DRAG in situ barcoding reveals an increased number of HSPCs contributing to myelopoiesis with age.

Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we present an in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis rather than the increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remains constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity.

Replicative history marks transcriptional and functional disparity in the CD8+ T cell memory pool.

Clonal expansion is a core aspect of T cell immunity. However, little is known with respect to the relationship between replicative history and the formation of distinct CD8+ memory T cell subgroups. To address this issue, we developed a genetic-tracing approach, termed the DivisionRecorder, that reports the extent of past proliferation of cell pools in vivo. Using this system to genetically 'record' the replicative history of different CD8+ T cell populations throughout a pathogen-specific immune response, we demonstrate that the central memory T (TCM) cell pool is marked by a higher number of prior divisions than the effector memory T cell pool, owing to the combination of strong proliferative activity during the acute immune response and selective proliferative activity after pathogen clearance. Furthermore, by combining DivisionRecorder analysis with single-cell transcriptomics and functional experiments, we show that replicative history identifies distinct cell pools within the TCM compartment. Specifically, we demonstrate that lowly divided TCM cells display enriched expression of stem-cell-associated genes, exist in a relatively quiescent state, and are superior in eliciting a proliferative recall response upon activation. These data provide the first evidence that a stem-cell-like memory T cell pool that reconstitutes the CD8+ T cell effector pool upon reinfection is marked by prior quiescence.

The precursors of CD8+ tissue resident memory T cells: from lymphoid organs to infected tissues.

CD8+ tissue resident memory T cells (TRM cells) are essential for immune defence against pathogens and malignancies, and the molecular processes that lead to TRM cell formation are therefore of substantial biomedical interest. Prior work has demonstrated that signals present in the inflamed tissue micro-environment can promote the differentiation of memory precursor cells into mature TRM cells, and it was therefore long assumed that TRM cell formation adheres to a 'local divergence' model, in which TRM cell lineage decisions are exclusively made within the tissue. However, a growing body of work provides evidence for a 'systemic divergence' model, in which circulating T cells already become preconditioned to preferentially give rise to the TRM cell lineage, resulting in the generation of a pool of TRM cell-poised T cells within the lymphoid compartment. Here, we review the emerging evidence that supports the existence of such a population of circulating TRM cell progenitors, discuss current insights into their formation and highlight open questions in the field.

Formation of Tissue-Resident CD8+ T-Cell Memory.

Resident memory CD8+ T (Trm) cells permanently reside in nonlymphoid tissues where they act as a first line of defense against recurrent pathogens. How and when antigen-inexperienced CD8+ T cells differentiate into Trm has been a topic of major interest, as knowledge on how to steer this process may be exploited in the development of vaccines and anticancer therapies. Here, we first review the current understanding of the early signals that CD8+ T cells receive before they have entered the tissue and that govern their capacity to develop into tissue-resident memory T cells. Subsequently, we discuss the tissue-derived factors that promote Trm maturation in situ. Combined, these data sketch a model in which a subset of responding T cells develops a heightened capacity to respond to local cues present in the tissue microenvironment, which thereby imprints their ability to contribute to the tissue-resident memory CD8+ T-cell pool that provide local control against pathogens.

A committed tissue-resident memory T cell precursor within the circulating CD8+ effector T cell pool.

An increasing body of evidence emphasizes the role of tissue-resident memory T cells (TRM) in the defense against recurring pathogens and malignant neoplasms. However, little is known with regard to the origin of these cells and their kinship to other CD8+ T cell compartments. To address this issue, we followed the antigen-specific progeny of individual naive CD8+ T cells to the T effector (TEFF), T circulating memory (TCIRCM), and TRM pools by lineage-tracing and single-cell transcriptome analysis. We demonstrate that a subset of T cell clones possesses a heightened capacity to form TRM, and that enriched expression of TRM-fate-associated genes is already apparent in the circulating TEFF offspring of such clones. In addition, we demonstrate that the capacity to generate TRM is permanently imprinted at the clonal level, before skin entry. Collectively, these data provide compelling evidence for early stage TRM fate decisions and the existence of committed TRM precursor cells in the circulatory TEFF compartment.

The Branching Point in Erythro-Myeloid Differentiation.

Development of mature blood cell progenies from hematopoietic stem cells involves the transition through lineage-restricted progenitors. The first branching point along this developmental process is thought to separate the erythro-myeloid and lymphoid lineage fate by yielding two intermediate progenitors, the common myeloid and the common lymphoid progenitors (CMPs and CLPs). Here, we use single-cell lineage tracing to demonstrate that so-called CMPs are highly heterogeneous with respect to cellular output, with most individual CMPs yielding either only erythrocytes or only myeloid cells after transplantation. Furthermore, based on the labeling of earlier progenitors, we show that the divergence between the myeloid and erythroid lineage develops within multipotent progenitors (MPP). These data provide evidence for a model of hematopoietic branching in which multiple distinct lineage commitments occur in parallel within the MPP pool.

T Cell factor 1 represses CD8+ effector T cell formation and function.

The Wnt-responsive transcription factor T cell factor 1 (Tcf1) is well known for its role in thymic T cell development and the formation of memory CD8(+) T cells. However, its role in the initial phases of CD8(+) T effector cell formation has remained unexplored. We report that high levels of Wnt signaling and Tcf1 are operational in naive and memory CD8(+) T cells, whereas Wnt signaling and Tcf1 were low in effector CD8(+) T cells. CD8(+) T cells deficient in Tcf1 produce IFN-γ more rapidly, coinciding with increased demethylation of the IFN-γ enhancer and higher expression of the transcription factors Tbet and Blimp1. Moreover, virus-specific Tcf1(-/-) CD8(+) T cells show accelerated expansion in acute infection, which is associated with increased IFN-γ and TNF production and lower viral load. Genetic complementation experiments with various Tcf1 isoforms indicate that Tcf1 dosage and protein stability are critical in suppressing IFN-γ production. Isoforms lacking the ß-catenin binding domain are equally effective in inhibiting CD8(+) effector T cell formation. Thus, Tcf1 functions as a repressor of CD8(+) effector T cell formation in a ß-catenin/Wnt-independent manner.

Single cell behavior in T cell differentiation.

Upon primary infection, naïve T cells that recognize their cognate antigen become activated, proliferate, and simultaneously differentiate into various subsets. A long-standing question in the field has been how this cellular diversification is achieved. Conceptually, diverse cellular output may either arise from every single cell or only from populations of naïve cells. Furthermore, such diversity may either be driven by cell-intrinsic heterogeneity or by external, niche-derived signals. In this review, we discuss how recently developed technologies have allowed the analysis of the mechanisms underlying T cell diversification at the single cell level. In addition, we outline the implications of this work on our understanding of the formation of immunological memory, and describe a number of unresolved key questions in this field.