Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Anti–4-1BB mAbs enhance or inhibit in vivo generation of  CTL responses during GVHD responses.  GVHD was established by intravenous injection of 5 × 107 C57BL/6 (H-2b) splenocytes into DBA/2 (H-2bd) mice as described in the Materials and Methods.  The administration of mAbs was carried  out as described earlier. On day 10,  spleens were collected from GVHD mice  and single cell suspensions prepared. CTL  activity in this population was measured  directly by assaying CTL-mediated killing  of 51Cr-labeled P815 cells. (B) Total splenocyte levels are markedly reduced in  mice receiving anti–4-1BB mAbs. The  total number of viable splenocytes obtained from groups of three spleens pooled  from each treatment group was assessed  by microscopy and trypan blue exclusion.  (C) Percentages of viable CD8+ T cells  were determined for each treatment group  using FITC-conjugated anti-murine CD8  mAb (PharMingen) and flow cytometry.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198949&req=5

Figure 6: (A) Anti–4-1BB mAbs enhance or inhibit in vivo generation of CTL responses during GVHD responses. GVHD was established by intravenous injection of 5 × 107 C57BL/6 (H-2b) splenocytes into DBA/2 (H-2bd) mice as described in the Materials and Methods. The administration of mAbs was carried out as described earlier. On day 10, spleens were collected from GVHD mice and single cell suspensions prepared. CTL activity in this population was measured directly by assaying CTL-mediated killing of 51Cr-labeled P815 cells. (B) Total splenocyte levels are markedly reduced in mice receiving anti–4-1BB mAbs. The total number of viable splenocytes obtained from groups of three spleens pooled from each treatment group was assessed by microscopy and trypan blue exclusion. (C) Percentages of viable CD8+ T cells were determined for each treatment group using FITC-conjugated anti-murine CD8 mAb (PharMingen) and flow cytometry.

Mentions: Because anti–4-1BB mAbs had such a profound effect upon the signaling and proliferative responses of CD8+ T cells, we questioned the importance of 4-1BB signaling in the generation of T cell effector functions such as cytotoxic T cell responses. Therefore, we measured the ability of a panel of anti–4-1BB mAbs of the IgG2A isotype including blocking (3E1, 22B6, and 3H3) and nonblocking (1D8) mAbs to affect either the generation of CTL during acute GVHD or in a murine cardiac allograft or MHC mismatched skin transplant model. To generate acute GVHD and assay CTL activity, splenic T cells were isolated from BDF1 (H-2db) mice 10 d after intravenous injection of 1–5 × 107 C57BL/6 (H-2b) splenocytes. Spleens removed from 1D8 and 22B6-treated GVHD mice were enlarged 3–5 times the size of normal spleens. Mice that received the isotype-matched negative control mAb 6E9 had spleens that were 2–3 times the size of normal mice. When assayed for CTL activity, 1D8 and 22B6, each known to bind to a different region of the 4-1BB molecule, enhanced CTL activity by nearly fourfold over that observed in control GVHD animals (Fig. 6 A). The powerful effect of anti–4-1BB mAbs on the enhanced development of CTL activity is further demonstrated in Fig. 6 B, which shows a marked reduction in the number of total viable splenocytes retrieved from mice treated with these antibodies. In addition, phenotypic analysis of splenocytes revealed that the percentage of CD8+ T cells increased to 30% of the total cell number in mice treated with anti–4-1BB mAbs (Fig. 6 C), whereas GVHD mice injected with 6E9, the isotyped-matched nonbinding control or those receiving no antibody had 5–8% CD8+ T cells. Epitope mapping studies of anti–4-1BB mAbs using 4-1BB fusion proteins in which domain swapping was carried out demonstrated that the 1D8 mAb was unique in that it bound to the membrane proximal region of the extracellular domain of the 4-1BB molecule not involved in 4-1BBL binding.


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 enhance or inhibit in vivo generation of  CTL responses during GVHD responses.  GVHD was established by intravenous injection of 5 × 107 C57BL/6 (H-2b) splenocytes into DBA/2 (H-2bd) mice as described in the Materials and Methods.  The administration of mAbs was carried  out as described earlier. On day 10,  spleens were collected from GVHD mice  and single cell suspensions prepared. CTL  activity in this population was measured  directly by assaying CTL-mediated killing  of 51Cr-labeled P815 cells. (B) Total splenocyte levels are markedly reduced in  mice receiving anti–4-1BB mAbs. The  total number of viable splenocytes obtained from groups of three spleens pooled  from each treatment group was assessed  by microscopy and trypan blue exclusion.  (C) Percentages of viable CD8+ T cells  were determined for each treatment group  using FITC-conjugated anti-murine CD8  mAb (PharMingen) and flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198949&req=5

Figure 6: (A) Anti–4-1BB mAbs enhance or inhibit in vivo generation of CTL responses during GVHD responses. GVHD was established by intravenous injection of 5 × 107 C57BL/6 (H-2b) splenocytes into DBA/2 (H-2bd) mice as described in the Materials and Methods. The administration of mAbs was carried out as described earlier. On day 10, spleens were collected from GVHD mice and single cell suspensions prepared. CTL activity in this population was measured directly by assaying CTL-mediated killing of 51Cr-labeled P815 cells. (B) Total splenocyte levels are markedly reduced in mice receiving anti–4-1BB mAbs. The total number of viable splenocytes obtained from groups of three spleens pooled from each treatment group was assessed by microscopy and trypan blue exclusion. (C) Percentages of viable CD8+ T cells were determined for each treatment group using FITC-conjugated anti-murine CD8 mAb (PharMingen) and flow cytometry.
Mentions: Because anti–4-1BB mAbs had such a profound effect upon the signaling and proliferative responses of CD8+ T cells, we questioned the importance of 4-1BB signaling in the generation of T cell effector functions such as cytotoxic T cell responses. Therefore, we measured the ability of a panel of anti–4-1BB mAbs of the IgG2A isotype including blocking (3E1, 22B6, and 3H3) and nonblocking (1D8) mAbs to affect either the generation of CTL during acute GVHD or in a murine cardiac allograft or MHC mismatched skin transplant model. To generate acute GVHD and assay CTL activity, splenic T cells were isolated from BDF1 (H-2db) mice 10 d after intravenous injection of 1–5 × 107 C57BL/6 (H-2b) splenocytes. Spleens removed from 1D8 and 22B6-treated GVHD mice were enlarged 3–5 times the size of normal spleens. Mice that received the isotype-matched negative control mAb 6E9 had spleens that were 2–3 times the size of normal mice. When assayed for CTL activity, 1D8 and 22B6, each known to bind to a different region of the 4-1BB molecule, enhanced CTL activity by nearly fourfold over that observed in control GVHD animals (Fig. 6 A). The powerful effect of anti–4-1BB mAbs on the enhanced development of CTL activity is further demonstrated in Fig. 6 B, which shows a marked reduction in the number of total viable splenocytes retrieved from mice treated with these antibodies. In addition, phenotypic analysis of splenocytes revealed that the percentage of CD8+ T cells increased to 30% of the total cell number in mice treated with anti–4-1BB mAbs (Fig. 6 C), whereas GVHD mice injected with 6E9, the isotyped-matched nonbinding control or those receiving no antibody had 5–8% CD8+ T cells. Epitope mapping studies of anti–4-1BB mAbs using 4-1BB fusion proteins in which domain swapping was carried out demonstrated that the 1D8 mAb was unique in that it bound to the membrane proximal region of the extracellular domain of the 4-1BB molecule not involved in 4-1BBL binding.

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