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Host Conditioning Is a Primary Determinant in Modulating the Effect of IL-7 on Murine Graft-versus-Host Disease¹

Maria Gendelman^*, Toby Hecht, Brent Logan, Sanja Vodanovic-Jankovic^*, Richard Komorowski and William R. Drobyski^2,*

Bone Marrow Transplant Program and Departments of ^* Medicine, Biostatistics, and Pathology, Medical College of Wisconsin, Milwaukee, WI 53226; and National Cancer Institute, Frederick, MD 21791

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin-7 has been shown to enhance T cell reconstitution after allogeneic bone marrow transplantation, in part, by expansion of mature donor T cells, but whether IL-7 also exacerbates graft-vs-host disease (GVHD) remains unresolved. To address this issue, we examined the effect of IL-7 on GVHD induction using a well-defined murine GVHD model (B6B6AF1/J). Administration of IL-7 to nonirradiated B6AF1/J recipients of B6 T cells resulted in expansion of splenic donor CD4⁺ and CD8⁺ T cells and increased GVHD mortality. In contrast, administration of IL-7 on the same schedule failed to increase GVHD mortality in either sublethally or lethally irradiated animals that received graded doses of T cells designed to induce varying degrees of GVHD severity. Moreover, IL-7 failed to increase the number of alloreactive T cells when examined in a murine model (B6BALB.B) that allowed for direct quantitation of graft-vs-host-reactive T cells. The combination of irradiation and transplantation of alloreactive donor T cells resulted in significantly increased levels of endogenous splenic IL-7 mRNA when compared with nonirradiated transplanted animals, providing a potential explanation for why exogenous IL-7 did not increase GVHD severity in these mice. We conclude that host conditioning modulates the ability of exogenous IL-7 to exacerbate GVHD and that this occurs through induction of endogenous IL-7 production.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Impaired T cell reconstitution is one of the major complications after allogeneic bone marrow (BM)³ transplantation (BMT), and is largely responsible for the increased risk of opportunistic infections that exists in this patient population (1, 2). T cell reconstitution posttransplantation has been shown to be dependent upon the generation of new donor T cells in the recipient thymus as well as the expansion of mature T cells in the periphery (3). Unfortunately, both pathways of T cell development are often adversely affected by the conditioning regimen itself or complications that arise in the post-BMT period. The generation of new donor T cells is impaired in the setting of an age-related decline in thymic function that is operative in the majority of transplant patients (4). Moreover, toxicity from the conditioning regimen causes direct damage to the thymus and inhibits the production of growth factors necessary for thymocyte development (5, 6). Finally, graft-vs-host (GVH) disease (GVHD) can directly target the thymus and further compromise thymic function (7, 8, 9). In the majority of patients, the T cell repertoire and effective T cell immunity are the consequence of mature T cells transferred from the marrow graft (10). Because these cells are not tolerant of host alloantigens, they are capable of inducing GVHD. Moreover, survival of both host-reactive and nonhost-reactive donor T cells can be compromised due to an increased susceptibility to undergo apoptotic cell death in the setting of a GVH reaction (11, 12, 13). The consequence of these events is that T cell immunity against endogenous and exogenous infectious agents is often compromised.

The clinical consequences of impaired T cell reconstitution have been the impetus for preclinical approaches designed to augment T cell immunity posttransplantation as a way to improve overall host immunity. One promising approach has focused on the use of IL-7 as a means to accelerate T cell reconstitution. IL-7 is a cytokine that is produced by both thymic and marrow stromal cells as well as dendritic cells (14, 15, 16, 17, 18), and has been shown to be critical for maintenance of T cell homeostasis (19, 20). IL-7 has also been shown to have a number of other effects on T cells, including the ability to provide a costimulatory signal for T cell activation (21), inhibit programmed cell death (22), and enhance Th1-type T cell responses (23, 24). A number of murine studies have demonstrated that administration of IL-7 can enhance lymphocyte recovery in murine recipients undergoing BMT (25, 26, 27). This has been attributable to augmentation of thymopoiesis as well as expansion of mature peripheral T cells (28). Although expansion of mature donor T cells may be beneficial with respect to augmenting immunity early after BMT, these same cells have the potential to exacerbate GVHD because they are not tolerant of recipient alloantigens. The ability of specific cytokines to exacerbate or inhibit GVHD, however, is dependent upon a number of variables such as timing of administration (29, 30) and the intensity of the conditioning regimen (30, 31). The purpose of this study was to examine how these variables affected the ability of IL-7 to modulate the severity of GVHD in murine allogeneic marrow transplant recipients.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6 (B6) (H-2^b), BALB.B (H-2^b), and (C57BL/6 x A/J)F₁ (B6AF1/J, H-2^b/a) mice were bred in the Animal Resource Center at the Medical College of Wisconsin or purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were housed in the American Association for Laboratory Animal Care-accredited Animal Resource Center of the Medical College of Wisconsin. Mice received regular mouse chow and acidified tap water ad libitum.

Reagents

Human rIL-7 was obtained from the National Cancer Institute (Frederick, MD). IL-7 was reconstituted in PBS and administered at a dose of 5 µg/day to murine transplant recipients.

CD4⁺ and CD8⁺ T cell subset enrichment

To obtain highly enriched populations of CD4⁺ T cells, B6 spleen cells were passed through nylon wool columns, and then CD4⁺ T cells were positively selected using the MACS magnetic cell separation system (Miltenyi Biotec, Auburn, CA). A similar procedure was done to isolate highly enriched CD8⁺ T cells. Cells were analyzed by flow cytometry to confirm purity before transplantation. Typically, >90% purity was obtained for the positively selected T cell subset with <2% contamination of the reciprocal subset.

BM transplantation

BM was flushed from donor femurs and tibias with DMEM and passed through sterile mesh filters to obtain single cell suspensions. BM was T cell depleted in vitro with anti-Thy-1.2 mAb plus low toxicity rabbit complement (C-SIX Diagnostics, Mequon, WI). The hybridoma for 30-H12 (anti-Thy-1.2, rat IgG2b) Ab was purchased from the American Type Culture Collection (Manassas, VA). BM cells were washed and resuspended in DMEM before injection. Naive donor T cells were obtained by passing erythrocyte-depleted spleen cells through nylon wool columns to remove non-T cells. Red cells were removed by hypotonic lysis using distilled water. Host mice were conditioned with total body irradiation (TBI) administered as a single exposure at a dose rate of 67 cGy using a Shepherd Mark I cesium irradiator (J.L. Shepherd and Associates, San Fernando, CA). Irradiated recipients received a single i.v. injection of T cell-depleted (TCD) BM (10⁷ cells) with or without added T cells. Nonirradiated recipients received T cells alone without BM.

Experimental design

GVHD was first assessed in a parentF₁ model to examine GVH reactivity in the absence of a conditioning regimen. In this model, nonirradiated B6AF1/J mice were transplanted with 25 x 10⁶ purified B6 T cells. In subsequent studies, the effect of a conditioning regimen on GVHD mortality was assessed using the same model in which B6AF1/J recipients were sublethally (500 cGy) or lethally (1000 cGy) irradiated and transplanted with TCD B6 BM alone or admixed with graded doses of B6 T cells.

Flow cytometric analysis and assessment of chimerism

mAbs conjugated to either FITC or PE were used to assess chimerism in marrow transplant recipients. PE anti-CD8 (clone CT-CD8a, rat IgG2a) was obtained from Caltag (San Francisco, CA). PE anti-TCR (clone H57-597, hamster IgG), PE anti-CD4 (clone GK1.5, rat IgG2b), CyChrome anti-CD3, and FITC anti-H-2K^k (clone AF3-12.1, mouse IgG1) were all purchased from BD PharMingen (San Diego, CA). Spleen cells were obtained from chimeras at defined intervals posttransplantation, processed into single cell suspensions, and stained for two- or three-color analysis. Cells were analyzed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA). The absolute number of splenic donor or host T cell subpopulations was determined by analyzing cells within a gate that included the entire spleen cell population after exclusion of debris and residual red cells. At least 10,000 cells were analyzed for each determination whenever possible.

Histological analysis

Representative samples of liver and colon were obtained from transplanted recipients and fixed in 10% neutral-buffered formalin. Samples were then embedded in paraffin, cut into 5-µm-thick sections, and stained with H&E. Semiquantitative scoring systems were used to account for histological changes consistent with GVHD. The degree of portal triad lymphocytic infiltration was assessed in the liver, while the severity of crypt cell apoptosis and overall crypt destruction was evaluated in the colon. Histological tissue damage compatible with GVHD in the liver was graded as 0 for normal, 1 for mild, 2 for moderate, and 3 for severe, while tissue injury in the colon was graded as 0 for normal, 1 for mild, and 2 for moderate. Scores were added to provide a composite score for each animal (maximal score 5/mouse). All slides were coded and read in a blinded fashion.

Quantitation of GVH-reactive T cells

Soluble H-2K^b Ig dimer (BD PharMingen) was incubated with the immunodominant peptides from H60 (LTFNYRNL) or OVA (SIINFEKL) for 48 h at 4°C, according to the manufacturer’s instructions. Peripheral blood or spleen cells were blocked with 1 µg of purified anti-mouse IgG Ab and 0.5 µg of Fc block for 20 min. Cells were then incubated with 4 µg of peptide-loaded H-2K^b:Ig fusion protein at 4°C for 1 h and then stained with PE anti-mouse IgG and FITC anti-CD8 Abs for 30 min at 4°C. At least 25,000 events were analyzed for each determination whenever possible. The percentage of H60-specific CD8⁺ T cells was determined by subtracting the percentage of CD8⁺OVA⁺ T cells (background) from the percentage of CD8⁺H60⁺ T cells for each individual animal.

Quantification of IL-7 gene expression by real-time PCR

Total RNA was isolated from spleen cells after extraction in TRIzol reagent (Invitrogen, Carlsbad, CA). Five hundred nanograms of RNA were reverse transcribed into cDNA using a Superscript II First-Strand RT-PCR system (Invitrogen), according to the manufacturer’s instructions. Primers and probes for 18S ribosomal RNA and IL-7 were purchased from Applied Biosystems (Foster City, CA). cDNA was suspended in 2x master mix along with 18S or IL-7 primer/probes, according to the manufacturer’s specifications. Real-time PCR was performed on a DNA Engine Opticon-2 from MJ Research (Watertown, MA). Reaction conditions were as follows: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The amount of IL-7 transcript, normalized to amount of 18S ribosomal RNA as an internal standard, was calculated by the comparative C_T method (ABI User Bulletin 2; Applied Biosystems).

Statistics

Group comparisons of the absolute number of splenic donor and host T cells were performed using the Mann-Whitney U test. The relative increase in IL-7 mRNA levels between groups was compared using the unpaired Student’s t test. Survival curves were constructed using the Kaplan-Meier product limit estimator and compared using the log rank rest. A value of p 0.05 was deemed to be significant in all experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Administration of IL-7 results in increased donor and host T cell expansion after BMT in nonirradiated mice

The effect of IL-7 administration on donor and host T cell expansion was first examined in the absence of a conditioning regimen by performing transplant experiments using a parentF₁ model in which there is no competing host-vs-graft response. B6AF1/J recipients were transplanted with 25 x 10⁶ B6 T cells and administered either PBS or IL-7 (5 µg/day) on days 0–9, unless terminated before completion of the regimen. Cohorts of mice were then sacrificed either 6 or 11–13 days after transplant to assess the kinetics of T cell reconstitution in the spleens of transplant recipients. Although host T cells were the predominant T cell population in both groups of animals 6 days post-BMT, IL-7 treatment resulted in a significant increase in the absolute number of host T cells compared with what was observed in PBS-treated mice (72 x 10⁶ vs 51 x 10⁶, p = 0.03) (Table I). Administration of IL-7 also increased the absolute numbers of donor CD4⁺ T cells (14 x 10⁶ vs 10 x 10⁶, p = 0.02), but there were no statistically significant differences in donor CD8⁺ or T cells. By days 11–13 posttransplantation, donor T cells were the primary T cell population in both cohorts of animals. IL-7-treated mice had a greater absolute number of splenic donor T cells when compared with animals treated with PBS (55 x 10⁶ vs 32 x 10⁶, p = 0.03) (Table I). This difference was observed for both CD4⁺ (18 x 10⁶ vs 11 x 10⁶, p = 0.002) and CD8⁺ (33 x 10⁶ vs 19 x 10⁶, p = 0.005) T cell populations. The absolute number of host and CD4⁺ T cells was also significantly greater in IL-7-treated animals, although there was no difference in total host CD8⁺ T cells. Due to the fact that IL-7 augmented expansion of both donor and host T cells, there was no statistically significant difference in the percentage of donor T cell engraftment in the spleens of PBS- vs IL-7-treated mice (55 vs 59%, respectively; p = 0.23). The ratio of donor:host CD8⁺ T cells, however, was higher in IL-7-treated vs PBS-treated animals (average 1.6 vs 1.3), whereas there was no difference in CD4⁺ T cells (average 1.1 vs 1.0). Overall, these data indicate that IL-7 was able to augment expansion of both donor and host T cell populations in nonirradiated transplant recipients. This effect was more pronounced later (i.e., 11–13 days post-BMT), in which there was preferential expansion of donor CD8⁺ T cells.

Because administration of IL-7 resulted in expansion of both donor and host T cells relative to PBS-treated control mice, we performed experiments to determine whether administration of IL-7 to transplanted animals affected the severity of GVHD. Nonirradiated B6AF1/J recipients were transplanted with 25 x 10⁶ B6 T cells and then treated with either PBS or IL-7 on days 0–9. Mice treated with PBS had 20% survival 60 days after BMT. In contrast, survival in IL-7-treated animals was significantly worse, with all mice dying by 33 days posttransplantation (Fig. 1A, p < 0.03). Weight loss was comparable in both groups of mice (Fig. 1B), with the exception that a percentage of PBS-treated animals survived for the duration of the experiment. To further characterize the severity of GVHD in both cohorts of mice, additional experiments were performed in which mice were sacrificed 13 days posttransplantation for histological analysis of GVHD target organs. Mice treated with IL-7 had significantly more GVHD pathology in both the liver and colon than PBS-treated mice (mean score 3.5 ± 0.4 vs 2.3 ± 0.4, p = 0.03) (Fig. 1C). Because IL-7 has been shown to enhance thymopoiesis after allogeneic BMT (25), we also examined the thymi of mice that were administered either PBS or IL-7. There was no difference in thymus size between PBS-treated (mean 21 x 10⁶ cells, n = 10) and IL-7-treated mice (mean 14 x 10⁶ cells, n = 13) when analyzed 11–13 days post-BMT (p = 0.23). The percentage of CD4⁺CD8⁺ thymocytes, however, was significantly lower in IL-7-treated vs PBS-treated animals (29 vs 48%, p = 0.008) consistent with more severe GVHD. Thus, administration of IL-7 exacerbated GVHD lethality and did not enhance thymopoiesis.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Administration of IL-7 exacerbates GVHD lethality in nonirradiated recipients. Nonirradiated B6AF1/J mice were transplanted with 25 x 106 B6 T cells and then treated with either PBS (, n = 15) or 5 µg/day IL-7 (, n = 15) on days 0–9 post-BMT. Actual survival is depicted in A, and serial weight curves are shown in B. Data are cumulative results from four separate experiments. In separate experiments (C), PBS- or IL-7-treated mice (n = 11/group) were sacrificed 13 days posttransplantation, and tissues (i.e., liver and colon) were analyzed for evidence of GVHD. A total GVHD score was calculated, as described in Materials and Methods. Data are from two separate experiments and are presented as the mean ± 1 SEM.

Irradiation is known to exacerbate GVHD through the production of inflammatory cytokines that directly or indirectly contribute to the pathophysiology of GVHD (32, 33, 34). Consequently, we questioned whether differences in survival between mice transplanted with or without IL-7 administration would exist under conditions in which GVHD severity was not solely dependent upon donor T cells. To examine this question, we performed studies in which mice were preconditioned with varying doses of TBI. Several donor T cell doses were also examined because an earlier study (35) had indicated that the ability of IL-7 to exacerbate GVHD was dependent, in part, upon T cell dose. In initial studies, B6AF1/J mice were lethally irradiated (1000 cGy) and then transplanted with TCD B6 BM alone or together with either 0.75 or 1.5 x 10⁶ B6 T cells. A dose of 1.5 x 10⁶ T cells resulted in death in all lethally irradiated recipients not administered IL-7, while 0.75 x 10⁶ cells caused fatal GVHD in 50% of transplanted animals (our unpublished observations). Similarly transplanted mice were then treated with either PBS or IL-7 on days 0–9 post-BMT using the same schedule that was used in nonirradiated recipients. There was no significant difference in survival between PBS- and IL-7-treated mice at donor T cell doses of either 1.5 x 10⁶ (Fig. 2A, p = 0.14) or 0.75 x 10⁶ (Fig. 2B, p = 0.59) cells. Because a significant percentage of mice transplanted with 0.75 x 10⁶ T cells did not die from GVHD, we also assessed weight loss and parameters of immune reconstitution as more sensitive indicators of GVHD. Serial weight curves demonstrated no significant difference between groups when a subthreshold number of donor T cells were transplanted into recipients (Fig. 2C). Both spleen (0.5 x 10⁶ vs 1.4 x 10⁶, p = 0.14) and thymus size (0.8 x 10⁶ vs 14 x 10⁶, p = 0.42) were not significantly different between PBS- and IL-7-treated mice (n = 5/group), respectively, when examined 60 days post-BMT. Thus, IL-7 did not exacerbate lethality under conditions in which GVHD was uniformly lethal or under conditions in which GVHD mortality was attenuated due to transplantation of a subthreshold number of donor T cells.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. Administration of IL-7 does not exacerbate GVHD lethality when administered early posttransplant to lethally irradiated recipients. A, Irradiated (1000 cGy) B6AF1/J mice were transplanted with 10 x 106 TCD B6 BM (•, n = 8) alone or together with 1.5 x 106 B6 T cells. Mice transplanted with T cells were then treated with PBS (, n = 10) or IL-7 (5 µg/day) (, n = 10) on days 0–9. B, Irradiated (1000 cGy) B6AF1/J mice were transplanted with 10 x 106 TCD B6 BM (•, n = 8) alone or together with 0.75 x 106 B6 T cells. Mice transplanted with T cells were then treated with PBS (, n = 12) or IL-7 (5 µg/day) (, n = 10) on days 0–9. Actual survival is depicted. Data are cumulative results from two separate experiments at each T cell dose. Mean serial weight measurements from mice transplanted in B are shown in C.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Delayed administration of IL-7 does not exacerbate GVHD mortality in lethally irradiated recipients. Irradiated (1000 cGy) B6AF1/J mice were transplanted with 10 x 106 TCD B6 BM (•, n = 9) alone or together with 0.75 x 106 B6 T cells. Mice transplanted with T cells were then treated with PBS (, n = 17) or IL-7 (5 µg/day) (, n = 17) on days 14–23. Actual survival is depicted. Data are cumulative results from three separate experiments.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Administration of IL-7 does not exacerbate GVHD lethality when administered early posttransplant to sublethally irradiated recipients. Irradiated (500 cGy) B6AF1/J mice were transplanted with 10 x 106 TCD B6 BM (•, n = 8) alone or together with 5 x 106 B6 T cells. Mice transplanted with T cells were then treated with PBS (, n = 10) or IL-7 (5 µg/day) (, n = 10) on days 0–9. Actual survival is depicted. Data are derived from two separate experiments.

In nonirradiated mice, we observed that exacerbation of GVHD mortality by IL-7 was associated with a significant increase in the absolute number of donor T cells. We therefore conducted a similar analysis in irradiated recipients to determine whether IL-7 enhanced expansion of donor T cells. Lethally irradiated B6AF1/J mice were transplanted with TCD B6 BM plus a limiting number (0.75 x 10⁶) of B6 T cells, and then administered either PBS or IL-7 on days 0–9 post-MT, unless terminated before completion of the regimen. Animals were sacrificed on either day 6 or 11 post-BMT, and the absolute numbers of donor splenic T cells were determined. There was a statistically significant, but minimal increase in the absolute number of donor CD4⁺ T cells on day 6 post-BMT. Otherwise, there were no other statistically significant differences in the absolute number of donor or host CD4⁺ or CD8⁺ T cells in the spleens of PBS- vs IL-7-treated animals at either 6 or 11–13 days posttransplantation (Table II).

View larger version (38K): [in this window] [in a new window]   FIGURE 5. H60-reactive B6 CD8+ T cells are detectable in the peripheral blood and spleen of PBS- and IL-7-treated BALB.B mice undergoing GVHD. Lethally irradiated (800 cGy) BALB.B mice were transplanted with 10 x 106 TCD B6 BM alone or together with 5 x 106 B6 T cells. Mice transplanted with T cells were then treated with either PBS or IL-7 (5 µg/day) on days 0–9 posttransplantation. Mice in each cohort were bled on day 12–13 before being sacrificed on day 13 post-BMT to sample both peripheral blood and spleen cells. Flow cytometric analysis of CD8+ T cells that recognized the H60 immunodominant peptide is shown from representative individual mice in each of the three cohorts.

We reasoned that production of endogenous IL-7, or lack thereof, by recipient animals after allogeneic BMT might be a factor that affected the ability of exogenous IL-7 to exacerbate GVHD mortality. To address this question, the level of IL-7 mRNA in the spleens of mice on day 7 posttransplantation was quantitated by real-time PCR. Groups of animals that were evaluated consisted of lethally irradiated (1000 cGy) B6AF1/J mice (no transplant), lethally irradiated B6AF1/J mice transplanted with TCD B6 BM alone, lethally irradiated B6AF1/J mice transplanted with TCD B6 BM plus 1.5 x 10⁶ B6 T cells, nonirradiated B6AF1/J mice transplanted with 25 x 10⁶ B6 T cells, and normal nontransplanted B6AF1/J mice. To control for the adequacy of template, 18S ribosomal RNA was also amplified from the same cDNA samples in individual mice, as detailed in Materials and Methods. There was no statistically significant difference in IL-7 levels between normal nontransplanted B6AF1/J mice and nonirradiated mice transplanted with B6 T cells (p = 0.66), indicating that transplantation of alloreactive donor T cells alone did not augment endogenous IL-7 production (Fig. 6). In contrast, IL-7 levels in all irradiated groups of mice were significantly greater than levels in normal B6AF1/J animals, demonstrating that host conditioning was a potent stimulus for induction of IL-7. In GVHD models, we observed that exogenous IL-7 administration exacerbated GVHD mortality in nonirradiated recipients (Fig. 1), but had no effect in irradiated animals (Figs. 2–4). Notably, in the absence of exogenous IL-7, the combination of TBI plus transplantation of alloreactive donor T cells resulted in a 72-fold higher level of IL-7 mRNA when compared with that observed in nonirradiated animals transplanted with donor T cells alone. Thus, irradiated mice undergoing GVHD had significantly more endogenous IL-7 production than nonirradiated mice with GVHD.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Host conditioning increases IL-7 mRNA levels in the spleens of mice transplanted with alloreactive donor T cells. RNA samples from spleen cells of individual mice (n = 3–4/group) were obtained on day 7 posttransplantation, cDNA generated, and amplified using 18S ribosomal RNA or IL-7-specific primers (see Materials and Methods). Data are presented as the relative increase for IL-7 mRNA levels ± 1 SD, with all groups normalized to normal nontransplanted B6AF1/J mice, which served as the calibrating group for the analysis. Normal B6AF1/J mouse (); nonirradiated B6AF1/J mice transplanted with 25 x 106 B6 T cells (No TBI, GVHD; ); lethally irradiated B6AF1/J mice transplanted with TCD B6 BM (TBI, No GVHD; stippled bar); lethally irradiated B6AF1/J mouse (no transplant) (TBI only; ); lethally irradiated B6AF1/J mice transplanted with TCD B6 BM plus 1.5 x 106 B6 T cells (TBI, GVHD; ). *, p < 0.01 relative to normal nontransplanted mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We observed that IL-7 administration resulted in a significant increase in the absolute number of both donor CD4⁺ and CD8⁺ T cells in nonirradiated animals transplanted with alloreactive donor T cells (Table I). This correlated with increased GVHD mortality in these animals, as determined by both survival and histological damage to GVHD target organs (Fig. 1). These results are consistent with in vitro studies, in which we have observed that IL-7 is able to augment the absolute numbers of both CD4⁺ and CD8⁺ T cells in standard mixed lymphocyte cultures, by both preventing apoptosis and enhancing T cell proliferation (our unpublished observations). Thus, in nonirradiated recipients, these dual effects of IL-7 appear to work in concert to increase the number of alloreactive donor T cells that are able to mediate a lethal GVH reaction. In that regard, we have previously shown that inhibition of T cell apoptosis alone can exacerbate GVHD lethality in nonirradiated recipients (37). In these studies, mice transplanted with donor T cells that were resistant to passive apoptotic cell death through overexpression of Bcl-x_L had more severe GVHD. The implication of these data was that any intervention that increased T cell survival in nonirradiated mice would exacerbate GVHD lethality. The results of the current study support this premise. Moreover, the present results provide further support for the interpretation that, under conditions in which GVHD severity is determined primarily by donor T cells as opposed to contributions from the conditioning regimen, factors that prolong donor T cell survival adversely affect GVHD. Thus, our data would predict that use of IL-7 in the setting of delayed donor lymphocyte infusions (38, 39, 40) or in instances in which IL-7 might be used to enhance the cytotoxic effector function of adoptively transferred donor T cells posttransplantation (41, 42) might be associated with an increase in the severity of GVHD.

Because the severity of GVHD is dependent upon a number of factors, including the conditioning regimen as well as the donor T cell dose (32, 33, 34), we examined the effect of IL-7 on GVHD severity in the presence of a TBI-based conditioning regimen as well as at several different donor T cell doses. In mice preconditioned with TBI, we were unable to distinguish any difference in survival between groups treated with and without IL-7, regardless of the T cell dose, intensity of the conditioning regimen, or IL-7 administration schedule. It should be pointed out that these results were observed under conditions in which GVHD was fatal in all recipients as well as conditions in which GVHD was not uniformly lethal. Furthermore, unlike what was observed in nonirradiated mice treated with IL-7, administration of IL-7 did not result in an increase in the absolute numbers of donor CD4⁺ and CD8⁺ T cells in the spleen early posttransplantation, even when assessed under conditions in which GVH-reactive donor T cells could be specifically identified and quantitated (Table III). These data indicated that the ability of IL-7 to effect donor T cell expansion and exacerbate GVHD was abrogated in mice that received pretransplant conditioning with TBI.

We hypothesized that a potential explanation for these discordant results was that the conditioning regimen might augment the production of endogenous IL-7 and thereby attenuate the effect of exogenous IL-7 administration. In support of this premise, Levy et al. (43) have shown that irradiated mice transplanted with either syngeneic or allogeneic BM with or without added T cells had detectable IL-7 mRNA in the spleen, although they did not examine whether a GVH reaction in the absence of host conditioning was sufficient to induce IL-7 transcription. Moreover, IL-7 levels have been demonstrated to be increased under conditions of T cell depletion (44, 45), as occurs after intensive conditioning regimens. We observed that there were no significant differences in IL-7 mRNA levels between normal mice and nonirradiated animals transplanted with donor T cells (Fig. 6). In contrast, irradiated mice, irradiated animals transplanted with TCD BM, and irradiated mice transplanted with TCD BM and alloreactive donor T cells all had significantly higher levels of splenic IL-7 mRNA. Notably, the combination of TBI and transplantation of alloreactive donor T cells resulted in a 72-fold higher IL-7 mRNA level than observed in nonirradiated mice transplanted with donor T cells alone. In light of these data, we believe the most likely interpretation for the discordant effects of IL-7 on GVHD mortality in these two groups of mice is that donor T cells in nonirradiated B6B6AF1/J chimeras were capable of a more robust response to exogenous IL-7 due to the limited endogenous production of the cytokine. Thus, IL-7 was able to expand the number of donor T cells in these animals, which led to worsening of GVHD. Conversely, T cells obtained from the spleens of irradiated GVHD animals were already exposed to significant levels of endogenous IL-7, and therefore the administration of exogenous IL-7 resulted in no further donor T cell expansion in vivo. This would explain why IL-7 administration did not exacerbate GVHD in these animals. The specific cells responsible for IL-7 secretion are not resolved by these studies, although dendritic cells located in the spleen are a prime candidate (17, 18, 46).

Prior studies that have examined the role of IL-7 on GVHD induction have been conducted exclusively in lethally irradiated recipients and have yielded discordant results. Sinha et al. (35) showed that IL-7 could exacerbate GVHD when a subthreshold dose of donor T cells was transplanted into lethally irradiated recipients. These investigators examined the effect of IL-7 at three donor T cell doses, all of which were insufficient to cause lethal GVHD in all recipients. IL-7 administered over a 28-day period exacerbated GVHD lethality at the highest T cell dose examined (7.5 x 10⁶), but did not worsen GVHD mortality at two lower doses. IL-7 did increase the degree of weight loss at all T cell doses examined. Histologic evidence of GVHD was also significantly worse in IL-7-treated animals at two of three T cell doses tested (i.e., 1 x 10⁶ and 10 x 10⁶, but not 5 x 10⁶) as well as in syngeneic recipients, suggesting that IL-7 may have some toxicity in the absence of alloreactive donor T cells. These data suggested that IL-7 administration could exacerbate GVHD under conditions in which the dose of donor T cells was insufficient to effect uniform lethality in transplanted recipients. These studies stand in juxtaposition to those of Alpdogan et al. (27), who reported that IL-7 administration did not exacerbate GVHD under conditions in which GVHD was uniformly lethal in untreated animals when examined in three different murine models. There were, however, differences between these two studies that may have affected the observed outcomes. First of all, the dose of IL-7 was significantly higher in the report by Sinha et al. (35) (i.e., 5 µg/day) than that used by Alpdogan et al. (27) (1 µg/day). Second, the duration of therapy was longer in the former study (28 vs 14 days). Finally, the more severe GVHD elicited in the latter models may have obscured effects of IL-7 on GVHD induction. In the current study, we used the higher dose of IL-7 (5 µg/day), as used by Sinha et al. (35), and examined GVHD induction under a variety of TBI and donor T cell doses designed to effect varying degrees of GVHD severity. Nonetheless, we were unable to uncover any scenario under which IL-7 increased GVHD mortality in irradiated animals. It should be noted that the duration of IL-7 administration was shorter in our study, and therefore we cannot exclude the possibility that this variable is responsible for the observed differences between the two studies.

In summary, these studies demonstrated that IL-7 has potent effects on the survival and expansion of T cells that are sufficient to significantly increase GVHD mortality in the absence of a conditioning regimen. In contrast, under inflammatory conditions resulting from TBI conditioning, the dose and administration schedule used in these experiments did not result in an exacerbation of GVHD mortality. These discordant results appear to be attributable, in part, to the increased endogenous production of IL-7 induced by the combination of TBI and transplantation of alloreactive donor T cells that serves to attenuate any detrimental effects on GVHD resulting from exogenous IL-7 administration. These data also establish that another manner in which the conditioning regimen can contribute to the pathophysiology of GVHD is by the induction of cytokines that can directly promote T cell survival and proliferation.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (HL64603 and HL55388) and the Midwest Athletes Against Childhood Cancer Fund.

² Address correspondence and reprint requests to Dr. William R. Drobyski, Bone Marrow Transplant Program, 9200 West Wisconsin Avenue, Milwaukee, WI 53226. E-mail address: bill{at}bmt.mcw.edu

³ Abbreviations used in this paper: BM, bone marrow; BMT, BM transplantation; GVH, graft-vs-host; GVHD, GVH disease; TBI, total body irradiation; TCD, T cell depletion.

Received for publication May 14, 2003. Accepted for publication December 30, 2003.

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