Type 1 Regulatory T Cell

A type 1 regulatory T cell (Tr1 cell) is an adaptive, peripherally induced subset of CD4⁺ regulatory T lymphocytes that lacks constitutive expression of the transcription factor FOXP3, co-expresses the cell-surface markers CD49b (integrin α₂) and LAG-3 (CD223), and enforces peripheral tolerance and infectious tolerance primarily through the antigen-triggered secretion of high concentrations of interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β) alongside perforin/granzyme B–mediated cytolysis of myeloid antigen-presenting cells. First discovered in 1997 by Hervé Groux, Anne O'Garra, René de Waal Malefyt, and Maria Grazia Roncarolo in severe combined immunodeficiency (SCID) patients who developed long-term mixed chimerism and transplantation tolerance without graft-versus-host disease (GvHD) after HLA-mismatched fetal liver hematopoietic stem cell transplantation, Tr1 cells constitute the primary FOXP3-independent arm of human peripheral immunoregulation. Unlike thymic CD4⁺CD25⁺FOXP3⁺ regulatory T cells (tT_reg), which arise during thymic selection to prevent systemic autoimmunity against self-antigens, Tr1 cells differentiate in peripheral tissues from naive or memory CD4⁺ T cells upon repeated antigen stimulation in the presence of IL-10 and IL-27 delivered by tolerogenic dendritic cells (such as human DC-10 cells).

For fifteen years after their discovery in 1997, purifying viable Tr1 cells was hindered by the absence of unique surface markers, forcing investigators to rely on intracellular cytokine staining (IL-10^high IL-4⁻ IFN-γ^+/int IL-2^low/−). In 2013, Nicola Gagliani, Silvia Gregori, Richard A. Flavell, and Maria Grazia Roncarolo demonstrated in Nature Medicine that co-expression of CD49b (the α₂ integrin subunit of the collagen receptor VLA-2) and lymphocyte-activation gene 3 (LAG-3 / CD223) uniquely identifies both human and murine functional Tr1 cells across peripheral blood, gut mucosa, and tolerant allografts.

Although Tr1 cells lack a single lineage-restricted master transcription factor analogous to FOXP3, work by Vijay K. Kuchroo, Francisco J. Quintana, Amit Awasthi, and Geoffrey R. Hill established the cooperative transcriptional circuit that activates the IL10 and GZMB loci while repressing IL2 and IL4:

1. Interleukin-27 (IL-27) Priming via STAT1/STAT3, IRF1, and BATF

While IL-10 was originally used by Groux and Roncarolo to induce Tr1 cells in vitro, T cells express low baseline levels of the IL-10 receptor α chain, and IL-10 alone cannot sustain robust Tr1 expansion in purified T-cell cultures. In 2007, Amit Awasthi, Vijay Kuchroo, and colleagues discovered that interleukin-27 (IL-27)—a heterodimeric IL-12-family cytokine composed of p28 (IL-27α) and Ebi3 secreted by tolerogenic dendritic cells—is the primary physiological initiator of Tr1 differentiation. Ligation of the IL-27Rα (WSX-1) / gp130 receptor complex on naive CD4⁺ T cells activates STAT1 and STAT3:

• Pioneer Chromatin Opening: Early STAT1 and STAT3 signaling induces the pioneer transcription factors IRF1 (interferon regulatory factor 1) and BATF (basic leucine zipper ATF-like transcription factor) (Karwacz et al., 2017), which remodel chromatin accessibility across the Il10 promoter and intronic enhancers.
• Induction of c-Maf, AhR, and IL-21 Autocrine Amplification: STAT3 activates transcription of the proto-oncogene c-Maf, the ligand-activated aryl hydrocarbon receptor (AhR), and the cytokine IL-21 (Pot et al., 2009; Apetoh et al., 2010). Secreted IL-21 acts as an autocrine growth and amplification factor via STAT3 to sustain c-Maf expression and drive clonal expansion of differentiating Tr1 cells in the absence of IL-2.

2. The c-Maf / AhR Complex and Blimp-1 / Eomes Cooperative Control

Once induced by IL-27 and TGF-β or IDO1-derived L-kynurenine, AhR physically dimerizes with c-Maf inside the nucleus (Apetoh et al., 2010). The c-Maf:AhR heterodimer binds directly to adjacent MARE (Maf recognition elements) and XRE (xenobiotic response elements) in the IL10 and IL21 promoters to drive high-output IL-10 transcription. Simultaneously:

• Egr-2 and Blimp-1 (PRDM1): Induced downstream of IL-27 and STAT3, Egr-2 activates Blimp-1, which collaborates with c-Maf at the IL10 locus while directly repressing IL2 transcription and upregulating coinhibitory receptors (LAG-3, PD-1, TIM-3, TIGIT).
• Eomesodermin (EOMES): In 2017, Geoffrey R. Hill, Ping Zhang, and colleagues demonstrated that the T-box transcription factor Eomes acts in concert with T-bet and Blimp-1 to drive both IL10 and granzyme B (GZMB) transcription in human and murine alloreactive Tr1 cells during bone marrow transplantation.

Importantly, Tr1 cells must be activated through their T-cell receptor (TCR) by their specific cognate peptide–MHC class II ligand to become suppressive; once triggered by cognate antigen, however, they mediate antigen-non-specific bystander suppression and linked suppression across the local microenvironment through four mechanisms:

1. Paracrine IL-10 and TGF-β Conditioning of Antigen-Presenting Cells: Tr1-derived IL-10 signals through STAT3 in neighboring dendritic cells and macrophages to downregulate MHC class II, CD80, CD86, and IL-12p70/IL-23/TNF-α while upregulating ILT3, ILT4, and HLA-G. This converts immunogenic APCs into secondary DC-10 tolerogenic dendritic cells that in turn differentiate new cohorts of naive CD4⁺ T cells into Tr1 cells—propagating infectious tolerance.
2. Granzyme B and Perforin Cytolysis of Myeloid APCs (Magnani et al., 2011): Human Tr1 cells selectively kill CD14⁺ monocytes and myeloid dendritic cells (while sparing B cells, T cells, and non-myeloid parenchymal cells). Lysis is independent of TCR–MHC recognition on the target myeloid cell and instead requires simultaneous engagement of Tr1 activating receptors CD226 (DNAM-1), CD2, and LFA-1 (CD11a/CD18) by myeloid ligands CD155 (PVR)/CD112, CD58, and ICAM-1 (CD54), triggering directional degranulation of perforin-1 and granzyme B.
3. Cell-Contact Coinhibition via LAG-3, CTLA-4, and PD-1: Surface LAG-3 binds with high affinity to stable peptide–MHC class II complexes on dendritic cells, delivering an ITAM-mediated inhibitory signal via SHP-1 that arrests DC maturation, while CTLA-4 strips CD80/CD86 via trogocytosis.
4. Purinergic Metabolic Disruption (CD39 / CD73): Driven by AhR signaling, Tr1 cells express high levels of ectonucleotidases CD39 and CD73, hydrolyzing pro-inflammatory extracellular ATP released by damaged tissue into adenosine, which binds A_2A receptors on effector T cells to elevate intracellular cAMP and suppress proliferation.

• Allogeneic Hematopoietic Stem Cell Transplantation (ALT-TEN & T-allo10 Trials — Bacchetta et al., 2014; Roncarolo et al., 2021): In haploidentical and mismatched allogeneic HSCT for hematological malignancies, donor CD4⁺ T cells stimulated ex vivo with host IL-10-conditioned dendritic cells (DC-10) generate an alloantigen-specific Tr1-enriched cell product (T-allo10, containing 10%–15% CD49b⁺LAG-3⁺ Tr1 cells). Infusion of T-allo10 promotes rapid immune reconstitution against viral and leukemic antigens while preventing acute and chronic graft-versus-host disease (GvHD).
• Refractory Crohn's Disease (The CATS1 Trial — Desreumaux et al., 2012): Ovalbumin-specific human Tr1 clones (Ova-Treg) infused intravenously into patients with refractory Crohn's disease (who ingested an ovalbumin-containing diet to trigger local gut TCR activation of the infused Tr1 cells) demonstrated safety and dose-dependent clinical response.
• Lentiviral IL10-Engineered CD4⁺ T Cells (LV-IL-10 / CD4^IL-10): To obtain homogeneous, high-purity human Tr1 populations at clinical scale, Maria Grazia Roncarolo, Rosa Bacchetta, and Silvia Gregori engineered a bidirectional lentiviral vector encoding human IL10 and a marker gene (ΔNGFR). Transduced human CD4⁺ T cells (CD4^IL-10) acquire a stable Tr1 phenotype (CD49b⁺LAG-3⁺GZMB⁺), lyse CD13⁺/CD33⁺HLA-class-I⁺ acute myeloid leukemia (AML) blasts via granzyme B (preserving the graft-versus-leukemia effect), and prevent xenogeneic GvHD.

How do Type 1 regulatory T cells (Tr1) differ from classical FOXP3⁺ regulatory T cells?

Classical thymic and peripheral T_reg cells depend on constitutive expression of the transcription factor FOXP3 (and demethylation of the FOXP3 CNS2/TSDR region) alongside high surface CD25 (IL-2Rα) for their lineage stability and suppressive function. By contrast, Tr1 cells are FOXP3-negative (human Tr1 cells may transiently express low levels of FOXP3 immediately upon TCR activation, just like conventional effector T cells, but never demethylate the TSDR or depend on FOXP3). Instead, Tr1 cells are defined by surface co-expression of CD49b and LAG-3, transcriptional control by c-Maf, AhR, Blimp-1, and Eomes, extremely high IL-10 secretion, and granzyme B–mediated killing of myeloid antigen-presenting cells.

Why can engineered Tr1 cells (CD4^IL-10) prevent graft-versus-host disease (GvHD) while still killing acute myeloid leukemia (AML) cells?

In allogeneic bone marrow transplantation, conventional FOXP3⁺ T_reg cells suppress both GvHD and the beneficial graft-versus-leukemia (GvL) response. Tr1 cells, however, uncouple GvHD from GvL against myeloid leukemias because they express high levels of CD226 (DNAM-1), LFA-1, and granzyme B/perforin. While Tr1-secreted IL-10 and TGF-β suppress alloreactive T cells that attack non-myeloid epithelial tissues (skin, gut, and liver, preventing GvHD), Tr1 cells directly recognize CD155/CD112, CD54 (ICAM-1), CD13, and HLA class I expressed on myeloid-lineage AML blasts, lysing the leukemic blasts via perforin and granzyme B.

Why is Interleukin-27 (IL-27) more effective than IL-10 alone at inducing Tr1 differentiation from naive CD4⁺ T cells?

Naive CD4⁺ T cells express very low baseline levels of the IL-10 receptor and do not proliferate strongly in response to IL-10 alone. IL-27 (secreted by activated dendritic cells) signals through IL-27Rα/gp130 to activate both STAT1 and STAT3, which simultaneously open chromatin at the IL10 locus (via IRF1 and BATF), induce the master Tr1 transcription factors c-Maf and AhR, and trigger autocrine secretion of IL-21, which drives clonal proliferation of the differentiating Tr1 population.

1. Groux, H., O'Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E., & Roncarolo, M. G. (1997). "A CD4⁺ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis." Nature, 389(6652), 737–742. https://doi.org/10.1038/39614
2. Gagliani, N., Magnani, C. F., Huber, S., Gianolini, M. E., Pala, M., Licona-Limon, P., Guo, B., Herbert, D. R., Bulfone, A., Trentini, F., Di Serio, C., Bacchetta, R., Andreani, M., Brockmann, L., Gregori, S., Flavell, R. A., & Roncarolo, M. G. (2013). "Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells." Nature Medicine, 19(6), 739–746. https://doi.org/10.1038/nm.3179
3. Apetoh, L., Quintana, F. J., Pot, C., Joller, N., Xiao, S., Kumar, D., Burns, E. J., Sherr, D. H., Weiner, H. L., & Kuchroo, V. K. (2010). "The aryl hydrocarbon receptor interacts with c-Maf to promote the differentiation of type 1 regulatory T cells induced by IL-27." Nature Immunology, 11(9), 854–861. https://doi.org/10.1038/ni.1912
4. Roncarolo, M. G., Gregori, S., Bacchetta, R., Battaglia, M., & Gagliani, N. (2018). "The biology of T regulatory type 1 cells and their therapeutic application in immune-mediated diseases." Immunity, 49(6), 1004–1019. https://doi.org/10.1016/j.immuni.2018.12.001