Early Microchimerism in Peripheral Blood Following Kidney Transplantation H. Kishikawa, T. Kinoshita, S. Yonemoto, M. Kawamura, S. Nakazawa, N. Ueda, T. Hirai, K. Nishimura, M. Hashimoto, and Y. Ichikawa ABSTRACT Background. The role of microchimerism found in the peripheral blood of renal transplant recipients remains a matter of debate. We assessed the frequency of microchimerism after kidney transplantation and examined its influence on clinical courses over a 12-month follow-up period. Patients and Methods. Ten single-kidney recipients underwent microchimerism detection at 2 days, 2 weeks, and 1, 3, 6, and 12 months after transplantation, with mismatch human leukocyte antigen (HLA)-A, -B, and -C used as markers. Results. Microchimerism was detected in 8 (80%) patients at 2 days after kidney transplantation. In 3 of those, microchimerism became negative within 3 months after transplantation, whereas it remained present for up to 12 months in 3 patients (33 %). There was 1 acute rejection episode in a patient in whom microchimerism became negative within 3 months. Protocol renal graft biopsy specimens obtained 3 months after transplantation revealed no acute cellular-mediated rejection (ACMR) or acute antibody-mediated rejection (AAMR) in the 5 patients positive for microchimerism at 3 months. Conclusions. Microchimerism was frequently detected after kidney transplantation. Microchimerism that remained for more than 3 months post-transplantation might be correlated with a lower incidence of rejection, thus its monitoring may help identify recipients with a low rejection risk.
S
TARZL et al were the first to report that persistence of donor-derived cells may be a key point for acceptance of grafted organs in cases of transplantation [1]. However, the role of microchimerism in the peripheral blood of renal transplant recipients remains a matter of debate. In some studies, microchimerism has been reported to be associated with graft tolerance or immunosuppressive therapy [2,3], whereas it was not found to be a sign of graft acceptance or graft rejection in another series of patients [4]. In a longitudinal follow-up study of recent transplant recipients, the amount of microchimerism was noted to have great variation in patients after different post-transplantation periods [5]. Thus, the relationship between microchimerism and rejection in recent transplant recipients is vague. In the present study, we assessed the serial frequency of microchimerism early after kidney transplantation and examined its influence on clinical course over a 12-month follow-up period. 0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.12.036 388
PATIENTS AND METHODS All 10 patients who underwent primary single-kidney transplantation in our hospital between June and December 2012 were enrolled in the study and clinically followed up in a prospective manner for a mean period of 13.2 months (range, 10e16 months). The independent ethics committee at our institution approved this study and written informed consent was obtained from each participant before enrollment. Demographic data are summarized in Table 1. All patients received standard immunosuppressive therapy consisting of a calcineurin inhibitor (CNI), mycophenolate mofetil (MMF), and steroid administration with basiliximab induction. Peripheral blood samples were collected 2 days, 2 weeks,
From the Department of Renal Transplantation Center, Hyogo Prefectural Nishinomiya Hospital, Nishinomiya, Hyogo, Japan. Address reprint requests to Hidefumi Kishikawa, MD, PhD, Department of Renal Transplantation Center, Hyogo Prefectural Nishinomiya Hospital, Rokutanji-cho 13-9, Nishinomiya, Hyogo, 662-0918, Japan. E-mail: [email protected] ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 388e390 (2014)
EARLY MICROCHIMERISM
389 Table 1. Patient Characteristics HLA-mismach
History of
Pt no.
Age
Gender
Original disease
Donor
A
B
DRB1
Pregnancy
Transfusion
CNI
Follow up period (m)
1 2 3 4 5 6 7 8 9 10
39 69 66 42 60 48 54 46 18 41
F F M M M F M M M M
IgAN DM CGN DM DM VUR nephropathy IgAN CGN IgAN CGN
mother daughter spouse spouse spouse mother deceased donor mother mother father
1 1 1 2 1 1 1 1 0 1
1 1 1 1 1 1 2 1 1 1
1 1 2 1 2 1 1 1 1 1
þ
þ þ þ þ þ e
CsA Tac CsA Tac CsA Tac Tac CsA Tac Tac
16 15 14 14 13 13 13 12 12 10
Abbreviations: F, female; M, male; IgAN, IgA nephropathy; DM, diabetes mellitus; CGN, chronic glomerulonephritis; VUR, vesicoureteral reflux; CsA, cyclosporine; Tac, tacrolimus.
and 1, 3, 6, and 12 months after transplantation. DNA was extracted from peripheral blood and microchimerism presence was analyzed using a nested polymerase chain reaction (PCR) using HLA class I (HLA-A, -B, or -C) locus specific primers for the first stage and donor-recipient mismatched allele-specific primers for the second stage. The first-stage template contained approximately 6 mg of genomic DNA, whereas the second contained 1 104e1 106 dilutions of the product obtained from the previous stage of amplification. Five microliters of each of the PCR-amplified products were analyzed on 2% NuSieve GTG and 1% SeaKem agarose ethidium bromide-stained gels. The sensitivity of this assay was confirmed by using samples collected from volunteers with known HLA and mixed at different proportions. This approach allowed us to investigate the presence of donor-mismatched alleles with a sensitivity of 104e105, independent of donor and recipient gender. The-donor specific anti-HLA antigen (DSA) was tested using flow cytometric panel-reactive antibody (FlowPRA) Single Antigen Class I and Class II beads before and 1 year after transplantation or at the last visit to our clinic. A renal allograft biopsy was performed 3 months after transplantation or when a rejection episode was suspected.
8 patients, microchimerism became undetectable within 3 months after transplantation, whereas it remained positive after 6 months in the other 5 (50%). Nine of the 10 patients were followed up for more than 1 year, and microchimerism was maintained in 3 (33%) throughout that period. Once microchimerism changed from positive to negative; it did not turn to positive again in any of our patients. There was 1 acute cellular-mediated rejection (ACMR) plus acute antibody-mediated rejection (AAMR) episode in a patient at 8 weeks after transplantation in whom microchimerism turned to negative within 3 months after the procedure. Protocol renal graft biopsy specimens obtained 3 months after transplantation revealed no ACMR or AAMR episodes in the 5 patients positive for microchimerism at 3 months after receiving the transplant, whereas there was 1 ACMR episode in a patient in whom microchimerism was not detected at 3 months. Therefore, among the patients who were microchimerism negative at 3 months, 40% showed biopsy-proven acute rejection, whereas there were no rejection episodes in the microchimerism-positive patients. DSA was tested at 1 year after transplantation or at the last visit (10e12 months post-transplantation). De novo DSA was detected in 1 patient who had a rejection episode and microchimerism was negative at 3 months.
RESULTS
Microchimerism was detected in 8 of 10 patients (80%) 2 days after kidney transplantation (Table 2). In 3 of those
Table 2. Serial Results of Microchimerism, Biopsy-Proven Rejection, De Novo DSA, and Graft Function After Kidney Transplantation Microchimerism Pt no.
1 2 3 4 5 6 7 8 9 10
Biopsy proven rejection
2 Days
2 Weeks
1 Month
3 Months
6 Months
12 Months
AAMR
ACMR
De novo DSA
eGFR at 12 Months
D D L D þ þ þ L D D
D D L D þ þ L L D D
D D L D þ L L L D D
D D L D L L L L D D
D D L D L L L L D D
D D L D L L L L L NT
L L L L þ L L L L L
L L L L þ L þ L L L
L L L L þ L L L L L
50.1 83.5 42.3 69.4 34.2 36.7 19.6 31.7 85.6 44.2*
Abbreviations: AAMR, acute antibody-mediated rejection; ACMR, acute cellular-mediated rejection. *10 months after transplantation.
390
Graft function at 1 year after transplantation was compared between patients who were microchimerism positive and negative at 3 months after transplantation. The estimated glomerular filtration rate (eGFR) value of the microchimerism-positive patients was higher (66.6 vs 32.9; P ¼ .0066). DISCUSSION
We used a nested PCR technique for detection of donorspecific HLA class I alleles to determine microchimerism in 10 patients following kidney transplantation. Advantages of this method include the ability to determine the presence of microchimerism regardless of a gender mismatch between donor and recipient, and revealed the serial rate of microchimerism early after kidney transplantation. The highest frequency of microchimerism in our patients appeared 2 days after transplantation. Thereafter, it gradually decreased and persistent microchimerism beyond 12 months was noted in 33%, as previously reported [3]. In patients with microchimerism that changed to negative later, this may have been from detached endothelial cells from the allograft, reflecting the ongoing repair mechanism after endothelial damage. However, it was not possible to further characterize these cells in this study. Regarding this point, it would be interesting to clarify the nature of allograft emigrating cells and further distinguish donor-specific cell subsets. We also found a relationship between the presence of microchimerism more than 3 months after transplantation and lower incidence of biopsy-proven acute rejection episodes, de novo DSA, and better kidney function. Maintenance of microchimerism more than 3 months after
KISHIKAWA, KINOSHITA, YONEMOTO ET AL
transplantation may have some immunologic relevance to clinical course, although it is not clear whether microchimerism is the origin or consequence of a tolerogenic state. Limitations of our study include the small number of patients and relatively short follow-up period. A greater number of patients and longer observation period would be helpful for a better understanding of the clinical effects of microchimerism following transplantation. In conclusion, microchimerism was frequently detected early after kidney transplantation. Microchimerism maintained more than 3 months post-transplantation might be correlated with low incidence of rejection, and its monitoring could help detection of low rejectionerisk recipients. Further studies should be undertaken with larger numbers of patients for a longer observation period to clarify the real biological significance of microchimerism.
REFERENCES [1] Starzl TE, Demetris AJ, Murase N, et al. Cell migration, chimerism, and graft acceptance. Lancet 1992;339(8809):1579e82. [2] Janes S, Dhaliwal P, Wood K. Tolerance in renal transplantation: is mixed chimerism the missing link? Nephrol Dial Transplant 2009;24(6):1726e9. [3] Pujal JM, Grinyo JM, Gil-Vernet S, et al. Early hematopoietic microchimerism predicts clinical outcome after kidney transplantation. Transplantation 2007;84:1103e11. [4] Curcio M, Cantarovich D, Barbuti S, et al. Association of donor-specific microchimerism with graft dysfunction in kidney transplant patients. Transpl Immunol 2012;26(2e3):151e5. [5] Elwood ET, Larsen CP, Maurer DH, et al. Microchimerism and rejection in clinical transplantation. Lancet 1997;349(9062): 1358e60.
S
TARZL et al were the first to report that persistence of donor-derived cells may be a key point for acceptance of grafted organs in cases of transplantation [1]. However, the role of microchimerism in the peripheral blood of renal transplant recipients remains a matter of debate. In some studies, microchimerism has been reported to be associated with graft tolerance or immunosuppressive therapy [2,3], whereas it was not found to be a sign of graft acceptance or graft rejection in another series of patients [4]. In a longitudinal follow-up study of recent transplant recipients, the amount of microchimerism was noted to have great variation in patients after different post-transplantation periods [5]. Thus, the relationship between microchimerism and rejection in recent transplant recipients is vague. In the present study, we assessed the serial frequency of microchimerism early after kidney transplantation and examined its influence on clinical course over a 12-month follow-up period. 0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.12.036 388
PATIENTS AND METHODS All 10 patients who underwent primary single-kidney transplantation in our hospital between June and December 2012 were enrolled in the study and clinically followed up in a prospective manner for a mean period of 13.2 months (range, 10e16 months). The independent ethics committee at our institution approved this study and written informed consent was obtained from each participant before enrollment. Demographic data are summarized in Table 1. All patients received standard immunosuppressive therapy consisting of a calcineurin inhibitor (CNI), mycophenolate mofetil (MMF), and steroid administration with basiliximab induction. Peripheral blood samples were collected 2 days, 2 weeks,
From the Department of Renal Transplantation Center, Hyogo Prefectural Nishinomiya Hospital, Nishinomiya, Hyogo, Japan. Address reprint requests to Hidefumi Kishikawa, MD, PhD, Department of Renal Transplantation Center, Hyogo Prefectural Nishinomiya Hospital, Rokutanji-cho 13-9, Nishinomiya, Hyogo, 662-0918, Japan. E-mail: [email protected] ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 388e390 (2014)
EARLY MICROCHIMERISM
389 Table 1. Patient Characteristics HLA-mismach
History of
Pt no.
Age
Gender
Original disease
Donor
A
B
DRB1
Pregnancy
Transfusion
CNI
Follow up period (m)
1 2 3 4 5 6 7 8 9 10
39 69 66 42 60 48 54 46 18 41
F F M M M F M M M M
IgAN DM CGN DM DM VUR nephropathy IgAN CGN IgAN CGN
mother daughter spouse spouse spouse mother deceased donor mother mother father
1 1 1 2 1 1 1 1 0 1
1 1 1 1 1 1 2 1 1 1
1 1 2 1 2 1 1 1 1 1
þ
þ þ þ þ þ e
CsA Tac CsA Tac CsA Tac Tac CsA Tac Tac
16 15 14 14 13 13 13 12 12 10
Abbreviations: F, female; M, male; IgAN, IgA nephropathy; DM, diabetes mellitus; CGN, chronic glomerulonephritis; VUR, vesicoureteral reflux; CsA, cyclosporine; Tac, tacrolimus.
and 1, 3, 6, and 12 months after transplantation. DNA was extracted from peripheral blood and microchimerism presence was analyzed using a nested polymerase chain reaction (PCR) using HLA class I (HLA-A, -B, or -C) locus specific primers for the first stage and donor-recipient mismatched allele-specific primers for the second stage. The first-stage template contained approximately 6 mg of genomic DNA, whereas the second contained 1 104e1 106 dilutions of the product obtained from the previous stage of amplification. Five microliters of each of the PCR-amplified products were analyzed on 2% NuSieve GTG and 1% SeaKem agarose ethidium bromide-stained gels. The sensitivity of this assay was confirmed by using samples collected from volunteers with known HLA and mixed at different proportions. This approach allowed us to investigate the presence of donor-mismatched alleles with a sensitivity of 104e105, independent of donor and recipient gender. The-donor specific anti-HLA antigen (DSA) was tested using flow cytometric panel-reactive antibody (FlowPRA) Single Antigen Class I and Class II beads before and 1 year after transplantation or at the last visit to our clinic. A renal allograft biopsy was performed 3 months after transplantation or when a rejection episode was suspected.
8 patients, microchimerism became undetectable within 3 months after transplantation, whereas it remained positive after 6 months in the other 5 (50%). Nine of the 10 patients were followed up for more than 1 year, and microchimerism was maintained in 3 (33%) throughout that period. Once microchimerism changed from positive to negative; it did not turn to positive again in any of our patients. There was 1 acute cellular-mediated rejection (ACMR) plus acute antibody-mediated rejection (AAMR) episode in a patient at 8 weeks after transplantation in whom microchimerism turned to negative within 3 months after the procedure. Protocol renal graft biopsy specimens obtained 3 months after transplantation revealed no ACMR or AAMR episodes in the 5 patients positive for microchimerism at 3 months after receiving the transplant, whereas there was 1 ACMR episode in a patient in whom microchimerism was not detected at 3 months. Therefore, among the patients who were microchimerism negative at 3 months, 40% showed biopsy-proven acute rejection, whereas there were no rejection episodes in the microchimerism-positive patients. DSA was tested at 1 year after transplantation or at the last visit (10e12 months post-transplantation). De novo DSA was detected in 1 patient who had a rejection episode and microchimerism was negative at 3 months.
RESULTS
Microchimerism was detected in 8 of 10 patients (80%) 2 days after kidney transplantation (Table 2). In 3 of those
Table 2. Serial Results of Microchimerism, Biopsy-Proven Rejection, De Novo DSA, and Graft Function After Kidney Transplantation Microchimerism Pt no.
1 2 3 4 5 6 7 8 9 10
Biopsy proven rejection
2 Days
2 Weeks
1 Month
3 Months
6 Months
12 Months
AAMR
ACMR
De novo DSA
eGFR at 12 Months
D D L D þ þ þ L D D
D D L D þ þ L L D D
D D L D þ L L L D D
D D L D L L L L D D
D D L D L L L L D D
D D L D L L L L L NT
L L L L þ L L L L L
L L L L þ L þ L L L
L L L L þ L L L L L
50.1 83.5 42.3 69.4 34.2 36.7 19.6 31.7 85.6 44.2*
Abbreviations: AAMR, acute antibody-mediated rejection; ACMR, acute cellular-mediated rejection. *10 months after transplantation.
390
Graft function at 1 year after transplantation was compared between patients who were microchimerism positive and negative at 3 months after transplantation. The estimated glomerular filtration rate (eGFR) value of the microchimerism-positive patients was higher (66.6 vs 32.9; P ¼ .0066). DISCUSSION
We used a nested PCR technique for detection of donorspecific HLA class I alleles to determine microchimerism in 10 patients following kidney transplantation. Advantages of this method include the ability to determine the presence of microchimerism regardless of a gender mismatch between donor and recipient, and revealed the serial rate of microchimerism early after kidney transplantation. The highest frequency of microchimerism in our patients appeared 2 days after transplantation. Thereafter, it gradually decreased and persistent microchimerism beyond 12 months was noted in 33%, as previously reported [3]. In patients with microchimerism that changed to negative later, this may have been from detached endothelial cells from the allograft, reflecting the ongoing repair mechanism after endothelial damage. However, it was not possible to further characterize these cells in this study. Regarding this point, it would be interesting to clarify the nature of allograft emigrating cells and further distinguish donor-specific cell subsets. We also found a relationship between the presence of microchimerism more than 3 months after transplantation and lower incidence of biopsy-proven acute rejection episodes, de novo DSA, and better kidney function. Maintenance of microchimerism more than 3 months after
KISHIKAWA, KINOSHITA, YONEMOTO ET AL
transplantation may have some immunologic relevance to clinical course, although it is not clear whether microchimerism is the origin or consequence of a tolerogenic state. Limitations of our study include the small number of patients and relatively short follow-up period. A greater number of patients and longer observation period would be helpful for a better understanding of the clinical effects of microchimerism following transplantation. In conclusion, microchimerism was frequently detected early after kidney transplantation. Microchimerism maintained more than 3 months post-transplantation might be correlated with low incidence of rejection, and its monitoring could help detection of low rejectionerisk recipients. Further studies should be undertaken with larger numbers of patients for a longer observation period to clarify the real biological significance of microchimerism.
REFERENCES [1] Starzl TE, Demetris AJ, Murase N, et al. Cell migration, chimerism, and graft acceptance. Lancet 1992;339(8809):1579e82. [2] Janes S, Dhaliwal P, Wood K. Tolerance in renal transplantation: is mixed chimerism the missing link? Nephrol Dial Transplant 2009;24(6):1726e9. [3] Pujal JM, Grinyo JM, Gil-Vernet S, et al. Early hematopoietic microchimerism predicts clinical outcome after kidney transplantation. Transplantation 2007;84:1103e11. [4] Curcio M, Cantarovich D, Barbuti S, et al. Association of donor-specific microchimerism with graft dysfunction in kidney transplant patients. Transpl Immunol 2012;26(2e3):151e5. [5] Elwood ET, Larsen CP, Maurer DH, et al. Microchimerism and rejection in clinical transplantation. Lancet 1997;349(9062): 1358e60.
