Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.

Background: Treatment options for previously treated metastatic triple-negative breast cancer (mTNBC) are limited. In cohort A of the phase II KEYNOTE-086 study, we evaluated pembrolizumab as second or later line of treatment for patients with mTNBC.Patients and methods: Eligible patients had centrally confirmed mTNBC, !1 systemic therapy for metastatic disease, prior treatment with anthracycline and taxane in any disease setting, and progression on or after the most recent therapy. Patients received pembrolizumab 200 mg intravenously every 3 weeks for up to 2 years. Primary end points were objective response rate in the total and PD-L1-positive populations, and safety. Secondary end points included duration of response, disease control rate (percentage of patients with complete or partial response or stable disease for !24 weeks), progression-free survival, and overall survival.Results: All enrolled patients (N ¼ 170) were women, 61.8% had PD-L1-positive tumors, and 43.5% had received !3 previous lines of therapy for metastatic disease. ORR (95% CI) was 5.3% (2.7-9.9) in the total and 5.7% (2.4-12.2) in the PD-L1-positive populations. Disease control rate (95% CI) was 7.6% (4.4-12.7) and 9.5% (5.1-16.8), respectively. Median duration of response was not reached in the total (range, 1.2þ-21.5þ) and in the PD-L1-positive (range, 6.3-21.5þ) populations. Median PFS was 2.0 months (95% CI, 1.9-2.0), and the 6-month rate was 14.9%. Median OS was 9.0 months (95% CI, 7.6-11.2), and the 6-month rate was 69.1%. Treatment-related adverse events occurred in 103 (60.6%) patients, including 22 (12.9%) with grade 3 or 4 AEs. There were no deaths due to AEs.Conclusions: Pembrolizumab monotherapy demonstrated durable antitumor activity in a subset of patients with previously treated mTNBC and had a manageable safety profile.

Sphingosine 1-phosphate (S1P) is a bioactive lysolipid with pleiotropic functions mediated through a family of G proteincoupled receptors, S1P 1,2,3,4,5 . Physiological effects of S1P receptor agonists include regulation of cardiovascular function and immunosuppression via redistribution of lymphocytes from blood to secondary lymphoid organs. The phosphorylated metabolite of the immunosuppressant agent FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol) and other phosphonate analogs with differential receptor selectivity were investigated. No significant species differences in compound potency or rank order of activity on receptors cloned from human, murine, and rat sources were observed. All synthetic analogs were high-affinity agonists on S1P 1 , with IC 50 values for ligand binding between 0.3 and 14 nM. The correlation between S1P 1 receptor activation and the ED 50 for lymphocyte reduction was highly significant (p Ͻ 0.001) and lower for the other receptors. In contrast to S1P 1 -mediated effects on lymphocyte recirculation, three lines of evidence link S1P 3 receptor activity with acute toxicity and cardiovascular regulation: compound potency on S1P 3 correlated with toxicity and bradycardia; the shift in potency of phosphorylated-FTY720 for inducing lymphopenia versus bradycardia and hypertension was consistent with affinity for S1P 1 relative to S1P 3 ; and toxicity, bradycardia, and hypertension were absent in S1P 3 Ϫ/Ϫ mice. Blood pressure effects of agonists in anesthetized rats were complex, whereas hypertension was the predominant effect in conscious rats and mice. Immunolocalization of S1P 3 in rodent heart revealed abundant expression on myocytes and perivascular smooth muscle cells consistent with regulation of bradycardia and hypertension, whereas S1P 1 expression was restricted to the vascular endothelium.

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)−/− mice that were also deficient in iNOS. ApoE−/− and iNOS−/− mice were cross-bred to produce apoE−/−/iNOS−/− mice and apoE−/−/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30–50% reduction in apoE−/−/iNOS−/− mice compared with apoE−/−/iNOS+/+ mice. Although the size of the lesions in apoE−/−/iNOS−/− mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45–70% reductions in both male and female apoE−/−/iNOS−/− mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.

1008 Background: In KEYNOTE-012, pembro showed durable activity and manageable safety in patients (pts) with PD-L1+ mTNBC. Cohort A of KEYNOTE-086 (NCT02447003) examined the efficacy/safety of pembro in previously treated mTNBC, regardless of PD-L1 expression. Methods: Pts with centrally confirmed mTNBC, ≥1 prior chemotherapy for metastatic disease, and ECOG PS 0-1 had pembro 200 mg Q3W for up to 24 mo; imaging q 9 wk for the first 12 mo, then q 12 wk. Clinically stable pts with PD could remain on pembro until PD confirmed on next assessment. Primary endpoints: ORR (RECIST v1.1, central review) in all pts and pts with PD-L1+ tumors, and safety. Secondary endpoints: DOR, disease control rate (DCR; CR + PR + SD ≥24 wk), PFS, and OS. Planned enrollment was 160 pts; analysis based on data as of Nov 10, 2016. Results: 60% of screened PD-L1-evaluable pts had PD-L1+ tumors (combined positive score ≥1%). Of 170 pts enrolled (100% women; median age 54 y), 44% had ≥3 prior lines of therapy, 51% had elevated LDH, 74% had visceral mets and 62% had PD-L1+ tumors. After a median follow-up of 10.9 mo, 9 (5%) pts remained on pembro. Treatment-related AEs (TRAEs) of any grade and grade 3-4 occurred in 60% and 12% of pts, respectively; 4% discontinued due to TRAEs. There were no deaths due to AE. Overall ORR was 5% regardless of PD-L1 expression (Table). Best overall response was 0.6% CR, 4% PR, 21% SD; not evaluable (3%). DCR was 8% (95% CI 4-13). Median DOR was 6.3 mo (range 1.2+ to 10.3+); 5 (63%) responders w/o PD at data cutoff. Median PFS and OS were 2.0 mo (95% CI 1.9-2.0) and 8.9 mo (95% CI 7.2-11.2), with 6-mo rates of 12% and 69%, respectively. ORR was numerically lower in pts with poor prognostic factors (e.g., high LDH, liver/visceral mets; Table). Conclusions: In KEYNOTE-086 Cohort A, pembro monotherapy showed manageable safety and durable responses in a subset of pts with heavily pretreated mTNBC. Randomized studies of monotherapy and combination therapy are ongoing. Clinical trial information: NCT02447003. [Table: see text]