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Supplementary Figure 1 from The Regulatory T Cell–Associated Transcription Factor FoxP3 Is Expressed by Tumor Cells

FoxP3 is a member of the forkhead family of transcription factors critically involved in the development and function of CD25+ regulatory T cells (Treg). Until recently, FoxP3 expression was thought to be restricted to the T-cell lineage. However, using immunohistochemistry and flow cytometric analysis of human melanoma tissue, we detected FoxP3 expression not only in the tumor infiltrating Treg but also in the melanoma cells themselves. FoxP3 is also widely expressed by established human melanoma cell lines (as determined by flow cytometry, PCR, and Western blot), as well as cell lines derived from other solid tumors. Normal B cells do not express FoxP3; however, expression could be induced after transformation with EBV in vitro and in vivo, suggesting that malignant transformation of healthy cells can induce FoxP3. In addition, a FOXP3 mRNA variant lacking exons 3 and 4 was identified in tumor cell lines but was absent from Treg. Interestingly, this alternative splicing event introduces a translation frame-shift that is predicted to encode a novel protein. Together, our results show that FoxP3, a key regulator of immune suppression, is not only expressed by Treg but also by melanoma cells, EBV-transformed B cells, and a wide variety of tumor cell lines. [Cancer Res 2008;68(8):3001–9]

Accurate and precise delineation of gross tumour volumes remains a barrier to radiotherapy dose escalation and boost dosing in the treatment of solid tumours, such as prostate cancer. Magnetic resonance imaging of tumour molecular targets has the power to enable focal dose boosting, particularly when combined with technological advances such as MRI-LINAC. Fibroblast activation protein (FAP) is a transmembrane protein overexpressed in stromal components of >90% of epithelial carcinomas. Herein we compare targeted MRI of gold standard PSMA with FAP in the delineation of orthotopic tumours in a mouse model of prostate cancer. Control (no ligand), FAP and PSMA-targeting iron oxide nanoparticles were prepared with modification of an MRI agent (FerroTrace). Mice with orthotopic LNCaP tumours underwent T2-weighted 3D MRI 24 hours after intravenous injection of contrast agents. FAP and PSMA nanoparticles produced contrast enhancement on MRI when compared to control nanoparticles, which was most pronounced on the tumour periphery. FAP-targeted MRI increased the proportion of tumour contrast enhancing black pixels by 13.37% when compared to PSMA. Furthermore, analysis of changes in R2 values between healthy prostates and LNCaP tumours indicated an increase in contrast enhancing pixels in the tumour border of 15%, when targeting FAP, in contrast to PSMA This study demonstrates preclinical feasibility of PSMA and FAP-targeted MRI which can enable targeted image-guided focal therapy of localized prostate cancer.

CAR-T cell therapies are being intensely investigated as a novel immunotherapy approach for glioblastoma (GBM), but so far clinical success has been limited. We recently described FAP as an ideal target antigen for GBM immunotherapy, with expression on tumor cells and tumor blood vessels occurring frequently in patients’ tumors but with very limited expression in healthy tissues. Here, we generated a novel FAP-targeting CAR with CD3ζ and CD28 signaling domains and tested the resulting CAR-T cells for their ability to lyse GBM cells in vitro and in vivo. FAP-CAR-T cells showed target specificity against model cell lines and exhibited potent cytotoxicity against patient-derived glioma neural stem (GNS) cells. Remarkably, complete destruction of tumor cells was observed even where the antigen was expressed by a minor subpopulation of cells, indicating a bystander killing mechanism. Using co-culture assays, we confirmed the ability of FAP-CAR-T cells to mediate bystander killing of antigen-negative tumor cells, but only after activation by antigen-expressing target cells. This bystander killing effect was at least partially mediated by soluble factors. We also observed that the non-transduced fraction of the CAR-T cell product could be activated via T cell-secreted IL-2 to mediate antigen-non-specific killing, further amplifying the bystander effect. FAP-CAR-T cells controlled without overt toxicity the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive GBM cells. Together, our findings advance FAP as a leading candidate for clinical CAR-T cell therapy of GBM and highlight under-recognized antigen non-specific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.

Die CAR-T-Zell-Therapie ist ein neuer Ansatz der Krebsbehandlung, bei dem körpereigene T-Zellen eines Patienten gentechnisch so verändert werden, dass sie ganz gezielt Krebszellen aufspüren und abtöten können. Dieser Ansatz hat bei verschiedenen Arten von Blutkrebs bemerkenswerte Wirksamkeit gezeigt; bei soliden Tumoren hingegen war er bisher weitaus weniger erfolgreich. Wir geben hier einen Überblick über die vielfältigen Auswirkungen von Chemotherapie-Wirkstoffen auf die verschiedenen Arten von Leukozyten im Blutkreislauf und gelangen zu der Schlussfolgerung, dass die Chemotherapie das Immunsysten zwar kurzfristig beeinträchtigen, auf lange Sicht aber günstig im Sinne des Krebspatienten beeinflussen kann. Da Blut das Ausgangsmaterial für die Herstellung von CAR-T-Zellen ist, stellen wir die These auf, dass dieser Effekt auch die erfolgreiche Herstellung und antitumorale Aktivität der CAR-T-Zellen erheblich beeinflussen könnte. Bei gutem Timing müssten demnach die Chemotherapie-bedingten Veränderungen der Immunzellen im Blutkreislauf dafür sorgen, dass die CAR-T-Zellen eine wirksamere antitumorale Reaktion entfalten können. Zusammenfassung Die adoptive T-Zell-Therapie mit autologen T-Lymphozyten, die gentechnisch so verändert sind, dass sie krebsspezifische chimäre Antigenrezeptoren (CAR) exprimieren, etabliert sich derzeit als neuartiger Ansatz der Krebstherapie. Die CAR-T-Zell-Therapie ist bereits in mehreren wichtigen Rechtsgebieten zur Anwendung bei therapierefraktären oder rezidivierenden Fällen von akuter lymphatischer Leukämie von B-Vorläuferzellen sowie diffusem großzelligem B-Zell-Lymphom zugelassen. Bei Patienten mit soliden Tumoren hingegen hat die CAR-T-Zell-Therapie in mehreren klinischen Studien minimale therapeutische Wirkung gezeigt, weshalb man sich nun mit der Frage beschäftigt, wie sich CAR-T-Zellen besser mit anderen Therapien wie z. B. einer konventionellen zytotoxischen Chemotherapie verbinden lassen. Immer mehr Evidenz spricht dafür, dass Chemotherapeutika nicht nur tumorizid wirken, sondern auch das Immunsystem in signifikantem Maße modulieren. Wir behandeln hier die immunmodulatorischen Wirkungen von Chemotherapeutika auf zirkulierende Leukozytenpopulationen, unter anderem ihr jeweiliges Vermögen, zytotoxische Effekte zu verstärken und die Häufigkeit von CD8+ T-Zellen zu erhalten und zugleich immunsuppressive Populationen wie regulatorische T-Zellen und Suppressorzellen myeloischen Ursprungs zu reduzieren. Durch diese Modulation der Häufigkeiten und Phänotypen der Leukozyten im Blut (die ja das «Ausgangsmaterial» für CAR-T-Zellen sind) könnte, so postulieren wir, die Herstellung eines möglichst wirkungsvollen CAR-T-Zellprodukts durch eine vorhergehende Chemotherapie begünstigt werden. Weitere Forschungsarbeiten sind erforderlich, um dieses Konzept zu prüfen und Strategien für die optimale Einbindung von CAR-T-Zell-Therapien und zytotoxische Chemotherapien in die Behandlung von Patienten mit soliden Tumoren zu entwickeln.

Histopathological whole slide images of haematoxylin and eosin (H&E)-stained biopsies contain valuable information with relation to cancer disease and its clinical outcomes. Still, there are no highly accurate automated methods to correlate histolopathological images with brain cancer patients’ survival, which can help in scheduling patients therapeutic treatment and allocate time for preclinical studies to guide personalized treatments. We now propose a new classifier, namely, DeepSurvNet powered by deep convolutional neural networks, to accurately classify in 4 classes brain cancer patients’ survival rate based on histopathological images (class I, 0–6 months; class II, 6–12 months; class III, 12–24 months; and class IV, >24 months survival after diagnosis). After training and testing of DeepSurvNet model on a public brain cancer dataset, The Cancer Genome Atlas, we have generalized it using independent testing on unseen samples. Using DeepSurvNet, we obtained precisions of 0.99 and 0.8 in the testing phases on the mentioned datasets, respectively, which shows DeepSurvNet is a reliable classifier for brain cancer patients’ survival rate classification based on histopathological images. Finally, analysis of the frequency of mutations revealed differences in terms of frequency and type of genes associated to each class, supporting the idea of a different genetic fingerprint associated to patient survival. We conclude that DeepSurvNet constitutes a new artificial intelligence tool to assess the survival rate in brain cancer. Graphical abstractA DCNN model was generated to accurately predict survival rates of brain cancer patients (classified in 4 different classes) accurately. After training the model using images from H&E stained tissue biopsies from The Cancer Genome Atlas database (TCGA, left), the model can predict for each patient, based on a histological image (top right), its survival class accurately (bottom right). Electronic supplementary material The online version of this article (10.1007/s11517-020-02147-3) contains supplementary material, which is available to authorized users.

Multiple myeloma (MM) is the second most common haematological malignancy and is an incurable disease of neoplastic plasma cells (PC). Newly diagnosed MM patients currently undergo lengthy genetic testing to match chromosomal mutations with the most potent drug/s to decelerate disease progression. With only 17% of MM patients surviving 10-years postdiagnosis, faster detection and earlier intervention would unequivocally improve outcomes. Here, we show that the cell surface protein desmoglein-2 (DSG2) is overexpressed in ˜ 20% of bone marrow biopsies from newly diagnosed MM patients. Importantly, DSG2 expression was strongly predictive of poor clinical outcome, with patients expressing DSG2 above the 70th percentile exhibiting an almost 3-fold increased risk of death. As a prognostic factor, DSG2 is independent of genetic subtype as well as the routinely measured biomarkers of MM activity (e.g. paraprotein). Functional studies revealed a nonredundant role for DSG2 in adhesion of MM PC to endothelial cells. Together, our studies suggest DSG2 to be a potential cell surface biomarker that can be readily detected by flow cytometry to rapidly predict disease trajectory at the time of diagnosis. Refereed/Peer-reviewed

BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of CAR-T cell therapy have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T cell therapy also remain limited.MethodsWe profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR-T cells from peripheral blood of glioblastoma and diffuse midline glioma patients and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control.ResultsHere we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite glioblastoma patients having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an IL-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Markers associated with tumor control included those related to T-cell homing, infiltration, and cytotoxicity.ConclusionsTargeting GD2 using a clinically deployed CAR-T therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.What is already known on this topicGD2 is a tumor antigen of significant interest for targeting immunotherapy. A single preclinical study has shown the effectiveness of GD2-CAR-T cell therapy in an orthotopic xenograft model of diffuse midline glioma. Similarly, there is one previous preclinical study of GD2-CAR-T therapy in a orthotopic glioblastoma xenograft model but tumor control was achieved only following intracranial injection of CAR-T cells. Given that GD2-CAR-T therapy is already being evaluated clinically for other tumor indications, it is important to establish whether there is an acceptable rationale for its use in brain tumors.What this study addsThis is the first description of a GD2-targeted CAR-T cell therapy that shows antitumor effectiveness in a preclinical model of human glioblastoma following intravenous administration. It is also the first study to investigate the potential effects that the immune profile of glioblastoma patients may have on the feasibility of CAR-T cell manufacturing.How this study might affect research, practice, or policyThe results of this study have led to the initiation of an Australian phase 1 clinical trial program aiming to test GD2-specific CAR-T cells for the treatment of childhood and adult primary brain tumors. The study provides valuable insights into the microenvironmental factors that influence the effectiveness of CAR-T cell therapy for this type of tumor, paving the way for further optimization of CAR-T cell technology for treatment of aggressive primary brain tumors such as glioblastoma.

Simple Summary CAR-T cell therapy is a new approach to cancer treatment that is based on manipulating a patient’s own T cells such that they become able to seek and destroy cancer cells in a highly specific manner. This approach is showing remarkable efficacy in treating some types of blood cancers but so far has been much less effective against solid cancers. Here, we review the diverse effects of chemotherapy agents on circulating leukocyte populations and find that, despite some negative effects over the short term, chemotherapy can favourably modulate the immune systems of cancer patients over the longer term. Since blood is the starting material for CAR-T cell production, we propose that these effects could significantly influence the success of manufacturing, and anti-cancer activity, of CAR-T cells. Thus, if timed correctly, chemotherapy-induced changes to circulating immune cells could allow CAR-T cells to unleash more effective anti-tumour responses. Abstract Adoptive T-cell therapy using autologous T cells genetically modified to express cancer-specific chimeric antigen receptors (CAR) has emerged as a novel approach for cancer treatment. CAR-T cell therapy has been approved in several major jurisdictions for treating refractory or relapsed cases of B-cell precursor acute lymphoblastic leukaemia and diffuse large B-cell lymphoma. However, in solid cancer patients, several clinical studies of CAR-T cell therapy have demonstrated minimal therapeutic effects, thus encouraging interest in better integrating CAR-T cells with other treatments such as conventional cytotoxic chemotherapy. Increasing evidence shows that not only do chemotherapy drugs have tumoricidal effects, but also significantly modulate the immune system. Here, we discuss immunomodulatory effects of chemotherapy drugs on circulating leukocyte populations, including their ability to enhance cytotoxic effects and preserve the frequency of CD8+ T cells and to deplete immunosuppressive populations including regulatory T cells and myeloid-derived suppressor cells. By modulating the abundance and phenotype of leukocytes in the blood (the ‘raw material’ for CAR-T cell manufacturing), we propose that prior chemotherapy could facilitate production of the most effective CAR-T cell products. Further research is required to directly test this concept and identify strategies for the optimal integration of CAR-T cell therapies with cytotoxic chemotherapy for solid cancers.

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6–9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microen-vironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cul-tures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma Refereed/Peer-reviewed

Chimeric antigen receptor (CAR)-T cell therapy has been clinically validated as a curative treatment for the difficult to treat malignancies of relapsed/refractory B-cell acute lymphoblastic leukaemia and lymphoma. Here, the CAR-T cells are re-directed towards a single antigen, CD19, which is recognised as a virtually ideal CAR target antigen because it has strong, uniform expression on cancer cells, and is otherwise expressed only on healthy B cells, which are ‘dispensable’. Notwithstanding the clinical success of CD19-CAR-T cell therapy, its single specificity has driven therapeutic resistance in 30% or more of cases with CD19-negative leukaemic relapses. Immune checkpoint blockade is also a highly successful cancer immunotherapeutic approach, but it will be less useful for many patients whose malignancies either lack a substantial somatic mutation load or whose tumours are intrinsically resistant. Although CAR-T cell therapy could serve this unmet medical need, it is beset by several major limitations. There is a lack of candidate antigens that would satisfy the requirements for ideal CAR targets. Biological properties such as clonal heterogeneity and micro-environmental conditions hostile to T cells are inherent to many solid tumours. Past clinical studies indicate that on-target, off-tumour toxicities of CAR-T cell therapy may severely hamper its application. Therefore, re-designing CARs to increase the number of antigen specificities recognised by CAR-T cells will broaden tumour antigen coverage, potentially overcoming tumour heterogeneity and limiting tumour antigen escape. Tuning the balance of signalling within bi-specific CAR-T cells may enable tumour targeting while sparing normal tissues, and thus minimise on-target, off-tumour toxicities. Refereed/Peer-reviewed

Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-xPlt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression.

Glioblastoma is the deadliest form of brain cancer. Aside from inadequate treatment options, one of the main reasons glioblastoma is so lethal is the rapid growth of tumour cells coupled with continuous cell invasion into surrounding healthy brain tissue. Significant intra- and inter-tumour heterogeneity associated with differences in the corresponding tumour microenvironments contributes greatly to glioblastoma progression. Within this tumour microenvironment, the extracellular matrix profoundly influences the way cancer cells become invasive, and changes to extracellular (pH and oxygen levels) and metabolic (glucose and lactate) components support glioblastoma growth. Furthermore, studies on clinical samples have revealed that the tumour microenvironment is highly immunosuppressive which contributes to failure in immunotherapy treatments. Although technically possible, many components of the tumour microenvironment have not yet been the focus of glioblastoma therapies, despite growing evidence of its importance to glioblastoma malignancy. Here, we review recent progress in the characterisation of the glioblastoma tumour microenvironment and the sources of tumour heterogeneity in human clinical material. We also discuss the latest advances in technologies for personalised and in vitro preclinical studies using brain organoid models to better model glioblastoma and its interactions with the surrounding healthy brain tissue, which may play an essential role in developing new and more personalised treatments for this aggressive type of cancer.

The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a "normal" human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening.

Background: Chimeric antigen receptor (CAR)-T cells are genetically engineered to recognize tumor-associated antigens and have potent cytolytic activity against tumors. Adoptive therapy with CAR-T cells has been highly successful in B-cell leukemia and lymphoma. However, in solid tumor settings, CAR-T cells face a particularly hostile tumor microenvironment where multiple immune suppressive factors serve to thwart the anti-cancer immune response. Clinical trials of solid tumor antigen-targeted CAR-T cells have shown limited efficacy, and issues for current CAR-T cell therapies include failures of expansion and persistence, tumor entry, deletion and functional exhaustion. Methods: We compared our standard protocol for CAR-T cell manufacturing, currently used to generate CAR-T cells for a phase 1 clinical trial, with two alternative approaches for T-cell activation and expansion. The resulting cultures were analyzed using multicolor flow cytometry, cytokine bead array and xCELLigence cytotoxicity assays. Results: We have found that by changing the method of activation we can promote generation of CAR-T cells with enhanced CD62L and CCR7 expression, increased interleukin (IL)-2 production and retention of cytolytic activity, albeit with slower kinetics. Discussion: We propose that these phenotypic characteristics are consistent with a central memory phenotype that will better enable CAR-T cell survival and persistence after activation in vivo, and we aim to test this in a continuation of our current phase 1 clinical trial of CAR-T cells in patients with advanced melanoma. Refereed/Peer-reviewed

Chimeric antigen receptor (CAR)‐T cell therapy is now approved in the United States and Europe as a standard treatment for relapsed/refractory B‐cell malignancies. It has also been approved recently by the Therapeutic Goods Administration in Australia and may soon be publicly reimbursed. This advance has accentuated scientific, clinical and commercial interest in adapting this exciting technology for the treatment of solid cancers where it is widely recognised that the challenges of overcoming a hostile tumor microenvironment are most acute. Indeed, CAR‐T cell technology may be of the greatest value for those cancers that lack pre‐existing immunity because they are immunologically ‘cold’, or have a low somatic tumor mutation load, or both. These cancers are generally not amenable to therapeutic immune checkpoint blockade, but CAR‐T cell therapy may be effective because it provides an abundant supply of autologous tumor‐specific T cells. This is achieved by using genetic engineering to re‐direct autologous T‐cell cytotoxicity towards a tumor‐associated antigen, bypassing endogenous T‐cell requirements for antigen processing, MHC‐dependent antigen presentation and co‐stimulation. One of the most challenging solid cancers is glioblastoma, which has among the least permissive immunological milieu of any cancer, and which is almost always fatal. Here, we argue that CAR‐T cell technology may counter some glioblastoma defences and provide a beachhead for furthering our eventual therapeutic aims of restoring effective antitumor immunity. Although clinical investigation of CAR‐T cell therapy for glioblastoma is at an early stage, we discuss three recently published studies, which feature significant differences in target antigen, CAR‐T cell phenotype, route of administration and tumor response. We discuss the lessons, which may be learned from these studies and which may guide further progress in the field., Glioblastoma is a deadly disease without effective treatment. Recent clinical data indicate that chimeric antigen receptor (CAR)‐T cell therapy may provide a useful treatment approach.

// BeeShin Tan 1 , Matthew Anaka 1 , Siddhartha Deb 2 , Claudia Freyer 1 , Lisa M. Ebert 3 , Anderly C. Chueh 1 , Sheren Al-Obaidi 1 , Andreas Behren 1 , Aparna Jayachandran 1 , Jonathan Cebon 1 , Weisan Chen 4 and John M. Mariadason 1 1 Ludwig Institute for Cancer Research Ltd. Melbourne-Austin Branch, Heidelberg, Victoria, Australia. 2 Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 3 Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, Australia. 4 School of Molecular Science, LaTrobe University, Bundoora, Victoria, Australia. Correspondence: John M. Mariadason, email: // Weisan Chen, email: // Keywords : FOXP3, melanoma, proliferation, apoptosis Received : November 12, 2013 Accepted : December 20, 2013 Published : December 20, 2013 Abstract The Forkhead box P3 (FOXP3) transcription factor is the key driver of regulatory T cell (Treg cells) differentiation and immunosuppressive function. In addition, FOXP3 has been reported to be expressed in many tumors, including melanoma. However, its role in tumorigenesis is conflicting, with both tumor suppressive and tumor promoting functions described. The aim of the current study was to characterize the expression and function of FOXP3 in melanoma. FOXP3 expression was detected by immunohistochemistry (IHC) in 12% (18/146) of stage III and IV melanomas. However expression was confined to fewer than 1% of cells in these tumors. Stable over-expression of FOXP3 in the SK-MEL-28 melanoma cell line reduced cell proliferation and clonogenicity in vitro , and reduced xenograft growth in vivo . FOXP3 over-expression also increased pigmentation and the rate of apoptosis of SK-MEL-28 cells. Based on its infrequent expression in human melanoma, and its growth inhibitory and pro-apoptotic effect in over-expressing melanoma cells, we conclude that FOXP3 is not likely to be a key tumor suppressor or promoter in melanoma.

Aim: Pilot clinical trial of NY-ESO-1 (ESO) protein in ISCOMATRIX™ adjuvant pulsed onto peripheral blood dendritic cells (PBDC), to ascertain feasibility, evaluate toxicity and assess induction of ESO-specific immune responses. Patients & methods: Eligible participants had resected cancers expressing ESO or LAGE-1 and were at high risk of relapse. PBDC were produced using CliniMACS®plus, with initial depletion of CD1c+ B cells followed by positive selection of CD1c+ PBDC. Patients received three intradermal vaccinations of ESO/IMX-pulsed PBDC at 4-week intervals. Results: The process was feasible and safe. No vaccine-induced immune responses were detected. Assays of immunomodulatory cells did not correlate with outcomes. One patient had a long lasting complete remission. Conclusion: This method was feasible and safe but was minimally immunogenic.

CD4+CD25+FoxP3+ naturally occurring regulatory T (Treg) cells play a crucial role in the maintenance of immune tolerance and in preventing autoimmune pathology. Interventions that expand Treg cells are highly desirable, as they may offer novel treatment options in a variety of autoimmune and transplantation settings. Paralleling previous preclinical studies, we demonstrate here that administration of the hematopoietic growth factor Flt3L to human subjects increases the frequency and absolute number of Treg cells, and reduces the ratio of CD8+ T cells to Treg cells in the peripheral blood. The increase in Treg cells was due to enhanced Treg-cell proliferation rather than release of Treg cells from the thymus. Further studies revealed that Flt3L-induced proliferation of Treg cells was an indirect effect that occurred via the interaction of Treg cells with the Flt3L-expanded pool of CD1c+ myeloid dendritic cells. On the basis of these findings, Flt3L may represent a promising agent for promoting immune tolerance in a variety of clinical settings.

The rigorous evaluation of cancer vaccination requires evidence of benefit to patients with cancer or those at risk of relapse from the disease. Clinical trials are expensive and require considerable human and clinical resources in order to demonstrate this benefit. In the era of defined cancer antigens, it is possible to evaluate immunogenic targets, and assess the quality and magnitude of immune responses against these antigens following vaccination. Analyzing these surrogate end points complements clinical assessment and provides a depth of understanding to better inform trial evaluation and design. We have used the immunogenic cancer testis antigen NY-ESO-1 as a model antigen. This article summarizes our experience in monitoring immunity against NY-ESO-1.

Purpose: Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known. Experimental Design: We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated. Results: Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A–specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A–specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A–expressing tumor cells. Conclusions: Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.

Purpose: NY-ESO-1 is a highly immunogenic antigen expressed in a variety of malignancies, making it an excellent target for cancer vaccination. We recently developed a vaccine consisting of full-length recombinant NY-ESO-1 protein formulated with ISCOMATRIX adjuvant, which generated strong humoral and T-cell–mediated immune responses and seemed to reduce the risk of disease relapse in patients with fully resected melanoma. This study examines the clinical and immunologic efficacy of the same vaccine in patients with advanced metastatic melanoma. Experimental Design: Delayed-type hypersensitivity responses, circulating NY-ESO-1–specific CD4+ and CD8+ T cells, and proportions of regulatory T cells (Treg) were assessed in patients. Results: In contrast to patients with minimal residual disease, advanced melanoma patients showed no clinical responses to vaccination. Although strong antibody responses were mounted, the generation of delayed-type hypersensitivity responses was significantly impaired. The proportion of patients with circulating NY-ESO-1–specific CD4+ T cells was also reduced, and although many patients had CD8+ T cells specific to a broad range of NY-ESO-1 epitopes, the majority of these responses were preexisting. Tregs were enumerated in the blood by flow cytometric detection of cells with a CD4+CD25+FoxP3+ and CD4+CD25+CD127− phenotype. Patients with advanced melanoma had a significantly higher proportion of circulating Treg compared with those with minimal residual disease. Conclusions: Our results point to a tumor-induced systemic immune suppression, showing a clear association between the stage of melanoma progression, the number of Treg in the blood, and the clinical and immunologic efficacy of the NY-ESO-1 ISCOMATRIX cancer vaccine.

As more tumor antigens are discovered and as computer-guided T cell epitope prediction programs become more sophisticated, many potential T cell epitopes are synthesized and demonstrated to be antigenic in vitro. However, it is estimated that about 50% of such tumor antigen-specific T cells have not been demonstrated to recognize the naturally presented epitopes due to either technical difficulties, such as T cell cloning which is still challenging for many laboratories; or the predicted T cell epitopes are not generated or not generated in sufficient amounts by the antigen processing machinery. However, to potentially identify clinically relevant vaccine candidate epitopes, it is essential to demonstrate natural antigen presentation. Here we combine the advantages of MHC tetramer and intracellular cytokine staining to sensitively detect natural antigen presentation by tumor cells for epitopes of interest. The novel method does not require T cell cloning or long-term T cell culture. Because the antigen-specific T cells are positively identified, this method is much less influenced by IFNgamma producing cells with unknown specificities and should be widely applicable.

FoxP3 is a member of the forkhead family of transcription factors critically involved in the development and function of CD25+ regulatory T cells (Treg). Until recently, FoxP3 expression was thought to be restricted to the T-cell lineage. However, using immunohistochemistry and flow cytometric analysis of human melanoma tissue, we detected FoxP3 expression not only in the tumor infiltrating Treg but also in the melanoma cells themselves. FoxP3 is also widely expressed by established human melanoma cell lines (as determined by flow cytometry, PCR, and Western blot), as well as cell lines derived from other solid tumors. Normal B cells do not express FoxP3; however, expression could be induced after transformation with EBV in vitro and in vivo, suggesting that malignant transformation of healthy cells can induce FoxP3. In addition, a FOXP3 mRNA variant lacking exons 3 and 4 was identified in tumor cell lines but was absent from Treg. Interestingly, this alternative splicing event introduces a translation frame-shift that is predicted to encode a novel protein. Together, our results show that FoxP3, a key regulator of immune suppression, is not only expressed by Treg but also by melanoma cells, EBV-transformed B cells, and a wide variety of tumor cell lines. [Cancer Res 2008;68(8):3001–9]

Tumors can develop a blood supply not only by promoting angiogenesis but also by forming vessel-like structures directly from tumor cells, known as vasculogenic mimicry (VM). Understanding mechanisms that regulate VM is important, as these might be exploitable to inhibit tumor progression. Here, we reveal the adhesion molecule desmoglein 2 (DSG2) as a novel mediator of VM in melanoma. Analysis of patient-derived melanoma cell lines and tumor tissues, and interrogation of The Cancer Genome Atlas (TCGA) data, revealed that DSG2 is frequently overexpressed in primary and metastatic melanomas compared to normal melanocytes. Notably, this overexpression was associated with poor clinical outcome. DSG2+ melanoma cells self-organized into tube-like structures on Matrigel, indicative of VM activity, which was inhibited by DSG2 knockdown or treatment with a DSG2-blocking peptide. Mechanistic studies revealed that DSG2 regulates adhesion and cell-cell interactions during tube formation, but does not control melanoma cell viability, proliferation or motility. Finally, analysis of patient tumors revealed a correlation between DSG2 expression, VM network density and expression of VM-associated genes. These studies identify DSG2 as a key regulator of VM activity in human melanoma and suggest this molecule might be therapeutically targeted to reduce tumor blood supply and metastatic spread. Refereed/Peer-reviewed

Antigen-driven T cell education and subsequent pathogen elimination present particular challenges for the immune system. Pathogens generally enter the body at peripheral sites such as the skin, gastrointestinal tract or lung, areas from which naïve T cells are largely excluded. Instead, naïve T cells constantly recirculate through secondary lymphoid organs, such as lymph nodes and Peyer's patches, in search for antigen brought to these locations by means of afferent lymphatic channels. Here, antigen-loaded dendritic cells present antigen-peptide-MHC complexes to clonotypic T cells and provide appropriate co-stimulatory signals for immune response initiation. As a result, short-lived effector T cells and long-lived memory T cells are generated that reach the peripheral tissue for participation in immune responses and immune surveillance. Effector and memory T cell relocation is non-random, due to tissue-specific "address codes" that allow proper tissue homing. This process involves adhesion molecules, including selectins, integrins, and corresponding vascular ligands as well as the large family of chemokines and their receptors. Here, we discuss the changes in chemokine receptor expression that occur during T cell activation and differentiation, and the ways in which these changes impact on the migration potential of naïve, effector, and memory T cells. We summarize our current understanding of T cell homing to the T zone and B cell follicles within secondary lymphoid tissues and highlight the two chemokine receptors CCR7 and CXCR5 that recognize chemokines constitutively present either in the T zone (CCR7 ligands CCL19/ELC and CCL21/SLC) or follicular compartment (CXCR5 ligand CXCL13/BCA-1). CCR7 is characteristic for naive and central memory T (T(CM)) cells whereas CXCR5 distinguishes follicular B helper T (T(FH)) cells. In addition, we further subdivide long-lived memory T cells into CCR7-negative effector memory T (T(EM)) cells and peripheral immune surveillance T (T(PS)) cells. The latter term designates the extraordinarily large subset of memory T cells with primary residence in normal (healthy) peripheral tissues. Our current understanding of T(PS) cell migration and function is highly fragmentary, but these cells are thought to provide immediate protection locally at the site of pathogen entry. Here, we propose that the tissue distribution of T(PS) cells is determined by a distinct set of chemokines and corresponding receptors that differs from those operating in secondary lymphoid tissues and inflammatory sites.

Effective immune surveillance is essential for maintaining protection and homeostasis of peripheral tissues. However, mechanisms controlling memory T cell migration to peripheral tissues such as the skin are poorly understood. Here, we show that the majority of human T cells in healthy skin express the chemokine receptor CCR8 and respond to its selective ligand I-309/CCL1. These CCR8(+) T cells are absent in small intestine and colon tissue, and are extremely rare in peripheral blood, suggesting healthy skin as their physiological target site. Cutaneous CCR8(+) T cells are preactivated and secrete proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma, but lack markers of cytolytic T cells. Secretion of interleukin (IL)-4, IL-10, and transforming growth factor-beta was low to undetectable, arguing against a strict association of CCR8 expression with either T helper cell 2 or regulatory T cell subsets. Potential precursors of skin surveillance T cells in peripheral blood may correspond to the minor subset of CCR8(+)CD25(-) T cells. Importantly, CCL1 is constitutively expressed at strategic cutaneous locations, including dermal microvessels and epidermal antigen-presenting cells. For the first time, these findings define a chemokine system for homeostatic T cell traffic in normal human skin.

Following infection, naive T cells are activated in the secondary lymphoid tissue, but then need to move to the infected tissue in the periphery to mediate their effector functions. The acquisition of inflammatory chemokine receptors, such as CCR5 and CCR6, may contribute to the efficient relocation of activated T cells to inflamed sites in the periphery. In keeping with this idea, the present study has demonstrated that CCR5 and CCR6 are up-regulated on CD4+ T cells upon activation in the MLR. The observed increase in expression correlated well with the acquisition of an activated/memory phenotype and was largely (CCR5) or completely (CCR6) separated temporally from the initiation of cell division. In contrast, the regulation of two other chemokine receptors, CXCR3 and CXCR4, occurred in close parallel with the cell division process. Increased mRNA levels are likely to contribute to the enhanced surface expression of CCR5 and CCR6, but in the case of CCR6, translocation of intracellular stores of protein to the cell surface may be an additional mechanism of regulation. The up-regulation of CCR5 was more extensive than that of CCR6, as only approximately half the activated CCR5+ T cells coexpressed CCR6. The increased expression of CCR5 resulted in enhanced chemotaxis toward the CCR5 ligand macrophage-inflammatory protein-1β/CCL4, but up-regulation of CCR6 did not result in altered chemotactic responsiveness to macrophage-inflammatory protein-3α/CCL20, suggesting an alternative function for this receptor.

Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133+ EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs), namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133+ cell expansion with clear pro-angiogenic properties (in vitro and in vivo) and thus may provide clinical utility for humans in the future. Refereed/Peer-reviewed

Upon activation, naive T cells alter their migratory patterns, acquiring the ability to move through peripheral tissues as well as the general lymphoid circulation. Although the mechanisms responsible for these alterations are not well understood, changes in chemokine receptor expression may play a critical role. To investigate these changes, the expression patterns of two chemokine receptors, CXCR3 and CXCR4, were compared on CD4+ T cells following activation in the MLR. By day 9 of activation, expression of the inflammatory chemokine receptor CXCR3 was up-regulated, while expression of the homeostatic chemokine receptor CXCR4 was down-regulated. Alterations in receptor expression occurred almost exclusively on a subpopulation of T cells that expressed higher levels of CD4. These CD4high T cells demonstrated many characteristics of activated T cells and had undergone division in the MLR. By day 9 of culture, the majority of CXCR3+ and CXCR4− cells had divided and had acquired an activated/memory phenotype (CD45RA− CD45RO+ CD69+ CD25+). The levels of transcripts for both CXCR3 and CXCR4 were increased upon allo-activation. The discrepancy between levels of CXCR4 mRNA and surface protein was not due to sequestration of the receptor in intracellular compartments, as CXCR4 was not detectable intracellularly. However, intracellular CXCR3 was readily detectable. Finally, cells from allogeneic cultures demonstrated enhanced migration toward IFN-inducible T cell α chemoattractant and reduced migration toward stromal cell-derived factor-1 compared with syngeneic controls, thus suggesting that the observed switch in receptor expression may at least partly contribute to the differential patterns of migration displayed by naive and memory T cells.

CD4(+) CD25(+) FoxP3(+) naturally occurring regulatory T (Treg) cells play a crucial role in the maintenance of immune tolerance and in preventing autoimmune pathology. Interventions that expand Treg cells are highly desirable, as they may offer novel treatment options in a variety of autoimmune and transplantation settings. Paralleling previous preclinical studies, we demonstrate here that administration of the hematopoietic growth factor Flt3L to human subjects increases the frequency and absolute number of Treg cells, and reduces the ratio of CD8(+) T cells to Treg cells in the peripheral blood. The increase in Treg cells was due to enhanced Treg-cell proliferation rather than release of Treg cells from the thymus. Further studies revealed that Flt3L-induced proliferation of Treg cells was an indirect effect that occurred via the interaction of Treg cells with the Flt3L-expanded pool of CD1c(+) myeloid dendritic cells. On the basis of these findings, Flt3L may represent a promising agent for promoting immune tolerance in a variety of clinical settings.

Antigenic epitopes recognized by FoxP3+ regulatory T cells (Treg) are poorly defined, largely due to a lack of assays for determining Treg specificity. We have developed a novel approach for detecting human Treg specific to peptide antigen, utilizing down-regulation of surface CD3 as a read-out of antigen recognition. Culture conditions and re-stimulation time have been optimized, allowing the detection of even very rare Treg, such as those specific to tumor antigens. Refereed/Peer-reviewed

The tumor antigen NY-ESO-1 is a promising cancer vaccine target. We describe here a novel HLA-B7–restricted NY-ESO-1 epitope, encompassing amino acids 60-72 (APRGPHGGAASGL), which is naturally presented by melanoma cells. The tumor epitope bound to HLA-B7 by bulging outward from the peptide-binding cleft. This bulged epitope was not an impediment to T-cell recognition, however, because four of six HLA-B7+ melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX vaccine generated a potent T-cell response to this determinant. Moreover, the response to this epitope was immunodominant in three of these patients and, unlike the T-cell responses to bulged HLA class I viral epitopes, the responding T cells possessed a remarkably broad TCR repertoire. Interestingly, HLA-B7+ melanoma patients who did not receive the NY-ESO-1 ISCOMATRIX vaccine rarely generated a spontaneous T-cell response to this cryptic epitope, suggesting a lack of priming of such T cells in the natural anti–NY-ESO-1 response, which may be corrected by vaccination. Together, our results reveal several surprising aspects of antitumor immunity and have implications for cancer vaccine design. [Cancer Res 2009;69(3):1046–54]

Normal (noninflamed) human skin contains a network of lymphocytes, but little is known about the homing and function of these cells. The majority of αβ T cells in normal skin express CCR8 and produce proinflammatory cytokines. In this study we examined other subsets of cutaneous lymphocytes, focusing on those with potential function in purging healthy tissue of transformed and stressed cells. Human dermal cell suspensions contained significant populations of Vδ1+ γδ T cells and CD56+CD16− NK cells, but lacked the subsets of Vδ2+ γδ T cells and CD56+CD16+ NK cells, which predominate in peripheral blood. The skin-homing receptors CCR8 and CLA were expressed by a large fraction of both cell types, whereas chemokine receptors associated with lymphocyte migration to inflamed skin were absent. Neither cell type expressed CCR7, although γδ T cells up-regulated this lymph node-homing receptor upon TCR triggering. Stimulation of cutaneous Vδ1+ γδ T cell lines induced secretion of large amounts of TNF-α, IFN-γ, and the CCR8 ligand CCL1. In contrast to cutaneous αβ T cells, both cell types had the capacity to produce intracellular perforin and displayed strong cytotoxic activity against melanoma cells. We therefore propose that γδ T cells and NK cells are regular constituents of normal human skin with potential function in the clearance of tumor and otherwise stressed tissue cells. Under conditions of homeostasis, human skin contains an extensive network of leukocytes, including dendritic cells (DCs),3 T cells, NK cells, mast cells, and macrophages (1, 2, 3). These leukocytes presumably function together in maintaining skin integrity by continuously surveying for the presence of foreign Ags or signs of cellular stress. The best-characterized mechanism of skin protection involves cutaneous DCs (Langerhans cells and dermal DCs), which migrate to skin-associated LNs for the initiation of tolerance or, in the case of skin infection, antimicrobial immune responses (4, 5). In this manner, microbe-specific T cells acquire cell surface molecules for guidance to affected skin to combat local infectious agents. Address cues for effector T cell migration to inflamed skin include E-selectin ligand (known as cutaneous lymphocyte-associated Ag/CLA in humans), as well as certain receptors for chemokines expressed in inflamed skin, including CCR4, CCR6, and CCR10 (6, 7, 8, 9). Thus, the mechanisms by which effector T cells are recruited from blood to inflamed skin are relatively well understood. In contrast, we are just beginning to understand the migration properties and function of T cells and other lymphocytes within healthy tissue (10, 11). We recently identified the chemokine receptor CCR8 as a specific marker of αβ T cells present in normal skin and demonstrated that CCL1/I-309, the unique ligand for CCR8, was constitutively produced at strategic sites in noninflamed skin (12). These cutaneous αβ T cells secreted large amounts of proinflammatory cytokines and may thus contribute to memory responses after contact with previously encountered Ags. However, αβ T cells are unlikely candidates for surveillance against tumors and stressed tissue cells (12). Therefore, we turned our attention to γδ T cells and NK cells. In contrast to αβ T cells, γδ T cells recognize Ags without the need for processing and presentation by classical MHC molecules (13, 14). The major subset of γδ T cells in human peripheral blood expresses the Vδ2 gene segment and recognizes nonpeptide Ags of mostly microbial origin. In contrast, most γδ T cells in epithelial tissues express the Vδ1 gene segment and respond to stress-associated factors, including heat shock proteins and the MHC-related molecules MHC-I-related protein A (MICA) and MHC-I-related protein B (MICB), which are induced on tumor and virus-infected cells (13, 14, 15, 16). NK cells similarly recognize abnormal cells using a repertoire of receptors that detect stress-associated molecules as well as the loss of surface MHC protein (17). Murine skin harbors a major population of Vδ1+ γδ T cells, known as dendritic epidermal T cells (DETCs), which appear to play a critical role in tumor surveillance, as evidenced in γδ T cell-deficient mice (18, 19). Surprisingly, however, a counterpart of DETCs does not exist in human epidermis (1), and it is thus unclear what types of lymphocyte mediate equivalent immune surveillance functions in human skin. In this study we demonstrate that normal human dermis contains distinct populations of γδ T cells and NK cells, both of which express receptors for homing to noninflamed skin and for recognition of allogeneic tumor cells. Together, these cells are likely to make a unique contribution to the elimination of cutaneous tumor and otherwise stressed cells.

Follicular B helper T (T(FH)) cells make up a CD4(+) memory T cell subset with unique migration properties, defined by the expression of the chemokine receptor CXCR5 and responsiveness to the CXCR5-selective chemokine BCA-1, which is produced in B cell follicles. The generation of T(FH) cells is closely associated with initiation of adaptive immunity. Here, we discuss the relationship of T(FH) cells with those T cells characterized by an inherent secondary lymphoid tissue homing program. Special emphasis is placed on the potential involvement of T(FH) cells in the humoral immune response within B cell follicles, as well as the differentiation potential of these non-polarized cells.

Cancer vaccines are designed to expand tumor antigen-specific T cells with effector function. However, they may also inadvertently expand regulatory T cells (Treg), which could seriously hamper clinical efficacy. To address this possibility, we developed a novel assay to detect antigen-specific Treg based on down-regulation of surface CD3 following TCR engagement, and used this approach to screen for Treg specific to the NY-ESO-1 tumor antigen in melanoma patients treated with the NY-ESO-1/ISCOMATRIXTM cancer vaccine. All patients tested had Treg (CD25bright FoxP3+ CD127neg) specific for at least one NY-ESO-1 epitope in the blood. Strikingly, comparison with pre-treatment samples revealed that many of these responses were induced or boosted by vaccination. The most frequently detected response was toward the HLA-DP4-restricted NY-ESO-1157-170 epitope, which is also recognized by effector T cells. Notably, functional Treg specific for an HLA-DR-restricted epitope within the NY-ESO-1115-132 peptide were also identified at high frequency in tumor tissue, suggesting that NY-ESO-1-specific Treg may suppress local anti-tumor immune responses. Together, our data provide compelling evidence for the ability of a cancer vaccine to expand tumor antigen-specific Treg in the setting of advanced cancer, a finding which should be given serious consideration in the design of future cancer vaccine clinical trials usc Refereed/Peer-reviewed