Modeling Chronic Graft-versus-Host Disease in MHC-Matched Mouse Strains: Genetics, Graft Composition, and Tissue Targets.

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD (aGVHD) results from direct damage by donor T cells, whereas the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood, mainly because of the paucity of representative preclinical models. We examined over an extended time period 7 MHC-matched, minor antigen-mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate aGVHD was observed and BALB.B survivors developed overt cGVHD at 6 to 12 months affecting eyes, skin, and liver. Naïve CD4+ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSCs) or HSC plus effector memory CD4+ or CD8+ cells. Furthermore, co-transferred naïve and effector memory CD4+ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor Th17 cells and the phenotype of aGVHD or cGVHD was observed in this model. Donor CD4+ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunologic events during the early post-transplant period.

Thymic Epithelial Cell Support of Thymopoiesis Does Not Require Klotho.

Age-related thymic involution is characterized by a decrease in thymic epithelial cell (TEC) number and function parallel to a disruption in their spatial organization, resulting in defective thymocyte development and proliferation as well as peripheral T cell dysfunction. Deficiency of Klotho, an antiaging gene and modifier of fibroblast growth factor signaling, causes premature aging. To investigate the role of Klotho in accelerated age-dependent thymic involution, we conducted a comprehensive analysis of thymopoiesis and peripheral T cell homeostasis using Klotho-deficient (Kl/Kl) mice. At 8 wk of age, Kl/Kl mice displayed a severe reduction in the number of thymocytes (10-100-fold reduction), especially CD4 and CD8 double-positive cells, and a reduction of both cortical and medullary TECs. To address a cell-autonomous role for Klotho in TEC biology, we implanted neonatal thymi from Klotho-deficient and -sufficient mice into athymic hosts. Kl/Kl thymus grafts supported thymopoiesis equivalently to Klotho-sufficient thymus transplants, indicating that Klotho is not intrinsically essential for TEC support of thymopoiesis. Moreover, lethally irradiated hosts given Kl/Kl or wild-type bone marrow had normal thymocyte development and comparably reconstituted T cells, indicating that Klotho is not inherently essential for peripheral T cell reconstitution. Because Kl/Kl mice have higher levels of serum phosphorus, calcium, and vitamin D, we evaluated thymus function in Kl/Kl mice fed with a vitamin D-deprived diet. We observed that a vitamin D-deprived diet abrogated thymic involution and T cell lymphopenia in 8-wk-old Kl/Kl mice. Taken together, our data suggest that Klotho deficiency causes thymic involution via systemic effects that include high active vitamin D levels.

Inactivation of the RB family prevents thymus involution and promotes thymic function by direct control of Foxn1 expression.

Thymic involution during aging is a major cause of decreased production of T cells and reduced immunity. Here we show that inactivation of Rb family genes in young mice prevents thymic involution and results in an enlarged thymus competent for increased production of naive T cells. This phenotype originates from the expansion of functional thymic epithelial cells (TECs). In RB family mutant TECs, increased activity of E2F transcription factors drives increased expression of Foxn1, a central regulator of the thymic epithelium. Increased Foxn1 expression is required for the thymic expansion observed in Rb family mutant mice. Thus, the RB family promotes thymic involution and controls T cell production via a bone marrow-independent mechanism, identifying a novel pathway to target to increase thymic function in patients.

Combined effects of interleukin-7 and stem cell factor administration on lymphopoiesis after murine bone marrow transplantation.

The decreased ability of the thymus to generate T cells after bone marrow transplantation (BMT) is a clinically significant problem. Interleukin (IL)-7 and stem cell factor (SCF) induce proliferation, differentiation, and survival of thymocytes. Although previous studies have shown that administration of recombinant human IL-7 (rhIL-7) after murine and human BMT improves thymopoiesis and immune function, whether administration of SCF exerts similar effects is unclear. To evaluate independent or combinatorial effects of IL-7 and SCF in post-BMT thymopoiesis, bone marrow (BM)-derived mesenchymal stem cells transduced ex vivo with the rhIL-7 or murine SCF (mSCF) genes were cotransplanted with T cell-depleted BM cells into lethally irradiated mice. Although rhIL-7 and mSCF each improved immune reconstitution, the combination treatment had a significantly greater effect than either cytokine alone. Moreover, the combination treatment significantly increased donor-derived common lymphoid progenitors (CLPs) in BM, suggesting that transplanted CLPs expand more rapidly in response to IL-7 and SCF and may promote immune reconstitution. Our findings demonstrate that IL-7 and SCF might be therapeutically useful for enhancing de novo T cell development. Furthermore, combination therapy may allow the administration of lower doses of IL-7, thereby decreasing the likelihood of IL-7-mediated expansion of mature T cells.

Prevention of graft-versus-host disease by anti IL-7Ralpha antibody.

Graft-versus-host disease (GVHD) continues to be a serious complication that limits the success of allogeneic bone marrow transplantation (BMT). Using IL-7-deficient murine models, we have previously shown that IL-7 is necessary for the pathogenesis of GVHD. In the present study, we determined whether GVHD could be prevented by antibody-mediated blockade of IL-7 receptor alpha (IL-7Ralpha) signaling. C57/BL6 (H2K(b)) recipient mice were lethally irradiated and underwent cotransplantation with T-cell-depleted (TCD) BM and lymph node (LN) cells from allogeneic BALB/c (H2K(d)) donor mice. Following transplantation, the allogeneic BMT recipients were injected weekly with either anti-IL-7Ralpha antibody (100 mug per mouse per week) or PBS for 4 weeks. Anti-IL-7Ralpha antibody treatment significantly decreased GVHD-related morbidity and mortality compared with placebo (30% to 80%). IL-7Ralpha blockade resulted in the reduction of donor CD4(+) or CD8(+) T cells in the periphery by day 30 after transplantation. Paradoxically, the inhibition of GVHD by anti-IL-7Ralpha antibody treatment resulted in improved long-term thymic and immune function. Blockade of IL-7R by anti-IL-7Ralpha antibody resulted in elimination of alloreactive T cells, prevention of GVHD, and improvement of donor T-cell reconstitution.

Sustained thymopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging.

Age-related thymopoietic insufficiency has been proposed to be related to either defects in lymphohematopoietic progenitors or the thymic microenvironment. In this study, we examined whether keratinocyte growth factor (KGF), an epithelial cell-specific growth factor, could increase thymopoietic capacity in aged mice by restoration of the function of thymic epithelial cells (TECs). The thymic cellularity in KGF-treated aged mice increased about 4-fold compared to placebo-treated mice, resulting in an equivalent thymic cellularity to young mice. Enhanced thymopoiesis was maintained for about 2 months after a single course of KGF, and sustained improvement was achieved by administration of monthly courses of KGF. With the enhanced thymopoiesis after KGF treatment, the number of naive CD4 T cells in the periphery and T-cell-dependent antibody production improved in aged mice. KGF induced increased numbers of TECs and intrathymic interleukin-7 (IL-7) production and reorganization of cortical and medullary architecture. Furthermore, KGF enhanced thymopoiesis and normalized TEC organization in klotho (kl/kl) mice, a model of premature degeneration and aging, which displays thymopoietic defects. The result suggests that TEC damage is pathophysiologically important in thymic aging, and KGF therapy may be clinically useful in improving thymopoiesis and immune function in the elderly.

Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation.

Decreased thymopoietic capacity contributes to the severe and clinically significant immune deficiency seen after bone marrow transplantation (BMT). One mechanism for thymopoietic failure is damage to the interleukin 7 (IL-7)-producing thymic epithelial cells (TECs) by irradiation and chemotherapy, which can be partially treated by IL-7 administration. Pretreatment of BMT recipients with keratinocyte growth factor (KGF, or Fgf7), an epithelial cell-specific growth factor, protects mucosal, cutaneous, and pulmonary epithelial cells from cytotoxic therapy-induced damage in experimental murine models. Like other epithelial cells, TECs specifically express KGF receptors. Because KGF specifically protects KGF receptor-bearing epithelial cells and post-BMT immune deficiency is caused by loss of TECs, we hypothesized that KGF pretreatment would improve post-BMT thymic function. To test the hypothesis, BMT recipient mice were given KGF or placebo prior to congenic or allogeneic BMT. Administration of KGF before murine BMT significantly increased the capacity of the thymus to generate donor-derived thymocytes. KGF pretreatment also normalized the proportion of thymic subpopulations, increased the number of naive T cells in the periphery, and improved the response to neoantigen immunization. KGF treatment caused increased production of intrathymic IL-7, and the thymopoietic effects of KGF required an intact IL-7 signaling pathway. These results demonstrate that KGF may have immunomodulatory effects by a unique mechanism of protection of TECs. Furthermore, thymic injury and prolonged posttransplantation immune deficiency in BMT recipients can be prevented by KGF administration.

Isolation and partial characterisation of the Triton X-100 solubilised protein antigen from Mycobacterium tuberculosis.

This report describes extraction of a new native antigen fraction from Mycobacterium tuberculosis without massive degradation of proteins by Triton X-100. The Triton X-100 solubilised protein (TSP) antigen showed a characteristic antigen profile and reproducible extraction pattern. To characterise the nature of their composition, the TSP antigen was fractionated by Triton X-114 phase partitioning. The TSP antigen contained a variety of lipids and glycoconjugates as well as diverse proteins. Most proteins were partitioned into the aqueous phase during phase fractionation, whereas non-protein molecules and lipoproteins were recovered in the detergent phase. The lymphoproliferative responses to the TSP aqueous fraction in healthy tuberculin reactors were significantly higher than those to the purified protein derivative (PPD) and unfractionated TSP. In contrast, the antibody responses to TSP aqueous fraction in tuberculosis patients showed weak reactivity. This study suggests that the TSP aqueous fraction can be used as a T-cell antigen associated with protective immunity against tuberculosis.