Trading places

Before trading begins, it is important to consider the legal form under which you will conduct business. There are four types of legal entity that can be set up in the UK, the most relevant of which is a limited liability company (Ltd). We will briefly discuss the other forms (a sole trader, partnership and cooperatives) for completeness.

The limited company

In this form of business your liability is limited to the amount you contribute by way of share capital. It is an independent legal entity and is separable from its owners, managers and employees. If the company fails therefore, any shareholders are not liable beyond the paid up value of their shares and creditors claims are only applicable to the assets of the company. In reality, this practice does not give enough security to lenders and you may be required to offer some guarantee (personal assets) to cover part of the loan. The limited liability company has a wider range of financing options when compared to other business forms. In particular, this can be by selling shares or creating a floating charge over its assets by selling debentures. Thus to summarise, the advantages of a limited liability company are: (a) it is a separate legal entity; (b) your personal assets will be protected (up to the value of your personal guarantees if you have given them); (c) there is a wider variety of security on offer; and (d) there are tax advantages upon which a specialist tax accountant will advise.

Introduction

The licensing option may or may not be suitable for your needs. Perhaps your aspirations and view of the market opportunity now dictate that you should ‘go it alone’ and consider starting your own company. It may be that you are in a position to set up a company and licence your technology through it, rather than your host institution. Whatever the circumstance, you will find that your business wants one thing more than any other – cash! The nature and type of financial arrangements that are available to you will be dictated by the purpose for which you require the money and the degree of risk involved for the investor. Investors are not in general of a charitable nature and will require a return on the investment. The more risk involved, the more justification and analyses you will be expected to provide. There are several mechanisms for financing the venture which are all dependent upon the degree of risk.

Short-term or long-term finance options

The time period of the investment is of significance, since all other factors being equal, shorter investments are likely to have a more accurate analysis associated with them. The variability of market conditions makes sales and cost predictions inaccurate beyond two years and thus an assessment of market risk will be necessary.

Introduction

A licence confers permission to do something which would otherwise be constrained. There are many forms of licence such as a fishing or driving licence and all have their own legal characteristics. However, licensing should be considered to be the tool of commercial practice and the route to mutual profit. To an organisation, licensing is a part of the overall activities of the company and cannot be separated from it. As such, licensing represents one of a series of options to a potential partner and this should be borne in mind once you have decided to licence your technology. Licences form an alternative to in-house R & D programmes and can be used to completely buy in a new technology or to augment existing programmes. Theoretically, the latter should maximise the use of internal resources without total dependence upon outsiders. Additionally, licensing can be used where direct capital investment in a project is difficult for political or other reasons. The action of licensing implies a degree of trust between the parties and could well be a long-term relationship, with the generation of a considerable amount of goodwill. Relationships can break down and you should take this into account when entering a licensing arrangement. Above all, when negotiating a licensing arrangement, proceed independent of trust.

Introduction

MHC antigens and the immune system

Since the report by Mitchison in the early 1950s demonstrating that cellmediated, rather than humoral, immunity played a greater role in tumour rejection (Mitchison, 1953), its primacy in tumour rejection has become an increasingly accepted mechanism by most immunologists. This has mainly been attributed to specific immunity involving MHC antigens as restriction element and to a lesser extent the non-specific immunity involving LAK/NK activity (Jabrane-Ferrat et al., 1990; Mule et al., 1984).

It has long been established that the MHC antigens are an individual's fingerprint and they exist in two forms, the class I and class II antigens. Zinkernagel & Doherty (1979) showed that they act as associative molecules for presentation of non-self antigens to CTLs and TH cells, respectively. The critical role of CTLs for regulating resistance to viral infection (McMichael et al., 1977) as well as in graft and tumour rejection in experimental models (Hui, Grosveld & Festenstein, 1984; Wallich et al., 1985) has previously been reported. TH cells have been shown to act mainly as an immune amplifier, since their stimulation results in the production of a series of immunoreactive cytokines, such as IL-2, which in turn are critical for initiating immune responses, including activation of CTLs (Greenberg et al., 1988).

Introduction

The growth cycles and oncogenic properties of the murine and human retroviruses are inextricably linked to the immune system. Those viruses that cause leukaemias, lymphomas or immunodeficiencies do so by infecting and often activating immune cells. Conversely, those retroviruses that induce solid tumours must evolve ways to aid the host cell in evading the cellular immune system. One major molecular mechanism by which these retroviruses can either activate or evade the immune system is by control of MHC class I antigen expression in the cells they infect. An effect of murine retrovirus infection on MHC antigen expression was first suspected in the late 1970s, when it was observed that thymocytes obtained from animals several weeks after infection with leukaemia viruses appeared to express higher levels of MHC class I antigens than thymic cells from control animals. Conversely, down-regulation of MHC expression on solid tumours induced by oncogene-containing (sarcoma) retroviruses had also been observed. Because of the experimental constraints of these in vivo systems, however, proof of a causal relationship between retrovirus infection and MHC regulation was lacking. More recent studies have demonstrated a direct action of retroviruses on MHC gene regulation and have begun to elucidate the ways in which these compact viruses, with only 6000–10 000 bases of coding sequence, regulate the histocompatibility antigen expression of their host cells.

Murine leukaemia virusesy

The murine retroviruses can be broadly divided into two classes: the leukaemia viruses and the sarcoma viruses.

While the association of particular major histocompatibility complex (MHC) haplotypes with human diseases has been extensively reviewed, we felt that the area of modulation of MHC antigen expression and its association with human and animal diseases was less well explored. These ideas were initially exchanged between the editors and some of the authors of this book at a most interesting meeting organized by the British Society for Immunology in Warwick in 1991 entitled ‘Viruses, Cytokines and the MHC’. Following this meeting, we realised that there was a need for a broad, interdisciplinary treatment of the area of MHC modulation that would be useful for both basic medical researchers and clinicians.

Expression of MHC class I and class II antigens follows a complex pathway from gene transcription to plasma membrane insertion and many steps can be stimulated or repressed leading to altered levels of cell surface MHC molecules. Therefore, to provide basic background information on the MHC, the genomic organization, antigen structure, biosynthesis and function and control of transcription of class I and II are considered first along with the important effects which cytokines and other extracellular agents can have on MHC antigen expression. Infection and oncogenic transformation of mammalian cells by viruses have provided powerful systems for analysing precise mechanisms of MHC antigen modulation and the relationship of this process to disease.

Introduction

To protect the individual from foreign agents, such as viruses and bacteria, mammals have evolved a sophisticated system that allows them to distinguish self from non-self. Self/non-self discrimination was first demonstrated in mammals by the rejection of foreign tissue grafts in mice (Gorer, 1936; Snell, 1958; Klein, 1975). The genetic loci involved in graft rejection were subsequently mapped to a region on chromosome 17 (Klein, 1975), which became known as the major histocompatibility complex (MHC). The human MHC, also known as the human leukocyte antigen (HLA) system, is located on chromosome 6 (van Someren et al, 1974).

The MHC occupies some 3.5 megabase pairs (Mb) of the genome and, in humans, approximately 75 genes (many still of unknown function) have been identified in this region (Trowsdale, Ragoussis & Campbell, 1991). The organization of the MHC is reviewed in detail in Chapter 2. The complex is often divided into three different classes of gene: I, II and III. There are multiple class I loci but the classical ‘transplantation antigens’ fall into three positions termed HLA-A, HLA-B and HLA-C in humans. The class II genes, encoded in the HLA-D region, encode proteins that help regulate the immune response to different antigens (Fig. 1.1). In early experiments that studied the genetic control of immunity to certain protein antigens, the immune response, whether strong or weak, was found to depend on particular alleles at loci within the MHC.

Introduction

Even before 1975, when the role of the MHC antigens as a ‘guidance mechanism’ for the immune system was discovered (Zinkernagel & Doherty, 1975), it was known that some tumour cells expressed abnormally low levels of MHC antigens and/or β2-m (Nilsson, Evsin & Welsh, 1974). In addition, there was ample experimental evidence that tumour cells could be ‘recognized’ and eliminated by the immune system, although early work had failed to take into account allogeneic recognition of transplantation antigens (Foley, 1953). By the 1950s it had been clearly shown that chemically induced, radiation-induced and spontaneously occurring tumours could express tumour antigens that could initiate and lead to ‘tumour rejection’, namely the tumour-associated transplantation antigens, TATAs (Gross, 1943). It was only in the late 1970s or early 1980s that these two strands of research could be put together and they have subsequently led to important advances in the clinical treatment of cancer.

These advances are related to the great insight of Paul Ehrlich, who postulated that the immune system acted as a surveillance system to detect changes within the body caused either by normal pathological events or by invading organisms (Ehrlich, 1909). It is now clear that the primary function of CTLs is to monitor cell surfaces for abnormal peptides presented by MHC class I antigens. T cells reactive to normal self peptides will have been made tolerant either by clonal deletion or clonal anergy.

Introduction

Infection with hepatitis B virus (HBV) can cause either an acute or a chronic hepatitis (Nielsen et al., 1971). Chronic infection is a major health problem affecting over 250 million people worldwide (Ganem & Varmus, 1987). Without treatment, chronic infection with HBV progresses to cirrhosis and/ or hepatocellular carcinoma in over 50% of infected patients. It is still not clear why some infected individuals develop a relatively mild, self-limiting hepatitis whilst others suffer from a prolonged, chronic infection. Recent research suggests that interferon-induced expression of MHC antigens on the surface of infected hepatocytes may play a key role in determining the outcome of infection with HBV.

Viral clearance in acute HBV infection

The majority of healthy adults infected with HBV develops a brief hepatitis, which resolves within a few months and is followed by elimination of the virus. A small percentage of infected adults (less than 5%) and the majority of infected neonates and children (greater than 90%) develop a chronic infection. Research in patients and chimpanzees acutely infected with HBV has provided an insight into the mechanisms underlying the normal eradication of HBV and these studies suggest that induction of MHC expression, by type I interferon (i.e. IFN-α/β), is a key factor in virus eradication.

Following infection with HBV, most adults show a marked increase in the serum concentration of circulating IFN (Ikeda, Lever & Thomas, 1986; Kato, Nakagawa & Kobayashi, 1986; Pignatelli et al., 1986).

Introduction: the CTL response to Epstein–Barr virus

Epstein–Barr virus (EBV) is a γ-herpesvirus that is carried by more than 90% of adults worldwide as an asymptomatic persistent infection. This virus is the causative agent of infectious mononucleosis, a benign lymphoproliferative disease and is also implicated in the pathogenesis of an increasing number of malignant diseases, including: Burkitt's lymphoma (BL), nasopharyngeal carcinoma and Hodgkin's disease (Miller, 1990). The importance of cellular immunity, and especially of CTLs, in controlling the potentially harmful consequences of EBV infection is underscored by the increased incidence of EBV-driven lymphoproliferations in organ transplant recipients receiving immunosuppressive therapy and in AIDS patients (Thomas, Allday & Crawford, 1991).

The pathogenic potential of EBV is well illustrated by the ease with which the virus can infect resting B lymphocytes in vitro, causing growth-transformation and the establishment of lymphoblastoid cell lines (LCLs) with infinite doubling capacity. This in vitro infection model provided the first clue as to how the virus is controlled in vivo. If circulating lymphocytes from EBV-seropositive donors are infected with EBV in vitro without first removing or inactivating the T lymphocytes, then the initial proliferation of EBV-infected B lymphocytes is followed by a regression at two to three weeks post-infection because of reactivation of EBV-specific memory T lymphocytes that eliminate the virus-infected cells (Rickinson et al, 1981). Activated T cells isolated from such cultures contain CTL populations that are HLA class I-restricted and specific for EBV-encoded antigens.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease with autoimmune features chiefly affecting the synovial joints. In common with many other autoimmune diseases, there is emerging evidence to suggest that susceptibility to RA is determined by several genes (Wordsworth & Bell, 1991). However, despite the evidence of a genetic predisposition (in particular HLA-DR4 or HLA-DR1), it is clear that the genetic component is probably relatively small since concordance rates in monozygotic twins are no more than 30%, suggesting that other factors are involved (Wordsworth & Bell, 1991; Rigby et al., 1991; Silman, 1991).

The target tissue for the autoimmune and inflammatory response in RA is the synovial joint. In healthy individuals, the synovial membrane is virtually acellular, but in RA it is infiltrated extensively by large numbers of cells from the blood, including activated T cells, macrophages and plasma cells (Janossy et al., 1981; Fϕrre, Dobloug & Natvig, 1982; Burmester et al., 1982). In addition, there is proliferation of fibroblasts in the lining layer of the synovial membrane. Many of these infiltrating and resident cells including endothelial cells, fibroblasts and T cells normally express little, if any, HLA class II antigens but are activated in the RA synovium and express abundant HLA class II antigens (Førre et al., 1982; Klareskog et al., 1982; Burmester et al., 1987). These cells may act as antigen-presenting cells and be involved in the stimulation of autoreactive T cells.

Introduction

Many animal viruses have the property of being able to modulate the expression of the MHC antigens of infected host cells and sometimes of uninfected cells of the host animal. Since the MHC is central to the immune system, this may be beneficial to the virus in evading an immune response and, therefore, have consequences for disease. The significance of such modulation can be seen from how widespread it is, its effects on pathogenicity and its prevalence in common and important human (and animal) infections. It will be shown here and below (Chapters 7 to 12) that modulation of MHC by viruses (and, briefly, other pathogens) is indeed widespread, does affect pathogenicity and is present in common and important infections. It will, therefore, be possible to conclude that modulation of MHC antigens is a highly important characteristic of viruses in general.

This chapter describes the different viruses for which modulation of MHC has been observed. Different mechanisms involved in modulation, both potential and actual, are summarized with suitable examples where known. This information is related to the pathogenicity of the virus where data are available. In the overall context of infectious disease, a brief note is made of other infectious organisms (i.e. microorganisms other than viruses) that are known to affect expression of functional MHC antigens with immunological consequences. Conclusions are drawn from these data on the importance of the modulation of MHC expression and on suitable directions for further research.

Introduction: immune recognition of tumour cells

In the late 1980s it became clear that cancer cells develop by multiple genetic alterations (reviewed in Weinberg, 1989; Bishop, 1991). These alterations include activation of proto-oncogenes as well as inactivation of tumour suppressor genes. Proto-oncogenes, more commonly called oncogenes, exert important functions in cell proliferation and differentiation and their activity is usually tightly controlled to ensure a minimal risk of inappropriate activity. Activation of oncogenes in animal and human tumours may occur through several mechanisms including amplification, elevated expression and point mutations. These genetic alterations usually result in an altered activity of the oncogene-encoded protein and this contributes to uncontrolled proliferation.

In the light of the crucial role that HLA class I antigens play in the interaction of altered self antigens or viral antigens with CTLs (see Chapter 1), one would expect that antigens specifically present in tumours are presented by HLA class I molecules. Such antigens might be viral antigens in the case of virally induced tumours or altered self proteins in the case of tumours induced by xenobiotics like carcinogens or radiation. In numerous animal and human tumours, mutations in oncogenes have been shown to be responsible for the tumorigenic properties of the tumour cell. These mutations are potential targets for recognition by the immune system of the host. Candidate oncogene and tumour suppressor proteins are ras and p53 proteins, respectively; these can be activated by various mutations and are involved in many forms of human cancer (reviewed in Bos, 1989; Levine, Momand & Finlay, 1991).

Introduction

Demyelinating disorders of the CNS, while bound together by a common process involving damage to or improper laying-down of the myelin sheath, do not share a common aetiology. This is demonstrated by recent advances, which have enabled the classification of these conditions into three groups based on disease aetiology: genetic, viral and autoimmune.

Pelizaeus–Merzbacher disease exemplifies the genetic component of certain demyelination conditions. This X-linked recessive disorder affecting CNS myelination in children is attributed to deficient biosynthesis of the proteolipid protein (PLP) (Koeppen et al., 1987). Lack of PLP, one of the most abundant components of the myelin sheath, contributes to physical symptoms such as spastic extremities and lack of purposeful movement and head control.

The viral aetiology of demyelinating diseases is seen in progressive multifocal leukoencephalopathy (PML). Originally described as a complication of leukaemia and Hodgkin's disease (Aström, Mancall & Richardson, 1958), PML develops mostly in immunocompromised patients with chronic diseases and, more recently, in individuals with AIDS (Levy, Bredsen & Rosenblum, 1985; Niedt & Schinelle, 1985). Symptoms in PML reflect the widespread destruction of the CNS white matter. Neurological deficits include dementia, confusion, aphasia, hemiparesis and ataxia. The viral aetiology of PML has been demonstrated by isolation of the papovavirus JC from the brains of diseased patients (Padgett et al., 1971) and by the demonstration that transgenic mice with part of the JC viral genome develop a neurological disorder that involves dysmyelination of the CNS (Small et al., 1986; Feigenbaum, Hinrichs & Jay, 1992).

Introduction

T lymphocytes play a central role in antigen-specific immune responses through their interactions with other cell types. These interactions are guided by specific T cell recognition of antigen associated with cell surface molecules of the MHC. The two main types of MHC molecule (class I and class II) are both involved in antigen presentation, but to different types of T cell (expressing CD8 or CD4 antigens, respectively). In addition, they normally show very different patterns of expression within tissues: most cell types of the body express MHC class I antigens, whereas the expression of MHC class II antigens is restricted primarily to cells of the immune system – macrophages, dendritic cells, B lymphocytes and (in humans) activated T lymphocytes. Teleologically, this accords with the usual functions of the T cells, which interact with antigen associated with the different types of MHC protein. Thus, class I-restricted, CD8 cytotoxic T cells may be required to interact with antigen presented by almost any cell of the body (for example, following viral infection). By contrast, a major role of class II-restricted, CD4+ helper T cells is to interact with, and help, other cells of the immune system.

Tissue distribution of MHC antigens

The distribution of MHC antigens is now known to be complex. The expression of class I antigens varies in intensity with, for example, a gradation of expression from strong to weak or negative through the following cell types: cells of the immune system, epithelial cells of the gastrointestinal and respiratory tracts, endocrine cells, striated muscle cells, hepatocytes and neurons (reviewed by Pujol-Borrell & Todd, 1987).

Introduction

The MHC is localized on the short arm of chromosome 6 in band 21.3. It extends over 4 Mb of DNA, which have been intensively studied for several decades. Detailed physical maps have been derived and the entire complex has been cloned in cosmid and yeast artificial chromosome (YAC) vectors. About 80 genes have been identified so far, which include the genes encoding the class I or classical transplantation antigens, the class II immune response genes, the class III genes originally defined by complement components and a number of novel genes involved in antigen processing or of unknown function (Trowsdale, Ragoussis & Campbell, 1991). It is intriguing that so many genes involved in immune responses are closely linked to each other in the genome. The class I and class II sequences have been maintained together through evolution and can be found on the same chromosome in species like chicken, mouse and humans. The organization and functional relationships between MHC genes will be presented in this chapter along with a description of MHC transcripts.

The class I region

The human class I region

The class I region is 2 Mb in length and contains the class I multigene family comprising about 20 non-allelic DNA sequences (Fig. 2.1).