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Cancer immunotherapy represents a significant breakthrough in cancer treatment mainly due to the ability to harness the activities of cancer-specific T cells. Despite this, most cancers remain resistant to T cell attack. Many reasons have been proposed to explain this, ranging from a lack of antigenicity through to the immunosuppressive effects of the tumour microenvironment. In this review, we examine the relationship between the immune system and a key component of the tumour microenvironment, namely the extracellular matrix (ECM). Specifically, we explore the reciprocal effects of immune cells and the tumour ECM and how the processes underpinning this relationship act to either promote or restrain tumour progression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: Asymptomatic SARS-CoV-2 infections were widely reported during the COVID-19 pandemic, acting as a hidden source of infection. Many existing studies investigating asymptomatic immunity failed to recruit true asymptomatic individuals. Thus, we conducted a longitudinal cohort study to evaluate humoral- and cell-mediated responses to infection and vaccination in well-defined asymptomatic young adults (the Asymptomatic COVID-19 in Education [ACE] cohort). Methods: Asymptomatic testing services located at three UK universities identified asymptomatic young adults who were subsequently recruited with age- and sex-matched symptomatic and uninfected controls. Blood and saliva samples were collected after SARS-CoV-2 Wuhan infection, and again after vaccination. 51 participant’s anti-spike antibody titres, neutralizing antibodies, and spike-specific T-cell responses were measured, against both Wuhan and Omicron B.1.1.529.1. Results: Asymptomatic participants exhibited reduced Wuhan-specific neutralization antibodies pre- and post-vaccination, as well as fewer Omicron-specific neutralization antibodies post-vaccination, compared to symptomatic participants. Lower Wuhan and Omicron-specific IgG titres in asymptomatic individuals were also observed pre- and post-vaccination, compared to symptomatic participants. There were no differences in salivary IgA levels. Conventional flow cytometry analysis and multi-dimensional clustering analysis indicated unvaccinated asymptomatic participants had significantly fewer Wuhan-specific IL-2 secreting CD4+ CD45RA+ T cells and activated CD8+ T cells than symptomatic participants, though these differences dissipated after vaccination. Conclusions: Asymptomatic infection results in decreased antibody and T cell responses to further exposure to SARS-CoV-2 variants, compared to symptomatic infection. Post-vaccination, antibody responses are still inferior, but T cell immunity increases to match symptomatic subjects, emphasising the importance of vaccination to help protect asymptomatic individuals against future variants. [ABSTRACT FROM AUTHOR]

Emerging studies have demonstrated the potential of PI3Kδ blockade as an immunotherapy for solid tumours. In pre-clinical models, we recently demonstrated that anti-LAG3 immune checkpoint blockade vastly potentiated PI3Kδ-based immunotherapy, enabling successful tumour control in all treated mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Recent studies have demonstrated that blocking the PI3Kδ signalling enzyme (by administering a small molecule inhibitor, PI-3065) can potently improve the anti-tumour T-cell response through direct inhibition of Tregs. This treatment also has a negative impact on MDSC numbers but the primary mechanism driving this effect has remained unclear.Methods: The 4T1 breast cancer mouse model was used in combination with PI-3065 to gain insights into the effect of PI3Kδ inhibition on MDSCs.Results: PI-3065 treatment resulted in a concomitant reduction in MDSC expansion and tumour size. However, targeting Tregs independent of PI-3065 was also associated with reduced tumour volume and MDSC numbers. Surgical removal of tumours resulted in a rapid and significant decline in MDSC numbers, whilst ex vivo studies using cells from PI-3065-treated mice demonstrated no direct effect of the inhibitor on MDSC activity.Conclusions: Our data suggest that MDSCs are not inhibited directly by PI-3065 treatment but that their reduced recruitment and immunosuppression within the tumour microenvironment is an indirect consequence of PI3Kδ-inhibition-driven tumour control. This indicates that PI3Kδ inhibition drives tumour immunity by breaking down multiple immunosuppressive pathways through both direct mechanisms (on Treg) and indirect mechanisms, secondary to tumour control (on MDSCs). [ABSTRACT FROM AUTHOR]

Defolliculated (Gsdma3Dfl/+) mice have a hair loss phenotype that involves an aberrant hair cycle, altered sebaceous gland differentiation with reduced sebum production, chronic inflammation, and ultimately the loss of the hair follicle. Hair loss in these mice is similar to that seen in primary cicatricial, or scarring alopecias in which immune targeting of hair follicle stem cells has been proposed as a key factor resulting in permanent hair follicle destruction. In this study we examine the mechanism of hair loss in GsdmA3Dfl/+ mice. Aberrant expression patterns of stem cell markers during the hair cycle, in addition to aberrant behavior of the melanocytes leading to ectopic pigmentation of the hair follicle and epidermis, indicated the stem cell niche was not maintained. An autoimmune mechanism was excluded by crossing the mice with rag1−/− mice. However, large numbers of macrophages and increased expression of ICAM-1 were still present and may be involved either directly or indirectly in the hair loss. Reverse transcriptase-PCR (RT-PCR) and immunohistochemistry of sebaceous gland differentiation markers revealed reduced peroxisome proliferator-activated receptor-γ (PPARγ), a potential cause of reduced sebum production, as well as the potential involvement of the innate immune system in the hair loss. As reduced PPARγ expression has recently been implicated as a cause for lichen planopilaris, these mice may be useful for testing therapies. [ABSTRACT FROM AUTHOR]

Oncolytic viruses possess the ability to infect, replicate and lyse malignantly transformed tumour cells. This oncolytic activity amplifies the therapeutic advantage and induces a form of immunogenic cell death, characterized by increased CD8 + T‐cell infiltration into the tumour microenvironment. This important feature of oncolytic viruses can result in the warming up of immunologically 'cold' tumour types, presenting the enticing possibility that oncolytic virus treatment combined with immunotherapies may enhance efficacy. In this review, we assess some of the most promising candidates that might be used for oncolytic virotherapy: immunotherapy combinations. We assess their potential as separate agents or as agents combined into a single therapy, where the immunotherapy is encoded within the genome of the oncolytic virus. The development of such advanced agents will require increasingly sophisticated model systems for their preclinical assessment and evaluation. In vivo rodent model systems are fraught with limitations in this regard. Oncolytic viruses replicate selectively within human cells and therefore require human xenografts in immune‐deficient mice for their evaluation. However, the use of immune‐deficient rodent models hinders the ability to study immune responses against any immunomodulatory transgenes engineered within the viral genome and expressed within the tumour microenvironment. There has therefore been a shift towards the use of more sophisticated ex vivo patient‐derived model systems based on organoids and explant co‐cultures with immune cells, which may be more predictive of efficacy than contrived and artificial animal models. We review the best of those model systems here. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although metastatic disease is responsible for the majority of cancer deaths, tests of novel immunotherapies in mouse tumour models often focus on primary tumours without determining whether these therapies also target metastatic disease. This study examined the impact of depleting Foxp3+ regulatory T cells (Treg), on lung metastases, using a mouse model of breast cancer. After Treg-depletion, generation of an immune response to the primary tumour was a critical determinant for limiting development of metastasis. Indeed, resection of the primary tumour abrogated any effect of Treg-depletion on metastases. In addition, whilst the immune response, generated by the primary tumour, prevented metastases development, it had little impact on controlling established disease. Collectively, the data indicate that metastatic cells in the lung are not controlled by immune responses induced by the primary tumour. These findings indicate that targeting Tregs alone will not suffice for treating lung metastases. [ABSTRACT FROM AUTHOR]

Edited by Peter Cresswell CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR-pHLA-II interactions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic "mimics" using subunits that do not exist in the natural world. We developed a platform based on D-amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus-specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery. [ABSTRACT FROM AUTHOR]

Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of 'Treg markers'. Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry. [ABSTRACT FROM AUTHOR]

Summary Background Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects. Results Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased “in situ transcriptomics” analysis—gene expression profiling of laser-captured TAMs to establish their activation signature in situ—we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma. Conclusions In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy. [ABSTRACT FROM AUTHOR]

Background There is indirect evidence that T cell responses can control the metastatic spread of colorectal cancer (CRC). However, an enrichment of CD4+Foxp3+ regulatory T cells (Tregs) has also been documented. Objective To evaluate whether CRC promotes Treg activity and how this influences anti-tumour immune responses and disease progression. Methods A longitudinal study of Treg activity on a cohort of patients was performed before and after tumour resection. Specific CD4+ T cell responses were also measured to the tumour associated antigens carcinoembryonic antigen (CEA) and 5T4. Results Tregs from 62 preoperative CRC patients expressed a highly significant increase in levels of Foxp3 compared to healthy age-matched controls (p¼0.007), which returned to normal after surgery (p¼0.0075). CD4+ T cell responses to one or both of the tumour associated antigens, CEA and 5T4, were observed in approximately two-thirds of patients and one third of these responses were suppressed by Tregs. Strikingly, in all patients with tumour recurrence at 12 months, significant preoperative suppression was observed of tumour-specific (p¼0.003) but not control CD4+ T cell responses. Conclusion These findings demonstrate that the presence of CRC drives the activity of Tregs and accompanying suppression of CD4+ T cell responses to tumour-associated antigens. Suppression is associated with recurrence of tumour at 12 months, implying that Tregs contribute to disease progression. These findings offer a rationale for the manipulation of Tregs for therapeutic intervention. [ABSTRACT FROM AUTHOR]

CD8+ T cells recognize immunogenic peptides presented at the cell surface bound to MHCI molecules. Ag recognition involves the binding of both TCR and CD8 coreceptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on Ag sensitivity. Anti-CD8 Abs have been used extensively to examine the role of CD8 in CD8+ T cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 Abs per se are capable of inducing effector function. In this article, we report on the ability of seven monoclonal anti-human CD8 Abs to activate six human CD8+ T cell clones with a total of five different specificities. Six of seven anti-human CD8 Abs tested did not activate CD8+ T cells. In contrast, one anti-human CD8 Ab, OKT8, induced effector function in all CD8+ T cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of Ag-specific CD8+ T cells. The anti-mouse CD8 Abs, CT-CD8a and CT-CD8b, also activated CD8+ T cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 Abs to trigger T cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of Ab-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CD8+ T cell signaling. [ABSTRACT FROM AUTHOR]

BACKGROUND: Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity. METHODS: A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity. RESULTS: Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice. CONCLUSIONS: Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection. [ABSTRACT FROM AUTHOR]

It is clear that CD4 CD25 Foxp3 regulatory T (Treg) cells inhibit chronic inflammatory responses as well as adaptive immune responses. Among the CD4 T-cell population in the skin, at least one-fifth express Foxp3. As the skin is constantly exposed to antigenic challenge and is a common site of vaccination, understanding the role of these skin-resident Treg cells is important. Although the suppressive effect of Treg cells on T cells is well documented, less is known about the types of innate immune cells influenced by Treg cells and whether the Treg cells suppress acute innate immune responses in vivo. To address this we used a mouse melanoma cell line expressing Fas ligand (B16FasL), which induces an inflammatory response following subcutaneous injection of mice. We demonstrate that Treg cells limit this response by inhibiting neutrophil accumulation and survival within hours of tumour cell inoculation. This effect, which was associated with decreased expression of the neutrophil chemoattractants CXCL1 and CXCL2, promoted survival of the inoculated tumour cells. Overall, these data imply that Treg cells in the skin are rapidly mobilized and that this activity serves to limit the amplification of inflammatory responses at this site. [ABSTRACT FROM AUTHOR]

Abstract: Naturally occurring regulatory T cells (Tregs) have been shown to suppress immune responses to self-antigens, thereby limiting autoimmunity. In the case of tumours, where immune responses to self-antigens are beneficial and lead to elimination of the tumour, such suppressive activity is actually detrimental to the host. Manipulation of Tregs holds great promise for the immunotherapy of cancer. Several studies performed using rodent models and indicate that Tregs cells inhibit effective anti-tumour immune responses and that their removal promotes tumour rejection. The increasing number of studies of Tregs in patients with cancer also point to a role for these cells in promoting disease progression. This review summarises the findings of these studies and addresses the advantages and potential pitfalls of manipulating Treg activity for the treatment of cancer. [Copyright &y& Elsevier]

Complement is not only part of the innate immune system, but has also been implicated in adaptive immunity. The role of complement and its regulatory proteins in modulating T cell activity has been the focus of several recent studies. These, which have included work on the membrane co-factor protein (MCP or CD46), decay accelerating factor (DAF or CD55) and CD59, indicate that complement regulators can influence the proliferative capacity of T cells and their ability to produce cytokines, influencing the outcome of a T cell response to a given antigen. Here we review these studies, which reveal another important link between the innate and the adaptive immune system. [Copyright &y& Elsevier]

Abstract: T cell responses are regulated by the affinity/avidity of the T cell receptor for the MHC/peptide complex, available costimulation and duration of antigenic stimulation. Altered peptide ligands (APLs) are usually recognized with a reduced affinity/avidity by the T cell receptor and are often able to only partially activate T cells in vitro or may even function as antagonists. Here we assessed the ability of APLs derived from peptide p33 of lymphocytic choriomeningitis virus (LCMV) to mediate lysis of target cells in vivo, confer anti-viral protection and cause auto-immune disease. In general, in vitro cross-reactivity between APLs was rather limited, and even strongly cross-reactive cytotoxic T lymphocytes were only able to mediate moderate anti-viral protection. Partial protection was observed for infection with LCMV or low doses of recombinant vaccinia virus, while no reduced viral titers could be seen upon infection with high dose of vaccinia virus. In a transgenic mouse model expressing LCMV glycoprotein in the islets of the pancreas, APLs induced a transient insulitis but failed to induce autoimmune diabetes. Thus, effector functions induced by even highly homologous APLs are rather limited in vivo. [Copyright &y& Elsevier]

Abstract: The complement system, a pillar of innate immunity, has belatedly become recognised as a key modulator of adaptive immunity, acting to direct, modulate and modify the responses of lymphocytes to stimuli. These effects are mediated by interactions between complement components or activation-derived fragments and specific binding proteins—complement receptors and regulators—on the target cells. This review will describe the current state of knowledge in this swiftly moving field. It is hoped that the recognition of these properties will help to establish complement in the role it richly deserves as the lynchpin of immunity. [Copyright &y& Elsevier]

CD4 + T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to "tune" CD4 + T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4 + and CD8 + T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4 + T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

CD4 + T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4 + T cell memory is finely poised at the level of peptide-HLA-II presentation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

High endothelial venules (HEVs) are specialized postcapillary venules that specifically serve to recruit circulating lymphocytes to secondary lymphoid organs (SLOs) where cognate antigens can be encountered, and immune responses can be initiated. The presence of HEV-like vessels in primary human solid tumours and their association with lymphocyte infiltration and favourable clinical outcomes and response to immunotherapy have provided a rationale for therapeutically inducing these vessels in tumours for immunotherapeutic benefit. Here we specifically discuss evidence for a link between T-cell activation and development of useful tumour-associated HEV (TA-HEV). We discuss the molecular and functional features of TA-HEV, highlighting the benefits for promoting tumour immunity and the important unanswered questions that need to be addressed before TA-HEV induction can be optimized for immunotherapeutic benefit., Competing Interests: Not applicable., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.)

High endothelial venules (HEV) are specialized post capillary venules that recruit naïve T cells and B cells into secondary lymphoid organs (SLO) such as lymph nodes (LN). Expansion of HEV networks in SLOs occurs following immune activation to support development of an effective immune response. In this study, we used a carcinogen-induced model of fibrosarcoma to examine HEV remodeling after depletion of regulatory T cells (Treg). We used light sheet fluorescence microscopy imaging to visualize entire HEV networks, subsequently applying computational tools to enable topological mapping and extraction of numerical descriptors of the networks. While these analyses revealed profound cancer- and immune-driven alterations to HEV networks within LNs, these changes did not identify successful responses to treatment. The presence of HEV networks within tumors did however clearly distinguish responders from nonresponders. Finally, we show that a successful treatment response is dependent on coupling tumor-associated HEV (TA-HEV) development to T-cell activation implying that T-cell activation acts as the trigger for development of TA-HEVs which subsequently serve to amplify the immune response by facilitating extravasation of T cells into the tumor mass.

T cells specific for SARS-CoV-2 are thought to protect against infection and development of COVID-19, but direct evidence for this is lacking. Here, we associated whole-blood-based measurement of SARS-CoV-2-specific interferon-γ-positive T cell responses with positive COVID-19 diagnostic (PCR and/or lateral flow) test results up to 6 months post-blood sampling. Amongst 148 participants donating venous blood samples, SARS-CoV-2-specific T cell response magnitude is significantly greater in those who remain protected versus those who become infected (P < 0.0001); relatively low magnitude T cell response results in a 43.2% risk of infection, whereas high magnitude reduces this risk to 5.4%. These findings are recapitulated in a further 299 participants testing a scalable capillary blood-based assay that could facilitate the acquisition of population-scale T cell immunity data (14.9% and 4.4%, respectively). Hence, measurement of SARS-CoV-2-specific T cells can prognosticate infection risk and should be assessed when monitoring individual and population immunity status., (© 2022. The Author(s).)

Improving the anti-tumour T cell response as a consequence of immunotherapy can result in eradication of tumour burden, however, the majority of patients fail with current treatment regimens and so novel immunotherapies with greater efficacy and improved tolerability are needed. The phosphoinositide-3-kinase (PI3K) family members that are directly involved in cell signalling comprise PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ, with the latter two isoforms expressed primarily by leukocytes. The survival and optimal function of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) is dependent on PI3Kδ, whereas tumour-associated macrophages (TAMs), use PI3Kγ. Blocking these signalling isoforms can boost development of effective anti-cancer immune responses and result in control of tumour burden. The dependence on different PI3K isoforms in immune cells makes targeting this pathway an attractive approach for tumour immunotherapy. Herein, we discuss how inhibiting specific PI3K isoforms in pro-tumoural Tregs, MDSCS and TAMs can unleash a powerful anti-tumour immune response, driven by CD8 + T cells, capable of controlling tumour burden and consider how the immune response to therapy needs careful investigation, to identify both the correlates of successful treatment and those that impede the generation of robust anti-tumour responses. Furthermore, we review how combination immunotherapy approaches with both PI3K inhibitors and subsequent immune checkpoint blockade can potentiate the efficacy of monotherapy. Finally, we discuss the recent advances in the use of PI3K isoform-specific inhibitors as an immunotherapy for solid tumours in clinical trials., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Despite three decades of research to its name and increasing interest in immunotherapies that target it, LAG-3 remains an elusive co-inhibitory receptor in comparison to the well-established PD-1 and CTLA-4. As such, LAG-3 targeting therapies have yet to achieve the clinical success of therapies targeting other checkpoints. This could, in part, be attributed to the many unanswered questions that remain regarding LAG-3 biology. Of these, we address: (i) the function of the many LAG-3-ligand interactions, (ii) the hurdles that remain to acquire a high-resolution structure of LAG-3, (iii) the under-studied LAG-3 signal transduction mechanism, (iv) the elusive soluble form of LAG-3, (v) the implications of the lack of (significant) phenotype of LAG-3 knockout mice, (vi) the reports of LAG-3 expression on the epithelium, and (vii) the conflicting reports of LAG-3 expression (and potential contributions to pathology) in the brain. These mysteries which surround LAG-3 highlight how the ever-evolving study of its biology continues to reveal ever-increasing complexity in its role as an immune receptor. Importantly, answering the questions which shroud LAG-3 in mystery will allow the maximum therapeutic benefit of LAG-3 targeting immunotherapies in cancer, autoimmunity and beyond., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Immunology.)

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., (© The Author(s) 2021. Published by Oxford University Press.)

Secondary lymphoid organs (SLOs) are important initiators and regulators of immunity. To carry out this function, the blood vasculature must deliver oxygen and nutrients and recruit circulating lymphocytes into the SLO parenchyma, where they encounter cognate antigen. High endothelial venules (HEVs) are specialised postcapillary venules that specifically serve this function and are found in all SLOs except spleen. It is becoming clear that alterations to HEV network density and/or morphology can result in immune activation or, as recently implicated, in providing an exit route for tumour cell dissemination and metastases. In this review, the structural plasticity of HEVs, the regulatory pathways underpinning this plasticity, and the relevance of these pathways to cancer progression will be discussed., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press.)

The nature of the tumor microenvironment (TME) influences the ability of tumor-specific T cells to control tumor growth. In this study, we performed an unbiased comparison of the TME of regulatory T-cell (Treg)-replete and Treg-depleted carcinogen-induced tumors, including Treg-depleted responding (regressing) and non-responding (growing) tumors. This analysis revealed an inverse relationship between extracellular matrix (ECM) and T-cell infiltrates where responding tumors were T-cell rich and ECM poor, whereas the converse was observed in non-responder tumors. For this reason, we hypothesized that the ECM acted as a barrier to successful T-cell infiltration and tumor rejection. However, further experiments revealed that this was not the case but instead showed that an effective T-cell response dramatically altered the density of ECM in the TME. Along with loss of ECM and high numbers of infiltrating T cells, responder tumors were distinguished by the development of lymphatic and blood vessel networks with specialized immune function. ECM-rich tumors exhibited a stem cell-like gene expression profile and superior tumor-initiating capacity, whereas such features were absent in responder tumors. Overall, these findings define an extended role for an effective immune response, not just in direct killing of tumor cells but in widescale remodeling of the TME to favor loss of ECM, elimination of cancer stem cells, and propagation of adaptive immunity., (©2020 American Association for Cancer Research.)

Background: Despite striking successes, immunotherapies aimed at increasing cancer-specific T cell responses are unsuccessful in most patients with cancer. Inactivating regulatory T cells (Treg) by inhibiting the PI3Kδ signaling enzyme has shown promise in preclinical models of tumor immunity and is currently being tested in early phase clinical trials in solid tumors., Methods: Mice bearing 4T1 mammary tumors were orally administered a PI3Kδ inhibitor (PI-3065) daily and tumor growth, survival and T cell infiltrate were analyzed in the tumor microenvironment. A second treatment schedule comprised PI3Kδ inhibitor with anti-LAG3 antibodies administered sequentially 10 days later., Results: As observed in human immunotherapy trials with other agents, immunomodulation by PI3Kδ-blockade led to 4T1 tumor regressor and non-regressor mice. Tumor infiltrating T cells in regressors were metabolically fitter than those in non-regressors, with significant enrichments of antigen-specific CD8 + T cells, T cell factor 1 (TCF1) + T cells and CD69 - T cells, compatible with induction of a sustained tumor-specific T cell response. Treg numbers were significantly reduced in both regressor and non-regressor tumors compared with untreated tumors. The remaining Treg in non-regressor tumors were however significantly enriched with cells expressing the coinhibitory receptor LAG3, compared with Treg in regressor and untreated tumors. This striking difference prompted us to sequentially block PI3Kδ and LAG3. This combination enabled successful therapy of all mice, demonstrating the functional importance of LAG3 in non-regression of tumors on PI3Kδ inhibition therapy. Follow-up studies, performed using additional cancer cell lines, namely MC38 and CT26, indicated that a partial initial response to PI3Kδ inhibition is an essential prerequisite to a sequential therapeutic benefit of anti-LAG3 antibodies., Conclusions: These data indicate that LAG3 is a key bottleneck to successful PI3Kδ-targeted immunotherapy and provide a rationale for combining PI3Kδ/LAG3 blockade in future clinical studies., Competing Interests: Competing interests: BV is a consultant for Karus Therapeutics (Oxford, UK), iOnctura (Geneva, Switzerland) and Venthera (Palo Alto, USA) and has received speaker fees from Gilead., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (©2020 American Association for Cancer Research.)

Epigenetic regulation plays a key role in the link between inflammation and cancer. Here we examine Mbd2, which mediates epigenetic transcriptional silencing by binding to methylated DNA. In separate studies the Mbd2 -/- mouse has been shown (1) to be resistant to intestinal tumourigenesis and (2) to have an enhanced inflammatory/immune response, observations that are inconsistent with the links between inflammation and cancer. To clarify its role in tumourigenesis and inflammation, we used constitutive and conditional models of Mbd2 deletion to explore its epithelial and non-epithelial roles in the intestine. Using a conditional model, we found that suppression of intestinal tumourigenesis is due primarily to the absence of Mbd2 within the epithelia. Next, we demonstrated, using the DSS colitis model, that non-epithelial roles of Mbd2 are key in preventing the transition from acute to tumour-promoting chronic inflammation. Combining models revealed that prior to inflammation the altered Mbd2 -/- immune response plays a role in intestinal tumour suppression. However, following inflammation the intestine converts from tumour suppressive to tumour promoting. To summarise, in the intestine the normal function of Mbd2 is exploited by cancer cells to enable tumourigenesis, while in the immune system it plays a key role in preventing tumour-enabling inflammation. Which role is dominant depends on the inflammation status of the intestine. As environmental interactions within the intestine can alter DNA methylation patterns, we propose that Mbd2 plays a key role in determining whether these interactions are anti- or pro-tumourigenic and this makes it a useful new epigenetic model for inflammation-associated carcinogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

High endothelial venules (HEVs) are structurally distinct blood vessels that develop during embryonic and neonatal life in all secondary lymphoid organs except the spleen. HEVs are critical for initiating and maintaining immune responses because they extract naïve and memory lymphocytes from the bloodstream, regardless of antigen receptor specificity, and deliver them to antigen-presenting cells inside lymph nodes under homeostatic conditions. HEVs also develop postnatally in nonlymphoid organs during chronic inflammation driven by autoimmunity, infection, allografts, and cancer. Extranodal HEVs are usually surrounded by dense lymphocytic infiltrates organized into lymph-node like, T- and B-cell-rich areas called tertiary lymphoid structures (TLS). HEV neogenesis is thought to facilitate the generation of tissue-destroying lymphocytes inside chronically inflamed tissues and cancers.We are studying the mechanisms underpinning HEV neogenesis in solid cancers and the role of homeostatic T-cell trafficking in controlling cancer immunity. In this chapter we describe methods for identifying HEV in tissue sections of cancerous tissues in humans and mice using immunohistochemical staining for the HEV-specific marker peripheral lymph node addressin (PNAd). L-selectin binding to PNAd is a necessary first step in homeostatic lymphocyte trafficking which is the defining function of HEV. We also describe methods to measure L-selectin-dependent homing of lymphocytes from the bloodstream into lymphoid tissues and tumors in preclinical cancer models.

The tumor microenvironment comprises newly formed blood and lymphatic vessels which shape the influx, retention and departure of lymphocytes within the tumor mass. Thus, by influencing the intratumoral composition of lymphocytes, these vessels affect the manner in which the adaptive immune system responds to the tumor, either promoting or impairing effective antitumor immunity. In our study, we utilized a mouse model of carcinogen-induced fibrosarcoma to examine the composition of tumor-infiltrating lymphocytes during tumor progression. In particular, we sought to determine whether CD4(+) Foxp3(+) regulatory T cells (Tregs) became enriched during tumor progression thereby contributing to tumor-driven immunosuppression. This was not the case as the proportion of Tregs and effector CD4(+) T cells actually declined within the tumor owing to the unexpected accumulation of naïve T cells. However, we found no evidence for antigen-driven migration of these T cells or for their participation in an antitumor immune response. Our data support the notion that lymphocytes can enter tumors via aberrantly formed blood and lymphatic vessels. Such findings suggest that targeting both the tumor vasculature and lymphatics will alter the balance of lymphocyte subpopulations that enter the tumor mass. A consideration of this aspect of tumor immunology may be critical to the success of solid cancer immunotherapies., (© 2013 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.)

During primary infection, murine cytomegalovirus (MCMV) spreads systemically, resulting in virus replication and pathology in multiple organs. This disseminated infection is ultimately controlled, but the underlying immune defense mechanisms are unclear. Investigating the role of the cytokine IL-22 in MCMV infection, we discovered an unanticipated function for neutrophils as potent antiviral effector cells that restrict viral replication and associated pathogenesis in peripheral organs. NK-, NKT-, and T cell-secreted IL-22 orchestrated antiviral neutrophil-mediated responses via induction in stromal nonhematopoietic tissue of the neutrophil-recruiting chemokine CXCL1. The antiviral effector properties of infiltrating neutrophils were directly linked to the expression of TNF-related apoptosis-inducing ligand (TRAIL). Our data identify a role for neutrophils in antiviral defense, and establish a functional link between IL-22 and the control of antiviral neutrophil responses that prevents pathogenic herpesvirus infection in peripheral organs., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The relationship between the adaptive CD4 + T cell response and human cancer is unclear. The oncofetal antigen 5T4 is expressed on many human carcinomas, including colorectal cancer (CRC) cells, but has limited expression on normal tissues. We previously identified anti-5T4 CD4 + T cells in a proportion of CRC patients, and we extended this study to examine whether the quality or quantity of the T cell response reflects tumor stage. An overlapping peptide library spanning 5T4 was used as a target to enumerate cognate IFN-γ + CD4 + T-cells (measured as spot forming cells [SFC]/10 5 cultured T cells) in peripheral blood-derived lymphocytes following a 12-day in vitro culture period comparing patients pre-operatively (n = 27) to healthy controls (n = 17). Robust 5T4-specific T cell responses were present in 100% of healthy donors. There was a steady loss of T cell responses with advancing tumors with a significant negative correlation from stage I to III (P = 0.008). The predictability of the decline meant < 200 SFC/10 5 was only found in subjects with stage III CRC. The mechanism of loss of T cell response is independent of HLA-DR type or patient age, but does correspond to increases in Foxp3 + regulatory T cells (Tregs). Using low-dose cyclophosphamide to reduce the proportion of Tregs in vivo resulted in increased anti-5T4 T cell responses in CRC patients. The selective loss of 5T4-specific IFN-γ + CD4 + T cell responses implies a link between tumor stage and antitumor Th1 effector function; depleting Tregs can enhance such responses.