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. 1998 Jun 1;187(11):1849-62.
doi: 10.1084/jem.187.11.1849.

CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand

K Saoulli  1 , S Y Lee, J L Cannons, W C Yeh, A Santana, M D Goldstein, N Bangia, M A DeBenedette, T W Mak, Y Choi, T H Watts
Affiliations

CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand

K Saoulli et al. J Exp Med. .

Abstract

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.

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Figures

Figure 1
Figure 1
Generation of a soluble form of 4-1BBL. (A) Schematic representation of the construct used to generate s4-1BBL. MAG SS, MAG signal sequence. (B) Coomassie blue–stained SDS-PAGE of s4-1BBL produced in baculovirus and isolated from insect cell supernatants using 12CA5 anti-HA affinity chromatograpy as described in Materials and Methods.
Figure 2
Figure 2
Immobilized s4-1BBL can costimulate proliferation and IL-2 production by CD28+ or CD28− T cells. (A) High density resting T cells isolated from the spleens of CD28+ or CD28− mice were incubated with various concentrations of anti-CD3 alone or with anti-CD3 plus immobilized s4-1BBL added to the plastic wells at 1 μg/ml as indicated in the figures. After 48 h, culture supernatants were removed and serial dilutions of supernatant were incubated with IL-2–dependent CTLL cells for 18–24 h. [3H]thymidine was added for the last 6 h of the CTLL culture. Data are shown for the first dilution of culture supernatant and are representative of four independent experiments. (B) CD28− T cells were cultured with immobilized anti-CD3 alone or with immobilized anti-CD3 plus s4-1BBL at 1 or 10 μg/ml as indicated in the figure. After 48 h, [3H]thymidine was added to the wells and thymidine uptake was determined after a further 6 h of culture. Similar results were obtained in three separate experiments. (C) CD28− cells were cultured with anti-CD3 (1 μg/ml) in the presence or absence of s4-1BBL (1 μg/ml) or in the presence of s4-1BBL (1 μg/ml) plus 4-1BB-AP (10 μg/ml). After 48-h incubation, culture supernatants were analyzed for induction of thymidine incorporation by CTLL. 4-1BB–AP alone had no effect on IL-2 production. Results are shown for serial dilutions of the culture supernatants and are representative of four independent experiments.
Figure 3
Figure 3
Effect of 4-1BBL titration on CD28+ and CD28− T cell responses. High density resting T cells isolated from the spleens of CD28+ or CD28− mice were incubated with anti-CD3 alone (1 μg/ml) or with anti-CD3 plus immobilized s4-1BBL added to the plastic wells at the concentration indicated in the figures. After 2 d, serial dilutions of the culture supernatant were incubated with IL-2– dependent CTLL cells overnight. [3H]thymidine was added to the CTLL cultures for the last 6 h of culture. Similar results were obtained in three independent experiments.
Figure 4
Figure 4
Kinetics of IL-2 production in response to 4-1BBL. Resting T cells isolated from the spleens of CD28− mice were incubated with anti-CD3 alone (1 μg/ml) or with anti-CD3 plus s4-1BBL immobilized at the concentrations indicated on the figure. After 12, 24, or 48 h, culture supernatants were removed and serial dilutions were analyzed for induction of proliferation of IL-2–dependent CTLL cells. Results are representative of two separate experiments.
Figure 5
Figure 5
Comparison of immobilized s4-1BBL with immobilized anti-CD28 for costimulation of IL-2 production by CD28+ and CD28− T cells. Resting T cells isolated from CD28+ or CD28− T cells were stimulated by immobilized anti-CD3 at the indicated concentrations plus or minus 4-1BBL immobilized at 5 μg/ml or anti-CD28 at 10 μg/ml or control Ig at 10 μg/ml. After 48 h, supernatants were removed and analyzed for the presence of IL-2 using CTLL cells. Results are representative of two independent experiments.
Figure 6
Figure 6
Association between the 4-1BB cytoplasmic tail-GST fusion protein and TRAF1 and TRAF2 proteins. As described in Materials and Methods, purified GST, GST-CD30CT, and GST–4-1BBCT proteins bound to glutathione beads were incubated with 35S-labeled in vitro– translated TRAF1 or TRAF2. After washing, samples were eluted and analyzed by SDS-PAGE followed by autoradiography. In vitro–translated TRAF1 and TRAF2 were also run directly on the gel (lanes marked TRAF1 and TRAF2) for comparison.
Figure 7
Figure 7
Association of TRAF1 and TRAF2 with 4-1BB in a T cell hybrid is induced by receptor aggregation at 37°C. C8.A3 T cells (2 × 107 cells/ lane) were stimulated with anti– 4-1BB or with anti–ICAM-1 on ice for 5 min and then cross-linked with anti–rat Ig for 15 min on ice or at 37°C. Lysates were immunoprecipitated with protein G–Sepharose. Immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with either anti-TRAF1 or anti-TRAF2. This experiment is representative of three separate experiments.
Figure 8
Figure 8
Decreased response to 4-1BBL by T cells isolated from mice expressing a dominant negative form of TRAF2. Resting T cells were isolated from spleen and lymph nodes TRAF2 DN mice or their transgene-negative littermates, as described in Materials and Methods. T cells were incubated with anti-CD3 immobilized on plastic at the concentrations indicated in the figures in the presence or absence of immobilized s4-1BBL (5 μg/ml) or immobilized anti-CD28 at 2 or 10 μg/ml as indicated in the figure. At 48 h of culture, supernatants were analyzed for IL-2 production using CTLL cells. Results shown are for a single representative mouse. Similar results were obtained in three independent experiments with two control and two mutant mice analyzed separately in each experiment.
Figure 9
Figure 9
Decreased response to 4-1BBL by T cells isolated from TRAF2− mice. Resting T cells were isolated from the lymph nodes of TRAF2− mice or their wild-type TRAF2+ littermates. T cells were incubated with anti-CD3 alone (immobilized at 1 μg/ml) or with anti-CD3 plus immobilized s4-1BBL (5 μg/ml) or immobilized anti-CD28 (2 μg/ml). Results are shown from one mouse and are representative of two independent experiments with two mice per experiment.
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References

    1. Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of T cell costimulation. Annu Rev Immunol. 1996;14:233–258. - PubMed
    1. Shahinian A, Pfeffer K, Lee KP, Kundig TM, Kishihara K, Wakeham A, Kawai K, Ohashi PS, Thompson CB, Mak TW. Differential T cell costimulatory requirements in CD28-deficient mice. Science. 1993;261:609–612. - PubMed
    1. Green JM, Noel PJ, Sperling AI, Walunas TL, Gray GS, Bluestone JA, Thompson CB. Absence of B7-dependent responses in CD28-deficient mice. Immunity. 1994;1:501–508. - PubMed
    1. Kawai K, Shahinian A, Mak TW, Ohashi PS. Skin allograft rejection in CD28-deficient mice. Transplantation. 1996;61:352–355. - PubMed
    1. Kwon BS, Weissman SM. cDNA sequences of two inducible T-cell genes. Proc Natl Acad Sci USA. 1989;86:1963–1967. - PMC - PubMed

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