Induction of I region-restricted hapten-specific cytotoxic T lymphocytes.

Murine cytotoxic T lymphocytes (CTL) reactive to TNP-conjugated syngeneic target cells do lyse to a moderate but significant extent TNP-conjugated, I region compatible but H-2K or H-2D region incompatible target cells. Antibody inhibition experiments and "cold inhibition" experiments indicate that some CTL clones recognize TNP-conjugated targets in association with syngeneic I region determinants independently of H-2K or H-2D gene products. The data suggest that besides TNP-conjugated H-2K or H-2D gene products, in principle, also TNP-conjugated I region determinants do stimulate TNP-specific CTL precursor cells and act as target antigens of TNP-specific CTL.     

Mechanism of killing by virus-induced cytotoxic T lymphocytes elicited in vivo.

The mechanism of lysis by in vivo-induced cytotoxic T lymphocytes (CTL) was examined with virus-specific CTL from mice infected with lymphocytic choriomeningitis virus (LCMV). LCMV-induced T cells were shown to have greater than 10 times the serine esterase activity of T cells from normal mice, and high levels of serine esterase were located in the LCMV-induced CD8+ cell population. Serine esterase was also induced in purified T-cell preparations isolated from mice infected with other viruses (mouse hepatitis, Pichinde, and vaccinia). In contrast, the interferon inducer poly(I.C) only marginally enhanced serine esterase in T cells. Serine esterase activity was released from the LCMV-induced T cells upon incubation with syngeneic but not allogeneic LCMV-infected target cells. Both cytotoxicity and the release of serine esterase were calcium dependent. Serine esterase released from disrupted LCMV-induced T cells was in the form of the fast-sedimenting particles, suggesting its inclusion in granules. Competitive substrates for serine esterase blocked killing by LCMV-specific CTL, but serine esterase-containing granules isolated from LCMV-induced CTL, in contrast to granules isolated from a rat natural killer cell tumor line, did not display detectable hemolytic activity. Fragmentation of target cell DNA was observed during the lytic process mediated by LCMV-specific CTL, and the release of the DNA label [125I]iododeoxyuridine from target cells and the accompanying fragmentation of DNA also were calcium dependent. These data support the hypothesis that the mechanism of killing by in vivo-induced T cells involves a calcium-dependent secretion of serine esterase-containing granules and a target cell death by a process involving nuclear degradation and DNA fragmentation.     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.     

Induction of feline immunodeficiency virus-specific cytotoxic T cells in vivo with carrier-free synthetic peptide.

The role of cellular immunity in the establishment and progression of immunosuppressive lentivirus infection remains equivocal. To develop a model system with which these aspects of the host immune response can be studied experimentally, we examined the response of cats to a hybrid peptide containing predicted T-and B-cell epitopes from the gag and env genes of feline immunodeficiency virus (FIV). Cats were immunized with an unmodified 17-residue peptide incorporating residues 196 to 208 (from gag capsid protein p24) and 395 to 398 (from env glycoprotein gp120) of the FIV Glasgow-8 strain by using Quil A as an adjuvant. Virus-specific lymphocytotoxicity was measured by chromium-51 release assays. The target cells were autologous or allogeneic skin fibroblasts either infected with recombinant FIV gag vaccinia virus or pulsed with FIV peptides. Effector cells were either fresh peripheral blood mononuclear cells or T-cell lines stimulated with FIV peptides in vitro. Cytotoxic effector cells from immunized cats lysed autologous, but not allogeneic, target cells when they were either infected with recombinant FIV gag vaccinia virus or pulsed with synthetic peptides comprising residues 196 to 205 or 200 to 208 plus 395. Depletion of CD8+ T cells, from the effector cell population abrogated the lymphocytotoxicity. Immunized cats developed an antibody response to the 17-residue peptide immunogen and to recombinant p24. However, no antibodies which recognized smaller constituent peptides could be detected. This response correlated with peptide-induced T-cell proliferation in vitro. This study demonstrates that cytotoxic T lymphocytes specific for FIV can be induced following immunization with an unmodified short synthetic peptide and defines a system in which the protective or pathological role of such responses can be examined.     

Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic approach for a number of diseases. To overcome the difficulty in generating specific CTLs, we established stable artificial antigen-presenting cells (AAPCs) that can be used to stimulate T cells of any patient of a given human leukocyte antigen (HLA) type. Mouse fibroblasts were retrovirally transduced with a single HLA-peptide complex along with the human accessory molecules B7.1, ICAM-1, and LFA-3. These AAPCs consistently elicit strong stimulation and expansion of HLA-restricted CTLs. Owing to the high efficiency of retrovirus-mediated gene transfer, stable AAPCs can be readily engineered for any HLA molecule and any specific peptide.     

Equilibrium binding of cytotoxic T lymphocytes to class I antigen

Cloned cytotoxic T lymphocytes specifically bind to purified alloantigen that has been immobilized on a surface. When the time course was examined, it was found that binding reached a plateau level within about 1 h at 37 degrees C, at which time about 30% of the CTL were tightly adhered to the surface. Analysis of the properties of binding demonstrated that this does not simply result because only a fraction of the cells in the clonal population are capable of binding. Instead, the binding is shown to result from an equilibrium involving tightly bound and unbound (or weakly bound) cells. Thus, the cells cycle between a tightly bound and unbound state, despite continuous contact with the Ag-bearing surface. The results suggest that dissociation of the bound cells may be an actively signaled event. A model that could account for these results based on activated CD8 binding is discussed.     

Efficiency and effectiveness of cloned virus-specific cytotoxic T lymphocytes in vivo

The efficiency of cloned class I MHC restricted CTL specific for the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus in either mediating virus clearance or immunopathologic disease in mice during acute infection was quantitated. Cloned CTL specific for either an internal (nucleoprotein) or surface (glycoprotein) protein of lymphocytic choriomeningitis virus, when administered intracerebrally 5 days after the initiation of infection induced fatal immunopathology, indicating that both internal and surface viral Ag play a role in immune mediated disease in vivo. Dose-response analysis indicated that only 10(2) to 10(3) cloned CTL injected intracerebrally were required to induce mortality in 50% of inoculated syngeneic mice. Thus relatively few virus-specific CTL are required to induce an acute immunopathologic disease in the central nervous system. In contrast, if cloned CTL are adoptively transferred at the time of initiation of viral infection they provide protection as demonstrated by their ability to eliminate virus from the brain and thus terminate the acute infection.     

Effects of cyclic AMP on in vivo cytotoxic T lymphocytes generation

Chronic schistosomiasis mansoni is associated with a modulation of schistosome egg-associated cell-mediated granuloma formation. In this study the injection of a cell-free sonicate from either spleen or thymus cells from chronic Schistosoma mansoni-infected mice (> 15 weeks) passively modulated granuloma size in acutely infected (6–8 weeks) syngeneic mice. Cell-free sonicates from age-matched uninfected mice did not suppress granuloma formation. The chronic extracts also suppressed the soluble schistosome egg antigenic preparation-elicited delayed skin test reactivity in 7–8 week infected animals, but failed to suppress the unrelated antigen-elicited reactivity to bovine serum albumin (BSA) of BSA-sensitized animals.     

The role of T cells in anti-herpes simplex virus immunity. I. Induction of antigen-specific cytotoxic T lymphocytes.

Mice infected with herpes simplex virus develop little or no cytotoxic T lymphocyte (CTL) response. However, in lymph nodes (LN's) draining a local site infected with HSV, antigen-specific CTL precursors are sensitized, which upon transfer to in vitro culture conditions develop within 72 hr into effective CTL. The in vivo blockade of CTL differentiation can be overcome by cyclophosphamide, suggesting that a cyclophosphamide-sensitive mechanism blocks the in vivo generation of HSV-immune CTL. The cytolytic activity of HSV-immune CTL is H-2 restricted and antigen specific. Thus CTL sensitized toward HSV type 1 discriminate between syngeneic targets infected with either the immunologic HSV variant type 1 or type 2 (and vice versa). H-2-matched target cells exposed for 30 min to infectious HSV are lysed within 60 min of contact with CTL. Since HSV replication is believed to require more than 4 to 5 hr, the data suggest that either the expression of HSV-dependent "early proteins" takes place within 30 to 90 min or cell membrane-integrated HSV virion represents the target antigen of CTL.     

Priming of influenza virus-specific cytotoxic T lymphocytes vivo by short synthetic peptides.

Influenza virus-specific CTL were primed in vivo by immunization with short synthetic peptides representing major CTL epitopes from the nucleoprotein of type A influenza virus. The resultant CTL after in vitro boosting of primed spleen cells recognized both virus-infected and peptide-pulsed target cells. The requirement of CD4+ T cell activation was investigated in several ways. First the addition of helper epitopes to the CTL epitope did not enhance CTL generation, suggesting that helper activity was either not limiting or not required. However, in vivo depletion of CD4+ T cells completely inhibited the generation of CTL by peptide immunization. The inclusion of anti-CD4 in the in vitro restimulation with peptide also prevented the generation of CTL, whereas in vitro reactivation of virus immune spleen cells with peptide was not inhibited by anti-CD4. Thus there appears to be heterogeneity in the requirement of CD4+ T cell proliferation in CTL generation. One possibility is that virus infected cells can stimulate higher affinity T cells that are less helper T cell dependent.     

Activation of cytotoxic function in T lymphocytes.

The requirements for activation of cytotoxic function in mouse T lymphocytes were investigated. Initial generation of cytotoxicity in normal lymphocytes was equal in magnitude with either Con A or specific alloantigen, and in either case required DNA synthesis. Cytotoxic function in MLC-primed cells could also be regenerated by Con A, the magnitude and target specificity of the cytotoxicity thus generated being indistinguishable from that recalled by specific alloantigen. Cytotoxicity could also be regenerated by third party-stimulating cells; however, the cytotoxicity evoked by third party cells was always specific only for target cells of the original stimulating cell H-2 genotype. The data presented suggest that there are a number of ways to activate cytotoxicity in effector T cells, and are most consistent with a model for T cell triggering that minimizes a strict informational function of antigen-receptor interactions.     

Mechanisms of in vivo generation of cytotoxic activity against allograft: 1. Local differentiation of mature cytotoxic T lymphocytes in rejection of allogeneic lymphocytes

Although cytotoxic activity was not detected within the spleen and regional lymph nodes from mice immunized sc with allogeneic lymphocytes, such activity was detected consistently in glass-nonadherent and anti-θ-sensitive peritoneal exudate cells (PE cells) from Day 5 after immunization and reached a maximum by Day 7. Immunized spleen cells developed cytotoxic T lymphocytes (CTLs) earlier and more effectively than normal spleen cells when transferred ip into X-irradiated syngeneic normal mice together with immunizing antigen, while they did not become cytotoxic when transferred without antigen. These results suggest that spleen and lymph node cells which may have differentiated into some transitional state by in vivo immunization may differentiate into mature CTLs, following direct contact with antigen at the site of graft. CTLs generated there appear to be responsible for the rejection of allogeneic lymphocytes. Cytotoxicity of PE cells was also generated in X-irradiated mice and augmented cytotoxicity was generated by treatment with cyclophosphamide.     

Cell surface glycoproteins of cytotoxic T lymphocytes induced in vivo and in vitro

CTL-blasts, generated in vitro or obtained from educated spleens in vivo, have previously been shown to display high levels of a unique membrane glycoprotein T145 (ca. 145,000 daltons), inferred to be a common CTL marker and implicated in CTL cytotoxicity. As shown here, however, these suggestions do not hold for all cytotoxic T lymphocytes. We have shown that T145 is not expressed on highly active CTL-induced in vivo by a standard alloimmunization protocol. The finding that several CTL populations are T145 positive, whereas others are T145 negative, may be due to the existence of functional CTL in 2 stages of differentiation or may represent different CTL lineages. In addition, no correlation was found between cytolytic activity and expression of T145. The functional significance of T145 on CTL is discussed.     

Cross-reactivity patterns of murine cytotoxic T lymphocytes.

Cross-reactivity of TNP-immune, virus-immune, and alloreactive murine cytotoxic thymus-derived (T_c) cells was investigated at the level of target cell lysis. Alloreactive T_c cells cross-reacted on TNP-modified and unmodified third-party targets and on syngeneic TNP-modified targets but did not cross-react on syngeneic virus-infected targets. TNP-immune T_c cells showed marked cross-reactivity on certain allogeneic targets modified by TNP (loss of H-2 restriction) and also on certain unmodified allogeneic targets but did not cross-lyse virus-infected syngeneic targets. Targets treated with TNP-Sendai virus were not lysed by TNP-immune T_c cells, but T_c cells stimulated by cells treated with TNP-Sendai virus lysed such targets readily. These results are consistent with the view that T_c-cell recognition of foreign H-2 antigens and TNP-modified self-H-2 antigens are mechanistically similar (possibly via one receptor), whereas recognition of viral plus H-2 antigens is different (possibly via two receptors).Virus-immune T_c cells ubiquitously exhibited strong cross-reactivity on syngeneic TNP-modified targets using pox-, arena-, alpha-, myxo-, and paramyxoviruses for T_c-cell induction. The lysis of virus-infected targets by virus-immune T_c cells could be inhibited by cold TNP-modified competitors, thus establishing that some individual virus-immune T_c cells could recognize both types of target cells. This genuine cross-reactivity at the effector level was not observed at the level of induction of secondary responses, since the cross-reactive subset of virus-immune memory T_c cells could not be activated by TNP-modified stimulator cells but could be activated by virus-infected stimulators. These results implied that requirements for stimulation of precursor T_c cells are sometimes different from antigenic requirements for recognition and lysis of effector T_c cells.     

In vivo generation of cytotoxic T cells from epitopes displayed on peptide-based delivery vehicles

The development of nonviral, peptide-based constructs able to elicit protective in vivo CTL responses represents a major challenge in the design of future vaccines. We report the design of branched peptide delivery vehicles, termed loligomers, that facilitate the import, processing, and presentation of CTL epitopes onto nascent MHC class I molecules. These complexes are then effectively displayed on the surface of APCs. The intracellular delivery of CTL epitopes by loligomers prolonged the expression of Ag-MHC class I complexes on the surface of APCs in comparison with free CTL epitope alone. Furthermore, the injection of CTL epitope-containing loligomers into mice led to the generation of in vivo CTL responses and the induction of autoimmune disease in an animal model. Synthetic epitope-carrying, peptide-based delivery vehicles may represent useful components to be included in the formulation of future vaccines.     

Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes

It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.     

Induction of TNP-specific cytotoxic T lymphocyte memory in vivo in the absence of T helper cell activity

The question of whether TH cells are required for the priming of CTL precursors (CTLp) in vivo was studied by using Txbm mice (Thymectomized, irradiated, and stem cell-reconstituted mice). In these mice, TNP-specific CTL could be induced in vitro with TNP-coupled spleen cells only if the cultures were supplemented with an IL 2-containing supernatant (ConAsup). In contrast to normal mice, TNP-specific Lyt-2-TH cells could not be induced by skin painting with trinitrochlorobenzene (TNCB) (as tested by the ability to help CTL formation from thymocyte or normal spleen precursors). These data confirm previous findings that Txbm mice possess CTLp but that their TH compartment is deficient. TNCB skin painting had, however, a clear priming effect on the CTLp population: spleen cells from TNCB-painted mice could give rise to specific CTL with a lower amount of ConAsup than spleen cells from unprimed mice. In addition to this, priming changed the CTLp so that stimulation with lightly coupled cells (0.1 mM trinitrobenzene sulfonic acid [TNBS] instead of 10 mM TNBS) became effective. These changes took place without a significant increase in the frequency of TNP-specific CTL precursors. The data obtained are consistent with the concept that at least with some antigens, CTLp proliferation (clonal expansion), which is probably caused by activated TH cells, is not required for the induction of immunologic memory in vivo.     

A T cell receptor-associated GTP-binding protein triggers T cell receptor-mediated granule exocytosis in cytotoxic T lymphocytes

To study the subcellular events occurring after T cell activation we used cloned human CTL permeabilized with alpha-toxin of Staphylococcus aureus. This method of permeabilization leads to stable transmembrane channels that permit the introduction of small molecules into the cell but preserves the cellular structures and macromolecular contents of the CTL. We used the exocytosis of CTL-specific serine esterases as a marker of T cell activation. The TCR-activated exocytosis is functioning in such permeabilized CTL. Introduction of the membrane impermeable guanosine nucleotide-binding protein (G-protein) activating GTP-analog GTP gamma S into CTL triggers exocytosis if Ca2+ is present. For optimal exocytosis ATP is required. The G-protein inactivating GDP-analog GDP beta S inhibited exocytosis triggered via the TCR-CD3 complex but not that triggered by activating the protein kinase C. If the protein kinase C was depleted in CTL by overnight incubation with phorbolester, the response to GTP-gamma S was reduced by more than 50%. These experiments demonstrate the presence of a G-protein involved in TCR-mediated CTL triggering. In the sequence of signaling steps this G-protein is localized after TCR-triggering but before the formation of the protein kinase C-activating phosphoinositol breakdown product diacylglycerol in the sequence of signaling steps.