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Allospecific human T suppressor cells in kidney transplantation
Allospecific Human T Suppressor Cells in Kidney Transplantation E. Renna Molajoni, P. Cinti, E. Ho, B. Evangelista, D. Peritore, L. Poli, R. Pretagostini, P. Berloco, N. Suciu Foca Cortesini, and R. Cortesini
T
HE EXCELLENT early results achieved by use of current immunosuppressive therapy are reflected in 1-year graft survival rates approaching 90%. However, late chronic rejection still occurs in a considerable segment of the patient population. Hence, to determine whether a new regimen provides superior results, large numbers of patients must be enrolled in long-term trials. Alternatively, the patient’s immune reactivity against the graft should be evaluated to ascertain the degree of unresponsiveness that can be achieved using different therapeutic strategies. We have previously shown that allograft rejection is mediated to a large extent by T cells that recognize donor HLA antigens via the indirect pathway. Allopeptide reactivity was found to occur prior to and during an acute rejection episode in both heart and liver allograft recipients.1,2 Furthermore, persistent allopeptide reactivity following an early acute rejection episode is predictive of chronic rejection in recipients of heart transplants.3 More recently, we have described the cellular and molecular basis of T-cell–mediated suppression. We showed that human suppressor cells are CD8⫹CD28⫺ T cells, which are oligoclonal, MHC-class I restricted, and capable of inhibiting the Th-mediated upregulation of costimulatory molecules on the APC used for priming.4 – 8 Based on these findings, we have used for immunologic monitoring of kidney allograft recipients the analysis of T helper cell reactivity to donor allopeptides and of T suppressor (CD8⫹CD28⫺ T) (Ts) cell capacity to inhibit T helper cell mediated upregulation of costimulatory molecules (CD80 and CD86) on donor APC. MATERIAL AND METHODS The presence of donor-specific Ts was monitored in a population of 14 recipients of kidney allografts from living donors. All patients received standard immunosuppressive therapy and were tested serially for Ts at 1, 3, 6, 9, 12, 16, 20, and 24 weeks posttransplantation. The method used for Ts testing consists of incubating (a) recipient CD4⫹ T cells, (b) CD8⫹ T cells, and (c) mixtures of CD4⫹ and CD8⫺ T cells with CD2⫺ depleted APC from the specific donor. After 24 hours, APC were stained with Annexin V to visualize apoptotic cells and with CD20 PE, CD80 FITC, and CD86 FITC to establish the level of expression of costimulatory B7 molecules. A 10% decrease in the mean channel of fluorescence of CD80 or CD86 on APC cultured with CD8⫹ T cells was considered indicative of T-cell–mediated suppression. In parallel with the Ts assay, limiting dilution analysis of allopeptide (HLA-DRB1) reactive Th was performed as previously described.1–3 The BMDP © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Table 1. Relationship Between T Cell Alloreactivity and Acute Rejection in Renal Allograft Recipients Acute Rejection LDA
⫹
⫺
⫹ ⫺
2 0 2
19 64 83
21 64 85
P ⬍ .05.
statistical software was used for statistical analysis. Chi-square analyses was used for establishing the relationship between allopeptide reactive T helper cells (Th), allospecific T suppressor cells (Ts), and rejection. The relationship between Ts and serum creatinine levels were computed using Student’s t test.
RESULTS AND DISCUSSION
Study of 85 samples of blood collected serially from 14 recipients of primary kidney allografts from a living donor showed a significant correlation between acute rejection and allopeptide reactivity (Table 1). Only 2 of these 14 patients showed an acute rejection episode during the first 6 months following transplantation. Allopeptide reactivity was found in conjunction with the rejection episode in both patients. One of these two patients showed persistent allopeptide reactivity following successful treatment of rejection. By analogy with the results seen in heart recipients, this persistent allopeptide reactivity may be predictive of chronic rejection. EBV infection also resulted in an increase in allopeptide reactivity in one patient. There was no clinical evidence of rejection in this patient indicating that viral infections may increase the inflammatory milieu and/or enhance processing of apoptotic donor cells by recipient APC which then elicit T cell allopeptide reactivity. No allopeptide reactivity was seen during quiescence in the other patients. Allospecific T suppressor cells were found in 59 samples obtained from 12 patients during quiescence From the University of Rome “La Sapienza” (P.C., B.E., L.P., R.P., P.B., R.C.), Rome, Italy; the ISMEDA, CNR (E.R.M., D.P.), Italy; and Columbia University (E.H., N.S.F.C.), New York, New York. Address reprint requests to Dr R. Cortesini, Caurportio peri Trafianti, Via G. Lauerti 23, 00161 Rome, Italy. 0041-1345/01/$–see front matter PII S0041-1345(00)02459-3 1129
Transplantation Proceedings, 33, 1129–1130 (2001)
1130
RENNA MOLAJONI, CINTI, HO ET AL
Table 2. Relationship Between Donor Specific T Suppressor Cells and Acute Rejection in Renal Allograft Recipients
Table 4. Correlation Between Allopeptide Reactivity and Donor-Specific T Suppressor Cells in Renal Allograft Recipients
Acute Rejection Ts
⫹ ⫺
⫹
0 2 2
LDA
⫺
59 23 82
59 25 84
P ⬍ .05. Note: One of the two rejections resulted a graft loss.
(Table 2). Ts were not found in the patients’ circulation during rejection being inversely related to rejection (P ⬍ .05). The mean serum creatinine level in patients with Ts was significantly lower than that seen in patients with rejection (Table 3). Acute rejection and indirect alloreactivity to synthetic peptides corresponding to donor HLADRB1 antigens showed an inverse correlation with the presence of donor specific Ts (P ⬍ .0001) (Table 4). This suggests that quiescence is maintained by Ts. Taken together, our data demonstrate for the first time that the outcome of organ allografts in human recipients depends Table 3. Comparison of Serum Creatinine Levels in Patients With and Without Donor-Specific T Suppressor Cells Donor-Specific T Suppressor Cells
Mean ISD Serum Creatinine Level (mg/dL)
Positive (n ⫽ 12) Negative (n ⫽ 12)
1.55 ⫾ 0.30 4.00 ⫾ 2.68
t-test: P ⫽ .002.
Ts
⫹
⫺
⫹ ⫺
7 13 20
51 12 63
58 25 83
P ⬍ .0001.
on the sensitive balance between effector and regulatory T cells. The discovery of the identity of Ts and of their mechanism of action opens new avenues to the development and design of new therapeutic strategies.
REFERENCES 1. Liu Z, Colovai AI, Tugulea S, et al: J Clin Invest 98:1150, 1996 2. Renna Molajoni E, Cinti P, Orlandini A, et al: Hum Immunol 53:57, 1997 3. Ciubotariu R, Liu Z, Colovai AI, et al: J Clin Invest 101:398, 1998 4. Liu Z, Tugulea S, Cortesini R, et al: Int Immunol 10:775, 1998 5. Jiang S, Tugulea S, Pennesi G, et al: Hum Immunol 59:690, 1998 6. Pennesi P, Liu Z, Ciubotariu R, et al: Hum Immunol 60:291, 1999 7. Li J, Liu Z, Jiang S, et al: J Immunol 163:6386, 1999 8. Colovai AI, Liu Z, Ciubotariu R, et al: Transplantation 69:1304, 2000
T
HE EXCELLENT early results achieved by use of current immunosuppressive therapy are reflected in 1-year graft survival rates approaching 90%. However, late chronic rejection still occurs in a considerable segment of the patient population. Hence, to determine whether a new regimen provides superior results, large numbers of patients must be enrolled in long-term trials. Alternatively, the patient’s immune reactivity against the graft should be evaluated to ascertain the degree of unresponsiveness that can be achieved using different therapeutic strategies. We have previously shown that allograft rejection is mediated to a large extent by T cells that recognize donor HLA antigens via the indirect pathway. Allopeptide reactivity was found to occur prior to and during an acute rejection episode in both heart and liver allograft recipients.1,2 Furthermore, persistent allopeptide reactivity following an early acute rejection episode is predictive of chronic rejection in recipients of heart transplants.3 More recently, we have described the cellular and molecular basis of T-cell–mediated suppression. We showed that human suppressor cells are CD8⫹CD28⫺ T cells, which are oligoclonal, MHC-class I restricted, and capable of inhibiting the Th-mediated upregulation of costimulatory molecules on the APC used for priming.4 – 8 Based on these findings, we have used for immunologic monitoring of kidney allograft recipients the analysis of T helper cell reactivity to donor allopeptides and of T suppressor (CD8⫹CD28⫺ T) (Ts) cell capacity to inhibit T helper cell mediated upregulation of costimulatory molecules (CD80 and CD86) on donor APC. MATERIAL AND METHODS The presence of donor-specific Ts was monitored in a population of 14 recipients of kidney allografts from living donors. All patients received standard immunosuppressive therapy and were tested serially for Ts at 1, 3, 6, 9, 12, 16, 20, and 24 weeks posttransplantation. The method used for Ts testing consists of incubating (a) recipient CD4⫹ T cells, (b) CD8⫹ T cells, and (c) mixtures of CD4⫹ and CD8⫺ T cells with CD2⫺ depleted APC from the specific donor. After 24 hours, APC were stained with Annexin V to visualize apoptotic cells and with CD20 PE, CD80 FITC, and CD86 FITC to establish the level of expression of costimulatory B7 molecules. A 10% decrease in the mean channel of fluorescence of CD80 or CD86 on APC cultured with CD8⫹ T cells was considered indicative of T-cell–mediated suppression. In parallel with the Ts assay, limiting dilution analysis of allopeptide (HLA-DRB1) reactive Th was performed as previously described.1–3 The BMDP © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Table 1. Relationship Between T Cell Alloreactivity and Acute Rejection in Renal Allograft Recipients Acute Rejection LDA
⫹
⫺
⫹ ⫺
2 0 2
19 64 83
21 64 85
P ⬍ .05.
statistical software was used for statistical analysis. Chi-square analyses was used for establishing the relationship between allopeptide reactive T helper cells (Th), allospecific T suppressor cells (Ts), and rejection. The relationship between Ts and serum creatinine levels were computed using Student’s t test.
RESULTS AND DISCUSSION
Study of 85 samples of blood collected serially from 14 recipients of primary kidney allografts from a living donor showed a significant correlation between acute rejection and allopeptide reactivity (Table 1). Only 2 of these 14 patients showed an acute rejection episode during the first 6 months following transplantation. Allopeptide reactivity was found in conjunction with the rejection episode in both patients. One of these two patients showed persistent allopeptide reactivity following successful treatment of rejection. By analogy with the results seen in heart recipients, this persistent allopeptide reactivity may be predictive of chronic rejection. EBV infection also resulted in an increase in allopeptide reactivity in one patient. There was no clinical evidence of rejection in this patient indicating that viral infections may increase the inflammatory milieu and/or enhance processing of apoptotic donor cells by recipient APC which then elicit T cell allopeptide reactivity. No allopeptide reactivity was seen during quiescence in the other patients. Allospecific T suppressor cells were found in 59 samples obtained from 12 patients during quiescence From the University of Rome “La Sapienza” (P.C., B.E., L.P., R.P., P.B., R.C.), Rome, Italy; the ISMEDA, CNR (E.R.M., D.P.), Italy; and Columbia University (E.H., N.S.F.C.), New York, New York. Address reprint requests to Dr R. Cortesini, Caurportio peri Trafianti, Via G. Lauerti 23, 00161 Rome, Italy. 0041-1345/01/$–see front matter PII S0041-1345(00)02459-3 1129
Transplantation Proceedings, 33, 1129–1130 (2001)
1130
RENNA MOLAJONI, CINTI, HO ET AL
Table 2. Relationship Between Donor Specific T Suppressor Cells and Acute Rejection in Renal Allograft Recipients
Table 4. Correlation Between Allopeptide Reactivity and Donor-Specific T Suppressor Cells in Renal Allograft Recipients
Acute Rejection Ts
⫹ ⫺
⫹
0 2 2
LDA
⫺
59 23 82
59 25 84
P ⬍ .05. Note: One of the two rejections resulted a graft loss.
(Table 2). Ts were not found in the patients’ circulation during rejection being inversely related to rejection (P ⬍ .05). The mean serum creatinine level in patients with Ts was significantly lower than that seen in patients with rejection (Table 3). Acute rejection and indirect alloreactivity to synthetic peptides corresponding to donor HLADRB1 antigens showed an inverse correlation with the presence of donor specific Ts (P ⬍ .0001) (Table 4). This suggests that quiescence is maintained by Ts. Taken together, our data demonstrate for the first time that the outcome of organ allografts in human recipients depends Table 3. Comparison of Serum Creatinine Levels in Patients With and Without Donor-Specific T Suppressor Cells Donor-Specific T Suppressor Cells
Mean ISD Serum Creatinine Level (mg/dL)
Positive (n ⫽ 12) Negative (n ⫽ 12)
1.55 ⫾ 0.30 4.00 ⫾ 2.68
t-test: P ⫽ .002.
Ts
⫹
⫺
⫹ ⫺
7 13 20
51 12 63
58 25 83
P ⬍ .0001.
on the sensitive balance between effector and regulatory T cells. The discovery of the identity of Ts and of their mechanism of action opens new avenues to the development and design of new therapeutic strategies.
REFERENCES 1. Liu Z, Colovai AI, Tugulea S, et al: J Clin Invest 98:1150, 1996 2. Renna Molajoni E, Cinti P, Orlandini A, et al: Hum Immunol 53:57, 1997 3. Ciubotariu R, Liu Z, Colovai AI, et al: J Clin Invest 101:398, 1998 4. Liu Z, Tugulea S, Cortesini R, et al: Int Immunol 10:775, 1998 5. Jiang S, Tugulea S, Pennesi G, et al: Hum Immunol 59:690, 1998 6. Pennesi P, Liu Z, Ciubotariu R, et al: Hum Immunol 60:291, 1999 7. Li J, Liu Z, Jiang S, et al: J Immunol 163:6386, 1999 8. Colovai AI, Liu Z, Ciubotariu R, et al: Transplantation 69:1304, 2000