Injection of tacrolimus just around the last day (C, row 2) was sufficient to suppress CD4+ T-cell proliferation to levels like the group that received the full 10-day course of tacrolimus (C, row 1), while missing just the last 2 doses of tacrolimus (C, row 3) produced no detectable suppression of T-cell proliferation, similar to what was seen in the control mice not receiving any tacrolimus (C, row 4)

Injection of tacrolimus just around the last day (C, row 2) was sufficient to suppress CD4+ T-cell proliferation to levels like the group that received the full 10-day course of tacrolimus (C, row 1), while missing just the last 2 doses of tacrolimus (C, row 3) produced no detectable suppression of T-cell proliferation, similar to what was seen in the control mice not receiving any tacrolimus (C, row 4). not statistically significant nor correlated with serum tacrolimus levels. We observed that cell processing and washing reduced the effects of tacrolimus on T-cell proliferation, as did discontinuation of tacrolimus treatment shortly before sampling. Conclusions T-cell proliferation is currently not suitable to measure immunosuppression because sample processing diminishes observable effects. Future immune function screening should focus on new samples with minimal washing actions. Our results also emphasize the importance of adherence to immunosuppressive treatment, because T-cell proliferation recovered substantially after even brief discontinuation of tacrolimus. Immunosuppression after solid organ transplantation should ideally achieve a balance between preventing allograft rejection and allowing the recipient to avoid contamination and malignancy. Acute and chronic rejections increase the risk of premature graft failure and sensitization against future transplants. 1 Rejection episodes and graft loss are frequently associated with nonadherence to immunosuppression. 2 Infections are also a significant complication after transplant, especially in children, where admission to the hospital for contamination is more common than for rejection.3,4 There is no reliable assay to measure the overall degree of immunosuppression after transplantation. Therefore, it is challenging to determine an individual patient’s risk for contamination or rejection, especially in the face of variable medication adherence. In current clinical practice, providers use proxy steps including drug levels, antihuman leukocyte antigen antibody assays, and the presence of opportunistic infections to estimate a patient’s degree of immunosuppression and adherence with medications.5,6 Prior attempts to directly measure a patient’s immune function have relied on nonspecific metabolic assays that have produced inconsistent results.7-12 We developed an assay quantifying the ability of T cells Rabbit Polyclonal to C-RAF to proliferate in response to fixed, exogenous stimulation as a readout of immunosuppression after transplantation. We first tested the methodology in a murine model and then piloted the assay in children who experienced received a kidney transplant, using healthy adult blood donors as controls. This report explains the initial findings of our assay and their implications for assessment of medication adherence and future efforts to measure an individual patient’s overall degree of immunosuppression. MATERIALS AND METHODS Patients and Healthy Donors We performed a cross-sectional study focusing on children and adolescents who experienced received a kidney transplant at the Children’s Hospital of Philadelphia. Patients were eligible for enrollment if they were 2 to 25 years of age; were on therapeutic immunosuppression, including prednisone, tacrolimus and mycophenolate; and were 6 months to 5 years after their first kidney transplant. Exclusion criteria were a history of renal allograft rejection, evidence of viral contamination (Epstein-Barr computer virus, BK, or cytomegalovirus) within 3 months before sample collection, history of malignancy or posttransplant lymphoproliferative disorder, or documented medication nonadherence. Comparison analyses were performed on healthy, deidentified adult donor peripheral blood mononuclear cell (PBMC) samples obtained through the Human Immunology Core at the University or college of Pennsylvania. All patients and guardians, as well as healthy adult blood donors, gave written informed consent as approved by the institutional evaluate boards at Childrens Hospital of Philadelphia (IRB 14-010784) and the University or college of Pennsylvania (IRB 70906). Murine Studies We purchased C57BL/6 mice from Jackson Laboratories. Mice were housed under specific pathogen-free conditions using protocols approved by the Institutional Animal Care and Use Dihydromyricetin (Ampeloptin) Committees of the Childrens Hospital of Philadelphia and University or college of Pennsylvania (13-000561). Sample Collection and Storage For murine studies, we obtained spleens and processed them into single-cell suspension after red blood cell lysis.13 For human studies, PBMC and T cells were isolated, cryopreserved, and recovered as previously described.14-16 Briefly, each human subject provided a single whole blood sample (1-10 mL) drawn in an ethylenediaminetetraacetic acid tube during a scheduled outpatient visit at the time of routine, 12-hour trough level testing. Blood samples were processed in SepMate tubes (Stemcell Technologies, Vancouver, Canada) to isolate PBMC. The PBMC were counted, and either stimulated or suspended in CryoStor answer (Sigma-Aldrich), and stored in cryotubes in liquid nitrogen. T-Cell Function Dihydromyricetin (Ampeloptin) Screening and Circulation Cytometry Our assay was designed to Dihydromyricetin (Ampeloptin) directly quantify the ability of T cells to proliferate. Specifically, in both murine and human studies, T cells were exogenously stimulated with a soluble, plate-bound, or bead-bound Dihydromyricetin (Ampeloptin) CD3 monoclonal antibodies (mAb) directed against the T-cell receptor in.