Lineage Stability Research Articles

Helios is a marker, not a driver, of human Treg stability.

Treg therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T-cells and, thus, exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Treg lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part, due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Heliosmid )-expressing population correlated with Treg destabilization. We used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-KO and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T-cells.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Coexpression of Helios in Foxp3+ Regulatory T Cells and Its Role in Human Disease.

Regulatory T cells (Tregs) expressing the Foxp3 transcription factor are indispensable for the maintenance of immune system homeostasis. Tregs may lose Foxp3 expression or be reprogrammed into cells that produce proinflammatory cytokines, for example, Th1-like Tregs, Th2-like Tregs, Th17-like Tregs, and Tfh-like Tregs. Accordingly, selective therapeutic molecules that manipulate Treg lineage stability and/or functional activity might have the potential to improve aberrant immune responses in human disorders. In particular, the transcription factor Helios has emerged as an important marker and modulator of Tregs. Therefore, the current review focuses on recent findings on the expression, function, and mechanisms of Helios, as well as the patterns of Foxp3+ Tregs coexpressing Helios in various human disorders, in order to explore the potential of Helios for the improvement of many immune-related diseases. The studies were selected from PubMed using the library of the Nanjing Medical University in this review. The findings of the included studies indicate that Helios expression stabilizes the phenotype and function of Foxp3+ Tregs in certain inflammatory environments. Further, Tregs coexpressing Helios and Foxp3 were identified as a specific phenotype of stronger suppressor immune cells in both humans and animal models. Importantly, there is ample evidence that Helios-expressing Foxp3+ Tregs are relevant to various human disorders, including connective tissue diseases, infectious diseases, solid organ transplantation-related immunity, and cancer. Thus, Helios+Foxp3+CD4+ Tregs could be a valuable target in human diseases, and their potential should be explored further in the clinical setting.

Foxp3 enhancers synergize to maximize regulatory T cell suppressive capacity.

T reg cells bearing a diverse antigen receptor repertoire suppress pathogenic T cells and maintain immune homeostasis during their long lifespan. How their robust function is determined genetically remains elusive. Here, we investigate the regulatory space of the cis-regulatory elements of T reg lineage-specifying factor Foxp3. Foxp3 enhancers are known as distinct readers of environmental cues controlling T reg cell induction or lineage stability. However, their single deficiencies cause mild, if any, immune dysregulation, leaving the key transcriptional mechanisms determining Foxp3 expression and thereby T reg cell suppressive capacity uncertain. We examined the collective activities of Foxp3 enhancers and found that they coordinate to maximize T reg cell induction, Foxp3 expression level, or lineage stability through distinct modes and that ablation of synergistic enhancers leads to lethal autoimmunity in young mice. Thus, the induction and maintenance of a diverse, stable T reg cell repertoire rely on combinatorial Foxp3 enhancers, suggesting broad, stage-specific, synergistic activities of cell-intrinsic factors and cell-extrinsic cues in determining T reg cell suppressive capacity.

192-LB: Human CD4+CD25+CD226- Regulatory T Cells Demonstrate High Purity and Lineage Stability following In Vitro Expansion for Adoptive Cell Therapy

Human regulatory T cells (Tregs) are essential for the suppression of autoimmunity and adoptive cell therapy has been proposed as a means for restoring durable immune tolerance in T1D. The rare nature of this subset requires the isolation and in vitro expansion of thymic-derived Tregs (tTregs) for adoptive cell therapies to attenuate autoimmunity or islet transplant rejection. Current approaches involve the isolation of a subset with the marker profile of CD4+CD25+CD127-/lo. This strategy yields a population of Tregs that contains both naïve and peripheral Tregs (pTregs), with peripheral Tregs tending to lose lineage stability over time. Here, a novel gating strategy is presented for obtaining tTregs with high purity and extended lineage stability involving the elimination of the effector costimulatory molecule CD226. Tregs were obtained by fluorescence-activated cell sorting (FACS). This allows for the isolation of CD4+CD25+CD226- Tregs for comparison to classically sorted CD4+CD25+CD127-/lo Tregs over a 28-day culture period. Throughout the expansion, assessment of the lineage’s canonical transcription factors (FOXP3 and Helios), along with functional characteristics and epigenetic markers of the lineage occurred. Isolation of CD226- Tregs as compared to CD127- Tregs yields a Treg population with a higher proportion of tTregs (9.35%, p=0.0016), increased expression of CD25 (1.13-fold, p=0.0023), Helios (1.21-fold, p=0.0419), and CD45RA (1.24-fold, p=0.0017), equally low expression of CD127, and decreased expression of CD226 (2.27-fold, p=0.0001). Overall, this study suggests CD226- Tregs possess an increased therapeutic potential for adoptive transfer immunotherapies, including a more naïve phenotype and increased suppressive capabilities - both of which are critical factors for suppressing autoimmune destruction in vivo. Disclosure M. E. Brown: None. S. R. Hanbali: None. L. Gomez rodriguez: None. J. M. Arnoletti: None. E. B. Carpenter: None. T. M. Brusko: Consultant; Self; OneVax, LLC. Funding National Institutes of Health (1R01DK106191-01A1, UG3DK122638)

Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

Regulatory T cell suppressive capacity is governed by synergistic Foxp3 enhancers

Abstract Regulatory T (Treg) cells bearing a diverse antigen receptor repertoire suppress pathogenic T cells and maintain immune homeostasis during their long lifespan. How robust Treg function is determined genetically remains elusive. Here, we investigate the regulatory space of the cis-regulatory elements of Treg lineage–specifying factor Foxp3 known as distinct modules promoting Treg cell induction or lineage stability. Their single deficiencies cause mild, if any, immune dysregulation, leaving the key transcriptional mechanisms of the Foxp3 gene determining Treg suppressive capacity uncertain. Therefore, we examined the collective activities of Foxp3 enhancers and found that they coordinate to maximize Treg cell induction and stability through distinct modes. Thus, the induction and maintenance of a diverse, stable Treg cell repertoire rely on combinatorial Foxp3 enhancers acting in a stage-specific manner, improving our understanding of the genetic determinants of Treg suppressive capacity.

The demethylase inhibitor GSK-J4 limits inflammatory colitis by promoting de novo synthesis of retinoic acid in dendritic cells

Dendritic cells (DCs) promote T-cell mediated tolerance to self-antigens and induce inflammation to innocuous-antigens. This dual potential makes DCs fundamental players in inflammatory disorders. Evidence from inflammatory colitis mouse models and inflammatory bowel diseases (IBD) patients indicated that gut inflammation in IBD is driven mainly by T-helper-1 (Th1) and Th17 cells, suggesting an essential role for DCs in the development of IBD. Here we show that GSK-J4, a selective inhibitor of the histone demethylase JMJD3/UTX, attenuated inflammatory colitis by reducing the inflammatory potential and increasing the tolerogenic features of DCs. Mechanistic analyses revealed that GSK-J4 increased activating epigenetic signals while reducing repressive marks in the promoter of retinaldehyde dehydrogenase isoforms 1 and 3 in DCs, enhancing the production of retinoic acid. This, in turn, has an impact on regulatory T cells (Treg) increasing their lineage stability and gut tropism as well as potentiating their suppressive activity. Our results open new avenues for the treatment of IBD patients.

Efficient IL-2R signaling differentially affects the stability, function, and composition of the regulatory T-cell pool

Signaling via interleukin-2 receptor (IL-2R) is a requisite for regulatory T (Treg) cell identity and function. However, it is not completely understood to what degree IL-2R signaling is required for Treg cell homeostasis, lineage stability and function in both resting and inflammatory conditions. Here, we characterized a spontaneous mutant mouse strain endowed with a hypomorphic Tyr129His variant of CD25, the α-chain of IL-2R, which resulted in diminished receptor expression and reduced IL-2R signaling. Under noninflammatory conditions, Cd25Y129H mice harbored substantially lower numbers of peripheral Treg cells with stable Foxp3 expression that prevented the development of spontaneous autoimmune disease. In contrast, Cd25Y129H Treg cells failed to efficiently induce immune suppression and lost lineage commitment in a T-cell transfer colitis model, indicating that unimpaired IL-2R signaling is critical for Treg cell function in inflammatory environments. Moreover, single-cell RNA sequencing of Treg cells revealed that impaired IL-2R signaling profoundly affected the balance of central and effector Treg cell subsets. Thus, partial loss of IL-2R signaling differentially interferes with the maintenance, heterogeneity, and suppressive function of the Treg cell pool.

Diversity of T Helper and Regulatory T Cells and Their Contribution to the Pathogenesis of Allergic Diseases.

T helper (Th) and regulatory T (Treg) cells represent important effectors of adaptive immunity. They mediate communication between the immune system and tissue sites and thereby coordinate effective defense against environmental threats or maintain tolerance, respectively. Since the discovery of two prototypic T helper cells, Th1 and Th2, additional phenotypic and functional distinct subsets have been described ranging from Th17, Th22, Th9, and T follicular helper cells. The same holds true for regulatory T cells that represent a family with functionally distinct subsets characterized by co-expression of the transcription factors T-bet, Gata3, or RORγt. Here, we summarize the current knowledge on differentiation and function of T helper and regulatory T cell subsets and discuss their lineage stability versus plasticity towards other subsets. In addition, we highlight the direct and indirect contribution of each subset to the pathology of allergies and indicate novel therapies for specific targeting the effector functions of T helper and regulatory T cells.

The Methyltransferase DOT1L Controls Activation and Lineage Integrity in CD4+ T Cells during Infection and Inflammation

CD4+ T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-γ under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-γ response, mice with a T-cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and are resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th2 cell lineage commitment and stability and suggest that inhibition of DOT1L may provide a therapeutic strategy to limit type 2 immune responses.

Cutting Edge: CRISPR-Based Transcriptional Regulators Reveal Transcription-Dependent Establishment of Epigenetic Memory of Foxp3 in Regulatory T Cells.

Transcription factor Foxp3 specifies and maintains regulatory T cell (Treg) identity. During Treg differentiation, a CpG-rich Foxp3 intronic enhancer, conserved noncoding sequence 2 (CNS2), is activated via DNA demethylation to establish epigenetic memory of Foxp3 expression to protect Treg identity. However, it is unclear how this epigenetic memory of Foxp3 expression is established, as CNS2 is thought to be demethylated independently of Foxp3 expression. In this article, we uncover an unexpected causal relationship between Foxp3-transcriptional activation and CNS2 demethylation in mice. CRISPR/dCas9-mediated Foxp3-transcriptional activation elicits CNS2 demethylation. Sustaining Foxp3-transcriptional activation in induced Tregs also promotes CNS2 demethylation, enhancing Treg lineage stability and suppressive function. Importantly, CRISPR-mediated silencing of Foxp3 transcription, but not protein expression, abolishes CNS2 demethylation. The novel finding that Foxp3-transcriptional activation promotes CNS2 demethylation may facilitate the development of Treg-based therapies and represent a general mechanism for the establishment of epigenetic memory of immune gene expression.

Maintenance DNA methylation is essential for regulatory T cell development and stability of suppressive function.

Tregs require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Tregs persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator ubiquitin-like with plant homeodomain and RING finger domains 1 (Uhrf1) is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3+ cells resulted in global yet nonuniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg lineage, and spontaneous inflammation. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg genome for both lineage establishment and stability of identity and suppressive function.

Enrichment of retinal ganglion and Müller glia progenitors from retinal organoids derived from human induced pluripotent stem cells - possibilities and current limitations.

BACKGROUNDRetinal organoids serve as excellent human-specific disease models for conditions affecting otherwise inaccessible retinal tissue from patients. They permit the isolation of key cell types affected in various eye diseases including retinal ganglion cells (RGCs) and Müller glia.AIMTo refine human-induced pluripotent stem cells (hiPSCs) differentiated into three-dimensional (3D) retinal organoids to generate sufficient numbers of RGCs and Müller glia progenitors for downstream analyses.METHODSIn this study we described, evaluated, and refined methods with which to generate Müller glia and RGC progenitors, isolated them via magnetic-activated cell sorting, and assessed their lineage stability after prolonged 2D culture. Putative progenitor populations were characterized via quantitative PCR and immunocytochemistry, and the ultrastructural composition of retinal organoid cells was investigated.RESULTSOur study confirms the feasibility of generating marker-characterized Müller glia and RGC progenitors within retinal organoids. Such retinal organoids can be dissociated and the Müller glia and RGC progenitor-like cells isolated via magnetic-activated cell sorting and propagated as monolayers.CONCLUSIONEnrichment of Müller glia and RGC progenitors from retinal organoids is a feasible method with which to study cell type-specific disease phenotypes and to potentially generate specific retinal populations for cell replacement therapies.

The role of RORγt at maternal-fetal interface during murine pregnancy.

Innate lymphoid cells (ILCs, including NK cells) and their subsets are the most frequent lymphocytes at the maternal-fetal interface (decidua). Recent recognition of extensive ILC subset diversity at mucosal sites and the possible role they might play at different stages of pregnancy poses questions about their composition and lineage stability. Namely, RORγt-dependent ILC3s have been recognized as a key cellular mediator of tissue organization in the gut and secondary lymphoid organs, prompting examination of their distribution and role in decidua during pregnancy. We employed highly polychromatic flow cytometry with conventional and machine learning-aided analysis to map ILC subsets and dissected the role of canonical transcription factor RORγt using fate-mapping animals and RORγt-/- animals. We demonstrate a comprehensive immunome map of ILCs/NKs, revealing a dynamic interface even in the absence of antigenic or allogeneic challenge. Strikingly, we demonstrate plasticity of RORγt expression in decidual ILCs with across gestation. However, gross reproductive efficiency is not affected in RORγt-/- animals. These results indicated that RORγt+ ILCs are highly plastic at the maternal-fetal interface, but dispensable for normal pregnancy, revealing a novel mechanism of transcriptional immunoregulation in pregnancy.

292-OR: Treg Cells Control Systemic Lipid and Iron Metabolism via the OGT-STAT5 Axis

Adipose tissue-resident regulatory T (Treg) cells have been proposed to exert metabolic roles in controlling inflammation, insulin sensitivity and thermogenesis. However, it is still largely unclear how Treg cells integrate immunological and metabolic signals to maintain systemic energy balance. Here we show that the glucose-sensitive O-linked N-Acetylglucosamine (O-GlcNAc) post-translational modification in Treg cells is activated by T cell receptor signaling. Through modifying the STAT5 protein and IL-2 signaling, protein O-GlcNAcylation supports the lineage stability and effector function of Treg cells, while loss of O-GlcNAc transferase (OGT) in Treg cells leads to systemic inflammation in mice. When fed with high fat diet, heterozygous Foxp3-OGT knockout females gain more fat mass as a result of reduced energy expenditure. On the other hand, constitutively active STAT5-mediated Treg-cell expansion and activation protect mice from diet-induced obesity and glucose intolerance. Mechanistically, Treg cells inhibit fatty acid uptake and regulate iron cycling in the adipose tissue. Taken together, we propose that the OGT-STAT5 axis enables Treg cells to maintain systemic lipid and iron homeostasis. Disclosure O.C. Salgado Barrero: None. H. Ruan: None. Funding American Diabetes Association (1-18-IBS-167 to H-B.R.); National Institutes of Health (R01AI139420)

1663-P: Gene Editing of the Human T-Cell Receptor Locus Enables Optimized In Vitro Testing of Autoreactive T-Cell Function in Type 1 Diabetes

Gene editing of the human T cell receptor locus enables optimized in vitro testing of autoreactive T cell function in type 1 diabetes. The generation of antigen specific T cells and subsequent implementation as “living drugs” has garnered great interest in the immunotherapy space. One of the main caveats to this approach is the potential for heterologous chain pairing between the α- and β-chains of the endogenous and de-novo T cell receptors (TCRs). The consequences of chain mispairing can range from reduced antigen specific functionality to off-target autoimmune reactivity. The latter is an important consideration when designing therapies for autoimmune and inflammatory diseases, specifically with regards to the introduction of regulatory T cells (Treg) adoptive cell therapy. To date, infusions of polyclonal Tregs have demonstrated safety but have failed to reverse established type 1 diabetes (T1D). This demonstrates the need for the generation of safe and effective antigen-specific Tregs capable of recognizing β-cells in T1D. We have previously shown the ability to generate GAD-specific Tregs which maintain lineage stability and suppressive phenotype. To extend this work, we deleted the endogenous TCR α-chain gene (TRAC) via CRISPR/cas9 in CD8+T cells. We achieved >90% editing efficiency through ribonucleoprotein (RNP) complex mediated single guide RNA delivery. Moreover, we found TRAC-KO CD8+ T cells displayed increased peptide MHC-dextramer staining, CD69 and CD25 upregulation after stimulation with cognate antigen, and increased antigen-specific killing of β-cell lines when compared to mock guide treated T cells. We believe this workflow can be applied to generate antigen specific Tregs, which we expect to provide increased suppressive capacity. Hence, we present a TCR-gene editing protocol in primary human T cells that results in efficient deletion of the endogenous TCR α-chain and robust enhancement of antigen-specific T cell function. Disclosure L.D. Peters: None. A.L. Posgai: None. T.M. Brusko: Board Member; Self; OneVax, LLC. Consultant; Self; Caladrius Biosciences, Inc., Sanofi-Aventis. Speaker’s Bureau; Self; CSL Behring. Funding National Institutes of Health (P01AI42288, R01DK106191, T325T32DK108736-03HIRN, UC4 P0063595)

Bcl6 Preserves the Suppressive Function of Regulatory T Cells During Tumorigenesis.

During tumorigenesis, tumor infiltrating regulatory T (Treg) cells restrict the function of effector T cells in tumor microenvironment and thereby promoting tumor growth. The anti-tumor activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of various types of human cancers. However, the immune suppressive function of Treg cells remains a major hurdle to broader effectiveness of tumor immunotherapy. In this article, we reported that the deletion of Bcl6 specifically in Treg cells led to stunted tumor growth, which was caused by impaired Treg cell responses. Notably, Bcl6 is essential in maintaining the lineage stability of Treg cells in tumor microenvironment. Meanwhile, we found that the absence of follicular regulatory T (Tfr) cells, which is a result of Bcl6 deletion in Foxp3+ cells, was dispensable for tumor control. Importantly, the increased Bcl6 expression in Treg cells is associated with poor prognosis of human colorectal cancer and lymph node metastasis of skin melanoma. Furthermore, Bcl6 deletion in Treg cells exhibits synergistic effects with immune checkpoint blockade therapy. Collectively, these results indicate that Bcl6 actively participates in regulating Treg cell immune responses during tumorigenesis and can be exploited as a therapeutic target of anti-tumor immunity.

Heat shock protein gp96 controls regulatory T cell lineage stability via IL-2/pSTAT5 signaling

Abstract Gp96 is an essential endoplasmic reticulum chaperone for various proteins including GARP, TLRs, integrins, Wnt co-receptor LRP6 and the platelet glycoprotein Ib/IX/V complex. Our laboratory has previously shown that loss of gp96 in murine Foxp3+ regulatory T cells (Tregs) results in their instability as determined by FOXP3 downregulation, impaired immunosuppressive function and production of effector cytokines (Zhang, et al., J Clin Invest., 2015, 125: 859–869). However, the mechanism underlying Treg dysfunction upon gp96 deletion remains unknown. Using tamoxifen-inducible Treg-specific gp96 gene (encoded by Hsp90b1) knockout (KO) mice (Foxp3-ERT2creHsp90b1f/f), we first phenotypically characterized gp96-deleted Tregs in mouse spleen and peripheral lymph nodes (pLNs). Gp96 deficient Tregs showed decreased frequency of TCF1−CD62L− effector population, but were enriched with short-lived KLRG1+ and Helios+ subsets, in both spleen and pLNs. We also confirmed that the expression of Foxp3 was decreased in splenic gp96-deleted Tregs, which correlated with a reduction of cell surface CD25. Intriguingly, both Foxp3 and surface CD25 levels stayed high in gp96-null Tregs in pLNs. In addition, gp96-null Tregs in mouse spleen, but not in pLNs, demonstrated compromised suppressive function. We further found that IL-2/CD25 signaling, as assessed by STAT5 phosphorylation, was more activated in gp96-null Tregs in pLNs, compared to that in spleen. In vitro IL-2 treatment was able to rescue the attenuated Foxp3 expression in gp96-null Tregs. Our work indicates that IL-2/pSTAT-5 signaling may be a key factor in maintaining Treg Foxp3 expression and its lineage stability upon gp96 deletion.

Human Regulatory T Cells From Umbilical Cord Blood Display Increased Repertoire Diversity and Lineage Stability Relative to Adult Peripheral Blood.

The human T lymphocyte compartment is highly dynamic over the course of a lifetime. Of the many changes, perhaps most notable is the transition from a predominantly naïve T cell state at birth to the acquisition of antigen-experienced memory and effector subsets following environmental exposures. These phenotypic changes, including the induction of T cell exhaustion and senescence, have the potential to negatively impact efficacy of adoptive T cell therapies (ACT). When considering ACT with CD4+CD25+CD127–/lo regulatory T cells (Tregs) for the induction of immune tolerance, we previously reported ex vivo expanded umbilical cord blood (CB) Tregs remained more naïve, suppressed responder T cells equivalently, and exhibited a more diverse T cell receptor (TCR) repertoire compared to expanded adult peripheral blood (APB) Tregs. Herein, we hypothesized that upon further characterization, we would observe increased lineage heterogeneity and phenotypic diversity in APB Tregs that might negatively impact lineage stability, engraftment capacity, and the potential for Tregs to home to sites of tissue inflammation following ACT. We compared the phenotypic profiles of human Tregs isolated from CB versus the more traditional source, APB. We conducted analysis of fresh and ex vivo expanded Treg subsets at both the single cell (scRNA-seq and flow cytometry) and bulk (microarray and cytokine profiling) levels. Single cell transcriptional profiles of pre-expansion APB Tregs highlighted a cluster of cells that showed increased expression of genes associated with effector and pro-inflammatory phenotypes (CCL5, GZMK, CXCR3, LYAR, and NKG7) with low expression of Treg markers (FOXP3 and IKZF2). CB Tregs were more diverse in TCR repertoire and homogenous in phenotype, and contained fewer effector-like cells in contrast with APB Tregs. Interestingly, expression of canonical Treg markers, such as FOXP3, TIGIT, and IKZF2, were increased in CB CD4+CD127+ conventional T cells (Tconv) compared to APB Tconv, post-expansion, implying perinatal T cells may adopt a default regulatory program. Collectively, these data identify surface markers (namely CXCR3) that could be depleted to improve purity and stability of APB Tregs, and support the use of expanded CB Tregs as a potentially optimal ACT modality for the treatment of autoimmune and inflammatory diseases.