BTLA-HVEM pathway. regulation by Teff cells and HVEM up-regulation by Tregs, and Given that the binding of HVEM and BTLA was identified in that as a result, the HVEM-BTLA pathway is important to Treg- 2005 (6, 9), the questions arise as to how the connection to Treg dependent control of T cell responses in vitro and in vivo. These function was not previously recognized, and what are the thera- data provide a new perspective on the BTLA-HVEM pathway and peutic implications of this expression? With regard to the first show that HVEM can be added to the armamentarium of Treg question, LIGHT knockout mice showed enhanced cardiac allo- effector molecules relevant to immune therapy. Several TNFR graft survival using the same model as used in the current studies, family members have costimulatory effects on T cells, including and use of HVEM-Ig in WT recipients also prolonged survival CD27, 4-1BB (CD137), OX40 (CD134), CD30 and GITR, and (25). These data were interpreted as showing the importance of the GITR and OX40 are also implicated in control of Treg function LIGHT/HVEM pathway in T cell costimulation, though in retro- (19–23). GITR is expressed at high levels on CD4ϩCD25ϩ Tregs spect a role for HVEM-Ig binding to BTLA and promoting neg- and its ligation by GITR ligand expressed by APC revokes Treg ative signals within T cells may also have contributed. Likewise, a suppression and provides a costimulatory signal for Ag-driven pro- previous report of an HVEM knockout mouse emphasized an un- liferation of naive T cells and polarized Th1 and Th2 clones (22, expected enhancement of T cell responses, with increased suscep- 23). Likewise, although both naive and activated Tregs express tibility to autoimmunity including Con A-induced hepatitis and OX40, triggering OX40 on Tregs using agonist Abs inhibited their myelin oligodendrocyte glycoprotein peptide-induced experimen- capacity to suppress, and restored Teff cell proliferation and IL-2 cy- tal autoimmune encephalopathy (11), though no data were gener- tokine production (24). However, in contrast to CTLA4, GITR, or ated relating to Treg functions. Clearly the complexity of interac- OX40, which are expressed by both Tregs and Teff cells after acti- tions involving HVEM and its ligands, as well as the ability of vation, the inhibitory receptor BTLA is up-regulated in Teff cells but LIGHT itself to bind HVEM, lymphotoxin-␤ receptor, and the The Journal of Immunology 6655 soluble TNFR , provide a considerable number of 13. Tao, R., L. Wang, R. Han, T. Wang, Q. Ye, T. Honjo, T. L. Murphy, permutations, even before taking into account differential expres- K. M. Murphy, and W. W. Hancock. 2005. Differential effects of B and T lym- phocyte attenuator and programmed death-1 on acceptance of partially versus sion upon cell activation. fully MHC-mismatched cardiac allografts. J. Immunol. 175: 5774–5782. With regard to therapeutic aspects, agonistic mAbs that cross- 14. Hancock, W. W., B. Lu, W. Gao, V. Csizmadia, K. Faia, J. A. King, S. T. Smiley, M. Ling, N. P. Gerard, and C. Gerard. 2000. Requirement of the chemokine link BTLA and suppress the proliferation and production of cyto- receptor CXCR3 for acute allograft rejection. J. Exp. Med. 192: 1515–1520. kines such as IL-2 and IFN-␥ by human (26) and murine (27) T 15. Chen, C., E. A. Rowell, R. M. Thomas, W. W. Hancock, and A. D. Wells. 2006. cells are reported, and administration of HVEM-Ig can also Transcriptional regulation by Foxp3 is associated with direct promoter occupancy dampen immune responses (25, 28–30). Our data suggest that the and modulation of histone acetylation. J. Biol. Chem. 281: 36828–36834. 16. Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, enhancement of HVEM expression by gene therapy or other ap- S. A. Yasayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, and F. 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