Single-Cell Transcriptomics Reveals Discrete Steps in Regulatory T Cell Development in the Human Thymus.

CD4+CD25+FOXP3+ regulatory T (Treg) cells control immunological tolerance. Treg cells are generated in the thymus (tTreg) or in the periphery. Their superior lineage fidelity makes tTregs the preferred cell type for adoptive cell therapy (ACT). How human tTreg cells develop is incompletely understood. By combining single-cell transcriptomics and flow cytometry, we in this study delineated three major Treg developmental stages in the human thymus. At the first stage, which we propose to name pre-Treg I, cells still express lineage-inappropriate genes and exhibit signs of TCR signaling, presumably reflecting recognition of self-antigen. The subsequent pre-Treg II stage is marked by the sharp appearance of transcription factor FOXO1 and features induction of KLF2 and CCR7, in apparent preparation for thymic exit. The pre-Treg II stage can further be refined based on the sequential acquisition of surface markers CD31 and GPA33. The expression of CD45RA, finally, completes the phenotype also found on mature recent thymic emigrant Treg cells. Remarkably, the thymus contains a substantial fraction of recirculating mature effector Treg cells, distinguishable by expression of inflammatory chemokine receptors and absence of CCR7. The developmental origin of these cells is unclear and warrants caution when using thymic tissue as a source of stable cells for ACT. We show that cells in the major developmental stages can be distinguished using the surface markers CD1a, CD27, CCR7, and CD39, allowing for their viable isolation. These insights help identify fully mature tTreg cells for ACT and can serve as a basis for further mechanistic studies into tTreg development.

GPA33 is expressed on multiple human blood cell types and distinguishes CD4+ central memory T cells with and without effector function.

The Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively determined GPA33 expression patterns on human blood leukocyte subsets, using mass and flow cytometry. We found that GPA33 was expressed on fractions of B, dendritic, natural killer and innate lymphoid cells. Most prominent expression was found in the CD4+ T cell compartment. Naïve and CXCR5+ regulatory T cells were GPA33high , and naïve conventional CD4+ T cells expressed intermediate GPA33 levels. The expression pattern of GPA33 identified functional heterogeneity within the CD4+ central memory T cell (Tcm) population. GPA33+ CD4+ Tcm cells were fully undifferentiated, bona fide Tcm cells that lack immediate effector function, whereas GPA33- Tcm cells exhibited rapid effector functions and may represent an early stage of differentiation into effector/effector memory T cells before loss of CD62L. Expression of GPA33 in conventional CD4+ T cells suggests a role in localization and/or preservation of an undifferentiated state. These results form a basis to study the function of GPA33 and show it to be a useful marker to discriminate between different cellular subsets, especially in the CD4+ T cell lineage.

Separating the wheat from the chaff: Making sense of Treg heterogeneity for better adoptive cellular therapy.

Regulatory T (Treg) cells are essential for immunological tolerance and can be used to suppress unwanted or excessive immune responses through adoptive cellular therapy. It is increasingly clear that many subsets of Treg cells exist, which have different functions and reside in different locations. Treg cell therapies may benefit from tailoring the selected subset to the tissue that must be protected as well as to characteristics of the immune response that must be suppressed, but little attention is given to this topic in current therapies. Here, we will discuss how three major axes of heterogeneity can be discerned among the Treg cell population, which determine function and lineage fidelity. A first axis relates to the developmental route, as Treg cells can be generated from immature T cells in the thymus or from already mature Tconv cells in the immunological periphery. Heterogeneity furthermore stems from activation history (naïve or effector) and location (lymphoid or peripheral tissues). Each of these axes bestows specific properties on Treg cells, which are further refined by additional processes leading to yet further variation. A critical aspect impacting on Treg cell heterogeneity is TCR specificity, which determines when and where Treg cells are generated as well as where they exhibit their effector functions. We will discuss the implications of this heterogeneity and the role of the TCR for the design of next generation adoptive cellular therapy with Treg cells.

Determining the extent of maternal-foetal chimerism in cord blood.

During pregnancy, maternal T cells can enter the foetus, leading to maternal-foetal chimerism. This phenomenon may affect how leukaemia patients respond to transplantation therapy using stem cells from cord blood (CB). It has been proposed that maternal T cells, primed to inherited paternal HLAs, are present in CB transplants and help to suppress leukaemic relapse. Several studies have reported evidence for the presence of maternal T cells in most CBs at sufficiently high numbers to lend credence to this idea. We here aimed to functionally characterise maternal T cells from CB. To our surprise, we could not isolate viable maternal cells from CB even after using state-of-the-art enrichment techniques that allow detection of viable cells in heterologous populations at frequencies that were several orders of magnitude lower than reported frequencies of maternal T cells in CB. In support of these results, we could only detect maternal DNA in a minority of samples and at insufficient amounts for reliable quantification through a sensitive PCR-based assay to measure In/Del polymorphisms. We conclude that maternal microchimerism is far less prominent than reported, at least in our cohort of CBs, and discuss possible explanations and implications.