Distinct Differences in the Expansion and Phenotype of TB10.4 Specific CD8 and CD4 T Cells after Infection with Mycobacterium tuberculosis

Background

Recently we and others have identified CD8 and CD4 T cell epitopes within the highly expressed M. tuberculosis protein TB10.4. This has enabled, for the first time, a comparative study of the dynamics and function of CD4 and CD8 T cells specific for epitopes within the same protein in various stages of TB infection.

Methods and Findings

We focused on T cells directed to two epitopes in TB10.4; the MHC class I restricted epitope TB10.4 _3–11 (CD8/10.4 T cells) and the MHC class II restricted epitope TB10.4 _74–88 (CD4/10.4 T cells). CD4/10.4 and CD8/10.4 T cells displayed marked differences in terms of expansion and contraction in a mouse TB model. CD4/10.4 T cells dominated in the early phase of infection whereas CD8/10.4 T cells were expanded after week 16 and reached 5–8 fold higher numbers in the late phase of infection. In the early phase of infection both CD4/10.4 and CD8/10.4 T cells were characterized by 20–25% polyfunctional cells (IL-2⁺, IFN-γ⁺, TNF-α⁺), but whereas the majority of CD4/10.4 T cells were maintained as polyfunctional T cells throughout infection, CD8/10.4 T cells differentiated almost exclusively into effector cells (IFN-γ⁺, TNF-α⁺). Both CD4/10.4 and CD8/10.4 T cells exhibited cytotoxicity in vivo in the early phase of infection, but whereas CD4/10.4 cell mediated cytotoxicity waned during the infection, CD8/10.4 T cells exhibited increasing cytotoxic potential throughout the infection.

Conclusions/Significance

Our results show that CD4 and CD8 T cells directed to epitopes in the same antigen differ both in their kinetics and functional characteristics throughout an infection with M. tuberculosis. In addition, the observed strong expansion of CD8 T cells in the late stages of infection could have implications for the development of post exposure vaccines against latent TB.     

Immunoprevalence and Immunodominance of HLA-Cw?0801-Restricted T Cell Response Targeting the Hepatitis B Virus Envelope Transmembrane Region

HLA-C-restricted T cells have been shown to play an important role in HIV control, but their impact on protection or pathogenesis in other viral infections remains elusive. Here, we characterized the hierarchy of HLA class I-restricted hepatitis B virus (HBV) epitopes targeted by CD8 T cells in HBV-infected subjects. The frequency of CD8 T cells specific for a panel of 18 HBV epitopes (restricted by HLA-A∗0201/03/07 [hereinafter HLA-A0201/03/07], -A1101, -A2402/07, -B5801, -B4001, -B1301, and -Cw0801) was quantified in a total of 59 subjects who resolved HBV infection. We found that the HLA-Cw0801-restricted epitope comprised of Env residues 171 to 180 (Env_171–180) is immunoprevalent in the Southeast Asian subjects (10/17 HLA-Cw0801-positive subjects) and immunodominant in the majority of HLA-Cw0801-positive subjects able to control HBV infection. HLA-Cw0801-restricted Env_171–180-specific CD8 T cells recognized endogenously produced HBV surface antigen (HBsAg) and tolerated amino acid variations within the epitope detected in HBV genotypes B and C. In conclusion, we demonstrate that the HLA-Cw0801-restricted Env_171–180 T cell response is an important component of the HBV-specific adaptive T cell immunity in Asians infected with HBV. Thus, HLA-C restricted T cells might play an important role in various viral infections.     

Expansion of human cytomegalovirus (HCMV) immediate-early 1-specific CD8+ T cells and control of HCMV replication after allogeneic stem cell transplantation

Recovery of human cytomegalovirus (HCMV)-specific T immunity is critical for protection against HCMV disease in the early phase after allogeneic stem cell transplantation (SCT). Using an enzyme-linked immunospot assay with overlapping 15-mer peptides spanning pp65 and immediate-early 1 HCMV proteins, we investigated which HCMV-specific CD8⁺ gamma interferon-positive (IFN-γ⁺) T-cell responses against pp65 and IE-1 were associated with control of HCMV replication in 48 recipients of unmanipulated HLA-matched allografts at 3 months (M3) and 6 months (M6) after SCT and in 23 donors. At M3 after SCT, the magnitude of the pp65-specific IFN-γ-producing CD8⁺ T-cell response was greater in recipients than in donors, regardless of HCMV status. In contrast, expansion of IE-1-specific CD8⁺ T cells at M3 was associated with protection against HCMV, and no patient with this expansion had HCMV replication at M3. At M6, the number of HCMV-specific CD8⁺ T cells against both pp65 and IE-1 had expanded in all recipients, regardless of their previous levels of HCMV replication. The recipients' HCMV-specific CD8⁺ T cells already detectable in related donors were predominantly targeting pp65. In contrast, in 40% of the cases, the HCMV-specific CD8⁺ T cells in recipients involved new CD8⁺ T-cell specificities undetectable in their related donors and preferentially targeting IE-1. Taken together, these results showed that the delay in reconstituting IE-1-specific CD8⁺ T cells is correlated with the lack of protection against HCMV in the first 3 months after SCT. They also show that IE-1 is a major antigenic determinant of the early restoration of protective immunity to HCMV after SCT.     

CD45RB Is a Novel Molecular Therapeutic Target to Inhibit Aβ Peptide-Induced Microglial MAPK Activation

Background

Microglial activation, characterized by p38 MAPK or p44/42 MAPK pathway signal transduction, occurs in Alzheimer''s disease (AD). Our previous studies demonstrated CD45, a membrane-bound protein tyrosine phosphatase (PTP), opposed β-amyloid (Aβ) peptide-induced microglial activation via inhibition of p44/42 MAPK. Additionally we have shown agonism of the RB isoform of CD45 (CD45RB) abrogates lipopolysaccharide (LPS)-induced microglial activation.

Methodology and Results

In this study, CD45RB modulation of Aβ peptide or LPS-activated primary cultured microglial cells was further investigated. Microglial cells were co-treated with “aged” FITC-Aβ_1–42 and multiple CD45 isoform agonist antibodies. Data revealed cross-linking of CD45, particularly the CD45RB isoform, enhances microglial phagocytosis of Aβ_1–42 peptide and inhibits LPS-induced activation of p44/42 and p38 pathways. Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-α and IL-6 release through p44/42 and/or p38 pathways. Moreover, inhibition of either of these pathways augmented CD45RB cross-linking induced microglial phagocytosis of Aβ_1–42 peptide. To investigate the mechanism(s) involved, microglial cells were co-treated with a PTP inhibitor (potassium bisperoxo [1,10-phenanthroline oxovanadate; Phen]) and Aβ_1–42 peptides. Data showed synergistic induction of microglial activation as evidenced by TNF-α and IL-6 release; both of which are demonstrated to be dependent on increased p44/42 and/or p38 activation. Finally, it was observed that cross-linking of CD45RB in the presence of Aβ_1–42 peptide, inhibits co-localization of microglial MHC class II and Aβ peptide; suggesting CD45 activation inhibits the antigen presenting phenotype of microglial cells.

Conclusion

In summary, p38 MAPK is another novel signaling pathway, besides p44/42, in which CD45RB cross-linking negatively regulates microglial Aβ phagocytosis while increasing potentially neurotoxic inflammation. Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Aβ lowering, and inflammation reducing, properties that are particularly targeted at microglial cells. Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.     

High CD8+ T Cell Activation Marks a Less Differentiated HIV-1 Specific CD8+ T Cell Response that Is Not Altered by Suppression of Viral Replication

Background

The relationship of elevated T cell activation to altered T cell differentiation profiles, each defining features of HIV-1 infection, has not been extensively explored. We hypothesized that anti-retroviral suppression of T cell activation levels would lead to alterations in the T cell differentiation of total and HIV-1 specific CD8+ T cell responses among recently HIV-1 infected adults.

Methodology/Principal Findings

We performed a longitudinal study simultaneously measuring T cell activation and maturation markers on both total and antigen-specific T cells in recently infected adults: prior to treatment; after the initiation of HAART; and after treatment was halted. Prior to treatment, HIV-1 Gag–specific CD8+ T cells were predominantly of a highly activated, intermediate memory (CD27+CD28−) phenotype, while CMV pp65-specific CD8+ T cells showed a late memory (CD27−CD28−), low activation phenotype. Participants with the highest fraction of late memory (CD27−CD28−) HIV-1-specific CD8+ T cells had higher CD4+ T cell counts (rho = +0.74, p = 0.004). In turn, those with the highest fraction of intermediate memory (CD27+ CD28−) HIV-1 specific CD8+ T cells had high total CD8+ T cell activation (rho = +0.68, p = 0.01), indicating poorer long-term clinical outcomes. The HIV-1 specific T cell differentiation profile was not readily altered by suppression of T cell activation following HAART treatment.

Conclusions/Significance

A more differentiated, less activated HIV-1 specific CD8+ T cell response may be clinically protective. Anti-retroviral treatment initiated two to four months after infection lowered T cell activation but had no effect on the differentiation profile of the HIV-1-specific response. Intervention during the first month of acute infection may be required to shift the differentiation phenotype of HIV-1 specific responses to a more clinically favorable profile.     

CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection

During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8⁺ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8⁺ T cells recognize the epitope YopE_69-77. The features of the interaction between pathogen and host that result in this large CD8⁺ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE_69-77-specific CD8⁺ T cells generated during the large response are polyclonal and are produced by a “translocation-dependent” pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE_69-77-specific CD8⁺ T cell response (~10% of the large expansion) can be generated in a “translocation-independent” pathway in which CD8α⁺ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE_69-77-specific CD8⁺ T cell expansion because this response was significantly reduced in Ccr2^-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE_69-77-specific CD8⁺ T cells ex vivo and promoted the expansion of YopE_69-77-specific CD8⁺ T cells in infected Ccr2^-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8⁺ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE_69-77-specific CD8⁺ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.     

Conservation and Diversity of Influenza A H1N1 HLA-Restricted T Cell Epitope Candidates for Epitope-Based Vaccines

Background

The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated.

Methodology/Principal Findings

HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007–2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes.

Conclusions/Significance

Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.     

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4⁺ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8⁺ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4⁺CD25^hiFOXP3⁺ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8⁺ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8⁺ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8⁺ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8⁺ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.     

Screening of human tumor antigens for CD4 T cell epitopes by combination of HLA-transgenic mice, recombinant adenovirus and antigen peptide libraries

Background

As tumor antigen-specific CD4⁺ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4⁺ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients.

Methods and Findings

To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4⁺ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4⁺ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4⁺ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4⁺ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-2_60–74 epitope and against the new epitope TRP-2_149–163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2_149–163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4⁺ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4⁺ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1_284–298 as a new HLA-DRB1*0301-restricted CD4⁺ T cell epitope.

Conclusions

Our screening approach identified new HLA-DRB1*0301-restricted CD4⁺ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives.     

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.     

Modification of One Epitope-Flanking Amino Acid Allows for the Induction of Friend Retrovirus-Specific CD8+ T Cells by Adenovirus-Based Immunization

While Friend retrovirus-infected mice readily mount a vigorous CD8⁺ T cell response to the leader-gag-derived peptide GagL_85–93, no GagL_85–93-specific T cells were detectable in mice immunized against Friend virus (FV) with viral vectors or DNA vaccines. By exchanging one epitope-flanking amino acid or using a scaffold protein we were able to demonstrate for the first time the induction of GagL_85–93-specific CD8⁺ T cells by genetic vaccination and show their high protective effect against FV challenge infection.     

Efficacious Early Antiviral Activity of HIV Gag- and Pol-Specific HLA-B*2705-Restricted CD8+ T Cells

The association between HLA-B*2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B*2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8⁺ T cells. In order to better define the mechanisms of the HLA-B*2705 immune control of HIV, we first characterized the CD8⁺ T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B*2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B*2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response was only marginally lower than that of the KK10-specific response (median, 695 versus 867 spot-forming cells [SFC]/million peripheral blood mononuclear cells [PBMCs]; not significant [NS]), and viral escape mutants were observed in both KY9 and KK10, resulting from selection pressure driven by the respective CD8⁺ T-cell response. By comparing inhibitions of viral replication by CD8⁺ T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B*2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early recognition of HIV-1-infected cells, within 6 h of infection, by KK10- and KY9-specific CD8⁺ T cells but not until 18 h postinfection by VL9-specific CD8⁺ T cells. There was no association between antiviral efficacy and proliferative capacity, cytotoxicity, polyfunctionality, or T-cell receptor (TCR) avidity. These data are consistent with previous studies indicating an important role for the B*2705-Gag KK10 response in the control of HIV but also suggest a previously unrecognized role played by the subdominant Pol-specific KY9 response in HLA-B*2705-mediated control of HIV and that the recognition of HIV-infected cells by CD8⁺ T cells early in the viral life cycle may be important for viral containment in HIV-infected individuals.Current human immunodeficiency virus (HIV) vaccine strategies are focused on emulating the protective effect observed for HIV-infected individuals carrying alleles such as B*2705 by inducing the virus-specific CD8⁺ T-cell responses that are thought to be responsible for delaying or preventing disease progression. Understanding why such alleles confer protection facilitates a rational approach to vaccine design. It has been hypothesized that the slow progression to AIDS exhibited by HLA-B*2705-positive (HLA-B*2705⁺) HIV-infected individuals is due to the immunodominant B*27-restricted CD8⁺ T-cell response toward the p24 Gag epitope KRWIILGLNK (KK10) (Gag residues 263 to 272). Escape from this epitope typically occurs late in infection and is associated with rapid progression to AIDS (14, 16). The commonly selected mutation R264K abrogates CD8⁺ T-cell recognition but also confers a substantial fitness cost to the virus, and the selection of compensatory mutations is required to restore viral replicative capacity (19, 29, 30). This has prompted the hypothesis that CD8⁺ T-cell responses that can drive escape mutations that reduce viral fitness are a contributing factor in the immune control of HIV, either by promoting the outgrowth of a viral quasispecies with a lower replicative capacity or by delaying the selection of escape mutations, both of which may slow the onset of AIDS (11, 21, 25).To better understand how CD8⁺ T cells can be most effective against HIV, recent studies have directly assessed the antiviral activity of CD8⁺ T cells via the viral suppression of HIV-infected CD4⁺ T cells during coculture. Such studies indicated that Gag-specific CD8⁺ T cells have a higher potency for viral suppression than Env-specific CD8⁺ T cells (10), supporting previous data indicating that broad CD8⁺ T-cell targeting of Gag epitopes was associated a with lower viral set point and, hence, slower progression to AIDS (20). A recent study of simian immunodeficiency virus (SIV) suggested that the protective effect of Gag-specific CD8⁺ T cells is mediated by the early presentation of Gag epitopes, processed from the viral Gag protein from incoming virions during infection, which can sensitize target cells for lysis by Gag-specific CD8⁺ T cells within 6 h of infection (26, 27). In addition, it was proposed previously that the ability of CD8⁺ T cells to secrete multiple cytokines may also be an important correlate of immune protection (6), and a further recent study demonstrated a more polyfunctional cytokine profile of Gag-specific B*2705-KK10 CD8⁺ T-cell responses than those of other HIV-specific CD8⁺ T-cell responses (1). The ability of CD8⁺ T cells to proliferate in response to the cognate epitope peptide has also been associated with immune control (1, 12). Other studies demonstrated the importance of lytic granule loading of CD8⁺ T cells for the effective elimination of HIV-infected cells (6, 22). However, the induction of a Gag KK10-specific CD8⁺ T-cell vaccine response in a B*2705-positive vaccinee did not protect against rapid progression following subsequent HIV-1 infection (5). This anecdotal case suggests the possibility that HLA-B*2705-associated immune control of HIV-1 may not be dependent on the Gag KK10-specific CD8⁺ T-cell response alone.Since current vaccine strategies hope to induce a protective effect, such as that observed for HLA-B*2705⁺ HIV-infected individuals, the study of the functional and phenotypic characteristics of B*2705-specific CD8⁺ T cells provides an opportunity to redefine the proposed correlates of immune protection essential for rational vaccine design. In this study we analyze three different specificities of HLA-B*2705-restricted CD8⁺ T cells from chronically HIV-infected individuals in order to directly compare antiviral activity with potential correlates of immune protection, including the kinetics of viral inhibition, cytokine profile, granzyme production, proliferative capacity, and cytotoxicity.     

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, “promiscuous” (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.     

Predominance of HLA-Restricted Cytotoxic T-Lymphocyte Responses to Serotype-Cross-Reactive Epitopes on Nonstructural Proteins following Natural Secondary Dengue Virus Infection

We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4⁺ and CD8⁺ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8⁺ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8⁺ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4⁺ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8⁺ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.     

Comparisons of CD8+ T Cells Specific for Human Immunodeficiency Virus,Hepatitis C Virus,and Cytomegalovirus Reveal Differences in Frequency,Immunodominance, Phenotype,and Interleukin-2 Responsiveness

To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8⁺ T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8⁺ T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP. In contrast to HIV-specific CD8⁺ T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8⁺ T cells were predominantly CD27⁺45RO⁺ for HIV and CD27⁻45RA⁺ for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8⁺ T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8⁺ T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8⁺ T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained.Gaining a better understanding of the immunologic control of human immunodeficiency virus type 1 (HIV-1) is among the most critical goals for the rational design of HIV vaccines and immunotherapies. Although most HIV-infected patients develop high-level viremia, CD4⁺ T-cell depletion, and progressive disease, a rare subgroup of patients variably termed long-term nonprogressors (LTNP) or elite controllers restrict HIV replication to below 50 copies of HIV RNA/ml plasma and remain disease free for up to 25 years without antiretroviral therapy (ART). Measurements of HIV-specific immune responses in these patients, in comparison with progressors, are providing insights into mechanisms that mediate immunologic control or loss of control in humans. Although the mechanisms of restriction of HIV replication remain incompletely understood, a number of lines of evidence suggest that it is mediated by HIV-specific CD8⁺ T cells (reviewed in reference 51). High frequencies of HIV-specific CD8⁺ T cells specific for the autologous virus are observed in both LTNP and untreated progressors, suggesting that differences in immunologic control are mediated not by quantitative but more likely by qualitative features of the immune response.A number of qualitative features of the HIV-specific CD8⁺ T-cell response of LTNP or progressors have recently been proposed as the cause of immunologic control or loss of control, respectively. HLA B*5701 is highly overrepresented in LTNP, and the HIV-specific CD8⁺ T-cell response is highly focused on B5701-restricted peptides in B*5701⁺ LTNP but not in B*5701⁺ progressors (19, 50). In addition, there is a difference in surface markers between HIV- and cytomegalovirus (CMV)-specific CD8⁺ T cells thought to represent differences in maturation of the T-cell response (8). The CD8⁺ T cells of progressors are diminished in proliferative capacity and perforin upregulation in response to autologous HIV-infected CD4⁺ T cells (49). Recently, it has been proposed that this diminished proliferative capacity is due to a lack of paracrine or autocrine interleukin-2 (IL-2) production by HIV-specific CD4⁺ T cells or CD8⁺ T cells (41, 42, 75). Interpretation of proliferation studies is complicated by the fact that the effects of IL-2 were measured on the basis of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution of major histocompatibility complex (MHC) tetramer-positive cells. Because cell division over 6 days is an exponential function, IL-2 may induce small increases in the percentage of cells dividing or in the number of cell divisions that can result in large changes in the percent CFSE^lo cells, and yet the majority of antigen-specific cells may not proceed through the cell cycle. In addition, there are very limited data regarding whether the features of immunodominance, surface phenotype, and IL-2 responsiveness of HIV-specific CD8⁺ T cells extend to other chronic virus infections.In the present study, we examined these qualitative features within the response to HIV, CMV, or hepatitis C virus (HCV) across patient groups. We observed that the high degree of focus upon B5701-restricted peptides found in LTNP does not extend to the HCV- or CMV-specific responses. The phenotype of HIV- or CMV-specific CD8⁺ T cells was highly variable and heavily influenced by the degree of viremia. In addition, when both the number of divisions and the percentage of cells dividing were analyzed, proliferation of HIV-specific CD8⁺ T cells was refractory to IL-2 stimulation, unlike that of CMV-specific cells. These results offer important insights into qualitative features of the HIV-specific CD8⁺ T-cell response, whether they extend to responses to other viruses, and whether they are associated with the presence or absence of immunologic control.     

TNFα and IFNγ but Not Perforin Are Critical for CD8 T Cell-Mediated Protection against Pulmonary Yersinia pestis Infection

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE_69–77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE_69–77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE_69–77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE_69–77-mediated protection. In contrast, YopE_69–77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.     

Highly Differentiated,Resting Gn-Specific Memory CD8+ T Cells Persist Years after Infection by Andes Hantavirus

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-γ ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3⁺CD8⁺ T cells were specific for the single HLA-B*3501-restricted epitope Gn_465–473 years after the acute infection. Remarkably, Gn_465–473–specific cells readily secreted IFN-γ, granzyme B and TNF-α but not IL-2 upon stimulation and showed a ‘revertant’ CD45RA⁺CD27⁻CD28⁻CCR7⁻CD127⁻ effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines.     

Physiological induction of regulatory Qa-1-restricted CD8+ T cells triggered by endogenous CD4+ T cell responses

T cell-dependent autoimmune diseases are characterized by the expansion of T cell clones that recognize immunodominant epitopes on the target antigen. As a consequence, for a given autoimmune disorder, pathogenic T cell clones express T cell receptors with a limited number of variable regions that define antigenic specificity. Qa-1, a MHC class I-like molecule, presents peptides from the variable region of TCRs to Qa-1-restricted CD8+ T cells. The induction of Vß-specific CD8+ T cells has been harnessed in an immunotherapeutic strategy known as the “T cell vaccination” (TCV) that comprises the injection of activated and attenuated CD4+ T cell clones so as to induce protective CD8+ T cells. We hypothesized that Qa-1-restricted CD8+ regulatory T cells could also constitute a physiologic regulatory arm of lymphocyte responses upon expansion of endogenous CD4+ T cells, in the absence of deliberate exogenous T cell vaccination. We immunized mice with two types of antigenic challenges in order to sequentially expand antigen-specific endogenous CD4+ T cells with distinct antigenic specificities but characterized by a common Vß chain in their TCR. The first immunization was performed with a non-self antigen while the second challenge was performed with a myelin-derived peptide known to drive experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We show that regulatory Vß-specific Qa-1-restricted CD8+ T cells induced during the first endogenous CD4+ T cell responses are able to control the expansion of subsequently mobilized pathogenic autoreactive CD4+ T cells. In conclusion, apart from the immunotherapeutic TCV, Qa-1-restricted specialized CD8+ regulatory T cells can also be induced during endogenous CD4+ T cell responses. At variance with other regulatory T cell subsets, the action of these Qa-1-restricted T cells seems to be restricted to the immediate re-activation of CD4+ T cells.     

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.     

Epitope Mapping of HIV-Specific CD8+ T cells in a Cohort Dominated by Clade A1 Infection

Background

CD8+ T cell responses are often detected at large magnitudes in HIV-infected subjects, and eliciting these responses is the central aim of many HIV-1 vaccine strategies. Population differences in CD8+ T cell epitope specificity will need to be understood if vaccines are to be effective in multiple geographic regions.

Methodology/Principal Findings

In a large Kenyan cohort, we compared responsive CD8+ T cell HIV-1 Env overlapping peptides (OLPs) to Best Defined Epitopes (BDEs), many of which have been defined in clade B infection. While the majority of BDEs (69%) were recognized in this population, nearly half of responsive OLPs (47%) did not contain described epitopes. Recognition frequencies of BDEs were inversely correlated to epitopic sequence differences between clade A1 and BDE (P = 0.019), and positively selected residues were more frequent in “new” OLPs (without BDEs). We assessed the impact of HLA and TAP binding on epitope recognition frequencies, focusing on predicted and actual epitopes in the HLA B7 supertype.

Conclusions/Significance

Although many previously described CD8 epitopes were recognized, several novel CD8 epitopes were defined in this population, implying that epitope mapping efforts have not been completely exhausted. Expansion of these studies will be critical to understand population differences in CD8 epitope recognition.