Cytokine release: A workshop proceedings on the state-of-the-science, current challenges and future directions.

In October 2013, the International Life Sciences Institute - Health and Environmental Sciences Institute Immunotoxicology Technical Committee (ILSI-HESI ITC) held a one-day workshop entitled, "Workshop on Cytokine Release: State-of-the-Science, Current Challenges and Future Directions". The workshop brought together scientists from pharmaceutical, academic, health authority, and contract research organizations to discuss novel approaches and current challenges for the use of in vitro cytokine release assays (CRAs) for the identification of cytokine release syndrome (CRS) potential of novel monoclonal antibody (mAb) therapeutics. Topics presented encompassed a regulatory perspective on cytokine release and assessment, case studies regarding the translatability of preclinical cytokine data to the clinic, and the latest state of the science of CRAs, including comparisons between mAb therapeutics within one platform and across several assay platforms, a novel physiological assay platform, and assay optimization approaches such as determination of FcR expression profiles and use of statistical tests.

Development of a squamous cell carcinoma mouse model for immunotoxicity testing.

An important component of safety assessment of new pharmaceuticals is evaluation of their potential to increase the risk of developing cancer in humans. The traditional 2-year rodent bioassay often is not feasible or scientifically applicable for evaluation of biotherapeutics. Additionally, it has poor predictive value for non-genotoxic immunosuppressive compounds.

Murine gammaherpesvirus-68 (MHV-68) is not horizontally transmitted amongst laboratory mice by cage contact.

Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Immunosuppressive agents are used for treatment of a variety of autoimmune diseases as well as for prevention of tissue rejection after organ transplantation and can result in recrudescence of latent herpesvirus infections. Prior to examination of MHV-68 as a suitable model for EBV, better characterization of the MHV-68 model was desirable.

Analysis of cytokine release assay data using machine learning approaches.

The possible onset of Cytokine Release Syndrome (CRS) is an important consideration in the development of monoclonal antibody (mAb) therapeutics. In this study, several machine learning approaches are used to analyze CRS data. The analyzed data come from a human blood in vitro assay which was used to assess the potential of mAb-based therapeutics to produce cytokine release similar to that induced by Anti-CD28 superagonistic (Anti-CD28 SA) mAbs.

Is murine gammaherpesvirus-68 (MHV-68) a suitable immunotoxicological model for examining immunomodulatory drug-associated viral recrudescence?

Immunosuppressive agents are used for treatment of a variety of autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosis (SLE), and psoriasis, as well as for prevention of tissue rejection after organ transplantation. Recrudescence of herpesvirus infections, and increased risk of carcinogenesis from herpesvirus-associated tumors are related with immunosuppressive therapy in humans. Post-transplant lymphoproliferative disorder (PTLD), a condition characterized by development of Epstein Barr Virus (EBV)-associated B-lymphocyte lymphoma, and Kaposi's Sarcoma (KS), a dermal tumor associated with Kaposi Sarcoma-associated virus (KSHV), may develop in solid organ transplant patients.

CNTO 530 increases expression of HbA and HbF in murine models of β-thalassemia and sickle cell anemia.

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g.

Immunotoxicologic effects of cyclosporine on tumor progression in models of squamous cell carcinoma and B-cell lymphoma in C3H mice.

Many immunosuppressive drugs are associated with an increased risk of neoplasia, principally non-melanoma skin cancers and B-cell lymphomas. However, only 6 of the 13 immunosuppressive drugs tested in 2 year bioassays increased the incidence of neoplasia. For example, the 2-year bioassays conducted with cyclosporine (CSA), an International Agency for Research on Cancer (IARC) Group 1 human carcinogen, were negative.

Development of a human whole blood assay for prediction of cytokine release similar to anti-CD28 superagonists using multiplex cytokine and hierarchical cluster analysis.

Anti-CD28 superagonist (SA) mediated cytokine release syndrome (CRS), an adverse event resulting in systemic release of cytokines, is an emergent issue in drug development. CRS is of potential concern for all monoclonal antibodies (mAbs) particularly those directed against cell surface targets on lymphocytes. Concern regarding patient safety requires development of novel methods to predict these adverse reactions.

Development of a murine model of lymph node metastases suitable for immunotoxicity studies.

Introduction: Immunosuppressive drugs are associated with an increased risk of infections and in some cases neoplasia, particularly non-melanoma skin cancers. This paper describes the development of a model to test the effects of immunosuppressive drugs on local invasion and metastases of a squamous cell carcinoma in syngeneic, immunocompetent mice.

Methods: SCC VII cells were labeled with 655 quantum dots (QDs), injected intramuscularly into C3H HEN mice and traffic and progressive growth in the draining popliteal lymph node were evaluated.

Critical review of preclinical approaches to evaluate the potential of immunosuppressive drugs to influence human neoplasia.

Many immunosuppressive drugs are associated with an increased risk of B-cell lymphoma, squamous cell carcinoma, and Kaposi sarcoma. Thirteen immunosuppressive drugs have been tested in 2-year carcinogenicity studies (abatacept; azathioprine; busulfan; cyclophosphamide; cyclosporine; dexamethasone; everolimus; leflunomide; methotrexate; mycophenolate mofetil; prednisone; sirolimus; and tacrolimus) and in additional models including neonatal and genetically modified mice; chemical, viral, ultraviolet, and ionizing radiation co-carcinogenesis, and in models with transplanted tumor cells. The purpose of this review is to outline the mechanisms by which immunosuppressive drugs can influence neoplasia, to summarize the available preclinical data on the 13 drugs, and to critically review the performance of the models.

Recent advances in the understanding of drug-mediated infusion reactions and cytokine release syndrome.

Infusion reactions and cytokine release syndrome (CRS) are an emerging issue in drug development and are of particular importance with the development of new therapeutic proteins. Increasing concerns regarding patient safety require a better understanding of the mechanism involved and the development of novel methods for preventing and predicting such reactions and CRS. This review discusses developments during the past few years in understanding the mechanisms that cause infusion reactions and CRS, advances in approaches to prevent CRS, the reason why preclinical animal models are unreliable predictors of CRS, and new developments in the design and analysis of in vitro screening systems for the prediction of CRS.

Ex vivo expanded human CD4+CD25+Foxp3+ regulatory T cells prevent lethal xenogenic graft versus host disease (GVHD).

Mouse studies demonstrated that infusion of CD4+CD25+ regulatory T cells (Tregs) prevented graft versus host disease (GVHD) lethality after bone marrow transplantation (BMT). But the potential impact of human Tregs on GVHD has not been well demonstrated. In this study, we demonstrated that human Tregs enriched from peripheral blood of healthy donors could be expanded ex vivo to clinically relevant cell numbers in 2-3 weeks while maintaining Foxp3, CD25, CTLA-4, and CD62L expression as well as in vitro suppressive function.

Functional islet-specific Treg can be generated from CD4+CD25- T cells of healthy and type 1 diabetic subjects.

CD4(+)CD25(+)FOXP3(+) Treg cells require TCR engagement for suppressive function, thus ensuring that suppression occurs only in the presence of specific antigens; however, to date no studies have addressed the function of self-antigen-specific Treg in humans. These studies were designed to determine whether peripheral generation and function of islet antigen-specific adaptive Treg are defective in human subjects with type 1 diabetes (T1D). Islet antigen-specific adaptive Treg were induced in vitro by activation of CD4(+)FOXP3(-) T cells with glutamic acid decarboxylase and islet-specific glucose-6-phosphate catalytic subunit-related protein peptides in the context of T1D-associated HLA-DRbeta alleles.

De novo generation of antigen-specific CD4+CD25+ regulatory T cells from human CD4+CD25- cells.

Antigen-specificity is a hallmark of adaptive T cell-mediated immune responses. CD4+CD25+FOXP3+ regulatory T cells (T(R)) also require activation through the T cell receptor for function. Although these cells require antigen-specific activation, they are generally able to suppress bystander T cell responses once activated.

MHC class II biosynthesis by activated rat CD4+ T cells: development of repression in vitro and modulation by APC-derived signals.

This study focused on synthesis of MHC class II glycoproteins (MHCII) by rat CD4(+) T-helper cells. During activation in Con A and IL-2, purified rat splenic CD4(+) T cells expressed abundant surface MHCII together with transcripts for I-A alpha/beta, invariant chain, and the type III and type IV MHC class II transactivator (CIITA). Activated thymic CD8(+)CD4(-) and CD8(+)CD4(+) T cells exhibited essentially the same phenotype.

Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25- T cells.

CD4+CD25+ regulatory T (TR) cells have been described in both humans and mice. In mice, TR are thymically derived, and lack of TR leads to organ-specific autoimmunity. Recently, the forkhead/winged helix transcription factor, FoxP3, has been shown to be important for the function of TR cells in mice.

Acquisition of functional MHC class II/peptide complexes by T cells during thymic development and CNS-directed pathogenesis.

This study provides evidence that both rat and mouse thymic and splenic T cells express significant levels of MHC class II glycoproteins (MHCII) in vivo. Derivation of rat and mouse chimeras revealed that a major source of MHCII on thymic T cells was acquired from radioresistant host APC. Expression of MHC on thymic T cells appeared physiologically relevant because presentation of rat myelin basic protein (RMBP) by nonadherent, radiosensitive thymic T cells was associated with the adoptive transfer of tolerance.