Donor Cell Composition and Reactivity Predict Risk of Acute Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Abstract

Background. Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). We designed a functional assay for assessment of individual risk for acute GVHD. Study Design and Methods. Blood samples were collected from patients and donors before HSCT. Two groups of seven patients each were selected, one in which individuals developed acute GVHD grades II-IV and one in which none showed any clinical signs of GVHD. Peripheral blood mononuclear cells (PBMCs) isolated from donors were incubated in mixed lymphocyte cultures (MLCs) with recipient PBMCs. The cells were characterized by flow cytometry before and after MLC. Results. Samples from donors in the GVHD group contained significantly lower frequencies of naïve γδ T-cells and T-cells expressing NK-cell markers CD56 and CD94. Donor samples in this group also exhibited lower frequencies of naïve CD95⁺ T-cells compared to controls. After MLC, there were dissimilarities in the CD4/CD8 T-cell ratio and frequency of CD69⁺ T-cells between the two patient groups, with the non-GVHD group showing higher frequencies of CD8⁺ and CD69⁺ T-cells. Conclusion. We conclude that a thorough flow cytometric analysis of donor cells for phenotype and allogeneic reactivity may be of value when assessing pretransplant risk for severe acute GVHD.

Figure 1

No significant differences between the non-GVHD and GVHD groups regarding major lymphocyte subsets or T-cell maturation subsets in unmanipulated donor samples. Flow cytometry-acquired phenotypic data analysed in blood samples from donors. The data were divided into two groups based on if patients did or did not develop acute GVHD grades II–IV. Each dot represents the cell-subset frequency of one donor and horizontal bars indicate the median of each group. Representative FACS plots are shown below each dot-plot of one non-GVHD and one GVHD patient. (a) Percentages of total T-cells (CD3⁺), NK-cells (CD3⁻CD56⁺), and B-cells (CD3⁻CD19⁺). No differences were observed for these cellular subsets between the non-GVHD and GVHD patient groups. (b) Proportions of T-cell subsets at different maturation states in the total T-cell population, expressed as median percentages. Terminal, terminally differentiated T-cells (CD45RO⁻CCR7⁻); effector, effector memory T-cells (CD45RO⁺CCR7⁻); central, central memory T-cells (CD45RO⁺CCR7⁺); naïve, naïve T-cells (CD45RO⁻CCR7⁺). No differences were observed.

Figure 2

The non-GVHD group had higher frequencies of CD94⁺, TCRγδ ⁺, CD56⁺, and CD95⁺ T-cell subsets than the GVHD group in unmanipulated donor samples. Each dot represents the cell-subset frequency of one donor and horizontal bars indicate the median of each group. Representative FACS plots are shown below each dot-plot of one non-GVHD and one GHVD patient. Statistical analysis was done with the Mann–Whitney U test. (a) Percentages of CD94⁺, TCRγδ ⁺, and CD95⁺ naïve T-cells were increased within the non-GVHD group. (b) CD94 was expressed to a higher degree on terminally differentiated T-cells within the non-GVHD group. (c) Percentages of CD56⁺ CD4⁺ T-cells were increased within the non-GVHD group.

Figure 3

The GVHD group showed dissimilarity in CD4/CD8 T-cell ratios and frequencies of CD69⁺ T-cells when responding donor cells were analysed after MLC. Flow cytometric analysis of responder cells (of donor origin) before and after MLC. Statistical analysis was done with the Mann–Whitney U test. (a) Frequencies of T-cells before and after MLC. T-cell frequencies did not differ between the non-GVHD and GVHD groups. (b) CD4/CD8 T-cell ratios before and after MLC. The CD4/CD8 T-cell ratio was similar between the two patient groups before MLC. After MLC, the CD4/CD8 ratio shifted towards an increase of CD4⁺ T-cells and a decrease of CD8⁺ T-cells in the GVHD group. (c) Frequencies of CD69⁺ T-cells before and after MLC. CD69 was expressed more on T-cells of non-GVHD patients after MLC for the unstimulated and 5 : 1 stimulated condition.