Differential expression of tissue-specific adhesion molecules on human circulating antibody-forming cells after systemic, enteric, and nasal immunizations. A molecular basis for the compartmentalization of effector B cell responses.

Expression of the adhesion molecules CD44, L-selectin (CD62L), and integrin alpha 4 beta 7 by antibody-secreting cells (ASC) was examined in human volunteers after oral, rectal, intranasal, or systemic immunization with cholera toxin B subunit. Almost all blood ASC, irrespective of immunization route, isotype (IgG and IgA), and immunogen, expressed CD44. On the other hand, marked differences were observed between systemically and intestinally induced ASC with respect to expression of integrin alpha 4 beta 7 and L-selectin, adhesion molecules conferring tissue specificity for mucosal tissues and peripheral lymph nodes, respectively. Thus, most ASC induced at systemic sites expressed L-selectin, whereas only a smaller proportion of ASC expressed alpha 4 beta 7. In contrast, virtually all IgA- and even IgG-ASC detected after peroral and rectal immunizations expressed alpha 4 beta 7, with only a minor fraction of these cells expressing L-selectin. Circulating ASC induced by intranasal immunization displayed a more promiscuous pattern of adhesion molecules, with a large majority of ASC coexpressing L-selectin and alpha 4 beta 7. These results demonstrate that circulating ASC induced by mucosal and systemic immunization express different sets of adhesion molecules. Furthermore, these findings provide for the first time evidence for differential expression of adhesion molecules on circulating ASC originating from different mucosal sites. Collectively, these results may explain the anatomical division of mucosal and systemic immune responses in humans as well as the compartmentalization of mucosal immune responses initiated in the upper vs. the lower aerodigestive tract.     

High frequency of spontaneous interferon-gamma-producing cells in human tonsils: role of local accessory cells and soluble factors.

The frequency of mononuclear cells (MNC) spontaneously secreting interferon-gamma (IFN-gamma) has been examined in freshly isolated cell suspensions from human palatine tonsils. Two-site reverse enzyme-linked immunospot (ELISPOT) analyses, involving short term (20 h) incubation of MNC in the absence of any added exogenous stimulus, revealed that tonsillar MNC suspensions contain exceptionally large numbers of cells secreting IFN-gamma. No significant differences were observed when comparing the frequency of IFN-gamma-producing cells between cell suspensions obtained from hyperplastic and tonsillitis specimens. Cell-sorting experiments disclosed that spontaneous tonsillar IFN-gamma production was essentially contributed by CD4+ T cells, and required the presence of accessory cells and/or soluble factors to be detected. Thus, depletion of plastic adherent cells or monocytes from the tonsillar MNC suspensions resulted in reduced numbers of detectable IFN-gamma-secreting cells. Addition of very small numbers of autologous monocytes restored spontaneous IFN-gamma production in tonsillar MNC cultures depleted of monocytes. Neutralization of endogenous IL-1 beta and IL-2, as well as blocking of the IL-2 receptor, also decreased IFN-gamma production from unfractionated tonsillar cells. Addition of exogenous IL-1 beta restored IFN-gamma production in cultures of tonsillar MNC depleted of plastic adherent cells. Furthermore, IL-1 beta synergized with IL-2 by tonsillar MNC depleted of plastic adherent cells. Furthermore, IL-1 beta synergized with IL-2 by increasing intracellular as well as cell-free levels of IFN-gamma in cultures of unfractionated tonsillar MNC. This study further establishes that the tonsils are highly active immunological organs containing large numbers of T cells spontaneously producing IFN-gamma whose detection is contingent upon the presence of functional accessory cells. It also demonstrates that concomitant production of IL-1 beta and IL-2 occurs in tonsils and is necessary to maintain ongoing synthesis and extracellular accumulation of IFN-gamma in these organs.     

alpha4 integrins and L-selectin differently orchestrate T-cell activity during diabetes prevention following oral administration of CTB-insulin

Oral administration of insulin conjugated to the B chain of cholera toxin (CTB-insulin) in non-obese diabetic (NOD) mice results in diabetes prevention. We investigated the respective contributions of L-selectin (CD62L) and alpha4-integrin pathways during CTB-driven tolerance. Purified CD62L+CD4+ cells from CTB-insulin fed mice significantly reduced the capacity of diabetogenic T cells to transfer diabetes in syngeneic recipients. In vivo antibody blockade of fed animals during adoptive co-transfer experiments indicated that both CD62L and alpha4-integrins pathways were necessary to develop a protective response after oral tolerance induction. In contrast, when antibodies were given to recipient mice, only CD62L was critical for the protection. In vitro stimulated CD62L+CD4+ cells from the spleen of fed animals secreted lower amounts of IL-4 and IL-10 but comparable levels of TGFbeta than CD62L-cells. A reduced IFN-gamma production between the two cell subsets was specifically observed in CTB-insulin fed mice. Furthermore, antibody treatments induced changes in T-cell migration to the spleen, mesenteric and pancreatic lymph nodes. The protective effect was also associated with migration of regulatory T cells into pancreatic islets. Taken together, our results suggest that L-selectin and alpha4-integrin have distinct but complementary roles in the generation and function of regulatory CD4+ T cells following CTB-insulin administration.     

Induction of compartmentalized B-cell responses in human tonsils.

The capacity of tonsillar and nasal mucosal lymphoid tissues to serve as induction sites of local and/or distant B-cell responses in humans has been examined. The frequencies of vaccine-specific antibody-secreting cells (ASC) in cell suspensions from palatine tonsils (PT) and adenoids were determined after local (intra-tonsillar [i.t.]) and regional (intranasal [i.n.]) immunizations as well as peroral and parenteral immunizations with cholera and tetanus toxoids. While peroral and parenteral immunizations evoked negligible ASC responses in PT, i.t. vaccination induced a substantial ASC response which consisted of immunoglobulin G (IgG) and IgA ASC. Responses were highly restricted to immunized tonsils. Primary immunization in one PT followed by a second immunization of both PT evoked a larger ASC response in the primed tonsil. The latter ASC response was associated with higher frequencies of ASC precursors in primed tonsils. Furthermore, two i.n. immunizations induced only modest ASC responses in PT, although such immunizations evoked high ASC responses in adenoids. However, both i.t. and i.n. routes of immunization induced specific peripheral blood ASC responses, suggesting that a fraction of B cells activated in tonsils or in nasal mucosa may enter the circulation and disseminate to distant organs. These blood ASC responses preceded increases in both IgA and IgG antibody titers in nasal washes and serum samples. However, vaccine-specific ASC were not detected in duodenal cell suspensions from volunteers who had received i.t. or i.n. immunizations. Collectively, these results indicate that tonsils can serve as expression sites of locally induced antibody responses and support the development of immunological memory. Furthermore, tonsils may serve as powerful inductive sites for immune responses expressed in the upper aerodigestive tract.     

Detection and specificity of antibodies secreted by spleen cells in mice immunized with Streptococcus mutans.

Immune responses of mice to Streptococcus mutans serotype c were analyzed by means of the enzyme-linked immunospot assay to determine the predominant specificities of the antibodies developed. In general, the numbers of splenic antibody-secreting cells correlated with serum antibody levels. A low dose (10(8) CFU) of killed whole cells injected twice intraperitoneally induced antibodies mainly against surface protein antigen I/II. A higher dose (10(9) CFU) given two to six times also resulted in a predominance of antigen I/II antibody-secreting cells and, in addition, antibody responses to surface protein antigen III and lipoteichoic acid occurred. Cells producing antibodies to serotype c polysaccharide were elicited only on repeated immunization. These results agreed with the development of antibodies in rabbits repeatedly immunized intravenously with killed whole cells of S. mutans, S. rattus, and S. sobrinus, which induced specific antibodies in accordance with the surface antigens that they express. Mice immunized twice with the same dose of purified antigens I/II and III developed greater numbers of antigen I/II splenic antibody-forming cells than antigen III splenic antibody-forming cells and higher serum antibody levels to antigen I/II than to antigen III. Furthermore, a single injection of antigen I/II but not of antigen III was sufficient to induce a strong specific-antibody response. Some evidence was also obtained for weak polyclonal stimulation of spleen cells by S. mutans cells and by antigen I/II, a result which could be relevant to the induction by S. mutans of antibodies reactive with mammalian tissues. It was concluded that for the antigens examined, S. mutans elicited the strongest antibody response against antigen I/II, which was also highly immunogenic in purified form.     

A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody-secreting cells

A novel assay system has been developed which is based on the ELISPOT methodology and employs a combination of two immunoenzyme visualization systems yielding distinct colour products. This variation permits the simultaneous enumeration of two different types of cell secreting antigenically distinct products. Optimal conditions for the concurrent detection of human mononuclear cells secreting IgG or IgA antibodies are described.     

Reverse enzyme-linked immunospot assay (RELISPOT) for the detection of cells secreting immunoreactive substances

A reverse modification of the recently described enzyme-linked immunospot assay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies. As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated. Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested.     

Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues.

Generation of local and systemic immune responses by the oral administration of antigens is frequently inefficient, requiring large quantities of immunogens and yielding only modest antibody responses. In this study, we have demonstrated that oral administration of microgram amounts of Streptococcus mutans protein antigen I/II covalently coupled to the B subunit of cholera toxin elicits vigorous mucosal as well as extramucosal immunoglobulin A and G antistreptococcal antibody responses in mice. These responses were manifested by the presence of large numbers of antibody-secreting cells in salivary glands, mesenteric lymph nodes, and spleens and by the development of high levels of circulating antibodies. This novel immunization strategy may find broad application in the construction of oral vaccines for the control of infectious diseases caused by pathogens encountered at mucosal and extramucosal sites.     

Human circulating specific antibody-forming cells after systemic and mucosal immunizations: differential homing commitments and cell surface differentiation markers

Circulating spontaneous antibody-secreting cells (ASC) induced by mucosal and systemic immunizations in human volunteers have been characterized with respect to differentiation stage and homing commitments. Irrespective of the immunization route, the large majority of ASC co-expressed CD19 and HLA-DR, which are normally lost during the transition of plasmablasts to plasmocytes, as well as CD38, a marker of activated B cell blasts, expressed also by plasmocytes. However, these cells expressed neither CD28, a molecule acquired by plasmocytes, nor CD22 and CD37, which are lost during the transition of plasmablasts to plasmocytes. Therefore, the large majority of ASC found in peripheral blood after oral and parenteral immunizations are terminally differentiated B cells, but not fully differentiated plasmocytes. As a whole, the mucosally derived ASC population seemed to be more homogenously differentiated. CD25 was detected on few ASC, whereas ASC expressing CD71 were more numerous, especially among systemically derived ASC. Almost all ASC expressed the adhesion molecules CD44 and α4-integrins, irrespective of immunization route. However, virtually all systemically derived ASC expressed L-selectin, recognizing the peripheral lymph node addressin, whereas only a minority of mucosally induced blood ASC expressed L-selectin. These studies are the first to demonstrate in humans that circulating precursors of mucosal B cell immunoblasts utilize organ-specific recognition mechanisms distinct from those of corresponding systemic B cells and appear to be more advanced in the B lineage maturation pathway. Specialization of receptor expression could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from non-mucosal immune mechanisms in humans.     

Amplified ELISPOT assay for the detection of HIV-specific antibody-secreting cells in subhuman primates.

A novel immunoenzyme amplification technique has been evaluated in an ELISPOT assay for the detection of antigen-specific antibody-secreting cells (ASC) in monkeys. In this assay, mononuclear cells containing putative ASC are incubated for a few hours in antigen-coated wells. Following removal of the cells, zones of solid phase bound antibodies secreted by individual ASC are visualized in four consecutive steps. First, a primary biotinylated anti-immunoglobulin (Ig) reagent is added followed by enzyme-labelled avidin. The amplification procedure comprises the addition of biotinylated anti-enzyme antibodies in the third stage, followed by enzyme-conjugated avidin and substrate. When evaluated in a modified ELISPOT assay for the detection of simian B cells secreting antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1), this amplification procedure proved to be suitable even when using anti-human Ig antisera as primary antibody reagents. This development should be useful for other ELISPOT assays where species specific anti-Ig reagents are not always available and, most importantly, for enumerating cells producing immunoreactive substances in such minute amounts that they may escape detection by conventional ELISPOT assays. Furthermore, a functional simian HIV-specific ELISPOT assay could prove valuable for assessing the humoral immunogenicity of future candidate vaccines against the acquired immunodeficiency syndrome (AIDS).