Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.

IL-33 expression in response to SARS-CoV-2 correlates with seropositivity in COVID-19 convalescent individuals

Our understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still developing. We perform an observational study to investigate seroprevalence and immune responses in subjects professionally exposed to SARS-CoV-2 and their family members (155 individuals; ages 5–79 years). Seropositivity for SARS-CoV-2 Spike glycoprotein aligns with PCR results that confirm the previous infection. Anti-Spike IgG/IgM titers remain high 60 days post-infection and do not strongly associate with symptoms, except for fever. We analyze PBMCs from a subset of seropositive and seronegative adults. TLR7 agonist-activation reveals an increased population of IL-6+TNF-IL-1β+ monocytes, while SARS-CoV-2 peptide stimulation elicits IL-33, IL-6, IFNa2, and IL-23 expression in seropositive individuals. IL-33 correlates with CD4+ T cell activation in PBMCs from convalescent subjects and is likely due to T cell-mediated effects on IL-33-producing cells. IL-33 is associated with pulmonary infection and chronic diseases like asthma and COPD, but its role in COVID-19 is unknown. Analysis of published scRNAseq data of bronchoalveolar lavage fluid (BALF) from patients with mild to severe COVID-19 reveals a population of IL-33-producing cells that increases with the disease. Together these findings show that IL-33 production is linked to SARS-CoV-2 infection and warrant further investigation of IL-33 in COVID-19 pathogenesis and immunity.

A novel whole-blood stimulation assay to detect and quantify memory T-cells in COVID-19 patients

SARS-CoV-2 specific T-cells responses are essential for virus clearance. We present a novel and simple whole-blood assay allowing the detection of interferon-gamma-producing antiviral T-cells following peptide stimulation. We show that unlike neutralizing antibodies, antiviral memory T-cells persist at least 6 months in convalescent Covid-19 individuals.

Cytotoxic CD8+ T Cells Expressing CXCR5 Are Detectable in HIV-1 Elite Controllers After Prolonged In Vitro Peptide Stimulation

Antiretroviral therapy (ART) is not curative as HIV-1 persists in long-lived viral reservoirs. Consequently, patients are dependent on life-long drug adherence with possible side effects. To overcome these limitations strategies of a functional cure aim at ART free viral remission. In this study, we sought to identify detailed subsets of anti-viral CD8+ T cell immunity linked to natural long-term control of HIV-1 infection. Here, we analyzed HIV controllers and ART suppressed progressors for in vitro viral suppressive capacity (VSC) at baseline and after peptide stimulation. Functional properties and phenotypes of CD8+ T cells were assessed by IFN-γ ELISPOT and 18 color flow cytometry. HIV controllers showed significantly increased suppression at baseline as well as after peptide stimulation. IFN-γ secretion and the proliferation marker Ki67 positively correlated with VSC. Moreover, the detailed phenotype of three distinct multifunctional memory CD8+ T cell subsets were specific traits of HIV controllers of which two correlated convincingly with VSC. Our results underline the importance of multifunctional CD8+ T cell responses during natural control. Especially the role of CXCR5 expressing cytotoxic subsets emphasizes potential surveillance in sites of reservoir persistence and demand further study.

The GPIbα intracellular tail - role in transducing VWF- and Collagen/GPVI-mediated signaling

Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GPIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail important for VWF-mediated signaling. GPIbαΔsig/Δsig platelets bound VWF normally under flow but formed fewer filopodia on VWF/botrocetin, demonstrating that the deleted region does not affect ligand binding but appreciably impairs VWF-dependent signaling. Notably, while haemostasis was normal in GPIbαΔsig/Δsig mice, GPIbαΔsig/Δsig platelets exhibited defective responses after collagen-related-peptide stimulation and formed smaller aggregates on collagen-coated microchannels at low and high shears. Flow assays performed with plasma-free blood or in the presence of αIIbβ3- or GPVI-blockers suggested reduced αIIbβ3 activation contributes to the phenotype of the GPIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF- GPIbα and collagen-GPVI signaling events in platelets.

825 Deep immune profiling of SARS-CoV-2 associated immune microenvironment in cancer tissues from recovered COVID-19 patients

BackgroundPersistence of SARS-CoV-2 virus particles in recovered COVID-19 patients remains a challenge as we continue to fight the ongoing pandemic. For instance, despite three negative consecutive nasopharyngeal swab PCR tests, residual SARS-CoV-2 was reported in the lungs of a deceased patient.1 Moreover, viral RNA could also be detected in rectal tissues that were obtained during incubation period.2 To date, there is no data regarding residual viral particles present in tissues from recovered COVID-19 patients. Hereby, we reported our findings of SARS-CoV-2 viral antigen in liver tissues from a recovered COVID-19 patient. These findings raise concern for potential transmissibility in recovered individuals.MethodsA 49-year-old South Asian male diagnosed with COVID-19 in June 2020, with incidental discovery of hepatitis B virus (HBV)-associated R0 Grade 2 hepatocellular carcinoma (HCC), was consented for our study. He did not develop significant acute respiratory symptoms throughout the course of the disease. He underwent curative resection of HCC 85 days after being tested COVID-19 negative where his blood, normal tissue and tumour samples were obtained for further analysis (figure 1). We performed deep immunopathological profiling on the specimens using multiplex immunohistochemistry and 25-colour flow cytometry to study SARS-CoV-2-elicited immune response.ResultsMultiplex immunohistochemistry detected SARS-CoV-2 nucleocapsid protein only in adjacent normal liver tissue but not within tumour core (figure 2). We also observed SARS-CoV-2 in some immune cells such as sinusoidal Kupffer cells (figure 2). Additionally, upon stimulation with SARS-CoV-2 peptides, we successfully elicited SARS-CoV-2-specific memory response which is distinct from the response upon challenge with HBV peptides. These findings were similar to our previous discovery in a patient with colorectal adenocarcinoma where we have shown viral antigen detection, validated with PCR to detect viral RNA, as well as the detection of SARS-CoV-2 memory-like T cells in situ (figure 2). Deep profiling of the samples is on-going with single-cell analysis and digital spatial profiling.Abstract 825 Figure 1Study design, methodology and brief summary of the findingsBlood, normal tissue and tumour samples were obtained from a 49-year-old South Asian male who was diagnosed with COVID-19 and hepatocellular carcinoma. Normal tissue and tumour samples were analysed with multiplex immunohistochemistry, while dissociated cells from blood and tissue samples were subjected to SARS-CoV-2 peptide stimulation and analysed with 25-colour flow cytometry. Multiplex immunohistochemistry detected SARS-CoV-2 proteins in both tumour and adjacent normal tissues, while flow cytometry identified distinct immune microenvironment involving memory-like T cells.Abstract 825 Figure 2Immunohistochemical staining of the SARS-CoV-2 nucleocapsid protein and immune profiling with 25-colour flow cytometry in normal colon and liver tissue a, Liver tissues were immunostained with SARS-CoV-2 nucleocapsid protein (NP), nuclei were counterstained with haematoxylin. Positive SARS-CoV-2 nucleocapsid staining in benign hepatocytes and sinusoidal Kupffer cells. Scale bar represents 50μm. b, Multiplex immunohistochemistry of normal liver tissue. From left to right, top to bottom: SARS-CoV-2 nucleocapsid (green), SARS-CoV-2 nucleocapsid (green) with CD14 (red), SARS-CoV-2 nucleocapsid (green) with CD68 (pink) and composite. Co-localisation were observed as shown by the white arrows. Scale bar represents 100μm. c, Colon tissues were immunostained with SARS-CoV-2 nucleocapsid protein, nuclei were counterstained with haematoxylin. Positive SARS-CoV-2 nucleocapsid staining in colonic crypts, with granular supranuclear cytoplasmic pattern. Scale bar represents 50μm. d, Multiplex immunohistochemistry of colon tissue. From left to right, top to bottom: DAPI (blue), CD3 (magenta), CD38 (green), granzyme B (yellow), interferon-gamma (red) and composite. Co-localisation was observed as shown by the white arrows. Scale bar represents 100μm, Magnification x200. e, Flow cytometry immune profiling of blood from colorectal cancer patient with COVID-19 following stimulation with SARS-CoV-2 peptides. Highlighted populations showed CD3 cells expressing CD38, supporting the CD3+ CD38+ co-localization findings observed in (c).ConclusionsWe believe this is the first immune profiling report of the in situ tumour microenvironment in a cancer patient with COVID-19. Our findings demonstrated the presence of viral proteins in the liver despite negative swab test result and the ability to elicit ex vivo SARS-CoV-2-specific immune responses through peptide stimulation assays. We also detected same immune cell phenotypes in situ in the cancer tissues. Taken together, we propose caution when handling tissues from patients who have a recent history of COVID-19, particularly during aerosol-generating procedures such as ultrasonic dissection surgery.Ethics ApprovalThis study was approved by Centralised Institutional Review Board of SingHealth, approval number 2019/2653.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.ReferencesYao XH, He ZC, Li TY, Zhang HR, Wang Y, Mou H, et al. Pathological evidence for residual SARS-CoV-2 in pulmonary tissues of a ready-for-discharge patient. Cell Res 2020;30(6):541-3.Qian Q, Fan L, Liu W, Li J, Yue J, Wang M, et al. Direct evidence of active SARS-CoV-2 replication in the intestine. Clin Infect Dis 2020.

IL-33 expression in response to SARS-CoV-2 correlates with seropositivity in COVID-19 convalescent individuals

Our understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still developing. We investigated seroprevalence and immune responses in subjects professionally exposed to SARS-CoV-2 and their family members (155 individuals; ages 5-79 years). Seropositivity for SARS-CoV-2 spike glycoprotein aligned with PCR results that confirmed previous infection. Anti-spike IgG titers remained high 60 days post-infection and did not associate with symptoms, but spike-specific IgM did associate with malaise and fever. We found limited household transmission, with children of infected individuals seldomly seropositive, highlighting professional exposure as the dominant route of infection in our cohort. We analyzed PBMCs from a subset of seropositive and seronegative adults. TLR7 agonist- activation revealed an increased population of IL-6+TNF-IL-1β+ monocytes, while SARS-CoV-2 peptide stimulation elicited IL-33, IL-6, IFNa2, and IL-23 expression in seropositive individuals. IL-33 correlated with CD4+ T cell activation in PBMCs from convalescent subjects, and was likely due to T cell-mediated effects on IL-33- producing cells. IL-33 is associated with pulmonary infection and chronic diseases like asthma and COPD, but its role in COVID-19 is unknown. Analysis of published scRNAseq data of bronchoalveolar lavage fluid (BALF) from patients with mild to severe COVID-19 revealed a population of IL-33-producing cells that increases with disease. Together these findings show that IL-33 production is linked to SARS-CoV- 2 infection and warrant further investigation of IL-33 in COVID-19 pathogenesis and immunity.