Evaluating frequency and quality of pathogen-specific T cells.

It is generally accepted that enumeration and characterization of antigen-specific T cells provide essential information about potency of the immune response. Here, we report a new technique to determine the frequency and potency of antigen-specific CD8 T cells. The assay measures changes of intracellular Ca(2+) in real time by fluorescent microscopy in individual CD8 T cells responding to cognate peptides. The T cells form continuous monolayer, enabling the cells to present the peptides to each other. This approach allows us to evaluate the kinetics of intracellular Ca(2+) signalling that characterizes the quality of T cell response. We demonstrate the usefulness of the assay examining the frequency and quality of cytomegalovirus-specific CD8 T cells from healthy donor and patient after haploidentical stem cell transplantation. The new assay has a potential to provide essential information determining the status of the immune system, disease morbidity, potency of therapeutic intervention and vaccine efficacy

Core-based lipid nanoparticles as a nanoplatform for delivery of near-infrared fluorescent imaging agents.

Pyropheophorbide a (Pyro) is a near-infrared (NIR) fluorescent dye and photosensitizer with high quantum yield that makes the dye suitable for tumor treatment both as an imaging and therapy agent. We have designed and synthesized a series of a Pyro-based NIR probes, based on the conjugation of Pyro with lipids. The nature of our probes requires the use of a lipophilic carrier to deliver the probes to cancer cell membranes. To address this, we have utilized lipid-based nanoparticles (LNPs) consisting of PEGylated lipids, which form the nanoparticle shell, and a lipid core. To endow the LNPs with targeting properties, nitrilotriacetic acid (NTA) lipids were included in the composition that enables the non-covalent attachment of His-tag targeting proteins preserving their functional activity. We found that the nature of the core molecules influence the nanoparticle size, shelf-life and stability at physiological temperature. Two different Pyro-lipid conjugates were loaded either into the core or shell of the LNPs. The conjugates revealed differential ability to be accumulated in the cell membrane of the target cells with time. Thus, the modular organization of the core-shell LNPs allows facile adjustment of their composition with goal to fine tuning the nanoparticle properties for in vivo application

The Immune Synapses Reveal Aberrant Functions of CD8 T Cells During Chronic HIV Infection

Chronic HIV infection causes persistent low-grade inflammation that induces premature aging of the immune system including senescence of memory and effector CD8 T cells. To uncover the reasons of gradually diminished potency of CD8 T cells from people living with HIV, here we expose the T cells to planar lipid bilayers containing ligands for T-cell receptor and a T-cell integrins and analyze the cellular morphology, dynamics of synaptic interface formation and patterns of the cellular degranulation. We find a large fraction of phenotypically naive T cells from chronically infected people are capable to form mature synapse with focused degranulation, a signature of a differentiated T cells. Further, differentiation of aberrant naive T cells may lead to the development of anomalous effector T cells undermining their capacity to control HIV and other pathogens that could be contained otherwise

Evidence that the density of self peptide-MHC ligands regulates T-cell receptor signaling.

Noncognate or self peptide-MHC (pMHC) ligands productively interact with T-cell receptor (TCR) and are always in a large access over the cognate pMHC on the surface of antigen presenting cells. We assembled soluble cognate and noncognate pMHC class I (pMHC-I) ligands at designated ratios on various scaffolds into oligomers that mimic pMHC clustering and examined how multivalency and density of the pMHCs in model clusters influences the binding to live CD8 T cells and the kinetics of TCR signaling. Our data demonstrate that the density of self pMHC-I proteins promotes their interaction with CD8 co-receptor, which plays a critical role in recognition of a small number of cognate pMHC-I ligands. This suggests that MHC clustering on live target cells could be utilized as a sensitive mechanism to regulate T cell responsiveness

Efficient killing of tumor cells by CAR-T cells requires greater number of engaged CARs than TCRs

Although CAR-T cells are widely used to treat cancer, efficiency of CAR-T cell cytolytic responses has not been carefully examined. We engineered CAR specific for HMW-MAA (highmolecular- weight melanoma-associated antigen) and evaluated potency of CD8+ CAR-T cells to release cytolytic granules and to kill tissue-derived melanoma cells, which express different levels of HMW-MAA. CAR-T cells efficiently killed melanoma cells expressing high level of HMW-MAA, but not melanoma cells with lower levels of HMW-MAA. The same melanoma cells presenting significantly lower level of stimulatory peptide- MHC ligand were readily lysed by T cells transduced with genes encoding α,β-TCR specific for the peptide-MHC ligand. The data suggest that higher level of targeted molecules is required to engage a larger number of CARs than TCRs to induce efficient cytolytic granule release and destruction of melanoma cells. Understanding the difference in molecular mechanisms controlling activation thresholds of CAR- versus TCR-mediated responses will contribute to improving efficiency of CAR T cells required to eliminate solid tumors presenting low levels of targeted molecules

Kinetics of Ca²⁺ flux in CTL stimulated with pMHC/QD or pMHC/Streptavidin containing cognate and noncognate or inactive pMHC ligands at various ratios.

<p>A. Time-dependent Ca²⁺ accumulation in the cytoplasm of Fluo-3-labeled 68A62 CTL induced by Streptavidin-based oligomers presenting IV9-HLA-A2 (cognate, red) and Tax-HLA-A2 (noncognate, yellow) ligands at various ratios, i.e., 4∶0, 2∶2, 1∶3, 0∶4, respectively. Corresponding Ca²⁺ flux traces are designated as 4 (4∶0, red), 2 (2∶2, blue), 1(1∶3, green), 0(0∶4, grey). B, C, D. Time-dependent Ca²⁺ accumulation in the cytoplasm of Fura Red-labeled 68A62 CTL induced by QD-based oligomers presenting IV9-HLA-A2 (cognate, red), Tax-HLA-A2 (noncognate, yellow) or Tax-HLA-A2_mut (inactive, grey) ligands at various ratios. One series of pHLA-A2/QD conjugates being tested displayed 4, 2, 1 or 0 cognate (red) ligands per dot and 6, 8, 9 or 10 noncognate (yellow) ligands per dot keeping the total number of pHLA-A2 molecules per dot to be 10 (B). In another series of tested pHLA-A2/QD conjugates, the number of the cognate (red) ligands was the same, i.e., 4, 2, 1 or 0 per dot and the number of the noncognate (yellow) ligands per dot was 0, 2, 3, or 4, respectively, while the number of the inactive (grey) ligands and the total number of pHLA-A2 ligands per dot was kept 6 and 10, correspondingly (C). In the latter series (C), the density of noncognate Tax-HLA-A2 ligands on the conjugates was lower as compared to the conjugates of the former series (B). In the third series, QD presenting 1 cognate (red) along with either 9 noncognate (yellow) or with 9 inactive (grey) pMHC-I per dot were tested. QD presenting 10 inactive GL9-HLA-A2_mut proteins per dot were used as a negative control (D). Corresponding Ca²⁺ flux profiles are designated as in A.</p

The dependence of cognate-noncognate pMHC-I cooperation on the nature of noncognate peptide. A, B.

<p>Ca²⁺ mobilization in Fura Red-labeled CER43 CTL stimulated with QD(520) that display different noncognate pMHC-I proteins (10 per dot) (<b>A</b>) or 1 cognate and various noncognate pMHC (9 per dot) (<b>B</b>) is presented. <b>C</b>. Normalized values of MFI of CER43 cells interacting with either cognate or various noncognate pMHC-I are shown. The nature of cognate and noncognate pMHC-I ligands is designated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041466#s4" target="_blank">Material and Methods</a> for details).</p

The dependence of TCR-mediated signaling kinetics upon the density of cognate and noncognate pMHC displayed on QD of different sizes.

<p>Time-dependent accumulation of intracellular Ca²⁺ in Fura Red-labeled 68A62 CTL in response to stimulation with 10 nM QD of different sizes presenting various combinations of cognate, noncognate and inactive pMHC ligands. The comparison of the stimulatory potency of (IV9-HLA-A2)₄₀/QD(620) (A) or (IV9-HLA-A2)₁₀(GL9-HLA-A2_mut)₃₀/QD(620) (B) or (IV9-HLA-A2)₁₀(GL9-HLA-A2)₃₀/QD(620) (C) versus (IV9-HLA-A2)₁₀/QD(520) is shown.</p