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Transplantation II

Immune Markers of Graft Dysfunction

 

Background

Graft survival for all solid organ transplantation procedures is restricted by acute and chronic rejections. The solution to this problem is induction of a state of donor-specific tolerance in the patient so rejections will not occur. Current methods of diagnosing allograft dysfunction are inadequate in that significant organ damage occurs prior to the establishment of a clinical diagnosis. Clinical tolerance remains an elusive goal despite success in animal models. One of the main hurdles in developing tolerance strategies is the lack of a clinical biomarker or a "tolerance assay." The development of assays or novel technologies that will enable detection of allograft dysfunction/rejection, monitor responses to therapy, and predict long-term

outcomes is vital for the success of transplantation clinical trials.

 

Objectives

•     Address the validation of histological evidence of graft dysfunction by immunological methods

•     Develop noninvasive techniques that use peripheral blood and urine to establish biomarkers that may be used as surrogate endpoints in transplantation clinical trials

•     Evaluate newer methods of functional prediction by genomic DNA typing

 

Agenda

Moderators:   Manikkam Suthanthirian, M.D., New York Hospital, Cornell Medical Center

Mohamed H. Sayegh, M.D., Brigham and Women’s Hospital, Harvard Medical School

Intragraft Cytokine and Cytotoxic T Lymphocyte Gene Expression

Margaret J. Dallman, D.Phil., Imperial College of Science, Technology, and Medicine, United Kingdom

Cytotoxic Lymphocyte Gene Expression Events and Allograft Rejection

Terry Strom, M.D., Harvard Medical School and Beth Israel Deaconess Medical Center

Cytotoxic T Lymphocyte Gene Expression in Urinary Cells During Acute Rejection

Manikkam Suthanthirian, M.D.

Cytokine Gene Polymorphisms

Ian V. Hutchinson, B.Sc., Ph.D., University of Manchester, United Kingdom

 

Break

Indirect Allorecognition:  A Predictor of Chronic Allograft Dysfunction?

Mohamed H. Sayegh, M.D.

Immune Parameters Correlating With Long-Term Graft Outcome

Nancy L. Reinsmoen, Ph.D., Duke University Medical Center

Open Discussion

Summary of Session Recommendations

 

ABSTRACTS

Intragraft Cytokine and Cytotoxic T Lymphocyte Gene Expression

Margaret J. Dallman, D.Phil.

Cytokines play a central role in directing both the magnitude and type of immune response generated to organ transplants.  Since they are normally expressed at low or undetectable levels but rapidly upregulated at the onset of an immune response, they should provide early predictors of graft dysfunction and in theory provide information about the mechanism of immune attack.  Difficulties have been encountered in (1) finding sensitive and easily performed methods of cytokine detection, (2) retrieving material from the transplant on a frequent basis for analysis, and (3) defining patterns of cytokine expression that are unique to rejection.  Genes associated with cytotoxic T lymphocyte function may aid in differentiating rejection from other immunological conditions.

 

Key References

Dallman MJ. Cytokines and transplantation: Th1/Th2 regulation of the immune response to solid organ transplants in the adult. Curr Opin Immunol 1995;7:632‑638.

McLean AG, Hughes D, Welsh KI, Gray DW, Roake J, Fuggle SV, Morris PJ, Dallman MJ. Patterns of graft infiltration and cytokkine gene expression during the first 10 days of kidney transplantation. Transplantation 1997;63:374‑380.

Piccotti JR, Chan SY, VanBuskirk AM, Eichwald EJ, Bishop DK. Are Th2 helper T lymphocytes beneficial, deleterious, or irrelevant in promoting allograft survival? Transplantation 1997;63:619‑624.

Strom TB, Roy‑Chaudhury P, Manfro R, Zheng XX, Nickerson PW, Wood K, Bushell A. The Th1/Th2 paradigm and the allograft response. Curr Opin Immunol 1996;8:688‑693.

 

Cytotoxic Lymphocyte Gene Expression Events and Allograft Rejection

Terry Strom, M.D.

Gene expression for granzyme B, perforin, and fas L are strongly linked to acute renal allograft rejection.  These events can be detected in allograft  biopsies, peripheral blood cells, or urinary leukocytes.  Expression of IL‑15 and IL‑10 are also closely linked to clinical or subclinical rejection.  We believe that a refined molecularly defined diagnostic strategy is ready for clinical deployment and may prevent fixed tissue injury and resultant chronic rejection.

 

Cytotoxic T Lymphocyte Gene Expression in Urinary Cells During

Acute Rejection

Manikkam Suthanthirian, M.D.

Acute rejection remains an important clinical challenge, and accurate diagnosis is contingent on the invasive procedure of renal allograft biopsy.  Core needle biopsies, while providing invaluable information, carry with them the risk of significant hematuria, arteriovenous fistulas, or graft loss.  Successful development of a noninvasive surrogate for allograft biopsies, therefore, has the significant potential for improving transplant patient management.  We have initiated studies investigating the correlation between histologic diagnosis of acute rejection and urinary cell cytotoxic attack molecule mRNA steady‑state levels.  RT‑quantitative polymerase chain reaction was used to quantify mRNA encoding perforin or granzyme B in urinary cells.  Renal allograft biopsies were classified on the basis of Banff criteria, and the mRNA steady‑state levels were correlated with the histologic diagnosis.  Our data suggest that the level of perforin transcripts or that of granzyme B transcripts in urinary cells is significantly higher during histologically validated acute rejection compared with no rejection.

 

Key References

Suthanthirian M. Molecular analyses of human renal allografts: Differential intragraft gene expression during rejection. Kidney Int 1997;51(Suppl 58):S‑15‑S21.

 

Cytokine Gene Polymorphisms

Ian V. Hutchinson, Ph.D.

The production of cytokines is under genetic control.  Alleles of the cytokine genes encode high or low production, and because these segregate independently, each one is a mosaic of higher or lower responses.  In the context of transplantation, the cytokine genes we have studied are those for the inflammatory cytokines tumor necrosis factor‑alpha (TNF‑alpha) and interferon‑gamma (IFN‑gamma) and the anti‑inflammatory/immunoregulatory cytokines interleukin‑10 (IL‑10) and transforming growth factor‑beta 1 (TGF‑beta 1).  TGF‑beta 1 also has potent fibrogenic activities.  We have used simple polymerase chain reaction‑based methods to genotype kidney, heart, lung, and liver transplant recipients for these cytokines.  Acute cellular rejection is strongly associated with high‑producer TNF‑alpha genotype in heart and kidney recipients, whereas IL‑10 and IFN‑gamma play modulating roles.  Chronic rejection (including declining graft function, transplant vasculopathy, graft loss, and patient death) is strongly associated with high‑producer TGF‑beta 1 genotype.  These results imply that TNF‑alpha and TGF‑beta 1 are pivotal cytokines in the acute and chronic transplant rejection.

 

Key References

Awad MR, El‑Gamel A, Simm E, Hasleton PS, Yonan N, Deiraniya AK, Sinnott PJ, Hutchinson IV. Genotypic variation in the transforming growth factor‑beta 1 gene: Association with TGF‑beta 1 production, fibrotic lung disease and graft fibrosis after lung transplantation. Transplantation 1998;67:1014‑1020.

Awad M, Pravica V, Perrey C, Sinnott PJ, Hutchinson IV. CA repeat allele polymorphism in the first intron of the human interferon‑gamma gene is associated with lung allograft fibrosis. Human Immunology, in press.

Crawley E, Kay R, Sillibourne J, Patel P, Hutcinson IV, Woo P. Polymorphic haplotypes of the IL‑10 5' flanking region determine variable IL‑10 transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis. Arthritis Rheum, in press.

Hutchinson IV, Pravica V, Sinnott PJ. Genetic regulation of cytokine synthesis: Consequences for acute and chronic transplant rejection. Graft 1998;1:186‑192.

Hutchinson IV, Turner DM, Sankaran D, Awad M, Pravica V, Sinnott PJ. Cytokine genotypes in allograft rejection: Guidelines for immunosuppression. Transpl Proc 1998;30:3991‑3992.

Perrey C, Pravica V, Sinnott PJ, Hutchinson IV. Genotyping for polymorphisms in interferon‑gamma, interleukin‑10, transforming growth factor‑beta 1 and tumour necrosis factor‑alpha genes: A technical report. Transpl Immunol 1998;6:193‑197.

Pravica V, Asderakis A, Perrey C, Hajeer A, Sinnott PJ, Hutchinson IV. In vitro production of IFN‑gamma correlates with CA repeat polymorphism in the human IFN‑gamma gene. Eur J Immunogen, in press.

Sankaran D, Ashraf S, Asderakis A, Roberts ISD, Short CD, Dyer PA, Sinnott PJ, Hutchinson IV. Tumour necrosis factor‑alpha and interleukin‑10 gene polymorphisms predict acute renal allograft rejection. Kidney Int, in press.

Turner DM, Grant SCD, Yonan N, Sheldon S, Dyer PA, Sinnott PJ, Hutchinson IV. Cytokine gene polymorphism and heart transplant rejection. Transplantation 1997;64:776‑779.

Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV. An investigation of polymorphism in the Interleukin‑10 gene promoter. Eur J Immunogen 1997;24:1‑8.

 

Indirect Allorecognition:  A Predictor of Chronic Allograft Dysfunction?

Mohamed H. Sayegh, M.D.

CD4+ T cell recognition of alloantigen is the key initial event that leads to graft rejection.  There are two nonmutually exclusive pathways of allorecognition.  In the direct allorecognition pathway, T cells recognize intact donor major histocompatibility complex (MHC) molecules on donor antigen‑presenting cells.  In the indirect pathway, T cells recognize processed donor alloantigen presented as allopeptides by recipient antigen‑presenting cells.  There is increasing evidence to suggest that both pathways play important roles in the rejection process but that indirect allorecognition may be the dominant role in chronic rejection.  Indeed, T cells from patients with chronic graft dysfunction exhibit specific alloreactivity to donor MHC peptides with epitope spreading.  The utility of these assays as a biomarker of allograft dysfunction will be discussed.

 

 

Immune Parameters Correlating With Long‑Term Graft Outcome

Nancy L. Reinsmoen, Ph.D.

Our long‑term goal is to identify immune parameters that predict graft outcome and use these parameters to identify recipients who are candidates for individualization of immunosuppression.  We have used the immune parameters of donor antigen‑specific hyporeactivity, peripheral blood allogeneic microchimerism, donor antigen‑specific anti‑HLA antibodies, and cytokine gene polymorphism to test solid organ recipients transplanted at two centers.  We have found that hyporeactivity of the donor antigen‑specific response at 1 year posttransplant, as determined by a decreased donor antigen‑specific proliferative response, is predictive of a chronic rejection‑free state in kidney, heart, and lung recipients (predictive value=96 percent for kidney, 100 percent for lung, and 91 percent for heart recipients).  Furthermore, kidney recipients who have experienced an acute rejection episode and remain responsive to donor antigen are at high risk for developing chronic rejection versus those who develop hyporeactivity (odds ratio=3.61, p=0.0042).  The presence of high levels of peripheral blood allogeneic microchimerism (>1:10,000) in lung recipients is also associated with a chronic rejection‑free state (BOS grade <1)(p=0.029 at 18 months posttransplant).  Recently, we have identified kidney recipients who, at the time of transplantation, had donor class II‑directed antibodies detectable only by ELISA or flow cytometry techniques but not by cytotoxicity, and were at high risk for acute and chronic rejection.  Of seven such kidney recipients, all had one or more acute rejection episodes, and three have developed biopsy‑proven chronic rejection.  We have also investigated whether cytokine (TNF‑(, TGF‑$ and IL‑10) genotype polymorphisms associated with high and low cytokine production contribute a greater propensity for acute or chronic rejection.  In heart recipients (n=28), four allelic patterns were found only in seven recipients with coronary artery disease (CAD); four other patterns were found only in eight recipients without CAD; and three other unique patterns were found in four recipients with and nine recipients without CAD (p<0.001).  We are currently genotyping lung and kidney recipients.  Thus, the use of multiple parameters to assess a recipient’s immune profile may more accurately predict graft outcome, thus permitting individualization and optimization of immunosuppression.