Higher Proportions of IFNg-Producing T Cells, CD56^bright NK Cells and Immature B Cells in the Lymphocytes of G-CSF Stimulated Donor Grafts Are Associated with Less Chronic Gvhd After HSCT: Results From the Canadian BMT Group 0601 Randomized, Phase III Trial

The insidious onset and heterogeneous nature of chronic GVHD (cGVHD) impedes its diagnosis and treatment. Donor and graft characteristics, including those which lead to variations in specific cell populations infused at the time of transplantation, may predict the onset of cGVHD.

This study evaluated graft lymphocyte populations of donors enrolled on the prospective Phase III Canadian BMT Group (CBMTG) clinical trial “A Randomized Multicentre Study Comparing G-CSF Mobilized Peripheral Blood and G-CSF Stimulated Bone Marrow in Patients Undergoing Matched Sibling Transplantation for Hematologic Malignancies (CBMTG 0601)”. Proportions of specific lymphocytes in donor grafts were associated with cGVHD in the recipient using logistic regression. Associations were significant if the corresponding p-value was less than 0.05 and the difference between the medians of the two groups was 50% lower than or twice as high as the control. Analyses were performed on G-CSF peripheral blood and bone marrow as a single population due to a data lock on the donor source randomization pending longer follow up. Recipients were considered cGVHD positive if diagnosed with extensive cGVHD and cGVHD negative if free of extensive cGVHD for a minimum of 12 months post transplant. Relapsed recipients and those who died without previous diagnosis of GVHD were excluded from analyses. Cell phenotypes and cytokine production of lymphocytes in the donor grafts were analyzed by flow cytometry in a set of panels that enabled identification of distinct NK, T and B cell subsets. After an initial analysis of 40 samples, a subset of immune populations, which significantly associated with recipient outcome, were analysed in an additional 20 samples.

Analyses of cGVHD^- vs. cGVHD⁺ showed the following associations: higher proportions of IFNg-producing T helper cells and CD56^bright NK cells in donor grafts were associated with a lack of cGVHD (p<0.005 and p<0.05, respectively). Higher graft proportions of a third population of cells, immature B cells (CD19⁺IgD⁻CD27⁻), were also associated with lack of cGVHD in the recipients (p<0.05). However, a multinomial logistic analysis which divided cGVHD+ outcomes into de novo and cGVHD developing after acute GVHD showed that this cell type actually associated with lack of de novo cGVHD only (see table for details).

We have identified an IFNg-producing CD4⁺ T cell, an NK cell, and an immature B cell population in donor grafts that impact the development of cGVHD in the recipient. CD56^bright NK cells are characteristically weakly cytotoxic but efficient producers of IFNg. The protective function of IFNg is supported by previous results from our lab showing that PBMCs of transplant recipients who did not develop cGVHD have higher levels of IFNg mRNA after stimulation (Rozmus et al, 2011). Donor IFNg polymorphisms, as well as graft source (bone marrow vs. peripheral blood), may cause differences in the immune cell proportions of donor grafts. The discovery that immature B cells are associated only with de novo cGVHD lends support to the concept that cGVHD following acute disease may differ biologically from de novo cGVHD. To better understand the role of these cells in disease pathology, more detailed functional assays of these lymphocyte subsets are required.

All values are expressed as medians (first – third percentile range). *acute GVHD present, chronic GVHD absent (A+C-) recipients were excluded from the multivariate analyses due to low numbers (n=2). The values are expressed as percentages of the following lymphocytes: CD56^bright NK cells; total CD3⁻, IFNg-producing T cells; total CD3⁺ and immature B cells; total CD19⁺.

No relevant conflicts of interest to declare.