Anti-CD3 antibody therapy induces long-term immune tolerance in animal models of autoimmunity and is clinically validated (teplizumab delays onset of stage-2 type 1 diabetes), yet how the treatment converts transient T-cell deletion into durable, organ-level tolerance in vivo remains incompletely resolved. Perruche et al. (Nature Medicine, 2008) established the core axis: intact anti-CD3 causes T-cell apoptosis; macrophages and immature dendritic cells ingesting the apoptotic T cells produce transforming growth factor-β (TGF-β), which induces Foxp3+ regulatory T cells; and depleting these phagocytes both abrogates anti-CD3 prevention of experimental autoimmune encephalomyelitis (EAE) and reverses therapy of established relapsing-remitting disease. Those findings left a coherent mechanistic question open: by which molecular and cellular machinery is the anti-CD3-induced rise in circulating latent TGF-β converted into locally active TGF-β in target tissues; how is this apoptotic-cell-clearance axis weighted and temporally ordered against apoptosis-independent mechanisms of anti-CD3 tolerance (partial T-cell receptor signaling, induction of the inhibitory receptor PD-1, regulatory T-cell niche expansion), particularly for nonmitogenic anti-CD3 formats whose efficacy shows only partial phagocyte dependence; whether Foxp3+ regulatory T cells accumulating in target organs such as the central nervous system are locally functional suppressors or peripheral correlates; and how tolerance induction and maintenance divide the labor of apoptotic-cell clearance and phagocyte-derived TGF-β. Later work defines general latent TGF-β activation routes and documents several auxiliary anti-CD3 mechanisms, but no study assigns the in vivo activation route, mechanism-dominance hierarchy, stage architecture, or organ-level regulatory T cell function within this axis; the problem remains open.

Background

Anti-CD3 antibodies bind the CD3 signaling chains of the T-cell receptor (TCR) complex on T lymphocytes and were among the first monoclonal antibodies shown to restore self-tolerance in established autoimmunity. Early intact (mitogenic) antibodies, such as the murine clone 145-2C11, strongly crosslink the TCR complex, causing transient cytokine release and widespread T-cell deletion by apoptosis — programmed cell death — whereas Fc-engineered nonmitogenic formats deliver a weaker, partial TCR signal without mitogenic cytokine release (Smith et al., J Exp Med 1997) and have reached the clinic: teplizumab delays onset of stage-2 type 1 diabetes (Herold et al., NEJM 2019). Experimental autoimmune encephalomyelitis (EAE) is the standard mouse model of multiple sclerosis; immunization with myelin antigens such as myelin oligodendrocyte glycoprotein (MOG) peptide produces acute disease in C57BL/6 mice and relapsing-remitting disease in SJL mice.

Perruche et al. (Nature Medicine, 2008) connected the two hallmark effects of intact anti-CD3 — depletion and tolerance — through efferocytosis, the phagocytic clearance of apoptotic cells. After antibody-induced T-cell apoptosis, macrophages and immature dendritic cells (iDCs, antigen-presenting cells that have not yet matured) ingest the apoptotic T cells and secrete transforming growth factor-β (TGF-β); iDCs that had ingested apoptotic T cells converted naive CD4+ T cells into Foxp3+ regulatory T cells (Tregs, suppressive T cells whose lineage is specified by the transcription factor Foxp3). Anti-CD3 raised circulating, largely latent TGF-β — TGF-β is always secreted as an inactive complex in which the mature cytokine is shielded by its latency-associated peptide (LAP) and must be activated to signal, typically by αv-integrins (cell-adhesion receptors that mechanically unfurl the latent complex, notably αvβ6 and αvβ8), thrombospondin-1, or proteases — and this rise required phagocytes. Depleting macrophages and iDCs with clodronate liposomes (a standard macrophage-depletion reagent) abrogated anti-CD3-mediated prevention of MOG-induced EAE and reversed the therapeutic effect on established relapsing-remitting EAE. The backdrop included TGF-β-dependence of anti-CD3 remission of diabetes (Belghith et al., Nat Med 2003) and the observation that myelin-specific Foxp3+ Tregs accumulate in the inflamed CNS yet fail to control it (Korn et al., Nat Med 2007). The study also deployed a nonmitogenic anti-CD3 IgG3 antibody, whose tolerogenic effect showed only partial dependence on phagocytes, implying additional apoptosis-independent mechanisms. The paper thus left a coherent set of mechanistic questions open, concerning local activation of the latent TGF-β, the dominance and timing of clearance-dependent versus clearance-independent mechanisms, the functional competence of organ-infiltrating Tregs, and the division of labor between tolerance induction and maintenance.

Problem Statement

Determine, by causal in vivo experimentation, how anti-CD3 antibody therapy converts T-cell apoptosis into durable systemic immune tolerance, resolving four source-faithful facets of one mechanism:

(1) Activation and location: by which molecular machinery (e.g., αv-integrins such as αvβ6/αvβ8, thrombospondin-1, proteases, pH/redox-dependent mechanisms) and in which cellular and anatomical contexts is the anti-CD3-induced rise in phagocyte-derived latent TGF-β converted into locally active TGF-β within target tissues, and which of these contexts are necessary for tolerance?

(2) Mechanism dominance and timing: what are the relative contributions and temporal ordering of the apoptotic-cell clearance/phagocyte-TGF-β axis versus apoptosis-independent mechanisms of anti-CD3 tolerance (partial TCR signaling/anergy, induction of the inhibitory receptor PD-1, regulatory T-cell niche expansion, altered trafficking)? In particular, for nonmitogenic anti-CD3 formats whose efficacy shows only partial phagocyte dependence, which mechanism(s) dominate tolerance induction and which sustain maintenance, relative in time to the clearance of apoptotic T cells?

(3) Organ-level Treg function: are the Foxp3+ regulatory T cells that increase in target organs such as the central nervous system after anti-CD3 treatment functionally competent local suppressors of inflammation in vivo, or a peripheral consequence of systemic tolerance?

(4) Stage architecture: how do apoptotic-T-cell clearance and phagocyte-derived TGF-β divide their contributions between the induction phase and the maintenance phase of tolerance, and can phagocyte subsets or their TGF-β production be manipulated therapeutically without deleterious systemic effects?

All four facets are jointly required: a proper subset is not sufficient, and both components of facet (4) — phase division and therapeutic manipulability — must be addressed.

References

  1. Perruche S, Zhang P, Liu Y, Saas P, Bluestone JA, Chen W. CD3-specific antibody–induced immune tolerance involves transforming growth factor-β from phagocytes digesting apoptotic T cells. Nature Medicine 2008;14(5):528–535. DOI 10.1038/nm1749. https://doi.org/10.1038/nm1749
  2. Korn T, Reddy J, Gao W, Bettelli E, Awasthi A, Petersen TR, Bäckström BT, Sobel RA, Wucherpfennig KW, Strom TB, Oukka M, Kuchroo VK. Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nature Medicine 2007;13(4):423–431. DOI 10.1038/nm1564. https://doi.org/10.1038/nm1564
  3. Belghith M, Bluestone JA, Barriot S, Mégret J, Bach JF, Chatenoud L. TGF-β–dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nature Medicine 2003;9(9):1202–1208. DOI 10.1038/nm924. https://doi.org/10.1038/nm924
  4. Perruche S, Zhang P, Maruyama T, Bluestone JA, Saas P, Chen W. Lethal effect of CD3-specific antibody in mice deficient in TGF-β1 by uncontrolled flu-like syndrome. Journal of Immunology 2009;183(2):953–961. DOI 10.4049/jimmunol.0804076. https://doi.org/10.4049/jimmunol.0804076
  5. Chen H, Kasagi S, Chia C, Zhang D, Tu E, Wu R, Zanvit P, Goldberg N, Jin W, Chen W. Extracellular Vesicles from Apoptotic Cells Promote TGFβ Production in Macrophages and Suppress Experimental Colitis. Scientific Reports 2019;9:5875. DOI 10.1038/s41598-019-42063-7. https://doi.org/10.1038/s41598-019-42063-7
  6. Worthington JJ, Kelly A, Smedley C, Bauché D, Campbell S, Marie JC, Travis MA. Integrin αvβ8-Mediated TGF-β Activation by Effector Regulatory T Cells Is Essential for Suppression of T-Cell-Mediated Inflammation. Immunity 2015;42(5):903–915. DOI 10.1016/j.immuni.2015.04.012. https://doi.org/10.1016/j.immuni.2015.04.012
  7. Travis MA, Sheppard D. TGF-β activation and function in immunity. Annual Review of Immunology 2014;32:51–82. DOI 10.1146/annurev-immunol-032713-120257. https://doi.org/10.1146/annurev-immunol-032713-120257
  8. Chen W. TGF-β Regulation of T Cells. Annual Review of Immunology 2023;41:483–512. DOI 10.1146/annurev-immunol-101921-045939. https://doi.org/10.1146/annurev-immunol-101921-045939
  9. Smith JA, Tso JY, Clark MR, Cole MS, Bluestone JA. Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy. Journal of Experimental Medicine 1997;185(8):1413–1422. DOI 10.1084/jem.185.8.1413. https://doi.org/10.1084/jem.185.8.1413
  10. Kohm AP, Williams JS, Bickford AL, McMahon JS, Chatenoud L, Bach JF, Bluestone JA, Miller SD. Treatment with nonmitogenic anti-CD3 monoclonal antibody induces CD4+ T cell unresponsiveness and functional reversal of established experimental autoimmune encephalomyelitis. Journal of Immunology 2005;174(8):4525–4534. DOI 10.4049/jimmunol.174.8.4525. https://doi.org/10.4049/jimmunol.174.8.4525
  11. Shiheido H, Aoyama T, Takahashi H, Hanaoka K, Abe T, Nishida E, Chen C, Koga O, Hikida M, Shibagaki Y, Morita A, Nikawa T, Hattori S, Watanabe T, Shimizu J. Novel CD3-specific antibody induces immunosuppression via impaired phosphorylation of LAT and PLCγ1 following T-cell stimulation. European Journal of Immunology 2014;44(6):1770–1780. DOI 10.1002/eji.201344146. https://doi.org/10.1002/eji.201344146
  12. Nishio J, Feuerer M, Wong J, Mathis D, Benoist C. Anti-CD3 therapy permits regulatory T cells to surmount T cell receptor–specified peripheral niche constraints. Journal of Experimental Medicine 2010;207(9):1879–1889. DOI 10.1084/jem.20100205. https://doi.org/10.1084/jem.20100205
  13. Wallberg M, Recino A, Phillips J, Howie D, Vienne M, Paluch C, Azuma M, Wong FS, Waldmann H, Cooke A. Anti-CD3 treatment up-regulates programmed cell death protein-1 expression on activated effector T cells and severely impairs their inflammatory capacity. Immunology 2017;151(2):248–260. DOI 10.1111/imm.12729. https://doi.org/10.1111/imm.12729
  14. Kuhn C, Weiner HL. Therapeutic anti-CD3 monoclonal antibodies: from bench to bedside. Immunotherapy 2016;8(8):889–906. DOI 10.2217/imt-2016-0049. https://doi.org/10.2217/imt-2016-0049
  15. Herold KC, Bundy BN, Long SA, Bluestone JA, DiMeglio LA, Dufort MJ, Gitelman SE, Gottlieb PA, Krischer JP, Linsley PS, Marks JB, Moore W, Moran A, Rodriguez H, Russell WE, Schatz D, Skyler JS, Tsalikian E, Wherrett DK, Ziegler AG, Greenbaum CJ; Type 1 Diabetes TrialNet Study Group. An Anti-CD3 Antibody, Teplizumab, in Relatives at Risk for Type 1 Diabetes. New England Journal of Medicine 2019;381(7):603–613. DOI 10.1056/NEJMoa1902226. https://doi.org/10.1056/NEJMoa1902226
  16. O'Connor RA, Anderton SM. Foxp3+ regulatory T cells in the control of experimental CNS autoimmune disease. Journal of Neuroimmunology 2008;193(1-2):1–11. DOI 10.1016/j.jneuroim.2007.11.016. https://doi.org/10.1016/j.jneuroim.2007.11.016
  17. Korn T. Foxp3+ regulatory T cells in the central nervous system and other nonlymphoid tissues. European Journal of Immunology 2023;53(8):e2250227. DOI 10.1002/eji.202250227. https://doi.org/10.1002/eji.202250227
  18. Marin-Rodero M, Cintado E, Walker AJ, Jayewickreme T, Pinho-Ribeiro FA, Richardson Q, Jackson R, Chiu IM, Benoist C, Stevens B, Trejo JL, Mathis D. The meninges host a distinct compartment of regulatory T cells that preserves brain homeostasis. Science Immunology 2025;10(104):eadu2910. DOI 10.1126/sciimmunol.adu2910. https://doi.org/10.1126/sciimmunol.adu2910
  19. Woodward KB, Zhao H, Shrestha P, Batra L, Tan M, Grimany-Nuno O, Bandura-Morgan L, Askenasy N, Shirwan H, Yolcu ES. Pancreatic islets engineered with a FasL protein induce systemic tolerance at the induction phase that evolves into long-term graft-localized immune privilege. American Journal of Transplantation 2020;20(5):1285–1295. DOI 10.1111/ajt.15747. https://doi.org/10.1111/ajt.15747
  20. Saas P, Daguindau E, Perruche S. Concise Review: Apoptotic Cell-Based Therapies—Rationale, Preclinical Results and Future Clinical Developments. Stem Cells 2016;34(6):1464–1473. DOI 10.1002/stem.2361. https://doi.org/10.1002/stem.2361
  21. Kuang R, Perruche S, Chen W. Apoptotic cell-linked immunoregulation: implications for promoting immune tolerance in transplantation. Cell & Bioscience 2015;5:27. DOI 10.1186/s13578-015-0019-9. https://doi.org/10.1186/s13578-015-0019-9
  22. Kasagi S, Wang D, Zhang P, Zanvit P, Chen H, Zhang D, Li J, Che L, Maruyama T, Nakatsukasa H, Wu R, Jin W, Sun L, Chen W. Combination of apoptotic T cell induction and self-peptide administration for therapy of experimental autoimmune encephalomyelitis. EBioMedicine 2019;44:50–59. DOI 10.1016/j.ebiom.2019.05.005. https://doi.org/10.1016/j.ebiom.2019.05.005
  23. Mathieu C, Sims EK, Chatenoud L, James EA, Atkinson MA, Herold KC. Toward Disease-Modifying Therapies in Type 1 Diabetes: Focus on Teplizumab. Diabetes Care 2026;49(3):365–374. DOI 10.2337/dci25-0066. https://doi.org/10.2337/dci25-0066