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HEMATOPOIESIS
From the Klinik für Viszeral- und
Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover,
Germany.
Anti-CD45 monoclonal antibodies (mAbs) are potentially
powerful tools for the depletion of mature leukocytes. As their
application for immunotherapy also depends on their effects on
bone marrow (BM) progeny, the in vivo effects of an anti-CD45 mAb
(anti-RT7a mAb) on BM precursor cells were analyzed in a
rat model. Anti-RT7a mAb treatment was performed in LEW.1W
(RT1u RT7a) rats with the use of different
dosages. In addition, major histocompatibility complex
(MHC)-congenic BM transplantation making use of a diallelic polymorphism (RT7a/RT7b) of rat CD45 was
applied. Following injection of anti-RT7a mAb into normal
LEW.1W rats, T cells were profoundly depleted in blood, lymph nodes,
and spleen, whereas B cells were coated only by the antibody. Single
injection of anti-RT7a mAb in a high dose induced a lethal
aplastic syndrome with severe thrombocytopenia. Rescue of
antibody-treated animals with BM from congenic LEW.1W-7B rats
(RT1u RT7b) and transplantation of BM from
LEW.1W rats pretreated with anti-RT7a mAb into sublethally
irradiated LEW.1W-7B recipients revealed a profound effect of
the mAb on progeny of myeloid and T-cell lineage. Following repeated
antibody treatment of stable mixed chimeras
(RT7b/RT7a), very few RT7a-positive
B cells were still detectable after 6 months and their number declined
during the subsequent year. These observations show that this
anti-RT7a mAb effectively depletes mature T cells as well
as BM precursor cells of myeloid, T-cell, and thrombocytic lineage
after in vivo application. In contrast, mature B cells are not
depleted, but precursors also appear to be eliminated. Overall, the
findings suggest that the anti-RT7a mAb efficiently
depletes early rat hematopoietic stem cells.
(Blood. 2002;99:3566-3572) Monoclonal antibodies (mAbs) can be highly
efficient tools for the systemic manipulation of specific cell
populations. Antibodies detecting a broad spectrum of hematopoietic
lineage cells represent an attractive approach in the therapy of
hematopoietic malignancies as well as for tolerance-induction strategies.
CD45, the leukocyte common antigen (LCA) (RT7 in the rat), is a
transmembrane tyrosine phosphatase1 expressed on nearly all hematopoietic lineage cells. At least 8 different isoforms of the
CD45 antigen are generated by alternative splicing, mainly of exons 4 through 6, of the CD45 gene product. Antibodies against epitopes on
several of these isoforms can target subpopulations of mononuclear
cells (isoform-specific antibodies). Some of these isoform-specific
antibodies have already been shown to be effective in transplantation
tolerance induction.2,3
In this study, a rat anti-rat CD45 (RT7) mAb that binds to all
leukocytes was examined for its in vivo effects on bone marrow (BM) cells as well as mature leukocytes. In the rat, 2 allomorphic forms of the RT7 antigen (RT7a and
RT7b) exist. In animals expressing the RT7a
allotype, the antibody (anti-RT7a mAb) binds to all
CD45+ cells. Initially, the RT7 system (previously
designated ART-1 or Ly-1) had been thought to represent a T
cell-specific antigen because rather selective in vitro lysis of T
cells by alloantisera against RT7 had been
observed.4,5 Subsequently, however, the RT7
antigen was revealed to be a common hematopoietic marker of the LCA
family (CD45).6,7
In previous experiments, the anti-RT7a mAb used in this
study was shown to cause massive T-cell depletion in vivo and to have an ability to induce tolerance to fully major histocompatibility complex (MHC)-mismatched grafts.8,9 It could also be
observed that the anti-RT7a mAb led to BM aplasia when
administered in combination with passenger leukocytes.10
It was the aim of this study to elucidate in detail the effect of the
anti-RT7a monoclonal antibody on BM precursor cells as well
as on mature leukocytes. Using congenic rat strains differing
in the RT7 allotype, we used MHC-identical BM transplantation as a tool
to evaluate the long-term effects of antibody-treatment on BM precursor
cells, including rat hematopoietic stem cells.
Animals
Anti-RT7a monoclonal antibody
Culture supernatants of the anti-RT7a-producing hybridoma were pooled and purified by affinity chromatography with the use of Protein-G 4 Fast Flow (Pharmacia, Erlangen, Germany) and dissolved in phosphate-buffered saline (PBS) (approximately 1.5 mg antibody protein per milliliter) for intravenous injection (tail vein or penis vein). The mAb concentration of each antibody charge was measured by a fluorescent-activated cell sorting (FACS) titration method. Mean channels of fluorescence of at least 10 different dilutions were compared with a standard curve originating from the standard pool of antibody. BM transplantation In the first group (rescue group), LEW.1W animals were treated with a lethal dose of anti-RT7a mAb. On day 2, they received al least 100 × 106 unmodified BM cells of native LEW.1W-7B rats. Peripheral blood was examined regularly for chimeric state by FACS. Lymphoid organs of animals killed on days 100 and 209 were analyzed by a 3-color FACS stain.In the second group (transfer group), LEW.1W-7B rats were treated with
7 Gy In the third group (elimination group), stable chimeras were generated
by lethally irradiating LEW.1A hosts by 11 Gy Donor animals were killed by CO2 or diethylether overdose. All long bones were flushed with TC 199 solution at 4°C. The cell suspension was centrifuged for 10 minutes at 990 rpm and brought to appropriate volumes before injection. No T-cell depletion or erythrocyte lysis was performed. Flow cytometric analysis Two- and 3-color antibody staining was carried out by standard protocols. Single cell suspensions of all examined organs (peripheral blood, spleen, thymus, lymph nodes, and BM) were produced by cutting the parenchyma into small pieces and pushing it through steel meshes. In addition, erythrocytes were lysed by NH4Cl. Immunostaining was performed at 4°C on ice with optimal staining concentrations of mAbs.The following antibodies were used: T cells/T-cell receptor
All samples were analyzed on a FACStar flow cytometer (Becton Dickinson, San Jose, CA). The cell counts (erythrocytes, leukocytes) were performed by standard procedures. Absolute cell numbers for specific subpopulations were calculated on the basis of total leukocyte FACS (open gate) and absolute cell counts. Immunohistochemistry Briefly, tissue samples were snap-frozen in liquid nitrogen and 5- to 7-µm kryostat sections were obtained. Samples were dried overnight and fixed in aceton at room temperature for 10 minutes. Normal goat serum in 5% PBS was used for blocking before slides were incubated with 50 µL of their respective mAbs (see above) at optimal staining concentrations. After being washed 3 times with PBS, slides were incubated with goat antimouse immunoglobulins coupled to horseradish peroxidase (Dianova, Hamburg, Germany) and 5% heat-inactivated normal rat serum for 60 minutes. Coloring of antibody-bound cells was achieved by 3-amino-9-ethyl-carbazole (Sigma, Deisenhofen, Germany) as a substrate.
In vitro staining patterns of the anti-RT7a mAb compared with OX1 mAb In vitro, all subtypes of peripheral blood mononuclear cells of LEW.1W animals were detected by both anti-RT7a mAb and OX1 mAb.11 For the anti-RT7a mAb, one could observe that binding was slightly more intense to T cells than to B cells, whereas OX1 mAb bound to T cells and B cells (labeled by OX33/CD45RA) with similar intensity. Staining of NK cells was stronger than staining of T cells with OX1, and about equal to that of anti-RT7a mAb. Staining intensity of granulocytes was lower than staining intensity of T cells with both of the mAbs (Figure 1).
LEW.1W mononuclear cells could also be labeled with both mAbs
simultaneously, irrespective of the order of staining ("correlated expression") (Figure 2). OX22 (CD45RC)
and OX33 (CD45RA) mAbs, which detect epitopes present on selective
isoforms of CD45 (isoform-specific mAbs) also showed correlated
expression on subpopulations of RT7a and OX1-positive
cells.
Anti-RT7a mAb and OX1 are anti-CD45 mAbs binding to 2 different epitopes of the constant region of the CD45 molecule. Although basically all mononuclear cells are labeled by both mAbs, staining intensities for specific subpopulations of blood mononuclear cells are somewhat distinct for each of the mAbs (Figure 1). In vivo depleting capacity of the
anti-RT7a mAb in
LEW.1W rats, complement-deficient
PVG.C6
In contrast to T cells, B cells were not depleted by the anti-RT7a mAb although they were intensively coated with antibody (not shown). In blood, spleen, and lymph nodes, a relative increase of B-cell numbers could be observed (Figure 3A-C). Granulocytes were strongly reduced in number at 1 day after antibody application. Until day 14, their numbers were relatively increased in blood and returned to normal by days 20 to 40. Depletion of NK cells was also a short-term effect. Their number was reduced to below 1% on day 1, but returned to normal by day 3 (not shown). Immunohistological staining of lymph nodes and spleen showed profound atrophy of the lymphatic tissues on day 3 with predominant depletion of T cells. B-cell counts (per field) were not reduced as compared with normal tissue (not shown). Therefore, the relative increase in B cells in the FACS analysis was considered to be due mainly to the lack of T cells. The most striking effect of anti-RT7a mAb therapy on cells in peripheral lymphoid organs was the profound and prolonged depletion of T cells, while mature B cells were not depleted. To elucidate the mechanism of cell death by anti-RT7a mAb
therapy, complement-deficient PVG.C6 Both native PVG and PVG.C6 In another set of experiments, splenectomized LEW.1W rats were treated with a standardized dose of anti-RT7a mAb one day after the operation. Results of FACS analysis were compared with those of normal LEW.1W rats. No significant differences between the 2 groups in the cell numbers of T and B cells could be observed. T cells were strongly depleted, whereas B cells were not affected. In conclusion, neither a properly functioning membrane attack complex nor the spleen as an organ for the degradation of antibody-coated cells is a necessary prerequisite for the depleting ability of the anti-RT7a mAb. In vivo titration of anti-RT7a mAb effects To assess the dose-to-effect ratio of the anti-RT7a mAb, in vivo LEW.1W rats were treated with different single doses of the mAb (1.25 mL/0.75 mL/0.4 mL/0.25 mL of standard pool per 100 g body weight [BW]). Figure 4 shows leukocyte and thrombocyte counts of animals treated with 1.25 mL per 100 g BW (high dose) and 0.75 mL per 100 g BW (low dose). All animals treated with 1.25 mL per 100 g anti-RT7a mAb died within 6 weeks (high dose = lethal dose), whereas all animals treated with lower doses of the mAb survived indefinitely (low dose is the same as a sublethal dose). In particular, thrombocyte counts turned out to be a sensitive marker for the antibody effect, as numbers fell to values below 100 000/µL approximately 2 to 3 weeks before animal death. Erythrocyte numbers were stable during the time of observation in all animals, except for animals treated with lethal doses of the mAb; these animals showed subsequent thrombocytopenic bleeding in the last days of life.
Effect of the anti-RT7a mAb on BM precursor cells (rescue group) To study the mAb effects on precursor cells, LEW.1W rats (n = 4) received a lethal dose of anti-RT7a mAb on day 1 (see
above). On day 0, 100 × 106 BM cells of congenic
LEW.1W-7B rats were transplanted by intravenous injection. These cells
were MHC-identical to the recipient but carried a different CD45
haplotype (RT7b) not detected by the anti-RT7a
mAb. As survival of animals in this experiment was guaranteed by the BM
transplantation, it was possible to study reconstitution of
BM-dependent cell lines of recipient and donor type, respectively.
All recipients of BM showed significant allogenic chimerism of
leukocytes in peripheral blood. The kinetics of leukocyte
subpopulations are shown in Figure 5.
Total granulocyte numbers were back to normal after at least 21 days.
Recipient granulocytes were almost totally replaced by donor cells
after 14 days (Figure 5C). T cells were strongly depleted in the
recipient circulation during the first weeks. Reconstitution occurred
by donor and recipient cells, resulting in a mixed allogenic chimerism
of T cells (Figure 5A). Mature B cells were not depleted in the
recipient, and the level of chimerism between recipient and donor B
cells reached a maximum of 40%. Analysis of animals killed on
days 100 and 209, respectively, showed total replacement of
CD90+(THY1+)/HIS48+ BM cells by
donor cells (100%) as well as an almost total shift toward donor type
within CD4+CD8+ thymic cells (99%) as
depicted in Figure 6.
Effect of anti-RT7a mAb donor pretreatment on the engraftment of RT7a-positive BM (transfer group) To analyze the effect of anti-RT7a pretreatment on BM engraftment potential, LEW.1W donors (n = 4) received a potentially lethal dose (see above) of the anti-RT7a mAb on day2. On
day 0, their unmodified BM was transplanted into LEW.1W-7B rats
sublethally irradiated with 7 Gy  -irradiation on day 1. Under
standard conditions, congenic LEW.1W BM easily engrafts in the
irradiated recipient (data not shown). Differences in engraftment and
survival of recipients were supposed to be the cause of donor antibody pretreatment.
The most impressive effect was again observed within the granulocytes.
No granulocytes of donor origin were found in the recipients, suggesting a lethal effect of the anti-RT7a mAb on cells of
myeloid lineage in the donor BM (Figure
7). Almost no T cells of donor origin
were found in the early phase, whereas donor B cells could already be
seen 10 days after BM transplantation. During the next weeks, stable
chimerism for B cells developed, whereas mature T cells and
granulocytes of donor origin were not found in significant numbers,
pointing toward damage of T-cell and granulocyte progeny or of a common
progenitor.
Treatment of stable mixed RT7a/RT7b chimeras by anti-RT7a mAb (elimination group) Stable irradiation chimeras were generated with the use of LEW.7B rats as donors and LEW.1A rats as recipients. Recipients were lethally irradiated (11 Gy) on day1 and transplanted with 100 × 106 donor and recipient cells on day 0 (50%
each). The resulting chimeras showed stable multilineage chimerism and
long-term survival. These animals (n = 3) were treated with a
"lethal" dose of anti-RT7a mAb on days 63, 66, 70, and
122 (Table 1). The
RT7a-positive fraction of the chimeric BM was depleted by
the anti-RT7a mAb application except for 4% to 8% of
total leukocytes. These cells, which were to be seen in FACS until day
300, were revealed to be mature B cells exclusively. Analysis
after 520 days, however, could not reveal persisting B cells of
RT7a allotype.
In this study, an anti-rat CD45 mAb (anti-RT7a mAb) was used, and its effects on mature leukocytes as well as on BM precursor cells were analyzed. In vitro, the antibody was shown to bind to all types of leukocytes,12-14 although staining intensity varied slightly among different leukocyte populations. This result was basically in keeping with the known expression pattern of the LCA as detected by OX1 antibody.11 The epitopes detected by OX1 and anti-RT7a are distantly located on the CD45 molecule, as antibodies did not block simultaneous binding of one another. The minor differences in binding patterns of OX1 and anti-RT7a mAb to leukocyte subpopulations may be due to steric interactions of the mAbs with sites of heavy glycosylation or by capping of epitopes. In vivo, profound T-cell depletion in peripheral blood, spleen, and lymph nodes was the most obvious effect observed in animals after a single injection of the anti-RT7a mAb. This is in contrast to the in vivo effects described for OX1, which does not lead to relevant leukocyte depletion. This functional difference is most likely due to the different species origin as well as the different isotype of the antibodies (rat IgG2b for anti-RT7a versus mouse IgG2c for OX1). Many previous studies have shown that the nonvariable parts of antibodies play an essential role in mediating their functional effects (opsonization, complement activation, triggering of killer cells, cross-linking of target structures). Since leukocyte depletion showed an identical pattern in normal and in complement-deficient rats, complement lysis is probably not important for the depleting effect of the anti-RT7a mAb. Also, splenectomy of the animals could not prevent leukocyte depletion, arguing against a relevant contribution of the reticuloendothelial system of spleen for elimination of the cells. Nevertheless, cell-mediated effects by Fc-receptor-mediated interactions are probably essential for destruction of the leukocytes since other anti-RT7a antibodies having different isotypes that had been tested in preliminary studies were markedly less effective (data not shown). Interestingly, mature B cells were not depleted by
anti-RT7a mAb in vivo, although their in vitro staining was
high (but lower than that of T cells; Figure 1). A similar
difference between T and B cells in susceptibility to lysis by the
antibody had already been shown by previously studying
complement-mediated lysis by anti-RT7 antisera in vitro; T
cells but not B cells were destroyed in the presence of the
antisera.5 Since T and B cells show different patterns of
CD45 isoform expression,12-14 it may be that morphological
distinctions of the CD45 molecule are responsible for the different
sensitivity toward antibody-mediated cell lysis. Another possible
explanation might be a different role of CD45 for activation of T and B
cells.15 In an in vitro model, CD45-deficient T-cell lines
could not respond to stimulation except for one cell line in which one
of the SYK protein kinases mediated signaling in absence of
CD45.16 As the same kinase (p72syk) led to
partial activation of CD45 Overall, our observations suggest that either certain morphological features of CD45 isoform molecules or signal transduction via the CD45 pathway (or both) play a role in the depletion mechanism of the anti-RT7a mAb. These aspects are currently being studied in more detail. In addition to the depletion of T cells from the circulation as well as from lymph nodes and spleen, a lethal outcome was frequently observed in animals treated with a high dose of the anti-RT7a mAb, generally following a transient recovery of the leukocyte numbers in the periphery. In these cases, injection of anti-RT7 mAb obviously led to BM failure22 associated with fatal bleeding disorders or lethal opportunistic infections. Histologically, the cellularity of the BM was strongly reduced. Particularly impressive was a progressive loss of megakaryocytes from the BM. While several noxas causing aplastic anemia are known,23,24 this is, to our knowledge, the first description of an antibody that leads to BM aplasia after a single injection. To analyze the effect of anti-RT7 mAb treatment on hematopoietic
progenitors in more detail, congenic BM transplanatation models were
used; the rationale for this was based on the diallelic polymorphism of CD45 (RT7a/RT7b) in the rat. In
a rescue model, recipient survival after a generally lethal
anti-RT7a mAb dose was secured by MHC-identical, but
RT7-mismatched, BM transplantation (RT7b These observations were confirmed by experiments in a transfer model,
in which BM transplantion was carried out shortly after lethal
antibody treatment of the BM donor (RT7a Owing to the lack of a suitable marker for rat hematopoietic stem cells
(HSCs), it is not yet clear whether very early rat HSCs are
CD45+. Wickenhauser et al25 described HSCs in
humans as weakly CD45+. In early human myeloid precursors,
CD45RA expression is altered during maturation,26 and
recently analysis of LSM-1 expression, a possible physiological
substrate for CD45, revealed low expression for
CD34+CD33 In conclusion, the study shows that an antibody against the rat CD45 homolog RT7 is highly effective in depleting not only peripheral leukocytes, but also BM precursor cells. The mechanism of cell depletion by the antibody is not clear since mature B cells are obviously protected from depletion. This suggests that either CD45 isoform expression or differential signal transduction triggered by antibody binding to the CD45 molecule contributes to the functional effects of the anti-RT7a mAb. The findings further suggest that early hematopoietic precursors strongly expressing CD45 give rise to granulocytes, thrombocytes, and T cells, and probably also B cells. For the future, there are 3 major fields in which depleting anti-CD45 mAbs might be of great use. At first, antibodies capable of depleting hematopoietic stem cells can be introduced into the therapy of hematopoietic malignancies or for conditioning for BM transplantation for other indications; studies on this issue are currently underway. For this purpose, native antibodies may be more specific than the respective isotope conjugates,32 which always have a nonspecific bystander effect on neighboring cells. Second, such antibodies might be useful for tolerance induction in the field of transplantation through elimination of the mature T-cell repertoire as has been demonstrated previously.9 Third, depleting anti-CD45 mAbs could be used as a tool in models studying differentiation (or trans-differentiation) of HSCs.
Submitted October 2, 2001; accepted January 7, 2002.
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: Hans J. Schlitt, Klinik für Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany; e-mail: schlitt.hans{at}mh-hannover.de.
1. Penninger JM, Irie-Sasaki J, Sasaki T, Oliveira-dos-Santos A. CD45: new jobs for an old acquaintance. Nat Immunol. 2001;2:389-396[Medline] [Order article via Infotrieve]. 2. Lazarovits AI, Visser L, Asfar S, et al. Mechanisms of induction of renal allograft tolerance in CD45RB-treated mice. Kidney Int. 1999;55:1303-1310[CrossRef][Medline] [Order article via Infotrieve]. 3. Lazarovits AI, Poppema S, Zhang Z, et al. Prevention and reversal of renal allograft rejection by antibody against CD45RB. Nature. 1996;380:717-720[CrossRef][Medline] [Order article via Infotrieve]. 4. Wonigeit K. Characterization of the RT-Ly-1 and RT-Ly-2 alloantigenic systems by congenic rat strains. Transplant Proc. 1979;11:1631-1635[Medline] [Order article via Infotrieve]. 5. Wonigeit K. Definition of lymphocyte antigens in rats: RT-Ly-1, RT-Ly-2, and a new MHC-linked antigen system. Transplant Proc. 1979;11:1334-1336[Medline] [Order article via Infotrieve]. 6. Kampinga J, Kroese FG, Pol GH, et al. RT7-defined alloantigens in rats are part of the leucocyte common antigen family. Scand J Immunol. 1990;31:699-710[CrossRef][Medline] [Order article via Infotrieve]. 7. Carter PB, Sunderland CA. Rat alloantisera ART and Ly-1 detect a polymorphism of a leukocyte-common antigen. Transplant Proc. 1979;11:1646-1647[Medline] [Order article via Infotrieve]. 8. Jäger MD, Tsui T, Aselmann H, et al. Features of tolerance achieved by antigen and a single injection of an anti-CD45 monoclonal antibody in rats. Transplant Proc. 2001;33:142[CrossRef][Medline] [Order article via Infotrieve]. 9. Ko S, Jäger MD, Tsui TY, et al. Long-term allograft acceptance induced by single dose anti-leukocyte common antigen (RT7) antibody in the rat. Transplantation. 2001;71:1124-1131[CrossRef][Medline] [Order article via Infotrieve]. 10. Ko S, Dahlke MH, Lauth O, et al. Bone marrow aplasia induced by passenger leukocytes from heart allografts. Exp Hematol. 2001;29:339-344[CrossRef][Medline] [Order article via Infotrieve]. 11. Fabre JW, Sunderland CA, Williams AF. Immunosuppressive properties of mouse monoclonal antibodies and rabbit antisera to a leukocyte-specific antigen in the rat. Transplant Proc. 1981;13:509-511[Medline] [Order article via Infotrieve]. 12. Thomas ML. The leukocyte common antigen family. Ann Rev Immunol. 1989;7:339-369[CrossRef][Medline] [Order article via Infotrieve]. 13. Thomas ML, Lefrancois L. Differential expression of the leucocyte-common antigen family. Immunol Today. 1988;9:320-326[CrossRef][Medline] [Order article via Infotrieve]. 14. Woollett GR, Barclay AN, Puklavec M, Williams AF. Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes. Eur J Immunol. 1985;15:168-173[Medline] [Order article via Infotrieve]. 15. Justement LB. The role of CD45 in signal transduction. Adv Immunol. 1997;66:1-65[Medline] [Order article via Infotrieve]. 16. Chu DH, Spits H, Peyron JF, Rowley RB, Bolen JB, Weiss A. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling. EMBO J. 1996;15:6251-6261[Medline] [Order article via Infotrieve].
17.
Pao LI, Cambier JC.
Syk, but not Lyn, recruitment to B cell antigen receptor and activation following stimulation of CD45 18. Kung C, Pingel JT, Heikinheimo M, et al. Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease. Nat Med. 2000;6:343-345[CrossRef][Medline] [Order article via Infotrieve].
19.
Tchilian EZ, Wallace DL, Wells RS, Flower DR, Morgan G, Beverley PC.
A deletion in the gene encoding the CD45 antigen in a patient with SCID.
J Immunol.
2001;166:1308-1313
20.
Byth KF, Conroy LA, Howlett S, et al.
CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation.
J Exp Med.
1996;183:1707-1718 21. Kishihara K, Penninger J, Wallace VA, et al. Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice. Cell. 1993;74:143-156[CrossRef][Medline] [Order article via Infotrieve]. 22. Ko S, Deiwick A, Dinkel A, Wonigeit K, Schlitt HJ. Passenger leukocytes from a heart allograft can induce lethal lymphoid aplasia. Transplant Proc. 1999;31:139-140[CrossRef][Medline] [Order article via Infotrieve]. 23. Schattenberg DG, Stillman WS, Gruntmeir JJ, Helm KM, Irons RD, Ross D. Peroxidase activity in murine and human hematopoietic progenitor cells: potential relevance to benzene-induced toxicity. Mol Pharmacol. 1994;46:346-351[Abstract].
24.
Watanabe KH, Bois FY, Daisey JM, Auslander DM, Spear RC.
Benzene toxicokinetics in humans: exposure of bone marrow to metabolites.
Occup Environ Med.
1994;51:414-420 25. Wickenhauser C, Thiele J, Drebber U, et al. CD34+ human hemopoietic progenitor cells of the bone marrow differ from those of the peripheral blood: an immunocytochemical and morphometric study. Acta Haematol. 1995;93:83-90[Medline] [Order article via Infotrieve].
26.
Fritsch G, Buchinger P, Printz D, et al.
Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis.
Blood.
1993;81:2301-2309 27. Shimizu Y, Sugiyama H, Fujii Y, et al. Lineage- and differentiation stage-specific expression of LSM-1 (LPAP), a possible substrate for CD45, in human hematopoietic cells. Am J Hematol. 1997;54:1-11[CrossRef][Medline] [Order article via Infotrieve]. 28. Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med. 1997;3:1337-1345[CrossRef][Medline] [Order article via Infotrieve].
29.
Cumming RC, Liu JM, Youssoufian H, Buchwald M.
Suppression of apoptosis in hematopoietic factor-dependent progenitor cell lines by expression of the FAC gene.
Blood.
1996;88:4558-4567 30. Gottlieb E, Haffner R, von Ruden T, Wagner EF, Oren M. Down-regulation of wild-type p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal. EMBO J. 1994;13:1368-1374[Medline] [Order article via Infotrieve]. 31. Selleri C, Sato T, Anderson S, Young NS, Maciejewski JP. Interferon-gamma and tumor necrosis factor-alpha suppress both early and late stages of hematopoiesis and induce programmed cell death. J Cell Physiol. 1995;165:538-546[CrossRef][Medline] [Order article via Infotrieve].
32.
Matthews DC, Martin PJ, Nourigat C, Appelbaum FR, Fisher DR, Bernstein ID.
Marrow ablative and immunosuppressive effects of 131I-anti-CD45 antibody in congenic and H2-mismatched murine transplant models.
Blood.
1999;93:737-745
© 2002 by The American Society of Hematology.
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  G. Westerhuis, W. G. E. Maas, R. Willemze, R. E. M. Toes, and W. E. Fibbe Long-term mixed chimerism after immunologic conditioning and MHC-mismatched stem-cell transplantation is dependent on NK-cell tolerance Blood, September 15, 2005; 106(6): 2215 - 2220. [Abstract] [Full Text] [PDF]
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 S. Gregori, P. Mangia, R. Bacchetta, E. Tresoldi, F. Kolbinger, C. Traversari, J. M. Carballido, J. E. de Vries, U. Korthauer, and M.-G. Roncarolo An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells J. Exp. Med., April 18, 2005; 201(8): 1293 - 1305. [Abstract] [Full Text] [PDF]
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C. Baum, J. Dullmann, Z. Li, B. Fehse, J. Meyer, D. A. Williams, and C. von Kalle Side effects of retroviral gene transfer into hematopoietic stem cells Blood, March 15, 2003; 101(6): 2099 - 2113. [Abstract] [Full Text] [PDF]
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G. G. Wulf, K.-L. Luo, M. A. Goodell, and M. K. Brenner Anti-CD45-mediated cytoreduction to facilitate allogeneic stem cell transplantation Blood, March 15, 2003; 101(6): 2434 - 2439. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
/
/
(R73, Pharmingen, San Diego, CA); B
cells/CD45RA (Ox33, Pharmingen); granulocytes (HIS 48, Pharmingen);
CD45 (OX1, Pharmingen)
-),
B-cell (-
-), and granulocyte (-
-) counts are shown as the
fraction of total leukocytes analyzed. In panel D, counts of total
thymic T-cell receptor-positive (TCR+) T cells (labeled
with R73 mAb) (-
RT7a), so that the progeny of the transplanted BM could not
be damaged by the antibody. Quite obviously, granulocytes were
progressively and completely replaced by donor-type cells within
several weeks, indicating that the progenitors of early myeloid lineage
had been destroyed by antibody treatment. In the lymphatic compartment, a mixed chimerism was observed in the later course with donor as well
as recipient T and B cells (approximately 50:50 for B cells and 75:25
for T cells), suggesting ongoing recipient lymphopoiesis. Long-term
analysis of T-cell progeny in the thymus, however, revealed almost no
CD4+CD8+ cells of recipient type. This finding
strongly suggests that antibody treatment may also have destroyed very
early T-cell precursors so that de novo lymphopoiesis is exclusively
due to transplanted RT7b-expressing cells. The recovering
and persisting T cells of recipient type may have been derived from the
few surviving mature T cells, which proliferate after the antibody disappears.
but detectable