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TRANSPLANTATION
From the Department of Pediatrics, University of South
Florida, All Children's Hospital, St Petersburg, FL; and the First
Department of Internal Medicine and First Department of Pathology,
Kansai Medical University, Osaka, Japan.
We examined whether mixed allogeneic transplantation with syngeneic
plus allogeneic peripheral blood stem cells (PBSCs) is sufficient to
interrupt autoimmune processes in BXSB mice and confer a potential
therapeutic option for the treatment of patients with autoimmune
diseases. Eight-week-old BXSB mice were lethally irradiated and
reconstituted with BALB/c (H-2d)+BXSB (H-2b)
PBSCs, in which the number of injected allogeneic progenitor cells was
5 times that of syngeneic progenitor cells. The survival of mixed PBSC
chimeras (BALB/c+BXSB Evidence is accumulating that a number of
autoimmune diseases are linked to abnormalities in the hematopoietic
stem cell (HSC) itself.1-5 Transplantation of bone marrow
cells (BMCs) from disease-resistant donors into autoimmune-prone and
autoimmune-expressing recipients has been shown to prevent and treat
autoimmunity. Conversely, BMCs from disease-prone donors have also been
shown to transfer and cause the relevant autoimmune disease process.
Bone marrow transplantation (BMT) has therefore been proposed as a
potential therapeutic strategy for autoimmune diseases, especially in
the presence of life-threatening diseases. Autologous transplantation is also being explored as a potential treatment for certain patients with life-threatening autoimmune diseases in a number of projects worldwide6-8 because the risk of morbidity and mortality
from this form of marrow transplantation may be low enough to permit use of large doses of chemotherapy as an approach to suppressing disease activity. However, the genetic susceptibility to the
development of autoimmune diseases continues even if the disease has
been put into remission. There have recently been reports of rapid recurrence or persistence of autoimmune diseases after autologous BMT.9 Although, from our many studies, allogeneic BMT
seems to be the most rational procedure for the treatment of autoimmune diseases, and although some patients with an autoimmune disease have
been reported to have long-term remissions (longer than 10 years) after
allogeneic BMT,10 the use of allogeneic BMT in nonmalignant disorders must be very carefully considered because allogeneic BMT is a procedure known to be quite toxic and
potentially lethal.
Murphy and Roths11 established the BXSB mouse strain
in 1976 after mating a C57BL/6J female with an SB/Le male. BXSB mice spontaneously develop a progressive and lethal autoimmune disease that
can be regarded as an experimental model for human systemic lupus
erythematosus.12 These mice manifest clinical and
immunological abnormalities, including moderate lymph node enlargement,
splenomegaly, impaired T-cell functions, B-cell hyperactivity reflected
in hypergammaglobulinemia and spontaneous polyclonal antibody
production, and the production of a variety of autoantibodies. This
disease regularly progresses to immune-complex deposition in the
kidney.13-15 These manifestations of the autoimmune
phenomena and renal disease are remarkably accelerated in male BXSB
mice, which exhibit a 50% mortality by 5 months of age as compared
with 50% mortality by 15 months in female BXSB mice. This disease can
be attributed to the influence of an unmapped single gene, designated
Y-chromosome-linked autoimmune accelerator (Yaa) gene.16
This gene is located on the Y chromosome. It has been shown that the
pace of BXSB disease is determined entirely by the donor stem cells but
not at all by the environment in which these cells
develop.17
In prior investigations, we have shown that mixed allogeneic BM cell
transplantation using BMCs from both allogeneic and syngeneic mice not only prevents and treats autoimmune diseases in BXSB mice but also establishes sufficient immunologic reconstitution, which
cannot be regularly observed in fully allogeneic
chimeras.18,19 This superior immunological function of the
mixed chimeras has likewise been observed after BMT between normal mice
without autoimmunities.20,21
The present investigation was designed to determine whether we might be
able to achieve a safe and reproducible prevention of autoimmune
diseases in BXSB mice using mixed peripheral blood stem cell
transplantation (PBSCT) instead of BMT. Very recently, we have shown
that one can use T-cell-depleted cells of an autoimmune- and renal
disease-free donor strain to produce, with PBSCs of the recipient, a
stable mixed chimerism and, thus, either prevent or cure the autoimmune
disease. Allogeneic PBSCT has also been carried out because the donor
could be spared the discomfort and risks of general anesthesia and the
recipient might also have shorter duration of posttransplantation
aplasia as in the case with autologous or syngeneic PBSCT. The merit of
allogeneic PBSCT is that it is likely to enhance donor
acceptance and lead to a high level of engraftment and acceptable
conditioning-related toxicity.22
In this paper, we describe our efforts to reconstitute irradiated BXSB
mice with PBSCs mobilized from BALB/c mice plus PBSCs from syngeneic
BXSB mice to produce a stable mixed chimerism as an approach to
preventing this severe autoimmune disease. We demonstrate here
that BXSB recipients of mixed PBSCs from BALB/c and BXSB mice have
significantly prolonged survival and reduced severity of lupus and
lupus nephritis. These mixed PBSC chimeras also produce the tolerance
of both donor and host major histocompatibility complex (MHC)
determinants, and establish a substantial population of both T and B
lymphocytes that cooperate effectively in antibody production. Since
increasing the number of BXSB PBSCs resulted in the transfer of
diseases to nonautoimmune recipients, it was necessary to increase the
normal allogeneic PBSCs by a factor of 5 to 1 to prevent a relapse of
the autoimmune diseases. Therefore, if we can clarify a minimum dose of
syngeneic PBSCs needed to produce these beneficial effects, this
approach might serve as a useful treatment for patients with
autoimmune diseases.
Mice
Monoclonal antibodies (mAbs)
Hematopoietic growth factors Recombinant murine interleukin-3 (mIL-3) and recombinant human erythropoietin (Epo) were used for colony-forming assay. Recombinant human granulocyte colony-stimulating factor (G-CSF) was kindly provided by Chugai Pharmaceutical (Tokyo, Japan) and mIL-3 and Epo were kindly provided by Kirin Brewery Co (Tokyo, Japan).Preparation of PBSCs We carried out splenectomy of donor mice (male BXSB, BALB/c, or B6 mice) at 5 weeks of age. The reason for this was to try to increase mobilization of PBSCs into circulation following treatment to foster mobilization of stem cells. At 7 weeks of age, splenectomized donor mice were intraperitoneally injected with cytosine-arabinoside (Ara-C) (Sigma Chemical, St Louis, MO) (200 mg/kg) twice with a 6-hour interval (10:00 AM and 4:00 PM) on day 0, followed by subcutaneous injection of 250 µg/kg/d of G-CSF on days 3 to 6. G-CSF was diluted in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) before subcutaneous injection. Injections were given at 9:00 AM and 5:00 PM. Since the use of cytotoxic chemotherapy followed by growth factor can enhance yields of progenitor cells into the peripheral blood, we administered these drugs to donor mice in this study. Peripheral blood was harvested from the orbital plexus under anesthesia. Approximately 1 mL blood was collected per mouse and centrifuged over Accuprep (density 1.077) (Accurate Chemical & Scientific, Westbury, NY) to remove erythrocytes and neutrophils. All nucleated cells from the interface were collected and washed once with PBS containing 2% fetal calf serum (FCS) (2% FCS-PBS). T cells were removed magnetically by treating the mobilized peripheral blood cells from donor mice with anti-Thy-1.2 monoclonal antibody (PharMingen) plus immunomagnetic beads (Dynabeads) (Dynal, Oslo, Norway) at a bead-to-T-cell ratio of 5:1. This resulted in a profound decrease in Thy-1.2+ T cells (fewer than 0.6%). We used these low-density and T-cell-depleted peripheral blood cells as PBSCs for the purposes of the present investigation.Mixed allogeneic PBSCT At 8 weeks of age, male BXSB and BALB/c recipient mice were lethally irradiated by means of a 137Cs source (0.70 Gy/min) with 9.5 Gy, and were injected intravenously with the PBSCs 1 day after irradiation. Different syngeneic and/or allogeneic donor PBSCs were used, as shown in Table 1. Since there was a greater difference in the activities of mobilized PBSCs among individual strains, transplantation was performed according to the number of hematopoietic progenitor cells with c-kit+Lin marker. We injected syngeneic
(1.9-4.6 × 106 cells) and/or allogeneic
(9.5-23.1 × 106 cells) PBSCs into recipients so that the
ratio of syngeneic to allogeneic c-kit+Lin
cells was 1:5. This ratio produced an average of 50% of chimerism after transplantation with the use of normal mice (see "Results"). These PBSCs contained c-kit+Lin cell counts
equivalent to 2 to 4 × 106 or 1 to
2 × 107 normal BMCs, respectively, these counts being
capable of reconstituting syngeneic or allogeneic recipients. At
various fixed time points, individual mice were characterized for
engraftment with syngeneic or allogeneic donor cells by means of flow
cytometry. The animals were also followed for clinical evidence of
renal disease, such as proteinuria.
Staining and cell surface analysis After depletion of red blood cells by an ammonium chloride-potassium buffer, cells (106) from the peripheral blood were stained with an optimal concentration of each mAb for 30 minutes on ice in 25 µL staining buffer (2% FCS-PBS and 0.1% NaN3), and were analyzed by means of an EPICS Elite flow cytometer (Beckman Coulter, Fullerton, CA). Unstained cells or cells stained with an isotype-matched mAb served as negative controls.Assessment of GVHD All chimeras were evaluated for evidence of graft-versus-host disease (GVHD) on a daily basis for the first month following reconstitution and weekly thereafter. The diagnosis of GVHD was based on the previously described manifestations of ruffled hair, hunchback, diarrhea, or weight loss. GVHD was diagnosed by the histologic analysis of the skin, liver, and intestine when we killed the chimeric mice for assays, usually 48 weeks after transplantation. Tissues were placed in 10% neutral buffered formalin and imbedded in paraffin, and 6-µm-thick sections were stained with hematoxylin and eosin (H&E).Pathological examination After the termination of the experiment, the following pathological examinations were regularly performed. Kidneys were excised and placed in 10% formalin and embedded in paraffin. Histological sections were stained either with the periodic acid-Schiff (PAS) reagent or with H&E. Glomerulonephritis was scored on a 0-4 scale based on the severity and extent of histopathological changes.23 A grade of 0 was given to kidneys that did not exhibit glomerular lesions; grade 1, to kidneys with minimal thickening of the mesangium; grade 2, to kidneys with noticeable increases in both mesangial and glomerular cellularity; grade 3, to kidneys with the preceding conditions plus superimposed inflammatory exudates and capsular adhesions; and grade 4, to kidneys with obliterated glomerular architecture in more than 70% of the glomeruli. Twenty glomeruli within one area were graded according to this classification, and the figures obtained were used to calculate the mean glomerular histopathological score for each mouse.Assay of anti-double-stranded DNA (anti-dsDNA) autoantibodies Serum antibodies specific for dsDNA were determined by using an enzyme-linked immunosorbent assay (ELISA). Immunoplates (Nunc, Roskilde, Denmark) were coated with heat-denatured calf thymus DNA (Sigma) at 20 µg/mL and postcoated with 1% BSA. Diluted sera (1:100) were added to wells and incubated for 1 hour at room temperature. After being washed in PBS-Tween, the plate was incubated for 1 hour with alkaline phosphatase-labeled antibody specific for mouse immunoglobulin G (Capple Laboratories, Malvern, PA). After the addition of the substrate, optimal density (OD) was determined at an absorbance at 405 nm with an automated spectrophotometer, and antibody activity was expressed as the mean OD of triplicate determinations.Assays for immunological functions Immunological functions of chimeric mice were determined by the following assays: (1) plaque-forming cell (PFC) responses to spleen red blood cells (SRBCs) (anti-SRBC PFC) assay and (2) mixed lymphocyte reaction (MLR). Spleen cells from BXSB or BALB/c control mice as well as various chimeric mice were used in all these assays when the mice were 48 weeks old. For anti-SRBC PFC assay, each treated or control mouse was injected intraperitoneally with 0.5 mL 2% SRBC. At 5 days later, the injected mice were killed, and a single-cell suspension of spleen cells was adjusted to a concentration of 5 × 106 cells per milliliter. A mixture of 500 µL 0.5% agarose (Sigma) solution, 100 µL spleen cell suspension, and 50 µL 1% SRBC suspension was poured onto a microslide coated with poly-L-lysine (Sigma). Dried slides were carefully inverted in a tray and incubated with guinea pig complement (Cappel, West Chester, PA). After these were incubated at 37°C for 3 hours, hemolytic plaques were counted and expressed as the number of plaques per 5 × 105 cells. MLR was carried out as follows: triplicate cultures were set up in a well containing 3 × 105 responder cells and 5 × 105 irradiated (15 Gy) stimulator cells in a total of 200 µL 5% FCS-RPMI. At 4 days later, the cells were pulsed with [3H]thymidine and incubated for another 16 hours.Statistics The statistical significance between groups was determined by one-way analysis of variance by means of the Student t test and was assumed for P < .05. Survival data were analyzed by the method of Kaplan-Meier, and the resulting survival curves were compared by means of the log-rank test.
Generation of mixed PBSC chimeras Low-density and T-cell-depleted peripheral blood cells were collected from splenectomized donor mice on day 7 of treatment with Ara-C plus G-CSF. Splenectomy was carried out to avoid pooling of PBSCs in the spleen during the mobilizing treatment. These mobilized PBSCs showed substantially high colony-forming unit (CFU) activities (Table 2). However, in contrast to normal BMCs from various strains, the concentrations of these progenitor cells, which were the decisive parameter to predict the engraftment potential of HSCs, were different among individual strains, probably owing to the difference in responses to the stem cell mobilizing treatment. To permit comparison among groups in an experiment, transplantation was performed according to the number of hematopoietic progenitor cells (c-kit+Lin cells). We injected syngeneic
(1.9-4.6 × 106 cells) and/or allogeneic
(9.5-23.1 × 106 cells) PBSCs into recipients so that the
ratio of syngeneic to allogeneic c-kit+Lin
cells was 1:5. These PBSCs contained
c-kit+Lin cell counts equivalent to 2 to
4 × 106 or 1 to 2 × 107 normal BMCs,
respectively, which are capable of reconstituting lethally irradiated
syngeneic or allogeneic recipients.
Our preliminary experiment using PBSCs of normal mice
BALB/c+B6 Survival rate after mixed PBSCT The survival of BXSB recipients reconstituted with a mixture of syngeneic and allogeneic PBSCs (BALB/c+BXSB BXSB) was excellent; they
achieved an 80% survival rate at 48 weeks of age (Figure 1). There was no statistically
significant difference in the survival from fully allogeneic chimeras
BALB/cBXSB which exhibited a 90% survival rate at 48 weeks of age.
Both types of chimeric mice appeared healthy and exhibited no evidence
of GVHD. In contrast, untreated BXSB mice or BXSB mice receiving
syngeneic PBSCs (BXSBBXSB) began to die from 17 or 24 weeks of age,
respectively, and then death became more and more frequent. Moreover,
B6+BXSBBXSB mice, both from donors with the same H-2
phenotype (H-2b), deteriorated and showed a poor survival
rate (35% at 48 weeks of age). Autopsies showed that all such deaths
of these mice were clearly attributable to lethal lupus
nephritis.
Flow cytometry analysis of chimerism H-2 typing revealed that the PB cells in BALB/cBXSB mice
were almost all of donor H-2d origin (at least 90%) from 5 to 40 weeks after transplantation (Table
3). Although the level of chimerism
varied from 2% to 80% for individual BALB/c+BXSBBXSB mice,
the average level of allogeneic chimerism was 46.7% at 40 weeks after
transplantation. Typing profiles showed that the proportion of
syngeneic versus allogeneic mixed chimerism was stable and fluctuated
little over time for individual animals. BXSB-derived cells did not
increase over time. Myelomonocytic cells (Gr-1+ and
Mac-1+), B cells (B220+), and T cells
(Thy1.2+) were also found to be derived from both the
syngeneic and allogeneic donors even 40 weeks after transplantation
(Figure 2). In 3 of 20 mice
BALB/c+BXSBBXSB, however, the percentage of BALB/c-derived cells was
fewer than 10% of the PB cells. These mice gradually developed
autoimmune disease. Similarly, of 20 mice BXSB+BALB/cBALB/c that
also showed various levels of allogeneic chimerism, 1 mouse did not
exhibit engraftment of BALB/c cells and 2 mice showed only
BALB/c-derived cells in the peripheral blood. These different levels of
engraftment were not seen in fully allogeneic chimeras such as
BALB/cBXSB or BXSBBALB/c mice. On the other hand, relatively invariable mixed chimerism was seen in B6+BXSBBXSB mice when the PB
cells were typed by Ly-5 mAb; the percentage of Ly-5.1 (phenotype of B6 mice) of peripheral blood cells was 39.8% ± 11.2% (mean ± SD) at 40 weeks after transplantation.
Histopathological findings of kidney The major cause of death in male BXSB mice is an exudative and proliferative glomerulonephritis. Animals were observed for 46 weeks following PBSCT for glomerulonephritis. Striking proteinuria was observed at 20 weeks of age in almost all untreated control male BXSB mice and BXSBBXSB mice, whereas only mild proteinuria was detected in fully allogeneic chimeras BALB/cBXSB or in mixed allogeneic chimeras (BALB/c+BXSBBXSB) during this period.
Histologic examination revealed that the glomeruli exhibited typical
advanced lesions, including wire-loop lesions and fibrinoid deposits
that were demonstrated with H&E or PAS staining in the kidneys of
BXSBBXSB mice (older than 20 weeks of age) as observed in male BXSB
mice (Figure 3). Conversely, glomerular
lesions of BALB/c+BXSBBXSB mice as well as BALB/cBXSB mice were
significantly less severe compared with those of BXSBBXSB mice. No
significant difference was seen in the degree of glomerulonephritis
between BALB/c+BXSBBXSB mice and BALB/cBXSB mice (Table
4).
Anti-dsDNA autoantibodies Anti-dsDNA antibodies in the sera were also examined when recipient mice were 48 weeks of age. As shown in Table 5, BALB/c+BXSBBXSB mice showed lower
levels of anti-dsDNA than untreated mice receiving BXSB and BXSBBXSB
syngeneic stem cell transplants, although the level was not as low as
that of BALB/cBXSB mice.
Immunologic reconstitution Prominent splenomegaly and increased spleen cell numbers were observed in untreated BXSB mice and BXSBBXSB mice (Table
6). Such mice also showed moderate
lymphadenopathy. The number of spleen cells in BALB/c+BXSBBXSB mice
decreased to near normal cellularity levels, although it was somewhat
higher than in BALB/cBXSB mice.
In an attempt to assess the in vivo immunocooperation of the mixed
allogeneic chimeras BALB/c+BXSB MLR of recipient mice was also assessed as an in vitro
measure of tolerance and immunocompetence. Spleen cells from
BALB/c+BXSB
Transfer of autoimmune disease We assessed whether PBSCs from autoimmune mice could transfer the disease to normal autoimmune-resistant allogeneic mice (Table 1). On flow cytometric analyses of the peripheral blood cells, syngeneic and/or allogeneic PBSCs were engrafted in recipients during the time period 5 to 40 weeks after transplantation. BXSBBALB/c and
BXSB+BALB/cBALB/c mice exhibited 95.1% ± 2.7% and
45.1% ± 25.9% (mean ± SD) allogeneic chimerism at 48 weeks
of age, respectively. The survival rates of BXSBBALB/c and
BXSB+BALB/cBALB/c mice were relatively high: 70% and 85%,
respectively, at 48 weeks of age. However, various levels of
proteinuria were detected in mice in these groups after 17 weeks of age
(9 weeks after transplantation). Mild to moderate glomerulonephritis
was also detected histologically (Figure 3), and serum levels of
anti-dsDNA antibodies increased (Table 5) in these chimeric mice at 48 weeks of age. Notably, the development of systemic lupus erythematosus
(SLE) in BXSB+BALB/cBALB/c mice was not different from that
in BXSBBALB/c mice (Tables 4 and 5). These findings showed that
PBSCs from BXSB mice could be sufficient to cause the expression of
autoimmune disease if recipients have been injected with larger numbers
of BXSB PBSCs than of BALB/c PBSCs, even when mixed
transplantation is carried out.
In recent years, immunosuppressive therapy for systemic autoimmune disease has, to some extent, improved survival and lowered morbidity. However, severe autoimmune disease can still be lethal, and both short- and long-term effects of current immunosuppressive therapy may be life threatening. Therefore, there is definitely room for improvement of current treatments, especially in the face of severe systemic autoimmune disease. Numerous murine studies have shown that allogeneic and, to a lesser extent, syngeneic and autologous transplantation of marrow after TBI, may control disease activity and are curative in many cases. There is no doubt that allogeneic stem cell transplantation is potentially a most rational treatment for autoimmune diseases, since it combines the administration of new, healthy HSCs with complete immune ablation. However, autoimmune diseases, regardless of severity, are nonmalignant disorders, and their prognoses are continuously improving because of early diagnosis and skillful management. Transplantation-related mortality and other late effects make such procedures still inadvisable in many patients, even though transplantation approaches appear to be more biologically appropriate than other approaches to treatment.24,25 In previous studies, we have shown that mixed transplantation using T-cell-depleted BM cells from both allogeneic and syngeneic autoimmune mice could effectively prevent and treat autoimmune diseases in BXSB mice as well as restore immune functions fully in these stable mixed chimeras.18,19 The present investigation is intended to develop this strategy of mixed transplantation further by using mixed PBCST. Recently, allogeneic PBSCT for clinical application has been expected to become a valuable strategy for the treatment of a number of diseases. Many theoretical and practical questions regarding the allogeneic PBSCT, however, remain unanswered.26-28 For example, what is the minimum dose of allograft cells? What, if any, are the risks of GVHD due to the vast amount of T cells present in the peripheral blood? Do the stem cells provide substantial graft-versus-leukemia activity? Nonetheless, owing to easy harvesting of PBSCs and the rapid hematopoietic recovery that occurs following transplantation, we presume that PBSCs provide a feasible and safe allogeneic transplantation for the treatment of patients with autoimmune diseases. In this study, our approach to the characterization of mixed chimeras
engrafted with PBSCs was not only to demonstrate the hematopoietic
potential of PBSCs to effectively function even in an allogeneic
situation, but also to assess the benefits and risks of this
alternative therapeutic strategy for clinical autoimmune disease. For
this purpose, we injected Ara-C and G-CSF into donors (male BXSB,
BALB/c, or B6 mice), resulting in remarkable increases in the number of
cells with CFU-C and CFU-S activities in the peripheral blood in
autoimmune-prone as well as autoimmune-resistant mice. We
demonstrated that the mixed PBSCs from BALB/c and BXSB mice could
successfully reconstitute most of the lethally irradiated BXSB mice, as
shown by H-2 typing of the peripheral blood cells. No evidence was
obtained for a gradual increase in the number of BXSB-derived cells in
the peripheral blood. These mixed PBSC chimeras (BALB/c+BXSB One major advantage of such mixed reconstitution would be the ability
to overcome the immunoincompetence of such fully allogeneic chimeras
while inducing and maintaining specific tolerance to the donor.
Lymphocytes from fully allogeneic chimeras are competent, but are
apparently restricted to their interactions with accessory cells that
express host, but not donor, MHC determinants.32 Numerous
studies have demonstrated that the transplantation of T-cell-depleted BMCs can lead to long-term survival of fully
allogeneic chimeras in specific pathogen-free facilities. However, the
survival of such animals is generally not as good as that of syngeneic chimeras owing to the presence of various degrees of
immunocompetence. Evidence has been reported for the
immunocompetence of such fully allogeneic chimeras in mice: their
survival in a conventional animal facility is inferior to that of mixed
chimeras, owing to endemic viral infections.20
Because reconstituted helper T cells collaborate only with B cells that
express host MHC determinants that are also reconstituted from
syngeneic PBSCs, the mixed allogeneically reconstituted mice
BALB/c+BXSB In our study, graft failure rarely occurred in spite of the use of T-cell-depleted PBSCs. This should be attributed to lethal irradiation and the use of high-dose HSCs, since HSCs induce anergy to CD8+ T cells of recipients34 and HSCs have natural suppressor activity.35 Non-stem cell components in the donor PBSCs may facilitate stem cell engraftment in allogeneic recipients. Studies are in progress to characterize facilitating cells of PBSCs, since our PBSCs contained not only HSCs but also progenitor cells and mature cells (natural killer [NK] cells, B cells, etc), as previously described.36 Despite reconstitution of the host with PBSCs containing 5 times more
allogeneic progenitor cells (c-kit+Lin Furthermore, as shown in our experiment of transfer of diseases, the increases in the number of BXSB PBSCs were sufficient to transfer lupus nephritis into BALB/c mice in spite of the simultaneous injection of PBSCs from BALB/c mice. It is most likely that the proliferation of BXSB PBSCs overcomes the effect of graft versus autoimmunity from BALB/c PBSCs, although these mice showed various levels of mixed chimerism. Thus, it is necessary to increase the number of normal allogeneic PBSCs to prevent a relapse of the autoimmune diseases in mixed transplantation. To obtain consistently good chimerism and prevent the development of autoimmune diseases, more than 10 times the number of normal allogeneic PBSCs as those of the autoimmune-prone mice might be required. It is unclear if there is a threshold dose of syngeneic PBSCs acceptable for reinfusion to autoimmune recipients. This disadvantage of the transfer of diseases must not surpass the superior immune reconstitution and the possible other advantages, such as fast engraftment of the mixed PBSC transplant. Therefore, further investigation is required not only for qualitative identification of PBSCs such as concomitant T cells in the mixed inoculum, but also for the quantitative relation of syngeneic to allogeneic PBSCs from the view of engraftment, GVHD, and graft versus autoimmunity in the mixed PBSCT for autoimmune diseases. If syngeneic plus allogeneic PBSC grafts can best be manipulated for a maximal effect of treatment without recurrence of autoimmune disease, the experiments reported here have encouraged us to continue with the clinical evaluation of mixed PBSCT.
We thank Ms Tazim Verjee for preparation of this manuscript and editorial assistance.
Submitted October 19, 2001; accepted April 5, 2002.
Supported by the US Public Health Service-National Institutes of Health, Institute on Aging grant no. 2R01 AG05628-14; the Suncoast Cardiovascular Research and Education Foundation; American Heart Association, Florida affiliate, grant no. AHA 9603017; and Pediatric Cancer Foundation to Children's Research Institute, All Children's Hospital, St Petersburg, FL.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yoshihisa Yamamoto, Kishiwada City Hospital, 1001 Gakuhara-cho, Kishiwada, Osaka 596-8501, Japan; e-mail: yama01{at}sc5.so-net.ne.jp; or Robert A. Good, University of South Florida, All Children's Hospital, 801 Sixth St S, St Petersburg, FL 33701; e-mail: goodr{at}allkids.org.
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© 2002 by The American Society of Hematology.
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  O. Jones, A Lacson, X Zeng, J. Jones, K Katti, R. Cahill, and A. Ahmed Long-term follow-up after non-myeloablative transplant of bone and marrow in BXSB mice Lupus, August 1, 2009; 18(9): 813 - 821. [Abstract] [PDF]
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Abstract
Introduction
, P < .05 compared with other
groups. * versus 
