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(Journal of Leukocyte Biology. 2002;72:898-912.)
© 2002 by Society for Leukocyte Biology

Donor T cell and host NK depletion improve the therapeutic efficacy of allogeneic bone marrow cell reconstitution in the nonmyeloablatively conditioned tumor-bearing host

Susanne Hummel*, Daniela Wilms*, Mario Vitacolonna* and Margot Zöller*,{dagger}

* Department of Tumor Progression and Tumor Defense, German Cancer Research Center, Heidelberg; and
{dagger} Department of Applied Genetics, University of Karlsruhe, Germany

Correspondence: Margot Zöller, Department of Tumor Progression and Immune Defense, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail: m.zoeller{at}dkfz.de


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ABSTRACT
 
Allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned host has the advantage that it can be tolerated in suboptimal health conditions. However, the problem of graft versus host disease (GvHD) remains. Also, graft acceptance may become delicate, and HvGD may arise. We report here on advantages/disadvantages of host natural killer (NK) depletion and graft T cell depletion in fully allogeneic, healthy and solid tumor-bearing mice. NK depletion of the "healthy" host improved the survival rate, whereas graft T cell depletion was disadvantageous. In the tumor-bearing host, graft T cell depletion was beneficial when the host was NK-depleted. Host NK depletion facilitated B lymphopoiesis, repopulation of the thymus, expansion of donor cells, and tolerance induction. The disadvantage of graft T cell depletion in the "healthy" host was a result of delayed engraftment. Because in tumor-bearing mice, host but not graft hematopoiesis was strongly impaired, donor hematopoiesis dominated. Graft T cell depletion reduced GvHD but hardly interfered with engraftment. Importantly, graft-mediated tumor reactivity appeared late and was unimpaired when the graft was T cell-depleted. Thus, concomitant depletion of host NK and donor T cells is advantageous when approaching therapeutic treatment of solid tumors by allogeneic reconstitution of the nonmyeloablatively conditioned host.

Key Words: rodent • graft versus host disease • transplantation • carcinoma


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INTRODUCTION
 
Allogeneic bone marrow cell (BMC) transplantation is considered a potentially curative option in patients with hematological malignancies and solid tumors after exhaustion of conventional therapies [1 2 3 4 ]. In the past, allogeneic BMC transplantation was only applied in young patients in good health condition because of the high toxicity of the myeloablative pretreatment regimen [5 6 7 8 ]. Recently, it has been documented that the transfer of allogeneic BMC after nonmyeloablative pretreatment does not essentially require hospitalization and is tolerated even in poor health condition and by elderly patients [9 10 11 12 13 14 15 ]. Yet, the problem of graft rejection becomes more severe, the problem of graft versus host disease (GvHD) remains, and the question of reducing the latter while maintaining graft versus tumor (GvT) reactivity is still a matter of concern [14 , 16 17 18 19 20 21 22 ].

GvHD was originally thought to be mainly T cell-mediated, and T cell depletion has been suggested as a convenient solution [16 , 19 , 23 , 24 ]. However, engraftment can be severely impaired [25 26 27 ]. Thus, alternative strategies are being explored such as selective depletion of CD4+ cells, which is supposed to reduce GvHD, and addition of a low dose of CD8+ cells to prevent graft rejection [28 ] or the selective depletion of alloreactive T cells [29 ]; various compositions of antibodies, e.g., anti-T cell receptor-{alpha}ß, anti-CD40L, or CTLA-4Ig [30 31 32 ]; or low molecular-weight substances such as a CD4-CDR3 peptide (ref. [33 ] and reviewed in ref. [34 ]).

Besides T cells, natural killer (NK)-mediated alloreactivity is important in allogeneic BMC reconstitution [35 36 37 38 ]. The phenomenon, known for a long time as hybrid resistance (reviewed in ref. [39 ]), has been partly unraveled by the recovery of killer-inhibitor receptors [40 41 42 43 ]. Killer-inhibitor receptors recognize certain major histocompatibility complex alleles [44 45 46 47 48 ]. Via binding, NK receives signals preventing activation of the lytic machinery [41 , 48 49 50 51 ] and detaches from the target [52 ]. Inhibitory receptors, which are also found on T cell subsets, dominate over activating receptors [53 54 55 56 57 58 59 ]. In the allogeneically reconstituted, myeloablatively pretreated host, NK transferred with the graft may not be inhibited and could display cytotoxic activity toward the host and the tumor [35 , 60 , 61 ]. In the allogeneically reconstituted, nonmyeloablatively pretreated host, alloreactive host NK will interfere, in addition, with engraftment by lysis of donor cells [22 , 62 , 63 ]. By this scenario, it is obvious that there is no straightforward solution to these opposing problems and that a balance has to be found that allows engraftment and minimizes GvH and HvG cytotoxic activity while preserving GvT reactivity. Particularly the latter may require an additional, supportive regimen [64 65 66 67 ].

Here, we describe a protocol aiming at improved reconstitution in the tumor-bearing host. We noted that host NK depletion is most important to avoid graft rejection. Furthermore, particularly in the tumor-bearing mouse, host NK depletion in combination with graft T cell depletion increased the engraftment rate and reduced the incidence of fatal GvHD, and tumor growth remained retarded, as after the transfer of T cell-nondepleted grafts. This regimen may provide an optimized platform for additional immunotherapeutic approaches, e.g., vaccination.


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MATERIALS AND METHODS
 
Mice and tumors
BALB/c (H-2d) and C57BL6 x 129SVEV (C57/129; H-2b) F1 mice were obtained from Charles River (Sulzfeld, Germany) or were bred at the central animal facilities of the German Cancer Research Center (Heidelberg). Mice were used for experiments at the age of 8–10 weeks. Where indicated, BALB/c mice were nonlethally irradiated (6.5 Gy). The BALB/c-derived renal cell carcinoma (RENCA) tumor line [68 ] was used in vivo and in vitro. The murine leukemia line YAC was used as NK target. Both lines were maintained in vitro in RPMI 1640, supplemented with 10% fetal calf serum (FCS). Confluent RENCA cultures were trypsinized and split.

Antibodies
The hybridomas 33D1 (DC-specific), 331.12 (anti-µ), 145-2C11 (anti-CD3), and PK136 (anti-NK1.1) were obtained from the American Type Culture Collection (Manassas, VA), and YTA3.2.1 (anti-CD4), YTS169 (anti-CD8), and YTS154.7.7.10 (anti-CD90) were from the European Collection of Animal Cell Cultures (United Kingdom). H-2Dd (K9-18)- and H-2Db-specific (K7-65) monoclonal antibodies (mAb) [69 ] were kindly provided by G. Hämmerling (German Cancer Research Center). mAb were purified by passage of culture supernatants over protein G Sepharose 4B. Where indicated, purified mAb were biotinylated or fluorescein isothiocyanate (FITC)-labeled. AntipanNK (DX5), biotinylated anticytokine antibodies, FITC- or phycoerythrin-labeled Streptavidin, and anti-mouse and anti-rat immunoglobulin G (IgG) and IgM were obtained commercially.

Preparation of hematopoietic cells
Mice were killed by cervical dislocation. Spleen, femura, tibiae, and thymus were removed. BMC were obtained by flushing the bones with 5 ml phosphate-buffered saline using a 21-G needle. Thymus, bone marrow, and spleen were teased through fine gauze. Tumor infiltrating leukocytes (TIL) were enriched by Ficoll gradient centrifugation of meshed tumor tissue. Where indicated, BMC were T cell-depleted by coating with a mixture of anti-CD4 and anti-CD8 and adherence of the antibody-coated cells to anti-rat IgG-coated Petri dishes [70 ]. The efficacy of T cell depletion was controlled by flow cytometry of the nonadherent population, which contained less than 2% CD4+ or CD8+ cells. For NK depletion of BMC (mentioned in the discussion), C57/129 F1 BMC were incubated with PK136 and after washing, were seeded for 2 h in anti-mouse IgG2a-coated Petri dishes. The nonadherent cells were collected.

Flow cytometry and immunohistology
BMC, spleen cells (SC), and thymocytes (TC; 5x105 cells) were stained according to routine procedures. For intracellular staining of cytokines, cells were fixed and permeabilized in advance. Negative controls were incubated with an isotype-matched control IgG and the secondary antibody. Analysis was performed with a FACSCalibur.

Immunohistology: Tumors were shock-frozen in liquid nitrogen. Tissue samples were sectioned at 6 µm and stained as described [71 ]. Briefly, cryostat sections were exposed to the primary antibodies (mouse or rat IgG for negative controls), biotinylated goat anti-mouse or goat anti-rat (Dianova, Hamburg, Germany), and alkaline phosphatase-conjugated avidin-biotin complex solutions in sequence. Sections were counter-stained with Mayer’s hematoxylin.

Colony-forming assays [72 ]
Colony-forming unit (CFU) activity was determined by plating 5 x 104 BMC in 24-well plates in 0.3% semisolid agar in Iscove’s modified Eagle’s medium containing 20% horse serum and keeping the cultures at 37°C, 5% CO2 in air. For the evaluation of granulocyte macrophage (GM)-CFU, 10 ng/ml GM-colony stimulating factor and 2 mM glutamine and for C-CFU (multilineage), 15% conditioned medium from WEHI-3B cells and 5% conditioned medium from L929 cells were added. Pre-B-CFU (pre-B cell) were cultured with 20% FCS, 15% supernatant of an interleukin (IL)-7 cDNA-transfected line (kindly provided by T. Rolink, Basel Institute for Immunology, Switzerland) [73 ], 2 mM L-glutamine, and 5 x 10-5 M 2-mercaptoethanol (2-ME). Colonies of at least 50 cells were counted after 7 days.

Long-term reconstitution, tumor implantation, and immunoreactivity
Irradiated BALB/c mice received an intravenous injection of 2 x 106 C57/129 BMC. Where indicated, the transferred BMC were T cell-depleted, or the host was treated with antiasialoGM1 (Wako Chemical Co., Kyoto, Japan) 24 h before reconstitution. The percentage of surviving mice, the repopulation with leukocytes, and the recovery of CFU in BMC were controlled. The percentage of donor and host cells was evaluated by flow cytometry. Evidence for GvHD and HvGD was obtained by macroscopic inspection, particularly of skin, gut, and liver, weight loss, the analysis of inflammatory cytokine expression via flow cytometry, and by determining the frequency of host- and donor-reactive-proliferating T cells via limiting dilution (LD). Cells (24 replicates) were titrated from 11200–100 cells per well and were cultured in RPMI 1640, supplemented with 10% FCS, 10-5 M 2-ME, and 10-3 M HEPES buffer in the presence of 104 irradiated stimulator lymphocytes. Under this culture condition, only T cell will proliferate. 3H-Thymidine incorporation was determined after 8 days. The frequency of proliferating cells was calculated as F0 (fraction of nonresponding cultures) = emsu, where u = c/w (number of cx cells distributed in wx wells) [74 ]. GvH, HvG, and anti-tumor reactivity was also evaluated by defining the cytotoxic potential of SC after cells had been cultured for 10 days in RPMI 1640, supplemented with 10% FCS, 10-5 M 2-ME, 10-3 M HEPES buffer, and 20 U/ml IL-2 in the presence of irradiated (30 Gy) host or donor lymphocytes or irradiated (350 Gy) tumor cells, respectively. Cytotoxic activity was evaluated using the JAM test [75 ]. Lymphoblasts from BALB/c and C57/129 mice and RENCA cells served as targets. NK activity was tested in freshly harvested BMC using YAC as target.

RENCA cells (5x104) were injected subcutaneously (s.c.) at the day of BMC reconstitution. Tumor growth was controlled twice per week.

Statistical analysis
Significance of differences was calculated according to the Wilcoxon rank sum test (in vivo assays) or the Student’s t-test (in vitro studies). Functional assays were repeated at least three times. Mean values and standard deviations of in vivo experiments are derived from 20–50 mice per group. Mean values of in vitro studies are based on three to four replicates.


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RESULTS
 
Controlling the experimental set-up
Allogeneic reconstitution of the nonmyeloablatively conditioned host could become a major option in cancer therapy. The optimal conditions to minimize GvHD and HvGD are unknown. We have explored here whether combining host NK and graft T cell depletion is advantageous in tumor-free and tumor-bearing mice.

We first evaluated the suitability of our experimental setting. BALB/c irradiated with 6.5 Gy or irradiated with 6.5 Gy and treated with antiasialoGM1 survived in over 95% of cases (data not shown), although only few cells were recovered from thymus and spleen 4 days after irradiation or irradiation and antiasialoGM1 treatment. NK depletion was efficient inasmuch as NK activity of BMC and SC as well as the number of NK were significantly reduced. Also, there was no decrease in the percentage of CD4+ and only a slight reduction in the percentage of CD8+ BMC in irradiated and antiasialoGM1-treated as compared with irradiated mice (Table 1 ).


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  		Table 1. Lymphocyte Subsets and NK Activity in Irradiated and AntiasialoGM1-Treated BALB/c Mice

Influence of graft and donor manipulation on the survival rate of allogeneically reconstituted, nonmyeloablatively conditioned mice
When BALB/c mice were irradiated with 6.5 Gy and received 2 x 106 allogeneic BMC the following day, 80% of mice survived. The survival rate was decreased when the graft was T cell-depleted. When the host was NK-depleted, the survival rate of mice receiving unmanipulated BMC was improved, and graft T cell depletion was no longer a disadvantage (Fig. 1 ). The advantage of graft NK depletion was confirmed by transferring titrated numbers of BMC. Host NK depletion was most efficient after the transfer of 3 x 104 and 1 x 105 BMC, i.e., 20% and 60% of animals, respectively, survived, whereas all NK-competent mice died. Finally, it should be noted that NK-depleted mice did not die before several months after reconstitution, i.e., when NK had recovered. Death during the starting 4 weeks after reconstitution was mainly a result of GvH reactions, which predominantly affected the gut. Animals that became moribund at later time points after reconstitution were anemic, and hardly any hematopoietic cell could be recovered from the bone marrow or the spleen; i.e., mice died as a result of a failure of engraftment.



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  		Figure 1. Influence of host NK depletion on survival of allogeneically reconstituted, nonmyeloablatively conditioned mice. BALB/c mice were irradiated with 6.5 Gy and received, where indicated, a single intraperitoneal injection of antiasialoGM1. (A) One day later, mice were reconstituted with 2 x 106 C57/129 BMC. BMC were unseparated or T cell-depleted as described in Materials and Methods. Survival time and survival rate of 30–50 mice/group are shown. Differences in comparison to the transfer of unmanipulated BMC and in comparison to the NK-competent host were borderline significant. (B and C) BALB/c mice received 3–300 x 104 C57/129 BMC. (B) The survival rate (20 weeks after grafting) and (C) the survival time of mice receiving 1 x 105 BMC are shown. *, Significance of differences in comparison to NK-competent mice.

Myelopoiesis and repopulation of the periphery in dependence on host NK depletion and graft T cell depletion
Recovery of myelopoiesis and lymphopoiesis was evaluated by determining the number of C-CFU, GM-CFU, pre-B-CFU, and repopulation of the thymus (Fig. 2A and 2B ). Three observations are of interest. First, graft T cell depletion led to a strong reduction in pre-B-CFU and a distinct decrease in C-CFU. Second, in mice receiving an unmanipulated graft, host NK depletion led to a reduced recovery of multilineage C-CFU early after reconstitution, but the number of B progenitor cells was significantly increased. However, third, host NK depletion did not result in an increased recovery of pre-B-CFU when the graft was T cell-depleted. As a parameter of T lymphopoiesis, repopulation of the thymus with CD4/CD8 double-positive cells was evaluated. Host NK depletion significantly accelerated repopulation of the thymus, yet graft T cell depletion was disadvantageous and was not efficiently corrected by host NK depletion. It should be mentioned that 80–90% of thymocytes were CD4/CD8 double-positive; i.e., the thymus was repopulated by newly emerging T cells.



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  		Figure 2. Impaired B and T lymphopoiesis after the transfer of an allogeneic T cell-depleted graft in the nonmyeloablatively conditioned host. BALB/c mice were conditioned and reconstituted as described in Figure 1 . Mice were killed 2–16 weeks after reconstitution to evaluate (A) the number of C-CFU, GM-CFU, and pre-B-CFU per femur, (B) the recovery of thymocytes, (C) the total number of SC, and (D) the distribution of subpopulations in the spleen 2 weeks after reconstitution. Values represent the mean ± SD of three to five experiments. *, Significant differences (P<0.01) by antiasioaloGM1 treatment and/or graft T cell depletion.

The recovery of SC (Fig. 2C and 2D) , as an example for mature hematopoietic cells, revealed an increased number of cells early after reconstitution in the NK-depleted host. When the graft was T cell-depleted, the number of SC remained low during the observation period of 12 weeks. No significant differences were seen in the relative percentages of leukocyte subpopulations depending on whether the graft was T cell-depleted. However, the influence of host NK depletion on B-lymphopoiesis became apparent 2 weeks after reconstitution by the highly increased percentage of sIgM+ cells. Eight weeks after reconstitution, leukocyte subpopulations in the spleen no longer differed significantly from age-matched mice (data not shown), with the exception of mice receiving a T cell-depleted graft where the percentage of CD8+ cells remained high.

Thus, host NK depletion was advantageous with respect to B lymphopoiesis, repopulation of thymus, and early after transplantation, cellularity of peripheral lymphoid organs. Graft T cell depletion had a negative impact on B lymphopoiesis and on repopulation of the thymus. The disadvantage of graft T cell depletion was partially mitigated in NK-depleted mice. Advantages of host NK depletion could have been a result of mitigated HvGD. Graft T cell depletion is known to impede engraftment but also to be accompanied by less-severe GvHD. Thus, it was mandatory to evaluate the underlying mechanisms and particularly GvH and HvG reactions in the allogeneically reconstituted, nonmyeloablatively conditioned host.

GvH/HvG reactions in allogeneically reconstituted, nonmyeloablatively conditioned mice
We started with analyzing chimerism in bone marrow, thymus, and spleen (Fig. 3 ). Two weeks after reconstitution, over 50% of BMC and SC were donor-derived. Thereafter, the number of donor-derived cells transiently decreased in both organs but reached 80–90% in the bone marrow and 70–80% in the spleen 8–12 weeks after reconstitution. In the thymus, only a low percentage of donor-derived cells was recovered 2 weeks after reconstitution. However, the percentage increased steadily, reaching 60–70% after 12 weeks. In bone marrow, thymus, and spleen, the percentage of donor-derived cells was decreased when the graft was T cell-depleted. Host NK depletion had no major impact on the recovery of donor-derived BMC and TC, yet the transient decrease in donor-derived SC was mitigated in the NK-depleted host.



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  		Figure 3. Mitigation of the transient reduction in donor-derived spleen cells by host NK depletion. BALB/c mice were conditioned and reconstituted as described in Figure 1 . Mice were killed 2–12 weeks after reconstitution. The percentage of donor and host cells was evaluated in bone marrow, thymus, and spleen by fluorescein-activated cell sorter analysis. The percentage of donor-derived cells (mean values±SD of at least three experiments) is shown. *, Significant differences (P<0.01) by antiasioaloGM1 treatment and/or graft T cell depletion.

The decrease in the number of donor-derived cells in the bone marrow of NK-depleted mice was unexpected and prompted us to evaluate expression of inflammatory cytokines (Fig. 4A ). A relatively high number of IL-6 and tumor necrosis factor (TNF) expressing BMC was observed 4 weeks after allogeneic reconstitution of the unmanipulated host. The percentage of cytokine-expressing BMC was not decreased in the NK-depleted host. Instead, when the graft was T cell-depleted, host NK depletion was accompanied by a further increase in cytokine expressing BMC. Thus, inflammatory reactions of BMC are not predominantly mediated by host NK or donor T cells. Nonetheless, the relatively high level of inflammatory cytokines in the bone marrow could well support the elimination of donor-derived cells.



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  		Figure 4. Influence of host NK depletion and graft T cell depletion on cytokine expression and cytotoxic effector cells. (A) BALB/c mice were conditioned and where indicated, reconstituted as described in Figure 1 . (A) Mice were killed 4 weeks after reconstitution. Expression of IL-6, TNF, and interferon-{gamma} (IFN-{gamma}) was evaluated by flow cytometry of fixed and permeabilized BMC and SC. (B) Mice were killed 2–8 weeks after reconstitution. Cytolytic NK activity was evaluated in freshly harvested BMC. (C) Alloreactivity of SC was evaluated after in vitro restimulation with irradiated donor or host cells, respectively. Cytotoxicity values at a ratio of BMC (NK):YAC (NK target) = 50:1 and of spleen effector cells:allogeneic targets (BALB/c or C57/129 blasts according to the in vitro stimulus) = 20:1 are shown. The experiments were repeated three times and revealed comparable values. Mean ± SD values are presented. *, Significance of differences (P<0.01) was evaluated in comparison to irradiated and reconstituted mice.

Surprisingly and distinct from BMC, the percentage of cytokine-expressing SC was decreased when the host was NK-depleted. Graft T cell depletion had no major impact. This cytokine pattern was in line with the improved recovery of donor-derived SC in the NK-depleted host. It did not provide an explanation for the low level of donor-derived SC in mice receiving a T cell-depleted graft.

The cytokine profile as an indicator of inflammatory reactions could only partly explain repopulation of central and peripheral lymphoid organs in the NK-competent versus the NK-depleted mouse reconstituted with allogeneic BMC. Alternatively, lytic effector cells of donor or graft could be of major importance.

Indeed, NK activity in the spleen of allogeneically reconstituted mice was extraordinarily high and mostly host-mediated; i.e., hardly any NK activity was recovered 2 weeks after reconstituting NK-depleted mice. Graft T cell depletion had no impact on NK activity (Fig. 4B) . Thus, host NK activity was apparently important in resistance of engraftment.

Besides NK, cytolytic T lymphocytes are major effectors in GvH and HvG reactivities, which can interfere with allogeneic reconstitution. The analysis of alloreactive, cytolytic activity (Fig. 4C) revealed increasing levels of antihost and antigraft cytotoxicity 2–4 weeks after transplantation. Thereafter, antidonor and more pronounced, antihost reactivity declined. These features were independent of host NK and graft T cell depletion. However, graft T cell depletion was characterized by a long-lasting reduction in GvH reactivity. Host NK depletion was accompanied by reduced antidonor reactivity, particularly early after reconstitution. Only when the NK-depleted host received a T cell-depleted graft, antidonor reactivity remained at a reduced level throughout the observation period.

Thus, the advantage of NK depletion relied mainly on the reduced NK activity itself, which was accompanied by an accelerated recovery of pre-B cells, B cells, and thymocytes and a donor-dominated chimerism in the spleen. Mitigation of the negative impact of graft T cell depletion on the reconstitution by NK depletion of the host could also be a result of the reduction in NK killing. Whether reduced levels of GvH cytotoxicity after the transfer of T cell-depleted grafts in the first weeks after transplantation were a result of the absence of mature T cells and later on were a result of a relative dominance of donor T cells, which had matured in the host thymus and accordingly, should be tolerant toward the host, was evaluated by determining the frequency of host- and donor-specific, proliferating T cells under LD conditions (Table 2 ).


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  		Table 2. Frequency of Donor- and Host-Specific, Proliferating Cells in the Allogeneically Reconstituted, Nonmyeloablatively Conditioned Host

Highest frequencies of host- and donor-specific, proliferating T cells were seen 4 weeks after reconstitution and declined thereafter. In the NK-depleted host, fewer donor-specific, proliferating T cells were recovered, and frequencies of host-specific, proliferating T cells were not significantly altered, except at 6 and 8 weeks after the transfer of a T cell-depleted graft. Furthermore, fewer host-specific, proliferating T cells were recovered after the transfer of T cell-depleted BMC. These observations accounted for SC and BMC.

The findings support the view that 10 weeks after reconstitution, a stable state of chimerism was established. Host NK depletion efficiently prevented NK-mediated lysis of the graft. Graft T cell depletion, although disadvantageous for donor lymphopoiesis, was accompanied by mitigated GvH reactions.

Influence of tumor growth on the process of allogeneic reconstitution
One particular application for allogeneic reconstitution is cancer patients after exhaustion of a conventional, therapeutic regimen. As tumor growth frequently has a negative impact on hematopoiesis and leukocyte activation, it became mandatory to search for potential tumor-mediated deviations in the fragile situation of an allogeneic reconstitution. The weakly immunogenic, BALB/c-derived renal cell carcinoma RENCA served as a model.

In fact, myelopoiesis was impaired to some degree and could not be restored toward normal levels by host NK depletion (Fig. 5A ). However, the negative impact of graft T cell depletion on B cell maturation was far less pronounced than in the tumor-free host. Furthermore, repopulation of the thymus was most severely impaired during the first 6 weeks after reconstitution (Fig. 5B) . Surprisingly, repopulation of the spleen proceeded well and was strikingly improved by host NK depletion. With respect to the different lymphoid lineages, we consistently observed a lower number of B cells but a significant increase in CD8+ cells in tumor-bearing as compared with tumor-free mice. Graft T cell and host NK depletion had no consistent impact on the subset distribution in early- and late-stage tumor-bearing mice (Fig. 5C and 5D) .



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  		Figure 5. Impaired myelopoiesis and lymphopoiesis in allogeneically reconstituted tumor-bearing mice. BALB/c mice were conditioned and reconstituted as described in Figure 1 . They received a s.c. injection of 5 x 104 RENCA cells. Mice were killed after 2–8 weeks. The number of C-CFU, GM-CFU, and pre-B-CFU per femur (A), the total recovery of TC (B) and of SC (C), as well as the composition of the major leukocyte subpopulations in the spleen (D) are presented. The degree of hematopoietic chimerism is presented as the percentage of donor-derived SC (E). For comparison, the corresponding values in tumor-free, NK-competent mice receiving unmanipulated BMC are indicated (—x—). Mean values ± SD of three experiments are presented. *, Significant differences (P<0.01) by antiasialoGM1 treatment and/or graft T cell depletion.

Donor hematopoiesis exceeded host hematopoiesis more strikingly in tumor-bearing than in tumor-free mice, such that 6 weeks at the latest after reconstitution, the vast majority of SC was donor-derived. Furthermore, only after reconstitution with T cell-depleted grafts, a minor decrease in the percentage of donor-derived SC was transiently observed. Host NK cell depletion had less impact than in the tumor-free host but sufficed to further stabilize donor hematopoiesis when the graft was T cell-depleted. Yet, despite impaired host hematopoiesis, we did not see full-donor chimerism. As in tumor-free mice, 5–20% host cells were recovered even at late stages of tumor growth. Thus, graft T cell depletion in combination with host NK depletion had no negative impact on graft acceptance even in the tumor-bearer.

In line with these features was the analysis of the frequencies of graft- and host-specific, proliferating T cells (Table 3 ). As a result of the poor recovery of host hematopoiesis, few graft-reactive T cells were recovered, and host-directed reactivities were indistinguishable from those in tumor-free mice (see Table 2 ); i.e., tumor growth had no negative impact on immune recognition.


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  		Table 3. Frequency of Donor- and Host-Specific, Proliferating Spleen Cells in the Allogeneically Reconstituted, Nonmyeloablatively Conditioned Tumor-Bearing Host

With respect to cytokine expression, it should be mentioned that distinct to SC of tumor-free mice, no decrease in cytokine-expression was seen in NK-depleted, tumor-bearing mice. This may have been a result of the overall dominance of donor cells in tumor-bearing mice (data not shown).

Also in line with the weakness of host hematopoiesis in tumor-bearing mice, antidonor cytotoxicity was comparably low and was further decreased by host NK depletion. Antihost cytotoxicity was slightly increased as compared with tumor-free mice and was lower after the transfer of T cell-depleted BMC. Different from tumor-free mice, it was slightly but consistently increased in the NK-depleted host. As in tumor-free mice, donor- and host-directed cytotoxicity decreased with time (Fig. 6 ).



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  		Figure 6. Reduction in donor-directed but not in host-directed cytotoxic activity in the reconstituted tumor-bearing host. BALB/c mice were conditioned and reconstituted as described in Figure 1 and received a s.c. injection of 5 x 104 RENCA cells. Mice were killed after 2–8 weeks. Cytotoxic activity was evaluated in SC after in vitro restimulation with irradiated donor or host cells, respectively. Cytotoxicity values at a ratio of effector:target (BALB/c or C57/129 blasts, according to the in vitro stimulus) = 20:1 are shown. The experiments were repeated two times and revealed comparable values. For comparison, the corresponding values in tumor-free, NK-competent mice receiving unmanipulated BMC are indicated (—x—). Mean values ± SD are presented. *, Significant differences (P<0.01) by antiasioaloGM1 treatment and/or graft T cell depletion.

Thus, the reconstitution process was slightly retarded in the tumor-bearing host but otherwise proceeded largely as in tumor-free mice. For reasons to be clarified, tumor growth had less impact on donor than host hematopoiesis. This could become an important parameter of tumor defense.

Therapeutic efficacy of allogeneic reconstitution of tumor-bearing mice
Mice received a s.c. injection of 5 x 104 RENCA cells, which is 20-fold the minimal dose required for tumor take. The reconstitution protocols followed the ones described for tumor-free mice.

As compared with the nonreconstituted host, the survival time of RENCA-bearing mice was significantly (P<0.001) prolonged by an allogeneic reconstitution, and 30–40% of mice completely rejected the tumor (Fig. 7 ). Reconstitution with a T cell-depleted graft diminished the curative effect as compared with an unmanipulated graft. Host NK depletion slightly prolonged the mean survival time. The negative impact of T cell depletion was less pronounced in the NK-depleted host. Furthermore, when differentiating between death as a result of tumor growth and death as a result of a failure of engraftment and/or GvHD, it became apparent that graft T cell depletion had no negative impact on tumor growth in the NK-depleted host. The tumor growth rate was in line with this interpretation. As shown in Figure 7C , the prolongation of the survival time, even of those mice that finally succumbed, was a result of rejection episodes. Tumors became partially necrotic and were shrinking in size before tumor growth restarted, setting a "new" tumor next to the necrotic remnant of the "primary" tumor. It is obvious from the mean tumor diameter of 10 mice per group (Fig. 7D) that partial tumor rejection was unimpaired when mice received a T cell-depleted graft, particularly when the host was NK-depleted. Furthermore, only in the nonreconstituted host was tumor growth accelerated when the host was NK-depleted. However, when reconstituted with allogeneic BMC, even the NK-depleted host partially rejected the tumor.



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  		Figure 7. Prolongation of survival times by allogeneic reconstitution of nonmyeloablatively conditioned, tumor-bearing mice: mainentance of the protective effect when transferring a T cell-depleted graft into NK-depleted mice. BALB/c mice were conditioned and reconstituted as described in Figure 1 and received a s.c. injection of 5 x 104 RENCA cells. (A) Survival time and survival rate of 20–50 mice/group are shown. (B) The death rate as a result of tumor growth was evaluated by subtracting those mice that died as a result of a failure of engraftment or GvHD. Significant differences in the death rate by NK depletion of the host or T cell depletion of the graft are indicated by an asterisk. (C and D) The process of partial and complete tumor rejection becomes apparent by stagnation and reduction in the mean tumor diameter, as demonstrated for 10 individual mice receiving a T cell-depleted graft (C) and the mean values of tumor diameters of 10 mice per group (D).

Taken together, NK depletion of the reconstituted host was favorable with respect to tumor rejection/growth retardation. Furthermore, tumor growth was not accelerated when NK-depleted mice received a T cell-depleted graft. The data support the interpretation that host NK depletion has a stronger impact on a reduction in GvH reactivities than on tumor defense and that newly emerging rather than transferred T cells account for tumor rejection.

Mechanisms of tumor defense in the allogeneically reconstituted host
We first evaluated the presence and composition of TIL. This was done by counting infiltrating leukocytes in 10 microscopic fields after immunohistological staining (Table 4 ). Nonetheless, as fully necrotic areas, which are most interesting with regard to lymphocyte infiltration, could not be evaluated, the numbers can only be considered in relation with one to the other and not as absolute. There were apparently fewer graft-derived TIL when the graft was T cell-depleted. When the host was NK-depleted, the number of donor-derived TIL was significantly increased. Thus, tumor protection was not weakened when NK-depleted mice received a T cell-depleted graft. Irrespective of the transplantation protocol, preferentially CD8+ cells, and only few CD4+ cells, were recovered. Besides CD8+ cells, predominantly monocytes infiltrated the tumor. B cells were rare, dispersed throughout the tumor, and obvious differences depending on the transfer protocol were not observed. Finally, flow cytometry of TIL revealed a clear dominance of host-derived T cells, whereas macrophages were host- or donor-derived (data not shown).


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  		Table 4. Donor and Host-Derived Tumor-Infiltrating Leukocytes

Most important was the analysis of tumor-specific, proliferating T cells and of tumor-directed cytotoxicity. Frequencies of T cells proliferating in response to irradiated RENCA cells were low but increased steadily. Importantly, the increase in tumor-specific, proliferating T cells was also seen when the graft was T cell-depleted. When, in addition, the host was NK-depleted, a further increase in "RENCA-specific", proliferating T cells was noted (Table 5 ). RENCA-directed cytotoxic activity also was low early after reconstitution but increased continuously. Graft T cell depletion had only a minor, negative impact on tumor-directed cytotoxicity. Antitumor cytotoxicity was hardly influenced by host NK depletion (Fig. 8 ).


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  		Table 5. Frequency of Tumor-Specific, Proliferating Spleen Cells in the Allogeneically Reconstituted, Nonmyeloablatively Conditioned Tumor-Bearing Host



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  		Figure 8. Continuous increase in tumor-directed cytotoxic activity after allogeneic reconstitution. BALB/c mice were conditioned and reconstituted as described in Figure 1 and received a s.c. injection of 5 x 104 RENCA cells. Mice were killed after 2–8 weeks. Cytotoxic activity was evaluated in SC after in vitro restimulation with irradiated RENCA cells. Cytotoxicity values at a ratio of effector:RENCA cells = 20:1 are shown. The experiments were repeated two times and revealed comparable values. *, Significant differences (P<0.01) by antiasialoGM1 treatment and/or graft T cell depletion.

Taken together, tumor-directed reactivity appeared late after reconstitution; i.e., it was probably mediated by newly emerging donor lymphocytes. Accordingly, graft T cell depletion exerted no negative impact. Host NK depletion, although initially accompanied by a reduced antitumor defense, was favorable, likely via the reduced cytolysis of grafted BMC.


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DISCUSSION
 
Allogeneic reconstitution of the nonmyeloablatively conditioned host is a promising therapeutic approach for leukemia and solid tumors, as it can be applied to patients irrespective of age and health condition [9 10 11 12 13 14 15 16 ]. Optimization of protocols to guarantee graft acceptance and to minimize GvHD while maintaining GvT reactivity remains a challenging task (reviewed in refs. [10 , 12 , 14 ]). We described here that host NK and graft T cell depletion may provide a solid platform for an additional vaccination regimen. Our findings are exemplified in the fully, allogeneically reconstituted mouse bearing a renal cell carcinoma. Although not shown, it should be mentioned that concordant results were obtained with the leukemic line YC8.

We briefly want to comment on three aspects of the model, i.e., the conditions for reconstitution and the number of transferred BMC, both differing from the clinical setting, and third, the route of tumor cell application. Why did we use a rather high dose of irradiation and a rather low number of cells for reconstitution? This setting facilitated the elaboration of several aspects of the reconstitution process. First, we noted that hematopoiesis was unimpaired in the nonreconstituted host despite the rather high dose of irradiation; i.e., bone marrow, thymus, and peripheral lymphoid organs were repopulated already 2 weeks after irradiation. Conversely, very few hematopoietic cells were recovered 2 weeks after irradiation and reconstitution. The finding provided direct evidence that donor leukocytes killed host leukocytes and/or that host leukocytes killed grafted leukocytes with high efficacy. As in our setting, reconstitution was severely retarded when transferring unseparated BMC into the nonlethally irradiated host, it became easy to elaborate the contribution of the individual subpopulations to GvH and HvG reactions by modulation of the host or the graft, such as NK and/or T cell depletion. Finally, as will be discussed below, the setting favors repopulation of the host by newly emerging T cells rather than by expansion of T cells transferred with the graft, a most important feature for elaborating concepts of vaccination.

To observe growth and rejection episodes of a nonleukemic tumor, the 20-fold dose of RENCA cells required for tumor growth was s.c.-injected at the time of reconstitution. The s.c. site was chosen, as carcinomas are mostly located separate from lymphoid organs. Finally, the tumor reached a reasonable size before the immune system had recovered. In this respect, the model reflected the clinical situation of a relatively large carcinoma burden, where it is most critical to see therapeutic effects. Finally, it should be mentioned that RENCA cells have been shown to secrete IL-10 and TNF, although not at a high level [76 ]. Nevertheless, this feature could well account for the poorer recovery of hematopoiesis in allogeneically reconstituted, tumor-bearing, as compared with tumor-free, mice—tumor-induced suppression of hematopoiesis also frequently being of concern in cancer patients.

In the vast majority of mice, tumor growth was retarded as a result of periods of partial tumor rejection. Some animals succeeded in complete rejection of the tumor. Thus, an allogeneic reconstitution of tumor-bearing mice, which were nonmyeloablatively conditioned, was beneficial in any case. This aspect will not be discussed further. However, the mechanisms underlying the superiority of a combined depletion of host NK and graft T cells require some comments.

The functional importance of NK in allogeneic reconstitution is well known [35 36 37 38 , 47 ] and was also recently confirmed for the interference of host NK with allogeneic stem-cell engraftment [22 , 38 , 62 , 63 ]. Our data confirm the negative influence of host NK on the engraftment of allogeneic BMC. Host NK depletion was advantageous in the allogeneically reconstituted host, which was not challenged by tumor cell inoculation, lethality decreasing from 20–9%. The efficacy of host NK depletion became most impressively overt when transferring low numbers of BMC, where, e.g., 60% of NK-depleted mice survived, and 100% of NK-competent mice died. Furthermore, the few NK-depleted mice that died survived significantly longer; i.e., they died when endogeneous NK had recovered. Thus, host NK cells are very efficient in eliminating grafted cells, and their depletion is favorable for allogeneic reconstitution.

When propagating host NK depletion, the question has to be addressed whether host NK may exert additional and potentially supporting effects on hematopoiesis, which would provide an argument against their depletion. Myelopoiesis and T cell maturation were not impaired, and B lymphopoiesis was improved in the NK-depleted host. Also, expression of inflammatory cytokines, which may be impaired after allogeneic reconstitution [26 , 77 78 79 ], was unaltered or even improved. We only noted one phenomenon, indicating a supporting contribution by host NK to the reconstitution process. We expected a rapid expansion of host cells in the bone marrow of the NK-depleted host. This was not the case early after reconstitution, at which time the recovery of C-CFU was also reduced. Whether the two phenomena, the retarded start of donor hematopoiesis and the reduced level of C-CFU, were linked and were a consequence of a reduction in hematopoietic growth factors, which have been described to be provided by NK cells [80 , 81 ], remains to be explored. Nonetheless, the delayed start in donor hematopoiesis was the only negative impact of host NK depletion, and it was transient; i.e., later on, donor cells expanded more readily in the NK-depleted as compared with the NK-competent host.

The lethality as a result of tumor growth was slightly increased (49% vs. 58%) in the NK-depleted host, indicating the involvement of host NK activity in the defense against the RENCA tumor. However, tumor-directed cytotoxic activity, appearing rather late after reconstitution, was not impaired. Thus, host NK depletion strongly improved graft acceptance. We did not observe serious disadvantages with respect to hematopoiesis or antitumor defense.

Before discussing the impact of graft T cell depletion on allogeneic reconstitution, it should be mentioned that the effect of graft NK depletion has been elaborated, in addition, in tumor-free and tumor-bearing mice. We did not observe any strong effects on engraftment, graft rejection, GvHD, or tumor growth. Therefore, these data have not been presented for the sake of clarity.

T cell depletion reduces GvHD [82 ], but T-depleted grafts frequently are not accepted, and recovery of immune responsiveness may be delayed [82 , 83 ]. As engraftment is the conditio sine qua non, the solution to the problem of GvHD was attempted by selective depletion of host-reactive T cells or by expansion of T cell subpopulations that regulate GvHD [25 , 84 85 86 87 ]. As host NK depletion efficiently prevented cytolysis of donor cells, we asked whether, in the NK-depleted host, the advantages of a T cell-depleted graft would be maintained without facing a higher graft rejection rate or a reduced antitumor reactivity. Although mice were only sublethally irradiated, a higher percentage of animals died when receiving a T cell-depleted graft, which may have been a result of alloreactive NK cells in the graft and the host. In fact, by concomitant host NK depletion, the survival rate was improved. In the tumor-bearing animal too, survival time and rate were impaired by graft T cell depletion but could be corrected by concomitant host NK depletion.

Ex vivo analyses confirmed the poor recovery of hematopoiesis when the NK-competent host received a T cell-depleted graft; i.e., early progenitor cell expansion, B lymphopoiesis, and T cell maturation were strongly reduced. These disadvantages could be overcome by host NK depletion. As expected, antihost reactivity was reduced when transferring T cell-depleted BMC. Yet, antitumor reactivity was not significantly impaired. Notably, tumor reactivity was low early after BMC transfer but increased irrespective of graft T cell depletion. Simultaneously, the frequency of host-reactive T cells declined. We interpret the finding in the sense that antitumor reactivity is not a result of expansion and activation of grafted T cells but will largely be mediated by graft-derived T cells, which matured in the host thymus.

Finally, it should be mentioned that in tumor-bearing mice, host hematopoiesis was more severely impaired than donor hematopoiesis. Particularly, a higher percentage of donor-derived CD8+ SC was recovered in tumor-bearing than in tumor-free animals, and the contribution of donor-derived SC was further augmented by host NK depletion. Although this feature requires further exploration, in the context of our questions of how to modulate host or graft to improve engraftment without deteriorating the antitumor response, it adds to the advantages of reconstituting the NK-depleted host with T cell-depleted BMC.

In conclusion, allogeneic reconstitution after nonmyeloablative conditioning sufficed in most instances for a significant prolongation of the survival time of tumor-bearing mice and was curative in some instances; the alloreactivity of host NK cells provided a major obstacle for graft acceptance, and host NK depletion improved engraftment reliably; the probability of acceptance of a T cell-depleted graft was significantly increased by host NK depletion; and the reduction in GvHD by T cell depletion was favorable, and importantly, the reduction in the late-appearing GvT reactivity was only minor. We interpret these findings in the sense that GvT reactivity is mainly mediated by graft-derived T cells, which matured in the allogeneic host, i.e., became host-restricted. Thus, reconstitution of the NK-depleted host with T cell-depleted BMC may provide an optimal vaccination platform to activate graft-derived, host-restricted T cells via host-derived tumor antigen(s)-presenting cells.


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ACKNOWLEDGEMENTS
 
This investigation was supported by the Deutsche Forschungsgemeinschaft grant Zo40/9-1 (M. Z.). We greatly appreciate the English editing by Gerard Devitt.

Received June 16, 2002; revised July 29, 2002; accepted August 1, 2002.


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M. Zoller
Tumor Vaccination after Allogeneic Bone Marrow Cell Reconstitution of the Nonmyeloablatively Conditioned Tumor-Bearing Murine Host
J. Immunol., December 15, 2003; 171(12): 6941 - 6953.
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