Virus-specific CD4+ T cells: ready for direct attack

CD4+ T cells are classically thought to orchestrate adaptive immune responses. But recent studies demonstrate that they can also kill infected cells directly. A new paper shows that highly efficient processing of Epstein Barr virus (EBV) glycoproteins for presentation on MHC class II makes virus-transformed B cells susceptible to lysis by CD4+ T cells. Thus, antiviral vaccines should aim to stimulate both helper and cytolytic CD4+ T cells

Covalent Modification of Human Immunodeficiency Virus Type 1 p6 by SUMO-1

The p6 domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein mediates virion budding from infected cells via protein-protein contacts with the class E vacuolar protein sorting factors, Tsg101 and AIP1/ALIX. Interaction with Tsg101 is strengthened by covalent attachment of monovalent ubiquitin to HIV-1 p6. To identify additional host factors that bind to HIV-1 p6, a human cDNA library was screened in the yeast two-hybrid system. HIV-1 p6 was found to interact with small ubiquitin-like modifier 1 (SUMO-1) as well as the E2 SUMO-1 transfer enzyme, Ubc9. Interaction with p6 was also detected with Daxx, a cellular protein to which SUMO-1 is sometimes covalently attached. SUMO-1 was incorporated into HIV-1 virions where it was protected within the virion membrane from digestion by exogenous protease. Of the two lysine residues in p6, lysine 27 uniquely served as a site of covalent SUMO-1 attachment. As previously reported, though, HIV-1 bearing the p6-K27R mutation replicated just like the wild type. Overproduction of SUMO-1 in HIV-1 producer cells had no apparent effect on virion release or on virion protein or RNA content. Infectivity of the resulting virions, though, was decreased, with the defect occurring after membrane fusion, at the time of viral cDNA synthesis. HIV-1 bearing the p6-K27R mutation was insensitive to SUMO-1 overexpression, suggesting that covalent attachment of SUMO-1 to p6 is detrimental to HIV-1 replication

ATPγS Disrupts Human Immunodeficiency Virus Type 1 Virion Core Integrity

Heat shock protein 70 (Hsp70) is incorporated within the membrane of primate lentiviral virions. Here we demonstrate that Hsp70 is also incorporated into oncoretroviral virions and that it remains associated with membrane-stripped human immunodeficiency virus type 1 (HIV-1) virion cores. To determine if Hsp70 promotes virion infectivity, we attempted to generate Hsp70-deficient virions with gag deletion mutations, Hsp70 transdominant mutants, or RNA interference, but these efforts were confounded, largely because they disrupt virion assembly. Given that polypeptide substrates are bound and released by Hsp70 in an ATP-hydrolytic reaction cycle, we supposed that incubation of HIV-1 virions with ATP would perturb Hsp70 interaction with substrates in the virion and thereby decrease infectivity. Treatment with ATP or ADP had no observable effect, but ATPγS and GTPγS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPγS-treated virions were competent for fusion with susceptible target cells, but viral cDNA synthesis was inhibited to an extent that correlated with the magnitude of decrease in infectivity. Intravirion reverse transcription by HIV-1, simian immunodeficiency virus, or murine leukemia virus was also inhibited by ATPγS. The effects of ATPγS on HIV-1 reverse transcription appeared to be indirect, resulting from disruption of virion core morphology that was evident by transmission electron microscopy. Consistent with effects on capsid conformation, ATPγS-treated viruslike particles failed to saturate host antiviral restriction activity. Our observations support a model in which the catalytic activity of virion-associated Hsp70 is required to maintain structural integrity of the virion core

Endogenous retroviral proteins provide an immunodominant but not requisite antigen in a murine immunotherapy tumor model

Clinical observations suggest that responses to cancer immunotherapy are correlated with intra-tumoral T cell receptor (TCR) clonality, tumor mutation burden (TMB) and host HLA genotype, highlighting the importance of host T cell recognition of tumor antigens. However, the dynamic interplay between T cell activation state and changes in TCR repertoire in driving the identification of potential immunodominant antigen(s) remains largely unexplored. Here, we performed single-cell RNA-sequencing on CD8+ tumor-infiltrating T cells (TILs) using the murine colorectal tumor model MC38 to identify unique TCR sequences and validate their tumor reactivity. We found that the majority of clonally expanded TILs are tumor-reactive and their TCR repertoire is unique amongst individual MC38 tumor-bearing mice. Our query identified that multiple expanded TCR clones recognized the retroviral epitope p15E as an immunodominant antigen. In addition, we found that the endogenous retroviral genome encoding for p15E is highly expressed in MC38 tumors, but not in normal tissues, due to epigenetic derepression. Further, we demonstrated that the p15E-specific TILs exhibit an activated phenotype and an increase in frequency upon treatment with anti-41BB and anti-PD-1 combination immunotherapy. Importantly, we showed that although p15E-specific TILs are not required to mount a primary anti-tumor response, they contributed to the development of strong immune memory. Overall our results revealed that endogenous retroviral antigens expressed by tumor cells may represent an important and underappreciated category of tumor antigens that could be readily targeted in the clinic

A novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production

Humanized mice are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, the existing models cannot support robust adaptive immune responses, especially the generation of class-switched, antigen-specific antibody responses. Here we describe a new mouse strain, in which human interleukin 6 (IL-6) gene encoding the cytokine that is important for B- and T-cell differentiation was knocked into its respective mouse locus. The provision of human IL-6 not only enhanced thymopoiesis and periphery T-cell engraftment, but also significantly increased class switched memory B cells and serum immunoglobulin G (IgG). In addition, immunization with ovalbumin (OVA) induced OVA-specific B cells only in human IL-6 knock-in mice. These OVA-specific antibodies displayed the highest frequency of somatic mutation, further suggesting that human IL-6 is important for efficient B-cell activation and selection. We conclude that human IL-6 knock-in mice represent a novel and improved model for human adaptive immunity without relying on complex surgery to transplant human fetal thymus and liver. These mice can therefore be used to exploit or evaluate immunization regimes that would be unethical or untenable in humans