Analysis of Regulatory Mechanisms for T Cell Activation

The primary function of the immune system is to seek and eliminate altered or unhealthy cells. T cells are a major component of the immune response and mediate their functions by recognizing specific antigens that allow elimination of infected or neoplastic cells. To avoid inappropriate activation and subsequent immune injuries, T cells have evolved strategies to discriminate self from non-self, or healthy from altered and infected. These strategies rely on the activation of receptors that restrict the T cell response. CD33rSiglecs are a family of mostly inhibitory receptors that bind to sialic acids. Siglecs respond to specific sialic acid patterns characteristic for healthy and self and trigger tolerogenic signaling pathways that prevent activation of the immune response. To date the expression of Siglecs in human T cells is not well appreciated. We found that one Siglec member, Siglec-5, is transiently expressed only on activated T cells. Using overexpression studies, we showed that Siglec-5 is a strong inhibitor of T cell activation. The pattern of expression, along with the functional studies, suggested that Siglec-5 is a checkpoint-like receptor that negatively regulates T cell activation. Using a previously described protein ligand, we found that Siglec-5 activation reduces the T cells effector functions, as measured by production of cytokines and cytolytic molecules. Knowing from the literature that cancers change their sialyation to evade immune recognition, we hypothesized that Siglec-5 is a mechanism to enable this. We found that soluble Siglec-5 binds to cancer cell lines from different tissues, suggesting the expression of putative Siglec-5 ligands. Using engineered melanoma specific T cells, we measured the T cell specific response when the Siglec-5 signaling axis is interrupted. We found that blocking the availability of Siglec-5 putative ligands on the cancer cells reinvigorates the T cells immune response against melanoma cancer cells. Altogether, this work identifies Siglec-5 as a novel checkpoint receptor that suppresses the activation of T cells. Several checkpoint receptors with similar functions to Siglec-5 already serve as successful targets for cancer immunotherapies. However, such therapies work in only a small fraction of cancer patients. Our work shows that single agent blockade of Siglec-5 strongly reinvigorates the T cell response. Alone, or in combination with other checkpoint targets, blockade of Siglec-5 can serve as a strategy to prevent cancer immune evasion

Alcohol exposure decreases osteopontin expression during fracture healing and osteopontin-mediated mesenchymal stem cell migration in vitro

Abstract Background Alcohol consumption is a risk factor for impaired fracture healing, though the mechanism(s) by which this occurs are not well understood. Our laboratory has previously shown that episodic alcohol exposure of rodents negatively affects fracture callus development, callus biomechanics, and cellular signaling which regulates stem cell differentiation. Here, we examine whether alcohol alters chemokine expression and/or signaling activity in the mouse fracture callus during early fracture healing. Methods A mouse model for alcohol-impaired tibia fracture healing was utilized. Early fracture callus was examined for alcohol-effects on tissue composition, expression of chemokines involved in MSC migration to the fracture site, and biomechanics. The effects of alcohol on MSC migration and cell adhesion receptors were examined in an in vitro system. Results Mice exposed to alcohol showed decreased evidence of external callus formation, decreased callus-related osteopontin (OPN) expression levels, and decreased biomechanical stiffness. Alcohol exposure decreased rOPN-mediated MSC migration and integrin β1 receptor expression in vitro. Conclusions The effects of alcohol exposure demonstrated here on fracture callus-associated OPN expression, rOPN-mediated MSC migration in vitro, and MSC integrin β1 receptor expression in vitro have not been previously reported. Understanding the effects of alcohol exposure on the early stages of fracture repair may allow timely initiation of treatment to mitigate the long-term complications of delayed healing and/or fracture non-union