iNKT cells at the interface between innate and adaptive immunity.

<p>iNKT cells recognize lipids presented by CD1d molecules. Upon activation, iNKT cells modulate the function of CD1d-expressing cells, such as APCs and B cells. This leads to the priming of antigen-specific T cells, induction of antibody responses, and activation of natural killer cells, a subset of cells acting in the innate immune response. iNKT cells can also inhibit the suppressive function of MDSCs.</p

iNKT-cell activation by lysosphospholipids.

<p>During inflammation cytoplasmic, membrane and secreted phospholipases (such as PLA2) produce lysophospholipids (such as LPC) from cellular phospholipids. Lysosphospholipids can be loaded onto CD1d molecules at the cell surface, in the lysosomes, or during intracellular trafficking through the ER and the Golgi. CD1d-LPC complexes elicit iNKT-cell activation in concert with IL-12, IL-18, and type I IFN secreted by APCs during inflammatory reactions.</p

Amide Analogues of CD1d Agonists Modulate <i>i</i>NKT-Cell-Mediated Cytokine Production

Invariant natural killer T (<i>i</i>NKT) cells are restricted by the non-polymorphic MHC class I-like protein, CD1d, and activated following presentation of lipid antigens bound to CD1d molecules. The prototypical <i>i</i>NKT cell agonist is α-galactosyl ceramide (α-GalCer). CD1d-mediated activation of <i>i</i>NKT cells by this molecule results in the rapid secretion of a range of pro-inflammatory (Th1) and regulatory (Th2) cytokines. Polarization of the cytokine response can be achieved by modifying the structure of the glycolipid, which opens up the possibility of using CD1d agonists as therapeutic agents for a range of diseases. Analysis of crystal structures of the T-cell receptor−α-GalCer–CD1d complex led us to postulate that amide isosteres of known CD1d agonists should modulate the cytokine response profile upon <i>i</i>NKT-cell activation. To this end, we describe the synthesis and biological activity of amide analogues of α-GalCer and its non-glycosidic analogue threitol ceramide (ThrCer). All of the analogues were found to stimulate murine and human <i>i</i>NKT cells by CD1d-mediated presentation to varying degrees; however, the thioamide and carbamate analogues of ThrCer were of particular interest in that they elicited a strongly polarized cytokine response (more interferon-gamma (IFN-γ), no interleukin-4 (IL-4)) in mice. While the ThrCer-carbamate analogue was shown to transactivate natural killer (NK) cells, a mechanism that has been used to account for the preferential production of IFN-γ by other CD1d agonists, this pathway does not account for the polarized cytokine response observed for the thioamide analogue

Amide Analogues of CD1d Agonists Modulate <i>i</i>NKT-Cell-Mediated Cytokine Production

Invariant natural killer T (<i>i</i>NKT) cells are restricted by the non-polymorphic MHC class I-like protein, CD1d, and activated following presentation of lipid antigens bound to CD1d molecules. The prototypical <i>i</i>NKT cell agonist is α-galactosyl ceramide (α-GalCer). CD1d-mediated activation of <i>i</i>NKT cells by this molecule results in the rapid secretion of a range of pro-inflammatory (Th1) and regulatory (Th2) cytokines. Polarization of the cytokine response can be achieved by modifying the structure of the glycolipid, which opens up the possibility of using CD1d agonists as therapeutic agents for a range of diseases. Analysis of crystal structures of the T-cell receptor−α-GalCer–CD1d complex led us to postulate that amide isosteres of known CD1d agonists should modulate the cytokine response profile upon <i>i</i>NKT-cell activation. To this end, we describe the synthesis and biological activity of amide analogues of α-GalCer and its non-glycosidic analogue threitol ceramide (ThrCer). All of the analogues were found to stimulate murine and human <i>i</i>NKT cells by CD1d-mediated presentation to varying degrees; however, the thioamide and carbamate analogues of ThrCer were of particular interest in that they elicited a strongly polarized cytokine response (more interferon-gamma (IFN-γ), no interleukin-4 (IL-4)) in mice. While the ThrCer-carbamate analogue was shown to transactivate natural killer (NK) cells, a mechanism that has been used to account for the preferential production of IFN-γ by other CD1d agonists, this pathway does not account for the polarized cytokine response observed for the thioamide analogue

Design, Synthesis, and Functional Activity of Labeled CD1d Glycolipid Agonists

Invariant natural killer T cells (<i>i</i>NKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) <b>1</b>, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to <i>i</i>NKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the <i>pro</i>-<i>S</i> hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the <i>pro</i>-<i>S</i>, and not the <i>pro</i>-<i>R</i>, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (<i>S</i>)-<b>10</b> displayed behavior comparable to that of ThrCer <b>5</b> itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 <b>4</b>, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (<b>11</b>) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching a label to a range of CD1d agonists. The flexibility of the synthetic strategy, and late-stage incorporation of the label, opens up the possibility of using this labeling approach to study the in vivo behavior of a wide range of CD1d agonists

Design, Synthesis, and Functional Activity of Labeled CD1d Glycolipid Agonists

Invariant natural killer T cells (<i>i</i>NKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) <b>1</b>, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to <i>i</i>NKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the <i>pro</i>-<i>S</i> hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the <i>pro</i>-<i>S</i>, and not the <i>pro</i>-<i>R</i>, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (<i>S</i>)-<b>10</b> displayed behavior comparable to that of ThrCer <b>5</b> itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 <b>4</b>, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (<b>11</b>) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching a label to a range of CD1d agonists. The flexibility of the synthetic strategy, and late-stage incorporation of the label, opens up the possibility of using this labeling approach to study the in vivo behavior of a wide range of CD1d agonists

Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity

CD8⁺ T-Cell Differentiation and Senescence

<div><p>(A) Expression of the replicative senescence-associated marker CD57 on antigen-experienced CD8⁺ T-cell subsets. The percentage and mean fluorescence intensity for the CD57⁺ cells are shown for one single donor. Data on several donors (HIV-1-infected or healthy) are also shown (<i>n</i> = 24).</p> <p>(B) Expression of CD57 on CD8⁺ T-cells (whole population or antigen-specific) from acute to postacute (on ART) HIV-1 infection.</p> <p>(C) CD69 expression and CFSE proliferation profile for CD8⁺ T-cell subsets gated on the basis of CD57 and CD27 expression following stimulation with anti-CD3 antibodies. PBMCs were analysed for CD69 expression after 18 h and CFSE labeling after 6 d. Percentages of proliferating cells (with background subtracted) are indicated. Representative results from three experiments (one HIV-infected and two healthy donors) are shown.</p> <p>(D) Telomere length measurement by flow FISH on naïve and antigen-experienced CD8⁺ T-cell subsets FACS-sorted on the basis of CD57, CD27, CCR7, and CD45RA expression. The average length of telomeres was obtained by substracting the mean fluorescence of the background control (no probe; open histogram) from the mean fluorescence obtained from cells hybridised with the FITC-labeled telomere probe (gray histogram). Representative results from two experiments (on healthy donors) are shown.</p> <p>(E) CD57 and perforin expression in the CD8⁺ T-cell population dissected into naïve (CD27^+high, perforin-negative), antigen-experienced CD27⁺ (perforin^low), and antigen-experienced CD27⁻ perforin^low or perforin^high subsets. The percentage and mean fluorescence intensity for the CD57⁺ cells are indicated.</p> <p>(F) Representative staining for perforin and CD57 in CD8⁺ T-cells from a HIV-1-infected or a healthy donor. Percentages of cells present in the top quadrants are shown.</p> <p>(G) Representative staining for perforin and CD57 in CD4⁺ T-cells from an HIV-1-infected or a healthy donor. Percentages of cells present in the top quadrants are shown.</p></div

CD8⁺ T-Cell Activation during Acute HIV-1 Infection

<div><p>(A) Percentages of activated CD38⁺ cells (gated on whole CD8⁺ T-cells, HIV tetramer-positive CD8⁺ T-cells, or whole CD4⁺ T-cells) in donors during acute HIV-1 infection and later postacute on ART (<i>n</i> = 12); healthy donors (<i>n</i> = 11) and untreated donors with nonprogressing chronic infection (<i>n</i> = 12) are also shown.</p> <p>(B and C) CD38 and Ki67 expression on CD8⁺ T-cell subsets defined by CD45RA/CD62L (B) or CD28/CD27 (C) expression, shown in one single donor from acute to postacute (on ART) HIV-1 infection. Percentages of positive cells are shown. Means (± SEM) of CD38⁺ and Ki67⁺ CD8⁺ T-cells for ten patients are also shown; statistics concern CD38 expression.</p> <p>(D) Staining for the activation marker CD38 on CMV-, EBV-, or influenza A virus-specific CD8⁺ T-cells during acute and postacute (on ART) HIV-1 infection in a single donor. Percentages of CD38⁺ tetramer-positive CD8⁺ T-cells are shown. Data on all donors (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020020#pbio-0020020-t001" target="_blank">Table 1</a>) are also shown.</p> <p>(E) Activation (CD38 and Ki67 staining) of CMV-specific CD8⁺ T-cells or whole CD8⁺ T-cell population during acute and postacute (on ART) HIV-1 infection in a single donor. Percentages of cells present in quadrants are shown.</p> <p>Statistics: * <i>p</i> < 0.002, ** <i>p</i> < 0.01, NS = nonsignificant, with the nonparametric Mann–Whitney test.</p></div

CD8⁺ T-Cell Differentiation and HIV-1 Disease Progression

<div><p>(A) Distribution of the CD8⁺ T-cell population in differentiated subsets (CD28⁺/CD27⁺ early, CD28⁻/CD27⁺ intermediate, and CD28⁻/CD27⁻ late) through the course of HIV-1 infection. Abbreviations: H, healthy (<i>n</i> = 15); A, acute HIV infection (<i>n</i> = 11); C, chronic HIV infection nonprogressor (no ART; <i>n</i> = 14); P, chronic HIV infection with signs of disease progression (no ART; <i>n</i> = 10). Statistics: * <i>p</i> < 0.0001 with the ANOVA test and <i>p</i> < 0.005 between each group.</p> <p>(B) Percentages of CD27⁻ CD8⁺ T-cells that are specific for HLA-B8 HIV (nef) or HLA-A2 CMV in HIV-1-infected individuals at different stages of infection. Statistics: ** <i>p</i> < 0.005 with the nonparametric Mann–Whitney test.</p> <p>(C) Inverse correlation between CD4⁺ T-cell counts and percentage of highly differentiated CD27⁻ cells in the whole CD8⁺ T-cell population of HIV-1-infected donors during chronic infection (untreated nonprogressors and progressors). The <i>p</i> value was obtained using the nonparametric Spearman rank correlation test.</p></div