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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. Significance: We used three-dimensional imaging methods with computational tools to analyze networks of specialized blood vessels called HEVs in LNs and tumors. By applying these techniques in a mouse model of carcinogen-induced tumors, we could identify network changes after depletion of Tregs.

High endothelial venules (HEVs) are specialised 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 optimised for immunotherapeutic benefit.

Naïve LN HEV Characterisation.

Supplementary Figures 1-8

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.Significance:We used three-dimensional imaging methods with computational tools to analyze networks of specialized blood vessels called HEVs in LNs and tumors. By applying these techniques in a mouse model of carcinogen-induced tumors, we could identify network changes after depletion of Tregs.

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.

Global overview of intra-tumoral HEVs in a 4T1 regressor tumour following PI-3065 treatment.

Global overview of intra-tumoral HEVs in a 4T1 regressor tumour following PI-3065 treatment.

Global overview of intra-tumoral HEVs in a 4T1 non-regressor tumour following PI-3065 treatment.

S1. Tumor HEV identified by PNAd staining are also positive for MAdCAM-1. S2. Efficacy of monoclonal antibody treatments to deplete immune cell subsets. S3. Splenic Marginal Zone B cells are profoundly decreased after treatment with LTbetaR.Fc. S4. Splenic Follicular Dendritic Cells and MAdCAM-1 staining are lost after treatment with LTbetaR.Fc or TNFRII.Ig. S5. Lymph Node architecture is disrupted following treatment with LTbetaR.Fc or TNFRII.Ig. S6. Tumor HEV identified by PNAd staining following blockade of LTbetaR or TNFR signalling are also positive for MAdCAM-1. S7. Agonism of LTβR induces formation of High Endothelial Venules in Treg replete tumors, but without concomitant increased T cell infiltration and reduced tumor growth. S8. Relative gene expression of TNF and LTalpha by intratumoral CD4+ and CD8+ T cells and dendritic cells (DC). Data are expressed as fold change in gene expression relative to splenic B cells, and represent two independent experiments.

T-cell infiltration into tumors represents a critical bottleneck for immune-mediated control of cancer. We previously showed that this bottleneck can be overcome by depleting immunosuppressive Foxp3+ regulatory T cells (Tregs), a process that can increase frequencies of tumor-infiltrating lymphocytes through promoting the development of specialized portals for lymphocyte entry, namely high endothelial venules (HEVs). In this paper, we used a carcinogen-induced tumor model that allows for coevolution of the tumor microenvironment and the immune response to demonstrate that Treg depletion not only results in widespread disruption to HEV networks in lymph nodes (LNs) but also activates CD8+ T cells, which then drive intratumoral HEV development. Formation of these vessels contrasts with ontogenic HEV development in LNs in that the process is dependent on the TNF receptor and independent of lymphotoxin β receptor–mediated signaling. These intratumoral HEVs do not express the chemokine CCL21, revealing a previously undescribed intratumoral blood vessel phenotype. We propose a model where Treg depletion enables a self-amplifying loop of T-cell activation, which promotes HEV development, T-cell infiltration, and ultimately, tumor destruction. The findings point to a need to test for HEV development as part of ongoing clinical studies in patients with cancer. Cancer Immunol Res; 5(11); 1005–15. ©2017 AACR.

PDF file - 10925K, S. Fig. 1. Cultured 5T4-specific CD4+ T cells can be cloned from CRC patients (A) and healthy donors (B). S. Fig. 2. A comparison of 5T4 responses and age amongst CRC patients. S. Fig. 3. 5T4 Immunohistochemistry staining of frozen sections. S. Fig. 4. CT scans demonstrating the growth of a single metastatic lung lesion in a patient who did not respond to low-dose cyclophosphamide treatment.

The relationship between the adaptive CD4+ T-cell response and human cancer is unclear. The oncofetal antigen 5T4 is expressed in many human carcinomas, including colorectal cancer cells, but has limited expression on normal tissues. We previously identified anti-5T4 CD4+ T cells in a proportion of patients with colorectal cancer, 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)/105 cultured T cells] in peripheral blood–derived lymphocytes following a 14-day in vitro culture period comparing patients preoperatively (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 5 were only found in subjects with stage III colorectal cancer. 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 (Treg). Using low-dose cyclophosphamide to reduce the proportion of Tregs in vivo resulted in increased anti-5T4 T-cell responses in patients with colorectal cancer. 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. Cancer Immunol Res; 1(6); 416–25. ©2013 AACR.

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Table S1

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.

Purpose: Anticancer T-cell responses can control tumors, but immunosuppressive mechanisms in vivo prevent their function. The role of regulatory T cells (Tregs) in metastatic colorectal cancer is unclear. We have previously shown depletion of Tregs enhances colorectal cancer–specific effector T-cell responses. Low-dose cyclophosphamide targets Tregs in animal models and some human studies; however, the effect of cyclophosphamide in metastatic colorectal cancer is unknown.Experimental Design: Fifty-five patients with metastatic colorectal cancer were enrolled in a phase I/II trial and randomly assigned to receive 2-week-long courses of low-dose (50 mg twice a day) cyclophosphamide or not. The absolute number, phenotype, and antitumor function of peripheral blood–derived lymphocyte subsets were monitored throughout treatment, as well as during 18-month follow-up.Results: Initially, cyclophosphamide reduced proliferation in all lymphocyte subsets; however, a rapid mobilization of effector T cells overcame this decrease, leading to increased absolute T-cell numbers. In contrast, a reduction in proportional and absolute Treg, B-cell, and NK-cell numbers occurred. The expansion and subsequent activation of effector T cells was focused on tumor-specific T cells, producing both granzyme B and IFNγ. Cyclophosphamide-treated patients demonstrating the most enhanced IFNγ+ tumor-specific T-cell responses exhibited a significant delay in tumor progression [HR = 0.29; 95% confidence interval (CI), 0.12–0.69; P = 0.0047), compared with nonresponders and no-treatment controls.Conclusions: Cyclophosphamide-induced Treg depletion is mirrored by a striking boost in antitumor immunity. This study provides the first direct evidence of the benefit of naturally primed T cells in patients with metastatic colorectal cancer. Our results also support the concept that nonmutated self-antigens may act as useful targets for immunotherapies. Clin Cancer Res; 23(22); 6771–80. ©2017 AACR.

Supplementary Figure 1. CONSORT diagram. Supplementary Figure 2. 5T4 peptide pools and matrix system. Supplementary Figure 3. B-cell number in response to cyclophosphamide. Supplementary Figure 4. Expression of activation markers in T-cell subsets during CPM treatment. Supplementary Figure 5. Immunological responses following the completion of CPM treatment. Supplementary Figure 6. Analysis of immunological responses over 8 courses of CPM in patient 102.

Supplementary Figures 1-7

Supplementary Figure 5 from Analysis of the T-Cell Receptor Repertoires of Tumor-Infiltrating Conventional and Regulatory T Cells Reveals No Evidence for Conversion in Carcinogen-Induced Tumors

Supplementary Figure 6 from Analysis of the T-Cell Receptor Repertoires of Tumor-Infiltrating Conventional and Regulatory T Cells Reveals No Evidence for Conversion in Carcinogen-Induced Tumors

Supplementary Figures 1-3 from Analysis of the T-Cell Receptor Repertoires of Tumor-Infiltrating Conventional and Regulatory T Cells Reveals No Evidence for Conversion in Carcinogen-Induced Tumors

Supplementary Figure 7 from Analysis of the T-Cell Receptor Repertoires of Tumor-Infiltrating Conventional and Regulatory T Cells Reveals No Evidence for Conversion in Carcinogen-Induced Tumors

Supplementary Figure 4 from Analysis of the T-Cell Receptor Repertoires of Tumor-Infiltrating Conventional and Regulatory T Cells Reveals No Evidence for Conversion in Carcinogen-Induced Tumors

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

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.

Immune checkpoint inhibitors (antibodies that block the T cell co‐inhibitory receptors PD‐1/PD‐L1 or CTLA‐4) have revolutionized the treatment of some forms of cancer. Importantly, combination approaches using drugs that target both pathways have been shown to boost the efficacy of such treatments. Subsequently, several other T cell inhibitory receptors have been identified for the development of novel immune checkpoint inhibitors. Included in this list is the co‐inhibitory receptor lymphocyte activation gene‐3 (LAG‐3), which is upregulated on T cells extracted from tumor sites that have suppressive or exhausted phenotypes. However, the molecular rules that govern the function of LAG‐3 are still not understood. Using surface plasmon resonance combined with a novel bead‐based assay (AlphaScreenTM), we demonstrate that LAG‐3 can directly and specifically interact with intact human leukocyte antigen class II (HLA‐II) heterodimers. Unlike the homologue CD4, which has an immeasurably weak affinity using these biophysical approaches, LAG‐3 binds with low micromolar affinity. We further validated the interaction at the cell surface by staining LAG‐3+ cells with pHLA‐II‐multimers. These data provide new insights into the mechanism by which LAG‐3 initiates T cell inhibition., We demonstrate that human LAG‐3 binds directly to peptide‐human leukocyte antigen class II complex (pHLA‐II) with low micromolar affinity. These findings provide new insights into the mechanism by which LAG‐3 initiates T cell inhibition and has implication for the development of novel immune checkpoint inhibitors for cancer immunotherapy.

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. Electronic supplementary material The online version of this article (10.1007/s00262-020-02603-x) contains supplementary material, which is available to authorised users.

High endothelial venules (HEVs) are specialized post-capillary venules that recruit naïve lymphocytes to lymph nodes. HEVs are essential for the development of adaptive immunity. HEVs can also develop in tumors where they are thought to be important for recruiting naïve T cells and B cells into the tumors and locally enhancing antitumor immunity by supporting the formation of tertiary lymphoid structures. Herein, we used comparative transcriptome analysis of human breast cancer to investigate genes differentially expressed between tumor-associated HEVs and the rest of the tumor vasculature. Tumor vessels highly expressing HEV-upregulated genes, such as the homeobox gene MEOX2 and the tetraspanin gene TSPAN7, were associated with extensive infiltration of T and B cells and the occurrence of tertiary lymphoid structures, which is known to predict therapeutic responses to immune checkpoint inhibitors. Moreover, high transcript counts of these genes in clinical tumor specimens were associated with a significant survival benefit in advanced breast cancer. The molecular signature of HEVs identified herein may be useful for guiding immunotherapies and provides a new direction for investigating tumor-associated HEVs and their clinical significance.

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.

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 TH1‐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, SARS‐CoV‐2 pandemic continues to cause morbidity and mortality especially in vulnerable patients. Monitoring individuals T and B cell responses to the virus is desirable in high risk populations after infection and/or vaccination. This paper describes a straightforward approach on a small whole blood sample to measure these responses.

SummaryEmerging 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.

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).

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., Oncolytic virotherapies have immense potential to ‘heat up’ otherwise immunologically ‘cold’ tumour types. In this review, the capacity of virotherapies to synergize with or express a range of types of immunotherapies is explored. Optimizing such combinations in preclinical models is complex, and we therefore review the most clinically relevant rodent and ex vivo human models available for such assessment.

Accurate assessment of SARS-CoV-2 immunity in the population is critical to 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 of the immune response that will likely form a key correlate of protection against COVID-19. Here, we developed and optimised a high-throughput whole blood-based assay to determine the T cell response associated with prior SARS-CoV-2 infection and/or vaccination amongst 156 healthy donors and 67 cancer patients. Following overnight in vitro stimulation with SARS-CoV-2-specific peptides, blood plasma samples were harvested and analysed for TH1-type effector cytokines (IFN-γ and IL-2). Amongst healthy donors, highly significant differential IFN-γ+/IL-2+ SARS-CoV-2-specific T cell responses were seen amongst vaccinated or previously infected COVID-19-positive individuals in comparison to unknown/naïve individuals (P < 0.0001). IL-2 production from T cells in response to SARS-CoV-2 derived antigens was a highly predictive diagnostic assay (P < 0.0001; 96.0% sensitivity, 93.9% specificity); measurement of IFN-γ+ SARS-CoV-2 specific T cell responses was equally effective at identifying asymptomatic (antibody and T cell positive) participants. A single dose of COVID-19 vaccine induced IFN-γ and/or IL-2 SARS-CoV-2-specific T cell responses in 28/29 (96.6%) of healthy donors, reducing significantly to 27/56 (48.2%) when measured in cancer patients (P = 0.0003). Overall, this cost-effective standardisable test ensures accurate and comparable assessments of SARS-CoV-2-specific T cell responses amenable to widespread population immunity testing.

Mouse models of cancer are essential in furthering our understanding both of the mechanisms that drive tumor development and the immune response that develops in parallel, and also in providing a platform for testing novel anti-cancer therapies. The majority of solid tumor models available rely on the injection of existing cancer cell lines into naive hosts which, while providing quick and reproducible model systems, typically lack the development of a tumor microenvironment that recapitulates those seen in human cancers. Administration of the carcinogen 3-methylcholanthrene (MCA), allows tumors to develop in situ, forming a tumor microenvironment with an established stroma and vasculature. This article provides a detailed set of protocols for the administration of MCA into mice and the subsequent monitoring of tumors. Protocols are also provided for some of the routinely used downstream applications that can be used for MCA tumors.

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 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.

BackgroundDespite 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.MethodsMice 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.ResultsAs 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.ConclusionsThese 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.

Summary: 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.

T-cell infiltration into tumors represents a critical bottleneck for immune-mediated control of cancer. We previously showed that this bottleneck can be overcome by depleting immunosuppressive Foxp3+ regulatory T cells (Tregs), a process that can increase frequencies of tumor-infiltrating lymphocytes through promoting the development of specialized portals for lymphocyte entry, namely high endothelial venules (HEVs). In this paper, we used a carcinogen-induced tumor model that allows for coevolution of the tumor microenvironment and the immune response to demonstrate that Treg depletion not only results in widespread disruption to HEV networks in lymph nodes (LNs) but also activates CD8+ T cells, which then drive intratumoral HEV development. Formation of these vessels contrasts with ontogenic HEV development in LNs in that the process is dependent on the TNF receptor and independent of lymphotoxin β receptor–mediated signaling. These intratumoral HEVs do not express the chemokine CCL21, revealing a previously undescribed intratumoral blood vessel phenotype. We propose a model where Treg depletion enables a self-amplifying loop of T-cell activation, which promotes HEV development, T-cell infiltration, and ultimately, tumor destruction. The findings point to a need to test for HEV development as part of ongoing clinical studies in patients with cancer. Cancer Immunol Res; 5(11); 1005–15. ©2017 AACR.

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.

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.

There have been substantial strides forward in our understanding of the contribution of regulatory T (Treg) cells to cancer immunosuppression. In this issue, we present a series of papers highlighting emerging themes on this topic relevant not only to our understanding of the fundamental biology of tumour immunosuppression but also to the design of new immunotherapeutic approaches. The substantially shared biology of CD4(+) conventional T (Tconv) and Treg cells necessitates a detailed understanding of the potentially opposing functional consequences that immunotherapies will have on Treg and Tconv cells, a prominent example being the potential for Treg‐mediated hyperprogressive disease following anti‐PD‐1 therapy. Such understanding will aid patient stratification and the rational design of combination therapies. It is also becoming clear, however, that Treg cells within tumours exhibit distinct biological features to both Tconv cells and Treg cells in other tissues. These distinct features provide the opportunity for development of targeted immunotherapies with greater efficacy and reduced potential for inducing systemic toxicity.

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. Electronic supplementary material The online version of this article (10.1007/s00262-018-2266-1) contains supplementary material, which is available to authorized users.