ABCB5 Identifies Immunoregulatory Dermal Cells

Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly defined immunomodulatory cell populations poses a barrier to this field. Here, we show that ATP-binding cassette member B5 (ABCB5) identifies dermal immunoregulatory cells (DIRCs) capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1). Purified Abcb5(+) DIRCs suppressed T cell proliferation, evaded immune rejection, homed to recipient immune tissues, and induced Tregs in vivo. In fully major-histocompatibility-complex-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5(+) DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy.

Multiple loci govern the bone marrow-derived immunoregulatory mechanism controlling dominant resistance to autoimmune orchitis.

The existence of immunoregulatory genes conferring dominant resistance to autoimmunity is well documented. In an effort to better understand the nature and mechanisms of action of these genes, we utilized the murine model of autoimmune orchitis as a prototype. When the orchitis-resistant strain DBA/2J is crossed with the orchitis-susceptible strain BALB/cByJ, the F1 hybrid is completely resistant to the disease. By using reciprocal radiation bone marrow chimeras, the functional component mediating this resistance was mapped to the bone marrow-derived compartment. Resistance is not a function of either low-dose irradiation- or cyclophosphamide (20 mg/kg)-sensitive immunoregulatory cells, but can be adoptively transferred by primed splenocytes. Genome exclusion mapping identified three loci controlling the resistant phenotype. Orch3 maps to chromosome 11, whereas Orch4 and Orch5 map to the telomeric and centromeric regions of chromosome 1, respectively. All three genes are linked to a number of immunologically relevant candidate loci. Most significant, however, is the linkage of Orch3 to Idd4 and Orch5 to Idd5, two susceptibility genes which play a role in autoimmune insulin-dependent type 1 diabetes mellitus in the nonobese diabetic mouse.

Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors

Background: Mast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats. Results: Two mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors. Conclusions: In response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.

Étude de l’expression de la molécule d’adhérence CD146 dans les lymphocytes T

Les tumeurs solides sont infiltrées par des cellules immunes (TIIC) dont la nature, la fonction et la composition varient d’un patient à l'autre. Ces cellules inflammatoires influencent l'invasion tumorale en contrôlant la croissance et le potentiel métastatique d’une tumeur. Ainsi, il est proposé d’utiliser cette infiltration comme outil diagnostic et pronostic de routine. Certaines cellules sont bien connues pour jouer un rôle important dans le contrôle de la progression tumorale, comme c’est le cas des lymphocytes T cytotoxiques CD8+ alors que d’autres possèdent un rôle contradictoire. Étant donné la dépendance des tumeurs sur l’équilibre entre ces différentes cellules, il est important d’identifier les fonctions précises des cellules immunes au sein de la tumeur. De nombreuses études sont réalisées afin d’identifier des marqueurs descriptifs du phénotype et la fonction des cellules immunes dans la tumeur. Ce projet de doctorat se divise en deux parties : 1- Identifier la méthode de désagrégation des tissus tumoraux altérant le moins la biologie des TIIC pour leur caractérisation. 2- Caractériser l’expression de la molécule d’adhérence CD146 dans les TIIC et en identifier l’origine. L’identification de marqueurs pour la caractérisation phénotypique et fonctionnelle des TIIC a été réalisée, entre autres, par la détection de protéines exprimées par la cellule. Dans la première partie de ce projet, nous avons démontré que les méthodes utilisées pour désagréger les tissus tumoraux dans le but d’isoler les TIIC induisent des changements dans la biologie de ces cellules ce qui peut fausser les conclusions qui en dérivent. Nous avons donc comparé l'impact de trois méthodes de désagrégation : une dissociation mécanique utilisant la MédimachineTM et deux digestions enzymatiques utilisant une collagénase de type I seule ou combinée à de la collagénase de type IV et de la DNase I de type II. Nous nous sommes intéressés à l'effet de ces méthodes sur des paramètres tels que la viabilité cellulaire, l’altération des protéines de surface et la capacité des cellules à proliférer. Nous avons démontré que ces méthodes affectent la viabilité des cellules de manière comparable, alors que la détection de certaines protéines de surface et la capacité de proliférer est réduite/inhibée par les traitements enzymatiques. Nous concluons qu’une méthode mécanique utilisant la MédimachineTM est mieux adaptée à la caractérisation des TIIC afin de conserver leurs propriétés. Dans la deuxième partie de notre projet, nous avons adapté cette méthode à la caractérisation des TIIC. Nous avons porté une attention particulière à la molécule d’adhérence CD146 dont l’implication dans la migration des cellules immunes à travers l’endothélium vers les sites d’inflammation est de plus en plus étudiée dans les maladies autoimmunes. Nous avons mis en évidence une augmentation des proportions de cellules immunes exprimant CD146 dans les tumeurs comparativement au sang de patients de cancers. Cette expression est induite par les cellules tumorales tout en étant accrue par la nécrose de celles-ci. Nous démontrons que ces cellules sont majoritairement des lymphocytes T CD4+ présentant un profil immunosuppressif. En conclusion, nos résultats suggèrent que CD146 participe à la mise en place du contexte immunitaire dans la tumeur et augmente la capacité de migration des lymphocytes T CD4+. L’induction par les cellules tumorales de cette molécule d’adhérence dans les cellules suppressives pourrait contribuer aux mécanismes immunorégulateurs mis en place par la tumeur. CD146 pourrait être un marqueur d’intérêt pour l’identification des cellules immunosuppressives et pour le développement de nouvelles thérapies.

Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4(+)CD25(+) T Cells by Phage Display

Thymic CD4(+)CD25(+) cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4(+)CD25(+) T regulatory cells we targeted thymic CD4(+)CD25(+) cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4(+)CD25(+) T cells. After four rounds of selection on CD4(+)CD25(+) enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4(+)CD25(+) cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells.

Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than CKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with CKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity. (C) 2009 Elsevier B.V. All rights reserved.

Function of the immunoregulatory CD4-CD8- T cells in the context of autoimmune diabetes

La tolérance immunitaire dépend de la distinction entre le soi et le non soi par le système immunitaire. Un bris dans la tolérance immunitaire mène à l'auto-immunité, qui peut provoquer la destruction des organes, des glandes, des articulations ou du système nerveux central. Le diabète auto-immun, également connu sous le nom diabète juvénile et diabète de type 1, résulte d'une attaque auto-immune sur les cellules β pancréatiques sécrétrices d’insuline, localisées au niveau des îlots de Langerhans du pancréas. Bien que le diabète auto-immun soit traitable par une combinaison d’injections quotidiennes d’insuline d’origine exogène, de régime et d'exercices, beaucoup de complications chroniques peuvent se manifester chez les patients, y compris, mais non limitées à, la cécité, les maladies cardiovasculaires, l’insuffisance rénale et l'amputation. En raison des nombreuses complications liées au diabète auto-immun à long terme, la recherche continue afin de mieux comprendre tous les facteurs impliqués dans la progression de la maladie dans le but de développer de nouvelles thérapies qui empêcheront, renverseront et/ou traiteront cette maladie. Un rôle primordial dans la génération et l'entretien de la tolérance immunitaire a été attribué au nombre et à la fonction des sous-populations de cellules régulatrices. Une de ces populations est constituée de cellules T CD4-CD8- (double négatives, DN), qui ont été étudiées chez la souris et l'humain pour leur contribution à la tolérance périphérique, à la prévention des maladies et pour leur potentiel associé à la thérapie cellulaire. En effet, les cellules de T DN sont d'intérêt thérapeutique parce qu'elles montrent un potentiel immunorégulateur antigène-spécifique dans divers cadres expérimentaux, y compris la prévention du diabète auto-immun. D’ailleurs, en utilisant un système transgénique, nous avons démontré que les souris prédisposées au diabète auto-immun présentent peu de cellules T DN, et que ce phénotype contribue à la susceptibilité au diabète auto-immun. En outre, un transfert des cellules T DN est suffisant pour empêcher la progression vers le diabète chez les souris prédisposées au diabète auto-immun. Ces résultats suggèrent que les cellules T DN puissent présenter un intérêt thérapeutique pour les patients diabétiques. Cependant, nous devons d'abord valider ces résultats en utilisant un modèle non-transgénique, qui est plus physiologiquement comparable à l'humain. L'objectif principal de cette thèse est de définir la fonction immunorégulatrice des cellules T DN, ainsi que le potentiel thérapeutique de celles-ci dans la prévention du diabète auto-immun chez un modèle non-transgénique. Dans cette thèse, on démontre que les souris résistantes au diabète auto-immun présentent une proportion et nombre absolu plus élevés de cellules T DN non-transgéniques, lorsque comparées aux souris susceptibles. Cela confirme une association entre le faible nombre de cellules T DN et la susceptibilité à la maladie. On observe que les cellules T DN éliminent les cellules B activées in vitro par une voie dépendante de la voie perforine et granzyme, où la fonction des cellules T DN est équivalente entre les souris résistantes et prédisposées au diabète auto-immun. Ces résultats confirment que l'association au diabète auto-immun est due à une insuffisance en terme du nombre de cellules T DN, plutôt qu’à une déficience fonctionnelle. On démontre que les cellules T DN non-transgéniques éliminent des cellules B chargées avec des antigènes d'îlots, mais pas des cellules B chargées avec un antigène non reconnu, in vitro. Par ailleurs, on établit que le transfert des cellules T DN activées peut empêcher le développement du diabète auto-immun dans un modèle de souris non-transgénique. De plus, nous observons que les cellules T DN migrent aux îlots pancréatiques, et subissent une activation et une prolifération préférentielles au niveau des ganglions pancréatiques. D'ailleurs, le transfert des cellules T DN entraîne une diminution d'auto-anticorps spécifiques de l'insuline et de cellules B de centres germinatifs directement dans les îlots, ce qui corrèle avec les résultats décrits ci-dessus. Les résultats présentés dans cette thèse permettent de démontrer la fonction des cellules T DN in vitro et in vivo, ainsi que leur potentiel lié à la thérapie cellulaire pour le diabète auto-immun.

Effects of oxidised low density lipoprotein on dendritic cells: a possible immunoregulatory component of the atherogenic micro-environment?

Objective: The objective of this study was to explore the relationship between low density lipoprotein (LDL) and dendritic cell (DC) activation, based upon the hypothesis that reactive oxygen species (ROS)-mediated modification of proteins that may be present in local DC microenvironments could be important as mediators of this activation. Although LDL are known to be oxidised in vivo, and taken up by macrophages during atherogenesis; their effect on DC has not been explored previously. Methods: Human DCs were prepared from peripheral blood monocytes using GM-CSF and IL-4. Plasma LDLs were isolated by sequential gradient centrifugation, oxidised in CuSO4, and oxidation arrested to yield mild, moderate and highly oxidised LDL forms. DCs exposed to these LDLs were investigated using combined phenotypic, functional (autologous T cell activation), morphological and viability assays. Results: Highly-oxidised LDL increased DC HLA-DR, CD40 and CD86 expression, corroborated by increased DC-induced T cell proliferation. Both native and oxidised LDL induced prominent DC clustering. However, high concentrations of highly-oxidised LDL inhibited DC function, due to increased DC apoptosis. Conclusions: This study supports the hypothesis that oxidised LDL are capable of triggering the transition from sentinel to messenger DC. Furthermore, the DC clustering–activation–apoptosis sequence in the presence of different LDL forms is consistent with a regulatory DC role in immunopathogenesis of atheroma. A sequence of initial accumulation of DC, increasing LDL oxidation, and DC-induced T cell activation, may explain why local breach of tolerance can occur. Above a threshold level, however, supervening DC apoptosis limits this, contributing instead to the central plaque core.

Colon cancer cells produce immunoregulatory glucocorticoids

Glucocorticoids (GC) have important anti-inflammatory and pro-apoptotic activities. Initially thought to be exclusively produced by the adrenal glands, there is now increasing evidence for extra-adrenal sources of GCs. We have previously shown that the intestinal epithelium produces immunoregulatory GCs and that intestinal steroidogenesis is regulated by the nuclear receptor liver receptor homolog-1 (LRH-1). As LRH-1 has been implicated in the development of colon cancer, we here investigated whether LRH-1 regulates GC synthesis in colorectal tumors and whether tumor-produced GCs suppress T-cell activation. Colorectal cancer cell lines and primary tumors were found to express steroidogenic enzymes and regulatory factors required for the de novo synthesis of cortisol. Both cell lines and primary tumors constitutively produced readily detectable levels of cortisol, as measured by radioimmunoassay, thin-layer chromatography and bioassay. Whereas overexpression of LRH-1 significantly increased the expression of steroidogenic enzymes and the synthesis of cortisol, downregulation or inhibition of LRH-1 effectively suppressed these processes, indicating an important role of LRH-1 in colorectal tumor GC synthesis. An immunoregulatory role of tumor-derived GCs could be further confirmed by demonstrating a suppression of T-cell activation. This study describes for the first time cortisol synthesis in a non-endocrine tumor in humans, and suggests that the synthesis of bioactive GCs in colon cancer cells may account as a novel mechanism of tumor immune escape.

Colon cancer cells produce immunoregulatory glucocorticoids

Expression or release of immunosuppressive molecules may protect tumor cells from the recognition and destruction by the immune system. New findings indicate that colorectal tumors produce immunoregulatory glucocorticoids and thereby suppress immune cell activation. The nuclear receptor LRH-1 plays a critical role in the regulation of colorectal tumor proliferation and glucocorticoid synthesis.

Resident tissue macrophages within the normal rat iris lack immunosuppressive activity and are effective antigen-presenting cells

Despite extensive study of the numerous immunoregulatory mechanisms that contribute to the immune-privileged nature of the anterior chamber (AC) of the eye, little is known of the functional nature of antigen-presenting cells (APC) present in the tissues adjoining the AC. In the present study, we have compared the antigen-presenting capacity of dendritic cells (DC) and macrophages isolated from the normal rat iris. Whereas iris DC exhibited a potent ability to stimulate resting allogeneic T cells in MLR cultures (an in-vitro correlate of the ability to induce primary T cell responses), resident iris macrophages displayed negligible MLR-stimulatory capacity. Significantly, iris macrophages could efficiently elicit proliferation of primed antigen-specific T cells (an in-vitro correlate of the ability to act as local APC in secondary responses). This antigen-presenting activity was approximately half that of fully mature iris DC and considerably greater than that of freshly isolated iris DC. A key contributor to the effectiveness of resident iris macrophage antigen presentation was considered to be the absence of lymphocytostatic control of T cell proliferation exerted by these cells. The results indicate dichotomous but complementary roles for DC (immune surveillance) and macrophages (local antigen presentation in secondary responses) in this tissue.

Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Lower numbers of circulating natural killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV-1) associated neurological disease

Human T lymphotropic virus type 1 (HTLV-1) infects 10-20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV-1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: Sao Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4(+) and fewer CD8(+) cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4(+) NK T subset are associated with HTLV-1 disease progression.

Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment

Oral squamous cell carcinoma (OSCC) is a cancerous lesion with high incidence worldwide. The immunoregulatory events leading to OSCC persistence remain to be elucidated. Our hypothesis is that regulatory T cells (Tregs) are important to obstruct antitumor immune responses in patients with OSCC. In the present study, we investigated the frequency, phenotype, and activity of Tregs from blood and lesions of patients with OSCC. Our data showed that > 80% of CD4(+)CD25(+) T cells isolated from PBMC and tumor sites express FoxP3. Also, these cells express surface Treg markers, such as GITR, CD45RO, CD69, LAP, CTLA-4, CCR4, and IL-10. Purified CD4(+)CD25(+) T cells exhibited stronger suppressive activity inhibiting allogeneic T-cell proliferation and IFN-gamma production when compared with CD4(+)CD25(+) T cells isolated from healthy individuals. Interestingly, approximately 25% of CD4(+)CD25(-) T cells of PBMC from patients also expressed FoxP3 and, although these cells weakly suppress allogeneic T cells proliferative response, they inhibited IFN-gamma and induced IL-10 and TGF-beta secretion in these co-cultures. Thus, our data show that Treg cells are present in OSCC lesions and PBMC, and these cells appear to suppress immune responses both systemically and in the tumor microenvironment.