High CD8 T-Cell Receptor Clonality and Altered CDR3 Properties Are Associated With Elevated Isolevuglandins in Adipose Tissue During Diet-Induced Obesity.
ABSTRACT: Adipose tissue (AT) CD4+ and CD8+ T cells contribute to obesity-associated insulin resistance. Prior studies identified conserved T-cell receptor (TCR) chain families in obese AT, but the presence and clonal expansion of specific TCR sequences in obesity has not been assessed. We characterized AT and liver CD8+ and CD4+ TCR repertoires of mice fed a low-fat diet (LFD) and high-fat diet (HFD) using deep sequencing of the TCRβ chain to quantify clonal expansion, gene usage, and CDR3 sequence. In AT CD8+ T cells, HFD reduced TCR diversity, increased the prevalence of public TCR clonotypes, and selected for TCR CDR3 regions enriched in positively charged and less polarized amino acids. Although TCR repertoire alone could distinguish between LFD- and HFD-fed mice, these properties of the CDR3 region of AT CD8+ T cells from HFD-fed mice led us to examine the role of negatively charged and nonpolar isolevuglandin (isoLG) adduct-containing antigen-presenting cells within AT. IsoLG-adducted protein species were significantly higher in AT macrophages of HFD-fed mice; isoLGs were elevated in M2-polarized macrophages, promoting CD8+ T-cell activation. Our findings demonstrate that clonal TCR expansion that favors positively charged CDR3s accompanies HFD-induced obesity, which may be an antigen-driven response to isoLG accumulation in macrophages.
Project description:The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCR?? sequencing of tetramer sorted CD8 T cells ex vivo, we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCR? chain. For the first time, we identify a CDR3? (complementarity determining region 3 ?) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCR? chains within the same donor (median 4; range: 1-9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3? motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3?. TCR cloning and site-directed mutagenesis of the CDR3? lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3? and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCR?, as well as TCR?, in understanding the CD8 T cell receptor repertoire.
Project description:BACKGROUND: Sepsis is a potentially deadly disease that often is caused by gram-positive bacteria, in particular Staphylococcus aureus (S. aureus). As there are few effective therapies for sepsis, increased basic knowledge about factors predisposing is needed. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this study was to study the effect of Western diet on mortality induced by intravenous S. aureus inoculation and the immune functions before and after bacterial inoculation. Here we show that C57Bl/6 mice on high-fat diet (HFD) for 8 weeks, like genetically obese Ob/Ob mice on low-fat diet (LFD), have increased mortality during S. aureus-induced sepsis compared with LFD-fed C57Bl/6 controls. Bacterial load in the kidneys 5-7 days after inoculation was increased 10-fold in HFD-fed compared with LFD-fed mice. At that time, HFD-fed mice had increased serum levels and fat mRNA expression of the immune suppressing cytokines interleukin-1 receptor antagonist (IL-1Ra) and IL-10 compared with LFD-fed mice. In addition, HFD-fed mice had increased serum levels of the pro-inflammatory IL-1beta. Also, HFD-fed mice with and without infection had increased levels of macrophages in fat. The proportion and function of phagocytosing granulocytes, and the production of reactive oxygen species (ROS) by peritoneal lavage cells were decreased in HFD-fed compared with LFD-fed mice. CONCLUSIONS: Our findings imply that chronic HFD disturb several innate immune functions in mice, and impairs the ability to clear S. aureus and survive sepsis.
Project description:Clonal CD8(+)/T-cell receptor (TCR)??(+) T-cell large granular lymphocyte (T-LGL) proliferations constitute the most common subtype of T-LGL leukemia. Although the etiology of T-LGL leukemia is largely unknown, it has been hypothesized that chronic antigenic stimulation contributes to the pathogenesis of this disorder. In the present study, we explored the association between expanded TCR-V? and TCR-V? clonotypes in a cohort of 26 CD8(+)/TCR??(+) T-LGL leukemia patients, in conjunction with the HLA-ABC genotype, to find indications for common antigenic stimuli. In addition, we applied purpose-built sophisticated computational tools for an in-depth evaluation of clustering of TCR? (TCRB) complementarity determining region 3 (CDR3) amino-acid LGL clonotypes. We observed a lack of clear TCRA and TCRB CDR3 homology in CD8(+)/TCR??(+) T-LGL, with only low level similarity between small numbers of cases. This is in strong contrast to the homology that is seen in CD4(+)/TCR??(+) T-LGL and TCR??(+) T-LGL and thus underlines the idea that the LGL types have different etiopathogenesis. The heterogeneity of clonal CD8(+)/TCR??(+) T-LGL proliferations might in fact suggest that multiple pathogens or autoantigens are involved.
Project description:The majority of human CD8 cytotoxic T lymphocytes express ?? T-cell receptors that recognize peptide-MHC class I complexes. Considerable attention has been devoted to TCR ? repertoires, but study of TCR ? chains has been limited. To gain a better understanding of the features of CDR3? and CDR3? in paired samples, we comprehensively analyzed 776 unique paired ?? TCR CDR3 regions in this study. We found that (I) the CDR3 length among paired ?? TCRs had a fairly narrow distribution due to random assortment of CDR3 length in alpha and beta chains; (II) nucleotide deletions among CDR3 regions were positively correlated with insertions in both ? and ? TCRs; (III) the CDR3 loops of both ? and ? chains contained an abundance of charged/polar residues and the CDR3 base regions contained a conserved motif; and (IV) the occurrence of Gly was CDR3 length- and position-dependent in both chains, whereas the frequency of Ser at positions 106 and 107 was positively correlated with CDR3 length in TCR ?. Overall, the amino acids in CDR3 loop regions were significantly different between TCR ? and ?, which suggests a distinct role for each chain in the recognition of antigen-MHC complexes. Here, we have provided detailed information on CDR3 in paired TCRs expressed on human CD8+ T cells and established the basis of a reference set for ?? TCR repertoires in healthy humans.
Project description:Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+ is poorly understood. To evaluate the influence of TCR sequence variation on CD4+/CD8+ lineage commitment, we sequenced rearranged TCRs for both ? and ? chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4+ and CD8+ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4+ and CD8+ subsets with some segments increasing the odds of being CD4+ (or CD8+) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4+ vs. CD8+ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both ? and ? chains, where a positively charged CDR3 is associated with CD4+ lineage and a negatively charged CDR3 with CD8+ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.
Project description:BACKGROUND:An influx of lipid-loaded macrophages characterizes visceral adipose tissue (VAT) inflammation, which is an important factor in the development of insulin resistance (IR) in obesity. Depletion of macrophage lipids accompanies increased whole body insulin sensitivity, but the underlying mechanism is unknown. Deficiency of autophagy protein ATG16L1 is associated with increases in inflammatory diseases and lipid metabolism, but the connection between ATG16L1, IR, and obesity remains elusive. We hypothesize that myeloid ATG16L1 contributes to lipid loading in macrophages and to IR. METHODS:Wild-type (WT) bone marrow derived macrophages (BMDMs) were treated with fatty acids and assessed for markers of autophagy. Myeloid-deficient Atg16l1 and littermate control male mice were fed high fat diet (HFD) or low fat diet (LFD) for 3 months starting at 8 weeks of age. Mice were assessed for body mass, fat and lean mass, glucose and insulin sensitivity, food consumption and adipose inflammation. Fluorescence-activated cell sorted VAT macrophages were assessed for lipid content and expression of autophagy related genes. RESULTS:VAT and VAT macrophages from HFD-fed WT mice did not show differences in autophagy protein and gene expression compared to tissue from LFD-fed mice. Fatty acid-treated BMDMs increased neutral lipid content but did not change autophagy protein expression. HFD-fed Atg16l1 myeloid-deficient and littermate mice demonstrated no differences in body mass, glucose or insulin sensitivity, food consumption, fat or lean mass, macrophage lipid content, or adipose tissue inflammation. CONCLUSION:ATG16L1 does not contribute to obesity, IR, adipose tissue inflammation or lipid loading in macrophages in mice fed HFD.
Project description:Increased animal fat consumption is associated with increased premenopausal breast cancer risk in normal weight, but not overweight, women. This agrees with our previous findings in obesity-resistant BALB/c mice, in which exposure to a high saturated animal fat diet (HFD) from peripuberty through adulthood promoted mammary tumorigenesis. Epidemiologic and animal studies support the importance of puberty as a life stage when diet and environmental exposures affect adult breast cancer risk. In this study, we identified the effects of peripubertal exposure to HFD and investigated its mechanism of enhancing tumorigenesis.Three-week-old BALB/c mice fed a low-fat diet (LFD) or HFD were subjected to 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis. At 9 weeks of age, half the mice on LFD were switched to HFD (LFD-HFD group) and half the mice on HFD were switched to LFD (HFD-LFD group). Tumor gene expression was evaluated in association with diet and tumor latency.The peripubertal HFD reduced the latency of DMBA-induced mammary tumors and was associated with tumor characteristics similar to those in mice fed a continuous HFD. Notably, short-latency tumors in both groups shared gene expression characteristics and were more likely to have adenosquamous histology. Both HFD-LFD and continuous HFD tumors showed similar gene expression patterns and early latency. Adult switch from HFD to LFD did not reverse peripubertal HFD tumor promotion. Increased proliferation, hyperplasia, and macrophages were present in mammary glands before tumor development, implicating these as possible effectors of tumor promotion. Despite a significant interaction between pubertal diet and carcinogens in tumor promotion, peripubertal HFD by itself produced persistent macrophage recruitment to mammary glands.In obesity-resistant mice, peripubertal HFD is sufficient to irreversibly promote carcinogen-induced tumorigenesis. Increased macrophage recruitment is likely a contributing factor. These results underscore the importance of early life exposures to increased adult cancer risk and are consistent with findings that an HFD in normal weight premenopausal women leads to increased breast cancer risk. Notably, short-latency tumors occurring after peripubertal HFD had characteristics similar to human basal-like breast cancers that predominantly develop in younger women.
Project description:A minor component (about 25%) of lymphocytes in Hodgkin's disease (HD) are CD8(+) T cells. It is unclear whether the presence of these cells reflects an antitumor cytotoxic response. The goal of the present study was to investigate clonal composition and the T cell receptor (TCR) beta repertoire of the CD8(+) T cell population in HD. Single CD8(+) cells were micromanipulated from frozen tissue sections of lymph nodes affected by primary HD and subjected to single target amplification of TCRbeta gene rearrangements. Sequence analysis of the V region genes revealed the presence of expanded CD8(+) T cell clones in all three cases analyzed. Most of these clonal expansions accounted for less than 10% of the CD8(+) T cell population. In one case, 30% of the CD8(+) T cells belonged to one or two clones. Comparison of V region sequences, however, did not provide evidence that the micromanipulated CD8(+) cells were sampled from a population that was selected for particular antigen specificities. No obvious biases in TCR Vbeta and Jbeta gene segment usage or CDR3 length distribution were found. Similarities of CDR3 amino acid sequences as found in selected CDR3 structures were rare. These results suggest that, like CD4(+) T cells, CD8(+) T cells may also be recruited into the tumor tissue in an antigen-nonspecific manner.
Project description:New massively parallel sequencing technology enables, through deep sequencing of rearranged T-cell receptor (TCR) V? complementarity-determining region 3 (CDR3) regions, a previously inaccessible level of TCR repertoire analysis. The CDR3 repertoire diversity reflects clonal composition, the potential antigenic recognition spectrum, and the quantity of available T-cell responses. In this context, T-large granular lymphocyte (T-LGL) leukemia is a chronic clonal lymphoproliferation of cytotoxic T cells often associated with autoimmune diseases and various cytopenias. Using CD8(+) T-LGL leukemia as a model disease, we set out to evaluate and compare the TCR deep-sequencing spectra of both patients and healthy controls to better understand how TCR deep sequencing could be used in the diagnosis and monitoring of not only T-LGL leukemia but also reactive processes such as autoimmune disease and infection. Our data demonstrate, with high resolution, significantly decreased diversity of the T-cell repertoire in CD8(+) T-LGL leukemia and suggest that many T-LGL clonotypes may be private to the disease and may not be present in the general public, even at the basal level.
Project description:Detailed assessment of how the structural properties of T cell receptors affect clonal repertoires of Ag-specific cells is a prerequisite for a better understanding of human antiviral immunity. Herein we examine the alpha TCR repertoires of CD8 T cells reactive against the influenza A viral epitope M1(58-66), restricted by HLA-A2.1. Using molecular cloning, we systematically studied the impact of alpha-chain usage in the formation of T cell memory and revealed that M1(58-66)-specific, clonally diverse VB19 T cells express alpha-chains encoded by multiple AV genes with different CDR3 sizes. A unique feature of these alpha TCRs was the presence of CDR3 fitting to an AGA(G(n))GG-like amino acid motif. This pattern was consistent over time and among different individuals. Further molecular assessment of human CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes led to the conclusion that the poly-Gly/Ala runs in CDR3alpha were a property of immune, but not naive, repertoires and could be attributed to influenza exposure. Repertoires of T cell memory are discussed in the context of clonal diversity, where poly-Gly/Ala runs in the CDR3 of alpha- and beta-chains might provide high levels of TCR flexibility during Ag recognition while gene-encoded CDR1 and CDR2 contribute to the fine specificity of the TCR-peptide MHC interaction.