Immunodominant CD8 Research Articles

CD8+ T Cells Specific for ImmunodominantTrans-Sialidase Epitopes Contribute to Control ofTrypanosoma cruziInfection but Are Not Required for Resistance

CD8(+) T cells are essential for controlling Trypanosoma cruzi infection. During Brazil strain infection, C57BL/6 mice expand parasite-specific CD8(+) T cells recognizing the dominant TSKB20 (ANYKFTLV) and subdominant TSKB74 (VNYDFTLV) trans-sialidase gene (TS)-encoded epitopes with up to 40% of all CD8(+) T cells specific for these epitopes. Although this is one of the largest immunodominant T cell responses described for any infection, most mice fail to clear T. cruzi and subsequently develop chronic disease. To determine if immunodominant TS-specific CD8(+) T cells are necessary for resistance to infection, we epitope-tolerized mice by high-dose i.v. injections of TSKB20 or TSKB74 peptides. Tolerance induction led to deletion of TS-specific CD8(+) T cells but did not prevent the expansion of other effector CD8(+) T cell populations. Mice tolerized against either TSKB20 or TSKB74, or both epitopes simultaneously, exhibited transient increases in parasite loads, although ultimately they controlled the acute infection. Furthermore, BALB/c mice tolerized against the TSKD14 peptide effectively controlled acute T. cruzi infection. These data are consistent with the hypothesis that development of high-frequency CD8(+) T cell populations focused on TS-derived epitopes contributes to optimal control of acute infection but is not required for the development of immune resistance.

Immediate-Early Expression of a Recombinant Antigen by Modified Vaccinia Virus Ankara Breaks the Immunodominance of Strong Vector-Specific B8R Antigen in Acute and Memory CD8 T-Cell Responses

Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediate-early protein, and remained higher over the first 6 h of infection than p7.5- or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5- and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigen-specific CD8 T-cell responses.

A membrane penetrating multiple antigen peptide (MAP) incorporating ovalbumin CD8 epitope induces potent immune responses in mice

Cell penetrating peptides (CPP) represent a novel approach to facilitate cytoplasmic delivery of macromolecules. The DNA binding domain of Drosophila Antennapedia contains 60 amino acids and consists of 3 α-helices, with internalizing activity mapped to a 16-amino acid peptide penetratin (Antp) within the third α-helix. Here, we report on the use of penetratin to deliver a multiple antigen peptide (MAP) incorporating the immunodominant CD8 epitope of ovalbumin, SIINFEKL (MAPOVACD8). We demonstrate that penetratin linked to the MAPOVACD8 construct either by a disulfide (SS) or thioether (SC) linkage promotes the uptake, cross presentation and subsequent in vivo proliferation and generation of OVACD8 (SIINFEKL)-specific T cells. The MAPOVACD8 construct without penetratin is not presented by MHC class I molecules nor does it generate an in vivo IFN-γ response in C57BL/6 mice. Moreover, we clearly define the uptake and intracellular processing pathways of AntpMAPOVACD8 SS and SC revealing the majority of AntpMAPOVACD8 is taken up by DC via an endocytic, proteasome and tapasin independent mechanism. We also show that the uptake mechanism of AntpMAPOVACD8 is dose dependent and uptake or intracellular processing is not altered by the type of chemical linkage.

Abstract 1923: Regulatory T-cell subsets suppress high avidity CD8 T-cell activation and trafficking

Abstract Cancer vaccines are one treatment modality that can prevent the development of some cancers. The advantage of cancer vaccines is their ability to target cancer proteins specifically, thereby resulting in minimal toxicities to normal tissue. Unfortunately, immune tolerance mechanisms provide a formidable barrier to effective treatment of cancer when vaccines are used as a single agent. One way to overcome these barriers is to develop vaccination strategies that combine vaccines with tolerance-reducing agents that allow the recruitment and activation of the most potent tumor-specific T cells. In fact, accumulating data suggest that the availability and activation of a diverse high avidity T cell repertoire is a key factor in the effectiveness of cancer vaccines. To further understand the role of high avidity T cells in the treatment of cancer, we developed TCR transgenic mice that are high avidity and specific for the immunodominant CD8+ T cell epitope, RNEU420–429, that derives from the HER-2/neu breast cancer antigen. By adoptively transferring these high avidity T cells into the tolerant environment of the neu-N mouse, we have studied their proliferation, trafficking, and tumor-clearing capacity. High avidity T cell responses were evaluated following vaccination using a neu-expressing, GM-CSF-secreting, whole cell vaccine. Vaccine was given either alone or in combination with two T regulatory cell (Treg) depleting agents: Cyclophosphamide (Cy) and the anti-CD25 antibody, PC61. Our data demonstrate that the Cy specifically targets a subset of Tregs and the depletion of this subset allows for more effective tumor clearance by high avidity T cells. While most tolerance reduction strategies focus on the depletion of CD4+CD25+ T regulatory cells, our studies reveal the existence of a population of CD25low effector Tregs that can effectively traffic to the tumor microenvironment and mediate tolerance. Through targeted removal of this CD25low effector Treg population, high avidity T cells undergo increased activation, T cell trafficking, and tumor clearance. Thus, we demonstrate that vaccination in combination with targeted, effector Treg depletion can activate higher avidity T cell populations to eradicate pre-existing tumor burdens. Through an in-depth understanding of the tolerance mechanisms that mediate high avidity T cells suppression, it will be possible to design more effective vaccination strategies to activate the small but potent high avidity T cell populations present in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1923.

A Novel HLA (HLA-A*0201) Transgenic Rabbit Model for Preclinical Evaluation of Human CD8+ T Cell Epitope-Based Vaccines against Ocular Herpes

We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8(+) T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8(+) T cell peptide epitopes from HSV-1 glycoprotein D (gD(53-61), gD(70-78), and gD(278-286)) were joined with a promiscuous human CD4(+) T cell peptide epitope (gD(49-82)) to construct three separate pairs of CD4-CD8 peptides. Each CD4-CD8 peptide pair was then covalently linked to an N(epsilon)-palmitoyl-lysine residue via a functional base lysine amino group to construct CD4-CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4-CD8 HSV-1 gD lipopeptides. The HSV-gD-specific T cell responses induced by the mixture of CD4-CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD(49-82)-specific CD4(+) T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD(53-61), gD(70-78), and gD(278-286)-specific CD8(+) T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8(+) T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8(+) T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4-CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed.

A novel transmembrane domain mediating retention of a highly motile herpesvirus glycoprotein in the endoplasmic reticulum

Gene m164 of murine cytomegalovirus belongs to the large group of 'private' genes that show no homology to those of other cytomegalovirus species and are thought to represent 'host adaptation' genes involved in virus-host interaction. Previous interest in the m164 gene product was based on the presence of an immunodominant CD8 T-cell epitope presented at the surface of infected cells, despite interference by viral immune-evasion proteins. Here, we provide data to reveal that the m164 gene product shows unusual features in its cell biology. A novel strategy of mass-spectrometric analysis was employed to map the N terminus of the mature protein, 107 aa downstream of the start site of the predicted open reading frame. The resulting 36.5 kDa m164 gene product is identified here as an integral type-I membrane glycoprotein with exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane with an outstandingly high lateral membrane motility, actually 100 times higher than those published for cellular ER-resident proteins. Notably, gp36.5/m164 does not contain any typical ER-retention/retrieval signals, such as the C-terminal motifs KKXX or KXKXX, and does not pass the Golgi apparatus. Instead, it belongs to the rare group of viral glycoproteins in which the transmembrane domain (TMD) itself mediates direct ER retention. This is the first report relating TMD usage of an ER-resident transmembrane protein to its lateral membrane motility as a paradigm in cell biology. We propose that TMD usage for ER retention facilitates free and fast floating in ER-related membranes and between ER subdomains.

Nasolacrimal Duct Closure Modulates Ocular Mucosal and Systemic CD4+T-Cell Responses Induced following Topical Ocular or Intranasal Immunization

Both topical ocular and topical intranasal immunizations have been reported to stimulate the ocular mucosal immune system (OMIS) and the systemic immune system. Nasolacrimal ducts (NLDs) are the connecting bridges between the OMIS and nasal cavity-associated lymphoid tissue (NALT). These ducts drain topical ocularly administrated solutions into the inferior meatus of the nose to reach the NALT. Inversely, NLDs also drain intranasally administrated solutions to the mucosal surface of the eye and thus the OMIS. This unique anatomical connection between the OMIS and NALT systems provoked us to test whether the OMIS and NALT are immunologically interdependent. In this report, we show that both topical ocular administration and topical intranasal administration of a mixture of immunodominant CD4(+) T-cell epitope peptides from herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) emulsified with the CpG(2007) mucosal adjuvant are capable of inducing local (in conjunctiva) as well as systemic (in spleen) HSV-peptide-specific CD4(+) T-cell responses. Interestingly, surgical closure of NLDs did not significantly alter local ocular mucosal CD4(+) T-cell responses induced following topical ocular immunization but did significantly enhance systemic CD4(+) T-cell responses (as measured by both T-cell proliferation and gamma interferon (IFN-gamma) production; P < 0.005). In contrast, NLD closure significantly decreased ocular mucosal, but not systemic, CD4(+) T-cell responses following intranasal administration of the same vaccine solution (P < 0.001). The study suggests that NALT and the OMIS are immunologically interconnected.

Assessment of Seasonal Influenza A Virus-Specific CD4 T-Cell Responses to 2009 Pandemic H1N1 Swine-Origin Influenza A Virus

Very limited evidence has been reported to show human adaptive immune responses to the 2009 pandemic H1N1 swine-origin influenza A virus (S-OIV). We studied 17 S-OIV peptides homologous to immunodominant CD4 T epitopes from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), M1 matrix protein (MP), and PB1 of a seasonal H1N1 strain. We concluded that 15 of these 17 S-OIV peptides would induce responses of seasonal influenza virus-specific T cells. Of these, seven S-OIV sequences were identical to seasonal influenza virus sequences, while eight had at least one amino acid that was not conserved. T cells recognizing epitopes derived from these S-OIV antigens could be detected ex vivo. Most of these T cells expressed memory markers, although none of the donors had been exposed to S-OIV. Functional analysis revealed that specific amino acid differences in the sequences of these S-OIV peptides would not affect or partially affect memory T-cell responses. These findings suggest that without protective antibody responses, individuals vaccinated against seasonal influenza A may still benefit from preexisting cross-reactive memory CD4 T cells reducing their susceptibility to S-OIV infection.

Differences in the source sequence of two HCV genotype 1b outbreaks within immunodominant CD8 epitopes are associated with differential outcome

Aims: Distinct HLA-class I alleles have a differential impact on the outcome of HCV infection. In the Irish Anti-D cohort HLA-B27 was associated with spontaneous control and HLA-B8 with viral persistence. Although the protective effect of HLA-B27 was confirmed in other cohorts, discrepant results have been obtained for HLA-B8. The aim of this study is to determine the role of sequence differences in the source virus for the differential outcome associated with these alleles.

Protective Effect of Human Leukocyte Antigen B27 in Hepatitis C Virus Infection Requires the Presence of A Genotype-Specific Immunodominant Cd8+ T-Cell Epitope

Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection.

The protective effect of HLA-B27 in hepatitis C virus infection requires presence of a genotype-specific immunodominant CD8+ T cell epitope

Introduction: HLA-B27 is associated with protection in HIV and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27 restricted CD8+ T cell epitopes in both infections.

Viral fitness implications of variation within an immunodominant CD8+ T-cell epitope of HIV-1

Cytotoxic T-lymphocyte (CTL) epitopes within the HIV genome are subject to negative and positive selective pressures, the balance of which influences CTL escape at a given epitope. We investigated whether viral fitness requirements dictate conservation of the HLA-A2 restricted immunodominant epitope SLYNTVATL (SL9). Viral clones incorporating changes throughout the SL9 epitope region were compared to consensus SL9 virus in terms of replication kinetics and relative viral fitness. Constructs recapitulating in vivo SL9-CTL escape variants showed markedly little effect on replication and fitness, as did non-natural conservative mutations targeting immunologically relevant positions of the epitope. Although certain residues of the epitope were constrained by viral requirements, our research reveals that there are multiple SL9 variants that are well tolerated virologically but fail to arise in vivo. In light of this data, assumptions regarding the balance of immune and viral selective pressures on this immunodominant epitope sequence need to be reassessed.

Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection.

Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8+ T cell populations in Mamu-A*01+ rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR β-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8+ T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181–189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8+ T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection.

A Protective Role for Dengue Virus-Specific CD8+ T Cells

Infection with one of the four serotypes of dengue virus (DENV1-4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8(+) T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8(+) T cell response, whereas viral replication and a robust CD8(+) T cell response were observed after infection of IFN-alpha/betaR(-/-) mice. Depletion of CD8(+) T cells from IFN-alpha/betaR(-/-) mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8(+) T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-alpha/betaR(-/-) mice. DENV-specific CD8(+) T cells produced IFN-gamma, TNF-alpha, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8(+) T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8(+) T cells in the host defense against DENV and demonstrate that the anti-DENV CD8(+) T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

Protective Response to Leishmania major in BALB/c Mice Requires Antigen Processing in the Absence of DM

Protection from the parasite Leishmania major is mediated by CD4 T cells. BALB/c mice are susceptible to L. major and show a nonprotective immunodominant CD4 T cell response to Leishmania homolog of activated receptor for c-kinase (LACK) 158-173. Host genes that underlie BALB/c susceptibility to L. major infections are poorly defined. DM, a nonclassical MHC class II molecule, due to its peptide editing properties has been shown to 1) edit the repertoire of peptides displayed by APC, and 2) focus the display of epitopes by APC to the immunodominant ones. We tested the hypothesis that deficiency of DM, by causing presentation of a different array of epitopes by infected APC than that presented by DM-sufficient APC, may change the course of L. major infection in the susceptible BALB/c mice. We show herein that unlike their susceptible wild-type counterparts, BALB/c mice deficient in DM are protected from infections with L. major. Furthermore, DM-deficient mice fail to display the immunodominant LACK 158-173 on infected APC. In its place, infected DM(-/-) hosts show elicitation of CD4 T cells specific for newer epitopes not presented by wild-type L. major-infected APC. Protection of BALB/c DM(-/-) mice is dependent on IFN-gamma. DM is thus a host susceptibility gene in BALB/c mice, and Ag processing in the absence of DM results in elicitation of a protective T cell response against L. major infections. This report suggests a novel mechanism to trigger host resistance against pathogens.

Identification of a dominant CD4 T cell epitope in the membrane lipoprotein Tul4 from Francisella tularensis LVS

Francisella tularensis is a Gram-negative intracellular bacterium that is the causative agent of tularemia. Small mammals such as rodents and rabbits, as well as some biting arthropods, serve as the main vectors for environmental reservoirs of F. tularensis. The low infectious dose, ability to aerosolize the organism, and the possibility of generating antibiotic resistant strains make F. tularensis a prime organism for use in bioterrorism. As a result, some strains of F. tularensis have been placed on the CDC category A select agent list. T cell immune responses are thought to be a critical component in protective immunity to this organism. However, investigation into the immune responses to F. tularensis has been hampered by the lack of molecularly defined epitopes. Here we report the identification of a major CD4 + T cell epitope in C57Bl/6 (B6) mice. The murine model of F. tularensis infection is relevant as mice are a natural host for F. tularensis LVS and exhibit many of the same features of tularemia seen in humans. Using T cell hybridomas derived from B6 mice that had either been inoculated with F. tularensis and allowed to clear the infection or which had been immunized by conventional means using purified recombinant protein in adjuvant, we have identified amino acids 86–99 of the lipoprotein Tul4 (RLQWQAPEGSKCHD) as an immunodominant CD4 T cell epitope in B6 mice. This epitope is a major component of both the acute and memory responses to F. tularensis infection and can constitute as much as 20% of the responding CD4 T cells in an acute infection. Reactive T cells can also effectively enter the long-term memory T cell pool. The identification of this epitope will greatly aid in monitoring the course of F. tularensis infection and will also aid in the development of effective vaccine strategies for F. tularensis.

A Comparative Study of HLA Binding Affinity and Ligand Diversity: Implications for Generating Immunodominant CD8+ T Cell Responses

Conventional CD8(+) T cell responses against intracellular infectious agents are initiated upon recognition of pathogen-derived peptides presented at the cell surface of infected cells in the context of MHC class I molecules. Among the major MHC class I loci, HLA-B is the swiftest evolving and the most polymorphic locus. Additionally, responses restricted by HLA-B molecules tend to be dominant, and most associations with susceptibility or protection against infectious diseases have been assigned to HLA-B alleles. To assess whether the differences in responses mediated via two major HLA class I loci, HLA-B and HLA-A, may already begin at the Ag presentation level, we have analyzed the diversity and binding affinity of their peptide repertoire by making use of curated pathogen-derived epitope data retrieved from the Immune Epitope Database and Analysis Resource, as well as in silico predicted epitopes. In contrast to our expectations, HLA-B alleles were found to have a less diverse peptide repertoire, which points toward a more restricted binding motif, and the respective average peptide binding affinity was shown to be lower than that of HLA-A-restricted epitopes. This unexpected observation gives rise to new hypotheses concerning the mechanisms underlying immunodominance of CD8(+) T cell responses.

Role of regulatory T cells in coronavirus-induced acute encephalitis

C57BL/6 mice infected with mouse hepatitis virus, strain JHM (JHMV) develop a rapidly fatal acute encephalitis. Previously, we showed that this disease is partially CD4 T cell-mediated since infection with a recombinant JHMV (rJ) mutated in only a single immunodominant CD4 T cell epitope (epitope M133, rJ.M Y135Q) results in a nonlethal disease. Increased mortality correlated with a greater number of JHMV-specific CD4 T cells in the brains of rJ compared to rJ.M Y135Q-infected mice. Here, we extend these results to show that the diminished number of virus-specific T cells correlates with a reduced cytokine/chemokine response in the infected brain. We also show that regulatory CD4 T cells (Tregs) are critical for mild disease in rJ.M Y135Q-infected mice because their depletion results in increased mortality. Further, a relative paucity of Tregs characterizes lethal infection because adoptive transfer of Tregs into rJ-infected mice increases survival from 0% to 50%. These results support the notion that clinical disease in coronavirus-induced acute encephalitis results from a balance between factors critical for virus clearance, such as virus-specific effector T cells and anti-inflammatory elements, such as Tregs. These findings also show that unlike chronic infections, in which an excessive number of Tregs contributes to pathogen persistence, Tregs in the setting of acute encephalitis may help to limit immunopathological disease without delaying virus clearance.

Generation of Combined CD8+ and CD4+ T Cell Lines with High Specificity for Adenovirus Hexon Epitopes for Adoptive Immunotherapy after Allogeneic Stem Cell Transplantation.

Human Adenovirus (HAdV) can cause serious morbidity in immunocompromised patients, in particular in pediatric recipients of allogeneic stem cell transplantation (alloSCT). Progression to disseminated adenoviral disease is associated with a high mortality, despite treatment with antiviral agents such as ribavirin and cidofovir. It has been demonstrated that reconstitution of HAdV-specific T cells is essential to control adenoviral infection after alloSCT. Adoptive transfer of donor-derived HAdV-specific T cells may therefore be a strategy to provide long-term protection from HAdV. In healthy individuals, T cells directed against HAdV are only detected at low frequencies and are predominantly directed to the HAdV hexon protein. Only recently, a number of immunodominant CD8+ and CD4+ epitopes of HAdV hexon have been defined. Since these epitopes are largely conserved between the different HAdV subgroups, T cells specific for these immunodominant epitopes may provide protection from a wide range of adenoviral serotypes. The aim of this study was to develop a method for the generation of combined CD8+ and CD4+ T cell lines with high and well defined specificity for the HAdV hexon protein. We first analyzed the frequencies of HAdV hexon-specific CD8+ and CD4+ T cells in healthy individuals using sensitive measurement by peptide-MHC tetramers, and intracellular cytokine staining combined with CD154 or peptide-MHC tetramer staining, after stimulation with defined MHC class I peptides, 30-mer peptides containing class II epitopes, or a HAdV hexon protein-spanning pool of overlapping 15-mer peptides (Miltenyi Biotec, Germany). We demonstrated that the frequencies of HAdV hexon-specific T cells were very low in most healthy individuals tested. HAdV hexon-specific CD8+ T cells were detectable in only 3/15 individuals (range 0.16–0.43% of CD8+ T cells), and hexon-specific CD4+ T cells were detected in all individuals with a median of 0.07% (range 0.004–0.38% of CD4+ T cells). The highest frequencies were found after stimulation with the hexon protein-spanning 15-mer peptide pool, indicating activation of both known and unknown epitopes. Kinetic analysis showed highest levels of IFNg production after 4–8 hours of stimulation for HAdV-specific CD8+ T cells, and after 4–48 hours of stimulation for HAdV-specific CD4+ T cells. The phenotype of these HAdV hexon-specific T cells corresponded to an early memory phenotype, CD27+, CD28+, CD62L+, CD45RO+. Despite these low or undetectable frequencies of HAdV-specific T cells, IFNg-based enrichment 4 hours after activation with the HAdV hexon protein-spanning peptide pool resulted in efficient isolation of CD8+ and CD4+ T cells recognizing both known and unknown HAdV hexon epitopes. Following a short culture period of 7 days, the T cell lines consisted of 49–80% CD8+ T cells and 13–15% CD4+ T cells. Restimulation by autologous EBV-LCL loaded with HAdV hexon peptide pool followed by intracellular IFNg staining showed that the frequency of HAdV-specific T cells was increased to 65–95% of CD8+ T cells, and 38–72% of CD4+ T cells. The frequency of HAdV-tetramer-positive cells was increased to 32–76% of CD8+ T cells, indicating that part of HAdV-specific CD8+ T cells recognized known epitopes. After 14 days, the frequency of HAdV-specific T cells had further increased to 89–94% of CD8+ T cells and 61–91% of CD4+ T cells. Starting with only 25x106 donor peripheral blood mononuclear cells, this strategy yielded T cell lines containing 1.3–2.7x106 HAdV-specific combined CD8+ and CD4+ T cells in 14 days. We conclude that we developed a GMP-grade method for the fast generation of highly HAdV-specific CD8+ and CD4+ T cell lines from all healthy donors tested, irrespective of HLA-restriction, for the treatment HAdV infection after alloSCT, with very limited risk of graft-versus-host disease.

A novel approach to evaluate the immunogenicity of viral antigens of clinical importance in HLA transgenic murine models

Transgenic (Tg) mice expressing HLA class I alleles and lacking murine MHC class I represent a useful model for the pre-clinical evaluation of human vaccines, which focus on induction of CD8 + T-cell responses. We have developed a platform to be used in Tg mice for exploring the immunogenicity of T-cell targets, whose immunologic epitopes have yet to be defined. To test the attributes of the evaluation system in the context of an important human pathogen, we have explored multiple antigens from cytomegalovirus (CMV). A panel of recombinant modified vaccinia Ankara (MVA) vectors, expressing various CMV proteins (CMV–MVA) was used to immunize HLA-A*0201, B*0702 and A*1101 Tg mice. Immune splenocytes were in vitro stimulated (IVS) either using syngeneic lipo-polysaccharide activated lymphoblasts or Tg HLA-I matched human EBV-transformed B-lymphoblastoid cells (LCL), both loaded with peptide libraries, encompassing the CMV protein under investigation. IVS performed with peptide library loaded lymphoblasts failed to provide a reliable stimulation. In contrast, the usage of LCL as antigen presenting cells (APC) of CMV peptide libraries resulted in a consistent and specific amplification of the Tg T-cell response in animals immunized with CMV–MVAs. The LCL IVS method reliably allowed defining the immunogenicity and immunodominant CD8 + T-cell regions of uncharacterized CMV antigens. The combination of CMV–MVA vectors, unbiased pools of CMV-specific peptide libraries presented by Tg HLA-I matched LCL constitutes a valid tool for the pre-clinical evaluation of model candidate vaccines. This convenient method could find application to investigate the immunogenicity profile of cancer antigens or proteins from infectious human pathogens.