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Brief Definitive Report |
blaza001{at}tc.umn.edu
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Key Words: transplantation • in vivo animal models • T cells • stem cells • immune tolerance
To determine whether CD47 ligation would facilitate T cell activation and expansion in vivo, we used an adoptive transfer system in which donor T cells expand and cause target tissue injury known as GVHD in allogeneic recipients 12. Allogeneic CD47–/– T cells had a markedly reduced capacity to cause GVHD and virtually no CD47–/– T cells were found in lymphoid tissues of CD47+/+ recipients. Donor CD47–/– bone marrow (BM) was unable to rescue irradiated congenic immunodeficient CD47+/+ recipients suggesting that the innate immune system might be eliminating CD47–/– cells. Splenic macrophages, CD11b+, and especially CD11b– DCs engulfed dye-labeled CD47–/– LN cells and CD47–/– BM cells clearing these cells within 24 h after infusion in marked contrast to CD47+/+ cells. The elimination of splenic macrophages partially restored the capacity of donor CD47–/– T cells to engraft in allogeneic recipients, indicating that donor CD47–/– T cells can expand in vivo. Thus, host DCs (and macrophages) can receive negative signals delivered via CD47 antigen expressed on donor BM cells or donor T cells. The downregulation of CD47 antigen expression on lymphohematopoietic cells during disease states may signal these cells for clearance by host DCs and macrophages.
GVHD Generation and Quantification of T Cell Expansion In Vivo.
MLR Response.
Long-Term Engraftment Studies.
Short-Term In Vivo BM Proliferation Assay.
Kinetic Assessment of CD47–/– Lymphohematopoietic Cell Clearance in the Spleen of CD47+/+ Recipients.
Statistical Analyses.
Introduction
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Introduction
Materials and Methods
Results
Discussion
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Integrin-associated protein (CD47) is a broadly distributed glycoprotein expressed on all hematopoietic cells 1. CD47 regulates leukocyte activation, chemotaxis, and migration 234. CD47 ligation can costimulate activated T cells 3456. Conversely, CD47 ligation can function as an immune inhibitory signal which limits T cell responses 67. Although CD47 ligation has been shown to deliver T cell costimulatory signals in in vitro assays, there are no reports of the potential role of CD47 costimulation in vivo. The counterreceptor for CD47 is the signal regulatory protein (SIRP)
, expressed on neutrophils and monocyte-derived cells including dendritic cells (DCs; references 8 and 9). The binding of SIRP to CD47 on T cells is associated with T cell activation and induction of antigen-specific CTL responses by DCs 9. Studies in CD47+/+ recipients infused with nonopsonized or opsonized RBCs obtained from CD47+/+ or CD47–/– 10 donors showed that RBC CD47 must deliver an inhibitory signal via macrophage SIRP to prevent clearance and phagocytosis by splenic macrophages 811. The effects of CD47 expression on the clearance of other cell types has not been reported.
Materials and Methods
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Materials and Methods
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Mice.
C57BL/6 severe combined immune deficient (B6 SCID), B6.129S7 recombination activating gene (Rag)1tm1Mom recombinase 1–deficient (B6-Rag1–/–), C.H2bm1 (bm1), C.H2bm12 (bm12), and B6.PL-Thy1a/Cy (termed B6-Thy1.1 congenic) mice were purchased from The Jackson Laboratory. C57BL/6 (termed B6:H2b), B6-CD45.1 congenic, Balb/c SCID (H2d), and Balb/c wild-type (CD47+/+) mice were purchased from the National Institutes of Health. B6-CD47 deletional mutant (B6-CD47–/–) and Balb/c CD47–/– mice were generated and backcrossed 
10 generations 810.
To determine the effects of CD47 expression on the separate contribution of CD4+ or CD8+ T cells to the GVHD response, bm12 or bm1 recipients were irradiated with 6.0 Gray (137Cesium) total body irradiation (TBI) and given CD4+ T cells (0.03–0.3 x 106 cells per recipient) or CD8+ T cells (0.6 x 106 cells per recipient) from CD47–/– or CD47+/+ donors 10. Hematocrit (Hct) values were obtained to assess the degree of GVHD manifested by BM destruction 13. Survival and weight curves were monitored 13. To determine the effects of CD47 expression on GVHD mediated by T cells administered under noninflammatory conditions, nonconditioned Balb/c SCID mice were depleted of NK cells by anti-asialo–GM1 antisera and given B6-CD47+/+ or CD47–/– T cells (0.5 or 2 x 106 cells per recipient). To quantify the number of donor T cells present in the spleen of allogeneic recipients, splenocytes from Balb/c SCID recipients of B6-CD47–/– or CD47+/+ T cells were analyzed on days 6–7 after transfer. Cohorts were treated intravenously with 0.2 ml of liposomes loaded with dichloromethylene diphosphonate (liposomal DMDP; clodronate) on days 2 and 1 before allogeneic T cell transfer to deplete splenic phagocytes 814. As an additional indicator of donor T cell expansion, cannulae were inserted in the thoracic duct of other cohorts of recipients at the time of peak proliferation (day 6) after BM transplantation (BMT) and lymphocytes were collected over 18 h 13.
B6-CD47+/+ or CD47–/– T cells were mixed with irradiated (30 Gray) T cell depleted Balb/c splenocyte stimulators, plated in replicates of six into 96-well round-bottomed plates containing 105 responders and 105 irradiated stimulators, and incubated for 2–8 d 13. Microtiter wells were pulsed with tritiated thymidine (1 µCi) for 18 h before harvesting and counted in the absence of scintillation fluid on a β-plate reader.
To determine whether CD47–/– recipients could reject donor BM grafts, B6-CD47–/– or B6-CD47+/+ recipients were irradiated (5.5 Gray TBI) and infused with Balb/c BM cells (107). To determine whether CD47–/– or CD47+/+ BM would engraft and provide long-term hematopoietic reconstitution, B6 recipients were irradiated (5.5–6.5 Gray TBI) on day 1 and were reconstituted with Balb/c CD47+/+ or CD47–/– T cell depleted BM (107 cells) on day 0 15. In other studies, B6-Thy1.1 recipients were irradiated (9.0 Gray TBI) and reconstituted with B6-CD45.2 CD47–/– and B6-CD45.1 CD47+/+ BM cells (5 x 106 each). A cohort was splenectomized 10 d pre-BMT. For chimerism assessment, peripheral blood mononuclear cells were stained with anti-H2b–PE and anti-H2d–FITC (BD PharMingen) and analyzed using a FACScaliburTM (Becton Dickinson). To preclude a host antidonor T cell response against donor CD47–/– BM cells, CD47+/+ B6-Rag1–/–, or B6-CD47–/– recipients were irradiated (9.5 Gray TBI) on day 0 and given 5 x 106 congenic B6-CD47–/– or CD47+/+ BM cells.
To determine the effect of CD47 expression on the short-term reconstituting capacity of CD47–/– BM, B6 recipients were irradiated (9.5 Gray TBI) to eliminate host hematopoiesis and given 3 x 106 CD47–/– or CD47+/+ BM cells on day 0 16. To preclude NK cell–mediated graft rejection, cohorts were injected with anti-NK1.1 (PK136) mAb (200 µg) on day 2 to deplete NK cells. Anti-NK1.1 mAb (100 µg) results in depletion by day 1 and lasts for >14 d. On day 5, 3 µCi of [125I]deoxyuridine and 10–11 molar fluorodeoxyuridine (Amersham Pharmacia Biotech) was given. Spleens were harvested on day 6, rinsed, and incorporated radioactivity determined using a 
counter.
Single cell suspensions of LN cells were prepared from B6-CD47+/+ and CD47–/– mice and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) according to the manufacturer's protocol. Cells were injected intravenously into unirradiated B6 recipients. Single cell splenocyte suspensions were prepared using 10 mM EDTA. Cells were incubated on ice with cychrome-labeled anti-B220, cychrome-labeled anti-CD3, PE-labeled anti-CD11c, and either allophycocyanin-labeled anti-CD11b or biotin-labeled anti-F4/80 followed by allophycocyanin-labeled streptavidin. Data were analyzed on a FACScanTM flow cytometer (Becton Dickinson). A portion of the spleens was frozen in precooled isopentane, sectioned (10 µ), dehydrated in acetone, rehydrated in PBS, and sequentially incubated with anti-FcR mAb (2.4G2), avidin/biotin solutions (Vector Laboratories), biotin-labeled anti-CD11c mAb (N418), streptavidin-labeled horseradish peroxidase (NEN Life Science Products), and Cy5-labeled tyramide (NEN Life Science Products). Confocal microscopy and image analysis were performed as described previously 17.
Group comparisons of continuous data were made by Student's t test. Survival data were analyzed by lifetable methods using the Mantel-Peto-Cox summary of chi-squared. P values < 0.05 were considered significant.
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CD47–/– T Cells Have a Reduced Capacity to Mediate GVH-induced Lethality and Fail to Expand In Vivo.
To determine whether the CD47 expression on donor T cells was required for optimal alloresponses in vivo, nonconditioned NK-depleted Balb/c SCID mice were given 0.5 or 2.0 x 106 purified LN T cells from B6-CD47+/+ or CD47–/– donors (Fig. 1 a). All recipients of CD47–/– T cells survived long-term, while recipients of CD47+/+ T cells died by 3 wk after transfer.
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To investigate the mechanism(s) responsible for impaired GVHD lethality observed with CD47–/– T cells, MLR responses were analyzed. As compared with CD47+/+ T cells, B6-CD47–/– T cells generated a comparable peak proliferative response to Balb/c stimulator cells (mean ± SD 6,409 ± 968 vs. 7,709 ± 1,309 cpm [day 3]; 5,175 ± 827 vs. 5,124 ± 512 cpm [day 4]; P values > 0.1]. These data indicate that CD47 expression was not critical for in vitro alloproliferation.
It is possible that reduced GVHD by CD47–/– T cells was due to a defect in the response of CD47–/– T cells to alloantigen-bearing cells in vivo which could not be uncovered by in vitro studies. As another indicator of in vivo alloresponses, we examined the capacity of host CD47–/– recipients to reject allogeneic donor BM grafts, a process which is predominantly T cell dependent. Sublethally irradiated (5.5 Gray TBI) B6-CD47+/+ or CD47–/– recipients were given Balb/c T cell–depleted BM grafts. Donor chimerism levels at 6 wk after BMT revealed no significant differences (P = 0.29) between CD47–/– versus CD47+/+ recipients (39 ± 8% vs. 27 ± 7%) (n = 18–20 per group). Therefore, CD47–/– host T cells do not have a major defect in allogeneic BM graft rejection.
Because MLR results were similar with CD47–/– versus CD47+/+ T cells, we sought to determine whether the impaired GVHD response was a result of poor T cell expansion in vivo. NK-depleted Balb/c SCID mice were given B6-CD47–/– or CD47+/+ T cells (106 cells per recipient). On day 6, we observed a mean of 2.6 x 106 donor CD47+/+ T cells as compared with <0.05 x 106 donor CD47–/– T cells in recipient spleens (n = three per group individually analyzed; P < 0.001). We found <0.05 x 106 CD47–/– T cells in the thoracic duct lymphatics of a separate cohort of mice that received CD47–/– T cells versus 0.6 x 106 T cells in mice given CD47+/+ T cells (n = 6 per group individually analyzed; P < 0.001). Thus, CD47–/– T cells do not appear to expand efficiently in or are eliminated by CD47+/+ allogeneic recipients.
CD47–/– BM Cannot Reconstitute Lethally Irradiated CD47+/+ Allogeneic or Congenic Recipients.
The data suggested that the reduction in GVHD by CD47–/– cells in CD47+/+ allogeneic recipients may not be due to a profound defect in generating an alloresponse. The reduced GVH response in irradiated recipients could be due to the clearance of CD47–/– T cells by CD47+/+ recipients. To determine whether CD47–/– BM cells would be eliminated by CD47+/+ recipients, irradiated (6.5 Gray TBI) B6 recipients were given Balb/c T cell–depleted BM grafts from CD47–/– or CD47+/+ donors. CD47+/+ BM infusion resulted in 87% survival and a mean of 79% donor cells in the peripheral blood at 2 mo after BMT. In marked contrast, recipients of CD47–/– BM cells died between days 12–19 after BMT (Fig. 2 a). Consistent with the presumed hematopoietic failure, recipients of CD47–/– BM cells experienced a progressive severe (28%) body weight loss. To determine whether the absence of CD47 expression was responsible for impaired hematopoietic reconstitution in the absence of an alloresponse, we infused congenic CD47–/– BM into CD47+/+ recipients. To preclude a T cell or B cell immune response to the donor graft, we used lethally irradiated (9.5 Gray TBI) CD47+/+Rag1–/– or Rag1+/+ mice as recipients of either CD47–/– or CD47+/+ BM. CD47–/– BM was able to rescue CD47–/– recipients from hematopoietic failure (Fig. 2 b). In contrast, CD47–/– BM was not able to rescue lethally irradiated CD47+/+ Rag1–/– recipients. Day 14 Hct values indicated that the Rag1–/– mice given CD47–/– but not CD47+/+ BM were dying of hematopoietic failure (13 ± 2% vs. 41 ± 1%; P < 0.001). Because congenic CD47–/– BM is able to rescue CD47–/– but not CD47+/+ recipients, these data are most consistent with a non-T cell, non-B cell immune–mediated resistance of CD47+/+ recipients to CD47–/– BM.
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Because long-term reconstitution could not be achieved with CD47–/– BM, we used a short-term in vivo assay to assess the proliferation of CD47–/– BM cells in CD47+/+ recipients early after infusion. We considered the possibility that NK cells were involved in graft resistance as has been shown for congenic MHC class I–/– BM cells. When CD47–/– BM is given to lethally irradiated (9.5 Gray TBI) CD47–/– recipients, we observed high proliferation by day 5 after BMT (Table ). In marked contrast, CD47–/– BM was unable to proliferate in CD47+/+ recipients regardless as to whether host NK cells were depleted. These data indicate that CD47–/– BM is able to home to the spleen and proliferate in CD47–/– but not in CD47+/+ recipients early after BMT.
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Host CD47+/+ Splenic DCs and Macrophages Rapidly Engulf Donor CD47–/– Lymphohematopoietic Cells In Vivo.
In contrast to the critical role of host macrophages in RBC clearance 811, these cells appeared to play only a partial role in lymphohematopoietic cell clearance. To directly examine which host cell populations would engulf CD47–/– lymphohematopoietic cells, B6-CD47+/+ recipients were given CFSE-labeled LN cells from B6-CD47–/– or CD47+/+ donors. Splenocytes were analyzed at various time periods after infusion to enumerate CD47–/– versus CD47+/+ cells and determine whether there was evidence that CD47–/– T cells were phagocytosed to a greater extent than CD47+/+ T cells. Flow cytometric analysis indicated that a higher proportion of splenic CD11b–CD11c+ DCs had phagocytosed CFSE-labeled CD47–/– than CD47+/+ congenic T cells by 1 h after infusion (5.5 vs. 1.0%, respectively; Fig. 3 a). Analysis of time kinetics revealed that CFSE-labeled CD47–/– versus CD47+/+ T cells had been engulfed by a higher proportion of CD11b–CD11c+ DCs (1, 2, and 4 h after infusion), CD11b+CD11c+ DCs (1 h), and F4/80+CD11c– macrophage populations (1 h; Fig. 3 b). Splenic sections obtained 1 h after infusion showed that CFSE-labeled T cells of both genotypes migrated to the periarteriolar sheath areas but approximately one-half of CFSE-labeled CD47–/– and virtually no CD47+/+ T cells could be found inside host CD11c+ DCs (Fig. 3 c). By 1 d after infusion, there were no detectable CFSE-labeled CD47–/– T cells present in the spleen as determined by either flow cytometry or splenic sections, in contrast to readily detectable CFSE-labeled CD47+/+ T cells which were still present (data not shown). Thus, CD11b– DCs and to a lesser extent CD11b+ DCs and macrophages preferentially engulfed CD47–/– versus CD47+/+ T cells.
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CD47–/– BM is able to proliferate and reconstitute lethally irradiated CD47–/– but not CD47+/+ recipients. These data are consistent with a clearing of CD47–/– hematopoietic progenitor cells by the host as further evidenced by an almost complete absence of BM proliferation early after transfer. Similarly, despite the pressure to generate effective alloresponses and for homeostatic expansion in vivo, B6-CD47–/– T cells could not be detected in allogeneic Balb/c SCID CD47+/+ recipients. Recently, we have reported that CD47–/– RBCs are cleared in CD47+/+ mice by splenic macrophages 811. The macrophage receptor engaged by CD47 is SIRP, a signal-regulatory protein which inhibits tyrosine-kinase signaling pathways 81119. NK inhibitory receptors also recruit and activate the same tyrosine-kinase signaling pathways so that NK cells won't eliminate lymphohematopoietic cells that express self-MHC class I antigens 2021. Conversely, tumor cells that have lost or downregulated MHC class I antigens could be removed by NK cells 19. In an analogous fashion, splenic macrophages clear RBCs which lack MHC antigens. Although an ovarian cancer cell line has been shown to be CD47 deficient, it is possible that CD47 expression is downregulated on other types of tumors or damaged lymphohematopoietic cells 22. In those instances, a low level of CD47 expression may cause the host to eliminate these cells.
Regulation of CD47 expression may be an important mechanism for host macrophages to remove damaged RBCs since the clearance of opsonized RBCs is controlled by CD47-SIRP. For other lymphohematopoietic cells such as T cells and BM cells, DCs appear to be more critical than macrophages in eliminating CD47–/– cells in a CD47+/+ microenvironment. We provide direct evidence that both CFSE-labeled CD47–/– and CD47+/+ T cells initially localized to the splenic periarteriolar sheath but that CD47–/– T cells were engulfed by CD11b– DCs and to a lesser extent by CD11b+ DCs and macrophages by 1 h after infusion, resulting in complete clearance of CD47–/– but not CD47+/+ T cells within 1 d after infusion. In contrast, RBCs normally stay within the macrophage-rich red pulp area of the spleen, which is why macrophages are the primary populations to eliminate CD47–/– RBCs. Consistent with CFSE-labeled T cell data, the elimination of host splenic CD11b+CD11c+ DCs and macrophages by clodronate only partially inhibited CD47–/– T cell clearance by CD47+/+ recipients. Because DCs are widely distributed throughout the body especially including DC-rich tissues such as the liver, lung, LN, and skin as well as the spleen, we hypothesize that the spleen may not be the major site of clearance for CD47–/– T cells or BM. In support of this hypothesis, irradiated, splenectomized recipients given equal numbers of congenic CD47–/– and CD47+/+ BM cells had exclusively congenic CD47+/+ BM-derived cells present in the peripheral blood when analyzed 1 mo later. Thus, CD47+/+ DCs and macrophages engulf and clear CD47–/– lymphohematopoietic cells, a process which can occur both within or outside the spleen. For lymphohematopoietic cells which express MHC antigens, CD47 expression may represent a safeguard for eliminating abnormal cells that have not downregulated MHC class I antigens.
We were unable to demonstrate a critical role for CD47 in regulating alloresponses. In vitro, MLR responses with CD47–/– T cells were similar to CD47+/+ T cells. In vivo, CD47–/– recipients could reject allogeneic donor BM grafts indicating that host antidonor responses by CD47–/– T cells were not markedly impaired. Several investigators have shown that CD47 can costimulate T cells that have received TCR signals 3456719. In nonirradiated or sublethally irradiated recipients, the donor T cells are rapidly cleared and therefore not available to mediate lethality. Because under lethal irradiation conditions, recipients of allogeneic CD47–/– T cells eventually succumbed to GVHD lethality, it is possible that heavy irradiation either eliminated SIRP-expressing host cells or inhibited their function. Regardless, CD47–/– T cells can cause lethality, providing evidence that CD47–/– T cell alloresponses are not markedly impaired in vivo.
In summary, we have found that CD47 expression is required to prevent clearance of lymphohematopoietic cells by DCs and macrophages. This rapid response mechanism may protect the host from damaged or defective cells that downregulate CD47 antigen in disease states. Approaches to reduce CD47 antigen expression on lymphohematopoietic cells may provide a means of eliminating abnormal or harmful cells in various disease settings.
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Acknowledgments |
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Submitted: 1 December 2000
Revised: 13 June 2001
Accepted: 28 June 2001
P.-A. Oldenborg's present address is Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University SE-901 87, Umeå, Sweden.
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| TABLE OF CONTENTS |
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26% except for CD11b+ DCs engulfing CD47+/+ T cells. At the indicated time points, there was a significantly (P