CD45 Congenic Bone Marrow Transplantation: Evidence for T Cell–Mediated Immunity - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:36 |只看该作者 |倒序浏览 |打印

a Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA;: t* _: S! @' g" J. W+ M

b Department of General and Thoracic Surgery, University of Kiel, Kiel, Germany

Key Words. Hematopoietic microenvironment ? Niche ? Nonmyeloablative conditioning ? CD45 ? Congenic bone marrow transplantation ? Chimerism& \+ R7 {; @# z3 X2 U! ?% d" X: l

Correspondence: Suzanne T. Ildstad, M.D., University of Louisville, 570 South Preston Street, Suite 404, Louisville, Kentucky 40202-1760, USA. Telephone: 502-852-2080; Fax: 502-852-2079; e-mail: stilds01@louisville.edu! e4 _3 ]; D* P. I5 ]0 H

ABSTRACT
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The morbidity associated with ablative conditioning has prevented the widespread application of bone marrow transplantation (BMT) to induce tolerance and treat a wide variety of autoimmune diseases and hemoglobinopathies. Nonmyeloablative conditioning has significantly reduced the risk-to-benefit ratio of BMT . Most nonmyeloablative conditioning approaches use a combination of immunosuppressive agents and low-dose irradiation , both of which act non-specifically. To further reduce the toxicity of conditioning, the mechanisms regulating engraftment must be understood. Whether the primary role of conditioning is to make space rather than control host-versus-graft alloreactivity or enhance competition remains controversial .
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Resistance to engraftment of transplanted bone marrow is mediated by immune responses. In previous studies, we characterized the cells in the recipient hematopoietic micro-environment that prevent allogeneic marrow engraftment using different T cell–deficient mice as recipients. Major histocompatibility complex (MHC)–disparate allogeneic bone marrow cells (BMCs) engrafted more readily in mice lacking ?-TCR , ?-TCR  plus -TCR , or CD8  cells, but not in mice lacking -TCR  or CD4  cells, suggesting that ?-TCR  and CD8  T cells in the host played critical and nonredundant roles in preventing engraftment of allogeneic bone marrow . Similarly, targeting host T cells with anti-?-TCR or CD8 decreased the requirement of total body irradiation (TBI) to establish allogeneic chimerism . Additionally, conditioning of mice with a single dose of cyclophosphamide (CyP) on day-2 enhanced engraftment, most likely by targeting alloreactive host cells responding to donor alloantigen . Taken together, these data support the fact that the major role of conditioning is to control host-versus-graft reactivity rather than to make space.

The CD45.1/CD45.2 model has been used extensively to characterize engraftment in the absence of alloreactivity . Notably, skin grafts from CD45.1 donors are not rejected by CD45.2 recipients , and this model was therefore used as a tool to study engraftment in the absence of alloreactivity. However, a recent report has challenged that paradigm and demonstrated that there is a weak immune response to BMC engraftment when the only disparity is between the CD45.1 and CD45.2 antigens. Although host T cells were hypothesized by the authors to be involved in the immunological resistance against the stem cells, this was not conclusively demonstrated . In the present studies, we have confirmed the findings of van Os et al.  and have specifically demonstrated that host CD8  and ?-TCR  cells are the effector cells for this reactivity. We first reproduced the work of others using the CD45.1/CD45.2 congenic model. Surprisingly, although high doses of BMCs established chimerism with little or no conditioning and there was a linear relationship between cell dose and level of chimerism, chimerism was durable only in recipients conditioned with  150 cGy TBI. Unconditioned knockout (KO) mice lacking ?-TCR  T cells or CD8  cells engrafted more readily and with significantly higher levels of donor chimerism compared with wild-type controls. In contrast, mice deficient in only CD4  or -TCR  T cells showed no increase in engraftment. Unlike wild-type recipients, the level of donor chimerism increased significantly over time in the mice that engrafted, with the most notable increases occurring in mice lacking ?-TCR  T cells and the CD4–/–/8–/– recipients. Because donor cells of all hematopoietic lineages were detected in recipient KO mice, durable chimerism was not merely attributable to homeostatic proliferation of the missing lineages. These results suggest that host CD8  and ?-TCR  cells play a significant role in the regulation of hematopoietic stem cell (HSC) engraftment in this congeneic model and confirm that low-level T cell–mediated reactivity against HSCs is present in this strain combination. As such, caution must be exercised in the interpretation of data using this model as it relates to mechanism of engraftment.

MATERIALS AND METHODS
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Level of Engraftment of CD45.1 BMCs Is Directly Correlated with Cell Dose and Amount of TBI Administered

To establish our model, increasing numbers of congenic (CD45.1) B6 BMCs were transplanted into unconditioned B6 (CD45.2) mice. A strict linear relationship between cell dose and percentage of donor chimerism was observed (Fig. 1). The level of chimerism was 0.4 ± 0.1% at 1 month after transplantation for animals receiving 15 x 106 BMCs. When the cell dose was increased to 50 x 106 (3.3-fold), the chimerism increased 3.3-fold. Similarly, when recipients were transplanted with 75 x 106 cells, a fivefold increase in level of donor chimerism occurred, compared with the level for 15 x 106 cells. The correlation factor between 15 x 106 and 100 x 106 cells was 6.7. The expected level of chimerism with a linear correlation between cell dose and level of engraftment would be 2.7% when 100 x 106 cells are transplanted, and the observed level was 2.6 ± 0.2%. When the cell dose was increased twofold, the level of chimerism doubled. This pattern for engraftment differs from that for nonmyeloablatively conditioned recipients of MHC-disparate bone marrow, in which an abrupt transition to 100% engraftment at significantly higher doses of TBI is observed ./ H& L. d7 ^1 P, N9 w/ y1 Y4 M7 ^

Figure 1. Cell dose and level of chimerism are directly correlated in the absence of conditioning. Unconditioned B6 recipients (CD45.2) were transplanted with increasing doses of congenic B6 (CD45.1) bone marrow cells. Engraftment was assessed by flow cytometric analyses of peripheral blood lymphocytes. Data represent average percent donor chimerism ± standard deviation from 6 to 12 recipients in each group 1 month after transplantation.
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Although significant levels of chimerism were present in the first months after transplantation, engraftment at levels >0.1% donor was not reliably maintained in any of the treatment groups, irrespective of cell dose. In recipients of 15 x 106 BMCs, only one of five of the animals maintained durable engraftment at 12 months after transplantation (0.7% donor). In the recipients of 50 x 106 BMCs, none (zero of six) of the recipients followed long-term maintained durable engraftment. Only two of six recipients of 100 x 106 BMCs were chimeric at 1 year.4 \: m% x% L1 E$ d$ Q, z  |

To evaluate the influence of low-dose TBI on engraftment, B6 (CD45.2) mice were conditioned with increasing amounts of TBI (50, 100, and 150 cGy) and transplanted with 5, 10, or 15 x 106 BMCs from B6 (CD45.1) donors. The percent donor engraftment was directly correlated with cell dose for a given dose of TBI, ranging from 0.4 ± 0.1% to 7.6 ± 1.5% when 5 x 106 cells with 50 cGy TBI versus 15 x 106 cells with 150 cGy TBI were infused. The degree of conditioning even more significantly influenced the level of chimerism for each given cell dose (Fig. 2). However, sustained long-term engraftment in 100% of recipients occurred in only those recipients given 150 cGy TBI and 15 x 106 BMCs. Only some animals from each of the other groups maintained their chimerism 8 months (Table 1)., x" {7 t: v  Q2 q) q# {

Figure 2. Low-dose TBI and cell number are directly correlated with engraftment. B6 (CD45.2) recipients were conditioned with increasing amounts of TBI (50, 100, or 150 cGy) and transplanted with 5, 10, or 15 x 106 bone marrow cells from CD45.1 donors. Data represent average donor chimerism ± standard deviation of six recipients in each group 1 month after bone marrow transplantation from three experiments. Abbreviation: TBI, total body irradiation.

Table 1. The influence of total body irradiation (TBI) dose on engraftment of syngeneic bone marrow when physiological cell doses are transplanteda

Cyclophosphamide Enhances the Engraftment of CD45.1 Congenic Bone Marrow
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The addition of a single dose of CyP (day 2) to a TBI-based partial conditioning regimen for allogeneic BMT allows the TBI dose to be significantly reduced , most likely by targeting alloreactive T cells in the host. We next evaluated whether CyP also enhances the engraftment of CD45 disparate bone marrow. Recipient mice were given 0, 100, or 200 cGy TBI and transplanted with 15 x 106 congenic BMCs. A significantly higher percentage of donor chimerism was achieved in animals conditioned with CyP (p
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Figure 3. The effect of CyP on engraftment in congenic BMT. Recipient CD45.2 mice were conditioned with 0, 100, or 200 cGy TBI and transplanted with 15 x 106 CD45.1 bone marrow cells. Recipients were treated with CyP 2 days after BMT. Controls were conditioned with TBI but did not receive CyP. Data represent average percent donor chimerism ± standard deviation of five to six recipients in each group 1 month after BMT from two experiments. Abbreviations: BMT, bone marrow transplantation; CyP, cyclophosphamide; TBI, total body irradiation.; J, f, ]7 \3 j; t+ O! O

Host ?-TCR T Cells Play an Important Role in Alloreactivity to CD454 ?7 f( I1 l* C! m- z! {0 _

To evaluate the relative contribution of specific host cellular subpopulations on engraftment, mice defective in the production of different T-cell subsets were used as recipients. Engraftment was significantly enhanced in recipients deficient in ?-TCR  T cells (Fig. 4). The levels of donor chimerism in TCR?–/– recipients and TCR?–/–/–/– recipients were 4.0 ± 0.8% and 3.9 ± 0.3% 1 month after transplantation, respectively. Both groups exhibited significantly higher (p 5 G# p$ C: E2 ]
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Figure 4. Congenic engraftment in B6 TCR?–/–, TCR–/–, or TCR?–/–/–/– mice. Unconditioned TCR?–/–, TCR–/–, and TCR?–/–/–/– (CD45.2) mice were transplanted with 15 x 106 CD45.1 bone marrow cells. (A): Level of donor chimerism at 1 month after transplantation. (B): Kinetics of donor chimerism at up to 8 months. Data represent average percent donor chimerism ± standard deviation from six to eight recipients in each group from two experiments. Abbreviation: BMT, bone marrow transplantation.
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Host CD8  Cells Are Important in Resistance
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We then evaluated engraftment patterns in recipients deficient in CD8, CD4, or CD8 plus CD4 cell populations. Engraftment was significantly enhanced in CD8–/– recipients (1.5 ± 0.5%; p = .002) and CD4–/–/8–/– recipients (3.2 ± 0.6%; p = .0005), but not in CD4–/– recipients (0.73 ± 0.5%; p = .40) compared with wild-type control mice 1 month after transplantation (Fig. 5A). These data suggest a role for host CD8  cells in resistance to CD45 congenic marrow engraftment.

Figure 5. CD45.1 BMC engraftment in CD45.2 CD4, CD8, or CD4–/–/8–/– mice. Unconditioned CD8–/–, CD4–/–, or CD4–/–/8–/–mice were transplanted with 15 x 106 CD45.1 BMCs. The level of chimerism was assessed by flow cytometric analysis. (A): Level of donor chimerism at 1 month. (B): Kinetics of syngeneic donor chimerism up to 10 months after transplant. Data represent average percent donor chimerism ± standard deviation from five to eight recipients in each group in three experiments. Abbreviations: BMC, bone marrow cell; BMT, bone marrow transplantation.- O  p5 m6 z* ]* |
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When we evaluated the distribution of T-cell subpopulations in B6 wild-type mice, the percent of marrow ?-TCR , -TCR , CD4 , and CD8  cells in the lymphoid gate was 3.4 ± 0.6%, 1.0 ± 0.2%, 1.9 ± 0.7%, and 1.5 ± 0.6%, respectively (Fig. 6). The percent of ?-TCR  T cells was significantly higher than -TCR  T cells in marrow (p   W5 t2 V3 L- O3 w5 l- w% q7 c) w

Figure 6. Phenotypic analysis of the T-cell populations in the BM microenvironment in wild-type B6 (CD45.2) mice. BM and PB were collected from 6- to 8-week-old unmanipulated wild-type B6 mice. The phenotypic analysis of the T-cell populations was performed by flow cytometry. The percentage of each T-cell population was analyzed from the lymphocyte gate of BM or PB. Data shown are average percent of T-cell subpopulations ± standard deviation of four individual wild-type B6 mice. Abbreviations: BM, bone marrow; PB, peripheral blood.$ ^% ?0 ?5 z: C9 K' ~2 E! |5 w
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Kinetics of Chimerism in CD8–/–, CD4–/–/8–/–, TCR?–/–, andTCR?–/–/–/– Recipients

Chimerism was durable for 8–10 months in the engrafted KO recipients. Strikingly, in contrast with B6 controls, the level of donor chimerism increased significantly with time, especially in the CD4–/–/8–/–, TCR?–/–, and TCR?–/–/–/– recipients (Figs. 4B, 5B). Although chimerism was reliably present in the CD8–/– recipients, the level remained significantly lower than the CD4–/–/CD8–/– group. In contrast, donor chimerism was only transient in TCR–/– and control B6 recipients, and 60% of CD4–/–recipients did not exhibit durable chimerism. The level of donor chimerism in CD8–/– recipients increased twofold at 2 months (2.7 ± 0.6%; p = .0009) compared with 1 month and remained stable for 8 months (Fig. 5B). The level of donor chimerism increased 3.2-fold from 1 month to 2 months after BMT in TCR?–/–, TCR?–/–/–/–, and CD4–/–/8–/–mice and then continued to increase (Figs. 4B, 5B). The level of donor chimerism at 1 month versus 2 months was 4.0 ± 0.8% versus 12.7 ± 1.5% for TCR?–/– mice (p

Multilineage Chimerism Occurs in Engrafted Mice

To evaluate whether the increase in donor chimerism was attributable to homeostatic normalization, multilineage typing was performed on PB in TCR?–/– (n = 3), TCR?–/–/–/– (n = 3), CD8–/– (n = 5), and CD4–/–/8–/– recipients (n = 5) 3–4 months after reconstitution. Donor-derived B cells, T cells, natural killer (NK) cells, macrophages, and granulocytes were present in all animals, making homeostatic expansion an unlikely explanation (Table 2).0 D, V9 j8 l+ O# b
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Table 2. Multilineage engraftment of donor hematopoietic stem cell in knockout mice after syngeneic engraftment
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Anti-?-TCR mAb Pretreatment Enhances Engraftment

To confirm a role for ?-TCR  and CD8  host cells in regulating engraftment, recipient wild-type mice were conditioned with 200, 100, or 0 cGy of TBI and pretreated with anti-?-TCR or anti-CD8 mAb in vivo. Notably, depletion of ?-TCR  T cells in wild-type recipients was also associated with enhanced engraftment (Fig. 7). Depletion of host CD8  cells did not enhance engraftment. However, the anti-?-TCR mAb provided a more efficient depletion of ?-TCR  cells in vivo (data not shown), whereas anti-CD8 treatment did not effectively remove the CD8dim cells from PB or marrow (Fig. 8A).

Figure 7. B6 mice (CD45.2) were preconditioned with anti-CD8 or anti-?-TCR monoclonal antibody in vivo and varying doses of TBI followed by infusion of CD45.1 bone marrow cells on day 0 relative to TBI. Engraftment was assessed at 28 days. Data represent five to six animals from two experiments. Abbreviation: TBI, total body irradiation.

Figure 8. Host CD8dim lymphocytes were not removed by host anti-CD8 treatment in vivo. B6 mice were preconditioned with anti-CD8 mAb in vivo. To document depletion, peripheral blood was obtained 3 days after mAb treatment from treated mice and stained with FITC-conjugated anti-CD8 (53–6.7). Staining was also performed with secondary mAb of mouse anti-rat immunoglobulin G2a–FITC to assure that cells were depleted or coated with mAb. The bright CD8  population was depleted, but there was a CD8dim population comprising 2% of the lymphoid gate that could not be removed by increasing amounts of mAb (A). These CD8dim cells were then stained for ?-TCR, CD4, B220, NK1.1, CD11b, or CD11c expression and analyzed by flow cytometry (B). Profile is representative of three experiments.Abbreviations: FITC, fluorescein-isothiocyanate; FSC, forward scatter; mAb, monoclonal antibody.

To define the composition of the residual CD8dim population, B6 mice were pretreated with anti-CD8 mAb and the phenotype of the CD8dim cells was determined. The residual cells were CD11c–, CD11b–, and NK1.1– but ?-TCR , CD4 , and B220  in both blood and marrow (Fig. 8B).
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CD4–/–/8–/– Mice Do Not Produce NK Cells

NK cells have been demonstrated to contribute to BMC rejection . We therefore examined the various strains for the production of NK cells with the rationale that this may be underlying the difference in chimerism between the strain combinations. PB lymphocyte from the CD8–/–, TCR?–/–, TCR?–/–/–/–, and CD4–/–/8–/– mice was analyzed for production of NK cells. Interestingly, CD4–/–/8–/– mice do not produce NK cells, whereas the others do at levels similar to wild-type controls (Fig. 9). Therefore, it is possible that the absence of NK cells in these mice may explain the enhanced advantage of wild-type donor marrow.! j1 [9 v! z" J" M7 C! @! j7 k; H
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Figure 9. Peripheral blood lymphocytes from CD4–/–/8–/–, CD8–/–, TCR?–/–, and TCR?–/–/–/– mice were analyzed for the presence of NK cells by flow cytometry (mean ± standard deviation from two experiments with three to six animals per group). Abbreviation: NK, natural killer.& B; y- f$ b/ x. e* Y3 n; K) N

DISCUSSION; e5 ^8 v' Z6 F2 V) R( k( }" L1 n  n" m

The authors thank Dr. Francine Rezzoug, Dr. Lee Grimes, and Dr. Isabelle Fugier for review of the manuscript and helpful comments; Carolyn DeLautre and Kim Nichols for manuscript preparation; and the staff of the animal facility for outstanding animal care. This research was supported in part by NIH DK52294 and R01 HL63442-01A2, The Commonwealth of Kentucky Research Challenge Trust Fund, The Jewish Hospital Foundation, and the University of Louisville Hospital.) u( s0 M% l- [; S( K

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