Stable Antigen Is Most Effective for Eliciting CD8+T-Cell Responses after DNA Vaccination and Infection with Recombinant Vaccinia VirusIn Vivo

The induction of strong CD8+T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8+T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VVin vitrowas enhanced in the presence of short-lived antigens.In vivo, however, the highest induction of NP-specific CD8+T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responsesin vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.

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NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-presentation and CD8 T Cell Responses

10.1101/570705 ◽

2019 ◽

Author(s):

Daniele Corridoni ◽

Seiji Shiraishi ◽

Thomas Chapman ◽

Tessa Steevels ◽

Daniele Muraro ◽

...

Keyword(s):

T Cell ◽

Mhc Class I ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Responses ◽

Class I ◽

Cross Presentation ◽

Cell Responses ◽

Direct Cross

AbstractNOD2 and TLR2 recognize components of bacterial cell wall peptidoglycan and direct defense against enteric pathogens. CD8+ T cells are important for immunity to such pathogens but how NOD2 and TLR2 induce antigen specific CD8+ T cell responses is unknown. Here, we define how these pattern recognition receptors (PRRs) signal in primary dendritic cells (DCs) to influence MHC class I antigen presentation. We show NOD2 and TLR2 phosphorylate PI31 via TBK1 following activation in DCs. PI31 interacts with TBK1 and Sec16A at endoplasmic reticulum exit sites (ERES), which positively regulates MHC class I peptide loading and immunoproteasome stability. Following NOD2 and TLR2 stimulation, depletion of PI31 or inhibition of TBK1 activity in vivo impairs DC cross-presentation and CD8+ T cell activation. DCs from Crohn’s patients expressing NOD2 polymorphisms show dysregulated cross-presentation and CD8+ T cell responses. Our findings reveal unidentified mechanisms that underlie CD8+ T cell responses to bacteria in health and in Crohn’s.

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Saccharomyces cerevisiae as delivery vehicle for a NY-ESO-1 protein vaccine

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.2578 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 2578-2578

Author(s):

C. Renner ◽

G. Held ◽

F. Neumann ◽

S. Kleber ◽

M. Thiel ◽

...

Keyword(s):

T Cell ◽

Mhc Class I ◽

T Cell Responses ◽

Full Length ◽

Class I ◽

Strong Positive Correlation ◽

Cell Recognition ◽

T Cell Recognition ◽

Cross Presentation ◽

Cell Responses

2578 Background: Vaccine strategies that target or activate dendritic cells in order to elicit potent cellular immunity are currently the subject of intense research. Here we report that genetically engineered yeast expressing the full-length tumor associated antigen NY-ESO-1 are a versatile host for protein production eliciting MHC class I and II T-cell responses. Methods: The pYD1 yeast display vector was chosen for full length NY-Eso-1 protein (pNY-ESO-1) expression. NY-ESO-1 and SSX-2 (as control) protein were affinity purified on. IFN-g ELISPOT assays were performed in triplicates on nitrocellulose-lined 96-well plates. MHC class I cross-presentation of peptide epitopes was demonstrated by blocking T-cell responses against DCs. For this purpose, antigen or peptide pulsed DCs were labeled with different doses (100, 10, 1 μg/ml) of antibodies specific for HLA-A2/peptide complexes (HLA-A2/ NY-Eso-1157–165; 3M4E5) or an irrelevant antibody (specific for HLA-A2/IMP58–66) as control. Results: Highest level of NY-ESO-1 expression was detected on the cell wall of wt EBY100 strain with lower expression levels on PMT deficient strains PMT-2 and PMT-4. After protein feeding of immature DCs, NY-ESO-1 157–165 peptide cross-presentation was detected by 3M4E5 and an antigen-specific CD8+ T cell clone. There was a strong positive correlation between the amount of Aga2p-NY-ESO-1 protein (0–15μg/ml) and peptide presentation. Specific T-cell recognition of NY-Eso-1 157–165/HLA-A2 complexes was validated by blocking experiments with Fab 3M4E5. Pre-incubation of protein fed DCs with the antibody at different concentrations (0–100 μg/ml) resulted in a significant reduction (p< 0.05) of spot numbers. Efficient presentation and T-cell recognition of epitope 157–165 was only adequately detectable when protein produced by EBY100 wt yeast strain was used (p < 0.05). MHC class II presentation was studied in an autologous setting using a T-cell line recognising the NY-ESO-1 157–170 in HLA-DP4 context revealing that NY-ESO-1 protein produced in yeast was efficiently taken up and presented. Conclusions: Together, these data add further evidence that yeast expressing recombinant proteins can be used for vaccine purposes and that antigen uptake in APC depends on glycoslation of yeast expressed antigens. No significant financial relationships to disclose.

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Sequence Dependent Efficiency of Cross-Presentation in MHC Class I Requires Rational Design of Long Synthetic Peptides for Vaccination or Ex Vivo Activation

Blood ◽

10.1182/blood.v112.11.3904.3904 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3904-3904

Author(s):

Maarten L. Zandvliet ◽

J.H. Frederik Falkenburg ◽

Michel G.D. Kester ◽

Arnoud H. de Ru ◽

Peter A. van Veelen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class I ◽

Cd8 T Cells ◽

Synthetic Peptides ◽

Ex Vivo ◽

Class I ◽

Cross Presentation ◽

Cell Responses

Abstract For the induction or boosting of antigen-specific CD8+ T cell responses, long synthetic peptides have been used in vaccination studies. Superior in vivo CD8+ T cell responses have been reported following vaccination with long peptides compared with minimal peptides, which was attributed to selective uptake and cross-presentation by professional antigen-presenting cells. Furthermore, to generate antigen-specific T cell lines for adoptive immunotherapy or to measure antigen-specific T cell responses, protein-spanning pools of overlapping long synthetic peptides can be used to simultaneously activate CD8+ and CD4+ T cells in peripheral blood mononuclear cells (PBMC) ex vivo. Although exogenous antigen is predominantly presented in MHC class II, it has been suggested that cross-presentation of long peptides in MHC class I can occur. However, the mechanism of cross-presentation of exogenous long peptides in MHC class I is not clear. Various models for cross-presentation have been described following uptake of soluble antigen in endosomes, among which antigen transport over the endosomal membrane followed by the classical proteasome- and TAP-dependent route, and entrance of MHC class I in the recycling endocytic MHC class II pathway where peptidase-trimmed exogenous antigens can exchange with peptides in the MHC class I molecules, resulting in TAP- and proteasome-independent cross-presentation. To improve the design of peptides for the in vivo or ex vivo activation of CD8+ T cells we investigated the mechanism and efficiency of cross-presentation of long peptides. We observed that antigen-presenting cells in peripheral blood, in particular monocytes, loaded with 15-mer peptides, 31-mer peptides or full length protein containing the NLV epitope were able to very efficiently induce IFNg production by cytomegalovirus (CMV) pp65 NLV-specific T cells. Specific T cells were most efficiently activated by N-terminally extended variants of the minimal epitope, while the use of C-terminally extended variants resulted in a 10-fold reduction of activation efficiency. Purification of these antigens by high performance liquid chromatography (HPLC) followed by mass spectrometry demonstrated that activation was not caused by contamination with the minimal epitope sequence. Also CD8+ T cells specific for other CMV and minor histocompatibility antigen (mHag) epitopes were activated by monocytes loaded with 15-mer or 20-mer peptides. Again N-terminally extended variants of minimal epitopes very efficiently induced activation, while the use of C-terminally variants or full length protein resulted in highly variable efficiency of activation, ranging from 10-fold reduction to complete absence of activation. Interestingly, TAP-deficient T2 cells loaded with CMV pp65 NLV antigens also efficiently activated NLV-specific T cells, indicating that the route of presentation was TAP-independent. Addition of lactacystin during loading of monocytes with CMV pp65 NLV 15-mer did not affect activation of specific T cells, suggesting that cross-presentation was proteasome-independent. Addition of primaquine reduced activation of specific T cells by the NLV 15-mer peptide, but not by the minimal NLV 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. To compare cross-presentation with presentation of endogenously synthesized antigen, TAP-competent T1 and TAP-deficient T2 cells were retrovirally transduced with the CMV pp65 gene. CMV pp65-specific T cells were activated by CMV pp65 transduced T1 but not T2 cells, indicating that endogenously synthesized CMV pp65 required processing and presentation by the classical proteasome- and TAP-dependent route. These data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling MHC class I molecules. Not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. As the efficiency of cross-presentation of long synthetic peptides may depend on the sequence of the C-terminal extension, a rational design of peptides is crucial for efficient activation of CD8+ T cells in approaches of vaccination, adoptive transfer and immune monitoring.

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Chemokine receptor targeting efficiently directs antigens to MHC class I pathways and elicits antigen-specific CD8+ T-cell responses

Blood ◽

10.1182/blood-2005-08-3207 ◽

2006 ◽

Vol 107 (12) ◽

pp. 4597-4605 ◽

Cited By ~ 44

Author(s):

Roberta Schiavo ◽

Dolgor Baatar ◽

Purevdorj Olkhanud ◽

Fred E. Indig ◽

Nicholas Restifo ◽

...

Keyword(s):

T Cell ◽

Mhc Class I ◽

T Cell Responses ◽

Class I ◽

Cell Trafficking ◽

Cross Presentation ◽

Cell Responses ◽

Inflammatory Conditions ◽

Processing Pathways ◽

Chemokine Ligand

Abstract Chemokines are key controllers of cell trafficking and are involved in numerous pathologic and inflammatory conditions. However, the fate of a chemokine ligand, once it is endocytosed with its receptor, remains obscure. Here, using chemokine–tumor antigen fusion constructs, we demonstrate for the first time that chemokines are internalized to early/late endosomal and lysosomal compartments through a clathrin-dependent process and subsequently delivered to the cytosol for proteasomal processing, facilitating efficient cross-presentation to the TAP-1–dependent MHC class I processing pathway. These data not only elucidate the intracellular fate of chemokine ligands upon receptor uptake, but also demonstrate the superior carrier potency of chemokines for delivering self-antigens to both class I and II processing pathways to induce CD8+ and CD4+ T-cell responses.

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608 Immunodominant listeria epitopes compete with vaccine-directed cd8+ t-cell responses rescued by peptide-MHC stabilizing modifications

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0608 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A644-A644

Author(s):

John Flickinger ◽

Jagmohan Singh ◽

Yanki Yarman ◽

Robert Carlson ◽

Scott Waldman ◽

...

Keyword(s):

Listeria Monocytogenes ◽

T Cell ◽

Cell Line ◽

Mhc Class I ◽

T Cell Responses ◽

Cd8 T Cell ◽

Class I ◽

Peptide Ligand ◽

Altered Peptide Ligand ◽

Cell Responses

BackgroundThe Gram-positive bacterium Listeria monocytogenes (Lm) is a promising vector for cancer immunotherapy due to its ability to directly infect antigen-presenting cells, induce potent CD8+ T-cell immunity, and remodel immunosuppressive tumor microenvironments.1 Recent clinical trials have demonstrated safety and immunogenicity of Lm-based cancer vaccines in lung, cervical, pancreatic, and other cancers. In colorectal cancer, the transmembrane receptor guanylyl cyclase C (GUCY2C) is an emerging target for immunotherapy.2 Here, we examined the immunogenicity of a recombinant strain of Listeria monocytogenes secreting GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination induced robust Lm-specific CD8+ T-cell immunity but failed to prime GUCY2C-specific CD8+ T-cell responses. These studies explore the hypothesis that immunodominant Lm antigens suppress primary immunity to subdominant GUCY2C epitopes in Lm-GUCY2CMethodsLm-GUCY2C expresses the extracellular domain of mouse GUCY2C23-429 downstream of an ActA promoter integrated into the genome of the live, attenuated delta actA delta inlB Lm strain. Altered peptide ligands were designed based on NetMHCpan 4.0 peptide-MHC binding algorithms and similarly cloned into Lm. Peptide-MHC class I complex stability was quantified by FACS-based surface peptide-MHC dissociation on the TAP-deficient cell line, RMA-S H-2Kd.3In vivo efficacy studies employed IFNγ-ELISpot quantification of T-cell responses and tumor challenge studies with the CT26 colorectal cancer cell line. Adenovirus expressing GUCY2C was used as a positive control.2 4ResultsLm-GUCY2C vaccination of BALB/c mice generated Lm-specific CD8+ T-cell responses but an absence of GUCY2C-specific immunity. Peptide-MHC stability studies revealed poor stability of the dominant GUCY2C254-262 epitope complexed with H-2Kd compared to H-2Kd-restricted Lm epitopes derived from the LLO and p60 Lm antigens. Mutation of the GUCY2C254-262 peptide at critical anchoring residues for binding H-2Kd revealed that the altered peptide ligand with an F255Y mutation significantly improved the stability of the GUCY2C254-262-H-2Kd complex. Similarly, vaccination of mice with recombinant Lm-GUCY2C expressing the altered peptide ligand (Lm-GUCY2CF255Y) restored GUCY2C immunogenicity and antitumor immunity.ConclusionsImmunodominant Lm antigens may interfere with immune responses directed to the vaccine target antigen GUCY2C by competing with GUCY2C epitope for MHC class I binding and presentation. Moreover, use of a substituted GUCY2C -peptide ligand with enhanced peptide-MHC class I stability restored GUCY2C-specific immunity in the context of Lm-GUCY2C, an approach that can be translated to patients. Importantly, these studies also suggest that ongoing Lm-based vaccine development programs targeting a variety of antigens in other cancer types may be similarly limited by the immunodominance of Lm epitopes.AcknowledgementsThe authors thank Dr. Peter Lauer for providing the pPL2 integration vector used in cloning Lm-GUCY2C and Dr. Sean Murphy for providing the RMA-S H-2Kd cell line.Ethics ApprovalStudies were approved by the Thomas Jefferson University IACUC (Protocol # 01956).ReferencesFlickinger JC, Rodeck U, Snook AE. Listeria monocytogenes as a Vector for Cancer Immunotherapy: Current Understanding and Progress. Vaccines (Basel) 2018;6. doi:10.3390/vaccines6030048.Snook AE, Baybutt TR, Xiang B, Abraham TS, Flickinger JC, Hyslop T, et al. Split tolerance permits safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients. J Immunother Cancer 2019;7:104. doi:10.1186/s40425-019-0576-2.Müllbacher A, Lobigs M, Kos FJ, Langman R. Alloreactive cytotoxic T-cell function, peptide nonspecific. Scand J Immunol 1999;49:563–9.Flickinger J. JC, Singh J, Carlson R, Leong E, Baybutt T, Barton J, et al. Chimeric Ad5.F35 vector evades anti-adenovirus serotype 5 neutralization opposing GUCY2C-targeted antitumor immunity. J Immunother Cancer 2020.

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