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CD8⁺, -TCR⁺, and -TCR⁺ Cells in the Recipient Hematopoietic Environment Mediate Resistance to Engraftment of Allogeneic Donor Bone Marrow¹

Hong Xu^2,*, Beate G. Exner^2,, Daniel E. Cramer^*, Michael K. Tanner^*, Yvonne M. Mueller and Suzanne T. Ildstad^3,*

^* Institute for Cellular Therapeutics, University of Louisville, Louisville, KY 40202; Department of General and Thoracic Surgery, University of Kiel, Kiel, Germany; and Department of Microbiology and Immunology, Medical College of Pennsylvania, Hahnemann University, Philadelphia, PA 19129

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of -TCR⁺, -TCR⁺, - plus -TCR⁺, CD8⁺, or CD4⁺ cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4⁺ T cells. Mice lacking both - and -TCR⁺ cells engrafted without conditioning, suggesting that both - and -TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8^- cell was targeted by the CyP conditioning. The CD8⁺ cell effector function is mechanistically different from that for conventional T cells, and independent of CD4⁺ T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The replacement of a failing organ with a functional one has become a clinical reality. However, organ transplantation currently relies upon the chronic use of nonspecific immunosuppressive agents to promote graft survival. The use of these agents is associated with opportunistic infection (1), an increased rate of malignancy (2), and end organ toxicities (3, 4), including nephrotoxicity and hepatotoxicity. Although these agents successfully prevent acute rejection, chronic rejection remains the primary cause of late graft loss (5). The five-year survival for renal allografts is 74% (6), cardiac transplants 68%, and pulmonary allografts 43% (7).

Mixed allogeneic chimerism has been suggested as a potential approach to induce tolerance to solid organ allografts (8). Mixed chimerism has the following advantages: 1) a relative resistance to graft-vs-host disease (GVHD)⁴ (3, 9); 2) superior immunocompetence (10, 11); 3) it can be established with partial myeloablative conditioning approaches (10, 12, 13); and 4) it prevents the development of chronic rejection in allografts (14).

Approaches to establish mixed chimerism with partial conditioning have been successfully pursued. Most conditioning approaches have used a combination of irradiation, other myelotoxic agents, and nonspecific immunosuppression (15, 16, 17, 18, 19, 20, 21, 22). We previously developed a total body irradiation (TBI)-based model to establish durable multilineage mixed chimerism in MHC plus minor Ag disparate recipients (12). Seven hundred centigray of TBI alone was required for 100% engraftment of MHC-disparate allogeneic bone marrow. The posttransplant administration of cyclophosphamide (CyP) (day +2) significantly reduced the minimum dose of TBI sufficient to establish mixed chimerism in 100% of recipients to 500 cGy (12). The addition of anti-lymphocyte globulin (ALG) (day -3) to the peritransplant-conditioning further reduced the minimum TBI dose to 200 cGy (23). We hypothesized that CyP and ALG removed activated alloreactive host cells from the repertoire. In the present study, we have characterized which host effector cells mediate alloresistance for engraftment of allogeneic bone marrow using mice genetically modified to lack production of specific cell types (knockout (KO) mice). We report for the first time that -TCR⁺ as well as -TCR⁺ T cells play critical and nonredundant roles in alloresistance to engraftment. Their mechanism of action was CyP, and to a slightly lesser extent, radiation-sensitive, because mice that did not produce - and -T cells did not require any conditioning, while mice lacking only -T cells or only -T cells required low-dose TBI plus CyP to engraft. In addition, CD8⁺ cells in the recipient exert a CD4-independent effect, as shown by requirement for significant conditioning for CD4 KO mice to engraft. A better understanding of the mechanism of engraftment and the role of host conditioning will enable development of cell-specific partial conditioning approaches associated with less toxicity than the nonspecific agents currently in use.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Transplant donor, B10.BR.SgSnJ (B10.BR; H-2^k), and various recipient mice: C57BL/6J (B6; H2^b), C57BL/6-Tcrb^tmlMom (TCR- KO, as -TCR T cell-deficient recipients), C57BL/6-Tcrd^tmlMom (TCR- KO, as -T cell-deficient recipients), C57BL/6-Tcrb^tmlMom Tcrd^tmlMom (TCR-/TCR- double KO, as - and -T cell deficient recipients), C57BL/6-Cd8^Tm/mak (CD8 KO), C57BL/10-Cd4^tml (CD4 KO), and C57BL/6J-CD4^tm/mak (CD4 KO) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a special pathogen-free barrier facility at the Institute for Cellular Therapeutics (Louisville, KY). Mice were cared for according to National Institutes of Health animal care guidelines.

Chimera preparation

Bone marrow was prepared from B10.BR donor mice as previously described (8). Briefly, B10.BR donor mice were euthanized and tibias and femurs were harvested. Bone marrow was expelled from the bones with Media 199 (Life Technologies, Grand Island, NY) containing 10 µg/ml Gentamicin (Life Technologies), referred to thereafter as chimera media (CM). The cells were filtered through sterile nylon mesh with 100 µm pores, centrifuged at 1000 rpm for 10 min at 4°C, and resuspended in CM. A cell count was performed, and the cells were diluted to a final concentration of 15 x 10⁶ bone marrow cells per 1 ml of CM.

Recipient mice were treated with 0–300 cGy TBI from a cesium source (Gamma-cell 40, Nordion, Ontario, Canada). Animals were transplanted with 1 ml CM containing 15 x 10⁶ B10.BR bone marrow cells via lateral tail vein injection within 4–6 h after irradiation. All animals in groups treated with CyP received a single i.p. injection of 200 mg/kg CyP (Sigma Aldrich, St. Louis, MO) 48 h after bone marrow transplantation (BMT). Each experiment was repeated at least three times.

Flow cytometric analysis

The level of hematopoietic chimerism was assessed monthly after BMT by flow cytometric analysis of PBL using mAb against MHC Ags of donor and host origin. Fifty microliters of whole blood obtained by tail-bleeding of the mice were incubated at room temperature for 8 min with lysing buffer (8.29 g of NH₄Cl, 1.0 g of KHCO₃, and 0.0372 g Na₂EDTA in 1 liter H₂O; prepared in our laboratory) to eliminate RBC. The leukocytes were then incubated with 10 µl diluted mAb for 30 min at 4°C in the dark. The appropriate dilution for the use of the mAb was determined in titration experiments before use. The cells were washed twice with 2 ml of FACS medium (0.36 g NaHCO₃, 1.0 g NaN₃, and 1.0 g BSA in 1 liter HBSS; prepared in our laboratory) and centrifuged at 1000 rpm for 10 min at 4°C. Finally, the cells were fixed with 1% paraformaldehyde in PBS (prepared in our laboratory). The analysis was conducted on a FACSCalibur (BD Biosciences, Mountain View, CA) with CellQuest software (BD Biosciences).

mAbs

Abs specific for MHC class I Ags of donor FITC-conjugated anti-H2K^k, (36-7-5, mouse IgG1) and recipient PE-conjugated anti-H2K^b (AF6-88.5, mouse IgG2a) origin were used to determine the percentage of donor cell chimerism in the recipient’s peripheral blood. Multilineage engraftment was assessed by staining with biotinylated anti-H2K^k (36-7-5, mouse IgG1), FITC-conjugated anti-H2K^b (AF6-88.5, mouse IgG2a) and PE-conjugated lineage markers 3 mo after BMT. The biotinylated Ab was counterstained with streptavidin-allophycocyanin. The following Abs were used as lineage markers: anti-GR-1 (RB6-8C5, rat IgG2b), anti-MAC-1 (M1/70, rat IgG2b), anti-CD4 (RM4-5, rat IgG2a), anti-CD8 (53-6.7, rat IgG2a), anti-B220 (RA3-6B2, rat IgG2a), anti-NK1.1 (PK136, mouse IgG2a), anti-TCR--chain (H57-597, hamster IgG), and anti--TCR (GL3, hamster IgG). NK subpopulations were assessed by four-color staining in naive B6, TCR- KO, TCR- KO, TCR-/TCR- KO, and CD8 KO mice with FITC-conjugated anti-TCR-/TCR-, PerCP-conjugated anti-CD3e (145-2C11, hamster IgG), APC-conjugated anti-CD8a, and PE-conjugated NK subpopulation markers. The following Abs were used as NK subpopulation markers: anti-NK1.1, anti-5E6 (5E6, mouse IgG2a), anti-2B4 (2B4, mouse IgG2b), and anti-DX5 (DX5, rat IgM). Nonspecific background staining was controlled by using isotype control Abs directed against irrelevant Ags conjugated with the same color as the experimental Ab (i.e., anti-TNP mouse IgG2a Ags conjugated with PE served as isotype control for PE-conjugated anti-H2K^b mouse IgG2a). All mAb were obtained from BD PharMingen (San Diego, CA). Streptavidin-allophycocyanin was purchased from BD Biosciences.

Assessment of GVHD

The primary diagnosis of GVHD was based on previously described clinical criteria, which consist of diffuse erythema (particularly of the ear), hyperkeratosis of the foot pads, hair loss, weight loss, unkempt appearance, or diarrhea (24). At the time of sacrifice, sections of skin, tongue, liver, and small intestine were fixed in 10% buffered formalin, stained with H&E, and processed for light microscopy.

Skin grafting

Skin grafting was performed by a modification of the method of Billingham (25). Full-thickness tail-skin grafts were harvested from the tails of B10.BR and nonobese diabetic (NOD) (H2K^d) mice. Recipient mice were anesthetized with Nembutal (pentobarbital sodium injection; Abbott Laboratories, Abbott Park, IL), and full-thickness graft beds were prepared surgically in the lateral thoracic wall, while preserving the panniculus carnosum. The grafts were covered with a double layer of Vaseline gauze and a plaster cast. Casts were removed on day 7, and grafts were scored by daily inspection for the first month, then weekly thereafter for percentage of rejection. Rejection was defined as complete when no residual viable graft could be detected.

Statistical analysis

Statistical significance was determined with a Student’s one-way t test. The difference between groups was considered to be significant if p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR-, TCR-/TCR- and CD8 KO mice require less TBI for engraftment

Conditioning with 500 cGy TBI followed by CyP treatment (day +2 relative to marrow infusion) is sufficient to establish durable chimerism in MHC-disparate as well as MHC- plus minor Ag-disparate recipients (12). The administration of CyP before transplantation is not sufficient to establish engraftment with 500 cGy TBI. We hypothesized that the administration of CyP on day +2 removes alloreactive host T-lymphocytes that have been activated by the marrow infusion. To evaluate which host cells play a role in engraftment, mice deficient in production of -TCR⁺, -TCR⁺, both -TCR⁺ and -TCR⁺, CD8⁺, and CD4⁺ cells were used as recipients. All are H-2^b in MHC (B6). A total of 15 x 10⁶ bone marrow cells from B10.BR donor mice were administered. One hundred percent of TCR-/TCR- KO recipients engrafted after conditioning with 300 cGy TBI plus CyP (n = 8) (Fig. 1A). The level of chimerism was 41.8 ± 1.2% 1 mo following BMT (Fig. 1B). Similarly, 100% of TCR- KO mice engrafted after conditioning with 300 cGy TBI plus CyP (n = 14; Fig. 1A). The level of chimerism was similar to that for the TCR-/TCR- KO recipients (42.5 ± 14%) (Fig. 1B). The engraftment was durable and multilineage. Similarly, engraftment occurred in 100% of CD8 KO mice conditioned with 300 cGy TBI plus CyP (n = 16; Fig. 1A). The level of chimerism was 48.7 ± 18.1% at 1 mo posttransplantation. At 3 mo, donor chimerism was 30.3 ± 8.4%, and remained stable for 6 mo. In striking contrast, only five of nine (56%) -TCR KO recipients engrafted, and the level of chimerism was significantly lower in mice that engrafted (14.5 ± 4.3%) compared with the TCR- KO (p < 0.005) and TCR-/TCR- double KO mice (p < 0.005) (Fig. 1B). CD4 KO mice conditioned in a similar fashion did not engraft (n = 20), suggesting that host CD8⁺ cells and - and -TCR⁺ T cells play a major role in alloresistance to engraftment, while CD4⁺ cells do not. Moreover, the effector cells in alloresistance did not require CD4⁺ cells, because CD4 KO mice require significant levels of conditioning for engraftment. As expected, B6 control mice did not engraft when conditioned, and transplanted in a similar fashion (n = 6; Fig. 1A).

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Characteristics of engraftment in KO mice conditioned with TBI plus CyP. B6 control mice and mice deficient in the production of -TCR+ cells (TCR- KO), -TCR+ cells (TCR- KO), both - and -TCR+ cells (TCR-/TCR- double KO), CD4 (CD4 KO), or CD8 (CD8 KO) were conditioned with 300 cGy TBI, transplanted with 15 x 106 bone marrow cells from B10. BR donors, and given 200 mg/kg CyP (i.p.) on day +2. This figure shows the frequency of engraftment (A) and level of chimerism in animals that engrafted (percentage of donor cells in PBL) 1 mo (B) and >3 mo after (C) as assessed by PBL typing. The results are the summary of three experiments.

Mice lacking - and -T cells do not require CyP conditioning for engraftment

It is hypothesized that CyP on day +2 relative to the marrow infusion removes alloreactive T lymphocytes that have been activated by alloantigen (17). To evaluate the contribution of CyP to the conditioning approach and further define which host cells were the target on the day +2 CyP infusion, TCR- KO, TCR- KO, TCR-/TCR- KO, and CD8 KO mice were conditioned with 300 cGy of TBI and transplanted with 15 x 10⁶ B10.BR bone marrow cells (Fig. 2). Neither the TCR- KO (n = 6), the TCR- KO (n = 6), nor CD8 KO (n = 4) mice engrafted. A total of 100% of the TCR-/TCR- KO mice engrafted (n = 6). The level of chimerism was 76.1 ± 10.4% 1 mo post-BMT and was durable for 6 mo. Therefore, these data suggest that recipient -TCR⁺ T cells as well as -TCR⁺ T cells contribute to alloresistance to engraftment, and support the hypothesis that alloreactive T cells are the primary target for the CyP conditioning in wild-type recipients.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Characteristics of engraftment in KO mice conditioned with TBI alone. To evaluate which cell subsets were sensitive to CyP in normal recipients, CD8 KO, TCR- KO, TCR- KO, and TCR-/TCR- KO mice were conditioned with 300 cGy TBI alone. Recipients were transplanted with 15 x 106 bone marrow cells from B10. BR donors on the same day with TBI. The figure shows the percentage of animals that engrafted 1 mo post-BMT. The results are the summary of three experiments.

TCR-/TCR- KO mice engraft with minimal conditioning

A dose-titration of TBI was performed to determine the minimal conditioning required in mice deficient in production of - and -TCR⁺ T cells. A total of 100% of mice conditioned with 200 cGy (n = 6) or 100 cGy (n = 7) of TBI engrafted (Fig. 3A). Eighty-six percent of TCR-/TCR- double KO mice engrafted without any conditioning (n = 7) (Fig. 3A). The level of chimerism was directly correlated with the degree of conditioning (Fig. 3, B–D). The engraftment in all groups was durable for 5 mo.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. The influence of TBI dose on engraftment and the level of chimerism in TCR-/TCR- double KO mice. TCR-/TCR- KO mice were transplanted with 15 x 106 bone marrow cells from B10. BR donors after conditioning with TBI doses ranging from 0 to 300 cGy on day 0. Animals were typed by flow cytometric analysis monthly for up to 5 mo after BMT. n = number of animals in each experiment. The percentage of animals with engraftment at 1 mo (A), and the level of donor chimerism at 1 mo (B), 3 mo (C), and 5 mo (D) post-BMT are shown. The results are the summary of three experiments.

Conditioning in CD8 KO mice requires CyP more than TBI

CD8 KO mice were transplanted with 15 x 10⁶ bone marrow cells from B10.BR donors on day 0. A total of 300, 200, 100, or 0 cGy TBI were administered 4–6 h before the marrow infusion on day 0. A single dose of CyP was administered on day +2. Engraftment was present at 1 mo in 100% of recipients including those that received no TBI (Fig. 4A). The level of chimerism was proportional to the dose of TBI (Fig. 4B). Engraftment was durable 6 mo in all animals conditioned with 300 (n = 16), 200 (n = 6), and 100 (n = 6) cGy of TBI. The engraftment was multilineage for donor-derived T cells, B cells, NK cells, macrophages, and granulocytes. Half of the CD8 KO mice conditioned with only CyP (n = 6) lost their chimerism by 4 mo. CD8 KO mice did not engraft with 300 cGy TBI only, but 50% exhibited durable engraftment without TBI. If CyP were used, these data suggest that the CD8⁺ cell population mediating alloresistance is more sensitive to TBI than to CyP. Therefore, these data also support that a CyP-sensitive host cell in CD8 KO mice that is probably not a conventional -TCR⁺ T cell also contributes to alloresistance.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. TBI dose-titration in CD8 KO mice conditioned with posttransplant CyP. CD8 KO mice were conditioned with TBI doses ranging from 0 to 300 cGy on day 0 and transplanted with 15 x 106 allogeneic B10. BR bone marrow cells 4–6 h after. A total of 200 mg/kg CyP was administered 2 days after BMT. Chimerism was analyzed by flow cytometry 1 mo (B) and 6 mo (C) after BMT. The results are the summary of three experiments.

The pluripotent hematopoietic stem cell (HSC) produces at least 11 different lineages. To confirm that the engraftment in TCR-/TCR- double KO recipients reflects engraftment of the pluripotent stem cell, animals were followed for 3 mo. The engraftment in TCR-/TCR- double KO mice conditioned with 300 cGy irradiation and administration of CyP on day +2 was durable (Fig. 1C) and multilineage. Three-color flow cytometric analysis showed the presence of multiple myeloid and lymphoid lineages of donor and host origin in all engrafted animals (Table I, group A). The T cell lineages deficient in the KO animals were restored after transplant recipients, and were all of donor origin. Similarly, TCR-/TCR- KO mice conditioned with 300 cGy TBI without CyP (Table I; group B) as well as CD8 KO recipients conditioned with 200 cGy TBI plus CyP (Table I; group C) were evaluated. In all groups, donor-derived T cells, B cells, NK cells, macrophages, and granulocytes were present, suggesting engraftment of the HSC itself rather than selected lineages.

View this table: [in this window] [in a new window]   Table I. Multilineage engraftment in TCR-/TCR- KO and CD8 KO mice1

Skin grafting was performed to assess donor-specific tolerance in vivo. Five unmanipulated TCR-/TCR- KO mice (H2^b) received full-thickness skin grafts of both B10/BR (H2^k) and NOD (H2^d) origin. All grafts survived >160 days, demonstrating that naive TCR-/TCR- KO mice do not reject skin allografts. Both donor-specific (B10.BR) and MHC-disparate third-party (NOD) skin grafts were placed on the chimeric TCR-/TCR- KO mice with different levels of donor chimerism (Table II). Grafts were assessed daily for the first 4 wk and weekly thereafter for evidence of rejection. The single nonchimeric mouse accepted the donor-specific and third party skin grafts in a fashion similar to that observed in naive TCR-/TCR- KO mice. In all other recipients, donor-specific allogeneic skin grafts were accepted by the mice with chimerism (range from 2.5 to 71.3%), while third-party skin grafts were promptly rejected. Therefore, these data demonstrate that TCR-/TCR- KO recipients that engraft as chimeras exhibit donor-specific tolerance, but are immunocompetent to reject MHC-disparate third party allografts.

Characterization of lymphoid populations in marrow and spleen of TCR- KO, TCR-, TCR-/TCR- KO, and CD8 KO mice

T cells, NK cells, and NK/T cells have all been implicated in alloresistance to engraftment. A number of NK subfamilies have been described, including 5E6, 2B4 (26), NK/T cells (27, 28), and CD8⁺ NK cells (27, 29). Marrow and splenocytes from CD8, TCR-/TCR-, TCR-, and TCR- KO mice were analyzed by four-color flow cytometry to enumerate which NK subfamilies might be absent (Fig. 5). All KO mice produced NK1.1⁺ and 5E6⁺ cells in marrow and spleen at levels similar to wild-type B6 controls. Marrow from the TCR-/TCR- KO mice contained a significantly lower number of NK/T cells than B6 mice (p < 0.0019). 2B4⁺ NK cells were also significantly reduced (p = 0.0005), and CD8⁺/NK cells were virtually absent (p = 0.0046). CD8 KO mice lack CD8⁺/NK cells (Fig. 5) as well as CD8⁺ T cells, as expected (data not shown). It could be hypothesized that the NK/T subfamily present in CD8 KO mice but lacking in TCR-/TCR- KO mice may represent the CyP-sensitive cell, and may explain why CyP is not required to achieve engraftment in TCR-/TCR- KO mice.

View larger version (30K): [in this window] [in a new window]   FIGURE 5. NK subset expression in bone marrow and spleen of KO mice. NK subset expression was enumerated for bone marrow and spleen from TCR- KO, TCR-, TCR-/TCR- KO, CD8 KO mice, and the B6 control mice using four-color cytometry. NK1.1 (), 5E6 (), NK/T (NK1.1 and TCR positive; ), CD8/NK1.1 (), and 2B4 (). Each bar represents the mean of three mice and their SDs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Until recently, it was believed that full ablation of the host hematopoietic microenvironment was a prerequisite to achieve engraftment of allogeneic marrow. The discovery that multiple hematopoietic systems can coexist and function in concert in the form of mixed hematopoietic chimerism (8, 31) led to the recognition that partially ablative conditioning may be sufficient. The observation that engraftment of physiologic numbers of sygeneic bone marrow cells required low-dose conditioning (32), and that substantially more TBI was required for MHC-matched but minor Ag-disparate (32, 12), and MHC-disparate marrow (12) led to the hypothesis that more than one host mechanism is probably operational in engraftment of allogeneic marrow (12). Down et al. (32) hypothesized that the differences in radiation dose for syngeneic vs allogeneic engraftment may represent removal of specific host cell populations with differing radiosensitivities.

A number of studies have demonstrated that engraftment can be achieved using partial conditioning (22, 33, 34). To date, most protocols have used nonspecific cytoreductive immunosuppressive and/or mAb treatment. Two stages occur in the early events in engraftment: host factors that control whether the donor marrow can engraft (alloresistance), and alloreactive host cells that can reject grafts that initially engrafted. One of three outcomes can occur: 1) the graft may never take; 2) the graft may take and then be rejected or be only transient due to failure of survival of self-renewing HSC; and 3) durable engraftment may occur. We and others have shown that CD8⁺ cells play an important role in host alloresistance to engraftment (22, 35, 36). Conditioning of recipients with a combination of both anti-CD4 and anti-CD8 depleting and nondepleting mAb allows engraftment of MHC-congenic marrow (35). The addition of 600–850 cGy of TBI (13) or 300 cGy of TBI plus 700 cGy of thymic irradiation (20) is required if the donor and recipient are MHC-disparate. It was concluded from those studies that conventional thymic-derived -TCR⁺ T cells were the primary effector cells being removed by this approach (20).

In our own model of partial conditioning to achieve stable mixed chimerism, the TBI dose could be reduced from 700 to 300 cGy TBI if ALG (day -3) plus a single dose of CyP were administrated on day +2 (12, 21). If the CyP was administrated before the TBI and BMT, engraftment did not occur. If ALG was given without CyP, engraftment occurred but was only transient. Moreover, if the ALG was replaced by in vivo pretreatment of the recipient with anti-CD8 mAb, a higher level of chimerism was consistently achieved. Anti-CD4 mAb pretreatment of the recipient did not replace the ALG. One caveat to preconditioning of normal recipients with mAb is that all putative effector cells may not be coated or depleted. The presence of immunocompetent residual CD8⁺ cells after mAb treatment has been demonstrated. Rosenberg et al. (37) demonstrated that CD8⁺ cells are the main effector cells in the rejection of MHC class I disparate skin grafts. Ichikawa et al. (38) reported that skin grafts with MHC class I disparities are rejected in CD8-depleted recipients. Rosenberg et al. (39) characterized the cell populations that mediated this rejection, and found it to be mediated by a small number of residual CD8⁺ cells, despite the fact that >99% of the CD8⁺ cells had been removed. To avoid the variable of residual effector cells in our studies, we used mice that were genetically deficient in the production of these cells. Thus, no residual cells are present in these animals.

T cells have been implicated as the primary effector cells in solid organ allograft rejection. Targeting -TCR⁺ T cells significantly prolongs survival of skin grafts (40). Although the same effect could be achieved by targeting CD3⁺ T cells, animals prepared by depletion of -TCR⁺ cells demonstrated superior immunocompetence (40). Similarly, TCR- KO mice do not reject MHC-disparate cardiac allografts (41). BMT from normal B10.BR donors restores the immunocompetence to reject third-party cardiac allografts in TCR- KO mice (41). In the present studies, we have demonstrated that TCR- KO mice do not accept allogeneic bone marrow without conditioning.

The role of T cells in rejection of bone marrow grafts has not been fully defined. When Kernan et al. (42) characterized the cells present in recipients of HLA-mismatched bone marrow grafts at the time of rejection, they found that graft failure was associated with the emergence of donor-reactive T cells. Other groups report that CD2⁺, CD3⁺, and CD8⁺ T cells of recipient origin in the peripheral blood of bone marrow recipients effectively inhibit the proliferation and differentiation of donor bone marrow cells in vitro (43). CyP has been shown to deplete activated T cells (17, 21, 44). Therefore, it is likely that the engraftment-enhancing effect of CyP on day +2 is due to the effect on activated donor-reactive cells. The results of the experiments described here confirm a critical role for host -TCR⁺ as well as -TCR⁺ T cells in the resistance to engraftment of allogeneic bone marrow. Animals deficient in the production of both - and -TCR⁺ T cells are significantly enhanced in their ability to accept allogeneic bone marrow grafts compared with wild-type controls. If both host - and -TCR⁺ T cells are lacking, allogeneic marrow is accepted with little or no conditioning. Mice lacking -TCR⁺ cells alone engraft only if CyP is administered 2 days after BMT after conditioning with 300 cGy TBI, suggesting that -T cells also contribute in a significant fashion to alloresistance. Mice lacking -TCR⁺ T cells exhibit enhanced engraftment, although to a lesser extent than those lacking -TCR⁺ cells, as evidenced by the fact that only 56% engrafted after conditioning with 300 cGy TBI plus CyP. Taken together, these data confirm that host -TCR⁺ T cells as well as -TCR⁺ T cells exert a critical influence on engraftment of allogeneic marrow. This finding is supported by the fact that only mice deficient in production of - plus -TCR⁺ T cells (TCR-/TCR- KO) reliably engraft with a low TBI dose alone or even no conditioning at all, confirming that both - and -TCR⁺ cells in the host contribute in a nonredundant and critical fashion in alloresistance to engraftment. These data also refute a role for conventional NK cells as the primary effector cells in allogeneic marrow graft rejection.

In the present studies, we defined a critical role for host CD8⁺ cells in regulating engraftment. Durable, multilineage engraftment occurred in all CD8 KO mice conditioned with CyP plus low dose TBI, and in 50% of CD8 KO mice conditioned with CyP alone. The level of chimerism was directly correlated with the dose of TBI. In striking contrast, none of the CD4 KO mice conditioned with as high as 300 cGy TBI plus CyP engrafted when transplanted in a similar fashion. These results demonstrate that TBI-sensitive CD8⁺ cells in the wild-type recipient hematopoietic microenvironment play a critical role in marrow rejection. Because CD8 KO mice produce -TCR⁺ T cells and CD4⁺ -TCR⁺ T cells, it could be hypothesized that in addition to -TCR⁺ T cells a separate CD8⁺ cell in normal recipients contributes to alloresistance. The fact that TCR- KO mice required TBI plus CyP to engraft while CD8 KO mice engraft with CyP conditioning alone supports this hypothesis. Moreover, a CD8^- cell in the CD8 KO recipient that is CyP-sensitive also contributes to alloresistance, because TCR-/TCR- KO mice engraft with no conditioning while TCR- KO mice require TBI plus CyP. However, the fact that NK/T cells are present in the marrow of CD8 KO mice, although not present in TCR-/TCR- KO mice, could support a role for a subfamily of NK cells contributing to alloresistance as well. If the sole effector cell were CD8⁺/TCR⁺ T cells, it would be expected for the CD8 KO mice to have similar conditioning requirements to the TCR- KO mice.

NK cells have been implicated as playing a major role in marrow rejection (24, 45). Several subfamilies of NK cells have been described, including 5E6, 2B4, and DX5. The 5E6⁺ NK cells comprise 50% of NK cells and have been demonstrated to influence engraftment and hematopoiesis (46, 47). The fact that the NK1.1⁺ and 5E6 NK subsets are present in TCR- KO, TCR- KO, TCR-/TCR-, and CD8 KO mice at levels similar to that for B6 control mice suggests that these subfamilies probably do not play a major role in graft rejection. The fact that mice which lack - and/or -TCR⁺ T cells engraft with less conditioning strongly supports a critical role for conventional T cells rather than NK cells in alloresistance. Moreover, the fact that TCR- KO mice have no NK/T cells and still require conditioning makes it unlikely that NK/T cells contribute significantly to alloresistance to engraftment.

It is of note that CD4 KO mice did not engraft when conditioned with 300 cGy TBI plus CyP, although CD8 KO, TCR- KO, and TCR- KO mice did. The classic pathway to initiate cytotoxicity mediated by CD8⁺ T cells requires the help of CD4⁺ cells (48). However, pathways of CD4⁺ cell-independent initiation of cytotoxicity have been described. Purified CD8⁺ cells can mount cytolytic responses without CD4 mediated help in vitro (49, 50) and in vivo (51). Another CD4-independent CD8-mediated mechanism of cytotoxicity is an NK-like mechanism of alloreactivity (52). A number of groups have described an overlap between T cells and NK cells. Dennert et al. (53) have suggested that CD3⁺ NK1.1⁺ cells can develop into CD8⁺ cytotoxic T cells during acute rejection of allogeneic bone marrow grafts. Although T cell-mediated cytotoxicity usually requires activation and takes 7–8 days to generate a cytotoxic response (54), rejection via NK cells occurs within 4–5 days (54). However, the early events of alloreactivity for T cell activation take only hours after exposure to Ag (55, 56). Our data demonstrate a critical role for a CD4-independent CD8-mediated mechanism that mediates resistance to engraftment in recipients of allogeneic bone marrow. Although this could be due to T cells or NK/T cells, the fact that -TCR⁺ T cells play a significant role in alloresistance to engraftment and that TCR-/TCR- KO mice produce NK cells strongly supports a role for conventional T cells.

CyP suppresses cell-mediated immunity and induces quantitative and qualitative changes in the lymphocyte repertoire (57). The administration of CyP results in leukopenia by depletion of mononuclear cell populations. At the same time, CyP can mediate a marked decrease in the cellular cytotoxic function of the remaining cells (57). We previously confirmed that the administration of CyP in the preparative regimen enhances allogeneic engraftment if it is administered 2 days after low dose TBI and bone marrow infusion. A similar effect does not occur if the CyP is administered before marrow infusion in this model (21). It was hypothesized that the mechanism for this effect involves elimination of alloreactive T cells from the recipient during the early stages of priming. In the present studies, the fact that mice lacking - and -TCR T cells engraft with very low doses of TBI alone suggests that these cell types are two major targets removed by CyP in wild-type recipients.

The level of engraftment in TCR-/TCR- double KO mice was higher in animals conditioned with 300 cGy TBI alone as compared with animals conditioned with 300 cGy TBI and CyP. We hypothesize that this is due to the fact that proliferation of donor reactive T cell clones is triggered within 2 days after BMT, rendering these cells an optimal target for CyP. At the same time, recipient-reactive T cell clones of donor origin that are proliferating against host alloantigens will be depleted by CyP as well. Thus, it would be expected for the level of engraftment to be higher in TCR-/TCR- double KO mice when CyP is not administered. Recipient-reactive donor T cells will not be depleted in animals conditioned without CyP. This could theoretically increase the level of donor chimerism, because T cells enhance engraftment through graft-vs-host reactivity. However, when - and -TCR⁺ T cells are present in the host, CyP is essential to neutralize their alloreactivity. Alternatively, there may be a CyP-sensitive mechanism which inhibits a residual rejection mechanism in - and -T cells.

The fact that CD8 KO mice do not require TBI for conditioning suggests that a TBI-sensitive population of CD8⁺ host marrow cells also contributes to alloresistance. The target of the TBI could be CD8⁺ T cells, or alternatively, a different cell type. TBI does not completely eliminate conventional T cells from the recipient microenvironment, even at high doses. Davenport et al. (58) described that CD8⁺ T cells of host origin capable of rejecting MHC mismatched donor bone marrow persist even in ablated mice (950 cGy). The importance of this phenomenon has been shown clinically in patients with graft rejection where donor-reactive T cell clones present before conditioning re-emerged after BMT (59). In the present studies, we observed that CyP is required to prevent rejection of allogeneic bone marrow grafts in mice conditioned with 300 cGy TBI, unless the animals lack both - and -TCR⁺ T cells, in which case the requirement for CyP but not TBI is eliminated to achieve significant levels of engraftment. These data therefore confirm that donor-reactive T cells in the recipient hematopoietic environment are not completely removed by low dose irradiation.

A low dose of TBI is required for conditioning if physiologic numbers of syngeneic bone marrow cells are administrated (32). It is important to note that CD8 KO mice engraft without TBI, but do not engraft if CyP is omitted from the conditioning. The level of engraftment is proportional to the irradiation dose and in this way resembles the characteristics of syngeneic engraftment. Therefore, our data confirm that host CD8⁺ cells as well as -T cells and -T cells each play a mechanistically different role in engraftment of MHC-disparate marrow.

Our data suggest that more than one cell type mediate the rejection of fully MHC and minor Ag disparate bone marrow grafts, showing a critical role for recipient -TCR⁺ and -TCR⁺ T cells, but also implicating an additional population CD8⁺ cells in the resistance to allogeneic bone marrow grafts. It is likely that the different cell types mediate rejection by different mechanisms. Interestingly, CD8⁺ host cells reject allogeneic bone marrow in the complete absence of CD4⁺ cells, suggesting a CD4 independent mechanism. Targeting - and -TCR⁺ T cells, as well as CD8⁺ cells in the recipient may allow a specific approach to the development of cell-specific conditioning strategies to establish mixed chimerism with less toxicity. If so, mixed chimerism could be more readily applied for tolerance induction, in gene therapy and treatment of nonmalignant diseases, autoimmune diseases, and hematological disorders, such as sickle cell disease and thalassemia.

	Acknowledgments

 
We thank H. Leighton Grimes, Geoffrey P. Herzig, Thomas C. Mitchell, and Paula M. Chilton for their careful review and helpful advice during the manuscript preparation, Mukunda B. Ray for the histopathologic analyses, Theresa Perry for technical support, Carolyn DeLautre for her superb administrative assistance in preparation of the manuscript, and the outstanding animal care team at the University of Louisville.

	Footnotes

 
¹ This work was supported in part by grants from the National Institutes of Health, DK 43901-09 (to S.T.I.), the Deutsche Forschungsgemeinschaft, EX 11/1-1 (to B.G.E.), the Jewish Hospital Foundation, and the Commonwealth of Kentucky Research Challenge Trust Fund.

² H.X. and B.G.E. contributed equally in this study.

³ Address correspondence and reprint requests to: Dr. Suzanne T. Ildstad, University of Louisville, 570 South Preston Street, Suite 404, Louisville, KY 40202-1760. E-mail address: stilds01{at}gwise.louisville.edu

⁴ Abbreviations used in this paper: GVHD, graft-vs-host disease; TBI, total body irradiation; CyP, cyclophosphamide; ALG, antilymphocyte globuln; KO, knockout; BMT, bone marrow transplantation; NOD, nonobese diabetic; HSC, hematopoietic stem cell; CM, chimeria media.

Received for publication July 13, 2001. Accepted for publication November 28, 2001.

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