Urinary FOXP3 Messenger RNA and Renal-Allograft Rejection
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《新英格兰医药杂志》
To the Editor: The observations of Muthukumar et al. (Dec. 1 issue)1 regarding the clinical usefulness of FOXP3 messenger RNA (mRNA) in predicting the outcome of renal-allograft rejection are thought-provoking, but we believe caution is needed. Although data from their study suggest that levels of FOXP3 mRNA in urine are the best prognostic indicator of reversal of acute rejection, there is substantial overlap in values between the patient groups (as shown in Figure 2 of the article). In addition, the overlapping confidence intervals for the areas under the receiver-operating-characteristic (ROC) curves (shown in Figure 3 of the article) suggest the need for larger group sizes to establish the superiority of this marker definitively.
It is not known whether FOXP3 mRNA levels in urine can be influenced by factors other than acute rejection (e.g., genetic variability and disease conditions). The authors note the need for reevaluation of the sensitivity and specificity of these markers in an independent population. When such confirmatory studies are performed, Standards for Reporting of Diagnostic Accuracy guidelines should be followed.2,3 Criteria for the selection of patients and approaches to the evaluation of results when the diagnosis is uncertain should be clearly specified.
Malek Kamoun, M.D., Ph.D.
University of Pennsylvania School of Medicine
Philadelphia, PA 19104
malekkam@mail.med.upenn.edu
James C. Boyd, M.D.
University of Virginia Health System
Charlottesville, VA 22908-0214
References
Muthukumar T, Dadhania D, Ding R, et al. Messenger RNA for FOXP3 in the urine of renal-allograft recipients. N Engl J Med 2005;353:2342-2351.
Bossuyt PM, Reitsma JB, Bruns DE, et al. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD Initiative. Ann Intern Med 2003;138:40-44.
Bossuyt PM, Reitsma JB, Bruns DE, et al. The STARD statement for reporting studies of diagnostic accuracy: explanation and elaboration. Clin Chem 2003;49:7-18.
To the Editor: Muthukumar et al. report that increased urinary FOXP3 transcripts are predictive of graft outcome in acute rejection. Enhanced expression of this T-cell "master switch" in acute rejection is a surprise that calls for validation on biopsy. We examined biopsy samples that had been obtained to rule out or confirm rejection in children with renal transplants. We also studied gene expression after obtaining informed consent and approval by the institutional review board of the Hannover Medical School in Hannover, Germany. Our data confirm high intragraft FOXP3 gene expression during acute rejection in 14 patients (FOXP3/GAPDH standard, 3.03±3.24x10–3 ), as compared with grafts without rejection in 16 patients (FOXP3/GAPDH standard, 0.95±1.4x10–3; P<0.02). In our series, FOXP3 gene expression correlated well with enhanced transcription of effector T cells (as indicated by Fas ligand, P=0.03), T-cell activation (nuclear factor-B, P<0.04), and the tumor necrosis factor receptor superfamily, member 4 (OX40) linking to B-cell activation (P<0.01). These findings occurred without exposure to sirolimus. However, intragraft FOXP3 levels did not correlate with creatinine levels either before or after rejection, nor did they correlate with the Banff classification or with later outcome. No allografts failed during the follow-up period.
Currently, the role and dynamics of intragraft FOXP3 remain unclear, and regulatory action may be far more complicated (Figure 1). Resolution of a CD20+ treatment-refractory rejection by rituximab is associated with enhanced FOXP3 expression, whereas all other gene transcripts were concurrently down-regulated.
Figure 1. Differential Intragraft Gene Expression in Treatment-Refractory Acute Rejection with CD20 Infiltrates and Imminent Graft Loss Resolved by Rituximab Therapy.
Rejection that was refractory to pulse methylprednisolone, tacrolimus, and antithymocyte globulin was resolved by rituximab, as analyzed by TaqMan polymerase-chain-reaction assay. The values shown for nuclear factor-B are 1/10 the actual values. OX40 denotes tumor necrosis factor receptor superfamily, member 4.
Jürgen Strehlau, M.D.
Anna Podolskaya
Jochen Ehrich, M.D.
Hannover Medical School
D-30623 Hannover, Germany
strehlau.juergen@mh-hannover.de
The authors reply: We agree with Kamoun and Boyd that our primary conclusion — that levels of FOXP3 mRNA in urinary cells serve as an informative biomarker of outcomes in transplants with acute rejection — should be further documented in a large, independent study. In this regard, only about 25 percent of distributions of FOXP3 mRNA in patients with reversible acute rejection overlap with those in patients with nonreversible acute rejection. The point estimate of the area under the ROC curve for FOXP3 exceeds the 95 percent confidence interval for each of the other ROC curves shown in Figure 3 of our article.
We previously reported that measurement of mRNA encoding cytotoxic proteins granzyme B and perforin in urine offers a noninvasive means of diagnosing acute rejection.1 The Cooperative Clinical Trial in Transplantation, sponsored by the National Institutes of Health and the National Institute of Allergy and Infectious Diseases, will test the hypothesis that these two biomarkers are an accurate, noninvasive predictor of acute rejection. The prognostic value of FOXP3 mRNA levels needs to be ascertained in a similar fashion.
We thank Strehlau et al. for extending our observation of heightened FOXP3 gene expression during acute rejection in urinary cells from adult recipients of renal allografts to pediatric recipients of renal allografts and for demonstrating high levels of FOXP3 transcripts in allograft-biopsy specimens showing acute rejection. Heightened expression of the FOXP3 gene has been reported in endomyocardial biopsy specimens of human cardiac allografts undergoing acute rejection.2
We hypothesize that the immune response directed at an allograft has at least two components — a graft-destructive one, as exemplified by cytotoxic T cells, and a graft-protective one, mediated by regulatory T cells — and that the balance between these two opposing forces influences allograft outcomes.3 An unresolved issue is whether the regulatory T cells are from the naturally arising CD4+CD25+ FOXP3+ cells that originate in the thymus4 or whether they are induced in the periphery after antigen encounter and originate from CD25–FOXP3– precursors in a tumor growth factor milieu.5 Nevertheless, our data and the findings of Strehlau et al. suggest that acute allograft rejection is associated with both graft-destructive and graft-protective immunity and that heightened expression of the FOXP3 gene is salutary in this context.
Thangamani Muthukumar, M.D.
Weill Medical College of Cornell University
New York, NY 10021
Joseph E. Schwartz, Ph.D.
State University of New York at Stony Brook
Stony Brook, NY 11794
Manikkam Suthanthiran, M.D.
Weill Medical College of Cornell University
New York, NY 10021
msuthan@med.cornell.edu
References
Li B, Hartono C, Ding R, et al. Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N Engl J Med 2001;344:947-954.
Baan CC, van der Mast BJ, Klepper M, et al. Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the induction of FOXP3 in human T cells. Transplantation 2005;80:110-117.
Zheng XX, Sanchez-Fueyo A, Sho M, Domenig C, Sayegh MH, Strom TB. Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance. Immunity 2003;19:503-514.
Sakaguchi S. Naturally arising Foxp3-expressing CD25+ CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005;6:345-352.
Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+CD25– naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 2003;198:1875-1886.
It is not known whether FOXP3 mRNA levels in urine can be influenced by factors other than acute rejection (e.g., genetic variability and disease conditions). The authors note the need for reevaluation of the sensitivity and specificity of these markers in an independent population. When such confirmatory studies are performed, Standards for Reporting of Diagnostic Accuracy guidelines should be followed.2,3 Criteria for the selection of patients and approaches to the evaluation of results when the diagnosis is uncertain should be clearly specified.
Malek Kamoun, M.D., Ph.D.
University of Pennsylvania School of Medicine
Philadelphia, PA 19104
malekkam@mail.med.upenn.edu
James C. Boyd, M.D.
University of Virginia Health System
Charlottesville, VA 22908-0214
References
Muthukumar T, Dadhania D, Ding R, et al. Messenger RNA for FOXP3 in the urine of renal-allograft recipients. N Engl J Med 2005;353:2342-2351.
Bossuyt PM, Reitsma JB, Bruns DE, et al. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD Initiative. Ann Intern Med 2003;138:40-44.
Bossuyt PM, Reitsma JB, Bruns DE, et al. The STARD statement for reporting studies of diagnostic accuracy: explanation and elaboration. Clin Chem 2003;49:7-18.
To the Editor: Muthukumar et al. report that increased urinary FOXP3 transcripts are predictive of graft outcome in acute rejection. Enhanced expression of this T-cell "master switch" in acute rejection is a surprise that calls for validation on biopsy. We examined biopsy samples that had been obtained to rule out or confirm rejection in children with renal transplants. We also studied gene expression after obtaining informed consent and approval by the institutional review board of the Hannover Medical School in Hannover, Germany. Our data confirm high intragraft FOXP3 gene expression during acute rejection in 14 patients (FOXP3/GAPDH standard, 3.03±3.24x10–3 ), as compared with grafts without rejection in 16 patients (FOXP3/GAPDH standard, 0.95±1.4x10–3; P<0.02). In our series, FOXP3 gene expression correlated well with enhanced transcription of effector T cells (as indicated by Fas ligand, P=0.03), T-cell activation (nuclear factor-B, P<0.04), and the tumor necrosis factor receptor superfamily, member 4 (OX40) linking to B-cell activation (P<0.01). These findings occurred without exposure to sirolimus. However, intragraft FOXP3 levels did not correlate with creatinine levels either before or after rejection, nor did they correlate with the Banff classification or with later outcome. No allografts failed during the follow-up period.
Currently, the role and dynamics of intragraft FOXP3 remain unclear, and regulatory action may be far more complicated (Figure 1). Resolution of a CD20+ treatment-refractory rejection by rituximab is associated with enhanced FOXP3 expression, whereas all other gene transcripts were concurrently down-regulated.
Figure 1. Differential Intragraft Gene Expression in Treatment-Refractory Acute Rejection with CD20 Infiltrates and Imminent Graft Loss Resolved by Rituximab Therapy.
Rejection that was refractory to pulse methylprednisolone, tacrolimus, and antithymocyte globulin was resolved by rituximab, as analyzed by TaqMan polymerase-chain-reaction assay. The values shown for nuclear factor-B are 1/10 the actual values. OX40 denotes tumor necrosis factor receptor superfamily, member 4.
Jürgen Strehlau, M.D.
Anna Podolskaya
Jochen Ehrich, M.D.
Hannover Medical School
D-30623 Hannover, Germany
strehlau.juergen@mh-hannover.de
The authors reply: We agree with Kamoun and Boyd that our primary conclusion — that levels of FOXP3 mRNA in urinary cells serve as an informative biomarker of outcomes in transplants with acute rejection — should be further documented in a large, independent study. In this regard, only about 25 percent of distributions of FOXP3 mRNA in patients with reversible acute rejection overlap with those in patients with nonreversible acute rejection. The point estimate of the area under the ROC curve for FOXP3 exceeds the 95 percent confidence interval for each of the other ROC curves shown in Figure 3 of our article.
We previously reported that measurement of mRNA encoding cytotoxic proteins granzyme B and perforin in urine offers a noninvasive means of diagnosing acute rejection.1 The Cooperative Clinical Trial in Transplantation, sponsored by the National Institutes of Health and the National Institute of Allergy and Infectious Diseases, will test the hypothesis that these two biomarkers are an accurate, noninvasive predictor of acute rejection. The prognostic value of FOXP3 mRNA levels needs to be ascertained in a similar fashion.
We thank Strehlau et al. for extending our observation of heightened FOXP3 gene expression during acute rejection in urinary cells from adult recipients of renal allografts to pediatric recipients of renal allografts and for demonstrating high levels of FOXP3 transcripts in allograft-biopsy specimens showing acute rejection. Heightened expression of the FOXP3 gene has been reported in endomyocardial biopsy specimens of human cardiac allografts undergoing acute rejection.2
We hypothesize that the immune response directed at an allograft has at least two components — a graft-destructive one, as exemplified by cytotoxic T cells, and a graft-protective one, mediated by regulatory T cells — and that the balance between these two opposing forces influences allograft outcomes.3 An unresolved issue is whether the regulatory T cells are from the naturally arising CD4+CD25+ FOXP3+ cells that originate in the thymus4 or whether they are induced in the periphery after antigen encounter and originate from CD25–FOXP3– precursors in a tumor growth factor milieu.5 Nevertheless, our data and the findings of Strehlau et al. suggest that acute allograft rejection is associated with both graft-destructive and graft-protective immunity and that heightened expression of the FOXP3 gene is salutary in this context.
Thangamani Muthukumar, M.D.
Weill Medical College of Cornell University
New York, NY 10021
Joseph E. Schwartz, Ph.D.
State University of New York at Stony Brook
Stony Brook, NY 11794
Manikkam Suthanthiran, M.D.
Weill Medical College of Cornell University
New York, NY 10021
msuthan@med.cornell.edu
References
Li B, Hartono C, Ding R, et al. Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N Engl J Med 2001;344:947-954.
Baan CC, van der Mast BJ, Klepper M, et al. Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the induction of FOXP3 in human T cells. Transplantation 2005;80:110-117.
Zheng XX, Sanchez-Fueyo A, Sho M, Domenig C, Sayegh MH, Strom TB. Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance. Immunity 2003;19:503-514.
Sakaguchi S. Naturally arising Foxp3-expressing CD25+ CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005;6:345-352.
Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+CD25– naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 2003;198:1875-1886.