Antigen-specific Recall Responses Research Articles

A bispecific antibody AP203 targeting PD-L1 and CD137 exerts potent antitumor activity without toxicity

BackgroundBispecific antibody has garnered considerable attention in the recent years due to its impressive preliminary efficacy in hematological malignancies. For solid tumors, however, the main hindrance is the suppressive tumor microenvironment, which effectively impedes the activation of infiltrating T cells. Herein, we designed a bispecific antibody AP203 with high binding affinity to PD-L1 and CD137 and assessed its safety and anti-tumor efficacy, as well as explored the mechanism of action.MethodsThe optimal antibody binders against PD-L1 and CD137 were screened from the OmniMab phagemid library. The binding affinity of the constructed AP203 were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-cell stimulatory capacity was assessed using the allogeneic mixed lymphocyte reaction (MLR), antigen-specific recall response, and coculture with PD-L1-expressing cells. In vivo antitumor efficacy was evaluated using two models of tumor-xenografted humanized mice with profiling of tumor infiltrating lymphocytes (TILs). The possible toxicity of AP203 was examined using in vitro cytokine release assay by human PBMCs.ResultsAP203, which simultaneously targeted PD-L1 and costimulatory CD137, elicit superior agonistic effects over parental antibodies alone or in combination in terms of T cell activation, enhanced memory recall responses, and overcoming Treg-mediated immunosuppression (P < 0.05). The agonistic activity of AP203 was further demonstrated PD-L1-dependent by coculturing T cells with PD-L1-expressing cells. In vivo animal studies using immunodeficient or immunocompetent mice both showed a dose-related antitumor efficacy superior to parental antibodies in combination (P < 0.05). Correspondingly, AP203 significantly increased tumor infiltrating CD8 + T cells, while decreased CD4 + T cells, as well as Treg cells (P < 0.05), resulting in a dose-dependent increase in the CD8 + /CD4 + ratio. Moreover, either soluble or immobilized AP203 did not induce the production of inflammatory cytokines by human PBMCs.ConclusionsAP203 exerts potent antitumor activity not only by blocking PD-1/PD-L1 inhibitory signaling, but also by activating CD137 costimulatory signaling in effector T cells that consequently counteracts Treg-mediated immunosuppression. Based on promising preclinical results, AP203 should be a suitable candidate for clinical treatment of solid tumors.

A 47-marker immune profiling flow cytometry assay to enable comprehensive antigen-specific immune analysis

Abstract The complex nature of the immune system requires deep interrogation at the single-cell level. Flow cytometry using CyTOF® technology employs antibodies tagged with isotopically enriched metals instead of fluorophores, resulting in discrete signals that facilitate highly multiparametric characterization on a single-cell level. CyTOF technology enables analysis of over 50 parameters simultaneously without the impediments of spectral overlap or autofluorescence, allowing for a reliable evaluation of immune responses. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, and validated 30-marker antibody cocktail for single-tube staining of human whole blood or PBMC and acquisition on CyTOF systems. Using the assay and Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations. In this study, we highlight the customizability of the Maxpar® Direct panel by adding an array of 17 surface and intracellular parameters with the Maxpar Direct™ T Cell Activation Expansion Panel and live-cell barcoding. The simultaneous detection of key surface and intracellular markers in this expanded panel enabled comprehensive analysis of immune cell activation and antigen-specific recall responses to PBMC stimulated with a pool of immunodominant microbial peptides. Taken together, this 47-marker panel and automated analysis unlocked deeper immunophenotyping and functional profiling of activated immune cells and provided insight into the cell-mediated adaptive immune response to foreign targets. Such phenomena are hallmarks for research on infection, vaccine development, and immunotherapy. For Research Use Only. Not for use in diagnostic procedures.

Duration of peripheral blood cellular immune responses induced in dogs in response to a modified live attenuated vaccine for Ehrlichia chaffeensis

Abstract Ehrlichia chaffeensis (ECH) is an obligate intracellular gram-negative bacterium and a frequent cause of severe and fatal tick-borne infection in people in North America. The canine is a common incidental host and a useful model for vaccine development and assessing immune responses to ECH infection. We recently established mutagenesis methods for ECH and demonstrated that immunization of dogs with an ECH mutant with a functional disruption in the ECH_0660 gene protected the vertebrate host from both tick-transmitted and intravenous wildtype ECH challenge when given 4 weeks after immunization. In the current study, we evaluated the duration of immunity offered by the ECH_0660 gene mutant live attenuated vaccine (MLAV) against wildtype infection challenge. Groups of 12 dogs were immunized with the ECH MLAV and then challenged 2, 4, 8 and 12 months later via tick-transmitted or blood-transmitted infection with wildtype ECH. Animals were monitored for 4 weeks after challenge and then sacrificed for evaluation of tissue pathology and bacterial burden. Immunization with the MLAV induced ECH-specific T cell responses, detectable by ELISPOT for IFNγ; and differentiation of IFNγ- and/or TNFα-producing ECH-specific T effector memory CD4 T cells (CD45RAneg CD45RO+ CD62Lneg). Antigen-specific recall responses were detectable in the peripheral blood of most (27/32) vaccinated dogs for 4 months after immunization, but only in 5/24 of the dogs after 8 months. This result suggests that a single MLAV vaccination induces cellular immune responses and differentiation of memory helper T cells. Future work will investigate the correlations between cellular immunity, humoral responses and bacterial load. Supported by grants from NIH (R01 AI152418)

A discrete subset of epigenetically primed human NK cells mediates antigen-specific immune responses.

Adaptive features of natural killer (NK) cells have been reported in various species with different underlying mechanisms. It is unclear, however, which NK cell populations are capable of mounting antigen-specific recall responses and how such functions are regulated at the molecular level. Here, we identify and characterize a discrete population of CD49a+CD16- NK cells in the human liver that displays increased epigenetic potential to elicit memory responses and has the functional properties to exert antigen-specific immunity in the skin as an effector site. Integrated chromatin-based epigenetic and transcriptomic profiling revealed unique characteristics of hepatic CD49a+CD16- NK cells when compared with conventional CD49a-CD16+ NK cells, thereby defining active genomic regions and molecules underpinning distinct NK cell reactivity. In contrast to conventional NK cells, our results suggest that adaptive CD49a+CD16- NK cells are able to bypass the KIR receptor-ligand system upon antigen-specific stimulation. Furthermore, these cells were highly migratory toward chemokine gradients expressed in epicutaneous patch test lesions as an effector site of adaptive immune responses in the skin. These results define pathways operative in human antigen-specific adaptive NK cells and provide a roadmap for harnessing this NK cell subset for specific therapeutic or prophylactic vaccine strategies.

Defining immune correlates during latent and active chlamydial infection in sheep

Ovine enzootic abortion (OEA) caused by the obligate intracellular bacterial pathogen Chlamydia abortus (C. abortus), is an endemic disease in most sheep-rearing countries worldwide. Following infection, C. abortus establishes a complex host–pathogen interaction with a latent phase in non-pregnant sheep followed by an active disease phase in the placenta during pregnancy leading to OEA. Improved knowledge of the host–pathogen interactions at these different phases of disease will accelerate the development of new diagnostic tests and vaccines to control OEA. Current evidence indicates that cellular immunity is essential for controlling C. abortus infection. We have previously described a model of mucosal (intranasal) infection of non-pregnant sheep with C. abortus that replicates the latent and active phases of OEA. We have investigated antigen-specific recall responses of peripheral blood mononuclear cells (PBMC) in sheep infected with C. abortus via the intranasal route to determine how these change during the latent and active phases of disease. By analysing cytokines associated with the major CD4+ve Thelper (Th) cell subsets (Interferon-gamma (IFN-γ)/Th1; Interleukin (IL)-4/Th2; IL-17A/Th17; IL-10/Tregulatory), we show that there is selective activation of PBMC producing IFN-γ and/or IL-10 during the latent phase following infection. These cytokines are also elevated during the active disease phase and while they are produced by sheep that are protected from OEA, they are also produced by sheep that abort, highlighting the difficulties in finding specific cellular immunological correlates of protection for complex intracellular pathogens.

Proof of long-term immunological memory in cartilaginous fishes

Immunological memory provides long-term protection against pathogen re-infection and is the foundation for successful vaccination. We have previously shown an antigen-specific recall response in nurse sharks almost one year after primary exposure. Herein, we extend the time between prime and successful recall to >8 years, the longest period for which immunological memory has been shown in any non-mammalian vertebrate. We confirm that antigen binding is mediated by monomeric IgM and IgNAR, but not pentameric IgM, in both the primary and recall phases. Our inability to find target-binding clones in recombinant VNAR expression libraries suggests that, at least in this instance, antigen-specific memory cells comprise a small fraction of the IgNAR-positive B cells in epigonal and spleen. Further, that the few memory cells present can generate a robust antigen-specific IgNAR titer following re-stimulation. Our results continue to challenge the long-held, but erroneous, belief that the shark adaptive immune system is ‘primitive’ when compared to that of mammals.

Microphysiologic Human Tissue Constructs Reproduce Autologous Age-Specific BCG and HBV Primary Immunization in vitro.

Current vaccine development disregards human immune ontogeny, relying on animal models to select vaccine candidates targeting human infants, who are at greatest risk of infection worldwide, and receive the largest number of vaccines. To help accelerate and de-risk development of early-life effective immunization, we engineered a human age-specific microphysiologic vascular-interstitial interphase, suitable for pre-clinical modeling of distinct age-targeted immunity in vitro. Our Tissue Constructs (TCs) enable autonomous extravasation of monocytes that undergo rapid self-directed differentiation into migratory Dendritic Cells (DCs) in response to adjuvants and licensed vaccines such as Bacille Calmette-Guérin (BCG) or Hepatitis B virus Vaccine (HBV). TCs contain a confluent human endothelium grown atop a tri-dimensional human extracellular matrix substrate, employ human age-specific monocytes and autologous non heat-treated plasma, and avoid the use of xenogenic materials and exogenous cytokines. Vaccine-pulsed TCs autonomously generated DCs that induced single-antigen recall responses from autologous naïve and memory CD4+ T lymphocytes, matching study participant immune-status, including BCG responses paralleling donor PPD status, BCG-induced adenosine deaminase (ADA) activity paralleling infant cohorts in vivo, and multi-dose HBV antigen-specific responses as demonstrated by lymphoproliferation and TCR sequencing. Overall, our microphysiologic culture method reproduced age- and antigen-specific recall responses to BCG and HBV immunization, closely resembling those observed after a birth immunization of human cohorts in vivo, offering for the first time a new approach to early pre-clinical selection of effective age-targeted vaccine candidates.

Chemokines: Critical Regulators of Memory T Cell Development, Maintenance, and Function.

Memory T cells are central to orchestrating antigen-specific recall responses in vivo. Compared to naïve T cells, memory T cells respond more quickly to cognate peptide:MHC with a shorter lag time for entering the cell cycle and exerting effector functions. However, it is now well established that this enhanced responsiveness is not the only mechanism whereby memory T cells are better equipped than naïve T cells to rapidly and robustly induce inflammation. In contrast to naïve T cells, memory T cells are composed of distinct subsets with unique trafficking patterns and localizations. Tissue-resident memory T cells persist in previously inflamed tissue and function as first responders to cognate antigen reexposure. In addition, a heterogeneous group of circulating memory T cells augment inflammation by either rapidly migrating to inflamed tissue or responding to cognate antigen within secondary lymphoid organs and producing additional effector T cells. Defining the mechanisms regulating T cell positioning and trafficking and how this influences the development, maintenance, and function of memory T cell subsets is essential to improving vaccine design as well as treatment of immune-mediated diseases. In this chapter, we will review our current knowledge of how chemokines, critical regulators of cell positioning and migration, govern memory T cell biology in vivo. In addition, we discuss areas of uncertainty and future directions for further delineating how T cell localization influences memory T cell biology.

Targeted Programming of the Lymph Node Environment Causes Evolution of Local and Systemic Immunity.

Biomaterial vaccines offer cargo protection, targeting, and co-delivery of signals to immune organs such as lymph nodes (LNs), tissues that coordinate adaptive immunity. Understanding how individual vaccine components impact immune response has been difficult owing to the systemic nature of delivery. Direct intra-lymph node (i.LN.) injection offers a unique opportunity to dissect how the doses, kinetics, and combinations of signals reaching LNs influence the LN environment. Here, i.LN. injection was used as a tool to study the local and systemic responses to vaccines comprised of soluble antigen and degradable polymer particles encapsulating toll-like receptor agonists as adjuvants. Microparticle vaccines increased antigen presenting cells and lymphocytes in LNs, enhancing activation of these cells. Enumeration of antigen-specific CD8+ T cells in blood revealed expansion over 7 days, followed by a contraction period over 1 month as memory developed. Extending this strategy to conserved mouse and human tumor antigens resulted in tumor antigen-specific primary and recall responses by CD8+ T cells. During challenge with an aggressive metastatic melanoma model, i.LN. delivery of depots slowed tumor growth more than a potent human vaccine adjuvant, demonstrating local treatment of a target immunological site can promote responses that are potent, systemic, and antigen-specific.

Antigenic peptide nanofibers elicit adjuvant-free CD8+ T cell responses

Vaccines that elicit robust CD8+ T cell responses are desirable for protection against infectious diseases and cancers. However, most vaccine adjuvants fail to elicit robust CD8+ T cell responses without inflammation and associated toxicity. We recently reported that self-assembling peptides that form nanofibers in physiological buffers elicited strong adjuvant-free and antigen-specific antibody responses in mice. However, whether or not such nanofibers likewise can elicit strong CD8+ T cell responses is unknown. Here, we demonstrate that the self-assembling peptide Q11 conjugated to a CD8+ T cell epitope of ovalbumin (Q11-OVA), elicits strong antigen-specific primary and recall responses, and in a vaccination regimen protects against subsequent infection. Importantly, we show that these antigenic peptide nanofibers do not persist as an inflammatory antigen depot at the injection site. Our results demonstrate for the first time that self-assembling peptides may be useful as carriers for vaccines where CD8+ T cell-mediated protection is needed.

Functional Testing of an Inhalable Nanoparticle Based Influenza Vaccine Using a Human Precision Cut Lung Slice Technique

Annual outbreaks of influenza infections, caused by new influenza virus subtypes and high incidences of zoonosis, make seasonal influenza one of the most unpredictable and serious health threats worldwide. Currently available vaccines, though the main prevention strategy, can neither efficiently be adapted to new circulating virus subtypes nor provide high amounts to meet the global demand fast enough. New influenza vaccines quickly adapted to current virus strains are needed. In the present study we investigated the local toxicity and capacity of a new inhalable influenza vaccine to induce an antigen-specific recall response at the site of virus entry in human precision-cut lung slices (PCLS). This new vaccine combines recombinant H1N1 influenza hemagglutinin (HAC1), produced in tobacco plants, and a silica nanoparticle (NP)-based drug delivery system. We found no local cellular toxicity of the vaccine within applicable concentrations. However higher concentrations of NP (≥103 µg/ml) dose-dependently decreased viability of human PCLS. Furthermore NP, not the protein, provoked a dose-dependent induction of TNF-α and IL-1β, indicating adjuvant properties of silica. In contrast, we found an antigen-specific induction of the T cell proliferation and differentiation cytokine, IL-2, compared to baseline level (152±49 pg/mg vs. 22±5 pg/mg), which could not be seen for the NP alone. Additionally, treatment with 10 µg/ml HAC1 caused a 6-times higher secretion of IFN-γ compared to baseline (602±307 pg/mg vs. 97±51 pg/mg). This antigen-induced IFN-γ secretion was further boosted by the adjuvant effect of silica NP for the formulated vaccine to a 12-fold increase (97±51 pg/mg vs. 1226±535 pg/mg). Thus we were able to show that the plant-produced vaccine induced an adequate innate immune response and re-activated an established antigen-specific T cell response within a non-toxic range in human PCLS at the site of virus entry.

CD4+TH2, not TH17 cells mount antigen specific recall response and drive allergic inflammation (P6029)

Abstract Allergic asthma is a heterogeneous inflammatory disease of the airway. In addition to CD4+ TH2 and TH17 cells of the adaptive immunity, recently described innate lymphoid cells (ILC2) also contribute to the pathogenesis of allergic asthma. However, it remains unclear that which cell type is responsible for mounting antigen-driven immune response at the recall phase of allergic inflammation. Repeated intranasal OVA re-challenges induced a significant expansion of TH2, not TH17 or ILC2 cells in rested mice that were previously sensitized with OVA plus papain. Consequently, the amount of IL-5 and IL-13, not IL-17 or IFN-γ production in the lung of mice re-exposed to OVA antigen were significantly elevated than those of mice sensitized only. Mice ablated of CD4+ T cells failed to respond to repeated OVA antigen re-challenges and exhibited ameliorated allergic airway inflammation, while their lung ILC2 remained intact. Furthermore, STAT6-deficient mice that lacked TH2 cells also failed to mount allergic recall response to OVA antigen, despite of their competency in eliciting TH17 immune response. Collectively, our results suggest that lung resident antigen specific CD4+TH2 cells, not TH17 or ILC2 cells, induced after sensitization are the primary cell type responsible for mounting allergen recall response that exacerbates allergic inflammation.

The Orchestrated Functions of Innate Leukocytes and T Cell Subsets Contribute to Humoral Immunity, Virus Control, and Recovery from Secondary Poxvirus Challenge

A pivotal role for antigen-specific recall responses to secondary virus infection is well established, but the contribution of innate immune cells to this process is unknown. Recovery of mice from a primary orthopoxvirus (ectromelia virus [ECTV]) infection requires the function of natural killer (NK) cells, granulocytes, plasmacytoid dendritic cells (pDC), T cells, and B cells. However, during a secondary challenge, resolution of infection is thought to be dependent on antibody but not T cell function. We investigated the contribution of NK cells, granulocytes, and pDC to virus control during a secondary virus challenge in mice that had been primed with an avirulent, mutant strain of ECTV. Mice depleted of NK cells, granulocytes, or pDC effectively controlled virus, as did mice depleted of both CD4 and CD8 T cell subsets. However, mice concurrently depleted of all three innate cell subsets had elevated virus load, but this was significantly exacerbated in mice also depleted of CD4 and/or CD8 T cells. Increased viral replication in mice lacking innate cells plus CD4 T cells was associated with a significant reduction in neutralizing antibody. Importantly, in addition to T-dependent neutralizing antibody responses, the function of CD8 T cells was also clearly important for virus control. The data indicate that in the absence of innate cell subsets, a critical role for both CD4 and CD8 T cells becomes apparent and, conversely, in the absence of T cell subsets, innate immune cells help contain infection.

P096 IFN-A protects against antigen induced arthritis through induction of antigen specific cellular tolerance

Type I interferons are pleiotropic cytokines having an important regulatory role in autoimmune diseases including rheumatoid arthritis. Our earlier data show that IFN α protects against mBSA induced arthritis (Type I IFN protects against arthritis. Eur J Immunol June 2011;41(6):1687–95) . This IFN α mediated protection was manifested by inhibition of antigen specific proliferation but antigen specific total IgG levels remained largely unaffected. To further define the altered cellular response associated with IFNa-mediated protection against arthritis, the circulating cytokines as well as cytokines produced upon antigen restimulation of leukocytes ex vivo during the development of arthritis (day 14, day 21 and on the day of termination, day 28) was determined. We also assessed the effect of IFN α on antigen-specific IgG isotypes, which may differ depending on the dominant T helper cell response. Arthritis was induced by two injections (day 1 and day 7) of mBSA and adjuvant, with or without 100–5000 U of IFNa. Arthritis was triggered by intra-articular injection of mBSA on day 21 and arthritis was scored on day 28. Presence of IFNaat the time of antigen-sensitization protected mice from arthritis in a dose-dependent manner. No significant differences were found on antigen specific serum IgG1, IgG2a and IgG2b levels regardless of IFN α treatment. The circulating cytokine analysis as measured by Luminex showed that IFN- α decreases the production of cytokines IL-6 and IL-13 in the initial phase (day 14) of arthritis induction. Furthermore, this inhibitory effect of IFNawas also apparent in antigen-specific cytokine release ex-vivo at day 14. In the arthritis phase (day 28), IFN α significantly reduced the levels of circulating, pro-inflammatory cytokines (IL-1 β , IL-17, TNF- α , IFN γ , and IL-12), which were clearly up-regulated in the control group. However, antigen-specific release of cytokines (IFN γ , IL-6, IL-17 and IL-10) after re-stimulation ex-vivo at the day of termination (day 28) showed that splenocytes from IFNatreated mice responded with higher cytokine levels than controls. Conclusion: The ability of IFNato prevent arthritis development is most likely due to its effects on cellular rather than humoral immunity. Presence of IFNaat the time of antigen sensitization (day 1 and day 7) inhibits both in vivo and ex vivo release of pro-inflammatory cytokines throughout the arthritis induction. This results in clearly lower serum concentrations of pro-inflammatory cytokines after intra-articular challenge (day 28) and likely explains the protective effect of IFNa. The inhibition of cytokine release at the time of arthritis manifestation in the IFN α treated group is in line with our earlier published data of inhibited antigen specific re-call responses upon rechallange with mBSA. The increase in ex-vivo release of cytokines from splenocytes at this time point in the IFNa-treated group may indicate that the inhibitory effect on cytokine release by IFNaadministrated on day 1 and day 7 has a time limit.

Foxp4 Is Dispensable for T Cell Development, but Required for Robust Recall Responses

Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4+ and CD8+ T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3+ T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.

Foxp4 is dispensable for T cell development, but required for Interferon gamma recall responses during chronic Toxoplasmosis (163.12)

Abstract Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are fundamental for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, another member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4+ and CD8+ T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore Foxp3+ T regulatory cells develop normally in Foxp4 deficient animals, and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii infection, IFNγ production during an antigen specific recall response is reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for T cell cytokine recall responses to antigen in chronically infected mice.

Complement regulatory protein Crry deficiency contributes to the antigen specific recall response in experimental autoimmune myasthenia gravis

BackgroundMyasthenia gravis (MG) and animal model of experimental autoimmune myasthenia gravis (EAMG) is the most common autoimmune disorder of neuromuscular transmission. The disease is caused by the breakdown of the acetylcholine receptor (AChR) which is largely due to complement activation at the neuromuscular junction (NMJ). Limited knowledge exists to the extent that complement receptor 1-related gene/protein y deficiency (Crry −/−) modulates the adaptive immune response and EAMG outcome.MethodsMouse EAMG was induced by s.c. administrations of purified acetylcholine receptor (AChR) to Crry −/− and age- matched WT (C57BL/6) mice. Disease severity was assessed by clinical score assessment and muscle grip strength measurements. Serum complement activity was determined by hemolytic assay. ELISA was used to detect the level of AChR specific antibodies. Splenic cells were analyzed for T and B cells subsets distribution, release of cytokines and AChR specific recall responses. Deposition of complement components at the NMJ was assessed by immunofluorescence staining.ResultsIn comparison to WT EAMG, Crry −/− EAMG mice showed signs of augmented muscle weakness but differences, except for one time point, were not statistically significant. Serum complement activity was reduced in Crry −/− EAMG mice and no substantial changes in deposition of C3, C3b/iC3b and C5b-9 (MAC) at the NMJ between WT EAMG and Crry −/− EAMG mice were detected. Lack of Crry affected adaptive immune response. Crry −/− EAMG mice showed increases in the number of AChR specific splenic T-cells secreting IFN-γ and IL-4. Production of complement fixing antibodies (IgG2b, IgG2c) was also augmented. More Th1, Th2 and Th17 cytokines were released into the bloodstream of Crry −/− EAMG mice.ConclusionsData suggest that Crry deficiency modulates the adaptive immune response in EAMG, but its effect on disease outcome is limited. This was due to the generally lower serum complement level caused by increased C3 turnover. Modulation of complement activity with soluble or membrane bound regulators of complement activity represents a potentially effective approach to modify autoimmune processes in MG and EAMG.

B7 Costimulation Molecules Encoded by Replication-Defective, vhs-Deficient HSV-1 Improve Vaccine-Induced Protection against Corneal Disease

Herpes simplex virus 1 (HSV-1) causes herpes stromal keratitis (HSK), a sight-threatening disease of the cornea for which no vaccine exists. A replication-defective, HSV-1 prototype vaccine bearing deletions in the genes encoding ICP8 and the virion host shutoff (vhs) protein reduces HSV-1 replication and disease in a mouse model of HSK. Here we demonstrate that combining deletion of ICP8 and vhs with virus-based expression of B7 costimulation molecules created a vaccine strain that enhanced T cell responses to HSV-1 compared with the ICP8−vhs− parental strain, and reduced the incidence of keratitis and acute infection of the nervous system after corneal challenge. Post-challenge T cell infiltration of the trigeminal ganglia and antigen-specific recall responses in local lymph nodes correlated with protection. Thus, B7 costimulation molecules expressed from the genome of a replication-defective, ICP8−vhs− virus enhance vaccine efficacy by further reducing HSK.

Role of Interleukin-23-Dependent Antifungal Immune Responses in Dendritic Cell-Vaccinated Mice

CD40 ligand (CD40L) transduction of antigen-pulsed dendritic cells (DCs) can result in antigen-specific humoral immune responses even in CD4(+) T-cell-depleted settings. Here, we show that CD40L transduction of DCs results in the induction of interleukin-12p40 (IL-12p40), IL-12p70, and IL-23. Using DCs that were deficient in IL-12p40, IL-12p35, or IL-23p19, we show that these molecules are dispensable for primary IgG1 responses to Pneumocystis, but IgG2c was dependent on IL-12p40 and IL-23p19 but not IL-12p35. Antigen-specific recall responses in CD4-deficient mice were critically dependent on IL-12p40 and IL-23p19 expression in DCs and were not affected by the lack of IL-12p35. To confirm that this defect in recall was due to IL-23, transduction of IL-12p40(-/-) DCs with a recombinant adenovirus expressing functional IL-23 restored recall responses in DC-vaccinated CD4-deficient mice. These data show that DC-produced IL-23 is critical for vaccine-induced antigen-specific IgG2c and recall antibody responses in the setting of CD4(+) T-cell depletion.

Adjuvant properties of a biocompatible thermo-responsive polymer of N -isopropylacrylamide in autoimmunity and arthritis

To evaluate the thermo-responsive poly(N-isopropylacrylamide) (PNiPAAm) polymer as an adjuvant, we synthesized PNiPAAm through free radical polymerization and characterized it both in vitro and in vivo. The polymer when mixed with collagen type II (CII) induced antigen-specific autoimmunity and arthritis. Mice immunized with PNiPAAm-CII developed significant levels of CII-specific IgG response comprising major IgG subclasses. Antigen-specific cellular recall response was also enhanced in these mice, while negligible level of IFN-γ was detected in splenocyte cultures, in vitro. PNiPAAm-CII-immunized arthritic mouse paws showed massive infiltration of immune cells and extensive damage to cartilage and bone. As determined by immunostaining, most of the CII protein retained its native configuration after injecting it with PNiPAAm in naive mice. Physical adsorption of CII and the high-molecular-weight form of moderately hydrophobic PNiPAAm induced a significant anti-CII antibody response. Similar to CII, mice immunized with PNiPAAm and ovalbumin (PNiPAAm-Ova) induced significant anti-ovalbumin antibody response. Comparable levels of serum IFN-γ, IL-1β and IL-17 were observed in ovalbumin-immunized mice with complete Freund, incomplete Freund (CFA and IFA) or PNiPAAm adjuvants. However, serum IL-4 levels were significantly higher in PNiPAAm-Ova and CFA-Ova groups compared with the IFA-Ova group. Thus, we show for the first time, biocompatible and biodegradable thermo-responsive PNiPAAm can be used as an adjuvant in several immunological applications as well as in better understanding of the autoimmune responses against self-proteins.