Fighting viral infections and virus-driven tumors with cytotoxic CD4+ T cells

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors

Three Immunoproteasome-Associated Subunits Cooperatively Generate a Cytotoxic T-Lymphocyte Epitope of Epstein-Barr Virus LMP2A by Overcoming Specific Structures Resistant to Epitope Liberation

The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A(222-230), from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A(222-230)-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A(222-230) epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A(222-230) is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen

Autophagy Creates a CTL Epitope That Mimics Tumor-Associated Antigens

<div><p>The detailed mechanisms responsible for processing tumor-associated antigens and presenting them to CTLs remain to be fully elucidated. In this study, we demonstrate a unique CTL epitope generated from the ubiquitous protein puromycin-sensitive aminopeptidase, which is presented via HLA-A24 on leukemic and pancreatic cancer cells but not on normal fibroblasts or EBV-transformed B lymphoblastoid cells. The generation of this epitope requires proteasomal digestion and transportation from the endoplasmic reticulum to the Golgi apparatus and is sensitive to chloroquine-induced inhibition of acidification inside the endosome/lysosome. Epitope liberation depends on constitutively active autophagy, as confirmed with immunocytochemistry for the autophagosome marker LC3 as well as RNA interference targeting two different autophagy-related genes. Therefore, ubiquitously expressed proteins may be sources of specific tumor-associated antigens when processed through a unique mechanism involving autophagy.</p> </div

Induced autophagy does not result in PSA epitope presentation in fibrobast cells.

<p>A. Immunofluorescence assays for endogenous LC3 in fibroblast cells after low nutrient culture or rapamycin treatment. In low nutrient conditions, fibroblast cells were cultured in medium supplemented with 10%, 5%, 0,5% FCS or in Hank’s Balanced Salt Solution (starved). B, CTL response to autophagy-induced fibroblast cells treated with low nutrient culture conditions or rapamycin. Target cells were treated with low nutrient culture conditions or rapamycin for 4h, washed twice and cultured with CTL overnight. Next day, supernatants were harvested and IFN-γ measured by ELISA. K562-A24 cells and K562-A2 cells were used as positive and negative control, respectively. The results show means ± SD of triplicates.</p

Autophagy is involved in the 16F3 epitope processing in cancer cells.

<p>A, K562 cells expressing both HLA-A2 or A24 and CMV pp65 were treated with an acid buffer for peptide stripping and incubated for 14 h in the presence or absence of 3-MA. Next, the cells were co-cultured with each clone for 4 h for IFN-γ secretion detected via the IFN-γ catch assay. The frequency of IFN-γ-secreting cells is shown as the percentage of the total living CD8⁺ T cells. 3-MA was not added during the co-culture period. In the lower panels, histograms of the IFN-γ signal and their mean fluorescence intensity are shown. B, RT-PCR analysis of scrambled, atg5- or atg7-specific siRNA-treated cells performed 70 h after transfection. The intensities of the bands were calculated using the ImageJ software. C, The CTL response against KP-3 and MIA PaCa-2 cells transfected with scrambled, atg5- or atg7-specific siRNA for 70 h was examined using an IFN-γ ELISA. Three different siRNA targets were chosen for each autophagy-associated gene. The results are the means ± SD of triplicates. Similar results were obtained in three separate experiments.</p

Degradation of full-length PSA protein is inhibited by CQ but not by lactacystin.

<p>A, Cells were cultured with or without CQ (50 µM) overnight, and then the cell lysates were subjected to Western blot analysis for PSA and p62 as a positive control for autophagic digestion. B, Cells were cultured with or without lactacystin (5 µM) overnight, and the cell lysates were subjected to Western blot analysis for PSA and HIF-2α as a positive control for proteasomal digestion. C, The intensities of the bands were calculated using the ImageJ software.</p