Selective autophagy in the maintenance of cellular homeostasis in aging organisms.

Altered cellular homeostasis, accumulation of damaged non-functional organelles and presence of protein inclusions are characteristics shared by almost all types of differentiated cells in aged organisms. Cells rely on quality control mechanisms to prevent the occurrence of these events and the subsequent cellular compromise associated with them. What goes wrong in aging cells? Growing evidence supports gradual malfunctioning with age of the cellular quality control systems. In this review, we focus on autophagy, a catabolic process that contributes to the maintenance of cellular homeostasis through the degradation of unwanted and damaged components in lysosomes. We describe recent advances on the molecular characterization of this process, its different variants and the multiplicity of functions attributed to them. Autophagic dysfunction has been identified in severe human disorders, many of which worsen with age. We comment on the contribution of an adequate autophagic function to longevity, and the negative impact on health-span of the age-dependent decline in autophagic function.

Viruses, autophagy genes, and Crohn's disease.

The etiology of the intestinal disease Crohn's disease involves genetic factors as well as ill-defined environmental agents. Several genetic variants linked to this disease are associated with autophagy, a process that is critical for proper responses to viral infections. While a role for viruses in this disease remains speculative, accumulating evidence indicate that this possibility requires serious consideration. In this review, we will examine the three-way relationship between viruses, autophagy genes, and Crohn's disease and discuss how host-pathogen interactions can mediate complex inflammatory disorders.

Ceacam1 separates graft-versus-host-disease from graft-versus-tumor activity after experimental allogeneic bone marrow transplantation.

BACKGROUND: Allogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models. METHODS: We studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1(-/-) T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25(hi), CD62L(lo)). Additionally, Ceacam1(-/-) CD8 T cells had greater expression of the gut-trafficking integrin α(4)ß(7), though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1(-/-) recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1(-/-) mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1(+) lymphoma model was improved in animals receiving Ceacam1(-/-) vs. control T cells. CONCLUSIONS: We conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation.

Macroautophagy regulates energy metabolism during effector T cell activation.

Macroautophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that macroautophagy is induced after effector T cell activation. Engagement of the TCR and CD28 results in enhanced microtubule-associated protein 1 light chain 3 (LC3) processing, increased numbers of LC3-containing vesicles, and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional KO mouse model where Atg7, a critical gene for macroautophagy, is specifically deleted in T cells, we have found that macroautophagy-deficient effector Th cells have defective IL-2 and IFN-γ production and reduced proliferation after stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T cells, where mitochondria and other organelles are selectively excluded. These results suggest that macroautophagy is an actively regulated process in T cells that can be induced in response to TCR engagement to accommodate the bioenergetic requirements of activated T cells.

Absence of P-selectin in recipients of allogeneic bone marrow transplantation ameliorates experimental graft-versus-host disease.

Alloreactive T cells are crucial for graft-versus-host disease (GVHD) pathophysiology, and modulating their trafficking patterns has been efficacious in ameliorating experimental disease. We report in this paper that P-selectin, a glycoprotein found on resting and inflamed endothelium, is important for donor alloreactive T cells trafficking into GVHD target organs, such as the intestines and skin. Compared with wild-type (WT) recipients of allogeneic bone marrow transplantation, P-selectin(-/-) recipients exhibit decreased GVHD mortality and decreased GVHD of the skin, liver, and small bowels. This was associated with diminished infiltration of alloactivated T cells into the Peyer's patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLOs). Surprisingly, however, donor T cells deficient for P-selectin glycoprotein ligand 1, the most well described P-selectin ligand, mediated GVHD similar to WT T cells and accumulated in SLO and target organs in similar numbers as WT T cells. This suggests that P-selectin may be required for trafficking into inflamed tissues but not SLO and that donor T cells may use multiple P-selectin ligands apart from P-selectin glycoprotein ligand 1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable strategy for GVHD prophylaxis or treatment.

STAT-3 and ERK 1/2 phosphorylation are critical for T-cell alloactivation and graft-versus-host disease.

Graft-versus-host disease (GVHD) is a serious complication of allogeneic bone marrow transplantation, and donor T cells are indispensable for GVHD. Current therapies have limited efficacy, selectivity, and high toxicities. We used a novel flow cytometry technique for the analysis of intracellular phosphorylation events in single cells in murine BMT models to identify and validate novel GVHD drug targets.(1-7) This method circumvents the requirement for large numbers of purified cells, unlike western blots. We defined a signaling profile for alloactivated T cells in vivo and identified the phosphorylation of ERK1/2 and STAT-3 as important events during T-cell (allo)activation in GVHD. We establish that interference with STAT-3 phosphorylation can inhibit T-cell activation and proliferation in vitro and GVHD in vivo. This suggests that phospho-specific flow cytometry is useful for the identification of promising drug targets, and ERK1/2 and STAT-3 phosphorylation in alloactivated T cells may be important for GVHD.

Rapidly proliferating CD44hi peripheral T cells undergo apoptosis and delay posttransplantation T-cell reconstitution after allogeneic bone marrow transplantation.

Delayed T-cell recovery is an important complication of allogeneic bone marrow transplantation (BMT). We demonstrate in murine models that donor BM-derived T cells display increased apoptosis in recipients of allogeneic BMT with or without GVHD. Although this apoptosis was associated with a loss of Bcl-2 and Bcl-X(L) expression, allogeneic recipients of donor BM deficient in Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- or Bax-, or BM-overexpressing Bcl-2 or Akt showed no decrease in apoptosis of peripheral donor-derived T cells. CD44 expression was associated with an increased percentage of BM-derived apoptotic CD4(+) and CD8(+) T cells. Transplantation of RAG-2-eGFP-transgenic BM revealed that proliferating eGFP(lo)CD44(hi) donor BM-derived mature T cells were more likely to undergo to apoptosis than nondivided eGFP(hi)CD44(lo) recent thymic emigrants in the periphery. Finally, experiments using carboxyfluorescein succinimidyl ester-labeled T cells adoptively transferred into irradiated syngeneic hosts revealed that rapid spontaneous proliferation (as opposed to slow homeostatic proliferation) and acquisition of a CD44(hi) phenotype was associated with increased apoptosis in T cells. We conclude that apoptosis of newly generated donor-derived peripheral T cells after an allogeneic BMT contributes to delayed T-cell reconstitution and is associated with CD44 expression and rapid spontaneous proliferation by donor BM-derived T cells.

Enhanced immune reconstitution by sex steroid ablation following allogeneic hemopoietic stem cell transplantation.

Delayed immune reconstitution in adult recipients of allogeneic hemopoietic stem cell transplantations (HSCT) is related to age-induced thymic atrophy. Overcoming this paucity of T cell function is a major goal of clinical research but in the context of allogeneic transplants, any strategy must not exacerbate graft-vs-host disease (GVHD) yet ideally retain graft-vs-tumor (GVT) effects. We have shown sex steroid ablation reverses thymic atrophy and enhances T cell recovery in aged animals and in congenic bone marrow (BM) transplant but the latter does not have the complications of allogeneic T cell reactivity. We have examined whether sex steroid ablation promoted hemopoietic and T cell recovery following allogeneic HSCT and whether this benefit was negated by enhanced GVHD. BM and thymic cell numbers were significantly increased at 14 and 28 days after HSCT in castrated mice compared with sham-castrated controls. In the thymus, the numbers of donor-derived thymocytes and dendritic cells were significantly increased after HSCT and castration; donor-derived BM precursors and developing B cells were also significantly increased. Importantly, despite restoring T cell function, sex steroid inhibition did not exacerbate the development of GVHD or ameliorate GVT activity. Finally, IL-7 treatment in combination with castration had an additive effect on thymic cellularity following HSCT. These results indicate that sex steroid ablation can profoundly enhance thymic and hemopoietic recovery following allogeneic HSCT without increasing GVHD and maintaining GVT.

DNA immunization against tissue-restricted antigens enhances tumor immunity after allogeneic hemopoietic stem cell transplantation.

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP --> B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation.

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

Remodeling specific immunity by use of MHC tetramers: demonstration in a graft-versus-host disease model.

Major histocompatibility complex (MHC) molecules carrying selected peptides will bind specifically to their cognate T-cell receptor on individual clones of reactive T cells. Fluorescently labeled, tetrameric MHC-peptide complexes have been widely used to detect and quantitate antigen-specific T-cell populations via flow cytometry. We hypothesized that such MHC-peptide tetramers could also be used to selectively deplete unique reactive T-cell populations, while leaving the remaining T-cell repertoire and immune response intact. In this report, we successfully demonstrate that a tetramer-based depletion of T cells can be achieved in a murine model of allogeneic bone marrow transplantation. Depletion of a specific alloreactive population of donor splenocytes (< 0.5% of CD8+ T cells) prior to transplantation significantly decreased morbidity and mortality from graft-versus-host disease. There was no early regrowth of the antigen-specific T cells in the recipient and in vivo T-cell proliferation was greatly reduced as well. Survival was increased more than 3-fold over controls, yet the inherent antitumor activity of the transplant was retained. This method also provides the proof-of-concept for similar strategies to selectively remove other unwanted T-cell clones, which could result in novel therapies for certain autoimmune disorders, T-cell malignancies, and solid organ graft rejection.

Absence of beta7 integrin results in less graft-versus-host disease because of decreased homing of alloreactive T cells to intestine.

The alpha4beta7 integrin plays a central role in the homing of T cells to the gut. We hypothesized that absence of the beta7 subunit would result in a reduction of intestinal graft-versus-host disease (GVHD) and an improvement in overall GVHD morbidity and mortality in recipients of hematopoietic stem cell transplantation (HSCT). Analysis of alloreactive beta7-/- T cells showed intact activation, proliferation, cytokine production, and cytotoxicity. However, recipients of beta7-/- donor T cells in murine HSCT models experienced less GVHD morbidity and mortality than recipients of wild-type (WT) T cells, associated with a decrease in donor T-cell infiltration of the liver and intestine and with an overall significant decrease in hepatic and intestinal GVHD. In graft-versus-tumor (GVT) experiments, we demonstrated intact or even enhanced GVT activity of beta7-/- donor T cells. In conclusion, beta7-/- donor T cells caused less GVHD morbidity and mortality than WT donor T cells because of selectively decreased T-cell infiltration of the liver and intestines. Our data suggest that strategies to target the beta7 integrin have the clinical potential to alleviate or prevent GVHD while sparing or potentiating GVT activity.

Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that mediates epithelial cell proliferation and differentiation in a variety of tissues, including the thymus. We studied the role of KGF in T-cell development with KGF-/- mice and demonstrated that thymic cellularity and the distribution of thymocyte subsets among KGF-/-, wildtype (WT), and KGF+/- mice were similar. However, KGF-/- mice are more vulnerable to sublethal irradiation (450 cGy), and a significant decrease was found in thymic cellularity after irradiation. Defective thymopoiesis and peripheral T-cell reconstitution were found in KGF-/- recipients of syngeneic or allogeneic bone marrow transplant, but using KGF-/- mice as a donor did not affect T-cell development after transplantation. Despite causing an early developmental block in the thymus, administration of KGF to young and old mice enhanced thymopoiesis. Exogenous KGF also accelerated thymic recovery after irradiation, cyclophosphamide, and dexamethasone treatment. Finally, we found that administering KGF before bone marrow transplantation (BMT) resulted in enhanced thymopoiesis and peripheral T-cell numbers in middle-aged recipients of an allogeneic BM transplant. We conclude that KGF plays a critical role in postnatal thymic regeneration and may be useful in treating immune deficiency conditions.

CCR2 is required for CD8-induced graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Migration of donor-derived T cells into GVHD target organs plays a critical role in the development of GVHD and chemokines and their receptors are important molecules involved in this process. Here, we demonstrate in murine bone marrow transplantation models that the expression of the inflammatory CC chemokine receptor 2 (CCR2) on donor-derived CD8+ T cells is relevant for the control of CD8+ T-cell migration and development of GVHD. Recipients of CCR2-deficient (CCR2-/-) CD8+ T cells developed less damage of gut and liver than recipients of wild-type CD8+ T cells, which correlated with a reduction in overall GVHD morbidity and mortality. Assessment of donor CD8+ T-cell target organ infiltration revealed that CCR2-/- CD8+ T cells have an intrinsic migratory defect to the gut and liver. Other causes for the reduction in GVHD could be excluded, as alloreactive proliferation, activation, IFN-gamma production and cytotoxicity of CCR2-/- CD8+ T cells were intact. Interestingly, the graft-versus-tumor effect mediated by CCR2-/- CD8+ T cells was preserved, which suggests that interference with T-cell migration by blockade of CCR2 signaling can separate GVHD from GVT activity.

Absence of inducible costimulator on alloreactive T cells reduces graft versus host disease and induces Th2 deviation.

Inducible costimulator (ICOS) is expressed on activated and memory T cells and is involved in the regulation of cytokine production. We studied the role of ICOS on alloreactive T cells in graft versus host disease (GVHD) and determined that ICOS expression was up-regulated on alloreactive T cells in recipients of an allogeneic hematopoietic stem cell transplantation (allo-HSCT) with GVHD. We compared ICOS-/- T cells with wild-type (WT) T cells in 2 GVHD models. In both models, recipients of ICOS-/- T cells demonstrated significantly less GVHD morbidity and mortality, which was associated with less intestinal and hepatic GVHD but increased cutaneous GVHD. In addition, recipients of ICOS-/- donor T cells displayed a slight decrease in graft versus leukemia (GVL) activity. Further analysis of alloreactive ICOS-/- T cells showed no defect in activation, proliferation, cytotoxicity, and target organ infiltration. Recipients of ICOS-/- T cells had decreased serum levels of interferon-gamma (IFN-gamma), while interleukin-4 (IL-4) and IL-10 levels were increased, suggesting that alloreactive ICOS-/- T cells are skewed toward T helper-2 (Th2) differentiation. These data suggest a novel role for ICOS in the regulation of Th1/Th2 development of activated T cells. In conclusion, alloreactive ICOS-/- donor T cells induce less GVHD due to a Th2 immune deviation while GVL activity is slightly diminished.

Interleukin-15 enhances immune reconstitution after allogeneic bone marrow transplantation.

Interleukin-15 (IL-15) is a gamma-common cytokine that plays an important role in the development, survival, and proliferation of natural killer (NK), NK T, and CD8+ T-cells. We administered IL-15 to recipients of an allogeneic bone marrow transplantation (allo BMT) to determine its effects on immune reconstitution. Posttransplantation IL-15 administration significantly increased donor-derived CD8+ T (mostly CD122(+)CD44(+)CD8+ T-cells), NK, and NK T-cells at day +28 in young and old recipients of allo BMT. This was associated with enhanced T-cell and NK-cell function. IL-15 stimulated homeostatic proliferation of donor CD8+ T-cells in recipients of carboxyfluorescein diacetate succinimidyl ester-labeled donor T-cell infusions. Posttransplantation IL-15 administration also resulted in a decrease in apoptotic CD8+ T-cells, an increase in Bcl-2-expressing CD8+ T-cells, and an increase in the fraction of Ki67+ proliferative NK and CD8+ T-cells in recipients of allo BMT. IL-15 did not exacerbate graft-versus-host disease (GVHD) in recipients of T-cell-depleted BMT but could aggravate GVHD in some cases in recipients of a T-cell-repleted BMT. Finally, we found that IL-15 administration could enhance graft-versus-leukemia activity. In conclusion, IL-15 can be administered safely to recipients of a T-cell-depleted allo BMT to enhance CD8+ T, NK, and NK T-cell reconstitution.

GITR activation induces an opposite effect on alloreactive CD4(+) and CD8(+) T cells in graft-versus-host disease.

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.