Reactivation of low avidity tumor-specific CD8 T cells associates with immunotherapeutic efficacy of anti-PD-1.

Background: CD8 T cells are a highly diverse population of cells with distinct phenotypic functions that can influence immunotherapy outcomes. Further insights on the roles of CD8 specificities and TCR avidity of naturally arising tumor-specific T cells, where both high and low avidity T cells recognizing the same peptide-major histocompatibility complex (pMHC) coexist in the same tumor, are crucial for understanding T cell exhaustion and resistance to PD-1 immunotherapy.

Methods: CT26 models were treated with anti-PD-1 on days 3, 6 and 9 following subcutaneous tumor implantation generating variable responses during early tumor development.

Tapasin-mediated editing of the MHC I immunopeptidome is epitope specific and dependent on peptide off-rate, abundance, and level of tapasin expression.

Tapasin, a component of the major histocompatibility complex (MHC) I peptide loading complex, edits the repertoire of peptides that is presented at the cell surface by MHC I and thereby plays a key role in shaping the hierarchy of CD8+ T-cell responses to tumors and pathogens. We have developed a system that allows us to tune the level of tapasin expression and independently regulate the expression of competing peptides of different off-rates. By quantifying the relative surface expression of peptides presented by MHC I molecules, we show that peptide editing by tapasin can be measured in terms of "tapasin bonus," which is dependent on both peptide kinetic stability (off-rate) and peptide abundance (peptide supply).

A Mechanistic Model for Predicting Cell Surface Presentation of Competing Peptides by MHC Class I Molecules.

Major histocompatibility complex-I (MHC-I) molecules play a central role in the immune response to viruses and cancers. They present peptides on the surface of affected cells, for recognition by cytotoxic T cells. Determining which peptides are presented, and in what proportion, has profound implications for developing effective, medical treatments.

Peptide-independent stabilization of MHC class I molecules breaches cellular quality control.

The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I molecules are characterized by their conformational mobility in the F-pocket region of the peptide-binding site. We have created a novel variant of an MHC-I protein, K(b)-Y84C, in which two α-helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound high-affinity peptide.

Differential suppression of tumor-specific CD8+ T cells by regulatory T cells.

In the CT26 BALB/c murine model of colorectal carcinoma, depletion of regulatory T cells (Tregs) prior to tumor inoculation results in protective immunity to both CT26 and other BALB/c-derived tumors of diverse histological origin. In this paper, we show that cross-protection can be conferred by adoptively transferred CD8(+) CTLs. Other schedules for inducing immunity to CT26 have been described, but they do not lead to cross-protection.

Absence of tapasin alters immunodominance against a lymphocytic choriomeningitis virus polytope.

Tapasin edits the peptide repertoire presented to CD8(+) T cells by favoring loading of slow off-rate peptides on MHC I molecules. To investigate the role of tapasin on T cell immunodominance we used poxvirus viral vectors expressing a polytope of lymphocytic choriomeningitis virus epitopes with different off-rates. In tapasin-deficient mice, responses to subdominant fast off-rate peptides were clearly favored.

Comparison of virus production in chicken embryo fibroblasts infected with the WR, IHD-J and MVA strains of vaccinia virus: IHD-J is most efficient in trans-Golgi network wrapping and extracellular enveloped virus release.

Modified vaccinia virus Ankara (MVA) is an attenuated strain derived from vaccinia virus (VV) Ankara that grows efficiently in primary chicken embryo fibroblasts (CEFs) and baby hamster kidney cells only. MVA produces significantly more of the enveloped forms of VV in infected CEFs compared with VV strain Copenhagen. In the present study, production of the different infectious forms of VV was compared in CEFs infected with MVA or with two well-characterized replication-competent VV strains, WR and IHD-J.

Generation of recombinant fowlpox virus using the non-essential F11L orthologue as insertion site and a rapid transient selection strategy.

Avipoxviruses show an abortive replication phenotype in mammalian cells and are under evaluation as safe vectors for vaccination. Non-essential gene sequences located in highly conserved regions of virus genomes are considered particularly useful to integrate heterologous DNA. Fowlpox virus F11L orthologue is described in this paper as a suitable locus for insertion into fowlpox virus genome.

Identification and characterization of three immunodominant structural proteins of fowlpox virus.

Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories.