L-selectin is essential for delivery of activated CD8+ T cells to virus-infected organs for protective immunity

Cytotoxic CD8+ T lymphocytes play a critical role in the host response to infection by viruses. The ability to secrete cytotoxic chemicals and cytokines is considered pivotal for eliminating virus. Of equal importance is how effector CD8+ T cells home to virus-infected tissues. L-selectin has not been considered important for effector T cell homing, because levels are low on activated T cells. We report here that, although L-selectin expression is downregulated following T cell priming in lymph nodes, L-selectin is re-expressed on activated CD8+ T cells entering the bloodstream, and recruitment of activated CD8+ T cells from the bloodstream into virus-infected tissues is L-selectin dependent. Furthermore, L-selectin on effector CD8+ T cells confers protective immunity to two evolutionally distinct viruses, vaccinia and influenza, which infect mucosal and visceral organs, respectively. These results connect homing and a function of virus-specific CD8+ T cells to a single molecule, L-selectin

Purity of transferred CD8+ T cells is crucial for safety and efficacy of combinatorial tumor immunotherapy in the absence of SHP-1

Adoptive transfer of tumor-specific cytotoxic T cells is a promising advance in cancer therapy. Similarly, checkpoint inhibition has shown striking clinical results in some patients. Here we combine adoptive cell transfer with ablation of the checkpoint protein Src homology 2-domain-containing phosphatase 1 (SHP-1, Ptpn6). Naturally occurring motheaten mice lack SHP-1 and do not survive weaning due to extensive immunopathology. To circumvent this limitation, we created a novel SHP-1(null) mouse that is viable up to 12 weeks of age by knocking out IL1r1. Using this model, we demonstrate that the absence of SHP-1 augments the ability of adoptively transferred CD8(+) T cells to control tumor growth. This therapeutic effect was only observed in situations where T-cell numbers were limited, analogous to clinical settings. However, adoptive transfer of non-CD8(+) SHP-1(null) hematopoietic cells resulted in lethal motheaten-like pathology, indicating that systemic inhibition of SHP-1 could have serious adverse effects. Despite this caveat, our findings support the development of SHP-1 inhibition strategies in human T cells to complement adoptive transfer therapies in the clinic

L-selectin enhanced T cells improve the efficacy of cancer immunotherapy

The T cell homing molecule, L-selectin (CD62L), is commonly used as a marker of T cell activation, as expression of L-selectin is downregulated following engagement of the T cell receptor. Furthermore, it is used to distinguish “central memory” T cells (TCM) from, “effector memory” T cells (TEM). It has been reported that CD8+ T cells with a CD62L+ TCM phenotype are better able to control tumour growth than CD62L- TEM CD8+ T cells, while L-selectin knockout T cells are poor at controlling tumour growth. Here, we test the hypothesis that T cells expressing a genetically modified form of L-selectin that is not downregulated following T cell activation (L-selectin enhanced T cells) are better able to control tumour growth than wild type T cells. Using mouse models of solid and disseminated tumours, we show that L-selectin enhancement improves the efficacy of CD8+ T cells in controlling tumour growth. Longitudinal tracking of Zirconium-89 (89Zr) labelled T cells using PET-CT showed that transferred T cells localised to tumours within 24 hours. Early T cell recruitment into tumours was not dependent on L-selectin, however, upregulation of the early activation marker CD69 was higher on L-selectin expressing T cells both inside tumours and in secondary lymphoid organs. Reduced growth of tumours by L-selectin enhanced T cells correlated with increased frequency of CD8+ tumour infiltrating T cells 21 days after commencing therapy. Ex vivo analysis showed that clonal expansion of L-selectin enhanced T cells was slower, and that L-selectin was linked to expression of the proliferation marker Ki67. Together these findings indicate that maintaining L-selectin expression on tumour-specific T cells offers an advantage in mouse models of cancer immunotherapy. The beneficial role of L-selectin may be unrelated to its’ well-known role in T cell homing and instead linked to activation, clonal expansion and retention of therapeutic T cells. These findings have implications both for the selection of T cell subsets for adoptive transfer immunotherapy, and for possible modifications of transgenic chimeric antigen receptor (CAR) T cells to broaden the clinical scope of these therapies

ADAM17-dependent proteolysis of L-selectin promotes early clonal expansion of cytotoxic T cells

L-selectin on T-cells is best known as an adhesion molecule that supports recruitment of blood-borne naïve and central memory cells into lymph nodes. Proteolytic shedding of the ectodomain is thought to redirect activated T-cells from lymph nodes to sites of infection. However, we have shown that activated T-cells re-express L-selectin before lymph node egress and use L-selectin to locate to virus-infected tissues. Therefore, we considered other roles for L-selectin proteolysis during T cell activation. In this study, we used T cells expressing cleavable or non-cleavable L-selectin and determined the impact of L-selectin proteolysis on T cell activation in virus-infected mice. We confirm an essential and non-redundant role for ADAM17 in TCR-induced proteolysis of L-selectin in mouse and human T cells and show that L-selectin cleavage does not regulate T cell activation measured by CD69 or TCR internalisation. Following virus infection of mice, L-selectin proteolysis promoted early clonal expansion of cytotoxic T cells resulting in an 8-fold increase over T cells unable to cleave L-selectin. T cells unable to cleave L-selectin showed delayed proliferation in vitro which correlated with lower CD25 expression. Based on these results, we propose that ADAM17-dependent proteolysis of L-selectin should be considered a regulator of T-cell activation at sites of immune activity

CD4 + T cells recognize conserved influenza A epitopes through shared patterns of V-Gene usage and complementary biochemical features

T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency

Background: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. Methods: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. Findings: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. Conclusions: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. Funding: Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366)

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

Enhancement of T Cell Responses as a Result of Synergy between Lower Doses of Radiation and T Cell Stimulation

As a side effect of cancer radiotherapy, immune cells receive varying doses of radiation. Whereas high doses of radiation (>10 Gy) can lead to lymphopenia, lower radiation doses (2–4 Gy) represent a valid treatment option in some hematological cancers, triggering clinically relevant immunological changes. Based on our earlier observations, we hypothesized that lower radiation doses have a direct positive effect on T cells. In this study, we show that 0.6–2.4 Gy radiation enhances proliferation and IFN-γ production of PBMC or purified T cells induced by stimulation via the TCR. Radiation with 1.2 Gy also lowered T cell activation threshold and broadened the Th1 cytokine profile. Although radiation alone did not activate T cells, when followed by TCR stimulation, ERK1/2 and Akt phosphorylation increased above that induced by stimulation alone. These changes were followed by an early increase in glucose uptake. Naive (CD45RA+) or memory (CD45RA−) T cell responses to stimulation were boosted at similar rates by radiation. Whereas increased Ag-specific cytotoxic activity of a CD8+ T cell line manifested in a 4-h assay (10–20% increase), highly significant (5- to 10-fold) differences in cytokine production were detected in 6-d Ag-stimulation assays of PBMC, probably as a net outcome of death of nonstimulated and enhanced response of Ag-stimulated T cells. T cells from patients receiving pelvic radiation (2.2–2.75 Gy) also displayed increased cytokine production when stimulated in vitro. We report in this study enhanced T cell function induced by synergistic radiation treatment, with potential physiological significance in a wide range of T cell responses