Biology of Blood and Marrow Transplantation
Volume 14, Issue 1 , Pages 16-27, January 2008

Importance of Interleukin-7 in the Development of Experimental Graft-Versus-Host Disease

  • Brile Chung

      Affiliations

    • Division of Stem Cell Transplantation, Department of Pediatrics, Stanford University School of Medicine, Stanford, California
    • ,
  • Eric Dudl

      Affiliations

    • Division of Research Immunology/Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, California
    • ,
  • Akira Toyama

      Affiliations

    • Division of Research Immunology/Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, California
    • ,
  • Lora Barsky

      Affiliations

    • Division of Research Immunology/Bone Marrow Transplantation, The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, California
    • ,
  • Kenneth I. Weinberg

      Affiliations

    • Division of Stem Cell Transplantation, Department of Pediatrics, Stanford University School of Medicine, Stanford, California
    • Corresponding Author InformationCorrespondence and reprint requests: Kenneth I Weinberg, MD, Division of Stem Cell Transplantation, Department of Pediatrics, Stanford University Medical Center, 1000 Welch Road, Suite 300, Palo Alto, CA 94304.

Received 12 July 2007; accepted 18 September 2007. published online 03 December 2007.

Article Outline

Abstract 

Interleukin (IL)-7 promotes both thymopoiesis and mature T lymphocyte survival and proliferation in experimental murine models of hematopoietic stem cell (HSC) transplantation. Because HSC products for transplantation also may contain IL-7–responsive mature T lymphocytes, we examined whether IL-7 is necessary for the induction of GVHD after allogeneic bone marrow transplantation (BMT). Lethally irradiated C57BL6J (B6) and B6.IL-7-/- (both H2Kb) recipient mice were co-transplanted with T cell–depleted (TCD) bone marrow cells and lymph nodes (LNs) from either congenic B6.SJL (CD45.1+) or allogeneic BALB/c (H2Kd) donor mice. After transplantation, the recipient mice were subcutaneously injected with either human recombinant IL-7 or phosphate-buffered saline (PBS) for 60 days. No evidence of GVHD was detected in the congenic recipients or in the allogeneic B6/IL-7-/- recipients treated with PBS; in contrast, significantly increased rates of GVHD-related mortality and morbidity were found in the allogeneic B6.IL-7-/- recipients treated with IL-7. The proliferation and number of donor T cells were significantly lower at day 30 post-BMT in the PBS-treated B6.IL-7-/- recipients compared with the IL-7–treated B6.IL-7-/- mice. These experiments demonstrate that IL-7 is an important factor in the development of GVHD, presumably by supporting the survival, proliferation, and possibly activation of alloreactive donor-derived T cells in the recipients.

Key Words: Allogeneic, Bone marrow transplantation, Graft-versus-host disease, Interleukin-7, T lymphocyte

 

Back to Article Outline

Introduction 

Although allogeneic bone marrow transplantation (BMT) is a potentially life-saving treatment for patients with hematologic, oncologic, or immunologic diseases, graft-versus-host disease (GVHD) continues to be a potentially serious complication that limits the efficacy of transplantation. GVHD is initiated when donor T lymphocytes are activated by host alloantigens. On activation, inflammatory cytokines produced by donor T cells induce proliferation and differentiation of various effector cells, including anti-host helper and cytotoxic T cells (CTLs), macrophages, and natural killer cells, which cause damage to target organs such as liver, gut, lung, and skin 1, 2, 3, 4, 5, 6. A number of therapeutic measures, including T cell depletion (TCD) and immunosuppressive therapies, have been used to prevent GVHD post-BMT [7].

The post-BMT period is marked by profound immunodeficiency, making newly transplanted patients susceptible to various bacterial, viral, and fungal infections 8, 9. GVHD, immunosuppressive procedures or therapies to prevent or treat GVHD, and the time required for donor stem cells to develop into T lymphocytes contribute to the post-BMT immunodeficiency. Although TCD can prevent GVHD, it increases the degree of post-BMT immune deficiency because of the additional time required for the development of new T lymphocytes from donor-derived progenitors. In addition, the nonspecific immunosuppressive effects of drugs to prevent or treat GVHD also may contribute to poor immune function. Immune reconstitution after BMT is further hindered by impaired function of the thymic microenvironment caused by age, pre-BMT conditioning, and GVHD itself 10, 11, 12. Methods to enhance the development of T lymphocytes and immune reconstitution are critical to solving the problem of post-BMT immune deficiency.

IL-7, along with Kit ligand (KL; stem cell factor), is the major lymphopoietic cytokine produced in the thymus and bone marrow (BM) compartment 13, 14. IL-7 induces proliferation, differentiation, and survival of immature T lymphocytes throughout development. During normal T cell development in the thymus, IL-7 produced by thymic epithelial cells binds to the cognate IL-7 receptors expressed on the surface of immature T lymphoid progenitor cells. IL-7 stimulates the differentiation of immature CD3-CD4-CD8- (triple-negative) thymocytes to later stages, ultimately resulting in the development of mature CD4+CD8- or CD4-CD8+ T cells. The importance of IL-7 for thymopoiesis is demonstrated by mice with targeted mutations of the IL-7, IL-7 receptor α, or common γ chain (γc) genes, dogs with X-SCID (γc mutations), or humans with X-SCID, all of which have defective thymopoiesis and impaired ability to produce T lymphocytes 15, 16, 17, 18. We and others have shown that administration of recombinant human IL-7 corrects the thymopoietic defects observed after histocompatible BMT, suggesting that post-BMT IL-7 administration may be a potential therapy for post-BMT immunodeficiency 19, 20.

Other effects of IL-7 have raised concerns about its safety in the context of allogeneic BMT, however. Besides immature lymphoid progenitors, such as common lymphoid progenitors and thymocytes, the IL-7 receptor is expressed by mature T lymphocytes [21]. The IL-7R expressed by mature T lymphocytes mediates several important biological effects that are likely to be clinically relevant. Homeostatic proliferation of naïve CD4+ and CD8+ T lymphocytes depends on combined T cell receptor (TCR) recognition of self-ligands and IL-7R signaling. IL-7Rα-/- T lymphocytes do not proliferate normally in a normal host, whereas, conversely, normal T lymphocytes do not undergo homeostatic proliferation in IL-7-/- hosts 22, 23, 24. Stimulation through IL-7R also increases expression of the bcl-2 anti-apoptotic protein, thereby increasing survival of mature T lymphocytes 24, 25, 26. Furthermore, IL-7 may function as a co-factor for T lymphocyte activation by stimulating production of Th1 cytokines such as IL-2, IFN-γ, and IL-12 27, 28, 29.

In allogeneic transplantation, the effects of IL-7 on mature T lymphocytes might cause or exacerbate GVHD by either promoting the proliferation or survival of alloreactive donor mature T cells, or by increasing their activation state. Studies of the effects of IL-7 on GVHD have yielded conflicting results 30, 31. Alpdogan et al. [30] using several mouse models of allogeneic BMT and IL-7 dosing schedules, reported that post-BMT administration of IL-7 did not cause GVHD, whereas Sinha et al. [31], using somewhat different models, showed that IL-7 treatment exacerbated GVHD.

To clarify the role of IL-7 in the pathogenesis of GVHD, we performed experiments to determine whether IL-7 is necessary for the development of GVHD after allogeneic BMT. We hypothesized that endogenous or exogenous IL-7 is required for maintenance, proliferation, and possibly activation of donor T lymphocytes can mediate GVHD. Using genetic models of IL-7 deficiency, we demonstrate that either endogenously produced or exogenously administered IL-7 is necessary for the development of GVHD, most likely by promotion of the survival and proliferation of alloreactive T lymphocytes. The results suggest that the therapeutic use of IL-7 to improve immune reconstitution in an allogeneic setting may exacerbate GVHD.

Back to Article Outline

Materials and Methods 

Mice 

Female C57BL/6J (H-2Kb, CD45.2), male B6.SJL (H-2Kb, CD45.1), male BALB/c (H-2Kd Thy 1.2), and male BALB/c (H-2Kd Thy 1.1) mice (age 8-10 weeks) were purchased from the Jackson Laboratory (Bar Harbor, ME). A breeding colony of IL-7-/- mice on a C57BL/6J background (H-2Kb, CD45.2, B6.IL-7-/-) was established from founder mice kindly provided by Dr Richard Murray (DNAX Research Institute, Palo Alto, CA). Protocols for animal care and BMT were approved by the Children's Hospital Los Angeles Research Institute Animal Care Committee and the University Committee on Use and Care of Laboratory Animals at Stanford University.

BMT Procedure 

Female C57BL/6J (H-2Kb) recipient mice were given 2 separate doses of radiation from a 137Cs source at 128 cGy/minute (700 cGy on day -1 and 600 cGy on day 0) before transplantation. In each experiment, control mice received irradiation without subsequent BMT, to verify that the doses of radiation were marrow-ablative. BM cells were harvested from the femur and tibia of male congenic (histocompatible) B6.SJL (H-2Kb, CD45.1), allogeneic BALB/c (H-2Kd Thy 1.2), or allogeneic BALB/c (H-2Kd Thy 1.1) donor mice by perfusion, after the mice were sacrificed by CO2 narcosis. The donor marrow was depleted of mature T lymphocytes by immunomagnetic depletion, using rat anti-mouse Thy 1, CD4, and CD8 monoclonal antibodies (mAb; Pharmingen, San Diego, CA) and sheep anti-rat antibodies conjugated to beads (Dynal, Great Neck, NY). The purity of the T lymphocyte–depleted cells was determined by flow cytometric analysis (FACS) to ensure a frequency of mature T lymphocytes in the infused marrow of < 0.1%.

GVHD was induced by administration of T lymphocytes derived from lymph nodes (LNs) of donor mice at the same time as BMT. Mesenteric, axillary, and inguinal LN were collected, minced, and filtered through nylon mesh to eliminate adherent cells. After completion of pre-BMT irradiation on day 0, 1 × 106 T cell–depleted (TCD) BM cells and 4 × 106 LN cells were resuspended in phosphate-buffered saline (PBS; Bio Whittaker, Walkerville, MD) and transplanted into each recipient through tail vein injection (0.3 mL total volume). After transplantation, mice were housed in sterilized micro-isolator cages and given normal chow and autoclaved water containing tetracycline HCl 20 μg/mL (Goldline, Miami, FL) for the first 2 weeks post-BMT and filtered water thereafter.

Administration of Recombinant Human IL-7 

Recombinant human IL-7 (rhIL-7; R&D Systems, Minneapolis, MN), resuspended in PBS, was aseptically administered to the BMT recipients by subcutaneous injection at a dose of 500 ng twice daily for 30 or 60 days or until sacrifice [19]. The control mouse groups were injected with normal saline according to the same schedule.

Assessment of GVHD 

The degree of GVHD severity was assessed using the clinical scoring system described by Cook et al. [33]. Each animal undergoing BMT animal was scored weekly for 5 parameters (weight loss, skin integrity, fur texture, mobility, and posture) on a scale of 0 to 2 (0, absent or normal; 1, mildly abnormal; 2, severely abnormal). The GVHD clinical index was the sum of the scores for individual criteria.

Histological Analysis 

Wax-coated tissue sections from small intestine and skin were cut into 5-μm-thick sections, mounted onto slides, fixed in 10% formalin, and stained with hematoxylin and eosin. The tissue sections were independently examined in a blinded manner for evidence of GVHD by 2 of the authors [5].

Flow Cytometry 

Peripheral blood lymphocytes or single-cell suspensions of splenocytes or LN cells were prepared by lysing the red blood cells with lysis buffer containing ammonium chloride (Gibco Life Technologies, Carlsbad, CA). Here, 1 × 105 cells were stained with optimal concentrations of fluorescein isothiocyanate, phycoerithrin, allophycocyanin, or PerCP-conjugated anti-Thy1.1, Thy 1.2, CD3, CD4, CD8, CD69, H2Kb, H2Kd, or isotype control monoclonal antibodies (Pharmingen, San Diego, CA). After staining, the cells were washed twice in PBS and analyzed with the FACSCalibur analyzer (Becton Dickinson, Franklin Lakes, NJ). In some experiments, donor LN cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) before transplantation to measure the proliferation in vivo of the cells after transplantation. Donor LN cell proliferation was assessed by measuring separate peaks of decreased intensity of CFSE fluorescence on successive cell division by FACS analysis of the labeled donor CD4 or CD8 T cell population.

Statistical Analyses 

Analyses of survival rates were performed using Wilcoxon's log-rank test. Comparisons of donor cell recovery, weight loss, and GVHD scoring were done using 2-way analysis of variance. Differences of between groups of different immunophenotypic populations of cells after transplant were analyzed by a 2-tailed t-test with unequal distributions. P values ≤ .05 were considered statistically significant.

Back to Article Outline

Results 

IL-7 Is Necessary for GVHD-Related Mortality After Allogeneic BMT 

To assess whether IL-7 is necessary for the development of GVHD, we measured the survival rate of recipient B6 or B6.IL-7-/- mice after transplantation of congenic or allogeneic BM and LN cells. Radiation-treated (1300 cGy) B6 and B6.IL-7-/- mice (H-2Kb) were co-transplanted with 1 × 106 TCD BM cells and 4 × 106 LN T lymphocytes from either congenic B6.SJL (H-2Kb) or allogeneic BALB/c mice (H-2Kd). After BMT, either PBS or rhIL-7 (500 ng twice a day subcutaneously) was administered to the transplant recipients for 60 days. A schedule of continuous administration of IL-7 was chosen to ensure that IL-7 was present at all times post-BMT, because it was unknown when IL-7 might exert effects on the co-transplanted mature T lymphocytes.

Figure 1 shows survival of the congenic and allogeneic B6 and B6.IL-7-/- recipients. The survival of the congenic recipients was 100%, regardless of whether they were normal B6 or B6.IL-7-/- mice. Administration of IL-7 to the congenic B6 or B6.IL-7-/- recipients did not decrease survival. Thus, neither the presence nor absence of endogenous IL-7, or the administration of exogenous IL-7, had any effect on survival in the congenic setting.

  • View full-size image.
  • Figure 1 

    Treatment with IL-7 increased GVHD-related mortality in B6.IL-7-/- recipient animals after allogeneic LN BMT. The B6 or B6.IL-7-/- recipients that had received 1300 cGy TBI received either 1 × 106 TCD BM and 4 × 106 LN cells from BALB/c donor mice, or similar cell numbers of LN and BM cells from congenic B6.SJL donors (CD45.1), and were then treated with recombinant human IL-7 (500 ng twice a day via subcutaneous injection) for 60 days. Survival over the 150 days after BMT is shown. Survival of all wild-type allogeneic B6 recipients was significantly lower than that of the congenic recipients (∗P < .002). Survival of the PBS-treated B6.IL-7-/- allogeneic recipients was significantly lower than that of congenic B6.IL-7-/- recipients (∗P < .03). The difference between the IL-7– and PBS-treated allogeneic B6.IL-7-/- recipients is significant (∗P < .002).

In contrast to the results observed in congenic recipients, the presence or absence of IL-7 affected the outcome of allogeneic BMT. In the first 25 days after allogeneic BMT, there was no difference between the B6 and B6.IL-7-/- recipients. As expected with the fully H2-incompatible BALB/c to B6 model, the mortality of either IL-7– or PBS-treated normal B6 recipients was approximately 70%, with deaths continuing to occur throughout the 150-day observation period. Although IL-7 injections induced higher mortality in normal allogeneic B6 recipients (75% vs 65%), the difference between the IL-7– and PBS-treated wild-type mice was not statistically significant. However, in the IL-7- recipients treated with PBS, GVHD-related mortality diminished to 41% at day 150. Similar to the normal B6 allogeneic recipients, some mortality occurred in the PBS-treated B6.IL-7-/- allogeneic recipient mice in the first 25 days after BMT. No further deaths were observed after this initial post-BMT period, however. In contrast, mortality of IL-7–treated allogeneic B6.IL-7-/- and B6 recipients continued throughout the 150-day observation period.

To further quantify the effects of IL-7 treatment on the clinical status of mice undergoing BMT, the mice were evaluated for GVHD based on a clinical scoring system for 6 weeks after transplantation [31]. As expected, both congenic B6 and B6.IL-7-/- recipients treated with either IL-7 or PBS showed no signs of GVHD-related morbidity (Figure 2). In contrast, allogeneic-normal B6 recipients displayed significant weight loss and other evidence of GVHD compared with the congenically transplanted controls (not shown). The overall clinical grading of GVHD in the IL-7–treated B6 mice was more severe (P < .05) (Figure 2A). Similar to the results observed in the congenic recipients, the allogeneic B6.IL-7-/- recipient mice treated with PBS demonstrated no clinical evidence of GVHD (Figure 2B). However, administration of IL-7 to the allogeneic B6.IL-7-/- recipients resulted in GVHD-related morbidity resembling that observed in the allogeneically transplanted B6 recipients (P < .012). The difference in mortality and GVHD score in the IL-7–treated and PBS-treated allogeneic B6.IL-7-/- recipients, combined with the lack of clinical toxicity of IL-7 after congenic transplantation, indicates that deaths occurring in the IL-7–treated allogeneic BMT recipients was GVHD-related.

  • View full-size image.
  • Figure 2 

    Treatment with IL-7 increased GVHD-related morbidity after allogeneic BMT. The severity of GVHD was determined by GVHD clinical grading system with scoring for 5 clinical criteria: percentage of weight loss, skin integrity, posture, mobility, and fur texture [31]. Clinical signs were graded on a scale of 0 to 2, (0, absent; 1, moderate; 2, severe), and the individual signs were summed. Shown are GVHD clinical index scores at 4 and 6 weeks for B6 recipients (A) and B6.IL-7-/- recipients (B). Differences between the IL-7–treated and PBS-treated allogeneic recipients are P < .05 in both B6 recipients and B6.IL-7-/- recipients (∗).

Absence of Histological GVHD in IL-7-/- Allogeneic BMT Recipients 

GVHD target organs were examined to determine whether IL-7 caused any histological damage. The irradiated animals received either IL-7 or PBS from day 1 to day 30 after allogeneic BMT. On day 30, sections from the small intestine and skin were analyzed for evidence of GVHD. The tissue samples from the PBS- or IL-7–treated congenic recipients showed no evidence of tissue inflammation. The results differ somewhat from those of Sinha et al. [31], who noted increased tissue inflammation in syngeneic BMT recipients treated with IL-7 compared with mice treated with IL-7 that did not undergo BMT. Compared with the congenic recipients, however, the IL-7 and PBS-treated allogeneic normal B6 recipients demonstrated histological evidence of GVHD, and the severity of the tissue inflammation did not appear to differ between the IL-7–treated and PBS-treated hosts (Figure 3A). The intestines exhibited villus blunting, lamina propria inflammation, crypt destruction, and mucosal atrophy in both the IL-7– treated and PBS-treated groups of allogeneic B6 recipients. The mice also had cutaneous GVHD, with infiltration of lymphocytes, hyperkeratosis, and hair loss. In the absence of IL-7 in the B6.IL-7-/- hosts, the histological samples from the allogeneic B6 recipients did not appear to be different from those of the congenic B6 recipients. The allogeneic B6.IL-7-/- allogeneic recipients treated with IL-7 displayed the same GVHD-related histological features seen in the allogeneic B6 recipients (Figure 3B). These findings demonstrate that IL-7 was important in GVHD-related tissue damage and inflammation, but was not intrinsically pathogenic in the histocompatible setting.

  • View full-size image.
  • Figure 3 

    Histological evidence of an increase in GVHD as a result of IL-7 administration. Skin and small intestine tissues from the recipients were analyzed at day 30 after BMT. Representative tissue samples from each group of mice were stained with hematoxylin and eosin. The tissue sections of skin and small intestine from the PBS-treated and IL-7–treated congenic BMT plus LN recipients and the PBS-treated allogeneic IL-7-/- recipients demonstrated normal histology. The tissue sections from allogeneic wild-type B6 recipients treated with PBS or IL-7 and allogeneic B6.IL-7-/- recipients treated with IL-7 showed evidence of GVHD, with lymphocytic infiltration and inflammation in the gut sections. Shown are hematoxylin and eosin staining of skin or gut sections from B6 (A) and B6.IL-7-/- (B) recipients of either allogeneic or congenic LN and TCD BM.

IL-7 Is Necessary for Maintenance of Donor T Cells in the Periphery of Recipient Mice 

Because the development of GVHD depends on donor-derived mature T cells, we investigated whether IL-7 treatment altered the number of donor-derived mature CD4 and CD8 T cells in the spleen, LN, and peripheral blood lymphocytes after allogeneic transplantation. IL-7 or PBS-treated allogeneic recipients were analyzed at days 10 and 30 after transplantation. Figure 4 shows the number of donor-derived CD4 and CD8 T cell populations at day 10 and day 30 in the peripheral blood, LN, and spleen of the mice treated with PBS and those treated with IL-7. Administration of IL-7 to normal B6 recipients increased the numbers of donor-derived mature T cells 1.2- to 3-fold in all sites (Figure 4A, C, E). The effects of IL-7 on mature T cell numbers were observed as early as day 10, before the generation of thymic-derived T cells would be expected. To distinguish newly generated donor T cells from adoptively transferred T lymphocytes, BALB/c TCD BM cells (H2Kd Thy1.1+) and BALB/c LN T cells (H2Kd Thy1.2+) were co-transplanted into allogeneic B6.IL-7-/- (H2Kb Thy1.2+) recipients treated with daily rhIL-7. The percentages of the donor-derived T cells generated from the transplanted marrow were significantly lower than that of adoptively transferred LN cells in the thymus, spleen, and LN of the recipient animals at day 18 posttransplantation (data not shown). Therefore, the increase in T cell numbers likely reflected IL-7–induced proliferation of the adoptively transferred mature T cells.

  • View full-size image.
  • View full-size image.
  • Figure 4 

    Administration of IL-7 resulted in increased number of donor CD4 and CD8 T cells in blood, LNs, and spleen after allogeneic BMT. Donor-derived peripheral lymphocyte numbers in B6 and B6.IL-7-/- recipients sacrificed 10 or 30 days after allogeneic transplantation with BALB/c LN and TCD BM cells were analyzed. Shown are numbers of donor CD4 and CD8 T cells in peripheral blood (A and B), LN (C and D), and spleen (E and F) of B6 and B6.IL-7-/- recipients at days 10 and 30. ∗Significant differences (P < .05) between the PBS- and IL-7–treated groups.

The PBS-treated B6.IL-7-/- recipients exhibited significantly lower donor T cell numbers in all 3 lymphoid compartments compared with the normal recipients (Figure 4B, D, F). Low numbers of donor CD4 and CD8 cells were detected in the peripheral blood of B6.IL-7-/- mice treated with PBS at day 10, but were 2- to 8-fold less frequent than those in normal B6 recipients (Figure 4A, B). The number of T cells detected in the lymph nodes of the B6.IL-7-/- recipients was 100- to 1000-fold lower than those in the normal B6 recipients (Figure 4C, D). Similarly, the number of splenic T cells was 10- to 30-fold lower in the B6.IL-7-/- recipients. By day 30 after transplantation, the number of donor-derived T cells in the PBS-treated B6.IL-7-/- mice was almost nil. Thus, in the absence of IL-7, adoptively transferred mature T lymphocytes did not survive (Figures 4B, D, F and 5).

  • View full-size image.
  • Figure 5 

    Progressive disappearance of donor-derived allogeneic T lymphocytes in the absence of IL-7. After allogeneic transplantation with BALB/c LN and TCD BM cells, donor T cells from the peripheral blood of B6.IL-7-/- recipient mice treated with IL-7 or PBS were gated and stained for CD4 and CD8 at days 10 and 30.

We then investigated whether the defect in lymphocyte survival could be rescued by administration of exogenous IL-7. IL-7 treatment significantly increased the numbers of donor-derived mature T cells in the B6.IL-7-/- recipients (Figures 4B, D, F and 5). The number of donor-derived T lymphocytes after IL-7 treatment was lower in the B6.IL-7-/- recipients than in the normal B6 recipients treated with either PBS or IL-7. This suggests that the bolus schedule of IL-7 administration is not as efficient at mediating T cell survival compared with endogenously produced IL-7. Overall, the data support the hypothesis that IL-7 is necessary to maintain transplanted mature T cells, including allogeneic cells, in the periphery 19, 23, 24, 25, 32.

Importance of IL-7 in Proliferation of Allogeneic T Lymphocytes 

Because naïve mature T lymphocytes are known to undergo homeostatic proliferation after interactions with self-peptides, we investigated whether there were phenotypic differences between T lymphocytes that proliferated after allogeneic or congenic transplantation 23, 24, 25. We analyzed the expression of CD69 as an activation marker, because up-regulation of CD69 has been reported during activation-induced T cell proliferation, including that of alloreactive cells 33, 34. Mice were co-transplanted with TCD BM and CFSE-labeled donor LN T cells. Using H2Kd to gate on the donor cells and CFSE to mark the proliferating T cells in the allogeneic recipients, we analyzed the expression of CD69. In the congenic recipients, expression of CD69 was low to absent in the proliferating donor-derived (CD45.1+) T cells (Figure 6A, B). The expression of CD69 in the dividing CD4 and CD8 LN-derived T cells was also analyzed in the allogeneic recipients (Figure 6A, B). Most proliferating donor CD4+ or CD8+ T cells in the allogeneic recipients had increased levels of CD69 expression. The differences in CD69 expression are consistent with CD69 expression marking proliferating alloreactive T cells, whereas CD69 expression was not induced during homeostatic proliferation of nonalloreactive cells, a concept proposed by Alpdogan et al. [32].

  • View full-size image.
  • Figure 6 

    IL-7 is required for IL-7 administration to maintain activated donor T cells in the LN during allogeneic proliferation at day 10. 1 ×106 TCD BM and 4 × 106 CFSE-labeled LN cells from either congenic or allogeneic donors were transplanted into either lethally irradiated B6 or B6.IL-7-/- recipients. At day 10, most of the proliferating CFSE-labeled congenic (CD45.1+) donor CD4 (A) and CD8 (B) T cells in lymph nodes of the B6 recipients did not express the CD69 activation marker, whereas the allogeneic (H2Kd) donor cells were predominantly CD69+. The frequency of donor-derived CD4 or CD8 T cells in the lymph nodes that expressed CD69 after allogeneic transplantation was not significantly changed by the IL-7 treatment of either B6 (C) or B6.IL-7-/- (D) recipients. (E) IL-7 treatment of the B6.IL-7-/- allogeneic recipients increased the absolute number of activated (CD69+) donor-derived CD4 and CD8 T lymphocytes (∗P < .02 for PBS vs IL-7).

IL-7 has been shown to both act as a co-factor for T cell activation and increase the survival of activated T lymphocytes [24]. We studied the effects of IL-7 on the survival of activated alloreactive T cells after transplantation to determine whether IL-7 enhanced the survival of activated allogeneic T cells in vivo. Allogeneic marrow and CFSE-labeled LN cells from BALB/c donors were transplanted into B6 and B6.IL-7-/- recipients, treated with either IL-7 or PBS. At day 10, donor-derived T cells in the lymph nodes of the recipient mice were analyzed by gating on H2Kd+ cells. The frequency of donor-derived T lymphocytes expressing CD69 was used to measure the effects of IL-7 on activation of T lymphocytes in vivo. No differences were observed in the frequency of activated CD4 or CD8 T lymphocytes in the B6 or B6.IL-7-/- recipients (Figure 6C, D). In normal B6 recipients, IL-7 treatment did not increase the frequency of activated T cells. The absolute number of activated T lymphocytes was higher in the IL-7–treated B6 recipients; although the difference was not statistically significant (data not shown). The total number of T lymphocytes was lower in the B6.IL-7-/- recipients treated with PBS compared with either the B6- or IL-7–treated B6.IL-7-/- recipients (Figure 4). However, the frequency of activated T lymphocytes in the PBS-treated B6.IL-7-/- recipients was similar to that of both the B6- and the IL-7–treated B6.IL-7-/- recipients (Figure 6D). The absolute number of activated T lymphocytes in the PBS-treated B6.IL-7-/- recipients was significantly lower than that in the IL-7–treated B6.IL-7-/- recipients (Figure 6E). These findings indicate that IL-7 is necessary for the survival and proliferation of the alloreactive T lymphocytes, but do not appear to be necessary for their activation.

Back to Article Outline

Discussion 

The present study investigated whether IL-7 is necessary for the development of GVHD. Based on previous data indicating that naïve T lymphocytes require IL-7 for proliferation and survival cells, we hypothesized that IL-7 is required for the maintenance of donor T lymphocytes that cause GVHD. We treated normal B6 and B6.IL-7-/- mice with IL-7 or PBS after myeloablative radiation and transplantation of either congenic or allogeneic BM and LN cells. Similar to the results observed in the congenic recipients, we found no evidence of GVHD in the B6.IL-7-/- allogeneic recipients that did not receive exogenous IL-7. On the other hand, administration of IL-7 to the B6.IL-7-/- recipients restored their sensitivity to the allogeneic cells, thereby decreasing their survival from 59% to 15%. IL-7 treatment also modestly decreased the survival of the normal B6 mice after allogeneic BMT. Although the survival rate was similar in all groups of mice for the first 25 days after transplantation, only the congenic recipients and the PBS-treated B6.IL-7-/- allogeneic recipients survived without GVHD afterward. Confounding our analysis is the possibility that T lymphopenia itself increased the susceptibility to GVHD [35]. Nevertheless, the absence of IL-7 protected allogeneic recipients from GVHD, as demonstrated by the increased survival rate of PBS-treated B6.IL-7-/- recipients compared with allogeneic B6 recipients. The very poor survival rate of B6.IL-7-/- allogeneic recipients treated with IL-7 compared with B6 recipients treated with IL-7 may reflect the combined effects of host lymphopenia plus post-BMT IL-7 treatment. Histological examination at day 30 showed that skin and gut tissues from IL-7– and PBS-treated congenic recipients and PBS-treated allogeneic B6.IL-7-/- recipients displayed no evidence of acute GVHD. The overall GVHD clinical index score of PBS-treated B6.IL-7-/- recipients was significantly lower than that of the IL-7–treated B6.IL-7-/- recipients. IL-7 deprivation prevented GVHD through decreased proliferation and survival of all transplanted CD4+ and CD8+ T lymphocytes, presumably including alloreactive T lymphocytes that cause GVHD. The death of the transplanted cells was slow, with complete disappearance of the donor-derived cells from the periphery not occurring until approximately day 25-30 post-BMT.

Almost all donor T lymphocytes disappeared from the PBS-treated B6.IL-7-/- recipients, indicating that both alloreactive and non–host reactive T lymphocytes were susceptible to cell death in the absence of IL-7. Alpdogan et al. [32] recently reported that alloreactive T lymphocytes express no or little IL-7Rα from hour 16 to day 8 posttransplantation; however, our experimental results indicate that the alloreactive T lymphocytes in the fully major histocompatibility complex–mismatched BALB/c to B6 model are dependent on IL-7. Although the discrepancy might reflect technical differences in the GVHD models used in the respective experiments, the results can be reconciled with those of Alpdogan et al. [32] if either IL-7Rαlow/- alloreactive T lymphocytes are still dependent on IL-7 or if a later differentiation stage of the alloreactive T lymphocytes is IL-7–dependent. It is possible that the mAb staining used to detect IL-7Rα+ cells is unable to detect low levels of biologically relevant IL-7Rα. Survival signals through IL-7R may require a smaller number of receptors than the limits of detection of the anti- IL-7Rα antibody used by Alpdogan et al [32]. It is also possible that IL-7Rα was reexpressed on alloreactive T cells at a later time point than the day 8 analyses reported by Alpdogan et al. [32].

Besides the pro-survival effects of IL-7 on naïve T lymphocytes, IL-7 also has been reported to be necessary for proliferation of naïve T cells in conditions of homeostatic expansion 36, 37. Adoptive transfer of congenic T cells to IL-7-/- mice resulted in decreased proliferation and eventual loss of the cells [23]. We hypothesized that IL-7 also may play a crucial role in the peripheral expansion of donor allogeneic mature T cells [38]. We found that the frequency of activated CD69+ T lymphocytes was similar in the allogeneically transplanted B6 and B6.IL-7-/- recipients. The absolute number of donor-derived T lymphocytes, including the activated CD69+ cells, declined over the first 25 days after transplantation in the absence of IL-7, at which point the T cell numbers were too low to allow further analysis. The recovery of proliferating activated donor T cells from the lymph nodes was significantly lower in PBS-treated B6 IL-7-/- mice than in the IL-7 treated B6/IL-7-/- recipients at day 10. Thus, the role of IL-7 in the development of the GVHD response is likely related to its proliferative and pro-survival effects, not to its co-activator function.

The experiments extend and contrast with the work of Alpdogan et al. [30] and Sinha et al. [31], who evaluated the effects of posttransplantation IL-7 administration on GVHD in various models of murine BMT recipients. Whereas the former observed no effects of IL-7 treatment on GVHD, the latter reported striking differences in the frequency of GVHD and increased sensitivity to differing doses of alloreactive T lymphocytes. There are potentially confounding technical differences in our study and those of Alpdogan et al. [30], Sinha et al. [31]. Whereas Alpdogan et al. administered IL-7 in their GVHD experiments from day -1 to day +13 via a mini-osmotic pump, Sinha et al. administered IL-7 intraperitoneally. We prefer subcutaneous injection of IL-7 to use of the mini-osmotic pump, because previous studies with mini-pump delivery of IL-7 failed to show even thymopoietic effects in a histocompatible BMT model, although such effects are readily observed after subcutaneous injection 19, 39. The discrepant results seen in analyses of thymopoiesis led to questions about whether the IL-7 can be stably delivered by a mini-pump over 14 days. Furthermore, in our experiments, we administered IL-7 every day post-transplantation, because we were uncertain when IL-7 dependence of alloreactive T lymphocytes might occur.

It is likely that the limited survival and expansion of alloreactive T cells in the absence of IL-7 can be overcome by transplantation of sufficient numbers of alloreactive cells. Sinha et al. [31] demonstrated that the increased severity of GVHD after IL-7 treatment was directly proportional to the number of allogeneic T cells transplanted. Although clinical and histological GVHD was not detected in PBS-treated IL-7-/- allogeneic recipients, their mortality rate (41%) was still significantly greater than that of the comparable congenic recipients. Similar to the results of the present experiments, Tan et al. [23] have demonstrated that the complete disappearance of transplanted T cells in an IL-7-/- recipient takes at least 2 weeks. Therefore, it is possible that the early mortality observed in the PBS-treated IL-7-/- allogeneic recipients was related to these animals' higher doses of alloreactive T cells, which overcame the decreased T cell survival caused by lack of IL-7. Consistent with this explanation, all PBS-treated IL-7-/- allogeneic recipients that survived the first 25 days became long-term survivors and exhibited no evidence of GVHD.

In the present experiments, the survival rate of IL-7–treated normal B6 allogeneic mice was lower than that of the PBS-treated control mice, but was not statistically significant. Nevertheless, IL-7 treatment increased the number of T lymphocytes and activated T lymphocytes in the normal B6 recipients. The relatively modest effect on mortality suggests that the endogenous production of IL-7 from the PBS-treated allogeneic B6 recipients was sufficient to maintain the survival of alloreactive T cells. Peripheral IL-7 levels due to endogenous production have been reported to be elevated after allogeneic BMT [40].

The experimental demonstration of the importance of IL-7 for both survival and proliferation of allogeneic T lymphocytes may allow synthesis of previous observations regarding IL-7 levels, lymphopenia, and GVHD. Several studies have demonstrated that peripheral IL-7 levels are significantly increased in lymphopenic hosts 38, 41, 42; for example, Bolotin et al. [41] found that patients with severe combined immunodeficiency syndrome undergoing BMT had high circulating levels of IL-7, which normalized after engraftment and development of normal donor T lymphocytes [41]. Subsequent studies by Fry et al. [38], Llano et al. [42], and Napolitano et al. [43] demonstrated that circulating IL-7 levels also were increased in lymphopenic patients with human immunodeficiency virus infection. The mechanisms by which lymphopenia result in increased circulating levels of IL-7 are unclear. Two possible mechanisms are either that IL-7 levels vary because of consumption by IL-7R–bearing lymphocytes, or that peripheral cells that regulate IL-7 production are actively inhibited by a factor produced by T lymphocytes. The variation in IL-7 levels is likely an important mechanism for regulation of homeostatic proliferation, which has been noted to occur under lymphopenic conditions, that is, when levels of IL-7 are high.

GVHD or GVHD-like autoimmune illnesses have been noted to occur more readily in lymphopenic hosts than in nonlymphopenic hosts [35]. Lymphocytic infiltration of organs has been observed in neonatally thymectomized mice and nude mice that have been engrafted with neonatal thymuses from a normal donor 44, 45. Assuming that the inverse relationship between T lymphocyte numbers and IL-7 levels can be generalized, IL-7 levels likely are higher in lymphopenic hosts than in nonlymphopenic hosts. The higher IL-7 levels in lymphopenic hosts may increase the proliferation of T lymphocytes and predispose to the expansion of alloreactive or autoreactive T cells.

The dependence of GVHD on the presence of IL-7 and the apparent increased risk of GVHD in recipients of extrinsic IL-7 suggest potential risks in the use of IL-7 to enhance immune reconstitution after hematopoietic stem cell transplantation. Although the positive effects of IL-7 on thymopoiesis and expansion of naïve T lymphocyte populations by homeostatic proliferation would be expected to increase immune function, our findings indicate that the use of rhIL-7 after allogeneic BM and mature T cell transplantation leads to maintenance of donor mature T cells and likely is responsible for the development of GVHD. The clinical testing of IL-7 therapies to enhance immune reconstitution in the allogeneic setting must be carefully designed to minimize the risk of GVHD, for example, by testing in the autologous transplant or TCD allogeneic settings 20, 33, 46. Conversely, blockade of the IL-7–signaling pathways may be a useful strategy for eliminating donor alloreactive T cells after transplantation [47].

Back to Article Outline

Acknowledgments 

This work was supported by grants from the National Institutes of Health (HL 54729, AI 50765, HL 73104, HL 70005, and CA 49605) and the T.J. Martell Foundation (to K.W.).

Back to Article Outline

References 

  1. Ferrara JML. Cytokine dysregulation as a mechanism of graft-versus-host disease. Curr Opin Immunol. 1993;5:794–799
  2. Antin JH, Verra JL. Cytokine dysregulation and acute graft-versus-host disease. Blood. 1992;80:2964–2968
  3. Sprent J, Schaefer M, Korngold R. Role of T-cell subsets in lethal graft-versus-host disease (GVHD) directed to class I versus class II H-2 differences. II. Protective effects of L3T4+ cells in anti-class II H-2 differences. J Immunol. 1990;144:2946–2954
  4. Korngold R, Sprent J. Bone Marrow Transplantation. London: Blackwell Scientific; 1994;p. 220–230
  5. Nikolic B, Lee S, Bronson RT, et al. Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets. J Clin Invest. 2000;105:1289–1298
  6. Sprent J, Schaefer J, Gao E, et al. Role of T cell subsets in lethal graft-versus-host disease (GVHD) directed to class I versus class II H-2 differences I. L3T4+ cells can either augment or retard GVHD elicited by Lyt-2+ cells in class I-different hosts. J Exp Med. 1988;167:556–560
  7. Ferrara JL, Deeg HJ. Graft-versus-host disease. N Engl J Med. 1991;324:667–674
  8. Weinberg KI, Annett G, Kashyap A, et al. The effect of thymic function on immunocompetence following bone marrow transplantation. Biol Blood Marrow Transplant. 1995;1:18–23
  9. Chung B, Barbra-Burnham L, Barsky L, et al. Radiosensitivity of thymic IL-7 production and thymopoiesis after bone marrow transplant (BMT). Blood. 2001;99:4592–4600
  10. Atkinson K. Reconstruction of the hematopoietic and immune systems after marrow transplantation. Bone Marrow Transplant. 1990;5:209–226
  11. Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood. 1987;69:369–380
  12. Miller RA, Daley J, Ghalie R, et al. Clonal analysis of T-cell deficiencies in autotransplant recipients. Blood. 1991;77:1845–1850
  13. Rodewald HR, Kretzschmar K, Swat W, et al. Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo. Immunity. 1995;3:313–319
  14. Murray R, Suda T, Wrighton N, et al. IL-7 is a growth and maintenance factor for mature and immature thymocyte subsets. Int Immunol. 1989;169:707–716
  15. Pechon JJ, Morrissey PJ, Grabstein KH, et al. Early lymphocyte expansion is severely impaired in interleukin-7 receptor–deficient mice. J Exp Med. 1994;180:1955–1960
  16. Von Freeden-Jeffry U, Vieira P, Lucian LA, et al. Lymphopenia in interleukin (IL)-7 gene–deleted mice identifies IL-7 as a nonredundant cytokine. J Exp Med. 1995;181:1519–1526
  17. Somberg R, Robinson JP, Felsburg P. T lymphocyte development and function in dogs with X-linked severe combined immune deficiency. J Immunol. 1994;153:4006–4015
  18. Noguchi M, Yi H, Rosenblatt HM, et al. Interleukin-2 receptor γ-chain mutation results in X-linked severe combined immune deficiency in humans. Cell. 1993;73:147–157
  19. Bolotin E, Smith S, Smogoreswka EM, et al. Enhancement of thymopoiesis after bone marrow transplant by in vivo IL-7. Blood. 1996;88:1887–1894
  20. Mackall CL, Fry TJ, Bare C, et al. IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation. Blood. 2001;97:1491–1497
  21. Sudo TS, Nishikawa N, Ohno N, et al. Expression and function of the interleukin-7 receptor in murine lymphocytes. Proc Natl Acad Sci U S A. 1993;90:9125–9129
  22. Maraskovsky E, Teepe M, Morrissey PJ, et al. Impaired survival and proliferation in IL-7 receptor–deficient peripheral T cells. J Immunol. 1996;157:5315–5323
  23. Tan JT, Dudl E, LeRoy E, et al. IL-7 is critical for homeostatic proliferation and survival of naïve T cells. Proc Natl Acad Sci U S A. 2001;98:8732–8737
  24. Schluns KS, Kieper WC, Jameson SC, et al. Interleukin-7 mediates the homeostasis of naïve and memory CD8 T cells in vivo. Nat Immunol. 2000;1:426–432
  25. Kondrack RM, Harbertson J, Tan JT, et al. Interleukin-7 regulates the survival and generation of memory CD4 cells. J Exp Med. 2003;198:1797–1806
  26. Vella AT, Dow S, Potter TA, et al. Cytokine-induced survival of activated T cells in vitro and in vivo. Proc Natl Acad Sci U S A. 1998;95:3810–3815
  27. Gringhuis SI, de Leij LF, Verschuren EW, et al. Interleukin-7 up-regulates the interleukin-2 gene expression in activated human T lymphocytes at the transcriptional level by enhancing the DNA binding activities of both nuclear factor of activated T cells and activator protein-1. Blood. 1997;90:2690–2700
  28. van Roon JA, Glaudemans KA, Bijlsma JW, et al. Interleukin-7 stimulates tumour necrosis factor alpha and Th1 cytokine production in joints of patients with rheumatoid arthritis. Ann Rheum Dis. 2003;62:113–119
  29. Fukui T, Katamura K, Abe N, et al. IL-7 proliferation, variable cytokine-producing ability and IL-2 responsiveness in naïve CD4+ T cells from human cord blood. Immunol Lett. 1997;59:21–28
  30. Alpdogan O, Schmaltz C, Muriglan SJ, et al. Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease. Blood. 2001;98:2256–2265
  31. Sinha ML, Fry TJ, Fowler DH, et al. Interleukin-7 worsens graft-versus-host disease. Blood. 2002;100:2642–2649
  32. Alpdogan O, Muriglan SJ, Eng JM, et al. IL-7 enhances peripheral T cell reconstitution after allogeneic hematopoietic stem cell transplantation. J Clin Invest. 2003;112:1095–1107
  33. Cooke KR, Kobzik L, Martin TR, et al. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation. I. The role of minor H antigens and endotoxin. Blood. 1996;88:3230–3239
  34. Maury S, Salomon B, Klatzmann D, et al. Division rate and phenotypic differences discriminate alloreactive and nonalloreactive T cells transferred in lethally irradiated mice. Blood. 2001;98:3156–3158
  35. Blazar BR, Lees CJ, Martin PJ, et al. Host T cells resist graft-versus-host disease by donor leukocyte infusions. J Immunol. 2000;165:4901–4909
  36. Tan JT, Ernst B, Kieper WC, et al. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J Exp Med. 2002;195:1523–1532
  37. Schluns KS, Lefrancois L. Cytokine control of memory T cell development and survival. Nat Rev Immunol. 2003;3:269–279
  38. Fry TJ, Connick E, Fallon J, et al. A potential role for interleukin-7 in T-cell homeostasis. Blood. 2001;97:2983–2990
  39. Mathur A, Vallera DA, Taylor PA, et al. Effect of IL-7 or IL-4 on reconstitution of donor lymphoid cells in congenic bone marrow transplantation. Bone Marrow Transplant. 1995;16:119–124
  40. Gendelman M, Hecht T, Logan B, et al. Host conditioning is a primary determinant in modulating the effect of IL-7 on murine graft-versus-host disease. J Immunol. 2004;172:3328–3336
  41. Bolotin E, Annett G, Parkman R, et al. Serum levels of IL-7 in bone marrow transplant recipients: relationship to clinical characteristics and lymphocyte count. Bone Marrow Transplant. 1999;23:783–788
  42. Llano A, Barretina J, Gutierrez A, et al. Interleukin-7 in plasma correlates with CD4 T-cell depletion and may be associated with emergence of syncytium-inducing variants in human immunodeficiency virus type1-positive individuals. J Virol. 2001;75:10319–10325
  43. Napolitano LA, Grant RM, Deeks SG, et al. Increased production of IL-7 accompanies HIV-1-mediated T-cell depletion: implications for T-cell homeostasis. Nat Med. 2001;7:73–79
  44. Sakajuchi S, Takahashi T, Nishizuka Y. Study on cellular events on post-thymectomy autoimmune oophoritis in mice. II. Requirement of Lyt-1 cells in normal female mice for the prevention of oophoritis. J Exp Med. 1982;156:1577–1586
  45. Sakaguchi S, Sakaguchi N. Thymus and autoimmunity: capacity of the normal thymus to produce pathogenic self-reactive T cells and conditions required for the induction of autoimmune disease. J Exp Med. 1990;172:537–545
  46. Alpdogan O, van den Brink MR. IL-7 and IL-15: therapeutic cytokines for immunodeficiency. Trends Immunol. 2005;26:56–64
  47. Chung B, Dudl EP, Min D, Barsky L, et al. Prevention of graft-versus-host disease by anti-IL-7Rα antibody. Blood. 2007;110:2803–2810

PII: S1083-8791(07)00463-6

doi:10.1016/j.bbmt.2007.07.015

Biology of Blood and Marrow Transplantation
Volume 14, Issue 1 , Pages 16-27, January 2008

Article Tools

* Image Usage & Resolution