HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Howland, K. C. Articles by Abbas, A. K. Search for Related Content PubMed PubMed Citation Articles by Howland, K. C. Articles by Abbas, A. K.

The Roles of CD28 and CD40 Ligand in T Cell Activation and Tolerance¹

Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The proliferation of naive T lymphocytes and their differentiation into effector cells require Ag recognition and additional signals provided by costimulators expressed on APCs. The best-defined costimulatory pathways are the B7:CD28 pathway and the CD40:CD40 ligand (CD40L)⁴ pathway (1, 2). The two known members of the B7 family, B7-1 (CD80) and B7-2 (CD86), are expressed on professional APCs, and their expression is enhanced by microbes and innate immune responses. Recognition of B7 by CD28, which is constitutively expressed on the majority of T lymphocytes, enhances Ag-stimulated cytokine production, clonal expansion of T cells, and their differentiation into effector cells (1). CD40 is constitutively expressed on APCs, and CD40L is induced on T cells upon Ag recognition. The interaction between CD40L and CD40 also enhances T cell responses (2, 3). Although the mechanism of this effect is unclear, it is currently believed that CD40:CD40L interactions serve to up-regulate the 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 CD40L:CD40 interactions in T cell responses, largely because of the potential for targeting these ligand:receptor pairs for the prevention of graft rejection and the treatment of various immunological diseases (5, 6, 7, 8, 9, 10, 11, 12, 13). However, many fundamental issues about the roles of these two costimulatory pathways remain unanswered. For instance, it is not known if both B7:CD28 and CD40L:CD40 interactions are required for initiating and/or sustaining T cell responses, especially in vivo. Perhaps more importantly, it is not clear if the absence of these ligand-receptor interactions results in reduction in T cell expansion and differentiation, or conversely, in an increased susceptibility to tolerance induction. A much more detailed understanding of the relative contributions of these two costimulatory pathways to T cell responses to Ag is critical for optimizing clinical protocols that employ costimulator blocking agents in an effort to modulate immune responses.

To evaluate the roles of B7:CD28 and CD40L:CD40 interactions in T cell responses to a cognate Ag, we have exploited TCR transgenic mice lacking either CD28 or CD40L. T cells from these mice can be exposed to immunogenic and tolerogenic forms of the same peptide Ag, and proliferation and differentiation of these cells can be followed quantitatively. Our results show that CD28 and CD40L play distinct and complimentary roles in T cell activation, and suggest why blocking both these costimulatory pathways may have additive effects on T cell responses to Ag.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

BALB/c mice, 6–8 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). Transgenic mice expressing the DO.11.10 TCR (DO.11), specific for the chicken OVA peptide (OVA_323–339) in the context of the MHC class II molecule I-A^d, were obtained from Dr. Dennis Loh (Hoffmann-LaRoche, Nutley, NJ). Mice deficient in CD40 ligand (14) on the BALB/c background were obtained from Dr. Richard Flavell (Yale University, New Haven, CT) and were bred with DO.11 mice. Crosses of DO.11 mice with CD28 knock-out mice were obtained from Dr. Jeff Bluestone (University of Chicago). All mice were bred and maintained in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (Washington, DC). The mice were typed for the DO.11 TCR by staining peripheral blood cells with Abs against CD4 and Vß8 (PharMingen, San Diego, CA). The CD40L-deficient mice were typed by genomic PCR for neomycin (5'-CATTGAACAAGATGGATTGCACGC-3'; 5'-CTCGATGCGATGTTTCGCTTGGTG-3') and CD40L (5'-GGATCCTCAAATTGCAGCACACG-3'; 5'-CCGAATGGCTTTGGAGTACCCAAC-3').

In vitro proliferation and cytokine assays

Naive CD4⁺ T cells were purified using Dynabeads (Dynal, Oslo, Norway) from wild-type, CD28^-/-, and CD40L^-/- DO.11 mice. The percentage of CD4⁺ DO.11⁺ cells was determined by flow cytometry using a mAb to the DO.11 TCR (KJ1-26). To measure T cell proliferation, CD4⁺ cells containing 2.5 x 10⁴ KJ1-26⁺ T cells were cultured with varying numbers of mitomycin C-treated BALB/c splenocytes as APCs in 0.2 ml of RPMI 1640 supplemented with 1 mM L-glutamine, penicillin, streptomycin, nonessential amino acids, sodium pyruvate, HEPES (all from Life Technologies, Grand Island, NY), 5 x 10^-5 M 2-ME, and 10% FBS (Sigma, St. Louis, MO) in 96-well plates. Cells were stimulated with 0–1 µg/ml of OVA_323–339 peptide. At the end of 48–96 h, cultures were pulsed for 6 h with 1 µCi [³H]thymidine (New England Nuclear, Boston, MA), and incorporated radioactivity was measured in a Betaplate scintillation counter (LBK Pharmacia, Piscataway, NJ). To determine cytokine production, 5 x 10⁴ CD4⁺ KJ1-26⁺ cells were cultured with 2.5 x 10⁶ mitomycin C-treated BALB/c splenocytes as APCs in 1 ml of medium in the presence of 0–1 µg/ml of OVA peptide. Supernatants were collected after 0, 24, 48, 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 secondary stimulation, CD4⁺ KJ1-26⁺ T cells were stimulated for 4 days with 1 µg/ml of OVA peptide. Viable cells were harvested, and rested for 1–2 days in 50 U/ml IL-2, and restimulated to assay for proliferative and cytokine responses as described above.

Adoptive transfers and FACS analysis

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 expressing the DO.11 TCR was measured by staining with the clonotypic Ab, KJ1-26, and flow cytometry. A total of 3–5 x 10⁶ DO.11 T cells were transferred into BALB/c recipients by tail vein injection. One day after transfer, recipients were either not immunized, immunized with 200 µg OVA peptide emulsified in IFA (Difco, Detroit, MI) by s.c. injection in four sites along the back, or tolerized with 300 µg OVA peptide in PBS injected in the tail vein. For the in vitro analyses of activation and tolerance, the axillary, brachial, and inguinal lymph nodes were collected from recipients 3–7 days after immunization or tolerization. Cell suspensions were blocked with anti-CD16/CD32 (mouse Fc receptor), then stained with cychrome c-labeled anti-CD4 mAb (both from PharMingen) and biotinylated KJ1-26 clonotypic Ab followed by streptavidin-PE (PharMingen) and analyzed by FACS. Proliferative responses of untreated, immunized, and tolerized T cells were assessed as above by culturing 5 x 10⁵ total lymph node cells in each well of a 96-well flat-bottom plate with 0–1 µg/ml OVA peptide without additional APCs. To measure cytokine responses of untreated, immunized, or tolerized T cells, 4 x 10⁶ total lymph node cells were cultured in 24-well plates with 0–1 µg/ml OVA peptide, and cytokine levels in the supernatants were assayed by ELISA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vitro primary responses of CD28^-/- and CD40L^-/- T cells

DO.11 TCR transgenic mice lacking either CD28 or CD40L have normal numbers of T cells expressing the transgenic TCR in the thymus and peripheral lymphoid tissues, indicating that neither molecule is required for T cell maturation (data not shown; and Refs. 14 and 15). To compare the primary responses of costimulator deficient T cells with their wild-type counterparts, naive CD4⁺ T cells were purified from wild-type, CD28^-/-, and CD40L^-/- DO.11 mice and stimulated in culture with OVA peptide and mitomycin C-treated syngeneic APCs. Assays of T cell proliferation demonstrated that at all ratios of T cells:APCs and all Ag concentrations tested, CD28^-/- T cells proliferated much less than wild-type T cells. In contrast, CD40L^-/- T cells proliferated less than wild-type T cells at low Ag and APC concentrations, but they showed normal levels of proliferation when stimulated with high Ag concentrations and high APC:T cell ratios (Fig. 1). Thus, under most conditions of in vitro T cell stimulation, T cell proliferation is more dependent on CD28:B7 interactions than on CD40:CD40L interactions. Assays for IL-2 production during primary in vitro responses gave similar findings, with CD28^-/- T cells showing reduced IL-2 production at all T cell:APC ratios, whereas CD40L^-/- T cells produced reduced levels of IL-2 in cultures with low numbers of APCs or low peptide concentrations (data not shown). None of the T cell populations produced IFN- or IL-4 upon primary stimulation (data not shown).

View larger version (12K): [in this window] [in a new window]   FIGURE 1. In vitro primary responses of wild-type (WT), CD28-/-, and CD40L-/- DO.11 T cells. CD4+ T cells from DO.11 mice, containing 2 x 104 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.

To evaluate the roles of CD28 and CD40L in the differentiation of T cells into effector Th1 and Th2 populations, DO.11 T cells were primed with Ag and APCs without added cytokines and restimulated, and cytokine production was assayed. These experiments showed that in the absence of CD28, T cells did not produce detectable IL-2 or IL-4 but secreted significant levels of IFN-. In contrast, in the absence of CD40L the T cells had a selective defect in IFN- production, and often produced even more IL-4 than did wild-type T cells (Fig. 2). Thus, the CD28 and CD40L pathways are required for Th2 and Th1 development, respectively.

View larger version (11K): [in this window] [in a new window]   FIGURE 2. Differentiation of wild-type (WT), CD28-/-, and CD40L-/- DO.11 T cells. CD4+ T cells from the DO.11 mice were primed for 4 days with 1 µg/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 x 105 KJ1-26+ T cells were restimulated using either no Ag or 1 µg/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.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. IL-12 corrects the defective Th1 response of CD40L-/- T cells. Wild-type (WT) and CD40L-/- 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.

The activation of T cells by Ag and APCs in culture may not accurately reflect their in vivo responses. The DO.11 TCR transgenic system is particularly useful for quantitative analyses of in vivo T cell responses (16). To examine the in vivo responses of CD28^-/- and CD40L^-/- T cells, 5 x 10⁶ naïve DO.11 T cells were transferred into syngeneic BALB/c mice and the recipients were immunized by s.c. administration of OVA_323–339 in IFA. Lymph node cells were isolated after 3 and 7 days, and examined for the expansion of DO.11 (CD4⁺ KJ1-26⁺) cells by staining and flow cytometry. Three days after immunization, the CD40L^-/- T cells increased in numbers to nearly the same extent as wild-type cells. Conversely, the CD28^-/- T cells exhibited markedly reduced expansion when compared with wild-type and CD40L^-/- T cells. By day 7, the numbers of wild-type T cells remained 3- to 5-fold more than the numbers without immunization, but at this time even the CD40L^-/- T cells were at baseline levels (Fig. 4). Thus, CD28 and CD40L play distinct roles in T cell expansion in vivo.

View larger version (49K): [in this window] [in a new window]   FIGURE 4. Expansion of wild-type (WT), CD28-/-, and CD40L-/- DO.11 T cells in response to immunization in vivo. Recipients of 5 x 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 (unimmunized) and immunized recipients after staining with anti-CD4 and KJ1-26. Numbers in FACS plots refer to CD4+ KJ1-26+ 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 x percent CD4+ KJ1-26+ cells. Data are pooled from two experiments. *, p < 0.02 by t test.

View larger version (27K): [in this window] [in a new window]   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 proliferation are corrected for numbers of CD4+ KJ1-26+ cells added to cultures. Cytokine secretion is shown in response to 0.1 µg/ml OVA323–339 peptide. Cells cultured without Ag did not produce any detectable cytokines. Results are from one representative experiment of four.

The role of costimulatory pathways in inducing tolerance in normal T cells can only be examined in vivo and is a major strength of the DO.11 T cell adoptive transfer system (17, 18). In the final set of experiments, recipients of DO.11 T cells were left untreated or injected with two doses of aqueous peptide Ag i.v. The T cells were assayed 3 and 7 days later for proliferation responses to Ag stimulation ex vivo. Exposure to aqueous (tolerogenic) Ag inhibited subsequent responses of both wild-type T cells and CD40L^-/- T cells (Fig. 6). It is not possible to examine the tolerance sensitivity of CD28^-/- T cells by this method, because, as demonstrated above, even untreated CD28^-/- T cells proliferate poorly in response to Ag.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Induction of tolerance in wild-type (WT), CD28-/- and CD40L-/- 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 x 105 cells were restimulated with Ag, and proliferation was assayed on day 3. Results of proliferation are corrected for numbers of CD4+ KJ1-26+ cells added to cultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our results also show that CD28 and CD40L serve opposing roles in the differentiation of CD4⁺ T cells into cytokine-producing effector cells. The absence of CD28 costimulation prevents Th2 differentiation when T cells are primed without exogenous cytokines, whereas the absence of CD40L prevents Th1 development (Fig. 2). The essential role of CD28 in Th2 differentiation has been described previously (27), although the operative mechanism is not known. It has also been suggested that the CD40 pathway is more important for Th1 differentiation (28), although there are few studies directly addressing this issue. The ability of IL-12 to overcome the lack of CD40L in Th1 differentiation (Fig. 3) is consistent with the idea that CD40L activates APCs to produce IL-12, and this is a mechanism by which it stimulates Th1 development (4). These results indicate that antagonists against B7:CD28 and CD40L:CD40 may have distinct effects on immune responses, and are therefore likely to be most useful for inhibiting different types of pathologic immunity. However, it should be pointed out that in vivo, CD28^-/- T cells show little differentiation into any effector subset (Fig. 5). This is probably because under the limiting conditions of in vivo Ag exposure, CD28^-/- T cells are unable to mount any functional responses.

The in vivo analysis of T cell responses also showed that CD28, but not CD40L, is required for initiating T cell expansion in response to immunogenic Ag. However, CD40L does play a role in sustaining the T cell response (Fig. 4). This is also consistent with an amplification function of the CD40L:CD40 pathway. Thus, with time after immunization, T cell expansion may be maintained by continuous stimulation by Ag released from its depot. As the 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 priming also fails to induce Th1 development in vivo (Fig. 5), as it does in vitro.

Finally, we have attempted to address the possibility that the absence of CD28 or CD40L increases the sensitivity of T cells to tolerance induction. Using an experimental system of tolerance induced by aqueous protein Ag, we find that CD40L-deficient T cells are as tolerance-sensitive as wild-type cells (Fig. 6). However, this experimental approach does not allow us to accurately and quantitatively compare the tolerance sensitivity of different cell populations, or to assay tolerance in CD28^-/- T cells (because these cells normally fail to respond to Ag).

The results in this paper provide a framework for explaining the additive or synergistic effects of antagonizing both the CD28:B7 and the CD40L:CD40 pathways in various immune responses, such as graft rejection (11, 12). Blocking the CD28:B7 pathway will inhibit the primary T cell response, whereas blocking the CD40L:CD40 pathway will inhibit Th1 differentiation and the maintenance of the response. The distinct but complimentary roles of CD28 and CD40L may provide new avenues for developing therapeutic agents for different types of immunologic diseases.

	Acknowledgments

 
We thank Nyree Bekarian and Michael Lodge for their expert technical assistance.

	Footnotes

 
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.).

² K.C.H. and L.J.A. contributed equally to the work.

³ Address correspondence and reprint requests to Dr. Abul K. Abbas at his current address: Department of Pathology, University of California, San Francisco, School of Medicine, 513 Parnassus Avenue, Room S-534c, San Francisco, CA 94143.

⁴ Abbreviation used in this paper: CD40L, CD40 ligand.

Received for publication September 24, 1999. Accepted for publication February 16, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Lenschow, D. J., T. L. Walunas, J. A. Bluestone. 1996. CD28/B7 system of T-cell costimulation. Annu. Rev. Immunol. 14:233.[Medline]
2. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[Medline]
3. Grewal, I. S., J. Xu, R. A. Flavell. 1995. Impairment of antigen-specific T-cell priming in mice lacking CD40 ligand. Nature 378:617.[Medline]
4. Shu, U., M. Kiniwa, C. Y. Wu, C. Maliszewski, N. Vezzio, J. Hakimi, M. Gately, G. Delespesse. 1995. Activated T cells induce interleukin-12 production by monocytes via CD40-CD40 ligand interaction. Eur. J. Immunol. 25:1125.[Medline]
5. Miller, S. D., C. L. Vanderlugt, D. J. Lenschow, J. G. Pope, N. J. Karandikar, M. C. Dal Canto, J. A. Bluestone. 1995. Blockade of CD28/B7-1 interaction prevents epitope spreading and clinical relapses of murine EAE. Immunity 3:739.[Medline]
6. Grewal, I. S., H. G. Foellmer, K. D. Grewal, J. Xu, F. Hardardottir, J. L. Baron, Jr C. A. Janeway, R. A. Flavell. 1996. Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 273:1864.[Abstract/Free Full Text]
7. Gerritse, K., J. D. Laman, R. J. Noelle, A. Aruffo, J. A. Ledbetter, W. J. Boersma, E. Claassen. 1996. CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc. Natl. Acad. Sci. USA 93:2499.[Abstract/Free Full Text]
8. Durie, F. H., R. A. Fava, T. M. Foy, A. Aruffo, J. A. Ledbetter, R. J. Noelle. 1993. Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science 261:1328.[Abstract/Free Full Text]
9. Balasa, B., T. Krahl, G. Patstone, J. Lee, R. Tisch, H. O. McDevitt, N. Sarvetnick. 1997. CD40 ligand-CD40 interactions are necessary for the initiation of insulitis and diabetes in nonobese diabetic mice. J. Immunol. 159:4620.[Abstract]
10. Lenschow, D. J., Y. Zeng, K. S. Hathcock, L. A. Zuckerman, G. Freeman, J. R. Thistlewaite, G. S. Gray, R. J. Hodes, J. A. Bluestone. 1995. Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies. Transplantation 60:1171.[Medline]
11. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434.[Medline]
12. Kirk, A. D., D. M. Harlan, N. N. Armstrong, T. A. Davis, Y. Dong, G. S. Gray, X. Hong, D. Thomas, J. H. J. Fechner, S. J. Knechtle. 1997. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc. Natl. Acad. Sci. USA 94:8789.[Abstract/Free Full Text]
13. Kenyon, N. S., M. Chatzipetrou, M. Masetti, A. Ranuncoli, M. Oliveira, J. L. Wagner, A. D. Kirk, D. M. Harlan, L. C. Burly, C. Ricordi. 1999. Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc. Natl. Acad. Sci. USA 96:8132.[Abstract/Free Full Text]
14. Xu, J., T. M. Foy, J. D. Laman, E. A. Elliott, J. J. Dunn, T. J. Waldschmidt, J. Elsemore, R. J. Noelle, R. A. Flavell. 1994. Mice deficient for the CD40 ligand. Immunity 1:423.[Medline]
15. Shahinian, A., K. Pfeffer, K. P. Lee, T. M. Kundig, K. Kishihara, A. Wakeham, K. Kawai, P. S. Ohashi, C. B. Thompson, T. W. Mak. 1993. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261:609.[Abstract/Free Full Text]
16. Pape, K. A., E. R. Kearney, A. Khoruts, A. Mondino, R. Merica, Z. M. I. Chen, E. J. White, J. G. Johnson, M. K. Jenkins. 1997. Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cells for the study of T-cell activation in vivo. Immunol. Rev. 156:67.[Medline]
17. Kearney, E. R., K. A. Pape, D. Y. Loh, M. K. Jenkins. 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1:327.[Medline]
18. Perez, V.L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 4:411.
19. Lucas, P. J., I. Negishi, K. Nakayama, L. E. Fields, D. Y. Loh. 1995. Naive CD28-deficient T cells can initiate but not sustain an in vitro antigen-specific immune response. J. Immunol. 154:5757.[Abstract]
20. Khoruts, A., A. Mondino, K. A. Pape, S. L. Reiner, M. K. Jenkins. 1998. A natural immunological adjuvant enhances T cell clonal expansion through a CD28-dependent, interleukin (IL)-2-independent mechanism. J. Exp. Med. 187:225.[Abstract/Free Full Text]
21. Clark, L. B., T. M. Foy, R. J. Noelle. 1996. CD40 and its ligand. Adv. Immunol. 63:43.[Medline]
22. Campbell, K. A., P. J. Ovendale, M. K. Kennedy, W. C. Fanslow, S. G. Reed, C. R. Maliszewski. 1996. CD40 ligand is required for protective cell-mediated immunity to Leishmania major. Immunity 4:283.[Medline]
23. Kamanaka, M., P. Yu, T. Yasui, K. Yoshida, T. Kawabe, T. Horii, T. Kishimoto, H. Kikutani. 1996. Protective role of CD40 in Leishmania major infection at two distinct phases of cell-mediated immunity. Immunity 4:275.[Medline]
24. Borrow, P., A. Tishon, S. Lee, J. Xu, I. S. Grewal, M. B. Oldstone, R. A. Flavell. 1996. CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8⁺ CTL response. J. Exp. Med. 183:2129.[Abstract/Free Full Text]
25. Ranhein, E. A., T. J. Kipps. 1993. Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal. J. Exp. Med. 177:925.[Abstract/Free Full Text]
26. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263.[Abstract/Free Full Text]
27. Rulifson, I. C., A. I. Sperling, P. E. Fields, F. W. Fitch, J. A. Bluestone. 1997. CD28 costimulation promotes the production of Th2 cytokines. J. Immunol. 158:658.[Abstract]
28. Stout, R., J. Stuttles. 1996. The many roles of CD40:CD40L interactions in cell-mediated inflammatory responses. Immunol. Today 17:487.[Medline]

This article has been cited by other articles:

				S. J. Jenkins, G. Perona-Wright, and A. S. MacDonald Full Development of Th2 Immunity Requires Both Innate and Adaptive Sources of CD154 J. Immunol., June 15, 2008; 180(12): 8083 - 8092. [Abstract] [Full Text] [PDF]

				D. Daoussis, I. Antonopoulos, A. P. Andonopoulos, and S.-N. C. Liossis Increased expression of CD154 (CD40L) on stimulated T-cells from patients with psoriatic arthritis Rheumatology, February 1, 2007; 46(2): 227 - 231. [Abstract] [Full Text] [PDF]

				B. Knoechel, J. Lohr, S. Zhu, L. Wong, D. Hu, L. Ausubel, and A. K. Abbas Functional and Molecular Comparison of Anergic and Regulatory T Lymphocytes. J. Immunol., June 1, 2006; 176(11): 6473 - 6483. [Abstract] [Full Text] [PDF]

				M. MacLeod, M. J. Kwakkenbos, A. Crawford, S. Brown, B. Stockinger, K. Schepers, T. Schumacher, and D. Gray CD4 memory T cells survive and proliferate but fail to differentiate in the absence of CD40 J. Exp. Med., April 17, 2006; 203(4): 897 - 906. [Abstract] [Full Text] [PDF]

				S. E. Feldon, D. J. J. Park, C. W. O'Loughlin, V. T. Nguyen, S. Landskroner-Eiger, D. Chang, T. H. Thatcher, and R. P. Phipps Autologous T-Lymphocytes Stimulate Proliferation of Orbital Fibroblasts Derived from Patients with Graves' Ophthalmopathy Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 3913 - 3921. [Abstract] [Full Text] [PDF]

				K. M. Pollard, M. Arnush, P. Hultman, and D. H. Kono Costimulation Requirements of Induced Murine Systemic Autoimmune Disease J. Immunol., November 1, 2004; 173(9): 5880 - 5887. [Abstract] [Full Text] [PDF]

				J. Lohr, B. Knoechel, E. C. Kahn, and A. K. Abbas Role of B7 in T Cell Tolerance J. Immunol., October 15, 2004; 173(8): 5028 - 5035. [Abstract] [Full Text] [PDF]

				C. Vasu, B. S. Prabhakar, and M. J. Holterman Targeted CTLA-4 Engagement Induces CD4+CD25+CTLA-4high T Regulatory Cells with Target (Allo)antigen Specificity J. Immunol., August 15, 2004; 173(4): 2866 - 2876. [Abstract] [Full Text] [PDF]

				L. Nagelkerken, I. Haspels, W. van Rijs, B. Blauw, J. L. Ferrant, D. M. Hess, E. A. Garber, F. R. Taylor, and L. C. Burkly FcR Interactions Do Not Play a Major Role in Inhibition of Experimental Autoimmune Encephalomyelitis by Anti-CD154 Monoclonal Antibodies J. Immunol., July 15, 2004; 173(2): 993 - 999. [Abstract] [Full Text] [PDF]

				M. Xu, A. J. Lepisto, and R. L. Hendricks CD154 Signaling Regulates the Th1 Response to Herpes Simplex Virus-1 and Inflammation in Infected Corneas J. Immunol., July 15, 2004; 173(2): 1232 - 1239. [Abstract] [Full Text] [PDF]

				D. Daoussis, A. P. Andonopoulos, and S.-N. C. Liossis Targeting CD40L: a Promising Therapeutic Approach Clin. Vaccine Immunol., July 1, 2004; 11(4): 635 - 641. [Full Text]

				P. G. Andres, K. C. Howland, D. Dresnek, S. Edmondson, A. K. Abbas, and M. F. Krummel CD28 Signals in the Immature Immunological Synapse J. Immunol., May 15, 2004; 172(10): 5880 - 5886. [Abstract] [Full Text] [PDF]

				K. Attanavanich and J. F. Kearney Marginal Zone, but Not Follicular B Cells, Are Potent Activators of Naive CD4 T Cells J. Immunol., January 15, 2004; 172(2): 803 - 811. [Abstract] [Full Text] [PDF]

				E. K. Deenick, A. V. Gett, and P. D. Hodgkin Stochastic Model of T Cell Proliferation: A Calculus Revealing IL-2 Regulation of Precursor Frequencies, Cell Cycle Time, and Survival J. Immunol., May 15, 2003; 170(10): 4963 - 4972. [Abstract] [Full Text] [PDF]

				C. Mueller and A. August Attenuation of Immunological Symptoms of Allergic Asthma in Mice Lacking the Tyrosine Kinase ITK J. Immunol., May 15, 2003; 170(10): 5056 - 5063. [Abstract] [Full Text] [PDF]

				U. M. Padigel and J. P. Farrell CD40-CD40 Ligand Costimulation Is Not Required for Initiation and Maintenance of a Th1-Type Response to Leishmania major Infection Infect. Immun., March 1, 2003; 71(3): 1389 - 1395. [Abstract] [Full Text] [PDF]

				W. J. Murphy, L. Welniak, T. Back, J. Hixon, J. Subleski, N. Seki, J. M. Wigginton, S. E. Wilson, B. R. Blazar, A. M. Malyguine, et al. Synergistic Anti-Tumor Responses After Administration of Agonistic Antibodies to CD40 and IL-2: Coordination of Dendritic and CD8+ Cell Responses J. Immunol., March 1, 2003; 170(5): 2727 - 2733. [Abstract] [Full Text] [PDF]

				U. Holzer, W. W. Kwok, G. T. Nepom, and J. H. Buckner Differential Antigen Sensitivity and Costimulatory Requirements in Human Th1 and Th2 Antigen-Specific CD4+ Cells with Similar TCR Avidity J. Immunol., February 1, 2003; 170(3): 1218 - 1223. [Abstract] [Full Text] [PDF]

				M. S. Vacchio and R. J. Hodes CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells J. Exp. Med., January 6, 2003; 197(1): 19 - 26. [Abstract] [Full Text] [PDF]

				J. E. Christensen, J. P. Christensen, N. N. Kristensen, N. J. V. Hansen, A. Stryhn, and A. R. Thomsen Role of CD28 co-stimulation in generation and maintenance of virus-specific T cells Int. Immunol., July 1, 2002; 14(7): 701 - 711. [Abstract] [Full Text] [PDF]

				G. Rajagopalan, M. K. Smart, E. V. Marietta, and C. S. David Staphylococcal enterotoxin B-induced activation and concomitant resistance to cell death in CD28-deficient HLA-DQ8 transgenic mice Int. Immunol., July 1, 2002; 14(7): 801 - 812. [Abstract] [Full Text] [PDF]

				A. L. Marzo, V. Vezys, K. Williams, D. F. Tough, and L. Lefrancois Tissue-Level Regulation of Th1 and Th2 Primary and Memory CD4 T Cells in Response to Listeria Infection J. Immunol., May 1, 2002; 168(9): 4504 - 4510. [Abstract] [Full Text] [PDF]

				S. Nierkens, P. van Helden, M. Bol, R. Bleumink, P. van Kooten, S. Ramdien-Murli, L. Boon, and R. Pieters Selective Requirement for CD40-CD154 in Drug-Induced Type 1 Versus Type 2 Responses to Trinitrophenyl-Ovalbumin J. Immunol., April 15, 2002; 168(8): 3747 - 3754. [Abstract] [Full Text] [PDF]

				C. M. Snyder, X. Zhang, and L. J. Wysocki Negligible Class II MHC Presentation of B Cell Receptor-Derived Peptides by High Density Resting B Cells J. Immunol., April 15, 2002; 168(8): 3865 - 3873. [Abstract] [Full Text] [PDF]

				H. L. Compton and J. P. Farrell CD28 Costimulation and Parasite Dose Combine to Influence the Susceptibility of BALB/c Mice to Infection with Leishmania major J. Immunol., February 1, 2002; 168(3): 1302 - 1308. [Abstract] [Full Text] [PDF]

				Y. Zaffran, O. Destaing, A. Roux, S. Ory, T. Nheu, P. Jurdic, C. Rabourdin-Combe, and A. L. Astier CD46/CD3 Costimulation Induces Morphological Changes of Human T Cells and Activation of Vav, Rac, and Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase J. Immunol., December 15, 2001; 167(12): 6780 - 6785. [Abstract] [Full Text] [PDF]

				M. Prlic, B. R. Blazar, A. Khoruts, T. Zell, and S. C. Jameson Homeostatic Expansion Occurs Independently of Costimulatory Signals J. Immunol., November 15, 2001; 167(10): 5664 - 5668. [Abstract] [Full Text] [PDF]

				M. Martin, D. J. Metzger, S. M. Michalek, T. D. Connell, and M. W. Russell Distinct Cytokine Regulation by Cholera Toxin and Type II Heat-Labile Toxins Involves Differential Regulation of CD40 Ligand on CD4+ T Cells Infect. Immun., July 1, 2001; 69(7): 4486 - 4492. [Abstract] [Full Text] [PDF]

				A. R. Hayward, M. Cosyns, M. Jones, and E. M. Ponnuraj Marrow-Derived CD40-Positive Cells Are Required for Mice To Clear Cryptosporidium parvum Infection Infect. Immun., March 1, 2001; 69(3): 1630 - 1634. [Abstract] [Full Text] [PDF]

				L. M. Howard and S. D. Miller Autoimmune Intervention by CD154 Blockade Prevents T Cell Retention and Effector Function in the Target Organ J. Immunol., February 1, 2001; 166(3): 1547 - 1553. [Abstract] [Full Text] [PDF]

				U. Grohmann, F. Fallarino, S. Silla, R. Bianchi, M. L. Belladonna, C. Vacca, A. Micheletti, M. C. Fioretti, and P. Puccetti CD40 Ligation Ablates the Tolerogenic Potential of Lymphoid Dendritic Cells J. Immunol., January 1, 2001; 166(1): 277 - 283. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Howland, K. C. Articles by Abbas, A. K. Search for Related Content PubMed PubMed Citation Articles by Howland, K. C. Articles by Abbas, A. K.