Showing papers in "Immunological Reviews in 1994"

Gal alpha (1,3)Gal, the major xenoantigen(s) recognised in pigs by human natural antibodies.

Abstract: The transplantation of pig organs to humans (xenotransplantation) is now receiving serious consideration because of the shortage of human donors for organ transplants of kidney, liver and heart, and of islet cell transplantation for diabetes. The problem with such xenografts would be hyperacute rejection--mediated by natural antibodies in humans to pig antigens, complement fixation to endothelial cells, and the rapid onset of intravascular coagulation. It is now clear that the major target of the natural IgM and IgG antibodies is the terminal carbohydrate epitope Gal alpha(1,3)Gal, formed by the alpha 1,3galactosyl transferase, which places a terminal galactose residue in an alpha-linkage to another galactose. The alpha 1,3galactosyl transferase in the pig gives rise to very high endothelial cell expression of Gal alpha(1,3)Gal, a ready explanation for the hyperacute rejection of vascularized organs. In addition the parenchuma of liver and kidneys have high levels of Gal alpha-(1,3)Gal. These tissues will all fail in a pig-to-human transplant in what can now be precisely defined in terms of antigen and antibody. We have already made some suggestions for removal of anti-Gal alpha(1,3)Gal antibodies and if the procedure were technically feasible xenotransplantation could be attempted now, especially in patients doomed to a certain death because of the absence of a donor (especially for liver where ex vivo perfusion could be performed). However, the immune system is far from simple, as is shown by the healthy status of mice lacking MHC Class I, Class II or both Class I & II molecules. Perhaps the curtain is about to go up to reveal a new scene! Islets differ from the other tissues and may well not undergo acute antibody-mediated hyperacute rejection--it will be of interest to see how these fare in xenotransplantation models or even in patients. Again, normal individuals do not have anti-islet antibodies; but a proportion of diabetic patients do have such antibodies--whether these will cause hyperacute or acute rejection or are markers of immunity of T-cell type, remains to be seen. Whatever, the area is exciting, is progressing rapidly and, as indicated elsewhere, within a few years we should know whether modified pig tissue can be grafted to some patients. The isolation of the cDNA clone encoding the pig alpha 1,3 galactosyl transferase is an essential first step in the production of a transgenic pig lacking the alpha 1,3Galactosyltransferase and therefore the Gal alpha(1,3)Gal epitope, and such animals could serve as donor for human transplantation.

Oligosaccharides and Discordant Xenotransplantation

Abstract: The initiating factor in the hyperacute rejection of pig organs by human or non-human primates is believed to be related to the presence of preformed "natural" antibodies in the host. In 1991, we demonstrated that human anti-pig antibodies were IgG, IgM and IgA and bound most strongly to oligosaccharides with an alpha galactose (alpha Gal) terminal residue. These included (i) alpha Gal-R (alpha galactose), (ii) alpha Gall-3 beta Gal-R (B disaccharide), (iii) alpha Gall-3 beta Gall-4 beta GlcNAc-R (linear B type 2 trisaccharide) and (iv) alpha Gall-3 beta Gall-4 beta Glc-R (linear B type 6 trisaccharide) where R is (CH2) 8COOCH3. In vitro studies using both the chromium release assay and a live/dead staining technique demonstrated that the cytotoxicity of human sera towards pig cells can be significantly reduced or abolished by immunoadsorption of the serum with immunoaffinity columns of an alpha Gal structure, particularly those with an alpha 1-3 linkage, and not by a large selection of other carbohydrates. Similarly, human anti-pig antibodies can be largely inhibited or "neutralized" by the addition of an alpha 1-3Gal di- or trisaccharide to the serum. Staining of pig vascular endothelium utilizing a panel of carbohydrate-specific lectins and immunoaffinity antibodies demonstrated the presence of three different carbohydrate epitopes, namely (i) alpha Gall-3 beta Gall-4 beta GlcNAc-R (linear B type 2 trisaccharide (ii) alpha NeuAc2-3 beta Gall-4 beta GlcNAc-R (sialyl-N-acetyllactosamine), and (iii) beta Gall-4 beta GlcNAc-R (N-acetyllactosamine). We have investigated organs from several breeds of pig and have concluded that the alpha Gal epitope is either monomorphic or at least has a high incidence in porcine species, since we have not found any pig negative for this antigen. Human vascular endothelial cells have at their surface the same lactosamine-ended precursor and sialylated chains as pigs, but instead of terminal alpha Gal they express the fucosylated polymorphic ABH histo-blood group epitopes. As we have found no evidence that human or baboon plasma contain antibodies directed against sialic acid or lactosamine, and as human tissues contain both of these carbohydrates, it seems unlikely that either of these epitopes plays a role in the vascular rejection that takes place when pig organs are transplanted into primates. Unfortunately, the alpha Gal disaccharide and trisaccharides were not available to us in the large quantities required for extracorporeal immunoadsorption or continuous intravenous infusion in adult baboons.(ABSTRACT TRUNCATED AT 400 WORDS)

The Role of APO-1-Mediated Apoptosis in the Immune System

Abstract: B and T lymphocytes contact antigen at different stages of maturation and in a different cellular context. Depending on these circumstances, their responses can be entirely different. Immature double-positive (CD4\"^/CD8^) thymocytes can be positively selected and exit the thymus to populate the peripheral immune system. Alternatively, autoreactive thymocytes are negatively selected and die within the thymus by programmed cell death, apoptosis. A similar process, albeit less well defined, might be operative for immature B cells in bone marrow. These events are the basis of self-tolerance and generate lymphocytes that react with foreign antigens. Under the appropriate conditions, antigen stimulation of resting mature lymphocytes in the peripheral lymphoid organs can induce expansion of the reactive lymphocyte pool or anergy in the absence of costimulatory signals. In previously activated lymphocytes, triggering of the antigen receptor may lead to proliferation and memory or to death by apoptosis. The molecular switches leading to these different responses are partially unknown. Apoptosis in the peripheral lymphoid system may serve several functions. It may be a second safeguard to assure self-tolerance and control autoreactive cells that arise in the periphery from cells that have escaped tolerization in the central lymphoid organs. In addition, apoptosis may prevent uncontrolled expansion of specific, antigen-reactive lymphocytes and may be one mechanism of immunosuppression (Krammer et

Endothelial cell activation and thromboregulation during xenograft rejection.

Abstract: Xenoreactive natural antibodies (XNA) and complement (C) are thought to be the two major inciting factors that result in hyperacute rejection (HAR) of an immediately vascularized, discordant xenograft within minutes to a very few hours, with destruction and infarction of the transplanted organ. If recipients are modified by various experimental modalities, such as removal and suppression of XNAand C-mediated responses, thus avoiding HAR, the process of delayed xenograft rejection (DXR) with a significant vascular component still occurs after a delay of several days or, at the most, a few weeks (Bach et al. 1993). The end result in both instances is the invariable and unacceptable loss of xenografts, which currently limits application of xenotransplantation beyond experimental protocols. The mechanisms underlying DXR are far from clear but appear not necessarily to involve XNAand C-mediated responses as those noted in HAR. Moreover, DXR can occur without the prominent participation of T lymphocytes. One of us (FHB) has suggested that the final common pathogenic mechanisms underly-

Role of lymphoid organs in the pathogenesis of human immunodeficiency virus (HIV) infection.

Abstract: The pathogenic mechanisms of HIV disease are multifactorial and multi-phasic The common denominator of the disease is the profound immunosuppression that occurs in the vast majority of infected patients Studies in lymphoid tissues in HIV disease have provided considerable insight into the pathogenic processes involved from the earliest phases of infection through the advanced stages Following primary infection, virus is disseminated throughout the body and seeds the lymphoid tissue where its replication is only incompletely suppressed and where a reservoir of virus is established Extracellular virus is trapped within the FDC of the lymph node germinal centers and serves as a source of infection for cells which reside in or migrate through the lymph node throughout the course of infection even during the early and often prolonged asymptomatic period Eventually, the architecture of the lymphoid tissue is destroyed, compounding the immune dysfunction that results from the depletion of CD4+ T cells In this regard, the lymphoid tissue of LTNPs is relatively intact and viral burden and replication is considerably lower in the peripheral blood and lymph node mono-nuclear cells of LTNPs than in individuals whose disease progresses Cytokines probably play a major role in the modulation of HIV expression in the milieu of the lymphoid tissue Further understanding of the pathogenic mechanisms operative in the lymphoid tissues of HIV-infected individuals will have important implications in the design of therapeutic strategies involving both antiretroviral and immunomodulatory approaches

Changing virus-host interactions in the course of HIV-1 infection.

Abstract: Acquired immunodeficiency syndrome (AIDS) patients may present with various clinical symptoms related to severe immunodeficiency resulting from persistent infection with the human immunodeficiency virus-l (HIV-1). CD4+ T-helper (Th) cells are an important target for HIV (Klatzmann et al. 1984, Dalgleish et al. 1984, McDougal et al. 1985), and loss of these eells in relatively late stages of HIV infection is well documented and known to be predictive for progression (Meibye et al. 1986, Fahey et al. 1984). In addition to depletion of Th eells, leukocytes from AIDS patients display a variety of functional defects finally resulting in a general disturbance of immune reactivity that includes almost all leukocyte functions. At that stage, the patient is extremely susceptible to diseases related to a variety of intracellular pathogens but also has a moderately increased risk for pyogenic infections particularly with encapsulated bacteria. Moreover, in addition to Kaposi's sarcomas, opportunistic neoplasia frequently develop (Fauci 1988). One feature of HIV-1 is its great variability with respect to biological properties such as syncytium inducing (SI) capacity, replication rate and cytotropism (ChengMayer et al. 1988, Asjo et al. 1986, Von Briesen et al. 1987, Evans et al. 1987, Tersmette et al. 1988). HIV-1 isolates recovered from peripheral blood mononuclear cells of asymptomatic subjects are able to grow in phytohemagglutinin (PHA)-stimulated primary blood lymphocytes (PBL) but, in contrast to isolates

Pooled human IgG modulates cytokine production in lymphocytes and monocytes.

Abstract: Human immunoglobulin preparations for intravenous use (i.v.Ig) have been successfully used in controlled clinical trials as substitution therapy for hypogammaglobulinemia (Ammann et al. 1982), but also in treatment of various conditions of unknown origin such as idiopathic thrombocytopenic purpura (Imbach et al. 1981), Kawasaki disease (Newburger et al. 1986) and dermatomyositis (Dalakas et al. 1993). The mechanisms by which i.v.Ig act in these diseases, apart from the antibody deficiencies, are not fully understood. Immunomodulatory effects of i.v.Ig include, among others, antigen neutralization, Fc-receptor blockade and anti-idiotypic interactions, all of which will be discussed in other reports of this volume. The aim of this work has been to examine whether i.v.Ig may influence cytokine production as an additional mode of immunoregulatory influence. Most of this presentation will be based on results obtained with studies of i.v.Igmediated effects on cytokine production in human cell cultures. Not many publications in the literature have addressed the question of whether i.v.Ig exposure of cells in vivo or in vitro will influence cytokine synthesis. Some examples of such studies will be summarized below. IL-1 receptor-antagonist (ILlra) was originally discovered in the supernatant of human monocytes cultured on adherent IgG (Arend et al. 1985). It was later confirmed that the co-culture of human monocytes and low doses of i.v.Ig also led to IL-1 ra formation (Pouts-

Anti-cytokine nature of natural human immunoglobulin: one possible mechanism of the clinical effect of intravenous immunoglobulin therapy.

Abstract: Natural human immunoglobulins are now widely used to treat many clinical diseases. Historically, this therapy began as a supplement for patients with agammaglobulinemia (Barandun et al. 1981/82). Pneumonia, bacterial meningitis and other infectious episodes often seen in patients with this disorder have been improved by intravenous administrations of natural immunoglobulin (Ersoy et al. 1992, Liese et al. 1992, Popescu et al. 1992). Rather than being just a supplement, immunoglobulin is now being used because of its therapeutic effects. One of its most prominent features is its antipyretic effects against infection (Iwata et al. 1987). Some specific antibody fractions against causative microorganisms may be present in natural immunoglobulin. However, it now appears that some unidentified mechanisms play important roles in the outcome of this therapy (Blanchette et al. 1992, Horiuchi et al. 1993, Ronda et al. 1993). Natural human immunoglobulin preparations have been widely used in cases of severe infection such as septic shock and bacterial meningitis {Ruiz et al. 1993, Saladino et al. 1992, Schedel et al. 1991, Wortel et al. 1993). Subsidence of an infection as evidenced by the alleviation of high-grade fever, and the improvement of survival rate have been noticed with the use of natural polyvalent immunoglobulin preparations from simple stock plasma, as well as IgM-enriched specific preparations (Ersoy et al. 1992, Iwata et al. 1987, Shimozato et al. 1990, Taylor et al. 1992). Another example ofthe clinically indicated disorders for this therapy is autoim-

V region-mediated selection of autoreactive repertoires by intravenous immunoglobulin (i.v.Ig).

Abstract: In addition to its use as substitute therapy for primary and secondary immunodeficiencies, intravenous immunoglobulin (i.v.Ig) is increasingly being used as an immunomodulating therapy in the treatment of patients with a variety of autoimmune and systemic inflammatory disorders (Schwartz 1990, Dietrich et al. 1992b, Dwyer 1992, Ronda et al. 1993). The use of i.v.Ig in these situations is supported by a few randomized clinical trials and a large number of uncontrolled and smaller studies. Of relevance to this chapter are that the reported beneficial effects of i.v.Ig include those in autoantibody-mediated autoimmune diseases (e.g., autoimmune cytopenias, anti-factor VIII autoimmune disease) as well as diseases in which autoaggressive T cells are primarily involved in the pathogenesis (e.g., autoimmune uveitis) (Imbach et al. 1981, Bussel et al. 1983, Sultan et al. 1984, McGuire et al. 1987, LeHoang et al. 1994). Where it is a feature of the disease, successful outcome of i.v.Ig therapy is associated with an improvement in the patient's systemic inflammatory condition. Modulation of B-cell and Tcell functions and of cytokine production has further been observed in animal models of autoimmune diseases following administration of human i.v.Ig or of normal homologous IgG (Forsgren et al. 1991, Rossi et al. 1991a, Saoudi et al. 1993, Hentati et al. 1994). The design of trials to establish the efficacy and appropriate therapeutic sched-

The Molecular Basis of X-Linked Severe Combined Immunodeficiency: The Role of the Interleukin-2 Receptor γ Chain as a Common γ Chain, γc

Abstract: X-linked severe combined immunodeficiency is characterized by severe and persistent infections from early life resulting from profound impairment of both cellular and humoral immune function. XSCID is characterized by an absence or diminished number of T cells and histologic evidence of hypoplastic and abnormal differention of the thymic epithelium. The discovery that this disease results from the mutations of the IL-2R gamma chain was surprising since IL-2-deficient mice and human SCID patients had milder phenotypes. This led to the speculation that IL-2R gamma would prove to be a common gamma chain, gamma c, which would play important roles in other cytokine receptors in addition to the IL-2 receptor. There is now compelling evidence to support a role in at least two other cytokine receptors, namely the IL-4 and IL-7 receptors. Thus, with inactivation of gamma c, multiple cytokine systems are simultaneously affected, resulting in the profoundly impaired phenotype of XSCID. It is possible and even likely that gamma c will be found to be a functional component of additional receptors as well. These findings have resulted in a significant improvement in our understanding of the pathophysiologic development of the defects in XSCID and also have important ramifications for prenatal and postnatal diagnosis, carrier female identification, and gene therapy for XSCID.

The Genetic Basis of Chronic Granulomatous Disease

Abstract: Chronic granulomatous disease is a serious clinical entity. The disease is caused by the failure of NADPH oxidase in phagocytic leukocytes to generate superoxide, needed for the killing of micro-organisms. The patients need careful management aimed at prevention and aggressive treatment of infections. CGD is a heterogeneous syndrome, both clinically and genetically. This disease is caused by a diversity of mutations, and multiple genes are affected. In fact, in the A22 and X91 subtypes of CGD, in which the alpha subunit and the beta subunit of cytochrome b558 are affected, respectively, the mutations are virtually unique for each CGD family tested. The results of these studies provide a better understanding of the mechanism of action of the various components of the superoxide-generating enzyme. Although treatment of CGD patients has improved considerably over the past 30 years, death caused by overwhelming infections is still a serious threat. Prenatal diagnosis now provides the relatives of a CGD patient with the possibility to choose for first-trimester abortion of an affected fetus. Moreover, genetic correction of the disease is now a goal within reach.

Distinctive developmental origins and specificities of murine CD5+B cells

Abstract: SUMMARY CD5+ B cells constitute a small fraction of cells in the spleen of adult mice that exhibit numerous features serving to distinguish them from the bulk of IgD++CD5−“conventional” B cells In this review we focus on two major questions relating to this population: 1) the relationship of CD5+ B cells to other B cells; and 2) the distinctive enrichment of particular autoreactive specificities in this subset The nature of their origins is clarified by a thorough analysis of intermediate stages of early B-cell development in both fetal and adult tissues The reactivity to bromeliad-treated mouse red blood cells serves as a prototype system for the investigation of biased specificities in CD5+ B cells These lines of investigation lead us to propose that CD5+ B cells in the adult are the remnant of a distinct fetal B-cell differentiation pathway wherein selection of cells from this fetal/neonatal population into the adult long-lived pool results in the over-expression of certain germline-encoded autoreactivities

Early B-cell development in the mouse: insights from mutations introduced by gene targeting.

Abstract: Whether a cell committed to the B-cell lineage finally succeeds in entering the pool of newly-generated B cells depends on the Ig gene rearrangements it acquires during its differentiation. During the past decade, the immunoglobulin (Ig) gene rearrangement program has heen analyzed with the help of Abelson virus-transformed pre-B cell lines that spontaneously undergo rearrangement of Ig loci in vitro. Based on these studies, the following mode! of B-cell development was formulated (Alt et al. 1984, 1987): B-cell precursors first assemble D^JH rearrangements. As soon as a productive VH-*DHJH joint is made, IgH rearrangement ceases and L (light) chain loci are rearranged. For IgL gene rearrangement again an order has been postulated: V Ĵ̂ . joints were suggested to be generated prior to V^J; joints. Once a L chain is expressed, the cell displays surface immunoglobulin (sig) and further Ig gene rearrangements are stopped. We have used gene-targeted mice to analyze the in vivo requirements for B-cell differentiation. While B-cell development in mice lacking membrane-bound /j.-

Defective Expression of CD40 Ligand on T Cells Causes “X‐Linked Immunodeficiency with Hyper‐IgM (HIGM1)”

Abstract: X-linked immunodeficiency with hyper-IgM (HIGM1) is a rare disorder, characterized by recurrent infections associated with very low or absent IgG and IgA, and normal to increased IgM serum levels. The disease has been earlier mapped to the q26-27 region of the X-chromosome. We have identified a novel molecule expressed on the surface of activated T cells, which was designated TRAP (Tumor necrosis factor Related Activation Protein), and could demonstrate that TRAP is a ligand for the CD40 receptor expressed on B cells. Our mapping of the TRAP gene to the Xq26.3-27.1 region suggested a causal relationship to HIGM1. Further work revealed that various mutations of the TRAP/CD40 ligand (CD40L) gene may lead to a defective expression of the TRAP/CD40L molecule on the T-cell surface in HIGM1 patients. A combination of structural and functional analyses finally demonstrated that the failure of TRAP/CD40L on T cells to interact with CD40 on B cells is responsible for the inefficient T-cell help for B cells observed in HIGM1. The observations made in HIGM1 allowed us to conclude that TRAP/CD40L is not required for IgM synthesis. In contrast, functional expression of TRAP is a prerequisite for effective immunoglobulin isotype switching and subsequent production of IgG, IgA and IgE by B cells in vivo. The interaction of TRAP/CD40L with CD40 thus provides a very critical link between the cellular and the humoral part of the immune system. The knowledge of TRAP/CD40L cDNA sequence, the availability of various reagents for the testing of expression and function of TRAP/CD40L, and our recent elucidation of the exon-intron structure of the TRAP/CD40L gene now provide all necessary tools for early diagnosis of affected patients and the detection of female carriers of HIGM1. The available information will also provide a basis for future attempts at gene therapy in this disease.

Role of TH1/TH2 Cytokines in HIV Infection

Abstract: Different experimental approaches were used to prove or disprove the "TH1/TH2 switch theory" of HIV-infection. No increase, or even a decrease, in the production of TH2-type cytokines (IL-4, IL-5, and IL-10) by either bulk circulating mononuclear cells or CD4+ T-cell clones generated by PHA stimulation of single T cells from HIV-infected individuals in all stages of disease compared to HIV-negative donors was observed. However, enhanced proportions of CD4+ T-cell clones able to produce both TH1-type and TH2-type cytokines (TH0 clones) were derived from either skin-infiltrating, in vivo-activated, T cells or in vitro antigen-stimulated peripheral blood T cells of HIV-infected individuals. Of note, TH1, TH2 and TH0 clones obtained from HIV-seronegative healthy donors showed different ability to support viral replication after infection with HIV in vitro. All TH2 and most TH0 clones supported HIV replication efficiently, whereas TH1 clones did not. These results suggest preferential HIV replication in T cells producing TH2-type cytokines rather than TH1/TH2 switch in HIV infection.

X‐Linked Agammaglobulinemia and Other Immunoglobulin Deficiencies

Abstract: Lack of immunoglobulins in a patient was described as early as 1952 by the American physician. Dr. Ogden C. Bruton (Bruton 1952). This was the ftrst immunodeficiency to be identified in any species and was later called X-linked agammaglobulinemia (XLA) or Bruton's agammaglobulinemia (MIM 30030). In 1952 lymphocyte differentiation was poorly understood and it was almost two decades before the defect was identified as a lack of B lymphocytes (Siegal et al. 1971, Cooper et al. 1971, Preud'Homme etal. 1973, Conley 1985, Campana et al. 1990). Fig. 1 is a schematic representation of B-lymphocyte differentiation and depicts the differentiation block/growth arrest seen in XLA. The first stages of B-cell progenitors have, as yet, not been identified, and the earliest lymphoid cells committed to becoming B cells are normally referred to as pro-B cells. These cells have not rearranged any immunoglobulin (Ig) genes, and the molecular nature of their commitment is not understood. Pre-B cells rearrange their Ig variable (V, D and J) segments and form complete regulatory mechanisms that involve transcription factors, signalling proteins, growth factors, apoptotic molecules, and surrogate Ig chains (for review see Kincade et al. 1989, Rolink & Melchers 1991, Banchereau & Rousset 1992). Following antigenic stimulation lymphocytes may undergo isotype switching, i.e. changing their isotype from IgM to a 'downstream' isotype. This event is

Sex Hormones as Negative Regulators of Lymphopoiesis

Abstract: B lymphocytes, together with cells of seven other lineages, are made in large numbers from precursors in the bone marrow. Using cell culture models and recombinant proteins, progress has been rapid in identifying cytokines which could potentially regulate the proliferation, differentiation and migration of B-cell precursors. However, we still know little about molecular mechanisms which are important for maintaining steady-state conditions in vivo. B lymphopoiesis is severely diminished during pregnancy in normal mice and this provided a clue that sex hormones might be important negative regulators. Administration of estrogens alone, or in combination with progesterone, preferentially suppressed IL-7 responding cells and their progeny in bone marrow. There is precedent for these observations in the thymus, which transiently involutes during pregnancy, and also atrophies following estrogen treatment. The actual mechanism(s) through which sex steroids influence lymphopoiesis remain unclear, but cell culture experiments should be informative about potential interactions between hormones, the bone marrow microenvironment, and lymphocyte precursors. These findings raise a number of other important issues. For example, we need to learn if sex steroids are produced and/or concentrated locally within the marrow, if human lymphopoiesis is sensitive to these hormones, and if production of lymphocytes can be augmented in aging and in immunodeficiency by hormone manipulation.

Bruton's Tyrosine Kinase is a Key Regulator in B‐Cell Development

Abstract: X-linked agammaglubulinemia (XLA) is the prototypic inherited humoral immunodeficiency described in 1952 by Bruton (Bruton 1952, Conley 1992, Cooper et al. 1993). Affected males develop recurrent bacterial infections early in life. Circulating immunoglobulin levels are profoundly decreased and there is a severe deficit in B cells and their plasma cell progeny. B cells in female obligate carriers of XLA exhibit nonrandom X-chromosome inactivation, implying that XLA results from an autonomous B-lineage defect (Conley et al. 1986). Despite the reduction in peripheral B cells, XLA bone marrow pre-B cell numbers are normal. The XLA phenotype is best described as a failure of these pre-B cells to thrive and undergo the dramatic clonal expansion responsible for the production of immature B-lineage cells. In XLA, proliferation of these c/x"*" pre-B cells is significantly reduced (Campana et al. 1990) and fewer c/z+ cells enter S phase (Pearl et al. 1978). For these reasons, it had been proposed that the XLA defect might interfere in a B lineage-specific signal transduction pathway critical to early Blineage growth and clonal expansion (Anker et al. 1989, Cooper et aL 1993). As will be demonstrated in this review, the novel cytoplasmic protein tyrosine kinase, Bruton's tyrosine kinase (Btk, formerly bpk or atk), fulfills these predictions and its involvement in the pathogenesis of XLA and xid (X-linked immunodeficiency) supports its role as a key regulator of normal B-cell development.

From AIDS to Parasite Infection: Pathogen-Mediated Subversion of Programmed Cell Death as a Mechanism for Immune Dysregulation

Abstract: Premature cell death can result either from cell injury or degeneration, leading to necrosis, or from the activation of a physiological cell-suicide process, termed programmed cell death or apoptosis, that is regulated by intercellular signalling. This process plays an essential role in the selection of developing lymphocytes, and is also involved in the function of the mature adaptative immune system. A growing number of experimental findings during the last 4 years has provided support to our hypothesis that inappropriate HIV-mediated dysregulation of programmed T-cell death is relevant to AIDS pathogenesis. A series of recent experimental results also supports the general concept that the persistence and pathogenesis of several infectious pathogens, ranging from retroviruses to parasites, may be related to their capacity to dysregulate programmed cell death in various cell populations including lymphocytes. Subversion by pathogens of the physiological control of programmed cell death provides a paradigm for the pathogenesis of a wide range of infectious diseases that involve immune dysregulation and suggests therapeutic potential for the in vivo modulation of cell signalling.

Analysis of the Role of bcl‐2 in Apoptosis

Abstract: Cells undergo apoptosis in response to a wide range of stimuli, and this response may represent an ancient defence mechanism against pathogens. Bcl-2 is able to prevent apoptosis in many cases. Although blocking cell suicide is not directly oncogenic, enforced bcl-2 expression can lead to cancer by lengthening the life-span of cells, during which time secondary changes, such as activation of additional oncogenes like c-myc, can occur. Bcl-2 cannot block apoptosis of target cells by cytotoxic T lymphocytes. Thus cytotoxic T cells are able to fight viruses that carry anti-apoptosis genes that resemble bcl-2. Genes involved in the regulation of mammalian apoptosis are similar to those that mediate programmed cell death in C. elegans. By studying cell death genes in viruses and worms as well as mammals, we will learn more about this fascinating process.

X-linked agammaglobulinemia: new approaches to old questions based on the identification of the defective gene.

Abstract: The identification of a cytoplasmic tyrosine kinase, Btk, as the defective protein in human XLA and xid in the mouse, supports the hypothesis that both disorders are due to defects in B-cell activation or differentiation. Phenotypic analysis of B-lineage cells and studies on X-chromosome inactivation patterns in both mice and human patients suggest that mutations in Bth do not affect entry of stem cells into the B-lineage pathway but they do inhibit progression at multiple steps along that pathway. Although the exact function of Btk in signal transduction is not yet known, it is probable that studies which correlate specific mutations in different patients with alterations in Btk function will provide clues about critical sites in the molecule. Diagnosis and genetic counseling for families at risk of carrying the gene for XLA will be improved almost immediately by the identification of the responsible gene. Improvements in therapy may come more slowly. The possibility of curative gene therapy is attractive; however, there are several features of Btk that suggest that this will be a challenging undertaking. Overexpression or expression in inappropriate cell lineages may carry unacceptable risks. Mutant proteins may interfere with the function of wild-type proteins provided by gene therapy. However, it is likely that a better understanding of Btk function and regulation will benefit not only patients with XLA but also other patients with defects in B-cell function.

The role of CD40 and its ligand in the regulation of the immune response.

Abstract: A successful immune response requires the coordinated interaction of multiple cell types. The interactioti between T-helper cells (Th) and antigen-presenting cells (APC) such as B cells, monocytes, and dendritic cells results from complex communications involving signals received through soluble cytokines or membrane-bound proteins as well as adhesive interactions. Many of these signals are not specific to a directed immune response and the proteins are broadly distributed. Through the preparation of monoclonal antibodies (mAb), several of the proteins unique to the cells involved in a targeted immune response have been identified and characterized. These mAb have proven critical for the biochemical characterization of the proteins they recognize and the isolation of cDNA clones which encode them. Studies of these proteins allow investigation of the mechanisms of immune response.

A Perspective on Xenograft Rejection and Accommodation1

Abstract: There is increasing interest in the potential clinical application of xenotransplantation. This interest derives in part from the need to identify a more abundant source of organs for transplantation and in part from rapid progress in understanding the cellular and molecular events that contribute to xenograft rejection. Recent areas of progress include the characterization of xenoreactive antibodies which would initiate the rejection of porcine organs transplanted into primates. These antibodies consist predominantly of IgM and their binding is characterized by high avidity and surprising uniformity. Xenoreactive antibodies recognize porcine glycoproteins of the integrin family; the determinants residing on N-linked substitutions. The predominant substitution has a terminal alpha Gal residue. Antibody binding initiates activation of complement through the classical pathway triggering a number of effector mechanisms. These mechanisms may include loss of heparan sulfate from endothelial cells mediated by C5a and xenoreactive antibody; a change in endothelial cell shape mediated by C5b-7 or the membrane-attack complex; procoagulant changes mediated by the membrane-attack complex; and neutrophil adhesion mediated by iC3b. If hyperacute rejection is prevented by the depletion of xenoreactive antibody and/or the inhibition of complement, acute vascular rejection may be seen some days later. Acute vascular rejection is characterized by prominent evidence of thrombosis and neutrophil infiltration. The cause of acute vascular rejection is unknown, but may reflect profound alterations in the function of endothelial cells lining blood vessels in the graft. In some cases, when recipients of xenografts are modified by depletion of xenoreactive antibodies, acute vascular rejection does not occur; rather, a process called accommodation allows the xenograft to survive despite the return to the circulation of xenoreactive antibodies and complement. The mechanism for accommodation is not known. New therapeutic strategies including the development of specific immunoabsorbants, identification of preferred donor animals expressing low levels of antigen and the development of transgenic donor animals expressing human complement regulatory proteins are among the strategies which may bring xenotransplantation closer to the clinical arena.

Apoptosis and macrophage-mediated cell deletion in the regulation of B lymphopoiesis in mouse bone marrow.

Abstract: Studies of cell population dynamics and microenvironmental organization of B lymphopoiesis in the bone marrow of normal mice and in various genetically modified states have shown that cell loss, involving processes of apoptosis and macrophage-mediated cell deletion, is a prominent feature of the primary genesis of B lymphocytes. Balanced against the influence of proliferative stimulants, the programmed death of precursor B cells provides a quantitative control, determining the magnitude of the final output of functional B lymphocytes to the peripheral immune system. The cell loss mechanisms can be readily set in motion by external or systemic influences, making the B-cell output particularly vulnerable to suppression by ionizing irradiation, stress or other systemic mediators. In addition, however, cell loss exerts an important quality control in the formation of the primary B-cell repertoire. The combination of apoptosis and macrophage-mediated deletion, acting at successive stages of B-cell differentiation, efficiently eliminates many precursors having non-productive Ig gene rearrangements, cell cycle dysregulations, and certain autoreactive Ig specificities. Outstanding areas of further work abound. Important questions concern the nature of mechanisms which underlie the processes of B-cell apoptosis and macrophage deletion in bone marrow, the microenvironmental signals involved in B-cell life or death decisions and genetic factors which may override these B-cell culling mechanisms. The answers will be relevant to problems of autoimmune disease, humoral immunodeficiency and B-cell neoplasia.

Early events in cell-mediated recognition of vascularized xenografts: Cooperative interactions between selected lymphocyte subsets and natural antibodies

Abstract: Cell-mediated early immune recognition of xenogeneic vascularized discordant grafts is poorly characterized. It has been the purpose of our studies to elucidate the role of lymphocytes in the recognition and rejection phenomena. To this end, we have utilized both ex vivo and in vitro model systems. We demonstrate that selected human lymphocyte subpopulations (mainly NK cells) rapidly and specifically adhere to xenogeneic endothelia. Efficient lysis of endothelial cells is subsequently mediated by such a population. Adhesion and cytotoxicity occur via at least two pathways, one dependent on and the other independent of the presence of human natural antibodies of the G class. Both IgG-dependent and IgG-independent adhesion and cytotoxicity can be partly inhibited by the use of anti-leukocyte integrin monoclonal antibodies. IgG-dependent adhesion and cytotoxicity can be also partly inhibited by carbohydrate structures that contain the alpha-galactosyl epitope. A possible role of these events in the eventual outcome of discordant vascularized xenogeneic transplants can be postulated.

Dimer formation in immunoglobulin preparations and speculations on the mechanism of action of intravenous immune globulin in autoimmune diseases

Abstract: IgG dimers occurring in therapeutic Ig preparations have been characterized as Id-anti-Id, in that the sites of interaction are localized to the distal tips of the Fab arms. The observation that such dimers are prevalent in Ig or Igi.v. prepared from large plasma pools, but absent in preparations from a single individual, supports the notion that the individual immune repertoire is small with respect to the species repertoire. A crude mathematical model that attempts to relate Id-dimer content to the number of donors is presented. This model suggests that Id-pairs may be much more prevalent in Ig than is reflected by the Id-dimer content, inasmuch as the concentrations of the individual Ids and anti-Ids may limit the equilibrium level of dimer. The model further suggests that the antibody diversity in Ig derived from thousands of donors may be representative of the species repertoire; hence, virtually all specificities, including anti-Id specificities, will be included. Because Ig is derived from normal healthy donors, it should be relatively free of pathogenic autoantibodies. However, there is no reason to suspect that anti-Ids to such autoantibodies would not occur, and indeed the presence of such anti-Ids has been demonstrated. Several mechanisms have been proposed by which such anti-Ids might ameliorate autoimmune disease. They may directly inhibit the binding of autoantibody to its target antigen, or they may target for destruction those cells expressing or secreting autoantibody. It may well be that anti-Ids play no role in the mechanism of action of Igi.v. in autoimmune disease. Such appears to have been demonstrated, for example, in the treatment of ITP. There is an obvious need for additional studies in order to elucidate the mechanism of action of Igi.v. in various autoimmune diseases. Experimental animal models of autoimmune disease, such as the mouse model for systemic lupus erythematosus (Mozes et al. 1993), might be very useful in this regard. Finally, it needs to be emphasized that the usefulness of high doses of Igi.v. in many autoimmune diseases remains to be established by controlled clinical trials. Because Igi.v. is a limited resource, and one which cannot be produced through biotechnological advances (at least in the foreseeable future), its widespread use should be restricted to the treatment of diseases for which efficacy has been demonstrated. To do otherwise might deprive appropriate patients of a valuable therapy.

Natural antibodies and human xenotransplantation.

Abstract: Landsteiner realised, in the early 1900's, that proteins regularly induce a specific response but that carbohydrates and other chemical groupings, although they regularly bind to antibodies, often do not induce them. He introduced the term 'hapten' for such molecules. He also considered the particular case of red cell agglutinins (now known to bind carbohydrates). Landsteiner's Law, or more properly Landsteiner's Theory, was formulated with respect to anti-ABO antibodies (Landsteiner 1945). It has been extended to include other similar systems including preformed anti-species antibodies. Each mammalian species has a repertoire of antibodies that are present without apparent immunization (so-called 'natural antibodies'). Each normal individual produces all the natural antibodies of the full species repertoire with only one exceptional state: there is no production of those antibodies which bind to ligands constitutively expressed within that individual in accessible cell membrane sites. Thus all humans have antibodies to blood group B except those who are themselves blood group B. This is in complete contrast to other antibodies which are critical to transplantation, viz. in a blood group O person there will invariably be antibodies to blood groups A and B, whereas antibodies to MHC antigens are only found in normal individuals following transfusion, transplant or pregnancy.

The biological basis of and strategies for clinical xenotransplantation.

Abstract: Recent discoveries have suggested that the exchange of multiple leukocyte lineages between grafts and host and subsequent long-term chimerism in both is the seminal mechanism of the acceptance of organs transplanted from the same (allografts) or different species (xenografts). This insight suggests new strategies which may allow xenotransplantation, the principal obstacle to which has been humoral rejection. We have defined humoral rejection as a family of complement activation syndromes afflicting allografts and xenografts in which there is a strong (but not invariable) association with preformed antigraft antibodies, invariable evidence of complement activation, histopathologic stigmas of vascular endothelial damage, and a concomitant local or systemic coagulopathy. The generic descriptive term hyperacute rejection is a misnomer because a slow-motion version of the same “humoral” process can occur with some allografts and is the rule with the so-called concordant species xenotransplantations. The pathway of experience and discovery leading to this conclusion shows clearly that the distinction frequently made between allograft versus xenograft humoral rejection does not actually exist in principle, but only in details and intensity. Breaking down this barrier to xenotransplantation, whether or not it is associated with antibodies, is unrealistic. However, the possibility of avoiding the barrier has been exposed by showing that animal organs can be humanized, with a mixed donor and recipient cell population similar to the chimerism seen in long surviving allografts or even with complete leukocyte replacement. Pilot experiments in rodents suggest that organs from fully xenogeneic chimeras can be made into xenogeneic targets that are no more provocative of complement activation than allografts when they are transplanted into the donor bone marrow species. Although the validity of this concept of organ xenograft preparation is only at the pilot stage of verification, there is reason to suspect that the complement trigger of humoral rejection can be thereby disarmed. If this can be accomplished, independent evidence suggests that cellular rejection can be controlled with conventional T-cell directed immunosuppression, perhaps even with surprising ease. The potential subtle liability of synthetic products of xenogeneic parenchymal cells is not yet known.

Differentiation of the secondary B-lymphocyte repertoire: the germinal center reaction.

Abstract: DifTerentiation of B lymphocytes can be divided into a pre-antigenic and a postantigenic phase, though the process of receptor editing following negative selection blurs the distinction somewhat (reviewed in Nossal 1993). Antigen-induced B-cell activation and differentiation can be further subdivided into events leading to antibody formation or to the production of a phenotypically and functionally separate populations of memory B cells, consisting mainly of cells with immunoglobulin receptors of isotypes other than IgM and IgD, and cells bearing somatic mutations in their immunoglobulin variable region (V) genes which confer on their antibody product a higher affinity for the immunogen in question. Many of the key events which result in memory B-cell formation occur in germinal centers (MacLennan et al. 1990, Jacob et al. 1991a, Nossal 1992) which thus play a critical role in the genesis of the secondary, antigenically experienced B-lymphocyte repertoire. Our laboratory has had an interest in germinal centers for 30 years, active for the first and last 5 years of this period and passive for the intervening 20! The purpose of this brief essay is to draw together the lessons learnt over this period and to speculate on the key features of this unique lymphoid microenvironment.

Signalling and chromatin fragmentation in thymocyte apoptosis.

Abstract: Apoptosis is a form of regulated cell deletion that plays a central role in immunity Processes such as negative cell selection, which eliminate autoreactive T and B lymphocytes during development, are mediated by apoptosis, and target cell killing by cytotoxic T lymphocytes and natural killer cells also proceeds via this mechanism. Furthermore, apoptosis is involved in the maintenance of immune tolerance in the periphery and is responsible for the elimination of expanded T cells at the termination of an immune response. Finally, the observation that 004\"^ T cells in HIV and other viral infections show increased tendency to undergo apoptosis, and the finding that thymocytes and other hematopoietic cell types exposed to various immunotoxins die by apoptosis suggests that it may be generally involved in the immune pathologies. For these reasons, much of what is currently known about the biochemical and molecular mechanisms that regulate the apoptotic response has come from work in hematopoietic model systems. Apoptosis is characterized by a series of morphological alterations including plasma and nuclear membrane blebbing, cell shrinkage, chromatin condensation, and dissolution of the nuclear lamina that are not observed in cells undergoing death by necrosis. Biochemical processes implicated in apoptosis include protease, transglutaminase, and endonuclease activation. Although the most characteristic biochemical feature of apoptosis is the cleavage of host chromatin into oligonucleosome-length DNA fragments, {\"DNA ladders\") by nucleases, more recent work suggests that a protease, or family of proteases, related to the nematode gene ced-3 and human interleukin-1/? converting enzyme (ICE) may be of particular importance in apoptosis. The biochemical machinery responsible for these