Fusion and biochemical expression of membrane receptors in foreign living cells

Successful transplantation of cell surface molecules from the membranes of one cell type to recipient cells of a different type is described. Plasma membranes purified from donor cells were fluoresceinated and fused to recipient cells using poly(ethylene glycol) and the fate of the transplanted membrane components was followed by fluorescence microscopy. In approximately 100 min the 'foreign' membrane components were seen to cluster and internalise. During this time, judged by the criteria of hormonal stimulation and immune cytotoxic killing, the cell surface of the recipient cell mimicked the cell surface phenotype of the donor cell.     

Antigen presentation

This paper reviews some of the cellular events involved in the immune recognition of foreign proteins. The recognition of an antigen by T lymphocytes is essential for its effective elimination by the host. T lymphocytes of the CD4 or CD8 subset recognize antigen but only after the antigen is handled by antigen-handling cells (antigen-presenting cells). Antigen molecules are recognized after an internal processing event by antigen-presenting cells that results in the generation of immunogenic peptides. Such peptides associate with histocompatibility molecules to form bimolecular complexes on the cell surface. The T cell receptors for antigen recognize the bimolecular complex and initiate the events that result in an inflammatory response. Antigen-presenting cells also produce molecules - termed costimulators - that stimulate the growth and differentiation of T lymphocytes.     

Modulation of Thy-1 alloantibody responses: donor cell-associated H-2 inhibition and augmentation without recipient Ir gene control

In this study we have found that in vivo PFC responses to Thy-1 are strongly modulated by H-2 gene products in at least 2 ways. First, a profound inhibition of primary PFC responses occurs when foreign H-2 antigens are expressed on Thy-1 incompatible donor cells. This interference effect does not reflect a requirement for H-2-restricted antigen presentation by donor cells, since it is also seen using semi-syngeneic antigenic cells that share a full H-2 haplotype with the recipient. Interference acts more profoundly on slower primary responses than on more rapid secondary responses and requires associative recognition of the H-2 and Thy-1 antigens. By contrast, strong augmentation of the anti-Thy-1 response was obtained when foreign H-2 antigens were expressed in the recipient, as shown by a poor response of an H-2k/k recipient to Thy-1.1 on AKR cells (H-2k) compared with high responses of H-2k/b recipients. A gene controlling this phenotype was mapped to the H-2IA or H-2K regions. However, subsequent experiments revealed that donor recognition of recipient H-2 antigens was required for these high responses; thus, an Ir-gene effect was excluded and an 'intimate form' of an allogeneic effect was postulated. Thus, the immune response to Thy-1 is regulated by at least 3 factors acting at the level of the donor cell, including non-H-2 helper alloantigens, H-2 interference, and H-2-associated allogeneic effects.     

Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD)

Haematopoietic stem cell transplantation (HSCT) offers promise for the treatment of haematological and immune disorders, solid tumours, and as a tolerance inducing regimen for organ transplantation. Allogeneic HSCTs engraftment requires immunosuppression and the anti-tumour effects are dependent upon the immune effector cells that are contained within or generated from the donor graft. However, significant toxicities currently limit its efficacy. These problems include: (i) graft-versus-host disease (GVHD) in which donor T cells attack the recipient resulting in multi-organ attack and morbidity, (ii) a profound period of immune deficiency following HSCT, and (iii) donor graft rejection. Currently available methods to prevent or treat GVHD with systemic immunosuppression can lead to impaired immune recovery, increased opportunistic infections, and higher relapse rates. This review will provide an overview of GVHD pathophysiology and discuss the roles of various cells, pathways, and factors in the GVHD generation process and in the preservation of graft-versus-tumour effects. Variables that need to be taken into consideration in attempting to extrapolate preclinical results to the clinical paradigm will be highlighted.     

New concepts of complement in allorecognition and graft rejection

In transplantation, activation of complement has largely been equated to antibody-mediated rejection, but complement is also important in recognition of apoptotic and necrotic cells as well as in modifying antigen presentation to T cells and B cells. As a part of the innate immune system, complement is one of the first responses to injury, and it can determine the direction and magnitude of the subsequent responses. Consequently, the effects of complement in allorecognition and graft rejection are increased when organs are procured from cadaver donors because these organs sustain a series of stresses from brain death, prolonged life support, ischemia and finally reperfusion that initiate proinflammatory processes and tissue injury. In addition, these organs are transplanted to patients, who frequently have been sensitized to histocompatibility antigens as the result of transfusions, pregnancies or transplants.Complement activation generates a series of biologically active effector molecules that can modulate graft rejection by directly binding to the graft or by modifying the response of macrophages, T and B cells of the recipient. However, complement is regulated and the process of regulation produces split products that can decrease as well as increase immune responses. Small animal models have been developed to test these variables. The guide for evaluating results from these models remains clinical findings because there are significant differences between the rodent and human complement systems.     

Biological aspects of limb transplantation: I. Migration of transplanted bone marrow cells into recipient

The transplanted limb contains bone marrow tissue. The hematopoietic cells contained in the bone of the graft normally differentiate after transplantation and can be released to the recipient. The cells migrate to the recipient bone marrow cavities and lymphoid organs. This causes the immune reaction between the donor and the recipient, which develops not only in the graft itself but also in the recipient immune organs where donor bone marrow cells home. The purpose of this study was to investigate the process of migration of the hematopoietic cells from the donor limb to the recipient bone marrow cavities and lymphoid tissues. The questions the authors asked were: what is the rate of release of bone marrow cells from the transplanted bone, where do the released bone marrow cells home in the recipient, how fast are donor bone marrow cells rejected by the recipient, and can some bone marrow cells homing in the recipient tissues survive and create a state of microchimerism. Experiments were performed on Brown Norway and Lewis inbred rat strains (n = 30). Limb donors received intravenous chromium-51-labeled bone marrow cells. Twenty-four hours later, the limb with homing labeled bone marrow cells was transplanted to an allogeneic or syngeneic recipient. The rate of radioactivity of bone marrow cells released from the graft and homing in recipient tissues was measured after another 24 hours. To eliminate factors adversely affecting homing such as the "crowding effect" and allogeneic elimination of bone marrow cells by natural killer cells, total body irradiation and antiasialo-GM1 antiserum were applied to recipients before limb transplantation. In rats surviving with the limb grafts for 7 and 30 days, homing of donor bone marrow cells was studied by specific labeling of donor cells and flow cytometry as well as by detecting donor male Y chromosome. The authors found that transplantation of the limb with bone marrow in its natural spatial relationship with stromal cells and blood perfusion brings about immediate but low-rate release of bone marrow cells and their migration to recipient bone marrow and lymphoid tissues. Large portions of allogeneic bone marrow cells are rapidly destroyed in the mechanism of allogeneic elimination by radioresistant but antiasialo-GM1-sensitive natural killer cells. Some transplanted bone marrow cells remain in the recipient's tissues and create a state of cellular and DNA microchimerism. A low number of physiologically released donor bone marrow cells do not seem to adversely affect the clinical outcome of limb grafting. Quite the opposite, a slight prolongation of the graft survival time was observed.     

Natural killer cell alloreactivity and haplo-identical hematopoietic transplantation

In haplo-identical hematopoietic transplantation, donor vs. recipient natural killer (NK) cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory killer cell Ig-like receptors (KIR) for self-HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. When faced with mismatched allogeneic targets, these NK clones sense the missing expression of self-HLA class I alleles and mediate alloreactions. KIR ligand mismatches in the GvH direction trigger donor vs. recipient NK cell alloreactions, which improve engraftment, do not cause GvHD and control relapse in AML patients . The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated in mouse models. The infusion of alloreactive NK cells ablates (i) leukemic cells, (ii) recipient T cells that reject the graft and (iii) recipient DC that trigger GvHD, thus protecting from GvHD.     

The evolution of vertebrate antigen receptors: a phylogenetic approach

Classical T cells, those with alpha beta T-cell receptors (TCRs), are an important component of the dominant paradigm for self-nonself immune recognition in vertebrates. alpha beta T cells recognize foreign peptide antigens when they are bound to MHC molecules on the surfaces of antigen-presenting cells. gamma delta T cells bear a similar receptor, and it is often assumed that these T cells also require specialized antigen-presenting molecules for immune recognition, which we term "indirect antigen recognition." B-cell receptors, or immunoglobulins, bind directly to antigens without the help of a specialized antigen-presenting molecule. Phylogenetically, it has been assumed that T-cell receptors and the genes that encode them are a monophyletic group, and that "indirect" antigen recognition evolved before the split into two types of TCR. Recently, however, it has been proposed that gamma delta-TCRs bind directly to antigens, as do immunoglobulins (Ig's). This calls into question the null hypothesis that indirect antigen recognition is a common characteristic of TCRs and, by extension, the hypothesis that all TCR gene sequences form a monophyletic group. To determine whether alternative explanations for antigen recognition and other historical relationships among TCR genes might be possible, we performed phylogenetic analyses on amino acid sequences of the constant and variable regions which encode the basic subunits of TCR and Ig molecules. We used both maximum-parsimony and genetic distance-based methods and could find no strong support for the hypothesis of TCR monophyly. Analyses of the constant region suggest that TCR gamma or delta sequences are the most ancient, implying that the ancestral immune cell was like a modern gamma delta T cell. From this gamma delta-like ancestor arose alpha beta T cells and B cells, implying that indirect antigen recognition is indeed a derived property of alpha beta-TCRs. Analyses of the variable regions are complicated by strong selection on antigen-binding sequences, but imply that direct antigen binding is the ancestral condition.     

Role of B cell antigen processing and presentation in the humoral immune response

In the 25 years since it was first indicated that lymphocyte subpopulations must interact during the generation of a humoral immune response, there has been an explosion of data on the molecular mechanism of this interaction. It has been demonstrated that T cells recognize a processed antigen fragment presented by a major histocompatibility complex molecule on the surface of an antigen-presenting cell. The minimal peptides required for T cell recognition of several proteins have been determined, the molecular genetics of many of the cell surface molecules involved have been defined, and the three-dimensional structure of the T cell receptor and the major histocompatibility antigens have been deduced. Several cell types have been found to act as antigen-presenting cells, although the roles of these populations in vivo remain unclear. However, it is clear that there must be a physical interaction between a B cell and a T cell before the B cell can respond to a T-dependent antigen. This interaction requires processing and presentation of the antigen by the B cell. Therefore this review focuses on antigen processing and presentation by resting B cells, one of the key steps in initiation of a humoral immune response.     

A reliable and safe T cell repertoire based on low-affinity T cell receptors

Antigens are presented to T cells as short peptides bound to MHC molecules on the surface of body cells. The binding between MHC/peptides and T cell receptors (TCRs) has a low affinity and is highly degenerate. Nevertheless, TCR-MHC/peptide recognition results in T cell activation of high specificity. Moreover, the immune system is able to mount a cellular response when only a small fraction of the MHC molecules on an antigen-presenting cell is occupied by foreign peptides, while autoimmunity remains relatively rare. We consider how to reconcile these seemingly contradictory facts using a quantitative model of TCR signalling and T cell activation. Taking into account the statistics of TCR recognition and antigen presentation, we show that thymic selection can produce a working T cell repertoire which will produce safe and effective responses, that is, recognizes foreign antigen presented at physiological levels while tolerating self. We introduce "activation curves" as a useful tool to study the repertoire's statistical activation properties.     

Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation

How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor–recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.     

Externalized glycolytic enzymes are novel, conserved, and early biomarkers of apoptosis

The intriguing cell biology of apoptotic cell death results in the externalization of numerous autoantigens on the apoptotic cell surface, including protein determinants for specific recognition, linked to immune responses. Apoptotic cells are recognized by phagocytes and trigger an active immunosuppressive response ("innate apoptotic immunity" (IAI)) even in the absence of engulfment. IAI is responsible for the lack of inflammation associated normally with the clearance of apoptotic cells; its failure also has been linked to inflammatory and autoimmune pathology, including systemic lupus erythematosus and rheumatic diseases. Apoptotic recognition determinants underlying IAI have yet to be identified definitively; we argue that these molecules are surface-exposed (during apoptotic cell death), ubiquitously expressed, protease-sensitive, evolutionarily conserved, and resident normally in viable cells (SUPER). Using independent and unbiased quantitative proteomic approaches to characterize apoptotic cell surface proteins and identify candidate SUPER determinants, we made the surprising discovery that components of the glycolytic pathway are enriched on the apoptotic cell surface. Our data demonstrate that glycolytic enzyme externalization is a common and early aspect of cell death in different cell types triggered to die with distinct suicidal stimuli. Exposed glycolytic enzyme molecules meet the criteria for IAI-associated SUPER determinants. In addition, our characterization of the apoptosis-specific externalization of glycolytic enzyme molecules may provide insight into the significance of previously reported cases of plasminogen binding to α-enolase on mammalian cells, as well as mechanisms by which commensal bacteria and pathogens maintain immune privilege.     

Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4CD25Foxp3 regulatory T cells and down-regulates cardiac allograft rejection

Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient’s autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-γ by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naïve T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4⁺CD25^highFoxp3⁺ regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.     

T cell microvilli simulations show operation near packing limit and impact on antigen recognition

T cells are immune cells that continuously scan for foreign-derived antigens on the surfaces of nearly all cells, termed antigen-presenting cells (APCs). They do this by dynamically extending numerous protrusions called microvilli (MVs) that contain T cell receptors toward the APC surface in order to scan for antigens. The number, size, and dynamics of these MVs, and the complex multiscale topography that results, play a yet unknown role in antigen recognition. We develop an anatomically informed model that confines antigen recognition to small areas representing MVs that can dynamically form and dissolve and use the model to study how MV dynamics impact antigen sensitivity and discrimination. We find that MV surveillance reduces antigen sensitivity compared with a completely flat interface, unless MV are stabilized in an antigen-dependent manner, and observe that MVs have only a modest impact on antigen discrimination. The model highlights that MV contacts optimize the competing demands of fast scanning speeds of the APC surface with antigen sensitivity. Our model predicts an interface packing fraction that corresponds closely to those observed experimentally, indicating that T cells operate their MVs near the limits imposed by anatomical and geometric constraints. Finally, we find that observed MV contact lifetimes can be largely influenced by conditions in the T cell/APC interface, with these lifetimes often being longer than the simulation or experimental observation period. This work highlights the role of MVs in antigen recognition.     

Gamete and immune cell recognition revisited

Fertilization is the result of a series of successful recognition and binding events mediated by gamete surface molecules. Recent advances in the identification and characterization of some of these recognition molecules provide extremely valuable information necessary to understand sperm-egg recognition and subsequent egg activation. We discuss these new data in the context of the model of gamete recognition first proposed by F.R. Lillie in the early part of the 20th century, and revisited periodically in the subsequent literature, which relates fortilization events to those of immune cell recognition and activation events. Here we discuss the principles underlying the molecular recognition and activation mechanisms of gametes and immune cells.     

Transplantation tolerance and autoimmunity after xenogeneic thymus transplantation

Successful grafting of vascularized xenografts (Xgs) depends on the ability to reliably induce both T cell-independent and -dependent immune tolerance. After temporary NK cell depletion, B cell suppression, and pretransplant infusion of donor Ags, athymic rats simultaneously transplanted with hamster heart and thymus Xgs developed immunocompetent rat-derived T cells that tolerated the hamster Xgs but provoked multiple-organ autoimmunity. The autoimmune syndrome was probably due to an insufficient development of tolerance for some rat organs; for example, it led to thyroiditis in the recipient rat thyroid, but not in simultaneously transplanted donor hamster thyroid. Moreover, grafting a mixed hamster/rat thymic epithelial cell graft could prevent the autoimmune syndrome. These experiments indicate that host-type thymic epithelial cells may be essential for the establishment of complete self-tolerance and that mixed host/donor thymus grafts may induce T cell xenotolerance while maintaining self-tolerance in the recipient.     

The regulation of hematopoiesis following bone marrow transplantation

Allogeneic bone marrow transplantation requires that donor stem cells home to the recipient bone marrow, proliferate and differentiate under normal physiologic regulatory mechanisms. Recent observations that T cell depletion of donor bone marrow leads to a greatly increased incidence of graft failure mandate a detailed understanding of the engraftment process. Post-transplant hematopoietic deficiencies appear to be related to several sources: decreased number of stem cells, activation of donor hematopoietic suppressor cells, rejection of donor stem cells by residual recipient lymphocytes and abnormal function of accessory cells that produce hematopoietic growth factors. A better understanding of the relative roles of these factors should lead to a better understanding of engraftment as well as graft failure and its prevention.     

Cytotoxic T cell activation by class I protein on cell-size artificial membranes: antigen density and Lyt-2/3 function

The role of antigen surface density, and its relationship to the function of the Lyt-2/3 complex, in recognition and triggering of allospecific cytotoxic T lymphocyte (CTL) precursors has been studied by using a novel type of Class I protein-bearing artificial membrane. The cell-size membranes, termed pseudocytes (artificial cells), can be handled like cells but have a well-defined and easily quantitated surface composition. Class I antigen on these membranes stimulated generation of secondary in vitro allogeneic CTL responses as effectively as allogeneic spleen cells, provided that lymphokines were added to the cultures. Antigen density on the pseudocyte surfaces could be varied over a wide range and quantitated by papain cleavage and fluorescence-activated cell sorter analysis. Recognition and triggering of precursor CTL was found to be dramatically dependent on the surface density of antigen and displayed a marked threshold density requirement, below which little or no response occurred. Examination of the effects of anti-Lyt-2 antibody on responses to pseudocytes provided direct evidence for a reciprocal relationship between antigen density and susceptibility to antibody blockade. The results strongly suggest that antigen density is likely to have important biological consequences in control of immune responses. They also show that if Lyt-2/3 functions by interaction with a ligand, then that ligand is the Class I protein.     

Medical applications of leukocyte surface molecules--the CD molecules

Leukocytes are the cells of the immune system and are centrally involved in defense against infection, in autoimmune disease, allergy, inflammation, and in organ graft rejection. Lymphomas and leukemias are malignancies of leukocytes, and the immune system is almost certainly involved in most other cancers. Each leukocyte expresses a selection of cell surface glycoproteins and glycolipids which mediate its interaction with antigen, with other components of the immune system, and with other tissues. It is therefore not surprising that the leukocyte surface molecules (CD molecules) have provided targets for diagnosis and therapy. Among the "celebrities" are CD20, a target for lymphoma therapeutic antibodies which earns $2 billion annually (and makes a significant difference to lymphoma patients), and CD4, the molecule used by the human immunodeficiency virus (HIV) as an entry portal into cells of the immune system. This short review provides a background to the CD molecules and antibodies against them, and summarizes research, diagnostic, and therapeutic applications of antibodies against these molecules.