Single-Cell Mass Profiling of Reconstituting Donor T Cells Following Stem Cell Transplantation to Predict Alloreactivity

Patients undergoing stem cell transplantation (SCT) are at risk for graft-versus-host disease (GVHD). It is not yet possible to discern in real-time which patients are at risk for GVHD as post-transplant immunosuppression is weaned. In this pilot study, we examined the utility of single-cell, T cell mass measurements as a biomarker for GVHD risk. Based on prior work demonstrating significant mass differences between activated and resting T cells, our group studied a model in which donor T cells exhibit a measurable increase in mass upon activation from exposure to recipient antigens. HLA-matched allogeneic (n = 14) and autologous SCT (n = 4) patients were enrolled. Myeloablative allogeneic SCT was performed with either HLA-matched unrelated (n = 11) or related (n = 3) donors. Samples (3 mL) were obtained from donor products and from the recipients (whole blood) at days 14, 30, 60 and 100 post-transplant. CD3+ cells were immunomagnetically isolated from each sample and analyzed (mean number of cells imaged per time point: nProduct = 1550, nD14 = 400, nD30 = 657, n60 = 605, nD100 = 709) by Live-Cell Interferometry (LCI), a novel, high-precision (<1% coefficient of variation) microscopy technique [LCI uses a specialized camera to measure local phase shifts, enabling the automated and non-invasive measurement of the masses of thousands of living cells with picogram (pg) sensitivity (Figure 1, Figure 2)].Figure 23D rendering of T cells from an LCI image.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Of the patients that underwent allogeneic SCT, 9 (69%) developed GVHD. Median onset of acute GVHD (n = 7) and chronic GVHD (n = 4) were at 58 and 126 days post-transplant, respectively. The mean mass of T cells from infused stem cell products was 68 pg for autologous SCT recipients and 58 pg for allogeneic SCT recipients. Post-transplant variability in T cell masses was observed in allogeneic SCT recipients. Patients who did not develop GVHD exhibited an initial (day 30) departure from, and later (day 60) restoration of T cell mass to levels similar to the infused stem cell product. Autologous SCT patients displayed similar kinetics. Together, autologous and allogeneic SCT patients free of GVHD exhibited no significant difference between infused product and day 100 normalized median T cell masses (p = .30). Conversely, patients with GVHD exhibited a significant (p = .006) difference between product and day 100 normalized median masses, showing a persistent elevation (~18% higher median T cell masses than infused stem cell product) (Figure 3). Of the 4 patients with a 20% or greater increase in mass at day 100, 2 developed grade 3-4 acute GVHD and one, severe chronic GVHD. LCI is an inexpensive and simple technique which provides a clinically-applicable assay that may help predict the risk of alloreactivity (GVHD) by measuring the departure from infused T cell mass. These data may be used to guide withdrawal of immunosuppression following SCT, and reduce the likelihood of GVHD or relapse occurring.