Cell Activation and Tolerance the Roles of CD28 and CD40 Ligand in T

The Roles of CD28 and CD40 Ligand in T Cell Activation and Tolerance Kimberly C. Howland, Lara J. Ausubel, Cheryl A. London and Abul K. Abbas This information is current as of September 27, 2021. J Immunol 2000; 164:4465-4470; ; doi: 10.4049/jimmunol.164.9.4465 http://www.jimmunol.org/content/164/9/4465 Downloaded from

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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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Roles of CD28 and CD40 Ligand in T Cell Activation and Tolerance1

Kimberly C. Howland,2 Lara J. Ausubel,2 Cheryl A. London, and Abul K. Abbas3

Costimulation of T cell activation involves both the B7:CD28 as well as the CD40 ligand (CD40L):CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28؊/؊ T cells showed a greater reduction in proliferative responses to Ag than did CD40L؊/؊ T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L؊/؊ T cells were defective in Th1 development. In vivo, CD28؊/؊ T cells failed to expand upon immunization, whereas CD40L؊/؊ T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7:CD28 and

CD40L:CD40 interactions has an additive effect in inhibiting T cell responses. The Journal of Immunology, 2000, 164: 4465–4470. Downloaded from

he proliferation of naive T lymphocytes and their differ- clear if the absence of these ligand-receptor interactions results in entiation into effector cells require Ag recognition and reduction in T cell expansion and differentiation, or conversely, in T additional signals provided by costimulators expressed on an increased susceptibility to tolerance induction. A much more APCs. The best-deﬁned costimulatory pathways are the B7:CD28 detailed understanding of the relative contributions of these two pathway and the CD40:CD40 ligand (CD40L)4 pathway (1, 2). costimulatory pathways to T cell responses to Ag is critical for http://www.jimmunol.org/ The two known members of the B7 family, B7-1 (CD80) and B7-2 optimizing clinical protocols that employ costimulator blocking (CD86), are expressed on professional APCs, and their expression agents in an effort to modulate immune responses. is enhanced by microbes and innate immune responses. Recogni- To evaluate the roles of B7:CD28 and CD40L:CD40 interaction of B7 by CD28, which is constitutively expressed on the ma- tions in T cell responses to a cognate Ag, we have exploited TCR jority of T lymphocytes, enhances Ag-stimulated cytokine produc- transgenic mice lacking either CD28 or CD40L. T cells from these tion, clonal expansion of T cells, and their differentiation into mice can be exposed to immunogenic and tolerogenic forms of the effector cells (1). CD40 is constitutively expressed on APCs, and same peptide Ag, and proliferation and differentiation of these CD40L is induced on T cells upon Ag recognition. The interaction cells can be followed quantitatively. Our results show that CD28

between CD40L and CD40 also enhances T cell responses (2, 3). and CD40L play distinct and complimentary roles in T cell acti- by guest on September 27, 2021 Although the mechanism of this effect is unclear, it is currently vation, and suggest why blocking both these costimulatory path- believed that CD40:CD40L interactions serve to up-regulate the ways may have additive effects on T cell responses to Ag. expression of B7 on APCs and to induce the production of cytokines, such as IL-12, which promote T cell differentiation (4). There has been great interest in the roles of B7:CD28 and Materials and Methods CD40L:CD40 interactions in T cell responses, largely because of Mice the potential for targeting these ligand:receptor pairs for the pre- BALB/c mice, 6–8 wk of age, were purchased from The Jackson Labo- vention of graft rejection and the treatment of various immuno- ratory (Bar Harbor, ME). Transgenic mice expressing the DO.11.10 TCR (DO.11), specific for the chicken OVA peptide (OVA ) in the context logical diseases (5–13). However, many fundamental issues about 323–339 of the MHC class II molecule I-Ad, were obtained from Dr. Dennis Loh the roles of these two costimulatory pathways remain unanswered. (Hoffmann-LaRoche, Nutley, NJ). Mice deficient in CD40 ligand (14) on For instance, it is not known if both B7:CD28 and CD40L:CD40 the BALB/c background were obtained from Dr. Richard Flavell (Yale interactions are required for initiating and/or sustaining T cell re- University, New Haven, CT) and were bred with DO.11 mice. Crosses of sponses, especially in vivo. Perhaps more importantly, it is not DO.11 mice with CD28 knock-out mice were obtained from Dr. Jeff Blue- stone (University of Chicago). All mice were bred and maintained in accordance with the guidelines of the Committee on Care and Use of Lab- oratory Animals of the Institute of Laboratory Resources, National Immunology Research Division, Department of Pathology, Brigham and Women’s Research Council (Washington, DC). The mice were typed for the DO.11 Hospital and Harvard Medical School, Boston, MA 02115 TCR by staining peripheral blood cells with Abs against CD4 and V␤8 Received for publication September 24, 1999. Accepted for publication February (PharMingen, San Diego, CA). The CD40L-deficient mice were typed by 16, 2000. genomic PCR for neomycin (5Ј-CATTGAACAAGATGGATTGCACGC -3Ј;5Ј-CTCGATGCGATGTTTCGCTTGGTG-3Ј) and CD40L (5Ј-GGAT The costs of publication of this article were defrayed in part by the payment of page CCTCAAATTGCAGCACACG-3Ј;5Ј-CCGAATGGCTTTGGAGTACCCA charges. This article must therefore be hereby marked advertisement in accordance Ј with 18 U.S.C. Section 1734 solely to indicate this fact. AC-3 ). 1 This work was supported by National Institutes of Health Grants PO1 AI35297 and R37 AI25022 (A.K.A.), T32 HL07627 (L.J.A.), and K01-RR00121 (C.A.L.). In vitro proliferation and cytokine assays 2 K.C.H. and L.J.A. contributed equally to the work. Naive CD4ϩ T cells were purified using Dynabeads (Dynal, Oslo, Norway) Ϫ Ϫ Ϫ Ϫ 3 Address correspondence and reprint requests to Dr. Abul K. Abbas at his current from wild-type, CD28 / , and CD40L / DO.11 mice. The percentage of address: Department of Pathology, University of California, San Francisco, School of CD4ϩ DO.11ϩ cells was determined by flow cytometry using a mAb to the Medicine, 513 Parnassus Avenue, Room S-534c, San Francisco, CA 94143. E-mail DO.11 TCR (KJ1-26). To measure T cell proliferation, CD4ϩ cells con- address: [email protected] taining 2.5 ϫ 104 KJ1-26ϩ T cells were cultured with varying numbers of 4 Abbreviation used in this paper: CD40L, CD40 ligand. mitomycin C-treated BALB/c splenocytes as APCs in 0.2 ml of RPMI

FIGURE 1. In vitro primary responses of wild-type (WT), CD28Ϫ/Ϫ, and CD40LϪ/Ϫ DO.11 T cells. CD4ϩ T cells from DO.11 mice, containing 2 ϫ 4 ϩ 10 KJ1-26 cells, were cultured in duplicate with OVA323–339 peptide and different numbers of mitomycin C-treated BALB/c splenocytes as APCs. Proliferation was assayed on day 4 by pulsing cultures with [3H]thymidine for the ﬁnal 6 h. Results are from one representative experiment of three. Downloaded from

5 1640 supplemented with 1 mM L-glutamine, penicillin, streptomycin, non- tolerized T cells were assessed as above by culturing 5 ϫ 10 total lymph essential amino acids, sodium pyruvate, HEPES (all from Life Technolo- node cells in each well of a 96-well ﬂat-bottom plate with 0–1 ␮g/ml OVA gies, Grand Island, NY), 5 ϫ 10Ϫ5 M 2-ME, and 10% FBS (Sigma, St. peptide without additional APCs. To measure cytokine responses of un- Louis, MO) in 96-well plates. Cells were stimulated with 0–1 ␮g/ml of treated, immunized, or tolerized T cells, 4 ϫ 106 total lymph node cells ␮ OVA323–339 peptide. At the end of 48–96 h, cultures were pulsed for 6 h were cultured in 24-well plates with 0–1 g/ml OVA peptide, and cytokine with 1 ␮Ci [3H]thymidine (New England Nuclear, Boston, MA), and in- levels in the supernatants were assayed by ELISA.

corporated radioactivity was measured in a Betaplate scintillation counter http://www.jimmunol.org/ (LBK Pharmacia, Piscataway, NJ). To determine cytokine production, 5 ϫ 4 ϩ ϩ ϫ 6 Results 10 CD4 KJ1-26 cells were cultured with 2.5 10 mitomycin C- Ϫ/Ϫ Ϫ/Ϫ treated BALB/c splenocytes as APCs in 1 ml of medium in the presence of In vitro primary responses of CD28 and CD40L T cells 0–1 ␮g/ml of OVA peptide. Supernatants were collected after 0, 24, 48, DO.11 TCR transgenic mice lacking either CD28 or CD40L have and 72 h, and levels of IL-2, IL-4, and IFN-␥ were assayed by ELISA according to instructions provided by the manufacturer (PharMingen). For normal numbers of T cells expressing the transgenic TCR in the secondary stimulation, CD4ϩ KJ1-26ϩ T cells were stimulated for 4 days thymus and peripheral lymphoid tissues, indicating that neither with 1 ␮g/ml of OVA peptide. Viable cells were harvested, and rested for molecule is required for T cell maturation (data not shown; and 1–2 days in 50 U/ml IL-2, and restimulated to assay for proliferative and Refs. 14 and 15). To compare the primary responses of costimu- cytokine responses as described above.

lator deficient T cells with their wild-type counterparts, naive by guest on September 27, 2021 ϩ Ϫ/Ϫ Adoptive transfers and FACS analysis CD4 T cells were purified from wild-type, CD28 , and CD40LϪ/Ϫ DO.11 mice and stimulated in culture with OVA pep- For adoptive transfer of naive cells into BALB/c recipients, lymph node and spleen cells were harvested from DO.11 mice. The number of T cells tide and mitomycin C-treated syngeneic APCs. Assays of T cell expressing the DO.11 TCR was measured by staining with the clonotypic proliferation demonstrated that at all ratios of T cells:APCs and all Ϫ Ϫ Ab, KJ1-26, and flow cytometry. A total of 3–5 ϫ 106 DO.11 T cells were Ag concentrations tested, CD28 / T cells proliferated much less transferred into BALB/c recipients by tail vein injection. One day after than wild-type T cells. In contrast, CD40LϪ/Ϫ T cells proliferated ␮ transfer, recipients were either not immunized, immunized with 200 g less than wild-type T cells at low Ag and APC concentrations, but OVA peptide emulsified in IFA (Difco, Detroit, MI) by s.c. injection in four sites along the back, or tolerized with 300 ␮g OVA peptide in PBS they showed normal levels of proliferation when stimulated with injected in the tail vein. For the in vitro analyses of activation and toler- high Ag concentrations and high APC:T cell ratios (Fig. 1). Thus, ance, the axillary, brachial, and inguinal lymph nodes were collected from under most conditions of in vitro T cell stimulation, T cell prolif- recipients 3–7 days after immunization or tolerization. Cell suspensions eration is more dependent on CD28:B7 interactions than on CD40: were blocked with anti-CD16/CD32 (mouse Fc receptor), then stained with CD40L interactions. Assays for IL-2 production during primary in cychrome c-labeled anti-CD4 mAb (both from PharMingen) and biotinyl- Ϫ/Ϫ ated KJ1-26 clonotypic Ab followed by streptavidin-PE (PharMingen) and vitro responses gave similar findings, with CD28 T cells show- analyzed by FACS. Proliferative responses of untreated, immunized, and ing reduced IL-2 production at all T cell:APC ratios, whereas

FIGURE 2. Differentiation of wild-type (WT), CD28Ϫ/Ϫ, and CD40LϪ/Ϫ DO.11 T cells. CD4ϩ T cells from the DO.11 mice were primed for 4 days ␮ ϫ 5 ϩ with 1 g/ml OVA323–339 peptide and APCs at a T cell:APC ratio of 1:20. Viable cells were rested for 2 days in IL-2 and then 1.25 10 KJ1-26 T cells were restimulated using either no Ag or 1 ␮g/ml OVA peptide and 1:20 APCs. Secreted cytokines in culture supernatants were collected on days 1, 2, and 3 and assayed by ELISA. No cytokines were seen in the absence of Ag. The Journal of Immunology 4467

FIGURE 3. IL-12 corrects the defective Th1 response of CD40LϪ/Ϫ T cells. Wild-type (WT) and CD40LϪ/Ϫ DO.11 T cells were primed as in Fig. 2, without or with 1 ng/ml IL-12, and restimulated with Ag and APCs without added IL-12. IFN-␥ in culture supernatants was measured by ELISA in the primary and secondary stimulations. No IFN-␥ was seen in the absence of Ag.

CD40LϪ/Ϫ T cells produced reduced levels of IL-2 in cultures APCs with and without IL-12, and IFN-␥ production was assayed with low numbers of APCs or low peptide concentrations (data not in primary cultures and upon restimulation. The addition of IL-12 shown). None of the T cell populations produced IFN-␥ or IL-4 enhanced IFN-␥ production by wild-type T cells, and restored the upon primary stimulation (data not shown). IFN-␥ response of CD40LϪ/Ϫ T cells to normal levels even upon restimulation in the absence of added IL-12 (Fig. 3). Differentiation of T cells into effector populations Downloaded from

To evaluate the roles of CD28 and CD40L in the differentiation of Ϫ/Ϫ Ϫ/Ϫ T cells into effector Th1 and Th2 populations, DO.11 T cells were In vivo responses of CD28 and CD40L T cells to primed with Ag and APCs without added cytokines and restimu- immunization lated, and cytokine production was assayed. These experiments The activation of T cells by Ag and APCs in culture may not showed that in the absence of CD28, T cells did not produce de- accurately reflect their in vivo responses. The DO.11 TCR trans- tectable IL-2 or IL-4 but secreted significant levels of IFN-␥.In genic system is particularly useful for quantitative analyses of in contrast, in the absence of CD40L the T cells had a selective defect vivo T cell responses (16). To examine the in vivo responses of http://www.jimmunol.org/ in IFN-␥ production, and often produced even more IL-4 than did CD28Ϫ/Ϫ and CD40LϪ/Ϫ T cells, 5 ϫ 106 naı¨ve DO.11 T cells wild-type T cells (Fig. 2). Thus, the CD28 and CD40L pathways were transferred into syngeneic BALB/c mice and the recipients are required for Th2 and Th1 development, respectively. were immunized by s.c. administration of OVA323–339 in IFA. It is possible that the inability of CD40LϪ/Ϫ T cells to differ- Lymph node cells were isolated after 3 and 7 days, and examined entiate into Th1 cells is because these T cells fail to activate APCs for the expansion of DO.11 (CD4ϩ KJ1-26ϩ) cells by staining and to produce the essential Th1-inducing cytokine, IL-12 (4). To test flow cytometry. Three days after immunization, the CD40LϪ/Ϫ T this, wild-type and CD40LϪ/Ϫ T cells were primed with Ag and cells increased in numbers to nearly the same extent as wild-type by guest on September 27, 2021

FIGURE 4. Expansion of wild-type (WT), CD28Ϫ/Ϫ, and CD40LϪ/Ϫ DO.11 T cells in response to immunization in vivo. Recipients of 5 ϫ 106 adoptively transferred DO.11 T cells

were immunized with OVA323–339 in IFA s.c. and assayed for number of DO.11 cells in draining lymph nodes by staining and ﬂow cytometry. A, Lymph nodes were isolated 3 days after immunization and FACS analysis was performed on lymph node cells from naive (un- immunized) and immunized recipients after staining with anti-CD4 and KJ1-26. Numbers in FACS plots refer to CD4ϩ KJ1-26ϩ cells as % of the total. Results are from one representative experiment of four. B, Numbers of DO.11 T cells recovered from inguinal, axillary and brachial lymph nodes were calculated as numbers of lymph node cells ϫ percent CD4ϩ KJ1-26ϩ cells. Data are pooled from two ex- .p Ͻ 0.02 by t test ,ء .periments 4468 CD28 AND CD40L IN T CELL RESPONSES Downloaded from http://www.jimmunol.org/

FIGURE 5. Responses of immunized T cells to Ag restimulation ex vivo. Lymph node cells recovered from adoptive transfer recipients 3 days after transfer, as in Fig. 4, were restimulated with Ag and assayed for proliferation on day 3, and for cytokine secretion on different days of culture. Results of ϩ ϩ ␮ proliferation are corrected for numbers of CD4 KJ1-26 cells added to cultures. Cytokine secretion is shown in response to 0.1 g/ml OVA323–339 peptide. Cells cultured without Ag did not produce any detectable cytokines. Results are from one representative experiment of four. by guest on September 27, 2021 cells. Conversely, the CD28Ϫ/Ϫ T cells exhibited markedly re- to immunization. Thus, after immunization, wild-type DO.11 T duced expansion when compared with wild-type and CD40LϪ/Ϫ T cells showed increased proliferation and secretion of IL-2 and cells. By day 7, the numbers of wild-type T cells remained 3- to IFN-␥ upon restimulation ex vivo, but all these enhanced recall 5-fold more than the numbers without immunization, but at this responses were reduced in the CD28Ϫ/Ϫ and CD40LϪ/Ϫ T cells time even the CD40LϪ/Ϫ T cells were at baseline levels (Fig. 4). (Fig. 5).

Thus, CD28 and CD40L play distinct roles in T cell expansion Ϫ/Ϫ Ϫ/Ϫ in vivo. Induction of tolerance in CD28 and CD40L T cells The T cells that had been exposed to Ag in vivo were recovered, The role of costimulatory pathways in inducing tolerance in nor- restimulated with Ag in culture, and assayed for proliferation and mal T cells can only be examined in vivo and is a major strength cytokine production. These experiments showed that both of the DO.11 T cell adoptive transfer system (17, 18). In the ﬁnal CD28Ϫ/Ϫ and CD40LϪ/Ϫ T cells were defective in their responses set of experiments, recipients of DO.11 T cells were left untreated

FIGURE 6. Induction of tolerance in wild-type (WT), CD28Ϫ/Ϫ and CD40LϪ/Ϫ DO.11 T cells. Adoptive transfer recipients, as in Fig. 4, were left untreated (naive), or given two doses of aqueous

OVA323–339 peptide i.v. (tolerogen, tol). Lymph node cells were recovered on day 3 or 7 after Ag administration, 5 ϫ 105 cells were restimulated with Ag, and proliferation was assayed on day 3. Results of proliferation are corrected for numbers of CD4ϩ KJ1-26ϩ cells added to cultures. The Journal of Immunology 4469 or injected with two doses of aqueous peptide Ag i.v. The T cells The in vivo analysis of T cell responses also showed that CD28, were assayed 3 and 7 days later for proliferation responses to Ag but not CD40L, is required for initiating T cell expansion in restimulation ex vivo. Exposure to aqueous (tolerogenic) Ag inhib- sponse to immunogenic Ag. However, CD40L does play a role in ited subsequent responses of both wild-type T cells and CD40LϪ/Ϫ sustaining the T cell response (Fig. 4). This is also consistent with T cells (Fig. 6). It is not possible to examine the tolerance sensi- an amplification function of the CD40L:CD40 pathway. Thus, tivity of CD28Ϫ/Ϫ T cells by this method, because, as demon- with time after immunization, T cell expansion may be maintained strated above, even untreated CD28Ϫ/Ϫ T cells proliferate poorly by continuous stimulation by Ag released from its depot. As the in response to Ag. quantity of available Ag decreases and the innate immune response to the adjuvant subsides, CD40L-mediated stimulation of APCs becomes increasingly important. In the absence of CD40L, T cell Discussion priming also fails to induce Th1 development in vivo (Fig. 5), as it does in vitro. The experiments described in this paper were designed to compare Finally, we have attempted to address the possibility that the the responses of T cells lacking either CD28 or CD40L to Ag both absence of CD28 or CD40L increases the sensitivity of T cells to in vitro and in vivo. Studies from numerous laboratories have tolerance induction. Using an experimental system of tolerance shown that CD28 is required for initiating and/or sustaining T cell induced by aqueous protein Ag, we find that CD40L-deficient T clonal expansion and differentiation in response to Ag recognition cells are as tolerance-sensitive as wild-type cells (Fig. 6). How- (19, 20). The CD40:CD40L pathway was first described for its role ever, this experimental approach does not allow us to accurately in T cell-dependent Ab production (reviewed in Refs. 2 and 21), and quantitatively compare the tolerance sensitivity of different Downloaded from but subsequent studies have shown that it is critically involved in cell populations, or to assay tolerance in CD28Ϫ/Ϫ T cells (because T cell-mediated immunity, including macrophage activation and these cells normally fail to respond to Ag). the generation of CTL (22–24). Few studies have directly exam- The results in this paper provide a framework for explaining the ined the role of CD40L in T cell clonal expansion and differenti- additive or synergistic effects of antagonizing both the CD28:B7 ation. It has been suggested that in the absence of CD40L, helper and the CD40L:CD40 pathways in various immune responses, T cells do not expand normally in response to Ag (6), but it is such as graft rejection (11, 12). Blocking the CD28:B7 pathway http://www.jimmunol.org/ unclear whether this reflects defective proliferation, survival, or will inhibit the primary T cell response, whereas blocking the maintenance. In our experiments, analysis of primary T cell re- CD40L:CD40 pathway will inhibit Th1 differentiation and the sponses in vitro showed that CD28 is much more important for maintenance of the response. The distinct but complimentary roles initiating the clonal expansion of T cells than is CD40L. Thus, of CD28 and CD40L may provide new avenues for developing Ϫ/Ϫ CD28 naive T cells are deficient in proliferation (and IL-2 therapeutic agents for different types of immunologic diseases. secretion) when stimulated with a wide range of Ag concentrations Ϫ/Ϫ and APC numbers. In contrast, CD40L T cells respond nor- Acknowledgments mally when stimulated in conditions of high Ag concentrations or high APC to T cell ratios, and show defects only when stimulated We thank Nyree Bekarian and Michael Lodge for their expert technical by guest on September 27, 2021 with low numbers of APCs or low concentrations of Ags (Fig. 1). assistance. These results are consistent with the hypothesis that B7:CD28 interactions are required for initiating T cell responses, whereas References CD40L:CD40 interactions serve mainly to amplify such responses. 1. Lenschow, D. J., T. L. Walunas, and J. A. Bluestone. 1996. CD28/B7 system of This amplification function would be most apparent when costimu- T-cell costimulation. Annu. Rev. Immunol. 14:233. 2. Grewal, I. S., and R. A. Flavell. 1998. CD40 and CD154 in cell-mediated im- lation is limiting; for instance, when T cells are stimulated with munity. Annu. Rev. Immunol. 16:111. low numbers of APCs. The likely mechanism of CD40L-induced 3. Grewal, I. S., J. Xu, and R. A. Flavell. 1995. Impairment of antigen-specific T-cell priming in mice lacking CD40 ligand. Nature 378:617. enhancement of T cell responses is that CD40L engagement of CD40 4. Shu, U., M. Kiniwa, C. Y. Wu, C. Maliszewski, N. Vezzio, J. Hakimi, M. Gately, on APCs increases the expression of B7 on these APCs (25, 26). and G. Delespesse. 1995. Activated T cells induce interleukin-12 production by Our results also show that CD28 and CD40L serve opposing monocytes via CD40-CD40 ligand interaction. Eur. J. Immunol. 25:1125. ϩ 5. Miller, S. D., C. L. Vanderlugt, D. J. Lenschow, J. G. Pope, N. J. Karandikar, roles in the differentiation of CD4 T cells into cytokine-produc- M. C. Dal Canto, and J. A. Bluestone. 1995. Blockade of CD28/B7-1 interaction ing effector cells. The absence of CD28 costimulation prevents prevents epitope spreading and clinical relapses of murine EAE. Immunity 3:739. 6. Grewal, I. S., H. G. Foellmer, K. D. Grewal, J. Xu, F. Hardardottir, J. L. Baron, Th2 differentiation when T cells are primed without exogenous C. A. Janeway, Jr., and R. A. Flavell. 1996. Requirement for CD40 ligand in cytokines, whereas the absence of CD40L prevents Th1 develop- costimulation induction, T cell activation, and experimental allergic encephalo- ment (Fig. 2). The essential role of CD28 in Th2 differentiation has myelitis. Science 273:1864. 7. Gerritse, K., J. D. Laman, R. J. Noelle, A. Aruffo, J. A. Ledbetter, W. J. Boersma, been described previously (27), although the operative mechanism and E. Claassen. 1996. CD40-CD40 ligand interactions in experimental allergic is not known. It has also been suggested that the CD40 pathway is encephalomyelitis and multiple sclerosis. Proc. Natl. Acad. Sci. USA 93:2499. more important for Th1 differentiation (28), although there are few 8. Durie, F. H., R. A. Fava, T. M. Foy, A. Aruffo, J. A. Ledbetter, and R. J. Noelle. 1993. Prevention of collagen-induced arthritis with an antibody to gp39, the studies directly addressing this issue. The ability of IL-12 to over- ligand for CD40. Science 261:1328. come the lack of CD40L in Th1 differentiation (Fig. 3) is consis- 9. Balasa, B., T. Krahl, G. Patstone, J. Lee, R. Tisch, H. O. McDevitt, and N. Sarvetnick. 1997. CD40 ligand-CD40 interactions are necessary for the initi- tent with the idea that CD40L activates APCs to produce IL-12, ation of insulitis and diabetes in nonobese diabetic mice. J. Immunol. 159:4620. and this is a mechanism by which it stimulates Th1 development 10. Lenschow, D. J., Y. Zeng, K. S. 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