Role of the H-2I region in the generation of an antiviral cytotoxic T-cell response in vitro

The participation of H-2I gene products in generating virus-specific proliferative and/or cytotoxic T-lymphocyte (CTL) responses was investigated. Spleen cells from mice infected with vaccinia virus were restimulated secondarily in vitro with syngeneic virus-infected peritoneal exudate cells (PEC) and then restimulated in tertiary cultures with virus-infected PEC from syngeneic and partially histoincompatible strains of mice. Based on the finding that comparable proliferative responses resulted when stimulating the responding cells were histocompatible at the H-2K, I, or D region of the major histocompatibility complex (MHC), the additively enhanced, but not potentiated, proliferation caused by identity at two or three H-2 regions was analyzed. Enhancement of proliferation followed when the H-2K/D components plus virus and the H-2I components plus virus were either on the same, or alternatively on two, stimulating cells. This suggests that H-2K, D, and I plus virus trigger distinct T-cell subsets. A virus-specific CTL response was generated in vitro when spleen cells from virus-primed mice and even unprimed mice were stimulated with cells sharing only H-2K and/or H-2D of the MHC. Identity at the H-2I region did not stimulate a CTL response, nor did it influence the magnitude of the $\text{K}\text{D}$ restricted response. Nevertheless, the presence of anti-Ia antiserum in cultures of syngeneic stimulators and responders inhibited the cytotoxic response to a great extent. Therefore, H-2I region products seem to participate in the generation of virus-specific CTL in vitro.     

Functional signatures in antiviral T-cell immunity for monitoring virus-associated diseases

At present, we only have indirect knowledge of the protective role of antigen-specific T cells in human viral infections, and it has been difficult to show a direct correlation between quantitative and qualitative measures of T-cell immunity and virus-associated diseases. However, as described in this Opinion article, recent advances in the characterization of T-cell functions and in the development of standardized T-cell assays have led to the identification of distinct functional signatures of T-cell responses that correlate with levels of viral replication and disease activity.     

Predicting the dynamics of antiviral cytotoxic T-cell memory in response to different stimuli: cell population structure and protective function

This paper examines the numerical and functional consequences of various stimuli on antiviral CD8+ T-cell memory using a mathematical model. The model is based upon biological evidence from the murine model of infection with lymphocytic choriomeningitis virus (LCMV) that the phenotype of immunological memory represents low-level responses driven by various stimuli, and the memory CTL population is partitioned between resting, cycling and effector cells. These subpopulations differ in their lifespan, their potential to mediate antiviral protection and in the stimuli needed for their maintenance. Three types of maintenance stimuli are examined: non-antigen-specific (bystander) stimulation, persisting antigen stimulation and reinfection-mediated stimulation. The modelling predicts that: (i) stable persistence of CTL memory requires the presence of either bystander or antigen-specific stimulation above a certain threshold depending on the sensitivity of memory CTL to stimulation and their life-span; (ii) a relatively low level of stimuli (approximately 10(4) fold less on a per CTL basis compared to acute infection) is needed to stabilize the expanded memory CTL population; (iii) the presence of CTL subsets in the memory pool of different activation states and lifespans ensures the robustness of memory persistence in the face of temporal variation in the low-level stimuli and; (iv) an 'optimal' population structure of the memory CTL pool, in terms of immediate protection, requires the presence of both activated cycling and effector CTL. For this, persisting antigen alone or synergistically with bystander signals provide the appropriate stimulation, so that the stimuli equivalent to approximately 30 p.f.u. of LCMV in the spleen are sufficient to maintain approximately 10(5)-10(6) specific CTL in the memory pool. These observations are relevant both to our understanding of natural protective immunity and to vaccine design.     

The requirement for biotin and fatty acids in the cytotoxic T-cell response.

Dialyzed fetal calf serum (FCS) was a poor source of serum supplement for in vitro cytotoxic T lymphocyte (CTL) generation. Serum dialysate or biotin fully restored dialyzed FCS to activities comparable to FCS. It was concluded that the active principal in serum dialysate was biotin because its further dialysis was prevented by addition of avidin, a biotin binding protein. Avidin inhibited CTL generation only when added during the early stages of mixed lymphocyte cultures, whereas biotin could restore activity even if added at a later time. When FCS enriched in a fatty acid mixture, or in palmitic acid alone, was used as the serum supplement, avidin-mediated inhibition of CTL generation was markedly reduced. Avidin also inhibited CTL generation in cultures containing killed macrophages as the stimulating cell, and supplemented with Con-A-induced spleen cell supernatant, a source of helper factor(s). These experiments suggest that fatty acid biosynthesis and the attendant synthesis of structural lipids of appropriate fatty acid composition play a prominent role in the generation of CTL     

Immunodominance of an antiviral cytotoxic T cell response is shaped by the kinetics of viral protein expression

Lymphocytic choriomeningitis virus (LCMV) infection induces a protective CTL response consisting of gp- and nucleoprotein (NP)-specific CTL. We find that a small load of LCMV led to immunodominance of NP-CTL, whereas a large viral load resulted in dominance of gp-CTL. This is the first study describing that immunodominance is not fixed after infection with a given pathogen, but varies with the viral load instead. We assumed higher Ag sensitivity for NP-CTL, which would explain their preferential priming at low viral load, as well as their overstimulation resulting in selective exhaustion at high viral load. The higher Ag sensitivity of NP-CTL was due to faster kinetics of NP-epitope presentation. Thus, we uncover a novel factor that impinges upon immunodominance and is related to the kinetics of virus protein expression. We propose that CTL against early viral proteins swiftly interfere with virus replication, resulting in efficient protection. If these "early" CTL fail in immediate virus control, they are activated in the face of higher viral load compared with "late" CTL and are therefore prone to be exhausted. Thus, the observed absence of early CTL in persistent infections might not be the cause, but rather the consequence of viral persistence.     

Antitumor cytotoxic T-cell response induced by a survivin peptide mimic

Survivin is a tumor-associated antigen with significant potential as a cancer vaccine target. We have identified a survivin peptide mimic containing human MHC class I epitopes and a potential class II ligand that induces a potent antitumor response in C57BL/6 mice with GL261 cerebral gliomas. This peptide is able to elicit both CD8+ CTL and T helper cell responses in C57BL/6 mice. The corresponding region of the human survivin molecule represented by peptide SVN53-67 is 100% homologous to the murine protein, but SVN53-67 is weakly immunogenic in man. We evaluated several amino acid substitutions in putative human MHC I anchor positions in SVN53-67 to identify potential peptide mimics that could provide an enhanced antitumor immune response against human glioma and primary central nervous system lymphoma (PCNSL) cells in culture. We evaluated survivin peptides with predicted binding to human HLA-A*0201 antigen using peptide-loaded dendritic cells from PBMC of patients with these malignancies. One alteration (M57) led to binding to HLA-A*0201 with significantly higher affinity. We compared the ability of autologous dendritic cells loaded with SVN53-67 peptide and SVN53-67/M57 in CTL assays against allomatched and autologous, survivin-expressing, human malignant glioma and PCNSL cells. Both SVN53-67 and SVN53-67/M57 produced CTL-mediated killing of malignant target cells; however, SVN53-67/M57 was significantly more effective than SVN53-67. Thus, SVN53-67/M57 may act as a peptide mimic to induce an enhanced antitumor CTL response in tumor patients. The use of SVN53-67/M57 as a cancer vaccine might have application for cancer vaccine therapy.     

Analysis of haplotype preference in the cytotoxic T-cell response to H-Y

The mechanism determining which parental haplotype is selected in (CBA × 1310) (k × b)F₁ female mice for major histocompatibility complex (H-2) restricted, male-specific (H-Y), immune, cytotoxic T-cell (Tc-cell) responses, was investigated. The data show that haplotype preference is variable, and may be directed towards one, both, or neither of the parental haplotypes. This preference is reflected in the precursor frequency of memory Tc cells as measured by limiting dilution assays. It was further shown that maternal influence, antigen dose, route of immunization, and a feedback mechanism on the stimulator cells in vivo could not influence haplotype preference or its observed variability. Evidence for cross-reactive killing by H-2^k and H-2^b H-Y immune Tc cells on H-2^b and H-2^k allogeneic targets, respectively, (i. e., the independent haplotype of the other parent of the F₁ mice), provide evidence for natural tolerance as a possible mechanism to explain haplotype preference.     

CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help

CD40 is essential in enabling antigen-presenting cells to process and present antigen effectively to T cells. We demonstrate here that when antibody against CD40 is used to treat mice with syngeneic lymphoma, a rapid cytotoxic T-cell response independent of T-helper cells occurs, with tenfold expansion of CD8+ T cells over a period of 5 days. This response eradicates the lymphoma and provides protection against tumor rechallenge without further antibody treatment. Thus, it seems that by treating mice with monoclonal antibody against CD40, we are immunizing against syngeneic tumors. The phenomenon proved reproducible with two antibodies against CD40 (3/23 and FGK-45) in three CD40+ lymphomas (A20, A31 and BCL1) and gave partial protection in one of two CD40- lymphomas (EL4 and Ten1). Although the nature of the target antigens on these lymphomas is unknown, CD8+ T cells recovered from responding mice showed powerful cytotoxic activity against the target B-cell lymphoma in vitro.     

The cytotoxic T-cell response to H-1 minor histocompatibility antigen differences

H-1-specific cytotoxic T cells were generated in in vitro secondary cultures. Effectors were assayed on H-2 compatible, peritoneal exudate cell targets in a ⁵¹Cr release assay. Target-cell lysis appeared to be specific for the H-1 type of the stimulator cells. Effector cells were T cells since they expressed Thy 1.2 alloantigen and required H-2 compatibility between donors of the stimulator cells, responder cells, and target cells for efficient lysis. Peritoneal exudate cells were found to be efficient specific competitors in the cytotoxicity assay. There appeared to be no strict correlation between in vitro cytotoxic T-cell activity and mean skin graft rejection times for a number of minor H and H-2D differences.     

T4+ T helper cell function in vivo: differential requirement for induction of antiviral cytotoxic T-cell and antibody responses.

This study documents the differential requirements of T4+ T helper cells in the induction of virus-specific cytotoxic T-lymphocyte (CTL) and antibody responses during acute lymphocytic choriomeningitis virus infection. Two monoclonal antibodies (GK1.5 and RL172.4) directed against the L3T4 (T4) molecule were used for depleting T helper cells from mice. Depletion of T4+ cells caused a pronounced suppression of antiviral antibody response (20-fold decrease) but had minimal effect on virus-specific CTL response (less than 2-fold reduction). Despite the elimination of greater than 90% of T helper cells, anti-L3T4-treated mice were able to generate a CTL response of sufficient magnitude to control the viral infection. In contrast, depletion of Lyt2+ T cells abrogated the CTL response and the ability to eliminate virus. Thus, our results underscore the importance of the Lyt2+ T-cell subset in controlling infection with this virus and show that a deficiency of T4+ T cells is likely to have a more severe effect on antibody production than on CTL responses.     

Transgenerational inheritance of an acquired small RNA-based antiviral response in C. elegans

Induced expression of the Flock House virus in the soma of C. elegans results in the RNAi-dependent production of virus-derived, small-interfering RNAs (viRNAs), which in turn silence the viral genome. We show here that the viRNA-mediated viral silencing effect is transmitted in a non-Mendelian manner to many ensuing generations. We show that the viral silencing agents, viRNAs, are transgenerationally transmitted in a template-independent manner and work in trans to silence viral genomes present in animals that are deficient in producing their own viRNAs. These results provide evidence for the transgenerational inheritance of an acquired trait, induced by the exposure of animals to a specific, biologically relevant physiological challenge. The ability to inherit such extragenic information may provide adaptive benefits to an animal.     

Rational design of cytotoxic T-cell inhibitors

This study describes the use of the CD8/major histocompatibility complex (MHC) class I crystal structure as a template for the de novo design of low-molecular-weight surface mimetics. The analogs were designed from a local surface region on the CD8 alpha-chain directly adjacent to the bound MHC class I, to block the protein associations in the T-cell activation cluster that occur upon stimulation of the cytotoxic T lymphocytes (CTLs). One small conformationally restrained peptide showed dose-dependent inhibition of a primary allogeneic CTL assay while having no effect on the CD4-dependent mixed lymphocyte reaction (MLR). The analog's activity could be modulated through subtle changes in its side chain composition. Administration of the analog prevented CD8-dependent clearance of a murine retrovirus in BALB/c mice. In C57BL/6 mice challenged with the same retrovirus, the analog selectively inhibited the antiviral CTL responses without affecting the ability of the CTLs to generate robust allogeneic responses.     

Murine cytotoxic T-cell response to alphavirus is associated mainly withH- 2D k

A secondary in vitro response to alphaviruses Bebaru, Sindbis, and Semliki Forest is described. Optimum response appears at day 5–6 of culture. The cells responsible for lytic activity are nonadherent, -positive, Ig^–, and mainly Ly-2.1 positive. Out of five haplotypes tested (H- 2 ^d ,H- 2 ^b ,H- 2 ^s ,H- 2 ^q , andH- 2 ^k ) onlyH- 2 ^k was a responder. Genetic mapping of the response located it solely in theD region of theH- 2 complex. The other four haplotypes responded with a high antiself activity after a second stimulation with viruses. This antiself response also maps in theD region of theH- 2 complex. No complementation was observed in F₁ hybrids between responder and nonresponder strains.     

Identification of human herpesvirus 8-specific cytotoxic T-cell responses.

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma-associated herpesvirus) is implicated in the etiopathogenesis of Kaposi's sarcoma (KS) and certain lymphoproliferations. The introduction of more effective therapies to treat human immunodeficiency virus infection has led to a decline in the incidence of KS and also in the resolution of KS in those already affected. This suggests that cellular immune responses including cytotoxic T lymphocytes (CTLs) could play a vital role in the control of HHV-8 infection and in KS pathogenesis. Here we elucidate HLA class I-restricted, HHV-8-specific cellular immune responses that could be important in the control of HHV-8 infection and subsequent tumor development. We show the presence of CTLs against HHV-8 latent (K12), lytic (K8.1), and highly variable (K1) proteins in infected individuals.     

Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus.

Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Optimisation of peptide-based cytotoxic T-cell determinants using

In this brief review we describe the methods that our group and others have developed in incorporating non-natural amino acids into peptide antigens, principally to increase protease resistance, for potential use in peptide-based vaccines. Peptide-based vaccination has the potential to generate protective immunity without the need for in situ antigen synthesis or further proteolytic processing of the antigen. The ability to deliver minimal T cell epitopes to the effector cells of the immune system also minimises unwanted side effects and simplifies clinical monitoring. The major hurdle in designing successful peptide-based vaccines resides in issues surrounding the delivery and stability of the peptide immunogen, as it is the form in which the epitope is delivered that will determine how it will be processed by the immune system and ultimately whether it will be capable of inducing an appropriate immune response. Thus, one of the confounding issues with peptide-based vaccines is their poor bioavailability, which is predominantly due to proteolysis and oxidative damage of the `naked' peptide. Strategies that stabilise peptide epitopes promise to overcome the current problems and make peptide-based immunogens more applicable in vaccine design.     

In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.