A Prospective Study of Donor ImmuKnow® as a Biomarker for Acute GvHD in Hematopoietic Cell Transplantation Recipients.

Graft versus host disease (GvHD) occurs in ∼40% of allogeneic hematopoietic cell transplantation (HCT) recipients and is associated with substantial morbidity and mortality. The exact mechanisms underlying GvHD remain to be defined, but donor immune cells are thought to play a major role in the pathogenesis of GvHD. Depletion of donor lymphocytes reduces the risk of GvHD but to the expense of disease relapse and lower engraftment. Immunological parameters of donor cells that predispose a recipient to GvHD will be of great value. The Cylex” ImmuKnow® assay determines the strength of immune function by quantifying the amount of ATP released from phytohemagglutinin-stimulated peripheral blood CD4⁺ cells. The amount of ATP production is a measure of lymphocyte activity. In our current study, we utilized the ImmuKnow® to assess whether a donor's immune response correlates with early outcomes in recipients post-HCT.

Twenty-six (26) donor-recipient pairs were included in our study (15 HLA identical sibling HCT and 11 haploidentical HCT). The median age of recipients at the time of transplantation was 49 years (range, 23-65). Twelve were female and 14 male. The median age of donors was 45 years (range, 18-67), and half of them were female. Recipients received an average cell-dose of 10.7 ± 4.9 × 10⁶ CD34⁺ cells/kg. Twenty-one patients received nonmyeloablative, reduced intensity conditioning consisting of Fluarabine, Busulfan or melphlan, and alemtuzumab, and mycophenolate mofetil was given for GvHD prophylaxis in these recipients. Five patients received myeloablative conditioning with total body irradiation or Busulfan, and cyclophosphamide. Methotrexate with/without cyclosporine or tacrolimus alone was used for GvHD prophylaxis in myeloablative patients. Stem cell source was G-CSF-mobilized peripheral blood. Blood samples obtained prior to G-CSF mobilization and prior to stem cell collection (approximately 2 weeks apart) were assayed for ImmuKnow values and cell counts (WBC, ANC, ALC & CD34⁺ count).

G-CSF mobilization led to a significant increase in ImmuKnow® ATP values from 342 to 728 ng/mL (p<0.001) along with an increase in all measured cell counts. Average ImmuKnow® values from recipients were 108 ± 116 at 2-3 weeks post-transplant, 231± 221 at 4-5 weeks, and 104 ± 95 ng/mL at 8-10 weeks. Grade ≥ II acute GvHD occurred in 27% of haploidentical HCT recipients (3/11 patients) and 20% of HLA identical HCT recipients (3/15 patients). We found no correlations between recipient post-transplant ImmuKnow® values and GvHD. No correlations were observed between various donor cell counts (WBC, ANC, ALC or CD34⁺ cell dose) and GvHD. In haploidentical HCT, mobilized donor blood ImmuKnow® ATP values did not correlate with GvHD. However, donor ImmuKnow® values correlated with increased risk of acute GvHD in HLA identical sibling HCT. ROC analysis of mobilized donor samples showed that in HLA identical sibling HCT ATP values in excess of 747 ng/mL predicted grade II or higher GvHD with a likelihood ratio of 4.00 (2.9-5.5, 95% confidence), sensitivity of 100%, and specificity of 75% (AUC=0.889, p=0.003) .

In HLA identical matched sibling transplantation, very strong ATP values (>747 ng/mL) following mobilization correlated with the risks of developing grade II or higher GvHD during the first 90 days post-transplant. If confirmed in larger studies, these data suggest that ImmuKnow can serve as an independent predictor/biomarker for the development of GvHD in HLA identical HCT.

No relevant conflicts of interest to declare.