(A) Flow cytometric analysis of surface L-selectin (mean fluorescence intensity; MFI) on splenic naïve CD4+CD44lo and CD8+CD44lo T cells of non-tumor bearing (NTB) and 4T1-bearing mice is shown (left). L-selectin mRNA expression in splenic CD4+ and CD8+ T cells from NTB and 4T1-bearing mice was determined by qRT-PCR with fold-change normalized with β-actin (right). (B) Soluble (s)L-selectin in serum of individual NTB and 4T1-bearing mice was assessed by ELISA. (A,B) Data are from three independent experiments (n ≥ 2 mice per group in each experiment; tumor volume >1500 mm3; average frequency of splenic CD11b+Gr-1+ cells (% CD45+ leukocytes) in tumor-bearing mice was ~40%). (C) Splenocytes were isolated from NTB wildtype (WT) C57BL/6 mice, L(E)-selectin transgenic mice, Adam17flox/flox/Vav1-Cre mice (Adam17−/−), or age-matched WT littermate controls. WT splenocytes were pretreated for 30 min with the ADAM17-specific inhibitor PF-5480090 (10 μM) or the ADAM17/10-specific inhibitor INCB7839 (20 μM). WT, L(E), and Adam17−/−splenocytes were then cultured 2 hr with or without phorbol myristate acetate (PMA, 100 ng/mL). Surface L-selectin (MFI) on viable naïve CD8+CD44lo T cells relative to untreated controls was determined by fluorocytometric analysis. (D) Splenic CD11b+Gr-1+ MDSC were purified from 4T1-bearing mice; splenocytes were from various NTB mice as described in (C). MDSC and WT splenocytes were both pretreated for 30 min with or without PF-5480090 or INC7839. MDSC and splenocytes from WT, L(E), or Adam17-/- mice were then co-cultured at a 10:1 ratio for 24 hr in media containing IFN-γ (20 U/mL) and LPS (100 ng/mL). L-selectin on viable naïve CD8+CD44lo T cells was assessed by flow cytometric analysis. (E) Fluorescently-labeled WT, L(E), and Adam17−/− splenocytes (i.e., from NTB mice) were adoptively transferred into NTB severe-combined immunodeficient (SCID) mice or 4T1-bearing SCID mice at 21 days-post tumor implantation (average tumor volume for all experiments, 1102 ± 191 mm3; average circulating CD11b+Gr-1+ frequencies in NTB SCID recipients, 75 ± 8 cells/µL blood, and 4T1-bearing SCID recipients, 4081 ± 876 cells/µL blood). After 24 hr post-ACT, L-selectin was assessed by flow cytometry on transferred splenocytes recovered from the blood of NTB and 4T1-bearing SCID mice. Representative flow histograms depict L-selectin expression on naïve CD8+CD44lo T cells (above); horizontal lines indicate positively stained cells, numbers are mean fluorescence intensity. Normalized data for L-selectin expression on B220+, CD4+CD44lo, CD8+CD44lo cells 24 hr post-adoptive transfer are for one representative experiment (n ≥ 2 mice per group) (below). (A–E) *p<0.05; ns, not significant; all data (mean±s.e.m.) were analyzed by unpaired two-tailed Student’s t-test. (C–E) Data are representative of ≥ two independent experiments (n ≥ 2 replicates or mice per group) and are normalized to untreated or NTB controls (indicated by dashed lines). NTB, non-tumor bearing; WT, wildtype; MFI, mean fluorescence intensity; sL-selectin, soluble L-selectin; ACT, adoptive cell transfer.