Damien Zanker

Unexpected Role for the Immunoproteasome Subunit LMP2 in Antiviral Humoral and Innate Immune Responses

Proteasomes are multisubunit proteases that initiate degradation of many Ags presented by MHC class I molecules. Vertebrates express alternate forms of each of the three catalytic proteasome subunits: standard subunits, and immunosubunits, which are constitutively expressed by APCs and are induced in other cell types by exposure to cytokines. The assembly of mixed proteasomes containing standard subunits and immunosubunits is regulated in a tissue specific manner. In this study, we report that the presence of mixed proteasomes in immune cells in LMP2−/− mice compromises multiple components that contribute to the generation of antiviral Ab responses, including splenic B cell numbers, survival and function of adoptively transferred B cells, Th cell function, and dendritic cell secretion of IL-6, TNF-α, IL-1β, and type I IFNs. These defects did not result from compromised overall protein degradation, rather they were associated with altered NF-κB activity. These findings demonstrate an important role for immunoproteasomes in immune cell function beyond their contribution to Ag processing.

A Cancer Vaccine Induces Expansion of NY-ESO-1-Specific Regulatory T Cells in Patients with Advanced Melanoma

Cancer vaccines are designed to expand tumor antigen-specific T cells with effector function. However, they may also inadvertently expand regulatory T cells (Treg), which could seriously hamper clinical efficacy. To address this possibility, we developed a novel assay to detect antigen-specific Treg based on down-regulation of surface CD3 following TCR engagement, and used this approach to screen for Treg specific to the NY-ESO-1 tumor antigen in melanoma patients treated with the NY-ESO-1/ISCOMATRIXTM cancer vaccine. All patients tested had Treg (CD25bright FoxP3+ CD127neg) specific for at least one NY-ESO-1 epitope in the blood. Strikingly, comparison with pre-treatment samples revealed that many of these responses were induced or boosted by vaccination. The most frequently detected response was toward the HLA-DP4-restricted NY-ESO-1157–170 epitope, which is also recognized by effector T cells. Notably, functional Treg specific for an HLA-DR-restricted epitope within the NY-ESO-1115–132 peptide were also identified at high frequency in tumor tissue, suggesting that NY-ESO-1-specific Treg may suppress local anti-tumor immune responses. Together, our data provide compelling evidence for the ability of a cancer vaccine to expand tumor antigen-specific Treg in the setting of advanced cancer, a finding which should be given serious consideration in the design of future cancer vaccine clinical trials.

The Exception that Reinforces the Rule: Crosspriming by Cytosolic Peptides that Escape Degradation

The nature of crosspriming immunogens for CD8(+) T cell responses is highly controversial. By using a panel of T cell receptor-like antibodies specific for viral peptides bound to mouse D(b) major histocompatibility complex class I molecules, we show that an exceptional peptide (PA(224-233)) expressed as a viral minigene product formed a sizeable cytosolic pool continuously presented for hours after protein synthesis was inhibited. PA(224-233) pool formation required active cytosolic heat-shock protein 90 but not ER g96 and uniquely enabled crosspriming by this peptide. These findings demonstrate that exceptional class I binding oligopeptides that escape proteolytic degradation are potent crosspriming agents. Thus, the feeble immunogenicity of natural proteasome products in crosspriming can be attributed to their evanescence in donor cells and not an absolute inability of cytosolic oligopeptides to be transferred to and presented by professional antigen-presenting cells.

Systematic identification of immunodominant CD8+ T-cell responses to influenza A virus in HLA-A2 individuals

Immunodominant T-cell responses are important for virus clearance. However, the identification of immunodominant T-cell peptide + HLA glycoprotein epitopes has been hindered by the extent of HLA polymorphism and the limitations of predictive algorithms. A simple, systematic approach has been used here to screen for immunodominant CD8+ T-cell specificities. The analysis targeted healthy HLA-A2+ donors to allow comparison with responses to the well-studied influenza matrix protein 1 epitope. Although influenza matrix protein 1 was consistently detected in all individual samples in our study, the response to this epitope was only immunodominant in three of eight, whereas for the other five, prominent CD8+ T-cell responses tended to focus on various peptides from the influenza nucleoprotein that were not presented by HLA-A2. Importantly, with the four immunodominant T-cell epitopes identified here, only one would have been detected by the current prediction programs. The other three peptides would have been either considered too long or classified as not containing typical HLA binding motifs. Our data stress the importance of systematic analysis for discovering HLA-dependent, immunodominant CD8+ T-cell epitopes derived from viruses and tumors. Focusing on HLA-A2 and predictive algorithms may be too limiting as we seek to develop targeted immunotherapy and vaccine strategies that depend on T cell-mediated immunity.

Mixed proteasomes function to increase viral peptide diversity and broaden antiviral CD8+ T cell responses.

The three proteasome subunits with proteolytic activity are encoded by standard or immunoproteasome genes. Many proteasomes expressed by normal cells and cells exposed to cytokines are “mixed”, that is, contain both standard and immunoproteasome subunits. Using a panel of 38 defined influenza A virus–derived epitopes recognized by C57BL/6 mouse CD8+ T cells, we used mice with targeted disruption of β1i, β2i, or β5i/β2i genes to examine the contribution of mixed proteasomes to the immunodominance hierarchy of antiviral CD8+ T cells. We show that each immunoproteasome subunit has large effects on the primary and recall immunodominance hierarchies due to modulating both the available T cell repertoire and generation of individual epitopes as determined both biochemically and kinetically in Ag presentation assays. These findings indicate that mixed proteasomes function to enhance the diversity of peptides and support a broad CD8+ T cell response.

Resident CD8 + and Migratory CD103 + Dendritic Cells Control CD8 T Cell Immunity during Acute Influenza Infection

The identification of the specific DC subsets providing a critical role in presenting influenza antigens to naïve T cell precursors remains contentious and under considerable debate. Here we show that CD8+ T lymphocyte (TCD8+) responses are severely hampered in C57BL/6 mice deficient in the transcription factor Batf3 after intranasal challenge with influenza A virus (IAV). This transcription factor is required for the development of lymph node resident CD8+ and migratory CD103+CD11b− DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+. Using a similar ex vivo approach, many publications on this subject matter excluded a role for resident, non-migratory CD8+ DC. We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes. Our results show that resident CD8+ DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes.

Flt3 ligand expands CD4+FoxP3+ regulatory T cells in human subjects

CD4+CD25+FoxP3+ naturally occurring regulatory T (Treg) cells play a crucial role in the maintenance of immune tolerance and in preventing autoimmune pathology. Interventions that expand Treg cells are highly desirable, as they may offer novel treatment options in a variety of autoimmune and transplantation settings. Paralleling previous preclinical studies, we demonstrate here that administration of the hematopoietic growth factor Flt3L to human subjects increases the frequency and absolute number of Treg cells, and reduces the ratio of CD8+ T cells to Treg cells in the peripheral blood. The increase in Treg cells was due to enhanced Treg‐cell proliferation rather than release of Treg cells from the thymus. Further studies revealed that Flt3L‐induced proliferation of Treg cells was an indirect effect that occurred via the interaction of Treg cells with the Flt3L‐expanded pool of CD1c+ myeloid dendritic cells. On the basis of these findings, Flt3L may represent a promising agent for promoting immune tolerance in a variety of clinical settings.

Immunodominant CD4+ T-Cell Responses to Influenza A Virus in Healthy Individuals Focus on Matrix 1 and Nucleoprotein

ABSTRACT Antigen-specific CD4+ T cells are essential for effective virus-specific host responses, with recent human challenge studies (in volunteers) establishing their importance for influenza A virus (IAV)-specific immunity. However, while many IAV CD4+ T cell epitopes have been identified, few are known to stimulate immunodominant CD4+ T cell responses. Moreover, much remains unclear concerning the major antigen(s) responded to by the human CD4+ T cells and the extents and magnitudes of these responses. We initiated a systematic screen of immunodominant CD4+ T cell responses to IAV in healthy individuals. Using in vitro expanded-multispecificity IAV-specific T cell lines and individual IAV protein antigens produced by recombinant vaccinia viruses, we found that the internal matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of CD4+ T cell responses. Ten epitopes derived from M1 and NP were definitively characterized. Furthermore, epitope sequence conservation analysis established that immunodominance correlated with an increased frequency of mutations, reflecting the fact that these prominent epitopes are under greater selective pressure. Such evidence that particular CD4+ T cells are important for protection/recovery is of value for the development of novel IAV vaccines and for our understanding of different profiles of susceptibility to these major pathogens. IMPORTANCE Influenza virus causes half a million deaths annually. CD4+ T cell responses have been shown to be important for protection against influenza and for recovery. CD4+ T cell responses are also critical for efficient CD8+ T cell response and antibody response. As immunodominant T cells generally play a more important role, characterizing these immunodominant responses is critical for influenza vaccine development. We show here that the internal matrix protein 1 (M1) and nucleoprotein (NP), rather than the surface proteins reported previously, are the immunodominant targets of CD4+ T cell responses. Interestingly, these immunodominant epitope regions accumulated many mutations over time, which likely indicates increased immune pressure. These findings have significant implications for the design of T cell-based influenza vaccines.

Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting

Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples.

Standard and immunoproteasomes show similar peptide degradation specificities

Vertebrates have evolved to express major histocompatibility complexes (MHCs) that present peptides of the intra‐cellular proteome for immunosurveillance against viruses and tumor. The MHC class I (MHC‐I) processing and presentation pathway allows for scrutiny of all cellular proteins and peptides by CD8+ cytotoxic T cells. The proteasome is part of the specialised machinery that degrades proteins down to peptides with the correct sequence for MHC‐I binding. However, much conjecture lies as to how the various proteasome isoforms and their active subunits create antigenic peptides, and if the specialised immunoproteasome solely performs this job. In this issue of the European Journal of Immunology, Mishto et al. [Eur. J. Immunol. 2014. 44: 3508‐3521] address this question through systematic biochemical peptide degradation studies and provide new insights into the functions of proteasome β‐subunits.