Specific in situ immuno-imaging of pulmonary-resident memory lymphocytes in human lungs.

Introduction: Pulmonary-resident memory T cells (TRM) and B cells (BRM) orchestrate protective immunity to reinfection with respiratory pathogens. Developing methods for the in situ detection of these populations would benefit both research and clinical settings. Methods: To address this need, we developed a novel in situ immunolabelling approach combined with clinic-ready fibre-based optical endomicroscopy (OEM) to detect canonical markers of lymphocyte tissue residency in situ in human lungs undergoing ex vivo lung ventilation (EVLV). Results: Initially, cells from human lung digests (confirmed to contain TRM/BRM populations using flow cytometry) were stained with CD69 and CD103/CD20 fluorescent antibodies and imaged in vitro using KronoScan, demonstrating it's ability to detect antibody labelled cells. We next instilled these pre-labelled cells into human lungs undergoing EVLV and confirmed they could still be visualised using both fluorescence intensity and lifetime imaging against background lung architecture. Finally, we instilled fluorescent CD69 and CD103/CD20 antibodies directly into the lung and were able to detect TRM/BRM following in situ labelling within seconds of direct intra-alveolar delivery of microdoses of fluorescently labelled antibodies. Discussion: In situ, no wash, immunolabelling with intra-alveolar OEM imaging is a novel methodology with the potential to expand the experimental utility of EVLV and pre-clinical models.

Pulmonary-Resident Memory Lymphocytes: Pivotal Orchestrators of Local Immunity Against Respiratory Infections.

There is increasing evidence that lung-resident memory T and B cells play a critical role in protecting against respiratory reinfection. With a unique transcriptional and phenotypic profile, resident memory lymphocytes are maintained in a quiescent state, constantly surveying the lung for microbial intruders. Upon reactivation with cognate antigen, these cells provide rapid effector function to enhance immunity and prevent infection. Immunization strategies designed to induce their formation, alongside novel techniques enabling their detection, have the potential to accelerate and transform vaccine development. Despite most data originating from murine studies, this review will discuss recent insights into the generation, maintenance and characterisation of pulmonary resident memory lymphocytes in the context of respiratory infection and vaccination using recent findings from human and non-human primate studies.

A Novel Outbred Mouse Model to Study Lung Memory Immunological Response Induced by Pertussis Vaccines.

Lung tissue resident memory (TRM) T cells can provide rapid and effective protective immunity against respiratory pathogens such as Bordetella pertussis We assessed an outbred CD1 mouse model and i.m. immunization to study vaccine-induced immune memory, using pertussis vaccines as an example. The phenotypes of cells from the lungs of CD1 mice that had been primed with either i.m. whole-cell B. pertussis (wP), acellular B. pertussis (aP) vaccines or buffer (unvaccinated) and challenged with B. pertussis were determined using flow cytometry and immunohistology. We observed a rapid and high increase of CD4+T cells expressing TRM markers by flow cytometry, supported by immunohistology observations, in lungs from wP-immunized mice. Priming mice with wP vaccine induced a more potent CD4+ response in lungs following B. pertussis challenge than priming with aP vaccine, although both were less potent than that observed in primoinfected mice. We also observed for the first time, to our knowledge, that CD8+ and γδ+ TRM-like T cell responses were induced in lungs of wP-primed mice postinfection. This novel outbred CD1 mouse model with i.m. immunization that enabled us to study vaccine-induced B. pertussis-specific memory T cells in lungs could be useful for evaluating candidate parenteral vaccines against B. pertussis or others pulmonary pathogens.

A single immunogenicity assay for testing potency of combination DTaP vaccines: Simultaneous quantitation of anti-DT, anti-TT, anti-PTxd and anti-FHA antibodies in Guinea-pig serum with a Luminex®-xMAP® bead-based serological assay.

The diphtheria toxoid (DT), tetanus toxoid (TT), and acellular pertussis (aP) single immunogenicity assay (DTaP SIA) is a Luminex®-xMAP®-bead-based multiplex immunoassay for estimating the potency of DTaP pediatric combination vaccines in guinea pigs. This manuscript describes the validation of this assay for the simultaneous quantitation of anti-diphtheria toxoid (anti-DT), anti-tetanus toxoid (anti-TT), anti-pertussis toxoid (anti-PTxd), and anti-filamentous hemagglutinin (anti-FHA) antibodies in guinea pig serum following injection of a DTaP vaccine formulation. The results were expressed in arbitrary units/mL (AU/mL) using reference serum for comparison. Specificity was demonstrated by ≥ 75% homologous and ≤25% heterologous inhibition for all the antigens. The results were linear for anti-DT, anti-TT, anti-PTxd and anti-FHA antibodies. Accuracy was demonstrated with recovery of between 80% and 120% for all four antibodies. The relative standard deviation of repeatability was ≤20%. The results demonstrate that this SIA can be used for the linear, accurate, and precise simultaneous detection of all four antibodies, based on both the ICH Q2 and the EMA guidelines on bioanalytical method validation.

Reactivating Immunity Primed by Acellular Pertussis Vaccines in the Absence of Circulating Antibodies: Enhanced Bacterial Control by TLR9 Rather Than TLR4 Agonist-Including Formulation.

Pertussis is still observed in many countries despite of high vaccine coverage. Acellular pertussis (aP) vaccination is widely implemented in many countries as primary series in infants and as boosters in school-entry/adolescents/adults (including pregnant women in some). One novel strategy to improve the reactivation of aP-vaccine primed immunity could be to include genetically- detoxified pertussis toxin and novel adjuvants in aP vaccine boosters. Their preclinical evaluation is not straightforward, as it requires mimicking the human situation where T and B memory cells may persist longer than vaccine-induced circulating antibodies. Toward this objective, we developed a novel murine model including two consecutive adoptive transfers of the memory cells induced by priming and boosting, respectively. Using this model, we assessed the capacity of three novel aP vaccine candidates including genetically-detoxified pertussis toxin, pertactin, filamentous hemagglutinin, and fimbriae adsorbed to aluminum hydroxide, supplemented-or not-with Toll-Like-Receptor 4 or 9 agonists (TLR4A, TLR9A), to reactivate aP vaccine-induced immune memory and protection, reflected by bacterial clearance. In the conventional murine immunization model, TLR4A- and TLR9A-containing aP formulations induced similar aP-specific IgG antibody responses and protection against bacterial lung colonization as current aP vaccines, despite IL-5 down-modulation by both TLR4A and TLR9A and IL-17 up-modulation by TLR4A. In the absence of serum antibodies at time of boosting or exposure, TLR4A- and TLR9A-containing formulations both enhanced vaccine antibody recall compared to current aP formulations. Unexpectedly, however, protection was only increased by the TLR9A-containing vaccine, through both earlier bacterial control and accelerated clearance. This suggests that TLR9A-containing aP vaccines may better reactivate aP vaccine-primed pertussis memory and enhance protection than current or TLR4A-adjuvanted aP vaccines.

G-protein based ELISA as a potency test for rabies vaccines.

The NIH test is currently used to assess the potency of rabies vaccine, a key criterion for vaccine release. This test is based on mice immunization followed by intracerebral viral challenge. As part of global efforts to reduce animal experimentation and in the framework of the development of Sanofi Pasteur next generation, highly-purified vaccine, produced without any material of human or animal origin, we developed an ELISA as an alternative to the NIH test. This ELISA is based on monoclonal antibodies recognizing specifically the native form of the viral G-protein, the major antigen that induces neutralizing antibody response to rabies virus. We show here that our ELISA is able to distinguish between potent and different types of sub-potent vaccine lots. Satisfactory agreement was observed between the ELISA and the NIH test in the determination of the vaccine titer and their capacity to discern conform from non-conform batches. Our ELISA meets the criteria for a stability-indicating assay and has been successfully used to develop the new generation of rabies vaccine candidates. After an EPAA international pre-collaborative study, this ELISA was selected as the assay of choice for the EDQM collaborative study aimed at replacing the rabies vaccine NIH in vivo potency test.

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis.

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.

Expression and activation of mitogen-activated protein kinase kinases-3 and -6 in rheumatoid arthritis.

The p38 mitogen-activated protein (MAP) kinase signal transduction pathway regulates the production of interleukin-1 and tumor necrosis factor-alpha. p38 kinase inhibitors are effective in animal models of arthritis and are currently being developed in rheumatoid arthritis (RA). However, little is known about the upstream kinases that control the activation of p38 in RA synovium. In vitro studies previously identified the MAP kinase kinases (MAPKKs) MKK3 and MKK6 as the primary regulators of p38 phosphorylation and activation. To investigate a potential role for MKK3 and MKK6 in RA, we evaluated their expression and regulation in RA synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that MKK3 and MKK6 are expressed in RA and osteoarthritis (OA) synovium. Digital image analysis showed no significant differences between OA and RA with regard to expression or distribution. However, phosphorylated MKK3/6 expression was significantly higher in RA synovium and was localized to the sublining mononuclear cells and the intimal lining. Actin-normalized Western blot analysis of synovial tissue lysates confirmed the increased expression of phosphorylated MKK3/6 in RA. Western blot analysis demonstrated constitutive expression of MKK3 and MKK6 in RA and OA FLS. Phospho-MKK3 levels were low in medium-treated FLS, but were rapidly increased by interleukin-1 and tumor necrosis factor-alpha, although phospho-MKK6 levels only modestly increased. p38 co-immunoprecipitated with MKK3 and MKK6 from cytokine-stimulated FLS and the complex phosphorylated activating transcription factor-2 in an in vitro kinase assay. These data are the first documentation of MKK3 and MKK6 activation in human inflammatory disease. By forming a complex with p38 in synovial tissue and FLS, these kinases can potentially be targeted to regulate the production of proinflammatory cytokine production in inflamed synovium.

Expression of the MAPK kinases MKK-4 and MKK-7 in rheumatoid arthritis and their role as key regulators of JNK.

OBJECTIVE: The mitogen-activated protein (MAP) kinase JNK is a key regulator of interleukin-1 (IL-1)-induced collagenase gene expression and joint destruction in arthritis. Two upstream kinases, MKK-4 and MKK-7, have been identified as potential activators of JNK. However, the role of MAP kinase kinases (MAPKKs) and their functional organization within fibroblast-like synoviocytes (FLS) have not been defined. We therefore evaluated the interactions between the various MAP kinase components and determined their subcellular localization. METHODS: MKKs were identified by immunohistochemistry of rheumatoid arthritis (RA) and osteoarthritis (OA) synovium. Western blotting was used to determine the expression of FLS. Immunoprecipitation experiments using antibodies specific for MKK-4, MKK-7, and JNK were performed. Phosphospecific antibodies and immunohistochemistry were used to evaluate the activation state of synovial MKK-4 and MKK-7. Confocal microscopy was used to determine the subcellular location of the kinases. RESULTS: Immunohistochemistry studies demonstrated abundant MKK-4 and MKK-7 in RA and OA synovium, but the levels of phosphorylated kinases were significantly higher in RA synovium. MKK-4 and MKK-7 were constitutively expressed by cultured RA and OA FLS, and IL-1 stimulation resulted in rapid phosphorylation of both kinases. JNK was detected in MKK-4 and MKK-7 immunoprecipitates. Furthermore, MKK-4 coprecipitated with MKK-7 and vice versa, indicating that the 3 kinases form a stable complex in FLS. Confocal microscopy confirmed that JNK, MKK-4, and MKK-7 colocalized in the cytoplasm, with JNK migrating to the nucleus after IL-1 stimulation. The signal complex containing MKK-4, MKK-7, and JNK was functionally active and able to phosphorylate c-Jun after IL-1 stimulation of FLS. CONCLUSION: These studies demonstrate that JNK, MKK-4, and MKK-7 form an active signaling complex in FLS. This novel JNK signalsome is activated in response to IL-1 and migrates to the nucleus. The JNK signalsome represents a new target for therapeutic interventions designed to prevent joint destruction.