Whole blood-based measurement of SARS-CoV-2-specific T cells reveals asymptomatic infection and vaccine immunogenicity in healthy subjects and patients with solid-organ cancers.

Accurate assessment of SARS-CoV-2 immunity is critical in evaluating vaccine efficacy and devising public health policies. Whilst the exact nature of effective immunity remains incompletely defined, SARS-CoV-2-specific T-cell responses are a critical feature that will likely form a key correlate of protection against COVID-19. Here, we developed and optimized a high-throughput whole blood-based assay to determine the T-cell response associated with prior SARS-CoV-2 infection and/or vaccination amongst 231 healthy donors and 68 cancer patients. Following overnight in vitro stimulation with SARS-CoV-2-specific peptides, blood plasma samples were analysed for TH 1-type cytokines. Highly significant differential IFN-γ+ /IL-2+ SARS-CoV-2-specific T-cell responses were seen amongst previously infected COVID-19-positive healthy donors in comparison with unknown / naïve individuals (p < 0·0001). IFN-γ production was more effective at identifying asymptomatic donors, demonstrating higher sensitivity (96·0% vs. 83·3%) but lower specificity (84·4% vs. 92·5%) than measurement of IL-2. A single COVID-19 vaccine dose induced IFN-γ and/or IL-2 SARS-CoV-2-specific T-cell responses in 116 of 128 (90·6%) healthy donors, reducing significantly to 27 of 56 (48·2%) when measured in cancer patients (p < 0·0001). A second dose was sufficient to boost T-cell responses in the majority (90·6%) of cancer patients, albeit IFN-γ+ responses were still significantly lower overall than those induced in healthy donors (p = 0·034). Three-month post-vaccination T-cell responses also declined at a faster rate in cancer patients. Overall, this cost-effective standardizable test ensures accurate and comparable assessments of SARS-CoV-2-specific T-cell responses amenable to widespread population immunity testing, and identifies individuals at greater need of booster vaccinations.

Prognostic significance of interleukin-17A-producing colorectal tumour antigen-specific T cells.

BACKGROUND: The T cell cytokine profile is a key prognostic indicator of post-surgical outcome for colorectal cancer (CRC). Whilst TH1 (IFN-γ+) cell-mediated responses generated in CRC are well documented and are associated with improved survival, antigen-specific TH17 (IL-17A+) responses have not been similarly measured. METHODS: We sought to determine the cytokine profile of circulating tumour antigen-(5T4/CEA) specific T cells of 34 CRC patients to address whether antigen-specific IL-17A responses were detectable and whether these were distinct to IFN-γ responses. RESULTS: As with IFN-γ-producing T cells, anti-5T4/CEA TH17 responses were detectable predominantly in early stage (TNM I/II) CRC patients. Moreover, whilst IL-17A was always produced in association with IFN-γ, this release was mainly from two distinct T cell populations rather than by 'dual producing' T cells. Patients mounting both tumour-specific TH1+/TH17+ responses exhibited prolonged relapse-free survival. CONCLUSIONS: Tumour antigen-specific TH17 responses play a beneficial role in preventing post-operative colorectal tumour recurrence.

Cancer Antigen Discovery Is Enabled by RNA Sequencing of Highly Purified Malignant and Nonmalignant Cells.

PURPOSE: Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit antitumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms. EXPERIMENTAL DESIGN: Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison with patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by IHC, and preexisting T-cell immunogenicity toward these antigens tested. RESULTS: The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFNγ+ response was markedly magnified by control of immunosuppression with cyclophosphamide in patients with cancer. CONCLUSIONS: This study highlights how prior methods that sequence whole tumor fractions (i.e., inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting.

The Ussing chamber system for measuring intestinal permeability in health and disease.

BACKGROUND: The relationship between intestinal epithelial integrity and the development of intestinal disease is of increasing interest. A reduction in mucosal integrity has been associated with ulcerative colitis, Crohn's disease and potentially could have links with colorectal cancer development. The Ussing chamber system can be utilised as a valuable tool for measuring gut integrity. Here we describe step-by-step methodology required to measure intestinal permeability of both mouse and human colonic tissue samples ex vivo, using the latest equipment and software. This system can be modified to accommodate other tissues. METHODS: An Ussing chamber was constructed and adapted to support both mouse and human tissue to measure intestinal permeability, using paracellular flux and electrical measurements. Two mouse models of intestinal inflammation (dextran sodium sulphate treatment and T regulatory cell depletion using C57BL/6-FoxP3DTR mice) were used to validate the system along with human colonic biopsy samples. RESULTS: Distinct regional differences in permeability were consistently identified within mouse and healthy human colon. In particular, mice showed increased permeability in the mid colonic region. In humans the left colon is more permeable than the right. Furthermore, inflammatory conditions induced chemically or due to autoimmunity reduced intestinal integrity, validating the use of the system. CONCLUSIONS: The Ussing chamber has been used for many years to measure barrier function. However, a clear and informative methods paper describing the setup of modern equipment and step-by-step procedure to measure mouse and human intestinal permeability isn't available. The Ussing chamber system methodology we describe provides such detail to guide investigation of gut integrity.

The nature of the human T cell response to the cancer antigen 5T4 is determined by the balance of regulatory and inflammatory T cells of the same antigen-specificity: implications for vaccine design.

The oncofoetal antigen 5T4 is a promising T cell target in the context of colorectal cancer, as demonstrated by a recent clinical study where 5T4-specific T cell responses, induced by vaccination or cyclophosphamide, were associated with a significantly prolonged survival of patients with metastatic disease. Whilst Th1-type (IFN-γ+) responses specific to 5T4, and other oncofoetal antigens, are often readily detectable in early stage CRC patients and healthy donors, their activity is suppressed as the cancer progresses by CD4+CD25hiFoxp3+ regulatory T cells (Treg) which contribute to the immunosuppressive environment conducive to tumour growth. This study mapped the fine specificity of Th1 and Treg cell responses to the 5T4 protein. Surprisingly, both immunogenic peptides and those recognised by Tregs clustered in the same HLA-DR transcending epitope-rich hotspots within the 5T4 protein. Similarly, regions of low Th1-cell immunogenicity also did not contain peptides capable of stimulating Tregs, further supporting the notion that Treg and Th1 cells recognise the same peptides. Understanding the rules which govern the balance of Th1 and Treg cells responding to a given peptide specificity is, therefore, of fundamental importance to designing strategies for manipulating the balance in favour of Th1 cells, and thus the most effective anti-cancer T cell responses.

Assessing the prognostic value of preoperative carcinoembryonic antigen-specific T-cell responses in colorectal cancer.

Current dogma suggests that tumor-reactive IFN-γ-producing (TH1-type) T-cells are beneficial to patient outcome; however, the clinical consequence of these responses with respect to long-term prognosis in colorectal cancer (CRC) is not understood. Here, we compared the utility of preoperative, peripheral blood-derived IFN-γ(+) T-cell responses specific to carcinoembryonic antigen (CEA), 5T4, or control antigens (n = 64) with tumor staging and clinical details (n = 87) in predicting five-year outcome of CRC patients who underwent resection with curative intent. Although disease recurrence was more likely in patients with stage III tumors, the presence of preoperative, CEA-specific IFN-γ-producing T-cells identified patients at a statistically significantly greater risk of tumor recurrence following surgical resection, irrespective of tumor stage (odds ratio = 5.00, 95% confidence interval = 1.96 to 12.77, two-sided P <.001). Responses to other antigens, including 5T4, did not reflect outcome. Whilst these results initially appear surprising, they could improve prognostication and help redirect adjuvant treatments.

Distinct bone marrow-derived and tissue-resident macrophage lineages proliferate at key stages during inflammation.

The general paradigm is that monocytes are recruited to sites of inflammation and terminally differentiate into macrophages. There has been no demonstration of proliferation of peripherally-derived inflammatory macrophages under physiological conditions. Here we show that proliferation of both bone marrow-derived inflammatory and tissue-resident macrophage lineage branches is a key feature of the inflammatory process with major implications for the mechanisms underlying recovery from inflammation. Both macrophage lineage branches are dependent on M-CSF during inflammation, and thus the potential for therapeutic interventions is marked. Furthermore, these observations are independent of Th2 immunity. These studies indicate that the proliferation of distinct macrophage populations provides a general mechanism for macrophage expansion at key stages during inflammation, and separate control mechanisms are implicated.