Shutdown of immunological priming and presentation after in vivo administration of adenovirus.

Adenoviral (Adv) vectors are widely used in both experimental and clinical trials for vaccination and gene therapy. Recombinant Adv can evoke potent innate immune responses and adaptive immune responses to encoded antigens. However, how Adv infection affects the response to subsequently encountered antigens is poorly understood. We show that intravenously administered replication defective (E1 and E3 deleted) Adv educes functional changes in dendritic cells (DC) resulting in impaired priming of cytotoxic T lymphocytes (CTL) more than 7 days after Adv treatment. Generalized DC activation was indicated by transient upregulation of CD86 and reduced endocytosis of fluorescent beads. It is known that CD8+ DC are predominantly responsible for uptake and presentation (cross-presentation) of exogenous antigens to CD8+ CTL. Hence, impaired endocytosis in CD8+, but not CD8-, DC at 7 days after Adv administration provided an explanation for the impaired CTL response to antigen at this time. Shutdown of cross-presentation was confirmed using cytochrome c (cytc), an agent that selectively depletes cross-presenting DC. Adv-infection rendered CD8+ DC resistant to depletion by cytc. As the cross-presentation pathway underlies CD8 T-cell responses to many cancers and to vaccines or viruses that do not directly infect DC, systemic Adv administration may impair these responses.

Changes in the repertoire of peptides bound to HLA-B27 subtypes and to site-specific mutants inside and outside pocket B.

HLA-B27 subtypes share many structural features, including their pocket B, which interacts with a conserved Arg residue at the second position of B*2705-bound peptides. Subtypes differ among each other at other locations in the peptide binding site. In this study, metabolic labeling and radiochemical pool sequencing were used to address the following issues: (a) presence of the Arg 2 (R2) motif among peptides bound to the various HLA-B27 subtypes; (b) influence of mutations inside and outside pocket B on this motif; and (c) the degree of similarity among the peptide pools bound to the various B27 subtypes. Sequencing of Arg-labeled peptide pools extracted from B*2701 to B*2706, and from two site-directed mutants of B*2705 with changes outside pocket B, indicated that all of these molecules bind peptides with Arg at position 2. Peptides from several mutants with changes altering the structure of pocket B, and from one mutant at the pocket B rim, also retained the R2 motif. However, this was absent in the peptide pool extracted from the M45 mutant, in which the negative charge of pocket B, conferred to HLA-B27 by Glu45, was canceled. These results indicate that alterations outside pocket B, and even disruption of the network of hydrogen bonds that stabilizes Arg binding in pocket B, do not impair binding of peptides bearing the R2 motif, but a nonconservative substitution at position 45 does. As a substantial fraction of anti-B*2705 cytotoxic T lymphocyte (CTL) clones crossreact with the M45 mutant (Villadangos, J., B. Galocha, D. López, V. Calvo, and J. A. López de Castro. 1992. J. Immunol. 149:505) this result suggest that determinant mimicry by nonidentical peptides may frequently account for unexpected CTL crossreactions. Metabolic labeling with various other amino acids and radiochemical sequencing revealed similarities, but also substantial differences, among the peptide pools from the various HLA-B27 subtypes. This strongly suggests that many peptides bind to multiple subtypes, but significant subsets of peptides bound to a given HLA-B27 subtype do not bind to other subtypes or do so with greatly altered efficiency. These results indicate the importance of polymorphism outside pocket B in modulating peptide binding to HLA-B27.

Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism.

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.

Organ-specific isoform selection of fatty acid-binding proteins in tissue-resident lymphocytes.

Tissue-resident memory T (TRM) cells exist throughout the body, where they are poised to mediate local immune responses. Although studies have defined a common mechanism of residency independent of location, there is likely to be a level of specialization that adapts TRM cells to their given tissue of lodgment. It has been shown that TRM cells in the skin rely on the uptake of exogenous fatty acids for their survival and up-regulate fatty acid-binding protein 4 (FABP4) and FABP5 as part of their transcriptional program. However, FABPs exist as a larger family of isoforms, with different members selected in a tissue-specific fashion that is optimized for local fatty acid availability. Here, we show that although TRM cells in a range of tissue widely express FABPs, they are not restricted to FABP4 and FABP5. Instead, TRM cells show varying patterns of isoform usage that are determined by tissue-derived factors. These patterns are malleable because TRM cells relocated to different organs modify their FABP expression in line with their new location. As a consequence, these results argue for tissue-specific overlays to the TRM cell residency program, including FABP expression that is tailored to the particular tissue of TRM cell lodgment.

Cathepsin S controls the trafficking and maturation of MHC class II molecules in dendritic cells.

Before a class II molecule can be loaded with antigenic material and reach the surface to engage CD4+ T cells, its chaperone, the class II-associated invariant chain (Ii), is degraded in a stepwise fashion by proteases in endocytic compartments. We have dissected the role of cathepsin S (CatS) in the trafficking and maturation of class II molecules by combining the use of dendritic cells (DC) from CatS(-/-) mice with a new active site-directed probe for direct visualization of active CatS. Our data demonstrate that CatS is active along the entire endocytic route, and that cleavage of the lysosomal sorting signal of Ii by CatS can occur there in mature DC. Genetic disruption of CatS dramatically reduces the flow of class II molecules to the cell surface. In CatS(-/-) DC, the bulk of major histocompatibility complex (MHC) class II molecules is retained in late endocytic compartments, although paradoxically, surface expression of class II is largely unaffected. The greatly diminished but continuous flow of class II molecules to the cell surface, in conjunction with their long half-life, can account for the latter observation. We conclude that in DC, CatS is a major determinant in the regulation of intracellular trafficking of MHC class II molecules.

Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus.

Degradation of invariant chain (Ii) is a critical step in major histocompatibility complex class II-restricted antigen presentation. Cathepsin L was found to be necessary for Ii degradation in cortical thymic epithelial cells (cTECs), but not in bone marrow (BM)-derived antigen-presenting cells (APCs). Consequently, positive selection of CD4+ T cells was reduced. Because different cysteine proteinases are responsible for specific Ii degradation steps in cTECs and BM-derived APCs, the proteolytic environment in cells mediating positive and negative selection may be distinct. The identification of a protease involved in class II presentation in a tissue-specific manner suggests a potential means of manipulating CD4+ T cell responsiveness in vivo.

Pathophysiological role of respiratory dysbiosis in hospital-acquired pneumonia.

Hospital-acquired pneumonia is a major cause of morbidity and mortality. The incidence of hospital-acquired pneumonia remains high globally and treatment can often be ineffective. Here, we review the available data and unanswered questions surrounding hospital-acquired pneumonia, discuss alterations of the respiratory microbiome and of the mucosal immunity in patients admitted to hospital, and explore potential approaches to stratify patients for tailored treatments. The lungs have been considered a sterile organ for decades because microbiological culture techniques had shown negative results. Culture-independent techniques have shown that healthy lungs harbour a diverse and dynamic ecosystem of bacteria, changing our comprehension of respiratory physiopathology. Understanding dysbiosis of the respiratory microbiome and altered mucosal immunity in patients with critical illness holds great promise to develop targeted host-directed immunotherapy to reduce ineffective treatment, to improve patient outcomes, and to tackle the global threat of resistant bacteria that cause these infections.

Cathepsin S activity regulates antigen presentation and immunity.

MHC class II molecules display antigenic peptides on cell surfaces for recognition by CD4(+) T cells. Proteolysis is required in this process both for degradation of invariant chain (Ii) from class II-Ii complexes to allow subsequent binding of peptides, and for generation of the antigenic peptides. The cysteine endoprotease, cathepsin S, mediates Ii degradation in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on antigen presentation and immunity. Specific inhibition of cathepsin S in A20 cells markedly impaired presentation of an ovalbumin epitope by interfering with class II-peptide binding, not by obstructing generation of the antigen. Administration of a cathepsin S inhibitor to mice in vivo selectively inhibited activity of cathepsin S in splenocytes, resulting in accumulation of a class II-associated Ii breakdown product, attenuation of class II-peptide complex formation, and inhibition of antigen presentation. Mice treated with inhibitor had an attenuated antibody response when immunized with ovalbumin but not the T cell-independent antigen TNP-Ficoll. In a mouse model of pulmonary hypersensitivity, treatment with the inhibitor also abrogated a rise in IgE titers and profoundly blocked eosinophilic infiltration in the lung. Thus, inhibition of cathepsin S in vivo alters Ii processing, antigen presentation, and immunity. These data identify selective inhibition of cysteine proteases as a potential therapeutic strategy for asthma and autoimmune disease processes.

Serpinb9 is a marker of antigen cross-presenting dendritic cells.

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (grB) that protects cytotoxic lymphocytes from grB-mediated death. In addition, Sb9 is also expressed in accessory immune cells, including dendritic cells (DCs), although its role is debated. Recently, we have demonstrated that Sb9 plays a grB-independent role in cross-presentation of antigens by CD8+ DCs. Here, using a mouse line expressing green fluorescent protein knocked in under the control of the Sb9 promoter, we demonstrate that Sb9 expression is highest in those tissue-resident and migratory DC subsets capable of cross-presentation. Further, we show that CD8+ DCs can be divided into two subsets based on Sb9 expression, and that only the subset expressing higher levels of Sb9 is capable of cross-presentation. These findings add support for role for Sb9 cross-presentation, and indicate that high Sb9 expression is a novel marker of cross-presentation capable DCs.

Presentation of antigens by MHC class II molecules: getting the most out of them.

The function of MHC class II molecules is to bind peptides derived from antigens that access the endocytic route of antigen presenting cells and display them on the plasma membrane for recognition by CD4(+) T cells. Formation of the MHC II-peptide complexes entails the confluence of the antigens and the MHC II molecules in the same compartments of the endocytic route. There, both the antigens and the MHC II molecules undergo a series of orchestrated changes that involve proteases, other hydrolases and chaperones, culminating in the generation of a wide repertoire of MHC II-peptide combinations. All the events that lead to formation of MHC II-peptide complexes show a considerable degree of flexibility; this lack of strict rules is advantageous in that it provides T cells with the maximum amount of information, ensuring that pathogens do not go undetected.

Antibody-targeted vaccination to lung dendritic cells generates tissue-resident memory CD8 T cells that are highly protective against influenza virus infection.

Influenza virus gains entry into the body by inhalation and initiates its replication cycle within the lung. The early stage of infection, while the virus is confined to the lung mucosa, provides the ideal window of opportunity for an effective immune response to control the infection. Tissue-resident memory (Trm) CD8 T cells, located in a variety of tissues including the lung, are ideally situated to act during this window and stall the infection. The factors involved in the differentiation of lung Trm cells remain poorly defined. We demonstrate that recognition of antigen presented locally by dendritic cells (DCs) and transforming growth factor-ß (TGFß) signaling are both required. We exploited this knowledge to develop an antibody-targeted vaccination approach to generate lung Trm cells. Delivering antigen exclusively to respiratory DCs results in the development of lung CD8 Trm cells that are highly protective against lethal influenza challenge. Our results describe an effective vaccination strategy that protects against influenza virus infection.

Proteolysis in MHC class II antigen presentation: who's in charge?

Antigen-presenting cells (APC) degrade proteins intracellularly to generate peptides, which are then bound by products of the major histocompatibility complex (MHC) and exposed on the surface of the APC for recognition by T cells. The supply of antigenic peptides and their association with MHC molecules requires the concerted action of a cohort of accessory molecules that includes chaperones, transporters of peptides, and the proteases that degrade the antigens.

Role of binding pockets for amino-terminal peptide residues in HLA-B27 allorecognition.

The peptide binding site of HLA-B27 and other class I Ag consists of a series of pockets that bind peptide side chains. Two of these pockets interact with the amino-terminal peptide residue (pocket A) and with the highly conserved second residue (pocket B). In this study, the role of pockets A and B in HLA-B27-specific T cell allorecognition has been analyzed. Four HLA-B27 mutants with single or double changes in pocket B (24T----A, 45E----M, 67C----V, and 24,67T,C----A,V) and three mutants with single changes in pocket A (163E----T, 167W----S, and 171Y----H) were constructed by site-directed mutagenesis and expressed in HMy2.C1R cells after DNA-mediated gene transfer. These transfectants were used as target cells in cytotoxicity assays with a series of HLA-B27-specific CTL. All the mutations analyzed affected allorecognition by a significant proportion of the CTL tested, but no single change abrogated recognition by all CTL. The global effects of each mutation on allorecognition were comparable to one another, except for the effect of the change at position 67, which was smaller. The behavior of individual CTL with the mutants was very diverse, ranging from CTL that did not recognize most of the mutants to CTL recognizing all of them. Thus, some alloreactive CTL can withstand drastic alterations in pockets A and B. Two CTL showed heteroclytic effects towards the V67 and M45 mutants. CTL behavior with the H171 mutant was closely parallel to that with the B*2703 subtype, having a single Y----H change at position 59. This parallelism correlates with the similar role of Tyr59 and Tyr171 in establishing hydrogen bonds with the amino termini of HLA-B27-bound peptides. The results demonstrate that altering the structure of pockets that interact with the amino-terminal first and second residues of HLA-B27-bound peptides significantly affects recognition by alloreactive CTL, and they strongly suggest widespread peptide involvement in HLA-B27 allorecognition.

Cross-reactive T cell clones from unrelated individuals reveal similarities in peptide presentation between HLA-B27 and HLA-DR2.

HLA-B27- responder cells were stimulated in vitro with B*2705+ lymphoblastoid cell lines and alloreactive CTL clones were obtained by limiting dilution. Of the CD3+ CD4-CD8+ HLA-B27-specific CTL clones obtained, two of them, possessing the same TCR, cross-reacted with HLA-DR2. The fine specificity of these CTL was established with HLA-B27 and HLA-DR2 subtypes. They recognized the B*2701 to B*2706 subtypes, but only DR2Dw2. Lysis of DR2+ target cells was specifically inhibited by anti-CD3, anti-class II, and anti-DR mAb, but not with an anti-CD8 antibody. The monoclonal nature of the cross-reaction was established by the mutual inhibition of HLA-B27 and DR2Dw2 cells in cold target competition experiments. The DR2 protein involved in the cross-reaction was the heterodimer carrying the B5*0101 product, as shown by using L cell transfectants expressing each of the two molecules encoded in the DR2Dw2 haplotype. A correlation between the fine specificity of these CTL clones and the amino acid sequences of HLA-B27 and HLA-DR2 subtypes revealed a shared structural motif between HLA-B27 and the DR2 B5*0101 chain, which could be related to the observed cross-reaction. This motif was contributed for by several residues located in adjacent beta strands, at the floor of the peptide-binding site. The contribution of two of these residues, as well as other beta-pleated sheet residues to HLA-B27 allorecognition by the cross-reactive CTL clones was directly demonstrated with site-directed mutants. These results suggest that the dual reactivity pattern reflects presentation of identical or structurally related peptide by HLA-B27 and HLA-DR2Dw2. As T cell cross-reactivity between HLA-B27 and HLA-DR2 was previously found in cells from an unrelated individual the results reported here are likely to reflect an intrinsic property of HLA-B27, rather than the fortuitous finding of a rare clonal reaction pattern. We speculate on the potential implications of these results for the pathogeny of HLA-B27-associated spondyloarthropathies.

Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H-2DM.

Major histocompatibility complex (MHC) class II molecules bind and present to CD4(+) T cells peptides derived from endocytosed antigens. Class II molecules associate in the endoplasmic reticulum with invariant chain (Ii), which (i) mediates the delivery of the class II-Ii complexes into the endocytic compartments where the antigenic peptides are generated; and (ii) blocks the peptide-binding site of the class II molecules until they reach their destination. Once there, Ii must be removed to allow peptide binding. The bulk of Ii-class II complexes reach late endocytic compartments where Ii is eliminated in a reaction in which the cysteine protease cathepsin S and the accessory molecule H-2DM play an essential role. Here, we here show that Ii is also eliminated in early endosomal compartments without the intervention of cysteine proteases or H-2DM. The Ii-free class II molecules generated by this alternative mechanism first bind high molecular weight polypeptides and then mature into peptide-loaded complexes.

Unusual topology of an HLA-B27 allospecific T cell epitope lacking peptide specificity.

Recognition of MHC + peptide complexes by TCRs is thought to involve a large surface formed by exposed residues from the bound peptide and from the alpha-helices of the MHC protein. This interaction appears to be essentially symmetrical in the positioning of the TCR relative to the MHC molecule. In this study the topology of HLA-B27 recognition by an alloreactive TCR, 64.8P, has been analyzed with a panel of site-specific mutants that have changes at multiple positions along the peptide binding site of HLA-B27. Abrogation of transfectant target cell lysis by CTL 64.8P was obtained only with some mutations in the peptide side chain binding pockets A and B, whereas little or no effect was observed with mutations outside these pockets. CTL 64.8P efficiently lysed murine transfectant cells, including HLA-B27+ RMA-S cells. Recognition of this latter transfectant was more efficient upon increased HLA-B27 expression at 26 degrees C. The uneven distribution of mutations affecting HLA-B27 allorecognition by CTL 64.8P strongly suggests an asymmetrical topology in the interaction of this TCR with HLA-B27, in which most of the binding energy is provided by contacts with HLA-B27 and/or peptide residues located close to pockets A and B, with little contribution from other areas of the MHC or peptide molecules. Its conservation in RMA-S cells further suggests that the epitope recognized by CTL 64.8P is either peptide-independent or requires any of a set of peptides having the same amino-terminal residues.

Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation.

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B-/- or Cat D-/- antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B-/- splenocytes, as it did in Cat D-/- cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.

Modulation of peptide binding by HLA-B27 polymorphism in pockets A and B, and peptide specificity of B*2703.

The results in this study address three aspects of peptide binding to the disease-associated antigen HLA-B27 and its modulation by polymorphism: the contribution of major anchor residues 2 and 9, the role of pocket B polymorphism in modulating peptide specificity, and the binding properties of B*2703, a subtype not found to be associated with spondyloarthropathy. Synthetic analogs of peptides naturally presented by B*2705 were used to demonstrate that residue 2 is essential, since Ala2 analogs bound marginally to B*2705, but the specificity of B*2705 for Arg2 is not absolute, and show that the contribution of basic residue 9 to binding was significant, but less than Arg2. The effect of single mutations in the B pocket was to decrease or--with the Glu > Met-45 mutation--totally shift pocket B specificity for Arg2 towards other residues at this position. This was shown by quantitating the relative binding of Gln2 and Ala2 analogs, and by pool-sequencing of the peptides bound in vivo to these mutants. Peptides naturally presented by B*2705 apparently bound with a lower affinity to pocket A variants with altered hydrogen bonding to the peptide N terminus, including B*2703. Binding of peptide analogs with changes at positions 2 or 9 suggested that in B*2703 pocket A, interactions are weaker and pocket B interactions are stronger than in B*2705. This can be explained by the effect of the unique His59 change in B*2703 in both pockets. Thus, B*2703 is probably the HLA-B27 sub-type with the most stringent specificity for the Arg2 peptide motif.

T-cell receptor usage in alloreactivity against HLA-B*2703 reveals significant conservation of the antigenic structure of B*2705.

B*2703 is an exceptional HLA-B27 molecule in that it differs from the most common B*2705 subtype by a unique amino acid change (His59) altering N-terminal peptide anchorage. To assess how this unusual feature affects the antigenic structure of HLA-B27, TCR usage by alloreactive CTL raised against B*2703 from two individuals was analyzed. Only few CTL recognized B*2703 from two individuals was analyzed. Only few CTL recognized B*2703 but nor ot at a lower level B*2705. Limited heterogeneity of these CTL was revealed by: 1) identity of TCR in two pairs of such CTL clones, 2) identity of beta chains, paired to distinct alpha chains, in two clonotypes, and 3) almost identical fine specificity of these two clonotypes with site-specific HLA-B27 mutants. These results indicate that B*2703 "private" epitopes are rare. TCR usage among anti-B*2703 CTL was analogous as in anti-B*2705 responses in the predominant and donor-independent usage of V beta segments from homology subgroup 4, more moderate and donor-dependent V alpha skewing, N+D beta diversity limited by motifs shared among clonotypes, and restricted J alpha heterogeneity. Homology of N+D beta motifs and J alpha segments of anti-B*2703 with anti-B*2705 TCR suggested significant sharing of peptide-associated epitopes between both subtypes. The results indicate that allospecific TCR are recruited by B*2703 following similar rules as in the anti-B*2705 response, and suggest that the B*2703 change keeps unaltered much of the antigenic structure of the molecule relative to B*2705. Therefore, most of the peptides bound to B*2703 should be the same and keep a similar conformation as in B*2705.