Published ahead of print on April 5, 2006
Clin J Am Soc Nephrol 1: 539-545, 2006
© 2006 American Society of Nephrology
doi: 10.2215/CJN.01741105
Immunophenotypic Analysis of Cellular Infiltrate of Renal Allograft Biopsies in Patients with Acute Rejection after Induction with Alemtuzumab (Campath-1H)
Lorenzo Gallon*,
,
Elena Gagliardini
,
Ariela Benigni,
Dixon Kaufman,
Ahmed Waheed*,
Marina Noris, and
Giuseppe Remuzzi
Divisions of * Nephrology and Solid Organ Transplantation, Northwestern University, Chicago, Illinois; and Department of Medicine and Transplantation, Ospedali Riuniti-Mario Negri Institute for Pharmacologic Research, Bergamo, Italy
Address correspondence to: Dr. Lorenzo Gallon, Northwestern University, 675 N. St. Clair, Galter Pavilion 17-200, Chicago, IL 60611. Phone: 312-695-4457; Fax: 312-695-9194; E-mail: l-gallon{at}northwestern.edu
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Abstract
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Alemtuzumab is a humanized anti-CD52 mAb that has emerged as a safe and effective lymphocyte-depleting agent for induction therapy in renal transplantation. Recent reports have suggested that acute cellular rejection (ACR) of renal allografts in patients who receive alemtuzumab induction may be mediated by an atypical population of monocytes and not through "classical" T cell-dependent pathways of allorecognition. However, more recently, T cells with memory phenotype have been described in renal biopsies that were taken from alemtuzumab-treated patients who were experiencing ACR. This study investigated the cellular basis of ACR after alemtuzumab induction as compared with ACR that was associated with nondepleting therapy. Twelve biopsies from patients who were treated with a single dose of alemtuzumab at the time of transplantation and subsequently developed ACR were stained for the following cell markers: CD3 (T cells), CD68 (monocytes), CD20 (B cells), and CD45RO and CD45RA (memory and naïve T cells). ACR biopsies from six patients who received no induction therapy were used as controls. In alemtuzumab-treated patients, ACR occurred despite profound lymphopenia. A consistent number of CD3+ T cells was found in all ACR biopsies, and the majority of infiltrating CD3+ T cells displayed a memory phenotype (CD45RO+, CD45RA–). The number of infiltrating CD3+ T cells and B cells (CD20+) was similar in the two groups of patients, whereas a higher number of monocytes (CD68+) were found in the alemtuzumab than in the control group. Despite profound peripheral T cell depletion by alemtuzumab, ACR occurs and is associated with T and B cell and monocyte infiltration of the kidney. Specifically, T cells express on their surface the memory phenotype, suggesting that memory T cells may have eluded the depleting agent.
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Introduction
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Induction and maintenance immunosuppression therapy in solid-organ transplantation have changed significantly in the past decade. Stronger and more specific immunosuppressive regimens have allowed the elimination of the chronic use of steroids, and new non-nephrotoxic immunosuppressive agents have helped us to minimize the overall dose of calcineurin inhibitors.
Substantial advances in the anti-T lymphocyte antibody preparations that are used for induction also were observed during this period. Drugs such as muromonab-CD3 and equine antithymocyte globulin, which were popular in the early 1990s, have been replaced by agents such as rabbit antithymocyte globulin (Thymoglobulin) and IL-2 RA (basiliximab and daclizumab) (1) and, more recently, by alemtuzumab (Campath-1H). Alemtuzumab is a humanized, complement-fixing antibody that reacts with the CD52 receptor, the most prevalent cell surface antigen on T and B lymphocytes, and to a lesser degree on natural killer cells and monocytes, inducing complement-mediated cell lysis not only in the peripheral blood but also in secondary lymphoid organs as well as in bone marrow (2–8). Such effect lasts several months in kidney transplant recipients (3,4). Its use in renal transplantation was first described by Calne et al. (2,3) in 1998 in a steroid-free and low-dose maintenance cyclosporine regimen with an acceptable rate of acute rejection and good patient and graft survival at 2 yr. In another study, the combination of alemtuzumab with a steroid-free regimen of low-dose tacrolimus and mycophenolate mofetil (MMF) lowered to 10% the incidence of acute rejection (4). The role of alemtuzumab induction in facilitating a steroid-free maintenance immunosuppression in kidney transplant recipients (7) has been confirmed by a subsequent trial with 30 mg intravenously of alemtuzumab on the day of transplantation followed by maintenance immunosuppression that consists of tacrolimus and MMF. The protocol was associated with >99% graft survival at 1 yr and a rejection rate of 14.3% (7).
Alemtuzumab also has been used in calcineurin-free protocols, with less encouraging results. In a pilot study, alemtuzumab was given to 29 kidney transplant recipients along with rapamycin monotherapy (6). This protocol generally was well tolerated, but it was associated with a high frequency (28%) of acute rejection episodes despite almost undetectable T cells in the peripheral blood (6). Similar results were observed in another pilot study in which high-dose alemtuzumab (0.9 to 1.2 mg/kg) plus deoxyspergualin was given to seven kidney transplant recipients without any maintenance immunosuppression. All patients developed acute rejection within the first month after transplant despite that no T lymphocytes were detected in the peripheral blood and lymph nodes (5). Monocyte depletion was more gradual and less sustained, and an immunophenotypic analysis of the cellular infiltrate of the graft biopsies showed a predominance of monocytes/macrophages without the detection of T cells, which leads to the hypothesis that monocytes play a key role in mediating the acute rejection episodes. In a more recent study, however, Pearl et al. (9) demonstrated that a population of T cells of effector memory surface phenotype were resistant to aggressive T cell depletion with either alemtuzumab or rabbit anti-thymocyte globulin. These cells expanded in the first month after transplant and were the prevalent T cells found in blood and in the allograft at the time of rejection (9).
Given these interesting yet thought-provoking findings, we sought to characterize the phenotype of the cellular infiltrates in renal biopsies of patients who received a transplant and developed acute cellular rejection (ACR) after profound T cell depletion with alemtuzumab to establish whether memory T cells do actually accumulate in the graft. Cellular infiltrates in renal allograft biopsies from patients who had received no induction also were studied to compare the cellular basis of ACR after alemtuzumab with those of ACR that occurred under a non-T cell-depleting regimen.
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Materials and Methods
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Patients
Since October 2001, all renal transplant patients have received intraoperative alemtuzumab induction at the Northwestern University Medical Center. After obtaining Institutional Review Board approval, we reviewed the medical charts of 220 patients who received kidney transplantation after alemtuzumab induction therapy. At 1 yr of follow-up, 28 of these patients had developed ACR. Paraffin-block biopsies that were obtained at the time of ACR from 12 patients were selected at random and used for this study. Biopsy specimens that were obtained at the time of ACR of patients who had received no induction (n = 6) were used as the control group. Demographic information was obtained from the two groups, including type of renal transplant (deceased or living donor), degree of HLA mismatch, percentage of peripheral lymphocytes at the time of ACR, and days after renal transplant when the ACR episode occurred (Table 1).
Immunosuppressive Therapy
Induction Therapy.
In the alemtuzumab-treated group, patients received alemtuzumab at the dose of 30 mg intravenously, which was administered on the day of renal transplant as a single dose (n = 12). Six patients did not receive induction therapy.
Maintenance Immunosuppression.
Tacrolimus and MMF were used as maintenance immunosuppression in the alemtuzumab-treated group. MMF was started on postoperative day 1 at 1000 mg twice daily. Tacrolimus dosage was adjusted for blood target levels between 6 and 8 ng/ml. The patients also received a 3-d course of intravenous corticosteroids: Methylprednisolone, 500 mg in the operating room, 250 mg on day 1, and 125 mg on day 2. No chronic prednisone was used in these two groups of patients (7).
Cyclosporine (n = 6) and MMF (n = 2) or azathioprine (n = 4) and chronic prednisone were used in patients who had not received any form of induction at the time of transplantation. MMF was started on postoperative day 1 at 1000 mg twice daily. Azathioprine was given at the dose of 75 mg/d. Cyclosporine dosage was adjusted for blood target levels of 300 to 400 ng/ml in the first month after transplantation, 200 to 300 ng/ml from 2 to 6 mo, and 150 to 250 ng/ml thereafter.
Diagnosis and Treatment of Acute Rejections
Patients were followed with weekly laboratory tests during the first 3 mo after transplantation and then every month thereafter. Renal biopsies were done as clinically indicated. All rejection episodes were biopsy proven. ACR were treated, on the basis of severity, with methylprednisolone 500 mg intravenously for 3 d followed by a 1-wk course of prednisone taper or with an antilymphocyte antibody therapy (Thymoglobulin or equine antithymocyte globulin) for 14 d.
Histology
Paraffin-embedded renal tissues were cut into 3- to 5-µm sections and underwent routine staining for renal biopsies, including three sections for hematoxylin-eosin staining, three sections for periodic acid-Schiff staining, and one section for Masson Trichrome staining. Immunoperoxidase staining was performed in paraffin-embedded kidney sections using antibodies against the following cell markers: CD3 (rabbit anti-human T cells), CD20 (mouse anti-human B cells), CD68 (mouse anti-human macrophages; DakoCytomation, Glostrup, Denmark), and CD45RO and CD45RA (mouse anti-human; Histo-Line Laboratories, Milan, Italy; to discriminate between naïve and memory T cells). The paraffin-embedded kidney sections (3 mm) were deparaffinized and rehydrated. For immunoperoxidase analysis of CD45RO and CD45RA antigens, the sections were incubated for 30 min with 0.3% H2O2 in methanol to quench endogenous peroxidase and permeabilized in 0.1% Triton X-100 in PBS 0.01 M (pH 7.2) for 30 min. For CD3, CD20, and CD68 antigens, before quenching endogenous peroxidase, kidney samples were treated with proteinase-K (20 mg/ml; Sigma-Aldrich, Milan, Italy) for 10 min at 37°C, instead of Triton X100, followed by microwave (twice for 5 min in citrate buffer 10 mM [pH 6] at operating frequency of 2450 MHz and 600-W power output) and citrate buffer (15 min) incubations. Primary antibodies were diluted (CD3, 1:400; CD20, 1:50; CD68, 1:100; CD45RO, 1:25; CD45RA, undiluted) and added overnight at 4°C. Subsequent steps included incubations with the secondary biotinylated antibodies (sheep anti-mouse IgG [Chemicon International, Temecula, CA] and goat anti-rabbit IgG [Vector Laboratories, Burlingame, CA]), avidin-biotin peroxidase complex solution, and finally development with diaminobenzidine. The sections then were counterstained with Harris hematoxylin (Biooptica, Milan, Italy). Negative controls were obtained by omitting the primary antibody on adjacent sections. For each marker, the number of cells was counted in at least 10 randomly selected high-power fields (HPF; x400) in the cortical region.
Statistical Analyses
Data, expressed as mean ± SD, were analyzed by the nonparametric Kruskal-Wallis test for multiple comparisons. Statistical significance level was defined as P < 0.05.
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Results
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Patients
Patient’s demographics, type of induction immunotherapy, maintenance immunosuppression, time of rejection onset, and percentage of peripheral lymphocytes at the time of ACR are shown in Table 1. The time of rejection in alemtuzumab-treated patients was not dependent on the number of peripheral lymphocytes to the extent that ACR occurred even in patients with no detectable lymphocytes in their blood (Table 1).
Immunohistology
Banff ’97 classification of the ACR in the two groups is reported (Table 2). Seven (58%) of 12 patients in the alemtuzumab-treated group were found to have grade IA ACR, three (25%) of 12 had grade IIA ACR, and two (16%) of 12 had grade IB ACR. In patients who received no induction, four (66%) of six were found to have grade IA ACR, one (16%) of six had grade IIA ACR, and one (16%) of six had grade IIB ACR. No patients in either group had evidence of associated glomerulitis at the time of ACR.
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Table 2. Percentage of peripheral lymphocytes and immunophenotypic analysis of cortical interstitial infiltrates in renal allograft biopsies at time of ACRa
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Immunophenotypic analysis of the cellular infiltrate revealed the presence of CD3+ T cells and CD68+ (monocytes) and CD20+ B cells in the two groups of patients (Table 2). Despite peripheral lymphopenia, a marked infiltration of CD3+ cells was observed in biopsies from alemtuzumab-treated patients so that mean CD3+ cell number per HPF was comparable to that found in biopsies of patients who received no induction. However, the number of infiltrating CD3+ cells varied greatly in the alemtuzumab group, and a direct correlation with the percentage of peripheral lymphocytes was found (Figure 1).
The numbers of infiltrating lymphocytes that expressed either the CD45RA or the CD45RO antigen were evaluated to differentiate subpopulations of naïve and memory T cells. In biopsies from patients who received either alemtuzumab induction or no induction, the number of CD45RO+ memory cells, which were localized mainly in the interstitium and occasionally in the glomerulus (Figure 2), greatly overwhelmed the number of CD45RA+ cells so that CD45RO/RA ratios higher than 5 were recorded in the two groups of patients (Table 3).
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Figure 2. Representative pictures of infiltrating CD3+ (A and D), CD45 RO+ (B and E), and CD45 RA+ (C and E) cells in renal allograft biopsies of patients with acute cellular rejection after alemtuzumab treatment (A through C) or nondepleting therapy (D through F).
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Table 3. Immunophenotypic analysis to identify the predominant cellular population (naïve, CD45RA+, or memory, CD45RO+) in renal allograft biopsies at time of ACR
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Naïve CD3+ T cells (CD45RO–, CD45RA+) were absent in biopsies from alemtuzumab-treated patients as documented by the numbers of CD45RO+ cells that were equal to or even exceeded the number of total infiltrating CD3+ T cells (Tables 2 and 3). A phenotype of CD45RO+ CD45RA+ recently activated cells could account for the subpopulation of CD45RA+ cells that was found in these biopsies.
CD68+ monocytes were largely present in all biopsies. In alemtuzumab-treated patients, the mean number of infiltrating monocytes was significantly higher (P < 0.01) than in patients with no induction (Table 2). However, a modest number of B cells (CD20+) were observed in biopsies from all renal allografts, and their mean number per field did not differ between the two groups of patients (Table 2). Representative biopsies at the time of ACR with staining for CD3, CD45RO, and CD45RA antigens from a patient who received alemtuzumab and from a patient who received no induction are displayed in Figure 2.
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Discussion
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Lymphocyte depletion reduces the requirement for maintenance immunosuppression after solid-organ transplantation. However, published (2–7) and our data show that despite profound peripheral T cell depletion that is induced with alemtuzumab, ACR still occurs. In addition, we have found that the acute rejection allografts have cellular infiltrates that are composed of consistent numbers of CD3+ T cells even in the context of very low peripheral lymphocytes measured at the time of ACR. In the alemtuzumab group, quantification of the infiltrating T cells, expressed in number of cells per HPF, showed a direct correlation with the number of peripheral T cells. Altogether, these data suggest that infiltrating CD3+ cells in rejected kidney grafts derive from a population of T cells that are resistant to alemtuzumab-induced depletion.
Pearl et al. (9) found that in renal transplant recipients who experienced ACR after receiving depletion induction with alemtuzumab/deoxyspergualin with no maintenance immunosuppression, postdepletion T cells were of a single phenotype (CD3+CD4+CD45RA–CD62L–CCR7–), consistent with depletion-resistant effector memory T cells (9). These cells expanded in the first month after transplantation and were the prevalent T cell population before and during allograft, both in the blood and in the allograft (9).
Immunohistochemical analysis of CD45RO and CD45RA antigens in infiltrating T cells of alemtuzumab-treated patients whose graft was rejected showed that the majority of CD3+ graft-infiltrating T cells in control groups displayed a phenotype of memory T cell (CD45RO+ CD45RA–), which confirms that memory T cells may have eluded the depleting agent or that rapid repopulation of memory T cells occurred after profound T cell depletion.
When compared with non-T cell-depleting strategy, the quality of the infiltrates in rejecting renal allografts did not differ substantially in patients who had received alemtuzumab. Similar numbers of T lymphocytes and B cells were found in rejecting renal allografts of alemtuzumab-treated patients and in allografts of patients with no induction at the time of transplantation, although the number of infiltrating monocytes was higher in the alemtuzumab group than in the no induction group. These data are consistent with a recent study by Zhang et al. (10), who found similar percentages of CD3+ lymphocytes and higher percentages of CD68+ monocytes in the rejected grafts from patients with alemtuzumab induction therapy as compared with cases of ACR in the absence of alemtuzumab induction. Intense monocyte infiltration in the graft can be interpreted as reflecting that blood monocyte depletion after alemtuzumab is gradual and transient and lymph nodal monocytes generally are not depleted (5).
These findings have important clinical implications. Indeed, the use of T cell-depleting agents as induction therapy with the goal of minimizing the exposure of "maintenance" immunosuppression and attempting induction of a protolerant state has garnered increasing popularity in recent years. However, animal studies have suggested that profound lymphocyte depletion does not eliminate immune memory to the extent that memory T cell proliferation can occur under the condition of lymphopenia (11,12). Memory T cells could orchestrate the immune response as they were found to mediate accelerated and early rejection in primed recipients (13–15). That lymphocyte depletion with alemtuzumab does not eliminate immune memory to defined antigens is in line with data showing that macaques retained the immune response to viral proteins after exposure to hepatitis B vaccine even after thymectomy and T cell depletion by anti-thymocyte globulin (16). In a lymphopenic environment, homeostatic proliferation of established memory cells begets fully functionally cells that are capable of vigorous response without the need for reexposure to antigen in secondary lymphoid environment (12). The data presented here extend to humans’ previous experimental findings and document that memory T cells do trigger acute rejection in the context of peripheral lymphopenia and highlight their primary role during ACR in solid-organ transplantation.
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Conclusion
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Here we found that despite profound peripheral T cell depletion by alemtuzumab, acute rejection still occurs and is associated with T and B cell as well as monocyte/macrophage infiltration of the kidney. Specifically, T cells express on their surface the memory phenotype, suggesting that memory T cells may have eluded the depleting agent or that rapid repopulation of memory T cells occurs after profound T cell depletion. It is tempting to speculate that memory T cells that reach the graft could initiate early responses that produce in situ effector cytokines that, by recruiting further immune cells, favor the progressive graft tissue damage, leading to allograft rejection. Further studies are needed to shed light on the immunologic mechanisms that are engaged by memory T cells and that could be instrumental to define future strategies to induce transplant tolerance.
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Acknowledgments
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This study was supported partially by ART (Transplant Research Association, Milan, Italy) and by the Negri-Weizmann Foundation.
These data were presented in abstract form at The American Transplant Congress; May 15 to 19, 2004; Boston, MA.
We thank Dr. Paolo Cravedi for help.
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Footnotes
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Published online ahead of print. Publication date available at www.cjasn.org.
Received November 16, 2005.
Accepted February 15, 2006.
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References
|
|---|
- Kaufman DB, Shapiro R, Lucey MR, Cherikh WS, T Bustami R, Dyke DB: Immunosuppression: Practice and trends.
Am J Transplant4[Suppl 9]
:38
–53,2004
- Calne R, Friend P, Moffatt S, Bradley A, Hale G, Firth J, Bradley J, Smith K, Waldmann H: Prope tolerance, perioperative campath 1H, and low-dose cyclosporin monotherapy in renal allograft recipients.
Lancet351
:1701
–1702,1998[Medline]
- Calne R, Moffatt SD, Friend PJ, Jamieson NV, Bradley JA, Hale G, Firth J, Bradley J, Smith KG, Waldmann H: Campath IH allows low-dose cyclosporine monotherapy in 31 cadaveric renal allograft recipients.
Transplantation68
:1613
–1616,1999[CrossRef][Medline]
- Ciancio G, Burke GW, Gaynor JJ, Mattiazzi A, Roohipour R, Carreno MR, Roth D, Ruiz P, Kupin W, Rosen A, Esquenazi V, Tzakis AG, Miller J: The use of Campath-1H as induction therapy in renal transplantation: Preliminary results.
Transplantation78
:426
–433,2004[CrossRef][Medline]
- Kirk AD, Hale DA, Mannon RB, Kleiner DE, Hoffmann SC, Kampen RL, Cendales LK, Tadaki DK, Harlan DM, Swanson SJ: Results from a human renal allograft tolerance trial evaluating the humanized CD52-specific monoclonal antibody alemtuzumab (CAMPATH-1H).
Transplantation76
:120
–129,2003[CrossRef][Medline]
- Knechtle SJ, Pirsch JD, H. Fechner JJ, Becker BN, Friedl A, Colvin RB, Lebeck LK, Chin LT, Becker YT, Odorico JS, D’Alessandro AM, Kalayoglu M, Hamawy MM, Hu H, Bloom DD, Sollinger HW: Campath-1H induction plus rapamycin monotherapy for renal transplantation: Results of a pilot study.
Am J Transplant3
:722
–730,2003[CrossRef][Medline]
- Kaufman DB, Leventhal JR, Axelrod D, Gallon LG, Parker MA, Stuart FP: Alemtuzumab induction and prednisone-free maintenance immunotherapy in kidney transplantation: Comparison with basiliximab induction—Long-term results.
Am J Transplant5
:2539
–2548,2005[CrossRef][Medline]
- Stuart FP, Leventhal JR, Kaufman DB, Stuart J, Abecassis M, Fryer JP, Koffron A, Spanier T, Gallon L: Alemtuzumab facilitates prednisone-free immunosuppression in kidney transplant recipients with no early rejection.
Am J Transplant2[Suppl 3]
:S397
,2002
- Pearl JP, Parris J, Hale DA, Hoffmann SC, Bernstein WB, McCoy KL, Swanson SJ, Mannon RB, Roederer M, Kirk AD: Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T-cell depletion.
Am J Transplant5
:465
–474,2005[Medline]
- Zhang PL, Malek SK, Prichard JW, Lin F, Yahya TM, Schwartzman MS, Latsha RP, Norfolk ER, Blasick TM, Lun M, Brown RE, Hartle JE, Potdar S: Acute cellular rejection predominated by monocytes is a severe form of rejection in human renal recipients with or without Campath-1H (alemtuzumab) induction therapy.
Am J Transplant5
:604
–607,2005[Medline]
- Adams AB, Williams MA, Jones TR, Shirasugi N, Durham MM, Kaech SM, Wherry EJ, Onami T, Lanier JG, Kokko KE, Pearson TC, Ahmed R, Larsen CP: Heterologous immunity provides a potent barrier to transplantation tolerance.
J Clin Invest111
:1887
–1895,2003[CrossRef][Medline]
- Wu Z, Bensinger SJ, Zhang J, Chen C, Yuan X, Huang X, Markmann JF, Kassaee A, Rosengard BR, Hancock WW, Sayegh MH, Turka LA: Homeostatic proliferation is a barrier to transplantation tolerance.
Nat Med10
:87
–92,2004[CrossRef][Medline]
- Cerny A, Ramseier H, Bazin H, Zinkernagel RM: Unimpaired first-set and second-set skin graft rejection in agammaglobulinemic mice.
Transplantation45
:1111
–1113,1988[Medline]
- Hall BM, Dorsch S, Roser B: The cellular basis of allograft rejection in vivo. II. The nature of memory cells mediating second set heart graft rejection.
J Exp Med148
:890
–902,1978[Abstract/Free Full Text]
- Heeger PS, Greenspan NS, Kuhlenschmidt S, Dejelo C, Hricik DE, Schulak JA, Tary-Lehmann M: Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes.
J Immunol163
:2267
–2275,1999[Abstract/Free Full Text]
- Pearl JP, Xu H, Christopher KL, Leopardi F, Preston E, Cendales LK, Hale DH, Kirk AD: Aggressive lymphocyte depletion does not eliminate immune memory.
Am J Transplant4
:302
,2004
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Abstract
Introduction
