Abstract
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.
Highlights
The human immune system undergoes dramatic changes over the course of a lifetime in order to maintain tissue and organism homeostasis
We sought to identify differences in the composition of native cord blood (CB) and adult peripheral blood (APB) Tregs at the single cell level that might contribute to non-Treg contaminants in a post-expansion cell product for use in ACT
After identification of 4842 high quality cells in CB (n = 1 subject) and 4320 in APB (n = 1 subject), datasets were normalized for cell-specific biases related to sequencing depth using the residuals of regularized negative binomial regression as described in [36]
Summary
The human immune system undergoes dramatic changes over the course of a lifetime in order to maintain tissue and organism homeostasis. Variable cellular dynamics abound during growth and development in early life This is apparent during the nascent perinatal period, as the periphery is actively seeded with innate and adaptive immune cells that quickly gain initial exposures to foreign antigens [1]. These early priming events must confer protection from pathogens, while maintaining peripheral tolerance to microbial commensals, inert environmental antigens, and self-tissues. Despite inconsistent immune profiles in umbilical cord blood (CB) and newborns, a recent report suggests individuals eventually converge on a common post-natal trajectory for healthy/normal immunological development [1]. TTregs are so essential for maintaining immune homeostasis that loss-offunction mutations in FOXP3, the canonical transcription factor that marks the Treg cell lineage, can result in the lethal multiorgan autoimmune disease referred to as immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome [reviewed in [4]]
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