Flow cytometric characterization of human umbilical cord blood lymphocytes: immunophenotypic features

Abstract

Background and objective: One of the most important potential advantages in the use of human umbilical cord blood (HUCB) for hematopoietic reconstitution after myeloablative therapy seems to be the lower occurrence of acute graft-versus-host-disease (GvHD) in recipients after allogeneic transplantation. Since mature T cells play an important role in GvHD pathogenesis, we tried to verify whether a different immunophenotypic pattern exists between HUCB and peripheral blood (PB) T cells.

Design and methods: An immunophenotypic study on 40 HUCB and 40 PB samples from healthy adult volunteers was performed, by means of flow cytometry using a large panel of monoclonal antibodies in double labeling.

Results: The absolute lymphocyte count was greater in HUCB (5233 +/- 1808 microL) than in adult PB (1941 +/- 378 microL). Significant differences in percentage were found between cord and adult T-cells, respectively (CD3+: 59.9 +/- 12 vs 74.9 +/- 4.6%), CD3- CD16+ and/or CD56+ natural killer (NK) cells (23.8 +/- 10.1 vs 10.8 +/- 5.3%) and CD3+CD16+ and/or CD56+ cytotoxic T lymphocyte subset (0.3 +/- 0.3 vs 10.7 +/- 4.1%). There was no difference in CD4/CD8 ratio (1.7 +/- 0.5 vs 1.6 +/- 0.2%) between the two groups. In absolute terms, HUCB contained an higher number of all lymphocyte subsets, with the exception of CD3+CD16+ and/or CD56+ T lymphocyte subpopulation, CD3+CD25+ and CD3+HLADR+ activated T-cells. CD38, a marker of activation and immaturity, was present on virtually all cord T cells and on approximately half of the adult T cells. The large majority of HUCB T cells co-expressed CD45RA naive antigen (CD4+CD45RA+: 87.6 +/- 5.2%, CD8+ CD45RA+: 93.5 +/- 7.8%; CD4+CD45RO+: 12.3 +/- 5.2%; CD8+CD45RO+: 6.4 +/- 7.8%) whereas in adult PB T cells an higher number of CD45RO+ memory cells was detected (CD4+CD45RA+: 44.8 +/- 9.6%; CD8+CD45RA: 71.5% +/- 8.1%; CD4+CD45RO+: 55.2 +/- 9.6%; CD8+ CD45RO+: 28.5 +/- 8.1%). Finally, less than 14% of lymphocytes were shown to belong to B lineage in both sources, while, in absolute terms, they were more represented in HUCB with respect to adult PB.

Interpretation and conclusions: In the present study we found significant difference between HUCB and adult PB lymphocytes in their immunophenotypic profile. In particular HUCB showed T lymphocytes that appeared to be phenotypically immature. Indeed, as a likely consequence of poor antigenic experience during pregnancy, the majority of HUCB cells were naive, expressing the RA isoform of the CD45 molecule. These findings could justify the previously reported reduced cord blood lymphocyte alloreactivity when allogeneic transplantation is performed and require further functional studies in order to confirm the impairment of HUCB immune system response to alloantigens.