Experimental extracorporeal photopheresis inhibits the sensitization and effector phases of contact hypersensitivity via two mechanisms: generation of IL-10 and induction of regulatory T cells.

Extracorporeal photopheresis (ECP) is used to treat immune-mediated diseases including transplant rejection and graft-vs-host-disease. An experimental murine model of ECP utilizing contact hypersensitivity (CHS) revealed that ECP inhibits the sensitization of CHS and induces regulatory T cells (Treg). In this study, we find that ECP inhibits not only the sensitization but also the effector phase of CHS, although Treg only inhibited sensitization. IL-10 was determined to be a critical component of the effector phase inhibition and also a driving force in developing Treg. Thus, we propose that the inhibition of the effector phase of CHS by ECP is a process that does not require Treg but may be mediated via enhanced IL-10 as suggested by the use of IL-10-deficient mice. This suggests that ECP has at least two mechanisms of action, one inhibiting the effector phase of CHS and one generating Treg, which in turn can inhibit CHS sensitization and is responsible for the transferable protection. Together, this may help explain the clinical benefits of ECP in prophylactic, acute, and therapeutic settings.

Inhibition of cytolytic T lymphocyte proliferation by autologous CD4+/CD25+ regulatory T cells in a colorectal carcinoma patient is mediated by transforming growth factor-beta.

Cancer patients often develop CTLs that lyse autologous tumor cells in culture. However, tumors can progress in vivo despite the presence of CTLs. Various mechanisms have been reported to down-modulate CTL functions. In this study, the role of CD4+/CD25+ regulatory T cells in CTL induction and proliferation of established CTLs was investigated in a patient with CRC. CD4+ cytotoxic and regulatory T-cell lines were derived from the peripheral blood mononuclear cells of the same patient in mixed-lymphocyte tumor culture. The cytotoxic T-cell line and a clonal derivative specifically lysed the autologous tumor cells but not the B lymphocytes. Only HLA-A1-matched allogeneic CRC cells were lysed by the CTL clone. The clone produced IFN-gamma and TNF-alpha. The regulatory CD4+/CD25+ T-cell line was tumor cell-dependent in its growth but did not lyse autologous tumor cells. This T-cell line suppressed pokeweed mitogen responses of allogeneic lymphocytes, proliferative activity of the established, autologous CTLs, and induction of CTLs in autologous, freshly isolated peripheral blood mononuclear cells. The immunosuppressive effect of the CD4+/CD25+ regulatory T cells was mediated by transforming growth factor-beta and did not require cell-to-cell contact. Thus, although CRC patients can develop specific CTLs against their tumors, the development of regulatory T cells may allow the escape of tumor cells from immune surveillance by the CTLs in vivo.

The Role of Protein Excipient in Driving Antibody Responses to Erythropoietin.

Human serum albumin (HSA) is an excipient present in formulations of several recombinant protein products that are approved for clinical use. We investigated the relative contributions of HSA and HSA particles to the generation of antibody responses against recombinant human erythropoietin (rhEPO) and the excipient HSA itself. Protein samples were characterized before injection for quantities of monomeric proteins, soluble protein aggregates, and nano- and micron-sized particles. rhEPO, containing various concentrations of HSA particles, were injected three times a week for 8 weeks into mice. Hematocrits and the production of anti-rhEPO and anti-HSA antibodies were determined at various time points. Levels of antibodies against rhEPO in mice injected with HSA-containing rhEPO were higher than those in mice treated with HSA-free rhEPO. Mice injected with formulations that contained particles of HSA produced strong anti-HSA antibody responses; whereas these responses were greatly reduced when particle-free formulations were administered. In contrast, anti-rhEPO antibody responses were not affected by the presence of particles.

A novel monoclonal antibody screening method using the Luminex-100 microsphere system.

We describe the development of a robust and sensitive assay system (detection limit <500 pg/ml biotin-IL-6, K(d)=75 ng/ml), using Luminex-100 microspheres, that could effectively screen for neutralizing antibody whenever a soluble form of the receptor for a target molecule is available. As an example, we coupled a recombinant human interleukin-6 soluble receptor to a Luminex carboxylated microsphere and used a biotin-labeled recombinant human interleukin-6 as a probe to assess binding competition. Three anti-human IL-6 monoclonal antibodies that bind distinct IL-6 epitopes were used as test articles to evince the stringency of the screen. Our assay was able to detect antibody concentration as low as 10 ng/ml without interference from hybridoma growth medium or cell supernatant. The time-saving benefits of this assay format make it ideal for high-throughput screening (HTS) applications for neutralizing monoclonal antibodies.

Discovery and mechanism of ustekinumab: a human monoclonal antibody targeting interleukin-12 and interleukin-23 for treatment of immune-mediated disorders.

Monoclonal antibody (mAb) therapy was first established upon the approval of a mouse antibody for treatment of human acute organ rejection. However, the high incidence of immune response against the mouse mAb restricted therapeutic utility. Development of chimeric, "humanized" and human mAbs broadened therapeutic application to immune-mediated diseases requiring long-term treatment. Indeed, mAb therapeutics targeting soluble cytokines are highly effective in numerous immune-mediated disorders. A recent example is ustekinumab, a first-in-class therapeutic human immunoglobulin G1 kappa mAb that binds to the interleukins (IL)-12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th17 cell subsets. Ustekinumab was generated via recombinant human IL-12 immunization of human immunoglobulin (hu-Ig) transgenic mice. Ustekinumab binds to the p40 subunit common to IL-12 and IL-23 and prevents their interaction with the IL-12 receptor ß1 subunit of the IL-12 and IL-23 receptor complexes. Ustekinumab is approved for treatment of moderate-to-severe plaque psoriasis and has demonstrated efficacy in Crohn disease and psoriatic arthritis. The clinical characterization of ustekinumab continues to clarify our understanding of human immune pathologies and may offer a novel therapeutic option for certain immune-mediated diseases.

Potential mechanisms of photopheresis in hematopoietic stem cell transplantation.

Immune tolerance describes specific unresponsiveness to antigens. In clinical situations such as graft-versus-host disease it may be useful to capitalize on these pre-existing tolerance mechanisms to treat patients. Extracorporeal photopheresis is a pheresis treatment whereby the approximately 5 x 10(9) leukocytes are treated with a photoactivatable compound (8-methoxypsoralen) and UVA light, and immediately returned to the patient in a closed-loop, patient-connected system. This therapy induces apoptosis of virtually all the treated leukocytes. There is growing evidence that infusion of apoptotic cells may trigger certain tolerance mechanisms and, thus, be of therapeutic use in graft-versus-host disease. These apoptotic cells are taken up by phagocytes (antigen-presenting cells) in the body of the patient. Apoptotic cell engagement has been reported to induce several changes and functional activities in the engulfing antigen-presenting cell. These antigen-presenting cells: (1) decrease production of proinflammatory cytokines; (2) increase production of anti-inflammatory cytokines; (3) lower ability to stimulate T-cell responses; (4) delete CD8 T effector cells; and (5) induce regulatory T cells. Any and all of these mechanisms could explain the noted effect in graft-versus-host disease. It is still unclear which one or ones are truly responsible. Ongoing studies in animals and human trials will ultimately unravel these details.

Intravenous infusion of syngeneic apoptotic cells by photopheresis induces antigen-specific regulatory T cells.

The basis of extracorporeal photopheresis is the reinfusion of leukocytes previously exposed to 8-methoxypsoralen (8-MOP) and UVA radiation. It has been approved for the palliative treatment of cutaneous T cell lymphoma and has reported benefits in autoimmune diseases, transplant rejection, and graft-vs-host disease. However, the underlying mechanism of photopheresis remains unresolved. Because UVB radiation can cause immune tolerance via induction of regulatory T cells, we studied whether photopheresis exerts a similar effect extracorporeally. Therefore, we established a model of photopheresis using a murine model of contact hypersensitivity. Splenocytes and lymph node cells of mice that were sensitized with dinitrofluorobenzene were exposed to 8-MOP plus UVA in vitro. Intravenous injection of these cells into naive mice caused inhibition of a hapten immune response, which was lost upon depletion of CD11c(+) cells but not T cells. Mice that received untreated cells or cells exposed to UVA or 8-MOP alone were not affected. Inhibition was cell-mediated and Ag-specific as demonstrated by transfer of tolerance from the primary recipients into naive animals, which could, however, properly respond to the unrelated hapten oxazolone. Transfer activity was lost when cells were depleted of CD4(+) or CD25(+) subpopulations. These data suggest that photopheresis exerts its immunomodulatory effects via the induction of Ag-specific regulatory T cells.

Prevention of experimental autoimmune encephalomyelitis in common marmosets using an anti-IL-12p40 monoclonal antibody.

The experimental autoimmune encephalomyelitis (EAE) model in the common marmoset approximates recognized features of the human disease multiple sclerosis (MS) with regard to its clinical presentation as well as neuropathological and radiological aspects of the lesions in brain and spinal cord. IL-12 is a proinflammatory cytokine that is produced by APC and promotes differentiation of Th1 effector cells. IL-12 is produced in the developing lesions of patients with MS as well as in EAE-affected animals. Previously it was shown that interference in IL-12 pathways effectively prevents EAE in rodents. In this study we report that in vivo neutralization of IL-12p40 using a novel Ab has beneficial effects in the myelin-induced EAE model in common marmosets. The Ab was injected i.v. at 7-day intervals starting well after immunization (day 14) and was continued until the end of the study (day 86). Stable levels of the Ab were measured 3 days after each injection throughout the study period. During this period anti-Ab responses could not be detected. We demonstrate that anti-IL-12p40 treatment has a protective effect on the neurological dysfunction as well as on neuropathological changes normally observed in the brain and spinal cord of EAE-affected individuals.

Induction of dendritic cell maturation by IL-18.

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18. However, the human myelomonocytic cell line KG-1 and monocyte-derived dendritic cells (generated by GM-CSF+IL-4) showed a marked increase in CD83, HLA-DR, and several costimulatory molecules upon stimulation with IL-18. Furthermore, IL-18 decreased pinocytosis of these cells and increased their ability to stimulate alloreactive T cell proliferation, all characteristics of mature dendritic cells. These results suggest that IL-18 is involved in the maturation of myeloid DCs, but not differentiation of monocytes into DCs. The finding that IL-18 is involved in the maturation of dendritic cells is both novel and unexpected and indicates another important role for IL-18 as a key regulator of immune responses.