https://doi.org/10.5061/dryad.t4b8gtjch

Description of the data and file structure

Heterotopic kidney transplants were performed between healthy 20-25g (approximately 12-16 weeks old) female C57BL/6J (donor) and Ly5.1 BALB/c (recipient) mice. Naïve controls were age- and weight-matched.

Kidney recipients received immunosuppression monotherapy or triple therapy (tacrolimus + mycophenolate + methylprednisolone). Tacrolimus (0.94mg/kg) and mycophenolate (34mg/kg) were injected 12 hourly intraperitoneally (i.p.), and methylprednisolone (16mg/kg) 72 hourly subcutaneously (s.c.), commencing on the morning of surgery. Abatacept (10mg/kg) was administered s.c. once only (day 0). Anti-CD3ε F(ab’) 2 (50μg) was administered intravenously (i.v.) daily for five days, commencing the day before surgery (days -1, 0,1, 2, 3). Control mice received kidney transplants only. Surgeons were blinded to treatment groups.

At day 7, blood was collected via cardiac puncture, and heparinized for flow cytometry. Thereafter, mice were perfused with saline via the aorta. Graft and native kidneys, liver, spleen, hind leg bones (for bone marrow), and abdominal lymph nodes were collected. Single cell suspensions of leukocytes were prepared from each organ. Cells were counted using a Countess II FL Automated Cell Counter (Thermo Fisher). These counts are provided in the Excel spreadsheet.

Single cell suspensions were then analyzed for lymphoid and myeloid subsets. Fc receptor blocking with CD16/32 (2.4G2, Becton Dickinson; BD) was performed prior to myeloid cell staining. All cells were stained with Zombie UV Fixable Viability Dye (BioLegend), before staining with surface marker antibodies diluted in Brilliant Stain Buffer (BD). For intracellular staining, the eBioscience FoxP3/Transcription Factor Staining Buffer set (Thermo Fisher) was used. Cells were fixed with Stabilizing Fixative (BD). Data was acquired on a 5-laser, 16-parameter BD Special Order Research Product LSRFortessa flow cytometer. Quality control checks were performed with Cytometer Setup and Tracking beads (BD), and Ultra Rainbow Calibration Particles (Spherotech) to adjust for laser drift. UltraComp eBeads (Thermo Fisher) were used for compensation, however manual compensation was also applied. Compensated data was subjected to FlowAI to clean data based on flow rate and signal acquisition. The provided fcs files are of this compensated, flowAI cleaned data.

Files and variables

File: Dryad_Upload_Additional_Information.xlsx

Description: Excel file linking mouse ID to treatment group, and providing original cell counts per organ for cell quantification

Variables

• Mouse ID: Matches fcs file name
• Group: Treatment group or control is listed
• Number of cells/organ: cell counts per organ
  • BM: bone marrow
  • Blood (count/mL)
  • LN: abdominal lymph nodes
  • Liver
  • Spleen
  • Kid Tx (Transplanted Kidney)
  • Kid NT (Native Kidney)

File: Dart_2026_Data_Upload.zip

Description: FCS files. Each File is labelled with the mouse ID, staining panel, and organ abbreviation (BM = hind leg bone marrow, LN = abdominal lymph nodes, Blood, Liver, Spleen, KTx = kidney transplant, KNT = native kidney)

Mouse IDs are connected to each treatment group as below:

Naïve Controls:

Staining Panels are as below:

Lymphoid:

Myeloid:

Code/software

FCS files may be analysed using FlowJo or https://floreada.io (free, web-based alternative).

Access information

Other publicly accessible locations of the data:

• N/A

Data was derived from the following sources:

• N/A