Epithelial barriers on mucosal surfaces at different sites in the body differ dramatically in their cellular organization, and antigen sampling strategies at diverse mucosal sites are adapted accordingly. In stratified and pseudostratified epithelia, dendritic cells migrate to the outer limit of the epithelium, where they sample antigens for subsequent presentation in local or distant organized lymphoid tissues. In simple epithelia, specialized epithelial M cells (a phenotype that occurs only in the epithelium over organized lymphoid follicles) deliver samples of foreign material by transepithelial transport from the lumen to organized lymphoid tissues within the mucosa. Certain pathogens exploit the M cell transport process to cross the epithelial barrier and invade the mucosa. Here we review the features of M cells that determine antigen and pathogen adherence and transport into mucosal lymphoid tissues.

Antigen sampling across epithelial barriers and induction of mucosal immune responses

Mucosal antibody defense depends on a complex cooperation between local B cells and secretory epithelia. Mucosa-associated lymphoid tissue gives rise to B cells with striking J-chain expression that are seeded to secretory effector sites. Such preferential homing constitutes the biological basis for local production of polymeric immunoglobulin A (pIgA) and pentameric IgM with high affinity to the epithelial pIg receptor that readily can export these antibodies to the mucosal surface. This ultimate functional goal of mucosal B-cell differentiation appears to explain why the J chain is also expressed by IgG- and IgD-producing plasma cells (PCs) occurring at secretory tissue sites; these immunocytes may be considered as 'spin-offs' from early effector clones that through class switch are on their way to pIgA production. Abundant evidence supports the notion that intestinal PCs are largely derived from B cells initially activated in gut-associated lymphoid tissue (GALT). Nevertheless, insufficient knowledge exists concerning the relative importance of M cells, major histocompatibility complex class II-expressing epithelial cells, and professional antigen-presenting cells for the uptake, processing, and presentation of luminal antigens in GALT to accomplish the extensive and sustained priming and expansion of mucosal B cells. Likewise, it is unclear how the germinal center reaction in GALT so strikingly can promote class switch to IgA and expression of J chain. Although B-cell migration from GALT to the intestinal lamina propria is guided by rather well-defined adhesion molecules and chemokines/chemokine receptors, the cues directing preferential homing to different segments of the gut require better definition. This is even more so for the molecules involved in homing of mucosal B cells to secretory effector sites beyond the gut, and in this respect, the role of Waldever's ring (including the palatine tonsils and adenoids) as a regional inductive tissue needs further characterization. Data suggest a remarkable compartmentalization of the mucosal immune system that must be taken into account in the development of effective local vaccines to protect specifically the airways, eyes, oral cavity, small and large intestines, and urogenital tract.

Mucosal B cells: phenotypic characteristics, transcriptional regulation, and homing properties.

▪ Abstract Infections often involve the mucosal surfaces of the body, which form a boundary with the outside world. This review focuses on immunoglobulin A (IgA) antibodies because IgA is the principal mucosal antibody class. IgA is synthesized by local plasma cells and has a specific polymeric immunoglobulin receptor-mediated transport mechanism for entry into the secretions. By serving as an external barrier capable of inhibiting attachment of microbes to the luminal surface of the mucosal epithelial lining, IgA antibodies form the first line of immune defense. In addition to this traditional mode of extracellular antibody function, recent evidence suggests that IgA antibodies can also function in a nontraditional fashion by neutralizing viruses intracellularly, if a virus is infecting an epithelial cell through which specific IgA antibody is passing on its way to the secretions. IgA antibodies are also envisaged as providing an internal mucosal barrier beneath the mucosal lining. Antigens intercepted b...

Interaction of antigens and antibodies at mucosal surfaces

The sensitivity of enzyme immunoassays, and of enzyme immunohistochemical techniques in general, depends mainly on the preparation of enzyme-antigen or enzyme-antibody conjugates possessing high enzymatic and immunological activity. The purpose of this paper is to review and to compare the various procedures described to date for the preparation of such conjugates. Coupling of small organic compounds with enzymes is a special procedure, and in this paper only the preparation of enzymelabelled proteins will be considered. The coupling of an enzyme with a protein involves the use of a cross-linking agent. The cross-linking agent reacts via its active groups (at least two) with the functional groups present in enzymes and proteins, and this is how coupling is achieved. Functional groups in proteins include amino, imino, hydroxyl, phenol, and thiol groups; their reactivity depends mainly on their microenvironment. These functional groups may be part of or contribute to the active site of enzymes, antibodies, and antigens. If this is the case, coupling via such a group can abrogate biological activity. These functional groups also contribute to the maintenance of the native conformation of the molecules; a crucial modification by interor intramolecular binding may lead to loss of biological activity. Furthermore, over-substitution can lead either to inaccessibility of the active site or to an increase in the nonspecific adherence of the conjugated macromolecules. Since functional groups present in proteins, including enzymes, are identical, it is evident that selective coupling producing an exclusive homogeneous enzyme-protein conjugate is too difficult to achieve. In fact, all coupling reactions reported so far have always produced a mixture of heterogeneous enzyme-protein conjugates and enzyme-enzyme and or protein-protein conjugates. Even under conditions where the preparation of a homogeneous 1:l molar ratio of peroxidase-antibody conjugate was highly favoured, the simultaneous preparation of minor amounts of other conjugates and of peroxidase dimers could not be avoided. From the above, it is evident that an effective coupling procedure is certainly not easy to devise and that even when a procedure has been shown to produce satisfactory results in any given system, its extrapolation to another system calls for circumspection. Two types of coupling reactions have been described. The first involves one-step reactions and the other two-step reactions. In one-step procedures the enzyme, the cross-linking agent and the protein are all mixed together and allowed to react. In this procedure the reaction is difficult to control, chiefly because the reaction rates of the functional group in enzyme and protein are different. This may lead to selective polymerization of either enzyme or protein. Conjugates prepared with one-step procedures are basically heterogeneous. Theoretically, a wider variety of enzyme-protein conjugates can be prepared with the one-step than with the two-step procedures. In two-step procedures the enzyme (or antigen or antibody) is first treated with the cross-linking agent and then the antigen or the antibody (or enzyme) is added. Alternatively, the enzyme is treated with an excess of cross-linking agent, then the excess reagent is removed, and finally the activated enzyme is allowed to react with the antigen or antibody. Two-step reactions make use of reagents possessing two potentially active groups, the reactivity of which can, however, be

Coupling of Enzymes to Antibodies and Antigens

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of COVID-19. Although potent immunoglobulin G (IgG) antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might affect the initial viral spread and transmissibility from the mucosa. Here, we characterize the IgA response to SARS-CoV-2 in a cohort of 149 convalescent individuals after diagnosis with COVID-19. IgA responses in plasma generally correlated with IgG responses. Furthermore, clones of IgM-, IgG-, and IgA-producing B cells were derived from common progenitor cells. Plasma IgA monomers specific to SARS-CoV-2 proteins were demonstrated to be twofold less potent than IgG equivalents. However, IgA dimers, the primary form of antibody in the nasopharynx, were, on average, 15 times more potent than IgA monomers against the same target. Thus, dimeric IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.

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Enhanced SARS-CoV-2 neutralization by dimeric IgA.

Immunization of mice with an attenuated Salmonella typhimurium strain (Phopc) carrying a plasmid encoding a hybrid form of the hepatitis B virus core antigen (HBc) induced specific antibody responses against the bacterial lipopolysaccharide (LPS) and HBc. Different mucosal routes of immunization, i.e., oral, nasal, rectal, and vaginal, were compared for their ability to induce a systemic as well as a mucosal response at sites proximal or distant to the site of immunization. Anti-LPS and anti-HBc immunoglobulin A (IgA) antibodies were measured in saliva, in feces, and in genital, bronchial, and intestinal secretions. Specific antibodies in serum and secretions were observed after immunization via all routes; however, the response to LPS was independent of that against HBc. In serum, saliva, and genital and bronchial secretions, high amounts of anti-HBc IgA were obtained by the nasal route of immunization. Vaginal immunization resulted in two different responses in mice: high and low. We observed a correlation between the level of specific immune response and the estrous status of these mice at the time of immunization. Rectal immunization induced high amounts of IgA against HBc and LPS in colonorectal secretions and feces but not at distant sites. These data suggest that S. typhimurium is able to invade different mucosal tissues and induce long-lasting local IgA responses against itself and a carried antigen after a single immunization.

https://iai.asm.org/content/iai/63/9/3279.full.pdf

A recombinant Salmonella typhimurium vaccine induces local immunity by four different routes of immunization.

The efficiency of small enzyme-labeled tracers for the demonstration of intracellular antigen was investigated in tissues fixed with picric acid-formaldehyde. The influence of fixation on the immunological activity was tested in vitro by radial immunodiffusion. The experimental model consisted of newborn pig jejunum after absorption of ferritin from the intestinal lumen. Ferritin was located after 1 hr in vacuoles scattered in the cytoplasm of the absorptive cells and represented an easily recognizable intracellular antigen. After immunohistochemical treatments with antiferritin preparations, the distribution of labeling enzyme reaction product was examined by morphometry. The ratio of the labeled volume to the total volume of vacuoles containing ferritin indicated the degree of specific labeling of the antigen. In both direct and indirect methods, the degree of labeling was low when enzyme-labeled immunoglobulin G was the tracer. With antigen binding fragments (Fab), the labeling was significantly increased. In the indirect method, the degree of labeling was influenced by the first-step reagents. Onlywhen the serum titer was optimum was a high degree of labeling obtained. With antigen binding fragments or papain-digested serum the effect of the titer was negligible and maximum labeling was achieved. In both methods, with peroxidase as the labeling enzyme, a diffuse nonspecific deposition of reaction product was observed. This could be avoided by using cytochrome c instead.

https://rupress.org/jcb/article-pdf/50/2/432/1385766/432.pdf

Ultrastructural localization of intracellular antigen using enzyme-labeled antibody fragments

Parafollicular cells in mammalian thyroid glands are thought to be responsible for the secretion of calcitonin. In this study, calcitonin was localized in pig thyroid gland by an indirect immunocytochemical technique using rabbit antiserum directed against synthetic porcine calcitonin for the first step, and sheep Fab fragments prepared against rabbit Fab and coupled to cytochrome c for the second step. The antigenic determinants of calcitonin were present only in the parafollicular cells, whose secretory granules were heavily labeled. Labeling of the cytoplasmic matrix is thought to indicate a possible leakage of the polypeptide from the granules. A striking observation was the complete absence of labeling in the cisternae of the rough-surfaced endoplasmic reticulum and of the Golgi apparatus. It is concluded that the secretory granules of parafollicular cells contain calcitonin; the mechanism of synthesis of this peptide is not clearly understood.

/pdf/ultrastructural-localization-of-calcitonin-in-the-3njtu4la2c.pdf

Ultrastructural localization of calcitonin in the parafollicular cells of pig thyroid gland with cytochrome c-labeled antibody fragments.

The silver nitrate technique developed by Grimelius was slightly modified in order to obtain a reproducible and simple method for the staining of C cells in the thyroid gland. The reliability of this method was demonstrated by comparing its results with those of immunofluorescent, cytochemical and ultrastructural studies. This staining procedure gives good and permanent results even when applied to tissues first fixed several hours after death. Its use allows the routine evaluation of the number and distribution of C cells in human and animal tissues.

P. B. De Grandi

Papers

A recombinant Salmonella typhimurium vaccine induces local immunity by four different routes of immunization.

Ultrastructural localization of intracellular antigen using enzyme-labeled antibody fragments

Ultrastructural localization of calcitonin in the parafollicular cells of pig thyroid gland with cytochrome c-labeled antibody fragments.

The routine demonstration of C cells in human and animal thyroid glands. Value of a simple silver stain.