Flashcards in Immunology Deck (57):
Innate immune defences consist of:
• physical barriers such as mucosal epithelium;
• secretions with antibacterial activity, including
• phagocytic cells: monocytes, macrophages and
• NK cells (lymphocytes capable of non-MHC restricted killing);
• soluble mediators which can enhance the activity
of innate and specifi c responses: C-reactive protein (CRP), mannose-binding lectin (MBL),
• soluble enzymic cascades such as the complement system, which is activated directly by exposure to pathogens and serves to directly lyse the pathogen, or to enhance and target the activity of innate and specifi c effector cells by opsonisation and activation via cell surface receptors for complement components.
Adaptive immune response (See diagram)
Specific (adaptive) immune responses are more effective than innate ones and are mediated by lymphocytes and antibodies which amplify and focus non-specific responses and provide additional effector functions. These cells are organised into lymphoid tissues. Humoral immunity often refers to the antibody arm of the specific immune response. Cellular (cellmediated)
immunity refers to lymphocyte-mediated
effector responses (T helper (Th) and cytotoxic cells) of the specific immune response. These two arms of the specific immune response are not really separable, since antibodies are usually not produced without some cell-mediated response to the same antigen and vice-versa. T and B lymphocytes possess infi nitely variable antigen receptors which can clonally expand.
Antigen receptors which can be secreted into interstitial fluid and onto mucosal surfaces are called antibodies. Antibodies can activate complement and also enhance opsonisation of antigen to facilitate phagocytosis. Both innate and adaptive mechanisms exponentially amplify the immune response, since clonal expansion of lymphocytes increases the number of cells reactive
with an antigen. Cytokines and complemen components recruit other immune effector mechanisms and antibodies activate complement and phagocytes.
The complement system is a soluble enzymic cascade which focuses and amplifi es the activity of the specific and innate immune systems as well as having lytic activity against bacteria (Fig. 6.3). It is part of the innate defences since it has no intrinsic antigen specificity.
The complement cascade has a fi nal common pathway which leads to the insertion of a multimeric poreforming structure (membrane associated complex (MAC) consisting of complement components C5-9) into bacterial cell membranes, leading to osmotic lysis.
The production of this lytic complex is achieved via
two mechanisms called the classical and alternative
pathways. Inability to generate the MAC complex
leads to particular susceptibility to infections with
Neisserial organisms causing recurrent meningitis.
The classical pathway is triggered by antigen-antibody immune complexes which bind circulating complement factor C1q to the Fc region of the antibody tail, which has undergone conformational changes as a result of antibody binding. The resultant sequential activation of complement proteins results in the formation of a C3 convertase (C4b2b) which cleaves C3, thus forming a C5-convertase (C4b2b3b) which catalyses the production of the C5-9 pore-forming complex.
In the process, C2, C3 and C4 are split into fragments, the smaller of which (C2a, C3a, C4a) are chemotactic and the larger of which (C3b, C4b) bind to immune complexes to opsonise or solubilise them, or to a pathogen surface to opsonise it. Thus multiple effects ensuing on other effector mechanisms are caused as a result of complement activation. CRP and MBL can directly activate the classical pathway of complement without the intervention of immune complexes.
The lectin pathway is very similar to classical pathway complement, with MBL binding to mannose on patho gens, which is then sequentially bound by MASP to form a C3 convertase.
Alternative pathway I
The alternative pathway is phylogenetically older than the classical pathway and is triggered by contact with exposed bacterial capsules without the need for prior antibody production. Factors B and D (analogous to the classical pathway C4 and C2) again lead to the production of a C3 convertase (C3bBb) and a C5 convertase (C3bBb3b), leading to opsonisation, chemotaxis and the final common pathway in a similar way to the classical pathway.
Complement activation is closely regulated by
various factors, because uncontrolled complement
activation would lead to tissue injury and infl ammation.
Alternative pathway II
C1-inh is the plasma inhibitor of fi rst component of complement. It is also the major plasma inhibitor of activated Hageman factor (the fi rst protease in the contact system) and of plasma kallikrein (the contact system protease that cleaves kininogen and releases bradykinin).
This diagnosis should be considered in patients presenting with recurrent abdominal pain where C4 levels are low. Acute management is with intravenous C1 inhibitor replacement, prophylaxis by increasing production with danzole,
or decreasing consumption by tranexamic acid. New inhibitors of bradykinin are in development.
Examples of diseases caused by abnormalities of complement control
Examples of diseases caused by abnormalities of complement control include:
C1 esterase inhibitor deficiency (hereditary angioedema)
C3 nephritic factor in type 2 MPGN, factor H deficiency in familial HUS.
Hereditary angioedema (HAE) is a rare autosomal
dominant disorder of C1 inhibitor (C1-INH) deficiency.
Deficiency leads to uncontrolled complement and
kallikrein activation resulting in edema of subcutaneous or submucosal tissues. Acute abdominal pain, nausea, and vomiting are the dominant symptoms in 25% of patients with HAE.
The presentation may mimic an acute abdomen
with peritonitis and effusions and many have had
invasive surgical investigation before diagnosis.
An antigen is any substance which can elicit a specific immune response. An antigen consists of many epitopes. An epitope is a specifi c sequence of a protein or carbohydrate recognised by the receptor molecules of the immune system (antibody or T cell receptor).
Antigens can be divided into foreign (non-self, allogeneic, xenogeneic, etc.) and self-antigens (autoantigens). Although an antigen usually elicits an immune response, if the antigen is encountered in appropriate circumstances the specific immune response may be switched off by a variety of mechanisms which will be important to consider when discussing the immunology of transplantation and autoimmune diseases.
An antibody is a soluble protein immune receptor produced by B lymphocytes, consisting of two identical antigen-binding sites (Fig. 6.4). The antigen specificity of the antibody resides in the antigen-binding variable regions (the fragment antigen-binding, Fab, portion).
Antibodies are divided into different isotypes (classes) which have different functional attributes due to the Fc (fragment constant) tails coded by the constant region genes of the heavy chain; thus different constant region genes produce different antibody classes.
Antibodies which bind to antigen or cells and activate complement via the Fc region thus recruit, activate, amplify and target non-specifi c defence mechanisms. Up to 1010 different antibody specifi cities may be produced in any individual. This is achieved by joining multiple different copies of genes encoding the variable regions of heavy and light chains of the immunoglobin.
Somatic recombination I
Somatic recombination of the gene segments (V, D and J region genes) leads to generation of diversity and broad repertoire of antibody specificities. The antigen binding variable regions are further (infi nitely) diversified by a combination somatic hypermutation which results from random mutations to the V genes in the hypervariable regions (mutation hotspots) and to the joins between V, D and J genes, enabling antibodies to be produced which can bind to virtually any natural or synthetic antigen encountered. Each cell producing antibody which binds an epitope of an antigen is stimulated to clonally reproduce, and thus further amplification of the immune response occurs with the progeny of each cell producing exactly the same antibody but many different clones expanding.
Somatic recombination II
Most antibody immune responses are polyclonal
(many cell clones expand, each recognising different, sometimes overlapping, epitopes on the antigen); oligoclonal responses occur when a limited number of clones expand for some reason (e.g. prolonged infl ammation); monoclonal proliferations are usually representative of malignant transformation of a single
clone of a B cell at some point in its differentiation
(early or late B cells = lymphoma, and often produce IgM; terminally differentiated plasma cells = myeloma and usually produce IgG/A isotypes).
The antigens recognised by antibodies are often
conformational (that is, they require a folded 3D structure for recognition), often bringing widely separated areas of a larger molecule together to form the epitope (which is, therefore, discontinuous in linear sequence, unlike the epitopes recognised by T cells). Antibodies thus tend to recognise native folded-3D structures.
Somatic recombination III
Most antibody production is ‘T cell dependent’ (i.e.
very inefficient in the absence of T cells, which recognise linear epitopes on the same antigen as that recognised by the antibody and provide ‘help’ (co-stimulation to amplify responses) to B cells,). A small number of relatively ‘T-independent’ B lymphocytes exist which bear the CD5 surface antigen. They tend to recognise conserved
carbohydrate epitopes on pathogens (including
human ABO blood groups), produce IgM and may represent a phylogenetically older type of B cell defence.
Isotypes and subclasses I
B lymphocytes initially produce IgM upon a primary
encounter with antigen; this is very efficient at complement fixation and opsonisation, but IgM circulates as a large pentameric (fi ve antibody molecules) structure with a short half-life (!fi ve days). Subsequently an individual B cell will undergo a class-switch to IgG, IgA, or IgE production, but class-switching depends
on effective T cell help following T cell recognition of an epitope on the same antigen.
Memory develops in parallel with the class switch. Both these processes require effective communication between B-cells, Antigen Presenting Cells (APC) and T cells (mediated by CD40–CD40L interaction).
Isotypes and subclasses II
IgG diffuses well into extracellular spaces and can neutralise circulating viruses and bacteria (prevent binding by blocking receptors), opsonise via complement or Fc receptors or lyse via complement activation. IgG is divided into four subclasses (IgG1, IgG2, IgG3, IgG4) which have different Fc regions (and thus are coded by different heavy chain constant region gene segments).
These classes and subclasses have different half lives and abilities to fi x complement, or bind Fc receptors (Table 6.3). There are several different types of Fc receptors (FcRI or CD64, FcRII or CD32, FcRIII or CD16) which bind some IgG subclasses better than others and are distributed differently on each effector cell type. IgG1 constitutes 60–70% of the circulating IgG in man;
IgG2 constitutes 20–40%. IgG3 constitutes 15–20%.
Isotypes and subclasses III
IgG4 circulates in trace amounts and its functional signifi cance is unknown, although it may be important in IgE-mediated antiparasite and allergic responses. IgG1 and IgG3 tend to be produced in response to protein antigens; IgG2 in response to polysaccharide antigens (such as those of bacterial capsules).
IgA is secreted preferentially onto mucosal surfaces and is important in prevention of initial adherence to epithelium or mucosal penetration (blocks interaction with cell surface receptors) of bacterial and viral pathogens spread via respiratory or gastrointestinal routes. IgA defi ciency thus predisposes to mucosal infections.
The gut contains peptidases which degrade IgG and IgM rapidly. IgA is protected from destruction by a remnant of the polyIg receptor (which selectively transports secretory IgA across epithelium to the outside of the mucosal surface) called the secretory component, and IgA is usually secreted as a dimer joined by a j(oining)-piece. Most secretory IgA is of the IgA2 subclass; most
circulating in serum is IgA1. The signifi cance of this is uncertain. Unlike most IgG subclasses or IgM, IgA does not effi ciently fi x complement via the classical pathway of complement activation.
Antigen presenting cells I
In contrast to antibodies, T cells can not recognize
native antigens. They recognise short linear peptides on the surface of APC which digest the whole antigen and present the fragments on the surface in the grooves of major histocompatibility complex (MHC) Class I or II molecules (MHC restriction). The initial interaction of T-lymphocytes with antigen is important in determining whether a specific immune response is promoted or suppressed.
The default pathway in unprimed ‘naive’ cells (which have not encountered specific antigen before) is either to become specifically unresponsive to the antigen (anergy) or to die (apoptosis) if the antigen is encountered in an insufficiently stimulating context. Naive T lymphocytes are relatively refractory to stimulation, and require potent signals to activate them to clonally proliferate and/or become effector cells. This usually occurs centrally in the lymph nodes, bone marrow or spleen, but can occur elsewhere. These extra signals are complex and multifactorial but act in addition to the recognition of antigenic peptide in the MHC groove by the T cell receptor (TCR) on the CD4 or the CD8 T cell.
Antigen presenting cells II
In addition to the recognition of antigenic peptide in the MHC groove by the T cell receptor (TCR) on the CD4 or the CD8 T cell.
This incorporates adhesion molecules which stabilise contact between lymphocyte and APC, and costimulator molecules which provide activation signals to the T cell from the APC (Immunological Synapse – cf. neurological synapse). Important interactions occurring at the immunological synapse are shown in Table 6.4.
APC of several different types provide these second signals while presenting a processed fragment of antigen to a lymphocyte. Primary stimulation of naive T cells requires a potent professional APC (such as the Dendritic cell (DC) or an activated B lymphocyte) with potent stimulatory capacity and ability to acquire and process (digest) antigen by phagocytosis or endocytosis. Secondary restimulation of recently activated or memory T cells is less stringent and can occur on non-professional APC which are not potent enough to stimulate naive cells effectively, e.g. activated endothelium or monocytes and other cells expressing MHC Class II molecules.
Antigen presenting cells III
‘Professional’ APC such as DC are resident as sentinels in the skin (Langerhans’ cells) or in the interstitium of most tissues (interstitial DC) including lymph nodes (interdigitiating DC). On encounter with antigen, DC become activated (mature) and migrate centrally via lymphatics to become resident in the T cell areas of lymph nodes (paracortical area) as interdigitating cells. There, T cells recirculating through lymph nodes via lymphatic drainage encounter antigen and clonally proliferate, if they carry the appropriate antigen-specifi c TCR. Subsequently they migrate back to the peripheral tissues and elicit a local immune response. Similar processes occur in the spleen and Peyer’s patches. B cells may also be stimulated directly by DC.
T cells recognise antigen fragments on the surface of APC which express MHC Class I and II molecules on the surface. MHC molecules have an antigen-binding groove on the surface which can bind antigen fragments of 9–11 amino acids (MHC Class I) or 14–20 amino acids (MHC Class II) in length. Thus they act as display platforms on which the TCR can recognise antigen, but because they bind antigen fragments themselves, the MHC molecules also influence the immune responses in any individual since each MHC type will bind some antigens better than others, and occasionally won’t bind some antigens at all.
T Lymphocytes: TCR
The TCR binds to a part of the lips of the groove as well as the antigen fragment. Thus the TCR is also self restricted (MHC Restriction), since it binds only to the combination of [self antigen (MHC) + foreign
antigen]. A T cell will not operate effectively with nonself APC which bears different MHC molecules. They can, however, co-operate with non-self cells provided they express the same MHC molecules (as they have to do in allogeneic bone marrow transplantation where the BM is donor-type and the recipient is host-type).
T Lymphocytes: MHC (i)
MHC Class I is bound by CD8, and MHC Class II by CD4 on the T cell surface (Fig. 6.5). Virtually every nucleated cell expresses MHC Class I on the surface, but MHC Class II expression is restricted to certain cell types (e.g. Professional APC, B lymphocytes) especially when the cell is activated. APC express MHC Class II in high density and thus are the major activators of CD4 positive lymphocytes. MHC Class I restricted CD8 positive T cells are also stimulated by APC, but they recognize foreign peptides (e.g. viral, intracellular
bacteria) on all nucleated cells by ‘seeing’ viral antigen in the surface groove of self-MHC Class I, and are activated to deliver a lethal attack on the cell.
Not surprisingly, viruses have adapted to reduce MHC Class I surface expression (e.g. adenovirus) and can partially evade their attentions (NB NK cells recognize this lack of MHC class I as a sign of an infected cell). Degraded intracellular antigens in the cytosol tend to get access to the MHC Class I groove in the process of MHC assembly in the endoplasmic reticulum, and thus responses to intracellular antigens tend to occur via the MHC Class I pathway (Fig. 6.6).
T Lymphocytes: MHC (ii)
Extracellular antigens from bacteria phagocytosed and digested in the lysosomes of APC tend to gain access to MHC Class II most (readily since the assembly pathway of MHC Class II molecules intersects with the lysosomal pathway). Thus degraded extracellular antigen gains access to ‘empty’ MHC Class II molecules after the invariant chain (which occupies the MHC groove prior to antigen binding in order to let the molecule pre-assemble without antigen) is displaced by alterations in the intralysosomal pH. All T cells have CD3 and TCR complex on their surface. The T cell receptor requires various co-receptor molecules (LFA-1, CTLA-4, CD28, CD40L) to be associated with it on the cell surface in order to enable
effi cient antigen recognition and signalling from
antigen-presenting cells. Therefore any T cells lacking these co-receptors will fail to function normally.
T Lymphocytes: CD4 and CD 8
CD4-bearing T cells generally have ‘helper’ functions; those which aid B cell antibody production are called Th2 and those which activate mononuclear phagocytes and promote cellular inflammatory activity are called Th1 (Table 6.5). These T cells types tend to produce different cytokines when activated by antigen; Th1 produce pro-infl ammatory IFNgamma, IL-1, IL-12,
TNF"; Th2 produce IL-4, IL-5, IL-13, TNF" and
others which promote antibody production. In any
particular immune response one type of T helper
activity will often dominate. This is important both
in defence against infection and in the pathogenesis of immunologically-mediated diseases. CD8 positive T cells in contrast often have cytotoxic effector properties and are critically important in defense against certain viral infections.
Once T and B lymphocytes have recognised their cognate antigen, they proliferate by producing clones of themselves and acquire effector functions which orchestrate the immune response. Some of these proliferating lymphocytes differentiate into long-lived cells which are called memory cells. B cells differentiate into long-lived plasma cells and some memory B cells are constantly re-stimulated by long-lasting reservoirs
of antigen on the follicular dendritic cells (FDC) in
lymph nodes. A secondary (memory) response thus involves the activation of an expanded pre-existing panel of antigen-reactive clones, which have differentiated to produce IgG or IgA rather than IgM, giving a response magnifi ed in both quantity and quality.
Previously activated cells have been ‘primed’ and thus are more readily activated by small amounts of antigen on APC, since they require less stimulation through their co-receptors. During an immune response, B cells are selected by competition for antigen on FDC in the lymph nodes; those binding strongly survive, others die through inability to compete for antigen and loss of a survival signal. Thus the antibody response undergoes a process of affi nity maturation (each generation of antibodies binds better to antigen). Antigen experienced T – cells differentiate into central memory (CM) T – cells and are primed to produce enhanced secondary immune responses on re-exposure to same pathogen.
Uncontrolled activaton of the immune sytem and
consequent infl ammatory processes is potentially
life threating if it is not tightly regulated. In order to
prevent an escalating cycle of destruction leading to inevitable death of the organism there are cellular
and humoral mechanisms which downregulate the
response of both innate and adaptive responses after activation. Down-regulation of cellular responses may involve cellular death as a result of apoptosis (programmed cell death) – either as a result of a cell completing its lifespan (approximately 20 clonal divisions for a T cell) or as a result of direct immunological interaction
with regulatory T cells. This apoptotic process
is non-inflammatory, unlike necrotic cell death.
T Cell stimulation: Signals 1 and 2
The principle of initiation of an immune response is
the same whether it is to an infection, to self-antigen in autoimmune diseases or to non-self MHC in an alloimmune response in transplantation. It requires both naïve and memory lymphocytes and the recognistion of antigen through the specifi c receptor (signal 1) and the simultaneous provision of additional co-stimulatory signals though other cell surface receptors (signal(s) 2). On internalizing the antigen, APCs become activated and move to the secondary lymphoid organs, bringing the antigen to the central lymphoid system where large numbers of T cells and B cells are present.
The antigen on the surface of dendritic cells triggers the T – cells with an appropriate T – cell receptor which recognizes the MHC-bound antigen fragment and this constitutes ‘Signal 1’, transduced through the TCR-CD3 complex. Co-stimulation or 'Signal 2’ which is delivered via CD80 (B7.1) and CD86 (B7.2) on APC to CD28 and other molecules on the T-cells.
T Cell stimulation: Signal 3
Signals 1 & 2 activate three internal signal transduction pathways:
• calcium – calcineurin pathway;
• RAS-mitogen activated protein-(MAP) kinase
• NF-kB pathway.
These pathways lead to activation of transcription
factors inducing expression of IL-2, CD-154 and
CD25. IL-2 and other cytokines activate the ‘target
of rapamycin’ (TOR) pathway to provide ‘Signal 3’,
which triggers cell proliferation. This results in the
clonal expansion of lymphocytes leading to generation of antibodies and of T-cell effector functions.
T cells recognise antigen together with self-MHC
epitopes in the antigen-binding groove of MHC
molecules. Strongly self-reactive cells are eliminated (deleted) by encounter with self-antigen on thymic APC (thymic epithelium and DC) in early fetal life.
Some self-antigens are probably not expressed in the thymus and remain hidden from the immune system (cryptic epitopes, e.g. intraocular antigens), and tolerance is not established. These antigens tend to reside in immunopriviledged sites, and an immune response does not occur unless released by trauma (e.g. sympathetic ophthalmitis). In adults any cells capable of some degree of self-reactivity which escape deletion in the thymus are probably actively suppressed or made unresponsive (anergised) by peripheral mechanisms which involve T – regulatory cells (CD4+, CD25+
FoxP3 positive T cells).
Self Tolerance and autoimmune diseases
Autoimmune disease may occur either by reactivation of anergised cells by encounter with potent APCs in certain circumstances which override their programmed unresponsiveness (e.g. where a strong immune response to another antigen results in bystander help sufficient to activate self-reactive T cells in the vicinity), by
cross-reactivity between self- and foreign antigens,
or as a result of inherited or acquired defects in molecules important in the control of immune responses and maintenance of anergy (e.g. Fas/FasL deficiencyleading to defective apoptosis).
The clinical phenotypes of the autoimmunity probably reflect the predominant effector mechanisms and the organ specificity of the antigen(s) and may result in direct damage or interference with normal function. The identity of many autoantigens is now known. Some are receptors, some enzymes.
Autoimmunity may also occur because of failure of induction of self tolerance in the thymus (e.g. auto immune regulator protein (AIRE) abnormality leading to autoimmune poly-endocrinopathy, candidiasis, ectodermal dysplasia – (APECED)) which results from an inability of the thymic APC to present self antigens to maturing T cells and, therefore, a failure of deletion of self-reactive T cells.
Self Tolerance and Organ-specific autoimmunity
Organ-specific autoimmunity manifests itself by
damage or malfunction of a single organ as a result
of a specific immune response, usually to multiple
antigens or to multiple organs on the basis of shared antigens (e.g. steroid cell antibodies linking Addison’s disease and premature ovarian failure, or the lung and kidney damage of Goodpasture’s syndrome). In some conditions (e.g. myasthenia gravis) humoral responses play a major role in many of the disease manifestations, but are unlikely to occur without cellular responses which may also be important.
In other diseases, cellular mechanisms may be the predominant pathogenic response: e.g. extrinsic allergic encephalomyelitis (EAE, a model for multiple sclerosis) to myelin basic protein (MBP) and other intracerebral autoantigens. In systemic autoimmunity such as SLE, pathogenesis
is multifactorial and involves multiple unrelated
antigens. Humoral and cellular responses to multiple nuclear (nucleosome) and cytoplasmic components are seen, particularly anti-double-stranded DNA antibodies (dsDNA) which can cause an immune complex nephritis.
Sometimes titres of antibodies or complement levels (reduced by immune complex deposition) reflect disease activity in an individual, but in others they do not. In some diseases, the autoantibodies or lymphocytes are pathogenic in models of disease (e.g. anti-dsDNA antibodies in SLE; anti-GBM antibodies
in Goodpasture’s). In other diseases they are not, and may be secondary markers of damage (e.g. many antinuclear antibodies (ANA) in SLE, antithyroid peroxidase antibodies in thyroid malignancy).
MHC antigens and autoimmunity I
MHC antigens are inherited (along with a package of minor antigens) as a haplotype consisting of an HLA-A, -B, -C (Class I); -DR, -DP, -DQ (Class II) allele from each parent (Fig. 6.7). Allogeneic immune responses (against a foreign MHC antigen from the same species) can be generated after transplantation.
The MHC molecule on the APC determines the type and composition of the peptide fragment that it
can present to the naïve T cell, and is an important fac- tor in predisposition, protection or disease expression. Certain alleles or haplotypes are associated with par- ticular diseases (Table 6.8). Both organ-specific and non-organ-specific autoimmune diseases are associated with similar MHC haplotypes in some cases, suggesting an inherited predisposition.
MHC antigens and autoimmunity II
Few MHC associations with diseases are very strong (most strongly seen between B27 and ankylosing spondyli- tis), since most conditions are multifactorial and are a result of a combination of genes and additional envir- onmental influences, perhaps including infection.
The apparent association of MHC Class I alleles (e.g. HLA-B27) and MHC Class II (e.g. DQB1) may also be due to molecular mimickry between pathogen and MHC, resulting in autoimmune attack. (Heat shock protein (HSP) 60 is widely conserved and gener- ates immune responses in bacterial infection and some autoimmune diseases.). In contrast, some of MHC haplotypes may actually confer protection from some infections and autoimmune disease.
Transplantation barrier I
Non-self antigens are subject to immune-mediated attack by adaptive humoral and cellular mechanisms. The most important antigens are those most widely expressed on the graft, e.g. ABO blood group antigens, and those eliciting strong responses, e.g. disparate MHC antigens (allogeneic response). Any other polymorphic cell surface molecule on the graft which is not expressed by the recipient will also elicit an immune response. In the case of cross-species grafting (xeno- geneic transplantation), the rejection response is even stronger as a result of increased disparity between the MHC molecules and the presence of broadly reactive antibodies which bind to the graft and cause hyperacute rejection.
The aim of immunosuppression is to depress the effector immune response to prevent graft rejection (at least initially). The hope is that subsequently either tolerance or graft acceptance will result from downregula- tion of the antigraft response and enable withdrawal of immunosuppression. The aim of ABO-matching and HLA-matching (tissue typing) is to reduce the anti-genic disparity between the graft and the recipient.
Transplantation barrier II
Other antigens clearly exist (e.g. endothelial antigens) but matching for these is not currently practicable; however, genetic linkage of genes means that related donors with a haplotype match are likely to share the same non-MHC genes. Cyclosporin A (CsA), a fungal metabolite, prolonged survival of renal transplants in man in the late 1970s.
By this time, however, graft survival from living related donors had reached a plateau, suggesting that early graft survival results from ABO matching and immunosuppressive drugs, with some contribution from HLA-DR matching (which is more effective than HLA-B or HLA-A matching). Some have, therefore, argued that the benefit of HLA match- ing is insignificant with modern immunosuppressive drug regimes; however, it appears that long-term graft survival appears more dependent on HLA-A and -B matching (Fig. 6.8).
Solid organ grafts contain passenger leucocytes, including lymphocytes and APC. The most important passenger leucocytes in the graft are dendritic cells expressing high levels of MHC Class II. Experimental depletion of these APC pre-transplant improves graft survival, but this strategy is not in routine use in human transplantation.
The object of tissue typing is to match the donor tissue to the recipient by the ABO blood group and the human leukocyte antigens (HLA) they express. In addition to assessing the degree of antigen mismatch between donor and recipient pairs, it is also necessary to ensure that the recipient does not have pre-formed anti-bodies to donor MHC antigens.
These may have arisen through blood transfusions or pregnancy, or from previous organ transplantation. A cross-match test is performed to ensure no anti-graft antibodies are present that could mediate rejection. Sensitization, is indicated by the presence of anti-donor antibodies prior to transplant, but the definition of high risk sensi- tization varies between centres from 50–90% of ‘panel reactivity’ (a panel a wide range of HLA alleles).
Currently, renal transplants are matched for ABO blood group, direct cross-match for anti-HLA alloanti- bodies and HLA matching (Table 6.9). Cross-matching is now usually performed using flow cytometry rather than lymphocytotoxicity assays for HLA class I react- ive IgG and IgM antibodies (predictive of antibody- mediated hyper-acute rejection) since it is easier to perform. Potential recipient sera are stored at inter- vals while awaiting a donor, for retrospective analysis. Flow cytometric cross-match may be more sensitive but some positivity is of uncertain significance and expert interpretation is required to determine the suitability for transplant in individual cases.
The accuracy of HLA typing depends on the tech- nology employed. HLA-DR matching confers better protection against graft loss in the first year than HLA-A or -B in the presence of cyclosporin. HLA- DR mismatch increases graft loss by five-fold, HLA-B mismatch by three-fold and HLA-A mismatch by two- fold. However this translates to only a minor (3–5%) increment in graft survival when immunosuppression with cyclosporin A is used, and it is often better to use a fresh but mismatched kidney locally, rather than endure prolonged ischaemic time in search of a better match elsewhere.
The technique for determining the HLA-type is important. The serological techniques for HLA class-I matching may be unable to distin- guish between certain alleles, and apparent identity on serology may miss minor differences in sequence which can be recognised by the immune system. In general, molecular techniques such as oligonucleotide probes are more specific and sensitive and used when matching for bone-marrow transplantation.
Ischaemia and reperfusion
The process of organ procurement and implantation results in severe physical stress on solid organs used in transplantation. Every transplant organ faces two insults – ischaemia, and later re-perfusion. Ischaemia results in build-up of toxic products of anaerobic res- piration (e.g. lactate), that contribute to free radical damage upon re-perfusion of the organ with recipient blood.
Severe ischaemia–reperfusion injury (I-RI) leads to delayed graft function post-transplant. I-RI may also make the transplanted organ more visible to the immune system of the recipient and promote activation of both innate and adaptive immunity against the donor organ. This is mediated by release of cytokines and chemokines from the damaged tis- sue leading to inflammation and facilitates a potent immune response.
Organ preservation I
To minimise the effects of I-RI, and to allow time for organ transportation and allocation to the most suit- able recipient, then surgery, the established method for organ procurement comprises an in-situ irrigation with a suitable cold-preservation solution, and hypo-thermic storage at 0–4oC.
The core components of these preservation solu- tions are the impermeants (usually sugars like glucose) that prevent fluid entry into cells and subsequent cellu- lar oedema (cell swelling), buffers to maintain pH, and ions. During warm ischaemia (Warm ischaemic time i.e. the time from cessation of circulation until per- fusion with cold preservative. In heart-beating donors this time is theoretically zero), active transport mech- anisms involving Na/K and Ca2/Mg2 ATPase are inhibited, which leads to a steady influx of Na, Cl and Ca2 into the cell with subsequent influx of water
causing cellular swelling.
Organ preservation II
This process is further accelerated during cold ischaemia (the time from per- fusion with ice cold preservative until circulation is re-established in the recipient), resulting in reperfusion injury. Current buffer systems include, phosphate, citrate, histidine and bicarbonate. It is thought that a high potassium concentration helps to prevent the build up of intracellulular calcium during ischaemia. Solutions having a high potassium content are classed as ‘intra-cellular’ type (Euro Collins (EC), University of Wisconsin (UW)) solutions. ‘extracellular’ type solutions contains only sodium and no potassium (Phosphate Buffered Sucrose-PBS140).
The cellular oedema caused by the influx of water is the primary event that damages ischaemic organs. In the human kidney, the proximal tubules appear most susceptible to I-RI. Cell volume is actively regulated in vivo but this regulation is lost in ischaemic tissue since the process is energy dependent and ATP is rap- idly depleted in an ischaemic organ. Advances in organ preservation will provide ways not only to improve the condition of a donated organ, but may lead to a reduction in the numbers of organs not used because of excessive ischaemic time.
A renal transplant is most likely to be lost in the first three months, but rejection is only one possible cause of graft loss. Most patients have at least one episode of acute rejection. Major immunologically mediated anti- transplant responses can be directed against several antigens, including A, B, O blood group antigens, MHC Class I and II molecules and cell-surface carbohydrates (e.g. alpha-gal in xenogeneic transplantation).
Anti-transplant responses can also occur against other cell-surface antigens which are poorly defined and for which matching is currently not performed (except serendipitously) in transplants from identical twins or close relatives. The presence of a non-self MHC on a cell surface will generate a strong allogeneic cellular and humoral immune response. 50% of renal grafts have at least one episode of acute rejection.
Hyperacute rejection is caused by pre-existing, complement-fixing antibodies. This should not happen with current tissue typing and matching strategies. Rapid allograft rejection (coagulopathy, infarction and neutrophil infiltrate mediated by antibody and complement) occurs within minutes or hours and is caused by IgG anti-HLA Class I (not IgM), or ABO antibodies (hence utility of cross-matching and ABO matching pretransplant). There is no effective therapy except prevention by screening allograft recipients and rapid graft excision once established.
Accelerated rejection (5 days)
Accelerated rejection is usually mediated by pre-existing non-complement-fixing anti-HLA antibodies in sensi- tised patients. Flow cytometry may pick up positive cross-matches missed by standard lymphocytoxicity testing. Early biopsy reveals interstitial cellular or vascular rejection. Some centres biopsy high risk grafts early to pick this up. High dose IVIG, plasmapheresis, rituximab and antilymphocyte agents such as OKT3 may be effective but less so than for acute rejection.
Acute rejection (100 days) I
Acute rejection probably represents a combination of T cell effector function (cellular rejection) and anti- body mediated endothelial damage (acute vascular rejection). The antibodies involved include IgM iso- haemagglutinins in ABO mismatch and IgG anti- HLA Class I antibodies in multiparous or previously transfused/transplanted patients. IgM anti-Class I antibodies do not appear to adversly affect graft survival, even if they can lyse in vitro.
Thus ABO cross-matching and pretransplant recipient screen- ing for anti-donor-HLA Class I is essential for renal and heart/lung recipients. Some centres also screen for anti-HLA Class II, but the significance of these antibodies is controversial. Peak antibody titres may wane with time while awaiting renal transplantation, but in view of the possibility of recrudescent immuno- logical memory, screening is often performed against this peak serum as well as the current serum.
Acute rejection (100 days) II
Anti-HLA antibodies are not looked for in all trans- plant types. Liver transplants are relatively insensitive to HLA Class I mismatches, and cross-matching is not practical in others because of constraints of time and limited donor availability. Hepatocytes may be protected by low level surface HLA expression or the secretion of soluble blocking Class I molecules. Acute rejection reflects major antigenic disparity between graft and recipient.
Renal cellular rejection is primarily driven by CD4 positive Th1 cells which recruit and activate effectors such as monocyte/macrophages, eosinophils, NK cells and cytotoxic T cells. There is usually tubulitis (inva- sion of tubules) with interstitial oedema and infiltra- tion. In antibody-mediated vascular rejection there is endothelial damage (fibrinoid necrosis, fibrin and plate- let thrombi if severe) with lymphocytic vascular inva- sion and peri-venous aggregates. Initially the rejection may be focal and be missed by a biopsy needle. The presence of anti-donor antibodies in serum or C4d deposition in intertubular capillaries on biopsy help to distinguish the presence of humoral rejection and have now been incorporated into the Banff criteria. Early treatment with high dose corticosteroids (pulsed doses of methyl prednisolone) is effective within 2–4 days in most cases. In steroid-resistant rejection ALG, ATG, rituximab or OKT3 may be effective.
Chronic rejection (100 days)
Chronic rejection (chronic allograft nephropathy) may reflect antibody responses to antigen mismatches whichare not effectively suppressed by immuno- suppressive agents, unlike acute rejection. The greater the mismatch (especially for HLA-DR) the more severe the chronic rejection, and number of acute rejection episodes correlates with the likelihood of chronic rejection (50% of renal recipients with one or more rejec- tion episodes show some chronic rejection within five years).
Reduction of immunosuppression accelerates
rejection, suggesting an immunological mechanism. Of allogeneic renal grafts, 3–5% are lost annually after the first year. Chronic renal rejection is a poorly under- stood vasculopathy of medium and small arterioles, to which hypertension, hyperlipidaemia and infection may contribute. Similar vascular changes occur in heart and lung transplantation. Vessels are thickened with elastic reduplication and intimal proliferation, medial necrosis and fibrin deposition. Cellular infiltrates are infrequent, but interstitial fibrosis occurs. There is no effective therapy.
A form of tolerance (antigen-specific immunological unresponsiveness) occurs in a few long-term human renal transplant recipients who can discontinue immu- nosuppressive drugs without graft loss. Although induction of tolerance would be a long-term goal for transplantation, there is as yet no reliable method to induce this.
In the past, blood transfusion was avoided if possible because of the risk of allosensitisation (in 20–30%) of the potential renal graft recipient which would restrict the number of suitable donors. However, transfusion paradoxically gave a survival benefit to the graft, per- haps as a result of specific induction of tolerance.
Early work suggested that transfusion can induce alloantigen-specific tolerance, improving graft survival in heart and renal transplantation (if there was a single DR match). However, the benefit is modest, and it has rarely been used since the introduction of cyclosporin (CsA), because a similar improvement in graft survival is achieved by the drug without the risk of allosensiti- sation, and there is no evidence that transfusion brings additional benefit to most patients.
In immune surveillance, the immune system is able to recognise variants from normal antigen expression and focus an immunologically mediated attack on them. The antigens recognised include overexpressed tissue-specific antigens, mutated self-antigens, or nor- mally repressed antigens to which tolerance has not been established (e.g. BCR/ABL, p53, C-myc, p21Ras, MAGE-1, MART-1, gp100). Since tumours are clonal cell populations, often with a high mutation rate, it is possible for immunological selection pressure to favour the evolution of less immunogenic variants (immuno- logical escape). Many tumours evade effective immune responses by a variety of mechanisms
Any soluble circulating antigen in serum or plasma, measurable by biochemical or immunoassay, can be used as a tumour marker. Most are proteins secreted in excess, and thus are not absolutely specific for any given tumour, interpretation depending on the abso- lute level. They are thus often more suited to moni- toring response to treatment rather than diagnostic screening. Some are cytokines or hormones secreted by the tumour (e.g. β human chorionic gonadotrophin, alpha-fetoprotein); others are cell surface antigens shed into the circulation (e.g. cell surface mucins in adenocarcinomas, CA125, CA159). Levels generally reflect tumour mass. Many also have non-neoplastic sources and can be affected by non-specific inflammation, liver disease, etc.
Attempts to use the immune system to treat tumours have utilised several approaches: (1) use of specific antibodies or cells to attack tumour cells, (2) induction of antitumour immune responses, or (3) enhancement of pre-existing antitumour responses, both innate and specific
Tumour immunotherapy: Passive antibody immunotherapy
Passive antibody immunotherapy
The detection of tumour-specific surface antigens may enable the use of targeted therapies where a monoclonal antibody is the carrier molecule which specifically directs and concentrates a therapeutic drug, prodrug, toxin, or isotope to the neoplasm. In practice the specifi- city of the toxin or isotope on the conjugate molecule is not absolute and there is some collateral damage to normal tissue. This technique has been used with some success in B cell lymphomas, with conjugates targeted to B cell specific surface molecules such as CD22. In addition, the antibodies may attach to Fc receptors of effector cells and recruit additional cellular effect- ors. This technique is also of use in radioimaging of tumours.
Tumour immunotherapy: Vaccination
Attempts to vaccinate with crude cell extracts and tumour specific antigens have been made with some success but depend on the existence and isolation of a relatively tumour specific antigen. Virally induced tumours can be reduced by preventing primary infec- tion by vaccination (e.g. hepatitis B).
Tumour immunotherapy: Cellular immunotherapy
Certain tumours are susceptible to the action of acti- vated CD8 positive cytotoxic T lymphocytes and NK cells, both in animal models and humans. Adoptive transfer or cellular immunotherapy is an attempt to activate or clonally expand pre-existing tumour specific T and NK cells in vitro. One form is called lymphokine activated killer cells (LAK) because IL-2 is used in their generation in vitro. Unfortunately this form of therapy requires the isolation and sterile in-vitro expansion of PBMC or T cells using IL-2 before re-infusion into the patient. It is a cumbersome, individualised, tumour spe- cific therapy, and impractical for general usage.
Tumour immunotherapy: Gene therapy
Animal models suggest that the direct conversion of poorly immunogenic tumours into potent APC may enhance effective tumour specific immunity. This can be accomplished by transfection of cytokine genes such as TNFα, IL-2, IL-4, IFNγ, GM-CSF or cos- timulatory molecules such as B-7 (CD80). This has yet to be shown to be of use in humans.
Another strategy to enhance the induction of anti- tumour responses is to transfect skeletal muscle with the DNA sequence of a tumour specific antigen. The skeletal muscle cell transiently expresses the antigen and acts as an APC. A costimulatory molecule may be transfected simultaneously. This is also a cumber- some, individualised therapy for each patient.
Tumour immunotherapy: APC enhancement
Another approach is to attempt to boost the immune response of the host by the use of potent autologous professional APC which have been pulsed with tumour antigen. This works well in animal models and is being developed for use in man. There is also the possibility of enhancing APC activity by using cytokines, or tar- geting gene transfection to APC using lineage-specific promotors.
Tumour immunotherapy: Cytokine immunotherapy
This is used in an attempt to boost cellular (T and NK) immune responses in the tumour host or to alter the immunogenicity of the tumour cells. IL-2 therapy has been used in melanoma and renal carcinoma with very limited success, and significant side effects. IL-12 and IL-7 are currently being assessed. The local release of IFNγ as a result of cytokine exposure may enhance the susceptibility of some tumour cells to lysis.