PTEN Loss Confers Resistance to Anti-PD-1 Therapy in Non-Small Cell Lung Cancer by Increasing Tumor Infiltration of Regulatory T Cells.

Immunotherapy resistance in non-small cell lung cancer (NSCLC) may be mediated by an immunosuppressive microenvironment, which can be shaped by the mutational landscape of the tumor. Here, we observed genetic alterations in the PTEN/PI3K/AKT/mTOR pathway and/or loss of PTEN expression in >25% of patients with NSCLC, with higher frequency in lung squamous carcinomas (LUSC). Patients with PTEN-low tumors had higher levels of PD-L1 and PD-L2 and showed worse progression-free survival when treated with immunotherapy. Development of a Pten-null LUSC mouse model revealed that tumors with PTEN loss were refractory to antiprogrammed cell death protein 1 (anti-PD-1), highly metastatic and fibrotic, and secreted TGFß/CXCL10 to promote conversion of CD4+ lymphocytes into regulatory T cells (Treg). Human and mouse PTEN-low tumors were enriched in Tregs and expressed higher levels of immunosuppressive genes. Importantly, treatment of mice bearing Pten-null tumors with TLR agonists and anti-TGFß antibody aimed to alter this immunosuppressive microenvironment and led to tumor rejection and immunologic memory in 100% of mice. These results demonstrate that lack of PTEN causes immunotherapy resistance in LUSCs by establishing an immunosuppressive tumor microenvironment that can be reversed therapeutically. SIGNIFICANCE: PTEN loss leads to the development of an immunosuppressive microenvironment in lung cancer that confers resistance to anti-PD-1 therapy, which can be overcome by targeting PTEN loss-mediated immunosuppression.

Human Oral Epithelial Cells Impair Bacteria-Mediated Maturation of Dendritic Cells and Render T Cells Unresponsive to Stimulation.

The oral mucosa is a first line of defense against pathogenic organisms and yet tolerates food antigens and resident bacteria. Mucosal epithelial cells are emerging as important regulators of innate and adaptive immune responses. However, the contribution of oral epithelial cells (OECs) determining oral immunity is understudied. Here, we evaluated the ability of H413 and TR146 cells, two OEC lines derived from human oral squamous cell carcinomas, and primary OECs to modulate immune responses to a cocktail of Gram+ and Gram- bacteria known as MV130. OECs expressed CD40 constitutively and class II major histocompatibility complex (MHC II) molecules when stimulated with IFNγ, but not CD80 or CD86. Dendritic cells (DCs) treated with bacteria in co-culture with OECs did not fully mature, as judged by the expression of MHC II, CD80 and CD86, and barely released IL-12 and TNFα, compared to control DCs. Furthermore, in the presence of OECs, DCs were unable to stimulate allogenic naive CD4 T cells to produce IFNγ and TNFα. Similarly, OECs in culture with total CD4 T cells or Th1 cells stimulated with anti-CD3 and anti-CD28 antibodies abrogated CD25 and CD69 expression, T cell proliferation and the release of IFNγ and TNFα. The inhibition on T cell activation by OECs was cell-contact dependent, TGFß independent and largely irreversible. Overall, this behavior of OECs is likely key to avoid immune system over-reaction against resident bacteria.

Evaluation of T cells in blood after a short gluten challenge for coeliac disease diagnosis.

BACKGROUND AND AIM: To diagnose coeliac disease (CD) in individuals on a gluten free diet (GFD), we aimed to assess the utility of detecting activated γδ and CD8 T cells expressing gut-homing receptors after a short gluten challenge. METHODS: We studied 15 CD patients and 35 non-CD controls, all exposed to three days of gluten when following a GFD. Peripheral blood was collected before and six days after starting gluten consumption, and the expression of CD103, ß7 and CD38 in γδ and CD8 T cells was assessed by flow cytometry. Determination of IFN-γ and IP-10 was performed by means of ELISPOT and/or Luminex technology. RESULTS: We observed both γδ and CD8 T cells coexpressing CD103, ß7hi and CD38 in every patient with CD on day six, but only in one control. The studied CD8 T subpopulation was easier to detect than the γδ subpopulation. Increased IFN-γ and IP-10 levels after challenge were observed in patients with CD, but not in controls. CONCLUSION: A short three-day gluten challenge elicits the activation of CD103+ ß7hi CD8+ T cells in CD. These cells can be detected by flow cytometry in peripheral blood, opening new possibilities for CD diagnosis in individuals on a GFD.

EPIPOX: Immunoinformatic Characterization of the Shared T-Cell Epitome between Variola Virus and Related Pathogenic Orthopoxviruses.

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

Customized predictions of peptide-MHC binding and T-cell epitopes using EPIMHC.

Peptide binding to major histocompatibility complex (MHC) molecules is the most selective requisite for T-cell recognition. Therefore, prediction of peptide-MHC binding is the main basis for anticipating T-cell epitopes. A very popular and accurate method to predict peptide-MHC binding is based on motif-profiles and here we show how to make them using EPIMHC (http://imed.med.ucm.es/epimhc/). EPIMHC is a database of T-cell epitopes and MHC-binding peptides that unlike any related resource provides a framework for computational vaccinology. In this chapter, we describe how to derive peptide-MHC binding motif-profiles in EPIMHC and use them to predict peptide-MHC binding and T-cell epitopes. Moreover, we show evidence that customization of peptide-MHC binding predictors can lead to enhanced epitope predictions.

Selection of conserved epitopes from hepatitis C virus for pan-populational stimulation of T-cell responses.

The hepatitis C virus (HCV) is able to persist as a chronic infection, which can lead to cirrhosis and liver cancer. There is evidence that clearance of HCV is linked to strong responses by CD8 cytotoxic T lymphocytes (CTLs), suggesting that eliciting CTL responses against HCV through an epitope-based vaccine could prove an effective means of immunization. However, HCV genomic plasticity as well as the polymorphisms of HLA I molecules restricting CD8 T-cell responses challenges the selection of epitopes for a widely protective vaccine. Here, we devised an approach to overcome these limitations. From available databases, we first collected a set of 245 HCV-specific CD8 T-cell epitopes, all known to be targeted in the course of a natural infection in humans. After a sequence variability analysis, we next identified 17 highly invariant epitopes. Subsequently, we predicted the epitope HLA I binding profiles that determine their potential presentation and recognition. Finally, using the relevant HLA I-genetic frequencies, we identified various epitope subsets encompassing 6 conserved HCV-specific CTL epitopes each predicted to elicit an effective T-cell response in any individual regardless of their HLA I background. We implemented this epitope selection approach for free public use at the EPISOPT web server.