MHC Class I-restricted Epitopes Research Articles

Targeting antigen-loss variant tumors through CD134 plus CD137 dual costimulation-mediated bystander programming (VAC10P.964)

Abstract Tumor-specific cytotoxic CD8 T cell-based therapies can be efficacious in cancer patients. Nevertheless, disease progression often resumes due to outgrowth of antigen-loss variants (ALVs) that lack expression of the targeted MHC class I-restricted epitopes. Co-administration of agonists to the TNF/TNFR costimulatory family members CD134 (OX40) and CD137 (4-1BB) can program antigen-stimulated CD4 T cells to differentiate into cytotoxic Th1 cells, which can directly target MHC II+ tumors and provide classical helper function that facilitates effector differentiation in CD8 T cells responding to a linked epitope. Interestingly, these specific cytotoxic CD4 Th1 cells also imprint antigen-non-responding T cells with a similar ability to express IFN-γ and cytotoxic molecules. Because these bystander-programmed effector T cells are polyclonal, we currently hypothesized that they contain specificities capable of targeting ALVs. In both lymphoma and melanoma models, dual costimulation therapy was markedly enhanced when specific CD4 T cells were engaged to differentiate into cytotoxic Th1 effectors. This augmentation occurred not only when the tumor expressed the MHC class II-restricted epitope recognized by the specific cytotoxic CD4 Th1 cells, but importantly also with tumors modeling ALVs that lack the targeted epitope. Taken together, dual-costimulated tumor-non-specific CD4 T cells provide an alternate form of help that facilitates targeting of ALVs.

High-throughput identification and dendritic cell-based functional validation of MHC class I-restricted Mycobacterium tuberculosis epitopes.

Emergence of drug-resistant strains of the pathogen Mycobacterium tuberculosis (Mtb) and the ineffectiveness of BCG in curtailing Mtb infection makes vaccine development for tuberculosis an important objective. Identifying immunogenic CD8+ T cell peptide epitopes is necessary for peptide-based vaccine strategies. We present a three-tiered strategy for identifying and validating immunogenic peptides: first, identify peptides that form stable complexes with class I MHC molecules; second, determine whether cytotoxic T lymphocytes (CTLs) raised against the whole protein antigen recognize and lyse target cells pulsed with peptides that passed step 1; third, determine whether peptides that passed step 2, when administered in vivo as a vaccine in HLA-A2 transgenic mice, elicit CTLs that lyse target cells expressing the whole protein antigen. Our innovative approach uses dendritic cells transfected with Mtb antigen-encoding mRNA to drive antigen expression. Using this strategy, we have identified five novel peptide epitopes from the Mtb proteins Apa, Mtb8.4 and Mtb19.

Induction of innate immune signatures following polyepitope protein-glycoprotein B-TLR4&9 agonist immunization generates multifunctional CMV-specific cellular and humoral immunity

Recent studies have suggested that a successful subunit human cytomegalovirus (CMV) vaccine requires improved formulation to generate broad-based anti-viral immunity following immunization. Here we report the development of a non-live protein-based vaccine strategy for CMV based on a polyepitope protein and CMV glycoprotein B (gB) adjuvanted with TLR4 and/or TLR9 agonists. The polyepitope protein includes contiguous multiple MHC class I-restricted epitopes with an aim to induce CD8+ T cell immunity, while gB is an important target for CD4+ T cell immunity and neutralizing antibodies. Optimal immunogenicity of this bivalent non-live protein vaccine formulation was dependent upon the co-administration of both the TLR4 and TLR9 agonist, which was associated with the activation of innate immune signatures and the influx of different DC subsets including plasmacytoid DCs and migratory CD8-DEC205+CD103-CD326- langerin-negative dermal DCs into the draining lymph nodes. Furthermore these professional antigen presenting cells also expressed IL-6, IL-12p70, TNFα, and IFNα which play a crucial role in the activation of adaptive immunity. In summary, this study provides a novel platform technology in which broad-based anti-CMV immune responses upon vaccination can be maximized by co-delivery of viral antigens and TLR4 and 9 agonists which induce activation of innate immune signatures and promote potent antigen acquisition and cross-presentation by multiple DC subsets.

Identification of Binding Affinity of TAP Peptides for Development of Fragment based Vaccine from Long neurotoxin 2 [N. naja

Long neurotoxin 2 protein from the venom of Naja naja shows a great affinity towards acetylcholine receptors, which produces peripheral paralysis by blocking neuromuscular transmission at the postsynaptic site. In this study, we use a cascade SVM based TAPPred method which found 26 High affinity TAP Transporter peptide regions. TAP is heterodimeric transmembrane protein which transports antigenic peptide proteins to the endoplasmic reticulum where it binds to MHC class I molecules, These MHC-peptide complexes will be translocated on the surface of antigen presenting cells (APCs), where they are recognized by T-cell receptors to elicit an immune response. Therefore, prediction of TAP-binding peptides is helpful in identifying the MHC class I-restricted T-cell epitopes and hence helpful in the designing of synthetic peptide vaccine.

Abstract 478: Strategy to elicit antigen-specific CD8+ T cell -mediated immune responses against a cryptic CTL epitope of merkel cell polyomavirus large T antigen.

Abstract Merkel cell carcinoma (MCC) is a relatively new addition to the expanding category of oncovirus-induced cancers. Although rare, the number of cases has risen dramatically in recent years. Furthermore, it is an extremely aggressive neoplasm that is correlated with poor patient prognosis. Exacerbating the situation is the limited availability of treatment options for advanced disease. The causative origin of MCC has been identified to be the merkel cell polyomavirus (MCPyV). The MCPyV-encoded large T (LT) antigen is an oncoprotein that is a necessary part of virus-mediated tumorigenesis and therefore, is an excellent MCC antigen for the generation of antitumor immune response. Since LT is a foreign antigen that faces no issue of immune tolerance that normally hinders the induction of antitumor immunity, it is an excellent target for anti-MCC immunotherapy. The present by Savings Sidekick study determined that LT-encoding DNA vaccine can be modified to favor the generation of LT-specific CD8+ T cells by linking the LT antigen to a damage-associated molecular pattern, calreticulin (CRT). This is relevant since the therapeutic effect of the pcDNA3-CRT/LT DNA vaccine was found to be mediated by the LT-specific CD8+ T cells. Since we have identified the MHC class I-restricted immunodominant LT epitope, the development of future modes of immunotherapy, such as peptide-based vaccines and adoptive transfer of LT-specific CD8+ T cells is a real possibility. Citation Format: Bianca P. Gomez, Connie Wang, Raphael Viscidi, Shiwen Peng, Liangmei He, T.-C WU, Chein-Fu Hung. Strategy to elicit antigen-specific CD8+ T cell -mediated immune responses against a cryptic CTL epitope of merkel cell polyomavirus large T antigen. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 478. doi:10.1158/1538-7445.AM2013-478

Abstract P4-04-08: A Potential non-viral vector to transfect dendritic cell and thereby MHC-Class I antigen presentation might be a potential use in carcinoma

Abstract To date almost no remarkable potential non-viral vector was developed to transfect promisingly primary dendritic cell like Bone Marrow Derived Dendritic Cell (BMDC) so that it might generate antigen presentation while epitopic-plasmid was delivered. Here we have introduced a potential non-viral vector named Stearyl-KALA MEND (100–130 nm, zeta potential 35–40 mV), which expressed to date the highest transgene expression while firefly luciferase was introduced (sub-cloned), and thereby promising antigen presentation in vitro while OVA plasmid containing MHC Class-I restricted SIINFEKL epitope was introduced and co-cultured with B3Z T-cell hybridoma. On the basis of these results, we used this vector to transfect BMDC and harvested the DC to chase the corresponding EG7-OVA induced tumor ex vivo. Later we further immunized mice directly with STR-KALA MEND containing OVA plasmid and challenged against the tumor (EG7-OVA induced). In this in vivo study we found also a significant antitumor activity. To evaluate the promptness of our vector we further sub-cloned Mart-1 gene, in our sub-cloned plasmid vector and immunized the mice as before. Thereafter inserting B16F10 melanoma cells to the immunized mice we found also a significant antitumor activity after 24 days of inoculation. Thus the vector, STR-KALA MEND might be a future potential use as DNA vaccine in anti-tumor methodology. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-08.

Steric recognition of T‐cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.

Inflammation-Associated Nitrotyrosination Affects TCR Recognition through Reduced Stability and Alteration of the Molecular Surface of the MHC Complex

Nitrotyrosination of proteins, a hallmark of inflammation, may result in the production of MHC-restricted neoantigens that can be recognized by T cells and bypass the constraints of immunological self-tolerance. Here we biochemically and structurally assessed how nitrotyrosination of the lymphocytic choriomeningitis virus (LCMV)-associated immunodominant MHC class I-restricted epitopes gp33 and gp34 alters T cell recognition in the context of both H-2Db and H-2Kb. Comparative analysis of the crystal structures of H-2Kb/gp34 and H-2Kb/NY-gp34 demonstrated that nitrotyrosination of p3Y in gp34 abrogates a hydrogen bond interaction formed with the H-2Kb residue E152. As a consequence the conformation of the TCR-interacting E152 was profoundly altered in H-2Kb/NY-gp34 when compared to H-2Kb/gp34, thereby modifying the surface of the nitrotyrosinated MHC complex. Furthermore, nitrotyrosination of gp34 resulted in structural over-packing, straining the overall conformation and considerably reducing the stability of the H-2Kb/NY-gp34 MHC complex when compared to H-2Kb/gp34. Our structural analysis also indicates that nitrotyrosination of the main TCR-interacting residue p4Y in gp33 abrogates recognition of H-2Db/gp33-NY complexes by H-2Db/gp33-specific T cells through sterical hindrance. In conclusion, this study provides the first structural and biochemical evidence for how MHC class I-restricted nitrotyrosinated neoantigens may enable viral escape and break immune tolerance.

Comparison of antibodies and carbohydrates to target vaccines to human dendritic cells via DC-SIGN

Vaccine efficacy is improved upon specific delivery to professional antigen (Ag) presenting cells, such as dendritic cells (DCs). Antigenicity and adjuvanticity of vaccine components can be enhanced by encapsulation within nanoparticle (NP) vaccine carriers that are targeted to the human DC-specific C-type lectin receptor DC-SIGN. Here we used two strategies to target vaccines components to DC-SIGN: 1) carbohydrates as natural receptor ligands and 2) receptor-specific antibodies (Abs). To determine the optimal targeting strategy, we coated NP vaccines harboring MHC class I or II-restricted Ags and the TLR ligands (TLRLs) poly I:C and resiquimod with either the DC-SIGN ligands Lewis-X (Lex), mannosylated lipoarabinomannan (ManLAM), glycosylated HIV protein gp120, or three distinct DC-SIGN Abs. Although, because of their lower MW, surface coating of NP vaccines with carbohydrates resulted in a higher number of surface molecules per NP than coating with Abs, NP vaccines carrying Abs were more effectively bound and internalized by human DCs than carriers harboring Lex, ManLAM or gp120. Furthermore, NP vaccines harboring TLRLs triggered significant induction of DC maturation markers when compared to those without TLRLs, irrespective of the targeting moiety. Ab- and gp120-mediated targeting induced equally high levels of proinflammatory cytokines and increased presentation of the MHC class I-restricted epitope. By contrast, presentation of the MHC class II-restricted epitope was more efficient upon Ab-mediated targeting than when using gp120, Lex or ManLAM. From these findings we conclude that receptor-specific Abs are more effective than carbohydrates for DC-targeted vaccination strategies.

Vaccination with ΔCD40L or Flagellin Gene-Modified T Cells Activates Dendritic Cells In Vivo and Induces a Potent Anti-Tumor Immune Response

Abstract 1909 Introduction:Cancer vaccines have shown promise in small animal models of cancer, but have thus far been disappointing in clinical settings. Successful induction of a systemic and long-term anti-tumor immune response following vaccination is dependent on delivery of tumor-associated antigens to lymphoid tissues, in combination with the activation of professional antigen presenting cells (APCs). Here we describe a novel live T cell vaccine (TCV) that delivers antigenic peptides to secondary lymph nodes while simultaneously activating endogenous dendritic cells (DCs) through transgenic expression of CD40L or bacterial flagellin (fliC). Methods:To generate TCVs, murine splenocytes were isolated from wild-type type C57BL/6 mice. Following activation with anti-CD3/anti-CD28 microbeads, splenocytes were transduced with pRV2011-luciferase-IRES-Thy1.1, pRV2011-CD40L-IRES-Thy1.1 or pRV2011-fliC-IRES-Thy1.1 retrovirus. Analysis of TCV migration to lymphoid organs was performed by bioluminescence imaging for firefly luciferase. Following transduction with CD40L and fliC molecules, TCVs were measured for transduction efficiency (Thy1.1) and transgene expression using FACS analysis of CD40L or by Western blot, respectively. TCVs were subsequently pulsed with MHC class I-restricted epitopes for ovalbumin257-264 (SIINFEKL) or Trp2180-188 (SVYDFFVWL) peptides and injected intravenously at a dose of 1×107 TCVs per vaccination. To test the protective effects of TCVs, C57BL/6 mice were immunized at days 0 and 14 and then challenged with either 5×105 B16-OVA (for TCV-SIIN) or parental B16.F10 (for TCV-SVYD) melanoma tumor cells. To examine the ability of TCVs to eliminate established tumors, mice received B16-OVA or B16.F10 tumor cells followed by vaccination with TCVs on days 3, 9 and 15. Immunological studies were performed on a subset of mice (n=5 per group) to analyze induction of tumor-specific T cells using tetramer and IFN-g ELIspot assays. In vivo activation of lymph node DCs was performed by FACS analysis for CD11c+ DC co-expressing CD86 and I-A/I-E mouse MHC class II antibodies. Results:Following activation, TCVs were efficiently transduced with retrovirus (>85% CD40L) or expressed high levels of fliC. Bioluminescent imaging showed that luciferase-expressing TCVs rapidly migrated to lymphoid organs including the spleen and cervical and inguinal lymph nodes demonstrating the capacity of TCVs to co-localize with professional APCs. Importantly, irradiation (30 Gy) of TCVs completely abrogated migration and persistence highlighting the requirement for live TCVs. Next we examined whether TCV-CD40L or TCV-fliC could induce a protective immune response against B16 tumors. Administration of TCV-fliC-SIIN (OVA) and TCV-CD40L-SIIN primed peptide-specific CD8+ T cells, and led to decreased tumor growth and increased survival in mice subsequently challenged with B16-Ova (p<0.05). This response corresponded with a statistically significant (p<.05) increase in SIIN-specific CD8+ T cells as measured by tetramer FACS analysis and IFN-g ELIspot assays. Vaccination of mice with established tumors showed similar tumor suppression with both TCV designs (p<05). As OVA is a xenogenic antigen, we next examined whether TCVs pulsed with Trp2 peptide (SVYD) could induce similar protective effects. While vaccination with SVYD-pulsed T cells alone (no gene modification) did not inhibit tumor growth, expression of CD40L or fliC by TCV pulsed with Trp2 peptide suppressed B16.F10 tumor proliferation and increased survival in mice with pre-established tumors (p<.05). As found in the B16.OVA experiments, immunological protection correlated with a dramatic increase in SVYD-specific CD8+ T cells in the spleen, tumor draining lymph nodes and tumor. Conclusions:The efficient delivery of tumor-associated antigens to lymphoid tissues by TCVs overcomes a major limitation of alternative vaccine strategies. Vaccination with peptide-pulsed TCVs primes antigen-specific T cell responses with anti-tumor capability, and endogenous DC maturation leads to the inhibition of established B16-Ova and B16-F10 tumors. This illustrates the role of endogenous DC as mediators of the vaccine response and demonstrates the effectiveness of using TCVs to deliver antigen in the context of DC activating molecules. Disclosures:No relevant conflicts of interest to declare.

Bis-(3′,5′)-cyclic dimeric adenosine monophosphate: Strong Th1/Th2/Th17 promoting mucosal adjuvant

New effective adjuvants are required to improve the performance of subunit vaccines. Here, we showed that bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP), a second messenger molecule in bacteria and archaea, exerts strong adjuvant activities when delivered by mucosal route. In vitro studies showed that c-di-AMP was able to both stimulate pre-activated murine macrophages and promote the activation and maturation of dendritic cells of murine and human origin. Co-administration of c-di-AMP with β-galactosidase (β-Gal) by intranasal route to BALB/c mice resulted in the elicitation of significantly higher serum antigen-specific IgG titres than in controls. The induction of local immune responses was shown by the production of antigen-specific secretory IgA in different mucosal territories. In addition, strong cellular immune responses were observed against both the β-Gal protein and a peptide encompassing its MHC class I-restricted epitope. The ratio of β-Gal-specific antibodies and the secreted cytokine profiles by in vitro re-stimulated splenocytes suggested that a balanced Th1/Th2/Th17 response pattern is promoted by c-di-AMP. When C57BL/6 mice were immunized with OVA and c-di-AMP, vigorous in vivo CTL responses were also observed. These results indicated that c-di-AMP exhibits a high potential as adjuvant for the development of mucosal vaccines, in particular when cellular immunity is needed.

An Early HIV Mutation within an HLA-B*57-Restricted T Cell Epitope Abrogates Binding to the Killer Inhibitory Receptor 3DL1

Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.

Recognition of a High Affinity MHC Class I-Restricted Epitope of Myelin Oligodendrocyte Glycoprotein by CD8+ T Cells Derived from Autoantigen-Deficient Mice

CD4+ T cells have a well-defined pathogenic role in experimental autoimmune encephalomyelitis, the rodent model of multiple sclerosis (MS), yet CD8+ T cells are commonly found in MS lesions. To determine whether immunological tolerance might impact differently on CD4+ versus CD8+ T cells, we studied T cell responses in mice genetically deficient for the central nervous system (CNS) autoantigen myelin oligodendrocyte glycoprotein (MOG) versus wild type (WT) C57BL/6 mice. We show that MOG−/− mice have enhanced sensitivity to immunization with the immunodominant peptide of MOG (35–55), as evidenced by increased expansion of both CD4+ and CD8+ T cell subsets. Most strikingly, CD8+ T cells from MOG−/− mice responded to a novel T cell epitope which binds to MHC class I with high affinity. Despite this, MOG-responsive CD8+ T cells sourced from either WT or MOG−/− mice failed to initiate CNS inflammation upon transfer to MOG-sufficient mice. In our hands, this capacity was only found in CD4+ T cells. However, MOG−/− CD4+ cells did not show greater pathogenic activity than their WT counterparts. Our data indicate that, in the presence of endogenous MOG, CD8+ T cells capable of responding to a MHC class I-restricted epitope that can be stably expressed are subject to rigorous control through central and/or peripheral tolerance.

Bryostatin-I: A dendritic cell stimulator for chemokines induction and a promising adjuvant for a peptide based cancer vaccine

Bryostatin-1 (Bryo-1), a PKC modulator, was previously shown to activate monocytes and lymphocytes to produce cytokines. In this report, we investigated the adjuvanticity of Bryo-1 both in vitro and in vivo. First, Bryo-1 was found to induce the release of CCL2 and CCL3 from mouse bone marrow-derived dendritic cells (BMDC) in a dose-dependent manner. As little as 0.1 nM Bryo-I induced release of chemokines from BMDC and the maximal induction could be achieved at 5–10 nM. Both PKC and ERK inhibitors attenuated the release of CCL2 and CCL3. Consistently, Western blot indicated that Bryo-I activated ERK in a dose- and time-dependent manner. Experiments with the NF-κB inhibitor, MG-132, demonstrated that NF-κB was involved in the induction of CCL2 but not CCL3. Because chemokines have been demonstrated to have profound effects on immune reactions by regulating the trafficking of DC and other lymphocytes into lymphoid organs, Bryo-I was tested as an adjuvant in an E7 peptide (MHC class I-restricted peptide epitope derived from human papillomavirus (HPV) 16 E7 protein)-based cancer vaccine. Mice immunized by s.c. injection with Bryo-I/E7 had enlarged draining lymph nodes and showed an antigen specific T-cell response demonstrated by the release of IFN-γ from isolated splenocytes and in vivo CTL activity. Finally, immunization with Bryo-I/E7 totally prevented the E7-expressing TC-1 tumor growth in mice. In conclusion, for the first time, we demonstrated that Bryo-I induced chemokine release from dendritic cell and was an effective adjuvant for peptide cancer vaccine.

Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein

BackgroundPlasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1.MethodsA recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes.ResultsFourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure.ConclusionsThis study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations.

Type‐1 polarized dendritic cells loaded with apoptotic prostate cancer cells are potent inducers of CD8+ T cells against prostate cancer cells and defined prostate cancer‐specific epitopes

In order to develop improved vaccines for patients with recurrent prostate cancer (PCa), we tested the feasibility of using type-1 polarized dendritic cells (αDC1s) to cross-present antigens from allogeneic PCa cells and to induce functional CD8(+) T cell responses against PCa cells and against defined MHC class I-restricted PCa-relevant epitopes. Monocyte-derived DCs from PCa patients were matured using the "standard" cytokine cocktail (IL-1β/TNFα/IL-6/PGE₂) or using the αDC1-polarizing cocktail (IL-1β/TNFα/IFNα/IFNγ/poly-I:C), loaded with UV-irradiated LNCaP cells, and used to sensitize autologous CD8(+) T cells. αDC1s from PCa patients secreted 10-30 times higher levels of IL-12p70 than sDCs. Importantly this elevated capacity for IL-12p70 secretion was not inhibited by loading with apoptotic tumor cells. Compared to standard DCs, αDC1s induced higher numbers of CD8(+) T cells capable of recognizing both the original PCa cells as well as another PCa cell line, DU145, in MHC class I-restricted fashion. Furthermore, αDC1s induced higher numbers of CD8(+) T cells recognizing defined PCa-specific class I-restricted peptide epitopes of prostate-specific antigen and prostatic acid phosphatase: PAP(135-143) (average 49-fold higher), PAP(112-120) (average 24-fold), PSA(141-150) (average 5.5-fold), and PSA(146-154) (average 11-fold). Type-1 polarization of GM-CSF/IL-4-generated DCs enhances their ability to present allogeneic tumor cells and to induce CD8(+) T cells recognizing different PCa cells and multiple defined PCa-specific epitopes. These observations help to develop improved immunotherapies of PCa for patients with different HLA types and lacking autologous tumor material.

Folding of Matrix Metalloproteinase-2 Prevents Endogenous Generation of MHC Class-I Restricted Epitope

BackgroundWe previously demonstrated that the matrix metalloproteinase-2 (MMP-2) contained an antigenic peptide recognized by a CD8 T cell clone in the HLA-A*0201 context. The presentation of this peptide on class I molecules by human melanoma cells required a cross-presentation mechanism. Surprisingly, the classical endogenous processing pathway did not process this MMP-2 epitope.Methodology/Principal FindingsBy PCR directed mutagenesis we showed that disruption of a single disulfide bond induced MMP-2 epitope presentation. By Pulse-Chase experiment, we demonstrated that disulfide bonds stabilized MMP-2 and impeded its degradation. Finally, using drugs, we documented that mutated MMP-2 epitope presentation used the proteasome and retrotranslocation complex.Conclusions/SignificanceThese data appear crucial to us since they established the existence of a new inhibitory mechanism for the generation of a T cell epitope. In spite of MMP-2 classified as a self-antigen, the fact that cross-presentation is the only way to present this MMP-2 epitope underlines the importance to target this type of antigen in immunotherapy protocols.

Superior Antitumor Response Induced by Large Stress Protein Chaperoned Protein Antigen Compared with Peptide Antigen

Our previous studies have demonstrated that the natural chaperone complexes of full-length tumor protein Ags (e.g., gp100) and large stress proteins (e.g., hsp110 and grp170) with exceptional Ag-holding capabilities augment potent tumor protective immunity. In this study, we assess the peptide-interacting property of these large chaperones and, for the first time, compare the immunogenicity of the recombinant chaperone vaccines targeting two forms of Ags (protein versus peptide). Both hsp110 and grp170 readily formed complexes with antigenic peptides under physiologic conditions, and the peptide association could be further stimulated by heat shock. The large chaperones displayed similar but distinct peptide-binding features compared with hsp70 and grp94/gp96. Immunization with hsp110- or grp170-tyrosinase-related protein 2 (TRP2(175-192)) peptide complexes effectively primed CD8(+) T cells reactive with TRP2-derived, MHC class I-restricted epitope. However, the tumor protective effect elicited by the TRP2(175-192) peptide vaccine was much weaker than that achieved by full-length TRP2 protein Ag chaperoned by grp170. Furthermore, immunization with combined chaperone vaccines directed against two melanoma protein Ags (i.e., gp100 and TRP2) significantly improved overall anti-tumor efficacy when compared with either of the single Ag vaccine. Lastly, treatment of tumor-bearing mice with these dual Ag-targeted chaperone complexes resulted in an immune activation involving epitope spreading, which was associated with a strong growth inhibition of the established tumors. Our results suggest that high m.w. chaperones are superior to conventional chaperones as a vaccine platform to deliver large protein Ags, and provide a rationale for translating this recombinant chaperoning-based vaccine to future clinical investigation.

MHC-I-restricted melanoma antigen specific TCR-engineered human CD4+ T cells exhibit multifunctional effector and helper responses, in vitro

MHC class I-restricted human melanoma epitope MART-1(27-35) specific TCR-engineered CD4+CD25- T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25- T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-gamma, TNF-alpha, IL-2, and MIP1ss), lyse target cells, and "help" the expansion of the MART-1(27-35) specific CD8+ T cells when stimulated by the MART-1(27-35) peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-1(27-35) specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.

Abstract 1925: MHC class I-restricted MART-127-35 specific TCR engineered human CD4+ and CD8+ T cells exhibit polyfunctional properties

Abstract The “polyfunctional” property of antigen specific T cells has turned out to be an important element in successful host immune responses in AIDS and in malignancies. We have shown that human CD4+ CD25- T cells engineered to express MHC Class I-restricted MART-127-35 epitope specific TCR show cytolytic activity, secret Th1 type cytokines. We have now carried out an extended analysis of the functional dimension of MART-127-35 epitope specific TCR engineered CD4+ and CD8+ T cells. Freshly isolated circulating CD4+ and CD8+ T cells, transduced with MART-127-35 epitope specific DMF5 TCR containing retrovirus, were tested for proliferative, cytokine secretory and for cytolytic capacity (polyfunctionality analyzed with intracytoplasmic immunofluorescence staining for IL-2, IFN-γ, TNF-α, MIP-1β and CD107a using the SPICE software). The TCR engineered CD4+ T cells were also tested for their potential for “helping” cytolytic T cells response by CD8+ T cells in in vitro CTL generation cultures. The TCR-engineered CD4+ as well as CD8+ T cells were found to be polyfunctional. For example, they underwent multiple rounds of division following stimulation by peptide pulsed dendritic cells and by melanoma cells, synthesized multiple cytokines (IFN-γ, TNF-α, IL-2, MIP1ß) and lysed target cells. The TCR engineered CD4+ T cells also amplified the CD8+ CTL burst size in vitro. Multiparametric analyses revealed that a single TCR-engineered CD4+ T cell could perform as many as five different functions. Nearly 100% MART-127-35 specific TCR expressing CD4+ as well as CD8+ T cells could be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR-transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies. 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 1925.