Biology subjects

McCall, J. L.

Publications and source records attributed to McCall, J. L..

4 recordsLinked to original sources

bioRxiv2024

A novel approach to digital characterisation of Tertiary Lymphoid Structures in colorectal cancer

Tertiary Lymphoid Structures (TLS) in cancer tissue are potential sites for the organisation of immune responses to cancer, and correlate positively with improved clinical outcomes for patients including in colorectal cancer (CRC). However it has proven challenging to standardise assessment of TLS due to the highly variable appearances of circumscribed domains of TLS within tissue sections. A recent three-dimensional reconstruction of TLS in CRC tissue showed that TLS are often large, multi-lobular structures, suggesting that assessing TLS across whole sections may be necessary to provide an accurate view of TLS activity in a patients tumour. In a pilot study we therefore used whole slide scans of multiplexed immunofluorescence images to characterise TLS from 22 subjects with CRC. Multiplexed staining for CD20, CD3, CD8, FoxP3 and Ki-67 enabled us to identify B-cells, CD8+ T cells, FoxP3- CD4 T-cells, and Foxp3+ CD4 T cells in all sections, and quantify both the presence of these cell subsets in lymphocytic clusters and their degree of proliferation within those clusters. In total we identified 524 lymphocytic clusters with morphology consistent with TLS. The count of TLS domains varied substantially between samples (from 4 to 100, mean 24) as did the proportion of total section area occupied by TLS (0.2%-7.8%) as well as size, morphology and cellular constituents; reflecting the intersection of the section plane with complex 3-dimensional structures. We quantified proliferation of B-cells and T-cell subsets within TLS domains across entire sections and compared to the canonical approach of counting and phenotyping individual TLS domains. The whole-slide approach proved far simpler than the canonical approach, generating digital summaries across our sample set clearly identifying patients with strikingly different levels of immune activity within their TLS. It also allowed us to demonstrate strong correlations between the proliferation of B-cells within CRC TLS and that of T-cell subsets. In summary we find that whole-section digital quantification of immune cell activity within TLS has major advantages over canonical approaches. Such whole-section approaches should accelerate research into correlations between TLS status and clinical outcomes, and ultimately enable standardised clinical tests based on automated analysis of multiplexed images.

cancer biology↗

bioRxiv2021

Prenatal cadmium exposure alters proliferation in mouse CD4+ T cells via LncRNA Snhg7

ObjectivePrenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA hostgene 7 (lncSnhg7) in T cell proliferation. MethodsRNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. ResultsWe identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. ConclusionPrenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

immunology↗

bioRxiv2021

Prenatal cadmium exposure does not induce greater incidence or earlier onset of autoimmunity in the offspring

We previously demonstrated that exposure of adult mice to environmental levels of cadmium (Cd) alters the immune cell development and function with increases in anti-streptococcal antibody levels, as well as decreases in splenic natural regulatory T cells (nTreg) in the adult female offspring. Based on these data, we hypothesized that prenatal Cd exposure could predispose an individual to developing autoimmunity as adults. To test this hypothesis, the effects of prenatal Cd on the development of autoimmune diabetes and arthritis were investigated. Non-obese diabetic (NOD) mice were exposed to Cd in a manner identical to our previous studies, and the onset of diabetes was assessed in the offspring. Our results showed a similar time-to-onset and severity of disease to historical data, and there were no statistical differences between Cd-exposed and control offspring. Numerous other immune parameters were measured and none of these parameters showed biologically relevant differences between Cd-exposed and control animals. To test whether prenatal Cd-exposure affected development of autoimmune arthritis, we used SKG mice. While the levels of arthritis were similar between Cd-exposed and control offspring of both sexes, the pathology of arthritis determined by micro-computed tomography (CT) between Cd-exposed and control animals, showed some statistically different values, especially in the female offspring. However, the differences were small and thus, the biological significance of these changes is open to speculation. Overall, based on the results from two autoimmune models, we conclude that prenatal exposure to Cd did not lead to a measurable propensity to develop autoimmune disease later in life.

immunology↗

bioRxiv2019

Type I interferons are important co-stimulatory signals during T cell receptor mediated MAIT cell activation

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells which can be stimulated either via their T cell receptor (TCR) or by innate cytokines. The MAIT cell TCR recognises a pyrimidine ligand, derived from riboflavin synthesising bacteria, bound to MR1. In infection, bacteria not only provide the pyrimidine ligand but also co-stimulatory signals, such as Toll-like receptor agonists, that can modulate TCR-mediated activation. Recently, type I interferons (T1-IFNs) have been identified as contributing to cytokine-mediated MAIT cell activation. However, it is unknown whether T1-IFNs also have a role during TCR-mediated MAIT cell activation. In this study, we investigated the co-stimulatory role of T1-IFNs during TCR-mediated activation of MAIT cells by the MR1 ligand 5-amino-6-D-ribitylaminouracil/methylglyoxal (5-A-RU/MG). We found that T1-IFNs were able to boost interferon-{gamma} and granzyme B production in 5-A-RU/MG-stimulated MAIT cells. Similarly, influenza virus-induced T1-IFNs enhanced TCR-mediated MAIT cell activation. An essential role of T1-IFNs in regulating MAIT cell activation by riboflavin synthesising bacteria was also demonstrated. The co-stimulatory role of T1-IFNs was confirmed using liver-derived MAIT cells. T1-IFNs acted directly on MAIT cells to enhance their response to TCR stimulation. Overall, our findings establish an important immunomodulatory role of T1-IFNs during TCR-mediated MAIT cell activation.

immunology↗