Lysis of infected cells in vivo by antiviral cytolytic T cells demonstrated by release of cell internal viral proteins

Immunocompetent adult mice infected with lymphocytic choriomeningitis virus (LCMV) generate a strong antiviral cytotoxic T cell response that clears virus from all organs. Although there is good evidence that immune cytotoxic T lymphocytes (CTL) kill target cells in vitro, in vivo it is debated whether antiviral activity of CD8⁺ T cells is mediated via direct target cell lysis or via soluble mediators. To demonstrate CD8⁺ T cell-mediated destruction of infected cells in vivo a specific cell-internal releasable marker was used as label, i.e. the nucleoprotein (NP) of LCMV. Since LCMV is non-cytopathic the viral NP will only be released in substantial amounts because of destruction of infected host cells by immune CTL. It is shown here that the amount of NP released from infected and ⁵¹Cr-labeled target cells in vitro correlated well with the amount of radioactivity released. Viral NP released in vivo by CTL is bound and masked by the anti-NP antibodies that are produced very early and efficiently. However, NP could readily be detected in sera of LCMV-infected CD8⁺ competent mice that could not generate antibodies specific for the NP because they were treated with a depleting anti-CD4 antibody. NP was also detected in the cerebrospinal fluid of mice suffering from CD8⁺ T cell-mediated lymphocytic choriomeningitis after intracerebral infection. NP titers in sera of anti-CD4-treated LCMV-infected mice exhibited a peak around day 7–8 when CTL activity was highest. When mice were CD8 T cell-depleted with anti-CD8 monoclonal antibody or in LCMV-carrier mice, no NP was detected in the serum. Highly activated LCMV-specific CTL adoptively transfused to LCMV-infected irradiated recipient mice also caused a time-dependent release of NP into serum. This confirms that the CD8⁺ population is responsible for the release of NP from infected host cells. These results represent an in vivo correlate of CTL-mediated cytolysis and evidence that antiviral cytotoxic T cells are cytolytic in vivo. They also suggest that antibody responses to internal antigens of non-cytopathic viruses may signal CD8⁺ Tcell-mediated destruction of infected host cells.     

Anti-viral immune responses in a primitive lung: Characterization and expression analysis of interferon-inducible immunoproteasome subunits LMP2, LMP7 and MECL-1 in a sarcopterygian fish,the Nigerian spotted lungfish (Protopterus dolloi)

Lungfishes (Dipnoi) represent the closest ancestor of tetrapods. Dipnoi have dual breathing modes extracting oxygen from water and air. The primitive lungs of lungfishes are exposed to external antigens including viruses. To date, the immune response of lungfishes against viruses has not been investigated. During viral immune responses, cell exposure to type I interferon induces the replacement of the constitutive proteasome with LMP2, LMP7 and MECL-1 beta subunits forming the immunoproteasome and enhancing antigen presentation to MHC class I molecules. In order to study the immune defense system of the lungfish lung, we have characterized for the first time the three immunoproteasome subunits in the sarcopterygian fish, the Nigerian spotted lungfish (Protopterus dolloi). LMP2, LMP7 and MECL-1 were identified in P. dolloi and their sequences encoded predicted proteins of 216, 275 and 278 amino acids, respectively. The mRNA of these three genes was expressed in multiple tissues, including the lung, with the highest abundance observed in kidney and post-pyloric spleen. In vitro stimulation of lungfish lung and kidney primary cell cultures with PolyI:C for 4 and 12 h resulted in increased LMP2, LMP7 and MECL-1 expression in both tissues. These results suggest a central role of these genes in the activation of an antiviral immune response in lungfish. Importantly, they indicate that the primitive lung of the common ancestor of all tetrapods is capable of inducing the expression of these genes in response to viral stimulation.     

Herpes simplex virus type I infection of mature dendritic cells leads to reduced LMP7-mRNA-expression levels

Mature dendritic cells (mDCs) are the most potent antigen presenting cells within the human immune system known today. However, several viruses, including herpes simplex virus type 1 (HSV-1) have developed numerous immune escape mechanisms, such as the avoidance of peptide presentation through the major histocompatibility complex (MHC) class I to CD8⁺ cytotoxic T-cells. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome, a multicatalytic protease complex. Upon exposure to IFN-γ, the constitutive proteasome is partially replaced by the immunoproteasome, which contains the IFN-γ-inducible subunits LMP2, MECL1 and LMP7.In this study, we report the downregulation of LMP7 on mRNA level in HSV-1 infected mDCs. Interestingly, this reduction was not vhs-mediated since using a virus strain lacking the vhs gene we obtained similar results. However, on protein level, LMP7-expression was not affected, which is probably due the high stability of the LMP7 protein. Also the incorporation of LMP7 into the immunoproteasome was not affected by HSV-1. However, for the in vivo situation, in which DC reside for a prolonged time period in peripheral tissues, the reduced LMP7-mRNA level could be of biological importance, since the virus could escape/hide from immune system of the host and establish latency processes.     

CD8+ T-cell auto-reactivity is dependent on the expression of the immunoproteasome subunit LMP7 in exposed to lipopolysaccharide antigen presenting cells and epithelial target cells

Abstract

Proteasome generates peptides for presentation to CD8+ T-cells by antigen presenting cells (APCs) and peripheral cells. Inflammation alters proteasome to immunoproteasome, producing a different pool of peptides. The effect of immunoproteasome expression in APCs and in peripheral cells on CD8+ T-cell self-tolerance was evaluated. Splenocytes (SP) were used as a source of APCs and CD8+ T-cells. Renal epithelial cells (RC) from the same mouse strain were used as peripheral cells. Lipopolysaccharide (LPS) was used for increasing immunoproteasome expression, whereas transduction with lentiviral particles encoding short-hairpin RNA targeting LMP7 (LnLMP7) were used for decreasing the expression of the LMP7 subunit of the immunoproteasome in SP and/or RC. LMP7 expression was tested with Western blotting, while the cytotoxicity of isolated CD8+ T-cells against RC with LDH release assay. LPS increased LMP7 expression in SP, while did not affect its expression in RC. LnLMP7 decreased LMP7 expression in both LPS-treated and untreated SP and RC. CD8+ T-cell auto-reactivity increased in case of LPS-treated APCs, further enhanced in the event of both APCs and RC treated with LPS. Transduction of APCs or RC with LnLMP7 decreased CD8+ T-cell auto-reactivity, which was further decreased in case of concurrent transduction of both APCs and RC. In conclusion, in controlled in vitro conditions, exposure to LPS of APCs and peripheral cells induces CD8+ T-cell auto-reactivity. Over-expression of the LMP7 subunit of the immunoproteasome in APCs due to LPS exposure, as well as LMP7 expression in peripheral cells, are required for CD8+ T-cell auto-reactivity.     

The immunoproteasome inhibitor ONX‐0914 regulates inflammation and expression of contraction associated proteins in myometrium

There are currently no effective treatments to prevent spontaneous preterm labor. The precise upstream biochemical pathways that regulate the transition between uterine quiescence during pregnancy and contractility during labor remain unclear. It is well known however that intrauterine inflammation, including infection, is commonly associated with preterm labor. In this study, we identified the immunoproteasome subunit low‐molecular‐mass protein (LMP)7 mRNA expression to be significantly upregulated in laboring human myometrium. Silencing LMP7 using siRNA‐targeted knockdown of LMP7 and its inhibitor ONX‐0914 in human myometrial cells and tissues decreased proinflammatory cytokines (IL‐6), cell chemotaxis (CXCL8, CCL2 expression, and THP‐1 migration), cell to cell adhesion (ICAM1 expression and myometrial adhesion), contraction‐associated proteins (PTGS2, FP, PGE2, and PGF2α), as well as suppressing contractions in myometrial cells and in myometrial tissues obtained from laboring women. In addition, LMP7 silencing reduced NF‐κB RelA activity. ONX‐0914 alleviated inflammation (CCL3, CXCL1, PTGS2, and IL‐6) in myometrium, placenta, fetal brain, amniotic fluid, and maternal serum induced by LPS in pregnant mice. Collectively, our data suggest a novel role for ONX‐014 to suppress uterine activation and contractility associated with preterm labor.     

The immunoproteasome subunit LMP7 is required in the murine thymus for filling up a hole in the T cell repertoire

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine‐inducible variant of the proteasome which has been implicated in regulating inflammatory responses and antigen presentation. In the thymus, medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs) do express different proteasome subunits exerting chymotrypsin‐like activities suggesting distinct functions in thymic T cell selection. Employing the lymphocytic choriomeningitis virus (LCMV) infection model, we could show that the immunoproteasome subunit LMP7 was absolutely required for the generation of LCMV GP_118‐125‐specific T cells although the class I mediated presentation of GP_118‐125 was not dependent on LMP7. Using bone marrow chimeras and adoptive transfer of LMP7‐deficient CD8⁺ T cells into RAG1‐deficient mice we show that LMP7‐deficient mice lacked GP_118‐125‐specific T cell precursors and that LMP7 was required in radioresistant cells – most likely thymic epithelial cells ‐ to enable their selection. Since LMP7 is strongly expressed in negatively selecting mTECs but barely in positively selecting cTECs our data suggest that LMP7 was required to avoid excessive negative selection of GP_118‐125‐specific T cell precursors. Taken together, this study demonstrates that the immunoproteasome is a crucial factor for filling up holes within the cytotoxic T cell repertoire.     

Hematopoietic and nonhematopoietic cells promote Type I interferon‐ and TLR7‐dependent monocytosis during low‐dose LCMV infection

Release of inflammatory monocytes from the bone marrow (BM) into the blood is an important physiological response to infection, but the mechanisms regulating this phenomenon during viral infection are not completely defined. Here, we show that low‐dose infection with lymphocytic choriomeningitis virus (LCMV) caused rapid, transient inflammatory monocytosis that required type I interferon (IFN) and Toll‐like receptor (TLR) 7 signaling. Type I IFN and TLR7 signals were critical for induction of IFN‐stimulated gene expression and CCR2 ligand upregulation in the BM microenvironment in response to LCMV infection. Experiments utilizing BM chimeric mice demonstrated that type I IFN and TLR7 signaling on either hematopoietic or nonhematopoietic cells was sufficient to initiate monocytosis in response to LCMV infection. BM plasmacytoid dendritic cells (pDCs) generated type I IFN directly ex vivo, suggesting that pDCs are a hematopoietic contributor of type I IFN in the BM early during LCMV infection. Overall, we describe novel roles for type I IFN and TLR7 signaling in nonhematopoietic cells and BM pDCs in directing IFN‐stimulated gene and CCR2 ligand expression in the BM to initiate an increase in blood inflammatory monocytes during viral infection.     

Critical role for the immunoproteasome subunit LMP7 in the resistance of mice to Toxoplasma gondii infection

Proteasome‐mediated proteolysis is responsible for the generation of immunogenic epitopes presented by MHC class I molecules, which activate antigen‐specific CD8⁺ T cells. Immunoproteasomes, defined by the presence of the three catalytic subunits LMP2, MECL‐1, and LMP7, have been hypothesized to optimize MHC class I antigen processing. In this study, we demonstrate that the infection of mice with a protozoan parasite, Toxoplasma gondii, induced the expression of LMP7 mRNA in APC and increased the capacity of APC to induce the production of IFN‐γ by antigen‐specific CD8⁺ T cells. In vitro infection of a DC cell line with T. gondii also induced the expression of LMP7 and resulted in enhanced proteasome proteolytic activity. Finally, mice lacking LMP7 were highly susceptible to infection with T. gondii and showed a reduced number of functional CD8⁺ T cells. These results demonstrate that proteasomes containing LMP7 play an indispensable role in the survival of mice infected with T. gondii, presumably due to the efficient generation of CTL epitopes required for the functional development of CD8⁺ T cells.     

A protective cytotoxic T cell response to a subdominant epitope is influenced by the stability of the MHC class I/peptide complex and the overall spectrum of viral peptides generated within infected cells

This study identifies instability of MHC class I/peptide complexes and intermolecular competition for MHC class I presentation as factors responsible for the subdominance of cyto toxic T lymphocyte (CTL) epitopes. This evidence is based on the characterization of a new CTL epitope derived from the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV). This epitope, peptide GP117-125 (GP117) is presented to T cells by the mouse MHC class I molecule, H-2D^b. In short-term experiments induction of GP117-specific CTL by vaccination rendered C57BL/6 mice only partially resistant to infection with wild-type LCMV (LCMV-WE) but completely resistant to challenge with a previously described LCMV variant. The variant virus, LCMV-8.7B23, bears point mutations within both known LCMV-GP, H-2 D^b-restricted epitopes GP33-41 (GP33) and GP276-286 (GP276) resulting in a valine to leucine change at position 35 in peptide GP33 (V35L) and an asparagine to serine change at position 280 in peptide GP276 (N280S). Although variant peptide GP33/V35L stimulates a weak CTL response, GP276/N280S does not. Elution of peptide GP117 from both LCMV-WE- and LCMV-8.7B23-infected cells revealed that the difference in the capacity of GP117-specific CTL to protect against LCMV-WE and the virus variant LCMV-8.7B23 was due to differences in the level of GP117 presentation on the surface of both types of cells. Thus, it appears that the protective capacity of CTL specific for the subdominant epitope GP117 is influenced by the extent of presentation of other immunodominant peptide epitopes present within infected cells.     

Immunoactivation induced by chronic viral infection inhibits viral replication and drives immunosuppression through sustained IFN‐I responses

Acute or chronic viral infections can lead to generalized immunosuppression. Several mechanisms, such as immunopathology of CD8⁺ T cells, inhibitory receptors, or regulatory T (Treg) cells, contribute to immune dysfunction. Moreover, patients with chronic viral infections usually do not respond to vaccination, a finding that has not been previously explained. Recently, we reported that CD169⁺ macrophages enforce viral replication, which is essential for guaranteeing antigen synthesis and efficient adaptive immune responses. In the present study, we used a chronic lymphocytic choriomeningitis virus infection mouse model to determine whether this mechanism is affected by chronic viral infection, which may impair the activation of adaptive immunity. We found that enforced viral replication of a superinfecting virus is completely blunted in chronically infected mice. This absence of enforced viral replication in CD169⁺ macrophages is not explained by CD8⁺ T‐cell‐mediated immunopathology but rather by prolonged IFN‐I responses. Consequently, the absence of viral replication impairs both antigen production and the adaptive immune response against the superinfecting virus. These findings indicate that chronic infection leads to sustained IFN‐I action, which is responsible for the absence of an antiviral immune response against a secondary viral infection.     

Virus infection-associated bone marrow B cell depletion and impairment of humoral immunity to heterologous infection mediated by TNF-alpha/LTalpha

We previously showed that influenza virus infection of mice induces a depletion of bone marrow B lineage cells due to apoptosis of early B cells mediated by a mechanism involving TNF-alpha/LTalpha. Here we demonstrate that this effect is also observed with acute lymphocytic choriomeningitis virus (LCMV) infection and resulted in a deficiency of both splenic transitional B cells and mature follicular B cells. To determine whether there was an associated impairment of humoral immunity, we infected mice with LCMV and 10 days later at the peak of the B cell depletion, inoculated them with influenza virus. We found that influenza virus-specific antibody titers were dramatically reduced in mice recovering from LCMV infection compared to those in mice infected with influenza virus alone. Further, we showed that there was no reduction of the influenza virus-specific antibody response in LCMV-infected TNF-alpha/LTalpha-deficient mice, suggesting that TNF-alpha/LTalpha-mediated effects on bone marrow and/or peripheral lymphocytes were responsible for the observed impairment in humoral immunity. These results show that the TNF-alpha/LTalpha production induced following infection with diverse viruses has detrimental effects on early B cells in the bone marrow, and may be among the factors that lead to the severely compromised humoral immunity observed to subsequent heterologous infections.     

HBV‐specific CD4+ cytotoxic T cells in hepatocellular carcinoma are less cytolytic toward tumor cells and suppress CD8+ T cell‐mediated antitumor immunity

In East Asia and sub‐Saharan Africa, chronic infection is the main cause of the development of hepatocellular carcinoma, an aggressive cancer with low survival rate. Cytotoxic T cell‐based immunotherapy is a promising treatment strategy. Here, we investigated the possibility of using HBV‐specific CD4⁺ cytotoxic T cells to eliminate tumor cells. The naturally occurring HBV‐specific cytotoxic CD4⁺ and CD8⁺ T cells were identified by HBV peptide pool stimulation. We found that in HBV‐induced hepatocellular carcinoma patients, the HBV‐specific cytotoxic CD4⁺ T cells and cytotoxic CD8⁺ T cells were present at similar numbers. But compared to the CD8⁺ cytotoxic T cells, the CD4⁺ cytotoxic T cells secreted less cytolytic factors granzyme A (GzmA) and granzyme B (GzmB), and were less effective at eliminating tumor cells. In addition, despite being able to secrete cytolytic factors, CD4⁺ T cells suppressed the cytotoxicity mediated by CD8⁺ T cells, even when CD4⁺CD25⁺ regulator T cells were absent. Interestingly, we found that interleukin 10 (IL‐10)‐secreting Tr1 cells were enriched in the cytotoxic CD4⁺ T cells. Neutralization of IL‐10 abrogated the suppression of CD8⁺ T cells by CD4⁺CD25^? T cells. Neither the frequency nor the absolute number of HBV‐specific CD4⁺ cytotoxic T cells were correlated with the clinical outcome of advanced stage hepatocellular carcinoma patients. Together, this study demonstrated that in HBV‐related hepatocellular carcinoma, CD4⁺ T cell‐mediated cytotoxicity was present naturally in the host and had the potential to exert antitumor immunity, but its capacity was limited and was associated with immunoregulatory properties.     

B7‐H1 and CD8+ Treg: The enigmatic role of B7‐H1 in peripheral tolerance

The interaction between B7‐H1 (PD‐L1) expressed on APC with PD‐1 expressed by T cells was shown previously to result in inhibition of T‐cell activation and autoimmune diseases. A paper in this issue of the European Journal of Immunology demonstrates that DC B7‐H1 expression can in fact enhance autoimmunity, rather than suppress it. Using a model of direct injection of self antigen‐loaded DC into the CNS, the authors demonstrate that DC with intact B7‐H1 expression exacerbate CNS autoimmune disease. Importantly, the improved disease outcome in animals treated with B7‐H1^?/? DC is a result of a population of CD8⁺ Treg cells that expand at the site of autoimmune inflammation.     

Genetic polymorphism in intron 6 of the LMP7 gene in Japanese and its association with sarcoidosis

Genetic polymorphism in intron 6 of the LMP7 gene was investigated using polymerase chain reaction-restriction fragment length polymorphism in 90 unrelated healthy Japanese controls and 66 Japanese patients with sarcoidosis. Four alleles, including two new ones recently identified in Koreans, LMP7*C and LMP7*D, were found in the Japanese population. The frequency of LMP7*C in the sarcoidosis patients was higher than in the healthy controls. However, this difference might be explained by a secondary association with HLA-DRB1*08 in the HLA-DRB1 gene, which is thought to be the gene primarily responsible for susceptibility to sarcoidosis.     

Inhibition of the LKB1–AMPK pathway by the Epstein–Barr virus‐encoded LMP1 promotes proliferation and transformation of human nasopharyngeal epithelial cells

The association of Epstein–Barr virus (EBV) infection with the development of nasopharyngeal carcinoma (NPC) is well established. Latent membrane protein 1 (LMP1), the major oncogene encoded by EBV, is believed to play a crucial role in NPC pathogenesis by virtue of its ability to constitutively activate multiple cell signalling pathways. The LKB1–AMPK pathway is a master regulator of cellular metabolism that, via modulation of energy metabolism, has tumour suppressor activity. In this study we identify a novel ability of LMP1 to inhibit the LKB1–AMPK pathway through phosphorylation of LKB1 at serine 428 with subsequent suppression of the phosphorylation of AMPK and its substrates, ACC and Raptor. We show that MEK/ERK–MAPK signalling, activated by the CTAR1 domain of LMP1, is responsible for LKB1–AMPK inactivation. In addition, reactivation of AMPK signalling by AMPK activator, AICAR, abolished LMP1‐induced cellular transformation (proliferation and anchorage‐independent growth) in nasopharyngeal epithelial cells. Immunohistochemical staining revealed that a low level of phosphorylated AMPK is common in primary NPC specimens, and that this correlated significantly with the expression of LMP1. AICAR treatment inhibited the proliferation and anchorage‐independent growth of NPC cells as well as potentiating the cytotoxic effect of the chemotherapeutic drug 5‐fluorouracil. The current findings demonstrate that LMP1‐mediated AMPK inactivation contributes to the proliferation and transformation of epithelial cells, thereby implicating the LKB1–AMPK pathway in the EBV‐driven pathogenesis of NPC. Our findings also suggest that AMPK activators could be used to enhance the efficacy of conventional chemotherapeutic agents in the treatment of local and metastatic NPC. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Vitronectin deficiency attenuates hepatic fibrosis in a non‐alcoholic steatohepatitis‐induced mouse model

Vitronectin (VN), an extracellular matrix protein, is a promising immune biomarker of non‐alcoholic steatohepatitis (NASH); however, its precise function remains unclear. This study investigated how VN deficiency contributes to the development of NASH. Towards this aim, wild‐type (WT) and VN?/? mice were fed with a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) for 6 and 10 weeks to induce NASH, and the livers were isolated. In WT mice fed with CDAHFD for 6 and 10 weeks, the expression of Vn mRNA and protein was up‐regulated compared with that in mice fed with the MF control diet, indicating that VN is regulated in NASH condition. VN?/? mice showed decreased picrosirius red staining in the liver area and Col1a2 mRNA expression levels, compared with WT mice, indicating that the severity of hepatic fibrosis is attenuated in the CDAHFD‐fed VN?/? mice. In addition, VN deficiency did not affect the area of lipid droplets in haematoxylin‐eosin staining and the mRNA expression levels of fatty acid synthases, Srebp, Acc and Fas in the CDAHFD‐fed mice. Moreover, VN deficiency decreased the inflammation score and the mRNA expression levels of Cd11b and F4/80, macrophage markers, as well as Tnf‐α and Il‐1β, inflammatory cytokines in the CDAHFD‐fed mice. Furthermore, VN deficiency decreased the protein and mRNA expression levels of α‐smooth muscle actin in the CDAHFD‐fed mice, suggesting that VN deficiency inhibits the activation of hepatic stellate cells (HSCs). Our findings indicate that VN contributes to the development of fibrosis in the NASH model mice via modulation of the inflammatory reaction and activation of HSCs.     

Bacteria and their cell wall components uniformly co‐activate interleukin‐17‐producing thymocytes

Interleukin (IL)‐17‐producing T cells play a critical role in the immune response against microbial pathogens. Traditionally, experimental studies have focused upon understanding the activity of IL‐17‐producing T cells which differentiate from naive T cells in the peripheral immune system. However, we have demonstrated previously that IL‐17‐producing T cells are also present in the thymus of naive wild‐type mice and can be co‐activated there by microbial stimuli. Other studies have supported the concept that IL‐17‐producing thymocytes have a specific role in the immediate defence against microbial pathogens, which is independent from the development of an adaptive immune response. Given an important role of the thymus in systemic bacterial infection and sepsis, in this study we investigate the effect of a broad spectrum of bacteria and cell wall components on thymocyte cytokine production. Surprisingly, we find that all types of bacteria investigated (including non‐pathogenic species) uniformly activate IL‐17‐producing thymocytes upon α‐CD3 stimulation. In contrast, there is a heterogeneous effect on IL‐6 and interferon (IFN)‐γ‐production with Gram‐negative bacteria inducing far higher frequencies of IL‐6‐ and IFN‐γ‐producing thymocytes than Gram‐positive bacteria. We conclude that IL‐17‐producing thymocytes constitute a ‘first line of recognition’, but not a ‘first line of defence’ against bacteria in general. Their activity might lead to immune activation, but not necessarily to a pathological inflammatory disease condition. The difference between these two states might be determined by other immunological effector molecules, such as IL‐6 and IFN‐γ.     

Defining the HLA class I‐associated viral antigen repertoire from HIV‐1‐infected human cells

Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High‐throughput definition of HLA class I‐associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T‐cell responses against pathogens such as HIV‐1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo‐assisted database searching to define the HLA class I‐associated immunopeptidome of HIV‐1‐infected human cells. We here report for the first time the identification of 75 HIV‐1‐derived peptides bound to HLA class I complexes that were purified directly from HIV‐1‐infected human primary CD4⁺ T cells and the C8166 human T‐cell line. Importantly, one‐third of eluted HIV‐1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T‐cell responses have previously been reported but for which the precise HLA class I‐binding sequences have not yet been defined. These results validate and expand the current knowledge of virus‐specific antigenic peptide presentation during HIV‐1 infection and provide novel targets for T‐cell vaccine development.