Browsing by Subject "T-cell"

Now showing 1 - 2 of 2

The influence of obesity and lipid metabolism on thymic function
(2011-05) Gulvady, Apeksha Ashok; Ciolino, Henry P.; Jolly, Christopher A.; Sanders, Bob G.; Kline, Kimberly; Richie, Ellen R.; Maynard, Jennifer A.
Approximately two-thirds of US adults are overweight or obese, and obesity is also becoming more prevalent in children and adolescents. Similar to adults, obese children are at a higher risk of developing health problems due in part to dysfunctional immune surveillance. Obesity has been shown reduce the generation of new T-cells by accelerating thymic aging in an adult mouse. This study therefore aimed at determining whether similar diet induced obesity (DIO) changes can be induced in a young mouse. Comparisons made between lean and DIO C57Bl/6 mice showed a significant increase in thymic weight, decrease in thymic cellularity and thymic output, and impaired T-cell development at the double negative stage. We associate these alterations with changes in thymic architecture and accumulation of lipid droplets within the thymic cortex and medulla of the obese mice. The above observations indicate that DIO can induce fat accumulation and reduce thymic function at a young age. Resveratrol, a natural polyphenolic compound, was then used to regulate fat metabolism in an attempt to reduce these DIO changes we observed. Resveratrol induces fat oxidation via 5' adenosine monophosphate-activated protein kinase (AMPK), and its reciprocal regulation of glycerol-3-phosphate acyltransferase-1 (GPAT-1) and carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting enzymes required for glycerophospholipid biosynthesis and oxidation, respectively. Through resveratrol feeding, we were able to prevent the effects of DIO on thymic architecture and thymic T-cell proliferation. This was achieved by manipulating AMPK into inhibiting GPAT-1 and enhancing CPT-1 activity. Since the expression of GPAT-1 was upregulated in the obese mice, we investigated whether deleting GPAT-1 altogether might prevent the thymic involution, by inhibiting synthesis of glycerophospholipids and triacylglycerol. Instead, we found that GPAT-1 deletion slowed thymic growth and reduced cellularity in young mice, which we associated with impaired thymic T-cell function and development, suggesting that the deleterious effects of GPAT-1 deficiency may be due to perturbations in thymic T-cell activation and signaling. These data provide a novel link between lipid metabolism and T-cell development, and identify the use of the naturally-occurring resveratrol to reduce lipid accumulation within the involution-prone thymus, thus providing a useful approach to preventing a decline in thymic function in childhood.
Linking TCR sequence, structure, and function in the DO11.10 mouse model
(2015-08) Frye, Zachary Paul; Maynard, Jennifer Anne, 1974-; Georgiou, George; Alper, Hal; Ehrlich, Lauren; Jiang, Ning
Thymus-maturated lymphocytes (T-cells) provide the immune system with the ability to appropriately detect and respond to diseases through a unique receptor. These T-cell receptors (TCRs) differentiate between self and foreign peptide antigens and form by hypervariable gene rearrangement and imprecise recombination, both of which can potentially generate upwards of 10¹⁸ unique TCRs in humans. Our work focused on characterizing the chicken ovalbumin (OVA) antigen specific parent hybridoma DO11.10 T-cell with (1) high-throughput sequencing, (2) structural analyses, and (3) TCR reconstitution into T-cells to determine the important factors linking TCR sequence, structure, and function. The repertoire of TCR sequences generated in mice immunized with OVA antigen were sequenced using a novel mouse TCR gene primer set. Using high-throughput sequencing, we determined the relative gene and hypervariable sequence frequencies in the repertoire. Based on our results in these experiments, no significant repertoire differences were found between mouse treatment groups. Additionally no sequences with similarity to previously studied OVA-specific T-cell clones were recovered. The antigen binding loops of TCRs have a high degree of structural similarity across diverse receptor sequences. Key residues in the complementarity determining, antigen-binding regions of TCRs form conserved canonical loop structures. Here we evaluated 249 TCRs and TCR-like antibodies to determine the prevalence of these key residues. Expanding on previous work, we found four new canonical class conformations and validated the key residues for these classes. Finally, we report our work to reconstitute engineered TCRs onto the TCR-deficient mouse 58 [superscript -/-] hybridoma line. We modified an existing antibody expression system to display the engineered TCRs in the hybridomas for activation assays. Expression of the TCR was found to be highly sensitive to the N-terminal signal sequence on each receptor chain. Additional work began to examine how these signal sequences alter stable expression and surface display of these TCRs. This work is important for the discovery of new methods and biological agents to target and respond to designed antigens, especially in the context of altering and engineering TCR specificity for therapeutic purposes.