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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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Cross-linking 4-1BB  initiates distinct patterns and kinetics of protein tyrosine phosphorylation in CD4+ and CD8+  T cell subsets. BALB/c splenocytes were activated as described  earlier. CD4+ (A) and CD8+ (B)  subsets were incubated with 10  ng/ml of anti-CD3, anti–4-1BB  (3E1), or both at 10 ng/ml each.  After 15-min incubation at room  temperature, the cells were  equilibrated to 37°C and cross-linked with 50 ng/ml of RG7, a  mouse anti–rat IgG that cross-reacts with hamster IgG. Where  both anti-CD3 and anti-4-1BB  were added together, RG7 was  added at 100 ng/ml. Untreated  (lane 1), 1 min (lane 2), 3 min  (lane 3), 9 min (lane 4).
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Figure 5: Cross-linking 4-1BB initiates distinct patterns and kinetics of protein tyrosine phosphorylation in CD4+ and CD8+ T cell subsets. BALB/c splenocytes were activated as described earlier. CD4+ (A) and CD8+ (B) subsets were incubated with 10 ng/ml of anti-CD3, anti–4-1BB (3E1), or both at 10 ng/ml each. After 15-min incubation at room temperature, the cells were equilibrated to 37°C and cross-linked with 50 ng/ml of RG7, a mouse anti–rat IgG that cross-reacts with hamster IgG. Where both anti-CD3 and anti-4-1BB were added together, RG7 was added at 100 ng/ml. Untreated (lane 1), 1 min (lane 2), 3 min (lane 3), 9 min (lane 4).

Mentions: In light of the observed selective effect that anti-4-1BB mAbs had on CD8+ T cell proliferation, we analyzed the ability to 3E1 (or 3H3; data not shown) to influence early signal transduction events in purified populations of 48-h anti-CD3–activated CD4+ and CD8+ T cells. After a 15-min incubation with 3E1, 145.2C11, or both (each mAb at 10 ng/ml) at room temperature, the cells were placed in a water bath equilibrated to 37°C and incubated for the indicated times after the addition of 50 ng/ml of a mouse anti– rat mAb, RG7, that cross-reacts with hamster mAb. When both anti-CD3 and anti–4-1BB were added together, the concentration of RG7 was doubled. In Fig. 5 A and Fig. 5 B, the results of a typical experiment are shown for CD4+ and CD8+ T cells, respectively. The most notable feature of this experiment is that previously activated CD8+ T cells are more responsive to anti-CD3 stimulation when compared with CD4+ T cells. This is not due to differential expression levels of CD3 within the two populations (our personal observations). Apart from the fact that CD8+ T cells were more activated than CD4+ T cells as judged by both the number and intensity of phosphoprotein bands on the gel, it is noteworthy that within the CD8+ subset, signals delivered by anti-CD3 and anti–4-1BB are virtually indistinguishable from each other in terms of phosphotyrosine species observed, intensity of phosphorylation, as well as the kinetics of phosphorylation. In contrast, this is less true of the CD4+ T cell population. In both figures, arrows delineate proteins undergoing significant changes in phosphorylation. The band with the slowest mobility also appears to undergo serine/threonine phosphorylation since its mobility is further retarded after 4-1BB cross-linking. Costimulation of CD8+ T cells through CD3 and 4-1BB receptors alters the magnitude, kinetics, and profile of phosphoproteins compared with stimulation with either one alone. It should be pointed out that the data in both figures were generated from equivalent cell numbers from the same mice and that the number of cell equivalents (lysates) used/lane were identical in both experiments, as were the exposure times used in generating the ECL Western blots.


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)

Cross-linking 4-1BB  initiates distinct patterns and kinetics of protein tyrosine phosphorylation in CD4+ and CD8+  T cell subsets. BALB/c splenocytes were activated as described  earlier. CD4+ (A) and CD8+ (B)  subsets were incubated with 10  ng/ml of anti-CD3, anti–4-1BB  (3E1), or both at 10 ng/ml each.  After 15-min incubation at room  temperature, the cells were  equilibrated to 37°C and cross-linked with 50 ng/ml of RG7, a  mouse anti–rat IgG that cross-reacts with hamster IgG. Where  both anti-CD3 and anti-4-1BB  were added together, RG7 was  added at 100 ng/ml. Untreated  (lane 1), 1 min (lane 2), 3 min  (lane 3), 9 min (lane 4).
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Related In: Results  -  Collection

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Figure 5: Cross-linking 4-1BB initiates distinct patterns and kinetics of protein tyrosine phosphorylation in CD4+ and CD8+ T cell subsets. BALB/c splenocytes were activated as described earlier. CD4+ (A) and CD8+ (B) subsets were incubated with 10 ng/ml of anti-CD3, anti–4-1BB (3E1), or both at 10 ng/ml each. After 15-min incubation at room temperature, the cells were equilibrated to 37°C and cross-linked with 50 ng/ml of RG7, a mouse anti–rat IgG that cross-reacts with hamster IgG. Where both anti-CD3 and anti-4-1BB were added together, RG7 was added at 100 ng/ml. Untreated (lane 1), 1 min (lane 2), 3 min (lane 3), 9 min (lane 4).
Mentions: In light of the observed selective effect that anti-4-1BB mAbs had on CD8+ T cell proliferation, we analyzed the ability to 3E1 (or 3H3; data not shown) to influence early signal transduction events in purified populations of 48-h anti-CD3–activated CD4+ and CD8+ T cells. After a 15-min incubation with 3E1, 145.2C11, or both (each mAb at 10 ng/ml) at room temperature, the cells were placed in a water bath equilibrated to 37°C and incubated for the indicated times after the addition of 50 ng/ml of a mouse anti– rat mAb, RG7, that cross-reacts with hamster mAb. When both anti-CD3 and anti–4-1BB were added together, the concentration of RG7 was doubled. In Fig. 5 A and Fig. 5 B, the results of a typical experiment are shown for CD4+ and CD8+ T cells, respectively. The most notable feature of this experiment is that previously activated CD8+ T cells are more responsive to anti-CD3 stimulation when compared with CD4+ T cells. This is not due to differential expression levels of CD3 within the two populations (our personal observations). Apart from the fact that CD8+ T cells were more activated than CD4+ T cells as judged by both the number and intensity of phosphoprotein bands on the gel, it is noteworthy that within the CD8+ subset, signals delivered by anti-CD3 and anti–4-1BB are virtually indistinguishable from each other in terms of phosphotyrosine species observed, intensity of phosphorylation, as well as the kinetics of phosphorylation. In contrast, this is less true of the CD4+ T cell population. In both figures, arrows delineate proteins undergoing significant changes in phosphorylation. The band with the slowest mobility also appears to undergo serine/threonine phosphorylation since its mobility is further retarded after 4-1BB cross-linking. Costimulation of CD8+ T cells through CD3 and 4-1BB receptors alters the magnitude, kinetics, and profile of phosphoproteins compared with stimulation with either one alone. It should be pointed out that the data in both figures were generated from equivalent cell numbers from the same mice and that the number of cell equivalents (lysates) used/lane were identical in both experiments, as were the exposure times used in generating the ECL Western blots.

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