Viral genetic determinants of T-cell killing and immunodeficiency disease induction by the feline leukemia virus FeLV-FAIDS.

Within the fatal immunodeficiency disease-inducing strain of feline leukemia virus, FeLV-FAIDS, are viruses which range in pathogenicity from minimally (clone 61E is the prototype) to acutely pathogenic, most of the latter of which are also replication defective (clone 61C is the prototype). Mixtures of 61E and 61C virus and chimeras generated between them, but not 61E alone, killed feline T cells. T-cell killing depended on changes within a 7-amino-acid region near the C terminus of the gp70 env gene or was achieved independently by changes within a 109-amino-acid region encompassing the N terminus of gp70. The carboxy-terminal change was also sufficient for induction of fatal immunodeficiency disease in cats. Other changes within the 61C gp70 gene enhanced T-cell killing, as did changes in the long terminal repeat, the latter of which also enhanced virus replication. T-cell killing correlated with high levels of intracellular unintegrated and proviral DNA, all of which were blocked by treatment of infected cells with sera from 61C-immune cats or with a neutralizing monoclonal antibody. These findings indicate that T-cell killing is a consequence of superinfection and that the mutations in env critical to pathogenicity of the immunosuppressive variant result in a failure to establish superinfection interference in infected cells.     

Inducer T-cell-mediated killing of antigen-presenting cells

L3T4+ inducer/helper T-cell clones, once activated by antigen-presenting cells (APC) expressing the appropriate Ia allele and antigen, autonomously kill their target APC. All 13 L3T4+ inducer T-cell clones tested demonstrated this cytolytic activity. In addition, 11 different target cells representing the three major APC types, namely, macrophages, B cells, and dendritic cells, were all sensitive to this cytolytic activity. Moreover, normal macrophages which were treated with interferon-gamma to increase Ia expression were also killed. These observations convincingly demonstrate that the cytolytic activity of L3T4+ inducer T-cell clones is a general phenomenon. In contrast to other reports, lysis of target APC could not be detected following 4-6 hr of incubation. Marginal lysis was observed after 9 hr and a 20-hr incubation period was required to achieve maximal killing. The kinetics of killing paralleled other parameters of T-cell activation such as IL-2 release and cell proliferation. Activation of T cells for cytolysis of APC requires the interaction of T-cell receptors with Ia and antigen. Monoclonal antibody to Ia, L3T4 and the T-cell receptor inhibited the cytolysis of APC. The ability to mediate nonspecific bystander killing was variable depending on both the T-cell clone and the target. The implications of these findings to immune regulation and autoimmunity are discussed.     

Comparison of cytotoxic T lymphocyte efficacy in acute and persistent lymphocytic choriomeningitis virus infection

Immune responses mediated by cytotoxic T lymphocytes (CTLs) have often been found to be functionally impaired in persistent infections. It is assumed that this impairment contributes to persistence of the infection. In this study, we compare the killing efficacy of CD8(+) T-cell responses in mice acutely and persistently infected with the lymphocytic choriomeningitis virus, using an in vivo CTL killing assay. To infer the killing efficacy of CTLs, we developed a new mathematical model describing the disappearance of peptide-pulsed cells from the blood of the mice over time. We estimate a lower half-life for peptide-pulsed cells in acute infection than in persistent infection, which indicates a higher killing efficacy of the CD8(+) T-cell response in acute infection. However, by controlling for the different levels of CTLs in acutely and persistently infected mice, we find that CTLs in persistent infection are only two times less efficacious than CTLs in acute infections. These results strongly suggest that the in vivo cytotoxicity of CD8(+) T-cell responses in persistent infection is modulated via the number of CTLs rather than their individual functionality.     

Cancer-induced defective cytotoxic T lymphocyte effector function: another mechanism how antigenic tumors escape immune-mediated killing

BACKGROUND: The notion that a deficit in immune cell functions permits tumor growth has received experimental support with the discovery of several different biochemical defects in T lymphocytes that infiltrate cancers. Decreased levels of enzymes involved with T-cell signal transduction have been reported by several laboratories, suggesting that tumors or host cells recruited to the tumor site actively down-regulate antitumor T-cell immune response. This permits tumor escape from immune-mediated killing. The possibility that defects in T-cell signal transduction can be reversed, which would potentially permit successful vaccination or adoptive immunotherapy, motivates renewed interest in the field. Summarizing the literature concerning tumor-induced T-cell dysfunction, we focus on the end stage of immune response to human cancer, that of defective cytotoxic T lymphocyte killing function. Based on the data from several laboratories, we hypothesize a biochemical mechanism that accounts for the unusual phenotype of antitumor T-cell accumulation in tumors, but with defective killing function.     

Predictive Models of Type 1 Diabetes Progression: Understanding T-Cell Cycles and Their Implications on Autoantibody Release

Defining the role of T-cell avidity and killing efficacy in forming immunological response(s), leading to relapse-remission and autoantibody release in autoimmune type 1 diabetes (T1D), remains incompletely understood. Using competition-based population models of T- and B-cells, we provide a predictive tool to determine how these two parametric quantities, namely, avidity and killing efficacy, affect disease outcomes. We show that, in the presence of T-cell competition, successive waves along with cyclic fluctuations in the number of T-cells are exhibited by the model, with the former induced by transient bistability and the latter by transient periodic orbits. We hypothesize that these two immunological processes are responsible for making T1D a relapsing-remitting disease within prolonged but limited durations. The period and the number of peaks of these two processes differ, making them potential candidates to determine how plausible waves and cyclic fluctuations are in producing such effects. By assuming that T-cell and B-cell avidities are correlated, we demonstrate that autoantibodies associated with the higher avidity T-cell clones are first to be detected, and they reach their detectability level faster than those associated with the low avidity clones, independent of what T-cell killing efficacies are. Such outcomes are consistent with experimental observations in humans and they provide a rationale for observing rapid and slow progressors of T1D in high risk subjects. Our analysis of the models also reveals that it is possible to improve disease outcomes by unexpectedly increasing the avidity of certain subclones of T-cells. The decline in the number of -cells in these cases still occurs, but it terminates early, leaving sufficient number of functioning -cells in operation and the affected individual asymptomatic. These results indicate that the models presented here are of clinical relevance because of their potential use in developing predictive algorithms of rapid and slow progression to clinical T1D.     

The immunodominant HLA-A2-restricted MART-1 epitope is not presented on the surface of many melanoma cell lines

Among the relatively large number of known tumor-associated antigens (TAA) which are recognized by human CD8 T-cells, Melan-A/MART-1 is one of the most—if not the most—frequently used target for anti-cancer vaccines in HLA-A2 + melanoma patients. In this study, we analyzed the killing of a large panel of melanoma cells by a high avidity, MART-1-specific T-cell clone or a MART-1-specific, polyclonal T-cell culture. Strikingly, we observed that the MART-1-specific T-cells only killed around half of the analyzed melanoma cell lines. In contrast a Bcl-2-specific T-cell clone killed all melanoma cell lines, although the T-cell avidity of this clone was significantly lower. The MART-1-specific T-cell clone expressed NKG-2D and was fully capable of releasing both perforin and Granzyme B. Notably, the resistance to killing by the MART-1-specific T-cells could be overcome by pulsing of the melanoma cells with the MART-1 epitope. Thus, the very frequently used MART-1 epitope was not expressed on the surface of many melanoma cell lines. Our data emphasize that the selected tumor antigens and/or epitopes are critical for the outcome of anti-cancer immunotherapy.     

Deterministic Mechanical Model of T-Killer Cell Polarization Reproduces the Wandering of Aim between Simultaneously Engaged Targets

T-killer cells of the immune system eliminate virus-infected and tumorous cells through direct cell–cell interactions. Reorientation of the killing apparatus inside the T cell to the T-cell interface with the target cell ensures specificity of the immune response. The killing apparatus can also oscillate next to the cell–cell interface. When two target cells are engaged by the T cell simultaneously, the killing apparatus can oscillate between the two interface areas. This oscillation is one of the most striking examples of cell movements that give the microscopist an unmechanistic impression of the cell's fidgety indecision. We have constructed a three-dimensional, numerical biomechanical model of the molecular-motor-driven microtubule cytoskeleton that positions the killing apparatus. The model demonstrates that the cortical pulling mechanism is indeed capable of orienting the killing apparatus into the functional position under a range of conditions. The model also predicts experimentally testable limitations of this commonly hypothesized mechanism of T-cell polarization. After the reorientation, the numerical solution exhibits complex, multidirectional, multiperiodic, and sustained oscillations in the absence of any external guidance or stochasticity. These computational results demonstrate that the strikingly animate wandering of aim in T-killer cells has a purely mechanical and deterministic explanation.     

Cloned protein antigen-specific, Ia-restricted T cells with both helper and cytolytic activities: mechanisms of activation and killing

Myoglobin-specific, Iad-restricted cloned helper T cells and T hybridomas were found to directly kill Iad-bearing, myoglobin-pulsed B lymphoma targets and could also kill bystander targets, but only in the presence of antigen-pulsed antigen presenting cells (APC). The induction of the killing requires recognition of processed antigen in the context of class II major histocompatibility complex (MHC) molecules. Despite the specificity of induction, the bystander killing suggests a nonspecific lytic mechanism. The direct killing can be inhibited only by cold specific targets, whereas the bystander killing can be blocked by both specific and nonspecific targets. The cold target inhibition seems to be due to interference with effector-to-target contact or proximity rather than due to high-dose suppression of T-cell activation. Experiments using T-cell supernatants or cyclosporin A suggested that the helper T cells kill targets by synthesizing short-range soluble factor(s) with nonspecific killing activity de novo during the effector phase, but only while antigen-specific signal transduction is occurring. The mechanism of cold target inhibition appears to be absorption or consumption of a short-acting cytotoxic lymphokine by cells which must be able to interact closely with the effector cell. Normal spleen B cells, despite their capability for activating the helper T cells, cannot inhibit specific killing or be killed by helper T cells, even after lipopolysaccharide stimulation. Thus, although killing by helper T cells may play a negative feedback role in the normal immune response, our data raise the possibility that the helper T-cell-mediated killing may contribute to the immune surveillance against malignancy by virtue of the preferential killing of tumor cells either directly or indirectly.     

Gene transfer of H-2 class II genes: antigen presentation by mouse fibroblast and hamster B-cell lines

We have transferred the mouse Ak alpha and Ak beta genes, which encode the class II I-Ak molecule, into mouse L-cell fibroblasts and hamster B cells. I-Ak molecules are expressed on the surface of both cell types. The L-cell and hamster B-cell I-Ak molecules appear normal by serological analyses and two-dimensional gel electrophoresis. Furthermore, the I-Ak molecules on L cells can act as targets for the allogenic T-cell killing of the transformed L cells. The I-Ak molecules in both mouse fibroblasts and hamster B cells can present certain antigens to T-cell helper hybridomas. Thus only class II molecules are required to convert the nonantigen-presenting cell. Accordingly, it will be possible to dissect the structure-function relationships existing between Ia molecules, foreign antigen, and T-cell receptor molecules by in vitro site-directed mutagenesis and gene transfer.     

A novel mechanism for HIV1-mediated bystander CD4+ T-cell death: neighboring dying cells drive the capacity of HIV1 to kill noncycling primary CD4+ T cells

CD4+ T-cell death is a crucial feature of AIDS pathogenesis, but the mechanisms involved remain unclear. Here, we present in vitro findings that identify a novel process of HIV1 mediated killing of bystander CD4+ T cells, which does not require productive infection of these cells but depends on the presence of neighboring dying cells. X4-tropic HIV1 strains, which use CD4 and CXCR4 as receptors for cell entry, caused death of unstimulated noncycling primary CD4+ T cells only if the viruses were produced by dying, productively infected T cells, but not by living, chronically infected T cells or by living HIV1-transfected HeLa cells. Inducing cell death in HIV1-transfected HeLa cells was sufficient to obtain viruses that caused CD4+ T-cell death. The addition of supernatants from dying control cells, including primary T cells, allowed viruses produced by living HIV1-transfected cells to cause CD4+ T-cell death. CD4+ T-cell killing required HIV1 fusion and/or entry into these cells, but neither HIV1 envelope-mediated CD4 or CXCR4 signaling nor the presence of the HIV1 Nef protein in the viral particles. Supernatants from dying control cells contained CD95 ligand (CD95L), and antibody-mediated neutralization of CD95L prevented these supernatants from complementing HIV1 in inducing CD4+ T-cell death. Our in vitro findings suggest that the very extent of cell death induced in vivo during HIV1 infection by either virus cytopathic effects or immune activation may by itself provide an amplification loop in AIDS pathogenesis. More generally, they provide a paradigm for pathogen-mediated killing processes in which the extent of cell death occurring in the microenvironment might drive the capacity of the pathogen to induce further cell death.     

Radiosensitization of human T-cell leukemia by thymidine and 41.8 degrees C hyperthermia in vitro

This study tested whether thymidine, a naturally occurring nucleoside metabolite, could increase the sensitivity of human T-cell neoplasms to ionizing irradiation and whether adding 41.8 degrees C hyperthermia (X 1 h) to the combination of thymidine and irradiation would further enhance killing of cells by radiation. The magnitude of cytotoxicity induced directly by thymidine was also evaluated. Finally, the ability of 2'-deoxycytidine to prevent thymidine-induced cytotoxicity and radiosensitization was studied. Using JM and MOLT3, two human T-cell acute lymphoblastic leukemias, it was found that thymidine itself was cytotoxic and the toxicity appeared rapidly upon exposure to the drug (i.e., at 4 h). Thymidine caused significant radiosensitization, and thymidine plus 1 h of 41.8 degrees C hyperthermia enhanced radiation-induced killing significantly more than did thymidine or hyperthermia separately. The addition of 2'-deoxycytidine only partly reversed thymidine-induced killing and did not prevent thymidine-induced radiosensitization. The applicability of these results to the clinical management of T- and null-cell malignancies is discussed, as is the presumed mechanistic basis for these observations relative to deoxyribonucleoside metabolism, NAD metabolism, and inhibition of poly(ADP-ribose) polymerase.     

Defense mechanisms in insects: Certain integumental proteins and tyrosinase are responsible for nonself-recognition and immobilization of Escherichia coli in the cuticle of developing Ceratitis capitata

A defense mechanism in the cuticle of developing C. capitata was demonstrated using an in vitro system consisting of isolated cuticular tyrosinase from C. capitata, cuticular tyrosinase-free proteins, tyrosine, and E. coli. The simultaneous presence of the above components resulted in the formation of large immobilized E. coli aggregates. By contrast, omission of any of the above components failed to produce such aggregates. In other words, E. coli retained their mobility and viability. The results indicate that certain cuticular proteins are responsible for the nonself-recognition, since they are able to bind to the E. coli surface in vitro, and a reactive tyrosine derivative is generated by the action of cuticular tyrosinase for the immobilization and probably killing of E. coli. Based on these studies the most likely explanation for the nonself-recognition and immobilization and/or killing of bacteria is the production of E. coli-protein complexes and their crosslinking through quinone intermediate. © 1993 Wiley-Liss, Inc.     

Distinct superinfection interference properties yet similar receptor utilization by cytopathic and noncytopathic feline leukemia viruses.

Cell killing by cytopathic retroviruses is often associated with a delay or failure in the establishment of superinfection interference. Superinfection has been observed during T-cell killing and fatal immunodeficiency disease induction by the feline leukemia virus (FeLV) chimera FeLV-FAIDS-EECC, containing the surface envelope glycoprotein (SU) of FeLV-FAIDS clone 61C. We demonstrate here that 61C SU has a defect that results in a nearly complete failure to establish superinfection interference against homologous virus challenge. This failure was evident only in feline T (FeT) cell clones expressing envelope protein, not in the rare cells that have survived cytopathic infection to become chronically infected. The regions of SU responsible for this defect were the same as those previously identified as responsible for T-cell killing. The superinfection interference properties of a noncytophatic molecular clone, FeLV-FAIDS-61E, were different in that 61E established interference to homologous virus challenge, both in SU-expressing cell clones and in chronically infected cells. Neither 61E nor EECC established interference against heterologous virus challenge. Viruses expressing chimeric SU proteins displayed varied and intermediate interference properties. Purified 61E and 61C SU competed for binding sites on FeT cell surfaces, and purified 61E SU blocked infection of virus bearing 61E or 61C SU. In addition, purified 61E and 61C SU each coprecipitated 70-kDa FeT cell surface proteins. Our data are consistent with the hypothesis that there are multiple cellular components necessary for 61E and 61C attachment to and penetration of FeT cells, a primary receptor that is utilized by both 61E and 61C, and secondary receptors that are likely to be virus specific.     

Diversification of CD1 Molecules Shapes Lipid Antigen Selectivity

Molecular studies of host–pathogen evolution have largely focused on the consequences of variation at protein–protein interaction surfaces. The potential for other microbe-associated macromolecules to promote arms race dynamics with host factors remains unclear. The cluster of differentiation 1 (CD1) family of vertebrate cell surface receptors plays a crucial role in adaptive immunity through binding and presentation of lipid antigens to T-cells. Although CD1 proteins present a variety of endogenous and microbial lipids to various T-cell types, they are less diverse within vertebrate populations than the related major histocompatibility complex (MHC) molecules. We discovered that CD1 genes exhibit a high level of divergence between simian primate species, altering predicted lipid-binding properties and T-cell receptor interactions. These findings suggest that lipid–protein conflicts have shaped CD1 genetic variation during primate evolution. Consistent with this hypothesis, multiple primate CD1 family proteins exhibit signatures of repeated positive selection at surfaces impacting antigen presentation, binding pocket morphology, and T-cell receptor accessibility. Using a molecular modeling approach, we observe that interspecies variation as well as single mutations at rapidly-evolving sites in CD1a drastically alter predicted lipid binding and structural features of the T-cell recognition surface. We further show that alterations in both endogenous and microbial lipid-binding affinities influence the ability of CD1a to undergo antigen swapping required for T-cell activation. Together these findings establish lipid–protein interactions as a critical force of host–pathogen conflict and inform potential strategies for lipid-based vaccine development.     

The virus-specific and allospecific cytotoxic T-lymphocyte response to lymphocytic choriomeningitis virus is modified in a subpopulation of CD8(+) T cells coexpressing the inhibitory major histocompatibility complex class I receptor Ly49G2

The role of negatively signaling NK cell receptors of the Ly49 family on the specificity of the acute CD8(+) cytotoxic T-lymphocyte (CTL) response was investigated in lymphocytic choriomeningitis virus (LCMV)-infected C57BL/6 mice. Activated CD8(+) T cells coexpressing Ly49G2 expanded during LCMV infection, and T-cell receptor analyses by flow cytometry and CDR3 spectratyping revealed a unique polyclonal T-cell population in the Ly49G2(+) fraction. These cells lysed syngeneic targets infected with LCMV or coated with two of three LCMV immunodominant peptides examined. Transfection of these sensitive targets with H2D(d), a ligand for Ly49G2, inhibited lysis. This was reversed by antibody to Ly49G2, indicating effective negative signaling. LCMV characteristically induces an anti-H2(d) allospecific T-cell response that includes T-cell clones cross-reactive between allogeneic and LCMV-infected syngeneic targets. The CD8(+) Ly49G2(+) population mediated no allospecific killing, nor was any NK-like killing observed against YAC-1 cells. This study shows that CD8(+) Ly49G2(+) cells participate in the virus-induced CTL response but lyse a more restricted range of targets than the rest of the virus-induced CTL population.     

Proliferative T-cell response to HIV envelope glycoprotein in immunized and infected primates and human beings

Human immunodeficiency virus (HIV)-specific helper T-cell response was studied in human subjects and nonhuman primates either infected with HIV or immunized with different HIV protein preparations. A strong group-specific T-cell response involving T-cell proliferation and lymphokine secretion was observed in immunized chimpanzees and rhesus monkeys as well as HIV-infected chimpanzees and gibbons. HIV-infected people demonstrated a low or no HIV-specific T-cell response. In contrast, five of 14 HIV antibody-negative sexual partners of HIV-infected men recognized one or more T-cell epitopes in the envelope glycoprotein of HIV.     

In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome,a disease with low mutational burden

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34⁺) and normal (CD3⁺) cells isolated from the patients’ blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4⁺ response and 11 (34%) induced a CD8⁺ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.     

Monoclonal antibodies against the NK cell-FcR and the T3-complex potentiate normal lymphocyte killing

Pretreatment of normal human lymphocytes with monoclonal IgG against the NK cell-FcR (IgG) or the T3 complex was found to potentiate killing of most NK sensitive target cells with the exception of T-cell derived cells. Anti-FcR IgM monoclonals were suppressive for all target cells. IgG anti-FcR mediated potentiation required minute amounts of antibody but was also seen at high anti-FcR concentrations that modulated FcR activity. Potentiated and FcR modulated cells retained anti-FcR IgG on the membrane and conjugated normally to target cells. Anti-FcR potentiation blocked antibody-dependent killing but did not influence lectin-dependent killing, with anti-T3 the opposed effect was seen. Combined anti-FcR and anti-T3 treatment resulted in decreased potentiation. The results suggest that the NK cell-FcR may be activated during normal NK cell killing (without the addition of antibody) as suggested for FcR in B cell triggering.     

Artificial T-cell receptors

Artificial T-cell receptors are generated by joining an Ag-recognizing domain (ectodomain) to the transmembrane and intracellular portion of a signaling molecule (endodomain). The ectodomain is most often derived from Ab variable chains, but may also be generated from T-cell receptor variable chains, as well as from other molecules. Various alternative ectodomain designs exist, with some comparative studies suggesting optimal forms. The endodomain most often used is the intracellular portion of CD-zeta. Although signaling by CD-zeta leads to IFN-n release and cell killing, it fails to transmit a full activation signal. Recently, unions of different signaling molecule segments have facilitated transmission of more potent signals, stimulating T-cell proliferation and overcoming this major limitation. Artificial T-cell receptors allow grafting of nearly any specificity to T cells. This allows generation of large numbers of specific T cells, without laborious selection and expansion procedures. Efficacy against tumors has been demonstrated in animal models. Phase I and II studies of T-cells transduced with artificial T-cell receptors as therapy for HIV infection have been performed. This rapidly advancing technology will make new strategies of adoptive immunotherapy possible.