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T regulatory lymphocytes are found in fetal blood at 29 weeks gestation, but do not express FOXP3

Presented at the Neonatal Society 2004 Summer Meeting (programme).

Lall A1,2, Yates J2, Duggan P1,2, Kennea N1, Butler M2, Lombardi G2, Lechler R2, Edwards AD1,3

1 Department of Paediatrics, Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London, UK
2 Department of Immunology, Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London, UK
3 MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London, UK

Introduction: In recent years, regulatory T (Treg) lymphocytes that constitutively express CD4 and the Interleukin-2 receptor-α chain (CD25) have been shown to modulate the inflammatory response to infection and prevent autoimmune disease. In mice the Treg subset appears only in postnatal life; however we have previously described a similar population of Treg cells in healthy, term, newborn infants (1) and suggested that these cells may play a role in the control of inflammation in the fetal and perinatal period. Expression of the FOXP3 gene has to date been found universally in rodent and human Treg cells and it is suggested that FOXP3 confers these cells with regulatory properties.

Aim: The aim of this study was to investigate whether Treg cells exist in the human fetus and to determine whether their phenotype and function was similar to that found in healthy newborn infants and adults. In particular we asked whether FOXP3 was expressed in this cell population.

Methods: Umbilical cord blood was obtained from 3 preterm infants who were delivered by caesarean section without labour or rupture of membranes, median gestational age 30 (range 29-32) weeks. Blood was also obtained from the umbilical cords of 3 healthy full-term infants, who were delivered by an elective caesarean section following a normal pregnancy and peripheral blood samples were obtained from healthy adult volunteers. Mononuclear cells (MCs) were obtained by density centrifugation using lymphoprep (Nygaard). Non-CD4+ cells were depleted by negative selection and CD4+CD25+ cells were then isolated by positive selection using CD25+ magnetic beads (Dynal) at 4oC. Isolated CD4+ cells were stained at a concentration of 104 cells in 100μl of 1% PBS for 20-30 minutes at 4oC in the dark with a range of antibodies and their surface phenotype assessed by flow cytometry. CD3CD28 beads (Dynal) were used as stimulators in proliferation assays (in vitro) with incorporation of 3H thymidine as the readout. RNA was extracted from CD4+CD25+ and CD4+CD25- cells, quantified and then incubated with reverse transcriptase to generate cDNA. FOXP3-specific primers were then used in the PCR reaction and the samples run on a 1% TAE gel and photographed under UV light.

Results: All term and preterm infants had Treg cells isolated from umbilical cord blood. These cells demonstrated typical T regulatory properties, being both hyporesponsive to stimuli and suppressing proliferation of T effector cells in co-culture. However mRNA for FOXP3 was only detected in term infants.

Conclusions: These results demonstrate that fully functional Treg cells arise in humans by 29 weeks gestation age. FOXP3 is not involved in the regulatory mechanisms of these cells in preterm infants and thus is not essential for regulatory function in humans.

1. Ng WF, Duggan PJ et al, (2001). Human CD4(+)CD25(+) cells: a naturally occurring population of regulatory T cells. Blood 98(9):2736-44.

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