Flow Cytometric Detection of the Expression of CXCR4 on CD34⁺ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34⁺ cells and to assess the distribution of lymphocyte subsets in each type allograft.

Methods: CD34⁺ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34⁺cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls.

Results:

1. The expression of CXCR4 of cord blood and mobilized peripheral blood CD34⁺ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05).

2. The CD3⁺CD8^low and CD3⁺CD4⁻CD8^low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01).

3. The relative frequencies of the naïve CD45RA⁺ CD45RO⁻ phenotype among CD4⁺ and CD8^high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01).

4. Naïve T cells (CD45RA⁺ CD45RO⁻) were mobilized preferentially compared to memory T cells (CD45RA⁻ CD45RO⁺)(P <0.01);

5. The relative frequencies of NKT (CD3⁺CD16+56⁺) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01).

Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34⁺ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.