Supplementary figure – IL2 purity and titration

(a) Size-exclusion chromatography elution of devil IL-2. The concentrated Ni chromatography eluate (1μL loaded in lane 1) was applied to a Superdex-75, 24mL column, eluted at 0.4mL/min in PBS, with 0.4mL fractions collected. 2μL of fractions 28-30 and 32-38 were resolved by reducing SDS-PAGE and visualized with SimplyBlue SafeStain. Fractions 35-38 were pooled, aliquoted and snap frozen for cell culture experiments. Based on the elution time of a 17.5kDa molecular weight standard (31.2 min) marked above the gel), we concluded that devil IL-2 eluted principally as a monomer. The protein migrated slower than the predicted molecular weight of devil IL-2 (14.8kDa) by reducing SDS-PAGE, suggestive of glycosylation of one of its two possible N-linked glycosylation sites.

(b) Representative figure showing two dilutions of IL2. In all cases 1/100 dilution gave the most consistent results, hence 1/100 was selected for cytotoxicity experiments. A limited number of ‘titrations’ were performed due to limited access to blood samples.

Supplementary table: Median Fluorescence Intensity (MFI) values of serum from captive and diseased devils

Devil MFI MFI /Average captive devil MFI*Positive (immunised) control 461 4.7

Cd1 126 1.3122 1.2

Cd16 71 0.7723 0.7

Cd38 74 0.874 0.8

Cd43 124 1.3120 1.2

Cd44 84 0.9Cd45 111 1.1

Average MFI of captive naive devils 98

Dd3 131 1.3134 1.4

Dd5 94 1.095 1.0

Dd7 75 0.874 0.8

Dd8 124 1.3124 1.3

Dd9 97 1.098 1.0

Dd10 141 1.4136 1.4

Dd11 87 0.987 0.9

Dd13 192 2.0165 1.7

Dd14 89 0.985. 0.9

Dd16 114 1.2110 1.1

Dd15 98 1.098 1.0

Dd17 124 1.3117 1.2

*MFI /Average captive devil MFI represents the ratio of the MFI of the serum from the captive (including immunised devil) or diseased devils divided by the average MFI of the serum from the captive devils.