Hematopoietic stem cell transplant (HSCT) therapy is limited by pulmonary infections. Mice with fully reconstituted hematopoietic compartments, including alveolar macrophages (AMs), post-bone marrow transplantation (BMT) have impaired host defense against Gram negative Pseudomonas aeruginosa. Impaired innate immunity is related to increased production of prostaglandin E2 (PGE2) by AMs. Cyclooxygenase (COX)-2 is the rate-limiting enzyme for synthesis of PGE2 from arachidonic acid, and COX-2 expression is elevated in AMs post-BMT. We hypothesized epigenetic mechanisms may be responsible for upregulation of COX-2 in AMs. Using bisulfite sequencing, we observed the 5’-untranslated region and exon 1 of the COX-2 gene is hypomethylated in the AMs of BMT mice compared to control. COX-2 expression was increased in primary AMs and in the AM cell line (MHS) following treatment with 5-aza-2-deoxycytidine (a methyltransferase inhibitor). Methylation by SssI methyltransferase of a 702 bp region of the COX-2 promoter including the beginning of exon 1 driving a luciferase reporter silenced luciferase expression. Because transforming growth factor beta (TGF-β1) is elevated in lungs post-BMT, we tested whether TGF-β1 could promote expression of COX-2 in a hypermethylated COX-2 vector, and observed TGF-β1 induced modest expression of COX-2, suggesting an ability to demethylate the promoter. Finally, BMTs performed with marrow from mice expressing a dominant negative form of the TGF-β receptor on CD11c-expressing cells (which includes AMs) demonstrated improved host defense and AM function. Our findings suggest impaired innate immunity and PGE2 elevation post-BMT are due to hypomethylation of the COX-2 gene which is at least partly regulated by TGF-β1.
Infectious pulmonary complications limit the success of hematopoietic stem cell transplantation (HSCT) as a therapy for malignant and non-malignant disorders. Susceptibility to pathogens in both autologous and allogeneic HSCT recipients persists despite successful immune reconstitution. As studying the causal effects of these immune defects in the human population can be limiting, a bone marrow transplant (BMT) mouse model can be used to understand the defect in mounting a productive innate immune response post-transplantation. When syngeneic BMT is performed, this system allows the study of BMT-induced alterations in innate immune cell function that are independent of the confounding effects of immunosuppressive therapy and graft-versus-host disease. Studies from several laboratories, including our own show that pulmonary susceptibility to bacterial infections post-BMT are largely due to alterations in the lung alveolar macrophages. Changes in these cells post-BMT include cytokine and eicosanoid dysregulations, scavenger receptor alterations, changes in micro RNA profiles, and alterations in intracellular signaling molecules that limit bacterial phagocytosis and killing. The changes that occur highlight mechanisms that promote susceptibility to infections commonly afflicting HSCT recipients and provide insight into therapeutic targets that may improve patient outcomes post-HSCT.
pulmonary complications; hematopoietic stem cell transplantation; eicosanoids; alveolar macrophage; polymorphonuclear leukocytes; scavenger receptors; microRNA; prostaglandins E
Graft-versus-host-disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host antigens as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT.
C57BL/6 (H2b; WT) and SP-A deficient mice on C57BL/6 background (H2b; SP-A−/−) mice underwent allogeneic (Allo) or syngeneic (Syn) BMT with cells from either C3HeB/FeJ (H2k; SP-A−/−alloBMT or WTalloBMT) or C57Bl/6 (H2b; SP-A−/−synBMT or WTsynBMT) mice. 5 weeks post BMT, mice were necropsied and lung and gastrointestinal (GI) tissue were analyzed.
SP-A−/−alloBMT or WTalloBMT had no significant differences in lung pathology however, SP-A−/−alloBMT mice developed marked features of GI GVHD including decreased body weight, increased tissue inflammation and lymphocytic infiltration. SP-A−/−alloBMT mice also had increased colon expression of IL-1β, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells, and diminished regulatory T (Treg) cells.
Our results demonstrate the first evidence of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an alloBMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased Tregs. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.
Graft-versus-host disease; Surfactant Protein A; Th17; Regulatory T cells
Patients receiving hematopoietic stem cell transplantation or bone marrow transplantation (BMT) as therapy for various malignancies or autoimmune diseases have an increased risk for infectious complications posttransplant, especially in the lung. We have used BMT in mice and murine gammaherpesvirus, γHV-68, to study the efficacy of adaptive immune responses post-BMT. Five weeks posttransplant, mice have fully reconstituted their hematopoietic lineages in both the lung and periphery. When challenged with virus, however, BMT mice have a reduced ability to clear lytic virus from the lung. Defective viral control in BMT mice is not related to impaired leukocyte recruitment or defective APC function. Rather, BMT mice are characterized by defective CD4 cell proliferation, skewing of effector CD4 T cells from a Th1 to a Th17 phenotype, and an immunosuppressive lung environment at the time of infection that includes overexpression of TGF-β1 and PGE2 and increased numbers of regulatory T cells. Neither indomethacin treatment to block PG synthesis nor anti-CD25 depletion of regulatory T cells improved antiviral host defense post-BMT. Transplanting mice with transgenic bone marrow expressing a dominant-negative TGF-βRII under the permissive CD4 promoter created mice in which effector CD4 and CD8 cells were unresponsive to TGF-β1. Mice with TGF-β1–nonresponsive effector T cells had restored antiviral immunity and improved Th1 responses post-BMT. Thus, our results indicate that over-expression of TGF-β1 following myeloablative conditioning post-BMT results in impaired effector T cell responses to viral infection.
There is an increased risk of failure of engraftment following nonmyeloablative conditioning. Sensitization resulting from failed bone marrow transplantation (BMT) remains a major challenge for secondary BMT. Approaches to allow successful retransplantation would have significant benefits for BMT candidates living with chronic diseases. We used a mouse model to investigate the effect of preparative regimens at primary BMT on outcome for secondary BMT. We found that conditioning with TBI or recipient T-cell lymphodepletion at primary BMT did not promote successful secondary BMT. In striking contrast, successful secondary BMT could be achieved in mice conditioned with anti-CD154 co-stimulatory molecule blockade at first BMT. Blockade of CD154 alone or combined with T-cell depletion inhibits generation of the humoral immune response after primary BMT as evidenced by abrogation of production of anti-donor Abs. The humoral barrier is dominant in sensitization resulting from failed BMT, as almost all CFSE-labeled donor cells were killed at 0.5 and 3 hr in sensitized recipients in in vivo cytotoxicity assay, reflecting antibody-mediated cytotoxicity. CD154:CD40 co-stimulatory blockade used at primary BMT promotes allogeneic engraftment in secondary BMT after engraftment failure at first BMT. The prevention of generation of anti-donor Abs at primary BMT is critical for successful secondary BMT.
Osteopetrosis is an inherited disorder characterized by bone sclerosis due to reduced bone resorption. Here we report that human osteopetrotic osteoblast-like (Ob) cells express a defective phenotype in primary cultures in vitro, and that bone marrow transplant (BMT) corrects osteoblast function. DNA analysis at polymorphic short-tandem repeat loci from donor, recipient, and primary Ob-like cells pre-BMT and 2 yr post-BMT revealed that Ob were still of recipient origin post-BMT. Osteopetrotic Ob-like cells obtained pre-BMT showed normal and abnormal 1,25(OH)2D3-induced alkaline phosphatase (ALPase) and osteocalcin production, respectively, and failed to produce macrophage colony-stimulating factor (M-CSF) in response to IL-1a and TNF-alpha. These parameters were all normalized in primary Ob-like cells prepared 2 yr post-BMT. X-linked clonality analysis at the human androgen receptor (HUMARA) locus revealed that osteoblasts showed a polyclonal and an oligoclonal derivation pre- and post-BMT respectively, indicating that a limited number of progenitor reconstituted this population. Because osteoblasts were still of recipient origin post-BMT, this suggests that functional osteoclasts, due to the replacement of hematopoeitic cells, provided a local microenvironment in vivo triggering the differentiation and/or recruitment of a limited number of functional osteoblasts.
The chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis.
Murine prostate epithelial cells from PTEN+/+, PTEN+/−, and PTEN−/− (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed.
Loss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth.
These results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases.
The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted on chromosome 10) is frequently mutated or deleted in various human cancers. PTEN localizes predominantly to the cytoplasm and functions as a lipid phosphatase, thereby negatively regulating the phosphatidylinositol 3-kinase-AKT signaling pathway. PTEN can also localize to the nucleus, where it binds and regulates p53 protein level and transcription activity. However, the precise function of nuclear PTEN and the factors that control PTEN nuclear localization are still largely unknown. In this study, we identified oxidative stress as one of the physiological stimuli that regulate the accumulation of nuclear PTEN. Specifically, oxidative stress inhibits PTEN nuclear export, a process depending on phosphorylation of its amino acid residue Ser-380. Nuclear PTEN, independent of its phosphatase activity, leads to p53-mediated G1 growth arrest, cell death, and reduction of reactive oxygen species production. Using xenografts propagated from human prostate cancer cell lines, we reveal that nuclear PTEN is sufficient to reduce tumor progression in vivo in a p53-dependent manner. The data outlined in this study suggest a unique role of nuclear PTEN to arrest and protect cells upon oxidative damage and to regulate tumorigenesis. Since tumor cells are constantly exposed to oxidative stress, our study elucidates the cooperative roles of nuclear PTEN with p53 in tumor suppression.
Chronic, low-grade inflammation, particularly in adipose tissue, is an important modulator of obesity-induced insulin resistance and the toll-like receptor 4 (Tlr4) is a key initiator of inflammatory responses in macrophages. We performed bone marrow transplantation (BMT) of Tlr4lps-del or control C57Bl/10J bone marrow cells into irradiated wild type C57Bl6 recipient mice to generate hematopoietic cell specific Tlr4 deletion mutant (BMT-Tlr4-/-) and control (BMT-wt) mice. When mice were fed a high-fat diet (HFD) for 16 weeks, BMT-wt mice developed obesity, hyperinsulinemia, glucose intolerance and insulin resistance. In contrast, BMT-Tlr4-/- mice became obese, but did not develop fasting hyperinsulinemia, and had improved hepatic and skeletal muscle insulin sensitivity during euglycemic clamp studies compared to HFD BMT-wt mice. The HFD BMT-Tlr4-/- mice showed markedly reduced adipose tissue inflammatory markers and macrophage content compared to HFD BMT-wt mice. In summary, our results indicate that Tlr4 signaling in hematopoietic-derived cells is important for the development of hepatic and adipose tissue insulin resistance in obese mice.
CD40 is a molecule present on multiple cell types including B lymphocyte lineage cells. CD40 has been shown to play an important role in B cell differentiation and activation in vitro, although little is known concerning the effects of CD40 stimulation in vivo. We therefore examined the effects of CD40 stimulation in mice using a syngeneic bone marrow transplantation (BMT) model in an effort to augment B cell recovery after high dose therapy with hematopoietic reconstitution. After the BMT, mice were treated with or without 2-6 microg of a soluble recombinant murine CD40 ligand (srmCD40L) given intraperitoneally twice a week. A significant increase in B cell progenitors (B220+/ surface IgM-) was observed in the bone marrow of mice receiving the srmCD40L. The treated recipients also demonstrated improved B-cell function with increases in total serum immunoglobulin and increased splenic mitogen responsiveness to LPS being noted. Additionally, srmCD40L treatment promoted secondary lymphoid organ repopulation, accelerating germinal center formation in the lymph nodes. Total B cell numbers in the periphery were not significantly affected even with continuous srmCD40L administration. Lymphocytes obtained from mice treated with the ligand also had increases in T cell mitogen and anti-CD3 mAb responsiveness and acquired the capability to produce IL-4. Surprisingly, treatment with srmCD40L also produced hematopoietic effects in mice, resulting in an increase of BM and splenic hematopoietic progenitor cells in the mice after BMT. Treatment with srmCD40L significantly increased granulocyte and platelet recovery in the peripheral blood. Incubation of BMC with srmCD40L in vitro also resulted in increased progenitor proliferation, demonstrating that the hematopoietic effects of the ligand may be direct. Thus, stimulation of CD40 by its ligand may be beneficial in accelerating both immune and hematopoietic recovery in the setting of bone marrow transplantation.
Many diseases associated with bone marrow transplantation (BMT) are caused by transplanted hematopoietic cells, and the onset of these diseases occurs after homing of donor cells in the initial phase after BMT. Noninvasive observation of donor cell homing shortly after transplantation is potentially valuable for improving therapeutic outcomes of BMT by diagnosing the early stages of these diseases.
Freshly harvested near-infrared fluorescence-labeled cells were noninvasively observed for 24 h after BMT using a photon counting device to track their homing process. In a congenic BMT model, the homing of Alexa Fluor 750-labeled donor cells in the tibia was detected less than 1 h after BMT. In addition, subsequent cell distribution in an intraBM BMT model was successfully monitored for the first time using this method. In the allogeneic BMT model, T-cell depletion decreased the near-infrared fluorescence (NIRF) signals of the reticuloendothelial system.
This approach in several murine BMT models revealed that the transplanted cells homed within 24 h after transplantation. NIRF labeling is useful for tracking transplanted cells in the initial phase after BMT, and this approach can contribute to in vivo studies aimed at improving the therapeutic outcomes of BMT.
Despite improvements in human leukocyte antigen matching and pharmacologic prophylaxis, acute graft-versus-host disease (GVHD) is often a fatal complication following hematopoietic stem cell transplant (HSCT). Older HSCT recipients experience significantly increased morbidity and mortality compared to young recipients. Prophylaxis with syngeneic regulatory dendritic cells (DCreg) in young bone marrow transplanted (BMT) mice has been shown to decrease GVHD-associated mortality. To evaluate this approach in older BMT recipients, young (3–4 months) and older (14–18 months) DCreg were generated using GM-CSF, IL-10, and TGFβ. Analysis of young versus older DCreg following culture revealed no differences in phenotype. The efficacy of DCreg treatment in older BMT mice was evaluated in a BALB/c→C57Bl/6 model of GVHD; on day 2 post-BMT (d +2), mice received syngeneic, age-matched DCreg. Although older DCreg-treated BMT mice showed decreased morbidity and mortality compared to untreated BMT mice (all of which died), there was a small but significant decrease in the survival of older DCreg-treated BMT mice (75% survival) compared to young DCreg-treated BMT mice (90% survival). To investigate differences between dendritic cells (DC) in young and older DCreg-treated BMT mice that may play a role in DCreg function in vivo, DC phenotypes were assessed following DCreg adoptive transfer. Transferred DCreg identified in older DCreg-treated BMT mice at d +3 showed significantly lower expression of PD-L1 and PIR B compared to DCreg from young DCreg-treated BMT mice. In addition, donor DC identified in d +21 DCreg-treated BMT mice displayed increased inhibitory molecule and decreased co-stimulatory molecule expression compared to d +3, suggesting induction of a regulatory phenotype on the donor DC. In conclusion, these data indicate DCreg treatment is effective in the modulation of GVHD in older BMT recipients and provide evidence for inhibitory pathways that DCreg and donor DC may utilize to induce and maintain tolerance to GVHD.
Nucleotide-binding oligomerization domain 2 (NOD2) polymorphisms are independent risk factors for Crohn's disease and graft-versus-host disease (GVHD). In Crohn's disease, the proinflammatory state resulting from NOD2 mutations have been associated with a loss of antibacterial function of enterocytes such as paneth cells. NOD2 has not been studied in experimental allogeneic bone marrow transplantation (allo-BMT). Using chimeric recipients with NOD2−/− hematopoietic cells, we demonstrate that NOD2 deficiency in host hematopoietic cells exacerbates GVHD. We found that proliferation and activation of donor T cells was enhanced in NOD-deficient allo-BMT recipients, suggesting that NOD2 plays a role in the regulation of host antigen-presenting cells (APCs). Next, we used bone marrow chimeras in an experimental colitis model and observed again that NOD2 deficiency in the hematopoietic cells results in increased intestinal inflammation. We conclude that NOD2 regulates the development of GVHD through its inhibitory effect on host APC function.
Macrophages play a central role in the development of atherosclerosis. However, the signaling pathways that regulate their function are not well understood. The Rho-associated coiled-coil containing kinases (ROCK1 and ROCK2) are serine-threonine protein kinases that are involved in the regulation of the actin cytoskeleton. Recent studies suggest that ROCK1 in macrophages and bone marrow (BM)-derived cells mediates atherogenesis. However, a similar role for ROCK2 in the pathogenesis of atherosclerosis has not been determined.
Methods and Results
The BMs from wild-type (WT), ROCK2+/− and ROCK2−/− mice were transplanted into irradiated recipient LDLr−/− mice and atherosclerosis was induced with a 16-week high-cholesterol diet. Compared to WT BM transplanted (BMT) mice, ROCK2+/− BMT and ROCK2−/− BMT mice showed substantially less lipid accumulation in the aorta (8.46 ± 1.42% and 9.80 ± 2.34% vs. 15.64 ± 1.89%, p<0.01 for both) and decreased atherosclerotic lesions in the subaortic sinus (158.1 ± 44.4 and 330.1 ± 109.5 ×103μm2 vs. 520.2 ± 125.7 ×103μm2, p<0.01 for both). These findings correlated with decreased foam cell formation (2.27 ± 0.57 vs. 4.10 ± 0.3, p<0.01) and increased cholesterol efflux (17.65 ± 0.6 vs. 9.75 ± 0.8, p<0.05) in ROCK2-deficient mice that are mediated, in part, through the PPARγ-LXR-ABCA-1 pathway in macrophages.
ROCK2 contributes to atherosclerosis, in part, by inhibiting PPARγ-mediated reverse cholesterol transport in macrophages, which contributes to foam cell formation. These findings suggest that inhibition of ROCK2 in macrophages may have therapeutic benefits in preventing the development of atherosclerosis.
atherogenesis; atherosclerosis; macrophage; Rho kinase; Rho-associated kinases
One of the greatest barriers against harnessing the potential of CD4+CD25+ Tregs as a cellular immunotherapy is their hypoproliferative phenotype. We have previously shown that the hypoproliferative response of Tregs to IL-2 is associated with defective downstream PI3K signaling. Here, we demonstrate that targeted deletion of the lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) regulates the peripheral homeostasis of Tregs in vivo and allows their expansion ex vivo in response to IL-2 alone. PTEN deficiency does not adversely affect either the thymic development or the function of Tregs, which retain their ability to suppress responder T cells in vitro and prevent colitis in vivo. Conversely, reexpression of PTEN in PTEN-deficient Tregs as well as in activated CD4+ T cells inhibits IL-2–dependent proliferation, confirming PTEN as a negative regulator of IL-2 receptor signaling. These data demonstrate that PTEN regulates the “anergic” response of Tregs to IL-2 in vitro and Treg homeostasis in vivo and indicate that inhibition of PTEN activity may facilitate the expansion of these cells for potential use in cellular immunotherapy.
Graft-versus-host disease (GVHD) remains the major complication of allogeneic bone marrow transplantation (allo-BMT). GVHD fundamentally depends upon the activation of donor T cells by host antigen-presenting cells (APCs), but the precise location of these interactions remains uncertain. We examined the role of secondary lymphoid organs (SLO) in the induction of GVHD by using homozygous aly/aly mice that are deficient in lymph nodes (LNs) and Peyer’s patches (PPs). Lethally irradiated, splenectomized, aly/ aly (LN/PP/Sp−/−) mice and sham-splenectomized, aly/+ (LN/PP/Sp+/+) mice received BMT from either syngeneic (aly/aly) or allogeneic, major histocompatibility complex (MHC) disparate donors. Surprisingly, although LN/PP/Sp−/− allo-BMT recipients experience a survival advantage, they developed significant systemic and target organ GVHD that is comparable to LN/PP/Sp+/+ controls. Early after allo-BMT, the activation and proliferation of donor T cells was significantly greater in the BM cavity of LN/PP/Sp−/− mice compared to LN/PP/Sp+/+ controls. Donor T cells in LN/PP/Sp−/− mice demonstrated cytolytic activity in vitro, but Graft vs Leukemia (GVL) activity could be overcome by increasing the tumor burden. These data suggest that SLO contribute to, but are not required for, allogeneic T cell responses, and suggest that the BM may represent an alternative, albeit less efficient site for T cell activation following allo-BMT.
Inflammation; Cytotoxicity; Immunity; Graft-versus-leukemia; Antigen presentation
AIMS--To compare the numbers of alkaline phosphatase positive reticulum cells (AL-RC) and macrophages in bone marrow transplant (BMT) recipients with numbers in normal subjects and to look for correlations with clinical features. METHODS--Sections of femoral marrow were obtained at necropsy from 18 BMT recipients and nine normal subjects who had died suddenly. AL-RC were visualised through their endogenous alkaline phosphatase activity. Macrophages were stained by an immunocytochemical technique using the antibody EBM/11 (CD68) and through their endogenous acid phosphatase activity. The numbers of stained cells were counted and expressed as a percentage of total nucleated cells. RESULTS--In both sets of marrow tissue, more macrophages stained for CD68 than for acid phosphatase, indicating macrophage heterogeneity. The percentage value for CD68 positive macrophages was higher among the transplant recipients (p < 0.01). At least in part this was caused by a reduction in haemopoietic cell numbers. Percentage values for acid phosphatase and alkaline phosphatase positive cells did not differ between the two groups. To exclude the effect of changes in marrow cellularity, stromal cell ratios were compared. The AL-RC: CD68 and acid phosphatase:CD68 ratios were both lower in BMT recipients, indicating that after BMT either the absolute number of AL-RC and acid phosphatase cells decreases, or CD68 cells increase, or there is a combination of the two. There was no correlation between the number of each cell type and cell dose given at transplantation, time after transplantation, presence of graft versus host disease or infection, marrow erythroid:myeloid ratio, or peripheral white cell count. The ratio of AL-RC to macrophages in our intact marrow was 0.43, considerably higher than that reported in cultured marrow. CONCLUSIONS--AL-RC and acid phosphatase positive cells may be most important for supporting haemopoiesis and their reduction after BMT may contribute to depression of haemopoiesis. CD68 positive cells include macrophages with a wide variety of functions and these may be increased in response to marrow damage.
Minor histocompatibility (mH) antigens appear to play a major role in bone marrow transplantation (BMT) using HLA-identical donors. Previously, we reported the isolation of major histocompatibility complex (MHC)-restricted mH antigen-specific cytotoxic T lymphocytes (CTL) from patients with graft-vs.-host disease or rejection after HLA- identical BMT. We have demonstrated that mH antigens can be recognized on hematopoietic progenitor cells, and residual recipient CTL specific for mH antigens expressed on donor hematopoietic progenitor cells may be responsible for graft rejection in spite of intensive conditioning regimens in HLA-identical BMT. Here, we investigated whether mH antigen- specific CTL directed against the mH antigens HA-1 to HA-5 and the male- specific antigen H-Y were capable of antigen-specific inhibition of in vitro growth of clonogenic leukemic precursor cells. We demonstrate that mH antigen-specific CTL against all mH antigens tested can lyse freshly obtained myeloid leukemic cells, that these mH antigen-specific CTL can inhibit their clonogenic leukemic growth in vitro, and that this recognition is MHC restricted. We illustrate that leukemic (precursor) cells can escape elimination by mH antigen-specific CTL by impaired expression of the relevant MHC restriction molecule. We suggest that mH antigen-specific MHC-restricted CTL may be involved in vivo in the graft-vs.-leukemia reactivity after BMT.
We recently showed that IL-11 prevents lethal graft-versus-host disease (GVHD) in a murine bone marrow transplantation (BMT) model of GVHD directed against MHC and minor antigens. In this study, we have investigated whether IL-11 can maintain a graft-versus-leukemia (GVL) effect. Lethally irradiated B6D2F1 mice were transplanted with either T cell–depleted (TCD) bone marrow (BM) alone or with BM and splenic T cells from allogeneic B6 donors. Animals also received host-type P815 mastocytoma cells at the time of BMT. Recipients were injected subcutaneously with recombinant human IL-11 or control diluent twice daily, from 2 days before BMT to 7 days after BMT. TCD recipients all died from leukemia by day 23. All control- and IL-11–treated allogeneic animals effectively rejected their leukemia, but IL-11 also reduced GVHD-related mortality. Examination of the cellular mechanisms of GVL and GVHD in this system showed that IL-11 selectively inhibited CD4-mediated GVHD, while retaining both CD4- and CD8-mediated GVL. In addition, IL-11 treatment did not affect cytolytic effector functions of T cells after BMT either in vivo or in vitro. Studies with perforin-deficient donor T cells demonstrated that the GVL effect was perforin dependent. These data demonstrated that IL-11 can significantly reduce CD4-dependent GVHD without impairing cytolytic function or subsequent GVL activity of CD8+ T cells. Brief treatment with IL-11 shortly after BMT may therefore represent a novel strategy for separating GVHD and GVL.
Glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) is a member of the tumor necrosis factor receptor (TNFR) family that is expressed at low levels on unstimulated T cells, B cells, and macrophages. Upon activation, CD4+ and CD8+ T cells up-regulate GITR expression, whereas immunoregulatory T cells constitutively express high levels of GITR. Here, we show that GITR may regulate alloreactive responses during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). Using a BMT model with major histocompatibility complex class I and class II disparity, we demonstrate that GITR stimulation in vitro and in vivo enhances alloreactive CD8+CD25− T cell proliferation, whereas it decreases alloreactive CD4+CD25− proliferation. Allo-stimulated CD4+CD25− cells show increased apoptosis upon GITR stimulation that is dependent on the Fas–FasL pathway. Recipients of an allograft containing CD8+CD25− donor T cells had increased GVHD morbidity and mortality in the presence of GITR-activating antibody (Ab). Conversely, recipients of an allograft with CD4+CD25− T cells showed a significant decrease in GVHD when treated with a GITR-activating Ab. Our findings indicate that GITR has opposite effects on the regulation of alloreactive CD4+ and CD8+ T cells.
transplantation immunology; in vivo animal models; immune regulation; lymphocyte activation; T lymphocyte subsets
Pseudomonas aeruginosa is an important pulmonary pathogen in cystic fibrosis, but the means by which it evades host defenses is understood poorly. Macrophages (M phi) are critical in protecting the lung and mucosal surfaces against infection and may need to perform their functions in the absence of opsonins before the evolution of an inflammatory response. The purpose of the present study was to define factors that regulate the capacity of macrophages to mediate nonopsonic phagocytosis. Phagocytosis of unopsonized P. aeruginosa by murine peritoneal and pulmonary alveolar M phi s was absolutely dependent upon the presence of glucose; only D-mannose could substitute. Glucose-dependent phagocytosis appears to be selective for P. aeruginosa by M phi s; ingestion of unopsonized zymosan, opsonized P. aeruginosa, EIgG, and E (IgM)C occurred in the presence or absence of glucose as did-ingestion of unopsonized P. aeruginosa by polymorphonuclear leukocytes. M phi binding and phagocytosis of unopsonized P. aeruginosa appeared to occur by a mechanism independent of complement receptor 3 and mannose receptors. Phagocytosis of P. aeruginosa killed by tobramycin or Formalin was glucose dependent, suggesting that the glucose exerted its effects on the M phi rather than the bacteria. The predilection of P. aeruginosa for lower airway disease in patients with cystic fibrosis might be explained in part by the unique dependency upon glucose for M phi phagocytosis.
When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform θ (PKCθ), a key regulator of TCR signaling. In contrast, PKCθ was required for alloreactivity and GVHD induction. Furthermore, absence of PKCθ raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCθ-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCθ is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.
PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTEN’s lipid phosphatase activity in regulating the PI3K signalling pathway is recognised, the significance of PTEN’s other mechanisms of action is currently unclear. Here, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP3 levels or the downstream protein kinase Akt/PKB. Finally, in 3D Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provides a novel tool to address the significance of PTEN’s separable lipid and protein phosphatase activities and suggest that both activities act to suppress proliferation and act together to suppress invasion.
PTEN; PI 3-kinase; phosphoinositide; cancer; TPIP; Akt
The authors compared efficiency of alveolar macrophage (AM) reconstitution from donor bone marrow post transplant following 4 chemotherapy conditioning regimens and 2 total body irradiation (TBI) regimens. TBI regimens are more effective in inducing AM reconstitution from donor marrow. However, mice conditioned with 13 Gy split-dose TBI or a dual-chemotherapy regimen (25 mg/kg busulfan × 4 days plus cyclophosphamide 100 mg/kg × 2 days) both demonstrate significant AM repopulation from donor marrow. Additionally, both protocols resulted in impaired pulmonary host defense associated with overproduction of prostaglandin E2 and I2 by AMs and impaired AM phagocytosis post bone marrow transplant.
phagocytosis; prostaglandin; P. aeruginosa
The present studies have evaluated the effect of age on the induction of syngeneic graft-versus-host disease (SGVHD) after syngeneic bone marrow transplantation (BMT) and cyclosporine (CsA) therapy. The results clearly document an inverse correlation of age with the incidence of SGVHD. Virtually a 100% incidence of SGVHD occurs in Lewis rats when syngeneic BMT and CsA therapy are started when the animals are 4 wk of age. Thereafter, there is a dramatic decline in the incidence of SGVHD with the increasing age of the animals. Although the age of the recipient was important, the most significant effect was the age of the marrow donor. Marrow from animals 6 mo of age was virtually incapable of eliciting SGVHD after BMT and CsA therapy. Furthermore, mixing the marrow from mature and immature animals resulted in a decreased incidence of SGVHD, implicating a regulatory effect present in the marrow from older rats. This regulatory effect was due to the presence of mature T cells in the marrow from animals 6 mo of age. Despite the fact that marrow from young animals possesses mature T lymphocytes, this regulatory activity was absent, suggesting that the host resistance mediated by T lymphocytes develops as the animal ages. These data further implicate the importance of a host resistance mechanism in preventing the induction of SGVHD with CsA, which appears to be mediated by the clonal inactivation of autoreactive cells.