Carolina Johnstone

Traffic of Proteins and Peptides across Membranes for Immunosurveillance by CD8+ T Lymphocytes: A Topological Challenge

Cytotoxic CD8+ T lymphocytes kill infected cells that display major histocompatibility complex (MHC) class I molecules presenting peptides processed from pathogen proteins. In general, the peptides are proteolytically processed from newly made endogenous antigens in the cytosol and require translocation to the endoplasmic reticulum (ER) for MHC class I loading. This last task is performed by the transporters associated with antigen processing (TAP). Sampling of suspicious pathogen‐derived proteins reaches beyond the cytosol, and MHC class I loading can occur in other secretory or endosomal compartments besides the ER. Peptides processed from exogenous antigens can also be presented by MHC class I molecules to CD8+ T lymphocytes, in this case requiring delivery from the extracellular medium to the processing and MHC class I loading compartments. The endogenous or exogenous antigen can be processed before or after its transport to the site of MHC class I loading. Therefore, mechanisms that allow the full‐length protein or processed peptides to cross several subcellular membranes are essential. This review deals with the different intracellular pathways that allow the traffic of antigens to compartments proficient in processing and loading of MHC class I molecules for presentation to CD8+ T lymphocytes and highlights the need to molecularly identify the transporters involved.

Exogenous, TAP-independent lysosomal presentation of a respiratory syncytial virus CTL epitope

Respiratory syncytial virus causes lower respiratory tract infections in infancy and old age, affecting also immunocompromised patients. The viral fusion protein is an important vaccine candidate eliciting antibody and cell‐mediated immune responses. CD8+ cytotoxic T lymphocytes (CTLs) are known to have a role in both lung pathology and viral clearance. In BALB/c mice, the fusion protein epitope F249‐258 is presented to CTLs by the murine major histocompatibility complex (MHC) class I molecule Kd. In cells infected with recombinant vaccinia viruses encoding the fusion protein, F249‐258 is presented by MHC class I molecules through pathways that are independent of the transporters associated with antigen processing (TAP). We have now found that F249‐258 can be generated from non‐infectious virus from an exogenous source. Antigen processing follows a lysosomal pathway that appears to require autophagy. As a practical consequence, inactivated virus suffices for in vivo priming of virus‐specific CTLs.

Human respiratory syncytial virus infects and induces activation markers in mouse B lymphocytes

Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. Although human airway epithelial cells are the main target of HRSV, it has been reported that this virus can also infect professional antigen‐presenting cells such as macrophages and dendritic cells, promoting upregulation of maturation markers. Here, we report that mouse spleen B220+ B lymphocytes were susceptible to HRSV infection in vitro, probably involving a glycosaminoglycan‐dependent mechanism. In contrast, neither CD4+ nor CD8+ T lymphocytes were infected. In B lymphocytes, HRSV infection upregulated major histocompatibility complex (MHC) class II but not MHC class I molecules and induced the expression of the activation marker CD86.

Concerted In Vitro Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

Relevance of viral context and diversity of antigen-processing routes for respiratory syncytial virus cytotoxic T-lymphocyte epitopes.

Antigen processing of respiratory syncytial virus (RSV) fusion (F) protein epitopes F85-93 and F249-258 presented to cytotoxic T-lymphocytes (CTLs) by the murine major histocompatibility complex (MHC) class I molecule Kd was studied in different viral contexts. Epitope F85-93 was presented through a classical endogenous pathway dependent on the transporters associated with antigen processing (TAP) when the F protein was expressed from either RSV or recombinant vaccinia virus (rVACV). At least in cells infected with rVACV encoding either natural or cytosolic F protein, the proteasome was required for epitope processing. In cells infected with rVACV encoding the natural F protein, an additional endogenous TAP-independent presentation pathway was found for F85-93. In contrast, epitope F249-258 was presented only through TAP-independent pathways, but presentation was brefeldin A sensitive when the F protein was expressed from RSV, or mostly resistant when expressed from rVACV. Therefore, antigen-processing pathways with different mechanisms and subcellular localizations are accessible to individual epitopes presented by the same MHC class I molecule and processed from the same protein but in different viral contexts. This underscores both the diversity of pathways available and the influence of virus infection on presentation of epitopes to CTLs.

The Viral Transcription Group Determines the HLA Class I Cellular Immune Response Against Human Respiratory Syncytial Virus

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design.

TLR4-Independent upregulation of activation markers in mouse B lymphocytes infected by HRSV

Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. In addition, HRSV poses a serious health risk in immunocompromised individuals and the elderly. It has been reported that this virus can infect mouse antigen-presenting cells, including B lymphocytes. In these B cells, HRSV infection upregulates the expression of activation markers, including MHC class II and CD86, but not MHC class I molecules. Here, we report that HRSV infection of spleen B lymphocytes downregulated TLR4. Either blocking with anti-TLR4 antibody or genetic deletion, but not functional deficiency of TLR4, moderately reduced the infectivity of HRSV in B lymphocytes. HRSV-infected B lymphocytes with deleted TLR4 upregulated MHC class II and CD86 molecules to the same levels as TLR4(+) wild type B cells. Since the activation of monocytes and macrophages by HRSV was previously reported to depend on TLR4, the current study indicates that these cells and B lymphocytes respond to HRSV infection with different activation pathways.

A Common Minimal Motif for the Ligands of HLA-B*27 Class I Molecules

CD8+ T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development.

Vaccination and the TAP-independent antigen processing pathways.

The cytotoxic CD8+ T lymphocyte-mediated cellular response is important for the elimination of virus-infected cells and requires the prior recognition of short viral peptide antigens previously translocated to the endoplasmic reticulum by the transporter associated with antigen processing (TAP). However, individuals with nonfunctional TAP complexes or infected cells with TAP molecules blocked by specific viral proteins, such as the cowpoxvirus, a component of the first source of early empirical vaccination against smallpox, are still able to present several HLA class I ligands generated by the TAP-independent antigen processing pathways to specific cytotoxic CD8+ T lymphocytes. Currently, bioterrorism and emerging infectious diseases have renewed interest in poxviruses. Recent works that have identified HLA class I ligands and epitopes in virus-infected TAP-deficient cells have implications for the study of both the effectiveness of early empirical vaccination and the analysis of HLA class I antigen processing in TAP-deficient subjects.