The induction of cytotoxic T-cell responses with H-2 antigens shed from viable lymphocytes.

Supernatant derived from the incubation of normal, unstimulated spleen cells was able to stimulate a strong specific in vitro cytotoxic lymphocyte (CTL) response in allogeneic spleen cells primed with the corresponding haplotype. The supernatant antigen (SA) was as efficient in inducing secondary CTLs as equivalent numbers of irradiated, adherent cell-depleted spleen cell stimulators present during the culture period, and the CTL stimulation with both was dependent on the presence of responder splenic adherent cells. SA obtained from P815 tumour cells was unable to stimulate a similar response under the same conditions. The stimulating fraction of the SA showed the characteristics of lipid-associated major histocompatibility (MHC) antigens shed from viable lymphocytes, in that it was removed with specific H-2 antiserum, it was sedimented at 100,000 g and its activity was reduced if spleen cells were incubated at 4 degrees instead of 37 degrees. These results indicate a possible role for the shedding of MHC antigens in the general induction of a cytotoxic response.     

T helper cells,IL-2 and the generation of cytotoxic T-cell responses

CD8 T-cell immunity is thought to require helper activity derived from CD4 T cells. Nevertheless, under some circumstances, effective CD8-dependent T-cell responses occur in vivo without CD4 T-cell help. Several recent papers help to explain this paradox and lead to a refined view concerning the role of T helper cells and interleukin-2 receptor signaling in the production of cytotoxic T lymphocytes.     

Cross-linking of Fc(gamma)-receptor on monocytes inhibits hepatitis C virus-specific cytotoxic T-lymphocyte induction in vitro.

In hepatitis C virus (HCV) infection, immune complex (IC)-type virus particles are frequently observed in circulation. The IC leads to cross-linking of Fcgamma receptors (FcgammaR) on monocytes and exerts immunoinhibitory function. To test the roles of IC in HCV-specific cytotoxic T lymphocyte (CTL) induction, we generated HCV CTL from peripheral blood mononuclear cells of chronic hepatitis C patients with or without HCV-IC- or immunoglobulin G (IgG)-coated culture plates and compared their lytic activities. HCV-IC or adherent IgG, which induces FcgammaR cross-linking, significantly reduced CTL activity. Expression of B7-1 on monocytes decreased on adherent IgG. In addition, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) production increased from cells on adherent IgG and their mRNA expression in monocytes was enhanced. Anti-TNF-alpha antibody during induction on adherent IgG inhibited lysis; however, anti-TGF-beta completely reversed its inhibitory effect. These results demonstrated that HCV-IC or adherent IgG impaired HCV-CTL induction in vitro. The FcgammaR-mediated CTL suppression occurred via decreased expression of monocyte B7-1 and/or enhanced production of TGF-beta1.     

A helper T-cell antigen enhances generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro

A T-cell help for generation of hepatitis C virus-specific cytotoxic T lymphocytes was studied in three patients with chronic hepatitis C. In all three, human leukocyte antigen B44-restricted cytotoxic T lymphocytes recognizing an epitope in hepatitis C virus nucleocapsid protein residues 81–100 were generated from the peripheral blood lymphocytes by repeated stimulation with a synthetic hepatitis C virus nucleocapsid pep-tide. The proliferative response of peripheral blood lymphocytes to hepatitis C virus nucleocapsid protein residues 1–120 was observed in one patient, and was ascribed to CD4⁺ T cells. The helper T cells recognized a major epitope in residues 21–40 and a minor epitope(s) in residues 81–110. They produced interferon γ, but interleukin 4 was not detectable in the T-helper cell culture supernatants. The hepatitis C virus nucleocapsid protein residues 1–120 and the major helper T-cell epitope enhanced generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro, although the protein alone did not generate them. In the other two patients, the protein did not enhance generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro. The results suggest that a hepatitis C virus-specific helper T-cell epitope is helpful for inducing a strong specific cytotoxic T-lymphocyte response. © 1995 Wiley-Liss, Inc.     

In vitro induction of cytotoxic effector cells against human neoplasms. I. Sensitization conditions and effect of cryopreservation on the induction and expression of cytotoxic responses to allogeneic leukemia cells.

Peripheral blood lymphocytes (PBL) from normal human donors were sensitized in vitro against allogeneic human acute myelocytic leukemia (AML) cells by means of an unidirectional mixed lymphocyte-tumor cell culture (MLTC) technique. The cytotoxic responsiveness of the sensitized lymphocytes, as determined in vitro by the 51Cr-release assay, varied among individual lymphocyte donors and was greatly dependent on the sensitization culture conditions. Induction of cytotoxic effector cells was augmented appreciably by adding to the cultures minute amounts of the immunopotentiating agent MER-BCG. Responding lymphocytes and stimulating leukemia cells cryopreserved for several weeks in liquid nitrogen were as effective as fresh cells in generating effector lymphocytes; the cytotoxic capacity of already sensitized lymphocytes was fully retained by cryopreservation. The implications of these findings for possible clinical employment of in vitro sensitized lymphocytes in adoptive immunotherapy of cancer are discussed.     

The effect of theophyllamine on T-lymphocyte activation in vitro

Theophyllamine in similar concentrations as the therapeutic serum level was found to inhibit the proliferative response in mixed lymphocyte cultures (MLC) and cultures stimulated with purified protein derivative of tuberculin (PPD). Theophyllamine inhibited interleukin 2 (IL-2) production of lymphocytes stimulated with phytohemaglutinin (PHA) and IL-2-dependent growth of T-cell lines, but had no effect on PPD pulsing of antigen-presenting cells.     

Characterization of a Db-specific helper factor required for the induction of cytotoxic responses to alloantigens with the use of monoclonal antibodies specific for the helper factor or the T-cell antigen receptor.

A T helper clone (clone 9), isolated from a H-2d anti-H-2b mixed lymphocyte culture, was previously found to produce an antigen-specific helper factor (ASHF) that could be specifically absorbed out with BIO.A(2R) (KkAkEkDb), but not B10.A (KkAkEkDd), spleen cells. In order to characterize this ASHF further, we have constructed T-cell hybridoma lines by fusing clone 9 cells with the AKR thymoma, BW5147. One of these hybridoma clones, referred to as clone 25, produced an ASHF that was specific for the Db alloantigen. Immunization of allogeneic C57BL/6 mice with clone 9 cells and subsequent fusion of these immune spleen cells with non-secreting myeloma cells led to the isolation of a monoclonal antibody (mAb) (clone 30 IgM) that was capable of neutralizing the helper activity of clone 25 ASHF. Clone 30 IgM affinity column was found to retain clone 25 ASHF; clone 30 IgM column eluates augmented the cytotoxic responses of CBA/J thymocytes to B6(H-2b), but not D2(H-2d), alloantigens. Preabsorption of clone 25 ASHF with Db-bearing spleen cells prior to affinity purification over a clone 30 IgM column resulted in the abrogation of Db-specific helper activity as well as the loss of a 50,000 molecular weight (MW) band in SDS-polyacrylamide gels run under reducing conditions. Clone 25 ASHF was also retained by immunoadsorbents made with an IgG2a mAb (F23.1) the reactivity of which is against the beta chain of the T-cell receptor. Furthermore, affinity purification of clone 25 ASHF over a F23.1 affinity column, but not an irrelevant mAb column, also yielded a 50,000 MW molecule. These findings suggest that this particular ASHF may be intimately related to the T-cell antigen receptor.     

Suppressive effect of cyclophosphamide on the T-cell system in chickens.

Neonatal administration of 16 mg of cyclophosphamide in inbred chickens resulted in a transient, but profound, deficiency in the in vitro proliferative response of spleen cells. Functional T-cell deficiency was accompanied by a marked morphological degeneration in the thymus and thymus-dependent areas in the spleen.     

Preservation of the helper and suppressor/cytotoxic T-lymphocyte phenotype in continuous culture

The objective of the present investigation was to culture helper or suppressor/cytotoxic T cell subsets after extensive purification in interleukin 2-containing medium to determine whether phenotype changes for OKT4/91d6- or OKT8/Leu-2a-defined antigens do occur with prolonged culture. Repeated and sequential analysis of such polyclonally activated cultured T cells documented stability of the OKT4/91d6+ and OKT8/Leu-2a+ phenotype. Both populations could be considerably expanded in culture. However, mixing experiments revealed a growth advantage of the OKT8/Leu-2a+ subset. A significant number of cells expressing both phenotypic markers was not detectable. While cultured T cells promoted on interleukin 2 were stable for expression of these functionally important differentiation markers, additional studies on activation antigens revealed high numbers of Tac+ cells at an early time of culture and decreasing numbers during further incubation. The expression of Tac antigen preceded the exponential expansion of the culture. In contrast, the number of cells bearing Ia determinants steadily increased with prolonged culture.     

Hierarchies of antigen-specific cytotoxic T-cell responses

Summary: Studies carried out using either mice or humans have shown that cytotoxic T-lymphocyte (CTL) responses to many different pathogenic organisms often comprise CTL specific for multiple class I-restricted pep-tide epitopes. Differences in the magnitude of epitope-specific CTL responses appear to arise mainly from differences in the expression level of the corresponding class I/peptide complex on the surface of the antigen-presenting cell. The size of the CTL response may be United by the frequency and possibly by the affinity of specific CTL precursors in the naive T-cell pool. Thus, both the efficiency of antigen processing and the composition of the peripheral T-cell pool impose direct limitations on the extent of a T-cell response to a given peptide epitope. Studies of CTL hierarchies have resulted in the identification of immunodominant epitopes i.e. peptide epitopes which stimulate the largest number of specific CTL and which are therefore generally believed to offer the best level of protection against the pathogen from which they were derived. It is also thought that CTL responses to non-dominant epitopes mediate protection against pathogenic challenge. These ideas are considered here with respect to experimental data collected following infection of mice with lymphocytic choriomeningitis virus.     

Monosaccharide inhibition of cytotoxic T-cell function: demonstration of clone-specific effects.

A range of monosaccharides has been tested for their capacity to influence the induction and effector function of alloreactive cytotoxic T (Tc) cells. Strain-specific differences in the capacity of monosaccharides to inhibit Tc cell induction have been demonstrated. Monosaccharides can also inhibit effector function of target cell lysis, but this could only be demonstrated by assessing the effect of sugars added to limiting dilution cultures of alloantigen-stimulated T cells. B10.A(4R) anti-BALB/c Tc cells have been reproducibly inhibited by D-glucosamine and D-galactosamine, as well as D-galacturonic acid, at both the induction and effector phases of the Tc cell response. Analysis of monosaccharide inhibition of cytotoxicity in limiting dilution cultures has confirmed that D-glucosamine is the most effective inhibitor of B10.A(4R) anti-BALB/c Tc cells, while D-galactosamine and D-galacturonic acid inhibit cytotoxicity in only some limiting dilution wells. Analysis of several B10.A(4R) anti-BALB/c Tc cell clones has revealed at least two different ''clone-specific'' patterns of inhibition by D-glucose, D-glucuronic acid and D-galacturonic acid. Since Tc cell recognition of antigen is generally specific for class I major histocompatibility complex (MHC) antigens, this data implicates a role for MHC-associated carbohydrate structures expressed by target cells in T-lymphocyte interactions with antigen.     

Primary proliferative and cytotoxic T-cell responses to HIV induced in vitro by human dendritic cells.

In earlier studies, primary proliferative and cytotoxic T-cell (CTL) responses to influenza virus were produced in vitro by using mouse dendritic cells (DC) pulsed with virus or viral peptide as the stimulus for syngeneic T cells in 20-microliters hanging-drop cultures. We have now adapted this system for producing primary responses with cells from non-immune donors to produce primary proliferative and CTL responses to human immunodeficiency virus I (HIV) and to HIV peptides in vitro using cells from normal human peripheral blood. All donors in this study were laboratory personnel with no history of HIV infection. DC enriched from peripheral blood were exposed to HIV in vitro and small numbers were added to T lymphocytes in 20-microliters hanging drops. Proliferative responses to virus-infected DC were obtained after 3 days in culture. After 6 days, CTL were obtained that killed virus-infected autologous--but not allogeneic--phytohaemagglutinin (PHA)-stimulated blast cells. Proliferative and CTL responses were obtained using cells from 14 random donors expressing a spectrum of major histocompatibility complex (MHC) types but the CTL, once produced, showed killing restricted by the MHC class I type. Treatment of cultures with monoclonal antibody (mAb) to CD4-positive cells at the beginning of culture blocked the development of both proliferative and CTL responses, but treatment after 5 days had no effect on the CTL activity. Treatment with MCA to CD8-positive cells at the beginning of culture did not block proliferation significantly, but treatment either before or after the 5-day culture period blocked CTL responses. Collaboration between proliferating CD4-positive cells and CD8-positive cells may thus be required to produce CTL of the CD8 phenotype. DC exposed to HIV also produced CTL that killed autologous blast cells pulsed with gp120 envelope glycoprotein. However, DC infected with whole virus did not produce CTL that lysed target cells pulsed with a synthetic peptide, which included a known T-cell epitope of gp120 (representing amino acids 111-126). DC pulsed with gp120 were a poor stimulus for the development of CTL. In contrast, DC pulsed with the peptide (111-126) stimulated both proliferative and CTL responses. The latter killed not only target cells pulsed with the peptide itself or with gp120 but also killed virus-infected autologous blast cells. CTL were again obtained reproducibly with this peptide using donors expressing a spectrum of MHC types.(ABSTRACT TRUNCATED AT 400 WORDS)     

The nonspecific helper effect of mixed lymphocyte reactions on the induction of T cell-mediated immunity in vitro

We compared induction of sensitization in the mixed lymphocyte reaction (MLR) and in a mouse anti-fibroblast reaction. Our study demonstrated that induction processes in these two reactions, both known to be T cell-mediated reactions, are governed by different factors. Moreover, these two reactions interact with each other and this interaction is manifested by the ability of a MLR occurring during the sensitization phase of the anti-fibroblast reaction to enhance specific cytolysis measured during the effector phase. This helper effect appears to be immunologically nonspecific, since it can be obtained by the use of third party, antigenically unrelated lymphocytes as stimulator cells in the MLR.     

The effect of nonviral liver damage on the T-lymphocyte helper/suppressor ratio

In the present investigation an attempt was made to ascertain whether nonviral liver impairment in rats affects the THelper/TSuppressor ratio. Two hepatotoxic agents were used: (i) galactosamine (GA), which causes a drug-induced hepatitis-like damage, and (ii) orotic acid (OA), which induces fatty changes. Since these two substances act as antidotes to one another they were administered to rats either separately or simultaneously. GA caused severe liver damage documented by a 104-, 48-, and 1.6- fold rise in the plasma concentrations of ALT, AST, and ALP and by multiple foci of hepatocyte necrosis. This was followed by a drop in TH/TS ratio from 2.25 observed in the controls to 0.89 in the GA-treated rats. All of these phenomena were prevented by concurrent administration of GA and OA. OA alone did not show an effect on the liver with respect to changes in plasma enzyme concentrations and by light microscopic analysis. However, OA caused a drop in the TH/TS ratio from 2.25 to 1.55. Neither GA nor OA produced a change in TH/TS ratios in in vitro experiments.     

Co-evolution of human immunodeficiency virus and cytotoxic T-lymphocyte responses

After more than a decade of intensive research, the precise of human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) in determining the course of the infection remains open to argument. It is established that HIV-specific CTL appear early in the infection and are temporally associated with the clearance of culturable virus from the blood; that CTL are generally detectable at very high levels throughout the asymptomatic phase and decline at the time of progression to AIDS; and that CTL-mediated killing is sufficiently fast to prevent production of new virus by HIV-infected cells. However, viral turnover is throughout the course of the infection, and –infected individuals progress inexorably to disease in spite of the CTL response. In order Co address the question of whether CTL play an active part in influencing the course of HIV infection, one approach has been to seek evidence for CTL-mediated selection pressure on the virus. Several clear examples of CTL epitope-specific mutations selected to fixation are described. We argue that CTL escape is a common event which occurs at all stages of the Infection. Detailed longitudinal studies are required to detect CTL escape and to nd the complexities contributed by factors SUC h as a polyvalent CTL response and the presence of epitope variants which antagonise the CTL response. In conclusion, there is strong evidence of a dynamic process in which CTL impose important selection constraints upon HIV fro m which the virus attempts to escape; ultimately, at the time of disease progression, the tenuous control of CTL over the virus is lost.     

T-T cell interactions during in vitro cytotoxic T lymphocyte responses. III. Antigen-specific T helper cells release nonspecific mediator(s) able to help induction of H-2-restricted cytotoxic T lymphocyte responses across cell-impermeable membranes

T helper cell induction and the specificity of T cell-mediated help as generated during alloreactive and H-2-restricted, virus- or hapten-specific cytotoxic T lymphocyte (CTL) responses have been compared. With the use of a double-chamber culture system, it was possible to dissect and separately analyze the induction phase of T helper cells from the T helper cell effector function. The data obtained revealed that during alloreactive as well as H-2-restricted T cell responses, antigen-specific T helper cells are induced. Upon specific restimulation of T helper cells, helper cell function is mediated across a cell-impermeable membrane via soluble products in an apparently nonspecific and nonrestricted manner. The data suggest that similar rules govern T-T cell interactions in alloreactive and H-2-restricted CTL responses.     

Effective induction of type 1 helper IgG2a and cytotoxic T-cell responses in mice following immunization with human papillomavirus type 16 E2 in MF59

Human papillomavirus (HPV) 16 E2-specific cell-mediated immunity to the early viral antigen E2 is associated with regression of natural infection in patients with cervical dysplasia. Vaccination strategies that activate this type of immune response may have application in the immunotherapeutic treatment of pre-existing HPV infections. The objective of this study was to test if cell-mediated immunity to E2 could be activated when delivered with the already licensed adjuvant MF 59.We found that immunization of mice with E2 in MF 59 stimulated T-cell responses when administered either intraperitoneally (IP) or subcutaneously (SC), and that the response was polarized to a Th-1 type IgG2a response in the IP immunized mice. The magnitude of the lymphoproliferative response was augmented by reducing the time interval between the primary and secondary immunizations from 12 to 4 wk. Stronger responses to the C-terminal third of E2 were detected, suggesting that one or more immunodominant epitopes were localized to this region. Significantly, immunization with E2 in MF 59 IP was sufficient to stimulate an E2-specific cytotoxic T-cell response.This immunization regimen activates the components of a cell-mediated immunity that are predicted to be efficacious in clearance of pre-existing infection, and supports its testing in a papillomavirus challenge model, as the next step in the progression toward its development as an immunotherapeutic vaccine for use in humans.     

Plasmid DNA vaccines against cancer: cytotoxic T-lymphocyte induction against tumor antigens

In recent years, a number of tumor vaccination strategies have been developed. Most of these rely on the identification of tumor antigens that can be recognized by the immune system. DNA vaccination represents one such approach for the induction of both humoral and cellular immune responses against tumor antigens. Studies in animal models have demonstrated the feasibility of utilizing DNA vaccination to elicit protective antitumor immune responses. However, most tumor antigens expressed by cancer cells in humans are weakly immunogenic, and therefore require the development of strategies to potentiate DNA vaccine efficacy in the clinical setting. This review focuses on recent advances in understanding of the immunology of DNA vaccines, as well as strategies used to increase DNA vaccine potency with respect to cytotoxic T-lymphocyte activity.     

The Mycobacterium bovis 32-kilodalton protein antigen induces human cytotoxic T-cell responses.

The 30-kDa protein (P32) is a mycobacterial secreted antigen which is homologous in Mycobacterium bovis and M. tuberculosis. In vitro, P32 induced T-cell proliferation. M. tuberculosis- or P32-stimulated T-cell lines lysed macrophages pulsed with P32 or M. tuberculosis, respectively. We conclude that P32 stimulates cytotoxic T cells specifically.