Supplementary Fig. 1. Time course of morphine analgesia (1–20 mg/kg) in CD-1 (A) and nude (B) mice over the 120-min testing period. Symbols represent mean ± SEM withdrawal latency (s) on the 47 °C tail-withdrawal test; group sample sizes are provided in parentheses. Both genotypes displayed highly significant dose x repeated measures interactions (p’s<0.001); asterisks are omitted here for clarity. Transformed data,
0 30 60 90 120
0 5 10 15 20 25 30
Time Post-Injection (min)
Withdrawal Latency (s)
1 mg/kg (8) 2.5 mg/kg (7) 5 mg/kg (6) 10 mg/kg (8) 20 mg/kg (6) A. CD-1
0 30 60 90 120
0 5 10 15 20 25 30
Time Post-Injection (min)
Withdrawal Latency (s)
B. nude
1 mg/kg (8) 2.5 mg/kg (8) 5 mg/kg (8) 10 mg/kg (8) 20 mg/kg (6)
Supplementary Fig. 2. Licking data in the late phase (10-60 min post-injection) of the 5% formalin test. Bars represent mean ± SEM samples featuring licking/biting behavior; group sample sizes are provided. **p<0.01, ***p<0.001 compared to 0 dose by Dunnett’s case-comparison (one-tailed) posthoc test. These data were converted to % analgesia data shown in Fig. 2B by comparing each dose to it’s own 0 dose group within-genotype.
0 5 10 20
0 10 20 30 40 50
Morphine Dose (mg/kg)
% Positive Samples
A. CD-1
** ***
16 8 11 ***
8
0 5 10 20 40 0
10 20 30 40 50
Morphine Dose (mg/kg)
% Positive Samples
B. nude
***
26 8 10 8
8
Supplementary Fig. 3. Time course of morphine analgesia (1–5 mg/kg) in C57BL/6, Cd4-/- and Rag1-/- mice over the 120-min testing period.
Symbols represent mean ± SEM withdrawal latency (s) on the 49 °C tail-withdrawal test; sample sizes are provided. All genotypes displayed highly significant effects of repeated measures (p’s<0.005); asterisks are omitted here for clarity. Transformed data, integrated over time, are provided in Fig. 2C.
0 30 60 90 120
0 5 10 15 20 25 30
Time Post-Injection (min)
Withdrawal Latency (s)
C57BL/6 (6) Cd4-/-(8) Rag1-/-(6)
C57BL/6 (6) (5 mg/kg)
1 mg/kg
Supplementary Fig. 4. FACS data confirming successful adoption transfer of T-cells into nude mice. Plots of CD3+CD4-CD8-, CD3+CD8+, CD3+CD4+, and CD3+CD4+CD8+ stained populations after flow cytometric sorting. Percentage of CD3+ (Alexa488) gated CD4+ (APC-H7) and CD8+
(PerCP-Cy5.5) cells from spenlocytes of naïve nude (a), naïve CD-1 (b), or nude mice 3 days after adoptive transfer of T cells (c). Results are expressed as percentage of total CD3+ cells.
a. b. c.
Supplementary Fig. 5. Expression of Th1- and Th2-related genes Ifng (interferon-g) and Tgfb1 (transforming growth factor, b1), respectively, in CD4+ T cells taken from untreated CD-1 mice (baseline) and CD-1 mice 30 minutes post-injection with morphine (5 mg/kg). Bars represent relative expression compared to the housekeeping gene, Gapdh, using the Ct method (average of three technical replicates); n=5–6 mice/condition). No main effects of sex nor interactions were observed.
Ifn g T g fb 1
0 .0 0 0 0 .0 0 5 0 .0 1 0 0 .0 1 5 0 .0 2 0
Ct
B a s e lin e P o s t- M o rp h in e
* *
