Current questions and possible controversies in autophagy.

Interest in autophagy has exploded over the last decade, with publications highlighting crosstalk with several other cellular processes including secretion, endocytosis, and cell suicide pathways including apoptosis. Autophagy proteins have also been implicated in other cellular processes independently of their roles in autophagy, creating complexities in the interpretation of autophagy (Atg) mutant gene data. Interestingly, this self-eating process is a survival mechanism that can also promote cell death, but when and how autophagy may 'switch' its function is still under debate. Indeed, there are currently many models of how autophagy actually influences cell death. In this review, we highlight some outstanding questions and possible controversies in the autophagy field.

Report from the National Institute of Allergy and Infectious Diseases workshop on drug allergy.

Allergic reactions to drugs are a serious public health concern. In 2013, the Division of Allergy, Immunology, and Transplantation of the National Institute of Allergy and Infectious Diseases sponsored a workshop on drug allergy. International experts in the field of drug allergy with backgrounds in allergy, immunology, infectious diseases, dermatology, clinical pharmacology, and pharmacogenomics discussed the current state of drug allergy research. These experts were joined by representatives from several National Institutes of Health institutes and the US Food and Drug Administration. The participants identified important advances that make new research directions feasible and made suggestions for research priorities and for development of infrastructure to advance our knowledge of the mechanisms, diagnosis, management, and prevention of drug allergy. The workshop summary and recommendations are presented herein.

New roles for autophagy and spermidine in T cells.

The conserved lysosomal degradation pathway autophagy is now recognised as an essential cog in immune function. While functionally widespread in the innate immune system, knowledge of its roles in adaptive immunity is more limited. Although autophagy has been implicated in naïve T cell homeostasis, its requirement in antigen-specific T cells during infection was unknown. Using a murine model where the essential autophagy gene Atg7 is deleted in the T cell lineage, we have shown that autophagy is dispensable for effector CD8+ T cell responses, but crucial for the formation of memory CD8+ T cells. Here, we suggest reasons why autophagy might be important for the formation of long-lasting immunity. Like in the absence of autophagy, T cell memory formation during ageing is also defective. We observed diminished autophagy levels in T cells from aged mice, linking autophagy to immunosenescence. Importantly, T cell responses to influenza vaccination could be significantly improved using the autophagy-inducing compound spermidine. These results suggest the autophagy pathway as a desirable target to improve aged immunity and modulate T cell function.

Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo.

Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7(-/-) erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119(+)/CD71(High) stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.

Depletion of CD4+CD25+ regulatory T cells enhances natural killer T cell-mediated anti-tumour immunity in a murine mammary breast cancer model.

Both invariant natural killer T (NK T) cells and CD4(+)CD25(+) T regulatory cells (T(regs)) regulate the immune system to maintain homeostasis. In a tumour setting, NK T cells activated by alpha-galactosylceramide (alpha-GalCer) execute anti-tumour activity by secreting cytokines. By contrast, T(regs) intrinsically suppress antigen-specific immune responses and are often found to be elevated in tumour patients. In this study, we have shown that T(regs) regulate NK T cell function negatively in vitro, suggesting a direct interaction between these cell types. In a murine mammary tumour model, we demonstrated that administration of either alpha-GalCer or anti-CD25 antibody alone markedly suppressed tumour formation and pulmonary metastasis, and resulted in an increase in the survival rate up to 44% (from a baseline of 0%). When treatments were combined, depletion of T(regs) boosted the anti-tumour effect of alpha-GalCer, and the survival rate jumped to 85%. Our results imply a potential application of combining T(reg) cell depletion with alpha-GalCer to stimulate NK T cells for cancer therapy.

Positive and negative influences of regulatory T cells on tumour immunity.

Clinicians and scientists have long questioned whether the immune system has a role in destroying cancerous tissue. Studies performed in animal models have, however, recently revealed that the immune system can, at least in principle, effectively control tumours. In parallel with these findings, a large body of evidence indicates that although the immune system has the capacity to control tumours, there are also regulatory mechanisms that subdue these responses. A major challenge of tumour immunotherapy, therefore, is to find ways of disabling these regulatory functions while restoring or priming any immune responses that are protective.

BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development.

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: sensitivity of human thymocytes.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

The lack of NF-kappa B transactivation and PKC epsilon expression in CD4(+)CD8(+) thymocytes correlates with negative selection.

Deletion of autoreactive thymocytes at the DP stage is the basis for tolerance to thymus-expressed self antigens. In this study we investigated whether distinct signalling pathways are induced in DP thymocytes as compared to mature T cells upon stimulation with antigen. Using triple transgenic mice expressing a TCR transgene, dominant negative ras/Mek proteins and a reporter gene construct with AP-1 or NF-kappa B binding sites, we showed a complete lack of transcriptional activity of NF-kappa B but not AP-1 in DP thymocytes, whereas both were transcriptionally active in mature T cells after antigenic stimulation. Lack of NF-kappa B induction correlated with increased death in response to antigen. AP-1 induction was dependent on the integrity of the ras/Mek pathway indicating that this pathway was activated in the DP thymocytes. In contrast, we found a complete lack of constitutive expression of the epsilon isoform of Protein Kinase C (PKC) in DP thymocytes, although it was present in mature thymocytes and peripheral T cells. Taken together the results suggest that the lack of PKC epsilon in DP thymocytes could lead to the absence of NF-kappa B activity after antigenic stimulation contributing to negative selection. Cell Death and Differentiation (2000) 7, 1253 - 1262.

Consequences of intrathymic TCR engagement by partial agonist on selection events and peripheral T cell activation program.

Functions elicited from mature T cells depend on the nature of the Ag. Thus, an agonist induces a larger set of cytokine responses than a partial agonist. Additionally, Ags present in the thymus influence both the selection of TCRs generated by gene rearrangement and the potential functional program of developing thymocytes. This can be approached by analysing the development of T cells in mice expressing the same transgenic TCR (tgTCR) under different conditions of intrathymic selection. H-2Kbm8 was found to act as a partial agonist for CD8+ T cells expressing a tgTCR specific for the H-2Kb alloantigen. Intrathymic exposure to full or to partial agonist affected the development of thymocytes at different stages, consistent with the respective CD8-independent and -dependent characteristic of the tgTCR/Ag interaction. The presence of the partial agonist led to the accumulation of a major population of thymocytes (tgTCR(high) CD4- CD8(low)) originating from TCR engagement at the immature single-positive CD8(low) stage as evidenced by: 1) results from reaggregated thymic organ culture in the presence of H-2(k/bm8) thymic stromal cells; 2) the absence of CD4+ thymocytes, the development of which depends on rearrangements of endogenous TCR alpha genes; and 3) the identification of the CD8(low) thymocytes as cycling cells. Peripheral CD8(low) T cells selected in an H-2(k/bm8) thymus expressed a partial functional program in response to H-2Kb, akin to the response of CD8(high) T cells to a partial agonist. The analysis of the molecular bases for partial reactivity revealed a correlation with inefficient AP-1, but efficient NF-kappaB transactivation.

Interferon-regulatory factors during development of CD4 and CD8 thymocytes.

Selection events in the thymus occur at the double-positive CD4+ CD8+ (DP) developmental stage leading either to further differentiation of the CD4+ and CD8+ lineages or to deletion. The interferon-regulatory factor IRF-1 has been implicated in signalling for T-cell death and also in CD8+ thymic differentiation. IRF-1 is an activator and IRF-2 a repressor of gene transcription regulated by type 1 interferons (IFN). To evaluate the role of IRF-1 and IRF-2 in the differentiation of CD4 and CD8 thymocytes, we analysed their DNA-binding activity before and after antigenic stimulation at different stages of thymic development and in peripheral T cells. Unseparated, double-positive and single-positive thymocytes as well as peripheral T lymphocytes from mice transgenic (tg) for a T-cell receptor (TCR), restricted either by major histocompatibility complex class I or class II, were stimulated by their nominal antigen. Our results demonstrate that the DNA-binding activity of IRF-2 and, weakly, that of IRF-1 are inducible in total thymocytes in response to antigen. There is no induction of IRF-1/IRF-2 binding activity at the double-positive stage of thymic development in the MHC class II-restricted model whereas in the MHC class I-restricted model IRF-1/IRF-2 activity is induced weakly. At the single-positive stage, antigen induces the IRF-1/IRF-2 DNA binding in both CD4+ and CD8+ thymocytes, but not in mature lymphocytes from the periphery. This pattern of expression suggests that IRF-1/IRF-2 binding activities resulting from antigen stimulation are developmentally regulated. No evidence for a selective role of IRF-1 in the development of the CD8+ lineage was found, however.

Developmental control of antigen-induced thymic transcription factors.

Antigen engagement of the TCR may lead to activation of mature T cells while inducing deletion or positive selection of immature thymocytes. Using thymocytes from TCR transgenic mice recognizing the allo-antigen H-2Kb we investigated whether double-positive CD4+CD8+ (DP) thymocytes constitute a particular developmental stage where signals originating from surface receptor engagement will lead to distinct nuclear signaling. We show that the developmental control of transcription factors is apparent, at least at two levels. First, NF-AT binding activity was not induced in response to either antigen or phorbol myristate acetate (PMA)/lonomycin in DP thymocytes, whereas it was induced in single-positive CD8 thymocytes. Second, antigen induced a different pattern of transcription factor binding activities than PMA/lonomycin in DP thymocytes, AP-1 activity being selectively induced by antigen and NF-kappa B by PMA/lonomycin. Further we show that the transcription factors found to be induced in the DP thymic population were not susceptible to the inhibitory effect of cyclosporin A.

Two signaling pathways can lead to Fas ligand expression in CD8+ cytotoxic T lymphocyte clones.

As shown previously, a given cytotoxic T lymphocyte (CTL) clone (KB5.C20) could be induced to express the Fas ligand (FasL) by either T cell receptor (TCR) engagement or phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation. In contrast, another CTL clone (BM3.3) has now been found to exert Fas-based cytotoxicity only after TCR engagement, but not after PMA/ionomycin stimulation. This suggested the existence of a PMA-insensitive, antigen-induced pathway leading to FasL expression. The inability of PMA to promote Fas-based cytotoxicity in BM3.3 cells was correlated with a defect in expression of the classical protein kinase C (PKC) isoforms alpha and beta I. In KB5.C20 cells depleted of PMA-sensitive PKC isoforms and thus no longer responsive to PMA, Fas-based cytotoxicity could still be induced via the TCR/CD3 pathway. On the other hand, a requirement for phosphatidylinositol-3 kinase (PI3K) selectively in this TCR/CD3-induced pathway was demonstrated by specific inhibition with wortmannin. These results suggest that FasL expression when induced via the TCR/CD3 involves PI3K, and when induced by PMA/ionomycin requires the expression of PMA-sensitive PKC isoforms absent in clone BM3.3. Additional data suggest that in neither case was NF-kappa B activation implicated in FasL expression.

Divergent T-cell cytokine patterns in inflammatory arthritis.

A major immunoregulatory mechanism in inflammatory infections and allergic diseases is the control of the balance of cytokines secreted by Th1/Th2 subsets of T helper (Th) cells. This might also be true in autoimmune diseases; a Th2 pattern that prevents an effective immune response in infections with intracellular bacteria may favor immunosuppression in autoimmune disease. The pattern of cytokine expression was compared in the synovial tissue from patients with a typical autoimmune disease, rheumatoid arthritis, and with a disorder with similar synovial pathology but driven by persisting exogenous antigen, reactive arthritis. We screened 12 rheumatoid and 9 reactive arthritis synovial tissues by PCR and in situ hybridization for their expression of T-cell cytokines. The cytokine pattern differs significantly between the two diseases; rheumatoid arthritis samples express a Th1-like pattern whereas in reactive arthritis interferon gamma expression is accompanied by that of interleukin 4. Studying the expression of cytokines by in situ hybridization confirmed the results found by PCR; they also show an extremely low frequency of cytokine-transcribing cells. In a double-staining experiment, it was demonstrated that interleukin 4 is made by CD4 cells. These experiments favor the possibility of therapeutic intervention in inflammatory rheumatic disease by means of inhibitory cytokines.

Analysis of cytokine profiles in synovial T cell clones from chlamydial reactive arthritis patients: predominance of the Th1 subset.

Subpopulations of human T cells (Th0, Th1 and Th2) can be distinguished by their cytokine-secretion pattern. Evidence is increasing from other studies that the outcome of a human disease may depend on the subpopulation of T cells that predominates at the site of inflammation. Reactive arthritis serves as a useful model of chronic inflammatory diseases, because the triggering antigen can be identified. Using this triggering antigen we raised 33 T cell clones reactive with Chlamydia trachomatis and 25 T cell clones that were not reactive, all from the synovial fluid of two patients suffering from Chlamydia-induced arthritis. Their cytokine secretion patterns for interferon-gamma (IFN-gamma), IL-2 and IL-4 were analysed, as also were mRNAs for IFN-gamma and IL-10 by in situ hybridization. Out of the 33 antigen-reactive clones 23 showed a Th1 pattern with IFN-gamma but not IL-4 secretion, while the remaining 10 exhibited a Th0 pattern. The clones that did not react with Chlamydia expressed all patterns of cytokine secretion, including a Th2 pattern, thus providing a control population that excludes bias in the sampling procedure. CD4 and CD8 clones displayed a similar cytokine-secretion pattern. In addition this study demonstrates for the first time the expression of IL-10 mRNA in T cell clones derived from synovial fluid, and this was not confined to the Th2 subset. The Th1 response that Chlamydia provoke can be regarded as appropriate for such an obligate intracellular pathogen.

In situ localization of HLA class I mRNA in human testis.

We have used a 0.35-kilobase (kb) antisense RNA probe complementary to the monomorphic regions of both classical and nonclassical HLA class I sequences to detect histocompatibility-class-I-antigen-specific mRNA in human testicular tissue. The message has been clearly detected in the interstitium while less intensive staining was revealed in the peribasal compartment of the seminiferous epithelium.