Human Cytotoxic T-Lymphocyte Repertoire to Influenza A Viruses

The murine CD8⁺ cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8⁺ CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4⁺ cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.     

Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2K^d-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8⁺ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.     

A mutation in the HLA-B*2705-restricted NP383-391 epitope affects the human influenza A virus-specific cytotoxic T-lymphocyte response in vitro

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in or adjacent to CTL epitopes. Recently, an amino acid substitution (R384G) in an HLA-B^*2705-restricted CTL epitope in the influenza A virus nucleoprotein (nucleoprotein containing residues 383 to 391 [NP_383-391]; SRYWAIRTR, where R is the residue that was mutated) was associated with escape from CTL-mediated immunity. The effect of this mutation on the in vitro influenza A virus-specific CTL response was studied. To this end, two influenza A viruses, one with and one without the NP_383-391 epitope, were constructed by reverse genetics and designated influenza viruses A/NL/94-384R and A/NL/94-384G, respectively. The absence of the HLA-B^*2705-restricted CTL epitope in influenza virus A/NL/94-384G was confirmed by using ⁵¹Cr release assays with a T-cell clone specific for the NP_383-391 epitope. In addition, peripheral blood mononuclear cells (PBMC) stimulated with influenza virus A/NL/94-384G failed to recognize HLA-B^*2705-positive target cells pulsed with the original NP_383-391 peptide. The proportion of virus-specific CD8⁺ gamma interferon (IFN-γ)-positive T cells in in vitro-stimulated PBMC was determined by intracellular IFN-γ staining after restimulation with virus-infected autologous B-lymphoblastoid cell lines and C1R cell lines expressing only HLA-B^*2705. The proportion of virus-specific CD8⁺ T cells was lower in PBMC stimulated in vitro with influenza virus A/NL/94-384G obtained from several HLA-B^*2705-positive donors than in PBMC stimulated with influenza virus A/NL/94-384R. This finding indicated that amino acid variations in CTL epitopes can affect the virus-specific CTL response and that the NP_383-391 epitope is the most important HLA-B^*2705-restricted epitope in the nucleoprotein of influenza A viruses.     

Establishment and Characterization of Japanese Encephalitis Virus-Specific, Human CD4+ T-Cell Clones: Flavivirus Cross-Reactivity, Protein Recognition, and Cytotoxic Activity

We analyzed the CD4⁺ T-lymphocyte responses of two donors who had received Japanese encephalitis virus (JEV) vaccine 6 or 12 months earlier. Bulk culture proliferation assays showed that peripheral blood mononuclear cells (PBMC) responded to JEV antigens (Ag) but also responded at lower levels to West Nile virus (WNV) and dengue virus type 1, 2, and 4 (D1V, D2V, and D4V, respectively) Ag. Five JEV-specific CD4⁺ human T-cell clones and one subclone were established from PBMC of these two donors. Two clones responded to WNV Ag as well as to JEV Ag, whereas the others responded only to JEV Ag. Three of five CD4⁺ T-cell clones had JEV-specific cytotoxic activity and recognized E protein. The HLA restriction of the JEV-specific T-cell clones was examined. Three clones were HLA-DR4 restricted, one was HLA-DQ3 restricted, and the HLA restriction of one clone was not determined. T-cell receptor analysis showed that these clones expressed different T-cell receptors, suggesting that they originated from different T lymphocytes. These results indicate that JEV vaccine induces JEV-specific and flavivirus-cross-reactive CD4⁺ T lymphocytes and that these T lymphocytes recognize E protein. The functions and HLA restriction patterns of these T lymphocytes are, however, heterogeneous.     

Differential Regulation of Effector- and Central-Memory Responses to Toxoplasma gondii Infection by IL-12 Revealed by Tracking of Tgd057-Specific CD8+ T Cells

Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-γ-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2K^b-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-γ production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-γ recall responses. Importantly, isolated CD62L^hi KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-γ-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses.     

Transduction of Human T Cells with a Novel T-Cell Receptor Confers Anti-HCV Reactivity

Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world''s population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073–1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4⁺ and CD8⁺ T cells recognized the HCV NS3:1073–1081 peptide-loaded targets and HCV⁺ hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-γ, IL-2, and TNF-α) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8⁻ Jurkat cells and CD4⁺ PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.     

Preferential CTL targeting of Gag is associated with relative viral control in long-term surviving HIV-1 infected former plasma donors from China

It is generally believed that CD8⁺ cytotoxic T lymphocytes (CTLs) play a critical role in limiting the replication of human immunodeficiency virus type 1 (HIV-1) and in determining the outcome of the infection, and this effect may partly depend on which HIV product is preferentially targeted. To address the correlation between HIV-1-specific CTL responses and virus replication in a cohort of former plasma donors (FPDs), 143 antiretroviral therapy naive FPDs infected with HIV-1 clade B'' strains were assessed for HIV-1-specific CTL responses with an IFN-γ Elispot assay at single peptide level by using overlapping peptides (OLPs) covering the whole consensus clade B proteome. By using a Spearman''s rank correlation analysis, we found that the proportion of Gag-specific CTL responses among the total virus-specific CTL activity was inversely correlated with viral loads while being positively correlated to CD4 counts, as opposed to Pol- and Env-specific responses that were associated with increased viral loads and decreased CD4 counts. In addition, Vpr-specifc CTL responses showed a similar protective effect with Gag responses, but with a much lower frequency of recognition. Significantly, we also observed an association between HLA-A^*30/B^*13/Cw^*06 haplotype and lower viral loads that was probably due to restricted Gag-specific CTL responses. Thus, our data demonstrate the prominent role of Gag-specific CTL responses in disease control. The advantage of HLA-A^*30/B^*13/Cw^*06 haplotype in viral control may be associated with the contribution of Gag-specific CTL responses in the studied individuals.     

A Mechanism To Explain the Selection of the Hepatitis e Antigen-Negative Mutant during Chronic Hepatitis B Virus Infection

Hepatitis B virus (HBV) expresses two structural forms of the nucleoprotein, the intracellular nucleocapsid (hepatitis core antigen [HBcAg]) and the secreted nonparticulate form (hepatitis e antigen [HBeAg]). The aim of this study was to evaluate the ability of HBcAg- and HBeAg-specific genetic immunogens to induce HBc/HBeAg-specific CD4⁺/CD8⁺ T-cell immune responses and the potential to induce liver injury in HBV-transgenic (Tg) mice. Both the HBcAg- and HBeAg-specific plasmids primed comparable immune responses. Both CD4⁺ and CD8⁺ T cells were important for priming/effector functions of HBc/HBeAg-specific cytotoxic T-lymphocyte (CTL) responses. However, a unique two-step immunization protocol was necessary to elicit maximal CTL priming. Genetic vaccination did not prime CTLs in HBe- or HBc/HBeAg-dbl-Tg mice but elicited a weak CTL response in HBcAg-Tg mice. When HBc/HBeAg-specific CTLs were adoptively transferred into HBc-, HBe-, and HBc/HBeAg-dbl-Tg mice, the durations of the liver injury and inflammation were significantly greater in HBeAg-Tg recipient mice than in HBcAg-Tg mice. Importantly, liver injury in HBc/HBeAg-dbl-Tg mice was similar to the injury observed in HBeAg-Tg mice. Loss of HBeAg synthesis commonly occurs during chronic HBV infection; however, the mechanism of selection of HBeAg-negative variants is unknown. The finding that hepatocytes expressing wild-type HBV (containing both HBcAg and HBeAg) are more susceptible to CTL-mediated clearance than hepatocytes expressing only HBcAg suggest that the HBeAg-negative variant may have a selective advantage over wild-type HBV within the livers of patients with chronic infection during an immune response and may represent a CTL escape mutant.     

Different abilities of escape mutant-specific cytotoxic T cells to suppress replication of escape mutant and wild-type human immunodeficiency virus type 1 in new hosts

There is much evidence that in human immunodeficiency virus type 1 (HIV-1)-infected individuals, strong cytotoxic T lymphocyte (CTL)-mediated immune pressure results in the selection of HIV-1 mutants that have escaped from wild-type-specific CTLs. If escape mutant-specific CTLs are not elicited in new hosts sharing donor HLA molecules, the transmission of these mutants results in the accumulation of escape mutants in the population. However, whether escape mutant-specific CTLs are definitively not elicited in new hosts sharing donor HLA molecules still remains unclear. A previous study showed that a Y-to-F substitution at the second position (2F) of the Nef138-10 epitope is significantly detected in HLA-A*2402⁺ hemophilic donors. Presently, we confirmed that this 2F mutant was an escape mutant by demonstrating strong and weak abilities of Nef138-10-specific CTL clones to suppress replication of the wild-type and 2F mutant viruses, respectively. We demonstrated the existence of the 2F-specific CTLs in three new hosts who had been primarily infected with the 2F mutant. The 2F-specific CTL clones suppressed the replication of both wild-type and mutant viruses. However, the abilities of these clones to suppress replication of the 2F virus were much weaker than those of wild-type-specific and the 2F-specific ones to suppress replication of the wild-type virus. These findings indicate that the 2F mutant is conserved in HIV-1-infected donors having HLA-A*2402, because the 2F-specific CTLs failed to completely suppress the 2F mutant replication and effectively prevented viral reversion in new hosts carrying HLA-A*2402.     

Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5

Broad, multispecific CD4⁺ and CD8⁺ T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8⁺ T-cell responses but low CD4⁺ T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4⁺ T helper responses but no CD8⁺ T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4⁺ T helper responses but no CD8⁺ T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4⁺ and CD8⁺ T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.     

Asymptomatic human CD4+ cytotoxic T-cell epitopes identified from herpes simplex virus glycoprotein B

The identification of “asymptomatic” (i.e., protective) epitopes recognized by T cells from herpes simplex virus (HSV)-seropositive healthy individuals is a prerequisite for an effective vaccine. Using the PepScan epitope mapping strategy, a library of 179 potential peptide epitopes (15-mers overlapping by 10 amino acids) was identified from HSV type 1 (HSV-1) glycoprotein B (gB), an antigen that induces protective immunity in both animal models and humans. Eighteen groups (G1 to G18) of 10 adjacent peptides each were first screened for T-cell antigenicity in 38 HSV-1-seropositive but HSV-2-seronegative individuals. Individual peptides within the two immunodominant groups (i.e., G4 and G14) were further screened with T cells from HLA-DR-genotyped and clinically defined symptomatic (n = 10) and asymptomatic (n = 10) HSV-1-seropositive healthy individuals. Peptides gB_161-175 and gB_166-180 within G4 and gB_661-675 within G14 recalled the strongest HLA-DR-dependent CD4⁺ T-cell proliferation and gamma interferon production. gB_166-180, gB_661-675, and gB_666-680 elicited ex vivo CD4⁺ cytotoxic T cells (CTLs) that lysed autologous HSV-1- and vaccinia virus (expressing gB)-infected lymphoblastoid cell lines. Interestingly, gB_166-180 and gB_666-680 peptide epitopes were strongly recognized by CD4⁺ T cells from 10 of 10 asymptomatic patients but not by CD4⁺ T cells from 10 of 10 symptomatic patients (P < 0.0001; analysis of variance posttest). Inversely, CD4⁺ T cells from symptomatic patients preferentially recognized gB_661-675 (P < 0.0001). Thus, we identified three previously unrecognized CD4⁺ CTL peptide epitopes in HSV-1 gB. Among these, gB_166-180 and gB_666-680 appear to be “asymptomatic” peptide epitopes and therefore should be considered in the design of future herpes vaccines.     

Strong Ability of Nef-Specific CD4+ Cytotoxic T Cells To Suppress Human Immunodeficiency Virus Type 1 (HIV-1) Replication in HIV-1-Infected CD4+ T Cells and Macrophages

A restricted number of studies have shown that human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic CD4⁺ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4⁺ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4⁺ T cells. The CD4⁺ T-cell clones specific for Nef187-203 showed strong gamma interferon production after having been stimulated with autologous B-lymphoblastoid cells infected with recombinant vaccinia virus expressing Nef or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous major histocompatibility complex class II processing pathways. Nef187-203-specific CD4⁺ T-cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4⁺ T cells from an HLA-DRB1*0803⁺ donor. In addition, these Nef-specific cytotoxic CD4⁺ T-cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and CD4⁺ T cells in vitro. Nef187-203-specific cytotoxic CD4⁺ T cells were detected in cultures of peptide-stimulated peripheral blood mononuclear cells (PBMCs) and in ex vivo PBMCs from 40% and 20% of DRB1*0803⁺ donors, respectively. These results suggest that HIV-1-specific CD4⁺ T cells may directly control HIV-1 infection in vivo by suppressing virus replication in HIV-1 natural host cells.Human immunodeficiency virus (HIV)-specific CD8⁺ cytotoxic T cells (CTLs) play a central role in the control of HIV type 1 (HIV-1) during acute and chronic phases of an HIV-1 infection (5, 29, 34). However, HIV-1 escapes from the immune surveillance of CD8⁺ CTLs by mechanisms such as mutations of immunodominant CTL epitopes and downregulation of major histocompatibility complex class I (MHC-I) molecules on the infected cells (9, 11, 12, 49). Therefore, most HIV-1-infected patients without highly active antiretroviral therapy (HAART) develop AIDS eventually.HIV-1-specific CD4⁺ T cells also play an important role in host immune responses against HIV-1 infections. An inverse association of CD4⁺ T-cell responses with viral load in chronically HIV-1-infected patients was documented in a series of earlier studies (8, 36, 39, 41, 48), although the causal relationship between them still remains unclear (23). Classically, CD4⁺ T cells help the expansion of CD8⁺ CTLs by producing growth factors such as interleukin-2 (IL-2) or by their CD40 ligand interaction with antigen-processing cells and CD8⁺ CTLs. In addition, CD4⁺ T cells provide activation of macrophages, which can professionally maintain CD8⁺ T-cell memory (17). On the other hand, the direct ability of virus-specific cytotoxic CD4⁺ T cells (CD4⁺ CTLs) to kill target cells has been widely observed in human virus infections such as those by human cytomegalovirus, Epstein-Barr virus (EBV), hepatitis B virus, Dengue virus, and HIV-1 (2, 4, 10, 19, 30, 31, 38, 50). Furthermore, one study showed that mouse CD4⁺ T cells specific for lymphocytic choriomeningitis virus have cytotoxic activity in vivo (25). These results, taken together, indicate that a subset of effector CD4⁺ T cells develops cytolytic activity in response to virus infections.HIV-1-specific CD4⁺ CTLs were found to be prevalent in HIV-1 infections, as Gag-specific cytotoxic CD4⁺ T cells were detected directly ex vivo among peripheral blood mononuclear cells (PBMCs) from an HIV-1-infected long-term nonprogressor (31). Other studies showed that up to 50% of the CD4⁺ T cells in some HIV-1-infected donors can exhibit a clear cytolytic potential, in contrast to the fact that healthy individuals display few of these cells (3, 4). These studies indicate the real existence of CD4⁺ CTLs in HIV-1 infections.The roles of CD4⁺ CTLs in the control of an HIV-1 infection have not been widely explored. It is known that Gag-specific CD4⁺ CTLs can suppress HIV-1 replication in a human T-cell leukemia virus type 1-immortalized CD4⁺ T-cell line (31). However, the functions of CD4⁺ T cells specific for other HIV-1 antigens remain unclear. On the other hand, the abilities of CD4⁺ CTLs to suppress HIV-1 replication in infected macrophages and CD4⁺ T cells may be different, as in the case of CD8⁺ CTLs for HIV-1-infected macrophages (17). In this study, we identified Nef-specific CD4⁺ T cells and investigated their ability to kill HIV-1 R5 virus-infected macrophages and HIV-1 X4 virus-infected CD4⁺ T cells and to suppress HIV-1 replication in the infected macrophages and CD4⁺ T cells. The results obtained in the present study show for the first time the ability of HIV-1-specific CD4⁺ CTLs to suppress HIV-1 replication in natural host cells, i.e., macrophages and CD4⁺ T cells.     

CD40 Ligand-Mediated Interactions Are Involved in the Generation of Memory CD8+ Cytotoxic T Lymphocytes (CTL) but Are Not Required for the Maintenance of CTL Memory following Virus Infection

CD8⁺ cytotoxic T lymphocytes (CTL) play a key role in the control of many virus infections, and the need for vaccines to elicit strong CD8⁺ T-cell responses in order to provide optimal protection in such infections is increasingly apparent. However, the mechanisms involved in the induction and maintenance of CD8⁺ CTL memory are currently poorly understood. In this study, we investigated the involvement of CD40 ligand (CD40L)-mediated interactions in these processes by analyzing the memory CTL response of CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV). The maintenance of memory CD8⁺ CTL precursors (CTLp) at stable frequencies over time was not impaired in CD40L-deficient mice. By contrast, the initial generation of memory CTLp was affected. CD40L-deficient mice produced lower levels of CD8⁺ CTLp during the primary immune response to LCMV than did wild-type controls, despite the fact that the LCMV-specific effector CTL response of CD40L-deficient mice was indistinguishable from that of control animals. The differentiation of naïve CD8⁺ T cells into effector and memory CTL thus involves pathways that can be discriminated from each other by their requirement for CD40L-mediated interactions. Expression of CD40L by CTLp themselves was not an essential step during their expansion and differentiation from naïve CD8⁺ cells into memory CTLp; instead, the reduction in memory CTLp generation in CD40L-deficient mice was likely a consequence of defects in the CD4⁺ T-cell response mounted by these animals. These results thus suggest a previously unappreciated role for CD40L in the generation of CD8⁺ memory CTLp, the probable nature of which is discussed.     

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

Background

The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.

Methodology/Principal Findings

We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor γ-chain knockout (NOD-scid IL2rγ^null) mice engrafted with human hematopoietic stem cells. Human CD45⁺ cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rγ^null mice with HLA-A2⁺ human cord blood hematopoietic stem cells, were able to secrete IFN-γ, IL-2 and TNF-α in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353_(111–119), NS4b 2423_(181–189), and NS4a 2148_(56–64).

Conclusions/Significance

This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections.     

CD4+ Cytotoxic T-Lymphocyte Activity against Macrophages Pulsed with Bovine Herpesvirus 1 Polypeptides

Bovine herpesvirus 1 (BHV-1) induces immune suppression, but the mechanisms for suppression are not well identified. We examined the induction and activity of BHV-1-specific cytolytic CD4⁺ T lymphocytes (CTL) by stimulating peripheral blood mononuclear cells (PBMC) of cattle immunized with attenuated live BHV-1. Cytolytic effector cells were primarily CD4⁺ T lymphocytes and lysed autologous, but not allogeneic, macrophages infected with BHV-1 or pulsed with BHV-1 polypeptides. Apoptosis of BHV-1-expressing target cells was observed in CD4⁺ CTL assays by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. To determine if apoptosis was mediated by a perforin- or Fas-mediated pathway, EGTA, a known selective inhibitor of the perforin pathway, was used. EGTA did not inhibit CD4⁺-T-cell-mediated cytotoxic activity, but it did limit the NK cell cytotoxicity of virus infected cells. These findings support the concept that CD4⁺ CTL lyse macrophages pulsed with BHV-1 polypeptides through a Fas-mediated lytic pathway by inducing apoptosis in the target cells. The prominent cytotoxicity mediated by CD4⁺ CTL suggests a mechanism of selective removal of viral antigen-associated antigen-presenting cells.     

Alphavirus-Specific Cytotoxic T Lymphocytes Recognize a Cross-Reactive Epitope from the Capsid Protein and Can Eliminate Virus from Persistently Infected Macrophages

Persistent alphavirus infections in synovial and neural tissues are believed to be associated with chronic arthritis and encephalitis, respectively, and represent likely targets for CD8⁺ αβ cytotoxic T lymphocytes (CTL). Here we show that the capsid protein is a dominant target for alphavirus-specific CTL in BALB/c mice and that capsid-specific CTL from these mice recognize an H-2K^d restricted epitope, QYSGGRFTI. This epitope lies in the highly conserved region of the capsid protein, and QYSGGRFTI-specific CTL were cross reactive across a range of Old World alphaviruses. In vivo the acute primary viraemia of these highly cytopathic viruses was unaffected by QYSGGRFTI-specific CTL. However, in vitro these CTL were able to completely clear virus from macrophages persistently and productively infected with the arthrogenic alphavirus Ross River virus.     

Self-Organized Criticality Theory of Autoimmunity

Background

The cause of autoimmunity, which is unknown, is investigated from a different angle, i.e., the defect in immune ‘system’, to explain the cause of autoimmunity.

Methodology/Principal Findings

Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not prone to spontaneous autoimmune diseases. Overstimulation of CD4⁺ T cells led to the development of autoantibody-inducing CD4⁺ T (aiCD4⁺ T) cell which had undergone T cell receptor (TCR) revision and was capable of inducing autoantibodies. The aiCD4⁺ T cell was induced by de novo TCR revision but not by cross-reaction, and subsequently overstimulated CD8⁺ T cells, driving them to become antigen-specific cytotoxic T lymphocytes (CTL). These CTLs could be further matured by antigen cross-presentation, after which they caused autoimmune tissue injury akin to systemic lupus erythematosus (SLE).

Conclusions/Significance

Systemic autoimmunity appears to be the inevitable consequence of over-stimulating the host''s immune ‘system’ by repeated immunization with antigen, to the levels that surpass system''s self-organized criticality.     

Targeting a Polyepitope Protein Incorporating Multiple Class II-Restricted Viral Epitopes to the Secretory/Endocytic Pathway Facilitates Immune Recognition by CD4+ Cytotoxic T Lymphocytes: a Novel Approach to Vaccine Design

The role of CD4⁺ and CD8⁺ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4⁺- and CD8⁺-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8⁺- or CD4⁺-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4⁺-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4⁺ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.There is now increasing evidence to suggest that both CD4⁺ and CD8⁺ T cells are critical for the generation of an effective immune response against intracellular pathogens. Although both CD4⁺ and CD8⁺ T cells recognize nonnative forms of the antigen in association with major histocompatibility complex (MHC) molecules, the presentation of antigen to these two types of T lymphocytes occurs through distinct pathways (24). In fact, the disparity in antigen presentation to these T cells is not due to processing differences but rather reflects the differences in the capacities of class I and class II molecules to bind antigenic determinants in an intracellular compartment. Indeed, earlier studies have shown that for processing and interaction with MHC class II molecules, antigen expressed de novo needs to be targeted to an endosomal or lysosomal compartment (5). There are two major pathways by which antigens are targeted to these compartments. The traditional pathway involves the phagocytosis or endocytosis of exogenous antigens, followed by degradation by acid proteases in the endosomal or lysosomal compartments (3, 8, 26, 41). On the other hand, class II-restricted presentation of endogenously synthesized proteins mainly involves membrane antigens which are thought to enter the endosomal or lysosomal pathway by internalization from the cell surface (11). Although, in certain experimental systems, cytoplasmic and nuclear proteins may also enter this endogenous pathway, generally these proteins are targets for the class I processing pathway (9, 14, 20, 27).One of the major limitations of the epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs (2, 35, 38). Based on these observations, we reasoned that a molecular approach that directly routes these epitopes into the MHC class II pathway, such as the endocytic or lysosomal compartments, might facilitate endogenous presentation to CD4⁺ T cells. The lysosome-associated membrane protein (LAMP-1) and the invariant chain (Ii) are transmembrane proteins which are localized predominantly in the lysosomes and endosomes, respectively. The cytoplasmic domains of these proteins contain specific targeting signals that mediate their translocation to the specific compartments. We therefore designed a chimeric polyepitope construct capable of encoding multiple class II-restricted CTL epitopes from Epstein-Barr virus (EBV) and influenza virus linked to the cytoplasmic and/or transmembrane domains of LAMP-1 and the Ii protein, with the aim of targeting the epitopes to the endosomal and lysosomal compartments. The data presented in this study clearly demonstrate that if the endogenously synthesized polyepitope protein is targeted to the endocytic/secretory pathway, processing and presentation of all the epitopes are dramatically enhanced. More importantly, minimal epitope sequences, without any natural flanking sequences, were adequate for efficient stimulation of the virus-specific memory CTL response, a result that has important implications for epitope-based vaccine design.     

Intestinal Intraepithelial Lymphocytes Are Primed for Gamma Interferon and MIP-1β Expression and Display Antiviral Cytotoxic Activity despite Severe CD4+ T-Cell Depletion in Primary Simian Immunodeficiency Virus Infection

Intraepithelial lymphocytes (IEL) are a critical effector component of the gut-associated lymphoid tissue (GALT) and play an important role in mucosal immunity as well as in the maintenance of the epithelial cell integrity and barrier function. The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of rhesus macaques would cause alterations in the immunophenotypic profiles of IEL and their mitogen-specific cytokine (gamma interferon [IFN-γ] and MIP-1β) responses (by flow cytometry) and virus-specific cytotoxic T-cell (CTL) activity (by the chromium release assay). Virally infected IEL were detected through the entire course of SIV infection by in situ hybridization. Severe depletion of CD4⁺ single-positive and CD4⁺CD8⁺ double-positive T cells occurred early in primary SIV infection, which was coincident with an increased prevalence of CD8⁺ T cells. This was in contrast to a gradual depletion of CD4⁺ T cells in peripheral blood. The CD8⁺ IEL were the primary producers of IFN-γ and MIP-1β and were found to retain their potential to produce both IFN-γ and MIP-1β through the entire course of SIV infection. SIV-specific CTL activity was detected in primary IEL at 1, 2, and 4 weeks post-SIV infection. These results demonstrated that IEL may be involved in generating antiviral immune responses early in SIV infection and in suppressing viral infection thereafter. Alterations in homeostasis in epithelia due to severe CD4⁺ T-cell depletion accompanied by changes in the cytokine and chemokine production by IEL may play a role in the enteropathogenesis of SIV infection.