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Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy.

Smith JA, Tso JY, Clark MR, Cole MS, Bluestone JA - J. Exp. Med. (1997)

Bottom Line: However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies.However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of zeta (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase.Finally, a bispecific anti-CD3 X anti-CD4 F(ab)'2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3.

View Article: PubMed Central - PubMed

Affiliation: Ben May Institute for Cancer Research, Department of Pathology, Chicago, Illinois 60637, USA.

ABSTRACT
Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonnmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including zeta chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of zeta (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cgamma-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 X anti-CD4 F(ab)'2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3.

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Stimulation with anti-CD3 × anti-CD4 heterodimer results  in increased phosphorylation of proteins associated with the TCR complex and reconstitutes a mitogenic stimulus. (A) 107 pGL10 T cells were  stimulated with the anti-CD3–Fos F(ab)′2, or the bispecific F(ab)′2 (antiCD3 × anti-CD4) for 5 min at 37°C. Samples were precipitated with  anti-ζ, and blots were probed with anti-phosphotyrosine as in Fig. 2 A.  (B) Whole spleen (top) or pGL10 T cells (bottom) were cultured with serial  log dilutions of anti-CD3–Fos homodimer (open diamonds) or bispecific  anti-CD3 × anti-CD4 (closed diamonds) for 48 h. Data is representative of  two (A) and three (B) separate experiments.
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Figure 7: Stimulation with anti-CD3 × anti-CD4 heterodimer results in increased phosphorylation of proteins associated with the TCR complex and reconstitutes a mitogenic stimulus. (A) 107 pGL10 T cells were stimulated with the anti-CD3–Fos F(ab)′2, or the bispecific F(ab)′2 (antiCD3 × anti-CD4) for 5 min at 37°C. Samples were precipitated with anti-ζ, and blots were probed with anti-phosphotyrosine as in Fig. 2 A. (B) Whole spleen (top) or pGL10 T cells (bottom) were cultured with serial log dilutions of anti-CD3–Fos homodimer (open diamonds) or bispecific anti-CD3 × anti-CD4 (closed diamonds) for 48 h. Data is representative of two (A) and three (B) separate experiments.

Mentions: The inability of nonmitogenic anti-CD3 to trigger specific downstream events and proliferation most likely stems from the defective proximal events observed involving ζ and ZAP- 70. Previous studies have suggested that the src family kinase, lck, plays a crucial role in the phosphorylation of ζ, which subsequently allows association and phosphorylation of ZAP-70 (26, 27). Thus, it was possible that the differences in ζ and ZAP-70 phosphorylation seen upon the addition of cross-linker to anti-CD3 may have reflected increased lck activation or enhanced recruitment to the TCR. Initial experiments examining lck activation by monitoring lck tyrosine phosphorylation revealed no differences between cross-linking and noncross-linking conditions. It is clear that CD4 associates with lck and can interact with the TCR complex inducibly upon TCR ligation of antigen– MHC. Thus, artificially bringing CD4–lck into the TCR complex might reconstitute a mitogenic anti-CD3 stimulus, even in the absence of a secondary cross-linking Ab. To test this hypothesis, we took advantage of a bispecific anti-CD3 × anti-CD4 reagent prepared by a molecular approach to insure the presence of monovalent arms specific for CD3 and CD4 (as described in Materials and Methods). T cells were incubated with anti-CD3–Fos homodimer or the anti-CD3 × anti-CD4 bispecific F(ab)′2, lysed, and the TCR–CD3 complex was then immunoprecipitated and analyzed. The bispecific construct induced significant p23 ζ, ZAP-70 phosphorylation, as well as association of the phosphoproteins between 30–46 kD (Fig. 7 A), even in the absence of a secondary cross-linking antibody. In contrast, the overall pattern induced by anti-CD3–Fos resembled the results seen in T cells stimulated with the anti-CD3– IgG3 mAb: specifically, a reduced association of phosphoproteins and barely detectable ZAP-70 phosphorylation. In the lysates of T cells stimulated with the bispecific antiCD3 × anti-CD4 construct, a large tyrosine-phosphorylated protein was observed that migrated just above the heavy chain. This phosphoprotein is likely to be p56 lck based on protein size. This band never appeared in the cross-linked anti-CD3 studies. One possible explanation for this difference is that in the absence of CD4 coaggregation, lck may dissociate from the TCR complex after lck phosphorylates its substrates. Whereas under stimulation conditions using the bispecific antibody, lck remains in the complex longer due to stable association with coaggregated CD4.


Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy.

Smith JA, Tso JY, Clark MR, Cole MS, Bluestone JA - J. Exp. Med. (1997)

Stimulation with anti-CD3 × anti-CD4 heterodimer results  in increased phosphorylation of proteins associated with the TCR complex and reconstitutes a mitogenic stimulus. (A) 107 pGL10 T cells were  stimulated with the anti-CD3–Fos F(ab)′2, or the bispecific F(ab)′2 (antiCD3 × anti-CD4) for 5 min at 37°C. Samples were precipitated with  anti-ζ, and blots were probed with anti-phosphotyrosine as in Fig. 2 A.  (B) Whole spleen (top) or pGL10 T cells (bottom) were cultured with serial  log dilutions of anti-CD3–Fos homodimer (open diamonds) or bispecific  anti-CD3 × anti-CD4 (closed diamonds) for 48 h. Data is representative of  two (A) and three (B) separate experiments.
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Related In: Results  -  Collection

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Figure 7: Stimulation with anti-CD3 × anti-CD4 heterodimer results in increased phosphorylation of proteins associated with the TCR complex and reconstitutes a mitogenic stimulus. (A) 107 pGL10 T cells were stimulated with the anti-CD3–Fos F(ab)′2, or the bispecific F(ab)′2 (antiCD3 × anti-CD4) for 5 min at 37°C. Samples were precipitated with anti-ζ, and blots were probed with anti-phosphotyrosine as in Fig. 2 A. (B) Whole spleen (top) or pGL10 T cells (bottom) were cultured with serial log dilutions of anti-CD3–Fos homodimer (open diamonds) or bispecific anti-CD3 × anti-CD4 (closed diamonds) for 48 h. Data is representative of two (A) and three (B) separate experiments.
Mentions: The inability of nonmitogenic anti-CD3 to trigger specific downstream events and proliferation most likely stems from the defective proximal events observed involving ζ and ZAP- 70. Previous studies have suggested that the src family kinase, lck, plays a crucial role in the phosphorylation of ζ, which subsequently allows association and phosphorylation of ZAP-70 (26, 27). Thus, it was possible that the differences in ζ and ZAP-70 phosphorylation seen upon the addition of cross-linker to anti-CD3 may have reflected increased lck activation or enhanced recruitment to the TCR. Initial experiments examining lck activation by monitoring lck tyrosine phosphorylation revealed no differences between cross-linking and noncross-linking conditions. It is clear that CD4 associates with lck and can interact with the TCR complex inducibly upon TCR ligation of antigen– MHC. Thus, artificially bringing CD4–lck into the TCR complex might reconstitute a mitogenic anti-CD3 stimulus, even in the absence of a secondary cross-linking Ab. To test this hypothesis, we took advantage of a bispecific anti-CD3 × anti-CD4 reagent prepared by a molecular approach to insure the presence of monovalent arms specific for CD3 and CD4 (as described in Materials and Methods). T cells were incubated with anti-CD3–Fos homodimer or the anti-CD3 × anti-CD4 bispecific F(ab)′2, lysed, and the TCR–CD3 complex was then immunoprecipitated and analyzed. The bispecific construct induced significant p23 ζ, ZAP-70 phosphorylation, as well as association of the phosphoproteins between 30–46 kD (Fig. 7 A), even in the absence of a secondary cross-linking antibody. In contrast, the overall pattern induced by anti-CD3–Fos resembled the results seen in T cells stimulated with the anti-CD3– IgG3 mAb: specifically, a reduced association of phosphoproteins and barely detectable ZAP-70 phosphorylation. In the lysates of T cells stimulated with the bispecific antiCD3 × anti-CD4 construct, a large tyrosine-phosphorylated protein was observed that migrated just above the heavy chain. This phosphoprotein is likely to be p56 lck based on protein size. This band never appeared in the cross-linked anti-CD3 studies. One possible explanation for this difference is that in the absence of CD4 coaggregation, lck may dissociate from the TCR complex after lck phosphorylates its substrates. Whereas under stimulation conditions using the bispecific antibody, lck remains in the complex longer due to stable association with coaggregated CD4.

Bottom Line: However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies.However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of zeta (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase.Finally, a bispecific anti-CD3 X anti-CD4 F(ab)'2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3.

View Article: PubMed Central - PubMed

Affiliation: Ben May Institute for Cancer Research, Department of Pathology, Chicago, Illinois 60637, USA.

ABSTRACT
Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonnmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including zeta chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of zeta (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cgamma-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 X anti-CD4 F(ab)'2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3.

Show MeSH
Related in: MedlinePlus