Exploring the Cord Blood T Cell Compartment with Mass Cytometry Reveals the Existence of Memory CD4, CD8 and Regulatory T Cell Pools in the Fetal Period

It has been long perceived that cord blood (CB) T cells are exclusively composed of antigen-inexperienced 'naïve' T cells under Th2 trophic and/or Th1 suppressive conditions, and that this is important for the maintenance of tolerance during pregnancy. However, a recent study reported the existence of a small fraction of memory CD4+ T cells with inflammatory cytokine profile within the sterile CB environment. This is in contrast to the common dogma challenging the presence of inflammatory T cells in the CB environment. To produce a more in-depth picture of the T cell compartment in CB, we assembled a panel of 40 antibodies to characterize the phenotypic and functional complexity of CB T cells. We interrogated the immune landscape of 12 CB donors vs 12 healthy adult peripheral blood (PB) donors to obtain single-cell profiles of the T-cell compartment. We used t-SNE algorithm to visualize the high dimensional data and the nearest-neighbor based clustering algorithm, PhenoGraph, to identify the potential clusters in high dimensional space. We observed significant segregation between PB and CB T cells. As expected, the majority of CD4+ and CD8+ T cells in CB were composed of naïve cells, while PB CD4+ and CD8+ T cells were characterized by a dominantly memory T cell phenotype. Within the CB CD4+ T cell compartment, in addition to the CD127⁺CD25^- naïve T cell subset, we observed two more populations, including CD127⁺CD25^int memory T cells (median 1.92%; range 0.69%-4.14%) and CD127^lowCD25^hi regulatory T-cells (Tregs) (median 1.9%; range 1.36%-2.98%). Similarly, a small subset of memory T cells, defined as CD45RA-CCR7+/-CD95+ (median 3.23%; range:1.53%-7.45%) was present in the CD8 T cell compartment in CB.

Each T-cell presents a diversity of surface phenotypes defined by distinct combinations of marker expression. Thus, we sought an overview of the similarities and differences between CB and PB T-cell subsets. Analysis of CB Tregs revealed a repertoire composed mainly of naïve and central memory subsets, the latter expressed CD95, a marker of activation and memory, suggesting the generation of memory Tregs during fetal life. Both CB and PB Tregs were characterized by expression of PD-1, CD39, TIGIT, ICOS, CD161, CCR4, DNAM-1, CD49f and CD26. Expression of TIGIT, PD-1, CD39, CD49f, ICOS and CCR4 was exclusively confined to the memory Treg subset. In contrast to PB Tregs, CB Tregs lacked CCR10 and CXCR5 expression, suggesting differential migratory properties for CB and PB Tregs. We next focused on the differences between the naïve and memory CD4+ T cell subsets in CB. Chemokine receptors can be used to classify T-cell subsets with distinct migratory capacities and effector function. Naïve CD4 T cells do not express chemokine receptors, while memory T cells express different combinations of chemokine receptors that give clues to their lineage commitment. We discovered CCR6, CCR4, CXCR3, CD161, DNAM-1, CD49f, KLRG1, Beta7, PD-1, and TIGIT as the set of combinatorial markers to identify the memory CD4+ T cell pool in CB. Notably, a significant proportion of memory CD4+ T cells in CB expressed CCR4, supporting the notion that the CB environment drives a Th2 rather than a Th1 or Th17 functional phenotype. Moreover, in CB, expression of CCR6, CXCR3 and CD161 was confined to the memory CD4 T cell pool, suggesting that Th1, Th2 and Th17 lineage commitment starts in the fetal period, although the exact mechanism leading to memory T cell formation is not known.

Taken together, interrogation of the T cell compartment in CB and PB in high dimensional space reveals well-defined and distinct memory signatures. Acquisition of memory phenotype instructs expression of unique sets of markers for each memory subset and varies among CD4, CD8 and Treg. Our data indicate the presence of a seemingly full-blown immune response, resulting in the generation of committed memory T cells and changes in the immune landscape of CB, although the initiating factor for this is yet to be determined. Mass cytometry proves to be a valuable tool in the characterization and discovery of novel T cell subsets, even if present at low frequencies. Studies to track the differentiation and diversification of T cells in CB transplant recipients and to link the impact of the T cell repertoire on clinical outcomes are under way.