Lesley Devine

Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.

CD8 Binding to MHC Class I Molecules Is Influenced by T Cell Maturation and Glycosylation

CD8 serves both as an adhesion molecule for class I MHC molecules and as a coreceptor with the TCR for T cell activation. Here we study the developmental regulation of CD8-mediated binding to noncognate peptide/MHC ligands (i.e., those not bound by the TCR). We show that CD8s ability to bind soluble class I MHC tetramers and to mediate T cell adhesion under shear flow conditions diminishes as double-positive thymocytes mature into CD8(+) T cells. Furthermore, we provide evidence that this decreased CD8 binding results from increased T cell sialylation upon T cell maturation. These data suggest that CD8s ability to interact with class I MHC is not fixed and is developmentally regulated through the T cells glycosylation state.

Genomic landscape of cutaneous T cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes. We performed exome and whole-genome DNA sequencing and RNA sequencing on purified CTCL and matched normal cells. The results implicate mutations in 17 genes in CTCL pathogenesis, including genes involved in T cell activation and apoptosis, NF-κB signaling, chromatin remodeling and DNA damage response. CTCL is distinctive in that somatic copy number variants (SCNVs) comprise 92% of all driver mutations (mean of 11.8 pathogenic SCNVs versus 1.0 somatic single-nucleotide variant per CTCL). These findings have implications for new therapeutics.

Direct Detection and Magnetic Isolation of Chlamydia trachomatis Major Outer Membrane Protein-Specific CD8+ CTLs with HLA Class I Tetramers

We recently identified HLA class I-presented epitopes in the major outer membrane protein (MOMP) of Chlamydia trachomatis that elicit CTL responses in human genital tract infections. T cells possessing cytolytic activities specific for these epitopes could be detected following in vitro stimulation of peripheral blood CD8+ T cells with peptides. In the present study we used HLA-A2 tetramers for detailed characterization of MOMP-specific CTL responses. Ex vivo tetramer analysis detected MOMP-specific T cells in the peripheral blood of infected individuals at significant frequencies (0.01–0.20% of CD8+ T cells). After in vitro stimulation with peptides, the frequencies of MOMP peptide-specific T cells increased up to 2.34% of CD8+ T cells in bulk cultures. In contrast, HLA-A2/MOMP tetramer-binding T cells were virtually undetectable in the peripheral blood from uninfected individuals, either ex vivo or after 3 wk of in vitro peptide stimulation of their T cells. Magnetically sorted, tetramer-bound T cells specifically lysed peptide-pulsed targets as well as C. trachomatis-infected epithelial cells with nearly 50-fold greater per cell efficiency than that of unsorted populations. This study provides conclusive evidence of in vivo induction of HLA class I-restricted CD8+ CTL responses to C. trachomatis MOMP. Direct detection of these cells with tetramers will allow their further characterization without prior manipulation and facilitate monitoring of CTL responses during infections and in immunization trials with MOMP-based vaccines.

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

The Complementarity-Determining Region-Like Loops of CD8α Interact Differently with β2-Microglobulin of the Class I Molecules H-2Kb and Thymic Leukemia Antigen, While Similarly with Their α3 Domains

The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8αα homodimers with a 10-fold higher affinity than H-2Kb class I molecules. To understand the molecular basis for this difference, we created a panel of CD8α mutants and tested the ability of the CD8αα homodimers to bind to H-2Kb tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the α3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2Kb class I sequences are highly conserved in the α3 domain of MHC class I, this suggests that CD8 contacts the α3 domain of TL and H-2Kb in a similar manner. In contrast, mutations in residues on the A and B β strands of CD8 that are involved in contact with β2-microglobulin affected interaction with the H-2Kb tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2Kb. The unique high affinity binding of TL with CD8αα is most likely a result of amino acid differences in the α3 domain between TL and H-2Kb, particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8αα-H-2Kb cocrystal.

Human CD8β, But Not Mouse CD8β, Can Be Expressed in the Absence of CD8α as a ββ Homodimer

The T cell coreceptor CD8 exists on mature T cells as disulfide-linked homodimers of CD8α polypeptide chains and heterodimers of CD8α- and CD8β-chains. The function of the CD8α-chain for binding to MHC class I and associating with the tyrosine kinase p56lck was demonstrated with CD8αα homodimers. CD8αβ functions as a better coreceptor, but the actual function of CD8β is less clear. Addressing this issue has been hampered by the apparent inability of CD8β to be expressed without CD8α. This study demonstrates that human, but not mouse, CD8β can be expressed on the cell surface without CD8α in both transfected COS-7 cells and murine lymphocytes. By creating chimeric proteins, we show that the murine Ig domain of CD8β is responsible for the lack of expression of murine CD8ββ dimers. In contrast to CD8αα, CD8ββ is unable to bind MHC class I in a cell-cell adhesion assay. Detection of this form of CD8 should facilitate studies on the function of the CD8 β-chain and indicates that caution should be used when interpreting studies on CD8 function using chimeric protein with the murine CD8ββ Ig domain. In addition, we demonstrate that the Ig domains of CD8α are also involved in controlling the ability of CD8 to be expressed. Mutation of B- and F-strand cysteine residues in CD8α reduced the ability of the protein to fold properly and, therefore, to be expressed.

Prolonged Proinflammatory Cytokine Production in Monocytes Modulated by Interleukin 10 After Influenza Vaccination in Older Adults

We evaluated in vivo innate immune responses in monocyte populations from 67 young (aged 21-30 years) and older (aged ≥65 years) adults before and after influenza vaccination. CD14(+)CD16(+) inflammatory monocytes were induced after vaccination in both young and older adults. In classical CD14(+)CD16(-) and inflammatory monocytes, production of tumor necrosis factor α and interleukin 6, as measured by intracellular staining, was strongly induced after vaccination. Cytokine production was strongly associated with influenza vaccine antibody response; the highest levels were found as late as day 28 after vaccination in young subjects and were substantially diminished in older subjects. Notably, levels of the anti-inflammatory cytokine interleukin 10 (IL-10) were markedly elevated in monocytes from older subjects before and after vaccination. In purified monocytes, we found age-associated elevation in phosphorylated signal transducer and activator of transcription-3, and decreased serine 359 phosphorylation of the negative IL-10 regulator dual-specificity phosphatase 1. These findings for the first time implicate dysregulated IL-10 production in impaired vaccine responses in older adults.

Bupropion pre-treatment of endotoxin-induced depressive symptoms.

Increased levels of inflammatory cytokines may play a role in depression. Depressive symptoms can be induced in humans with administration of low-dose lipopolysaccharide (LPS; endotoxin), which activates the innate immune system and causes release of inflammatory cytokines. We previously found that pre-treatment with the serotonin reuptake inhibitor citalopram reduced LPS-induced fatigue and anhedonia. This is a follow-up study to determine whether LPS-induced symptoms could be reduced by pre-treatment with bupropion, a norepinephrine and dopamine reuptake inhibitor. In this double-blind, randomized, placebo-controlled, cross-over study, 10 healthy subjects received intravenous LPS (0.8 ng/kg) after oral pre-treatment with bupropion (75 mg twice a day) or placebo for 7 days. The Montgomery-Åsberg Depression Rating Scale (MADRS), the Profile of Mood States (POMS), and a visual analog scale (VAS) were used to measure depressive symptoms. Serum levels of inflammatory cytokines and chemokines were measured with electrochemiluminescence assays. The results of this study, which must be considered preliminary, showed that LPS administration was associated with (1) increase in serum levels of all cytokines and chemokines assayed; (2) increase in total MADRS score, mostly due to items 7 (lassitude) and 8 (anhedonia); (3) increase in fatigue; (4) decrease in vigor; and (5) decrease in social interest. Bupropion pre-treatment had no statistically significant effect on the innate immune response to LPS or on LPS-induced behavioral changes, suggesting that 1-week pre-treatment with bupropion does not inhibit LPS-induced fatigue and anhedonia, contrary to what was found previously with citalopram.

Molecular Basis for the High Affinity Interaction between the Thymic Leukemia Antigen and the CD8αα Molecule

The mouse thymic leukemia (TL) Ag is a nonclassical MHC class I molecule that binds with higher affinity to CD8αα than CD8αβ. The interaction of CD8αα with TL is important for lymphocyte regulation in the intestine. Therefore, we studied the molecular basis for TL Ag binding to CD8αα. The stronger affinity of the TL Ag for CD8αα is largely mediated by three amino acids on exposed loops of the conserved α3 domain. Mutant classical class I molecules substituted with TL Ag amino acids at these positions mimic the ability to interact with CD8αα and modulate lymphocyte function. These data indicate that small changes in the α3 domain of class I molecules potentially can have profound physiologic consequences.