Human Papillomavirus Deregulates the Response of a Cellular Network Comprising of Chemotactic and Proinflammatory Genes

Despite the presence of intracellular pathogen recognition receptors that allow infected cells to attract the immune system, undifferentiated keratinocytes (KCs) are the main targets for latent infection with high-risk human papilloma viruses (hrHPVs). HPV infections are transient but on average last for more than one year suggesting that HPV has developed means to evade host immunity. To understand how HPV persists, we studied the innate immune response of undifferentiated human KCs harboring episomal copies of HPV16 and 18 by genome-wide expression profiling. Our data showed that the expression of the different virus-sensing receptors was not affected by the presence of HPV. Poly(I:C) stimulation of the viral RNA receptors TLR3, PKR, MDA5 and RIG-I, the latter of which indirectly senses viral DNA through non-self RNA polymerase III transcripts, showed dampening in downstream signalling of these receptors by HPVs. Many of the genes downregulated in HPV-positive KCs involved components of the antigen presenting pathway, the inflammasome, the production of antivirals, pro-inflammatory and chemotactic cytokines, and components downstream of activated pathogen receptors. Notably, gene and/or protein interaction analysis revealed the downregulation of a network of genes that was strongly interconnected by IL-1β, a crucial cytokine to activate adaptive immunity. In summary, our comprehensive expression profiling approach revealed that HPV16 and 18 coordinate a broad deregulation of the keratinocyte's inflammatory response, and contributes to the understanding of virus persistence

Identification of TRDV-TRAJ V domains in human and mouse T-cell receptor repertoires

Here, we describe the identification of two T-cell receptors (TRs) containing TRDV genes in their TRA chains, the first one in human and the second one in mouse. First, using 5’RACE on a mixed lymphocyte-tumor cell culture (MLTC), we identified TRDV1 5’-untranslated region (UTR) and complete coding sequence rearranged productively to TRAJ24. Single-cell TR RNA sequencing (RNA-seq) of the MLTC, conducted to identify additional clonotypes, revealed that the analysis software detected the hybrid TRDV-TRAJ TRA (TRA) chain but excluded it from the final results. In a separate project, we performed TR sequencing of tumor-infiltrating lymphocytes (TILs) in a murine tumor model. Here, the predominant clonotype contained a TRA chain with a TRDV2-2-TRAJ49 rearrangement. Again, the hybrid TRA chain was not reported in the final results. Transfection of both TR cDNAs resulted in cell surface localization of TR together with CD3, suggesting a productive protein in both cases. Tumor recognition of the Homo sapiens (Homsap) TRDV1-containing TR could be demonstrated by IFN Gamma ELISA ELISpot kit, whereas the Mus musculus (Musmus) TR did not recognize a tumor-derived cell line. To determine whether the TRDV-containing TRA chains we detected were rare events or whether TRDV genes are commonly incorporated into TRA chains, we queried the NCBI Sequence Read Archive for TR single-cell RNA-seq data and analyzed 21 human and 23 murine datasets. We found that especially Homsap TRDV1, Musmus TRDV1, and to some extent Musmus TRDV2-2 are more commonly incorporated into TRA chains than several TRAV genes, making those TRDV genes a relevant contribution to TRA diversity. TRDV-containing TRA chains are currently excluded from the final results of V-(D)-J dataset analyses with the CellRanger software. We provide a work-around to avoid exclusion of those hybrid TRA chains from the final analysis results

The nonpolymorphic MHC Qa-1b mediates CD8+ T cell surveillance of antigen-processing defects

The nonclassical major histocompatibility complex (MHC) Qa-1b accommodates monomorphic leader peptides and functions as a ligand for germ line receptors CD94/NKG2, which are expressed by natural killer cells and CD8+ T cells. We here describe that the conserved peptides are replaced by a novel peptide repertoire of surprising diversity as a result of impairments in the antigen-processing pathway. This novel peptide repertoire represents immunogenic neoantigens for CD8+ T cells, as we found that these Qa-1b–restricted T cells dominantly participated in the response to tumors with processing deficiencies. A surprisingly wide spectrum of target cells, irrespective of transformation status, MHC background, or type of processing deficiency, was recognized by this T cell subset, complying with the conserved nature of Qa-1b. Target cell recognition depended on T cell receptor and Qa-1b interaction, and immunization with identified peptide epitopes demonstrated in vivo priming of CD8+ T cells. Our data reveal that Qa-1b, and most likely its human homologue human leukocyte antigen-E, is important for the defense against processing-deficient cells by displacing the monomorphic leader peptides, which relieves the inhibition through CD94/NKG2A on lymphocytes, and by presenting a novel repertoire of immunogenic peptides, which recruits a subset of cytotoxic CD8+ T cells

Proimmunogenic impact of MEK inhibition synergizes with agonist anti-CD40 immunostimulatory antibodies in tumor therapy

Cancer types with lower mutational load and a non-permissive tumor microenvironment are intrinsically resistant to immune checkpoint blockade. While the combination of cytostatic drugs and immunostimulatory antibodies constitutes an attractive concept for overcoming this refractoriness, suppression of immune cell function by cytostatic drugs may limit therapeutic efficacy. Here we show that targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) does not impair dendritic cell-mediated T cell priming and activation. Accordingly, combining MEK inhibitors (MEKi) with agonist antibodies (Abs) targeting the immunostimulatory CD40 receptor results in potent synergistic antitumor efficacy. Detailed analysis of the mechanism of action of MEKi shows that this drug exerts multiple pro-immunogenic effects, including the suppression of M2-type macrophages, myeloid derived suppressor cells and T-regulatory cells. The combination of MEK inhibition with agonist anti-CD40 Ab is therefore a promising therapeutic concept, especially for the treatment of mutant Kras-driven tumors such as pancreatic ductal adenocarcinoma. Immune checkpoint inhibitors have limited efficacy in tumors with lower mutational burden and non-permissive microenvironment. Here, the authors show that combining MEK inhibition with an agonist anti-CD40 immunostimulatory antibody improves antitumor treatment by inducing immunogenic changes in the tumor microenvironment

Expression of the Serpin Serine Protease Inhibitor 6 Protects Dendritic Cells from Cytotoxic T Lymphocyte–Induced Apoptosis: Differential Modulation by T Helper Type 1 and Type 2 Cells

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis