ABSTRACT
Mass spectrometry (MS) has advanced greatly and many of its applications are ready for utilization within regulatory procedures and could significantly contribute to overcome challenges in standardization of allergen products. It seems sensible to discuss MS within the regulatory framework, before addressing technical questions. While the application to purified proteins is well established from product development to manufacturer's release analytics, its application to complex products such as allergen products is still under development. It needs to be determined where it can complement or replace established methods or where MS offers limited improvement. Despite its technical appeal and versatility, currently MS is mentioned in regulatory guidelines only as one possible measurement method. For example, no specific MS method is given in the European Pharmacopoeia. We discuss applications of MS within the EU regulatory framework. This includes their advantages and disadvantages and their positioning between research, characterization, manufacturer's release analytics and official batch testing. We discuss the qualitative detection of single and multiple allergens as proof of identity, qualitative to semi-quantitative protein profiles for batch to batch consistency testing, and quantification of allergens to state mass units of allergens. MS may also facilitate standardization of allergen products, reference products and reference standards.
Subject(s)
Allergens , European Union , Mass Spectrometry , Quality Control , Allergens/analysis , Mass Spectrometry/methods , Mass Spectrometry/standards , Humans , Reference StandardsABSTRACT
Patch testing is the only clinically applicable diagnostic method for Type IV allergy. The availability of Type IV patch test (PT) allergens in Europe, however, is currently scarce. This severely compromises adequate diagnostics of contact allergy, leading to serious consequences for the affected patients. Against this background, the European Society of Contact Dermatitis (ESCD) has created a task force (TF) (i) to explore the current availability of PT substances in different member states, (ii) to highlight some of the unique characteristics of Type IV vs. other allergens and (iii) to suggest ways forward to promote and ensure availability of high-quality patch testing substances for the diagnosis of Type IV allergies throughout Europe. The suggestions of the TF on how to improve the availability of PT allergens are supported by the ESCD, the European Academy of Allergy and Clinical Immunology, and the European Academy of Dermatology and Venereology and intend to provide potential means to resolve the present medical crisis.
Subject(s)
Allergens , Dermatitis, Allergic Contact , Dermatitis, Occupational , Patch Tests , Humans , Patch Tests/methods , Europe , Dermatitis, Allergic Contact/diagnosis , Dermatitis, Allergic Contact/etiology , Allergens/adverse effects , Dermatitis, Occupational/diagnosis , Dermatitis, Occupational/etiology , Societies, Medical , Advisory CommitteesABSTRACT
The Biological Standardization Project BSP090 has been successfully concluded in 2021. As a result, two standard methods for quantification of the major allergens Bet v 1 and Phl p 5 will be implemented in the European Pharmacopoeia (Ph. Eur.). The General Chapter describing the protocol of the respective Bet v 1-specific ELISA has already been adopted by the Ph. Eur. Commission and will become an official part of the Ph. Eur. in the beginning of 2023. As this will be the first allergen-specific standard method in the EU, this paper intends to summarize the preceding process and outline the measures necessary to comply with the new regulatory requirement.
Subject(s)
Allergens , Humans , Allergens/analysis , Enzyme-Linked Immunosorbent Assay/methodsABSTRACT
Clinical studies demonstrate that efficacy and safety in allergen immunotherapy (AIT) are linked to a multiplicity of factors decisively influencing success or failure. In recent years, numerous trials were performed with correspondent study results published. Yet, the number of AIT products successfully obtaining licensure in the analogous time frame is comparably limited. Essential for licensure is that the AIT product investigated remains comparable in its qualitative and quantitative composition throughout the clinical development. Verification of efficacy is not solely demonstrated by a statistically significant difference between the test and control populations; it must also be shown to be clinically relevant. Choice of meaningful inclusion and end-point criteria is critical. Post hoc or subgroup analysis can be supportive but needs verification as predefined criteria in additional studies. Data analysis may be presented on varying analysis populations, while it should be based on the intention-to-treat population for regulatory review to allow objective assessment of the treatment effect on the overall study population. Apparently conflicting interpretations of clinical data between publications and regulatory review are frequently based on their inherently different objectives, with regulatory review taking into considerations the full data sets of all relevant clinical studies for the concerned AIT product to allow an informed decision on licensure.
Subject(s)
Allergens , Desensitization, Immunologic , Allergens/therapeutic use , Desensitization, Immunologic/methods , Europe , Humans , United StatesABSTRACT
PURPOSE OF REVIEW: Medicinal products for allergen immunotherapy (AIT) of food allergies have gained enormous momentum in recent years. With this new class of products entering marketing authorization procedures, compliance to regulatory requirements becomes a critical element. Here, an overview is provided on specific requirements and aspects concerning the quality control and manufacturing of these products. RECENT FINDINGS: Recent developments in the field of AIT for food allergies are divers, including products for oral, epicutaneous, and subcutaneous application, most notably targeting egg, milk, and peanut allergy. As the source materials for food AIT product are typically produced for food consumption and not for medicinal purposes, unique challenges arise in the manufacturing processes and controls of these medicinal products. Individual approaches are needed to assure acceptable quality, including control of relevant quantitative and qualitative characteristics. Major characteristics for quality verification include determination of protein content, total allergenic activity, and major allergen content. The applied manufacturing processes need to be established such that relevant process parameters are kept within justified limits and consistency of produced batches is assured. Allergen products for food AIT present specific challenges with respect to quality aspects that differentiate them from other commonly available AIT products. While established regulation is available and provides clear guidance for most aspects, other issues require consideration of new and individual settings relevant here. Consequently, as experience grows, respective amendments to currently available guidance may be needed.
Subject(s)
Allergens/therapeutic use , Desensitization, Immunologic/standards , Drug Industry/standards , Drug and Narcotic Control/legislation & jurisprudence , Food Hypersensitivity/therapy , Quality Control , Drug Industry/legislation & jurisprudence , European Union , Food/adverse effects , Food Hypersensitivity/immunology , Government Regulation , Humans , Peanut Hypersensitivity/immunology , Peanut Hypersensitivity/therapyABSTRACT
Diagnostic allergens are defined as medicinal products in the EU. Marketing authorization by national authorities is necessary; however, diagnostic allergens are not homogeneously regulated in different EU member states. Allergen manufacturers argue with increasing costs forcing them to continuously reduce the diagnostic allergen portfolios offered to allergists. In contrast, EAACI and national European Allergy Societies see the need for the availability of a wide range of high-quality diagnostic allergens for in vivo diagnosis of IgE-mediated allergies not only covering predominant but also less frequent allergen sources. In a recent EAACI task force survey, the current practice of allergy diagnosis was shown to rely on skin tests as first option in almost 2/3 of all types of allergic diseases and in 90% regarding respiratory allergies. With the need to ensure the availability of high-quality diagnostic allergens in the EU, an action plan has been set up by EAACI to analyse the current regulatory demands in EU member states and to define possible solutions stated in this document: (a) simplification of authorization for diagnostic allergens; (b) specific regulation of special types of diagnostic allergens; (c) new models beyond the current model of homologous groups; (d) simplification of pharmacovigilance reporting; (e) reduction of regulation fees for diagnostic allergens; (f) reimbursement for diagnostic allergens. Joining forces of allergists, manufacturers and authorities are of high importance to ensure remaining relevant allergens in the EU markets to facilitate a sustainable and comprehensive service for the diagnosis and treatment of allergic diseases.
Subject(s)
Allergens , Hypersensitivity , Diagnostic Tests, Routine , Europe , Humans , Hypersensitivity/diagnosis , Hypersensitivity/epidemiology , Skin TestsABSTRACT
The European Academy of Allergy and Clinical Immunology (EAACI) organized the first European Strategic Forum on Allergic Diseases and Asthma. The main aim was to bring together all relevant stakeholders and decision-makers in the field of allergy, asthma and clinical Immunology around an open debate on contemporary challenges and potential solutions for the next decade. The Strategic Forum was an upscaling of the EAACI White Paper aiming to integrate the Academy's output with the perspective offered by EAACI's partners. This collaboration is fundamental for adapting and integrating allergy and asthma care into the context of real-world problems. The Strategic Forum on Allergic Diseases brought together all partners who have the drive and the influence to make positive change: national and international societies, patients' organizations, regulatory bodies and industry representatives. An open debate with a special focus on drug development and biomedical engineering, big data and information technology and allergic diseases and asthma in the context of environmental health concluded that connecting science with the transformation of care and a joint agreement between all partners on priorities and needs are essential to ensure a better management of allergic diseases and asthma in the advent of precision medicine together with global access to innovative and affordable diagnostics and therapeutics.
Subject(s)
Asthma/epidemiology , Capital Financing , Hypersensitivity/epidemiology , Research , Translational Research, Biomedical , Asthma/diagnosis , Asthma/therapy , Big Data , Bioengineering , Disease Management , Drug Development , Environmental Health , Europe/epidemiology , Health Policy , Humans , Hypersensitivity/diagnosis , Hypersensitivity/etiology , Hypersensitivity/therapy , Implementation Science , Information Technology , Patient Participation , Translational Research, Biomedical/economics , Translational Research, Biomedical/legislation & jurisprudence , Translational Research, Biomedical/methods , Translational Research, Biomedical/organization & administrationABSTRACT
In order to ensure valid diagnostics for occupational test allergen solutions despite the ongoing reduction in the availability of commercial test extracts, a plan B was initiated for the possible production of skin prick test (SPT) solutions in public pharmacies. For important occupational allergen sources (wheat and rye, storage mites, animal epithelia, mold material) laboratory extraction methods were analyzed in comparison to pharmacy compatible extraction methods regarding protein quantity and quality in SDS-PAGE combined with silver staining. Subsequently, using the example of bovine epithelia, adapted extraction procedures as well as in-process and final product controls were transferred to a public pharmacy. Allergen sources with a high protein content, such as wheat and rye grains as well as storage mites, showed good comparability of the extractable protein quantity and protein pattern, regardless of the applied extraction method. In contrast, allergen source materials with a low total protein content, such as animal epithelia and molds, can benefit from laboratory extraction conditions such as mechanical disruption and specific buffer additives. In the qualitative protein silver staining, characteristic protein patterns were identified for each allergen source. Depending on the extraction method, only minor differences in total protein patterns were observed in animal epithelia and molds. Using source materials from two suppliers, the resulting allergen extracts displayed clear differences in protein content in storage mites and quantitative and qualitative differences in molds. A practical preparation attempt of SPT solutions in a public pharmacy was successful. SPT solutions prepared with adapted pharmacy extraction methods showed a comparable protein and Bos d 2 allergen content and equivalent qualities in the protein pattern compared to a previously available commercial SPT solution. Accordingly, it can be assumed that standardized SPT solutions with sufficient allergen quality for occupational allergen sources can be prepared in public pharmacies if certified allergen sources with appropriate protein content are available.
ABSTRACT
The availability of high-quality skin test allergens is a prerequisite for the reliable diagnosis of occupational type I allergies. Due to the withdrawal of existing marketing authorizations (MAs) by pharmaceutical companies and the lack of new MAs for commercial test allergens, there is an increasing diagnostic gap in Germany and other EU member states, which makes it necessary to investigate alternative ways of providing in vivo diagnostics. The German Medicinal Products Act (Arzneimittelgesetz = AMG) allows for the possibility of preparing medicinal products in pharmacies without the need for an MA or a manufacturing authorization pursuant to Section 13 (2) No. 1 in conjunction with Section 13 (2a) Sentence 2 No. 3 AMG. This also includes test allergens. In addition to the AMG, the requirements of the German Ordinance on the Operation of Pharmacies (Apothekenbetriebsordnung - ApBetrO) and the European Pharmacopoeia apply in particular. Medicolegal and practical challenges, as well as potentials of manufacturing skin prick test solutions in public pharmacies are presented based on examples of different allergen source materials.
ABSTRACT
T-regulatory cells (Treg) and mast cells (MC) are abundant in colorectal cancer (CRC) tumors. Interaction between the two is known to promote immune suppression or loss of Treg functions and autoimmunity. Here, we demonstrate that in both human CRC and murine polyposis the outcome of this interaction is the generation of potently immune suppressive but proinflammatory Treg (DeltaTreg). These Treg shut down IL10, gain potential to express IL17, and switch from suppressing to promoting MC expansion and degranulation. This change is also brought about by direct coculture of MC and Treg, or culture of Treg in medium containing IL6 and IL2. IL6 deficiency in the bone marrow of mice susceptible to polyposis eliminated IL17 production by the polyp infiltrating Treg, but did not significantly affect the growth of polyps or the generation of proinflammatory Treg. IL6-deficient MC could generate proinflammatory Treg. Thus, MC induce Treg to switch function and escalate inflammation in CRC without losing T-cell-suppressive properties. IL6 and IL17 are not needed in this process.
Subject(s)
Colorectal Neoplasms/immunology , Mast Cells/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Coculture Techniques , Humans , Interleukins/deficiency , Interleukins/immunology , Mice , Mice, Knockout , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
Allergen immunotherapy (AIT) has been performed for 112 years. In this article we summarize regulatory standards and challenges based on scientific evidence on AIT. Most crucial and timely aspects concerning AIT are addressed from the regulatory perspective of the authors as employees of a national competent authority in Europe: (1) product specificity; (2) clinical efficacy; (3) treatment for adults and children (needs for extrapolation); (4) allergen exposure chambers; (5) biomarkers; (6) standardization; (7) real-world evidence; (8) independent official batch release (benefit and challenges); (9) harmonization on the EU level. The Paul-Ehrlich-Institut (PEI), the Federal Institute for Vaccines and Biomedicines, in Langen near Frankfurt/Main in Germany, examines and evaluates the benefits and risks of AIT products within the course of clinical development, marketing authorization, and subsequently throughout their entire life cycle to ensure high-quality, safe, and effective AIT products.
ABSTRACT
A roundtable discussion on February 10, 2023 between the German Society for Allergology and Clinical Immunology (DGAKI) and the Paul-Ehrlich-Institut (PEI) aimed to discuss in detail current aspects of allergen immunotherapy (AIT), its regulatory framework under the transitional provision of the Therapy Allergen Ordinance (TAO), and the consequences for the planned guideline work of the DGAKI, regulatory challenges in the approval of AIT products for children and adolescents as well as allergy diagnostics. The content and discussion points of this dialogue are summarized and are set in context with the current literature.
ABSTRACT
Mast cells (MC) are a bone marrow-derived, long-lived, heterogeneous cellular population that function both as positive and negative regulators of immune responses. They are arguably the most productive chemical factory in the body and influence other cells through both soluble mediators and cell-to-cell interaction. MC are commonly seen in various tumors and have been attributed alternatively with tumor rejection or tumor promotion. Tumor-infiltrating MC are derived both from sentinel and recruited progenitor cells. MC can directly influence tumor cell proliferation and invasion but also help tumors indirectly by organizing its microenvironment and modulating immune responses to tumor cells. Best known for orchestrating inflammation and angiogenesis, the role of MC in shaping adaptive immune responses has become a focus of recent investigations. MC mobilize T cells and antigen-presenting dendritic cells. They function as intermediaries in regulatory T cells (Treg)-induced tolerance but can also modify or reverse Treg-suppressive properties. The central role of MC in the control of innate and adaptive immunity endows them with the ability to tune the nature of host responses to cancer and ultimately influence the outcome of disease and fate of the cancer patient.
Subject(s)
Mast Cells/pathology , Neoplasms/pathology , Animals , Antigen Presentation/immunology , Disease Models, Animal , Humans , Immunity, Cellular , Mast Cells/immunology , Mice , Neoplasm Invasiveness/immunology , Neoplasms/diagnosis , Neoplasms/immunology , Prognosis , T-Lymphocytes, Regulatory/immunologyABSTRACT
BACKGROUND & AIMS: Chronic pancreatitis is characterized by alternating phases of acute inflammation and quiescent disease. Involvement of T-cell responses has been suggested, but pancreatitis-specific T cells have not been described. METHODS: We characterized T-cell responses against pancreatitis, pancreatic carcinoma-associated antigens, and tetanus toxoid in the bone marrow, blood, and/or pancreatitis lesions of patients with pancreatitis, pancreatic cancer, and healthy individuals. T cells were functionally characterized by antigen-dependent secretion of interferon (IFN)-gamma, interleukin (Il)-4, and IL-10, which indicate type 1, type 2, or regulatory T-cell responses, respectively. Regulatory T cells were characterized by multicolor flow cytometry. Isolated regulatory T cells were tested for their capacity to recognize pancreatitis-associated antigens and to suppress conventional T cells in an antigen-dependent manner. T cell-derived cytokines in tissue lesions were quantified by enzyme-linked immunosorbent assay. RESULTS: Chronic pancreatitis patients showed similar to pancreatic cancer patients and healthy individuals type 1 T-cell responses against tetanus toxoid; however, they exhibited strong IL-10-based T-cell responses against pancreatitis-associated but not pancreatic carcinoma-associated antigens. T cells from pancreatic cancer patients responded to pancreatic cancer-associated but not pancreatitis-associated antigens with IFN-gamma secretion. Pancreatitis-specific IL-10 responses were mediated by IL-10(+)IFN-gamma(-)FoxP3(+) regulatory T cells, which were expanded in the blood, bone marrow, and pancreatitis lesions and possessed the potential to suppress the proliferation of autologous conventional T cells in an antigen-specific manner. Pancreatitis lesions, in comparison with pancreatic carcinomas, contained increased concentrations of IL-10 and reduced levels of IFN-gamma, suggesting pancreatitis-specific activity of regulatory T cells in situ. CONCLUSIONS: Chronic pancreatitis is associated with disease-specific regulatory T-cell responses.
Subject(s)
Bone Marrow Cells/immunology , Carcinoma/immunology , Immunologic Memory , Pancreas/immunology , Pancreatic Neoplasms/immunology , Pancreatitis, Chronic/immunology , T-Lymphocytes, Regulatory/immunology , Antigens, Neoplasm/immunology , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Forkhead Transcription Factors/metabolism , Humans , Immunohistochemistry , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-4/metabolism , Tetanus Toxoid/immunologyABSTRACT
BACKGROUND: The efficiencies of T cell based immunotherapies are affected by insufficient migration and activation of tumor specific effector T cells in the tumor. Accumulating evidence exists on the ability of ionizing radiation to modify the tumor microenvironment and generate inflammation. The aim of this phase I/II clinical trial is to evaluate whether low dose single fraction radiotherapy can improve T cell associated antitumor immune response in patients with pancreatic cancer. METHODS/DESIGN: This trial has been designed as an investigator initiated; prospective randomised, 4-armed, controlled Phase I/II trial. Patients who are candidates for resection of pancreatic cancer will be randomized into 4 arms. A total of 40 patients will be enrolled. The patients receive 0 Gy, 0.5 Gy, 2 Gy or 5 Gy radiation precisely targeted to their pancreatic carcinoma. Radiation will be delivered by external beam radiotherapy using a 6 MV Linac with IMRT technique 48 h prior to the surgical resection. The primary objective is the determination of an active local external beam radiation dose, leading to tumor infiltrating T cells as a surrogate parameter for antitumor activity. Secondary objectives include local tumor control and recurrence patterns, survival, radiogenic treatment toxicity and postoperative morbidity and mortality, as well as quality of life. Further, frequencies of tumor reactive T cells in blood and bone marrow as well as whole blood cell transcriptomics and plasma-proteomics will be correlated with clinical outcome. An interim analysis will be performed after the enrollment of 20 patients for safety reasons. The evaluation of the primary endpoint will start four weeks after the last patient's enrollment. DISCUSSION: This trial will answer the question whether a low dose radiotherapy localized to the pancreatic tumor only can increase the number of tumor infiltrating T cells and thus potentially enhance the antitumor immune response. The study will also investigate the prognostic and predictive value of radiation-induced T cell activity along with transcriptomic and proteomic data with respect to clinical outcome.
Subject(s)
Immunomodulation/radiation effects , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/radiotherapy , Humans , Neoplasm Staging , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/surgery , Radiotherapy DosageABSTRACT
PURPOSE OF REVIEW: Key aspects and outcomes from the recently published guidance on the regulation of allergen products are summarized. RECENT FINDINGS: A new regulatory guideline has been published to enhance harmonized national approaches on the regulation of allergen products and thereby strengthen the availability of high-quality products across the European Union (EU). As the guideline was developed, critical aspects for allergen products regulation were identified and are discussed in the document, including recommendations on the regulatory procedures to be applied for diagnostics, allergen immunotherapy products and named-patient products. SUMMARY: The new guidance is expected to provide clarifications on and support harmonization of the regulation of allergen products in the EU.
Subject(s)
Allergens/chemistry , Desensitization, Immunologic/methods , Social Control, Formal/methods , Allergens/immunology , Animals , European Union , Humans , Practice Guidelines as TopicABSTRACT
PURPOSE OF REVIEW: Cancer immunology became scientifically credible only some 20 years ago with the demonstration of the existence of human tumor antigens. In this short time span, outcomes of cancer vaccine trials have raised hopes and also surfaced disappointments. This review focuses on the prospects of peptide-based vaccines in cancer immunotherapy. RECENT FINDINGS: Accurate descriptions of the natural immune responses to cancer allow for a more precise targeting of such tumors by boosting preexisting antitumor immune responses in patients. The development of synthetic long-peptide vaccines avoids many of the pitfalls of previous vaccination trials through the presence of multiple epitopes that may elicit memory antitumor immune responses. Furthermore, the combination of standard therapy with newly developed immunomodulating agents, such as antibodies blocking cytotoxic T lymphocyte-associated antigen-4 or programmed death receptor-1, and more efficient immune adjuvants has shown promising results. SUMMARY: Immunotherapy is becoming an effective means of targeting human cancers, and the application of such approaches in combination with current standard schemes of treatment can lead to a significant benefit in survival and quality of life for cancer patients.
Subject(s)
Antigens, Neoplasm/immunology , Cancer Vaccines/immunology , Neoplasms/immunology , Vaccines, Subunit/immunology , Antigens, CD/immunology , CTLA-4 Antigen , Cancer Vaccines/administration & dosage , Humans , Immunotherapy/methods , Immunotherapy/trends , Neoplasms/therapy , Treatment Outcome , Vaccines, Subunit/administration & dosageABSTRACT
PURPOSE OF REVIEW: The recent developments in the manufacturing and quality assessment of allergenic extracts in Europe are summarized. RECENT FINDINGS: Quality assessment has always been a fundamental part of allergen product evaluation. New analytical methods have been reported that fill currently existing gaps in the characterization of commonly used allergen products. New types of products require innovative considerations and concepts for their assessment. Advanced standardization efforts aim at increasing reliability and comparability of analytical tools applied for allergen product characterization. In consequence, regulatory requirements are updated in line with such developments. SUMMARY: Current demands on the quality of allergen products ensure production of well characterized products of consistent quality. While experience with manufacturing processes and successful product characterization approaches increase, accompanying and continuous re-evaluation of underlying quality control and assessment concepts is being performed.
Subject(s)
Allergens/immunology , Cell Extracts/standards , Desensitization, Immunologic , Europe , Humans , Quality Assurance, Health Care , Reproducibility of ResultsABSTRACT
Endogenous antitumor effector T-cell responses and immune-suppressive regulatory T cells (Treg) critically influence the prognosis of patients with cancer, yet many of the mechanisms of how this occurs remain unresolved. On the basis of an analysis of the function, antigen specificity, and distribution of tumor antigen-reactive T cells and Tregs in patients with breast cancer and transgenic mouse tumor models, we showed that tumor-specific Tregs were selectively activated in the bone marrow (BM) and egressed into the peripheral blood. The BM was constantly depleted of tumor-specific Tregs and was instead a site of increased induction and activity of tumor-reactive effector/memory T cells. Treg egress from the BM was associated with activation-induced expression of peripheral homing receptors such as CCR2. Because breast cancer tissues express the CCR2 ligand CCL2, the activation and egress of tumor antigen-specific Tregs in the BM resulted in the accumulation of Tregs in breast tumor tissue. Such immune compartmentalization and redistribution of T-cell subpopulations between the BM and peripheral tissues were achieved by vaccination with adenoviral vector-encoded TRP-2 tumor antigen in a RET transgenic mouse model of spontaneous malignant melanoma. Thus, the BM simultaneously represented a source of tumor-infiltrating Tregs and a site for the induction of endogenous tumor-specific effector T-cell responses, suggesting that both antitumor immunity and local immune suppression are orchestrated in the BM.
Subject(s)
Breast Neoplasms/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antigens, Neoplasm/immunology , Bone Marrow/immunology , Cell Line, Tumor , Female , Humans , Melanoma/immunology , Mice, Transgenic , Proto-Oncogene Proteins c-ret/geneticsABSTRACT
The composition of tumor-targeted T cell infiltrates is a major prognostic factor in colorectal cancer (CRC) outcome; however, the functional role of these populations in prolonging patient survival remains unclear. Here, we evaluated 190 patients with CRC for the presence of functionally active tumor-infiltrating lymphocytes (TILs), the tumor specificity of these TILs, and the correlation between patient TILs and long-term survival. Using intracytoplasmic cytokine staining in conjunction with HLA multimers loaded with tumor peptide and antigen-specific cytokine secretion assays, we determined that TNF-α expression delineates a population of tumor antigen-specific (TA-specific) cytotoxic T lymphocytes (CTLs) present within tumors from patients with CRC. Upregulation of TNF-α expression in TILs strongly correlated with an increase in the total amount of intratumoral TNF-α, which is indicative of tumor-specific CTL activity. Moreover, a retrospective multivariate analysis of 102 patients with CRC, which had multiple immune parameters evaluated, revealed that increased TNF-α concentration was an independent prognostic factor. Together, these results indicate that the prognostic impact of T cell infiltrates for CRC maybe largely based on subpopulations of active TA-specific T cells within the tumor, suggesting causal implication for these cells in patient survival. Additionally, these results support the use of intratumoral TNF-α, which is indicative of T cell function, as a prognostic parameter for CRC.