The soluble viral glycoprotein of vesicular stomatitis virus efficiently sensitizes target cells for lysis by CD4+ T lymphocytes

The soluble glycoprotein Gs of vesicular stomatitis virus (VSV), at approximately 10(4) molecules per cell, sensitized target cells for lysis by clones of CD4+ cytolytic T lymphocytes (CTL). In addition to lysis, the clones responded by proliferation and interleukin-2 release. Targets sensitized by Gs competed effectively with VSV-infected cells for recognition. Immune cytolysis by these CD4+ CTLs was restricted by class II major histocompatibility complex (MHC) antigens and was specific to VSV. The specific class II MHC antigen which was restricting for each clone remained the same whether the targets were sensitized by infection with VSV or by exogenously added soluble antigen. Sensitization by Gs appeared to require prior processing because the antigen-presenting cells that were fixed prior to exposure to Gs failed to be recognized by the CTL clones. The high efficiency of this uptake and processing was suggested by the inability of Gs at concentrations up to 10(7) per cell to block superinfection by VSV or to effect the RNA-synthetic machinery of uninfected cells. Also, Gs failed to hemolyze sheep erythrocytes when there was hemolysis by virions or an amino-terminal peptide of the VSV glycoprotein. Extrapolation of these results to viral diseases was possible because soluble viral glycoproteins were naturally synthesized during many viral infections and class II MHC antigens were inducible in cells of nonlymphoid origin. Therefore, CD4+ CTLs may be important participants in increasing virus-induced pathology, especially among adjacent uninfected cells.     

Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.

Artificial receptors provide a promising approach to target T lymphocytes to tumor antigens. However, the receptors described thus far produce either an activation or a co-stimulatory signal alone, thus limiting the spectrum of functions accomplished by the genetically modified cells. Here we show that human primary T lymphocytes expressing fusion receptors directed to prostate-specific membrane antigen (PSMA) and containing combined T-cell receptor-zeta (TCRzeta), and CD28 signaling elements, effectively lyse tumor cells expressing PSMA. When stimulated by cell-surface PSMA, retrovirally transduced lymphocytes undergo robust proliferation, expanding by more than 2 logs in three weeks, and produce large amounts of interleukin-2 (IL-2). Importantly, the amplified cell populations retain their antigen-specific cytolytic activity. These data demonstrate that fusion receptors containing both TCR and CD28 signaling moieties are potent molecules able to redirect and amplify human T-cell responses. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of tumor cells that fail to express major histocompatibility complex antigens and co-stimulatory molecules.     

Cytolytic T-lymphocyte responses to respiratory syncytial virus: effector cell phenotype and target proteins.

Cytolytic T-lymphocyte (CTL) activity specific for respiratory syncytial (RS) virus was investigated after intranasal infection of mice with RS virus, after intraperitoneal infection of mice with a recombinant vaccinia virus expressing the F glycoprotein, and after intramuscular vaccination of mice with Formalin-inactivated RS virus or a chimeric glycoprotein, FG, expressed from a recombinant baculovirus. Spleen cell cultures from mice previously infected with live RS virus or the F-protein recombinant vaccinia virus had significant CTL activity after one cycle of in vitro restimulation with RS virus, and lytic activity was derived from a major histocompatibility complex-restricted, Lyt2.2+ (CD8+) subset. CTL activity was not restimulated in spleen cells from mice that received either the Formalin-inactivated RS virus or the purified glycoprotein, FG. The protein target structures for recognition by murine CD8+ CTL were identified by using target cells infected with recombinant vaccinia viruses that individually express seven structural proteins of RS virus. Quantitation of cytolytic activity against cells expressing each target structure suggested that 22K was the major target protein for CD8+ CTL, equivalent to recognition of cells infected with RS virus, followed by intermediate recognition of F or N, slight recognition of P, and no recognition of G, SH, or M. Repeated stimulation of murine CTL with RS virus resulted in outgrowth of CD4+ CTL which, over time, became the exclusive subset in culture. Murine CD4+ CTL were highly cytolytic for RS virus-infected cells, but they did not recognize target cells infected with any of the recombinant vaccinia viruses expressing the seven RS virus structural proteins. Finally, the CTL response in peripheral blood mononuclear cells of adult human volunteers was investigated. The detection of significant levels of RS virus-specific cytolytic activity in these cells was dependent on at least two restimulations with RS virus in vitro, and cytolytic activity was derived primarily from the CD4+ subset.     

Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins.

By using target cells that expressed isolated env, gag, p27nef, or p23vif molecules introduced by recombinant vaccinia viruses containing genes encoding these polypeptides, it was possible to identify env, gag, p27nef, and p23vif as cytolytic target antigens for freshly isolated blood cells from human immunodeficiency virus 1 (HIV-1) seropositive patients. Most of the patients tested (95%) manifested a specific cytotoxic activity against vaccinia virus-env-infected target cells. The env-specific cytotoxic activity was not restricted by the major histocompatibility complex and was not mediated by T lymphocytes, as shown by the absence of blocking effect with an anti-CD3 monoclonal antibody and by the inefficiency of CD3+, CD8+, or CD4+ and CD8+ depletion to reduce the cytotoxic activity against the env-expressing target cells. In the same conditions, the cytotoxic activity specific for gag was abrogated and gag major histocompatibility complex-restricted cytotoxic T lymphocytes were detected in 85% of the subjects tested. Therefore, in a HIV-1 seropositive subject, distinct types of effector cells mediate the lysis of target cells expressing gag and env proteins.     

Phosphorylation of the CD8 antigen on cytotoxic human T cells in response to phorbol myristate acetate or antigen-presenting B cells

We have used the monoclonal antibody OKT8 to demonstrate that the cluster designation (CD)8 antigen on cytotoxic human T cells undergoes a previously unreported protein modification. Immunoprecipitation of CD8 with OKT8 from a CD8+ and CD4+ T cell line prelabeled with [32P]phosphate demonstrates that CD8 can be constitutively labeled with phosphate. CD8 undergoes rapid and intense phosphorylation in serine upon addition of phorbol myristate acetate to the cells. CD8 phosphorylation is induced upon addition of heterologous, Epstein-Barr virus-transformed B cells, which cause proliferation and are target cells for a cytotoxic CD8+CD4+ T cell line and a CD8+CD4- T cell clone. Phosphorylation induced by targets is dose-dependent, rapid, and followed by a fast dephosphorylation. Epstein-Barr virus-transformed B cells that do not induce proliferation of and are not targets for the CD8+CD4+ line and the CD8+CD4- clone do not induce CD8 phosphorylation. Cloned CD8+CD4- cells that proliferate in response to target cells, but lyse them only in the presence of lectin do not undergo target cell-induced CD8 phosphorylation. These data suggest that induction of CD8 phosphorylation is antigen-specific and is coincident with the cytotoxic response. Finally, preincubation of effector and target cells with an antibody to a monomorphic determinant of major histocompatibility complex class I antigens reduces target-induced CD8 phosphorylation to a greater extent than antibody to a major histocompatibility complex class II subregion (DR) monomorphic determinant, reinforcing the notion that major histocompatibility complex class I antigens interact with CD8+ cells.     

CD4(+) T cells and gamma interferon in the long-term control of persistent friend retrovirus infection

We have used the Friend virus model to determine the basic mechanisms by which the immune system can control persistent retroviral infections. Previously we showed that CD4(+) T cells play an essential role in keeping persistent retrovirus in check. The present in vitro experiments with a Friend virus-specific CD4(+) T-cell clone revealed that these cells produce gamma interferon (IFN-gamma), which acts with two distinct mechanisms of antiviral activity. First, IFN-gamma had a direct inhibitory effect on virus production. This inhibitory effect was noncytolytic and, interestingly, was not associated with decreased cell surface expression of viral antigens. The second mechanism of IFN-gamma-mediated antiviral activity was an enhancement of CD4(+) T-cell-mediated cytolytic activity. We also found an in vivo role for IFN-gamma in the control of persistent Friend virus infections. Neutralization of IFN-gamma in persistently infected mice resulted in significantly increased levels of virus in the spleen, and a significant percentage of IFN-gamma-deficient mice were unable to maintain long-term control over Friend virus infections.     

A single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector

Live attenuated vaccine vectors based on recombinant vesicular stomatitis virus (VSV) are effective in several viral disease models. In this study, we asked if a VSV vector capable of only a single cycle of replication might be an effective alternative to replication-competent VSV vectors. We compared the cellular immune responses to human immunodeficiency virus (HIV) envelope protein (Env) expressed by replication-competent and single-cycle VSV vectors and also examined the antibody response to Env. The single-cycle vector was grown by complementation with VSV G protein and then tested initially for immunogenicity when given by four different routes. When given by the intramuscular route in mice, we found that the single-cycle vector was equivalent to the replication-competent VSV vector in generating high-level primary and memory CD8 T-cell responses as well as antibody responses to Env. Cellular responses were analyzed using major histocompatibility complex class I tetramers and direct measurement of cytotoxic T-lymphocyte activity in vivo. We also found that the recall responses after boosting were equivalent in animals vaccinated with replication-competent or single-cycle vectors. Additionally, we observed recall and heightened memory responses after boosting animals with a single-cycle vector complemented with G protein from a different vesiculovirus. Because expression of HIV Env by G-deleted VSV might allow replication in human cells expressing CD4, we generated a single-cycle VSV recombinant expressing a secreted form of the HIV Env protein. This virus was just as effective as the recombinant expressing the membrane-anchored Env protein at producing CD8 T cells and antibody responses.     

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.Abbreviations used in this paper are as follows CMC cell-mediated cytolysis - CTL cytolytic T lymphocyte - LCM lymphocytic choriomeningitis - MHC major histocompatibility complex - MST mean survival time - T cell thymus-derived cell - TCID₅₀ 50 percent tissue culture infective dose     

The origins of alloreactivity: differentiation of prekiller cells to viral infection results in alloreactive cytolytic T lymphocytes

Mice maintained in our animal colony become primed to Sendai virus. This "environmental" priming is reflected in a shift in prekiller activity from the Ly 123 to Ly 23 T cell set and in increased virus-specific cytolytic activity. This transition is accompanied by the development of cytolytic activity against allogeneic targets (not expressing Sendai antigens). These findings are consistent with the view that continued stimulation of Ly 123 cells by autologous MHC antigens, associated with foreign antigens such as a virus, generate Ly 23 prekiller cells that respond to alloantigens as well as autologous cells infected with the relevant virus.     

Kaposi's sarcoma as a model for cancer immunotherapy

Physicians have, for over a century, attempted to harness the potential therapeutic power of the immune system to treat patients with cancer. The discovery that cancer regression can be achieved by immune rejection of tumour antigens theoretically allows the eradication of neoplastic cells without toxicity to normal tissues. An understanding of the mode of presentation of tumour antigens, including those complexed to heat shock proteins by major histocompatibility complex (MHC) class I and class II molecules, and their recognition by CD8(+) and CD4(+) T cells, respectively, has further delineated the potential cancer rejection pathways involved. This also enables the sustained induction and expansion of specific anti-tumour T cells with cytolytic activity.     

Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay

Successful adoptive T-cell therapy has been demonstrated in viral disease and selected forms of cancer. However, it is limited by the difficulty to efficiently isolate and amplify autologous tumor-reactive T-cell clones. Tetramers of major histocompatibility complex (MHC) class I and peptide have facilitated the characterization of CD8+ T cells specific for tumor-associated antigens. However, for adoptive T-cell therapy, MHC-tetramers have limitations: they require knowledge of tumor antigens, which is often not available; they select T cells with a single specificity, thereby posing risk for selection of tumor escape variants; they do not select for function, so that T cells may be anergic when isolated from cancer patients; and they do not allow the isolation of CD4+ T cells that can be essential for tumor rejection. Because interferon (IFN)-gamma is essential for tumor rejection, we isolated live T cells based on their IFN-gamma production. IFN-gamma secreted by previously activated T cells is retained on the cell surface, allowing their specific isolation and expansion. We show here that IFN-gamma+ but not IFN-gamma- T cells from tumor-immunized mice are cytolytic and mediate tumor rejection upon adoptive transfer. Importantly, tumor-specific T cells can be enriched from lymphocytes infiltrating human renal cell carcinoma by the IFN-gamma capture assay.     

Effect of Vesicular Stomatitis Virus Infection on the Histocompatibility Antigen of L Cells

When mouse L cells are infected for 22 hr with vesicular stomatitis virus (VSV), a ribonucleic acid-containing enveloped virus, greater than 70% of the major histocompatibility antigen (H-2), is no longer detectable by the method of inhibition of immune cytolysis. Infected cells prelabeled with (14)C-glucosamine also show a correspondingly greater loss of trichloroacetic acid-insoluble radioactivity than uninfected cells. The loss of H-2 antigenic activity is not due to the viral inhibition of host cell protein synthesis since cells cultured for 18 hr in the presence of cycloheximide have the same amount of H-2 activity as untreated controls. Also, cells infected with encephalomyocarditis virus, a picornavirus, show no loss of H-2 activity at a time when host cell protein synthesis is completely inhibited. VSV structural proteins associated in vitro with uninfected L-cell plasma membranes do not render H-2 sites inaccessible to the assay. Although antibodies may not combine with all the H-2 antigenic sites on the plasma membrane, anti-H-2 serum reacted with L cells before infection does not prevent a normal infection with VSV. H-2 activity can be detected in virus samples purified from the medium of infected L cells; this virus purified after being mixed with L-cell homogenates shows greater H-2 activity than virus purified after being mixed with HeLa cell homogenates. However, VSV made in HeLa cells shows no H-2 activity when mixed with L-cell homogenates.     

The role of T8 in the cytotoxic activity of cloned cytotoxic T lymphocyte lines specific for class II and class I major histocompatibility complex antigens

It is reported here that most cytotoxic T lymphocytes (CTL), which recognize class I major histocompatibility complex (MHC) loci, express the T cell differentiation antigen T8. However, a minority of T8+ CTL clones was found to recognize class II MHC antigens. To test the hypothesis that T8 is involved only in T cell recognition of class I MHC antigens, we studied the role of T8 in the cytotoxic activity of class II MHC-specific CTL. Monoclonal antibodies specific for T8 blocked the activity of most class I MHC-specific CTL clones but did not affect the activity of class II MHC-specific CTL clones. Moreover, a mild trypsin treatment of the clones, which removed and T8 determinant, affected the activity of class I MHC but not that of class II MHC-specific CTL clones. These findings indicate that the class II-specific MHC CTL clones described here did not require T8 for their cytolytic activity. The activity of one T8+ class I MHC-specific (HLA-B27) CTL clone (HG-61) against the B cell line JY, which was used to raise this CTL clone, was not blocked by trypsin treatment of this clone. However, the activity of CTL clone HG-61 against target cells different from JY but carrying the appropriate HLA specificity was blocked by anti-T8 antibodies and trypsin treatment. The implications of these findings for the hypothesis that T8 is involved only in the activity of CTL with a relatively low avidity for class I MHC antigens are discussed.     

Limitation of VSV infection by the host's response to VSV-associated cellular antigens

It has been reported that during the maturation of enveloped viruses, host proteins such as H-2 antigens of the mouse associate with the budding viruses. This finding led us to investigate the possible biologic significance of this association. In our studies, we examined, with purified vesicular stomatitis virus (VSV) from various sources, the in vivo infection of mice immunized with allogeneic tumors. Immunization of H-2k mice with an H-2d tumor caused the limitation of replication, within the spleen, of VSV derived from an H-2d cell line compared with the replication of VSV derived from an H-2k line. Conversely, immunization of H-2d mice with an H-2k tumor caused the limitation of replication of VSV derived from an H-2k cell line. Viral mixture experiments ruled out indirect inactivation or inhibition of virus replication by nonspecific factors, such as immune interferon, as having a major role in the observed limitation of VSV replication. We conclude that virus infections can be limited by an immune response directed against the specific host surface antigen that the virus carries in its envelope.     

T cell exhaustion and Interleukin 2 downregulation

T cells reactive to tumor antigens and viral antigens lose their reactivity when exposed to the antigen-rich environment of a larger tumor bed or viral load. Such non-responsive T cells are termed exhausted. T cell exhaustion affects both CD8+ and CD4+ T cells. T cell exhaustion is attributed to the functional impairment of T cells to produce cytokines, of which the most important may be Interleukin 2 (IL2). IL2 performs functions critical for the elimination of cancer cells and virus infected cells. In one such function, IL2 promotes CD8+ T cell and natural killer (NK) cell cytolytic activities. Other functions include regulating naïve T cell differentiation into Th1 and Th2 subsets upon exposure to antigens. Thus, the signaling pathways contributing to T cell exhaustion could be linked to the signaling pathways contributing to IL2 loss. This review will discuss the process of T cell exhaustion and the signaling pathways that could be contributing to T cell exhaustion.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes

We have recently described a novel method for the production and characterization of mAb reactive with T cell-restricted intracellular antigens. From a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells, we have selected one, designated TIA-1, that reacts with 20 to 36% of digitonin permeabilized peripheral blood T lymphocytes. Flow cytometric analysis of purified CD4+ and CD8+ subsets showed TIA-1 to recognize a subpopulation of 49 to 64% of CD8+ lymphocytes. Little or no reactivity with CD4+ resting T lymphocytes was observed. TIA-1 did not react with any of a panel of T cell lines, B cell lines, or monocytoid cell lines. TIA-1 reacted strongly with NK cell clones and CD8+ cytolytic T cell clones, and less strongly with CD4+-activated T cell clones, suggesting a preferential expression in cells possessing cytolytic potential. Cell fractionation experiments showed TIA-1 to be membrane associated. Furthermore, Percoll gradient fractionation of a cytolytic T cell clone (T4T8C1) showed the majority of TIA-1 to be contained in a low density membrane fraction that also contained serine protease activity. Immunoelectron microscopy showed TIA-1 to decorate the membranes of electron lucent and electron dense cytoplasmic granules in this same cytolytic T cell clone. Biochemical analysis showed TIA-1 to be a 15-kDa protein in unstimulated T cells. Upon activation with Con A or anti-CD3 antibodies. TIA-1 was induced to form disulfide linked dimers, trimers, and tetramers of the basic 15-kDa unit. Taken together, our data suggest that TIA-1 is a cytolytic granule associated protein that may define a subpopulation of resting CD8+ T lymphocytes possessing cytolytic potential.     

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)