Immune subset differences by flow cytometry between paediatric kidney transplant recipients and healthy matching-age paediatric and adult controls
Elvira Jimenez Vera1, Haina Wang1, Patricia Anderson1, Catherine Lai2, Shounan Yi1, Wayne Hawthorne1, Natasha Rogers1, Peter Hsu2, Philip O'Connell1, Stephen Alexander3, Min Hu1.
1Centre for Transplant and Renal Research, Westmead Institute for Medical Research, Sydney, Australia; 2Allergy & Immunology lab, Children's Hospital at Westmead, Sydney, Australia; 3Centre for Kidney Research, Children's Hospital at Westmead, University of Sydney, Sydney, Australia
Aim: Determine differences in immunophenotype of paediatric kidney transplant recipients vs. healthy matching-age paediatric and adult controls by minimum blood volume.
Methods: Absolute cell-count and leukocyte-profiling panels (46 fluorochrome-conjugated mouse-anti-human antibodies) for T cell, Treg, B, NK, DCs, and monocyte subsets were used to quantify immune-cell populations for 8 paediatric kidney transplant (PKTx) recipients, 5 healthy matching-age paediatric and 8 adult controls. Whole-blood sample (1050µl) were used for flow-cytometry analysis.
Results: Absolute number of immune-cell subsets were in normal range for all groups. Age has correlation to CD45RO+memory T cells, but not to CD27+IgD-memory B cells. Healthy children had higher proportions of CD4-CD8-T (13.7±5.8 vs 7±4.3%, p=0.03) and γδT-cells (12±5.7vs 5.4±4.6%, p=0.04), CD4+CD25+CD127-Tregs (10±1.4 vs 7±1.6%, p<0.01), lower proportion of HLADR+CD45RA-CD4+ (0.03±0.04vs 7.42±5.64%, p<0.01), HLADR+CD45RA-CD8+ (0.0012±0.0018 vs 8±5.1%, p =0.04) T-cells compared to adults. HLA-DR was upregulated on both CD4+CD45RA- and CD8+CD45RA-T cells in PKTx, whilst CD183 (CXCR3) on CD4+CD45RO+ T-cells was downregulated. Compared to their healthy counterparts, PKTx had lower proportions of CD27+CD38lowclass-switch memory B-cells (38±7.6 vs 53±5.3%, p<0.01), non-classical monocytes (1.9±0.9 vs 5.8±1.1%, p<0.001), Tregs [CD4+CD25+CD127-Tregs (4.8±1.7% vs 10±1.4%, p<0.001) and CD4+FOXP3+Tregs (2.8±0.85% vs 6.3±1.3%, p<0.01)]. A small proportion of CD127+CD45RO+FOXP3 (2-5%) were observed in all groups. There was no difference in proportions of NK and DC between transplant vs control population.
Conclusion: Immune profiling by multi-colour flow cytometry revealed differences by ages and after transplantation and offers valuable insight into unique cell subset changes present in the transplant population that could be targeted clinically or to monitor patient condition.
