- Email: [email protected]
Primate renal transplants using immunotoxin
Primate renal transplants using immunotoxin Stuart J. Knechtle, MD, John H. Fechner, Jr, MS, Yinchen Dong, MD, Scott Stavrou, BS, David M. Neville, Jr, MD, Terry Oberley, MD, Patrick Buckley, MD, Nicholas Armstrong, MD, Kristin Rusterholz, BS, Xuening Hong, MD, Masahiro Tsuchida, MD, and Majed M. Hamawy, PhD, Madison, Wis, and Bethesda, Md
Background. T-lymphocyte depletion 7 days before transplantation with immunotoxin FN 18-CRM9 has resulted in tolerance to subsequent renal allografts. We tested the effect of giving immunotoxin on the day of the transplantation and evaluated its effect on rhesus monkey renal allograft survival, on antibody production, and on T-cell recovery. Methods. Major histocompatibility complex mismatched renal allografts were performed in rhesus monkeys. Immunotoxin was given starting on the day of transplantation, with and without prednisone and mycophenolate mofetil for 3 days. T-cell subsets and alloantibody levels were measured by flow cytometry. The ability of treated monkeys to develop antibody to tetanus, diphtheria, and xenoantibody was measured. Histology of renal transplants was read in a blinded manner. Results. Immunotoxin started on the day of transplantation resulted in prolonged allograft survival in all treatment groups. Graft loss between days 50 and 135 was most often due to interstitial nephritis. Later graft loss was due to chronic rejection. Monkeys had intact antibody responses to alloantigen, tetanus, diphtheria, and xenoantibody. Their CD4 cells recovered gradually over 6 months. Conclusions. Immunotoxin reliably prolongs renal allograft survival when started on the day of transplantation, but interstitial nephritis and chronic rejection limit the development of long-term tolerance. Tcell-dependent B-cell responses remain intact after treatment. (Surgery 1998;124:438-47.) From the Departments of Surgery and Pathology, University of Wisconsin, Madison, Wis, and Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Md
PRECLINICAL MODELS of allograft tolerance have the goal of devising a clinically relevant approach to promoting long-term allograft acceptance in the absence of continuous drug therapy. This elusive goal has been met in a small number of patients undergoing transplantation1-3 and in nonhuman primates using strategies that are not yet in clinical use.4-6 A transient but profound decrease in circulating and lymph node T-lymphocyte populations produced by FN18-CRM9 immunotoxin has been shown to reliably result in long-term tolerance to major histocompatibility complex (MHC) mismatched renal allografts in rhesus monkeys.7 This
Supported by the University Surgical Associates, University of Wisconsin, Madison, Wis, Novartis Pharmaceuticals Corporation, East Hanover, NJ, and by National Institutes of Health, Bethesda, Md. Presented at the Fifty-ninth Annual Meeting of the Society of University Surgeons, Milwaukee, Wis, Feb 12-14, 1998. Reprint requests: Stuart J. Knechtle, MD, Department of Surgery, University of Wisconsin Hospital, 600 Highland Avenue, Madison, WI 53792-7375. Copyright © 1998 by Mosby, Inc. 0039-6060/98/$5.00 + 0
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immunotoxin developed by Neville et al8 consists of a conjugate of a murine anti-rhesus monkey CD3 monoclonal antibody (FN18) and a mutated diphtheria toxin, CRM9. The advantage of CRM9 relative to wild-type diphtheria toxin (DT) is that it maintains its potent ability to inhibit protein synthesis while reducing its toxicity to 1/300th of the wild-type DT. We, as well as Thomas et al,9 have shown that immunotoxin is capable of inducing unresponsiveness in a preclinical model of renal allografting and that immunotoxin is well tolerated. Immunotoxin reduces CD3 T lymphocytes in the peripheral blood lymphocytes (PBLs) of rhesus monkeys by 2 to 3 logs and in the lymph nodes by 2 logs, when measured 1 to 2 days after a 3-day course of immunotoxin. This ability differentiates immunotoxin from monoclonal antibody alone, which has not been shown to substantially deplete lymphocytes in lymphoid tissues. Immunotoxin reduces mixed lymphocyte culture (MLC) proliferation and cytotoxic T-lymphocyte precursor (CTLp) frequency to donor and thirdparty alloantigen when measured 3 to 4 months after treatment. However, long-surviving immunotoxin-treated renal allograft recipients are able to
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Fig. 1. Renal transplant histology of a monkey killed on day 58, with creatinine level of 8.6 mg/dL and histologic characteristics of severe interstitial nephritis. A prominent infiltrate occupies the interstitial spaces without tubular invasion. (Original magnification ×20.)
Fig. 2. Changes in lymphocyte subset populations after immunotoxin treatment on the day of transplantation. Percentage of T cells (CD3, filled triangle), T cells and NK cells (CD2, open square), and B cells (CD20, open circle) in PBLs was determined by single color flow cytometry. Each time point is the mean ± SD of 5 to 11 monkeys treated with immunotoxin on the day of transplantation with or without other treatments. Later time points have few animals in the group, as animals were lost for analysis because of death.
generate new CTLp against third-party skin graft donors 6 months after renal transplantation, but PBLs of these same recipients have no increase in the number of CTLp against the donor kidney.10 In vivo correlation of this result comes from the observation that long-surviving monkeys accept donor skin grafts placed 200 days after treatment but reject third-party skin grafts. To test the relevance of immunotoxin to cadaveric organ transplantation in which immunosuppression must be started on the day of the transplantation (and pretreatment is not feasible), we used an altered treatment regimen giving immunotoxin on days 0, 1, and 2 with respect to the renal transplantation. The influence of immunotoxin on T- and B-cell function after transplantation was
evaluated, and graft outcome was assessed histologically and functionally. We report herein the longterm follow-up of monkeys treated with immunotoxin to induce allograft unresponsiveness. METHODS Animals and surgery. Male juvenile rhesus monkeys weighing 2.5 to 3.5 kg (age range 11⁄2 to 3 years) were purchased from either the University of Wisconsin Primate Center or LABS (Yemassee, SC) and were screened for herpes B, simian immunodeficiency virus, simian T-lymphotropic virus, and simian retrovirus and skin tested for tuberculosis. Monkeys were fed and maintained according to National Institutes of Health and US Department of Agriculture guidelines. Donor-recipient pairs were
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Fig. 3. Ratio of T-helper cells to all T cells after immunotoxin treatment on the day of transplantation. Percentage of PBLs that was T-cell (CD3) and T-helper cell (CD4) was determined by single color flow cytometry in a panel of 6 monkeys. Ratio was determined by using the percentage of CD3 cells as the denominator and the percentage of CD4 cells as the numerator. Two more color flow analyses demonstrated that 100% of CD4 lymphocytes are also CD3.
selected based on CTL and MLC responses and on MHC class I and MHC class II typing as previously described7,11 to maximize disparity. Recipients were screened for a low titer of antibody to DT as previously described.12 Renal transplantations were performed by transplanting the donor left kidney to recipients that had undergone bilateral native nephrectomy. Graft function was monitored by measuring serum creatinine level, and monkeys were killed when the creatinine rose to >8 mg/dL, when weight loss >20% occurred, or by other criteria determined by the veterinary staff and investigators. All protocols were approved by the University of Wisconsin Animal Use Committee. Immunosuppressive agents. FN18-CRM9 was administered either on days -7, -6, -5 (initial experiments) or on days 0, 1, 2 (later groups) with respect to renal transplantation at a total dose of 0.2 mg/kg given intravenously in 3 equally divided doses. Mycophenolate mofetil (CellCept, Roche, Nutley, NJ) 250 mg/day was given orally on days 0, 1, and 2. Methylprednisolone was administered at a dose of 125 mg intravenously on days 0, 1, and 2. The day 0 doses of drugs were administered intraoperatively. Flow cytometry analysis. A total of 200 µL of whole blood was diluted with 100 µl phosphatebuffered saline solution/1% fetal calf serum and stained with fluorescein isothiocyanate labeled FN18 (anti-CD3ε), T11 (anti-CD2; Coulter Immunology, Hialeah, Fla), B1 (anti-CD20; Coulter), OKT4 (anti-CD4; Ortho Diagnostics, Raritan, NJ), or isotype control antibody (Sigma, St Louis, Mo) according to the manufacturer. Red blood cells
were removed from whole blood by ACK solution (155 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L ethylenediaminetetraacetic acid) treatment after staining. Cells were then either subjected to flow cytometry immediately or fixed in 1% paraformaldehyde. Flow cytometry was performed on a Becton Dickinson FACSCAN. Lymphocytes were gated on the basis of forward and side scatters and 10,000 events were analyzed. Detection of antidonor immunoglobulin. Antidonor immunoglobulin G (IgG) antibodies were detected by flow cytometry as previously described.10 The presence of antidonor IgG was monitored by observing the shift in mean channel fluorescence of goat antihuman IgG binding in CD3 positive lymphocytes. Relative changes in titer were measured by taking the ratio of the mean channel fluorescence of posttransplantation serum to the mean channel fluorescence of pretransplantation serum. Complement-fixing alloantibody was detected by antibody-complement cytotoxicity assay. 13 Target cells were 51Cr labeled donor and thirdparty PHA blasts in RPMI/10% fetal calf serum. A total of 10 µL of target cells (1 × 106/mL) were incubated with 20 µL diluted serum in triplicate for 45 minutes in 96-well round bottom plates at room temperature. Rabbit complement, diluted 1:8, was then added to each well (20 µL) and plates were incubated for 45 minutes at 37° C. After 100 µL of cold medium was added to each well, the supernatant was harvested on Skatron filters (Skatron, Sterling, Va). Controls included spontaneous lysis (no complement added), lysis
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A
B Fig. 4. Monkeys immunized with tetanus toxoid after immunotoxin treatment are capable of making an antitetanus IgG response. Presence of antitetanus IgG in the sera of animals was measured by ELISA. (A) Humoral response of animal AKJ after primary tetanus toxoid immunization (TT) 2 months after transplantation/immunotoxin treatment with a subsequent secondary immunization 1 month later. Serum was taken just before immunization (Pre TT), 3 weeks after the primary immunization (Post TT), and 1 week after the secondary immunization (post TT boost). (B) Response to primary and secondary TT in 1 month (XV3), 3 months (2CP), 4 months (AXG), and 5 months (BED) after transplantation/immunotoxin treatment.
due to complement alone (always less than spontaneous lysis), and maximum lysis (2% TritonX100). Percent specific 51Cr release was calculated and the end point titer was determined as the reciprocal of the last dilution to obtain cpm greater than 3 standard deviations above the mean spontaneous release. Tetanus antigen immunization. Monkeys were immunized intramuscularly with alum adsorbed tetanus toxoid at either 1 or 5 months after transplantation. Animals were bled 1 week before immunization and 3 weeks after the primary immunization. A secondary immunization was done 4 weeks after the primary and sera were then collected 1 week later. Sera were measured for antitetanus IgG by enzyme-linked immunosorbent assay (ELISA).
ELISA for anti-mIgG, anti-CRM9 and antitetanus toxoid antibodies. Costar 96-well flat bottom ELISA plates (Costar, Cambridge, Mass) were coated overnight at 4° C with 25 µL appropriate antigen (3 mg/mL). After plates were blocked with phosphate-buffered saline/2% bovine serum albumin and washed, 25 µL diluted serum was added to each well. After incubation for 1 hour at room temperature, plates were washed and appropriately diluted horseradish peroxidase-conjugated goat antihuman IgG was added to each plate. Again, after incubation for 1 hour at room temperature, substrate was added and, after adding 1 M H2SO4, the plate was read by an automated ELISA plate reader. Antibodies against CRM9 were detected by ELISA as previously described.12
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Fig. 5. Antibody response to mouse IgG of anti-CD3-immunotoxin. Open symbols are individual animals at 3 months to 1 year after immunotoxin treatment in either day -7 or day 0 treatment groups. Closed symbol is the mean value of 4 untreated animals..
Table I. Graft survival* Group
Anti-CD3-CRM9 (total 0.2 mg/kg)
Additional therapy
1 2 3
None Days -7,-6,-5 Days -7,-6,-5
4
Days -7,-6,-5
5 6 7
Days 0, 1, 2 Days 0, 1, 2 Days 0, 1, 2
None None Intrathymic injection-normal saline Intrathymic injection-donor cell None Steroid MMF + steroid
Graft survival time (days)
P value †
Reason for graft loss
5, 7, 7, 8, 9 51, 84, 203,302‡, 728 36, 68, 72, 887, >921
NS vs 2
ar, ar, ar, ar, ar ar, ar, 1, 1&2, cr ar, ar, ar, cr, cgf
41, 45‡, 181, 846‡, >1011
NS vs 2
ar, 3, ar§, cr, cgf
51‡, 57, 79, 184, 287 35‡, 105, >333 133, 172, 307, >416, 435
NS vs 5 NS vs 5
2, in, in, cr, cr 4, in, cgf in, cr, cr, cgf,cr
ar, acute rejection; in, chronic interstitial nephritis; cr, chronic rejection; 1, weight loss > 20%; 2, hematocrit < 25% anemia; 3, small bowel hemorrhage; 4, skin burn; cgf, continued graft function with a creatinine < 2.0 mg/dL. *Some of the data reported in this table has been previously published.7 †Comparison
between Kaplan-Meier graft survival estimates of groups by log-rank test; NS = not significant.
‡Killed
with a creatinine level < 2.0 mg/dL.
§Acute
rejection 40 days after donor skin graft placed.
Histology. At autopsy all tissues were examined grossly, and transplanted kidneys were evaluated histologically in a blinded manner by an experienced renal transplant pathologist (T.O.) and graded according to modified Banff criteria (1997). RESULTS Long-term follow-up of monkeys pretreated on days -7, -6, and -5 with immunotoxin, initially reported by our group,7 are shown in Table I. The pathology of rejecting allografts showed acute cellular rejection for untreated controls, whereas the long-surviving allografts ultimately failed because of either rejection or death with a functioning graft. Wasting was seen in 3 monkeys and was characterized
by slow, progressive weight loss with loss of appetite despite normal liver function, renal function, and serum chemistries. Two of these monkeys became anemic, but bone marrow biopsy specimens showed normal erythrocyte precursors. The histology of interstitial nephritis is shown in Fig. 1. The results of monkeys receiving immunotoxin starting on the day of transplantation rather than 1 week before transplantation are summarized in Table I. If the immunotoxin was used alone (group 5), graft survival was consistently prolonged but ultimately limited by chronic rejection in 2 of 5 recipients. One monkey in this group was killed because of early weight loss, and this monkey had a normal creatinine level of 0.7 at the time of death
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Surgery Volume 124, Number 2 Table II. Alloreactive antibody and graft outcome* Animal AXF 1347 AXG WF6 AKJ AXA XV3 BED 2CP DFW EEF
GST 51 57 184 287 133 172 307 >411 435 105 >328
Test† 51 57 110 98 133 118 120 125 120 105 112
ABX‡ 1.3 2.3 0.9 6.3 1.2 8.4 1 2.5 1.6 2.9 0.8
ACC§ ND ND 1 243 1 81 1 >729 1 1 1
Outcome Creatinine < 2.0 mg/dL in cr cr cr cr cr Creatinine < 2.0 mg/dL cr in Creatinine < 2.0 mg/dL
in, chronic interstitial nephritis; cr, chronic rejection; ND, not determined. *Some of the data reported in this table has been previously published.14 †Days
after transplant from which sera were tested.
‡Ratio
of the mean channel fluorescence of posttransplantation anti-human IgG reactive serum (1:100 dilution) binding to donor CD3+ lymphocytes to the mean channel fluorescence of pretransplantation antihuman IgG reactive serum (1:100 dilution) binding to donor CD3+ lymphocytes. §Reciprocal
end point titer of antibody complement cytotoxicity assay.
on day 51. When steroids were added to immunotoxin in a group of 3 monkeys (group 6), one graft was lost at day 105 as a result of interstitial nephritis and 1 monkey was still surviving at 333 days. A third monkey was killed at day 35 because of skin infection after an electrocautery grounding pad burn (renal histology was normal at autopsy). Five monkeys were treated with immunotoxin, prednisone, and mycophenolate on days 0, 1, and 2 (group 7). All 5 members of this group had graft survival greater than 100 days, with 1 monkey still surviving more than a year after transplantation. Nevertheless, 4 monkeys in this group lost their grafts, 3 from chronic rejection and 1 from focal interstitial nephritis. Flow cytometric analysis of lymphocyte subsets. Fluorescein isothiocyanate–labeled anti-CD2, CD3, CD4, and CD20 monoclonal antibodies were used to evaluate the effect of day 0 immunotoxin on lymphocyte subsets. A summary of analysis of 11 monkeys is shown in Fig. 2. At 1 to 7 days after treatment, both CD2 and CD3 cells were severely depleted, with rapid recovery of CD2 cells and gradual recovery of CD3 cells over 3 months. Preliminary analysis of a few immunotoxin-treated animals indicates that natural killer (NK) cells expressing the CD16 marker accounted for the relatively rapid repopulation of CD2 cells (data not shown). The early relative rise in B lymphocytes (CD20) reflected a compensatory increase because B cells are spared destruction by immunotoxin. Fig. 3 shows the ratio of CD4 to CD3 T lymphocytes after treatment. Initially, T-helper cells (CD4) accounted for the vast majority of the repopulating T cells in the PBLs. However, by 5 weeks after trans-
Table III. Serum anti-DT IgG response in antiCD3-immunotoxin treated monkeys
Monkey
Preimmunotoxin serum (1:100) OD
92023 P0F P0J P1P P1N WF6 AXG AXA 2CP
.065 .065 .073 .065 .072 .085 .075 .077 .072
Postimmunotoxin serum (1:100) OD .299 .854 .428 .761 .346 .131 .634 .128 .070
Days after immunotoxin treatment 431 251 223 113 48 99 77 65 50
OD, Optical density.
plantation, CD4 cells were in the minority and remained so for more than 150 days after transplantation. Later analysis of the recovering T cells showed that T-helper cells remain the minority population, with the CD8 phenotype predominating (data not shown). Antibody responses after immunotoxin and renal transplantation. Antidonor alloantibody was measured using a flow cytometric crossmatch test to measure antidonor IgG in recipient monkeys. Relative to pretransplantation serum, antidonor/IgG increased in 8 of 11 monkeys when tested 2 to 4 months after transplantation (Table II). Five of these 8 monkeys also had chronic rejection histologically. One monkey (BED), that currently has a creatinine level of 1.1 mg/dL and continues to do well more than 411 days after transplantation, had a 21⁄2-fold increase in the mean
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channel fluorescence owing to the presence of antidonor antibody. This monkey also developed a transient but severe interstitial nephritis with later resolution spontaneously. Of the 11 monkeys with antidonor antibody present by flow crossmatch, 9 were tested for presence of complement-fixing antibody; 3 of 9 had detectable titers of complement fixing antibody. Antitetanus antibody. As a measure of T-celldependent B-cell responses after immunotoxin treatment and renal transplantation, monkeys were immunized between 1 and 5 months after immunotoxin treatment with tetanus toxoid and boosted 4 weeks later. ELISA testing for antitetanus IgG was performed before immunization, 3 weeks after initial immunization, and 1 week after boosting. Fig. 4, A shows a representative monkey treated with combined immunotoxin, mycophenolate mofetil (MMF), and prednisone that developed antitetanus antibody after immunization and boosting at 2 months after transplantation. Data from 8 monkeys treated with immunotoxin confirms an intact antitetanus response when tested 1 to 5 months after treatment in 6 of 8 monkeys (Fig. 4, B). Antibody response to FN18-CRM9. The ability of recipient monkeys to develop an antibody response to the xenogeneic mouse monoclonal antibody (FN18) and the mutant DT (CRM9) component of the immunotoxin was evaluated. An IgG response against the mouse IgG portion of immunotoxin was detected in 4 of 6 animals at 2 weeks after immunotoxin treatment, 5 of 5 tested at 4 weeks, and 9 of 9 tested at 2 months or longer after immunotoxin treatment. Fig. 5 shows titration curves for 6 animals tested 2 weeks to 1 year after immunotoxin treatment. Table III summarizes the anti-CRM9 antibody level of monkeys before and after immunotoxin treatment. Number values are expressed as optical density derived from the ELISA assay at a 1:100 serum dilution. Eight of 9 monkeys had an increase in anti-CRM9 antibody when tested 6 to 60 weeks after immunotoxin treatment. DISCUSSION The most reliable and consistent observation about immunotoxin with regard to rhesus monkey renal allografts is that acute rejection is prevented, often indefinitely, by T-cell ablation using this agent starting on day 0. This result is highly reproducible, and given the difficulty of preventing acute rejection in this non-human primate outbred model, the result is startling when compared with other immunosuppressive strategies. Nevertheless, long-
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term follow-up has revealed 3 unexpected longterm consequences: interstitial nephritis, chronic rejection, and wasting disease. Unconjugated FN18 antibody (without toxin), when administered to monkeys receiving renal allografts, resulted in no long-term survivors, with the same peritransplantation deoxyspergualin and steroid immunosuppression that led to a majority of long survivors in the immunotoxin group (Judith Thomas, personal communication, February 1998).14 These results suggest that the immunotoxin construct lends a substantial advantage over antibody alone. Interstitial nephritis is generally associated with autoimmune injury of the native kidneys and has not been associated with renal transplantation. The exact cause and nature of the infiltrate in the autoimmune type disease are unclear as it is in the immunotoxin transplant model. Interestingly, many of our long-term surviving monkeys with excellent kidney function have nests of interstitial infiltrating cells scattered throughout the graft but without causing any tubular or glomerular injury. In addition, interstitial nephritis has been present in some monkeys with spontaneous resolution over time and without apparent graft injury. However, some monkeys develop such an intense interstitial nephritis that it causes a rise in serum creatinine level. As with autoimmune disease, this may be mediated by interstitial edema, causing compromise of capillary blood flow and resulting in ischemic injury. We have not tested whether this process responds to steroid therapy, which might be expected to reverse it. If interstitial nephritis did cause graft destruction in our series, it generally occurred between 50 and 135 days after transplantation. Most of these animals were treated with immunotoxin alone, and graft survival time was similar to that seen by Thomas et al,9 who used similar therapy. Later graft losses were usually attributable to chronic rejection. Chronic rejection of renal allografts was typified by hyalinization of glomeruli with reduplication of the glomerular capillary basement membrane, vasculopathy with neointimal hyperplasia, and by relative absence of graft infiltrating cells as has been reported in detail by Armstrong et al.15 Although most monkeys tested did develop alloantibody, there was not a perfect correlation between development of alloantibody and chronic rejection. In fact, one monkey with high levels of alloantibody capable of complement fixation continues to have excellent graft function without histologic evidence of graft injury. The etiology of chronic allograft rejection may involve both antibody-mediated injury and nonspecific mediators of inflam-
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mation such as cytokine-mediated injury. If tolerance strategies are to become attractive for clinical implementation, it would be highly desirable to prevent not only acute but also chronic rejection. To achieve this in the absence of daily drug therapy would be ideal. Although an initial 3-day course of steroids and MMF added to immunotoxin prevented graft loss in the first 100 days, 4 of the 5 monkeys eventually succumbed to chronic rejection. Better strategies, perhaps including a longer course of MMF, need to be developed to prevent chronic rejection in this model. The 3 monkeys that developed wasting disease in the setting of normal renal function and normal serum chemistries are of obvious concern, but to date they have defied precise explanation. Two possible explanations of their unremitting weight loss are an as yet undetermined viral infection occurring as a result of lymphocyte depletion, and cachexia resulting from a CD4 depletion. The latter has been described in a mouse model,16 and we are currently performing viral screening to rule out the former. The depletion and repopulation of T cells in PBLs after day 0 immunotoxin treatment were similar to that previously reported for day -7 immunotoxin-treated monkeys7,10 and when used with donor bone marrow infusion.9 In the 3 months after treatment with immunotoxin, CD4 cells repopulated at a slower rate than CD8 T cells, with a relative ratio of CD4/CD8 equal to 1:2. The altered peripheral T-lymphocyte ratio observed in this model parallels that reported in both bone marrow transplant patients17 and in patients treated with total lymphoid irradiation.18 Given that we have seen a nonspecific suppression of MLC and CTL responses during the first 6 months after immunotoxin treatment, we hypothesize that the skewed CD4/CD8 repopulation may be a significant factor favoring graft prolongation in our model. The rate of CD4 repopulation may also be age dependent.19 The precise nature of the repopulating T cells is the subject of ongoing investigation. The ability of rhesus monkeys to mount an antibody response is intact after immunotoxin treatment. With few exceptions, monkeys developed responses to tetanus, diphtheria antigen, xenoantibody, and alloantigen. Because isotype switching in B cells is generally T-cell dependent, this aspect of T-cell function does not appear to be impaired by immunotoxin treatment. In summary, immunotoxin appears to be a safe and reliable means of preventing acute allograft rejection when used as an induction agent. Further work is necessary to determine the mechanism by
which it may produce long-term tolerance, and new strategies must be developed to overcome the problems of interstitial nephritis within the first 4 months after transplantation and chronic rejection occurring even later. REFERENCES 1. Strober S, Dhillon M, Schubert M, Holm B, Engleman E, Benike C, et al. Acquired immune tolerance to cadaveric renal allografts: a study of t h r e e patients treated with total lymphoid irradiation. N Engl J Med 1989;321:28-33. 2. Mazariegos GV, Reyes J, Marino IR, Demetris AJ, Flynn B, Irish W, et al. Weaning of immunosuppression in liver transplant recipients. Transplantation 1997;63:243-9. 3. Burlingham WJ, Grailer AP, Fechner JH Jr, Kusaka S, Trucco M, Kocova M, et al. Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient. Transplantation 1995; 59:1147-55. 4. Thomas FT, Carver FM, Foil MB, Pryor WH, Larkin EW, Hall WR, et al. Long-term incompatible kidney survival in outbred higher primates without chronic immunosuppression. Ann Surg 1983;198:370-8. 5. Kawai T, Cosimi AB, Colvin RB, Powelson J, Eason J, Kozlowski T, et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 1995;59:256-62. 6. Kirk AD, Harlan DM, Armstrong NN, Davis TA, Dong Y, Gray GS, et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci U S A 1997;94:8789-94. 7. Knechtle SJ, Vargo D, Fechner J, Zhai Y, Wang J, Hanaway MJ, et al. FN18-CRM9 immunotoxin p r o m o t e s tolerance in primate renal allografts. Transplantation 1997;63:1-6. 8. Neville DM Jr, Scharff J, Srinivasachar K. In vivo T-cell ablation by holo-immunotoxin directed at human CD3. Proc Natl Acad Sci U S A 1992;89:2585-9. 9. Thomas JM, Neville DM, Contreras JL, Eckhoff DE, Meng G, Lobashevsky AL, et al. Preclinical studies of allograft tolerance in rhesus monkeys: a novel anti-CD3immunotoxin given peritransplant with donor bone marrow induces operational tolerance to kidney allografts. Transplantation 1997;64:124-35. 10. Fechner JH Jr, Vargo DJ, Geissler EK, Graeb C, Wang J, Hanaway MJ, et al. Split tolerance induced by immunotoxin in a rhesus monkey allograft model. Transplantation 1997;63:1339-45. 11. Knapp LA, Cadavid LF, Eberle ME, Knechtle SJ, Bontrop RE, Watkins DI. Identification of new mamu-DRB alleles using DGGE and direct sequencing. Immunogenetics 1997;45:171-9. 12. Neville DM, Scharff J, Hu HZ, Rigaut K, Shiloach J, Slingerland W, et al. A new reagent for the induction of T-cell depletion, anti-CD3-CRM9. J Immunother 1996;19:85-92. 13. Ohkawa S, Xu K, Wilson LA, Montelaro R, Martin LN, Murphey-Corb M. Analysis of envelope glycoproteinspecific antibodies from SIV-infected and gp110-immunized monkeys in ACC and ADCC assays. AIDS Res Hum Retroviruses 1995;11:395-403. 14. Eckhoff DE, Contreras JL, Wang PX, Lobashevsky AL, Meng G, Jiang Y, et al. Immunologic mechanisms favoring operational transplant tolerance induced by deoxyspergualin
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in rhesus monkeys given anti-CD3 immunotoxin. Transplantation 1998 In press. 15. Armstrong N, Buckley P, Oberley T, Fechner J Jr, Dong Y, Hong X, et al. Analysis of primate renal allografts following T cell depletion with anti-CD3-CRM9. Transplantation 1998 In press. 16. Morrissey PJ, Charrier K, Braddy S, Liggitt D, Watson JD. CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice: disease development is prevented by cotransfer of purified CD4+ T cells. J Exp Med 1993;178:237-44. 17. Leblond V, Othman TB, Blanc C, Theodorou I, Choquet S, Sutton L, et al. Expansion of CD4+CD7- T cells, a memory subset with preferential interleukin-4 production, after bone marrow transplantation. Transplantation 1997; 64:1453-9. 18. Halperin EC. Total lymphoid irradiation as an immunosuppressive agent for transplantation and the treatment of ‘autoimmune’ disease: a review. Clin Radiol 1985;36:125-30. 19. Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC, Feuerstein IM, et al. Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. N Engl J Med 1995;332:143-9.
DISCUSSION Dr Stephen T. Bartlett (Baltimore, Md). You have demonstrated that the G protein FN18 CRM9 markedly prolongs the survival of these MLC mismatched rhesus monkey allografts. One of the side effects was a wasting phenomenon combined with a prolonged CD4 depletion. Was there a relationship between the animals that had wasting and the prolonged CD4 depletion? Would you speculate on the pharmacologic mechanism of the immunotoxin? At least with such prolonged wasting, one would imagine that the immunotoxin would have been long gone. My second question relates to those cases in which the drug eventually proved ineffective and interstitial nephritis and chronic rejection developed. Did you look at the phenotype of the graft infiltrating cells? Dr Knechtle. With regard to the wasting, we have a small number of monkeys, 3 in this series, that had significant weight loss at variable times after treatment. The etiology of this wasting syndrome has eluded diagnosis to date. One possibility is that this is a virally mediated infection that we have not been able to diagnose. We have screened these monkeys for retroviruses and all are negative. The other possibility is that this may be related in some way to CD4 depletion. CD4 depletion and repopulation in mouse models have been associated with a wasting phenomenon. This is worrisome because it suggests that perhaps tolerance induction, at least with a depleting approach, may be associated with a systemic problem such as wasting. In terms of the mechanism of wasting, we do not have any evidence that it is related to the toxin component itself. The toxin itself is metabolized early and is gone within a few days. So the toxicity typically caused by DT is hepatotoxicity, and we are seeing this wasting syndrome months later. We are in the process of looking at the phenotype of the graft infil-
Surgery August 1998 trating cells using immunocytochemical techniques. Dr Bartlett. One of the reasons I wanted to follow up on the question of the graft infiltrating cells is that, in the slide you showed, there is a fair amount of interstitial nephritis. However, there is no particular direct cellular attack. It could be that there is cytokine release, particularly if some of those cells are B cells, that may occur with B-cell disregulation with prolonged CD4 and CD3 depression. This is not necessarily a posttransplantation lymphoproliferative disorder (PTLD) but a PTLD-like state. Dr. Knechtle. There is no evidence that these are tumor cells at all. They have a benign phenotype. They are a combination of CD4, CD8, and CD20 cells. We are using a large number of markers to look at them. We are also doing a polymerase chain reaction to evaluate cytokine expression within the grafts. So far, no clear patterns are emerging in control animals compared with experimental animals in terms of the cytokine pattern of tolerant monkeys. Dr Carl E. Haisch (Greenville, NC). I am concerned about what might be called a cytokine release syndrome. As I recall, your previous studies have had the native kidneys in place. Do you think that there is some relationship between the transplanted kidney and removing the native kidneys and the cytokine release syndrome? Also, do you think that there may be some relationship between interstitial cells in chronic rejection and cytokine release syndrome? Dr Knechtle. Although it is true that in the first group of experiments the recipient monkeys had their native kidneys in place when they received immunotoxin, in all of the experiments that I just showed you, given on day 0, there was no native kidney in place. Cytokine release syndrome has not been a clinical problem in our monkeys. We are not seeing hypertension, flushing, fever, or the pulmonary problems typically associated with cytokine release. We have not done measurements of peripheral blood cytokines. Judy Thomas, who also is using the same immunotoxin, has done that and tumor necrosis factor levels are elevated after administration of the immunotoxin as expected, but it has not been an overwhelming problem for her since initiating new protocols. Dr Mark H. Deierhoi (Birmingham, Ala). You describe rechallenging the animals with skin grafts 6 months after the transplantation. In the animals with chronic rejection, does their cellular tolerance or their tolerance to skin grafts continue through the period when the kidneys are failing? What is the mechanism for chronic rejection in this situation? The second question relates to the prolonged T-cell depletion. Presumably these are relatively juvenile animals with intact thymic function. What will be the effect of this sort of T-cell depletion in more mature animals or, for example, adult human beings, where thymic function may be much more depressed? Dr Knechtle. One of 13 monkeys has developed acute rejection after skin grafting. That skin graft was placed at 180 days, which is the earliest I have placed a skin graft. I believe that there is a metastable situation early on that
Surgery Volume 124, Number 2 becomes more robust tolerance later. If a skin graft, which is the most potent immunogen, is given too early, we will probably break tolerance. None of the other animals have had acute rejection induced by skin grafting. They have gone on to develop chronic rejection, but in some cases 300 days later. That has not differed from the animals who have not undergone skin grafting, so I do not have any evidence to suggest that the skin grafts cause chronic rejection. In terms of T-cell depletion, I would agree with your presumption that Tcell repopulation, in particularly CD4 cell repopulation, will be dependent on developmental age and thymic maturity. Data published by Mackall in the New England Journal of Medicine19 showed that CD4 repopulation was dependent on age. One would presume that the older the patient the less well CD4 cells will return. I don’t know whether that is clinically significant. Dr M. Wayne Flye (St. Louis, Mo). The depletion of pan-T cells is impressive. Do you think there is a different mechanism involved by using the toxin than is obtained with antilymphocyte serum or monoclonal antibodies? Specifically, did you look at thymic depletion and bone marrow and the repopulation there? Following up on the previous question, does this have a greater effect centrally on the thymic mechanism than distally in the periphery? Also, you can decrease the nonspecific response to tetanus and other antigens. Do you get specific depression that is facilitated by the graft being in place? Is that allospecific? When you looked at the anti-
Knechtle et al. 447
body formation, did you characterize it in terms of isotypes or even IgG, IgM? Could you be getting enhancing antibodies in some of these animals? That is a real possibility, particularly in the animal that had prolonged survival and a high titer. Dr Knechtle. We have tried to look at the repopulation of the thymus after immunotoxin therapy. We were unable to find thymic tissue at autopsy in these monkeys. There appears to be complete involution of the thymus. Unfortunately, that has also been true of control, untreated monkeys. The amount of thymic involution in these monkeys is substantial even in the absence of immunotoxin. So we have not really been able to determine thymic histology and what happens to the CD3 cells in the thymus. In terms of the antibody, IgG was measured, but unfortunately IgG subclass specificity cannot be determined in monkeys because reagents are not available to differentiate. There is the possibility, as you suggest, that the antibodies that we see are enhancing antibodies; however, we are not seeing suppression of allospecific antibodies. Dr Flye. Is there any degree of allospecificity in terms of your suppression after the graft is in place by in vitro, MLC, or CTL testing? Dr Knechtle. CTL is suppressed in an allospecific manner after skin grafting and in a nonspecific manner early on. In the first 200 days, CTSs are suppressed to donor and third-party. After skin grafting, the monkeys develop an excellent CLT response to the third-party but not to the donor. So the CTL suppressiveness takes time. Presumably the presence of the donor alloantigen is critical in inducing that.
Surgery is abstracted and/or indexed in Index Medicus, Science Citation Index, Current Contents/ Clinical Medicine, Current Contents/Life Sciences, and MEDLINE. This Journal has been registered with Copyright Clearance Center, Inc., 222 Rosewood Dr., Danvers, MA 01923. Consent is given for the copying of articles for personal or internal use of specific clients. This consent is given on the condition that the copier pay directly to the Center the per-copy fee stated on the first page of each article for copying beyond that permitted by U.S. Copyright Law. This consent does not extend to other kinds of copying, such as for general distribution, resale, advertising and promotional purposes, or for creating new collective works. All inquiries regarding copyrighted material from this publication other than those that can be handled through Copyright Clearance Center should be directed to Associate Journal Publisher, Permissions, Mosby, Inc., 11830 Westline Industrial Dr., St. Louis, MO 63146; phone (314)453-4129; fax (314)432-1380.
Background. T-lymphocyte depletion 7 days before transplantation with immunotoxin FN 18-CRM9 has resulted in tolerance to subsequent renal allografts. We tested the effect of giving immunotoxin on the day of the transplantation and evaluated its effect on rhesus monkey renal allograft survival, on antibody production, and on T-cell recovery. Methods. Major histocompatibility complex mismatched renal allografts were performed in rhesus monkeys. Immunotoxin was given starting on the day of transplantation, with and without prednisone and mycophenolate mofetil for 3 days. T-cell subsets and alloantibody levels were measured by flow cytometry. The ability of treated monkeys to develop antibody to tetanus, diphtheria, and xenoantibody was measured. Histology of renal transplants was read in a blinded manner. Results. Immunotoxin started on the day of transplantation resulted in prolonged allograft survival in all treatment groups. Graft loss between days 50 and 135 was most often due to interstitial nephritis. Later graft loss was due to chronic rejection. Monkeys had intact antibody responses to alloantigen, tetanus, diphtheria, and xenoantibody. Their CD4 cells recovered gradually over 6 months. Conclusions. Immunotoxin reliably prolongs renal allograft survival when started on the day of transplantation, but interstitial nephritis and chronic rejection limit the development of long-term tolerance. Tcell-dependent B-cell responses remain intact after treatment. (Surgery 1998;124:438-47.) From the Departments of Surgery and Pathology, University of Wisconsin, Madison, Wis, and Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Md
PRECLINICAL MODELS of allograft tolerance have the goal of devising a clinically relevant approach to promoting long-term allograft acceptance in the absence of continuous drug therapy. This elusive goal has been met in a small number of patients undergoing transplantation1-3 and in nonhuman primates using strategies that are not yet in clinical use.4-6 A transient but profound decrease in circulating and lymph node T-lymphocyte populations produced by FN18-CRM9 immunotoxin has been shown to reliably result in long-term tolerance to major histocompatibility complex (MHC) mismatched renal allografts in rhesus monkeys.7 This
Supported by the University Surgical Associates, University of Wisconsin, Madison, Wis, Novartis Pharmaceuticals Corporation, East Hanover, NJ, and by National Institutes of Health, Bethesda, Md. Presented at the Fifty-ninth Annual Meeting of the Society of University Surgeons, Milwaukee, Wis, Feb 12-14, 1998. Reprint requests: Stuart J. Knechtle, MD, Department of Surgery, University of Wisconsin Hospital, 600 Highland Avenue, Madison, WI 53792-7375. Copyright © 1998 by Mosby, Inc. 0039-6060/98/$5.00 + 0
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immunotoxin developed by Neville et al8 consists of a conjugate of a murine anti-rhesus monkey CD3 monoclonal antibody (FN18) and a mutated diphtheria toxin, CRM9. The advantage of CRM9 relative to wild-type diphtheria toxin (DT) is that it maintains its potent ability to inhibit protein synthesis while reducing its toxicity to 1/300th of the wild-type DT. We, as well as Thomas et al,9 have shown that immunotoxin is capable of inducing unresponsiveness in a preclinical model of renal allografting and that immunotoxin is well tolerated. Immunotoxin reduces CD3 T lymphocytes in the peripheral blood lymphocytes (PBLs) of rhesus monkeys by 2 to 3 logs and in the lymph nodes by 2 logs, when measured 1 to 2 days after a 3-day course of immunotoxin. This ability differentiates immunotoxin from monoclonal antibody alone, which has not been shown to substantially deplete lymphocytes in lymphoid tissues. Immunotoxin reduces mixed lymphocyte culture (MLC) proliferation and cytotoxic T-lymphocyte precursor (CTLp) frequency to donor and thirdparty alloantigen when measured 3 to 4 months after treatment. However, long-surviving immunotoxin-treated renal allograft recipients are able to
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Knechtle et al. 439
Fig. 1. Renal transplant histology of a monkey killed on day 58, with creatinine level of 8.6 mg/dL and histologic characteristics of severe interstitial nephritis. A prominent infiltrate occupies the interstitial spaces without tubular invasion. (Original magnification ×20.)
Fig. 2. Changes in lymphocyte subset populations after immunotoxin treatment on the day of transplantation. Percentage of T cells (CD3, filled triangle), T cells and NK cells (CD2, open square), and B cells (CD20, open circle) in PBLs was determined by single color flow cytometry. Each time point is the mean ± SD of 5 to 11 monkeys treated with immunotoxin on the day of transplantation with or without other treatments. Later time points have few animals in the group, as animals were lost for analysis because of death.
generate new CTLp against third-party skin graft donors 6 months after renal transplantation, but PBLs of these same recipients have no increase in the number of CTLp against the donor kidney.10 In vivo correlation of this result comes from the observation that long-surviving monkeys accept donor skin grafts placed 200 days after treatment but reject third-party skin grafts. To test the relevance of immunotoxin to cadaveric organ transplantation in which immunosuppression must be started on the day of the transplantation (and pretreatment is not feasible), we used an altered treatment regimen giving immunotoxin on days 0, 1, and 2 with respect to the renal transplantation. The influence of immunotoxin on T- and B-cell function after transplantation was
evaluated, and graft outcome was assessed histologically and functionally. We report herein the longterm follow-up of monkeys treated with immunotoxin to induce allograft unresponsiveness. METHODS Animals and surgery. Male juvenile rhesus monkeys weighing 2.5 to 3.5 kg (age range 11⁄2 to 3 years) were purchased from either the University of Wisconsin Primate Center or LABS (Yemassee, SC) and were screened for herpes B, simian immunodeficiency virus, simian T-lymphotropic virus, and simian retrovirus and skin tested for tuberculosis. Monkeys were fed and maintained according to National Institutes of Health and US Department of Agriculture guidelines. Donor-recipient pairs were
440 Knechtle et al.
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Fig. 3. Ratio of T-helper cells to all T cells after immunotoxin treatment on the day of transplantation. Percentage of PBLs that was T-cell (CD3) and T-helper cell (CD4) was determined by single color flow cytometry in a panel of 6 monkeys. Ratio was determined by using the percentage of CD3 cells as the denominator and the percentage of CD4 cells as the numerator. Two more color flow analyses demonstrated that 100% of CD4 lymphocytes are also CD3.
selected based on CTL and MLC responses and on MHC class I and MHC class II typing as previously described7,11 to maximize disparity. Recipients were screened for a low titer of antibody to DT as previously described.12 Renal transplantations were performed by transplanting the donor left kidney to recipients that had undergone bilateral native nephrectomy. Graft function was monitored by measuring serum creatinine level, and monkeys were killed when the creatinine rose to >8 mg/dL, when weight loss >20% occurred, or by other criteria determined by the veterinary staff and investigators. All protocols were approved by the University of Wisconsin Animal Use Committee. Immunosuppressive agents. FN18-CRM9 was administered either on days -7, -6, -5 (initial experiments) or on days 0, 1, 2 (later groups) with respect to renal transplantation at a total dose of 0.2 mg/kg given intravenously in 3 equally divided doses. Mycophenolate mofetil (CellCept, Roche, Nutley, NJ) 250 mg/day was given orally on days 0, 1, and 2. Methylprednisolone was administered at a dose of 125 mg intravenously on days 0, 1, and 2. The day 0 doses of drugs were administered intraoperatively. Flow cytometry analysis. A total of 200 µL of whole blood was diluted with 100 µl phosphatebuffered saline solution/1% fetal calf serum and stained with fluorescein isothiocyanate labeled FN18 (anti-CD3ε), T11 (anti-CD2; Coulter Immunology, Hialeah, Fla), B1 (anti-CD20; Coulter), OKT4 (anti-CD4; Ortho Diagnostics, Raritan, NJ), or isotype control antibody (Sigma, St Louis, Mo) according to the manufacturer. Red blood cells
were removed from whole blood by ACK solution (155 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L ethylenediaminetetraacetic acid) treatment after staining. Cells were then either subjected to flow cytometry immediately or fixed in 1% paraformaldehyde. Flow cytometry was performed on a Becton Dickinson FACSCAN. Lymphocytes were gated on the basis of forward and side scatters and 10,000 events were analyzed. Detection of antidonor immunoglobulin. Antidonor immunoglobulin G (IgG) antibodies were detected by flow cytometry as previously described.10 The presence of antidonor IgG was monitored by observing the shift in mean channel fluorescence of goat antihuman IgG binding in CD3 positive lymphocytes. Relative changes in titer were measured by taking the ratio of the mean channel fluorescence of posttransplantation serum to the mean channel fluorescence of pretransplantation serum. Complement-fixing alloantibody was detected by antibody-complement cytotoxicity assay. 13 Target cells were 51Cr labeled donor and thirdparty PHA blasts in RPMI/10% fetal calf serum. A total of 10 µL of target cells (1 × 106/mL) were incubated with 20 µL diluted serum in triplicate for 45 minutes in 96-well round bottom plates at room temperature. Rabbit complement, diluted 1:8, was then added to each well (20 µL) and plates were incubated for 45 minutes at 37° C. After 100 µL of cold medium was added to each well, the supernatant was harvested on Skatron filters (Skatron, Sterling, Va). Controls included spontaneous lysis (no complement added), lysis
Knechtle et al. 441
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A
B Fig. 4. Monkeys immunized with tetanus toxoid after immunotoxin treatment are capable of making an antitetanus IgG response. Presence of antitetanus IgG in the sera of animals was measured by ELISA. (A) Humoral response of animal AKJ after primary tetanus toxoid immunization (TT) 2 months after transplantation/immunotoxin treatment with a subsequent secondary immunization 1 month later. Serum was taken just before immunization (Pre TT), 3 weeks after the primary immunization (Post TT), and 1 week after the secondary immunization (post TT boost). (B) Response to primary and secondary TT in 1 month (XV3), 3 months (2CP), 4 months (AXG), and 5 months (BED) after transplantation/immunotoxin treatment.
due to complement alone (always less than spontaneous lysis), and maximum lysis (2% TritonX100). Percent specific 51Cr release was calculated and the end point titer was determined as the reciprocal of the last dilution to obtain cpm greater than 3 standard deviations above the mean spontaneous release. Tetanus antigen immunization. Monkeys were immunized intramuscularly with alum adsorbed tetanus toxoid at either 1 or 5 months after transplantation. Animals were bled 1 week before immunization and 3 weeks after the primary immunization. A secondary immunization was done 4 weeks after the primary and sera were then collected 1 week later. Sera were measured for antitetanus IgG by enzyme-linked immunosorbent assay (ELISA).
ELISA for anti-mIgG, anti-CRM9 and antitetanus toxoid antibodies. Costar 96-well flat bottom ELISA plates (Costar, Cambridge, Mass) were coated overnight at 4° C with 25 µL appropriate antigen (3 mg/mL). After plates were blocked with phosphate-buffered saline/2% bovine serum albumin and washed, 25 µL diluted serum was added to each well. After incubation for 1 hour at room temperature, plates were washed and appropriately diluted horseradish peroxidase-conjugated goat antihuman IgG was added to each plate. Again, after incubation for 1 hour at room temperature, substrate was added and, after adding 1 M H2SO4, the plate was read by an automated ELISA plate reader. Antibodies against CRM9 were detected by ELISA as previously described.12
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Fig. 5. Antibody response to mouse IgG of anti-CD3-immunotoxin. Open symbols are individual animals at 3 months to 1 year after immunotoxin treatment in either day -7 or day 0 treatment groups. Closed symbol is the mean value of 4 untreated animals..
Table I. Graft survival* Group
Anti-CD3-CRM9 (total 0.2 mg/kg)
Additional therapy
1 2 3
None Days -7,-6,-5 Days -7,-6,-5
4
Days -7,-6,-5
5 6 7
Days 0, 1, 2 Days 0, 1, 2 Days 0, 1, 2
None None Intrathymic injection-normal saline Intrathymic injection-donor cell None Steroid MMF + steroid
Graft survival time (days)
P value †
Reason for graft loss
5, 7, 7, 8, 9 51, 84, 203,302‡, 728 36, 68, 72, 887, >921
NS vs 2
ar, ar, ar, ar, ar ar, ar, 1, 1&2, cr ar, ar, ar, cr, cgf
41, 45‡, 181, 846‡, >1011
NS vs 2
ar, 3, ar§, cr, cgf
51‡, 57, 79, 184, 287 35‡, 105, >333 133, 172, 307, >416, 435
NS vs 5 NS vs 5
2, in, in, cr, cr 4, in, cgf in, cr, cr, cgf,cr
ar, acute rejection; in, chronic interstitial nephritis; cr, chronic rejection; 1, weight loss > 20%; 2, hematocrit < 25% anemia; 3, small bowel hemorrhage; 4, skin burn; cgf, continued graft function with a creatinine < 2.0 mg/dL. *Some of the data reported in this table has been previously published.7 †Comparison
between Kaplan-Meier graft survival estimates of groups by log-rank test; NS = not significant.
‡Killed
with a creatinine level < 2.0 mg/dL.
§Acute
rejection 40 days after donor skin graft placed.
Histology. At autopsy all tissues were examined grossly, and transplanted kidneys were evaluated histologically in a blinded manner by an experienced renal transplant pathologist (T.O.) and graded according to modified Banff criteria (1997). RESULTS Long-term follow-up of monkeys pretreated on days -7, -6, and -5 with immunotoxin, initially reported by our group,7 are shown in Table I. The pathology of rejecting allografts showed acute cellular rejection for untreated controls, whereas the long-surviving allografts ultimately failed because of either rejection or death with a functioning graft. Wasting was seen in 3 monkeys and was characterized
by slow, progressive weight loss with loss of appetite despite normal liver function, renal function, and serum chemistries. Two of these monkeys became anemic, but bone marrow biopsy specimens showed normal erythrocyte precursors. The histology of interstitial nephritis is shown in Fig. 1. The results of monkeys receiving immunotoxin starting on the day of transplantation rather than 1 week before transplantation are summarized in Table I. If the immunotoxin was used alone (group 5), graft survival was consistently prolonged but ultimately limited by chronic rejection in 2 of 5 recipients. One monkey in this group was killed because of early weight loss, and this monkey had a normal creatinine level of 0.7 at the time of death
Knechtle et al. 443
Surgery Volume 124, Number 2 Table II. Alloreactive antibody and graft outcome* Animal AXF 1347 AXG WF6 AKJ AXA XV3 BED 2CP DFW EEF
GST 51 57 184 287 133 172 307 >411 435 105 >328
Test† 51 57 110 98 133 118 120 125 120 105 112
ABX‡ 1.3 2.3 0.9 6.3 1.2 8.4 1 2.5 1.6 2.9 0.8
ACC§ ND ND 1 243 1 81 1 >729 1 1 1
Outcome Creatinine < 2.0 mg/dL in cr cr cr cr cr Creatinine < 2.0 mg/dL cr in Creatinine < 2.0 mg/dL
in, chronic interstitial nephritis; cr, chronic rejection; ND, not determined. *Some of the data reported in this table has been previously published.14 †Days
after transplant from which sera were tested.
‡Ratio
of the mean channel fluorescence of posttransplantation anti-human IgG reactive serum (1:100 dilution) binding to donor CD3+ lymphocytes to the mean channel fluorescence of pretransplantation antihuman IgG reactive serum (1:100 dilution) binding to donor CD3+ lymphocytes. §Reciprocal
end point titer of antibody complement cytotoxicity assay.
on day 51. When steroids were added to immunotoxin in a group of 3 monkeys (group 6), one graft was lost at day 105 as a result of interstitial nephritis and 1 monkey was still surviving at 333 days. A third monkey was killed at day 35 because of skin infection after an electrocautery grounding pad burn (renal histology was normal at autopsy). Five monkeys were treated with immunotoxin, prednisone, and mycophenolate on days 0, 1, and 2 (group 7). All 5 members of this group had graft survival greater than 100 days, with 1 monkey still surviving more than a year after transplantation. Nevertheless, 4 monkeys in this group lost their grafts, 3 from chronic rejection and 1 from focal interstitial nephritis. Flow cytometric analysis of lymphocyte subsets. Fluorescein isothiocyanate–labeled anti-CD2, CD3, CD4, and CD20 monoclonal antibodies were used to evaluate the effect of day 0 immunotoxin on lymphocyte subsets. A summary of analysis of 11 monkeys is shown in Fig. 2. At 1 to 7 days after treatment, both CD2 and CD3 cells were severely depleted, with rapid recovery of CD2 cells and gradual recovery of CD3 cells over 3 months. Preliminary analysis of a few immunotoxin-treated animals indicates that natural killer (NK) cells expressing the CD16 marker accounted for the relatively rapid repopulation of CD2 cells (data not shown). The early relative rise in B lymphocytes (CD20) reflected a compensatory increase because B cells are spared destruction by immunotoxin. Fig. 3 shows the ratio of CD4 to CD3 T lymphocytes after treatment. Initially, T-helper cells (CD4) accounted for the vast majority of the repopulating T cells in the PBLs. However, by 5 weeks after trans-
Table III. Serum anti-DT IgG response in antiCD3-immunotoxin treated monkeys
Monkey
Preimmunotoxin serum (1:100) OD
92023 P0F P0J P1P P1N WF6 AXG AXA 2CP
.065 .065 .073 .065 .072 .085 .075 .077 .072
Postimmunotoxin serum (1:100) OD .299 .854 .428 .761 .346 .131 .634 .128 .070
Days after immunotoxin treatment 431 251 223 113 48 99 77 65 50
OD, Optical density.
plantation, CD4 cells were in the minority and remained so for more than 150 days after transplantation. Later analysis of the recovering T cells showed that T-helper cells remain the minority population, with the CD8 phenotype predominating (data not shown). Antibody responses after immunotoxin and renal transplantation. Antidonor alloantibody was measured using a flow cytometric crossmatch test to measure antidonor IgG in recipient monkeys. Relative to pretransplantation serum, antidonor/IgG increased in 8 of 11 monkeys when tested 2 to 4 months after transplantation (Table II). Five of these 8 monkeys also had chronic rejection histologically. One monkey (BED), that currently has a creatinine level of 1.1 mg/dL and continues to do well more than 411 days after transplantation, had a 21⁄2-fold increase in the mean
444 Knechtle et al.
channel fluorescence owing to the presence of antidonor antibody. This monkey also developed a transient but severe interstitial nephritis with later resolution spontaneously. Of the 11 monkeys with antidonor antibody present by flow crossmatch, 9 were tested for presence of complement-fixing antibody; 3 of 9 had detectable titers of complement fixing antibody. Antitetanus antibody. As a measure of T-celldependent B-cell responses after immunotoxin treatment and renal transplantation, monkeys were immunized between 1 and 5 months after immunotoxin treatment with tetanus toxoid and boosted 4 weeks later. ELISA testing for antitetanus IgG was performed before immunization, 3 weeks after initial immunization, and 1 week after boosting. Fig. 4, A shows a representative monkey treated with combined immunotoxin, mycophenolate mofetil (MMF), and prednisone that developed antitetanus antibody after immunization and boosting at 2 months after transplantation. Data from 8 monkeys treated with immunotoxin confirms an intact antitetanus response when tested 1 to 5 months after treatment in 6 of 8 monkeys (Fig. 4, B). Antibody response to FN18-CRM9. The ability of recipient monkeys to develop an antibody response to the xenogeneic mouse monoclonal antibody (FN18) and the mutant DT (CRM9) component of the immunotoxin was evaluated. An IgG response against the mouse IgG portion of immunotoxin was detected in 4 of 6 animals at 2 weeks after immunotoxin treatment, 5 of 5 tested at 4 weeks, and 9 of 9 tested at 2 months or longer after immunotoxin treatment. Fig. 5 shows titration curves for 6 animals tested 2 weeks to 1 year after immunotoxin treatment. Table III summarizes the anti-CRM9 antibody level of monkeys before and after immunotoxin treatment. Number values are expressed as optical density derived from the ELISA assay at a 1:100 serum dilution. Eight of 9 monkeys had an increase in anti-CRM9 antibody when tested 6 to 60 weeks after immunotoxin treatment. DISCUSSION The most reliable and consistent observation about immunotoxin with regard to rhesus monkey renal allografts is that acute rejection is prevented, often indefinitely, by T-cell ablation using this agent starting on day 0. This result is highly reproducible, and given the difficulty of preventing acute rejection in this non-human primate outbred model, the result is startling when compared with other immunosuppressive strategies. Nevertheless, long-
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term follow-up has revealed 3 unexpected longterm consequences: interstitial nephritis, chronic rejection, and wasting disease. Unconjugated FN18 antibody (without toxin), when administered to monkeys receiving renal allografts, resulted in no long-term survivors, with the same peritransplantation deoxyspergualin and steroid immunosuppression that led to a majority of long survivors in the immunotoxin group (Judith Thomas, personal communication, February 1998).14 These results suggest that the immunotoxin construct lends a substantial advantage over antibody alone. Interstitial nephritis is generally associated with autoimmune injury of the native kidneys and has not been associated with renal transplantation. The exact cause and nature of the infiltrate in the autoimmune type disease are unclear as it is in the immunotoxin transplant model. Interestingly, many of our long-term surviving monkeys with excellent kidney function have nests of interstitial infiltrating cells scattered throughout the graft but without causing any tubular or glomerular injury. In addition, interstitial nephritis has been present in some monkeys with spontaneous resolution over time and without apparent graft injury. However, some monkeys develop such an intense interstitial nephritis that it causes a rise in serum creatinine level. As with autoimmune disease, this may be mediated by interstitial edema, causing compromise of capillary blood flow and resulting in ischemic injury. We have not tested whether this process responds to steroid therapy, which might be expected to reverse it. If interstitial nephritis did cause graft destruction in our series, it generally occurred between 50 and 135 days after transplantation. Most of these animals were treated with immunotoxin alone, and graft survival time was similar to that seen by Thomas et al,9 who used similar therapy. Later graft losses were usually attributable to chronic rejection. Chronic rejection of renal allografts was typified by hyalinization of glomeruli with reduplication of the glomerular capillary basement membrane, vasculopathy with neointimal hyperplasia, and by relative absence of graft infiltrating cells as has been reported in detail by Armstrong et al.15 Although most monkeys tested did develop alloantibody, there was not a perfect correlation between development of alloantibody and chronic rejection. In fact, one monkey with high levels of alloantibody capable of complement fixation continues to have excellent graft function without histologic evidence of graft injury. The etiology of chronic allograft rejection may involve both antibody-mediated injury and nonspecific mediators of inflam-
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mation such as cytokine-mediated injury. If tolerance strategies are to become attractive for clinical implementation, it would be highly desirable to prevent not only acute but also chronic rejection. To achieve this in the absence of daily drug therapy would be ideal. Although an initial 3-day course of steroids and MMF added to immunotoxin prevented graft loss in the first 100 days, 4 of the 5 monkeys eventually succumbed to chronic rejection. Better strategies, perhaps including a longer course of MMF, need to be developed to prevent chronic rejection in this model. The 3 monkeys that developed wasting disease in the setting of normal renal function and normal serum chemistries are of obvious concern, but to date they have defied precise explanation. Two possible explanations of their unremitting weight loss are an as yet undetermined viral infection occurring as a result of lymphocyte depletion, and cachexia resulting from a CD4 depletion. The latter has been described in a mouse model,16 and we are currently performing viral screening to rule out the former. The depletion and repopulation of T cells in PBLs after day 0 immunotoxin treatment were similar to that previously reported for day -7 immunotoxin-treated monkeys7,10 and when used with donor bone marrow infusion.9 In the 3 months after treatment with immunotoxin, CD4 cells repopulated at a slower rate than CD8 T cells, with a relative ratio of CD4/CD8 equal to 1:2. The altered peripheral T-lymphocyte ratio observed in this model parallels that reported in both bone marrow transplant patients17 and in patients treated with total lymphoid irradiation.18 Given that we have seen a nonspecific suppression of MLC and CTL responses during the first 6 months after immunotoxin treatment, we hypothesize that the skewed CD4/CD8 repopulation may be a significant factor favoring graft prolongation in our model. The rate of CD4 repopulation may also be age dependent.19 The precise nature of the repopulating T cells is the subject of ongoing investigation. The ability of rhesus monkeys to mount an antibody response is intact after immunotoxin treatment. With few exceptions, monkeys developed responses to tetanus, diphtheria antigen, xenoantibody, and alloantigen. Because isotype switching in B cells is generally T-cell dependent, this aspect of T-cell function does not appear to be impaired by immunotoxin treatment. In summary, immunotoxin appears to be a safe and reliable means of preventing acute allograft rejection when used as an induction agent. Further work is necessary to determine the mechanism by
which it may produce long-term tolerance, and new strategies must be developed to overcome the problems of interstitial nephritis within the first 4 months after transplantation and chronic rejection occurring even later. REFERENCES 1. Strober S, Dhillon M, Schubert M, Holm B, Engleman E, Benike C, et al. Acquired immune tolerance to cadaveric renal allografts: a study of t h r e e patients treated with total lymphoid irradiation. N Engl J Med 1989;321:28-33. 2. Mazariegos GV, Reyes J, Marino IR, Demetris AJ, Flynn B, Irish W, et al. Weaning of immunosuppression in liver transplant recipients. Transplantation 1997;63:243-9. 3. Burlingham WJ, Grailer AP, Fechner JH Jr, Kusaka S, Trucco M, Kocova M, et al. Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient. Transplantation 1995; 59:1147-55. 4. Thomas FT, Carver FM, Foil MB, Pryor WH, Larkin EW, Hall WR, et al. Long-term incompatible kidney survival in outbred higher primates without chronic immunosuppression. Ann Surg 1983;198:370-8. 5. Kawai T, Cosimi AB, Colvin RB, Powelson J, Eason J, Kozlowski T, et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 1995;59:256-62. 6. Kirk AD, Harlan DM, Armstrong NN, Davis TA, Dong Y, Gray GS, et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci U S A 1997;94:8789-94. 7. Knechtle SJ, Vargo D, Fechner J, Zhai Y, Wang J, Hanaway MJ, et al. FN18-CRM9 immunotoxin p r o m o t e s tolerance in primate renal allografts. Transplantation 1997;63:1-6. 8. Neville DM Jr, Scharff J, Srinivasachar K. In vivo T-cell ablation by holo-immunotoxin directed at human CD3. Proc Natl Acad Sci U S A 1992;89:2585-9. 9. Thomas JM, Neville DM, Contreras JL, Eckhoff DE, Meng G, Lobashevsky AL, et al. Preclinical studies of allograft tolerance in rhesus monkeys: a novel anti-CD3immunotoxin given peritransplant with donor bone marrow induces operational tolerance to kidney allografts. Transplantation 1997;64:124-35. 10. Fechner JH Jr, Vargo DJ, Geissler EK, Graeb C, Wang J, Hanaway MJ, et al. Split tolerance induced by immunotoxin in a rhesus monkey allograft model. Transplantation 1997;63:1339-45. 11. Knapp LA, Cadavid LF, Eberle ME, Knechtle SJ, Bontrop RE, Watkins DI. Identification of new mamu-DRB alleles using DGGE and direct sequencing. Immunogenetics 1997;45:171-9. 12. Neville DM, Scharff J, Hu HZ, Rigaut K, Shiloach J, Slingerland W, et al. A new reagent for the induction of T-cell depletion, anti-CD3-CRM9. J Immunother 1996;19:85-92. 13. Ohkawa S, Xu K, Wilson LA, Montelaro R, Martin LN, Murphey-Corb M. Analysis of envelope glycoproteinspecific antibodies from SIV-infected and gp110-immunized monkeys in ACC and ADCC assays. AIDS Res Hum Retroviruses 1995;11:395-403. 14. Eckhoff DE, Contreras JL, Wang PX, Lobashevsky AL, Meng G, Jiang Y, et al. Immunologic mechanisms favoring operational transplant tolerance induced by deoxyspergualin
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in rhesus monkeys given anti-CD3 immunotoxin. Transplantation 1998 In press. 15. Armstrong N, Buckley P, Oberley T, Fechner J Jr, Dong Y, Hong X, et al. Analysis of primate renal allografts following T cell depletion with anti-CD3-CRM9. Transplantation 1998 In press. 16. Morrissey PJ, Charrier K, Braddy S, Liggitt D, Watson JD. CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice: disease development is prevented by cotransfer of purified CD4+ T cells. J Exp Med 1993;178:237-44. 17. Leblond V, Othman TB, Blanc C, Theodorou I, Choquet S, Sutton L, et al. Expansion of CD4+CD7- T cells, a memory subset with preferential interleukin-4 production, after bone marrow transplantation. Transplantation 1997; 64:1453-9. 18. Halperin EC. Total lymphoid irradiation as an immunosuppressive agent for transplantation and the treatment of ‘autoimmune’ disease: a review. Clin Radiol 1985;36:125-30. 19. Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC, Feuerstein IM, et al. Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. N Engl J Med 1995;332:143-9.
DISCUSSION Dr Stephen T. Bartlett (Baltimore, Md). You have demonstrated that the G protein FN18 CRM9 markedly prolongs the survival of these MLC mismatched rhesus monkey allografts. One of the side effects was a wasting phenomenon combined with a prolonged CD4 depletion. Was there a relationship between the animals that had wasting and the prolonged CD4 depletion? Would you speculate on the pharmacologic mechanism of the immunotoxin? At least with such prolonged wasting, one would imagine that the immunotoxin would have been long gone. My second question relates to those cases in which the drug eventually proved ineffective and interstitial nephritis and chronic rejection developed. Did you look at the phenotype of the graft infiltrating cells? Dr Knechtle. With regard to the wasting, we have a small number of monkeys, 3 in this series, that had significant weight loss at variable times after treatment. The etiology of this wasting syndrome has eluded diagnosis to date. One possibility is that this is a virally mediated infection that we have not been able to diagnose. We have screened these monkeys for retroviruses and all are negative. The other possibility is that this may be related in some way to CD4 depletion. CD4 depletion and repopulation in mouse models have been associated with a wasting phenomenon. This is worrisome because it suggests that perhaps tolerance induction, at least with a depleting approach, may be associated with a systemic problem such as wasting. In terms of the mechanism of wasting, we do not have any evidence that it is related to the toxin component itself. The toxin itself is metabolized early and is gone within a few days. So the toxicity typically caused by DT is hepatotoxicity, and we are seeing this wasting syndrome months later. We are in the process of looking at the phenotype of the graft infil-
Surgery August 1998 trating cells using immunocytochemical techniques. Dr Bartlett. One of the reasons I wanted to follow up on the question of the graft infiltrating cells is that, in the slide you showed, there is a fair amount of interstitial nephritis. However, there is no particular direct cellular attack. It could be that there is cytokine release, particularly if some of those cells are B cells, that may occur with B-cell disregulation with prolonged CD4 and CD3 depression. This is not necessarily a posttransplantation lymphoproliferative disorder (PTLD) but a PTLD-like state. Dr. Knechtle. There is no evidence that these are tumor cells at all. They have a benign phenotype. They are a combination of CD4, CD8, and CD20 cells. We are using a large number of markers to look at them. We are also doing a polymerase chain reaction to evaluate cytokine expression within the grafts. So far, no clear patterns are emerging in control animals compared with experimental animals in terms of the cytokine pattern of tolerant monkeys. Dr Carl E. Haisch (Greenville, NC). I am concerned about what might be called a cytokine release syndrome. As I recall, your previous studies have had the native kidneys in place. Do you think that there is some relationship between the transplanted kidney and removing the native kidneys and the cytokine release syndrome? Also, do you think that there may be some relationship between interstitial cells in chronic rejection and cytokine release syndrome? Dr Knechtle. Although it is true that in the first group of experiments the recipient monkeys had their native kidneys in place when they received immunotoxin, in all of the experiments that I just showed you, given on day 0, there was no native kidney in place. Cytokine release syndrome has not been a clinical problem in our monkeys. We are not seeing hypertension, flushing, fever, or the pulmonary problems typically associated with cytokine release. We have not done measurements of peripheral blood cytokines. Judy Thomas, who also is using the same immunotoxin, has done that and tumor necrosis factor levels are elevated after administration of the immunotoxin as expected, but it has not been an overwhelming problem for her since initiating new protocols. Dr Mark H. Deierhoi (Birmingham, Ala). You describe rechallenging the animals with skin grafts 6 months after the transplantation. In the animals with chronic rejection, does their cellular tolerance or their tolerance to skin grafts continue through the period when the kidneys are failing? What is the mechanism for chronic rejection in this situation? The second question relates to the prolonged T-cell depletion. Presumably these are relatively juvenile animals with intact thymic function. What will be the effect of this sort of T-cell depletion in more mature animals or, for example, adult human beings, where thymic function may be much more depressed? Dr Knechtle. One of 13 monkeys has developed acute rejection after skin grafting. That skin graft was placed at 180 days, which is the earliest I have placed a skin graft. I believe that there is a metastable situation early on that
Surgery Volume 124, Number 2 becomes more robust tolerance later. If a skin graft, which is the most potent immunogen, is given too early, we will probably break tolerance. None of the other animals have had acute rejection induced by skin grafting. They have gone on to develop chronic rejection, but in some cases 300 days later. That has not differed from the animals who have not undergone skin grafting, so I do not have any evidence to suggest that the skin grafts cause chronic rejection. In terms of T-cell depletion, I would agree with your presumption that Tcell repopulation, in particularly CD4 cell repopulation, will be dependent on developmental age and thymic maturity. Data published by Mackall in the New England Journal of Medicine19 showed that CD4 repopulation was dependent on age. One would presume that the older the patient the less well CD4 cells will return. I don’t know whether that is clinically significant. Dr M. Wayne Flye (St. Louis, Mo). The depletion of pan-T cells is impressive. Do you think there is a different mechanism involved by using the toxin than is obtained with antilymphocyte serum or monoclonal antibodies? Specifically, did you look at thymic depletion and bone marrow and the repopulation there? Following up on the previous question, does this have a greater effect centrally on the thymic mechanism than distally in the periphery? Also, you can decrease the nonspecific response to tetanus and other antigens. Do you get specific depression that is facilitated by the graft being in place? Is that allospecific? When you looked at the anti-
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body formation, did you characterize it in terms of isotypes or even IgG, IgM? Could you be getting enhancing antibodies in some of these animals? That is a real possibility, particularly in the animal that had prolonged survival and a high titer. Dr Knechtle. We have tried to look at the repopulation of the thymus after immunotoxin therapy. We were unable to find thymic tissue at autopsy in these monkeys. There appears to be complete involution of the thymus. Unfortunately, that has also been true of control, untreated monkeys. The amount of thymic involution in these monkeys is substantial even in the absence of immunotoxin. So we have not really been able to determine thymic histology and what happens to the CD3 cells in the thymus. In terms of the antibody, IgG was measured, but unfortunately IgG subclass specificity cannot be determined in monkeys because reagents are not available to differentiate. There is the possibility, as you suggest, that the antibodies that we see are enhancing antibodies; however, we are not seeing suppression of allospecific antibodies. Dr Flye. Is there any degree of allospecificity in terms of your suppression after the graft is in place by in vitro, MLC, or CTL testing? Dr Knechtle. CTL is suppressed in an allospecific manner after skin grafting and in a nonspecific manner early on. In the first 200 days, CTSs are suppressed to donor and third-party. After skin grafting, the monkeys develop an excellent CLT response to the third-party but not to the donor. So the CTL suppressiveness takes time. Presumably the presence of the donor alloantigen is critical in inducing that.
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