Mixed chimerism — the coexistence of donor and recipient hematopoietic cells — can induce donor-specific transplant tolerance, and co-transferred regulatory T cells (Treg) enable it with markedly reduced recipient conditioning in mice. However, the mechanisms that maintain this tolerant state remain incompletely characterized. The founding murine protocol (retinoic-acid-induced alloantigen-specific Treg plus non-myeloablative bone-marrow transplantation, Ruiz et al. 2015) left four linked questions open: (1) what is the origin, tissue distribution, and functional contribution of the donor-derived Tregs that appear in tolerant chimeras; (2) why donor myeloid (macrophage/granulocyte) chimerism declines over time while lymphoid chimerism is stable, and whether this lineage shift affects long-term tolerance; (3) whether performing the organ transplant earlier after bone-marrow transplantation, when chimerism is higher, increases tolerance incidence; and (4) how to differentiate human CD4+ T cells into stable, functional, donor-specific Tregs suitable for a clinically translatable chimerism-induction protocol. Later literature (reviews through 2023 and first-in-human donor-antigen-specific Treg dosing) advances parts of the translational agenda but resolves none of the mechanistic cores, which remain open.

Background

Transplantation tolerance — durable acceptance of a donor graft without chronic immunosuppression — is a central goal of transplant immunology. The most reproducible route in both mice and humans is mixed chimerism: transplanting donor hematopoietic stem cells so donor and recipient blood cells coexist, which extends the recipient's own self-tolerance machinery (deletion of alloreactive T cells, anergy, and active regulation by regulatory T cells) to donor antigens. Regulatory T cells (Tregs) are CD4+ T lymphocytes expressing the transcription factor FoxP3 that suppress other immune cells; induced Tregs (iTregs) can be generated from naive CD4+ T cells in culture. Ruiz et al. (Frontiers in Immunology, 2015) built a murine protocol in which iTregs generated with all-trans retinoic acid (RA-iTregs) and specific for donor alloantigens were infused together with a non-myeloablative bone-marrow transplant (NM-BMT: low-dose total-body irradiation, rapamycin, and the costimulation blocker CTLA4-Ig) into MHC-mismatched mice (donor C57BL/6, H-2b; recipient BALB/c, H-2d; MHC molecules are the major histocompatibility complex antigens that trigger graft rejection). This produced stable multilineage mixed chimerism and greatly prolonged donor-specific skin allograft survival without further immunosuppression, though not 100% graft survival. Tolerant chimeras contained donor-derived (H-2Kb+, i.e. positive for H-2Kb, the MHC class I molecule of the H-2b donor strain, used as a donor-cell marker) CD4+FoxP3+ Tregs in blood and spleen that correlated with graft acceptance without reaching statistical significance, and donor myeloid cells (monocytes/granulocytes) declined over 16 weeks while donor lymphoid cells persisted — two observations whose mechanistic meaning the authors could not settle. The paper closes by asking whether transplanting skin earlier after BMT, when chimerism levels are higher, would improve tolerance rates, and states that translating the protocol to induce donor-specific tolerance in humans is possible since several protocols to differentiate human CD4+ T cells into Tregs have been described, while the donor-specific human Treg application needed for a clinical chimerism protocol remains to be achieved. Subsequent field reviews (Pilat et al. 2017; Mahr and Wekerle 2017; Pathak and Meyer 2021; Lowsky and Strober 2022; Weijler and Wekerle 2023; Mengrelis et al. 2023) confirm that chimerism-based tolerance works clinically but that the mechanisms maintaining it — particularly the relative roles of clonal deletion versus regulation, host versus donor Tregs, and transient versus persistent chimerism — are still being delineated; clinical tolerance failure despite persistent chimerism is attributed to insufficient regulation, and donor-antigen-specific human Tregs have only just entered early trials.

Problem Statement

For donor-specific transplant tolerance established in MHC-mismatched mice by RA-induced alloantigen-specific Treg-facilitated non-myeloablative bone-marrow transplantation (Ruiz et al. 2015), determine the mechanisms that induce and maintain the tolerant state, and enable its translation, by answering four linked questions: (1) Donor-derived Tregs: what is the origin of the donor-derived CD4+FoxP3+ Tregs found in tolerant chimeras (de novo generation from donor hematopoietic precursors, peripheral conversion of mature donor T cells), what is their distribution across graft and lymphoid/non-lymphoid tissues in mice that accept versus reject their allografts, and what is their functional contribution — for example, as demonstrated by lineage-specific depletion or deficiency — to maintaining tolerance, as distinct from the contribution of host Tregs? (2) Lineage dynamics of chimerism: why do donor-derived myeloid cells (monocytes/macrophages and granulocytes) decline over time while donor lymphoid cells remain stable, and does this lineage-specific shift in chimerism causally affect, or merely accompany, the long-term stability of tolerance? (3) Protocol timing: does performing the allogeneic skin transplant at an earlier time after BMT, when mixed-chimerism levels are higher, increase the incidence of durable graft acceptance — i.e., is chimerism level at the time of transplantation a causal determinant of tolerance induction, and over what time window? (4) Human translation: can human CD4+ T cells be differentiated into donor-antigen-specific Tregs that are phenotypically stable and functionally suppressive under conditions compatible with clinical-grade manufacture and capable of substituting for murine RA-iTregs in a reduced-conditioning chimerism protocol? All four questions are jointly required, and the common objective uniting them is a mechanistic account of how Treg-facilitated mixed chimerism establishes and maintains donor-specific tolerance: the task is complete when each of the four questions is answered specifically with a proposed mechanism, causal determination, or feasibility finding that is grounded in the cited literature and accompanied by the discriminating experiment or observation that would confirm or refute it.

References

  1. Ruiz P, Maldonado P, Hidalgo Y, Sauma D, Rosemblatt M, Bono MR. Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance. Frontiers in Immunology 2015;6:596. DOI: 10.3389/fimmu.2015.00596, https://doi.org/10.3389/fimmu.2015.00596
  2. Weijler A, Wekerle T. Combining Treg Therapy With Donor Bone Marrow Transplantation: Experimental Progress and Clinical Perspective. Transplantation 2023;108(5):1100-1108. DOI: 10.1097/TP.0000000000004814, https://doi.org/10.1097/TP.0000000000004814
  3. Mengrelis K, Muckenhuber M, Wekerle T. Chimerism-based Tolerance Induction in Clinical Transplantation: Its Foundations and Mechanisms. Transplantation 2023;107(12):2473-2485. DOI: 10.1097/TP.0000000000004589, https://doi.org/10.1097/TP.0000000000004589
  4. Lowsky R, Strober S. Establishment of Chimerism and Organ Transplant Tolerance in Laboratory Animals: Safety and Efficacy of Adaptation to Humans. Frontiers in Immunology 2022;13:805177. DOI: 10.3389/fimmu.2022.805177, https://doi.org/10.3389/fimmu.2022.805177
  5. Pilat N, Granofszky N, Wekerle T. Combining Adoptive Treg Transfer with Bone Marrow Transplantation for Transplantation Tolerance. Current Transplantation Reports 2017;4(4):253-261. DOI: 10.1007/s40472-017-0164-7, https://doi.org/10.1007/s40472-017-0164-7
  6. Mahr B, Wekerle T. Murine models of transplantation tolerance through mixed chimerism: advances and roadblocks. Clinical and Experimental Immunology 2017;189(2):181-189. DOI: 10.1111/cei.12976, https://doi.org/10.1111/cei.12976
  7. Pathak S, Meyer EH. Tregs and Mixed Chimerism as Approaches for Tolerance Induction in Islet Transplantation. Frontiers in Immunology 2021;11:612737. DOI: 10.3389/fimmu.2020.612737, https://doi.org/10.3389/fimmu.2020.612737
  8. Candia E, Reyes P, Covian C, Rodriguez F, Wainstein N, Morales J, Mosso C, Rosemblatt M, Fierro JA. Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells. PLoS One 2017;12(7):e0182009. DOI: 10.1371/journal.pone.0182009, https://doi.org/10.1371/journal.pone.0182009
  9. Guinan EC, Contreras-Ruiz L, Crisalli K, Rickert C, Rosales I, Makar R, Colvin R, Geissler EK, Sawitzki B, Harden P, Tang Q, Blancho G, Turka LA, Markmann JF. Donor antigen-specific regulatory T cell administration to recipients of live donor kidneys: A ONE Study consortium pilot trial. American Journal of Transplantation 2023;23(12):1872-1881. DOI: 10.1016/j.ajt.2023.06.012, https://doi.org/10.1016/j.ajt.2023.06.012
  10. Tonsho M, O JM, Ahrens K, Robinson K, Sommer W, Boskovic S, Patel PM, Becerra DC, Huh KH, Miller CL, Dehnadi A, Hanekamp I, Rosales IA, Colvin RB, Sachs DH, Alessandrini A, Cosimi A, Fairchild RL, Cravedi P, Bin S, Heeger PS, Allan JS, Kawai T, Benichou G, Madsen JC. Cardiac allograft tolerance can be achieved in nonhuman primates by donor bone marrow and kidney cotransplantation. Science Translational Medicine 2025;17(782):eads0255. DOI: 10.1126/scitranslmed.ads0255, https://doi.org/10.1126/scitranslmed.ads0255
  11. Bono MR, Tejon G, Flores-Santibañez F, Fernandez D, Rosemblatt M, Sauma D. Retinoic Acid as a Modulator of T Cell Immunity. Nutrients 2016;8(6):349. DOI: 10.3390/nu8060349, https://doi.org/10.3390/nu8060349