Virus-specific Cytotoxic T-lymphocyte Response Research Articles

Differential Recognition of Influenza A Viruses by M158-66 Epitope-Specific CD8+ T Cells Is Determined by Extraepitopic Amino Acid Residues.

Natural influenza A virus infections elicit both virus-specific antibody and CD4(+) and CD8(+) T cell responses. Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M158-66) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M158-66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8(+) T cell recognition of the M158-66 epitope. These data indicate that human influenza A viruses can impair recognition by M158-66-specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8(+) cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M158-66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M158-66 epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

Recombinant measles viruses expressing respiratory syncytial virus proteins induced virus-specific CTL responses in cotton rats

Respiratory syncytial virus (RSV) is a common cause of serious lower respiratory tract illnesses in infants. Natural infections with RSV provide limited protection against reinfection because of inefficient immunological responses that do not induce long-term memory. RSV natural infection has been shown to induce unbalanced immune response. The effective clearance of RSV is known to require the induction of a balanced Th1/Th2 immune response, which involves the induction of cytotoxic T lymphocytes (CTL). In our previous study, recombinant AIK-C measles vaccine strains MVAIK/RSV/F and MVAIK/RSV/G were developed, which expressed the RSV fusion (F) protein or glycoprotein (G). These recombinant viruses elicited antibody responses against RSV in cotton rats, and no infectious virus was recovered, but small amounts of infiltration of inflammatory cells were observed in the lungs following RSV challenge. In the present study, recombinant AIK-C measles vaccine strains MVAIK/RSV/M2-1 and MVAIK/RSV/NP were developed, expressing RSV M2-1 or Nucleoprotein (NP), respectively. These viruses exhibited temperature-sensitivity (ts), which was derived from AIK-C, and expressed respective RSV antigens. The intramuscular inoculation of cotton rats with the recombinant measles virus led to the induction of CD8+ IFN-γ+ cells. No infectious virus was recovered from a lung homogenate following the challenge. A Histological examination of the lungs revealed a significant reduction in inflammatory reactions without alveolar damage. These results support the recombinant measles viruses being effective vaccine candidates against RSV that induce RSV-specific CTL responses with or without the development of an antibody response.

FoxO1 directly regulates the expression of PD-1 and sustains viral specific CD8+ T cell responses during chronic infection (IRM4P.501)

Abstract Understanding the signaling and transcriptional pathways that regulate the function, survival and homeostasis of CD8+ cytotoxic T lymphocytes (CTLs) during chronic infection is fundamental to the development of therapies to treat chronic infections such as HIV, HSV, and HCV. Of these, the PI3K/AKT/mTOR signaling pathway is of significant interest because it controls T cell differentiation, proliferation, metabolism and survival. We show that during murine chronic lymphocytic choriomeningitis virus (LCMV) infection, chronic antigen stimulation greatly impairs the ability of virus-specific CD8 T cells to activate PI3K/AKT/mTOR signaling in response to TCR and cytokine stimulation, resulting in enhanced nuclear retention of the transcription factor, FoxO1. Therapeutic blockade of the inhibitory PD-1 receptor, which restores antiviral CTL responses, was dependent on increased PI3K/AKT/mTOR activity. FoxO1 expression was critical to sustain virus-specific CTL responses and control viral infection. Lastly, we demonstrate that FoxO1 was both necessary and sufficient to induce the expression of the inhibitory receptor PD-1 on virus-specific CD8 T cells, and therefore represents a critical regulatory factor in balancing effector and exhausted CTL states during chronic infection. These findings highlight a novel role for AKT-FoxO1 pathway in the regulation of exhaustion CTL responses during chronic infection.

Understanding MHC class I presentation of viral antigens by human dendritic cells as a basis for rational design of therapeutic vaccines.

Effective viral clearance requires the induction of virus-specific CD8+ cytotoxic T lymphocytes (CTL). Since dendritic cells (DC) have a central role in initiating and shaping virus-specific CTL responses, it is important to understand how DC initiate virus-specific CTL responses. Some viruses can directly infect DC, which theoretically allow direct presentation of viral antigens to CTL, but many viruses target other cells than DC and thus the host depends on the cross-presentation of viral antigens by DC to activate virus-specific CTL.Research in mouse models has highly enhanced our understanding of the mechanisms underlying cross-presentation and the dendritic cells (DC) subsets involved, however, these results cannot be readily translated toward the role of human DC in MHC class I-antigen presentation of human viruses. Here, we summarize the insights gained in the past 20 years on MHC class I presentation of viral antigen by human DC and add to the current debate on the capacities of different human DC subsets herein. Furthermore, possible sources of viral antigens and essential DC characteristics for effective induction of virus-specific CTL are evaluated.We conclude that cross-presentation is not only an efficient mechanism exploited by DC to initiate immunity to viruses that do not infect DC but also to viruses that do infect DC, because cross-presentation has many conceptual advantages and bypasses direct immune modulatory effects of the virus on its infected target cells.Since knowledge on the mechanism of viral antigen presentation and the preferred DC subsets is crucial for rational vaccine design, the obtained insights are very instrumental for the development of effective anti-viral immunotherapy.

A Safe Bacterial Microsyringe for In Vivo Antigen Delivery and Immunotherapy

The industrial development of active immunotherapy based on live-attenuated bacterial vectors has matured. We developed a microsyringe for antigen delivery based on the type III secretion system (T3SS) of P. aeruginosa. We applied the "killed but metabolically active" (KBMA) attenuation strategy to make this bacterial vector suitable for human use. We demonstrate that attenuated P. aeruginosa has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild-type vector. In a mouse model of cancer, we demonstrate that this KBMA strain, which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigen. The attenuated strain effectively induces a cellular immune response to the cancerous cells while lowering the systemic inflammatory response. Hence, a KBMA P. aeruginosa microsyringe is an efficient and safe tool for in vivo antigen delivery.

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.

The route of immunization with adenoviral vaccine influences the recruitment of cytotoxic T lymphocytes in the lung that provide potent protection from influenza A virus

Virus-specific cytotoxic T lymphocytes (CTLs) in the lung are considered to confer protection from respiratory viruses. Several groups demonstrated that the route of priming was likely to have an implication for the trafficking of antigen-specific CTLs. Therefore, we investigated whether the route of immunization with adenoviral vaccine influenced the recruitment of virus-specific CTLs in the lung that should provide potent protection from influenza A virus. Mice were immunized with recombinant adenovirus expressing the matrix (M1) protein of influenza A virus via various immunization routes involving intraperitoneal, intranasal, intramuscular, or intravenous administration as well as subcutaneous administration in the hind hock. We found that the immunization route dramatically impacted the recruitment of M1-specific IFN-γ + CD8 + T cells both in the lung and the spleen. Surprisingly, hock immunization was most effective for the accumulation in the lung of IFN-γ-producing CD8 + T cells that possessed M1-specific cytolytic activity. Further, antigen-driven IFN-γ + CD8 + T cells in the lung, but not in the spleen, were likely to be correlated with the resistance to challenge with influenza A virus. These results may improve our ability to design vaccines that target virus-specific CTL responses to respiratory viruses such as influenza A virus.

Cytotoxic T Cells Are the Predominant Players Providing Cross-Protective Immunity Induced by γ-Irradiated Influenza A Viruses

We previously demonstrated that a single dose of nonadjuvanted intranasal gamma-irradiated influenza A virus can provide robust protection in mice against both homologous and heterosubtypic challenges, including challenge with an H5N1 avian virus strain. We investigated the mechanism behind the observed cross-protection to define which arms of the adaptive immune response are involved in mediating this protection. Studies with gene knockout mice showed the cross-protective immunity to be mediated mainly by T cells and to be dependent on the cytolytic effector molecule perforin. Adoptive transfer of memory T cells from immunized mice, but not of memory B cells, protected naïve recipients against lethal heterosubtypic influenza virus challenge. Furthermore, gamma-irradiated influenza viruses induced cross-reactive Tc-cell responses but not cross-neutralizing or cross-protective antibodies. In addition, histological analysis showed reduced lung inflammation in vaccinated mice compared to that in unvaccinated controls following heterosubtypic challenge. This reduced inflammation was associated with enhanced early recruitment of T cells, both CD4(+) and CD8(+), and with early influenza virus-specific cytotoxic T-cell responses. Therefore, cross-protective immunity induced by vaccination with gamma-irradiated influenza A virus is mediated mainly by Tc-cell responses.

Induction of cross‐protective immunity against influenza A virus H5N1 by an intranasal vaccine with extracts of mushroom mycelia

The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co-administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti-A/PR8 HA-specific IgA and IgG response in nasal washings and serum, respectively. Virus-specific cytotoxic T-lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross-protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow-derived dendritic cells. These results suggest that mycelial extract-adjuvanted vaccines can confer cross-protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection.

The regulatory T-cell response during acute retroviral infection is locally defined and controls the magnitude and duration of the virus-specific cytotoxic T-cell response

AbstractCytotoxic CD8+ T cells control acute viremia in many viral infections. However, most viruses that establish chronic infections evade destruction by CD8+ T cells, and regulatory T cells (Treg) are thought to be involved in this immune evasion. We have infected transgenic mice, in which Treg can be selectively depleted, with Friend retrovirus (FV) to investigate the influence of Treg on pathogen-specific CD8+ T-cell responses in vivo. We observed that Treg expansion during acute infection was locally defined to organs with high viral loads and massive activation of virus-specific effector CD8+ T cells. Experimental ablation of Treg resulted in a significant increase of peak cytotoxic CD8+ T-cell responses against FV. In addition, it prevented the development of functional exhaustion of CD8+ T cells and significantly reduced FV loads in lymphatic organs. Surprisingly, despite the massive virus-specific CD8+ T-cell response after temporary Treg depletion, no evidence of immunopathology was found. These results demonstrate the important role of Treg in controlling acute retrovirus-specific CD8+ T-cell responses, and suggest that temporary manipulation of Treg might be a possible therapeutic approach in chronic infectious diseases.

Influenza virus CTL epitopes, remarkably conserved and remarkably variable

Virus-specific cytotoxic T lymphocytes (CTL) contribute to the control of virus infections including those caused by influenza viruses. Especially under circumstances when antibodies induced by previous infection or vaccination fail to recognize and neutralize the virus adequately, CTL are important and contribute to protective immunity. During epidemic outbreaks caused by antigenic drift variants and during pandemic outbreaks of influenza, humoral immunity against influenza viruses is inadequate. Under these circumstances, pre-existing CTL directed to the relatively conserved internal proteins of the virus may provide cross-protective immunity. Indeed, most of the known human influenza virus CTL epitopes are conserved. However, during the evolution of influenza A/H3N2 viruses, the most important cause of seasonal influenza outbreaks, variation in CTL epitopes has been observed. The observed amino acid substitutions affected recognition by virus-specific CTL and the human virus-specific CTL response in vitro. Examples of variable epitopes and their HLA restrictions are: NP383–391/HLA-B*2705, NP380–388/HLA-B*0801, NP418–426/HLA-B*3501, NP251–259/HLA-B*4002, NP103–111/HLA-B*1503. In some cases amino acid substitutions occurred at anchor residues and in other cases at T cell receptor contact residues. It is of special interest that the R384G substitution in the NP383–391 epitope was detrimental to virus fitness and was only tolerated in the presence of multiple functionally compensating co-mutations. In contrast, other epitopes, like the HLA-A*0201 restricted epitope from the matrix protein, M158–66, are highly conserved despite their immunodominant nature and the high prevalence of HLA-A*0201 in the population. A mutational analysis of this epitope indicated that it is under functional constraints. Also in influenza A viruses of other subtypes, including H5N1, the M158–66 is highly conserved.

Human immunodeficiency virus type 1-specific CD8+ T-cell responses during primary infection are major determinants of the viral set point and loss of CD4+ T cells.

Primary HIV-1 infection (PHI) is marked by a flu-like syndrome and high levels of viremia that decrease to a viral set point with the first emergence of virus-specific CD8+ T-cell responses. Here, we investigated in a large cohort of 527 subjects the immunodominance pattern of the first virus-specific cytotoxic T-lymphocyte (CTL) responses developed during PHI in comparison to CTL responses in chronic infection and demonstrated a distinct relationship between the early virus-specific CTL responses and the viral set point, as well as the slope of CD4+ T-cell decline. CTL responses during PHI followed clear hierarchical immunodominance patterns that were lost during the transition to chronic infection. Importantly, the immunodominance patterns of human immunodeficiency virus type 1 (HIV-1)-specific CTL responses detected in primary, but not in chronic, HIV-1 infection were significantly associated with the subsequent set point of viral replication. Moreover, the preservation of the initial CD8+ T-cell immunodominance patterns from the acute into the chronic phase of infection was significantly associated with slower CD4+ T-cell decline. Taken together, these data show that the specificity of the initial CTL response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.

Conjugation of Latent Membrane Protein (LMP)-2 Epitope to Gold Nanoparticles as Highly Immunogenic Multiple Antigenic Peptides for Induction of Epstein−Barr Virus-Specific Cytotoxic T-Lymphocyte Responses in Vitro

Nasopharyngeal carcinoma is a neoplasm with a high incidence in Southeast Asia, and it is strongly associated with Epstein-Barr virus (EBV) activation involving the expression of a weakly immunogenic protein, namely, latent membrane protein (LMP)-2. Previous immunological studies already identified the human leukocyte antigen (HLA)-A11 restricted peptide epitope (SSCSSCPLSK) in the LMP-2 antigen. In this work, we prepared gold nanoparticle (AuNP)-peptide conjugate 1 by treating the nanoparticles with the N-cysteinated LMP-2 epitope. The AuNP-peptide conjugates have been characterized by TEM (15-24 nm in diameter) and UV-vis spectroscopy (surface plasmon resonance absorption band at lambda(max) = 520 nm). In the presence of a CALNN capping peptide, the AuNP-peptide conjugates are stable in solution without aggregation at room temperature for at least 48 h. By ELIspot studies, AuNP-peptide conjugate 1 was found to elicit a significantly stronger INF-gamma response [number of spot forming cells (SPC) = 727 +/- 198] from peripheral blood mononuclear cells of healthy HLA-A11 donors when compared to that induced by the unconjugated LMP-2 peptides (SFC = 73 +/- 28). Further studies showed that dendritic cells treated with conjugate 1 can effect CD8+ T-cell activation leading to epitope-specific cytotoxic T lymphocyte killing responses in vitro.

Enhancement of Cytotoxic T-lymphocyte Response in Aged Mice by a Novel Treatment with Recombinant AdIL-12 and Wild-type Adenovirus in Rapid Succession

A decrease in the expression of Th1 cytokines has been associated with age-related decrease in cytotoxic T-lymphocyte (CTL) function. We utilized an E1-deleted adenovirus (Ad) vector to deliver the murine interleukin-12 (IL-12) gene in order to enhance the antivirus CTL response. Wild-type (WT) Ad was administered 3 days after AdIL-12 treatment, when IL-12 production was at its peak and the anti-Ad antibody response had not yet begun to develop. Before receiving AdIL-12 treatment, aged (18 month old) mice exhibited a 58% decrease in the number of virus-specific CTLs, and a 30% decrease in in vivo CTL activity as compared to young (2 month old) mice. After AdIL-12 treatment, aged mice displayed a greater increase in IL-12 expression and endogenous production of interferon-gamma than observed in young mice. When infected with WT Ad, these AdIL-12-treated aged mice exhibited an increased in vivo CTL response and an in vitro proliferative response that was similar to those in young mice. The frequencies of occurrence of D(b)-E1Bp(+)CD8(+) T cells in the spleen, liver, and lung in aged mice were higher than the corresponding values in young mice. These results indicate that IL-12 treatment significantly promotes the virus-specific CTL response in aged mice and, more importantly, specifically targets the virally infected organs, such as the liver and lung, promoting enhanced CTL activity against the virus.

HIV-1-Specific Cytotoxic T Lymphocyte (CTL) Responses Against Immunodominant Optimal Epitopes Slow the Progression of AIDS in China

To assess the immunodominance patterns of HIV-1-specific cytotoxic T lymphocyte (CTL) responses and the contribution of these responses against the peptides scanning optimal epitopes in chronic infection, we test the HIV-1-specific CTL responses against a panel of 413 overlapping peptides spanning HIV-1 Asian B sequence, including 147 peptides corresponding to optimal clade B epitopes in 49 chronically HIV-1 infected individuals by interferon-gamma Elispot assay. A large variation in the recognition of peptides restricted by the same HLA class I allele is presented. Some epitopes are targeted frequently by individuals while other epitopes restricted by the same allele are rarely recognized in our research. HLA-B35 and HLA-A03 rather than other HLA alleles contribute greatly to total virus-specific CTL responses. Furthermore, there is a significant inverse correlation between the total contribution of HIV-1-specific CTL responses restricted by different HLA alleles to virus-specific immune responses and viral load in the individuals during advanced infection (P=0.002, r=-0.549). The peptides targeted by individuals have significantly lower entropy compared with those not targeted but restricted by the same HLA class I alleles (P<0.05) in 49 individuals infected by HIV-1, especially the advanced infection subgroup (P=0.044). These data demonstrate that the consistent immunodominance patterns of HIV-1-specific CTL responses of Chinese HIV-1 infected individuals and an inverse correlation between the relative contribution of responses restricted by HLA alleles and viral load, which indicates the important protective effect of optimal epitopes against slow disease progression even in advanced infection.

Cross-Recognition of Avian H5N1 Influenza Virus by Human Cytotoxic T-Lymphocyte Populations Directed to Human Influenza A Virus

Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there is considerable interest in immune correlates of protection against these viruses, which could be a target for the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1 analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2 influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive T-cell immunity in the human population may dampen the impact of a next pandemic.

Acute Resolving Woodchuck Hepatitis Virus (WHV) Infection Is Associated with a Strong Cytotoxic T-Lymphocyte Response to a Single WHV Core Peptide

Woodchucks infected with woodchuck hepatitis virus (WHV) are an excellent model for studying acute, self-limited and chronic hepadnaviral infections. Defects in the immunological response leading to chronicity are still unknown. Specific T-helper cell responses to WHV core and surface antigens (WHcAg and WHsAg, respectively) are associated with acute resolving infection; however, they are undetectable in chronic infection. Up to now, cytotoxic T-lymphocyte (CTL) responses could not be determined in the woodchuck. In the present study, we detected virus-specific CTL responses by a CD107a degranulation assay. The splenocytes of woodchucks in the postacute phase of WHV infection (18 months postinfection) were isolated and stimulated with overlapping peptides covering the whole WHcAg. After 6 days, the cells were restimulated and stained for CD3 and CD107a. One peptide (c96-110) turned out to be accountable for T-cell expansion and CD107a staining. Later, we applied the optimized degranulation assay to study the kinetics of the T-cell response in acute WHV infection. We found a vigorous T-cell response against peptide c96-110 with peripheral blood cells beginning at the peak of viral load (week 5) and lasting up to 15 weeks postinfection. In contrast, there was no T-cell response against peptide c96-110 detectable in chronically WHV-infected animals. Thus, with this newly established flow cytometric degranulation assay, we detected for the first time virus-specific CTLs and determined one immunodominant epitope of WHcAg in the woodchuck.

Differential Outcome of Tolerance Induction in Naive versus Activated Theiler's Virus Epitope-Specific CD8+Cytotoxic T Cells

Tolerance induced by the intravenous injection of peptide-pulsed, ethylene carbodiimide (ECDI)-fixed splenic antigen-presenting cells (Ag-SP) is a safe and effective method of inducing specific unresponsiveness in CD4+ T cells for the prevention and treatment of a variety of autoimmune diseases. We determined whether Ag-SP tolerance could also be used to tolerize CD8+ T cells. We show in the Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease model of multiple sclerosis that CD8+ T cells specific for both dominant and subdominant epitopes can be rendered tolerant. Interestingly, although virus clearance was delayed, lack of the virus-specific cytotoxic T-lymphocyte response did not result in the conversion of normally TMEV-resistant C57BL/6 mice to a susceptible phenotype. Importantly, we found that Ag-SP tolerance may not be a practical treatment for human diseases in which CD8+ T cells play a major role in pathogenesis, as tolerance induction in mice previously infected with TMEV led to a severe, often fatal reaction.

The loss of immunodominant epitopes affects interferon-γ production and lytic activity of the human influenza virus-specific cytotoxic T lymphocyte responsein vitro

In the present study, we examined the effect of the loss of the human leucocyte antigen (HLA)-B*3501-restricted nucleoprotein (NP)(418-426) epitope on interferon (IFN)-gamma-production and lytic activity of the human cytotoxic T lymphocyte (CTL) response in vitro. Extensive amino acid variation at T cell receptor contact residues of the NP(418-426) epitope has led to repeated evasion from specific CTL. We generated recombinant influenza viruses with variants of the NP(418-426) epitope, which were used to stimulate peripheral blood mononuclear cells obtained from six HLA-B*3501-positive study subjects in order to expand virus-specific CTL. Loss of the NP(418-426) epitope resulted in a significant reduction of IFN-gamma-expressing CD8+ T cells, similar to that observed previously after the loss of the HLA-B*2705-restricted NP(383-391) epitope. In addition, the effect of the loss of the NP(418-426) epitope on the lytic activity of the virus-specific CTL response was assessed. Also this functional property of the virus-specific CTL response was affected significantly by the loss of this and the NP(383-391) epitope, as determined using the newly developed fluorescent antigen-transfected target cell (FATT)-CTL assay. These findings indicate that the loss of single immunodominant epitopes affects the functionality of the virus-specific CTL response significantly.

Assessment of the extent of variation in influenza A virus cytotoxic T-lymphocyte epitopes by using virus-specific CD8+ T-cell clones

The influenza A virus nucleoprotein (NP) and matrix protein are major targets for human virus-specific cytotoxic T-lymphocyte (CTL) responses. Most of the CTL epitopes that have been identified so far are conserved. However, sequence variation in CTL epitopes of the NP has recently been demonstrated to be associated with escape from virus-specific CTLs. To assess the extent of variation in CTL epitopes during influenza A virus evolution, 304 CTL clones derived from six study subjects were obtained with specificity for an influenza A/H3N2 virus isolated in 1981. Subsequently, the frequency of the CTL clones that failed to recognize a more recent influenza virus strain isolated in 2003 was determined. In four of six study subjects, CTLs were found to be specific for variable epitopes, accounting for 2.6 % of all CTL clones. For some of these CTL clones, the minimal epitope and the residues responsible for abrogation of T-cell recognition were identified.