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4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses.

Shuford WW, Klussman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, Brown TJ, Emswiler J, Raecho H, Larsen CP, Pearson TC, Ledbetter JA, Aruffo A, Mittler RS - J. Exp. Med. (1997)

Bottom Line: Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB.In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice.The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.

ABSTRACT
The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-gamma production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

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(A) Anti–4-1BB mAbs differentially costimulate in vitro proliferation of CD4+ and CD8+ T  cell subsets. Purified resting CD4+ or CD8+ BALB/c  splenic T cells were cultured for 72 h in 96-well microtiter plates at 1 × 105/well with 0.5% APC ± anti– 4-1BB mAb at 10 μg/ml and the indicated concentration of anti-CD3 mAb. Cells were pulsed with 0.5  μCi/well [3H]thymidine for the final 12 h of culture,  harvested, and counted by liquid scintillation spectroscopy. (B) 4-1BB–Ig fusion protein inhibits anti-CD3– induced T cell proliferation. CD4+ and CD8+ T cells  with APC were stimulated with anti-CD3 mAb at 300  ng/ml in the absence or presence of soluble 4-1BB–Ig fusion protein. Cells were pulsed during the final 12 h of culture with [3H]thymidine, harvested,  and counted by liquid scintillation spectroscopy.
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Figure 3: (A) Anti–4-1BB mAbs differentially costimulate in vitro proliferation of CD4+ and CD8+ T cell subsets. Purified resting CD4+ or CD8+ BALB/c splenic T cells were cultured for 72 h in 96-well microtiter plates at 1 × 105/well with 0.5% APC ± anti– 4-1BB mAb at 10 μg/ml and the indicated concentration of anti-CD3 mAb. Cells were pulsed with 0.5 μCi/well [3H]thymidine for the final 12 h of culture, harvested, and counted by liquid scintillation spectroscopy. (B) 4-1BB–Ig fusion protein inhibits anti-CD3– induced T cell proliferation. CD4+ and CD8+ T cells with APC were stimulated with anti-CD3 mAb at 300 ng/ml in the absence or presence of soluble 4-1BB–Ig fusion protein. Cells were pulsed during the final 12 h of culture with [3H]thymidine, harvested, and counted by liquid scintillation spectroscopy.

Mentions: 4-1BB expression on activated CD4+ and CD8+ T cells occurs with the same kinetics and degree of expression (data not shown). Although both subsets express 4-1BB at equivalent levels it is not known whether the 4-1BB receptor preferentially or differentially regulates the activation or proliferative potential of either subset. To test this possibility, we isolated highly purified populations of resting splenic BALB/c CD4+ or CD8+ T cells (>98% purity) by negative selection procedures described earlier. T cell subsets were then incubated with soluble 145.2C11 mAb in a range of 10–1,000 ng/ml ± 10 μg/ml anti–4-1BB mAb for 3 d in the presence of 0.5% APC. Proliferation was monitored by [3H]thymidine incorporation during the final 12 h of culture. In Fig. 3 A we show the results of one of four proliferation experiments carried out on isolated CD4+ T cells or CD8+ T cells obtained from the same group of mice. From these experiments, it is clear that costimulation of CD4+ T cells with anti–4-1BB mAbs such as 3E1 and 3H3 led to a two- to threefold enhancement of anti-CD3–induced proliferation, whereas others such as 1D8, a nonligand-blocking antibody, had little effect upon T cell proliferation. mAb 6E9 is an isotype-matched negative control that is reactive with human gp-39. In contrast, the same set of antibodies markedly enhanced CD8+ T cell proliferative responses by lowering the threshold of anti-CD3 concentration 10- (1D8) to 100-fold (3H3) and increased the incorporation of [3H]thymidine ∼100-fold.


4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses.

Shuford WW, Klussman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, Brown TJ, Emswiler J, Raecho H, Larsen CP, Pearson TC, Ledbetter JA, Aruffo A, Mittler RS - J. Exp. Med. (1997)

(A) Anti–4-1BB mAbs differentially costimulate in vitro proliferation of CD4+ and CD8+ T  cell subsets. Purified resting CD4+ or CD8+ BALB/c  splenic T cells were cultured for 72 h in 96-well microtiter plates at 1 × 105/well with 0.5% APC ± anti– 4-1BB mAb at 10 μg/ml and the indicated concentration of anti-CD3 mAb. Cells were pulsed with 0.5  μCi/well [3H]thymidine for the final 12 h of culture,  harvested, and counted by liquid scintillation spectroscopy. (B) 4-1BB–Ig fusion protein inhibits anti-CD3– induced T cell proliferation. CD4+ and CD8+ T cells  with APC were stimulated with anti-CD3 mAb at 300  ng/ml in the absence or presence of soluble 4-1BB–Ig fusion protein. Cells were pulsed during the final 12 h of culture with [3H]thymidine, harvested,  and counted by liquid scintillation spectroscopy.
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Related In: Results  -  Collection

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Figure 3: (A) Anti–4-1BB mAbs differentially costimulate in vitro proliferation of CD4+ and CD8+ T cell subsets. Purified resting CD4+ or CD8+ BALB/c splenic T cells were cultured for 72 h in 96-well microtiter plates at 1 × 105/well with 0.5% APC ± anti– 4-1BB mAb at 10 μg/ml and the indicated concentration of anti-CD3 mAb. Cells were pulsed with 0.5 μCi/well [3H]thymidine for the final 12 h of culture, harvested, and counted by liquid scintillation spectroscopy. (B) 4-1BB–Ig fusion protein inhibits anti-CD3– induced T cell proliferation. CD4+ and CD8+ T cells with APC were stimulated with anti-CD3 mAb at 300 ng/ml in the absence or presence of soluble 4-1BB–Ig fusion protein. Cells were pulsed during the final 12 h of culture with [3H]thymidine, harvested, and counted by liquid scintillation spectroscopy.
Mentions: 4-1BB expression on activated CD4+ and CD8+ T cells occurs with the same kinetics and degree of expression (data not shown). Although both subsets express 4-1BB at equivalent levels it is not known whether the 4-1BB receptor preferentially or differentially regulates the activation or proliferative potential of either subset. To test this possibility, we isolated highly purified populations of resting splenic BALB/c CD4+ or CD8+ T cells (>98% purity) by negative selection procedures described earlier. T cell subsets were then incubated with soluble 145.2C11 mAb in a range of 10–1,000 ng/ml ± 10 μg/ml anti–4-1BB mAb for 3 d in the presence of 0.5% APC. Proliferation was monitored by [3H]thymidine incorporation during the final 12 h of culture. In Fig. 3 A we show the results of one of four proliferation experiments carried out on isolated CD4+ T cells or CD8+ T cells obtained from the same group of mice. From these experiments, it is clear that costimulation of CD4+ T cells with anti–4-1BB mAbs such as 3E1 and 3H3 led to a two- to threefold enhancement of anti-CD3–induced proliferation, whereas others such as 1D8, a nonligand-blocking antibody, had little effect upon T cell proliferation. mAb 6E9 is an isotype-matched negative control that is reactive with human gp-39. In contrast, the same set of antibodies markedly enhanced CD8+ T cell proliferative responses by lowering the threshold of anti-CD3 concentration 10- (1D8) to 100-fold (3H3) and increased the incorporation of [3H]thymidine ∼100-fold.

Bottom Line: Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB.In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice.The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.

ABSTRACT
The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-gamma production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

Show MeSH
Related in: MedlinePlus