Endogenous Ags Research Articles

Transplanting mitochondria from gastric epithelial cells reduces the malignancy of gastric cancer

Mitochondria are essential for energy supplementation and metabolic homeostasis of cancer cells. Using mitochondria transplantation to reduce the malignancy of gastric cancer (GC) cells is herein proposed. In our study normal human gastric mucous epithelium cell line (GES-1) showed a lower mitochondrial membrane potential (MMP) compared to immortalized human vascular endothelial cell line (EAhy 926) and human gastric adenocarcinoma cell line (AGS). The transplantation of GES-1 mitochondria to AGS were confirmed both by confocal microscopy and flow cytometry. After transplanting GES-1 mitochondria, the AGS showed a reduced cell migration, and invasion without affecting cell viability and apoptosis. Investigating the expression of proteins involved in epithelial-mesenchymal-transition (EMT), transplanted GES-1 mitochondria reduced the expression of mesenchymal markers α-SMA, MMP-9, snail, vimentin and N-cadherin, whereas the epithelial markers E-cadherin and clauding-1 were not changed. The proteins implicated in the cell cycle such as cyclin B1 and D1 were decreased. In mice, inoculation with AGS carrying the transplanted GES-1 mitochondria resulted in smaller sized tumors. Further investigating the mitochondrial balance, the transplanted GES-1 mitochondria were more stably preserved compared to endogenous AGS mitochondria. The MMP, ATP production and mitochondrial mass decreased in GES-1 mitochondria and the mitophagic proteins LC3 II and PINK1 were up-regulated. In conclusion the decreased malignancy of AGS was a result of exogenous GES-1 mitochondria transplantation. This suggests for a therapy with low efficiency mitochondria transplantation in the treatment of cancer cells.

PD-L1 siRNA–hyaluronic acid conjugate for dual-targeted cancer immunotherapy

"Foreignization" of tumor cells via delivery of a non-self foreign antigen (Ag) into tumors is an appealing strategy to initiate anti-tumor immunity that can facilitate tumor rejection by pre-existing foreign-Ag-reactive T cells. However, the immune-suppressive factors in the tumor microenvironment (TME) limit the durable and potent immune response of these cells against tumor antigens, stressing the need for improved tumor-foreignization strategies. Here, we demonstrate that blockade of programmed cell death ligand 1 (PD-L1) on both tumor cells and dendritic cells (DCs) can markedly potentiate the induction of tumor-reactive T cells, thereby strengthening the anti-tumor immunity ignited by tumor-foreignization. Specifically, we developed a polymeric nanoconjugate (PEG-HA-OVA/PPLs), consisting of siPD-L1-based polyplexes, PEGylated hyaluronic acid as the CD44-targeting moiety, and ovalbumin (OVA) as a model foreign antigen. Notably, PEG-HA-OVA/PPLs were simultaneously delivered into CD44high tumor cells and CD44high DCs, leading to efficient cross-presentation of OVA and downregulation of PD-L1 in both cell types. Importantly, the nanoconjugate not only allowed OVA-specific T cells to vigorously reject the foreignized tumor cells but also reprogrammed the TME to elicit robust T-cell responses specific to the endogenous tumor Ags, eventually generating long-lasting protective immunity. Thus, our combination strategy represents an innovative approach for the induction of potent tumor immunity via a two-step consecutive immune boost against exogenous and endogenous tumor Ags.

Optimal Development of Mature B Cells Requires Recognition of Endogenous Antigens.

It has long been appreciated that highly autoreactive BCRs are actively removed from the developing B cell repertoire by Ag-dependent receptor editing and deletion. However, there is persistent debate about whether mild autoreactivity is simply tolerated or positively selected into the mature B cell repertoire as well as at what stage, to what extent, under what conditions, and into which compartments this occurs. In this study, we describe two minor, trackable populations of B cells in B1-8i Ig transgenic mice that express the VH186.2 H chain and recognize a common foreign Ag (the hapten 4-hydroxy-3-nitrophenylacetyl) but differ in L chain expression. We use the Nur77-eGFP reporter of BCR signaling to define their reactivity toward endogenous Ags. The less autoreactive of these two populations is strongly counterselected during the development of mature B1a, follicular, and marginal zone B cells. By genetically manipulating the strength of BCR signal transduction via the titration of surface CD45 expression, we demonstrate that this B cell population is not negatively selected but instead displays characteristics of impaired positive selection. We demonstrate that mild self-reactivity improves the developmental fitness of B cell clones in the context of a diverse population of B cells, and positive selection by endogenous Ags shapes the mature B cell repertoire.

Age-Associated B Cells Express a Diverse Repertoire of VH and Vκ Genes with Somatic Hypermutation.

The origin and nature of age-associated B cells (ABCs) in mice are poorly understood. In this article, we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for 1 mo in the absence of external Ag. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors but not from MHC class II- or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what Igs ABCs express, we sequenced VH and Vκ rearranged genes from unimmunized 22-mo-old C57BL/6 mice and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting that ABCs have undergone mild stimulation from endogenous Ags over time. These observations show that quiescent ABCs are Ag-experienced cells that accumulate during T cell-dependent responses to diverse Ags during the life of an individual.

HIV-Infected Dendritic Cells Present Endogenous MHC Class II–Restricted Antigens to HIV-Specific CD4+ T Cells

It is widely assumed that CD4(+) T cells recognize antigenic peptides (epitopes) derived solely from incoming, exogenous, viral particles or proteins. However, alternative sources of MHC class II (MHC-II)-restricted Ags have been described, in particular epitopes derived from newly synthesized proteins (so-called endogenous). In this study, we show that HIV-infected dendritic cells (DC) present MHC-II-restricted endogenous viral Ags to HIV-specific (HS) CD4(+) T cells. This endogenous pathway functions independently of the exogenous route for HIV Ag presentation and offers a distinct possibility for the immune system to activate HS CD4(+) T cells. We examined the implication of autophagy, which plays a crucial role in endogenous viral Ag presentation and thymic selection of CD4(+) T cells, in HIV endogenous presentation. We show that infected DC do not use autophagy to process MHC-II-restricted HIV Ags. This is unlikely to correspond to a viral escape from autophagic degradation, as infecting DC with Nef- or Env-deficient HIV strains did not impact HS T cell activation. However, we demonstrate that, in DC, specific targeting of HIV Ags to autophagosomes using a microtubule-associated protein L chain 3 (LC3) fusion protein effectively enhances and broadens HS CD4(+) T cell responses, thus favoring an endogenous MHC-II-restricted presentation. In summary, in DC, multiple endogenous presentation pathways lead to the activation of HS CD4(+) T cell responses. These findings will help in designing novel strategies to activate HS CD4(+) T cells that are required for CTL activation/maintenance and B cell maturation.

T Cell Priming by Activated Nlrc5-Deficient Dendritic Cells Is Unaffected despite Partially Reduced MHC Class I Levels.

NLRC5, a member of the NOD-like receptor (NLR) protein family, has recently been characterized as the master transcriptional regulator of MHCI molecules in lymphocytes, in which it is highly expressed. However, its role in activated dendritic cells (DCs), which are instrumental to initiate T cell responses, remained elusive. We show in this study that, following stimulation of DCs with inflammatory stimuli, not only did NLRC5 level increase, but also its importance in directing MHCI transcription. Despite markedly reduced mRNA and intracellular H2-K levels, we unexpectedly observed nearly normal H2-K surface display in Nlrc5(-/-) DCs. Importantly, this discrepancy between a strong intracellular and a mild surface defect in H2-K levels was observed also in DCs with H2-K transcription defects independent of Nlrc5. Hence, alongside with demonstrating the importance of NLRC5 in MHCI transcription in activated DCs, we uncover a general mechanism counteracting low MHCI surface expression. In agreement with the decreased amount of neosynthesized MHCI, Nlrc5(-/-) DCs exhibited a defective capacity to display endogenous Ags. However, neither T cell priming by endogenous Ags nor cross-priming ability was substantially affected in activated Nlrc5(-/-) DCs. Altogether, these data show that Nlrc5 deficiency, despite significantly affecting MHCI transcription and Ag display, is not sufficient to hinder T cell activation, underlining the robustness of the T cell priming process by activated DCs.

MHC/Peptide-Specific Interaction of the Humoral Immune System: A New Category of Antibodies.

Abs bind to unprocessed Ags, whereas cytotoxic CD8(+) T cells recognize peptides derived from endogenously processed Ags presented in the context of class I MHC complexes. We screened, by ELISA, human sera for Abs reacting specifically with the influenza matrix protein (IMP)-derived peptide(58-66) displayed by HLA-A*0201 complexes. Among 653 healthy volunteers, blood donors, and women on delivery, high-titered HLA-A*0201/IMP(58-66) complex-specific IgG Abs were detected in 11 females with a history of pregnancies and in 1 male, all HLA-A*0201(-). These Abs had the same specificity as HLA-A*0201/IMP(58-66)-specific cytotoxic T cells and bound neither to HLA-A*0201 nor the peptide alone. No such Abs were detected in HLA-A*0201(+) volunteers. These Abs were not cross-reactive to other self-MHC class I alleles displaying IMP(58-66), but bound to MHC class I complexes of an HLA nonidentical offspring. HLA-A*0201/IMP(58-66) Abs were also detected in the cord blood of newborns, indicating that HLA-A*0201/IMP(58-66) Abs are produced in HLA-A*0201(-) mothers and enter the fetal blood system. That Abs can bind to peptides derived from endogenous Ags presented by MHC complexes opens new perspectives on interactions between the cellular and humoral immune system.

Cutting Edge: Caspase-11 Limits the Response of CD8+ T Cells to Low-Abundance and Low-Affinity Antigens.

Inflammatory caspases, including caspase-11, are upregulated in CD8(+) T cells after Ag-specific activation, but little is known about their function in T cells. We report that caspase-11-deficient (Casp11(-/-)) T cells proliferated more readily in response to low-affinity and low-abundance ligands both in vitro and in vivo due to an increased ability to signal through the TCR. In addition to increased numbers, Casp11(-/-) T cells had enhanced effector function compared with wild-type cells, including increased production of IL-2 and reduced expression of CD62L. Casp11(-/-) T cells specific for endogenous Ags were more readily deleted than wild-type cells. These data indicate that caspase-11 negatively regulates TCR signaling, possibly through its ability to regulate actin polymerization, and inhibiting its activity could enhance the expansion and function of low-affinity T cells.

Positional identification of RT1-B (HLA-DQ) as susceptibility locus for autoimmune arthritis.

Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.

Comparison of Vaccine-Induced Effector CD8 T Cell Responses Directed against Self- and Non–Self-Tumor Antigens: Implications for Cancer Immunotherapy

It is generally accepted that CD8 T cells play a major role in tumor control, yet vaccination aimed at eliciting potent CD8 T cell responses are rarely efficient in clinical trials. To try and understand why this is so, we have generated potent adenoviral vectors encoding the endogenous tumor Ags (TA) tyrosinase-related protein-2 (TRP-2) and glycoprotein 100 (GP100) tethered to the invariant chain (Ii). Using these vectors, we sought to characterize the self-TA-specific CD8 T cell response and compare it to that induced against non-self-Ags expressed from a similar vector platform. Prophylactic vaccination with adenoviral vectors expressing either TRP-2 (Ad-Ii-TRP-2) or GP100 (Ad-Ii-GP100) had little or no effect on the growth of s.c. B16 melanomas, and only Ad-Ii-TRP-2 was able to induce a marginal reduction of B16 lung metastasis. In contrast, vaccination with a similar vector construct expressing a foreign (viral) TA induced efficient tumor control. Analyzing the self-TA-specific CD8 T cells, we observed that these could be activated to produce IFN-γ and TNF-α. In addition, surface expression of phenotypic markers and inhibitory receptors, as well as in vivo cytotoxicity and degranulation capacity matched that of non-self-Ag-specific CD8 T cells. However, the CD8 T cells specific for self-TAs had a lower functional avidity, and this impacted on their in vivo performance. On the basis of these results and a low expression of the targeted TA epitopes on the tumor cells, we suggest that low avidity of the self-TA-specific CD8 T cells may represent a major obstacle for efficient immunotherapy of cancer.

The Context of Gene Expression Defines the Immunodominance Hierarchy of Cytomegalovirus Antigens

Natural immunity to CMV dominates the CD4 and CD8 memory compartments of the CMV-seropositive host. This property has been recently exploited for experimental CMV-based vaccine vector strategies, and it has shown promise in animal models of AIDS and Ebola disease. Although it is generally agreed that CMV-based vaccine vectors may induce highly protective and persistent memory T cells, the influence of the gene expression context on Ag-specific T cell memory responses and immune protection induced by CMV vectors is not known. Using murine CMV (MCMV) recombinants expressing a single CD8 T cell epitope from HSV-1 fused to different MCMV genes, we show that magnitude and kinetics of T cell responses induced by CMV are dependent on the gene expression of CMV Ags. Interestingly, the kinetics of the immune response to the HSV-1 epitope was paralleled by a reciprocal depression of immune responses to endogenous MCMV Ags. Infection with a recombinant MCMV inducing a vigorous initial immune response to the recombinant peptide resulted in a depressed early response to endogenous MCMV Ag. Another recombinant virus, which induced a slowly developing "inflationary" T cell response to the HSV-1 peptide, induced weaker long-term responses to endogenous CMV Ags. Importantly, both mutants were able to protect mice from a challenge with HSV-1, mediating strong sterilizing immunity. Our data suggest that the context of gene expression markedly influences the T cell immunodominance hierarchy of CMV Ags, but the immune protection against HSV-1 does not require inflationary CD8 responses against the recombinant CMV-expressed epitope.

Competition for Antigen at the Level of the APC Is a Major Determinant of Immunodominance during Memory Inflation in Murine Cytomegalovirus Infection

The unique ability of CMV to drive the expansion of virus-specific T cell populations during the course of a lifelong, persistent infection has generated interest in the virus as a potential vaccine strategy. When designing CMV-based vaccine vectors to direct immune responses against HIV or tumor Ags, it becomes important to understand how and why certain CMV-specific populations are chosen to inflate over time. To investigate this, we designed recombinant murine CMVs (MCMVs) encoding a SIINFEKL-enhanced GFP fusion protein under the control of endogenous immediate early promoters. When mice were infected with these viruses, T cells specific for the SIINFEKL epitope inflated and profoundly dominated T cells specific for nonrecombinant (i.e., MCMV-derived) Ags. Moreover, when the virus encoded SIINFEKL, T cells specific for nonrecombinant Ags displayed a phenotype indicative of less frequent exposure to Ag. The immunodominance of SIINFEKL-specific T cells could not be altered by decreasing the number of SIINFEKL-specific cells available to respond, or by increasing the number of cells specific for endogenous MCMV Ags. In contrast, coinfection with viruses expressing and lacking SIINFEKL enabled coinflation of T cells specific for both SIINFEKL and nonrecombinant Ags. Because coinfection allows presentation of SIINFEKL and MCMV-derived Ags by different cells within the same animal, these data reveal that competition for, or availability of, Ag at the level of the APC determines the composition of the inflationary response to MCMV. SIINFEKL's strong affinity for H-2K(b), as well as its early and abundant expression, may provide this epitope's competitive advantage.

Induction of Endoplasmic Reticulum–Endosome Fusion for Antigen Cross-Presentation Induced by Poly (γ-Glutamic Acid) Nanoparticles

We previously reported that poly (γ-glutamic acid)-based nanoparticles (γ-PGA NPs) are excellent vaccine carriers for inducing efficient cross-presentation in dendritic cells, thereby producing strong antitumor immunity in vivo. Analyzing the mechanism of cross-presentation induced by γ-PGA NPs will be useful toward designing novel vaccine carriers. In this study, we show an intracellular mechanism of efficient cross-presentation induced by OVA-loaded γ-PGA NPs. Cross-presentation induced by γ-PGA NPs depended on cytoplasmic proteasomes and TAP, similar to the classical MHC class I presentation pathway for endogenous Ags. Intracellular behavior analyzed by confocal laser scanning microscopy revealed that encapsulated OVA and γ-PGA accumulated in both the endoplasmic reticulum (ER) and endosome compartments within 2 h. At the same time, electron microscopy analysis clearly showed that intracellular γ-PGA NPs and encapsulated Au NPs were enveloped in endosome-like vesicles, not in the ER. These findings strongly suggest that γ-PGA NPs enhance ER-endosome fusion for cross-presentation. Moreover, inhibition of ER translocon sec61 significantly decreased the γ-PGA NP/OVA-mediated cross-presentation efficiency, indicating that sec61 is important for transporting Ags from the fused ER-endosome to the cytoplasm. These findings imply that the ER-endosome complex is key for the efficient cross-presentation of Ags encapsulated in γ-PGA NPs.

TRAIL/DR5 Plays a Critical Role in NK Cell-Mediated Negative Regulation of Dendritic Cell Cross-Priming of T Cells

Dendritic cells (DCs) are critical in initiating immune responses by cross-priming of tumor Ags to T cells. Previous results showed that NK cells inhibited DC-mediated cross-presentation of tumor Ags both in vivo and in vitro. In this study, enhanced Ag presentation was observed in draining lymph nodes in TRAIL(-/-) and DR5(-/-) mice compared with that of wild-type mice. NK cells inhibit DC cross-priming of tumor Ags in vitro, but not direct presentation of endogenous Ags. NK cells lacking TRAIL, but not perforin, were not able to inhibit DC cross-priming of tumor Ags. DCs that lack expression of TRAIL receptor DR5 were less susceptible to NK cell-mediated inhibition of cross-priming, and cross-linking of DR5 receptor led to reduced generation of MHC class I-Ag peptide complexes, followed by attenuated cross-priming of CD8(+) T cells. In addition, key molecules involved in the TRAIL/DR5 pathway during DC/NK cell interactions were determined. In summary, these data indicate a novel alternative pathway for DC/NK cell interactions in antitumor immunity and may reflect homeostasis of both DCs and NK cells for regulation of CD8(+) T cell function in physiological conditions.

Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens, presenting endogenous Ags, and regulating T lymphocyte survival and proliferation. In this study, we have investigated the role of autophagy in regulating the endoplasmic reticulum (ER) compartment in T lymphocytes. We found that ER content is expanded in mature autophagy-related protein (Atg) 7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the TCR display impaired influx, but not efflux, of calcium, and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes, suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore, we found that the stimulation-induced redistribution of stromal interaction molecule-1, a critical event for the store-operated Ca(2+) release-activated Ca(2+) channel opening, is impaired in Atg7-deficient T cells. Together, these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores, and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes.

Naive CD4+ T Lymphocytes Circulate through Lymphoid Organs To Interact with Endogenous Antigens and Upregulate Their Function

Naive T lymphocytes recirculate through the lymph-vascular system and enter and exit lymphoid organs. Using mice expressing the photoconvertible fluorescence protein Kaede, we demonstrated that naive T cells seek to interact with endogenous Ags after migrating to the lymphoid organs. The interaction with endogenous Ags transiently induces CD69 expression on T cells, which prolongs retention in the lymphoid organs. Cells that fail to express CD69 or lose CD69 expression migrate to other lymphoid organs. Functionally, CD69(+)-naive CD4(+) T cells exhibit faster and greater cytokine production than do CD69(-) naive CD4(+) T cells. These results indicate that CD4(+) T cells continuously migrate to interact with endogenous Ags, and such an interaction plays an important role in the Ag reactivity of naive CD4(+) T cells.

Human Plasmacytoid Dendritic Cells Phagocytose, Process, and Present Exogenous Particulate Antigen

Plasmacytoid dendritic cells (pDCs) play a major role in shaping both innate and adaptive immune responses, mainly via their production of large amounts of type I IFNs. pDCs are considered to primarily present endogenous Ags and are thought not to participate in the uptake and presentation of Ags from the extracellular environment, in contrast to their myeloid counterparts, which efficiently endocytose extracellular particulates. In this study, we show that human pDCs are able to phagocytose and process particulate forms of Ag entrapped in poly(lactic-coglycolic acid) microparticles. Furthermore, pDCs were also able to sense TLR ligands (TLR-Ls) incorporated in these particles, resulting in rapid pDC activation and high IFN-alpha secretion. Combining a tetanus toxoid peptide and TLR-Ls (CpG C and R848) in these microparticles resulted in efficient pDC activation and concomitant Ag-specific T cell stimulation. Moreover, particulate Ag was phagocytosed and presented more efficiently than soluble Ag, indicating that microparticles can be exploited to facilitate efficient delivery of antigenic cargo and immunostimulatory molecules to pDCs. Together, our results show that in addition to their potency to stimulate innate immunity, pDCs can polarize adaptive immune responses against exogenous particulate Ag. These results may have important consequences for the development of new immunotherapeutic strategies exploiting Ag and TLR-Ls encapsulated in microparticles to target APC subsets.

Substrain Differences Reveal Novel Disease-Modifying Gene Candidates That Alter the Clinical Course of a Rodent Model of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis that is executed in animals by immunization with myelin Ag in adjuvant. The SJL/J autoimmune-prone strain of mouse has been used to model relapsing-remitting multiple sclerosis. However, significant variations in peak scores, timing of onset, and incidence are observed among laboratories, with the postacute (relapse) phase of the disease exhibiting significant inconsistency. We characterized two substrains of SJL/J mice that exhibit profoundly different EAE disease parameters. Induction of EAE in the first SJL/J substrain resulted in many cases of chronic EAE that was dominated by an aggressive B cell response to the immunizing Ag and to endogenous CNS Ags. In contrast, the other SJL/J substrain exhibited a relapsing-remitting form of EAE concomitant with an elevated number of cytokine-producing CD4(+) T cells in the CNS. Exploiting these interstrain differences, we performed a genome-wide copy number analysis on the two disparate SJL/J substrains and discovered numerous gene-dosage differences. In particular, one inflammation-associated gene, Naip1, was present at a higher copy number in the SJL/J substrain that exhibited relapsing-remitting EAE. These results demonstrate that substrain differences, perhaps at the level of genomic copy number, can account for variability in the postacute phase of EAE and may drive chronic versus relapsing disease.

Application of the modified vaccination technique for the prevention and cure of chronic ailments

Over the years vaccination has proven to be the most successful health protection program for large populations, to prevent them from acquiring serious infectious and contagious diseases caused by exogenous antigens (ags) such as bacteria and viruses. Protection is generally achieved by an active immunization program, though passive immunization has also been employed, especially in the past, to combat diseases caused by certain bacterial infections (e.g. tetanus, diphtheria, etc.). Most recently, encouraging research data suggests that therapeutic approaches employing vaccination techniques can also be used to correct or deal with mishaps induced by or involving endogenous ags. However, most attempts at employing conventional vaccination techniques to do so have proven less than successful. In the case of cancer, one of the reasons for this is that the presentation of cancer related ags in presently available immunization frameworks is unable to evoke a powerful, specific cancer killing response. Therefore, drug treatments have been required in order to achieve additional beneficial effects. Recently, the Barabas group has developed a new vaccination technique (the third vaccination method, after active and passive immunization) called Modified Vaccination Technique (MVT). In experiments the MVT has been able to prevent-and with equal effectiveness, terminate-mishaps induced by or involving endogenous ags, e.g. in an experimental autoimmune kidney disease called slowly progressive Heymann nephritis (SPHN). The MVT is safe, and is able to initiate a specific immune response in the injected host (provided the injected components are in pure form). The MVT promises to provide the next generation of vaccines for the prevention, treatment, and termination of chronic disorders in humans, such as autoimmune diseases, cancer, and chronic infections.

Efficient Cross-Priming of Antiviral CD8+ T Cells by Antigen Donor Cells Is GRP94 Independent

Cross-priming, the activation of naive CD8+ T cells by dendritic cells presenting Ags synthesized by other cells, is believed to play an important role in the generation of antiviral and antitumor responses. The molecular mechanism(s) underlying cross-priming remain poorly defined and highly controversial. GRP94 (gp96), an abundant endoplasmic reticulum chaperone with innate immune-activating capacity, has been widely reported to play a major role in cross-priming. In this study, we show that cells whose expression of GRP94 is silenced via transient or stable transfection with GRP94-directed small interfering RNAs demonstrate no reduction in their abilities to generate class I peptide complexes in cultured cells or to prime antiviral CD8+ T cell responses in vivo. In demonstrating the dispensability of GRP94, our finding points to the importance of alternative mechanisms for generation of class I peptide complexes from endogenous and exogenous Ags and immunogens.