Print version ISSN 1516-8484On-line version ISSN 1806-0870
Rev. Bras. Hematol. Hemoter. vol.23 no.3 São José do Rio Preto Sept./Dec. 2001
Immunological aspects of unrelated bone marrow transplantation - Alloreactivity and immunoreconstitution
J. Alejandro Madrigal
The main complications after bone marrow transplantation (BMT) are graft versus host disease (GvHD), post-transplant viral infections and disease relapse. The underlying causes of these problems are the degree of HLA matching between donor and patient and the rate of immune reconstitution.
Keywords: Bone marrow transplantations, HLA matching, alloreativity, immune reconstrution
Impact of HLA Mismatches on the outcome of BMT
Selection of volunteer unrelated donors (VUD) for unrelated BMT is primarily based on matching for HLA-A, B and DR molecules. Studies have shown that recipients of transplants from unrelated donors that are matched for these HLA antigens have a lower risk of acute GvHD than recipients of marrow from mismatched VUD. However, the incidence of GvHD is still much higher than that observed for HLA-identical sibling transplants and there is no improvement in overall survival of HLA-A, B, DR matched unrelated marrow recipients when compared with mismatched related marrow recipients. Thus, the benefits of HLA matching on survival after transplantation with unrelated donor marrow have been controversial. It is clear that additional factors must influence the success of unrelated BMT. These include presence of HLA-A, B and DR disparities that are not detected by low resolution HLA typing techniques currently employed, mismatches at other loci such as HLA-C, DQ and DP, and mismatches for minor transplantation antigens.
HLA typing of unrelated individuals for class I antigens is often still performed by serology and class II typing is performed by a combination of serology and cellular and molecular assays. DNA sequencing analysis has identified over one thousand HLA alleles and many more remain to be discovered. It is now evident that the complexity of the HLA system can not be resolved by serology because each serologic specificity actually comprises many different molecular subtypes. For example, there are currently more than one hundred recognised HLA-A alleles but only a fraction can be identified by serology. DNA-based methods are therefore required to achieve high resolution HLA typing for better matching of unrelated donors and patients.
We have developed a novel DNA-based typing technique that offers a high level of resolution similar to direct sequencing techniques but without the problems of interpretation associated with heterozygous samples. The method, known as Reference Strand mediated Conformation Analysis (RSCA), is simple and easy to use and is based on detection of the conformation of HLA DNA duplexes. Chimeric duplexes are formed between locus-specific, fluorescent labelled, reference strands (FLR) and the HLA alleles from the sample to be typed. Mobilities measured by gel electrophoresis are characteristic for each HLA allele.
A retrospective study of unrelated patient and donor pairs by RSCA has revealed that transplants initially classified as matched, based on serology, were actually performed across at least one HLA mismatch. A patient's risk of GvHD increases proportionate to the number of HLA differences between the patient and donor and mismatches at all loci, including class I HLA-C and class II HLA-DP, contribute to increased risk of GvHD.
With knowledge of the enormous diversity of HLA genes within the human population revealed by high resolution typing methods comes realisation that finding an exact HLA matched donor for a patient is a formidable task. We now appreciate that often the best that can be achieved is a close but not perfect match. Most patients therefore receive immunosuppressive treatments to control GvHD that results from recognition of the mismatched 'foreign' HLA molecules by alloreactive T cells. Although an effective treatment, non-specific continuous immunosuppression increases susceptible to infectious diseases and cancer.
Methods are required for rapid and easy monitoring of alloresponses in patients that will enable appropriate levels of immunosuppression to be administered only when needed rather than continuous treatment currently given to patients based on perceived risk. Also needed are new immunosuppressive therapies that selectively inhibit alloresponses but preserve a functional immune response to foreign pathogens and tumours. These goals require a detailed knowledge of the immuological basis of the T cell alloresponse. Studies are in progress using HLA tetramers to identify the precise molecular nature of the allogeneic MHC / peptide complexes that stimulate alloreactive T cells. Preliminary results indicate the vigorous alloresponse is caused by recognition of numerous novel self-peptides derived from normal cellular proteins presented by foreign HLA molecules. Knowledge of alloligands should ultimately provide a basis for design of new methods for monitoring alloresponses in transplant patients and novel therapeutic strategies for controlling GvHD.
After BMT, T-cells are reconstituted via two distinct routes. The thymus-dependent pathway eventually produces a diverse T cell repertoire able to mount an effective immune response to infectious agents and tumours. The thymus-independent pathway relies on peripheral expansion of graft-derived T cells. Because the thymus is the major site for production and generation of T cells, age-related thymic involution has an adverse effect on the rate of T cell reconstitution in older patients. Reconstitution rate is also slowed by transplantation with a T cell depleted graft, episodes of GvHD, immunosuppressive treatments, and infections such as CMV or EBV.
Three parameters are important to consider when assessing T cell reconstitution; total T cell numbers, their diversity and their antigen specificity. We have shown that detection and quantification of T cells containing T cell receptor excision circles (TRECs) can be used to accurately measure output of T cells from the thymus and peripheral expansion of T cell numbers. As expected, TREC and naive T-cells numbers increase more rapidly in children than adult BMT patients, and those who experience episodes of GvHD have low TRECs and low numbers of naive T cells indicating virtually no thymic production of T cells.
We assess diversity of the T cell repertoire by screening for variety among T cell receptor gene rearrangements using DNA-based techniques including spectrotyping and RSCA. Detection of T cells with defined antigen specificities is achieved using fluorescent-labelled HLA tetramers as probes. Tetramers containing known antigenic epitopes from CMV are being used to detect emergence of a protective immune response to this opportunistic infection. We have found that an anti-CMV response is present in BMT patients as early as three weeks post-transplant. Patients who receive a transplant from a CMV positive donor have a better prognosis because they produce a more rapid and larger anti-CMV response that is maintained for at least one year post-transplant. A sustained decrease in specific T cells, such as occurs after treatment for GvHD, is predictive of CMV reactivation. HLA tetramers are being used to monitor patients at risk of CMV reactivation and to evaluate the efficacy of anti-viral treatments. Work is in progress to exploit these reagents to isolate antigen-specific T cells for use in adoptive immunotherapy.
The data described here represents the work of all the members of The Anthony Nolan Research Institute and I am very grateful to them for their important contributions.
Aspectos imunológicos do transplante de medula óssea não relacionado - aloreatividae e imunoreconstituição
J. Alejandro Madrigal
Neste relato são discutidos aspectos que interferem nos resultados dos transplantes de medula óssea não relacionados. As principais complicações devem-se à Doença Enxerto contra o Hospedeiro e infeções virais pós-transplante. A seleção de doadores, baseada na compatibilidade entre as moléculas HLA-A,B e DR por métodos sorológicos tem se mostrado insuficiente para evitar o estabelecimento da Doença Enxerto contra o Hospedeiro, indicando que doadores aparentemente compatíveis, quando aplicadas técnicas mais refinadas, na realidade não o são. Outro aspecto refere-se à sobrevida global em que transplantes compatíveis ou com pequenas diferenças nos resultados não diferem significativamente, indicando a existência de outros fatores importantes nos transplantes de medula óssea não relacionados. Dentre os fatores devem ser incluídos os lócus HLA- C, DQ, DP, antígenos menores de histocompatibilidade ale da aloreatividade das células e peculiaridades observadas na imunoreconstituição após o TMO.
Palavras-chave: Transplantes de medula óssea, compatibilidade de HLA, aloreatividade, imunoreconstituição
The Anthony Nolan Research Institute,
The Royal Free Hospital, London
Correspondence: Professor J.A Madrigal
Anthony Nolan Research Institute
Pond St, The Royal Free Hospital, Hampstead NW3 2QG United Kingdom
Fone: (44) 20 7284-8315 e-mail:email@example.com