The HLA system in blood transfusion

BaillieÁre's Clinical Haematology Vol. 13, No. 4, pp. 511±532, 2000

doi:10.1053/beha.2000.0097, available online at http://www.idealibrary.com on

2 The HLA system in blood transfusion Cristina V. Navarrete*

BSc, PhD, MRCPath

National Head and Lead Scientist Department of Histocompatibility & Immunogenetics, National Blood Service, North London Centre, Colindale Avenue, London NW9 5BG, UK

The human leukocyte antigen (HLA) system, originally discovered as the result of a transfusion reaction, is now known to play a crucial role in many areas of clinical medicine. The main function of the HLA molecules is to present antigenic peptides to the immune system and in this way regulate the induction of immune responses. This is a highly regulated process which requires a close interaction between the HLA molecules, the antigenic peptide and the T cell receptor. HLA molecules are also known to be associated with a variety of autoimmune, nonautoimmune and infectious diseases and to restrict the antibody response to certain antigens and to vaccines. It is likely that the mechanism responsible for this restriction is the preferential presentation of antigen-derived peptides to T cells. Furthermore, HLA antigens, in contrast to most polymorphic molecules, have the ability to activate the immune system using two di€erent pathways of T cell activation, the direct and indirect pathways. As a result of these features, HLA antigens and antibodies are responsible for some of the serious clinical complications of blood transfusion, and have an important in¯uence on the outcome of solid organ and haemopoietic stem cell transplantation. Key words: human leukocyte antigen; human platelet antigen; panel reactive antibodies; haemopoietic stem cell transplantation; non-haemolytic febrile transfusion reaction; transfusionrelated acute lung injury; transfusion-associated graft-versus-host disease; neonatal alloimmune thrombocytopenia; hereditary haemochromatosis.

The major histocompatibility complex (MHC) spans some 4±6 megabases on the short arm of chromosome 6 in humans and contains over 200 genes, of which approximately 100 are expressed and 40 have an immune-related function.1 The most important genes in this region code for the human leukocyte antigens (HLA) whose main function is to present antigenic peptides, including those derived from HLA molecules, to the immune system. The HLA molecules are highly polymorphic and immunogenic and the antibodies produced as the result of HLA alloimmunization are responsible for some of the serious clinical complications of blood transfusion. In addition, HLA genes have an important in¯uence on the outcome of haemopoietic stem cell and solid organ transplantation and are also known to be associated with a variety of autoimmune, *The author is Honorary Senior Lecturer at the Department of Immunology, Royal Free & University College Medical School, London NW3 2PF, UK. 1521±6926/00/040511‡22 $12.00/00

c 2000 Harcourt Publishers Ltd. *

512 C. V. Navarrete

non-autoimmune and infectious diseases and to restrict the antibody response to certain antigens and to vaccines. It is likely that the mechanism responsible for this restriction is the preferential presentation of antigen-derived peptides to T cells. This chapter gives an account of some basic aspects of the structure and function of the HLA genes, and describes the main characteristics of the HLA antibodies produced as the result of HLA alloimmunization and the techniques currently used for their detection. The role of the HLA molecules in the development of transfusion-related complications and in the outcome of haemopoietic stem cell and solid organ transplantation is also presented. Finally, the role of the HLA genes in disease is also discussed. HLA GENES AND MOLECULES The HLA molecules are encoded by a cluster of tightly linked genes located on the short arm of chromosome 6. Based on some of the structural and functional characteristics of the genes, the region has been divided into three. The class I region contains genes encoding the heavy chain of the HLA class I molecules. Between the genes for class I and II lies the class III region which contains genes encoding a diverse group of proteins which include complement components (C4, Bf), tumour necrosis factor (TNF) and heat shock proteins.2 A current map of the HLA region is shown in Figure 1. One of the main features of the HLA genes is their high degree of polymorphism. This polymorphism is, however, di€erentially expressed across human populations, and certain HLA alleles, for example, HLA-A2, are expressed with similar frequency in most populations studied so far whereas others are found only in certain groups, for example, B53 is found predominantly in blacks. Another important feature of the HLA genes is that all the genes on one chromosome segregate in strong linkage disequilibrium, and recombination rates between the various alleles are low (51%). This linkage pattern of segregation de®nes characteristic HLA haplotypes, some of which are found across di€erent ethnic groups, for example,

MHC-class III subregion MHC-class II subregion

MHC-class I subregion MICB

DMB DP

DOA

DMA

LMP7 TAP2 TAP1 DOB LMP2

DQ

DR

B1 A1

B1 A1

class II genes ABC transporter genes Proteasome-like genes

Centromere

B1

HSP70 TNF

B3 B5 B4 A

HSP70 TNF MICA & MICB

MICA B

C

E

A

G F

HFE

A B

Classical class I genes Non-classical class I genes HFE

4Mb

Figure 1. Map of the human major histocompatibility complex.

Telomere

4Mb

The HLA system 513

HLA-A2-B44-DR4 or DR7. Other haplotypes are unique to a particular population, for example, HLA-A30-B42-DR18 in African negroids. HLA class I genes and molecules The HLA class I genes have been classi®ed according to their structure, expression and function as classical (HLA-A, B and C) and non-classical (HLA-E, F and G). Both classical and non-classical HLA class I genes encode a heavy (a) chain, of approximately 43 kDa, non-covalently linked to a non-polymorphic light chain, the b2m which is encoded by a gene on chromosome 15. The extracellular portion of the heavy chain has three domains (a1, a2 and a3) and is about 90 amino acids in length. The a1 and a2 domains are the most polymorphic, and they form a peptide-binding groove which can accommodate antigenic peptides of eight or nine amino acids in length. A schematic representation of the HLA class I gene and molecule is shown in Figure 2. Although the molecular structure of the HLA-E, F and G genes is very similar to that of the classical class I genes, the non-classical class I genes have a more restricted polymorphism. In addition to the class I genes, two MHC class I chain-related genes (MIC-A and MICB) have been described, located centromeric to HLA-B. Unlike classical or non-classical class I genes, MIC-A and MIC-B do not bind to b2m nor do they require peptide for their expression.3 The main function of the HLA-A, B and C molecules is to present antigenic peptides, derived primarily but not exclusively from endogenous proteins, to CD8‡ cytotoxic T cells. These molecules (together with HLA-E and MIC) are also known to interact with a new family of receptors present on natural killer (NK) cells.4 Some NK receptors, which are polymorphic and di€erentially expressed, have an inhibitory role, whereas others are involved in NK cell activation.5 MIC-A and MIC-B molecules also interact with gd T cells.6

α1

PEPTIDEBINDING REGION IMMUNOGLOBULINLIKE REGION

β2M

α2

N S

S

S

S

α3

C

TRANSMEMBRANE REGION CYTOPLASMIC REGION

HLA-CLASS A GENE EXONS

Regulatory Sequences

C

P P P

α1

α2

α3

TM

2

3

4

5

CYT

3’UT

L 1

6

7

8

Figure 2. HLA class I molecule/gene. L ˆ leader sequence; TM ˆ transmembrane region; CYT ˆ cytoplasmic region; 30 UT ˆ 30 untranslated region.

514 C. V. Navarrete

The HLA-A, B and C molecules are expressed on the majority of tissues and cells, including T- and B-lymphocytes, dendritic cells, granulocytes and platelets. Low levels of expression have been detected in endocrine tissue, skeletal muscle and on cells of the central nervous system. HLA-E and F are also expressed on most tissues tested, but HLA-G shows a more restricted tissue distribution and, to date, the HLA-G product has been detected only on extravillous cytotrophoblasts of the placenta and mononuclear phagocytes.7 The expression of MIC-A and MIC-B is largely restricted to ®broblasts and intestinal epithelial cells but they are also expressed on tumours as the result of stress.4 HLA class II genes and molecules The HLA class II, DR, DQ and DP A and B genes encode a heterodimer formed by two non-covalently associated a and b chains of approximately 34 and 28 kDa respectively. The expressed a and b chains consist of four domains: two extracellular, two extracellular one transmembrane and one cytoplasmic. The majority of the polymorphism is located in the b1 domain of the DR molecules and in the a1 and b1 domains of the DQ and DP molecules. Like class I molecules, these domains form a peptide-binding groove. However, for class II molecules, the groove is open at both sides and can accommodate antigenic peptides of 13±25 amino acids in length. A schematic representation of the HLA class II genes and molecule is shown in Figure 3. There is one non-polymorphic DRA gene and nine DRB genes, of which B1, B3, B4 and B5 are polymorphic and B2, B6 and B9 are pseudogenes. The main DR speci®cities (DR1±DR18) are determined by the highly polymorphic DRB1 gene, and

PEPTIDEBINDING REGION

β1

α1

IMMUNOGLOBULINLIKE REGION

β2

α2 Papain cleavage sites

Papain cleavage sites

TRANSMEMBRANE REGION

EXONS HLA-CLASS A GENE HLA-CLASS B GENE EXONS

CYTOPLASMIC REGION

1 L

2 α1

3 α2

4 TM/CYT

5 3’UT

Regulatory Sequences L

β1

β2

1

2

3

TM CYT 3’UT 4

5

6

Figure 3. HLA class II molecule genes. L ˆ leader sequence; TM ˆ transmembrane region; CYT ˆ cytoplasmic region; 30 UT ˆ 30 untranslated region.

The HLA system 515

DR Specificities DR 1, DR10, DR103, DR15

DRB1

DRB6 Ψ

DRB1

DRB6 Ψ

DRB1

DRB2 Ψ

DR 15, DR16, DR1 DR17, DR18, DR11, DR12, DR13, DR14, DR1403, DR1404

DRB9 Ψ

DRA

DRB5

DRB9 Ψ

DRA

DRB3

DRB9 Ψ

DRA

DRB9 Ψ

DRA

DRB9 Ψ

DRA

DRB1

DR 8 DR 4, DR7, DR9

DRB1

DRB7 Ψ

DRB8 Ψ

DRB4

Figure 4. Expression of HLA-DRB genes. C ˆ pseudo genes. DRB5 encodes DR51; DRB3 encodes DR52; DRB4 encodes DR53.

the number of DRB genes expressed varies according to the DRB1 allele expressed (see Figure 4). However, a DRB5 gene has been found to be expressed with some DR1 alleles. In addition, non-expressed or null genes, and genes determining low expression of the HLA class II (and class I) molecules, have also been identi®ed.8 There are two DQA genes and three DQB genes, of which only the polymorphic DQA1 and DQB1 are expressed. Similarly, there are two DPA genes and two DPB genes of which only the polymorphic DPA1 and DPB1 are expressed. This class II region also contains the LMP2 and LMP7 genes, which are involved in the processing of endogenous antigenic peptides, and the TAP1 and TAP2 genes involved in the transport of antigenic peptides which bind to the class I molecules. HLA-DR, DQ and DP molecules are constitutively expressed on antigen-presenting cells (APC) such as B-lymphocytes, monocytes and dendritic cells but can also be detected on activated T-lymphocytes and activated granulocytes.9 It is not clear whether they are also present on activated platelets. HLA class II expression can also be induced on cells and tissues such as ®broblasts and endothelial cells as the result of activation and/or by certain cytokines such as g-IFN, TNF, IL-10.10 The main function of the HLA-DR, DQ and DP molecules is to present antigenic peptides, mostly of exogenous origin, to CD4‡ helper T cells. Recently, the DMA/DMB and DOA/DOB genes, encoding non-classical class II molecules, have been described. The DMA/DMB and DOA/DOB molecules appear to function at an intracellular level by promoting peptide binding to the class II molecules.11 HLA-DM seems to be expressed in those cells which express HLA-DR, DQ and DP, whereas HLA-DO is expressed mainly in B cells.11 CHARACTERIZATION OF HLA POLYMORPHISM HLA polymorphism has traditionally been characterized using serological and cellular techniques, but DNA-based techniques for its de®nition are now relatively well established in most laboratories. DNA sequence data have revealed that nucleotide sequences may be shared between alleles of the same and/or di€erent loci, and that locus-speci®c nucleotide sequences are found in both the coding (exons) and non-coding (introns) regions of the genes.

516 C. V. Navarrete

Furthermore, the majority of nucleotide variability is located in the a1 and a2 domains of the class I molecules and the a1 and b1 domains of the class II molecules, in the socalled hypervariable (HV) regions.12 Accordingly, most of the DNA techniques currently available use the polymerase chain reaction (PCR) to amplify the gene or region to be analysed. These techniques include PCR-SSOP (sequence-speci®c oligonucleotide probing) and PCR-SSP (sequencespeci®c priming). Sequencing-based typing (SBT) and conformational methods such as reference strand conformational analysis (RSCA) are also available but are not used routinely.13,14 The number of recognized serologically de®ned speci®cities and DNA identi®ed alleles are shown in Table 1. HLA ANTIBODIES HLA antibodies may be produced in any situation that exposes the host to HLA antigens, including pregnancy, transplantation, blood transfusions and planned immunizations. The anity, avidity and class of the antibody produced will depend on various factors, including the route of immunization, the persistence and type of cells present in the immunological challenge and the immune status of the host. Cytotoxic HLA antibodies, which can be identi®ed in some 15±20% of multiparous women, are normally multispeci®c, high titre, high anity and of the IgG class. Although they can cross the placenta, these antibodies have not been shown to be harmful to the fetus. Antibodies produced following transplantation are largely dependent on the degree of HLA mismatch between donor and recipient, and most of these antibodies are IgG, although few IgM antibodies have also been identi®ed. In contrast, the majority of HLA antibodies produced as the result of blood transfusion are multispeci®c IgM and IgG

Table 1. Number of recognized HLA speci®cites/alleles. Alleles

Speci®ties

HLA class I HLA-A HLA-B HLA-C HLA-E HLA-F HLA-G

188 385 92 5 1 14

24 50 9

HLA class II HLA-DRB1 HLA-DRA1 HLA-DRB3 HLA-DRB4 HLA-DRB5 HLA-DQB1 HLA-DQA1 HLA-DPB1 HLA-DPA1

257 2 30 10 15 45 20 89 19

17 ± 1 1 1 6 ± 6* ±

Alleles ˆ DNA de®ned; speci®cities ˆ serologically or cellularly* de®ned. (Steve Marsh, personal communication).

The HLA system 517

and are directed mainly at public epitopes. Some 30±50% of multitransfused patients receiving non-leukodepleted blood components, particularly those on long-term prophylactic platelet transfusion support, develop HLA antibodies. In the past, healthy individuals were deliberately immunized with HLA mismatched cells in order to produce potent HLA-speci®c reagents, but nowadays this is dicult to justify ethically. Planned HLA immunization is still carried out, however, as a form of treatment for women with a history of recurrent spontaneous abortion (RSA). These women are immunized with lymphocytes from their partners or a third party, and this is thought to induce an immunomodulatory response that results in maintenance of the pregnancy. Detection of HLA antibodies HLA antibodies have traditionally been detected and characterized using the complement-dependent lymphocytotoxicity test (LCT), developed by Terasaki and McClelland.17 More recently, the enzyme-linked immunoabsorbent assay (ELISA)18 and ¯ow cytometry techniques have been used.19 The relative advantages and disadvantages of these techniques are described in Table 2.

Table 2. HLA antibody screening techniques. Advantages

Disadvantages

LCT …lymphocytotoxicity test† . Viable cells required . Needs separated T and B cells for class I and class II antibody screening . Requires small amount of serum . Large and selected panel of cells required . Used for antibody screening and . Detects only cytotoxic antibodies cross-matching . Low cost . Does not discriminate between HLA and non-HLA cytotoxic antibodies, for example, autoantibodies

. Well established . Robust

ELISA …enzyme linked immunosorbent assay† Easy to standardize . Large amounts of serum required Can distinguish class I from class II antibodies . Cannot be used for cross-matching Objective read-out . Expensive to test individual samples Suitable for bulk testing Detects cytotoxic and non-cytotoxic HLA-speci®c antibodies . More sensitive than LCT . Medium cost

. . . . .

Flow …flow cytometry† . Highly sensitive . Not well standardized . Detects weak and early sensitization . Large panel of cells required to establish antibody speci®city . Detects cytotoxic and non-cytotoxic . Expensive antibodies . Can de®ne class I and class II antibodies . Does not discriminate between HLA and simultaneously non-HLA cytotoxic antibodies, for example, leukocyte antibodies

518 C. V. Navarrete

CLINICAL RELEVANCE OF THE HLA SYSTEM Blood transfusion Donor HLA antigens which are not identical to those expressed by the recipient will be recognized as non-self and will activate T cells which will initiate a cascade of events leading to the destruction of the donor cells. This process, known as direct allorecognition, is particularly e€ective for the activation of naive T cells. Dendritic cells (DCs) present in transfused blood or in the transplanted organ and expressing HLA and co-stimulatory molecules play a crucial role in antigen presentation in this pathway. Furthermore, antigenic peptides derived from HLA molecules present on transfused blood products can also be recognized by T cells when presented by the patient's own APC. This mechanism, known as indirect allorecognition, operates in previously sensitized individuals who carry memory T-cells that rapidly respond to the antigenic challenge. Thus, HLA antigens present on transfused cells can induce strong cellular and antibody responses in the recipient. HLA alloantibodies are responsible for some of the serious clinical complications observed following blood transfusion, including non-haemolytic febrile transfusion reaction (NHFTR), transfusion-related acute lung injury (TRALI) and immunological refractoriness to random platelet transfusions. On the other hand, immunocompetent cells present in blood and blood products can recognize HLA and other minor histocompatibility (mH) antigens present on the host cells and induce transfusionassociated graft-versus-host disease (TA-GVHD) similar to that seen following haemopoietic stem cell transplantation (HSCT). Viable white cells (and possibly soluble HLA) also seem to be involved in the induction of blood transfusion-induced immunomodulation. Non-haemolytic febrile transfusion reactions (NHFTR) This condition occurs in some 5% of transfused patients and is characterized by a rise in temperature of more than 1 or 28C and chills in patients during or shortly after transfusion of non-leukoreduced/non-leukodepleted cellular blood components.30 Most NHFTR are mediated by HLA and sometimes granulocyte-speci®c antibodies reacting with the infused leukocytes. It is likely that the release of C3a and C5a following complement activation as well as the antibody/antigen complexes can directly activate mast cells and monocytes to produce pyrogenic cytokines leading to the febrile reaction.31 Cytokines, including TNF-a, IL-6 and IL-8, present in some transfused products, are also pyrogenic. The amount of these soluble mediators increases with duration of storage, leukocyte content and bacterial contamination.32 In future, the transfusion of fresh leukodepleted products should prevent the majority of these reactions by the direct removal of leukocytes and/or by reducing the amount of cytokines present in the blood product. Transfusion-related acute lung injury (TRALI) This reaction, characterized by acute respiratory distress, pulmonary oedema, and severe hypoxia, is a rare but serious complication of transfusion of blood products containing plasma. The reaction normally develops within 6 hours, but cases developing after 48 hours have also been reported. The episode tends to resolve within 96 hours, and the majority of patients will respond to respiratory support therapy. Patients who recover have no residual damage but mortality remains at 6%.33

The HLA system 519

The development of TRALI is associated with the presence of leukocyte antibodies in the transfused plasma or blood product, but antibodies present in recipients and reacting with the transfused cells have been found in a few cases. TRALI reactions due to inter-donor antigen/antibody reactions have also been documented.34 In most TRALI cases HLA class I and granulocyte-speci®c antibodies such as anti-NA1, anti-NA2, anti-NB1 and NB2 and anti-5b have been identi®ed.35 Recently it has been suggested that transfusion of biologically active lipids in stored blood could also cause TRALI.36 This could explain why leukocyte antibodies are not always found in patients with TRALI reactions. However, such reactions might also be caused by antibodies other than HLA class I or granulocyte speci®c, not normally tested for, for example, HLA class II or leukocyte-speci®c. The patho-physiological mechanism of TRALI is not well understood, but in animal models such a reaction is initiated when granulocytes are activated by transfused antibodies and/or biologically active lipids. This activation induces the release of anaphylatoxins, cytokines and chemokines which promote neutrophil chemotaxis and aggregation in the lungs. The result is endothelial damage and, hence, increased pulmonary vascular permeability and ¯uid leakage into the alveoli, causing noncardiogenic pulmonary oedema. Blood components from donors implicated in TRALI reactions have often been transfused into other patients after previous and subsequent donations, apparently with no serious clinical consequences, suggesting that other factors may in¯uence the reaction, such as the predisposing clinical (and perhaps genetic) condition of the recipient. The type, titre and speci®city of the antibody, the nature and distribution of the related antigen and the extent of complement activation may also be relevant. To determine the relevance of leukocyte antibodies in implicated donors, serological crossmatch studies should be performed between donor serum and recipient granulocytes and/or lymphocytes. Patients and implicated donors should also be typed for granulocyte and HLA antigens, as appropriate.

Immunological refractoriness to random platelet transfusions Some 30±50% of transfusion-dependent patients become refractory to platelet transfusion, de®ned by the failure to gain adequate increments (i.e. 10  109/l, 1 hour posttransfusion), following at least two platelet transfusions from random donors. Platelet refractoriness may be due to immunological or non-immunological causes: in the majority of patients both causes are implicated.37 HLA and, to a lesser extent, human platelet antigen (HPA) and high-titre ABO alloantibodies are the main causes of immunological refractoriness. However, although HLA antibodies are found in approximately 50% of multitransfused patients, only 30% of them are immunologically refractory. Patients with a low percentage panel reactivity (PRA) may not necessarily be immunologically refractory, indicating that the titre and speci®city of the antibodies present are important. As platelet destruction occurs via the monocyte/macrophage system and the FcR expressed on monocytes preferentially binds IgG3 and IgG1, these antibodies could be more relevant. A number of patients never develop HLA antibodies neither do they become refractory, despite multiple platelet transfusions. The production of antibodies in these patients may be in¯uenced by the level of mismatch with the infused product, previous sensitization, the immune status of the patient and/or the original disease.

520 C. V. Navarrete

Individual cases of refractoriness due to high-titre ABO have been reported in patients receiving HLA matched platelets. The transfusion of HLA matched but ABO mismatched platelets into alloimmunized patients results in a 20% reduction in the platelet increments post-transfusion.38 Circulating immune complexes involving the ABO system have also been shown to a€ect the platelet survival.39 An independent e€ect of HPA antibodies has not been established because most cases occurred in highly HLA immunized patients.40 Other antibodies, such as those induced by drugs, have also been implicated but their occurrence is rare. Finally, secondary thrombocytopenia may be due to the presence of platelet autoantibodies (PAIg), but these antibodies have not been shown to a€ect platelet survival.41 Non-immunological factors involved in the destruction of transfused platelets include the use of old and/or badly stored platelets, or of products containing small platelet doses, or clinically related conditions such as splenomegaly, hepatomegaly, disseminated intravascular coagulation (DIC), septicaemia, fever, infections (e.g. CMV) and malignancies. Drugs used as part of the treatment, including antibiotics, for example, amphotericin B, vancomycin and cipro¯oxacin, can also contribute. Patients refractory to random platelet transfusions are initially screened for the presence of both cytotoxic and non-cytotoxic HLA-speci®c antibodies, and if either of these are present HLA matched or crossmatch-negative platelets are provided. HLA class I typing is performed as soon as HLA-speci®c antibodies are detected in order to identify the most suitable HLA matched platelet unit. Platelets express HLA-A and -B and, to a lesser extent, -Cw antigens. At present, most laboratories perform only HLA-A and -B typing on these patients and on platelet donors, so the clinical signi®cance of HLA-Cw antibodies to immunological refractoriness has not been established, but this could change with the development of DNA-based techniques to de®ne them. By the same techniques, it is also possible to HLA-type patients with low cell counts, including patients requiring HLA-matched platelets, in whom serological methods cannot be used. The techniques can also provide a higher level of resolution of the HLAA, -B and -Cw alleles, and this may help to identify immunodominant serological epitopes relevant to alloimmunization. Provision of HLA-matched platelets. For refractory patients, in whom non-immune causes have been excluded, and HLA speci®c antibodies are detected, the provision of HLAmatched platelets should be considered. However, the HLA system is highly polymorphic, so that large panels of HLA-typed platelet donors are required to provide HLA-matched platelets. In most laboratories the matching is based on the serological de®nition of the HLA antigens at the pertinent speci®city levels, including serological splits. Matching is also based on the known serological cross-reactivity between di€erent antigens of the HLA-A and -B loci as described by Duquesnoy et al.42 This reduces the problems associated with rare HLA types and the scarcity of large HLA-typed platelet panels. However, patients with antibodies to antigens within the cross-reactive groups (CREG) have been described. In the UK, the degrees of matching used are de®ned as follows43: (i) A grade matching. Patient and donor are serologically compatible for the four HLAA and -B locus antigens, for example: Patient ˆ HLA-A1,A2/B8,B44

The HLA system 521

Donor ˆ HLA-A1,A2/B8,B44 or *homozygous donor ˆ HLA-A1,A1/B8,B8 or HLA-A2,A2/B44,B44 *The provision of HLA-matched platelets for people with relatively common HLA types can be facilitated by the use of donors homozygous for common HLA haplotypes. (ii) B grade matching (B1±B4). Patient and donor are mismatched for one to four serologically cross-reactive{ antigens, for example, a B1 match would be: Patient ˆ HLA-A1,A2{/B8,B44 Donor ˆ HLA-A1,A28{/B8,B44 (iii) C grade matching (C1±C4). In this grade the patient and donor are mismatched for one to four serologically non-cross-reactive} antigens, for example, a C1 match would be: Patient ˆ HLA-A1,A2}/B8,B44 Donor ˆ HLA-A1,A31}/B8,B44 Similar matching criteria are used in most countries, although the nomenclature varies slightly in that B (B1±B4) grade matching may be used to mean cross-reactive donor antigen mismatch which is either unknown (B1U) or cross-reactive (B1X). Similar criteria are used for C grade matching.42 Provision of cross-matched platelets. If no A, -B or -C matches are available, and the speci®city of the antibody is known, single-donor (SD) platelets negative for the relative antigen are sometimes issued. If this is not possible, available apheresis platelets are cross-matched with the patient's serum. However, this approach requires large numbers of platelets to be cross-matched for each individual patient. Ideally, both HLAmatched and cross-match-negative platelets should be provided. The relative bene®ts of these two approaches are still controversial and are generally determined by the infrastructure available to support each type of treatment rather than by sound clinical and scienti®c evidence. Several platelet cross-match techniques have been described but most laboratories use a commercially available solid-phase technique: (CAPTURE-P1, Immucor Inc., Norcross, GA, USA). The limitation of this technique is that it does not distinguish HLA from non-HLA antibodies. Cross-match-negative platelets are also the only alternative for patients with low cell counts and in whom no HLA typing is available. Other approaches have been used , mostly for patients who fail to respond to HLA-matched or cross-matched-compatible platelets and who are bleeding. These include massive transfusions of ABO-identical platelets, intravenous immunoglobulin (IVIG) and plasma exchange but with less success. More recently, a method has been described for eliminating the HLA class I antigens from the membrane of platelets using acid treatment.44 Transfusion of acid-treated platelets achieved increments in an alloimmunized thrombocytopeneic patient. Of these approaches, massive platelet transfusion seems to be the most successful. Nevertheless, if immunologically refractory patients fail to respond to HLA-matched platelets, in the absence of bleeding, prophylactic platelet transfusions should be discontinued. Correlation between platelet support with increments following platelet transfusion. An important aspect in the management of these patients is the correlation between the grade of HLA-matched platelet support and the increment obtained. In our centre, following the issue of HLA-matched or cross-matched platelets, clinicians are asked to obtain a pre-transfusion and a 1-hour post-transfusion platelet count. If the response to

522 C. V. Navarrete

a grade A matched or solid-phase cross-match-negative platelets is poor, the e€ect of HPA or high titre (HT) ABO alloantibodies should be considered. If HT ABO antibodies are present, both ABO and HLA compatible platelets are provided whenever possible. Providing HLA and HPA compatible platelets is not impossible, but it requires a large panel of HLA and HPA typed platelet donors. The majority of patients requiring HLA-matched platelets have HLA types that are relatively common in the UK and for whom HLA-matched platelets are readily found from the panel of donors. For patients with uncommon HLA types, particularly among the panel of platelet donors in Southeast England which is largely composed of people of Northern European background, an active recruitment of donors from ethnic minority groups has been established within the UK National Blood Service.45 Prevention of HLA alloimmunization There are various ways to prevent HLA alloimmunization, including the use of ultraviolet (UV) light to reduce the immunogenicity of the transfused cells.46 Data from animal models have shown that 92% of recipients of UV-irradiated platelets do not become platelet-refractory following eight transfusions of single-donor platelets in contrast to only 14% of recipients of unmodi®ed platelets.47 It is not proven whether the use of platelets from single apheresis donors should also decrease alloimmunization by reducing donor exposure. However, if such platelet concentrates are leukodepleted, HLA alloimmunization is obviously signi®cantly reduced. The recent implementation of universal leukodepletion of blood and blood products may lead to a reduction in alloimmunization. However, this may not be very e€ective in preventing alloimmunization in already-sensitized recipients, i.e. women who have become immunized as a result of pregnancy or previously multitransfused patients. Furthermore, a recent publication has indicated that extreme leukodepletion of HLA class II-positive B cells may enhance rather than reduce alloimmunization.48 Transfusion-associated graft-versus-host disease (TA-GVHD) Graft-versus-host disease is a rare but severe and often fatal disorder which occurs following the transfusion of blood or blood components containing viable, immunocompetent lymphocytes into immunosuppressed, and sometimes immunocompetent, patients. The condition typically occurs 1 to 2 weeks after transfusion with fever, skin rash, biochemical hepatitis and diarrhoea. Immunocompetent donor T cells reacting with HLA and mH antigens present on patients' cells and tissues are known to be involved in the pathogenesis, which is similar to that occurring following HSCT. However, unlike GVHD following HSCT, there is evidence of bone marrow failure due to hypoplasia.49 Relevant factors in the pathogenesis of TA-GVHD include the degree of recipient immunosuppression (congenital or acquired, but not HIV infection), the lymphocyte cell dose and the degree of HLA sharing between the blood donor and recipient. The last is particularly relevant in cases where the recipients share one haplotype with an HLA homozygous donor and are therefore unable to recognize the donor lymphocytes as foreign.50 CD4‡ and CD8‡ cytotoxic as well as CD4‡ T cell clones lacking direct cytotoxicity but with lytic supernatants containing tumour necrosis factor (TNF-b), have been

The HLA system 523

isolated from the lesion of a patient with TA-GVHD.51 In addition, the levels of proin¯ammatory cytokines, such as interleukin IL-1, IL-2, TNF and g-interferon, are greatly increased in TA-GVHD as the result of tissue injury due to infection, chemo/ radiotherapy or tumour invasion. These cytokines can, in turn, upregulate HLA expression, and recruit and activate other donor-derived T cells and macrophages. A positive feedback loop is thus established, leading to a full clinical picture similar to the one seen following HSCT.52 Diagnosis Diagnosis depends on ®nding evidence of donor-derived cells, chromosomes or DNA in the blood and/or a€ected tissues of the recipient. The detection of chimaerism is an important aid in the diagnosis, which may otherwise be delayed in view of the severe but initially non-speci®c clinical features. Because donor leukocytes remain in the circulation of otherwise healthy recipients for periods ranging from days to even weeks, the demonstration of donor lymphocytes in a recipient on one occasion, indicating a state of chimaerism, does not in itself de®ne TA-GVHD. Evidence of chimaerism, preferably on more than occasion, within the appropriate clinical context, is necessary to substantiate the diagnosis. Detection of chimaerism using DNA analysis allows a wider range of markers to be employed, including HLA genes or other genetic markers. However, for any selected pair of donor and recipient, these DNA techniques at a particular locus may fail to be informative, for example, if the donor is HLA homozygous for a haplotype found in the recipient. It may be necessary to look at multiple polymorphic sites in a recipient to identify the presence of multiple alleles such as the variable-number tandem repeat (VNTR) pro®le. Ideally, samples for DNA extraction should be obtained from the recipient (before and after transfusion) and from the implicated donors. Post-transfusion samples may be dicult to obtain because the recipient is pancytopenic. Alternative tissues for DNA extraction include skin (both a€ected and una€ected areas), hair follicles and nail clippings. Post-mortem samples from the spleen or bone marrow, if available, can also be used as a source of DNA. Follow-up samples can provide valuable information about temporal changes to the DNA pro®le, particularly when a pre-transfusion sample is not available. Patients at risk The type of patients at risk include fetuses and infants receiving exchange transfusions (especially those given inter-uterine transfusions) and patients with evidence of an underlying immunode®ciency state (except chronic mucocutaneous candidiasis and HIV). Because of the importance of HLA haplotype sharing as a risk factor for TAGVHD, all transfusions between family members, and all HLA-matched platelets, should be irradiated. Rare cases of GVHD following transplantation of liver or heart/ lung have been shown to be due to the presence of passenger lymphocytes in the transplanted organ. Prevention The critical number of T cells known to cause GVHD following HSCT is between 1  105 and 1  106/kg.53 The precise number of lymphocytes required to initiate

524 C. V. Navarrete

TA-GVHD is not known. In the mouse, a minimum of 107 lymphocytes are required54, and although most reported clinical cases received more than 1010 cells, a child with immunode®ciency apparently developed the condition after only 104 cells/kg.55 Leukocyte depletion of blood is therefore not an adequate precaution as the residual lymphocyte numbers may still be above the threshold dose, and there has been at least one reported case of TA-GVHD associated with leukocyte-depleted red cells. All cellular blood products should therefore be irradiated, at a minimum of 25 Gy, before being transfused into any of the at-risk patients described above. Samples of fresh frozen plasma, shown to contain haemopoietic precursors, gave no cellular responses with phytohaemagglutinin stimulation. Therefore, irradiation of fresh frozen plasma is not currently recommended.56±58 Treatment Most patients receive immunosuppressive therapy, including high-dose steroids in addition to other agents such as methotrexate, cyclosporin and anti-lymphocyte globulin, but the response to treatment is poor. Cambridge Pathology Department (CAMPATH) antibodies, G-CSF, anti-CD3 monoclonal antibody and haemopoietic stem cell transplantation have all been unsuccessful.49 Two new therapeutic strategies, the protease inhibitor nafomostat mesilate (NM), and the anti-malarial drug chloroquine, have been described. Both drugs inhibit CD4 ‡ and CD8‡ cytotoxic T cell responses against HLA-expressing EBV-transformed B cell lines.59 Immunomodulation The possible immunomodulatory role of blood transfusion was ®rst described by Billingham et al in 195360 and has been a topic for debate for the last 25 years. During this period, a number of studies have been published demonstrating bene®cial clinical e€ects of blood transfusion-induced immunomodulation in kidney transplant patients, and in women with recurrent spontaneous abortions. A similar number of studies have, at the same time, shown a detrimental e€ect of blood transfusions on the survival of patients with tumours and on the risk of infectious complications in patients undergoing major abdominal, cardiac and orthopaedic surgery. Despite a large number of reports, the mechanism underlying the immunomodulatory e€ect is still largely unknown.61 Immunomodulation in renal transplantation A bene®cial immunomodulatory role of blood transfusion in renal transplantation was ®rst reported by Opelz et al in 1973.62 However, with the advent of modern immunosuppressive drugs, for example, cyclosporin A, such an e€ect was no longer apparent, but recent publications have con®rmed improved 1-year and 5-year renal allograft survival in previously transfused patients. The bene®t is greatest when there is one HLA haplotype or one HLA-DR sharing between the patient and the donor but there is also an advantage for HLA-matched sibling allografts.63 A further study on renal dialysis patients showed that transfusion of one haplotype matched or HLA mismatched blood had no e€ect on the cytotoxic T cell alloresponses in this group of patients, but the numbers of speci®c helper T lymphocyte precursors were signi®cantly decreased 3 months after transfusion in both groups of patients.64

The HLA system 525

The down-regulation of helper T cell responses may thus contribute to the immunosuppressive e€ect of allegeneic blood transfusion. However mediated, the transfusion e€ect in renal allografting appears to require viable leukocytes, in that patients transfused with leukocyte-poor, washed or frozen/ thawed red cells receive less immunological bene®t from their transfusions.65 Now that a policy of universal leukocyte depletion is being introduced in a number of countries its impact on potential renal transplant recipients remains to be seen. Recurrent spontaneous abortion (RSA) RSA occurs in approximately 1% of couples, and in half of the cases the causes are unknown. During pregnancy there is maternal recognition of the fetus, as demonstrated by maternal antibodies against paternal antigens. However, a certain degree of immunomodulation of the mother occurs during pregnancy. Initial studies in women with RSA showed an increased degree of parental HLA haplotype sharing and a reduced cellular reactivity against paternal HLA antigens in the standard mixed lymphocyte culture assay. On the basis of these observations, women with RSA have been deliberately immunized with their partners' or third party cells in order to induce an immunoregulatory response leading to the acceptance of the fetus. The majority of alloimmunization programmes have been uncontrolled open studies, and have reported successful pregnancies in 50±90% of cases. However, a recent randomized trial indicated that immunization with paternal MNCs did not improve pregnancy outcome in women with RSA.66 Immunomodulation and tumour growth A number of well-controlled experimental studies in animals have demonstrated a deleterious e€ect of allogeneic transfusion on tumour growth. This e€ect appears to be immunologically mediated in that the tumour-promoting e€ect can be adoptively transferred using spleen cells.67 The e€ect is reduced by the pre-storage, but not poststorage, removal of leukocytes from the transfusate, suggesting that cytokines released during the storage period may also contribute to the immunomodulatory e€ect. Recently a real and independent clinical e€ect of transfusion on colorectal cancer recurrence has been shown in a randomized trial.68 Post-operative infection The evidence for transfusion as an independent risk factor for the infections commonly seen after elective surgery is increasing. Non-randomized studies in both clean orthopaedic and potentially contaminated abdominal surgery have all shown signi®cantly fewer infections in the post-operative period in non-transfused patients69 and in patients receiving autologous blood. The implementation of universal leukodepletion in most countries will provide a unique opportunity to assess the impact of this policy on the development of such infections. Mechanisms involved The immunological interactions leading to induction of this immunomodulatory e€ect are still not fully understood but white blood cells are certainly involved. Cells present in the transfusate can directly activate the mechanisms postulated for

526 C. V. Navarrete

immunomodulation, including induction of suppressor cells or veto cells, anergy or the secretion of immunoregulatory cytokines or prostaglandins. Storage time, which in¯uences this e€ect, can allow the accumulation of soluble mediators in the plasma. Plasma, or a temperature-sensitive factor present in plasma, can suppress mitogeninduced T cell responses in vitro.70 Animal studies have shown that blood transfusion can lead to the preferential secretion of Th2 type immunoregulatory cytokines such as IL-4 and IL-10 which favour humoral immunity.71 Th2-type responses can also down-regulate the secretion of Th1 derived cytokines, such as IL-2, and IFN-g, involved in promoting cellular immunity. Decreased IL-2 secretion may also contribute to the development of T cell anergy. Similar results were obtained in renal recipients in whom a decrease of TNF-a, IL-2 and IFN-g secretion was observed following a single transfusion of whole blood.72 The cellular content of the transfusate may also contribute to the immunomodulatory e€ect because activation of T cells requires two signals, one provided by the MHC/peptide complex interacting with the T cell receptor and the other provided by the co-stimulatory molecules in the APC, interacting with their respective ligands on the T cells.59 In this context, the failure to activate T cells may be due to the lack of cells providing co-stimulatory molecules such as CD40, B7.1 (CD80), B7.2 (CD86) in the transfused product. Alternatively, this e€ect may be mediated by the types of APC present in blood and delivering the signals required for T cell activation, for example, B cells, myeloid or lymphoid dendritic cells or monocytes. B cells could be tolerogenic whereas dendritic cells and monocytes could be more immunogenic. Recent data suggest that soluble HLA molecules and Fas ligand may play a role in the immunomodulatory e€ect of blood transfusion.73 Soluble HLA class I molecules can induce apoptosis in alloreactive cytotoxic T lymphocytes74 and can also block NK recognition of class I molecules.75 However, these various possibilities do not explain why HLA-DR or one HLA haplotype sharing should be needed for the induction of immunomodulation by blood transfusion. SOLID ORGAN AND HSC TRANSPLANTATION HLA compatibility is one of the important factors in determining the outcome of solid organ and haemopoietic stem cell transplantation (HSCT). Studies on the e€ect of HLA matching in renal transplantation have shown that 1-year graft survival in recipients of fully HLA-A, B and DR matched kidneys is signi®cantly better than that of recipients receiving HLA haplotype matched kidneys from a parent or a sibling (94%, as opposed to 89 and 90% respectively). When grafts from cadaver donors are analysed, the 1-year graft survival rate is 88% for HLA-A, B and DR matched and 79% for mismatched kidneys: HLA-DR has the strongest e€ect. The di€erences between match grades, which are minimal at 1 year post-transplant, become more apparent with increased follow-up survival ®gures.20 DNA-based techniques in this ®eld have shown that when recipients and donors are HLA-DR identical by serological and molecular techniques the graft survival rate is higher than when serological DR matching alone has been used (87 versus 69%).21 The presence of circulating HLA-speci®c antibodies directed against donor antigens in renal and cardiac recipients before transplantation is associated with hyperacute

The HLA system 527

rejection of the graft. These antibodies, which greatly limit the possibility of ®nding a compatible organ, should be clearly identi®ed as soon as patients are registered on the transplant waiting list to ensure that incompatible donors are not considered for crossmatching. Similarly, the appearance of donor-speci®c antibodies post-transplantation seems to correlate with signs of chronic rejection, so antibodies should be monitored post-transplantation.22 In haemopoietic stem cell transplantation the probability of developing acute GVHD is directly related to the degree of HLA incompatibility. Although transplantation between HLA-identical siblings ensures matching for the HLA-A, B, Cw, DR and DQ genes, acute GVHD still develops in 20±30% of these patients. These results are probably due to the e€ect of untested antigens, for example, HLA-DP or minor histocompatibility (mH) antigens in the activation of donor T cells. Patients receiving grafts from HLA-matched unrelated donors have a higher risk of developing GVHD than those transplanted using an HLA-identical sibling.23 However, increased GVHD results in lower relapse rates probably due to a graft-versus-leukaemia (GVL) response associated with the GVH response. Again, the use of DNA-based methods provides a unique opportunity to improve HLA matching of patients and unrelated donors and should result in a reduced incidence of GVHD in these patients.24 HLA-DR incompatibility is one of the main risk factors associated with the development of GVHD, but mismatches at other HLA loci are independent risk factors in patients following HSC transplantation.25,26 Recently cord blood has been successfully used as a source of HSC for patients requiring marrow reconstitution. Clinical data have shown a reduced risk and severity of GVHD following HLA-matched and mismatched cord blood transplantation.27 This may result from the immunological naivety of CB cells, together with more stringent requirements for the activation of CB T cells compared to cells present in adult bone marrow.28 The impact of reduced GVHD on relapse rates is not yet clear. Graft failure in HSCT is thought to be mediated by residual recipient T and/or NK cells reacting with major or mH antigens present in the donor stem cell harvest. However, antibodies reacting with HLA antigens present on donor cells have also been implicated. Studies on patients with leukaemia or aplastic anaemia undergoing HSCT have shown that patients with donor-speci®c antibodies and with a positive cytotoxic cross-match have a higher incidence of graft failure than those with a negative cross-match.29 In spite of these reports, HLA antibodies seem to be more relevant in the post-transplant setting, where patients receiving multiple transfusions can experience immunological refractoriness to random platelet transfusions due to the presence of HLA antibodies. These patients require transfusions of HLA-matched platelets as described above. HLA AND DISEASES HLA antigens are associated with the development or outcome of a variety of diseases. Di€erent mechanisms have been put forward to explain the nature of these associations but very few have been shown to be relevant or con®rmed.76 Among those best studied are (a) molecular mimicry between certain pathogenic peptides and host-derived peptides, for example, ankylosing spondylitis (AS) and Klebsiella77; (b) linkage disequilibrium between the HLA and the relevant disease susceptibility

528 C. V. Navarrete

genes, for example, haemochromatosis78; and (c) the preferential presentation, by certain HLA molecules, of a pathogenic peptide, resulting in the activation of cellular e€ectors, for example, coeliac disease79 or the development of an antibody response, for example, neonatal alloimmune thrombocytopenia (NAITP).80

Hereditary haemochromatosis (HH) HH is a common genetic disorder in Northern Europe and its clinical manifestations include cirrhosis of the liver, diabetes and cardiomyopathy. Detection of asymptomatic iron overload is important because removal of excess iron by phlebotomy can prevent organ damage. Previous screening methods relied on the measurement of iron saturation con®rmed with a fasting sample, with de®nitive diagnosis by liver biopsy. Approximately 1/200 to 1/400 individuals su€er from the disease, with an estimated carrier frequency of between 1/8 and 1/10. A close association between HLA-A3 and HH has long been known and until recently HLA-A3 was the only immunological test available to assist diagnosis, but was a very limited value because the majority of HLAA3-positive individuals do not have HH. A class I-related gene, HFE, located 4 Mb telomeric of the HLA region, has been found to be responsible for HH (see Figure 1). Two mutations (C282Y and H63D) have been identi®ed in the HFE gene.81 Over 90% of HH patients in the United Kingdom are homozygous for the C allele at codon 282. The second mutation (H63D), is thought to be less important, although it may have an additive e€ect if inherited with the ®rst mutation. Recent studies on blood donors have shown that approximately 1/280 donors are homozygous for the mutations. A DNA-based PCR-SSP technique for detecting both mutations simultaneously provides a simple, rapid and unambiguous de®nition of these mutations.

Neonatal alloimmune thrombocytopenia (NAITP) NAITP is characterized by isolated neonatal thrombocytopenia present at birth and reaching a nadir within the ®rst hours of life. More than 80% of NAITP cases occur in women who are homozygous for the HPA-1b allele. An amino acid substitution of leucine for proline (L/P) at position 33 on the GPIIIa chain, de®ning the HPA-1a and b alleles respectively, is responsible for the production of alloantibodies.80 Early studies indicated that the production of HPA-1a antibodies is strongly associated with the HLA-DRB3* 0101 allele. The mechanism responsible for this restriction is probably the preferential presentation of HPA-1a-derived peptides to T cells.82 Recently T cells responding to a peptide derived from HPA-1a have been identi®ed in an HPA1b/b mother with an a€ected child. Some NAITP cases are associated with HPA-5b antibodies. Demonstration of platelet-speci®c IgG antibodies in the mother is essential for the diagnosis. However, only some 35% of HPA-1a negative/DRB3* 0101-positive women develop antibodies on exposure to the antigen, suggesting that other genes or factors, as yet unknown, may be involved in the development of full clinical NAITP. HLA genes have also been reported to be associated with the antibody response to hepatitis B surface antigen (HBsAg)83 vaccine and with the clearance of circulating hepatitis C virus.84 A list of diseases associated with both HLA class I and class II is shown in Table 3.

The HLA system 529 Table 3. HLA-associated and HLA-linked diseases. Birdshot chorioretinopathy BehcËet's disease Ankylosing spondylitis Malaria

I. HLA associated diseases HLA-A29 HLA-B51 HLA-B27 HLA-B53

IDDM

HLA-DQ8

Rheumatoid arthritis

Amino acids 70±74 on the DRB1 gene (QKRAA or QRRAA)

Narcolepsy Coeliac disease Selective IgA de®ciency

HLA-DQB1*0602/DQA1*0102 HLA-DQB1*0201/DQA1*0501 HLA-DRB1*0301/-DQB1*02

Development of HPA1a abs in NAITP Non-ab respond to HBV vaccine

HLA-DRB3*0101 HLA-B44-DR7-DQ2 (in Caucasoids) HLA-B8-DR-DQ2 (in Caucasoids) HLA-B564-DR4-DQ4 (in Japanese)

Clearance of circulating HCV Haemochromatosis 21 OH de®ciency

HLA-DRB1*11-DQB1*0301

II. HLA linked diseases (HLA-A3) HFE gene C282Y and H63D (HLA-B27) 21 OH gene

( ) ˆ associated allele.

Acknowledgement I would like to thank Denny Williams for her expert secretarial assistance in the preparation of this manuscript, and my thanks also to Colin Brown for his helpful advice and critical reading of this chapter.

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