Cholesterol Lowering Modulates T Cell Function In Vivo and In Vitro

T cells are key immune cells involved in the pathogenesis of several diseases, rendering them important therapeutic targets. Although drug delivery to T cells is the subject of continuous research, it remains challenging to deliver drugs to primary T cells. Here, we used a peptide-based drug delivery system, AP, which was previously developed as a transdermal delivery peptide, to modulate T cell function. We first identified that AP-conjugated enhanced green fluorescent protein (EGFP) was efficiently delivered to non-phagocytic human T cells. We also confirmed that a nine-amino acid sequence with one cysteine residue was the optimal sequence for protein delivery to T cells. Next, we identified the biodistribution of AP-dTomato protein in vivo after systemic administration, and transduced it to various tissues, such as the spleen, liver, intestines, and even to the brain across the blood–brain barrier. Next, to confirm AP-based T cell regulation, we synthesized the AP-conjugated cytoplasmic domain of CTLA-4, AP-ctCTLA-4 peptide. AP-ctCTLA-4 reduced IL-17A expression under Th17 differentiation conditions in vitro and ameliorated experimental autoimmune encephalomyelitis, with decreased numbers of pathogenic IL-17A+GM-CSF+ CD4 T cells. These results collectively suggest the AP peptide can be used for the successful intracellular regulation of T cell function, especially in the CNS.

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CD3 limits the efficacy of TCR gene therapy in vivo

Blood ◽

10.1182/blood-2011-04-346338 ◽

2011 ◽

Vol 118 (13) ◽

pp. 3528-3537 ◽

Cited By ~ 64

Author(s):

Maryam Ahmadi ◽

Judith W. King ◽

Shao-An Xue ◽

Cécile Voisine ◽

Angelika Holler ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Function ◽

Cell Receptor ◽

Surface Expression ◽

T Cell Function ◽

Engineered T Cells ◽

Rate Limiting

Abstract The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/β heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

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Exendin-4 Exacerbates Burn-Induced Morbidity in Mice by Activation of the Sympathetic Nervous System

Mediators of Inflammation ◽

10.1155/2019/2750528 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 1

Author(s):

Xiao-Jing Ji ◽

Ji-Wei Hao ◽

Guang-Lei Li ◽

Ning Dong ◽

Xin-Qi Wang ◽

...

Keyword(s):

Nervous System ◽

T Cell ◽

Sympathetic Nervous System ◽

Cell Function ◽

Burn Injury ◽

Total Body Surface Area ◽

T Cell Function ◽

Sympathetic Nervous

Background. Although glucagon-like peptide 1- (GLP-1-) based therapy of hyperglycemia in burn injury has shown great potential in clinical trials, its safety is seldom evaluated. We hypothesize that exendin-4, a GLP-1 analogue, might affect the immune response via the activation of the sympathetic nervous system in burn injury. Methods. Male Balb/c mice were subjected to sham or thermal injury of 15% total body surface area. Exendin-4 on T cell function in vitro was examined in cultured splenocytes in the presence of β-adrenoceptor antagonist propranolol (1 nmol/L) or GLP-1R antagonist exendin (9-39) (1 μmol/L), whereas its in vivo effect was determined by i.p. injection of exendin-4 (2.4 nmol/kg) in mice. To further elucidate the sympathetic mechanism, propranolol (30 mg/kg) or vehicle was applied 30 min prior to injury. Results. Although the exacerbated burn-induced mortality by exendin-4 was worsened by propranolol pretreatment, the inhibition of T cell proliferation by exendin-4 in vitro could be restored by propranolol instead of exendin (9-39). However, a Th2 switch by exendin-4 in vitro could only be reversed by exendin (9-39). Likewise, the inhibition of splenic T cell function and NFAT activity by exendin-4 in vivo was restored by propranolol. By contrast, the increased splenic NF-κB translocation by exendin-4 in vivo was potentiated by propranolol in sham mice but suppressed in burn mice. Accordingly, propranolol abrogated the heightened inflammatory response in the lung and the accelerated organ injuries by exendin-4 in burn mice. On the contrary, a Th2 switch and higher serum levels of inflammatory mediators by exendin-4 were potentiated by propranolol in burn mice. Lastly, exendin-4 raised serum stress hormones which could be remarkably augmented by propranolol. Conclusions. Exendin-4 suppresses T cell function and promotes organ inflammation through the activation of the sympathetic nervous system, while elicits Th2 switch via GLP-1R in burn injury.

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Discovery and characterization of llama VHH targeting the RO form of human CD45

10.1101/2020.09.01.278853 ◽

2020 ◽

Author(s):

Deepti Rokkam ◽

Patrick J. Lupardus

Keyword(s):

T Cell ◽

Cell Function ◽

Tyrosine Phosphatase ◽

Surface Protein ◽

Cell Receptor ◽

Phage Display Library ◽

Signaling Complex ◽

T Cell Function

AbstractCD45 is an abundant and highly active cell-surface protein tyrosine phosphatase (PTP) found on cells of hematopoietic origin. CD45 is of particular importance for T-cell function, playing a key role in the activation/inactivation cycle of the T-cell receptor signaling complex. The extracellular domain of CD45 is comprised of an N-terminal mucin-like domain which can be alternatively spliced to a core domain (RO) consisting of four domains with fibronectin 3 domain (FN3)-like topology. The study of CD45 has been hampered by a small set of publicly available antibodies, which we characterized as specific to the N-terminal FN3 domains of CD45 RO. To broaden the human CD45 reagent set, we identified anti-CD45 single domain VHH antibodies from a post-immune llama phage display library. Using a yeast display domain mapping system and affinity measurement we characterized seven unique clonotypes specific for CD45 RO, including binders that target each of the four FN3-like domains. These VHH molecules are important new tools for studying the role of CD45 in T-cell function in vitro and in vivo.

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