Factors contributing to the potency of CD8+ T cells.

CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in targeting virus-infected and cancer cells. Although other cytotoxic lymphocytes such as CD4+ T and natural killer (NK) cells, as well as chimeric antigen receptor (CAR)-T cells, can also identify and destroy aberrant cells, they seem to be significantly less potent based on available experimental data. Here, I contemplate the molecular mechanisms controlling the sensitivity and kinetics of granule-mediated CD8+ T cell cytolytic responses. I posit that the clustering of MHC-I molecules and T cell receptors (TCRs) on the cell surface, as well as the contribution of the CD8 co-receptor, are major factors driving exceptionally potent cytolytic responses. I also contend that CD8+ T cells with known specificity and engineered TCR-T cells might be among the most efficient cytolytic effectors for treating patients suffering from viral infections or cancer.

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.

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 (high-molecular-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,ß-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.

The DISC1-Girdin complex - a missing link in signaling to the T cell cytoskeleton.

In this study, using Jurkat cells, we show that DISC1 (disrupted in schizophrenia 1) and Girdin (girders of actin filament) are essential for typical actin accumulation at the immunological synapse. Furthermore, DISC1, Girdin and dynein are bound in a complex. Although this complex initially forms as a central patch at the synapse, it relocates to a peripheral ring corresponding to the peripheral supramolecular activation cluster (pSMAC). In the absence of DISC1, the classic actin ring does not form, cell spreading is blocked, and the dynein complex fails to relocate to the pSMAC. A similar effect is seen when Girdin is deleted. When cells are treated with inhibitors of actin polymerization, the dynein-NDE1 complex is lost from the synapse and the microtubule-organizing center fails to translocate, suggesting that actin and dynein might be linked. Upon stimulation of T cell receptors, DISC1 becomes associated with talin, which likely explains why the dynein complex colocalizes with the pSMAC. These results show that the DISC1-Girdin complex regulates actin accumulation, cell spreading and distribution of the dynein complex at the synapse.This article has an associated First Person interview with the first author of the paper.

Extent of MHC Clustering Regulates Selectivity and Effectiveness of T Cell Responses.

MHC proteins that present peptide ligands for recognition by TCR form nanoscale clusters on the cell membrane of APCs. How the extent of MHC clustering controls productive TCR engagement and TCR-mediated signaling has not been systematically studied. To evaluate the role of MHC clustering, we exploited nanoscale discoidal membrane mimetics (nanolipoprotein particles) to capture and present peptide-MHC (pMHC) ligands at various densities. We examined the binding of these model membrane clusters to the surface of live human CD8+ T cells and the subsequent triggering of intracellular signaling. The data demonstrate that the proximity of pMHC ligands, high association rate of CD8-MHC interactions, and relatively long lifetime of cognate TCR-pMHC complexes emerge as essential parameters, explaining the significance of MHC clustering. Rapid rebinding of CD8 to MHC suggests a dual role of CD8 in facilitating the T cells' hunt for a rare foreign pMHC ligand and the induction of rapid T cell response. Thus, our findings provide a new understanding of how MHC clustering influences multivalent interactions of pMHC ligands with CD8 and TCR on live T cells that regulate Ag recognition, kinetics of intracellular signaling, and the selectivity and efficiency of T cell responses.

Protective Allele for Multiple Sclerosis HLA-DRB1*01:01 Provides Kinetic Discrimination of Myelin and Exogenous Antigenic Peptides.

Risk of the development of multiple sclerosis (MS) is known to be increased in individuals bearing distinct class II human leukocyte antigen (HLA) variants, whereas some of them may have a protective effect. Here we analyzed distribution of a highly polymorphous HLA-DRB1 locus in more than one thousand relapsing-remitting MS patients and healthy individuals of Russian ethnicity. Carriage of HLA-DRB1*15 and HLA-DRB1*03 alleles was associated with MS risk, whereas carriage of HLA-DRB1*01 and HLA-DRB1*11 was found to be protective. Analysis of genotypes revealed the compensatory effect of risk and resistance alleles in trans. We have identified previously unknown MBP153-161 peptide located at the C-terminus of MBP protein and MBP90-98 peptide that bound to recombinant HLA-DRB1*01:01 protein with affinity comparable to that of classical antigenic peptide 306-318 from the hemagglutinin (HA) of the influenza virus demonstrating the ability of HLA-DRB1*01:01 to present newly identified MBP153-161 and MBP90-98 peptides. Measurements of kinetic parameters of MBP and HA peptides binding to HLA-DRB1*01:01 catalyzed by HLA-DM revealed a significantly lower rate of CLIP exchange for MBP153-161 and MBP90-98 peptides as opposed to HA peptide. Analysis of the binding of chimeric MBP-HA peptides demonstrated that the observed difference between MBP153-161, MBP90-98, and HA peptide epitopes is caused by the lack of anchor residues in the C-terminal part of the MBP peptides resulting in a moderate occupation of P6/7 and P9 pockets of HLA-DRB1*01:01 by MBP153-161 and MBP90-98 peptides in contrast to HA308-316 peptide. This leads to the P1 and P4 docking failure and rapid peptide dissociation and release of empty HLA-DM-HLA-DR complex. We would like to propose that protective properties of the HLA-DRB1*01 allele could be directly linked to the ability of HLA-DRB1*01:01 to kinetically discriminate between antigenic exogenous peptides and endogenous MBP derived peptides.

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease.

CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine ß-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and ß-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of ß-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and ß-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues.

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 Ca2+ 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 Ca2+ 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.

Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner.

Integrin engagement on lymphocytes initiates "outside-in" signaling that is required for cytoskeleton remodeling and the formation of the synaptic interface. However, the mechanism by which the "outside-in" signal contributes to receptor-mediated intracellular signaling that regulates the kinetics of granule delivery and efficiency of cytolytic activity is not well understood. We have found that variations in ICAM-1 expression on tumor cells influence killing kinetics of these cells by CD16.NK-92 cytolytic effectors suggesting that changes in integrin ligation on the effector cells regulate the kinetics of cytolytic activity by the effector cells. To understand how variations of the integrin receptor ligation may alter cytolytic activity of CD16.NK-92 cells, we analyzed molecular events at the contact area of these cells exposed to planar lipid bilayers that display integrin ligands at different densities and activating CD16-specific antibodies. Changes in the extent of integrin ligation on CD16.NK-92 cells at the cell/bilayer interface revealed that the integrin signal influences the size and the dynamics of activating receptor microclusters in a Pyk2-dependent manner. Integrin-mediated changes of the intracellular signaling significantly affected the kinetics of degranulation of CD16.NK-92 cells providing evidence that integrins regulate the rate of target cell destruction in antibody-dependent cell cytotoxicity (ADCC).

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.

Integrin receptors on tumor cells facilitate NK cell-mediated antibody-dependent cytotoxicity.

NK cells that mediate ADCC play an important role in tumor-specific immunity. We have examined factors limiting specific lysis of tumor cells by CD16.NK-92 cells induced by CNTO 95LF antibodies recognizing αV integrins that are overexpressed on many tumor cells. Although all tested tumor cells were killed by CD16.NK-92 effectors in the presence of the antibodies, the killing of target cells with a low level of ICAM-1 expression revealed a dramatic decrease in their specific lysis at high antibody concentration, revealing a dose limiting effect. A similar effect was also observed with primary human NK cells. The effect was erased after IFN-γ treatment of tumor cells resulting in upregulation of ICAM-1. Furthermore, killing of the same tumor cells induced by Herceptin antibody was significantly impaired in the presence of CNTO 95Ala-Ala antibody variant that blocks αV integrins but is incapable of binding to CD16. These data suggest that αV integrins on tumor cells could compensate for the loss of ICAM-1 molecules, thereby facilitating ADCC by NK cells. Thus, NK cells could exercise cytolytic activity against ICAM-1 deficient tumor cells in the absence of proinflammatory cytokines, emphasizing the importance of NK cells in tumor-specific immunity at early stages of cancer.

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.

Enteric alpha defensins in norm and pathology.

Microbes living in the mammalian gut exist in constant contact with immunity system that prevents infection and maintains homeostasis. Enteric alpha defensins play an important role in regulation of bacterial colonization of the gut, as well as in activation of pro- and anti-inflammatory responses of the adaptive immune system cells in lamina propria. This review summarizes currently available data on functions of mammalian enteric alpha defensins in the immune defense and changes in their secretion in intestinal inflammatory diseases and cancer.

Mechanisms controlling granule-mediated cytolytic activity of cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) play a critical role in immunity against viruses and cancer. The antigen receptor or T-cell receptor (TCR) on CTL determines the specificity toward target cells. The CD8 co-receptor functions in concert with the TCR to enhance TCR-mediated signaling, accounting for the remarkable sensitivity and swift signaling kinetics of the CTL response. The latter ensures efficient delivery and release of lytic granules, resulting in sensitive and rapid destruction of target cells.

T cell receptor signaling kinetics takes the stage.

It has been long surmised that the strength of stimulation of the T cell receptor (TCR) determines the robustness of TCR-mediated signaling and the magnitude of a T cell response. However, it is becoming evident that the signal from the TCR develops over time to approach its steady-state, affinity-determined maximal extent and that variations in this time have a substantial effect on the responsiveness of T cells. Here, I discuss data that show that the kinetics of signal propagation in various segments of the TCR signaling network can influence the spatiotemporal regulation of the effector functions of T cells and the quality of the T cell response.

A novel composite immunotoxin that suppresses rabies virus production by the infected cells.

Using strepavidin as a scaffold, we have assembled a composite immunotoxin that consists of recombinant Pseudomonas exotoxin A subunit (PE38) and recombinant 25-D1.16 Fab fragment which recognizes the SIINFEKL (pOV8) peptide from ovalbumin in association with H-2K(b) MHC class I protein. The composite immunotoxin exercises cytotoxicity against H-2K(b+) cells sensitized with pOV8 peptide but not with irrelevant peptide. Specific binding of the immunotoxin to H-2K(b+) cells infected with recombinant rabies virus (RV) expressing pOV8 epitope (RV-pOV8) resulted in the suppression of the production of virus particles by the infected cells. This strategy allows readily produce different immunotoxins with desired specificity by combining various targeting and toxin molecules. The results provide a proof of concept that composite immunotoxins can be utilized as novel immunotherapeutics to stop virus spread in the acute phase of the infection allowing winning time for the development of protective immune response.

Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.

Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response.