Immunodominant CD8 Research Articles

High Frequency of CD4+T Cells Specific for the TB10.4 Protein Correlates with Protection againstMycobacterium tuberculosisInfection

TB10.4 is a newly identified antigen of Mycobacterium tuberculosis recognized by human and murine T cells upon mycobacterial infection. Here, we show that immunization with Mycobacterium bovis BCG induces a strong, genetically controlled, Th1 immune response against TB10.4 in mice. BALB/c and C57BL/6 strains behave as high and low responders to TB10.4 protein, respectively. The TB10.4:74-88 peptide was identified as an immunodominant CD4+ T-cell epitope for H-2d mice. Since recent results, as well as the present study, have raised interest in TB10.4 as a subunit vaccine, we analyzed immune responses induced by this antigen delivered by a new vector, the adenylate cyclase (CyaA) of Bordetella pertussis. CyaA is able to target dendritic cells and to deliver CD4+ or CD8+ T-cell epitopes to the major histocompatibility complex class II/I molecule presentation pathways, triggering specific Th1 or cytotoxic T-lymphocyte (CTL) responses. Several CyaA harboring either the entire TB10.4 protein or various subfragments containing the TB10.4:20-28 CTL epitope were shown to induce TB10.4-specific Th1 CD4+ and CD8+ T-cell responses. However, none of the recombinant CyaA, injected in the absence of adjuvant, was able to induce protection against M. tuberculosis infection. In contrast, TB10.4 protein administered with a cocktail of strong adjuvants that triggered a strong Th1 CD4+ T-cell response induced significant protection against M. tuberculosis challenge. These results confirm the potential value of the TB10.4 protein as a candidate vaccine and show that the presence of high frequencies of CD4+ T cells specific to this strong immunogen correlates with protection against M. tuberculosis infection.

New perspectives for T-cell-based HCV vaccines

A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Folgori A, Capone S, Ruggeri L, Meola A, Sporeno E, Ercole BB, Pezzanera M, Tafi R, Arcuri M, Fattori E, Lahm A, Luzzago A, Vitelli A, Colloca S, Cortese R, Nicosia A. Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here, we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses. [Abstract reproduced by permission of Nat Med 2006;12(2):190–197]

Induction of Multifunctional Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T Cells Capable of Proliferation in Healthy Subjects by Using a Prime-Boost Regimen of DNA- and Modified Vaccinia Virus Ankara-Vectored Vaccines Expressing HIV-1 Gag Coupled to CD8+T-Cell Epitopes

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.

Inhibition of Interferon-γ Signaling in Oligodendroglia Delays Coronavirus Clearance without Altering Demyelination

Infection of the central nervous system (CNS) by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces an acute encephalomyelitis associated with demyelination. To examine the anti-viral and/or regulatory role of interferon-γ (IFN-γ) signaling in the cell that synthesizes and maintains the myelin sheath, we analyzed JHMV pathogenesis in transgenic mice expressing a dominant-negative IFN-γ receptor on oligodendroglia. Defective IFN-γ signaling was associated with enhanced oligodendroglial tropism and delayed virus clearance. However, the CNS inflammatory cell composition and CD8+ T-cell effector functions were similar between transgenic and wild-type mice, supporting unimpaired peripheral and CNS immune responses in transgenic mice. Surprisingly, increased viral load in oligodendroglia did not affect the extent of myelin loss, the frequency of oligodendroglial apoptosis, or CNS recruitment of macrophages. These data demonstrate that IFN-γ receptor signaling is critical for the control of JHMV replication in oligodendroglia. In addition, the absence of a correlation between increased oligodendroglial infection and the extent of demyelination suggests a complex pathobiology of myelin loss in which infection of oligodendroglia is required but not sufficient.

Experimental autoimmune diabetes is caused by IFN-γ+/CD8+ T cell immunity against a single insulin A-chain epitope

Insulin and its primary gene product, preproinsulin (PPI), are considered central autoantigens in human type 1 diabetes (T1D), with potential value for antigen-specific immune therapy. We have previously demonstrated (Diabetes 51:3237–44) that PPI can adversely induce hyperglycemia and experimental autoimmune diabetes (EAD). Here we identify a C-terminal insulin A-chain epitope as the cause of EAD. RIP-B7.1 transgenic mice were immunized by DNA vaccination using PPI-expressing plasmid vectors, resulting in CD4+/CD8+ insulitis and hyperglycemia in >95% animals after only 2–3 weeks. Adoptive spleen cell transfer shows that autoreactive PPI-specific CD8 T cell clones can be induced in both transgenic and wild type mice, but beta cell co-stimulation (B7.1) is required for diabetes manifestation. Using gene-targeted B7.1 mice we demonstrate that interferon-γ, but not perforin, is required for autoimmune beta cell damage in EAD. Using PPI minigene constructs and a PPI peptide library, we localize and fine-map an immunodominant CD8 T cell epitope to the C-terminus of the A chain of insulin. We identify its MHC restriction as class I Db by a stabilization experiments in TAP-deficient H2 cell lines. After i.m. injection as peptide-DNA complex, this peptide is sufficient to induce insulitis and hyperglycemia in mice. Our data show that experimental autoimmune diabetes results from CD8+ T cell immunity against a Db-restricted T cell epitope of insulin, located in the C-terminal moiety of the insulin A-chain.

Effect of tolerance induction to immunodominant T-cell epitopes of Sendai virus on gene expression following repeat administration to lung

Sendai virus (SeV) is able to transfect airway epithelial cells efficiently in vivo. However, as with other viral vectors, repeated administration leads to reduced gene expression. We have investigated the impact of inducing immunological tolerance to immunodominant T-cell epitopes on gene expression following repeated administration. Immunodominant CD4 and CD8 T-cell peptide epitopes of SeV were administered to C57BL/6 mice intranasally 10 days before the first virus administration with transmission-incompetent F-protein-deleted DeltaF/SeV-GFP. At 21 days after the first virus administration, mice were again transfected with DeltaF/SeV. To avoid interference of anti-GFP antibodies, the second transfection was carried out with DeltaF/SeV-lacZ. At 2 days after the final transfection lung beta-galactosidase expression, T-cell proliferation and antibody responses were measured. A state of 'split tolerance' was achieved with reduced T-cell proliferation, but no impact on antiviral antibody production. There was no enhancement of expression on repeat administration; instead, T-cell tolerance was, paradoxically, associated with a more profound extinction of viral expression. Multiple immune mechanisms operate to eradicate viruses from the lung, and these findings indicate that impeding the adaptive T-cell response to the immunodominant viral epitope is not sufficient to prevent the process.

The Impairment of CD8 Responses Limits the Selection of Escape Mutations in Acute Hepatitis C Virus Infection

Evasion from protective CD8 responses by mutations within immunodominant epitopes represents a potential strategy of HCV persistence. To investigate the pathogenetic relevance of this mechanism, a careful search for immunodominant CD8 epitopes was conducted in six patients with chronic evolution of HCV infection by analyzing their global CD8 response with a panel of overlapping synthetic peptides covering the overall HCV sequence and by studying the CD8 frequency by tetramer staining. Immunodominant responses were followed longitudinally from the time of acute onset in relation to the evolution of the epitopic sequences. Although intensity of CD8 responses and frequency of HCV-specific CD8 cells declined over time in all patients, mutations emerged in only three of the six acute patients studied. Variant sequences were less efficiently recognized by CD8 cells than parental epitopes and were poorly efficient in inducing a CD8 response in vitro. CD8 epitopes undergoing mutations were targeted by high avidity CD8 cells more efficient in effector function. Our data support the view that immunodominant CD8 responses are affected by inhibitory mechanisms operating early after infection and that the emergence of escape mutations represents an additional mechanism of virus evasion from those CD8 responses that are functionally preserved.

Suppression of Inhibitor Formation to F.IX in Gene Transfer through Immune Deviation Induced by Mucosal Peptide Administration.

Gene therapy is a promising novel strategy for treatment of the X-linked bleeding disorder hemophilia B (deficiency in factor IX, F.IX). However, immune responses to the F.IX transgene product pose a concern. Previously, we demonstrated induction of immune tolerance to human F.IX (hFIX) by hepatic, adeno-associated (AAV) vector-mediated gene transfer. However, not all strains of mice can be tolerized by this protocol (JCI 111:1347). Hemophilia B mice (with a large F.IX gene deletion) on a C3H genetic background form inhibitory antibodies to hF.IX after hepatic gene transfer. Using a peptide library spanning the entire hF.IX protein, we identified a peptide containing an immunodominant CD4+ T cell epitope for hemophilia B C3H/HeJ mice. Since inhibitor formation is dependent on activation of CD4+ T cells, we attempted to induce tolerance by mucosal administration of this hF.IX-derived peptide. We chose nasal administration, which does not need to overcome the proteolytic barriers present in the digestive system. The hF.IX peptide (25 μl of PBS containing 100 μg peptide, n=10) or saline control (n=16) was administered into both nostrils of hemophilia B C3H/HeJ mice twice a week, starting 2 weeks before hepatic gene delivery of AAV-hFIX vector (1x1011 vg/mouse). Mouse plasma samples were collected up to 20 weeks after vector administration. All control mice formed inhibitors (40% had Bethesda titers of 1–2 BU, 60% showed 2–6 BU). Antibodies were mostly IgG1, while IgG2a and IgG2b responses were also measured. In contrast, peptide-treated mice did not develop anti-hF.IX (50%) or had only low-titer antibodies (1–2 BU, 50%). Interestingly, 70–75% of both experimental groups had circulating hF.IX antigen following gene transfer. However, aPTT coagulation times of control mice were either uncorrected, 87% (>50 sec) or mildly corrected, 13% (48–50 sec), while 60% of peptide-treated mice clearly had partial correction of coagulation (40–46 sec compared to 20–30 sec in normal mice). These data suggest that anti-hF.IX inhibited hF.IX activity in mice that did not receive peptide therapy. Furthermore, cytokine release assay indicated that lymphocytes isolated either from nasal draining lymph nodes (superficial cervical lymph nodes) or spleen showed increased production of cytokine IL-10 and TGF-β in peptide treated animals, while levels of interferon-γ production were decreased compared to mice that were not treated with peptide. In vivo adoptive T cell transfer experiments showed that CD4+CD25+ cells from peptide tolerized mice, but not CD4+CD25- cells suppressed anti-hF.IX IgG formation following immunization. Compared to controls, antibody titers were 3-fold lower, with statistical significance of p<0.014 (n=4–5/group). In summary, nasal administration of hF.IX-derived peptide antigen substantially reduced the risk of an inhibitor response in the context of gene transfer-based treatment of hemophilia B. Nasal antigen administration results in an immune deviation from an adaptive, Th1/2 type immune response to a suppressive, Th2/3 type response. Inhibitor formation is likely reduced through cytokine and regulatory T cell-mediated mechanisms.

Contrasting Effects of Post-Transplant Lymphopenia on Proliferation and Degranulation in Antigen-Specific CD8 Memory T Cells.

Lymphopenia following bone marrow transplant (BMT) can induce expansion of donor T cells which help contribute to reconstitution in recipients. We are interested in elucidating the responses of transplanted CD8 T memory (TM) to lymphopenic signals in the recipient splenic and marrow environments. To compare proliferative responses of marrow and spleen populating antigen-specific CD8 TM to lymphopenic signals, purified (95% pure) CFSE labeled B6 CD8 TM were co-transplanted with syngeneic or allogeneic TCD-BM to ablatively conditioned (9.0 Gy) syngeneic B6 recipients. Antigen-specific CD8 TM were obtianed following priming B6 mice against BALB.B antigens. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to identify immunodominant H60-specific CD8+ cells which expressed a central memory (TM) phenotype (CD44+, Ly6C+). Three days post-transplant of these CD8 TM, recipient spleen and marrow cells were analyzed for CFSE dilution in gated H60+CD8+ T cells. Transferred H60+ CD8+ cells in the marrow compartment consistently showed greater (5 – 6 cell divisions) proliferative activity compared to those populating the spleen (1 – 2 divisions) in recipients of syngeneic (i.e. homeostatic) as well as allogeneic TCD-BM. Thus, the marrow compartment apparently provided greater support for the early homeostatic proliferation of these MiHA-specific CD8 TM. Furthermore, the presence of antigen in B6 recipients of allogeneic BM did not significantly enhance the proliferation of transferred CD8+ TM, indicating that homeostatic signals induced greater overall proliferation than antigen activation in the simultaneous presence of both signals early post-transplant. To examine for non-proliferative functional activity in transplanted antigen-specific CD8 TM, degranulation was assessed by surface expression of LAMP-1 (CD8 107a) which was analyzed 3 days after the transfer of purified splenic CD8 T cells from B6BALB.B into lethally irradiated (9.0 Gy) syngeneic B6 mice. Analysis of recipient spleeen and marrow showed that transferred antigen-specific in these tissues markedly upregulated surface LAMP-1 expression. Interestingly, there was greater frequency (~90%) of CD8+ TM in the spleen expressing LAMP-1 than in the bone marrow (56%). To determine if this degranulation response was limited to transplanted TM, a homogeneous population of naive T cells (TN) from OT-1 RAG−/− TCR tg mice was prepared and transplanted in an identical manner. LAMP-1 expression was marginally detectable in these cells. In total, these results suggest that transplanted CD8 TM and TN respond differently in the homeostatic environment post-transplant. Moreover the observations may suggest that CD8 TM can exhibit effector-like activity in the lymphopenic milieu immediately post-transplant. These findings have implications for the relative contribution of hematopoietic compartments during immune reconstitution after HCT.

Tat 28-35 SL8-Specific CD8 + T Lymphocytes Are More Effective than Gag 181-189 CM9-Specific CD8 + T Lymphocytes at Suppressing Simian Immunodeficiency Virus Replication in a Functional In Vitro Assay

Epitope-specific CD8+ T lymphocytes may play an important role in controlling human immunodeficiency virus (HIV)/simian immunodeficiency virus replication. Unfortunately, standard cellular assays do not measure the antiviral efficacy (the ability to suppress virus replication) of CD8+ T lymphocytes. Certain epitope-specific CD8+ T lymphocytes may be better than others at suppressing viral replication. We compared the antiviral efficacy of two immunodominant CD8+ T lymphocyte responses--Tat(28-35)SL8 and Gag(181-189)CM9--by using a functional in vitro assay. Viral suppression by Tat-specific CD8+ T lymphocytes was consistently greater than that of Gag-specific CD8+ T lymphocytes. Such differences in antigen-specific CD8+-T-lymphocyte efficacy may be important for selecting CD8+ T lymphocyte epitopes for inclusion in future HIV vaccines.

Minimal T-Cell-Stimulatory Sequences and Spectrum of HLA Restriction of Immunodominant CD4 + T-Cell Epitopes within Hepatitis C Virus NS3 and NS4 Proteins

The hepatitis C virus (HCV)-specific CD4+ T-cell response against nonstructural proteins is strongly associated with successful viral clearance during acute hepatitis C. To further develop these observations into peptide-based vaccines and clinical immunomonitoring tools like HLA class II tetramers, a detailed characterization of immunodominant CD4+ T-cell epitopes is required. We studied peripheral blood mononuclear cells from 20 patients with acute hepatitis C using 83 overlapping 20-mer peptides covering the NS3 helicase and NS4. Eight peptides were recognized by > or = 40% of patients, and specific CD4+ T-cell clones were obtained for seven of these and three additional, subdominant epitopes. Mapping of minimal stimulatory sequences defined epitopes of 8 to 13 amino acids in length, but optimal T-cell stimulation was observed with 10- to 15-mers. While some epitopes were presented by different HLA molecules, others were presented by only a single HLA class II molecule, which has implications for patient selection in clinical trials of peptide-based immunotherapies. In conclusion, using two different approaches we identified and characterized a set of CD4+ T-cell epitopes in the HCV NS3-NS4 region which are immunodominant in patients achieving transient or persistent viral control. This information allows the construction of a valuable panel of HCV-specific HLA class II tetramers for further study of CD4+ T-cell responses in chronic hepatitis C. The finding of immunodominant epitopes with very constrained HLA restriction has implications for patient selection in clinical trials of peptide-based immunotherapies.

During Viral Infection of the Respiratory Tract, CD27, 4-1BB, and OX40 Collectively Determine Formation of CD8+ Memory T Cells and Their Capacity for Secondary Expansion

Independent studies have shown that CD27, 4-1BB, and OX40 can all promote survival of activated CD8+ T cells. We have therefore compared their impact on CD8+ memory T cell formation and responsiveness within one, physiologically relevant model system. Recombinant mice, selectively lacking input of one or two receptors, were challenged intranasally with influenza virus, and the immunodominant virus-specific CD8+ T cell response was quantified at priming and effector sites. Upon primary infection, CD27 and (to a lesser extent) 4-1BB made nonredundant contributions to accumulation of CD8+ virus-specific T cells in draining lymph nodes and lung, while OX40 had no effect. Interestingly though, in the memory response, accumulation of virus-specific CD8+ T cells in spleen and lung critically depended on all three receptor systems. This was explained by two observations: 1) CD27, 4-1BB, and OX40 were collectively responsible for generation of the same memory CD8+ T cell pool; 2) CD27, 4-1BB, and OX40 collectively determined the extent of secondary expansion, as shown by adoptive transfers with standardized numbers of memory cells. Surprisingly, wild-type CD8+ memory T cells expanded normally in primed OX40 ligand- or 4-1BB ligand-deficient mice. However, when wild-type memory cells were generated in OX40 ligand- or 4-1BB ligand-deficient mice, their secondary expansion was impaired. This provides the novel concept that stimulation of CD8+ T cells by OX40 and 4-1BB ligand during priming imprints into them the capacity for secondary expansion. Our data argue that ligand on dendritic cells and/or B cells may be critical for this.

Contributions of the Viral Genetic Background and a Single Amino Acid Substitution in an Immunodominant CD8+T-Cell Epitope to Murine Coronavirus Neurovirulence

The immunodominant CD8+ T-cell epitope of a highly neurovirulent strain of mouse hepatitis virus (MHV), JHM, is thought to be essential for protection against virus persistence within the central nervous system. To test whether abrogation of this H-2Db-restricted epitope, located within the spike glycoprotein at residues S510 to 518 (S510), resulted in delayed virus clearance and/or virus persistence we selected isogenic recombinants which express either the wild-type JHM spike protein (RJHM) or spike containing the N514S mutation (RJHM(N514S)), which abrogates the response to S510. In contrast to observations in suckling mice in which viruses encoding inactivating mutations within the S510 epitope (epitope escape mutants) were associated with persistent virus and increased neurovirulence (Pewe et al., J Virol. 72:5912-5918, 1998), RJHM(N514S) was not more virulent than the parental, RJHM, in 4-week-old C57BL/6 (H-2b) mice after intracranial injection. Recombinant viruses expressing the JHM spike, wild type or encoding the N514S substitution, were also selected in which background genes were derived from the neuroattenuated A59 strain of MHV. Whereas recombinants expressing the wild-type JHM spike (SJHM/RA59) were highly neurovirulent, A59 recombinants containing the N514S mutation (SJHM(N514S)/RA59) were attenuated, replicated less efficiently, and exhibited reduced virus spread in the brain at 5 days postinfection (peak of infectious virus titers in the central nervous system) compared to parental virus encoding wild-type spike. Virulence assays in BALB/c mice (H-2d), which do not recognize the S510 epitope, revealed that attenuation of the epitope escape mutants was not due to the loss of a pathogenic immune response directed against the S510 epitope. Thus, an intact immunodominant S510 epitope is not essential for virus clearance from the CNS, the S510 inactivating mutation results in decreased virulence in weanling mice but not in suckling mice, suggesting that specific host conditions are required for epitope escape mutants to display increased virulence, and the N514S mutation causes increased attenuation in the context of A59 background genes, demonstrating that genes other than that for the spike are also important in determining neurovirulence.

Anatomical and cellular requirements for the activation and migration of virus-specific CD8+ T cells to the brain during Theiler's virus infection.

Theiler's murine encephalomyelitis virus (TMEV) infection of the brain induces a virus-specific CD8(+) T-cell response in genetically resistant mice. The peak of the immune response to the virus occurs 7 days after infection, with an immunodominant CD8(+) T-cell response against a VP2-derived capsid peptide in the context of the D(b) molecule. The process of activation of antigen-specific T cells that migrate to the brain in the TMEV model has not been defined. The site of antigenic challenge in the TMEV model is directly into the brain parenchyma, a site that is considered immune privileged. We investigated the hypothesis that antiviral CD8(+) T-cell responses are initiated in situ upon intracranial inoculation with TMEV. To determine whether a brain parenchymal antigen-presenting cell is responsible for the activation of virus-specific CD8(+) T cells, we evaluated the CD8(+) T-cell response to the VP2 peptide in bone marrow chimeras and mutant mice lacking peripheral lymphoid organs. The generation of the anti-TMEV CD8(+) T-cell response in the brain requires priming by a bone marrow-derived antigen-presenting cell and the presence of peripheral lymphoid organs. Although our results show that activation of TMEV-specific CD8(+) T cells occurs in the peripheral lymphoid compartment, they do not exclude the possibility that the immune response to TMEV is initiated by a brain-resident, bone marrow-derived, antigen-presenting cell.

Prevention of Gene Transfer-Induced Inhibitor Formation by Nasal Administration of Human F.IX T Cell Epitope in a Murine Model of Hemophilia B.

Hemophilia B is the X-linked bleeding disorder caused by absence of functional coagulation factor IX (F. IX). Gene therapy is a promising novel treatment strategy that can provide a continuous supply of F.IX through stable gene transfer. However, immune responses to the F.IX transgene product pose a concern. In particular, formation of inhibitory antibodies against F.IX represents a serious complication of treatment. In published work, we demonstrated induction of immune tolerance to human F.IX (hFIX) by hepatic, adeno-associated (AAV) vector-mediated gene transfer. However, not all strains of mice can be tolerized by this protocol (JCI 111:1347). Hemophilia B mice (with a large F.IX gene deletion) on a C3H genetic background form inhibitors to hF.IX after hepatic gene transfer. Antibody formation is dependent on activation of hF.IX-specific CD4+ T cells. Using a peptide library spanning the entire hF.IX protein, we identified peptide TEQKRNVIRIIPHHNYNAAI to contain an immunodominant CD4+ T cell epitope for hemophilia B C3H/HeJ mice. T cells can be specifically tolerized by giving antigen through specific routes, such as oral or nasal administration. Such procedures have been shown to prevent or treat T cell-mediated autoimmune diseases, and can induce T cell anergy, deletion, and activation of regulatory Th3 cells, which suppress T cell responses through secretion of the TGF- β cytokine. Short T epitope sequence may be safer for T cell tolerance procedures, since peptide-specific antibodies rarely cross-react with the cognate native Ag. Nasal administration does not need to overcome the proteolytic barriers present in the digestive system, and peptides cross epithelia more easily than large proteins. The hF.IX peptide described above (25 μl of PBS containing 100 μg peptide, n=10) or saline control (n=16) was administered into both nostrils of hemophilia B C3H/HeJ mice twice a week, starting 2 weeks before hepatic gene delivery of AAV-hFIX vector (1x1011 vg/mouse) in C3H hemophilia B mice. Mouse plasma samples were collected 2 and 4 weeks after vector administration. All control mice formed inhibitors (40% had Bethesda titers of 1–2 BU, 60% showed 2–6 BU). Antibodies were mostly IgG1, while IgG2a and IgG2b responses were also measured. In contrast, peptide-treated mice did not develop anti-hF.IX (50%) or had only low-titer antibodies (1–2 BU, 50%). Interestingly, 70–75% of both experimental groups had circulating hF.IX antigen following gene transfer. However, aPTT coagulation times of control mice were either uncorrected, 87%(>50 sec) or mildly corrected, 13%(48–50 sec), while 60% of peptide-treated mice clearly had partial correction of coagulation (40–46 sec compared to 20–30 sec in normal mice). These data indicate that anti-hF.IX, while often not eliminating systemic expression, inhibited hF.IX activity in mice that did not receive peptide therapy. Successfully treated animals expressed 27–333 ng hF.IX/ml plasma. In summary, nasal administration of hF.IX-derived peptide antigen substantially reduced the risk of an inhibitor response in the context of gene transfer-based treatment of hemophilia B. Therefore, mucosal antigen presentation could be a promising new tool for improved safety of gene therapy or other types of treatments for hemophilia in the context of gene deletions or other settings of increased risk for immune responses to coagulation factors.

Rapid T Cell Response to Vaccination Can Occur without Antibody Response in Children Post HSCT.

Vaccination is widely used to improve pathogen-specific immunity in patients post HSCT, but it is not known whether patients can mount an effective T cell response to vaccine antigens (vAg). Moreover the relationship between T and B cell response to vAg has not been studied. We hypothesized that a sufficiently sensitive assay of T cell response to vAg would allow vaccination to be used as a tool to measure immune recovery post HSCT and improve vaccine design. We therefore: (1) developed a flow-cytometry-based approach to quantify and characterize T cells specific for vAg; (2) validated it by measuring T cell immunity to influenza A in normal donors; and (3) characterized the T and B cell response to influenza vaccination in pediatric HSCT patients. PBMC were labeled with CFSE and stimulated in vitro with whole influenza Ag. Ag-specific T cells were sensitively detected by their proliferation (loss of CFSE fluorescence) and simultaneous expression of the activation marker HLA-DR. Proliferating/active T cells could be readily detected after stimulation with influenza A Ag in healthy adult (n=4) and pediatric (n=19) donors but were absent in control conditions. Both CD4+ and CD8+ T cell proliferation was detected in all donors but one, and in children as young as 6mo. Staining with MHC I- and MHC II-tetramers confirmed that the proliferating/active population contained T cells specific for immunodominant CD8+ and CD4+ epitopes, demonstrating that vAg were processed and presented to epitope-specific T cells. To characterize the phenotype of influenza-specific T cell memory, we separated memory and naive CD4+ cells prior to antigen-stimulation. Antigen-experienced (CD45RA−/CCR7−) but not naive (CD45RA+/CCR7+) T cells proliferated to vAg confirming that the assay detected pre-existing influenza-A-specific T cell memory. We next assessed Influenza-A-specific T cell immunity before and after influenza vaccination in five pediatric HSCT recipients (mean age 10.6y, range 5–15y; mean time from transplant 13m, range 3–21m). Prior to vaccination the CD4 proliferation to influenza-A was a mean of 3.3% (range 0.04–11%). Following vaccination CD4 proliferation increased significantly in all patients (mean 19.0%, range 6.9%–31.8%, p=0.02). This increase was specific as proliferation to control Ag was unchanged. Influenza-A CD8+ proliferation also increased in 3 of 5 patients but was not statistically significant for the group consistent with the limited efficacy of soluble vAg in inducing CD8+ T cell response. All patients had detectable influenza-A-specific IgG levels prior to vaccination but despite a T cell response to vaccination in all patients, none had a significant increase in IgG level following vaccination. Only one patient had an IgM response; this patient also had the highest influenza-A-specific CD4 proliferation before and after immunization suggesting that there may be a threshold of T cell response required for a B cell response. Using a novel assay we demonstrate that a T cell response to vaccination can occur without an accompanying B cell response. This assay provides a more sensitive measure of immunity to vaccination and allows vaccine response to be used as a benchmark of strategies to accelerate post-HSCT T cell reconstitution.

Identification of murine H2-Dd- and H2-Ab-restricted T-cell epitopes on a novel protective antigen, MPT51, of Mycobacterium tuberculosis.

Both CD4(+) type 1 helper T (Th1) cells and CD8(+) cytotoxic T lymphocytes (CTL) play pivotal roles in protection against Mycobacterium tuberculosis infection. Here, we identified Th1 and CTL epitopes on a novel protective antigen, MPT51, in BALB/c and C57BL/6 mice. Mice were immunized with plasmid DNA encoding MPT51 by using a gene gun, and gamma interferon (IFN-gamma) production from the immune spleen cells was analyzed in response to a synthetic overlapping peptide library covering the mature MPT51 sequence. In BALB/c mice, only one peptide, p21-40, appeared to stimulate the immune splenocytes to produce IFN-gamma. Flow cytometric analysis with intracellular IFN-gamma and the T-cell phenotype revealed that the p21-40 peptide contains an immunodominant CD8(+) T-cell epitope. Further analysis with a computer-assisted algorithm permitted identification of a T-cell epitope, p24-32. In addition, a major histocompatibility complex class I stabilization assay with TAP2-deficient RMA-S cells transfected with K(d), D(d), or L(d) indicated that the epitope is presented by D(d). Finally, we proved that the p24-32/D(d) complex is recognized by IFN-gamma-producing CTL. In C57BL/6 mice, we observed H2-A(b)-restricted dominant and subdominant Th1 epitopes by using T-cell subset depletion analysis and three-color flow cytometry. The data obtained are useful for analyzing the role of MPT51-specific T cells in protective immunity and for designing a vaccine against M. tuberculosis infection.

Interferon-$alpha; for hepatitis C: antiviral or immunotherapy?

Interferon-$alpha; for hepatitis C: antiviral or immunotherapy?

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis.

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.

Elimination of an immunodominant CD4+ T cell epitope in human IFN-beta does not result in an in vivo response directed at the subdominant epitope.

The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4(+) T cell epitope peptide region in human IFN-beta, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-beta molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-beta responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-beta molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.