Table 1.
Patient Characteristics.
Fig 1.
Comparison of soluble biomarkers between patients with mixed and donor chimerism.
Concentrations of cytokines, IgG and IgG subclasses were determined in the plasma of 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients at median 10 years post-HSCT. Asterisks indicate significant P-values (* = P < .05 and ** = P < .01), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group. (A) A higher IgG3 concentration was seen in MC patient plasma (P = .027). (C-D) A lower concentration of IL-4 (B), IL-12 (p40) (C) and G-CSF (D) was observed in MC patients (P = .016, P = .003 and P = .022 respectively).
Fig 2.
Phenotypic comparison of cellular subsets between patients with mixed and donor chimerism.
For most cellular subsets no significant differences were observed between 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients (A-C). Asterisks indicate significant P-values (* = P < .05 and ** = P < .01), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group. (A) The white blood cell (i), platelet (ii) and neutrophil (iii) count in the two patient groups. Platelet counts were higher in MC patients (P = .041). K/mL = 1 000 cells/mL. (B) Radar graphs depicting relative distribution of T, B and NK-cells (i) and T-cell subsets (ii) for DC and MC patient groups. (C) Differentiation status of total T-cells (CD3+), as defined by naïve memory (CCR7+ CD45RO-), central memory (CCR7+ CD45RO+), effector memory (CCR7- CD45RO+) and terminally differentiated memory (CCR7- CD45RO-), was found to be similar between the DC and MC patient groups. (D) Representative FACS plots of potential NKT-cells (CD56+ (i-ii) or CD94+ (iv-v)) gated on CD8+ T-cells. In the corresponding graphs (iii, vi)), individual ratios of the subsets for each group are shown (P = .004 and P = .035 respectively).
Fig 3.
Protein expression of molecules involved in lymphocyte signalling between patients with mixed and donor chimerism.
Protein expression of ZAP-70, LCK and actin was assessed in lymphocytes of 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients. Asterisks indicate significant P-values (* = P < .05), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group. (A) Representative blots displaying 3 DC (UPN 1167, 887 and 1065) and 3 MC patients (UPN 921, 652 and 527). (B) The individual values for LCK and ZAP-70 of all patients, with regards to their respective actin intensity. A difference was observed for ZAP-70 expression (P = .013) between the DC and MC patient groups.
Fig 4.
Higher frequency of IL-2-producing cells for steady state lymphocytes in mixed chimerism patients.
The frequency of IL-2 producing cells after a 4 hour mitogenic stimulation of 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients. (A) Median frequency of IL-2 and IFNγ producing lymphocytes of several T-cell (CD3+) subsets (total T-cells, CD4+ T-cells, CD8+ T-cells and CD45RO+ T-cells) in the mixed chimerism (MC, n = 9) and donor chimerism (DC, n = 10) groups after a 4-hour mitogenic stimulation with PMA and Ionomycin. No statistical difference was observed between the patient groups. (B) Pie charts displaying the median results for the same T-cell subsets after a 4-hour non-stimulation (simulating steady-state production of IL-2 and IFNγ). A higher frequency of IL-2 producing cells could be observed. (Ci-iv) The dot plots display a difference in the frequency of IL-2 producing cells between the DC and MC patient groups in the non-stimulation condition for the same T-cell subsets. The frequency was higher for total T cells (i), P = .017; CD4 T cells (ii), P = .034; CD8 T cells (iii), P = .034; and memory (CD45RO+) T cells (iv), P = .022. Asterisks indicate significant P-values (* = P < .05), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group.
Fig 5.
Recipient derived cells present in several cellular subsets in mixed chimerism patients.
Each graph depicts percentages of chimerism for different cell subsets for each mixed chimerism (MC, n = 9) patient. Chimerism was analysed for T-cells (CD3+), CD4+ T-cells, CD8+ T-cells, B-cells (CD19+ CD3-), NK-cells (CD56+ CD3-), myeloid cells (CD33+) and TCRγδ+ T-cells. A black column represents the percentage of recipient derived cells, while the white column represents donor derived cells. ND depicts the subsets where chimerism analysis was unsuccessful due to insufficient DNA.
Fig 6.
Recipient derived cells capable of cytokine production.
(A) Each graph depicts percentages of chimerism for cytokine producing lymphocytes after a 4-hour incubation with PMA and Ionomycin. Chimerism was analysed for IFNγ-producing lymphocytes and IL-2 and IFNγ producing lymphocytes. A black column represents the percentage of recipient derived cells, while the white column represents donor-derived cells. ND depicts the subsets where chimerism analysis was unsuccessful due to insufficient DNA. (B) A representative plot from patient UPN 615 demonstrates the gating strategy used on varying cellular subsets. Sorting was done on total lymphocytes.