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The establishment of robust T cell memory is critical for the development of novel vaccines for infections and cancers. Classical memory generated by CD8(+)T cells is characterized by contracted populations homing to lymphoid organs. T cell memory inflation, as seen for example after CMV infection, is the maintenance of expanded, functional, tissue-associated effector memory cell pools. Such memory pools may also be induced after adenovirus vaccination, and we recently defined common transcriptional and phenotypic features of these populations in mice and humans. However, the rules that govern which epitopes drive memory inflation compared with classical memory are not fully defined, and thus it is not currently possible to direct this process. We used our adenoviral model of memory inflation to first investigate the role of the promoter and then the role of the epitope context in determining memory formation. Specifically, we tested the hypothesis that conventional memory could be converted to inflationary memory by simple presentation of the Ag in the form of minigene vectors. When epitopes from LacZ and murine CMV that normally induce classical memory responses were presented as minigenes, they induced clear memory inflation. These data demonstrate that, regardless of the transgene promoter, the polypeptide context of a CD8(+)T cell epitope may determine whether classical or inflating memory responses are induced. The ability to direct this process by the use of minigenes is relevant to the design of vaccines and understanding of immune responses to pathogens., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The critical role of IFN-α in the pathogenesis of human systemic lupus erythematosus has been highlighted in recent years. Exposure of young lupus-prone NZB/W F1 mice to IFN-α in vivo leads to an accelerated lupus phenotype that is dependent on T cells and is associated with elevated serum levels of BAFF, IL-6, and TNF-α, increased splenic expression of IL-6 and IL-21, formation of large germinal centers, and the generation of large numbers of short-lived plasma cells that produce IgG2a and IgG3 autoantibodies. In this study, we show that both IgG2a and IgG3 autoantibodies are pathogenic in IFN-α-accelerated lupus, and their production can be dissociated by using low-dose CTLA4-Ig. Only high-dose CTLA4-Ig attenuates both IgG2a and IgG3 autoantibody production and significantly delays death from lupus nephritis. In contrast, BAFF/APRIL blockade has no effect on germinal centers or the production of IgG anti-dsDNA Abs but, if given at the time of IFN-α challenge, delays the progression of lupus by attenuating systemic and renal inflammation. Temporary remission of nephritis induced by combination therapy with cyclophosphamide, anti-CD40L Ab, and CTLA4-Ig is associated with the abrogation of germinal centers and depletion of short-lived plasma cells, but relapse occurs more rapidly than in conventional NZB/W F1 mice. This study demonstrates that IFN-α renders NZB/W F1 relatively resistant to therapeutic intervention and suggests that the IFN signature should be considered when randomizing patients into groups and analyzing the results of human clinical trials in systemic lupus erythematosus.

We previously reported the strong immunostimulatory effects of a CpG oligodeoxynucleotide (ODN), designated MsST, from the lacZ gene of Streptococcus (S.) thermophilus ATCC19258. Here we show that 24h of stimulation with MsST in mouse splenocytes and peritoneal macrophages strongly induces expression of interleukin (IL)-33, a cytokine in the IL-1 superfamily. Other IL-1 superfamily members, including IL-1alpha, IL-1beta and IL-18, are down-regulated after 24h of stimulation of MsST. We also found that MsST-induced IL-33 mRNA expression is inhibited by the suppressive ODN A151, which can inhibit Toll-like receptor 9 (TLR9)-mediated responses. This is the first report to show that IL-33 can be induced by CpG ODNs. The strong induction of IL-33 by MsST suggests that it may be a potential therapeutic ODN for the treatment of inflammatory disease. The presence of a strong CpG ODN in S. thermophilus also suggests that the bacterium may be a good candidate as a starter culture for the development of new physiologically functional foods., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.

LacZ (Gal)-reactive immune cells were transferred into athymic nu/nu mice inoculated with Gal-expressing syngeneic tumour cells (ESbL-Gal) in order to study tumour-protective T-cell memory. This transfer prevented tumour outgrowth in recipients and resulted in the persistence of a high frequency of Gal-specific CD8(+) T cells in the bone marrow and spleen. In contrast, such Ag-specific memory CD8(+) T cells were not detectable by peptide-major histocompatibility complex (MHC) multimer staining in animals that had not previously received an antigenic challenge. Even though CD44(hi) memory T cells from the bone marrow showed a significantly higher turnover rate, as judged by bromodeoxyuridine (BrdU) incorporation, than respective cells from spleen or lymph nodes, as well as in comparison to CD44(lo) naïve T cells, these findings suggest that tumour-associated antigen (TAA) from residual dormant tumour cells are implicated in maintaining high frequencies of long-term surviving Gal-specific memory CD8(+) T cells. Memory T cells could be recruited to the peritoneal cavity by tumour vaccination of immunoprotected nu/nu mice and exhibited ex vivo antitumour reactivity. Long-term immune memory and tumour protection could be maintained over four successive transfers between tumour-inoculated recipients, which involved periodic antigenic restimulation in vivo prior to reisolating the cells for adoptive transfer. Using a cell line (ESbL-Gal-BM) that was established from dormant tumour cells isolated from the bone marrow of immunoprotected animals, it could be demonstrated that the tumour cells had up-regulated the expression of MHC class I molecules and down-regulated the expression of several adhesion molecules during the in vivo passage. Our results suggest that the bone marrow microenvironment has special features that are of importance for the maintenance of tumour dormancy and immunological T-cell memory, and that a low level of persisting antigen favours the maintenance of Ag-specific memory T cells over irrelevant memory T cells.

This study describes a tumor vaccine-induced secondary in vivo T cell response to an immunodominant epitope of beta-galactosidase (Gal) as a model tumor-associated antigen. DBA/2 mice are primed with lacZ transfected live ESbL tumor cells (ESbL-Gal) in the ear pinna, a site which had previously been shown to be non-tumorigenic and immunogenic. Intraperitoneal challenge of such mice with tumor vaccine (i.e., 10(7) irradiation-inactivated ESbL-Gal cells) leads to the production of a powerful CD8+ CTL response and to the establishment of immune memory. Using peptide/MHC-tetrameric complexes, clonal expansion of antigen-specific T cells could be detected during the primary response in bone marrow (BM) and during the secondary response in the peritoneal cavity and BM. The secondary response in the peritoneal cavity involved a >80-fold enrichment of epitope specific CD8+ T cells and the release of various cytokines, including IL-12 and TNF-alpha. The recruitment and/or expansion of Gal specific T cells within the peritoneal cavity could be inhibited by anti-IL-12 and anti-TNF-alpha monoclonal antibody (mAb) treatment. Interestingly, the secondary CTL response was inhibited by anti-IL-12 but not by anti-TNF-alpha mAb. The results characterize a strong systemic CD8+ memory T cell response to a cell bound antigen without the use of adjuvant.

Naked circular plasmid DNA containing the cytomegalovirus (CMV)-promoter-driven lacZ reporter gene (pCMV-LacZ) was injected in the epaxial muscle of gilthead sea bream (Sparus aurata). A mosaic pattern of expression of beta-galactosidase (beta-gal) in the myofibres at the site of injection was visualised by in situ histochemical staining using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. As measured by o-nitrophenyl-beta-D-galactopyranoside assay, beta-gal enzymatic activity was found to steadily increase for at least 50 days post injection (p.i.) in pCMV-LacZ-injected muscle. In parallel, foreign DNA was detected by polymerase chain reaction in injected muscles (but not in other tissues) up to 60 days p.i., persisting most probably in an extrachromosomal, non-replicative, circular form. Neither beta-gal activity nor pCMV-LacZ-related amplification products were found 90 days p.i. Antibodies against beta-gal were demonstrated in pCMV-LacZ-injected fish sampled 45 days p.i. The results suggest that intramuscular delivery of foreign genes represents a realistic approach for DNA vaccine technology for the prevention of infectious diseases in gilthead sea bream.

One component of the protective host response against mucosal pathogens includes the local production and increased expression of certain neuropeptides and their receptors. The present study further demonstrates this fact by investigating the contribution that substance P receptor expression makes toward immunity against a gamma-herpesvirus infection. Following intragastric inoculation with murine gamma-herpesvirus 68 (gamma HV-68), expression of substance P and its receptor was increased in mucosal and peripheral lymphoid organs in wild-type strains of mice. These results suggested that this receptor/ligand pair might be an important component of the host response against this viral infection. Such a hypothesis was supported by the demonstration that mice, genetically deficient in substance P receptor expression, showed an increased viral burden when compared with syngeneic C57BL/6 mice. Furthermore, substance P receptor-deficient mice showed a reduced CTL response against gamma HV-68, suggesting a mechanism to explain this increased viral burden. Such limitations in the Ag-specific CTL response in substance P receptor-deficient mice could result from lowered expression of IL-12 during viral infection. Consistent with this hypothesis, increases in mRNA encoding IL-12 and secretion of this cytokine into sera of infected, wild-type animals were markedly reduced in substance P receptor-deficient mice. These studies demonstrate that genetic elimination of substance P receptors in mice results in an increased gamma-herpesvirus burden and an altered host response.

Although some animal models suggest an involvement of CD4 T cells reactive to luminal microbial antigen(s) for the pathogenesis of inflammatory bowel diseases (IBD), direct linkage between microflora-driven clonal expansion of CD4 T cells and the development of colitis has not been well studied. Here, BALB/c and SCID mice were given CD4 T cells purified from Rag-2(-/-) mice crossed to transgenic mice expressing TCR specific to ovalbumin (OVA) then administered with antibiotic-resistant Escherichia coli producing OVA (ECOVA) or LacZ (ECLacZ) via the rectum. The ECOVA-inoculated BALB/c and SCID mice developed a subacute colitis with microscopic features of distortion of crypt architecture, loss of goblet cells, and focal infiltration by mononuclear cells in the lamina propria (LP) and submucosa. Expanding OVA-specific CD4 T cells were detected in colonic follicles of mice with ECOVA. Early in colitis, OVA-specific CD4 T cells producing IFN-gamma predominate in the LP of the colon, which was followed by an emergence of OVA-specific CD4 T cells producing IL-4 and IL-10 at a later time point. Co-transfer of an IL-10-secreting OVA-specific CD4 T cell line prevented colitis. Thus, an expansion of CD4 T cells monospecific to OVA, an antigen non-cross-reactive to colonic tissue, can mediate both induction and inhibition of the colitis which was associated with hyperplasia of lymph follicles.

Clinical success in tumor vaccination frequently does not reach expectation. Since vaccination protocols are quite variable, we used the murine renal cell carcinoma line RENCA transfected with the lacZ gene (RENCA-beta-gal) to compare the efficacy of two different vaccination strategies or their combination and to elaborate on the underlying mechanisms. BALB/c mice were vaccinated either with naked lacZ DNA or with attenuated Salmonella typhimurium transformed with lacZ DNA or with dendritic cells (DC) loaded with the beta-galactosidase protein or mice were vaccinated with both DNA and protein. Although all regimens led to a prolongation of survival time, oral vaccination with transfected S. typhimurium followed by i.v. transfer of protein-loaded DC provided the optimal schedule. In this setting, >50% of mice remained tumor free after challenge with 10 times the lethal tumor dose of RENCA-beta-gal. As explored in transfer experiments, the superior efficacy of combining DNA and protein vaccination is due to the facts that 1) optimal protection depends on both activated CD4(+) and CD8(+) cells and 2) CD8(+) CTL are most strongly activated by vaccination with transformed Salmonella, whereas vaccination with protein-loaded DC is superior for the activation of Th. The latter induced sustained activation of CTL and recruitment of nonadaptive defense mechanisms. The data demonstrate the strength of DNA vaccination, particularly by the oral route, and provide evidence that a combined treatment with protein-loaded DC can significantly increase the therapeutic efficacy.

Recombinant E1-deleted adenoviral vectors (DeltaE1-Ad) are promising tools for in vivo gene transfer into the mammalian CNS including the retina. However, the duration of transgene expression is limited, and this limitation has partly been attributed to an immune response directed against vector-derived proteins. Here, we employed immunocytochemistry to assess the immune response to intravitreously injected DeltaE1-Ad encoding the lacZ gene or various neurotrophins (NTs). beta-Galactosidase was expressed by retinal cells for up to 4 weeks. Following intravitreal inoculation of AdCMV-lacZ, microglial and T cells were detected with a panel of antibodies in the retinal cell layers after 2 days (D2). The inflammatory response reached a maximum between D7 and D14. In contrast, no immune response was seen following injection of Ad encoding NTs. Yet, like with Ad-CMV-lacZ, their expression was also limited to approximately 4 weeks. Thus, beta-galactosidase seems to trigger a host immune response following intravitreal adenoviral lacZ gene transfer, but immune responses are not the cause of limited NT transgene expression from the CMV promoter in the inner retina.

To optimize polynucleotide vaccinations for protective antitumor immunity we used a self-replicating RNA vaccine in which Semliki Forest virus replicase drives RNA expression of the lacZ gene coding for beta-galactosidase as model tumor-associated antigen (TAA). This was compared with replicase-deficient control RNA and with lacZ DNA plasmids with respect to gene expression in vitro and in vivo and for vaccination using the mouse ear pinna as an optimal immunization site. In vitro, the highest expression was observed with self-replicating RNA. Gene expression following pinna inoculation of either non-replicating DNA plasmids or self-replicating RNA was similar, lasting for 2-3 weeks. Higher antibody responses were obtained with RNA than with DNA. beta-Gal peptide specific CTL memory responses to lacZ DNA or RNA lasted for more than 6 weeks while respective responses induced by lacZ-transfected tumor cells lasted for only 2 weeks. To achieve a protective response against lacZ tumor cells with self-replicating RNA about a 100-fold lower dose of polynucleotide was sufficient in comparison to DNA. The extent of protective antitumor immunity not only depended on the gene dose used for vaccination, but also on the aggressiveness of the lacZ-transfected tumor line used for challenge. In comparison to lacZ-transfected tumor cells as vaccines, polynucleotide vaccination also demonstrated superiority with regard to cross-protection. Protective antitumor immunity could be strongly increased upon co-inoculation of lacZ DNA with IL-2 DNA or IL-12 RNA. IL-2 DNA, but not IL-12 RNA, also augmented the CTL response while IL-12 RNA, but not IL-2 DNA, reduced the antibody response. These results demonstrate efficient protective antitumor immunity after intra-pinna lacZ TAA polynucleotide vaccination and show additional immunomodulatory effects by co-administration of cytokine polynucleotides.

To enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors that contained replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, Semliki Forest virus. When transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating RNA vector (replicon) which in turn directs the expression of a model tumor antigen. Immunization with plasmid DNA replicons elicited immune responses at doses 100 to 1000-fold lower than conventional DNA plasmids and effectively treated mice bearing an experimental tumor expressing the model antigen. Significantly, replicon-based DNA plasmids did not produce a greater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA replicons mediated antigen production that was homogeneous in all transfected cells and associated with the apoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA replicons may be useful in the development of recombinant vaccines for infectious diseases and cancer.

Murine herpesvirus-68 (MHV-68) is a type 2 gamma herpesvirus that productively infects alveolar epithelial cells during the acute infection and establishes long-term latency in B cells and lung epithelial cells. In C57BL/6 mice, T cells specific for lytic cycle MHV-68 epitope p56/Db dominate the acute phase of the infection, whereas T cells specific for another lytic cycle epitope, p79/Kb, dominate later phases of infection. To further understand this response, we analyzed the kinetics of Ag presentation in vivo using a panel of lacZ-inducible T cell hybridomas specific for several lytic cycle epitopes, including p56/Db and p79/Kb. Two distinct peaks of Ag presentation were observed. The first peak was at day 6 in the mediastinal lymph nodes and correlated with the initial pulmonary lytic infection. The second peak was at day 18 in both the mediastinal lymph nodes and spleen and correlated with the peak of latent infection. Interestingly, the p56 epitope was detected only in the mediastinal lymph nodes at day 6 after infection whereas the p79 epitope was predominantly presented in the spleen at day 18, suggesting that differential epitope presentation drives the switch in T cell responses to this virus. Phenotypic analysis of APCs at day 18 postinfection revealed that dendritic cells, macrophages, and B cells all presented lytic cycle epitopes. Taken together, the data suggest that there is a resurgence of lytic cycle Ags in the spleen, which explains the kinetics and specificity of the T cell response to MHV-68.

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.

The origin of follicular dendritic cells (FDC) is unresolved, and as such, remains controversial. Based on the migration of Ag-transporting cells (ATC) into lymphoid follicles and the phenotypic similarity between FDC and ATC, one hypothesis is that ATC may represent emigrating FDC precursors. This contrasts with the view that FDC originate from local stromal cells in the secondary lymphoid tissues. Mice homozygous for the severe combined immunodeficiency (prkdc(scid)) mutation (scid) lack FDC. Thus, they provide a powerful tool for assessing de novo generation of FDC. To test whether FDC precursors could be found in bone marrow or fetal liver, scid/scid mice were reconstituted with either: 1) bone marrow cells from (BALB/c x C57BL/6)F1 donors, 2) bone marrow cells from ROSA BL/6 F1 (lacZ-transfected) mice, 3) rat bone marrow cells, or 4) rat fetal liver cells. Six to eight weeks after reconstitution with F1 bone marrow, cells reactive with the FDC-labeling mAb, FDC-M1, also expressed donor class I molecules on their surfaces. Similarly in mice reconstituted with lacZ-transfected bone marrow cells, these cells were also positive for the lacZ gene product. Furthermore, in spleens of animals reconstituted with either rat bone marrow or rat fetal liver, rat FDC were identified using the specifically labeling mAb, ED5. In all cases, host FDC were also present, indicating that scid/scid mice have FDC precursors that will mature in the presence of allogeneic or xenogeneic lymphoid cells. In summary, FDC can be derived from progenitor cells present in primary lymphoid tissues.

We have investigated the immune response to E1-deleted adenovirus vectors encoding the lacZ gene introduced into the brains of adult mice. Injection of these nonreplicating vectors caused a marked inflammatory response in the brain as assessed by immunocytochemistry and flow cytometry of leukocytes. Infiltrating leukocytes were detectable within 2 days of injection and reached a maximum by 9 days. Thereafter, the number of infiltrating cells decreased, but a small number persisted in the brain until day 60. Between 2 and 4 days after injection, the percentage of CD8+ cells detectable increased whereas the percentage of CD4+ cells present in the infiltrating population did not significantly increase until day 6, peaking on day 15. Activated CD25+ T cells were detectable between days 6 and 15. beta-Galactosidase (beta-Gal), the product of the lacZ gene encoded by the vector, was also detected, both at the injection site in the striatum and also in the substantia nigra. Expression peaked between 4 and 6 days but a small number of beta-Gal+ cells was still seen at 60 days after injection. This study demonstrates that a quantitative analysis of the immune responses caused by a nonreplicating adenovirus vector is possible in the brain. E1-deleted adenoviral vectors trigger a strong inflammatory response in the brain, but this immune response is not sufficient to eliminate completely expression of genes encoded by the adenoviral construct.

The major cause of upper respiratory tract disease in cats in the feline herpesvirus type 1 (FHV-1). FHV-1 replicates predominantly in the mucosal epithelium of the oral and nasal cavities, local immunity should therefore be the key target for vaccine development. Recombinant DNA technology enables accurate manipulation of the genetic content of the FHV-1 genome, hopefully resulting in a next generation of safe vaccine strains that can be used intranasally in cats. Integration of a reporter gene into the glycoprotein I (gI) homologous gene of FHV-1, resulted in strain C4-1-4-1 which displayed reduced replication not only in cell culture but also in the respiratory tract of infected cats. Oronasal application of strain C4-1-4-1 caused less severe clinical signs than local administration of the parent virus. In addition, oronasally vaccinated cats were better protected against the clinical signs of a challenge infection than cats vaccinated subcutaneously.

Retroviral vectors have been used in most human gene therapy trials that have been undertaken. Many of these therapies have focused on the introduction of genes into hematopoietic stem cells with the goal of obtaining expression in the mature T lymphocytic progeny. It has proven difficult to achieve expression in the lymphoid lineage, although several groups have demonstrated low expression of transduced genes in the myeloid lineage. In this study we used an in vitro thymic organ culture in which stem/progenitor cells can develop into T cells and all intermediate stages can be studied and manipulated to investigate the fate of a retrovirally introduced Escherichia coli LacZ gene in this system. Here we show that certain conditions can transduce Jurkat T cells, three different antigen-specific T cell clones and CD34+CD3-CD4-CD8- thymocytes (progenitor T cells) with high (> 80%) efficiency. Moreover, retroviral transduction with the LacZ gene does not inhibit T and NK cell differentiation of progenitor cells in fetal thymic organ cultures (FTOC). The LacZ gene also is functionally expressed at all stages of development, although the expression decreases somewhat during differentiation. This experimental system, combining FTOC and retroviral transduction, provides a genetic tool for the study of human T cell development.