Therapeutic vaccination with p24-VLP and zidovudine augments HIV-specific cytotoxic T lymphocyte activity in asymptomatic HIV-infected individuals

This study evaluates the impact of therapeutic vaccination with p24-VLP and zidovudine on the induction or maintenance of HIV-specific cytotoxic lymphocyte activity in a cohort of asymptomatic patients with CD4 counts greater than 400 cells/microl. In a dummy, randomized, phase II clinical trial of the therapeutic vaccine, participants were randomized to one of three arms for 6 months: p24-VLP (500 microg) in alum monthly plus zidovudine 200 mg tds, alum adjuvant plus zidovudine, or p24-VLP plus placebo. Subjects were studied for a total of 52 weeks from baseline. Monitoring included viral load, CD4 and CD8 counts, markers of immune activation, delayed-type hypersensitivity (DTH) skin testing, and cytotoxic T lymphocyte (CTL) measurement. The nine subjects who received p24-VLP and zidovudine had an augmentation and/or broadening of their CTL response compared with baseline (p = 0.004). The eight subjects receiving p24-VLP and seven subjects receiving zidovudine did not have a statistically significant increase or broadening of CTL activity. The augmentation of the CTL response in the subjects who received p24-VLP and zidovudine was not associated with a decline in viral load or an increase in CD8 counts. This study suggests that HIV-specific CTL activity can be augmented in HIV-infected individuals receiving p24-VLP and zidovudine, supporting the hypothesis of therapeutic vaccination in the presence of antiretroviral therapy.     

HIV-specific cytotoxic T lymphocyte precursors exist in a CD28-CD8+ T cell subset and increase with loss of CD4 T cells.

OBJECTIVES: To determine whether the CD28-CD8+ T cells that develop during HIV infection contain HIV-specific cytotoxic precursor cells. DESIGN: CD8 subpopulations from six asymptomatic HIV-positive adults, with varying degrees of CD4 T cell loss, were sorted by flow cytometry and HIV-specific precursor cytotoxic T lymphocyte frequencies were measured. Three populations of CD8 T cells were tested: CD28+CD5-- T cells, CD28-CD57+ T cells (thought to be memory cells) and CD28-CD57- T cells (function unknown). METHODS: Sorted CD8 subsets were stimulated with antigen presenting cells expressing HIV-1 Gag/Pol molecules. Cytotoxic T cell assays on Gag/Pol expressing 51Cr-labeled Epstein-Barr virus transformed autologous B cells lines or control targets were performed after 2 weeks. Specific lysis and precursor frequencies were calculated. RESULTS: Both CD28 positive and CD28-CD57+ populations contained appreciable numbers of precursors (9-1720 per 10(6) CD8+ T cells). However, the CD28-CD57- population had fewer precursors in five out of six people studied. More CD28 positive HIV-specific cytotoxic T lymphocyte precursors were found in patients with CD4:CD8 ratios > 1, whereas more CD28-CD57+ precursors were found in patients whose CD4:CD8 ratios were < 1 (r2, 0.68). CONCLUSIONS: Memory HIV-specific precursor cytotoxic T lymphocytes are found in both CD28 positive and CD28-CD8+ cells, however, a CD28-CD57- subpopulation had fewer. Because CD28-CD57+ cells are antigen-driven with limited diversity, the loss of CD28 on CD8 T cells during disease progression may reduce the response to new HIV mutations; this requires further testing.     

Antiviral cytotoxic T lymphocyte induction and vaccination

In developing vaccines to counteract viral infections, it is important to produce reagents that engender the type of immune response best suited to provide protection and avoid immunopathology. Recovery from infection is the principal role of T cell immunity, and cells that recognize virus-infected cells (cytotoxic T lymphocytes) and kill them--often before new virus is replicated--represent a crucial component of the recovery process. The cytotoxic T lymphocytes are particularly important in situations where the load of infection is high. In this review the cells and mediators, as well as the viral and host cell antigens involved in antiviral induction of cytotoxic T lymphocytes, are discussed. Particular attention is paid to the importance of the intracellular pathway of processing taken by viral antigens and the outcome in terms of the recognition of cytotoxic T lymphocytes. The new types of vaccines--especially subunits, antiidiotypes, and recombinant viral vectors--are discussed in terms of their likely effectiveness at inducing cytotoxic T lymphocytes.     

Quantifiable cytotoxic T lymphocyte responses and HLA-related risk of progression to AIDS

There are significant associations between possession of certain HLA class I alleles and rate of progression to AIDS. Immunological data provide an explanatory mechanism for this relationship. Patients with HLA types associated with rapid disease progression recognize a significantly smaller fraction of their known repertoire of viral epitopes than do patients with HLA types associated with slow progression. Population frequency of HLA types (or supertypes) and their capacity to elicit cytotoxic T lymphocyte responses are also negatively correlated. These data provide an immunological mechanism to explain HLA-related risk of progression to AIDS and emphasize the central role of viral evolution in the pathogenesis of HIV.     

Hepatitis B surface antigen specific cytotoxic T lymphocyte activity in hepatitis B virus infection

Cytotoxic T lymphocyte (CTL) activity against HBs antigen (HBsAg)-coated autologous mononuclear cells as target cells was examined in hepatitis B virus infection. Target cells were prepared by coincubation of mononuclear cells with purified HBsAg in 0.5 mM CrCl3. The distribution and uniformity amounts of HBsAg on target cells was shown by immune fluorescence and by analyzing the fluorescence intensity with a fluorescent activated cell sorter. CTL activity was detected in 7 of 9 patients with acute hepatitis type B, in 4 of 11 chronic active hepatitis type B, in none of 8 healthy HBsAg carriers, and in none of 22 healthy volunteers. The antigen specificity of the cytotoxicity was confirmed by a blocking experiment with purified HBsAg and by cold target inhibition with HBsAg or bovine serum albumin (irrelevant antigen) coupled cold target cells. CTL lysed HBsAg coupled allogeneic target cells that shared HLA-A or B locus antigens. This finding suggests that HLA restriction may be involved in the killing mechanism. This HBsAg specific CTL clone may participate in the immunopathogenesis of hepatitis B virus infection.     

Prevention of cytotoxic T lymphocyte responses to factor IX-expressing hepatocytes by gene transfer-induced regulatory T cells

Treatment of genetic disease such as the bleeding disorder hemophilia B [deficiency in blood coagulation factor IX (F.IX)] by gene replacement therapy is hampered by the risk of immune responses to the therapeutic gene product and to the gene transfer vector. Immune competent mice of two different strains were tolerized to human F.IX by hepatic gene transfer mediated by adenoassociated viral vector. These animals were subsequently challenged by systemic administration of an E1/E3-deleted adenoviral vector, which is known to induce a cytotoxic T lymphocyte response to the transgene product. Immune tolerance prevented cytotoxic T lymphocyte activation to F.IX and CD8(+) cellular infiltrates in the liver. Moreover, a sustained and substantial increase in hepatic F.IX expression from the adenoviral vector was achieved despite in vitro T cell responses to adenoviral antigens. Cytolytic responses to therapeutic and to viral vector-derived antigens had been prevented in vivo by activation of regulatory CD4(+) T cells, which mediated suppression of inflammatory lymphocyte responses to the liver. This result suggests that augmentation of regulatory T cell activation should provide new means to avoid destructive immune responses in gene transfer.     

Distinct role of lymphocyte function-associated antigen-1 in mediating effective cytolytic activity by cytotoxic T lymphocytes

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecules (ICAMs) facilitates T cell antigen receptor (TCR)-mediated killing. To dissect TCR and LFA-1 contributions, we evaluated cytolytic activity and granule release by cytotoxic T lymphocytes (CTL) as well as intracellular granule redistribution and morphology of CTL stimulated with natural TCR ligand in the presence or absence of LFA-1 engagement. Although other adhesion mechanisms, e.g., CD2-CD58 interaction, could substitute for LFA-1 to trigger CTL degranulation, productive LFA-1 ligation was indispensable for effective target cell lysis by the released granules. LFA-1-mediated adhesion to glass-supported bilayers containing intercellular adhesion molecule-1 was characterized by a much larger junction area, marked by LFA-1 segregation, and a more compact cell shape compared with those observed for CD2-mediated adhesion to bilayers containing CD58. A larger contact induced by intercellular adhesion molecule 1 determined a unique positioning of granules near the interface. These data provide evidence that LFA-1 delivers a distinct signal essential for directing released cytolytic granules to the surface of antigen-bearing target cells to mediate the effective destruction of these cells by CTL.     

Dendritic cells pulsed with unfractionated helminthic proteins to generate antiparasitic cytotoxic T lymphocyte

Dendritic cells (DC) are sentinels of immunity. We determined their role in the induction of immunity against alveolar echinococcosis, caused by the larval stage of the cestode Echinococcus multilocularis. Furthermore, we evaluated if unfractionated protein from E. multilocularis (Em-Ag) can be used as loading agent for DC (comparable to unfractionated tumour proteins) in order to generate antiparasitic cytotoxic T lymphocyte (CTL). Interestingly, immature DC did not mature in the presence of 1 microg/ml Em-Ag as analysed by FACS and mixed leucocyte reactions. Yet, their capacity to take up dextran was markedly reduced. Further maturation of immature Em-Ag pulsed DC could be induced by proinflammatory cytokines. These mature DC were slightly better inducers of T cell proliferation when compared with unpulsed mature DC. Importantly, by repetetive stimulation of autologous CD8+ lymphocytes with the Em-Ag pulsed mature DC, we were able to generate specifically proliferating CTL lines. Thus, immunotherapy with ex vivo generated Em-Ag pulsed DC might be of benefit for patients inheriting this incurable disease.     

Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2D^b-restricted murine cytotoxic T lymphocyte clone (F5) and D^b-transfected L929 mouse fibroblasts (LD^b) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LD^b target cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds D^b, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LD^b adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity.     

Defective cytotoxic granule-mediated cell death pathway impairs T lymphocyte homeostasis

Hemophagocytic syndrome is a severe and often fatal syndrome resulting from excessive activation and proliferation of T lymphocytes and macrophages. Onset of a hemophagocytic syndrome characterized the course of several human inherited immune disorders, all of them resulting from molecular defects of the perforin-dependent cytotoxic process exerted by both T and Natural Killer (NK) lymphocytes. These disorders highlight the determinant role of this lytic pathway in the control of lymphocyte expansion and homeostasis. New effectors of this secretory pathway have been thus identified.     

An algorithm for evaluating human cytotoxic T lymphocyte responses to candidate AIDS vaccines.

Development of an effective vaccine against HIV-1 will likely require the induction of a broad array of immune responses, including virus-specific CTLs and neutralizing antibodies. One promising vaccine approach involves live recombinant canarypox (CP)-based vectors (ALVAC) containing multiple HIV-1 genes. In phase I clinical trials in HIV-1-seronegative volunteers, the cumulative rate of detection of HIV-1-specific CTLs has been as high as 60-70%. In the present study, the factors associated with CTL responsiveness were evaluated in a subset of vaccinees immunized with a CP vector expressing portions of the gag, pro, and env genes of HIV-1 (ALVAC-HIV). CTL responses were detected in one of seven examined. While the responding individual had both CD4+ and CD8+ CTLs directed at multiple HIV-1 antigens, this response was not detectable 1 year after the last vaccination. In-depth characterization of "CTL nonresponders" showed that nonresponsiveness was not associated with defects in antigen processing or presentation. A generalized defect in CTL responsiveness was ruled out by parallel assays to detect CMV-specific CTLs from these same volunteers. Furthermore, HIV-1-specific memory CTLs were not detectable by peptide stimulation or by a novel technique for flow cytometric visualization of Gag epitope-specific T lymphocytes while HIV-1-seropositive donors frequently had 0.1-3% of CD8+ cells stain positively for this epitope (SLYNTVATL). Taken together, these results suggest that the lack of detectable HIV-1 CTLs in these volunteers was not due to classic MHC-linked nonresponsiveness.     

Regulation of cytotoxic T lymphocyte triggering by PIR-B on dendritic cells

Priming of cytotoxic T lymphocytes (CTLs) by dendritic cells (DCs) is crucial for elimination of pathogens and malignant cells. To activate CTLs, DCs present antigenic peptide-complexed MHC class I molecules (MHC-I) that will be recognized by the CTLs with T cell receptors and CD8 molecules. Here we show that paired Ig-like receptor (PIR)-B, an MHC-I receptor expressed on antigen-presenting cells, can regulate CTL triggering by blocking the access of CD8 molecules to MHC-I. PIR-B-deficient DCs evoked CTLs more efficiently, leading to accelerated graft and tumor rejection. PIR-B(+) non-DC transfectant cells served as less efficient stimulators and targets for CTLs than PIR-B(-) cells at the effector phase in vitro. On surface plasmon resonance analysis, PIR-B and CD8alpha alpha were revealed to compete in binding to MHC-I. Our results may provide a novel strategy for regulating CTL-mediated immunity and diseases in a sterical manner.     

Honokiol-enhanced cytotoxic T lymphocyte activity against cholangiocarcinoma cells mediated by dendritic cells pulsed with damage-associated molecular patterns

BACKGROUND Cholangiocarcinoma or biliary tract cancer has a high mortality rate resulting from late presentation and ineffective treatment strategy. Since immunotherapy by dendritic cells (DC) may be beneficial for cholangiocarcinoma treatment but their efficacy against cholangiocarcinoma was low. We suggest how such antitumor activity can be increased using cell lysates derived from an honokioltreated cholangiocarcinoma cell line (KKU-213L5). AIM To increase antitumour activity of DCs pulsed with cell lysates derived from honokiol-treated cholangiocarcinoma cell line (KKU-213L5). METHODS The effect of honokiol, a phenolic compound isolated from Magnolia officinalis, on choangiocarcinoma cells was investigated in terms of the cytotoxicity and the expression of damage-associated molecular patterns (DAMPs). DCs were loaded with tumour cell lysates derived from honokiol-treated cholangiocarcinoma cells their efficacy including induction of T lymphocyte proliferation, proinflammatory cytokine production and cytotoxicity effect on target cholangiocarcinoma cells were evaluated. RESULTS Honokiol can effectively activate cholangiocarcinoma apoptosis and increase the release of damage-associated molecular patterns. DCs loaded with cell lysates derived from honokiol-treated tumour cells enhanced priming and stimulated T lymphocyte proliferation and type I cytokine production. T lymphocytes stimulated with DCs pulsed with cell lysates of honokiol-treated tumour cells significantly increased specific killing of human cholangiocarcinoma cells compared to those associated with DCs pulsed with cell lysates of untreated cholangiocarcinoma cells. CONCLUSION The present findings suggested that honokiol was able to enhance the immunogenicity of cholangiocarcinoma cells associated with increased effectiveness of DC-based vaccine formulation. Treatment of tumour cells with honokiol offers a promising approach as an ex vivo DC-based anticancer vaccine.     

Quantification of simian immunodeficiency virus cytotoxic T lymphocyte escape mutant viruses

Escape from cytotoxic T-lymphocyte (CTL) pressure is common in HIV-1 infection of humans and simian immunodeficiency virus (SIV) infections of macaques. CTL escape typically incurs a fitness cost as reversion back to wild-type can occur upon transmission. We utilized sequence-specific primers and DNA probes with real-time polymerase chain reaction (PCR) to sensitively and specifically track wild-type and escape mutant viremia at the Mane-A*17-restricted SIV Gag(371379) epitope AF9 in pigtail macaques. The generation of minor escape mutant populations is detected by the real-time PCR 2 weeks earlier than observed using standard sequencing techniques. We passaged the AF9 CTL escape mutant virus into two na?ve Mane-A*17-negative pigtail macaques and showed that reversion to wild-type was rapid during acute infection and then slowed considerably at later stages of the infection. These data help refine our understanding of how CTL escape mutant viruses evolve.     

Frequency analysis of cytotoxic T lymphocyte precursors--possible relevance to HLA-matched unrelated donor bone marrow transplantation

HLA-matched unrelated donor (MUD) bone marrow transplants and transplants between HLA mismatched family members are associated with an increased incidence and severity of graft-versus-host disease (GVHD) in comparison with HLA-identical sibling transplants. A limiting dilution analysis system was set up to measure the frequency of alloreactive cytotoxic T lymphocyte precursors (CTL-p) in normal individuals and in potential donor/patient pairs selected for bone marrow transplantation. The donor/recipient pairs were divided into four groups depending on their degree of HLA disparity. A distinct range of CTL-p frequencies was obtained for each group and these showed a hierarchy of response related to the degree of HLA disparity between donors and recipients in that particular group. This assay system may be of value in selecting potential matched unrelated and mismatched family donor/patient pairs for those at lower risk of GVHD.     

Persistent numbers of tetramer+ CD8(+) T cells, but loss of interferon-gamma+ HIV-specific T cells during progression to AIDS

Although CD8(+) T cells initially suppress human immunodeficiency virus (HIV) replication, cytotoxic T-cell precursor frequencies eventually decline and fail to prevent disease progression. In a longitudinal study including 16 individuals infected with HIV-1, we studied both the number and function of HIV-specific CD8(+) T cells by comparing HLA-peptide tetramer staining and peptide-induced interferon-gamma (IFN-gamma) production. Numbers of IFN-gamma-producing T cells declined during progression to acquired immunodeficiency syndrome (AIDS), whereas the number of tetramer+ T cells in many individuals persisted at high frequencies. Loss of IFN-gamma-producing T cells correlated with declining CD4(+) T-cell counts, consistent with the need of CD4(+) T-cell help in maintaining adequate CD8(+) T-cell function. These data indicate that the loss of HIV-specific CD8(+) T-cell activity is not due to physical depletion, but is mainly due to progressively impaired function of HIV-specific CD8(+) T cells.     

Generation of Aspergillus-specific T lymphocytes with cytotoxic activity

Dendritic cells (DC) are highly specialized antigen presenting cells with the unique capacity to initiate and direct immune responses. The superior ability of DC to present antigens to T cells has led to the development of DC-based strategies for the purpose of enhancing the immune response against tumors and infectious agents. In this study Aspergillus (Asp)-pulsed DC were used to generate Asp-specific cytotoxic T-lymphocytes (CTL). Two different Asp-antigen preparations were used here. Asp-specific CTL were generated by four stimulations with autologous, mature, monocyte (Mo)-derived DC that are pulsed with either Aspergillus crude extract (CE) or culture filtrate (CF) antigens. The cytolytic activity of the generated CTL was assessed one week after the 4th stimulation by chromium release assay. No significant difference (p > 0.05) was found between the proliferative responses induced by either CF or CE Asp-pulsed DC. Both types of Asp-pulsed mature DC were capable of priming Asp-specific CTL responses. Analysis of the Asp-CTL effectors revealed that they are mixed of CD3+/CD4+ and CD3+/CD8+ populations and that they secrete IFN-gamma in response to Asp-pulsed mature DC and specifically kill autologous DC pulsed with the same Aspantigen. The killing was higher in bulk-cultures generated using Asp-CF pulsed DC. The percentage of CD3+/CD8+ cytotoxic T cells was significantly increased (p < 0.001) in Asp-CF bulk-culture when compared with Asp-CE bulk-culture (31.55 +/- 1.96% versus 9.70 +/- 1.84%, respectively). In conclusion, Asp-specific T cell lines with cytotoxic activity can be generated from healthy donors. These cells may be used as prophylaxis in high-risk immunocompromised patients.