Purpose: We engineered activity nanosensors that sense anti-allograft T cell activity in vivo to noninvasively detect the onset of acute cellular rejection from urine.

Methods: Granzyme (GzmB) substrate peptides labelled with FITC was synthesized (TUCF) and conjugated to iron oxide nanoparticles (IONPs). Donor BALB/c skin was grafted onto recipient B6 mice. On day 7, splenocytes and lymphocytes are harvested and stained for GzmB for flow cytometry analysis. Surface-labelled nanoparticles were administered to skin graft mice and imaged by IVIS Imaging System. Nanosensors were administered prior to, and 7 days after transplant, and urine samples were collected and analyzed by fluorimetry. All animal works were approved by IACUC.

Results: To develop an activity nanosensor to sense T cell killing, we conjugated FITC-labelled GzmB substrate (IEFDSG) to the surface of IONPs. In a T cell killing assay, we observed activation of our nanosensors when transgenic OT1 T cells were coincubated with ovalbumin-expressing EG7-OVA but not EL4 target cells (Fig 1a,b). To test our nanosensors in the BALB/c donor to B6 recipient transplantation model, we first verified that GzmB is upregulated in activated splenocytes and lymphocytes at the onset of rejection (Fig 1c,d). At day 7, we intravenously administered nanosensors and detected preferential accumulation in skin allografts, and significant increases in urine signal in allograft mice but not in isograft mice or CD8-depleted mice (Fig 1e,f). Collectively, these experiments showed that GzmB activity nanosensors produce detection signals in urine during acute T cell rejection.

Conclusions: The “gold standard” for monitoring graft health remains the core biopsy despite its invasiveness and limited predictive power. We developed activity nanosensors that detect the onset of rejection noninvasively from urine, and this platform may be broadly useful for monitoring T cell activity in cancer immunotherapy and other autoimmune diseases.

CITATION INFORMATION: Mac Q., Mathews D., Kahla J., Stoffers C., Delmas O., Holt B., Adams A., Kwong G. Activity Nanosensors for Early and Noninvasive Detection of Acute Organ Transplant Rejection Am J Transplant. 2017;17 (suppl 3).

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