Search

Vaccine development for Plasmodium vivax, an important human relapsing malaria, is lagging behind. In the case of the most deadly human malaria P. falciparum, unprecedented high levels of protection have been obtained by immunization with live sporozoites under accompanying chemoprophylaxis, which prevents the onset of blood-stage malaria. Such an approach has not been fully evaluated for relapsing malaria. Here, in the P. cynomolgi-rhesus macaque model for relapsing malaria, we employ the parasites’ natural relapsing phenotype to self-boost the immune response against liver-stage parasites, following a single-shot high-dose live sporozoite vaccination. This approach resulted in sterile protection against homologous sporozoite challenge in three out of four animals in the group that was also exposed for several days to blood stages during primary infection and relapses. One out of four animals in the group that received continuous chemoprophylaxis to abort blood-stage exposure was also protected from sporozoite challenge. Although obtained in a small number of animals as part of a Proof-of-Concept study, these results suggest that limited blood-stage parasite exposure may augment protection in this model. We anticipate our data are a starting point for further research into correlates of protection and extrapolation of the single-shot approach to develop efficacious malaria vaccines against relapsing human malaria.

It has recently become clear that spatial organization contributes to cellular function and that expanding our knowledge on cellular organization is essential to further our understanding of processes in health and disease. Imaging mass cytometry enables high dimensional imaging of tissue while preserving spatial context and is therefore a suitable tool to unravel spatial relationships between cells. As availability of human tissue collected over the course of disease or infection is limited, preclinical models are a valuable source of such material. Non-human primate models are used for translational research as their anatomy, physiology and immune system closely resemble those of humans due to close evolutionary proximity. Tissue from non-human primate studies is often preserved large archives encompassing a range of conditions and organs. However, knowledge on antibody clones suitable for FFPE tissue of non-human primate origin is very limited. Here, we present an imaging mass cytometry panel development pipeline which enables the selection and incorporation of antibodies for imaging of non-human primate tissue. This has resulted in an 18-marker backbone panel which enables visualization of a broad range of leukocyte subsets in rhesus and cynomolgus macaque tissues. This high-dimensional imaging mass cytometry panel can be used to increase our knowledge of cellular organization within tissues and its effect on outcome of disease.

Vaccination through the natural route of infection represents an attractive immunization strategy in vaccinology. In the case of tuberculosis, vaccine delivery by the respiratory route has regained interest in recent years, showing efficacy in different animal models. In this context, respiratory vaccination triggers lung immunological mechanisms which are omitted when vaccines are administered by parenteral route. However, contribution of mucosal antibodies to vaccine- induced protection has been poorly studied. In the present study, we evaluated in mice and non-human primates (NHP) a novel whole cell inactivated vaccine (MTBVAC HK), by mucosal administration. MTBVAC HK given by intranasal route to BCG-primed mice substantially improved the protective efficacy conferred by subcutaneous BCG only. Interestingly, this improved protection was absent in mice lacking polymeric Ig receptor (pIgR), suggesting a crucial role of mucosal secretory immunoglobulins in protective immunity. Our study in NHP confirmed the ability of MTBVAC HK to trigger mucosal immunoglobulins. Importantly, in vitro assays demonstrated the functionality of these immunoglobulins to induce M. tuberculosis opsonization in the presence of human macrophages. Altogether, our results suggest that mucosal immunoglobulins can be induced by vaccination to improve protection against tuberculosis and therefore, they represent a promising target for next generation tuberculosis vaccines.

Tuberculosis (TB) causes 1.6 million deaths annually. Early differential diagnosis of active TB infection is essential in optimizing treatment and reducing TB mortality, but is hampered by a lack of accurate and accessible diagnostics. Previously, we reported on complement component C1q, measured in serum by ELISA, as a candidate biomarker for active tuberculosis. In this work we further examine the dynamics of C1q as a marker of progressive TB disease in non-human primates (NHP). We assessed systemic and pulmonary C1q levels after experimental infection using high or low single dose as well as repeated limiting dose Mycobacterium tuberculosis (Mtb) challenge of macaques. We show that increasing C1q levels, either peripherally or locally, correlate with progressive TB disease, assessed by PET-CT imaging or post-mortem evaluation. Upregulation of C1q did not precede detection of Mtb infection by a conventional interferon-gamma release assay, confirming its association with disease progression. Finally, pulmonary vaccination with Bacillus Calmette Guérin also increased local production of C1q, which might contribute to the generation of pulmonary protective immunity. Our data demonstrate that NHP modelling of TB can be utilized to study the role of C1q as a liquid biomarker in TB protection and disease, complementing findings in TB patients.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

While tuberculosis continues to afflict mankind, the immunological mechanisms underlying TB disease development are still incompletely understood. Advanced preclinical models for TB research include both rhesus and cynomolgus macaques (Macaca mulatta and Macaca fascicularis, respectively), with rhesus typically being more susceptible to acute progressive TB disease than cynomolgus macaques. To determine which immune mechanisms are responsible for this dissimilar disease development, we profiled a broad range of innate and adaptive responses, both local and peripheral, following experimental pulmonary Mycobacterium tuberculosis (Mtb) infection of both species. While T-cell and antibody responses appeared indistinguishable, we identified anti-inflammatory skewing of peripheral monocytes in rhesus and a more prominent local pro-inflammatory cytokine release profile in cynomolgus macaques associated with divergent TB disease outcome. Importantly, these differences were detectable both before and early after infection. This work shows that inflammatory and innate immune status prior to and at early stages after infection, critically affects outcome of TB infection.

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a ‘challenge’ experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

Summary BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis-derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces., Graphical Abstract, Highlights Nonhuman primates recapitulate trained immunity upon live attenuated TB vaccination Intravenous BCG induces changes in H3K27 acetylation and enhances cytokine production Mucosal BCG improves induction of trained immunity of monocytes over intradermal BCG The M. tuberculosis-derived candidate vaccine MTBVAC appears equally potent as BCG, Vierboom et al. demonstrate the induction of trained immunity in blood and bone marrow monocytes after vaccination with live attenuated TB vaccines in nonhuman primates. Mucosal respiratory delivery of BCG or MTBVAC induces trained immunity more efficiently compared to standard intradermal vaccination.

Background: Tuberculosis (TB) remains a major threat to global health. Currently, diagnosis of active TB is hampered by the lack of specific biomarkers that discriminate active TB disease from other (lung) diseases or latent TB infection (LTBI). Integrated human gene expression results have shown that genes encoding complement components, in particular different C1q chains, were expressed at higher levels in active TB compared to LTBI. Methods: C1q protein levels were determined using ELISA in sera from patients, from geographically distinct populations, with active TB, LTBI as well as disease controls. Results: Serum levels of C1q were increased in active TB compared to LTBI in four independent cohorts with an AUC of 0.77 [0.70; 0.83]. After 6 months of TB treatment, levels of C1q were similar to those of endemic controls, indicating an association with disease rather than individual genetic predisposition. Importantly, C1q levels in sera of TB patients were significantly higher as compared to patients with sarcoidosis or pneumonia, clinically important differential diagnoses. Moreover, exposure to other mycobacteria, such as Mycobacterium leprae (leprosy patients) or BCG (vaccinees) did not result in elevated levels of serum C1q. In agreement with the human data, in non-human primates challenged with Mycobacterium tuberculosis, increased serum C1q levels were detected in animals that developed progressive disease, not in those that controlled the infection. Conclusions: In summary, C1q levels are elevated in patients with active TB compared to LTBI in four independent cohorts. Furthermore, C1q levels from patients with TB were also elevated compared to patients with sarcoidosis, leprosy and pneumonia. Additionally, also in NHP we observed increased C1q levels in animals with active progressive TB, both in serum and in broncho-alveolar lavage. Therefore, we propose that the addition of C1q to current biomarker panels may provide added value in the diagnosis of active TB.

DC-SIGN (CD209/CLEC4L) is a C-type lectin receptor (CLR) that serves as a reliable cell-surface marker of interleukin 4 (IL-4)-activated human macrophages [M(IL-4)], which historically represent the most studied subset within the M2 spectrum of macrophage activation. Although DC-SIGN plays important roles in Mycobacterium tuberculosis (Mtb) interactions with dendritic cells, its contribution to the Mtb-macrophage interaction remains poorly understood. Since high levels of IL-4 are correlated with tuberculosis (TB) susceptibility and progression, we investigated the role of DC-SIGN in M(IL-4) macrophages in the TB context. First, we demonstrate that DC-SIGN expression is present both in CD68+ macrophages found in tuberculous pulmonary lesions of non-human primates, and in the CD14+ cell population isolated from pleural effusions obtained from TB patients (TB-PE). Likewise, we show that DC-SIGN expression is accentuated in M(IL-4) macrophages derived from peripheral blood CD14+ monocytes isolated from TB patients, or in macrophages stimulated with acellular TB-PE, arguing for the pertinence of DC-SIGN-expressing macrophages in TB. Second, using a siRNA-mediated gene silencing approach, we performed a transcriptomic analysis of DC-SIGN-depleted M(IL-4) macrophages and revealed the upregulation of pro-inflammatory signals in response to challenge with Mtb, as compared to control cells. This pro-inflammatory gene signature was confirmed by RT-qPCR, cytokine/chemokine-based protein array, and ELISA analyses. We also found that inactivation of DC-SIGN renders M(IL-4) macrophages less permissive to Mtb intracellular growth compared to control cells, despite the equal level of bacteria uptake. Last, at the molecular level, we show that DC-SIGN interferes negatively with the pro-inflammatory response and control of Mtb intracellular growth mediated by another CLR, Dectin-1 (CLEC7A). Collectively, this study highlights a dual role for DC-SIGN as, on the one hand, being a host factor granting advantage for Mtb to parasitize macrophages and, on the other hand, representing a molecular switch to turn off the pro-inflammatory response in these cells to prevent potential immunopathology associated to TB.

DC-SIGN (CD209/CLEC4L) is a C-type lectin receptor (CLR) that serves as a reliable cell-surface marker of interleukin 4 (IL-4)-activated human macrophages [M(IL-4)], which historically represent the most studied subset within the M2 spectrum of macrophage activation. Although DC-SIGN plays important roles in Mycobacterium tuberculosis (Mtb) interactions with dendritic cells, its contribution to the Mtb–macrophage interaction remains poorly understood. Since high levels of IL-4 are correlated with tuberculosis (TB) susceptibility and progression, we investigated the role of DC-SIGN in M(IL-4) macrophages in the TB context. First, we demonstrate that DC-SIGN expression is present both in CD68+ macrophages found in tuberculous pulmonary lesions of non-human primates, and in the CD14+ cell population isolated from pleural effusions obtained from TB patients (TB-PE). Likewise, we show that DC-SIGN expression is accentuated in M(IL-4) macrophages derived from peripheral blood CD14+ monocytes isolated from TB patients, or in macrophages stimulated with acellular TB-PE, arguing for the pertinence of DC-SIGN-expressing macrophages in TB. Second, using a siRNA-mediated gene silencing approach, we performed a transcriptomic analysis of DC-SIGN-depleted M(IL-4) macrophages and revealed the upregulation of pro-inflammatory signals in response to challenge with Mtb, as compared to control cells. This pro-inflammatory gene signature was confirmed by RT-qPCR, cytokine/chemokine-based protein array, and ELISA analyses. We also found that inactivation of DC-SIGN renders M(IL-4) macrophages less permissive to Mtb intracellular growth compared to control cells, despite the equal level of bacteria uptake. Last, at the molecular level, we show that DC-SIGN interferes negatively with the pro-inflammatory response and control of Mtb intracellular growth mediated by another CLR, Dectin-1 (CLEC7A). Collectively, this study highlights a dual role for DC-SIGN as, on the one hand, being a host factor granting advantage for Mtb to parasitize macrophages and, on the other hand, representing a molecular switch to turn off the pro-inflammatory response in these cells to prevent potential immunopathology associated to TB.

Clinical trials of novel tuberculosis (TB) vaccines are expensive, while global resources for TB vaccine development are limited. Therefore, there is a need for robust and predictive preclinical data to support advancement of candidate vaccines into clinical trials. Here, we provide a rationale for using the nonhuman primate as an essential component of these efforts, as well as guidance to the TB community for standardizing experimental design and aligning endpoints to facilitate development of new TB vaccines.

Interleukin-7 (IL-7) and the IL-7 receptor (IL-7R) have been shown to be alternatively spliced in infectious diseases. We tested IL-7 and IL-7R splicing in a tuberculosis (TB)-vaccine/Mycobacterium tuberculosis (Mtb)-challenge model in non-human primates (NHPs). Differential IL-7 splicing was detected in peripheral blood mononuclear cells (PBMCs) from 15/15 NHPs showing 6 different IL-7 spliced isoforms. This pattern did not change after infection with virulent Mtb. We demonstrated increased IL-7 (6 exon) and IL-17 protein production in lung tissue along with concomitant decreased transforming growth factor-β (TGF-β) from NHPs (vaccinated with a recombinant BCG (rBCG)) who showed increased survival after Mtb challenge. IL-7 increased IL-17 and interferon-γ (IFN-γ) gene and protein expression in PBMCs. Mtb-infected NHPs showed differential IL-7R splicing associated with the anatomical location and tissue origin, that is, in lung tissue, hilus, axillary lymph nodes (LNs) and spleen. Differential splicing of the IL-7R was typical for healthy (non-Mtb infected) and for Mtb-infected lung tissue with a dominant expression of soluble IL-7R (sIL-7R) receptor lacking exon 6 (9:1 ratio of sIL-7R/cell-bound IL-7R). Differential ratios of cell-bound vs sIL-7R could be observed in hilus and axillary LNs from Mtb-infected NHPs with an inversed ratio of 1:9 (sIL-7R/cell-bound IL-7R) in spleen and PBMCs. Soluble IL-7R is exclusively present in lung tissue.

The human CD14+ monocyte compartment is composed by two subsets based on CD16 expression. We previously reported that this compartment is perturbed in tuberculosis (TB) patients, as reflected by the expansion of CD16+ monocytes along with disease severity. Whether this unbalance is beneficial or detrimental to host defense remains to be elucidated. Here in the context of active TB, we demonstrate that human monocytes are predisposed to differentiate towards an anti-inflammatory (M2-like) macrophage activation program characterized by theCD16+CD163+MerTK+pSTAT3+ phenotype and functional properties such as enhanced protease-dependent motility, pathogen permissivity and immunomodulation. This process is dependent on STAT3 activation, and loss-of-function experiments point towards a detrimental role in host defense against TB. Importantly, we provide a critical correlation between the abundance of the CD16+CD163+MerTK+pSTAT3+ cells and the progression of the disease either at the local level in a non-human primate tuberculous granuloma context, or at the systemic level through the detection of the soluble form of CD163 in human sera. Collectively, this study argues for the pathogenic role of the CD16+CD163+MerTK+pSTAT3+ monocyte-to-macrophage differentiation program and its potential as a target for TB therapy,and promotes the detection of circulating CD163 as a potential biomarker for disease progression and monitoringof treatment efficacy. Fil: Lastrucci, Claire. Centre National de la Recherche Scientifique; Francia Fil: Bénard, Alan. Centre National de la Recherche Scientifique; Francia Fil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Pingris, Karine. Centre National de la Recherche Scientifique; Francia Fil: Souriant, Shanti. Centre National de la Recherche Scientifique; Francia Fil: Poincloux, Renaud. Centre National de la Recherche Scientifique; Francia Fil: Al Saati, Talal. Inserm; Francia Fil: Rasolofo, Voahangy. Pasteur Institute in Antananarivo; Madagascar Fil: González Montaner, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; Argentina Fil: Inwentarz, Sandra. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; Argentina Fil: Moraña, Eduardo José. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; Argentina Fil: Kondova, Ivanela. Biomedical Primate Research Centre; Países Bajos Fil: Verreck, Franck A. W.. Biomedical Primate Research Centre; Países Bajos Fil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Neyrolles, Olivier. Centre National de la Recherche Scientifique; Francia Fil: Maridonneau Parini, Isabel. Centre National de la Recherche Scientifique; Francia Fil: Lugo Villarino, Geanncarlo. Centre National de la Recherche Scientifique; Francia Fil: Cougoule, Celine. Centre National de la Recherche Scientifique; Francia

Summary Infection with Mycobacterium tuberculosis results in disease in 5–10% of exposed individuals, whereas the remainder controls infection effectively. Similar inter-individual differences in disease susceptibility are characteristic features of leprosy, typhoid fever, leishmaniasis and other chronic infectious diseases, including viral infections. Although the outcome of infection is influenced by many factors, it is clear that genetic host factors play an important role in controlling disease susceptibility to intracellular pathogens. Knowledge of the genes involved and their downstream cellular pathways will provide new insights for the design of improved and rationalized strategies to enhance host-resistance, e.g. by vaccination. In addition, this knowledge will aid in identifying better biomarkers of protection and disease, which are essential tools for the monitoring of vaccination and other intervention trials. The recent identification of patients with deleterious mutations in genes that encode major proteins in the type-1 cytokine (IL-12/IL23-IFN- γ ) axis, that suffered from severe infections due to otherwise poorly pathogenic mycobacteria (non-tuberculous mycobacteria (NTM) or M. bovis Bacille Calmette-Guerin (BCG)) or Salmonella species has revealed the major role of this system in innate and adaptive immunity to mycobacteria and salmonellae. Clinical tuberculosis has now been described in a number of patients with IL-12/IL23-IFN- γ system defects. Moreover, unusual mycobacterial infections were reported in several patients with genetic defects in NEMO, a key regulatory molecule in the NFκB pathway. These new findings will be discussed since they provide further insights into the role of type-1 cytokines in immunity to mycobacteria, including M. tuberculosis .

The remarkable advances in TB vaccinology over the last decade have been driven by a pragmatic approach to moving candidates along the development pipeline to clinical trials, fuelled by encouraging data on protection in animal models. Efficacy data from Phase IIb trials of the first generation of new candidates are anticipated over the next 1-2 years. As outlined in the TB Vaccines Strategic Blueprint, to exploit this information and to inspire design of next generation candidates, it is important that this empirical approach is complemented by progress in understanding of fundamental immune mechanisms and improved translational modalities. Current trends towards improved experimental and computational approaches for studying biological complexity will be an important element in the developing science of TB vaccinology.

The immune system plays a central role in the defense against environmental threats - such as infection with viruses, parasites or bacteria - but can also be a cause of disease, such as in the case of allergic or autoimmune disorders. In the past decades the impressive development of biotechnology has provided scientists with biological tools for the development of highly selective treatments for the different types of disorders. However, despite some clear successes the translation of scientific discoveries into effective treatments has remained challenging. The often-disappointing predictive validity of the preclinical animal models that are used in the selection of the most promising vaccine or drug candidates is the Achilles heel in the therapy development process. This publication summarizes the relevance and usage of non-human primates as pre-clinical model in infectious and autoimmune diseases, in particular for biologicals, which due to their high species-specificity are inactive in lower species. (C) 2015 Elsevier B.V. All rights reserved.

HLA-DM is known to catalyze the exchange of class II-associated invariant chain (Ii) peptide (CLIP) for cognate peptide during biosynthesis. In DM-negative cells HLA-DR3 molecules have been shown to predominantly present CLIP and to lack the DR3-specific mAb epitope 16.23, which has led to the assumption that CLIP prevents binding of mAb 16.23. In the present study we show that CLIP does not prohibit 16.23 epitope expression, but that the formation of this epitope is directly influenced by interactions of the DR molecule with Ii and DM. Detergent solubilized DR3 from wild-type as well as DM(-) cells bound CLIP in a 16.23(+) mode. On cells, however, neither CLIP nor antigenic peptide bound to DR3 in a 16.23(+) conformation, unless HLA-DM was expressed. Thus, HLA-DM appears to alter the conformation of DR3 in a peptide-independent fashion. Since in DM-deficient cells that also lack Ii, DR3 molecules assembled in a 16.23(+) conformation, we conclude that during biosynthesis Ii and DM exert opposing conformational constraints, characterized by suppressing or releasing 16.23 epitope expression. These results imply that DR3/peptide complexes, including DR3/ CLIP, can exist in two conformations depending on previous interaction with DM, but independent of the nature of the peptide bound. We show that these naturally occurring class II conformers can be selectively recognized by T cells.

Rheumatoid arthritis (RA) occurs more frequently in HLA-DR4+ individuals than in those who do not express this MHC class II molecule. Although the role of this genetic factor in the immunopathology of this autoimmune disease is unclear, the association of RA with HLA-DR4 may indicate that DR4 molecules present autoantigen(s) to T cells. Here we report the analysis of naturally processed peptides, eluted from a mixture of HLA-DR4Dw4 (DRB1*0401) and DR53 (DRB4*0101) molecules isolated from an RA patient-derived EBV-transformed B cell line. Several (size variants of) self-peptides originating from the autologous molecules HLA-A2, HLA-Cw9, HLA-B62, HLA-DR4Dw4 and HLA-DR53, were identified. We also found a sequence that has no homology to any protein in the SwissProt protein sequence databank, and a peptide identical to an internal fragment of the autoantigen calreticulin. The association of the identified peptides with cells expressing HLA-DR4Dw4/DR53 was confirmed by peptide binding analysis. In agreement with previously described peptide binding motifs for DR4Dw4, most peptides contained an aromatic residue (Phe, Tyr, Trp) at relative position i and a small hydroxyl-containing residue (Ser, Thr) at i + 5. Our findings indicate that in RA patient-derived EBV-transformed B cells DR4Dw4/DR53 molecules present a peptide from the autoantigen calreticulin. Interestingly, autoantibodies against calreticulin have been found in various rheumatic diseases, including rheumatoid arthritis. Thus, the analysis of HLA class II-bound peptides can lead to the identification of putative T helper epitopes, which might be involved in the immunopathology of autoimmune diseases.

Non-human primate models aid to design novel vaccine strategies. We analyzed therefore the dynamics of cellular immune responses induced by a bacille Calmette-Guerin (BCG) / recombinant BCG (rBCG, perfringolysin plus Ag85A/B, TB10.4) prime, subsequent adenoviral (Ad35 expressing Ag85A/B and TB10.4) boosts followed by a challenge with M. tuberculosis (Mtb, Erdman strain) in peripheral blood mononuclear cells from rhesus macaques. T-cell compartment mobilization was defined by CD45RA/CCR7/CD27/CD28 expression on CD4+, CD8αα+ and CD8αβ+ T-cells and functional analysis (STAT-5 phosphorylation) was carried out using multicolor flow cytometry. CD4+ as well as CD8+ T-cells showed a marked decrease in IL-7 receptor alpha-chain (IL-7Rα) expression in vaccinated animals (n=12/12) after the first adenoviral boost, but not in control animals (who received saline). BCG- and rBCG- vaccinated animals showed a dramatic decrease of precursor (CD45RA+CCR7+) and subsequent increase of activated central memory (CD45RA-CCR7+) T-cells in the CD4+, CD8αα+ and CD8αβ+ T-cell compartments 2 weeks after Mtb challenge. In contrast, this pattern was observed in control animals only 7 weeks after Mtb challenge. Expression of IL-7Rα was markedly reduced in CD4+ T-cell subsets two weeks after Mtb challenge in BCG-vaccinated animals, yet not in rBCG-vaccinated animals or control animals. These data show that vaccination profoundly shapes the dynamics of immune memory T-cells associated with Mtb infection.

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogeneous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I-restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guerin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8+ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3+ and CD8+ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8+ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals.

Major histocompatibility complex class II molecules exist in two forms, which can be distinguished on the basis of their stability in sodium dodecyl sulfate (SDS) as SDS-stable and SDS-unstable alpha beta dimers. The ratio of stable vs. unstable alpha beta dimers varies between murine H-2 alleles and isotypes, but the molecular basis for this observation is unknown. Here we show that for the human HLA-DRB1 and HLA-DRB3 gene products this ratio is controlled by the valine/glycine dimorphism at position 86. Haplotypes coding for DR beta chains with a valine at position 86 express higher numbers of stable dimers compared to similar haplotypes expressing DR beta chains with a glycine at that position. Reverse-phase high-performance liquid chromatography analysis of iodinated peptides, which were eluted from DR dimers with either a DRB1*1101 or a DRB1*1104 beta chain which differ only at position 86, indicated that these DR dimers contain (partially) distinct sets of peptides. The valine/glycine dimorphism is highly conserved, present in most HLA-DR alleles and influences peptide-binding. Analysis of the occurrence of the Val86 and the Gly86 gene products revealed that these are not equally present in the population. Depending on the DR specificity either the Val86 of Gly86 allelic variant is favored. Thus, the natural, highly conserved dimorphism at HLA-DR beta chain position 86 influences peptide selection. The dimorphism is therefore likely to influence antigen presentation and forms the molecular basis for the observed differences in stability of Val86- and Gly86-containing DR dimers in the presence of SDS.

The 25th Dageraad Symposium on The Molecular Mechanisms of Host–Pathogen Interactions in Infectious Disease: Towards Better Intervention Strategies? was held in Enkhuizen, The Netherlands, from 29 June to 3 July 2001.

BACKGROUND: Continuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection. METHODS AND FINDINGS: Here, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNgamma responses. Antigen 85A-specific IFNgamma secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNgamma levels post-infection as the strongest immunocorrelate for protection (spearman's rho: -0.60). CONCLUSIONS: Both the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates.

Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP againstMycobacterium tuberculosisto identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated withM. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP.

Contains fulltext : 52114.pdf (Publisher’s version ) (Closed access) Unusual susceptibility to mycobacterial infections can be caused by deleterious mutations in genes that encode the interferon-gamma receptor 1 chain. Such mutations hamper the activation of macrophages by a type 1 immune response and result in enhanced survival of intracellular pathogens. We here report two patients with unusual mycobacterial infections, both diagnosed with homozygous deleterious interferon-gamma receptor 1 gene mutations. Patient 1 became ill after Bacillus Calmette-Guerin vaccination at the age of 9 months and died at the age of 18 months. She carried a homozygous C71Y mutation in the extracellular part of the mature interferon-gamma receptor 1 protein, resulting in the lack of detectable protein expression and absence of interferon-gamma dependent signaling. Patient 2 became ill at the age of 3 years, is still alive at 19 years of age, and has suffered from five successive infection episodes with atypical mycobacteria. A homozygous splice-site mutation in intron 3 was identified, resulting in the deletion of exon 3 at the mRNA level and consequently a truncated interferon-gamma receptor 1 protein with absence of the transmembrane domain. Protein expression and interferon-gamma dependent signaling were not detectable.

IL-23 is regarded as a major pro-inflammatory mediator in autoimmune disease, a role which until recently was ascribed to its related cytokine IL-12. IL-23, an IL-12p40/p19 heterodimeric protein, binds to IL-12Rbeta1/IL-23R receptor complexes. Mice deficient for p19, p40 or IL-12Rbeta1 are resistant to experimental autoimmune encephalomyelitis or collagen-induced arthritis. Paradoxically, however, IL-12Rbeta2- and IL-12p35-deficient mice show remarkable increases in disease susceptibility, suggesting divergent roles of IL-23 and IL-12 in modulating inflammatory processes. IL-23 induces IL-17, which mediates inflammation and tissue remodeling, but the role of IL-12 in this respect remains unidentified. We investigated the roles of exogenous (recombinant) and endogenous (macrophage-derived) IL-12 and IL-23, on IL-17-induction in human T-cells. IL-23 enhanced IL-17 secretion, as did IL-2, IL-15, IL-18 and IL-21. In contrast, IL-12 mediated specific inhibition of IL-17 production. These data support the role of IL-23 in inflammation through stimulating IL-17 production by T lymphocytes, and importantly indicate a novel regulatory function for IL-12 by specifically suppressing IL-17 secretion. These data therefore extend previous reports that had indicated unique functions for IL-23 and IL-12 due to distinct receptor expression and signal transduction complexes, and provide novel insights into the regulation of immunity, inflammation and immunopathology.

MHC class I molecules expressed on cell surfaces are composed of H chain, β2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.

Epstein-Barr virus (EBV) resides as a persistent infection in human leukocyte antigen (HLA) class II+B lymphocytes and is associated with a number of malignancies. The EBV lytic-phase protein gp42 serves at least two functions: gp42 acts as the coreceptor for viral entry into B cells and hampers T-cell recognition via HLA class II molecules through steric hindrance of T-cell receptor-class II-peptide interactions. Here, we show that gp42 associates with class II molecules at their various stages of maturation, including immature αβIi heterotrimers and mature αβ-peptide complexes. When analyzing the biosynthesis and maturation of gp42 in cells stably expressing the viral protein, we found that gp42 occurs in two forms: a full-length type II membrane protein and a truncated soluble form. Soluble gp42 is generated by proteolytic cleavage in the endoplasmic reticulum and is secreted. Soluble gp42 is sufficient to inhibit HLA class II-restricted antigen presentation to T cells. In an almost pure population of Burkitt's lymphoma cells in the EBV lytic cycle, both transmembrane and soluble forms of gp42 are detected. These results imply that soluble gp42 is generated during EBV lytic infection and could contribute to undetected virus production by mediating evasion from T-cell immunity.

IFN-gamma and IL-12 are crucial cytokines for cell-mediated immunity against intracellular pathogens. We have previously shown that human IL-12Rbeta1-deficiency leads to impaired IL-12 responsiveness and unusual susceptibility to infections due to mycobacteria and salmonellae. IL-23 is a cytokine with functions that partially overlap with those of IL-12. IL-23 consists of IL-12p40 and a novel p19 protein, and binds to a receptor complex comprising IL-12Rbeta1 and IL-23R. Thus, IL-12Rbeta1-deficiency may impair both IL-12- and IL-23 signaling, and both may contribute to the immunological phenotypes. To examine whether IL-12Rbeta1 is essential for IL-23 signaling in human T cells, we have studied IL-23 responsiveness of four IL-12Rbeta1-deficient individuals. Whereas IL-23 promoted IFN-gamma production by CD4(+) and CD8(+) T cells in controls, IL-12Rbeta1-deficient T cells lacked IL-23-induced IFN-gamma secretion, but responded normally to IL-2, IL-4, IL-15 and IL-18. We also show that induction of IFN-gamma production by IL-23 depends upon TCR-ligation and is enhanced by CD28-costimulation. Furthermore, IL-23 cooperates with IL-12 and IL-18 in promoting IFN-gamma production in controls, but not in patients. We conclude that IL-12Rbeta1-deficiency impairs IL-12- and IL-23-dependent signaling in human T cells. The syndrome caused by IL-12Rbeta1-deficiency thus needs to be reinterpreted as resulting from defective IL-12-as well as IL-23-mediated immunity.

Epstein–Barr virus (EBV) persists lifelong in infected hosts despite the presence of antiviral immunity. Many viral antigens are expressed during lytic infection. Thus, for EBV to spread, it must have evolved effective ways to evade immune recognition. Here, we report that HLA class II-restricted antigen presentation to T helper cells is hampered in the presence of the lytic-phase protein gp42. This interference with T cell activation involves association of gp42 with class II peptide complexes. Using HLA-DR tetramers, we identify a block in T cell receptor (TCR)–class II interactions imposed by gp42 as the underlying mechanism. EBV gp42 sterically clashes with TCR Vα-domains as visualized by superimposing the crystal structures for gp42–HLA-DR1 and TCR–MHC class II complexes. Blocking TCR recognition provides a previously undescribed strategy for viral immune evasion.

Cell mediated immunity plays a critical role in human host defence against intracellular bacteria. In patients with unusual, severe infections caused by poorly pathogenic species of mycobacteria and salmonellae, genetic deficiencies have been identified in key genes in the type-1 cytokine pathway, especially in IFNGR1 and IL12RB1. Here, we analyzed 11 patients originating from Turkey and suffering from unusual Mycobacterium bovis Bacille Calmette-Guerin infections following vaccination, and found that most patients (n=8) are deficient in IL-12Rbeta1 expression and function. No defects were found in patients' IFN-gammaR or IL-18R. In addition, a first patient suffering from partial IL-12Rbeta1 deficiency is described. This patient presented with an intermediate cellular and immunological phenotype: a consistent, low response to IL-12 was found, which could be further augmented by IL-18. Despite a lack of cell surface IL-12Rbeta1 expression, normal levels of intracellular IL-12Rbeta1 protein were detectable, which was not seen in the other, completely IL-12Rbeta1 deficient patients examined. Moreover, this patient had a relatively mild clinical phenotype and was the only individual with a single homozygous amino acid substitution in IL-12Rbeta1 (C198R). Collectively, our findings indicate that idiopathic, unusually severe infections due to M. bovis BCG can be caused by complete as well as partial IL-12Rbeta1 deficiency.

Protective immunity to intracellular bacteria such as mycobacteria and salmonellae depends on an effective cell-mediated immune response (CMI). A major effector mechanism of CMI is the activation of infected macrophages by type-1 cytokines, particularly interferon-gamma (IFN-y). IFN-y is produced by natural killer (NK) and T helper-1 (Thl) cells and its production is regulated by interleukin-12 (IL-12) and interleukin-18 (IL-18), both of which are released by activated macrophages and dendritic cells (Fig. 1) [Ottenhoff and Mutis, 1995; Kumararatne et al., 1990]. IFN-y together with monokines such as TNF-a, activates microbicidal mechanisms of macrophages that are thought to be responsible for the control and elimination of the intracellular infectious pathogen. Moreover, IFN-y induces and enhances MHC class I and II expression, and modulates expression of other molecules involved in antigen presentation, such as proteasomes and TAP, thus resulting in increased immune T cell activation. Patients are likely to benefit from supplementary IFN-y therapy and in the case of IL-12 deficiency, IL-12 supplementation may be an option as well. As a prophylactic measure, live bacterial vaccines such as M. bovis BCG and typhoid vaccines, as well as live viral vaccines, such as yellow fever, should preferably be avoided. Exposure to infectious diseases, such as tuberculosis, listeriosis, salmonellosis and leishmaniasis should obviously be minimized, given the increased risk to develop severe infections. Genetic correction strategies may be developed in the nearby future, including transplantation with gene transduced autologous stem cells.

Genetic lack of interleukin 12 receptor β1 (IL-12Rβ1) surface expression predisposes to severe infections by poorly pathogenic mycobacteria or Salmonella and causes strongly decreased, but not completely abrogated, interferon (IFN)-γ production. To study IL-12Rβ1–independent residual IFN-γ production, we have generated mycobacterium–specific T cell clones (TCCs) from IL-12Rβ1–deficient individuals. All TCCs displayed a T helper type 1 phenotype and the majority responded to IL-12 by increased IFN-γ production and proliferative responses upon activation. This response to IL-12 could be further augmented by exogenous IL-18. IL-12Rβ2 was found to be normally expressed in the absence of IL-12Rβ1, and could be upregulated by IFN-α. Expression of IL-12Rβ2 alone, however, was insufficient to induce signal transducer and activator of transcription (Stat)4 activation in response to IL-12, whereas IFN-α/IFN-αR ligation resulted in Stat4 activation in both control and IL-12Rβ1–deficient cells. IL-12 failed to upregulate cell surface expression of IL-18R, integrin α6, and IL-12Rβ2 on IL-12Rβ1–deficient cells, whereas this was normal on control cells. IL-12–induced IFN-γ production in IL-12Rβ1–deficient T cells could be inhibited by the p38 mitogen-activated protein kinase (MAP) kinase inhibitor SB203580 and the MAP kinase kinase (MEK) 1/2 inhibitor U0126, suggesting involvement of MAP kinases in this alternative, Stat4-independent, IL-12 signaling pathway. Collectively, these results indicate that IL-12 acts as a partial agonist in the absence of IL-12Rβ1. Moreover, the results reveal the presence of a novel IL-12Rβ1/Stat4–independent pathway of IL-12 responsiveness in activated human T cells involving MAP kinases. This pathway is likely to play a role in the residual type 1 immunity in IL-12Rβ1 deficiency.

MHC class II molecules can exist in two conformations which can be distinguished on the basis of their stability in SDS. The formation of SDS-stable dimers has been shown to correlate with persistent expression of antigen MHC class II-peptide complexes by murine antigen-presenting cells. HLA DR molecules contain either a Val or a Gly at position 86 of the beta chain, which is located in a conserved and prominent hydrophobic pocket in the peptide binding site. Here we show that Val86-containing DR molecules more frequently select peptides which induce the formation of SDS-stable dimers than Gly86 variants. Using analogues of the influence virus haemagglutinin epitope 307-319 we found that the replacement of the aromatic hydrophobic anchor residue (Tyr)at position 309 by amino acids with an aliphatic hydrophobic side chain resulted in the specific formation of high numbers of SDS-stable Val86-DR but not Gly86-DR dimers. These results indicate that the fit between the first anchor residue and the hydrophobic pocket around Dr beta 86 plays a critical role in the formation of SDS-stable DR dimers. Synthetic analogues of naturally processed DR-associated peptides displayed promiscuity in their capacity to bind to several DR specificities and in their ability to induce the SDS-stable conformation. However, no correlation was observed between binding capacity and the ability to induce the SDS-stable conformation. Since it has been shown that SDS stability can relate to the kinetics of peptide-MHC class II interactions, the definition of the requirements for the formation of SDS-stable HLA class II molecules may be important for the design of effective peptide-based immunomodulation protocols.

Purified HLA-A2.1 molecules obtained by affinity chromatography of 6 x 10(10) Epstein Barr virus (EBV)-transformed B lymphocytes were used in an attempt to isolate the human HLA-A2.1-restricted minor histocompatibility (H) peptides H-Y and HA-2. Fraction 18 of the high-performance liquid chromatography (HPLC)-separated HLA-A2.1 peptide pool was found to contain the natural HA-2 peptide. An HA-2-specific, HLA-A2.1-restricted cytotoxic T lymphocyte clone lysed HLA-A2.1+ HA-2- EBV-transformed B lymphocyte cell lines reproducibly and in a concentration-dependent fashion in the presence of fraction 18, but not in the presence of other HPLC fractions. By contrast, H-Y sensitizing activity was not found in any fraction. Amino acid sequencing of peptide fraction 18 revealed a mixture of peptides with maximal length of nine amino acids, in which the presence of Leu at positions 2 and 9 was dominant. Surprisingly, the HA-2 peptide could not be mimicked by any of the peptide mixtures synthesized according to the amino acid sequences found in fraction 18. Our failure to obtain the actual amino acid sequence of the human minor H peptide HA-2 from a peptide pool with the established pattern for binding to HLA-A2.1 may indicate that this CTL defined minor H peptide does not represent an abundant HLA-A2.1 binding peptide.

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to γ-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.

Expression of MHC class II genes by epithelial cells is induced in inflammatory conditions such as autoimmunity and organ transplantation. Class II ligands generated by the epithelial cell processing mechanisms are unknown, although some unique epitopes have been described in epithelial cells that B cells could not generate. Epithelial cells are the targets of autoreactive T cell responses in autoimmune diseases and of transplant rejection processes, which may involve recognition of cell type-specific epitopes. In the present report, we have compared the DR4-associated repertoire and the intracellular distribution of class II, invariant chain (Ii), and DM molecules between a human DR4-, Ii-, and DM-transfected rat neuroendocrine epithelial cell line and a homozygous DR4 (DRB1*0401) lymphoblastoid B cell line, by mass spectrometry sequencing techniques, and immunoelectron microscopy. The epithelial cells chosen for transfection, RINm5F, are rat insular cells widely used for human studies of autoimmune diabetes. The results revealed a remarkably heterogeneous pool of self protein-derived peptides from the cell surface and various intracellular compartments, including the cytosol and secretory vesicles in epithelial cells, compared with a very restricted homogeneous repertoire in lymphoblastoid B cell lines, where few epitopes from surface molecules were predominant. The generation of distinct DR4-associated peptide repertoires in these two cell types could be due to the effect of several factors including differences in subcellular location of Ii and DM molecules, differential DO expression, and cell type-specific mechanisms of class II ligand generation. This is specially relevant to processes involving epithelial T cell interactions such as organ-specific autoimmunity and transplant rejection.

Summary To fight tuberculosis, better vaccination strategies are needed. Live attenuated Mycobacterium tuberculosis-derived vaccine, MTBVAC, is a promising candidate in the pipeline, proven to be safe and immunogenic in humans so far. Independent studies have shown that pulmonary mucosal delivery of Bacillus Calmette-Guérin (BCG), the only tuberculosis (TB) vaccine available today, confers superior protection over standard intradermal immunization. Here we demonstrate that mucosal MTBVAC is well tolerated, eliciting polyfunctional T helper type 17 cells, interleukin-10, and immunoglobulins in the airway and yielding a broader antigenic profile than BCG in rhesus macaques. Beyond our previous work, we show that local immunoglobulins, induced by MTBVAC and BCG, bind to M. tuberculosis and enhance pathogen uptake. Furthermore, after pulmonary vaccination, but not M. tuberculosis infection, local T cells expressed high levels of mucosal homing and tissue residency markers. Our data show that pulmonary MTBVAC administration has the potential to enhance its efficacy and justifies further exploration of mucosal vaccination strategies in preclinical efficacy studies., Graphical Abstract, Highlights Pulmonary MTBVAC delivery confers immune signature correlating with TB protection This signature spreads through the lung without a recall response in the skin Vaccine-induced T cells have increased mucosal homing and tissue residency markers Vaccine-induced antibodies enhance phagocytosis of M. tuberculosis, Dijkman et al. show that pulmonary immunization with the M. tuberculosis-derived vaccine candidate MTBVAC confers a local mucosal antigen-specific signature—polyfunctional Th1/Th17 cells exhibiting increased homing and tissue residency marker expression, IL-10, and phagocytosis-promoting immunoglobulins—that has been associated previously with protection from TB infection and disease in rhesus macaques.