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Blood, Vol. 89 No. 3 (February 1), 1997:
pp. 1100-1109
Donor   T Lymphocytes Promote Allogeneic Engraftment Across the Major Histocompatibility Barrier in Mice
By
William R. Drobyski and
David Majewski
From The Department of Medicine and The Bone Marrow Transplant Program, Medical College of Wisconsin, Milwaukee.
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ABSTRACT |
T cells that express the   T-cell receptor are thought to be the T-cell population primarily responsible for facilitating alloengraftment. The role of   + T cells that comprise only a minority of mature T cells in promoting allogeneic engraftment, however, has not been extensively studied. The purpose of this study was to determine whether T cells were capable of facilitating alloengraftment in murine recipients of major histocompatibility complex-mismatched marrow grafts. We developed a model where engraftment of C57BL/6 × 129/F2 (H-2b) marrow in sublethally irradiated (800 cGy) recipients (AKR/J, H-2k) is dependent on the presence of mature donor T cells in the marrow graft. In this model, donor T-cell engraftment was significantly augmented by as few as 1 × 105 T cells. The role of T cells was then investigated using transgenic donors (C57BL/6 × 129 background) in which a portion of the T-cell receptor- chain gene was deleted by gene targeting so that these mice lack T cells. Addition of 10 × 106 naive T cells to T-cell depleted marrow grafts was required to significantly increase alloengraftment, although donor T cells averaged <50% of total splenic T cells. To determine whether higher doses of T cells would improve donor engraftment and eradicate residual host T cells, T cells were ex vivo expanded with a T-cell-specific monoclonal antibody and interleukin-2 and then transplanted into irradiated recipients. Transplantation of  160 × 106 activated T cells was necessary to consistently and significantly augment donor cell chimerism and enhance hematopoietic reconstitution when compared to control mice, but host T cells persisted in these chimeras. Addition of 2.5 × 104 mature T cells, which alone were incapable of facilitating engraftment, to T-cell depleted marrow grafts containing 160 × 106 activated T cells resulted in long-term (<100 day) complete donor engraftment, indicating that limiting numbers of T cells were required in the marrow graft for the eradication of residual host T cells. Using serial weight curves and B-cell reconstitution as end points, clinically significant graft-versus-host disease was not observed in these chimeras under these experimental conditions. These data show that, whereas less potent than T cells, T cells are able to promote engraftment and enhance hematopoietic reconstitution in allogeneic marrow transplant recipients.
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INTRODUCTION |
THE PROCESS of engraftment of allogeneic bone marrow (BM) can be conceptualized as the dynamic interplay between residual host immunity and transplanted donor immune effector cell populations, which must either eradicate or inactivate host cells for engraftment of donor hematopoietic stem cells to occur. A significant body of evidence indicates that donor-derived T cells play a critical role in this process. The most compelling evidence derives from the observation that T-cell depletion (TCD) of the donor marrow has, in most instances, led to higher rates of nonengraftment in human transplantation.1 This premise is also supported by murine models of transplantation where the addition of graded doses of donor T cells to the marrow inoculum has been able to overcome graft resistance in major histocompatibility complex (MHC)-incompatible donor/recipient strain combinations.2 Furthermore, in suboptimally conditioned murine recipients, increasing the number of donor T cells in the graft has been able to compensate for a less intense preparative regiment.3 Collectively, these data are evidence that donor T cells play a pivotal role in overcoming graft resistance across the MHC.
Donor T cells that express the T-cell receptor (TCR) are thought to be primarily responsible for facilitating alloengraftment in both human and animal models because of the fact that these cells comprise the vast majority of mature T cells. In contrast, the role of T cells that express the TCR and constitute a minor population of mature T cells in alloengraftment has not be extensively studied. and T cells share similarities in that both differentiate primarily in the thymus, possess common cell surface markers, and have a diversity of clonotypic receptors associated with the CD3 complex.4-6 Both and T cells are also able to secrete a variety of lymphokines7,8 and have cytotoxic capability.9,10 However, while having certain similarities, and T cells also have significant differences. Unlike T cells, the majority of T cells lack the functional expression of CD4 and CD8 molecules and the manner in which T cells recognize alloantigen appears to be different from that of T cells.11-16 These observations suggest that and T cells may each have unique roles. Moreover, from a clinical perspective, we have previously shown that patients transplanted with TCD marrow grafts in which T cells are preferentially spared from the depletion procedure have high rates of engraftment, suggesting that T cells may be important in facilitating engraftment.17 Therefore, the purpose of this study was to determine whether T cells were capable of facilitating alloengraftment of rigorously TCD bone marrow in the absence of supplemental mature T cells. We evaluated this question in a murine model where donor (C57BL/6 × 129/F2, H-2b) and recipient (AKR/J, H-2k) are mismatched at the major histocompatibility complex and where alloengraftment is dependent on the addition of mature donor T cells to the marrow graft.
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MATERIALS AND METHODS |
Mice.
AKR/J (H-2k, Thy1.1+), C57BL/6 × 129/F2 (H-2b, Thy 1.2+), TCR -/- (C57BL/6 × 129/J background, T-cell deficient), TCR -/- (C57BL/6 × 129/J background, T-cell deficient), TCR -/- (pure C57BL/6 background) and normal C57BL/6 (H-2b, Thy1.2+) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in the American Association for Lab Animal Care (AALAC)-accredited Animal Resource Center of the Medical College of Wisconsin. The characteristics of the transgenic mice have been previously described by Mombaerts et al.18 Mice received regular mouse chow and acidified tap water ad libitum.
Flow cytometric analysis.
Monoclonal antibodies (MoAbs) conjugated to either fluoroscein isothiocyanate (FITC) or phycoerythrin (PE) were used to assess chimerism in marrow transplant recipients. FITC-anti-Thy1.2 (CD90, rat IgG2b), and PE anti-L3T4 (CD4, rat IgG2b) were purchased from Collaborative Biomedical Products (Bedford, MA). FITC-anti-Ly5 (B220, rat IgG2a) and PE anti-Lyt2 (CD8, rat IgG2a) were obtained from Caltag (San Francisco, CA). PE anti-H57-597 (TCB , hamster IgG), PE hamster IgG (isotype control), and FITC-anti-H-2Kb (Class I, mouse IgG2a) were all purchased from Pharmingen (San Diego, CA). T cells were distinguished using a biotinylated antibody specific for the TCR (3A10, hamster IgG, kindly provided by Dr S. Tonegawa, Massachusetts Institute of Technology, Cambridge)19 followed by secondary conjugation to PE-streptavidin. Spleen cells were obtained from chimeras at defined intervals posttransplant and stained for two-color analysis. Cells were analyzed on a FACS analyzer (Becton Dickinson, Mountain View, CA) with Consort 32 computer support and Lysis II software. Red cells and nonviable cells were excluded using forward and side scatter settings before analysis of spleen cell populations. Ten thousand cells were analyzed for each determination whenever possible.
Ex vivo expansion of T cells.
Spleen cells were obtained from TCR -/- donor animals and passed through nylon wool columns to remove B cells. The resulting population was typically comprised of approximately 50% cells expressing the TCR. Cells were then resuspended in CDMEM plus 10% fetal bovine serum (FBS) and cultured in flasks precoated with an immobilized T-cell-specific MoAb (GL4, hamster IgG; Pharmingen) at a concentration of 5 to 10 µg/mL. Twenty-four hours after the initiation of culture, human interleukin-2 (IL-2; Cetus Corp, Norwalk, CT) was added at a concentration of 20 U/mL (Cetus units). All cultures were split into fresh flasks as needed to maintain a cell concentration of 0.5 to 1.5 × 106 cells/mL. Cells were exposed to immobilized MoAb for the first 3 to 4 days of culture and thereafter grown only in medium plus IL-2 to allow for reexpression of the TCR. After a total of 7 to 8 days in culture, cells were counted and the percentage of T cells was analyzed by flow cytometry. Routinely, at total of 5 to 10 × 109 cells from C57BL/6 × 129/J donors was obtained with 95% to 99% of cells expressing the TCR and <1% expressing the NK1.1 antigen. When T cells were expanded from pure B6 background -/- mice, similar results were obtained for T-cell percentages. However, a minority of activated T cells (~30%) from these animals coexpress NK1.1 and thus natural killer (NK) cells could not be independently quantified.
Assessment of hematopoietic reconstitution.
Peripheral blood (PB) was obtained by retroorbital venipuncture and collected in EDTA-containing blood tubes. PB counts (ie, white blood cells, hematocrit, and platelet counts) were determined on a Coulter STK-S machine (Hialeah, FL). Because platelet counts greater 1 × 106/mm3 could not be accurately quantitated, mice whose platelets exceeded 1 million were arbitrarily defined as having a platelet count of exactly 1 × 106. All PB counts were subsequently corrected for the amount of diluent contained in the collection tube. In three instances, white blood cell (WBC) and/or platelet counts were not measurable on the Coulter machine because of technical problems. In these cases, the WBC count was estimated by counting contiguous fields on the blood smear under 40× magnification, averaging the number of cells/field, and multiplying by 2,500/mm3. Platelet counts were calculated similarly under oil (100× magnification) and multiplying the average by 15,000/mm3.
BM transplantation.
BM was flushed from donor femurs and tibias with complete Dulbecco's modified essential medium (CDMEM) and passed through sterile mesh filters to obtain single cell suspensions. BM was TCD in vitro with anti-Thy1.2 MoAb plus low toxicity rabbit complement (C6 Diagnostics, Mequon, WI). The hybridoma for 30-H12 (anti-Thy1.2, rat IgG2b) antibody was obtained from the American Tissue Culture Center (Rockville, MD) and grown in CDMEM plus 5% FBS. The culture supernatants were then harvested, precipitated in ammonium sulfate, and dialyzed against phosphate-buffered saline before use in in vitro depletion experiments. The efficacy of TCD was confirmed with a cytolytic limiting dilution assay (LDA), which indicated precursor T-cell frequencies of 1/13, 777, and >1/400,000 before and after TCD, respectively. Each succeeding lot of antibody that was used in these studies was similarly screened in an LDA before in vitro depletion with equivalent results. BM cells were then washed and resuspended in DMEM before injection.
Spleen cell suspensions were obtained by pressing spleens through wire mesh screens. Erythrocytes were removed from spleen cell suspensions by hypotonic lysis with sterile distilled water. T cells (either or T cells) for admixture with TCD BM before transplantation were then obtained by passing spleen cells once or twice through nylon wool columns (Robbins Scientific, Sunnyvale, CA) to remove B cells. The percentage of + T cells from B6129 or B6 donors and + T cells from -/- donors was quantified by flow cytometry. + T cells were defined as Thy1.2+ + and T cells as Thy1.2+ +. The average number of naive or T cells in the spleen cell suspensions after nylon wool depletion is indicated in the respective Table legends. The remaining cells as assessed by flow cytometry consisted of NK cells (NK1.1+) and residual B cells not removed by the nylon wool columns.
AKR recipient mice were given varying doses of total body irradiation (TBI) as a single exposure at a dose rate of 79 cGy using a Shepherd Mark I Cesium Irradiator (J.L. Shepherd and Associates, San Fernando, CA). Irradiated AKR/J recipients then received a single intravenous injection of TCD BM (10 × 106) with or without added naive or ex vivo activated spleen cells.
Experimental design.
Analysis of the role of T cells on alloengraftment is dependent on the ability to obtain sufficient numbers of T cells and to insure that these cells are not contaminated by T cells. To this end, we employed transgenic mice (129/J × C57BL/6, H-2b), in which a portion of the chain had been deleted by gene targeting, as donors for these experiments. Henceforth, these mice are referred to as 0, reflecting the fact that they do not make T cells. Normal 129 × C57BL/6/F2 mice in which there are no qualitative or quantitative abnormalities in T cells were used as comparable donor control animals, and are referred to as B6129. In latter experiments, availability of pure background C57BL/6 transgenic mice from Jackson Labs allowed us to validate our findings with a genetically homogenous donor strain. In these studies, normal C57BL/6 (B6) mice were employed as comparable donor control animals. AKR/J (H-2k) mice served as recipients in all these studies. Thus, donor and recipient differed at both class I and class II, as well as at multiple minor histocompatibility loci.
Statistics.
Group comparisons of mean donor T-cell chimerism, donor cell chimerism, overall spleen cell content, splenic B-cell content, and PB counts were performed using the unpaired Student's t-test. The correlation between the percentage of donor T-cell engraftment and the number of splenic B cells was assessed using Pearson's correlation coefficient. A two-tailed P value  .05 was deemed to be significant.
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RESULTS |
AKR mice reject TCD B6129 marrow grafts when conditioned with < 900 cGy.
Initial experiments were conducted to determine the dose of TBI, which would allow engraftment of TCD B6129 BM in AKR recipients. Recipient animals were conditioned with doses of TBI ranging from 700 to 1,000 cGy in 100 cGy increments and then transplanted with 10 × 106 TCD B6129 marrow cells. The rationale for using B6129 as opposed to 0 BM was to allow for the normal reconstitution of BM-derived T cells, analogous to what occurs in clinical marrow transplantation. At 45 to 62 days posttransplant, mice were sacrificed and the extent of donor T-cell chimerism in the spleen was determined by two-color immunofluorescence (Table 1). All recipients were reconstituted with donor T cells when conditioned with 1,000 cGy. At a dose of 900 cGy, only one-half of the recipients had evidence of donor T-cell engraftment, whereas all mice conditioned with 700 to 800 cGy rejected their grafts. Based on these data, a dose of 800 cGy was determined to be the maximal dose that would result in uniform rejection of TCD marrow grafts from B6129 donors.
Engraftment is dependent on the presence of donor T cells in the marrow graft.
Having defined the maximal dose of TBI, which resulted in uniform rejection of TCD B6129 BM in AKR recipients, we then evaluated whether addition of donor T cells to the marrow graft was able to facilitate alloengraftment. Irradiated recipients were transplanted with TCD B6129 BM with or without graded doses of T cells from B6129 donors (Table 2). Because the majority of T cells in the spleens of these mice are + (>98%), this was essentially a functional assessment of the capability of T cells to promote engraftment. As expected, animals in group I that received TCD B6129 BM only all rejected their grafts, as evidenced by the absence of donor T cells. Recipients transplanted with 1 × 104 T cells (P = .44) or 5 × 104 T cells (P = .17) had levels of T-cell engraftment that were not significantly different from control animals (Table 2). However, when mice were transplanted with 1 × 105 T cells, donor T-cell engraftment was significantly enhanced (P < .02). Although there was a trend toward increased donor T-cell chimerism with higher doses of T cells, this was not statistically significant (P = .17, group IV v VII). We then evaluated whether there was a correlation between donor T-cell engraftment and engraftment of other non T-cell populations in the spleens of transplanted recipients to determine whether donor T cells facilitated hematopoietic reconstitution. We employed B-cell reconstitution as an end point because this cell constitutes the major cell population in the spleen. A statistically significant correlation between the percentage of splenic donor T cells and the magnitude of B-cell repopulation in the spleen of these animals was observed (Table 2) (r = .66, P < .00001), indicating that donor T-cell engraftment promotes B-cell reconstitution in this model.
Facilitation of alloengraftment by T-cell-enriched spleen cells.
Studies were then conducted to determine if T cells were capable of preventing graft rejection in this model. Irradiated (800 cGy) AKR recipients were transplanted with TCD B6129 BM with or without graded doses of T-cell enriched spleen cells. Animals transplanted with TCD BM only all rejected their grafts (6% mean donor T cells, n = 18) (Table 3). The addition of spleen cells containing 10 × 106 T cells to the marrow graft was required to significantly enhance donor T-cell engraftment (P < .02), whereas doses below this threshold were ineffective in promoting alloengraftment. However, there was some variability in the degree of donor T-cell engraftment observed between animals transplanted with 10 × 106 T cells as half (7/14) of these mice had 50% donor T-cell chimerism in the spleen whereas 3 of 14 had < 10% donor T cells. As was observed in mice transplanted with + T cells, there was a significant correlation between the percentage of donor T cells and the number of splenic B cells (Pearson's correlation coefficient r = .72, P < .00001). These data suggested that T cells were capable of facilitating engraftment of donor-derived B cells.
Activated T cells facilitate alloengraftment.
The administration of higher doses of naive T cells (ie, > 20 × 106) to recipients was limited by constraints pertaining to the number of cells, which could be feasibly obtained from transgenic donors. Therefore, we examined an alternative strategy to obtain larger numbers of T cells. Specifically, we evaluated the ability of T cells that were activated ex vivo with a T-cell specific MoAb and then selectively expanded in recombinant IL-2 to facilitate alloengraftment. This also allowed us to assess the role of a purer population of T cells in facilitating engraftment as well as determine the effect of activation of T cells on alloengraftment. Transplantation of 20 × 106 activated T cells failed to enhance donor T-cell engraftment in sublethally irradiated recipients (Table 4), in contrast to what was observed with naive T cells. The addition of 160 × 106 T cells to TCD BM was required before a significant increase in donor T cell (P .02) and overall donor cell (P .002) engraftment was observed. Seven of eleven animals in these groups (VII and VIII) had 90% H-2b cells and 8 of 11 50% donor T cells. The extent of donor T-cell chimerism was also significantly correlated with the magnitude of splenic B-cell reconstitution in these chimeras as individual mice with higher percentages of splenic donor T cells also had greater numbers of B cells (r = .88, P < .00001). These data indicated that activated T cells can prevent graft rejection and that activation of T cells per se does not preclude these cells from facilitating engraftment.
Limiting numbers of mature T cells are required in the marrow graft in addition to activated T cells for complete allogeneic engraftment.
Despite the administration of large numbers of activated T cells, residual host T cells persisted in these chimeras. Since prior studies by other investigators20 have indicated that T cells may require the presence of T cells for optimal function, experiments were performed to determine whether the addition of limiting numbers of T cells to the marrow graft was required to eradicate remaining host cells. Because T cells themselves can facilitate alloengraftment when present in sufficient numbers (Table 2), the dose of T cells had to be low enough so as not to be able to facilitate measurable engraftment. We had previously shown that AKR mice consistently rejected TCD B6129 BM if less than 50,000 T cells were present in the marrow graft. Therefore, we added 25,000 T cells to these grafts to determine if donor T-cell chimerism could be further enhanced. As expected, mice transplanted with TCD B6129 only all rejected their grafts (Table 5). When AKR recipients were transplanted with TCD B6129 BM and 160 × 106 T cells, both donor T cell and overall donor cell engraftment were significantly enhanced (P < .0001). We then assessed whether the addition of limiting numbers of mature T cells to the marrow inoculum could further enhance donor cell engraftment. T cells were obtained from 0 donors (B6129 background) and contained no T cells. Animals transplanted with TCD BM plus 2.5 × 104 T cells rejected their grafts indicating that this number of T cells could not facilitate measurable engraftment. The addition of 2.5 × 104 T cells to 160 × 106 T cells, however, enhanced donor T-cell chimerism and overall donor cell engraftment when compared to mice transplanted with activated T cells alone (P .03, Group II v IV). These data indicated that to achieve complete (>98% H-2b+ cells) allogeneic engraftment after transplantation with activated T cells, small numbers of mature T cells were also required to be present in the graft.
T cells facilitate long-term alloengraftment.
Because activated T cells were able to facilitate complete allogeneic engraftment when animals were sacrificed at ~30 days posttransplant, we assessed whether engraftment was durable when tested more than three months post BMT. To permit a comparative analysis of the relative potency of and T cells in facilitating long-term engraftment, groups of mice were also transplanted with either TCD BM alone or TCD BM plus graded doses of T cells that had been shown to promote short-term alloengraftment (Table 2). The majority of animals transplanted with TCD BM or TCD BM plus 1 × 105 T cells rejected their grafts after 100 days (Table 6). A minimum dose of 5 × 105 T cells was required in this model for durable donor engraftment (P < .002 relative to TCD BM only). The addition of 20 × 106 naive T cells was ineffective in promoting long-term engraftment. Only one of four mice evaluable after 86 days had donor cell engraftment; however, this animal also had evidence of graft-versus-host disease (GVHD) (~30% loss of body weight). One additional animal died 43 days posttransplant because of GVHD. In contrast, mice transplanted with 160 × 106 activated T cells had augmented donor T-cell chimerism and donor cell repopulation in the spleen (P < .02 v group I). The addition of limiting numbers of T cells (2.5 × 104) to grafts containing 160 × 106 activated T cells (group VIII) further enhanced donor T cell (P < .0001) and overall donor cell chimerism (P < .05), and resulted in complete allogeneic engraftment (ie, 100% H-2b+ cells), when compared to mice transplanted with 160 × 106 activated T cells only (group VI).
To further assess the efficacy of T cells in promoting long-term engraftment, we evaluated B-cell reconstitution in mice transplanted with activated T cells (Table 6, groups VI and VIII). No significant differences were observed in B-cell reconstitution between animals transplanted with activated T cells (mean 61 × 106, n = 7) versus those receiving TCD BM only (mean 36 × 106, n = 6, P = .19). Transplantation with activated T cells, however, did result in an increased overall spleen size (mean 130 × 106, n = 7) when compared to animals transplanted with TCD marrow only (mean 66 × 106, n = 6, P < .05). These data suggested that T cells enhanced overall splenic reconstitution.
T cells facilitate alloengraftment in a pure background B6 AKR model.
In the previous studies, we evaluated the role of T cells using mixed background (B6129) donors. Because there is some donor-to-donor genetic variability in these F2 animals that could theoretically be a confounding factor, we subsequently performed experiments employing T cells derived from pure background (C57BL/6) 0 donors. In the initial experiment, similar to the experimental design in Table 4, irradiated (800 cGy) AKR recipients were transplanted with or without graded doses of activated T cells. PB counts were also measured in these chimeras 5-weeks posttransplant to assess the effect of T cells on hematopoietic reconstitution. Sublethally irradiated recipients transplanted with TCD B6 BM all rejected their grafts, similar to what was observed with TCD B6129 BM. Although supplementation of 40 × 106 T cells to the graft failed to augment these parameters of engraftment, the addition of 80 × 106 activated T cells to TCD marrow grafts significantly enhanced donor T cell and overall donor chimerism (P < .03) (Table 7). Hematopoietic reconstitution was also augmented as both WBC and platelet counts were greater in these animals (group III) than in mice that rejected their grafts (group I) (P < .03).
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Table 7.
Activated T Cells From Pure Background 0 Donors Facilitate Alloengraftment and Enhance Hematopoietic Reconstitution
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In a second experiment, we evaluated whether the presence of a limiting number of mature T cells was required for complete allogeneic engraftment, using an experimental design identical to that in Table 5. Mice transplanted with TCD BM plus 160 × 106 activated T cells had significantly enhanced donor T cell (P < .001) and donor cell engraftment (P < .0001), although host T cells persisted in these chimeras (Table 8). The addition of 2.5 × 104 T cells to the same marrow graft further augmented engraftment (P < .05, group IV v group III, Table 8) with 99% overall donor cell chimerism. Engraftment parameters in mice transplanted with TCD BM plus 2.5 × 104 T cells did not differ from those of animals receiving TCD BM only (P .50, group I versus II for donor T cell and donor cell chimerism). Collectively, these data validate the results of prior experiments performed using mixed background donors, substantiate that activated T cells can facilitate alloengraftment, and confirm that limiting numbers of mature T cells are required for complete allogeneic engraftment.
Transplantation with activated T cells does not result in clinically significant GVHD.
Given the large doses of activated T cells required to facilitate alloengraftment, we evaluated whether these cells were able to induce GVHD under the conditions used to evaluate engraftment. Weight curves of engrafting mice transplanted with 160 × 106 activated T cells with or without 2.5 × 104 T cells were virtually indistinguishable from mice transplanted with TCD BM only, consistent with a lack of clinically significant GVHD (Fig 1). No mortality was noted in these animals. As another parameter of GVHD, we observed in this model (B6129AKR) that B-cell reconstitution was a sensitive indicator of GVH reactivity. Specifically, AKR recipients transplanted with either 2.2 × 106 (n = 14) or 5 × 106 T cells (n = 5) had a mean of 7.2 × 106 splenic B cells/mouse when evaluated 26 to 35 days posttransplant (data not shown). This was significantly less than engrafting animals transplanted with 1 × 106 T cells that averaged 19 × 106 splenic B cells (n = 17, Table 3) (P = .0001) when assessed at similar time points posttransplant. We inferred from these data that the reduced number of splenic B cells in these mice was likely due to GVHD, as has been shown in other murine models.21,22 The subsequent observation that mice transplanted with 160 × 106 activated T cells with or without 2.5 × 104 T cells averaged 61 × 106 splenic B cells/mouse (n = 7) (Table 6) provided further evidence that these chimeras lacked clinically significant GVHD.
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| Fig 1.
Transplantation of activated T cells does not result in clinically significant GVHD in long-term chimeras. Irradiated (800 cGy) AKR mice were transplanted with 10 × 106 TCD B6129 BM alone (n = 3,  ) or TCD B6129 BM plus 160 × 106 activated T cells (from 0 donors, B6129 background) with or without 2.5 × 104 naive T cells (n = 7,  ). The mean weights of animals for the first 100 days posttransplant are shown. Weight curves are from mice shown in Table 6.
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DISCUSSION |
This study was designed to examine the role of T cells in facilitating engraftment across the MHC under experimental conditions where donor T cells in the marrow graft are required for alloengraftment. To this end, we employed transgenic donors which lacked the ability to make T cells as a source of T cells. This allowed us to examine the role of T cells without the confounding presence of mature T cells and also to dissect the relative contributory role of both and T cells in this model.
Addition of spleen cells containing 10 × 106 naive T cells to TCD marrow grafts significantly enhanced donor T-cell engraftment in sublethally irradiated recipients. These results indicated that engraftment of a thoroughly TCD marrow graft could occur in the absence of mature splenic T cells. We concluded that T cells were most likely responsible for facilitating donor engraftment although our data do not conclusively support this interpretation. This is based on the fact that approximately 50% of the spleen cells administered to animals consisted of T cells, whereas the remaining cells consisted of NK and residual B cells still present after nylon wool depletion. Thus, mice did not receive a pure population of T cells. Based on prior studies by Blazar et al23 who failed to show a facilitory role for NK cells in allogeneic engraftment, we believe it unlikely that NK cells prevented graft rejection in this model and consider the most plausible interpretation to be that alloengraftment was facilitated by T cells. However, these data do not exclude the possibility that NK cells may have played a graft facilitory role.
Transplantation with doses of naive T cells as high as 20 × 106, while enhancing donor engraftment, still failed to eradicate all host T cells in this model. Although it is possible that higher numbers of naive T cells may have further enhanced engraftment, dose escalation was constrained by limitations in the number of cells that could be feasibly obtained from 0 donors, since T cells represent only 3% to 4% of all spleen cells in transgenic donors. Therefore, we examined the efficacy of activated T cells in facilitating engraftment, because this was a clinically feasible approach and allowed us to examine the effect of transplanting augmented numbers of T cells. This also served as a means of obtaining a relatively pure population of T cells since after ex vivo activation greater than 95% to 99% of all B6129 cells expressed the TCR and less than 1% expressed the NK 1.1 antigen. The use of activated T cells therefore allowed the graft facilitory role of T cells to be more definitively addressed.
Results from several different experiments using both mixed background and pure background transgenic donors confirmed that addition of large numbers of activated T cells (in excess of 40 to 80 × 106) significantly enhanced engraftment. These data strongly suggested that T cells could facilitate alloengraftment. The relative purity of the T-cell population used in these experiments is important in light of studies by Murphy et al24 who showed that IL-2 activated NK cells can promote allogeneic engraftment in mice. In their work, transplantation of 20 to 30 × 106 activated NK cells plus the in vivo administration of exogenous IL-2 were required for donor cell engraftment to be observed. In the present studies, because NK1.1 expressing cells represented less than 1% of the B6129 activated cell population, these cells would have constituted no more than 2.4 × 106 NK cells at the highest dose administered (ie, 240 × 106) and no more than 0.4 × 106 cells at the lowest dose (ie, 40 × 106), which was shown to enhance donor engraftment. Therefore, we believe the likelihood that IL-2 activated NK cells promoted engraftment in this model was small and the preponderance of data support a direct role for activated T cells in facilitating alloengraftment.
Our conclusion that T cells can facilitate engraftment of H-2 disparate TCD BM is supported by prior studies of Blazar et al25 who also showed that T cells could enhance alloengraftment of TCD scid BM. In their experiments, T cells expressing a single TCR were able to promote engraftment by recognition of H-2Tb antigens presumably on host hematopoietic cells. Significant differences between the two studies, however, were that we examined the graft facilitory effect of a heterogeneous population of activated T cells and observed that a much higher dose of T cells was required for engraftment. The relative significance of each of these studies for human marrow transplantation is presently unknown.
Paradoxically a greater number of activated than naive T cells were necessary in this model for an equivalent degree of donor cell engraftment. Since activation of T cells in vitro has been shown to augment cytolytic activity,26 we would have surmised that fewer cells would have been required to facilitate engraftment. That this was not the case suggested that either the life span of these cells was reduced in vivo, their homing properties altered, or their functional capabilities affected by activation with TCR-specific antibody and IL-2. Prior studies with lymphokine activated killer cells have shown early cell death and altered trafficking,27,28 raising the possibility that activated T cells may have shared a similar fate. Alternatively, other studies have showed that activated T cells are more susceptible than naive cells to apoptosis after religation of the T-cell receptor in vitro.29,30 Experiments are currently underway to explain the requirement for larger cell doses of activated T cells in this model.
One of the most striking aspects of this study was that very high doses of both naive and activated T cells were required to augment engraftment. Specifically, on a cell to cell comparison, T cells were 100- to 500-fold less potent than T cells at preventing graft rejection in this model. Because we had shown that engraftment in this model was dependent on the presence of donor T cells in the marrow graft (Table 2), this was evidence that donor T cells were required to interact with the host microenvironment to eradicate or inactivate residual host T-cell populations capable of rejecting the graft. These data indicated that T cells are functionally more competent to perform this role than T cells. There are several possible explanations for this observation. First of all, if one assumes that donor T-cell recognition and destruction of host T cells is one mechanism by which engraftment is facilitated,2 then this would require that donor T cells have the capability to be cytolytic. The relative ability of and T cells to facilitate engraftment would therefore be contingent, in part, on their cytotoxic capabilities. This question has been indirectly examined by Kabelitz et al31 who showed that the frequency of allocytotoxic T-cell clones in the PB in man is significantly lower than for T-cell clones. In their study, the majority of T-cell clones did not discriminate between autologous and allogeneic target cells. The authors concluded that most T cells lacked specific allocytotoxicity. Given the broader reactivity and lower allocytotoxic frequency of T cells at the clonal level, this would predict that higher doses of these cells would be required to facilitate equivalent degrees of donor cell engraftment.
A second possibility is that T cells are inherently competent to promote engraftment either by destruction or inactivation of host T cells but require help from T cells for optimal function. We observed that transplantation of T cells in the absence of mature T cells were limited in their ability to eradicate residual host T cells and facilitate complete allogeneic engraftment. Subsequent studies indicated that limiting numbers of T cells were required for complete engraftment and eradication of host T cells. The manner by which limiting numbers of T cells enhanced engraftment in this model is not known, but the data suggest that T cells play an important facilitory role. Several possible mechanisms can be advanced. First of all, prior studies have shown that the CD4+ + T-cell subset that secretes IL-2 represents only 0% to 5% of all T cells.20 This is a much lower percentage than found for T cells, and indicates that the T-cell subpopulation that is capable of providing "help" is markedly diminished. Similarly, T cells may secrete other cytokines necessary for T-cell proliferation.32 This would presume that the inability of lower numbers of T cells to inactivate or eradicate host T-cell populations is caused by a deficit of appropriate T-cell help that is cytokine-mediated. Alternatively, small numbers of T cells may be necessary to compensate for T cells, which are relatively deficient in generating a cytolytic response against host target cells.33 In this instance, T cells that are cytotoxic may be required to eradicate residual host T cells, which are not completely removed by T cells.
Although + T cells are thought to play a primary role in the pathophysiology of GVHD,34,35 the role of T cells has not been defined. Blazar et al36 showed that transgenic T cells could cause GVHD by specific recognition of nonclassical class Ib antigens in mice. Meanwhile, Ellison et al37 have postulated a role for a subset of T cells, which coexpress the NK1.1 antigen in mediating GVH reactivity. In the present study, which employed a heterogeneous population of T cells, high doses of naive T cells (20 × 106) were able to cause GVHD in chimeras, but since most of these animals failed to engraft, GVHD was not further assessable. In contrast, clinically significant GVHD was not observed when animals were transplanted with large numbers of activated T cells. Weight curves over 3 months in these engrafting mice were similar to those obtained from TCD BM controls. Moreover, B-cell reconstitution that appeared to be a more sensitive indicator of GVH reactivity in this model was increased relative to control animals. Since in this engraftment model recipients were sublethally irradiated, it remains to be seen whether more intensively conditioned recipients would develop GVHD under similar circumstances. Although additional studies are needed, these data do provide evidence that, under certain conditions, T cells may be able to facilitate alloengraftment without escalating toxicity from GVHD. Whether activation of T cells per se functionally alters these cells in such a way as to minimize their propensity to cause GVH reactivity will require further study.
The results of this study may have implications for human marrow transplantation. Although obtaining large numbers of naive T cells in humans is problematic, transplantation of ex vivo activated T cells is a clinically feasible approach. If one assumes that T cells are 500-fold less potent on a cell to cell basis than T cells, the required number of T cells for engraftment would still be well within the range of what has been shown to be technically feasible in adoptive immunotherapy studies using in vitro polyclonally expanded anti-CD3 stimulated T cells.38-40 Furthermore, because limiting numbers of T cells are graft facilitory, it is conceivable that the requisite number of T cells for complete allogeneic engraftment may be lower than the dose of 160 × 106 activated cells which we tested in this model. This would commensurately reduce the T-cell requirement in humans. It is also worth noting that even if activated T cells are only able to facilitate mixed T-cell chimerism in humans, this may still be of potential clinical benefit, if there is a corresponding reduction in GVHD in transplanted recipients.41,42 Specifically, in nonmalignant disorders amenable to BM transplantation (eg, immunodeficiency states, BM failure syndromes, metabolic, and red blood cell disorders), the establishment of partial yet durable donor T-cell chimerism might allow for correction of the underlying disorder.43 Thus, the supplementation of the marrow graft with activated T cells may represent a unique approach for the facilitation of BM engraftment in humans.
| |
FOOTNOTES |
Submitted May 30, 1996;
accepted September 13, 1996.
Supported by Grant No. CA01534 from the National Institutes of Health, The Ralph and Marian Falk Medical Research Trust, and The Florence Carter Fellowship from The American Medical Association.
Address reprint requests to William R. Drobyski, MD, Bone Marrow Transplant Program, Froedtert East Hospital, 9200 W Wisconsin Ave, Milwaukee, WI 53226-3596.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hearly marked
``advertisment'' in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Paul Martin for helpful discussions. This manuscript was prepared with the assistance of Deanna Cherubini.
| |
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Abstract
Introduction
Abstract