Photopheresis efficacy in the treatment of rheumatoid arthritis: a pre-clinical proof of concept.

BACKGROUND: Despite major advances in rheumatoid arthritis outcome, not all patients achieve remission, and there is still an unmet need for new therapeutic approaches. This study aimed at evaluating in a pre-clinical murine model the efficacy of extracorporeal photopheresis (ECP) in the treatment of rheumatoid arthritis, and to provide a relevant study model for dissecting ECP mechanism of action in autoimmune diseases. METHODS: DBA/1 mice were immunized by subcutaneous injection of bovine collagen type II, in order to initiate the development of collagen-induced arthritis (CIA). Arthritic mice received 3 ECP treatments every other day, with psoralen + UVA-treated (PUVA) spleen cells obtained from arthritic mice. Arthritis score was measured, and immune cell subsets were monitored. RESULTS: ECP-treated mice recovered from arthritis as evidenced by a decreasing arthritic score over time. Significant decrease in the frequency of Th17 cells in the spleen of treated mice was observed. Interestingly, while PUVA-treated spleen cells from healthy mouse had no effect, PUVA-treated arthritic mouse derived-spleen cells were able to induce control of arthritis development. CONCLUSIONS: Our results demonstrate that ECP can control arthritis in CIA-mice, and clarifies ECP mechanisms of action, showing ECP efficacy and Th17 decrease only when arthritogenic T cells are contained within the treated sample. These data represent a pre-clinical proof of concept supporting the use of ECP in the treatment of RA in Human.

In vitro PUVA treatment triggers calreticulin exposition and HMGB1 release by dying T lymphocytes in GVHD: New insights in extracorporeal photopheresis.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective therapy for graft vs host disease (GVHD), based on infusion of UVA-irradiated and 8 methoxy-psoralen (PUVA)-treated leukocytes. Reinfusion of these apoptosing cells affects the functionality of pathogenic T cells through poorly understood immunomodulatory mechanisms. Apoptosis is usually a silent, tolerance-associated process, but can also be immunogenic, depending on death-inducers and environmental context. METHODS: To understand ECP mechanisms of action, human alloreactive T cells generated in an in vitro model mimicking GVHD were used, as well as primary cells from GVHD patients. Cells were submitted to PUVA treatment and their phenotype and immunogenicity were analyzed, using cell culture and flow cytometry. RESULTS: In vitro PUVA treatment induced the expression of several damage-associated molecular patterns (DAMPs) by dying T cells (calreticulin, high-mobility group box-1, and to a lesser extent heat shock proteins 70 and 90), especially upon T cell activation, leading to their phagocytosis by macrophages and dendritic cells (DCs). Allogeneic DCs preincubated with PUVA treated T cells induced comparable naive T cell proliferation and polarization as control allogeneic DC. CONCLUSION: Altogether, in our experimental settings, in vitro PUVA-treatment induces a partially immunogenic phenotype allowing phagocytosis of apoptotic cells by macrophages and DC, however not sufficient to induce dendritic cell maturation and T cell activation. These data refine current models of ECP-mediated immune modulation and emphasize the need to further analyze PUVA-treated cell interactions with immune cells.

Photochemotherapy induces the apoptosis of monocytes without impairing their function.

BACKGROUND: Extracorporeal photopheresis (ECP) is a powerful therapy currently used to treat various hematological disorders as in graft versus host disease. Clinical data clearly demonstrate its efficacy and immunomodulation toward the pathogenic T cells. However, ECP mechanism of action is still poorly understood. Monocytes represent up to 30% of the total amount of treated cells and are known to play an important role in adaptive immunity. However, data from previous reports analyzing the effect of psoralen and UV-A irradiation (PUVA) on their functions are heterogeneous. In this study, we focused on the effect of PUVA on human monocytes functions in adaptive immunity. DESIGN AND METHODS: Purified human monocytes were treated in vitro by PUVA. We measured their kinetic of apoptosis after the treatment. We also determine whether their phenotype and functionalities were modified. Finally, we assessed the functionalities of PUVA-treated monocytes-derived dendritic cells (DC). RESULTS: PUVA treatment sentenced purified monocytes to die in 6 days and immediately altered their migratory capacities without impairing their ability of endocytosis. It also up-regulated co-stimulatory molecules and production of inflammatory cytokines on activation and consequently stimulated allogeneic or autologous T cells as efficiently as untreated monocytes. Moreover, PUVA-treated monocytes retained their ability to differentiate into fully functional DC that maturated and stimulated T cells as well as normal DC. CONCLUSIONS: Our data demonstrate that monocytes undergo apoptosis and loose a part of their migratory capacity after ECP and the surviving cell functionalities are not impaired, suggesting that monocytes have a minor effect on ECP-mediated immunomodulation.

Photochemotherapy induces a faster apoptosis of alloreactive activated T cells than of nonalloreactive resting T cells in graft versus host disease.

BACKGROUND: Graft versus host disease (GvHD) is the main complication after hematopoietic stem-cell transplantation.Extracorporeal photochemotherapy (ECP) is a cell therapy currently used for the treatment of T-cell­mediated diseases and seems as a valuable second-line therapy for patients suffering from steroid-refractory acute or chronic GvHD. ECP induces the apoptosis of treated cells and is believed to elicit a specific immune regulation of alloreactive T cells through repeated apoptotic T-cell infusions. However, its mechanisms of action have not yet been elucidated. In GvHD,alloreactive but not non alloreactive T cells are continuously activated by their environment. We hypothesized that ECP has a differential apoptotic effect on activated compared with resting T cells. METHOD: The ECP-induced apoptosis of resting and activated T cells from patients with chronic GvHD was assessed.The kinetic of apoptosis was also evaluated using several triggers of T-cell activation such as mitogenic or antigen specific activation. The influence of survival cytokines (interleukin-2, -7, and -15) was also evaluated. RESULTS: Activated T cells from patients with chronic GvHD underwent apoptosis faster than resting T cells. This phenomenon was confirmed using mitogenic and antigen-specific activated T cells from healthy donors and cannot be delayed by protective cytokines. CONCLUSIONS: ECP induces a faster apoptosis of alloreactive activated T cells than of non alloreactive resting T cells in GvHD and more generally of activated T cells than of resting T cells. These novel findings provide new insights about the ECP-induced specific control of pathogenic T cells in GvHD.

C3b complexation diversifies naturally processed T cell epitopes.

In addition to its well-established role in innate immunity, the complement component C3 is of critical importance in modulating the humoral response. In this study, we examined the effect of C3b linkage to tetanus toxin (TeNT) in the production of antigenic peptides inside human APC. We purified HLA-DR associated peptides isolated either from TeNT or TeNT-C3b pulsed cells. This study revealed that TeNT-C3b derived antigenic peptides are different and more numerous than TeNT derived peptides. This increased production of antigenic peptides correlated with a C3b-induced TeNT modification of proteolysis. These findings argue in favour of a new role for C3b in the modulation of T cell epitope during antigen processing and presentation.

Increased proteolysis of diphtheria toxin by human monocytes after heat shock: a subsidiary role for heat-shock protein 70 in antigen processing.

The expression of heat-shock proteins (hsp) increases after exposure to various stresses including elevated temperatures, oxidative injury, infection and inflammation. As molecular chaperones, hsp have been shown to participate in antigen processing and presentation, in part through increasing the stability and expression of major histocompatibility complex molecules. Heat shock selectively increases human T-cell responses to processed antigen, but does not affect T-cell proliferation induced by non-processed antigens. Here, we have analysed the mechanisms by which stress such as heat shock, and the ensuing hsp over-expression affect the processing of diphtheria toxin (DT) in peripheral blood monocytes. We found that heat shock increased DT proteolysis in endosomes and lysosomes while the activities of the cathepsins B and D, classically involved in DT proteolysis, were decreased. These effects correlated with the heat-shock-mediated increase in hsp 70 expression observed in endosomes and lysosomes. Actinomycin D or blocking anti-hsp 70 antibodies abolished the heat-shock-mediated increase in DT proteolysis. These data indicate that the increased expression of hsp 70 constitutes a subsidiary mechanism that facilitates antigen proteolysis in stressed cells. Confirming these data, presentation by formaldehyde-fixed cells of DT proteolysates that were obtained with endosomes and lysosomes from heat-shocked peripheral blood monocytes showed higher stimulation of T cells than those generated with endosomes and lysosomes from control peripheral blood monocytes.

B cell receptors and complement receptors target the antigen to distinct intracellular compartments.

The processing of exogenous Ags is an essential step for the generation of immunogenic peptides that will be presented to T cells. This processing relies on the efficient intracellular targeting of Ags, because it depends on the content of the compartments in which Ags are delivered in APCs. Opsonization of Ags by the complement component C3 strongly enhances their presentation by B cells and increases their immunogenicity in vivo. To investigate the role of C3 in the targeting of Ags, we compared the intracellular traffic of proteins internalized by complement receptor (CR) and B cell receptor (BCR) in B lymphocytes. Whereas both receptors are able to induce efficient Ag presentation, their intracellular pathways are different. CR ligand is delivered to compartments containing MHC class II molecules (MHC-II) but devoid of transferrin receptor and Lamp-2, whereas BCR rapidly targets its ligand toward Lamp-2-positive, late endosomal MHC-II-enriched compartments through intracellular vesicles containing transferrin receptor. CR and BCR are delivered to distinct endocytic pathways, and the kinetic evolution of the protein content of these pathways is very different. Both types of compartments contain MHC-II, but CR-targeted compartments receive less neosynthesized MHC-II than do BCR-targeted compartments. The targeting induced by CR toward compartments that are distinct from BCR-targeted compartments probably participates in C3 modulation of Ag presentation.