Inflammatory bowel disease (IBD) is a generic term typically used to describe a group of idiopathic inflammatory intestinal conditions in humans that are generally divided into Crohn's disease and ulcerative colitis. Although the etiology of these diseases remains unknown, a number of rodent models of IBD have recently been identified, all sharing the concept that the development of chronic intestinal inflammation occurs as a consequence of alterations in the immune system that lead to a failure of normal immunoregulation in the intestine. On the basis of these models, it has been hypothesized that the development of IBD in humans may be related to a dysregulated immune response to normal flora in the gut. Immunodeficient scid mice injected with CD4+ CD45RB(high) T cells and mice deficient in interleukin (IL)-10 (IL-10-/-) are among the rodent models of IBD. In both models, there is inflammation and evidence of a Th1-like response in the large intestine, characterized by CD4+ T-cell and macrophage infiltrates, and elevated levels of interferon-gamma. Because IL-10 is an immunomodulatory cytokine that is capable of controlling Th1-like responses, the role of IL-10 was investigated in these models. IL-10 was shown to be important in regulating the development of intestinal inflammation in both models. These results provided key data that supported initiation of clinical trials evaluating the efficacy of IL-10 in patients with IBD.
Immunomodulatory therapeutics such as monoclonal antibodies (mAb) carry an inherent risk of undesired immune reactions. One such risk is cytokine release syndrome (CRS), a rapid systemic inflammatory response characterized by the secretion of pro-inflammatory cytokines from immune cells. It is crucial for patient safety to correctly identify potential risk of CRS prior to first-in-human dose administration. For this purpose, a variety of in vitro cytokine release assays (CRA) are routinely used as part of the preclinical safety assessment of novel therapeutic mAbs. One of the challenges for the development and comparison of CRA performance is the lack of availability of standard positive and negative control mAbs for use in assay qualification. To address this issue, the National Institute for Biological Standards and Control (NIBSC) developed a reference panel of lyophilised mAbs known to induce CRS in the clinic: human anti-CD52, mouse anti-CD3 and human superagonistic (SA) anti-CD28 mAb manufactured according to the respective published sequences of Campath-1H® (alemtuzumab, IgG1) , Orthoclone OKT-3® (muromonab, IgG2a) and TGN1412 (theralizumab, IgG4), as well as three isotype matched negative controls (human IgG1, mouse IgG2a and human IgG4, respectively). The relative capacity of these control mAbs to stimulate the release of IFN-γ, IL-2, TNF-α and IL-6 in vitro was evaluated in eleven laboratories in an international collaborative study mediated through the HESI Immuno-safety Technical Committee Cytokine Release Assay Working Group. Participants tested the NIBSC mAbs in a variety of CRA platforms established at each institution. This paper presents the results from the centralised cytokine quantification on all the plasma/supernatants corresponding to the stimulation of immune cells in the different CRA platforms by a single concentration of each mAb. Each positive control mAb induced significant cytokine release in most of the tested CRA platforms. There was a high inter-laboratory variability in the levels of cytokines produced, but similar patterns of response were observed across laboratories that replicated the cytokine release patterns previously published for the respective clinical therapeutic mAbs. Therefore, the positive and negative mAbs are suitable as a reference panel for the qualification and validation of CRAs, comparison of different CRA platforms (e.g. solid vs aqueous phase), and intra- and inter-laboratory comparison of CRA performance. Thus, the use of this panel of positive and negative control mAbs will increase the confidence in the robustness of a CRA platform to identify a potential CRS risk for novel immunomodulatory therapeutic candidates.
Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (β-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and β-arrestin endpoints and a specificity of 93% (β-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.
Abstract Our studies have elucidated, in part, the mechanism whereby persistent stimulation by normal enteric antigens leads to the development of chronic enterocolitis in interleukin 10-deficient (IL-10-/-) mice. This disease is mediated by IL-10-/-CD4+ T cells as evidenced by their ability to transfer colitis to immunodeficient RAG-2-/-mice. Furthermore, the CD4+ T cells recovered from the affected colons of IL-10-/- mice consisted of a highly polarized Th1-like population because they produced interferon-γ (IFN-γ) but not IL-4. We found that enterocolitis could be prevented if 3-week-old mutants were treated for 6–8 weeks with either anti-IL-12 or anti-IFN-γ monoclonal antibodies (mAb). These results were consistent with the findings of in vitro studies suggesting that IFN-γ and, in particular, IL-12 direct the differentiation of naive T cells toward a Th1 phenotype. Apparently, the uncontrolled production of IL-12 and IFN-γ by accessory cells and T cells, respectively, in IL-10-/- mice ultimately resulted in the excessive generation and activation of Th1 cells, hence, immunopathology. IL-10-/- mice have also been used to evaluate the importance of IL-10 in regulating immune responses outside of the gastrointestinal (GI) tract. In these studies, IL-10-/-mice were challenged with a variety of foreign antigens using different routes of administration. In general, the results repeatedly demonstrated that a major function of IL-10 is to protect the host from the harmful side effects of an overly zealous immuneinflammatory response. However, other studies have confirmed speculations that the potent immunosuppressive activities of IL-10 may, under certain circumstances, increase the host's susceptibility to infection with certain types of pathogenic organisms.
Regulatory T cells (Tregs) constitute a subset of lymphocytes that have the capability of suppressing immune responses in vivo and in vitro both directly by cell-cell contact and indirectly through the production of anti-inflammatory cytokines, such as interleukin-10 and tumor growth factor-β. Tregs constitute a small subset of T lymphocytes, yet their presence can prevent and control autoimmune disease and organ transplant rejection and contribute to maternal tolerance of fetal alloantigens, whereas their absence results in uncontrolled inflammation. But Treg function may not always be considered beneficial: There is growing evidence that the immunosuppressive effects of Tregs are also associated with growth of tumor cells. Thus, Tregs are of considerable medical interest as targets for the treatment of both inflammatory diseases and cancer. In this review of published literature, we describe some well-characterized immunomodulatory drugs and environmental toxicants that can either positively or negatively affect the number and/or function of Tregs in animal models and/or human patients. The targeted suppression or enhancement of Treg function needs to be carefully considered in immunotoxicity evaluations as manipulation of this immune cell population could result in undesired consequences, including decreased host resistance, decreased fertility, or increased incidence of inflammatory disease.
Abstract IL-4 is associated with Th2-type immune responses and can either inhibit or, in some cases, promote Th1-type responses. We tested the effect of IL-4 treatment on the development of inflammation in the CD4+CD45RBhigh T cell transfer model of colitis, which has been characterized as a Th1-dependent disease. IL-4 treatment significantly accelerated the development of colitis in immunodeficient recipients (recombinase-activating gene-2 (Rag2)−/−) of CD4+CD45RBhigh T cells. Quantitative analysis of mRNA expression in the colons of IL-4-treated mice showed an up-regulation of both Th1- and Th2-associated molecules, including IFN-γ, IP-10, MIG, CXCR3, chemokine receptor-8, and IL-4. However, cotreatment with either IL-10 or anti-IL-12 mAb effectively blocked the development of colitis in the presence of exogenous IL-4. These data indicate that IL-4 treatment exacerbates a Th1-mediated disease rather than induces Th2-mediated inflammation. As other cell types besides T cells express the receptor for IL-4, the proinflammatory effects of IL-4 on host cells in Rag2−/− recipients were assessed. IL-4 treatment was able to moderately exacerbate colitis in Rag2−/− mice that were reconstituted with IL-4Rα-deficient (IL-4Rα−/−) CD4+CD45RBhigh T cells, suggesting that the IL-4 has proinflammatory effects on both non-T and T cells in this model. IL-4 did not cause colitis in Rag2−/− mice in the absence of T cells, but did induce an increase in MHC class II expression in the lamina propria of the colon, which was blocked by cotreatment with IL-10. Together these results indicate that IL-4 can indirectly promote Th1-type inflammation in the CD4+CD45RBhigh T cell transfer model of colitis.
Regulatory T cells (Tregs) are a rare subset of lymphocytes that inhibit the activation and effector functions of T cells and are important regulators of immune responses. Although Tregs are well characterized in humans and rodents, little is known about their immunophenotyping (IP) profile in cynomolgus macaques (Macaca fascicularis), which is an important species for pharmacological and toxicological evaluation of potential immune modulators because of their similar physiologic, genetic, and metabolic response patterns to humans. The authors have developed an immunophenotyping panel using a high-throughput 96-well microtiter plate-based assay to detect circulating Tregs (CD3(+)CD4(+)CD25(hi)FoxP3(+)) and have determined the normal range for the number of Tregs in naive healthy cynomolgus macaques to be 56.4 to 179.7 cells/µL (mean ± SEM = 113.6 ± 5.1 cells/µL; n = 25). Furthermore, the authors compared the resulting FoxP3(+) Treg profiles with a CD127(lo) cell-surface panel (CD3(+)CD4(+)CD25(hi) CD127(lo)) and found a close correlation between the absolute numbers of CD3(+)CD4(+)CD25(hi)FoxP3(+) and CD3(+)CD4(+)CD25(hi)CD127(lo) cells (mean ± SD = 120 ± 8.0 cells/µL). Quantification of circulating Tregs in cynomolgus macaques in this high-throughput assay may help to identify drug candidates that affect this rare, but critical, immunoregulatory cell population.
