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Keywords:

  • Spleen colony formation;
  • Major histocompatibility complex;
  • Restriction

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To clarify major histocompatibility complex (MHC) restriction between hematopoietic stem cells (HSCs) and microenvironments, T cell-depleted bone marrow cells (BMCs) were transplanted into MHC-compatible and MHC-incompatible recipients. A significantly large number of spleen colony-forming units (CFU-S) on day 12 were noted in MHC-compatible recipients, while only a small number were observed in MHC-incompatible recipients. There was, however, no significant difference in CFU-S counts on day 8 between the two groups. A large number of CFU-S counts on day 12 were also observed in F1 hybrid recipients, as seen in syngeneic recipients. The decrease in CFU-S counts on day 12 in MHC-incompatible recipients was also observed even after in vivo abrogation of T and NK cells. The difference in CFU-S counts on day 12 became more prominent when HSC-enriched cells were transferred. These results suggest that an MHC restriction exists between pluripotent HSCs (P-HSCs) and spleen microenvironments. Furthermore, experiments using B10.A recombinant strains revealed that H-2D and S loci play a crucial role in the MHC restriction. The experiments of serial transplantation suggest that the differentiation and proliferation of P-HSCs are inhibited in MHC-incompatible microenvironments. It is therefore likely that the MHC-compatible microenvironment is essential to the differentiation and proliferation of P-HSCs.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Stromal cells, which create the hematopoietic microenvironment, support the proliferation and differentiation of hematopoietic cells by direct cell-to-cell interaction with adherent molecules [1-4] and/or hematopoietic factors [4-8]. Bone marrow (BM) stromal cells are reported to generate from the endosteal layer [9, 10]. Indeed, the engraftment of bone without bone marrow cells (BMCs) provides a hematopoietic microenvironment [10, 11]. We have recently found that hematolymphoid reconstitution of BMCs in major histocompatibility complex (MHC)-incompatible recipients is significantly enhanced when bones of the BM donor are engrafted simultaneously with BM transplantation (BMT) [12, 13]. More recently, we have also found that significant numbers of hematopoietic progenitors accumulate in the engrafted bones which had the same MHC phenotype as that of the transplanted BMCs [14]. From these results, we conclude that hematopoietic stem cells (HSCs) prefer the “self” microenvironment for their proliferation and differentiation.

The colony-forming unit-spleen (CFU-S) assays devised by Till and McCulloch [15] have been considered a means of measuring HSC contents; CFU-S counts on day 12 are thought to reflect the number of P-HSCs, while CFU-S counts on day 8 are thought to more closely reflect the number of progenitor cells committed to the erythroid lineage. Since the colonies derive from single colony-forming cells (CFCs) and since the stromal cells in the spleen are found to maintain long-term hematopoiesis [16], spleen colony formation seems to be a useful tool for analyzing the interaction between hematopoietic progenitors and stromal microenvironments in vivo. In the present study, to obtain further detailed information of the MHC restriction between HSCs and stromal microenvironments, we examine spleen colony formation in MHC-compatible and -incompatible mouse combinations.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mice

C57BL/6 (B6) and C3H/He (C3H) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and Charles River Japan, Inc. (Yokohama, Japan). C57BL/10 (B10), B10.BR/SgSn (B10.BR), B10.D2/nSn (B10.D2), and B10.A/SgSn (B10.A) mice were purchased from The Jackson Laboratory and Japan SLC, Inc. (Hamamatsu, Japan). (B10 × B10.BR) F1 (B10/B10.BRF1) mice were purchased from The Jackson Laboratory. (B6 × DBA/2) F1 (BDF1) mice were purchased from Japan SLC, Inc. B10.A (4R) /SgSn and B10.A (5R)/SgSn mice were bred in our colony at Kansai Medical University. The original breeding pairs of B10.A (5R) mice were provided by Dr. Kazuo Moriwaki at National Institute of Genetics, (Tokyo, Japan), and B10.A (4R) mice were kindly provided by Dr. Kazunori Onoe at the Institute of Immunological Science, Hokkaido University. Ten- to 12-week-old mice were used as recipients, and 6- to 8-week-old mice as donors. All recipient mice were kept in specific pathogen-free conditions and given acidic water (pH 2.5) supplemented with oxytetracyclin hydrochloride.

Preparation of BMCs

Whole BMCs were collected from the femurs and tibias. T cells were depleted from the whole BMCs by treatment with the monoclonal antibody (mAb) against Thy1.2 (clone HO-13-4, ATCC; Rockville, MD) and rabbit complement (Perfreeze; Brown Deer, WI). The wheat germ agglutinin (WGA)-positive (HSC-enriched) BMC fraction was prepared as previously described [17, 18] with a minor modification. In brief, low-density BMCs ([LDBMCs]; 1.060-1.073 g/ml) were separated from whole BMCs by equilibrium density centrifugation (1,000 g, 30 min, 4°C) on discontinuous density gradients of Percoll (Pharmacia-LKB; Uppsala, Sweden) solution. T cells, B cells, macrophages, and mature granulocytes were depleted from LDBMCs using mAbs (rat-anti-mouse Thy 1.2, clone 30H-12; rat-anti-mouse B220, clone RA3-6B2; rat-anti-mouse Mac1α chain, clone M1/70.15; ATCC, and rat-anti-mouse Gr-1, clone RA3-8C5, Pharmingen; San Diego, CA); and anti-rat IgG immunomagnetic beads (Dynal; Oslo, Norway). Resultant cells (Lin LDBMCs) were further separated into WGA-positive (high affinity to WGA) and WGA-negative (low-affinity) cells by sorting on a flow cytometer (Epics C, Coulter; Hialeah, FL). The sorted WGA+ Lin LDBMCs were 1% of whole BMCs.

Assays of CFU-S

Recipient mice were exposed to 137Cs gamma irradiation at a dose rate of 0.96-1.14 Gy/min. The exposure doses were 9.0 Gy for B6, B10, B10.BR, B10.D2, B10.A, B10.A (4R), and B10.A (5R) mice, and 9.5 Gy for C3H and B10/B10.BRF1 mice. Eighteen to 24 hours after the irradiation, recipient mice were intravenously injected with T cell-depleted BMCs or WGA+ Lin LDBMCs. The recipient mice were killed either 8 or 12 days after the injection, and their spleens were removed and fixed in Bouin's solution. Visible surface colonies were counted. More than three mice of each recipient strain were exposed to irradiation but not given any cell preparations (irradiation control) in each experiment. The irradiation control had less than one colony per spleen. Recipient mice in each experiment were composed of age- and sex-matched mice.

Analyses of MHC Phenotypes

MHC phenotypes of CFCs in the recipient spleen were analyzed using flow cytometry and the antibody-dependent complement-mediated cytotoxicity test (cytotoxicity test). Single spleen colonies were excised and minced to obtain a single-cell suspension. In the flow cytometric analyses, colony contents were suspended in phosphate-buffered saline (PBS) supplemented with fetal bovine serum ([FBS]: 2%) and sodium azide (0.1%) and stained with fluorescein isothiocyanate (FITC)-conjugated anti-H-2Kd, anti-H-2Kb, or anti-H-2Kk (Meiji Milk Products; Tokyo, Japan) mAbs. After staining, H-2 phenotypes were analyzed on an Epics C. Analyses of MHC-phenotypes by the cytotoxicity test were performed as previously described [19]. In brief, a cell suspension (5 × 105/100 μl) of the colony contents was incubated with purified anti-H-2Kd, anti-H-2Kb or anti-H-2Kk mAb (final 1:50 dilution: Meiji Milk Products) for 30 min on ice. After washing two times, the cells were suspended in 100 μl of a 1:20 dilution of rabbit complement (Perfreeze) previously absorbed with mouse spleen cells. After a 30-min incubation at 37°C, the viability of the cells was determined by the trypan blue dye exclusion test. In some experiments, frozen sections were stained with biotinylated anti-H-2Kd, anti-H-2Kb or anti-H-2Kk (Meiji Milk Products) mAb. After washing in Tris buffer (0.1M, pH7.2), the frozen sections were incubated with avidin DH and biotinylated horseradish peroxidase H (Vectastain ABC Kit, Vector Laboratories; Burlingame, CA). After incubation, the antigen-antibody complexes in the sections were made visible by incubation with hydrogen peroxide (0.01%) and diaminobenzidine tetrahydrochloride (0.05%) in Tris buffer.

Pretreatment of Mice

In some experiments, T cells and NK cells in the recipient mice were depleted in vivo by injection of 0.3 ml GK1.5 (rat-anti-mouse CD4 mAb) ascites (cytotoxic titer 1:20,000) and 0.3 ml 2.43 (rat-anti-mouse CD8 mAb) ascites (cytotoxic titer 1:20,000) and 0.4 ml PK136 (murine anti-NK1.1 mAb) ascites. The mAbs were injected one day before and seven days after irradiation. As previously described [20], treatment with GK1.5 and 2.43 ascites completely depleted the CD4+ and CD8+ cells, respectively. The treatment with PK136 ascites decreased NK activity in the spleen cells from 24% to 3% in lysis of YAC-1 cells at the target:effector ratio of 1:100. To deplete myeloid cells in vivo, some recipient mice were injected with dimethyl myleran ([DMM], 10 mg/kg), a myeloablative agent, one day before irradiation, as reported by Lapidot et al. [21].

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Decreases in CFU-S Counts on Day 12 (not on day 8) in MHC-Incompatible Recipients

We compared CFU-S counts between MHC-compatible and -incompatible combinations. As shown in Table 1, when BMCs from B10.BR mice were injected into irradiated recipients, a significant number of CFUs-S on day 12, which are formed by multipotent progenitors [22], were noted in MHC-compatible C3H recipients, whereas the CFU-S counts were significantly lower in the MHC-incompatible B10 recipients. Also, when B10 BMCs were injected, a large number of CFUs-S on day 12 were observed in MHC-compatible B6 recipients, whereas only a few were noted in MHC-incompatible C3H or B10.BR recipients. The number of CFUs-S on day 8, which are formed by progenitors more committed to erythroid lineage [22], was also smaller in the MHC-incompatible recipients. However, the decrease in the CFU-S counts on day 8 was not statistically significant. The decrease in CFU-S counts on day 12 was also observed when B10.D2 (H-2d, Mlsb) BMCs were employed: a large number of CFUs-S were observed in the MHC-compatible BALB/c (H-2d, Mlsb; 19.9 ± 5.1) or DBA/2 (H-2d, Mlsa; 22.9 ± 9.9) recipients, whereas far fewer were noted in the MHC-incompatible B10BR (1.4 ± 1.6) or B10 (1.5 ± 2.1) recipients.

Table Table 1.. Decreases in CFU-S counts on day 12 in MHC-incompatible recipients
 DonorRecipientsCFU-S counts per 105 cells injected
   Day 8Day 12
Exp. 1B10BRB10.BR (H-2k)6.0 ± 2.018.4 ± 2.3
  B10 (H-2b)4.5 ± 1.02.5 ± 2.2*
  C3H (H-2k)12.7 ± 2.716.3 ± 3.1
  B10/B10.BRF18.2 ± 3.317.7 ± 3.7
Exp. 2 B10.BR18.3 ± 2.616.0 ± 3.9
  B1018.3 ± 4.13.5 ± 2.9*
  C3H16.7 ± 3.815.1 ± 3.7
  B10/B10.BRF116.7 ± 5.113.2 ± 4.0
  1. a

    Recipients were irradiated and injected with T cell-depleted BMCs (1 × 105) from donor mice. Seven recipients in each strain were used for CFU-S counts on days 8 and 12 after injection. CFU-S counts in mice that received irradiation alone (four mice in each strain) were less than one. *p < 0.001 compared with syngeneic recipients.

Exp. 1B10B10.BR24.5 ± 5.37.6 ± 4.1*
  B1032.4 ± 5.931.2 ± 4.8
  C3H28.0 ± 7.63.9 ± 4.1*
  B10/B10.BRF132.0 ± 2.933.6 ± 3.6
  B6 (H-2b)34.0 ± 6.329.8 ± 6.4
Exp. 2 B10.BR12.4 ± 2.36.3 ± 2.4*
  B109.7 ± 3.619.1 ± 3.3
  C3H9.3 ± 1.25.0 ± 1.1*
  B10/B10.BRF113.2 ± 4.421.5 ± 1.1
  B611.9 ± 6.218.8 ± 5.7

To examine the MHC phenotype of CFUs-S in MHC-incompatible recipients, 47 colonies were randomly collected from 30 spleens of recipients that had received MHC-incompatible BMCs and analyzed using flow cytometry, cytotoxicity tests, or immunohistochemical staining using frozen sections. Analyses using these three methods revealed that the cells in the colonies show the MHC phenotypes of BMC donors (data not shown). To examine whether the MHC incompatibility directly influences the CFCs, hematopoietic progenitors were enriched and injected into either MHC-compatible or -incompatible recipients. In comparison with WBMCs, the WGA+ Lin LDBMCs elicited 51-fold more CFUs-S on day 8 and 31-fold more CFU-S counts on day 12. As shown in Figure 1, the CFU-S counts on day 12 formed by the WGA+ Lin LDBMCs of the B10.BR were significantly reduced in the MHC-incompatible recipients (B10 mice).

thumbnail image

Figure Figure 1.. Decreases in CFU-S counts on day 12 in the MHC-incompatible recipients after injection of HSC-enriched population.WGA+LinLDBMCs of B10.BR mice were injected into recipients that had been irradiated one day before. Seven recipients in each strain were used for CFU-S counts on day 8 and on day 12 after injection. Data are presented as CFU-S counts by injection of 5×103WGA+LinLDBMCs. *p < 0.001 compared with syngeneic recipients (B10.BR).

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No Rejection Mechanism in Decreased CFU-S Counts on Day 12 in MHC-Incompatible Recipients

To clarify the causes of decreased CFU-S counts on day 12 in the MHC-incompatible recipients, B10 congenic mouse strains were used. In this system, F1 mice (B10/B10.BRF1) that received BMCs from a parent strain (B10 or B10.BR) showed many CFU-S counts on day 12, as did syngeneic recipients (Table 1 and Fig. 1). It seems, therefore, unlikely that hybrid resistance [23], which is mediated by natural killer (NK) cells [24], is involved in the decreased CFU-S counts on day 12. To further determine whether NK-cell- or T cell-mediated rejection is involved in the decrease, mAbs against CD4, CD8, or NK1.1 were injected [20, 25]. However, as shown in Figures 2A and 2B, the treatments using these mAbs did not restore the decreased CFU-S counts on day 12 in the MHC-incompatible recipients. To rule out the possibility of BMC exclusion by phagocytosis, we injected DMM, a myeloablative drug, which has been reported to prevent rejection of BMC grafts in MHC-incompatible recipients [21]. However, the treatment with DMM had no effect (data not shown ). Finally, we examined the CFU-S counts on day 12 in [F1 [RIGHTWARDS ARROW] parent]. In this combination, graft rejection should occur if any responding cells survive after irradiation. As shown in Figure 3, BMCs from B10/B10.BRF1 formed many CFU-S counts on day 12 in recipients with the MHC phenotype of the parents (B6 for B10 and C3H for B10.BR). We obtained the same results in [BDF1 [RIGHTWARDS ARROW] B6] or [BDF1 [RIGHTWARDS ARROW] DBA/2] combinations (data not shown).

thumbnail imagethumbnail image

Figure Figure 2.. Effects of pretreatment with mAb(s) on CFU-S counts on day 12 in MHC-incompatible recipients.C3H (A) or B6 (B) recipient mice were injected with indicated mAbs, irradiated, and injected with T cell-depleted BMCs, as described inMaterials and Methods. The recipient mice in each group were used for CFU-S counts on day 12. *p < 0.001 compared with MHC-compatible counterparts.

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Figure Figure 3.. No significant difference in CFU-S counts on day 12 between [F1[RIGHTWARDS ARROW]P] and [F1[RIGHTWARDS ARROW]F1] combinations.T cell-depleted BMCs from donor mice were injected into recipients that had been irradiated one day before. The recipient mice in each group were used for CFU-S counts on day 12. *p < 0.001 compared with MHC-compatible combinations.

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MHC Loci Involved in MHC Restriction

To determine which loci in the MHC are important for CFU-S on day 12, we injected BMCs from B10, B10.BR or B10.D2 mice into irradiated B10.A, B10.A(4R), and B10.A(5R) mice (Table 2). BMCs from B10.A, B10.A(4R), or B10.A(5R) mice were also injected into B10 congenic mice (Table 3). As shown in Tables 3 and 4, significant numbers of CFU-S on day 12 were observed when BMC donors and recipients were compatible in the H-2S and D loci, whereas the compatibility in the K and I loci seems not to be crucial for the colony counts. MHC class IB antigens such as Qa-1, Qa-2, or Tla do not seem to affect the CFU-S counts. A minimum number of CFUs-S were observed when BMCs and recipients were incompatible in any class IA and class II loci: the combinations of [B10 [RIGHTWARDS ARROW] B10.A] and [B10.D2 [RIGHTWARDS ARROW] B10.A(4R)]. In contrast to hybrid resistance, BMCs from B10.A(4R) mice formed many numbers of colonies in B10/B10.BRF1 recipients, in which one of two phenotypes is compatible with that of B10.A (4R) in all H-2 loci.

Table Table 2.. Phenotypes of alleles at H-2 loci of B10.A recombinant mice
MiceClass IA and Class IIClass IB
 KI-AβI-AαI-EβI-EαSDLQa-2TlaQa-1
  1. a

    Δ = deleted.

B10.Akkkkkdddaaa
B10.A (4R)kkkk/bbbbΔabb
B10.A (5R)bbbbkdddaaa
Table Table 3.. CFU-S counts on day 12 in recipients partially compatible in H-2 loci - I
DonorRecipients(n)CFU-S countsCompatible alleles at H-2 loci
    Class IA and Class IIClass IB
B10.AB10.A(5)18.1 ± 1.8AllQa-2, Tla, Qa-1
B10B10.A(7)0.0NoneQa-2
 B10(6)14.8 ± 3.8AllQa-2, Tla, Qa-1
B10.BRB10.A(8)2.8 ± 1.2K, Aβ, Aα, Eβ, EαTla, Qa-1
 B10.BR(6)14.2 ± 3.8AllQa-2, Tla, Qa-1
B10.D2B10.A(8)12.9 ± 3.3S, D, LQa-2
 B10.D2(5)13.0 ± 5.5AllQa-2, Tla, Qa-1
B10.A (4R)B10.A (4R)(6)15.4 ± 2.8AllQa-2, Tla, Qa-1
B10B10.A (4R)(7)10.4 ± 3.9Eβ, Eα, S, DQa-2, Tla, Qa-1
 B10(5)15.3 ± 0.9AllQa-2, Tla, Qa-1
B10.BRB10.A (4R)(7)4.8 ± 1.9K, Aβ, Aα, EβNone
 B10.BR(6)12.4 ± 3.1AllQa-2, Tla, Qa-1
B10.D2B10.A (4R)(7)0.7 ± 0.9NoneQa-2, Qa-1
 B10.D2(5)13.1 ± 3.7AllQa-2, Tla, Qa-1
  1. a

    Recipient mice were irradiated (9Gy) one day before the injection of T cell-depleted BMCs (1 × 105). Twelve days after the injection, recipient mice were killed. Numbers in parentheses represent numbers of recipient mice. CFU-S counts in mice that received irradiation alone (four mice in each strain) were less than one. Experiments were carried out twice and reproducible results obtained. Representative data are therefore shown.

B10.A (5R)B10.A (5R)(5)11.0 ± 2.6AllQa-2, Tla, Qa-1
B10B10.A (5R)(8)1.1 ± 1.7K, Aβ, Aα, EβQa-2
 B10(5)13.6 ± 1.1AllQa-2, Tla, Qa-1
B10.BRB10.A (5R)(8)3.5 ± 3.1Tla, Qa-1
 B10.BR(6)13.2 ± 5.3AllQa-2, Tla, Qa-1
B10.D2B10.A (5R)(8)13.0 ± 5.8S, D, LQa-2
 B10.D2(6)13.8 ± 3.3AllQa-2, Tla, Qa-1
Table Table 4.. CFU-S counts on day 12 in recipients partially compatible in H-2 loci - II
DonorRecipients(n)CFU-S countsCompatible alleles at H-2 loci
    Class IA and Class IIClass IB
B10.AB10.A(6)12.6 ± 1.5AllQa-2, Tla, Qa-1
 B10(6)0.6 ± 0.6NoneQa-2
 B10.BR(6)0.3 ± 0.4K, Aβ, Aα, Eβ, EαTla, Qa-1
 B10.D2(6)11.9 ± 1.2S, D, LQa-2
B10.A (4R)B10.A (4R)(9)19.6 ± 3.9AllQa-2, Tla, Qa-1
 B10/B10.BRF1(9)20.1 ± 2.4AllQa-2, Tla, Qa-1
 B10(9)23.8 ± 4.3Eβ, Eα, S, DQa-2, Tla, Qa-1
 B10.BR(9)6.1 ± 4.3K, Aβ, Aα, EβNone
 B10.D2(8)1.9 ± 2.0NoneQa-2, Qa-1
  1. a

    Recipient mice were irradiated (9Gy) one day before the injection of T cell-depleted BMCs (1 × 105). Twelve days after the injection, recipient mice were killed. Numbers in parentheses represent numbers of recipient mice. CFU-S counts in mice that received irradiation alone (four mice in each strain) were less than one. Experiments were carried out twice and reproducible results obtained. Representative data are therefore shown.

B10.A (5R)B10.A (5R)(5)13.1 ± 1.1AllQa-2, Tla, Qa-1
 B10(8)1.6 ± 0.8K, Aβ, Aα, EβQa-2
 B10.BR(8)0.5 ± 0.4Tla, Qa-1
 B10.D2(9)15.9 ± 2.5S, D, LQa-2

Survival of Primitive Progenitors in Spleens of MHC-Incompatible Recipients

Wolf and Priestley have reported that more primitive progenitors exist in “intercolonial space” than “colonial space” [26]. If this is true, we can expect that more primitive progenitors survive in the spleens of MHC-incompatible recipients, since there is much more “intercolonial space” in the spleens of MHC-incompatible recipients than MHC-compatible recipients. We therefore collected spleen cells from MHC-compatible or -incompatible recipients 12 days after BMC injection and transferred them into second recipients to examine the CFU-S counts and marrow-repopulating ability. As shown in Table 5, a small but significant number (3.2 ± 1.1) of CFUs-S on day 12 (not day 8) were observed in the second recipients which had been injected with the spleen cells collected from the MHC-incompatible recipients (C3H); the number was more than that (0.8 ± 0.4) formed in the MHC-incompatible first recipients (C3H). In contrast, when the spleen cells from the MHC-compatible first recipients (B6) were injected, the number decreased in the second recipients (20.6 ± 2.3[RIGHTWARDS ARROW]12.2 ± 6.5). It is noted that a small but significant number (2.52 ± 4.1) of CFUs-S on day 12 were also observed in the third recipients when BMCs collected from the second recipients which had received the spleen cells from the MHC-incompatible first recipients were injected into the third recipients, and that 83% (five of six) of the third recipients survived for 30 days. In contrast, the BMCs of the second recipients which had received the spleen cells from the MHC-compatible recipients neither formed CFUs-S on day 12 nor survived for 30 days.

Table Table 5.. CFU-S counts on day 12 in second and third recipients
 1st recipients (n = 17)2nd recipients (n = 26)3rd recipients (n = 12)
DonorCFU-S counts on day 12 in 105 BMCsCells in spleen (× 105)CFU-S counts in 106 spleen cells of 1st recipientsCells in BM (× 104)CFU-S counts on day 12 (n = 6) in 105 BMCs of 2nd recipients30-day survival
   Day 8 (n = 8) Day 12 (n = 18)   
B6C3HB6B6
 0.8 ± 0.419 ± 503.2 ± 1.114.3 ± 4.12.52 ± 4.15/6
  1. a

    All recipient mice were irradiated (9 Gy) one day before injection. First recipients were treated with anti-NK1.1 mAb and injected with T cell-depleted BMCs (1×105) of donor mice. Second recipients were injected with cells (1×106) collected from the spleens of first recipients on day 12 after the first injection. Third recipients were injected with cells (1×105) collected from the BM of second recipients on day 12 after the second injection.

B6B6B6B6
 20.6 ± 2.31242 ± 21612.2 ± 3.412.2 ± 6.554.1 ± 15.900/6

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Inhibition of hematopoietic colony formation in spleens of MHC-incompatible hosts have been found by Lengerová et al. [27] They proposed involvement of non-immunological mechanisms for the inhibition. Since discovery of NK cell activity, such inhibition in the initiation of hematopoiesis and the hematopoietic reconstitution in allogeneic or hybrid hosts has been explained as rejection exerted by NK cells [28-32], which are genetically controlled by the hematopoietic histocompatibility-1 genetic system [33, 34]. However, we have recently found that hematolymphoid reconstitution of BMCs in MHC-incompatible recipients is significantly enhanced when bones of the BM donor are engrafted simultaneously with BM transplantation [12, 13]. Furthermore, we have found that significant numbers of hematopoietic progenitors accumulate in grafted bones with the same MHC phenotype as the transplanted BMCs [14]. From this evidence, it is suggested that the inhibition in the hematopoietic reconstitution is not due to rejection by NK or T cells but may be due to missing interaction between HSCs and the H-2-incompatible microenvironment (stromal cells). In the present study, to examine whether our hypothesis is true in the initiation of hematopoiesis, we investigated the MHC restriction between hematopoietic progenitors and spleen stromal cells employing the CFU-S system. In agreement with the previous study, the proliferation and differentiation of hematopoietic progenitors (spleen colony formation) was inhibited in the MHC-incompatible microenvironment. Furthermore, we have found that the CFU-S counts on day 12 were more strongly influenced by the MHC restriction whereas the CFU-S counts on day 8 were not. Since CFU-S counts on day 12 are thought to reflect the number of P-HSCs while CFU-S counts on day 8 are thought to more closely reflect the number of committed progenitor cells, these findings suggest that more primitive progenitors are more strongly influenced by the MHC restriction in the interaction with the stromal microenvironment, whereas the growth of committed progenitors is not influenced by the restriction but rather independent of the interaction with the microenvironment. This hypothesis is supported by reports of El Badri et al. [35], van den Berg et al. [36] and Dexter et al. [37]. El Badri and Good found that the isolated primitive progenitor population failed lymphohematopoietic reconstitution in the MHC-incompatible recipients but succeeded in the MHC-compatible recipients. On the other hand, van den Berg et al. have reported that the growth of committed progenitor cells is relatively independent of stromal influence, and Dexter at al. observed CFUs-S by committed progenitor cells collected from the five-week culture with the MHC-incompatible stromal cells. In our experiments using B10 congenic strains, B10/B10.BRF1 mice receiving BMCs from B10, B10.BR or B10.A(4R) donors showed many CFU-S counts on day 12, as did syngeneic recipients. This is in contrast to the hybrid resistance [23] which is exerted by NK cells. Furthermore, the inhibition of colony formation in the MHC-incompatible recipients was not prevented by in vivo depletion of NK1.1+, CD4+ and CD8+ cells. Taken together, these results suggest that the decreased CFU-S counts on day 12 are not due to rejection but to the MHC restriction between multipotent CFCs and spleen stromal cells.

Recently, it has been found that receptors for insulin or insulin-like growth factor (IGF) internalize in the form binding with H-2D molecules [38-40]. The addition of the binding peptide of the H-2D molecules prevents the internalization of the receptors and enhances the activity of the ligands. IGF has been reported to promote the proliferation and differentiation of hematopoietic progenitors [41-45] or to stimulate stromal cells to produce hematopoietic cytokines [46]. Therefore, the trapping of H-2D molecules on the surface of progenitor (or stromal cells) by MHC-class-I-binding proteins on stromal cells (or progenitor cells) should inhibit the internalization of IGF receptors and enhance hematopoiesis. In this connection, it is of interest that in the experiments using B10.A recombinant strains, larger numbers of CFUs-S on day 12 were observed in the recipients compatible with H-2 loci, including the D locus, when compared with CFU-S counts in the recipients compatible with the other loci.

From our experiments on CFU-S and marrow repopulation in the second and third recipients, it is suggested that hematopoietic progenitor cells survive in a quiescent state for short periods in the MHC-incompatible microenvironment but should be activated when they are transferred into an MHC-compatible environment (Table 4). Van Zant et al. have reported that the eclipse of DBA/2 (H-2d) hematopoiesis is neither preceded nor accompanied by the demise of genotypically compatible stromal progenitor cells in the allophenic chimera (C57BL/6[LEFT RIGHT ARROW]DBA/2). They therefore conclude that the quiescence/activation of HSCs is independent of the stromal cell genotype [47]. However, the DBA/2 hematopoiesis reappeared when the DBA/2-eclipsed BMCs of the allophenic chimera had been transplanted into irradiated B6/D2F1 mice, which express H-2d on all kinds of stromal cells. Our present study provides a reasonable explanation for the reappearance of the DBA/2 hematopoiesis after BM transplantation.

The generation of signals after stimulation of MHC class I molecules on T cells has been demonstrated [48-50]. Recently, we have found that an MHC restriction exists between purified HSCs (5-FU-resistant, Lin, CD71, H-2Khigh, LDBMCs) [51] and stromal cells in in vitro culture (manuscript in preparation). Our experiments are therefore under way to demonstrate the generation of differentiation/proliferation signals in pluripotent HSCs after the stimulation of MHC class I molecules in vitro.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank Yuki Matsui and Roberta Hill for their expert technical assistance and Keiko Ando for her help in the preparation of the manuscript. This work was supported in part by a grant for Experimental Models for Intractable Diseases from the Ministry of Health and Welfare of Japan, and grants-in-aid for Scientific Research (02152117, 02670162, 03454177) from the Japanese Ministry of Education, Science and Culture and the Research Aid of Inoue Foundation for Science, and National Institute of Health grants AGO5627 and AI22360.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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