Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help

The unique ether glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

Phosphatidylserine receptor-mediated recognition of archaeosome adjuvant promotes endocytosis and MHC class I cross-presentation of the entrapped antigen by phagosome-to-cytosol transport and classical processing

Archaeal isopranoid glycerolipid vesicles (archaeosomes) serve as strong adjuvants for cell-mediated responses to entrapped Ag. We analyzed the processing pathway of OVA entrapped in archaeosomes composed of Methanobrevibacter smithii lipids, high in archaetidylserine (OVA-archaeosomes). In vitro, OVA-archaeosomes stimulated spleen cells from OVA-TCR-transgenic mice, D011.10 (CD4(+) cells expressing OVA(323-339) TCR) or OT1 (>90% CD8(+) OVA(257-264) cells), indicating both MHC class I and II presentations. In vivo, when naive (Thy1.2(+)) CFSE-labeled OT1 cells were transferred into OVA-archaeosome-immunized Thy 1.1(+) recipient mice, there was profound accumulation and cycling of donor-specific cells, and differentiation of H-2K(b)Ova(257-264) CD8(+) T cells into CD44(high)CD62L(low) effectors. Both macrophages and dendritic cells (DCs) efficiently cross-presented OVA-archaeosomes on MHC class I. Blocking phagocytosis by phosphatidylserine-specific receptor agonists strongly inhibited MHC class I presentation of OVA-archaeosomes, whereas blocking mannose receptors or FcRs lacked effect, indicating specific recognition of the archaetidylserine head group of M. smithii lipids by APCs. In addition, inhibitors of endosomal acidification blocked MHC class I processing of OVA-archaeosomes, whereas endosomal protease inhibitors lacked effect, suggesting acidification-dependent phagosome-to-cytosol diversion. Proteasomal inhibitors blocked OVA-archaeosome MHC class I presentation, confirming cytosolic processing. Both in vitro and in vivo, OVA-archaeosome MHC class I presentation required TAP. Ag-free archaeosomes also activated DC costimulation and cytokine production, without overt inflammation. Phosphatidylserine-specific receptor-mediated endocytosis is a mechanism of apoptotic cell clearance and DCs cross-present Ags sampled from apoptotic cells. Our results reveal the novel ability of archaeosomes to exploit this mechanism for cytosolic MHC class I Ag processing, and provide an effective particulate vaccination strategy.     

Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease

Alloantigen expression on host APCs is essential to initiate graft-vs-host disease (GVHD); however, critical APC subset remains to be elucidated. We compared the ability of dendritic cells (DCs) and B cells to initiate acute GVHD by an add-back study of MHC class II-expressing APCs (II(+/+)) into MHC class II-deficient (II(-/-)) mice that were resistant to CD4-dependent GVHD. Injection of host-derived, but not donor-derived, II(+/+) DCs or host-derived II(+/+) B cells, was sufficient to break GVHD resistance of II(-/-) mice and induced lethal acute GVHD. By contrast, host-derived II(+/+) B cells, both naive and LPS stimulated, failed to induce activation or tolerance of donor CD4(+) T cells. Similarly, in a model of CD8-dependent GVHD across MHC class I mismatch injection of allogeneic DCs, but not B cells, induced robust proliferation of donor CD8(+) T cells and broke GVHD resistance of chimeric recipients in which APCs were syngeneic to donors. These results demonstrate that host-derived DCs are critical in priming donor CD4(+) and CD8(+) T cells to cause GVHD, and selective targeting of host DCs may be a promising strategy to prevent GVHD.     

Presentation of soluble antigens to CD8+ T cells by CpG oligodeoxynucleotide-primed human naive B cells

Naive B lymphocytes are generally thought to be poor APCs, and there is limited knowledge of their role in activation of CD8(+) T cells. In this article, we demonstrate that class I MHC Ag presentation by human naive B cells is enhanced by TLR9 agonists. Purified naive B cells were cultured with or without a TLR9 agonist (CpG oligodeoxynucleotide [ODN] 2006) for 2 d and then assessed for phenotype, endocytic activity, and their ability to induce CD8(+) T cell responses to soluble Ags. CpG ODN enhanced expression of class I MHC and the costimulatory molecule CD86 and increased endocytic activity as determined by uptake of dextran beads. Pretreatment of naive B cells with CpG ODN also enabled presentation of tetanus toxoid to CD8(+) T cells, resulting in CD8(+) T cell cytokine production and granzyme B secretion and proliferation. Likewise, CpG-activated naive B cells showed enhanced ability to cross-present CMV Ag to autologous CD8(+) T cells, resulting in proliferation of CMV-specific CD8(+) T cells. Although resting naive B cells are poor APCs, they can be activated by TLR9 agonists to serve as potent APCs for class I MHC-restricted T cell responses. This novel activity of naive B cells could be exploited for vaccine design.     

B cell lipid rafts regulate both peptide-dependent and peptide-independent APC-T cell interaction

Formation of an immunological synapse (IS) between APCs and T CD4(+) lymphocytes is a key event in the initiation and the termination of the cognate immune response. We have analyzed the contribution of the APC to IS formation and report the implication of the actin cytoskeleton, the signaling proteins and the lipid rafts of B lymphocytes. Recruitment of MHC class II molecules to the IS is concomitant with actin cytoskeleton-dependent B cell raft recruitment. B cell actin cytoskeleton disruption abrogates both IS formation and T cell activation, whereas protein kinase C inhibition only impairs T cell activation. Pharmacological B cell lipid raft disruption inhibited peptide-dependent T lymphocyte activation and induced peptide-independent but HLA-DR-restricted APC-T cell conjugate formation. Such peptide-independent conjugates did not retain the ability to activate T cells. Thus, B cell lipid rafts are bifunctional by regulating T cell activation and imposing peptide stringency.     

Temporal development of autoreactive Th1 responses and endogenous presentation of self myelin epitopes by central nervous system-resident APCs in Theiler's virus-infected mice

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a chronic-progressive, immune-mediated CNS demyelinating disease and a relevant model of multiple sclerosis. Myelin destruction is initiated by TMEV-specific CD4(+) T cells targeting persistently infected CNS-resident APCs leading to activation of myelin epitope-specific CD4(+) T cells via epitope spreading. We examined the temporal development of virus- and myelin-specific T cell responses and acquisition of virus and myelin epitopes by CNS-resident APCs during the chronic disease course. CD4(+) T cell responses to virus epitopes arise within 1 wk after infection and persist over a >300-day period. In contrast, myelin-specific T cell responses are first apparent approximately 50-60 days postinfection, appear in an ordered progression associated with their relative encephalitogenic dominance, and also persist. Consistent with disease initiation by virus-specific CD4(+) T cells, CNS mononuclear cells from TMEV-infected SJL mice endogenously process and present virus epitopes throughout the disease course, while myelin epitopes are presented only after initiation of myelin damage (>50-60 days postinfection). Activated F4/80(+) APCs expressing high levels of MHC class II and B7 costimulatory molecules and ingested myelin debris chronically accumulate in the CNS. These results suggest a process of autoimmune induction in which virus-specific T cell-mediated bystander myelin destruction leads to the recruitment and activation of infiltrating and CNS-resident APCs that process and present endogenous myelin epitopes to autoreactive T cells in a hierarchical order.     

Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry

Immune responses appropriate for control of an intracellular pathogen are generated in mice infected with Brucella abortus, shown by the ability of T cells to adoptively transfer resistance to naive mice. The infection nevertheless persists for months. It was hypothesized that one factor in maintaining the infection despite the presence of immune T cells was suboptimal expression of major histocompatibility complex (MHC) molecules on macrophages containing brucellae. This would allow B. abortus to elude detection by the host's immune system. To test this, B. abortus organisms expressing green fluorescent protein (GFP-Brucella) were constructed and three-color flow cytometry used to evaluate MHC expression on macrophages following in vitro or in vivo infection. When infected in vitro, the levels of MHC class I and class II expression on J774 macrophages containing GFP-Brucella were the same or higher than on macrophages without GFP-Brucella in the same cultures. Similarly, the MHC expression was higher on GFP(+) peritoneal exudate cells following infection or phagocytosis of heat-killed GFP-Brucella than it was on uninfected peritoneal exudate cells. Following in vivo infection of mice the level of MHC class I and II expression on GFP(+) cells in their spleens (the main site of infection) also tended to be as high as or higher than that on the GFP-negative cells. The only in vivo GFP(+) cells that showed a decreased MHC expression was a population of splenic Mac1(+) cells recovered from interferon-gamma gene-disrupted mice at the time of their death due to an overwhelming number of bacteria per spleen. Overall, it was concluded that decreased MHC expression is not a general principle associated with brucella infection of macrophages and thus not likely to contribute to maintenance of the chronic infection.     

Zwitterionic polysaccharides stimulate T cells by MHC class II-dependent interactions

Polysaccharides of pathogenic extracellular bacteria commonly have negatively charged groups or no charged groups at all. These molecules have been considered classic T cell-independent Ags that do not elicit cell-mediated immune responses in mice. However, bacterial polysaccharides with a zwitterionic charge motif (ZPSs), such as the capsular polysaccharides of many strains of Bacteroides fragilis, Staphylococcus aureus, and Streptococcus pneumoniae type 1 elicit potent CD4(+) T cell responses in vivo and in vitro. The cell-mediated response to ZPS depends on the presence of both positively charged and negatively charged groups on each repeating unit of the polysaccharide. In this study, we define some of the requirements for the presentation of ZPS to CD4(+) T cells. We provide evidence that direct interactions of T cells with APCs are essential for T cell activation by ZPS. Monocytes, dendritic cells, and B cells are all able to serve as APCs for ZPS-mediated T cell activation. APCs lacking MHC class II molecules do not support this activity. Furthermore, mAb to HLA-DR specifically blocks ZPS-mediated T cell activation, while mAbs to other MHC class II and class I molecules do not. Immunoprecipitation of lysates of MHC class II-expressing cells following incubation with ZPS shows binding of ZPS and HLA-DR. Electron microscopy reveals colocalization of ZPS with HLA-DR on the cell surface and in compartments of the endocytic pathway. These results indicate that MHC class II molecules expressing HLA-DR on professional APCs are required for ZPS-induced T cell activation. The implication is that binding of ZPS to HLA-DR may be required for T cell activation.     

Rejection of mouse cardiac allografts by costimulation in trans

The activation of T cells by B7 costimulation in trans has been demonstrated in vitro, but the in vivo relevance is unknown. To study costimulation in trans of CD4(+) T cells in vivo, we performed cardiac transplants from B7-1/B7-2-deficient mice to recipients that do not express MHC class II molecules on peripheral APCs, but do have functional CD4(+) T cells (II(-)/4(+) mice). This model restricts the B7-dependent activation of CD4(+) T cells to costimulation in trans and excludes any contribution from indirect Ag presentation. We find that II(-)/4(+) recipients reject B7-deficient grafts as rapidly as wild-type grafts, suggesting that costimulation in trans can mediate rejection as potently as costimulation in cis. Treatment of II(-)/4(+) recipients of B7-deficient grafts with depleting Abs to CD4 or CD8 demonstrates that indirect Ag presentation to CD8(+) cells does not significantly contribute to rejection. This is the first demonstration that costimulation in trans can mediate an immune response in vivo and has important therapeutic implications.     

Peripheral tolerance via the anterior chamber of the eye: role of B cells in MHC class I and II antigen presentation

Ags introduced into the anterior chamber (AC) of the eye induce a form of peripheral immune tolerance termed AC-associated immune deviation (ACAID). ACAID mitigates ocular autoimmune diseases and promotes corneal allograft survival. Ags injected into the AC are processed by F4/80(+) APCs, which migrate to the thymus and spleen. In the spleen, ocular APCs induce the development of Ag-specific B cells that act as ancillary APCs and are required for ACAID induction. In this study, we show that ocular-like APCs elicit the generation of Ag-specific splenic B cells that induce ACAID. However, direct cell contact between ocular-like APCs and splenic B cells is not necessary for the induction of ACAID B cells. Peripheral tolerance produced by ACAID requires the participation of ACAID B cells, which induce the generation of both CD4(+) regulatory T cells (Tregs) and CD8(+) Tregs. Using in vitro and in vivo models of ACAID, we demonstrate that ACAID B cells must express both MHC class I and II molecules for the generation of Tregs. These results suggest that peripheral tolerance induced through the eye requires Ag-presenting B cells that simultaneously present Ags on both MHC class I and II molecules.     

Brucella abortus lipopolysaccharide in murine peritoneal macrophages acts as a down-regulator of T cell activation

Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4(+) T cells but not those of OVA to specific CD8(+) T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4(+) T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.     

Microbial and T cell-derived stimuli regulate antigen presentation by dendritic cells in vivo

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4(+) T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8alpha(+) DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.     

Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II

Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcgammaRgamma and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.     

On the role of APC-activation for in vitro versus in vivo T cell priming

Professional antigen-presenting cells take up antigens for processing and presentation in association with MHC class I and II molecules. When APCs receive the right stimuli, they undergo a maturation process and migrate to secondary lymphoid organs to trigger T cell activation. In this study, we compared side-by-side in vivo and in vitro activation of T cells. Transgenic CD8(+) T cells specific for the p33 epitope, derived from the lymphocytic choriomeningitis virus glycoprotein, were labeled with CFSE and injected into syngeneic mice or alternatively, co-cultured in vitro with APCs. The p33 epitope was delivered as free peptide or genetically fused to virus-like particles. Whereas proliferation of specific T cells was comparable in both systems, the production of IFN-gamma and the expression of CD25 showed important differences. Induction of effector function and expression of activation markers were strongly enhanced in vitro by both the free peptide and VLPs. Surprisingly, addition of CpG-containing immune-stimulating DNA for activation of APCs dramatically increased effector T cell differentiation in vitro, whereas no enhancement could be observed in vitro. Thus, activation of professional APCs was mandatory for induction of effector CD8(+) T cell responses in vivo, while this step was largely dispensable in vitro.     

B lymphocytes participate in cross-presentation of antigen following gene gun vaccination

Although endocytosed proteins are commonly presented via the class II MHC pathway to stimulate CD4(+) T cells, professional APCs can also cross-present Ags, whereby these exogenous peptides can be complexed with class I MHC for cross-priming of CD8(+) T cells. Whereas the ability of dendritic cells (DCs) to cross-present Ags is well documented, it is not known whether other APCs may also play a role, or what is the relative contribution of cross-priming to the induction of acquired immunity after DNA immunization. In this study, we compared immune responses generated after gene gun vaccination of mice with DNA vaccine plasmids driven by the conventional CMV promoter, the DC-specific CD11c promoter, or the keratinocyte-specific K14 promoter. The CD11c promoter achieved equivalent expression in CD11c(+) DCs in draining lymph nodes over time, as did a conventional CMV-driven plasmid. However, immunization with DC-restricted DNA vaccines failed to generate protective humoral or cellular immunity to model Ags influenza hemagglutinin and OVA, despite the ability of CD11c(+) cells isolated from lymph nodes to stimulate proliferation of Ag-specific T cells directly ex vivo. In contrast, keratinocyte-restricted vaccines elicited comparable T and B cell activity as conventional CMV promoter-driven vaccines, indicating that cross-priming plays a major role in the generation of immune responses after gene gun immunization. Furthermore, parallel studies in B cell-deficient mu-MT mice demonstrated that B lymphocytes, in addition to DCs, mediate cross-priming of Ag-specific T cells. Collectively, these data indicate that broad expression of the immunogen is required for optimal induction of protective acquired immunity.     

CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.     

Vascular endothelium does not activate CD4+ direct allorecognition in graft rejection

Expression of MHC class II by donor-derived APCs has been shown to be important for allograft rejection. It remains controversial, however, whether nonhemopoietic cells, such as vascular endothelium, possess Ag-presenting capacity to activate alloreactive CD4(+) T lymphocytes. This issue is important in transplantation, because, unlike hemopoietic APCs, allogeneic vascular endothelium remains present for the life of the organ. In this study we report that cytokine-activated vascular endothelial cells are poor APCs for allogeneic CD4(+) T lymphocytes in vitro and in vivo despite surface expression of MHC class II. Our in vitro observations were extended to an in vivo model of allograft rejection. We have separated the allostimulatory capacity of endothelium from that of hemopoietic APCs by using bone marrow chimeras. Hearts that express MHC class II on hemopoietic APCs are acutely rejected in a mean of 7 days regardless of the expression of MHC class II on graft endothelium. Alternatively, hearts that lack MHC class II on hemopoietic APCs are acutely rejected at a significantly delayed tempo regardless of the expression of MHC class II on graft endothelium. Our data suggest that vascular endothelium does not play an important role in CD4(+) direct allorecognition and thus does not contribute to the vigor of acute rejection.     

Acquisition of CD80 by human T cells at early stages of activation: functional involvement of CD80 acquisition in T cell to T cell interaction

The interaction between CD28 on T cells and CD80 on APCs intensifies the linkage between TCR and MHC at the site of contact between T cells and APCs. In this study, we demonstrate that during human T cell/human APC interaction, the autologous or allogeneic human CD4(+) T cells become positive for the detection of CD80 at an early stage of activation (24 h). This detection of CD80 is attributable to the acquisition of CD80 from APCs, as opposed to the up-regulation of endogenous CD80, as demonstrated by CD4(+) T cells treated with cyclohexamide. Furthermore, no CD80 mRNA could be detected at 24 h in T cells that had acquired CD80 from APCs. CD80 acquisition by T cells from APCs was enhanced upon TCR engagement. The amount of CD80 acquisition by CD4(+) T cells was shown to be related to the expression of CD80 on APCs. Using soluble fusion proteins (soluble CTLA-4, CD28, and CD80) to block either CD28 on the surface of T cells or CD80 on the surface of APCs, it was demonstrated that CD80 acquisition by T cells is mediated through its receptors, possibly CD28 interaction. Moreover, we demonstrate that T cells that have acquired CD80 have the ability to stimulate other T cells. These data thus suggest that CD80 acquisition by human T cells might play a role in the immunoregulation of T cell responses.