Presented at the Neonatal Society Spring Meeting 2019

Authors: T Sproat^1,2, J Spegarova¹, R Payne¹, CJ Stewart¹, J Berrington^1,2, N Embleton^1,2, S Hambleton^1,2

Corresponding author e-mail address: T.D.R.Sproat2@ncl.ac.uk

Institution(s)

1. Institute of Cellular Medicine, Newcastle University
2. Newcastle Upon Tyne Hospitals NHS Foundation Trust

Introduction
Enteral diet in preterm babies is associated with diseases such as necrotising enterocolitis (NEC) and late onset sepsis (LOS). NEC and LOS are both associated with an inappropriate immune response, as well as changes in the gut microbiota. 1
We aimed to determine the impact of an exclusive human milk diet on gut microbiota and development of T lymphocytes. We hypothesised that there will be an association between diet and mucosal-associated immune cell (MAIT, iNK T, Treg) populations, and that T cell populations change over time.

Methods
Extremely preterm (<30 weeks gestation) infants were recruited and randomised within 72 hours of age to a dietary intervention until 34w corrected age. All infants received mother’s own milk but were randomised to supplementation with either human (Prolacta Biosciences) or bovine milk products (Nutriprem C&G). Daily stool samples, as well as 500ul blood were collected (2 time points). Lymphocytes were isolated and stained with a 39 antibody mass cytometry (CyTOF) panel as well as a conventional 7 channel flow cytometry panel (defining major lymphocyte subsets). ISRCTN doi.org/10.1186/ISRCTN16799022 Funding: Prolacta Biosciences US

Results
We present preliminary data on 25 lymphocyte profiles from 17 preterm infants (mean; gestational age 27w, birthweight 973g). An average of 186,000 leucocytes were analysed using mass cytometry. As expected, the proportion of major lymphocyte populations were similar between flow and mass cytometry. Conventional T cells were mostly naïve, therefore we focused on the innate-like lymphocyte subsets. Our data shows an increased abundance of V-alpha 7.2 CD161- cells (CD4+ and CD8+) as % of T cells in neonates compared to adults (mean 2.9% vs 1.13% (p<0.001)). Further characterisation of these cells may improve the understanding of the development of mucosa-associated invariant T (MAIT) cells (V-alpha 7.2+CD161+). Furthermore, the population of invariant NK (iNK) T cells is heterogenous, expanded in neonates compared to adults, and has a predominance of chemokine receptor 4 expression in neonates.

Conclusions
Mass cytometry enables a broader description of immune development and is a valuable tool to characterise immune cell populations in preterm babies. Our data is some of the first to clearly identify circulating mucosal associated T cells in preterm infants, suggesting major differences in the size and composition of this compartment. Further study will enable correlation with clinical, dietary, and microbiome data.

References

1. M. A. Mara et al. Innate and Adaptive Immunity in Necrotising Enterocolitis. Seminars in Fetal and Neonatal Medicine 23 (2018) 394–399 a prospective case-control study