Since its humble beginnings with donor-specific transfusion therapies in the 1970s, the development of cell-based therapies to promote antigen (Ag)-specific immunosuppression/tolerance aiming to prevent or to treat transplant rejections or autoimmune disorders has mainly focused on the ex vivo generation of polyclonal or Ag-specific regulatory T cells (Tregs), regulatory dendritic cells, and more recently on regulatory B cells.1,2 The introduction of chimeric Ag receptors via genetic engineering of in vitro–generated Tregs has opened a new horizon in search of Ag-specific cell-based therapies.3 However, the potential therapeutic use of immunoregulatory properties of monocyte/macrophages, including M2 and regulatory types (regulatory macrophages [Mregs]), has not received the same level of attention.4
In a recent issue of Nature Communications, Riquelme and colleagues from Ed Geissler’s and James Hutchinson’s group published a remarkable study on the immunosuppressive capacity of in vitro–generated human Mregs and their direct effects on alloreactive T cells. The extensive study included in vitro, experimental, and clinical models.5 Mregs were generated from purified CD14 peripheral blood monocytes cultured over 6 days in medium supplemented with human AB serum and macrophage colony-stimulating factor, followed by a 1-day pulse with interferon-γ. Fully developed Mregs retained the expression of generic myeloid-lineage markers and phagocytic ability, expressed HLA-DR, the chemokine receptor CX3CR1, and markers of tissue-resident macrophages, however, lacked molecules typically present in human conventional dendritic cell (DC)1 or DC2. When used as Ag-presenting cells in mixed leukocyte cultures, human Mregs increased the percentage of CD25 FoxP3 CD4 T cells, the main source of interleukin (IL)-10 in the mixed leukocyte culture supernatants. The assessment of Treg-associated transcription factors, Treg-specific epigenetic patterns, and T-cell subset phenotypes suggested that Mreg-induced Tregs (iTregs) originated from FoxP3neg CD4 T cells, rather than from expansion of naturally occurring Tregs (nTregs). Mreg-mediated generation of Tregs required Ag-presenting cell:T-cell contact, and was dependent on the signaling of T-cell receptor, CD28 and Notch; Mreg-produced indoleamine 2,3-dioxygenase and transforming growth factor-β1, and required the presence of IL-2 and retinoid acid in the culture medium. Moreover, Treg generation by Mregs was also dependent on the presence of progestogen-associated endometrial protein or glycodelin-A, a glycoprotein that plays a key role in fetomaternal tolerance selectively upregulated by Mregs.6 By contrast, the increase in Tregs was IL-10 independent, consistent with the finding that resting and lipopolysaccharide-stimulated Mregs secreted less IL-10 than activated macrophages.
Importantly, T cells previously cocultured with allogeneic Mregs suppressed CD3 antibody (Ab)–induced proliferation of allospecific T cells and inhibited maturation of allogeneic monocyte–derived DCs, although both phenomena occurred at relatively high suppressor:responder cell ratios. Although the initial activation of alloreactive T cells by Mregs is allospecific, Mreg cocultured T cells also exert in vitro short-term nonspecific bystander suppression of T-cell proliferation.
Clinically, the authors demonstrated that Mreg-iTregs can be expanded exponentially by restimulation with CD3 and CD28 agonistic Abs plus IL-2. Nevertheless, the percentage of FoxP3pos cells declined considerably (from 69.6 ± 14.2% to 36.6 ± 5.2%) over the 3-week period of cell expansion.
In Riquelme’s study, analysis of Treg-associated markers revealed that Mreg-iTregs differ from freshly isolated nTregs and iTregs expanded by other methods, a phenomenon that could be linked to the specific phenotype and transcriptomic profiles of Mregs. The authors propose that molecules expressed by Mreg-iTregs may also serve as biomarkers to assess the interaction between recipient T cells and donor-derived Mregs. The B7-related immunoregulatory molecule ~37-kD isoform of “butyrophilin-like protein 8” and the coinhibitor receptor of the CD28 family “T-cell immunoreceptor with Ig and ITIM domains” (TIGIT) have identified as potential biomarkers of Mreg-iTregs before.7–9 Importantly, upregulation of butyrophilin-like protein 8 and TIGIT by human T cells after intravenous administration of allogeneic Mregs was confirmed in vivo in humanized mice.
The study also refers to the Mreg-containing cell product Mreg_UKR. This product has been developed to reduce maintenance immunosuppression following kidney transplantation and is currently tested in the ONEmreg12 trial. In this phase I/II trial, patients received donor-derived Mregs 7 days before transplantation; patients did not receive an induction treatment and were kept on a triple maintenance immunosuppression. Four of 5 patients treated with donor Mregs (2 from the ONEmreg12 trial and 3 from a previous trial) exhibited a significant increase in the percentage of circulating TIGIT Tregs. Serial T-cell receptor spectratyping conducted in 1 kidney recipient infused with donor Mregs revealed expansion and persistence (up to day 168) of oligoclonal or monoclonal TIGIT Tregs. On the basis of these promising results, it will be important to ascertain whether donor Mreg-based therapies will further decrease (1) antidonor responses, (2) if the effect is donor specific, and (3) if administration of Mreg_UKR allows reducing maintenance immunosuppression in kidney transplant patients. The survival of systemically administered donor-derived Mregs may be another issue to consider for long-term therapeutic effects, since regulatory cells of myeloid lineage exhibit shorter lifespans than their lymphoid counterparts, such as Tregs and regulatory B cells.10 Thus, repetitive administration of donor Mregs may be required to achieve maximal effects.
In summary, the meticulous analysis of the immunoregulatory properties of human Mregs by Riquelme and coworkers has opened a new area of research in the already complex arena of tolerogenic cell-based therapies.
1. Wood KJ, Bushell A, Hester J. Regulatory immune cells in transplantation. Nat Rev Immunol. 2012;12(6):417–430.
2. Morelli AE, Thomson AW. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol. 2007;7(8):610–621.
3. MacDonald KG, Hoeppli RE, Huang Q, et al. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor. J Clin Invest. 2016;126(4):1413–1424.
4. Hutchinson JA, Riquelme P, Sawitzki B, et al. Cutting edge: immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients. J Immunol. 2011;187(5):2072–2078.
5. Riquelme P, Haarer J, Kammler A, et al. TIGIT+ itregs elicited by human regulatory macrophages control T cell immunity. Nat Commun. 2018;9(1):2858.
6. Lee CL, Lam KK, Vijayan M, et al. The pleiotropic effect of glycodelin-A in early pregnancy. Am J Reprod Immunol. 2016;75(3):290–297.
7. Rhodes DA, Reith W, Trowsdale J. Regulation of immunity by butyrophilins. Annu Rev Immunol. 2016;34:151–172.
8. Anderson AC, Joller N, Kuchroo VK. Lag-3, tim-3, and TIGIT: co-inhibitory receptors with specialized functions in immune regulation. Immunity. 2016;44(5):989–1004.
9. Kurtulus S, Sakuishi K, Ngiow SF, et al. TIGIT predominantly regulates the immune response via regulatory T cells. J Clin Invest. 2015;125(11):4053–4062.
10. Divito SJ, Wang Z, Shufesky WJ, et al. Endogenous dendritic cells mediate the effects of intravenously injected therapeutic immunosuppressive dendritic cells in transplantation. Blood. 2010;116(15):2694–2705.
The meticulous analysis of the immuno-regulatory properties of human Mregs by Riquelme and coworkers has opened a new area of research in the already complex arena of tolerogenic cell-based therapies.