Immune surveillance via self digestion

The adaptive immune system is orchestrated by CD4+ T cells. These cells detect peptides presented on Major Histocompatibility Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4+ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4+ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.     

Immune Surveillance via Self Digestion

The adaptive immune system is orchestrated by CD4⁺ T cells. These cells detect peptides presented on Major Histocompatiblity Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4⁺ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4⁺ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.

Addendum to:

MHC Class II Antigen Loading Compartments Continuously Receive Input from Autophagosomes

Dorothee Schmid, Marc Pypaert and Christian Münz

Immunity 2006; In press     

A dendritic cell population generated by a fusion of GM-CSF and IL-21 induces tumor-antigen-specific immunity

We have previously shown that the fusion of GM-CSF and IL-21 (GIFT-21) possesses a potent immune stimulatory effect on myeloid cells. In this study, we define the effect of GIFT-21 on naive murine monocytes (GIFT-21 dendritic cells [DCs]), which express increased levels of Gr-1, CD45R, MHC class I, CD80, CD86, and CXCR4 and suppress CD11c and MHC class II. Compared with conventional dendritic cells, GIFT-21 DCs produced substantially more CCL2, IL-6, TNF-α, and IFN-α and induced significantly greater production of IFN-γ by CD8(+) T cells in MHC class I-restricted Ag presentation assays. B16 melanoma and D2F2 Neu breast cancer growth was inhibited in mice treated with Ag-naive GIFT-21 DCs. This effect was lost in CD8(-/-) and CCR2(-/-) mice and when mice were treated with β(2)-microglobulin-deficient GIFT-21 DCs, indicating that GIFT-21 DCs migrated to and sampled from the tumors to present tumor Ags to CCL2 recruited CD8(+) T cells via MHC class I. We propose that autologous GIFT-21 DCs may serve as a cell therapy platform for the treatment of cancer.     

Immunization with live and dead Chlamydia muridarum induces different levels of protective immunity in a murine genital tract model: correlation with MHC class II peptide presentation and multifunctional Th1 cells

Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ(+)-producing CD4(+) T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4(+) T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

Macroautophagy as a pathomechanism in sporadic inclusion body myositis

Skeletal muscle fibers show a high level of constitutive and starvation-induced macroautophagy. Sporadic Inclusion Body Myositis (sIBM) is the most common acquired skeletal muscle disease in patients above the age of 50 years and is characterized by inflammation and intracellular accumulation of aggregate-prone proteins such as amyloid precursor protein (APP)/beta-amyloid, hyperphosphorylated tau, and presenilin. In a recent study, we found that muscle fibers of sIBM patients show increased frequencies of Atg8/LC3(+) autophagosomes and that intracellular APP/beta-amyloid colocalized with Atg8/LC3 in degenerating fibers. Colocalization of APP/beta-amyloid with LC3(+) autophagosomes was further associated with upregulation of major histocompatibility complex (MHC) class I and class II molecules and T cell infiltration. These findings indicate that APP/beta-amyloid is a substrate for autophagy in skeletal muscle fibers and suggest that degradation of aggregate-prone proteins via macroautophagy can be linked with both immune-mediated and degenerative tissue damage. A better understanding of this pathway in skeletal muscle and in the inflammatory environment of sIBM might provide a rationale for novel therapeutic strategies targeting pathogenic protein aggregation.     

ATGs help MHC class II,but inhibit MHC class I antigen presentation

We have recently shown that the LC3/Atg8 lipidation machinery of macroautophagy is involved in the internalization of MHC class I molecules. Decreased internalization in the absence of ATG5 or ATG7 leads to MHC class I surface stabilization on dendritic cells and macrophages, resulting in elevated CD8⁺ T cell responses during viral infections and improved immune control. Here, we discuss how the autophagic machinery supports MHC class II restricted antigen presentation, while compromising MHC class I presentation via internalization and degradation.     

CD137 differentially regulates innate and adaptive immunity against Mycobacterium tuberculosis

Protective immunity against Mycobacterium tuberculosis is primarily mediated by the interaction of antigen-specific T cells and antigen presenting cells, which often depends on the interplay of cytokines produced by these cells. Costimulatory signals represent a complex network of receptor-ligand interactions that qualitatively and quantitatively influence immune responses. Thus, here we investigated the function of CD137 and CD137L, molecules known to have a central role in immune regulation, during human tuberculosis (TB). We demonstrated that M. tuberculosis antigen stimulation increased both CD137 and CD137L expression on monocytes and NK cells from TB patients and healthy donors, but only up-regulated CD137 on T lymphocytes. Blockage of the CD137 pathway enhanced the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α produced by monocytes and NK against M. tuberculosis. In contrast, CD137 blockage significantly decreased the specific degranulation of CD8(+) T cells and the percentage of specific IFN-γ and TNF-α producing lymphocytes against the pathogen. Furthermore, inhibition of the CD137 pathway markedly increased T-cell apoptosis. Taken together, our results demonstrate that CD137:CD137L interactions regulate the innate and adaptive immune response of the host against M. tuberculosis.     

The immune system utilizes two distinct effector mechanisms of T cells depending on two different life cycle stages of a single pathogen,Toxoplasma gondii,to control its cerebral infection

Toxoplasma gondii takes two different life cycle stages within intermediate hosts including humans. Tachyzoites proliferate during the acute stage, and they transform into cysts to establish a chronic infection preferentially in the brain. IFN-γ production by infiltrated CD4⁺ and CD8⁺ T cells is required for the prevention of cerebral tachyzoite growth. IFN-γ production by brain-resident cells, most likely microglia, plays a key first line defense role to facilitate both innate and T cell-mediated protective immunity to control the tachyzoite growth. IFN-γ produced by brain-resident cells activates cerebral expression of IFN-dependent effector molecules to suppress tachyzoite growth during the early stage of infection. Their IFN-γ production also induces an expression of CXCL9 and CXCL10 chemokines to recruit immune T cells into the brain, and upregulates cerebral expression of MHC class I and II molecules for antigen presentation to the recruited T cells to activate their IFN-γ production. CD8⁺ T cells also have the activity to remove T. gondii cysts from the brains of infected hosts. Of interest, the anti-cyst activity of CD8⁺ T cells does not require their IFN-γ but does require perforin. Notably, we discovered that CD8⁺ cytotoxic T cells penetrate in the cysts in a perforin-mediated manner, which induces morphological deterioration and destruction of the cysts and an accumulation of microglia and macrophages for their elimination. Thus, the immune system employs two distinct effector mechanisms mediated by IFN-γ or perforin depending on two different life cycle stages of a single pathogen, T. gondii, to control its cerebral infection.     

Cutting edge: impaired MHC class I expression in mice deficient for Nlrc5/class I transactivator

MHC class I and class II are crucial for the adaptive immune system. Although regulation of MHC class II expression by CIITA has long been recognized, the mechanism of MHC class I transactivation has been largely unknown until the recent discovery of NLRC5/class I transactivator. In this study, we show using Nlrc5-deficient mice that NLRC5 is required for both constitutive and inducible MHC class I expression. Loss of Nlrc5 resulted in severe reduction in the expression of MHC class I and related genes such as β(2)-microglobulin, Tap1, or Lmp2, but did not affect MHC class II levels. IFN-γ stimulation could not overcome the impaired MHC class I expression in Nlrc5-deficient cells. Upon infection with Listeria monocyogenes, Nlrc5-deficient mice displayed impaired CD8(+) T cell activation, accompanied with increased bacterial loads. These findings illustrate critical roles of NLRC5/class I transactivator in MHC class I gene regulation and host defense by CD8(+) T cell responses.     

Baculovirus activates murine dendritic cells and induces non-specific NK cell and T cell immune responses

We previously reported that the baculovirus induced a strong host immune response against infections and malignancies. Among the immune cells, the dendritic cells were most strongly infected and activated by the baculovirus, although the exact mechanism remained unclear. Here, we evaluated the non-specific immune responses of bone marrow-derived dendritic cells (BMDCs) after infection by a wild-type baculovirus. MHC class I and II molecules and co-stimulation molecules (CD40, CD80, and CD86) on BMDCs were up-regulated by baculovirus infection. At the same time, the BMDCs produced pre-inflammatory cytokines (IL-6, IL12p70, and TNF-α) and IFN-α. NK cells showed IFN-γ production, CD69 up-regulation, and enhanced cytotoxicity when they were co-cultured with baculovirus-infected BMDCs. T cells showed IFN-γ production, CD69 up-regulation, and cell proliferation. Ex vivo analysis performed in vitro produced similar results. These findings suggested that baculovirus-infected dendritic cells induce non-specific immune responses and can be used as an immunotherapeutic agent against viral infections and malignancies, together with present therapeutic drug regimens.     

Single-cell analysis of costimulation by B cells, endothelial cells, and fibroblasts demonstrates heterogeneity in responses of CD4(+) memory T cells.

Human endothelial cells (EC) express MHC class II molecules in vivo and are likely to be involved in presentation of antigens to CD4(+) T cells. We examined, at the single-cell level, EC presentation of superantigens to resting CD4(+) memory T cells. Within 2 h of adherence to class II+ EC early T cell activation is evidenced by translocation of nuclear factor of activated T cells (NFAT), surface expression of CD69, and synthesis of IFN-gamma and IL-2. Naive T cells are not activated. T cell activation is dependent on the prior induction of MHC class II molecules on EC and is blocked by antibodies to LFA-3 (CD58). Our data place EC along a spectrum of antigen-presenting ability. Activated B cells and macrophages trigger more cells to express cytokines than do EC and at lower antigen concentrations; EC are in turn, superior to fibroblasts or smooth muscle cells. Furthermore, the concept of activation thresholds for cytokine synthesis within T cells also extends to earlier activation events: NFAT translocation is relatively easy to trigger, as is CD69 expression; fewer cells can be triggered to express IFN-gamma and fewer still to express IL-2. EC may, therefore, contribute to a graded immune response by inducing qualitatively and quantitatively different responses than professional APC.     

The spleen CD4+ T cell response to blood-stage Plasmodium chabaudi malaria develops in two phases characterized by different properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-A(b-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-α and IFN-γ production depend mostly on conventional CD4(+) T cells. IFN-γ is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-γ and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.     

Phenotypic changes in CD8+ peripheral blood lymphocytes in cats infected with feline immunodeficiency virus

It is well documented that several cell surface molecules of T lymphocytes are altered by immune activation. We previously reported that feline immunodeficiency virus (FIV) infection induces a reduction in CD8beta chain expression of peripheral blood lymphocytes (PBLs) in cats. In this study, we performed three-color flow-cytometric analysis for activation-associated cell surface molecules (CD2, CD11a, CD45RA-like and major histocompatibility complex antigen class II (MHC II)) and light scatters (cellular size and complexity) to examine whether phenotypic changes also occurred in CD4(+) PBLs in addition to CD8(+) PBLs, of five FIV-infected cats and one uninfected cat. It was shown that (i) CD8alpha(+) PBLs, but not CD4(+) PBLs, had a distinct subpopulation with increased CD11a expression accompanying a reduced CD8beta chain and increased intracellular granules (ii) CD8alpha(+) PBLs, but not CD4(+) PBLs, expressed CD45RA-like antigen with diverse expression levels and (iii) MHC II expression was greater in CD8alpha(+) PBLs than CD4(+) PBLs, and the CD8beta chain reduction was correlated with the MHC II decrease within CD8alpha(+) PBLs. These results suggest that FIV infection induces phenotypically heterogeneous subpopulations in CD8(+) PBLs, including activated phenotypes, rather than in CD4(+) PBLs.     

Expression of invariant chain (CD 74) and major histocompatibility complex (MHC) class II antigens in the human fetus.

During the initiation of an immune response, antigen-presenting cells employ MHC class II antigens as key molecules to present small peptides to CD4-positive lymphocytes. The invariant chain (Ii; CD74) plays a critical role in this process by influencing the expression and peptide loading of the MHC class II molecules. Therefore, coordinate expression of these molecules is believed to play an important role in antigen presentation. This study explores the expression of these molecules in fetal tissues. Formalin-fixed, paraffin-embedded multi-organ tissue blocks from aborted fetuses (age range 7-22 weeks) were immunostained for Ii/CD74 and MHC class II antigens using commercially available monoclonal antibodies for Ii/CD74 (LN2) and MHC class II antigens (LN3), respectively. Coordinate staining for Ii/CD74 and MHC class II antigens was seen in the skin, proximal renal tubules, tips of small intestinal mucosa, and cells of the reticuloendothelial system, including the spleen and thymus. Expression of Ii/CD74, but not of MHC class II antigens, was seen in pulmonary alveolar epithelium in all cases and in testicular Leydig cells (11 of 11 testes examined). The distribution and intensity of staining did not change significantly with age. In conclusion, this study describes distribution of Ii/CD74 and MHC class II antigens in human fetal tissues. Coordinate expression of Ii/CD74 and MHC class II antigens was identified in most fetal tissues, but there were also notable exceptions. In all cases this took the form of expression of Ii/CD74 in the absence of MHC class II expression. Discordance was particularly striking in pulmonary alveolar epithelium and testicular Leydig cells. This suggests that the Ii/CD74 molecule has functional roles in addition to its role in antigen presentation.     

Functional macroautophagy induction by influenza A virus without a contribution to major histocompatibility complex class II-restricted presentation

Major histocompatibility complex (MHC) class II-presented peptides can be derived from both exogenous (extracellular) and endogenous (biosynthesized) sources of antigen. Although several endogenous antigen-processing pathways have been reported, little is known about their relative contributions to global CD4(+) T cell responses against complex antigens. Using influenza virus for this purpose, we assessed the role of macroautophagy, a process in which cytosolic proteins are delivered to the lysosome by de novo vesicle formation and membrane fusion. Influenza infection triggered productive macroautophagy, and autophagy-dependent presentation was readily observed with model antigens that naturally traffic to the autophagosome. Furthermore, treatments that enhance or inhibit macroautophagy modulated the level of presentation from these model antigens. However, validated enzyme-linked immunospot (ELISpot) assays of influenza-specific CD4(+) T cells from infected mice using a variety of antigen-presenting cells, including primary dendritic cells, revealed no detectable macroautophagy-dependent component. In contrast, the contribution of proteasome-dependent endogenous antigen processing to the global influenza CD4(+) response was readily appreciated. The contribution of macroautophagy to the MHC class II-restricted response may vary depending upon the pathogen.     

Reactive oxygen species are essential mediators in antigen presentation by Kupffer cells

Kupffer cells (KC) act as APC in the liver and play a major role in the clearance of gut-derived antigens and pathogens entering the liver with portal venous blood. Antigen presentation by KC has been implicated in regulation of the local and systemic immune responses. In this study, modulation of KC antigen presentation by antioxidants and the role of reactive oxygen species (ROS) as essential mediators of antigen presentation in KC were investigated. Co-culture of KC with ovalbumin (OVA) antigens resulted in upstream intracellular endogenous ROS generation and increased expression of MHC class II and costimulator molecules, and consequent OVA-specific CD4(+) T-cell proliferation in response to antigen presentation by KC. Scavenging of KC ROS by antioxidants, or blocking of KC ROS generation by specific inhibitors of NADPH oxidase and/or xanthine oxidase, or by specific inhibitors of the mitochondrial electron transport chain, significantly decreased OVA-specific T-cell proliferation in response to antigen presentation by KC. Increased expression of MHC class II and costimulatory molecules in KC pulsed with OVA antigens was blocked by inhibiting ROS generation enzymatically. Intracellular endogenous ROS generation during antigen processing may therefore provide essential secondary signalling for KC antigen presentation.