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Cutting Edge: Stromal Cell-Derived Factor-1 Is a Costimulator for CD4 + T Cell Activation Toshihiro Nanki and Peter E. Lipsky This information is current as J Immunol 2000; 164:5010-5014; ; of September 28, 2021. doi: 10.4049/jimmunol.164.10.5010 http://www.jimmunol.org/content/164/10/5010 Downloaded from References This article cites 29 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/164/10/5010.full#ref-list-1

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Cutting Edge: Stromal Cell-Derived Factor-1 Is a Costimulator for CD4؉ T Cell Activation1

Toshihiro Nanki,2 and Peter E. Lipsky3

Mice lacking SDF-1 or CXC chemokine receptor-4 (CXCR4), Stromal cell-derived factor (SDF)-1 is a chemoattractant for T the unique ligand of SDF-1, exhibited cardiovascular, vascular, ␣ cells, precursor B cells, monocytes, and neutrophils. SDF-1 and neurologic defects as well as defective B cell lymphopoiesis was also found to up-regulate expression of early activation and a severe impairment of bone marrow myelopoiesis (7–9). markers (CD69, CD25, and CD154) by anti-CD3-activated SDF-1 is thought to attract progenitor B cells into the microenvi- Downloaded from ؉ CD4 T cells. In addition, SDF-1␣ costimulated proliferation ronment of stromal cells where growth and differentiation factors ؉ of CD4 T cells and production of IL-2, IFN-␥, IL-4, and IL- are released (4, 7, 10, 11). These results suggest that in certain 10. Stimulation with SDF-1␣ alone did not induce activation circumstances SDF-1 and CXCR4 interactions may have other marker expression, proliferation, or cytokine production by functions than merely their chemoattractant activities. -the CD4؉ T cells. SDF-1␣-mediated costimulation was blocked To examine other specific activities of SDF-1/CXCR4 interac by anti-CXC chemokine receptor-4 mAb. RANTES also in- tions, we examined the capacity of SDF-1␣ to stimulate T cell http://www.jimmunol.org/ creased activation marker expression by anti-CD3-stimulated activation. Costimulation with SDF-1␣ up-regulated the expres- peripheral CD4؉ T cells, but less effectively than SDF-1␣ did, sion of activation markers, proliferation, and cytokine production ϩ and did not up-regulate IL-2 production and proliferation. by anti-CD3-activated CD4 T cells. These findings indicate that These results indicate that SDF-1 and CXC chemokine recep- besides a role in chemoattraction, SDF-1␣ may play an important tor-4 interactions not only play a role in T cell migration but role in costimulating activation of Ag-reactive T cells. also provide potent costimulatory signals to Ag-stimulated T Materials and Methods cells. The Journal of Immunology, 2000, 164: 5010–5014. Sample by guest on September 28, 2021 PBMCs were isolated by Ficoll-Hypaque (Pharmacia Biotech, Piscataway, tromal cell-derived factor-1 (SDF-1),4 which is one of the NJ) gradient centrifugation from healthy donors. CD4ϩ T cells were isolated by negative selection using StemSep columns (Stem Cell Technolo- CXC chemokines, was originally identified as a growth ϩ factor for murine pre-B cells (1, 2). It occurs in two alter- gies, Vancouver, Canada). Purity of the separated CD4 T cells was more S than 95%. native splice variants, SDF-1␣ and SDF-1␤ (1). SDF-1 is expressed constitutively by various cells and tissues (1, 3) and has Culture chemoattractive activity for monocytes, bone marrow neutrophils, The purified peripheral CD4ϩ cells (2 ϫ 106 cells/ml) were cultured in and early-stage B cell precursors (4, 5). In addition, SDF-1 is a RPMI 1640 with 10% FCS for 6 h because this induced spontaneous ex- highly efficient and potent chemoattractant for T cells (5). Further- pression of CXCR4, the ligand for SDF-1 on the T cells. For blocking CXCR4, 50 ␮g/ml anti-CXCR4 mAb (12G5; R&D Systems, Minneapolis, more, SDF-1 induces adhesion of T cells to ICAM-1 (CD54) (6) MN) or isotype-matched control mAb (20102.1; R&D Systems) was added by up-regulating the binding activity of LFA-1 (CD11a/CD18). As for the last hour. Afterward, the cells were incubated in medium supple- a result of these activities, SDF-1 is thought to play an important mented where indicated with SDF-1␣ or RANTES (R&D Systems) for 2 h. ϩ role in the attraction of T cells into specific sites. Subsequently, the CD4 T cells were transferred to 96-well microtiter plates with or without anti-CD3 mAb OKT3 (500 ng/microwell (American Type Culture Collection, Manassas, VA)). After incubation at 37°C for 8 h, surface activation marker expression was analyzed, cytokine production Department of Internal Medicine and Harold C. Simmons Arthritis Research Center, was analyzed after 8–12 h, and proliferation was determined after University of Texas Southwestern Medical Center, Dallas, TX 75235 20–60 h. Received for publication December 22, 1999. Accepted for publication March 16, 2000. FACS analysis The costs of publication of this article were defrayed in part by the payment of page PE-conjugated anti-CXCR4 mAb (12G5; R&D Systems), FITC- or PE- charges. This article must therefore be hereby marked advertisement in accordance conjugated anti-CD69 mAb (L78; Becton Dickinson, San Jose, CA), FITC- with 18 U.S.C. Section 1734 solely to indicate this fact. conjugated anti-CD25 mAb (ACT-1; Dako, Carpinteria, CA), and FITC- or 1 This work was supported by National Institutes of Health Grant AR-39169. PE-conjugated anti-CD154 mAb (TRAP1, PharMingen, San Diego, CA; 2 Current address: National Institute of Arthritis and Musculoskeletal and Skin Dis- and 89h76, Becton Dickinson; respectively) were used. The stimulated ϩ eases, Bethesda, MD 20892. peripheral CD4 T cells were stained with the above mAbs and were 3 Address correspondence and reprint requests to Dr. Peter E. Lipsky at the current analyzed with a FACScan (Becton Dickinson). address: National Institute of Arthritis and Musculoskeletal and Skin Diseases, Build- Proliferation assay ing 10, Room 9N228, 10 Center Drive MSC 1820, Bethesda, MD 20892-1820. E-mail address: [email protected] To analyze CD4ϩ T cell proliferation, the MTT assay (Cell Proliferation 4 Abbreviations used in this paper: SDF, stromal cell-derived factor; CXCR, CXC Kit I (MTT); Roche, Indianapolis, IN) was used according to the manu- chemokine receptor; MIP, macrophage inflammatory protein. facturer’s protocol, and the data reported as OD units.

● The Journal of Immunology 5011

ϩ FIGURE 1. CXCR4 expression of peripheral CD4 T cells and cultured FIGURE 3. SDF-1␣ up-regulates CD4ϩ T cell proliferation. Peripheral ϩ CD4 T cells. CXCR4 expression was analyzed by FACS. The data shown CD4ϩ T cells were cultured with medium for 6 h, and then the T cells were ϩ are from CD4 T cells stained with an isotype-matched control mAb (A), stimulated in medium supplemented with SDF-1␣ for 2 h. Subsequently, ϩ stained with anti-CXCR4 mAb (B), from CD4 T cells cultured with me- the T cells were transferred to microtiter plates with or without coating dium for6h(C), or stimulated with anti-CD3 for8h(D) and stained with with anti-CD3 mAb and were cultured for 20 (A), 40 (B), and 60 h (C). The anti-CXCR4 mAb. Representative data from one of three independent ex- proliferation was measured by MTT assay and reported as OD units. Rep- periments are shown. resentative mean data from one of three independent experiments analyzed in triplicate are shown. Concentration of SDF-1␣ (ng/ml) and the presence .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .of anti-CD3 mAb are indicated

ELISA Downloaded from

Cytokine concentrations in the culture supernatant were assayed with SDF-1␣ increased expression of all activation markers beyond that ELISA kits for IL-2 (R&D Systems), IFN-␥ (R&D Systems), IL-4 (R&D Systems), and IL-10 (BioSource International, Camarillo, CA). Cells were induced by anti-CD3 alone. cultured for8htoassess IL-2 and IFN-␥ production and for 12 h to Similarly, anti-CD3 stimulation induced proliferation of periph- determine IL-4 and IL-10 production. eral CD4ϩ T cells, whereas SDF-1␣ alone did not (Fig. 3). How- ␣ ever, SDF-1 significantly enhanced proliferation of anti- http://www.jimmunol.org/ Statistics CD3-stimulated CD4ϩ T cells in a concentration-dependent To compare proliferation and cytokine expression, the Student t test manner. Finally, SDF-1␣ costimulated cytokine production by anti- was used. CD3-stimulated CD4ϩ T cells (Fig. 4). Although SDF-1␣ alone ϩ Results failed to induce cytokine production by CD4 T cells, it caused ϩ significant concentration-dependent increases in production of all CXCR4 expression by peripheral CD4 T cells and cultured ϩ cytokines measured by anti-CD3-stimulated CD4 T cells. CD4ϩ T cells The SDF-1␣-mediated enhancement of CD154, CD69, and ϩ Purified peripheral CD4 T cells expressed low levels of CXCR4 CD25 expression by anti-CD3-stimulated peripheral CD4ϩ T cells

(Fig. 1). Culture with medium for 6 h up-regulated surface CXCR4 was blocked by anti-CXCR4 mAb (Fig. 5A and data not shown). by guest on September 28, 2021 expression. Stimulation with anti-CD3 mAb for 8 h did not further In addition, the enhancement of IL-2 production was blocked by up-regulate CXCR4 expression beyond that induced by culture in anti-CXCR4 mAb (Fig. 5B). medium alone. RANTES also increased CD154, CD69, and CD25 expression by anti-CD3-stimulated peripheral CD4ϩ T cells (Fig. 6A and data ϩ ␣ Costimulation of CD4 T cell function by SDF-1 not shown). However, the degree of the enhancement by RANTES Anti-CD3 induced expression of CD69, CD25, and CD154, was smaller than that by SDF-1␣. Moreover, costimulation with whereas SDF-1␣ did not (Fig. 2). The combination of anti-CD3 ϩ RANTES did not up-regulate IL-2 production and proliferation by

FIGURE 2. SDF-1␣ up-regulates activation marker expression by anti-CD3-stimulated CD4ϩ T cells. Peripheral CD4ϩ T cells were cultured with medium for 6 h and then were stimulated in medium supplemented with 500 ng/ml SDF-1␣ for 2 h. Subsequently, the T cells were transferred to microtiter plates with or without coating with anti- CD3 mAb and were cultured for 8 h. Expression of CD69 (A), CD25 (B), and CD154 (C) was measured by FACS. Representative data from one of three independent experiments are shown. Pres- ence of SDF-1␣ or anti-CD3 mAb is indicated. 5012 CUTTING EDGE

FIGURE 4. SDF-1␣ up-regulates cytokine production. Peripheral CD4ϩ T cells were cultured with medium for 6 h and then were stimulated in medium supplemented with SDF-1␣ for 2 h. Subsequently, the T cells were transferred to microtiter plates with or without coating with anti-CD3 mAb and were cultured for 8 h for IL-2 and IFN-␥ production or for 12 h for IL-4 and IL-10 secretion. IL-2 (A), IFN-␥ (B), IL-4 (C), and IL-10 (D) concentration in the culture supernatant was analyzed by ELISA. Representative mean data from one of three independent experiments analyzed in triplicate are shown. .p Ͻ 0.005 ,ءء ;p Ͻ 0.05 ,ء .Concentration of SDF-1␣ (ng/ml) and the presence of anti-CD3 mAb are indicated

ϩ ϩ anti-CD3-stimulated peripheral CD4 T cells (Fig. 6B and data not anti-CD3-stimulated CD4 T cells. Stimulation with SDF-1␣ Downloaded from shown). alone did not up-regulate any of these functions. It is important to note that resting peripheral CD4ϩ T cells express CXCR4, the Discussion ligand for SDF-1 (36–53% positive; n ϭ 3), and can respond to SDF-1␣ by migration (data not shown). However, SDF-1␣ en- The current data show that SDF-1␣ is a costimulator of peripheral ϩ ϩ hanced activation of CD4 T cells only when they were also stim- CD4 T cell activation. SDF-1␣ up-regulated cell surface activa-

ulated with anti-CD3. These results suggest that SDF-1 may play http://www.jimmunol.org/ tion marker expression, proliferation, and cytokine productions by an important role not only in T cell migration but also in the ef- fective activation of Ag-stimulated T cells. Recently, it was shown that SDF-1 also binds a non-CXCR4 receptor (12) and could associate with heparan sulfate (13), suggesting that CXCR4 may not be a unique ligand for SDF-1. How- ever, the current data showed that anti-CXCR4 mAb blocked the by guest on September 28, 2021

FIGURE 5. Anti-CXCR4 mAb blocks enhancement of CD154 expression and IL-2 production by SDF-1␣. Peripheral CD4ϩ T cells were cul- FIGURE 6. Comparison of costimulatory efficacy of SDF-1␣ and tured with medium for5htoup-regulate expression of CXCR4 and then RANTES. Peripheral CD4ϩ T cells were cultured with medium for6hand were incubated with isotype-matched control mAb or anti-CXCR4 mAb then were stimulated in medium supplemented with 50 or 500 ng/ml for 1 h. Subsequently, they were stimulated in medium supplemented with SDF-1␣ or RANTES for 2 h. Subsequently, the T cells were transferred to 100 ng/ml SDF-1␣ for 2 h. The T cells were transferred to microtiter plates microtiter plates coated with anti-CD3 mAb. After an 8-h incubation, ex- coated with anti-CD3 mAb and cultured for 8 h. A, Expression of CD154 pression of CD154 was assessed by FACS (A). The data obtained from the was assessed by FACS. The data obtained from the cells incubated with cells incubated without chemokines (left panel), from the cells costimu- isotype-matched control mAb (upper panels) and from the cells incubated lated with SDF-1␣ (center panel), and from the cells costimulated with with anti-CXCR4 mAb (lower panels) are depicted. Representative data RANTES (right panel) are depicted. Representative data from one of two from one of two independent experiments are shown. B, IL-2 concentration independent experiments are shown. After 60 h of culture, proliferation in the culture supernatant was analyzed by ELISA. Representative mean was measured by MTT assay and reported as OD units (B). Representative data from one of two independent experiments analyzed in triplicate are mean data from one of two independent experiments analyzed in triplicate p Ͻ ,ء .shown. Presence of SDF-1␣, control mAb, or anti-CXCR4 mAb is are shown. Concentrations of SDF-1␣ and RANTES are indicated indicated. 0.01. The Journal of Immunology 5013 costimulation of anti-CD3-activated CD4ϩ T cells by SDF-1, es- liferation of anti-CD3-stimulated peripheral CD3ϩ T cells (27), tablishing that CXCR4 is necessary for the costimulation by and high concentrations of RANTES stimulated T cells without SDF-1. anti-CD3 stimulation (28). The current data confirmed that RAN- In the periphery, mainly naive CD4ϩ T cells express CXCR4 TES enhanced early activation marker expression by anti-CD3- (14–16), although a substantial percentage (28–37%; n ϭ 3) of stimulated CD4ϩ T cells. However, the effect of RANTES ap- memory T cells also express CXCR4. SDF-1 and CXCR4 inter- peared to be considerably weaker than that of SDF-1␣ in that actions have been thought to be primarily involved in normal ho- SDF-1␣ but not RANTES also enhanced proliferation and IL-2 meostasis by playing a role in the homing of naive T cells to production by anti-CD3-activated CD4ϩ T cells. Therefore, SDF-1 secondary lymphoid organs (17). The current data suggest that appears to have a greater potential as a costimulator of peripheral SDF-1 and CXCR4 interaction could also play a role in costimu- CD4ϩ T cells. It is unlikely that the results reflect the frequency of lating Ag-activated CD4ϩ T cells during immune responses in RANTES ligand expressing peripheral CD4ϩ T cells because secondary lymphoid organs. Cytokine productions by the CD4ϩ T RANTES can bind to numerous receptors, including C-C chemo- cells along with CD154 (CD40 ligand) expression were up-regu- kine receptors 1, 3, 5, and 9. Although previous studies reported lated by SDF-1 costimulation. The expressed cytokines and CD40 that RANTES enhanced IL-2 production and proliferation by anti- ligand could activate B cells and DCs in lymphoid organs. Thus, CD3-stimulated peripheral CD3ϩ T cells (27), the current data SDF-1 may not only attract T cells to secondary lymphoid organs showed that RANTES did not enhance IL-2 production and pro- but also may play an important role in costimulating their ability to liferation by anti-CD3-stimulated peripheral CD4ϩ T cells. There- effect a successful immune response. fore, it is possible that CD8ϩ T cells or ␥␦ T cells are necessary for

Recently, we found that SDF-1 and CXCR4 interaction might be up-regulation of IL-2 production and proliferation by RANTES. Downloaded from important for accumulation of CD4ϩ T cells in rheumatoid arthritis Recently, it was reported that MIP-3␤ up-regulated IL-10 produc- synovium.5 The current data showed that SDF-1 could also be a tion by peripheral T cells stimulated with anti-CD3 and anti-CD28 costimulator of CD4ϩ T cell activation. Thus, SDF-1 and CXCR4 (29). However, MIP-3␤ did not up-regulate CD69 and CD25 ex- interactions might act not only to stimulate accumulation of CD4ϩ pression. These results suggest that other chemokines may also T cells in the rheumatoid arthritis synovium but also to costimulate have the potential to costimulate T cell activation, although the

their activation and thereby the capacity to inﬂuence the ongoing pattern of activation may differ for different chemokine-chemokine http://www.jimmunol.org/ inﬂammation in the tissue. receptor interactions. The mechanism by which SDF-1 costimulates anti-CD3- induced T cell activation is not known. Previous data suggested that SDF-1 inhibited anti-CD3-stimulated phosphorylation of the References TCR signaling molecules ZAP-70, SLP-76, and pp36 in Jurkat 1. Tashiro, K., H. Tada, R. Heilker, M. Shirozu, T. Nakano, and T. Honjo. 1993. cells, suggesting that SDF-1 could down-regulate T cell activation Signal sequence trap: a cloning strategy for secreted proteins and type I mem- (18). However, the current data showed that SDF-1 stimulation brane proteins. Science 261:600. up-regulated anti-CD3-mediated activation of peripheral CD4ϩ T 2. Nagasawa, T., H. Kikutani, and T. Kishimoto. 1994. Molecular cloning and struc-

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