Oriana Hawkins

Two MHC Class I Molecules Associated with Elite Control of Immunodeficiency Virus Replication, Mamu-B*08 and HLA-B*2705, Bind Peptides with Sequence Similarity

HLA-B27- and -B57-positive HIV-infected humans have long been associated with control of HIV replication, implying that CD8+ T cell responses contribute to control of viral replication. In a similar fashion, 50% of Mamu-B*08-positive Indian rhesus macaques control SIVmac239 replication and become elite controllers with chronic-phase viremia <1000 viral RNA copies/ml. Interestingly, Mamu-B*08-restricted SIV-derived epitopes appeared to match the peptide binding profile for HLA-B*2705 in humans. We therefore defined a detailed peptide-binding motif for Mamu-B*08 and investigated binding similarities between the macaque and human MHC class I molecules. Analysis of a panel of ∼900 peptides revealed that despite substantial sequence differences between Mamu-B*08 and HLA-B*2705, the peptide-binding repertoires of these two MHC class I molecules share a remarkable degree of overlap. Detailed knowledge of the Mamu-B*08 peptide-binding motif enabled us to identify six additional novel Mamu-B*08-restricted SIV-specific CD8+ T cell immune responses directed against epitopes in Gag, Vpr, and Env. All 13 Mamu-B*08-restricted epitopes contain an R at the position 2 primary anchor and 10 also possess either R or K at the N terminus. Such dibasic peptides are less prone to cellular degradation. This work highlights the relevance of the Mamu-B*08-positive SIV-infected Indian rhesus macaque as a model to examine elite control of immunodeficiency virus replication. The remarkable similarity of the peptide-binding motifs and repertoires for Mamu-B*08 and HLA-B*2705 suggests that the nature of the peptide bound by the MHC class I molecule may play an important role in control of immunodeficiency virus replication.

Identification of breast cancer peptide epitopes presented by HLA-A*0201

Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.

Differential MHC class I expression in distinct leukocyte subsets

BackgroundMHC class I proteins are partly responsible for shaping the magnitude and focus of the adaptive cellular immune response. In humans, conventional wisdom suggests that the HLA-A, -B, and -C alleles are equally expressed on the majority of cell types. While we currently have a thorough understanding of how total MHC class I expression varies in different tissues, it has been difficult to examine expression of single MHC class I alleles due to the homogeneity of MHC class I sequences. It is unclear how cDNA species are expressed in distinct cell subsets in humans and particularly in macaques which transcribe upwards of 20 distinct MHC class I alleles at variable levels.ResultsWe examined MHC gene expression in human and macaque leukocyte subsets. In humans, while we detected overall differences in locus transcription, we found that transcription of MHC class I genes was consistent across the leukocyte subsets we studied with only small differences detected. In contrast, transcription of certain MHC cDNA species in macaques varied dramatically by up to 45% between different subsets. Although the Mafa-B*134:02 RNA is virtually undetectable in CD4+ T cells, it represents over 45% of class I transcripts in CD14+ monocytes. We observed parallel MHC transcription differences in rhesus macaques. Finally, we analyzed expression of select MHC proteins at the cell surface using fluorescent peptides. This technique confirmed results from the transcriptional analysis and demonstrated that other MHC proteins, known to restrict SIV-specific responses, are also differentially expressed among distinct leukocyte subsets.ConclusionsWe assessed MHC class I transcription and expression in human and macaque leukocyte subsets. Until now, it has been difficult to examine MHC class I allele expression due to the similarity of MHC class I sequences. Using two novel techniques we showed that expression varies among distinct leukocyte subsets of macaques but does not vary dramatically in the human cell subsets we examined. These findings suggest pathogen tropism may have a profound impact on the shape and focus of the MHC class I restricted CD8+ T cell response in macaques.

Direct discovery and validation of a peptide/MHC epitope expressed in primary human breast cancer cells using a TCRm monoclonal antibody with profound antitumor properties

The identification and validation of new cancer-specific T cell epitopes continues to be a major area of research interest. Nevertheless, challenges remain to develop strategies that can easily discover and validate epitopes expressed in primary cancer cells. Regarded as targets for T cells, peptides presented in the context of the major histocompatibility complex (MHC) are recognized by monoclonal antibodies (mAbs). These mAbs are of special importance as they lend themselves to the detection of epitopes expressed in primary tumor cells. Here, we use an approach that has been successfully utilized in two different infectious disease applications (WNV and influenza). A direct peptide-epitope discovery strategy involving mass spectrometric analysis led to the identification of peptide YLLPAIVHI in the context of MHC A*02 allele (YLL/A2) from human breast carcinoma cell lines. We then generated and characterized an anti-YLL/A2 mAb designated as RL6A TCRm. Subsequently, the TCRm mAb was used to directly validate YLL/A2 epitope expression in human breast cancer tissue, but not in normal control breast tissue. Moreover, mice implanted with human breast cancer cells grew tumors, yet when treated with RL6A TCRm showed a marked reduction in tumor size. These data demonstrate for the first time a coordinated direct discovery and validation strategy that identified a peptide/MHC complex on primary tumor cells for antibody targeting and provide a novel approach to cancer immunotherapy.

Functional analysis of frequently expressed Chinese rhesus macaque MHC class I molecules Mamu-A1*02601 and Mamu-B*08301 reveals HLA-A2 and HLA-A3 supertypic specificities

The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques’ major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC–peptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses.

An HLA-Presented Fragment of Macrophage Migration Inhibitory Factor Is a Therapeutic Target for Invasive Breast Cancer

This report describes a novel HLA/peptide complex with potential prognostic and therapeutic roles for invasive breast cancer. Macrophage migration inhibitory factor (MIF) mediates inflammation and immunity, and MIF overexpression is observed in breast cancer. We hypothesized that the HLA class I of cancerous breast epithelial cells would present MIF-derived peptides. Consistent with this hypothesis, the peptide FLSELTQQL (MIF19–27) was eluted from the HLA-A*0201 (HLA-A2) of breast cancer cell lines. We posited that if this MIF19–27/HLA-A2 complex was exclusively found in invasive breast cancer, it could be a useful prognostic indicator. To assess the presentation of MIF peptides by the HLA of various cells and tissues, mice were immunized with the MIF19–27/HLA-A2 complex. The resulting mAb (RL21A) stained invasive ductal carcinoma (IDC) but not ductal carcinoma in situ, fibroadenoma, or normal breast tissues. RL21A did not stain WBCs (total WBCs) or normal tissues from deceased HLA-A2 donors, substantiating the tumor-specific nature of this MIF/HLA complex. As this MIF/HLA complex appeared specific to the surface of IDC, RL21A was tested as an immunotherapeutic for breast cancer in vitro and in vivo. In vitro, RL21A killed the MDA-MB-231 cell line via complement and induction of apoptosis. In an in vivo orthotopic mouse model, administration of RL21A reduced MDA-MB-231 and BT-20 tumor burden by 5-fold and by >2-fold, respectively. In summary, HLA-presented MIF peptides show promise as prognostic cell surface indicators for IDC and as targets for immunotherapeutic intervention.

The Immune Response Under Stress: Class I HLA Presentation of Host-Derived Peptides

Major histocompatibility complex (MHC) class I molecules are found at the surface of all nucleated cells. Class I molecules function as immune sentries by scanning the intracellular proteome and then reflecting the proteome at the cell surface. Through class I presented peptides, T lymphocytes and other immune effector cells can continuously survey the intracellular proteome. Viral infection and cancerous transformation results in the presentation of peptides not found on healthy cells. Class I presented peptides therefore act to distinguish infected and cancerous cells in the eyes of the immune response. Here, we review how class I molecules reflect host cell stress resulting from infection and cancerous transformation. Class I molecules display peptides derived from heat shock proteins on both cancerous and virus-infected cells, and these peptides are clearly recognized by the immune response. The class I of diseased cells also reveal less obvious stress-related signals: Peptide fragments of proteins involved in cell homeostasis act to distinguish infected or cancerous cells. Finally, peptides derived from particular host proteins act as broad indicators of cellular stress, distinguishing both cancerous and virus-infected cells. These class I presented peptides are positioned to influence adaptive and innate immune responses to cellular stress