Cytokine genotypes in allograft rejection: guidelines for immunosuppression

Cytokine Genotypes in Allograft Rejection: Guidelines for Immunosuppression I.V. Hutchinson, D. Turner, D. Sankaran, M. Awad, V. Pravica, and P. Sinnott

T

HE immune system is regulated by the release of cytokines which influence cellular activation, differentiation, and function. The production of cytokines is under genetic control. Individual differences in the inheritance of polymorphic cytokine genes1–3 lead to individual variation in the immune response. Hence, a definition of cytokine genotypes can indicate how recipients will respond to their transplants and may provide an approach to tailor immunosuppression in individuals. The particular focus of this presentation concerns two important questions: (1) Can cytokine genotypes be used to indicate optimal immunosuppression in individual recipients? (2) Can cytokine genotypes be used to select patient subgroups for clinical trials, thereby increasing the power of such studies?

INDIVIDUAL VARIATION IN CYTOKINE RESPONSES IS ASSOCIATED WITH POLYMORPHISM IN CYTOKINE GENES

We have made a study of allelic variation in cytokine genes, and we have attempted to associate differences in the DNA sequence with the ability of cultured leukocytes to produce cytokines. For the cytokines we studied, there are considerable differences in the amounts produced when peripheral blood leukocytes are stimulated in vitro with mitogens. In most cases, we have found a difference or differences in the gene sequence which may be either directly functional or closely linked to such a functional polymorphism. For example, a directly functional polymorphism may alter the DNA sequence to create or abrogate a binding motif for a transcription factor in the gene promoter region. Here we discuss the relationship between acute and

chronic graft rejection and alleles of four cytokine genes, namely tumor necrosis factor-alpha (TNF-a), an inflammatory molecule, interferon-gamma (IFN-g) which is proinflammatory, interleukin-10 (IL-10) which is anti-inflammatory and may also promote antibody responses, and transforming growth factor-beta 1 (TGF-b1) which is antiinflammatory and immunosuppressive but has a range of other functions including the promotion of fibrogenesis and wound healing. The genetic variants of these cytokines are summarized in Table 1. In each case we can associate gene sequence with cytokine phenotype, so that individuals homozygous for a high producer allele are the highest producers of that cytokine. Homozygotes for the low producer allele are the lowest cytokine producers. Those who are heterozygous, having inherited one high and one low producer allele, are intermediate cytokine producers. All of the cytokines are independently regulated so that each person is a mosaic of high and low cytokine-producing genes. RELATIONSHIP BETWEEN CYTOKINE GENOTYPE AND GRAFT REJECTION

The inheritance of cytokine gene alleles seems to influence the outcome of kidney,4 heart,5,6 and lung7 transplantation. Acute cellular rejection of heart and kidney transplants is most prevalent in the high TNF-a producers. Death of From the School of Biological Sciences, University of Manchester, Manchester, United Kingdom. Address reprint requests to I.V. Hutchinson, School of Biological Sciences, University of Manchester, Stopford Bldg 3.239, Oxford Road, Manchester M13 9PT, UK.

Table 1. Polymorphisms in Cytokine Genes Cytokine

Cytokine Function

Polymorphism (Position)

Alleles

Cytokine Production

Reference

A G 12 repeats Other alleles G A Leucine Proline Argine Proline

High Low High Low High Low High Low High Low

1

TNF-a

Inflammatory

Promoter (2308)

IFN-g

Proinflammatory “Th1”-like functions Anti-inflammatory “Th2”-like functions Anti-inflammatory Immunosuppressive Profibrogenic

First intron (CA)n repeat Promoter (21082)

IL-10 TGF-b1

Codon 10 Codon 25

Unpublished data 2 3

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Transplantation Proceedings, 30, 3991–3992 (1998)

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heart graft recipients due to acute cellular rejection is strongly associated with TNF-a genotypes as well (unpublished data). In heart graft recipients, the high IL-10 producer genotype appears to protect against early acute rejection. However, in kidney graft recipients, high IL-10 production may exacerbate rejection, perhaps by promoting vascular rejection processes. The genotype for IFN-g is also associated with acute kidney transplant rejection,8 and the development of fibrosis in lung transplants.9 The most dramatic association with chronic rejection is with the TGF-b1 genotype. Many patients requiring a lung transplant have fibrosis of their lungs. After transplantation, the development of fibrosis is associated with TGF-b1 in the graft and with the TGF-b1 genotype. The survival of patients of the homozygous high TGF-b1 is very poor (100% mortality within 3.5 years) compared to the survival of patients with intermediate or low TGF-b1 producer genotypes (about 65% at 5 years). IMPLICATIONS OF THE ASSOCIATION BETWEEN CYTOKINE GENOTYPE AND REJECTION

We may be able to predict which of our patients are most likely to suffer acute rejection episodes. In some cases, this may be used to rationalize organ allocation, because it is clear that HLA-DR mismatched grafts fare poorly in recipients of the high TNF-a producer genotype. After transplantation, those recipients who can be classified in a broad sense as potentially high responders to their transplant will probably need to be started on higher levels of immunosuppression, or more potent combinations of agents, than those who are of low responder status. Indeed, it may be possible to omit the administration of steroids in the low responder group and to manage immunosuppression with moderate doses of cyclosporine (CyA) or tacrolimus-based therapy. Since it is necessary to avoid the side effects of immunosuppressive therapy (toxicity, infection, and malignancy), determination of the low responder may be as important as the identification of the high responder. We are also interested in the cytokine gene profile of those who seemingly become tolerant of their grafts and have minimal or no immunosuppression, as we may be able to predict those in whom it is safe to withdraw therapy. The choice between CyA and tacrolimus may depend on the genetically endowed predisposition of individuals to produce high or low amounts of TGF-b1, since there is clear genetic evidence that high TGF-b1 production is detrimental to long-term graft outcome. CyA increases TGF-b1 production in vitro10 and in vivo.11 It can be argued that patients already inclined to produce high levels of this cytokine should not be treated with agents which exacerbate this propensity. Conversely, TGF-b1 is immunosuppressive and anti-inflammatory, so a case can be made that certain low TGF-b1 genotype patients should receive CyA, at least in the early transplant period. The place of rapamycin and its derivatives will be made clear by clinical trials. It is predicted that the combination of

HUTCHINSON, TURNER, SANKARAN ET AL

rapamycin with CyA or tacrolimus will be synergistic and may particularly benefit high responder recipients. Mycophenolate and tacrolimus are thought to be good at regulation of B-cell responses. So far we have not analyzed the genotype of those prone to “antibody-mediated” rejection, other than to note that the subgroup of kidney transplant recipients with a high IL-10 genotype appears to be at greater risk of vascular rejection. Again, we may speculate that certain combinations of agents may be especially effective in those prone to humoral responses. The conduct of clinical trials of new immunosuppressive agents becomes ever more difficult as two potent agents are compared in a limited patient pool. If the acute rejection rate of a transplanted population is only 25% then to show a statistically significant reduction of 5% of one agent over another would require testing in nearly 2000 patients. However, if such trials were confined to high TNF-a genotyped recipients in whom there is, approximately, a 75% acute rejection rate, then only 200 patients would be need to show a difference, if one existed, between agents. THE FUTURE

Clearly, the use of cytokine genotype profiles to guide immunosuppression requires greater refinement, especially in prospective trials. However, the determination of cytokine genotype appears to be a reliable way to predict outcome in retrospective analyses. The methodology is simple for any tissue typing laboratory to perform. We suggest that genotyping for high and low cytokine responses will become routine in many transplant centers. In addition, selected patient subgroups may be used to compare the efficacy of new immunosuppressive agents or new formulations of old ones. REFERENCES 1. Wilson AG, Symons JA, McDowell T, et al: Proc Natl Acad Sci USA 94:3195, 1997 2. Turner DM, Williams DM, Sankaran D, et al: Eur J Immunogenet 24:1, 1997 3. Awad MR, Turner DM, Sinnott PJ, et al: J Immunogenet 24:45, 1997 4. Sankaran D, Turner DM, Johnson RW, et al: Eur J Immunogenet 24:65, 1997 5. Turner DM, Sankaran D, Grant SCD, et al: Eur J Immunogenet 24:67, 1997 6. Turner DM, Grant SCD, Yonan N, et al: Transplantation 64:776, 1997 7. El Gamel A, Awad MR, Yonan NA, et al: Int J Heart Lung Transplant 16:65, 1997 8. Asderakis A, Pravica V, Sankaran D, et al: Presented at the XVII World Congress of the Transplantation Society Montreal (Abstract 290), 1998 9. Awad MR, Pravica V, El Gamel A, et al: Immunology 92(suppl 1):49, 1997 10. Ahuja SS, Shrivastav S, Danielpour D, et al: Transplantation 60:718, 1995 11. Suthanthiran M, Shin GT, Khanna A, et al: Presented at the XVI International Congress of the Transplantation Society, Barcelona (Abstract 185), 1996