STAT4-associated natural killer cell tolerance following liver transplantation.

OBJECTIVE: Natural killer (NK) cells are important mediators of liver inflammation in chronic liver disease. The aim of this study was to investigate why liver transplants (LTs) are not rejected by NK cells in the absence of human leukocyte antigen (HLA) matching, and to identify a tolerogenic NK cell phenotype. DESIGN: Phenotypic and functional analyses on NK cells from 54 LT recipients were performed, and comparisons made with healthy controls. Further investigation was performed using gene expression analysis and donor:recipient HLA typing. RESULTS: NK cells from non-HCV LT recipients were hypofunctional, with reduced expression of NKp46 (p<0.05) and NKp30 (p<0.001), reduced cytotoxicity (p<0.001) and interferon (IFN)-γ secretion (p<0.025). There was no segregation of this effect with HLA-C, and these functional changes were not observed in individuals with HCV. Microarray and RT-qPCR analysis demonstrated downregulation of STAT4 in NK cells from LT recipients (p<0.0001). Changes in the expression levels of the transcription factors Helios (p=0.06) and Hobit (p=0.07), which control NKp46 and IFNγ expression, respectively, were also detected. Hypofunctionality of NK cells was associated with impaired STAT4 phosphorylation and downregulation of the STAT4 target microRNA-155. Conversely in HCV-LT NK cell tolerance was reversed, consistent with the more aggressive outcome of LT for HCV. CONCLUSIONS: LT is associated with transcriptional and functional changes in NK cells, resulting in reduced activation. NK cell tolerance occurs upstream of major histocompatibility complex (MHC) class I mediated education, and is associated with deficient STAT4 phosphorylation. STAT4 therefore represents a potential therapeutic target to induce NK cell tolerance in liver disease.

Immunomodulatory effects of imatinib and second-generation tyrosine kinase inhibitors on T cells and dendritic cells: an update.

The discovery of new drugs has occasionally led to a better understanding of biologic processes and unforeseen therapeutic applications. One such example is the new group of tyrosine kinase inhibitors, exemplified by the Bcr-Abl inhibitor imatinib (Glivec). In the last 10 years, these so-called 'small molecules' have started to enter the clinic with the promise of cancer treatments targeted at the underlying molecular changes that are responsible for specific malignant phenotypes. The aim of these small molecules has been to avoid the side-effects of systemic chemotherapies and the high morbidity/mortality risks associated with hematopoietic stem cell transplantation. Concurrently, however, increasing evidence has emerged to indicate that these drugs exert profound immunomodulatory effects on T cells and antigen-presenting cells, such as dendritic cells, which play major roles in immune tumor surveillance and the outcome of hematopoietic stem cell transplantation. Targeted tyrosine kinase inhibitor therapy may thus control cancer cell growth both directly and indirectly by changing the immunologic microenvironment. Furthermore, such molecules might help to unravel the complexities of the human immune system and could find therapeutic application in conditions as diverse as autoimmune diseases and certain infectious processes. In this brief review, we discuss recent developments in this fast evolving field.

Sensory adaptation in naive peripheral CD4 T cells.

T cell receptor interactions with peptide/major histocompatibility complex (pMHC) ligands control the selection of T cells in the thymus as well as their homeostasis in peripheral lymphoid organs. Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56(lck). Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca(2)+ responses to subsequent TCR engagement by anti-CD3 or nominal antigen. Adaptation to peripheral pMHC contact may be important for regulating naive CD4 T cell responsiveness.

The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain.

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.

A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex.

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and FasL-mediated CTL apoptosis. Blocking CD8 binding using alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, FasL expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

Specificity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent.

Tetrameric peptide-MHC class I complexes ("tetramers") are proving invaluable as reagents for characterizing immune responses involving CTLs. However, because the TCR can exhibit a degree of promiscuity for binding peptide-MHC class I ligands, there is potential for cross-reactivity. Recent reports showing that the TCR/peptide-MHC interaction is dramatically dependent upon temperature led us to investigate the effects of incubation temperature on tetramer staining. We find that tetramers rapidly stain CTLs with high intensity at 37 degrees C. We examine the fine specificity of tetramer staining using a well-characterized set of natural epitope variants. Peptide variants that elicit little or no functional cellular response from CTLs can stain these cells at 4 degrees C but not at 37 degrees C when incorporated into tetramers. These results suggest that some studies reporting tetramer incubations at 4 degrees C could detect cross-reactive populations of CTLs with minimal avidity for the tetramer peptide, especially in the tetramer-low population. For identifying specific CTLs among polyclonal cell populations such as PBLs, incubation with tetramers at 37 degrees C improves the staining intensity of specific CTLs, resulting in improved separation of tetramer-high CD8+ cells. Confocal microscopy reveals that tetramers incubated at 37 degrees C can be rapidly internalized by specific CTLs into vesicles that overlap with the early endocytic compartment. This TCR-specific internalization suggests that coupling of tetramers or analogues with toxins, which are activated only after receptor internalization, may create immunotoxins capable of killing CTLs of single specificities.

Antagonism of cytotoxic T-lymphocyte activation by soluble CD8.

The CD8 co-receptor is important in the differentiation and selection of class I MHC-restricted T cells during thymic development, and in the activation of mature T lymphocytes in response to antigen. Here we show that soluble CD8alphaalpha receptor, despite an extremely low affinity for MHC, inhibits activation of cytotoxic lymphocytes by obstructing CD3 zeta-chain phosphorylation. We propose a model for this effect that involves interference of productive receptor multimerization at the T-cell surface. These results provide new insights into the mechanism of T-cell activation and evidence that CD8 function is exquisitely sensitive to disruption, an effect that might be exploited by molecular therapeutics.

The influence of antigenic variation on cytotoxic T lymphocyte responses in HIV-1 infection.

The propensity of HIV-1 for genetic variation, a consequence of error-prone reverse transcription combined with high rates of replication, is thought to contribute to the establishment of persistent infection in the host despite the presence of a vigorous antiviral immune response. Protective immunity to viruses is mediated primarily by cytotoxic T lymphocytes, which recognize viral peptides of 8-11 amino acids bound to major histocompatibility complex class I molecules on the surface of infected cells. In this review we examine the mechanisms by which mutation within peptide antigen-encoding regions of the viral genome enables HIV-1 to evade recognition by virus-specific cytotoxic T lymphocytes. The discussion is relevant to other genetically unstable viruses and more generally to intracellular pathogens of variable antigenicity.

Copresentation of natural HIV-1 agonist and antagonist ligands fails to induce the T cell receptor signaling cascade.

It is not known how human immunodeficiency virus type 1 (HIV-1)-derived antagonist peptides interfere with intracellular activation of cytotoxic T lymphocytes (CTL). We identified Gag epitope variants in HIV-1-infected patients that act as antagonists of CTL responses to unmutated epitopes. We then investigated the effect that presentation of each variant has on the early events of T cell receptor (TCR) signal transduction. We found that altered peptide ligands (APL) failed to induce phosphorylation of pp36, a crucial adaptor protein involved in TCR signal transduction. We further investigated the effect that simultaneous presentation of APL and native antigen at low, physiological, peptide concentrations (1 nM) has on TCR signal transduction, and we found that the presence of APL can completely inhibit induction of the protein tyrosine phosphorylation events of the TCR signal transduction cascade.