Combined PD-L1 and TIM3 blockade improves expansion of fit human CD8+ antigen-specific T cells for adoptive immunotherapy.

Antigen-specific T cell expansion ex vivo followed by adoptive transfer enables targeting of a multitude of microbial and cancer antigens. However, clinical-scale T cell expansion from rare precursors requires repeated stimulation, which may lead to T cell dysfunction and limited therapeutic potential. We used a clinically compliant protocol to expand Epstein-Barr virus (EBV) and Wilms tumor 1 (WT1) antigen-specific CD8+ T cells, and leveraged T cell exhaustion-associated inhibitory receptor blockade to improve T cell expansion. Several inhibitory receptors were expressed early by ex vivo-expanded antigen-specific CD8+ T cells, including PD-1 and TIM3, with co-expression matching evidence of T cell dysfunction as the cultures progressed. Introduction of anti-PD-L1 and anti-TIM3 blockade in combination (but not individually) to the culture led to markedly improved antigen-specific T cell expansion without inducing T cell dysfunction. Single-cell RNA sequencing (RNA-seq) and T cell receptor (TCR) repertoire profiling revealed that double blockade does not impart specific transcriptional programs in T cells or alterations in TCR repertoires. However, combined blockade may affect gene expression in a minority of clonotypes in a donor-specific fashion. We conclude that antigen-specific CD8+ T cell manufacturing can be improved by using TIM3 and PD-L1/PD-1 axis blockade in combination. This approach is readily applicable to several adoptive immunotherapy strategies.

Downregulation of low-density lipoprotein receptor mRNA in lymphatic endothelial cells impairs lymphatic function through changes in intracellular lipids.

Rationale: Impairment in lymphatic transport is associated with the onset and progression of atherosclerosis in animal models. The downregulation of low-density-lipoprotein receptor (LDLR) expression, rather than increased circulating cholesterol level per se, is involved in early atherosclerosis-related lymphatic dysfunction. Enhancing lymphatic function in Ldlr-/- mice with a mutant form of VEGF-C (VEGF-C 152s), a selective VEGFR-3 agonist, successfully delayed atherosclerotic plaque onset when mice were subsequently fed a high-fat diet. However, the specific mechanisms by which LDLR protects against lymphatic function impairment is unknown. Methods and results: We have thus injected wild-type and Pcsk9-/- mice with an adeno-associated virus type 1 expressing a shRNA for silencing Ldlr in vivo. We herein report that lymphatic contractility is reduced upon Ldlr dowregulation in wild-type mice only. Our in vitro experiments reveal that a decrease in LDLR expression at the mRNA level reduces the chromosome duplication phase and the protein expression of VEGFR-3, a membrane-bound key lymphatic marker. Furthermore, it also significantly reduced the levels of 18 lipid subclasses, including key constituents of lipid rafts as well as the transcription of several genes involved in cholesterol biosynthesis and cellular and metabolic processes. Exogenous PCSK9 only reduces lymphatic endothelial-LDLR at the protein level and does not affect lymphatic endothelial cell integrity. This puts forward that PCSK9 may act upon lymphatic muscle cells to mediate its effect on lymphatic contraction capacity in vivo. Conclusion: Our results suggest that treatments that specifically palliate the down regulation of LDLR mRNA in lymphatic endothelial cells preserve the integrity of the lymphatic endothelium and sustain lymphatic function, a prerequisite player in atherosclerosis.

Early rescue of lymphatic function limits atherosclerosis progression in Ldlr-/- mice.

BACKGROUND AND AIMS: Our previous data showed that lymphatic function impairment occurs before the onset of atherosclerosis in mice and is precociously associated with a defect in the propelling capacity of the collecting lymphatic vessels. Concomitantly, we found that lymphatic transport can be restored in mice by systemic injections of a mutant form of VEGF-C (VEGF-C 152s), a growth factor known to increase mesenteric collecting lymphatic vessel pumping through a VEGFR-3-dependent mechanism in rats. In the present study, we aimed to determine whether and how early modulation of collecting lymphatic vessel function could restrain atherosclerosis onset and limit its progression. METHODS: Before the administration of a pro-atherosclerotic regimen, Ldlr-/- mice at 6 weeks of age were injected intraperitoneally with VEGF-C 152s or PBS every other day for 4 weeks, fed on high fat diet (HFD) for an additional 8 weeks to promote plaque progression, and switched back on chow diet for 4 more weeks to stabilize the lesion. RESULTS: Early treatment with VEGF-C first improved lymphatic molecular transport in 6-week-old Ldlr-/- mice and subsequently limited plaque formation and macrophage accumulation, while improving inflammatory cell migration through the lymphatics in HFD-fed mice. The contraction frequency of the collecting lymphatic vessels was significantly increased following treatment throughout the whole atherosclerotic process and resulted in enhanced plaque stabilization. This early and maintained rescue of the lymphatic dysfunction was associated with an upregulation of VEGFR3 and FOXC2 expression on lymphatic endothelial cells. CONCLUSIONS: These results suggest that early treatments that specifically target the lymphatic contraction capacity prior to lesion formation might be a novel therapeutic approach for the prevention and treatment of atherosclerosis.