Human chorionic gonadotropin beta (beta-hCG) is an oncofetal antigen expressed by trophoblast cells of the placenta, with minimal expression in adult somatic tissues. Numerous studies have demonstrated that beta-hCG-encoding genes are expressed in various cancers, but expression of these genes (CGB3, CGB5, CGB7, and CGB8) across diverse cancers has not been systematically evaluated. Here, we report that CGB genes are more widely expressed across diverse cancer types than previously appreciated and that secreted beta-hCG is readily detected. In particular, CGB genes are expressed in the majority of urothelial bladder cancers, where CGB7 is most frequently expressed and significantly associated with an immunosuppressed tumor microenvironment, including decreased CD8+ T cell infiltration. Multiple CGB genes are associated with failure to respond to immune checkpoint inhibitor (ICI) therapy, and CGB7 is particularly strongly predictive of poor prognosis. Overall, our findings indicate that beta-hCG is a clinically accessible, predictive biomarker of immunotherapeutic response.

Data associated with an original research study examining T cell responses to mRNA vaccination in COVID-19 recovered individuals. 10X Cell Ranger outputs, bulk TCR sequencing data, and T cell functional (ICS) data in this study have been deposited. Authors KMB and HR contributed equally to this effort. Address correspondence to EWN.

Background Cervical cancer (CC) is the most common cancer in women living with HIV (WLWH) and the leading cause of cancer mortality in women in Uganda.1,2 CC occurs in a fraction of unresolved high-risk human papilloma virus (hrHPV) infections. HIV is a risk factor for hrHPV infection, however, infection and early stage, low grade squamous epithelial lesions (LSIL), can be resolved by a competent immune response. Nevertheless, WLWH with immune function restored by antiretroviral therapy (ART) remain at higher risk for persistent hrHPV infection, LSIL and progression to high grade squamous epithelial lesions (HSIL).3 Therefore, we hypothesize that the immune response differs between WLWH, on ART, who progress from LSIL to HSIL/ICC, compared to WLWH, on ART, who experience LSIL regression. Methods Recently, a cohort (n=304) of Ugandan WLWH and HIV seronegative counterparts was established to categorize hrHPV status and examine associations with the immune response.4 When stratified by degree of dysplasia, hrHPV infection was more prevalent in WLWH with LSIL (cervical intraepithelial neoplasia (CIN) 0/1), compared to seronegative women, than the same comparison in HSIL (CIN2/3).4 Due to the scale of this study, immune parameters analyzed were limited to CD4 and CD8 counts and ratio. This prompted us to design a pilot study, subsampling the cohort, for in-depth interrogation of the immune response in LSIL. The study involves analysis of formalin fixed paraffin embedded (FFPE) cervical tissue and peripheral blood mononuclear cells (PBMC) from WLWH, on ART, diagnosed with LSIL associated with hrHPV infection with progression (n=4) or regression (n=4) or WLWH with no dysplasia diagnosis (n=4). Results We developed a mass cytometry panel incorporating markers of T cell dysregulation and mucosal homing and leveraged combinatorial tetramer technology to analyze PBMC-derived T cell responses to antigens from various hrHPV types. Further functional analysis is being performed using cytokine intracellular staining (ICS) flow cytometry. FFPE-derived DNA and RNA are subject to TCR sequencing and immunology gene focused-Nanostring analysis, respectively, to complement peripheral immune response data. Owing to complexities of sample procurement, data analysis is ongoing. Conclusions LSIL represents a reversible stage in the development of CC, with recent data3 suggesting this may be associated with poorer resolution of hrHPV infection in WLWH, compared to seronegative women. In this pilot study, we expect to identify features of immune dysregulation underlying increased risk of malignant progression from LSIL, which could propel larger scale high dimensional analysis of the cohort. References C. de Martel, M. Plummer, J. Vignat, and S. Franceschi, Worldwide burden of cancer attributable to HPV by site, country and HPV type, Int. J. Cancer, vol. 141, no. 4, pp. 664–670, Aug. 2017, doi: 10.1002/ijc.30716. F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre, and A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA. Cancer J. Clin., vol. 68, no. 6, pp. 394–424, 2018, doi: 10.3322/caac.21492. S. Blitz et al., Evaluation of HIV and highly active antiretroviral therapy on the natural history of human papillomavirus infection and cervical cytopathologic findings in HIV-positive and high-risk HIV-negative women, J. Infect. Dis., vol. 208, no. 3, pp. 454–462, Aug. 2013, doi: 10.1093/infdis/jit181. C. Nakisige et al., Multiple High-Risk HPV Types Contribute to Cervical Dysplasia in Ugandan Women Living With HIV on Antiretroviral Therapy, J. Acquir. Immune Defic. Syndr. 1999, vol. 90, no. 3, pp. 333–342, Jul. 2022, doi: 10.1097/QAI.0000000000002941. Ethics Approval This study including the informed consent process and consent forms in English and Luganda was approved by the Fred Hutchinson Cancer Research Center Institutional Review Office, the UCI Research Ethics Committee and the Uganda National Council for Science and Technology. All participants gave informed consent. Consent De-identified patient information from this cohort was obtain through approval by the institutional review board at the Fred Hutchinson Cancer Center (IR File#: 10496)

Abstract Destabilization of balanced immune cell numbers and frequencies is a common feature of viral infections. This occurs due to, and further enhances, viral immune evasion and survival. Since the discovery of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which manifests in coronavirus disease 2019 (COVID-19), a great number of studies have described the association between this virus and pathologically increased or decreased immune cell counts. In this review, we consider the absolute and relative changes to innate and adaptive immune cell numbers, in COVID-19. In severe disease particularly, neutrophils are increased, which can lead to inflammation and tissue damage. Dysregulation of other granulocytes, basophils and eosinophils represents an unusual COVID-19 phenomenon. Contrastingly, the impact on the different types of monocytes leans more strongly to an altered phenotype, e.g. HLA-DR expression, rather than numerical changes. However, it is the adaptive immune response that bears the most profound impact of SARS-CoV-2 infection. T cell lymphopenia correlates with increased risk of intensive care unit admission and death; therefore, this parameter is particularly important for clinical decision-making. Mild and severe diseases differ in the rate of immune cell counts returning to normal levels post disease. Tracking the recovery trajectories of various immune cell counts may also have implications for long-term COVID-19 monitoring. This review represents a snapshot of our current knowledge, showing that much has been achieved in a short period of time. Alterations in counts of distinct immune cells represent an accessible metric to inform patient care decisions or predict disease outcomes.

Abstract COVID-19 is characterized by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterized by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients’ long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2-specific T cells to elucidate markers that may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that while some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation.

Cytotoxic CD8+ T lymphocytes (CTLs) recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathological conditions such as cancer. Difficulty in predicting HLA class I ligands is attributed to the complexity of the Ag processing pathway across the cytosol and the endoplasmic reticulum. By means of HLA ligandome analysis using mass spectrometry, we collected natural HLA class I ligands on a large scale and analyzed the source-protein sequences flanking the ligands. This comprehensive analysis revealed that the frequency of proline at amino acid positions 1-3 upstream of the ligands was selectively decreased. The depleted proline signature was the strongest among all the upstream and downstream profiles. Experiments using live cells demonstrated that the presence of proline at upstream positions 1-3 attenuated CTL responses against a model epitope. Other experiments, in which N-terminal-flanking Ag precursors were confined in the endoplasmic reticulum, demonstrated an inability to remove upstream prolines regardless of their positions, suggesting a need for synergistic action across cellular compartments for making the proline signature. Our results highlight, to our knowledge, a unique role and position of proline for inhibiting downstream epitope presentation, which provides a rule for defining natural peptide-HLA class I repertoire formation and CTL responses.

Introduction: Localized prostate cancer (PCa) can be successfully treated by androgen deprivation, radiotherapy and surgery, however these may not be sufficient to eradicate cancer stem cells (CSCs). CSCs are more resistant to such treatments than the bulk of the tumor; and can contribute to disease relapse. PCa patients who relapse have a poor prognosis. We hypothesize that CSCs could be killed by T-cells in an antigen specific way, thus preventing the possibility of relapse. In this study we identified novel PCa CSC antigens by HLA ligandome analysis and isolated antigen specific CD8+ T-cells. Methods: We identified CSCs using aldehyde dehydrogenase (ALDH) activity as a CSC marker. ALDH high and low cells from the DU145 PCa cell line and from prostate adenocarcinoma primary tissue were characterized in vitro; DU145 CSCs and non-CSCs were additionally characterized in vivo. We isolated peptide-HLA complexes from DU145 cells by immunoprecipitation and analyzed the eluted peptides by mass spectrometry. We identified CSC antigens based on the gene expression in ALDH high and low DU145 cells (measured by qPCR). To select antigens for further analysis we performed homology modelling of the HLA-peptide interface using COOT software and the YASARA server for energy minimization. The interface interactions were quantified using PISA software. We additionally confirmed antigen expression in the primary cells by fluorescence microscopy and PCR. Tetramers were produced to isolate T-cells which recognized a selection of these antigens. Results: The ligandome analysis identified over 1900 peptides. We selected antigens with low gene expression in healthy tissues (www.GTexportal.org) and high predicted binding to DU145 HLA alleles (http://tools.immuneepitope.org/mhci/). ALDH high DU145 cells were more tumorigenic in vivo than ALDH low cells. ALDH high DU145 and primary prostate cancer cells grew larger colonies and spheres in vitro. We identified 11 CSC antigens by qPCR; 6 upregulated in ALDH high DU145 cells (e.g., TACSTD2) and 5 abundant in both ALDH high and ALDH low DU145 cells (e.g., XPO1). Relevant 9-mer epitopes from three antigens* induced CD8+ T-cell responses in vitro. Antigen-specific CD8+ T-cells were identified by tetramer staining at a frequency of approx. 15 per 100000 cells. These cells are currently being expanded to use in CTL assays. Conclusion: We have identified CSC antigens which could lead to specific targeting by T-cells and prevention of PCa relapse. Further epitopes restricted to the more frequent HLA alleles could additionally be predicted in silico from the novel antigens we identified. We are also investigating prediction of viral epitopes highly aligned with the self-peptides (determined in silico) to boost the immune response against CSCs. *Subject of an ongoing patent application. Citation Format: Amy S. Codd, Saly Al-Taei, Serina Tokita, Emi Mizushima, Pierre J. Rizkallah, Tom Whalley, Barbara Szomolay, Kristin Ladell, James E. McLaren, Sian Llewellyn-Lacey, David A. Price, Takayuki Kanaseki, Toshihiko Torigoe, Stephen Man, Zsuzsanna Tabi. Identification of prostate cancer stem cell antigens for T-cell immunotherapy by HLA ligandome analysis [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B007.

Introduction Cancer stem cells (CSCs) are more resistant to chemo- and radiation therapy than the bulk of the tumour. CSCs, surviving traditional therapies, possess self-renewal capacity and contribute to tumour re-growth. We are investigating the susceptibility of CSCs to antigen-specific killing by T cells. We generated a dataset incorporating all HLA Class I epitopes of the DU145 prostate cancer cell line. We aim to identify novel CSC epitopes which may serve as T cell targets. Material and methods We isolated HLA Class I:peptide complexes from DU145 cells by immunoaffinity precipitation and analysed the epitope sequences by liquid chromatography tandem-mass spectrometry (LC-MS/MS). We selected epitopes in the dataset which showed low gene expression in healthy tissues (www.GTexportal.org) and high predicted HLA-binding affinity (http://tools.immuneepitope.org/mhci/). DU145 CSCs were isolated based on aldehyde dehydrogenase (ALDH) activity using FACS. ALDHhigh and ALDHlow DU145 cells were determined to have CSC or non-CSC characteristics, respectively, by in vivo tumour initiation. Gene expression of the epitopes was measured by qPCR in ALDHhigh and ALDHlow DU145 cells. Protein expression was determined by immunofluorescence. We performed homology modelling of the HLA:peptide interfaces and quantified the binding interactions using PISA interface analysis software. We measured T cell activation by selected peptides analysing cytokine release by flow cytometry. Results and discussions We selected 42 epitopes from the DU145 dataset with low global tissue expression and predicted HLA binding percentile rank Conclusion We have identified prostate CSC epitopes from proteins involved in vascular regulation, calcium signalling and nuclear export. This highlights the potential for targeting CSC epitopes from proteins other than those used for CSC isolation and maybe shared with non cancer stem cells. Our ongoing work involves tetramer based isolation of CSC epitope-specific cytotoxic T cells.

Current cancer therapies target the bulk of the tumour, while a population of highly resistant tumour cells may be able to repopulate the tumour and metastasize to new sites. Cancer cells with such stem cell-like characteristics can be identified based on their phenotypical and/or functional features which may open up ways for their targeted elimination. In this review we discuss potential off-target effects of inhibiting cancer stem-cell self-renewal pathways on immune cells, and summarize some recent immunological studies specifically targeting cancer stem cells based on their unique antigen expression.

Current cancer therapies target the bulk of the tumour, while a population of highly resistant tumour cells may be able to repopulate the tumour and metastasize to new sites. Cancer cells with such stem cell-like characteristics can be identified based on their phenotypical and/or functional features which may open up ways for their targeted elimination. In this review we discuss potential off-target effects of inhibiting cancer stem-cell self-renewal pathways on immune cells, and summarize some recent immunological studies specifically targeting cancer stem cells based on their unique antigen expression.

Cancer initiating cells (CIC) or cancer stem-like cells (CSC) represent a small, distinct population of cancer cells best characterized by their high tumorigenicity. They undergo asymmetric cell division that results in repopulation of the bulk of the tumor and self-renewal. CICs are likely responsible for treatment failure and tumor recurrence, as they are highly resistant to traditional therapies, such as chemo- and radiotherapy (1). This resistance is partly due to their proliferative quiescence and increased anti-apoptotic features. Immune targeting is an emerging alternative approach, which may override these resistance mechanisms. However, relatively little is known about the interaction between CICs and immune cells that have the capacity to recognise and destroy not only the bulk of the tumor but also CICs.