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The Journal of Immunology, 2007, 178: 671-682.
Copyright © 2007 by The American Association of Immunologists, Inc.

A Costimulatory Function for T Cell CD401

Melissa E. Munroe* and Gail A. Bishop2,*,{dagger},{ddagger}

* Department of Microbiology and {dagger} Department of Internal Medicine, University of Iowa, Iowa City, IA 52242; and {ddagger} Veterans Affairs Medical Center, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD40 plays a significant role in the pathogenesis of inflammation and autoimmunity. B cell CD40 directly activates cells, which can result in autoantibody production. T cells can also express CD40, with an increased frequency and amount of expression seen in CD4+ T lymphocytes of autoimmune mice, including T cells from mice with collagen-induced arthritis. However, the mechanisms of T cell CD40 function have not been clearly defined. To test the hypothesis that CD40 can serve as a costimulatory molecule on T lymphocytes, CD40+ T cells from collagen-induced arthritis mice were examined in parallel with mouse and human T cell lines transfected with CD40. CD40 served as effectively as CD28 in costimulating TCR-mediated activation, including induction of kinase and transcription factor activities and production of cytokines. An additional enhancement was seen when both CD40 and CD28 signals were combined with AgR stimulation. These findings reveal potent biologic functions for T cell CD40 and suggest an additional means for amplification of autoimmune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The 50-kDa membrane receptor of the TNFR superfamily, CD40, is expressed by APC, including dendritic cells, macrophages, and B lymphocytes (1, 2). The ligand for CD40, CD154, is expressed on activated T cells and allows for interactions with APC during the cognitive phase of the immune response, as well as directing effector T cell-dependent B cell activation (3). Such interactions have been directly implicated in autoimmunity. Blocking CD40-CD154 interactions has been shown to either prevent or alleviate such diseases as insulin dependent diabetes mellitus (4), arthritis (5), and systemic lupus erythematosus (6), the latter two benefiting from abrogated T cell-dependent autoantibody production (7, 8).

The role of CD40 as a direct signal receptor has now been expanded to T cells. Shortly after CD154 was cloned, it was demonstrated that CD154 can augment mitogen and TCR-mediated proliferation of CD4+ and CD8+ T cells (9), although the lack of CD154-specific Abs at that time precluded further investigation. Although a specific biologic role for CD40 on CD8+ T cells remains undefined (10, 11, 12, 13), it has been shown that autoimmune-prone strains of mice have increased numbers of CD40+CD4+ T cells compared with normal strains (14). The most extensively studied of these is the NOD mouse (15, 16, 17).

To date, the physiologic role(s) of CD40 on T cells has not been characterized, nor have the mechanisms by which CD40 affects T cell function been defined. We have previously studied CD40 as an important signaling molecule on B lymphocytes, delivering signals alone and synergistically with the BCR (18), leading to NF-{kappa}B and JNK pathway activation and subsequent proliferation, secretion of cytokines, Ig production and isotype switching (reviewed in Refs. 19 and 20). Like B cells, T cells have been shown to use TNFR family members as costimulatory molecules for Ag receptor stimulation (reviewed in Refs. 21 and 22). We hypothesized that costimulation is a plausible role for CD40 on T cells. In the studies presented here, we determined that T cell CD40 augmented CD3 and CD3 plus CD28-mediated cytokine production in T cells from mice which have developed collagen-induced arthritis (CIA),3 as well as in T cell lines stably transfected with CD40. Although CD40 signals alone did not activate NFAT or IL-2 secretion, CD40 ligation markedly augmented CD3 and CD3 plus CD28 responses. As in B cells, T cell CD40 was able to efficiently bind the adaptor proteins TNFR-associated factors (TRAF), activate NF-{kappa}B and AP-1 pathways, and stimulate TNF-{alpha} secretion. Taken together, these findings reveal that CD40 can act as a powerful signaling receptor on T as well as B lymphocytes, a function that may have important implications for T-B interactions in autoimmune diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

The mouse T cell line 2B4.11 (23) and human T cell line Jurkat (24) have been described previously. Cell lines and their stable transfectants expressing hCD40 were maintained in RPMI 1640 containing 10% FCS (HyClone), 10 µM 2-ME, and antibiotics. These subclones are referred to as 2B4.hCD40 and J.hCD40. Hi5 insect cells expressing hCD154 have been described and characterized previously (25, 26). These cells grow at 26°C and rapidly die to form membrane fragments at 37°C and therefore do not overgrow cell cultures.

Stable transfections

Cell lines were stably transfected with a previously reported hCD40 expression plasmid (27) as described previously (28). G418-resistant clones were analyzed for expression of hCD40 using a FACScan flow cytometer (BD Biosciences) and mean channel fluorescence (MCF) determined using FlowJo software.

Reagents

Recombinant mouse TNF-{alpha}, IL-2, and IFN-{gamma} were purchased from PeproTech. Streptavidin-HRP was purchased from Jackson ImmunoResearch Laboratories. ELISA TMB peroxidase substrate was purchased from KPL. Tosylactivated Dynabeads for Ab conjugation (per manufacturer’s instructions) were purchased from Dynal Biotech. PMA and ionomycin were purchased from Sigma-Aldrich.

Antibodies

The 1C10 (anti-mCD40, rat IgG2a), 72-2 (rat IgG2a isotype control), and G28-5 (anti-hCD40, mouse IgG1) hybridomas were purchased from the American Type Culture Collection. MOPC-31c (mouse IgG1 isotype control) was from Sigma-Aldrich. Polyclonal rabbit anti-TRAF2 Ab was from MBL. Polyclonal mouse anti-yy1 and polyclonal rabbit anti-TRAF3, anti-TRAF1, anti-TRAF6, and anti-hCD40 Abs were from Santa Cruz Biotechnology. Polyclonal rabbit anti-I{kappa}B{alpha}, anti-phosphorylated I{kappa}B{alpha}, anti-NF{kappa}B2 p100/p52, anti-JNK, and anti-phosphorylated JNK Abs were from Cell Signaling Technology. Mouse anti-actin Ab (C4) was from Chemicon International. Peroxidase-labeled goat anti-rabbit and goat anti-mouse IgG Abs were from Jackson ImmunoResearch Laboratories. Anti-mouse CD3{epsilon} (145-2C11; Armenian hamster IgG1), anti-human CD3 (OKT3; mouse IgG2a), anti-mouse CD28 (37.51; hamster IgG), anti-human CD28 (CD28.2; mouse IgG1), and relevant isotype control Abs were purchased from eBioscience. PE-labeled anti-mouse CD3 (145-2C11; Armenian hamster IgG1), FITC labeled anti-mouse CD40 (HM40-3; Armenian hamster IgM), anti-human CD40 (5C3; mouse IgG3), anti-human CD154 (TRAP1, mouse IgG1), anti-mouse CD80 (16-10A1; Armenian hamster IgG2), anti-mouse CD86 (GL1; rat IgG2a), anti-mouse CD95 (Jo2; Armenian hamster IgG2), and relevant isotype control Abs were purchased from BD Pharmingen. FITC labeled anti-mouse CD25 (PC61.5; rat IgG1), anti-mouse CD54 (YN1/1.7.4; rat IgG2b), and anti-mouse CD11{alpha} (M17/4; rat IgG2a) Abs were purchased from eBioscience. Biotin-labeled anti-mouse CD154 (MR1; Armenian hamster IgG) and relevant isotype control Abs were purchased from eBioscience. Alexa Fluor 488-labeled streptavidin was purchased from Molecular Probes/Invitrogen Life Technologies. Anti-mouse IL-2 and IFN-{gamma} (coating and biotinylated) ELISA Abs were purchased from Caltag Laboratories. Anti-human IL-2 and anti-mouse TNF-{alpha} (coating and biotinylated) ELISA Abs were purchased from eBioscience.

Mice/CIA induction

Female C57BL/6 mice were purchased at 5–8 wk of age from the National Cancer Institute. Mice were housed in a specific pathogen-free barrier facility with restricted access, and all procedures were performed as approved by the University of Iowa Animal Care and Use Committee. CIA was induced based on the methods of Campbell et al. (29). Briefly, mice were either left naive, immunized in the tail s.c. with 100 µg of type II chicken collagen (CII; Sigma-Aldrich) dissolved in 10 mM acetic acid and emulsified in IFA (Sigma-Aldrich) containing 5 mg/ml H37 RA heat-killed mycobacteria (CFA; Difco Laboratories), or immunized with 10 mM acetic acid emulsified in CFA. Mice were monitored for limb erythema and swelling and paws measured (each paw recorded individually; four measurements per mouse) with calipers two to three times per week (4026F; Mitutoyo) (29, 30). All mouse studies were reviewed and approved by the University of Iowa Animal Care and Use Committee.

T cell isolation

T cells were isolated from mouse spleens 70 days postimmunization. Briefly, spleens from euthanized mice were teased apart with forceps, erythrocytes lysed in ACK buffer, and remaining cells placed over a T cell enrichment column per manufacturer’s protocol (R&D Biosystems). T cells were enriched to ~90% purity as determined by flow cytometry (see Table I). Primary mouse T cells were cultured in Click’s medium containing 1% nutridoma-SP (Roche), 10 µM 2-ME, and antibiotics, or stained for CD3{epsilon} (PE) and CD40 (FITC) and analyzed by flow cytometry.


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  		Table I. Increased number of CD40+ T cells in CIA micea

 
NF-{kappa}B/NFAT/AP-1 dual luciferase reporter assays

2B4.hCD40 or J.hCD40 cells (1.5 x 107) were transiently transfected with 20 µg of 4x NF-{kappa}B, 40 µg of 4x NFAT, or 40 µg of 7x AP-1 luciferase reporter plasmid (31), and 1 µg of Renilla luciferase vector (pRL-null; Promega) by electroporation. Cells were rested on ice for 15 min, then stimulated (2 x 106 cells/ml) for 6 h (NF-{kappa}B) or 24 h (NFAT/AP-1) with 10 µg/ml anti-hCD40 or isotype controls, and/or 5 x 105 beads/ml anti-CD3 or anti-CD3+CD28-coated Dynabeads. After stimulation, cells were pelleted, lysed, and assayed for relative luciferase activity (NF-{kappa}B, NFAT, or AP-1:Renilla) per manufacturer’s protocol (Promega) using a Turner Designs 20/20 luminometer, with settings of a 2-s delay followed by a 10-s read.

I{kappa}B{alpha}/JNK assays

2B4.hCD40 or J.hCD40 cells (2 x 106) were stimulated for indicated times with culture medium, 10 µg of anti-hCD40 Ab (or respective isotype controls) and/or 5 x 105 beads/ml anti-CD3 or anti-CD3 plus CD28-coated Dynabeads) to induce phosphorylation and degradation of the proteins blotted. The cells were pelleted by centrifugation, lysed and analyzed by SDS PAGE and Western blotting. Peroxidase-labeled Abs were visualized on Western blots using a chemiluminescent detection reagent (Pierce).

NF-{kappa}B2 activation

2B4.hCD40 or J.hCD40 cells (2 x 106) were stimulated for indicated times with culture medium, 10 µg of anti-hCD40 Ab (or respective isotype controls) and/or 5 x 105 beads/ml anti-CD3 or anti-CD3 plus CD28-coated Dynabeads) to induce RelB activation, and processing of p100 to p52. The cells were pelleted by centrifugation and cytoplasmic and nuclear fractions isolated as described previously (32). Samples were analyzed by SDS-PAGE and Western blotting. Peroxidase-labeled Abs were visualized on Western blots using a chemiluminescent detection reagent.

Cytokine ELISA

Primary mouse T cells or 2B4.hCD40 or J.hCD40 cells (4 x 105) were stimulated at optimal, empirically derived time points with culture medium, 1 µg/ml anti-hCD40 Ab and/or anti-CD28, or plate-bound anti-CD3 (or respective isotype controls). Cytokine concentrations in culture supernatants were determined by ELISA, using cytokine-specific coating Abs and biotinylated detection Abs. Streptavidin-HRP binding to biotinylated detection Abs was visualized with TMB substrate and the reaction was stopped with 0.18 M H2SO4. Plates were read at 450 nm by a SpectraMax250 Reader (Molecular Devices). Data were analyzed with SoftMax Pro software (Molecular Devices); unknowns were compared with a standard curve containing at least five to seven dilution points of the relevant recombinant cytokine on each assay plate. In all cases, the coefficient of determination for the standard curve (r2) was >0.98. ELISA unknowns were diluted to fall within the standard values.

TRAF recruitment to receptors in detergent-insoluble microdomains (Rafts) and immunoprecipitation.

2B4.hCD40 or J.hCD40 (1 x 107) cells were stimulated with 10 µg of anti-hCD40 Ab (or isotype control Abs) or Hi5 cells expressing hCD154 (or Hi5 cells expressing WT baculovirus; 1:4 Hi5 cells:lymphocytes) for 15 min at 37°C to induce recruitment of TRAFs to membrane rafts and allow formation of CD40 signaling complexes, as described previously (33). Detergent (1% Brij 58)-soluble and insoluble fractions were separated as described previously (34). Samples of soluble and insoluble lysates were reserved for SDS-PAGE separation and analysis by Western blotting. The remainder of the lysates were immunoprecipitated with protein G-Sepharose beads (Amersham Biosciences) prewarmed with anti-hCD40 Ab for 3 h at 4°C. The immunoprecipitation complexes were washed four times with lysis buffer before separation by SDS-PAGE and analysis by Western blot.

Up-regulation of cell surface proteins

In experiments evaluating activation-induced up-regulation of surface proteins, 2B4.11 or 2B4.hCD40 cells were incubated with the indicated stimuli in 96-well plates (1–2 x 105 cells/well). After 48–72 h, the cells were washed, then incubated for 20 min on ice in PBS-0.5% FCS-0.02% sodium azide containing 2.5 mM EDTA. EDTA treatment helped to dissociate cell aggregates formed upon CD40 stimulation. Following the EDTA incubations, cells were washed and stained (in the absence of EDTA) with Abs for analysis by flow cytometry.

Statistical analyses

Analyses were performed with GraphPad Instat software. A two-tailed paired Student’s t test was used to determine significance between groups in CIA experiments, for cytokine ELISA, surface molecule up-regulation experiments, and luciferase reporter assays.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD40 expression on T cells from mice with CIA

CIA is a frequently used mouse model of inflammatory rheumatoid arthritis that is both Ab and T cell dependent (35, 36). It has been previously demonstrated that mouse strains prone to autoimmune diabetes have an increased number of T cells expressing CD40 that correlates with development of pathology (14). We examined whether this was also true during the inflammatory process of CIA development. C57BL/6 mice injected with CII/CFA developed significant paw swelling (p < 0.001) compared with mice given CFA only or naive controls (Fig. 1A). Splenic T cells from these mice were isolated and evaluated for dual expression of CD3{epsilon} and CD40. A representative FACS plot of isolated T cells is presented in Fig. 1B; quantitation is presented in Table I. Small numbers of cells in the FACS samples that were CD3{epsilon}– were also CD40– and therefore unlikely to be APC (Fig. 1B and Table I, line 5). Data in Table I demonstrate that there are more than twice as many CD40+ T cells in the spleens of mice that received CII/CFA compared with mice that received CFA only or naive controls (p ≤ 0.01 for percentage, line 3, or absolute number, line 4). This is true whether evaluating the percentage or absolute numbers of CD3+CD40+ cells as a part of the whole spleen (Table I, lines 3 and 4) or after enrichment of T lymphocytes (Table I, lines 7 and 8). This phenomenon was not due to an alteration in the splenic T cell population as a whole, or post-T cell isolation after CII/CFA immunization. There was no significant difference between immunization groups in the percentage (first line) or absolute numbers (second line) of CD3+ T cells, whether evaluating mixed splenocyte, or isolated T cell populations (Table I).


Figure 1
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  		FIGURE 1. CD40 expressed on T cells from mice with CIA. A, C57BL/6 mice were immunized with CFA only, CII plus CFA, or remained naive, as described in Materials and Methods. Paws were measured two to three times per week from days 21–70 postimmunization. Mice receiving CII plus CFA had significant (p < 0.001) paw swelling compared with CFA or naive controls. Data represent two experiments (4 mice/group/experiment; 8 mice/32 paws/data point total). B, Spleens from mice in A were pooled (2 spleens/pool, 4 pools/group), and T cells were isolated as described in Materials and Methods. Cells were stained with anti-mouse CD40 (FITC) and anti-mouse CD3{epsilon} (PE) or isotype control Abs and analyzed by flow cytometry. Quadrants were drawn based on staining with isotype control Abs.

 
T cell CD40 as a costimulatory molecule

The findings discussed above raise the possibility that CD40 expressed by T cells may play a role in T cell activation. Experiments presented in Fig. 2 explored whether CD40 engagement could augment CD3 or CD3 plus CD28-mediated cytokine production by splenic T cells isolated from mice immunized with CII/CFA. Because minimal cytokine production was detected from cells stimulated with medium alone, isotype control Abs, or anti-CD3 Ab, it is unlikely that the small amounts of residual non-T cells found after T cell enrichment (Fig. 1 and Table I) are APC. As expected, anti-CD3 Ab induced a modest amount of IL-2 (Fig. 2A), and anti-CD28 Ab significantly enhanced CD3-mediated IL-2 production in all three experimental groups (CD3 vs CD3 plus CD28: naive, p = 0.02; CFA only, p = 0.01; CII/CFA, p < 0.0001). CD40 stimulation alone did not induce any appreciable IL-2 production. However, in T cells from mice immunized with CII/CFA, CD40 significantly enhanced the level of CD3 (p = 0.002)- and CD3 plus CD28 (p = 0.009)-mediated IL-2 production. This enhancement did not occur in the largely CD40-negative T cells from naive or CFA-treated mice because there was no significant difference in IL-2 produced between T cells treated with agonists for CD3 vs CD3 plus CD40 or CD3 plus CD28 vs CD3 plus CD28 plus CD40.


Figure 2
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  		FIGURE 2. CD40 enhances CD3 and CD3 plus CD28-mediated cytokine production in T cells from mice with CIA. Splenic T cells from C57BL/6 mice immunized with CII plus CFA, CFA only, or remaining naive were isolated as described in Materials and Methods. Cells were stimulated with plate-bound anti-CD3 ± anti-CD28 and/or anti-mouse CD40 Abs (compared with medium (Med)/isotype control (IC)). Culture supernatants were collected and assayed for IL-2 (A, 48 h), IFN-{gamma} (B, 72 h), or TNF-{alpha} (C, 24 h) by ELISA. Data represent the mean ± SEM of duplicate samples of three independent experiments. There was no difference between medium and isotype control samples.

 
In addition to IL-2 production, we also evaluated the ability of CD40 to contribute to the production of the proinflammatory cytokines IFN-{gamma} (Fig. 2B) and TNF-{alpha} (Fig. 2C) by T cells from CIA mice compared with controls. As with IL-2 secretion, CD28 enhanced CD3-mediated IFN-{gamma} (Fig. 2B, p ≤ 0.001 for all groups) and TNF-{alpha} (Fig. 2C, p ≤ 0.001 for all groups) production in T cells from all three mouse groups. Unlike IL-2, anti-CD40 alone induced a significant amount of both IFN-{gamma} (Fig. 2B, p = 0.001) and TNF-{alpha} (Fig. 2C, p < 0.0001), but only in T cell cultures from mice immunized with CII/CFA and not in cultures from control mice. CD40 significantly enhanced CD3 and CD3 plus CD28-mediated IFN-{gamma} (Fig. 2B, p ≤ 0.02 for both stimuli) and TNF-{alpha} (p ≤ 0.01 for both stimuli) production, but only in mice immunized with CII/CFA. These data indicate that CD40 can act as a TCR costimulator and that it can cooperate in a nonredundant manner with CD28 to further enhance T cell cytokine production.

The above ex vivo experiments contained a mixed population of CD40-expressing and nonexpressing T cells (Fig. 2), with a relatively small percentage of T cells expressing CD40 (Fig. 1 and Table I). This small percentage limited detailed molecular characterization of CD40 function on CD40-expressing T cells, although the data presented in Fig. 2 indicate that this population has significant biologic activity distinct from that of CD4+ T cells that do not express CD40. We thus wanted to complement these experiments with stimulation of homogeneous populations of CD40+ T cells, as well as explore CD40 signaling pathways. Because of limiting numbers of CD40+ T cells in CIA mice that would require potentially function-altering positive selection for isolation, CD40+ T cell lines were a desirable alternative. We thus stably transfected mouse 2B4.11 (2B4.hCD40) and human Jurkat (J.hCD40) T cell lines with hCD40 (Fig. 3, A and B; MCF for 2B4.hCD40 = 704.66 vs 147.48 for 2B4.11; MCF for J.hCD40 = 245.72 vs 199.11 for Jurkat) and evaluated the ability of CD40 to activate IL-2 production (Fig. 4, A and B). Interestingly, even with PMA/ionomycin stimulation, there was no detectable CD154 expression by either 2B4.hCD40 or J.hCD40 cells (Fig. 3, C and D), although we see CD154 expression by Hi5 insect cells infected with a baculovirus encoded to express CD154 (data not shown). Because these clones do not express CD154, autocrine CD40 stimulation does not contribute to subsequent findings.


Figure 3
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  		FIGURE 3. Expression of hCD40 and CD154 on 2B4.11 and Jurkat cell lines. A and B, 2B4.11 (A) or Jurkat (B) cells were transfected with DNA encoding for hCD40. Cells were stained with anti-human CD40 and analyzed by flow cytometry. Gray profiles represent anti-hCD40 mAb staining of untransfected cells; black profiles represent staining of transfected cells. There was no significant staining of cells with an isotype control Ab. C and D, 2B4.hCD40 (C) or J.hCD40 (D) cells remained untreated or were treated for 6 h with 1 µg/ml PMA plus 200 µM ionomycin, then stained with anti-CD154 and analyzed by flow cytometry. Dashed profiles represent staining of untreated cells with isotype control mAb; gray profiles represent anti-CD154 staining of untreated cells; black profiles represent anti-CD154 staining of PMA-ionomycin-treated cells. There was no significant staining of PMA-ionomycin-treated cells with an isotype control Ab. Similar results were seen at 24 and 48 h (data not shown).

 

Figure 4
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  		FIGURE 4. CD40 enhances CD3 and CD3 plus CD28-mediated cytokine production in 2B4.hCD40 and J.hCD40 lines. 2B4.11 ± 2B4.hCD40 (A, C, and D) or Jurkat ± J.hCD40 (B) cells were stimulated with plate-bound anti-CD3 ± anti-CD28 and/or anti-CD40 Abs (compared with medium (Med)/isotype control (IC)). Culture supernatants were collected after 24 (TNF-{alpha}), 48 (IL-2), or 72 (IFN-{gamma}) h and assayed for IL-2 (A and B), TNF-{alpha} (C), or IFN-{gamma} (D) by ELISA. Data represent the mean ± SEM of triplicate samples from two independent experiments. There was no difference between medium and isotype control samples.

 
We first evaluated the ability of the transfected 2B4.hCD40 and J.hCD40 to secrete cytokines in response to CD3 ± CD28 and/or CD40 stimulation (Fig. 4). To closely mimic the design of our mouse ex vivo experiments, in which biologic responses to CD40 were made by small percentages of CD40+ T cells, CD40 transfected T cells were cocultured with their untransfected parent cell lines at various ratios ranging from 6 to 100% transfected cells. CD40 stimulation alone of cells stably expressing the transfected receptor did not induce IL-2 production (Fig. 4, A and B). While CD28 ligation appropriately enhanced CD3-mediated IL-2 production in all T cell clones, CD40 stimulation enhanced IL-2 production via CD3 or CD3 plus CD28 only if cells stably expressing CD40 were present in the culture well, similar to responses of primary T cell cultures from CIA mice (Fig. 2B). CD40 was able to act in costimulatory fashion with CD3 with only 6% of CD40+ cells in the coculture (p = 0.01 when 6% 2B4.hCD40 present, p < 0.0001 at 100% 2B4.hCD40; p < 0.001 when 6% J.hCD40 present, p < 0.0001 at 100% J.hCD40), just as was seen in primary T cells from CIA mice (Fig. 2B). Enhancement of CD3 plus CD28-mediated IL-2 production by CD40 increased with greater numbers of transfected cells in the well (p = 0.02 at 100% 2B4.hCD40; p = 0.005 when 6% J.hCD40 present, p < 0.0001 at 100% J.hCD40). Of particular interest is that CD40 could ultimately enhance CD3-mediated IL-2 production to a degree similar to that of CD28 in 2B4.hCD40 cells (Fig. 4A) and significantly better in J.hCD40 cells (Fig. 4B, p < 0.0001 at 100% J.hCD40). Thus, the response of both 2B4.hCD40 and J.hCD40, even when only 6% of the T cells in each culture were CD40+ cells, closely paralleled the responses seen in freshly isolated T cells, validating these cell lines as useful experimental models for the study of CD40 function in T cells.

In addition to IL-2 production, we evaluated the ability of CD40 to contribute to the production of proinflammatory cytokines TNF-{alpha} (Fig. 4C) and IFN-{gamma} (Fig. 4D). These cytokines were readily detected in culture supernatants of stimulated 2B4.11 or 2B4.hCD40 cells, but not Jurkat cells, which have been propagated as an IL-2-producing human leukemia T cell line (24) and which require overexpression of other proteins to induce TNF-{alpha} (37) and IFN-{gamma} (38) secretion. Unlike IL-2, CD40 stimulation alone was able to stimulate both production of TNF-{alpha} (p = 0.02 when 6% 2B4.hCD40 cells were present, p = 0.001 at 100%) and IFN-{gamma} (p = 0.02 when 6% 2B4.hCD40 cells were present, p < 0.0001 at 100%) in those cells stably expressing CD40. Similar to IL-2, CD40 stimulation of T cells expressing CD40 was able to augment both CD3 and CD3 plus CD28-mediated cytokine production.

The role of T cell CD40 in up-regulation of cell surface molecules

CD40 signaling is known to up-regulate a number of cell surface molecules (27), including CD11{alpha} (LFA-1), CD54 (ICAM-1), and CD95 (Fas), all of which play significant roles in T cell activation (39, 40, 41). We also evaluated the ability of CD40 to contribute to CD80 (B7-1) and CD86 (B7-2) up-regulation, an important component of activation by other CD40-expressing cells (42), as well as CD25, a marker of T cell activation (43). We compared baseline expression of these cell surface molecules on 2B4.11 and 2B4.hCD40 cells, as well as receptor-specific up-regulation of these molecules postactivation (Fig. 5). Both 2B4.11 and 2B4.hCD40 expressed basal CD80 (Fig. 5C), CD86 (Fig. 5D), and CD11{alpha} (LFA-1; Fig. 5E), with higher expression in 2B4.hCD40 cells. CD3 plus CD28 stimulation induced up-regulation of all surface molecule tested in both 2B4.11 and 2B4.hCD40 cells (right graph in each panel, p ≤ 0.01 for all groups). CD40 signals alone, and in conjunction with CD3 or CD3 plus CD28 stimulation, induced up-regulation of all surface molecules tested in 2B4.hCD40 cells, but not the nontransfected 2B4.11 cell line. CD40 signals augmented CD3-mediated up-regulation of cell surface molecules similar to CD28 stimulation in 2B4.hCD40 cells (p < 0.01 CD3 vs CD3 plus CD28 or CD3 plus CD40 for all groups, no significant difference in response between CD3 plus CD28 and CD3 plus CD40 stimulation), with a maximal response achieved when triple CD3 plus CD28 plus CD40 stimulation was given. Interestingly, CD40 signaling induced a maximal enhancement of the costimulatory response in 2B4.hCD40 cells in conjunction with CD3 plus CD28 to up-regulate CD25 expression (Fig. 5A).


Figure 5
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  		FIGURE 5. CD40 enhances CD3 and CD3 plus CD28-mediated cell surface molecule up-regulation. 2B4.11 or 2B4.hCD40 cells were stimulated with plate-bound anti-CD3 ± anti-CD28 and/or anti-CD40 Abs (compared with isotype control) for 48–72 h, then analyzed by flow cytometry to determine expression levels of surface proteins. Histograms (gray = isotype control; black = surface molecule) represent baseline expression (stimulated with isotype control Ab), with corresponding median channel fluorescence ({Delta}MCF = MCF of surface molecule – MCF of isotype control staining) represented in the left graph of each panel. Changes in MCF for each surface molecule tested (poststimulation minus baseline) is represented in the right graph of each panel. A–F, Data represent the mean ± SEM of MCF from two independent experiments.

 
The role of T cell CD40 in activation of cytokine signaling pathways

Experiments presented above demonstrate that CD40 can act as a costimulatory molecule to enhance CD3 and CD3 plus CD28-mediated T cell activation. NFAT, AP-1, and NF-{kappa}B are known to be involved in the activation of several T cell proinflammatory cytokine genes, including those encoding IL-2 (44, 45, 46), IFN-{gamma} (47, 48), and TNF-{alpha} (45, 49). We and others have demonstrated that CD40-mediated activation of B lymphocytes involves AP-1 (50, 51) and NF-{kappa}B (31, 52) signaling pathways, and there is evidence of CD40-mediated NFAT activation (51, 53). We asked if this is also the case for T cell CD40.

CD3 or CD40 signals alone induced minimal NFAT reporter gene activation compared with control stimuli in 2B4.hCD40 cells (Fig. 6A), while CD28 in conjunction with CD3 signals induced an increased response (p = 0.001) compared with medium/isotype controls or CD3 single stimulation in 2B4.hCD40 cells (Fig. 6A; p < 0.0001 compared with medium/isotype controls or CD3 in J.hCD40 cells, Fig. 6B). While CD40 signaling was able to enhance CD3-mediated NFAT activation (p < 0.001 in both 2B4.hCD40 and J.hCD40 cells), it was not to the same degree as CD28-mediated enhancement. The greatest NFAT response was achieved via engagement of all three receptors: CD3 plus CD28 plus CD40 (p = 0.007 compared with CD3 plus CD28, p < 0.001 compared with CD3 plus CD40 in 2B4.hCD40 cells, p = 0.017 compared with CD3 plus CD28, p = 0.003 compared with CD3 plus CD40 in J.hCD40 cells). Although CD3 signals alone were able to trigger increased NFAT activity in J.hCD40 cells (p = 0.001; Fig. 6B), CD28 and CD40 signals augmented this response in a manner similar to their effects in 2B4.hCD40 cells.


Figure 6
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  		FIGURE 6. CD40 enhances CD3 and CD3 plus CD28-mediated NFAT activation. 2B4.hCD40 (A) or J.hCD40 (B) cells were transiently transfected with 4x NFAT-luciferase and Renilla-luciferase reporter plasmids, rested on ice for 30 min, then stimulated for 24 h. Cells were stimulated with anti-hCD40 or isotype control (IC) Abs or Dynal beads armed with anti-CD3 or anti-CD3 plus anti-CD28 Abs. Relative luciferase activity (NFAT:Renilla) of stimulus vs control was calculated as the mean ± SEM of duplicate samples from three independent experiments. There was no difference between medium and isotype control samples.

 
We next investigated the ability of CD40 to signal via AP-1 in T cells (Fig. 6). Unlike NFAT (Fig. 6), CD40 signals alone activated AP-1 ~2-fold over control stimuli in 2B4.hCD40 cells (Fig. 7A; p = 0.005). While CD40 signals enhanced CD3-mediated AP-1 activation in both cell lines (p < 0.001 in 2B4.hCD40 cells; p = 0.009 in J.hCD40 cells), CD40 was a less efficient costimulator in this response than CD28 (CD3 vs CD3 plus CD28: p = 0.007 in 2B4.hCD40 cells; p = 0.005 in J.hCD40), and the maximal response was again achieved with simultaneous engagement of CD3 plus CD28 plus CD40 (p = 0.02 compared with CD3 plus CD28, p = 0.03 compared with CD3 plus CD40 in 2B4.hCD40 cells, p = 0.01 compared with CD3 plus CD28, p = 0.002 compared with CD3 plus CD40 in J.hCD40 cells). CD40 signals in B cells strongly activate phosphorylation of the AP-1 family member c-jun, via activation of JNK (54, 55, 56). We evaluated the ability of T cell CD40 to contribute to JNK activation alone or in combination with CD3 or CD3 plus CD28. Strikingly, in both 2B4.hCD40 and J.hCD40 cells (Fig. 8, A and B), only when CD40 was engaged was strong phosphorylation of JNK observed, with no phosphorylation seen via CD3 signaling and minimal phosphorylation via CD3 plus CD28 signaling.


Figure 7
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  		FIGURE 7. CD40 activates and enhances CD3 and CD3 plus CD28-mediated AP-1 activation. 2B4.hCD40 (A) or J.hCD40 (B) cells were transiently transfected with 7x AP-1-luciferase and Renilla-luciferase reporter plasmids, rested on ice for 30 min, then stimulated for 24 h. Cells were stimulated with anti-hCD40 or isotype control (IC) Abs or Dynal beads armed with anti-CD3 or anti-CD3/anti-CD28 Abs. Relative luciferase activity (AP-1:Renilla) of stimulus vs control cell groups was calculated as the mean ± SEM of duplicate samples from two independent experiments. There was no difference between medium and isotype control samples.

 

Figure 8
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  		FIGURE 8. CD40 activates and enhances CD3 and CD3 plus CD28-mediated JNK phosphorylation. 2B4.hCD40 (A) or J.hCD40 (B) cells were rested at 37°C for 30 min, then stimulated for 5–60 min with anti-hCD40 or isotype control (IC) Abs or Dynal beads armed with anti-CD3 or anti-CD3 plus anti-CD28 Abs. Cells were pelleted, lysed, and lysates analyzed by SDS-PAGE and Western blot for phosphorylated (P.) and total (T.) JNK. Similar results were obtained in two independent experiments.

 
It is well established that CD40 signals lead to NF-{kappa}B activation in B cells (52, 56, 57, 58) and we asked if this was also true for T cell CD40. CD40 alone initiated a strong NF-{kappa}B response in 2B4.hCD40 (Fig. 9A, p < 0.001) and J.hCD40 (Fig. 9B, p < 0.001) cells, while CD3 evoked a minimal response compared with controls. CD3 plus CD40 activated NF-{kappa}B more than CD40 alone (CD40 vs CD3 plus CD40: p = 0.03 in 2B4.hCD40 cells, p = 0.02 in J.hCD40 cells) and 2.5- to 5-fold greater than CD3 plus CD28 (CD3 plus CD28 vs CD3 plus CD40: p = 0.005 in 2B4.hCD40 cells; p = 0.004 in J.hCD40 cells), while the combination of CD3 plus CD28 plus CD40 signals provided an additional 20% increase in NF-{kappa}B activation (p < 0.001 compared with CD3 plus CD28, p = 0.03 compared with CD3 plus CD40 in 2B4.hCD40 cells; p = 0.01 compared with CD3 plus CD28, p = 0.04 compared with CD3 plus CD40 in J.hCD40 cells).


Figure 9
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  		FIGURE 9. CD40 activates and enhances CD3 and CD3 plus CD28-mediated NF-{kappa}B activation. 2B4.hCD40 (A) or J.hCD40 (B) cells were transiently transfected with 4x NF-{kappa}B-luciferase and Renilla-luciferase reporter plasmids, rested for 15 min on ice, then stimulated for 6 h. Cells were stimulated with anti-hCD40 or isotype control (IC) Abs or Dynal beads armed with anti-CD3 or anti-CD3 plus anti-CD28 Abs. Relative luciferase activity (NF-{kappa}B:Renilla) of stimulus vs control was calculated as the mean ± SEM of duplicate samples from two independent experiments. There was no difference between medium and isotype control samples.

 
Early events in NF-{kappa}B activation have been shown to include phosphorylation and degradation of I{kappa}B{alpha} (59), and recent studies suggest that an alternate pathway for activating NF-{kappa}B (NF-{kappa}B2), in which p100 is processed to p52 and shuttled to the nucleus by RelB, is also used by some TNFR family members, including CD40 (56, 58). To test which NF-{kappa}B activation pathways are used by CD40 in T cells, we assayed for I{kappa}B{alpha} phosphorylation and degradation (NF-{kappa}B1; Fig. 10) or processing of p100 to p52 with nuclear translocation of p52 and RelB (NF-{kappa}B2; Fig. 11). Using densitometry to normalize I{kappa}B{alpha} values to the loading control actin (Fig. 10B), CD40 engagement alone stimulated phosphorylation and degradation of I{kappa}B{alpha} with up to 100% greater efficiency than CD3 or CD3 plus CD28, with an even greater increase when CD40 signals were combined with CD3 or CD3 plus CD28. Similar results were seen in J.hCD40 cells (data not shown). With respect to the NF-{kappa}B2 pathway, in both 2B4.hCD40 (Fig. 11) and J.hCD40 (data not shown), CD40 stimulation alone, but not CD3, resulted in processing of p100 to p52 (Fig. 11, A and B) and translocation of p52 and RelB to the nucleus (Fig. 11, C and D). Using densitometry to normalize p100, p52, and RelB values to the loading control actin (Fig. 11B, cytoplasmic fraction) or yy1 (Fig. 11D, nuclear fraction), we observed that CD40 and CD28 augmented CD3-mediated activation of NF-{kappa}B2 to a similar degree, whereas the combination of CD3 plus CD28 plus CD40 gave maximal stimulation.


Figure 10
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  		FIGURE 10. CD40 activates and enhances I{kappa}B{alpha} phosphorylation/degradation. A, 2B4.hCD40 cells were stimulated with anti-hCD40 or isotype control (IC) Abs or Dynal beads armed with anti-CD3 or anti-CD3 plus anti-CD28 Abs. Cells were pelleted, lysed, and lysates analyzed by SDS-PAGE and Western blot for phosphorylated and total I{kappa}B{alpha}. B, Density of bands as a proportion of cells treated with medium (med) alone and normalized to the value of the actin bands shown in B. Similar results were obtained in two independent experiments.

 

Figure 11
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  		FIGURE 11. CD40 activates and enhances CD3 and CD3 plus CD28-mediated nuclear translocation of p52 and RelB in the NF-{kappa}B2 pathway. 2B4.hCD40 (A–D) cells were stimulated with anti-hCD40 or isotype control (IC) Abs or Dynal beads armed with anti-CD3 or anti-CD3/anti-CD28 Abs. Cells were pelleted, cytoplasmic and nuclear fractions isolated, and lysates analyzed by SDS-PAGE and Western blot for p100/p52 and RelB. Blots were stripped and reblotted for actin (cytoplasmic fractions; A and B) or yy1 (nuclear fractions; C and D) as loading controls. B, Density of bands as a proportion of cells treated with medium (med) alone and normalized to the value of the actin bands shown in A. D, Density of bands as a proportion of cells treated with medium (med) alone, normalized to the value of the yy1 bands shown in C. Similar results were obtained in two independent experiments.

 
TRAF association of T cell CD40

CD40, like other members of the TNFR superfamily, relies on the association of adaptor molecules, TRAFs, for downstream signaling events, including activation of kinases and transcription factors, production of cytokines, up-regulation of surface molecules, and various aspects of the humoral response (20). However, the characteristics of TRAF association with CD40 have been shown to differ between B cells, macrophages, dendritic cells, and epithelial cells (19). It was thus important to determine CD40-TRAF associations in T cells. In mouse B cells, we have previously demonstrated that TRAFs 1, 2, 3, and 6 associate with either endogenous mouse CD40 or transfected hCD40, following receptor ligation (19, 60, 61, 62). We therefore compared the ability of TRAFs to move into membrane lipid rafts (Fig. 12, A and B, left panel) and associate with hCD40 (Fig. 12, A and B, right panel) in Brij soluble (cytoplasmic) and insoluble (lipid raft) fractions in T cells. TRAF2 and TRAF3 moved efficiently into the lipid raft fraction (left panel) and associated with CD40 (right panel) in both 2B4.hCD40 (Fig. 12A) and J.hCD40 (Fig. 12B) cells, similar to their recruitment in B cells (61). This was true if hCD40 was engaged by agonistic Abs or by CD154, although more efficient movement (left panel) and receptor association (right panel) of TRAF1 and TRAF6 occurred when hCD154 was the stimulus, as previously reported (63, 64).


Figure 12
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  		FIGURE 12. CD40-mediated TRAF recruitment. 2B4.hCD40 (A) or J.hCD40 (B) cells were stimulated for 15 min with anti-hCD40 Ab (vs isotype control Ab; IC) or Hi5 insect cells expressing hCD154 (vs WT baculovirus; WT). Detergent soluble (S) and insoluble (I) lysates were prepared as described in Materials and Methods. Seventy percent of the lysates were immunoprecipitated with protein G-Sepharose armed with anti-hCD40. Lysates and immunoprecipitates were separated by SDS-PAGE and analyzed by Western blot. Similar results were obtained in two identical experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
All B cells express CD40, and its functions on B cells have been the subject of much study. Much less well appreciated is that activated T cells can also express CD40, and the roles and mechanisms of action of T cell-expressed CD40 still represent a significant knowledge gap. Various TNFR family members have been shown to act as coregulatory molecules on T cells, some possibly contributing to inflammatory disease (65). This role has been previously established for CD40 on B cells (66), and we demonstrate here that this is also true for T cell CD40. We see a reproducible increase in CD40 expression on T cells from mice with the chronic inflammatory disorder CIA (Fig. 1 and Table I). Although the increase in CD40+ cells in CIA vs control mice is modest compared with genetically predisposed autoimmune-prone strains of mice (14, 17), it is consistent with the FACS profile of T cells from normal mice (9, 17, 67). Strains of mice that are prone to spontaneous autoimmune disease have robust expression of CD40 by T cells, whereas nondisease prone strains do not (14), so it is not surprising that C57BL/6 mice do not have as robust proportions of CD40+ T cells as strains that develop autoimmune diseases relatively early in life. A normal mouse strain can induce functional CD40 on T cells when encountering an Ag/danger signal (67), but this level still does not reach that of mouse strains prone to spontaneous autoimmune disease, which have likely been receiving chronic stimulation since early in life. However, findings presented here show that T cell CD40 can contribute to enhanced T cell activation even in individuals who do not have strong genetic predisposition to autoimmunity.

Importantly, this CD40 is capable of T cell stimulation, indicating that it may have significant functional consequences (9, 14, 15, 67). Initially, this was a surprising finding. However, the Ag-specific precursor frequency for naive T cells is ~1/1 x 105 (100 cells/spleen) (68, 69), a small population capable of significantly expanding and producing a sufficient protective adaptive immune response upon antigenic stimulation. This response ultimately leads to the survival of ~5% of the activated T cells (~1 x 105 cells) to serve as a memory pool after the contraction phase (69). Interestingly, in the CIA model, at 70 days postimmunization, we can isolate a population of 3–4 x 105 CD40+ T cells per spleen (Table I) that are capable of CD40-mediated activation of cytokine production (Fig. 2), despite only representing ~7% of the isolated T cell population (Table I). This finding was recapitulated in experiments whereby CD40+ T cells were mixed with CD40– T cells in a controlled fashion and a significant CD40-mediated response was seen with as few as 6% CD40+ T cells in culture (Fig. 4).

Like other costimulatory TNFR family members expressed on T cells (70), while CD40 itself cannot induce IL-2 production, it augments the CD3 response and gives maximal stimulation together with CD3 plus CD28 signals (Figs. 2 and 4). This is true in both T cells from CIA mice (Fig. 2) and in T cell lines expressing CD40 (Fig. 4, A and B), even when transfected CD40+ cells are mixed with CD40– (untransfected) T cells to give a similar small percentage of CD40+ cells as that observed in CIA mice. Importantly, anti-CD40 stimulation does not yield a positive cytokine response in T cells lacking CD40, although their response to CD3 plus CD28 stimulation is similar to that of CD40+ T cells (Figs. 2 and 4). Activation of the transcription factor NFAT is critical to IL-2 production (44). Also consistent with a role as a costimulator, CD40 itself cannot induce NFAT activation (Fig. 6), but can augment these responses to CD3 and CD3 plus CD28 ligation. It is likely that the TCR complex provides calcium-mediated signaling that is necessary, but not sufficient, for T cell activation and IL-2 production (71), while CD40 provides costimulatory signaling via NF-{kappa}B and AP-1, necessary to activate NFAT and subsequent IL-2 production (44, 72, 73).

Both CD40 (74) and CD28 (75) contribute to cell activation via association with lipid rafts. CD40 signals use TRAF adaptor molecules in B cells (61, 64), and we demonstrate in Fig. 12 that the same is true for T cell CD40. TRAFs 1, 2, 3, and 6 bind CD40 within the Brij insoluble (raft) fraction upon engagement with agonistic anti-CD40 Ab or membrane-bound CD154. This suggests that TRAFs are a critical component of T cell CD40 signaling, providing a key difference from CD28-mediated signaling. As in B cells (50), T cell CD40 efficiently activates up-regulation of cell surface molecules (Fig. 5), both canonical and noncanonical NF-{kappa}B pathways (Figs. 10 and 11), AP-1 (Fig. 7), and the AP-1 activator JNK (Fig. 8). CD40 as a costimulatory molecule is as effective as CD28 at signaling via AP-1 and severalfold more efficient at signaling via NF-{kappa}B, with maximal increase in both responses when stimulating T cells with agonists for CD3 plus CD28 plus CD40. This suggests that CD40 and CD28 may have different molecular mechanisms leading to activation of AP-1 and NF-{kappa}B, as has been proposed when comparing CD28 and other TNFR family members on T cells (76). Importantly, it shows that CD40 can provide more powerful enhancement of NF-{kappa}B, a transcription factor that induces cytokine genes with particular potency, than other T cell costimulators. This is also seen in the activation of JNK, which in the two T cell lines tested, was seen only when a CD40 costimulus was included.

The above findings suggest that CD40 can increase the potency and number of signaling pathways available to T cells that express it. This is important when considering threshold requirements for developing autoimmune disease and associated chronic inflammation. The presence of CD40-expressing T cells or autoantibodies is not enough to develop autoimmunity. NOR mice, like their NOD counterparts, have CD40-expressing T cells, yet do not develop disease (17). Similarly, the presence of autoantibodies does not necessarily indicate pathogenesis (77, 78, 79). Cooperation between cell types and signals from the environment to the immune response determine development of disease (80, 81).

Like many autoimmune diseases, including diabetes (82), arthritis (83), and lupus (7), CIA requires cooperation between T and B cells in its pathogenesis (84), and CD40 maybe a potent costimulator in this process. CD40 expressed by B and T cells may use similar molecular mechanisms, described above, to contribute to the pathogenesis of inflammation in autoimmune disease. CD40 on both B cells (18, 85, 86, 87, 88, 89) and T cells (this study) synergizes with AgRs to enhance lymphocyte activation, cytokine production (Figs. 2 and 4), and up-regulation of cell surface molecules (Fig. 5). CD40 signaling leads to isotype switching and autoantibody production in B cells (90). In T cells, it has been demonstrated that CD40 engagement leads to TCR revision within germinal centers (67), skewing the T cell population further toward autoimmunity (14, 15, 16).

A proinflammatory cytokine environment is critical for reaching the threshold of autoimmune disease development (65, 91). CD40 engagement in either T or B cells leads to TNF-{alpha} secretion, as shown in Figs. 2C and 4C and (56, 92). We have previously reported that CD40 signaling to B cells is partially mediated by TNF-{alpha} binding to TNFR2 (56, 92) and hypothesize this also to be true of T cell CD40, as it has been demonstrated that TNF-{alpha} acts via TNFR2 to lower the threshold of T cell activation and IL-2 production (93, 94, 95). Taken together, the presence of increased numbers of CD40+ T cells in autoimmune mice and the demonstration that CD40 can act as an effective costimulatory receptor on T cells suggest that blockade of CD40-CD154 interactions can abrogate the pathogenesis of autoimmune disease on several levels. This has been demonstrated in the experimental autoimmune encephalomyelitis model of multiple sclerosis, whereby disease development is dependent on CD40 signaling, particularly in the absence of CD28 (96). In the HgCl2-induced autoimmunity model, CD28 is unable to overcome the lack of CD40 signaling to induce disease (97).

CD40 may not just be a TCR costimulatory molecule on T cells but may make these cells effective APC by up-regulating cell surface molecules (Fig. 5). Increased levels of not only CD40, but other costimulatory molecules such as CD80, CD86, ICAM-1, and LFA-1, may also be pivotal in autoimmune disease development (97). Although beyond the scope of this study, it would be interesting to investigate the costimulatory capacity of activated CD40-expressing T cells for CD40-negative T cells, as well as for other cell types, including APC. It is quite possible that activation of CD40 on T cells lowers the threshold of disease development but is not sufficient for it to occur, requiring CD40 activation on APC for cytokine production and on B cells for autoantibody secretion. In this way, CD40 would prove central to autoimmune disease development and pathogenesis, influencing not only the cognitive activation of APC but also T cells. As seen in Fig. 2, results in enhanced effector proinflammatory cytokine production that along with CD40 activation on B cells would lead to autoantibody production and, ultimately, chronic inflammation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health and the Veterans’ Administration (to G.A.B.) and postdoctoral fellowship support provided by the American Heart Association and the American Cancer Society (to M.E.M.). Back

2 Address correspondence and reprint requests to Dr. Gail A. Bishop, 2193B MERF, Department of Microbiology, University of Iowa, Iowa City, IA 52242. E-mail address: gail-bishop{at}uiowa.edu Back

3 Abbreviations used in this paper: CIA, collagen-induced arthritis; CII, type II chicken collagen; MCF, mean channel fluorescence; TRAF, TNFR-associated factor. Back

Received for publication June 21, 2006. Accepted for publication October 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Grewal, I. S., R. A. Flavell. 1996. The role of CD40 ligand in costimulation and T cell activation. Immunol. Rev. 153: 85-106. [Medline]
  2. Rodriguez-Pinto, D., J. Moreno. 2005. B cells can prime naive CD4+ T cells in vivo in the absence of other professional antigen-presenting cells in a CD154-CD40-dependent manner. Eur. J. Immunol. 35: 1097-1105. [Medline]
  3. Bishop, G. A., B. S. Hostager. 2003. The CD40-CD154 interaction in B cell-T cell liaisons. Cytokine Growth Factor Rev. 14: 297-309. [Medline]
  4. 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: 4620-4627. [Abstract]
  5. 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: 1328-1330. [Abstract/Free Full Text]
  6. Toubi, E., Y. Shoenfeld. 2004. The role of CD40-CD154 interactions in autoimmunity and the benefit of disrupting this pathway. Autoimmunity 37: 457-464. [Medline]
  7. Datta, S. K.. 1998. Production of pathogenic antibodies: cognate interactions between autoimmune T and B cells. Lupus 7: 591-596. [Abstract/Free Full Text]
  8. Kyburz, D., M. Corr, D. C. Brinson, A. Von Damm, H. Tighe, D. A. Carson. 1999. Human rheumatoid factor production is dependent on CD40 signaling and autoantigen. J. Immunol. 163: 3116-3122. [Abstract/Free Full Text]
  9. Fanslow, W. C., K. N. Clifford, M. Seaman, M. R. Alderson, M. K. Spriggs, R. J. Armitage, F. Ramsdell. 1994. Recombinant CD40 ligand exerts potent biologic effects on T cells. J. Immunol. 152: 4262-4269. [Abstract]
  10. Bourgeois, C., B. Rocha, C. Tanchot. 2002. A role for CD40 expression on CD8+ T cells in the generation of CD8+ T cell memory. Science 297: 2060-2063. [Abstract/Free Full Text]
  11. Koschella, M., D. Voehringer, H. Pircher. 2004. CD40 ligation in vivo induces bystander proliferation of memory phenotype CD8 T cells. J. Immunol. 172: 4804-4811. [Abstract/Free Full Text]
  12. Lee, B. O., L. Hartson, T. D. Randall. 2003. CD40-deficient, influenza-specific CD8 memory T cells develop and function normally in a CD40-sufficient environment. J. Exp. Med. 198: 1759-1764. [Abstract/Free Full Text]
  13. Sun, J. C., M. J. Bevan. 2004. Cutting edge: long-lived CD8 memory and protective immunity in the absence of CD40 expression on CD8 T cells. J. Immunol. 172: 3385-3389. [Abstract/Free Full Text]
  14. Wagner, D. H., Jr, E. Newell, R. J. Sanderson, J. H. Freed, M. K. Newell. 1999. Increased expression of CD40 on thymocytes and peripheral T cells in autoimmunity: a mechanism for acquiring changes in the peripheral T cell receptor repertoire. Int. J. Mol. Med. 4: 231-242. [Medline]
  15. Wagner, D. H., Jr, G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, K. Haskins. 2002. Expression of CD40 identifies a unique pathogenic T cell population in type 1 diabetes. Proc. Natl. Acad. Sci. USA 99: 3782-3787. [Abstract/Free Full Text]
  16. Vaitaitis, G. M., M. Poulin, R. J. Sanderson, K. Haskins, D. H. Wagner, Jr. 2003. Cutting edge: CD40-induced expression of recombination activating gene (RAG) 1 and RAG2: a mechanism for the generation of autoaggressive T cells in the periphery. J. Immunol. 170: 3455-3459. [Abstract/Free Full Text]
  17. Waid, D. M., G. M. Vaitaitis, D. H. Wagner, Jr. 2004. Peripheral CD4loCD40+ auto-aggressive T cell expansion during insulin-dependent diabetes mellitus. Eur. J. Immunol. 34: 1488-1497. [Medline]
  18. Haxhinasto, S. A., B. S. Hostager, G. A. Bishop. 2002. Cutting edge: molecular mechanisms of synergy between CD40 and the B cell antigen receptor: role for TNF receptor-associated factor 2 in receptor interaction. J. Immunol. 169: 1145-1149. [Abstract/Free Full Text]
  19. Bishop, G. A., B. S. Hostager. 2001. Molecular mechanisms of CD40 signaling. Arch. Immunol. Ther. Exp. 49: 129-137.
  20. Bishop, G. A., B. S. Hostager. 2001. Signaling by CD40 and its mimics in B cell activation. Immunol. Res. 24: 97-109. [Medline]
  21. Croft, M.. 2003. Co-stimulatory members of the TNFR family: keys to effective T cell immunity?. Nat. Rev. Immunol. 3: 609-620. [Medline]
  22. Watts, T. H.. 2005. TNF/TNFR family members in costimulation of T cell responses. Ann. Rev. Immunol. 23: 23-68. [Medline]
  23. Ashwell, J. D., R. E. Cunningham, P. D. Noguchi, D. Hernandez. 1987. Cell growth cycle block of T cell hybridomas upon activation with antigen. J. Exp. Med. 165: 173-194. [Abstract/Free Full Text]
  24. Gillis, S., J. Watson. 1980. Biochemical and biological characterization of lymphocyte regulatory molecules. V. Identification of an interleukin 2-producing human leukemia T cell line. J. Exp. Med. 152: 1709-1719. [Abstract/Free Full Text]
  25. Warren, W. D., M. T. Berton. 1995. Induction of germ-line {gamma}1 and {epsilon} Ig gene expression in murine B cells—IL-4 and the CD40 ligand-CD40 interaction provide distinct but synergistic signals. J. Immunol. 155: 5637-5646. [Abstract]
  26. Bishop, G. A., W. D. Warren, M. T. Berton. 1995. Signaling via major histocompatibility complex class II molecules and antigen receptors enhances the B cell response to gp39/CD40 ligand. Eur. J. Immunol. 25: 1230-1238. [Medline]
  27. Hostager, B. S., Y. Hsing, D. E. Harms, G. A. Bishop. 1996. Different CD40-mediated signaling events require distinct CD40 structural features. J. Immunol. 157: 1047-1053. [Abstract]
  28. Bishop, G. A., J. A. Frelinger. 1989. Haplotype-specific differences in signaling by transfected class II molecules to a Ly-1+ B cell clone. Proc. Natl. Acad. Sci. USA 86: 5933-5937. [Abstract/Free Full Text]
  29. Campbell, I. K., J. A. Hamilton, I. P. Wicks. 2000. Collagen-induced arthritis in C57BL/6 (H-2b) mice: new insights into an important disease model of rheumatoid arthritis. Eur. J. Immunol. 30: 1568-1575. [Medline]
  30. Rosloniec, E. F., A. H. Kang, L. K. Myers, M. A. Cremer. 1997. Collagen-Induced Arthritis Wiley & Sons, New York.
  31. Berberich, I., G. L. Shu, E. A. Clark. 1994. Cross-linking CD40 on B cells rapidly activates nuclear factor {kappa}B. J. Immunol. 153: 4357-4366. [Abstract]
  32. Schreiber, E., P. Matthias, M. M. Muller, W. Schaffner. 1989. Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells. Nucleic Acids Res. 17: 6419[Free Full Text]
  33. Moore, C. R., G. A. Bishop. 2005. Differential regulation of CD40-mediated TNF receptor-associated factor degradation in B lymphocytes. J. Immunol. 175: 3780-3789. [Abstract/Free Full Text]
  34. Xie, P., B. S. Hostager, G. A. Bishop. 2004. Requirement for TRAF3 in signaling by LMP1 but not CD40 in B lymphocytes. J. Exp. Med. 199: 661-671. [Abstract/Free Full Text]
  35. Myers, L. K., E. F. Rosloniec, M. A. Cremer, A. H. Kang. 1997. Collagen-induced arthritis, an animal model of autoimmunity. Life Sci. 61: 1861-1878. [Medline]
  36. Schulze-Koops, H., J. R. Kalden. 2001. The balance of Th1/Th2 cytokines in rheumatoid arthritis. Best Pract. Res. Clin. Rheumatol. 15: 677-691. [Medline]
  37. Ballester, A., A. Velasco, R. Tobena, S. Alemany. 1998. Cot kinase activates tumor necrosis factor {alpha} gene expression in a cyclosporin A-resistant manner. J. Biol. Chem. 273: 14099-14106. [Abstract/Free Full Text]
  38. Kashiwakura, J., N. Suzuki, H. Nagafuchi, M. Takeno, Y. Takeba, Y. Shimoyama, T. Sakane. 1999. Txk, a nonreceptor tyrosine kinase of the Tec family, is expressed in T helper type 1 cells and regulates interferon {gamma} production in human T lymphocytes. J. Exp. Med. 190: 1147-1154. [Abstract/Free Full Text]
  39. Chung, D. H., K. C. Jung, W. S. Park, I. S. Lee, W. J. Choi, C. J. Kim, S. H. Park, Y. Bae. 2000. Costimulatory effect of Fas in mouse T lymphocytes. Mol. Cell 10: 642-646.
  40. Tibbetts, S. A., C. Chirathaworn, M. Nakashima, D. S. Jois, T. J. Siahaan, M. A. Chan, S. H. Benedict. 1999. Peptides derived from ICAM-1 and LFA-1 modulate T cell adhesion and immune function in a mixed lymphocyte culture. Transplantation 68: 685-692. [Medline]
  41. Moulian, N., J. Bidault, C. Planche, S. Berrih-Aknin. 1998. Two signaling pathways can increase fas expression in human thymocytes. Blood 92: 1297-1307. [Abstract/Free Full Text]
  42. Bhatia, S., M. Edidin, S. C. Almo, S. G. Nathenson. 2006. B7-1 and B7-2: similar costimulatory ligands with different biochemical, oligomeric and signaling properties. Immunol. Lett. 104: 70-75. [Medline]
  43. Adler, S. H., E. Chiffoleau, L. Xu, N. M. Dalton, J. M. Burg, A. D. Wells, M. S. Wolfe, L. A. Turka, W. S. Pear. 2003. Notch signaling augments T cell responsiveness by enhancing CD25 expression. J. Immunol. 171: 2896-2903. [Abstract/Free Full Text]
  44. Cianferoni, A., M. Massaad, S. Feske, M. A. de la Fuente, L. Gallego, N. Ramesh, R. S. Geha. 2005. Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome. J. Allergy Clin. Immunol. 116: 1364-1371. [Medline]
  45. Palanki, M. S.. 2002. Inhibitors of AP-1 and NF-{kappa}B mediated transcriptional activation: therapeutic potential in autoimmune diseases and structural diversity. Curr. Med. Chem. 9: 219-227. [Medline]
  46. Jain, J., C. Loh, A. Rao. 1995. Transcriptional regulation of the IL-2 gene. Curr. Opin. Immunol. 7: 333-342. [Medline]
  47. Kaminuma, O., C. Elly, Y. Tanaka, A. Mori, Y. C. Liu, A. Altman, S. Miyatake. 2002. Vav-induced activation of the human IFN-