Antigen-specific Plasma Cells Research Articles

Is There Evidence That the Gut Contributes to Mucosal Immunity in Humans?

Our understanding of the common mucosal immune system derives from animal studies. Antigen-sensitized lymphocytes in the gut-associated lymphoid tissue (GALT) migrate via the blood to mucosal tissues to generate the mucosal-associated lymphoid tissue (MALT). In these sites, B cells differentiate into plasma cells and produce antigen-specific secretory IgA, the principal specific immune antiviral and antibacterial defense of moist mucosal surfaces. Responses to oral intake seem necessary to actively maintain this system in health. Experimentally, lack of enteral stimulation with parenteral feeding alters GALT and MALT size and function. These alterations disturb intestinal and extraintestinal mucosal immunity. This review is an overview of current and classical studies demonstrating the human mucosal immune system and interactions with nutrition. Human evidence of the mucosal immune system exists, although most data are indirect. Gut stimulation after oral intake induces a generalized immune response in the human MALT through a mucosal-immune network. Examples include neonatal development of GALT influenced by enteral feeding, the presence of antigen-specific IgA and antigen-specific IgA-secreting plasma cells in distant mucosal effector sites such as the breast after gut luminal antigen exposure, and isolation of IgA-producing cells from circulating blood. It is unlikely that clinical studies will ever completely define the effect of route of feeding in all patient populations. This may be possible, however, if investigators understand, define and characterize nutrition-dependent immunologic mechanisms, allowing clinicians to examine clinical responses to nutrition in specific patient populations. This might allow generation of new approaches to protect mucosal immunity.

Immunization-Induced Perturbation of Human Blood Plasma Cell Pool: Progressive Maturation, IL-6 Responsiveness, and High PRDI-BF1/BLIMP1 Expression Are Critical Distinctions between Antigen-Specific and Nonspecific Plasma Cells

The present study shows that reimmunization with tetanus toxoid (tet) caused a transient increase of the human blood plasma cell (PC) pool, detectable from 6th to 15th day postboost, as well as the temporal alteration of several PC features. Labeling of specific PC with FITC-tet C fragment (tetC) allowed kinetics analysis of the tetC(+) and tetC(-) PC, and revealed remarkable differences between them: 1) the kinetics of tetC(+) PC occurrence was exponential, and most of them appeared in a narrow time frame (5th to 8th day postboost), whereas the tetC(-) PC increase was lower (three to five times) and more prolonged (4th to 15th day postboost). 2) The tetC(+) PC subset contained a fraction of cycling cells, expressed high levels of DR, CD138, and CD126, and responded to IL-6 by improving their survival and Ig secretion; in contrast, the tetC(-) PC showed higher CXCR4 and lower DR and CD138, did not respond to IL-6, and contained a fraction of apoptotic cells. 3) Sequential phenotypic analysis revealed maturational changes within the tetC(+), but not tetC(-), PC subset; sequencing of tetC(+) PC IgVH genes showed clear features of Ag selection. 4) The tetC(+) PC expressed several times more positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1 transcription factor than the tetC(-) PC. 5) The tetC(-) PC and bone marrow resident PC similarly expressed low DR and high CXCR4, but differed in that the latter exhibited higher levels of CD31, CD138, and positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1. These findings support the view that tetC(+) PC contain bone marrow PC precursors, and tetC(-) PC probably belong to a removable compartment of aged PC.

Dendritic cell-based therapies in the bench and the bedsides.

Antigen-presenting dendritic cells (DCs) represent trace population of leukocytes that are widely distributed over the whole body. DCs are regarded as inducer of antigen-specific adaptive immune responses. However, various types of functions of DCs are now exposing. In the steady-state, DCs induce immunogenic tolerance to self antigens and harmless entities and thus maintain normal homeostasis. On the other hand, in presence of non-self and dangerous entities, DCs play a cardinal role in the induction of innate immunity by producing type-1 interferons. DCs are also essential for the development of antigen-specific B-lymphocytes and plasma cells. Infection, depletion and dysfunction of DCs have been reported from patients with chronic microbial infections. Impaired functions of DCs have also been shown from patients with autoimmune diseases and allergic diseases. Patients with cancers also have phenotypic and functional impairment of DCs. These observations inspired optimism of using DC-based therapy for treating different pathological conditions. DC-based therapies showed excellent therapeutic potential in animal model of human diseases. The efficacy of DC-based therapy has not been properly evaluated in patients with different diseases. However, some clinical trials indicate that administration of antigen-pulsed DCs might be safe and possibly effective. In this review, we would first provide a description about the nature and functions of DCs that have been taught from the laboratory benches. Next, we would discuss how the information taught in the laboratory benches has been applied in patient's bedsides.

Loss of CD154 impairs the Th2 extrafollicular plasma cell response but not early T cell proliferation and interleukin-4 induction.

Ligation of CD40 by CD4 T cells through CD154 is key both to germinal centre induction and follicular T-dependent Ig class switching, but its requirement for aspects of T cell priming and extrafollicular antibody responses is less clear. Here comparison of the T helper (Th) type 2 response in lymph nodes from wild-type mice and CD154-deficient mice after immunization with alum-precipitated antigen reveals selective effects of this immunodeficiency. The timing and magnitude of the early interleukin (IL)-4 induction and proliferation in T cells of the T zone were unaltered by CD154 deficiency. As expected, germinal centres were not induced. Additionally the T-dependent extrafollicular antibody response, which initially requires T cell help but expands without further T cell involvement, was severely curtailed. The median number of extrafollicular antigen-specific plasma cells was 370-fold lower in CD154-deficient mice. Of these plasma cells the median proportion that had switched to IgG1 was <5%, while in wild-type mice the proportion was 89%. Surprisingly, some CD154-deficient lymph nodes showed substantial switching to IgG1. Commensurately, increases in gamma1 germline transcripts and Blimp-1 mRNA were observed, albeit significantly lower than in controls, but activation-induced cytidine deaminase mRNA was undetectable in CD154-deficient mice. These experiments demonstrate that the acquisition of some T cell priming characteristics can be CD154-independent; in contrast, T-dependent extrafollicular responses require CD154. Thus functional CD154 ligation during the first encounter of T cells and B cells in the T zone is critical for follicular and extrafollicular antibody responses.

Developmentally Distinct Th Cells Control Plasma Cell Production In Vivo

Differential Ly6C expression identifies a major phenotypic division in CD44loCD62LhiCD4+ Th cells. Using two separate models of single subset adoptive transfer, we demonstrate the unique capacity of Ly6Chi Th cells to promote antigen-specific plasma cell production in vivo. In contrast, both compartments support germinal center formation and proliferate to equivalent levels upon TCR triggering in vivo and in vitro. Developmentally, CD4+CD8- thymocytes leave the thymus expressing low levels of Ly6C; 3 days later approximately 50% stably upregulate Ly6C without cell division or TCR engagement in the periphery. Interestingly, antigen-specific Th cell clonotypes unevenly assort into these peripheral compartments, creating separate TCR repertoires that underpin peripheral functional diversity. Taken together, these data reveal a developmentally distinct Ly6Chi naive Th cell compartment subspecialized to regulate plasma cell production in vivo.

Analysis of the germinal center reaction and in vivo long-lived plasma cells.

This chapter describes the analysis of long- and short-lived plasma cells on tissue sections. Mice are immunized with 4-hydroxy-3-nitrophenyl acetyl (NP) coupled to a T-cell-dependent carrier. Antigen-specific germinal center cells and extrafollicular plasma cells are identified by immunohistology on tissue sections. Plasma cells labeled with 5-bromo-2'-deoxyuridine (BrdU) pulses given during different phases of B-cell response are identified on spleen sections. To identify mutated cells originating from cells in germinal centers, antigen-specific cells from spleen sections are isolated by microdissection. From these NP-specific recombined VDJ genes are amplified with family-specific primers by polymerase chain reaction (PCR) for deoxyribonucleic acid (DNA) sequencing. The methods described can be used to characterize origins and life-span of plasma cells in vivo.

In vivo manipulation of endogenous metallothionein with a monoclonal antibody enhances a T-dependent humoral immune response.

Metallothionein (MT) is a small stress response protein that can be induced by exposure to heavy metal cations, oxidative stressors, and acute phase cytokines that mediate inflammation. In previous experiments, we have shown that exogenous MT can affect cell proliferation, macrophage and cytotoxic T lymphocyte function, and humoral immunity to T-dependent antigens. In the studies described here, we have explored the effect of a monoclonal anti-MT antibody (clone UC1MT) on the role that endogenous MT plays in the humoral immune response. In vivo injection of UC1MT significantly increased the humoral response to simultaneous challenge with ovalbumin (OVA). In contrast, mice immunized with OVA in the presence of an isotype-matched antibody control (MOPC 21) showed no change in the anti-OVA humoral response. The predominant anti-OVA response that was enhanced by UC1MT treatment was the IgG(1) response; the IgG(2a) anti-OVA response was not altered by UC1MT treatment. UC1MT treatment increased the numbers of IgG anti-OVA secreting cells as measured by ELISPOT assay, suggesting that blocking the effects of MT synthesized during the immune response augments the differentiation of antigen-specific plasma cells. The percentages of T and B cells in the spleens of animals from each treatment group were not significantly different, suggesting that this regimen of UC1MT treatment does not significantly affect hematopoiesis, but rather alters antigen-induced differentiation of lymphocytes. These observations are compatible with previous results from our laboratory that suggest that endogenous MT synthesized during the normal immune response or as a consequence of toxicant exposure suppresses in vivo immune function. In light of the fact that significant amounts of MT can be synthesized during toxicant exposure, manipulation of MT levels with an anti-MT antibody may ultimately represent an important therapeutic approach to the treatment of immune dysfunctions that result from toxicant exposure.

Immunohistochemical Study of Interstitial Keratitis in an Animal Model

Purpose: We performed an immunohistochemical study on the development of interstitial keratitis in a rabbit model sensitized by ovalbumin (OA). Methods: A mixture of OA (5 μg/mL) and Freund's complete adjuvant was initially injected subcutaneously (0.4 mL) into the rabbit's back and footpad (0.2 mL). After a week, the rabbits were sensitized by an injection of the same solution into the back (0.5 mL) again. After 4 weeks the rabbits underwent the same treatment as a booster shot. Then a week later OA solution (0.05 mL) was injected into the corneal stroma. We observed the cornea on days 1–3, 7, and 10 after the corneal treatment. Corneas on days 3 and 10 were examined by immunohistological methods using hematoxylin-eosin stain, methylgreen pyronin stain, and by immunohistochemical methods with anti-CD 4, anti-CD 8, and anti-OA antibodies. The localization of specific antibodies was identified by fluorescent-antigen method. Electron-microscopic observation was also done. Results: Rabbits developed corneal opacity and edema on day 1, corneas had on immune ring with a peak intensity on day 3, and vascularization on day 7 after corneal treatment. Microscopic examination revealed infiltration of neutrophils into the area of the immune ring on day 3 and many plasma cells at the limbus and stroma on day 10. CD 4 positive cells were found at the limbus and stroma on days 3 and 10. CD 8 positive cells were found at the limbus and stroma only on day 10. OA positive findings were observed at the immune ring and its inner area. In the fluorescent-antigen method, specific antibodies were found in plasma cells at the limbus on days 3 and 10. Conclusions: In our experimental model of interstitial keratitis with immune ring and vascularization, the Arthus reaction was dominant. Infiltration of antigen specific plasma cells in the stroma with vascularization caused a modification of pathologic reaction in keratitis.

Selective in vivo suppression of T lymphocyte responses in experimental measles virus infection.

During and after measles virus (MV) infection humans are highly susceptible to opportunistic infections because of a marked immunosuppressive effect of the virus. The mechanisms by which the virus induces this phenomenon is not well understood. In particular, detailed information is missing on the targets of suppression in relation to antigen-specific T and B cell responses. Because such studies require animal experiments, we used the cotton rat model, in which the MV causes a respiratory tract infection. Primary as well as secondary T cell responses were impaired in vivo and ex vivo by MV infection. The proliferation of T cells was greatly reduced, but their effector functions, such as cytolysis or cytokine secretion, were not. In contrast, primary and secondary B cell responses in vivo as measured by the frequency of antigen-specific plasma cells in an enzyme-linked immunospot (ELISPOT) assay were not altered by MV infection. Only the secretion of immunoglobulins was reduced slightly in animals primarily infected with MV after 2 weeks. These data demonstrate that MV-induced immunosuppression acts primarily on the T cell responses in vivo.

Multidrug resistance of a continuously differentiating monoclonal B lineage in the blood and bone marrow of patients with multiple myeloma.

Although multiple myeloma has long been regarded as a malignancy localized to the bone marrow, recent evidence clearly shows a monoclonal population of B cells in the blood of patients with myeloma both at diagnosis and during therapy. It is difficult however to determine whether or not these circulating tumor-related B cells are in fact malignant or if they are non-malignant relatives of the plasma cells in the bone marrow. Our working hypothesis predicts that circulating monoclonal B lineage cells are the precursors of the bone marrow plasma cells and that, contrary to well established dogma, traffic of myeloma cells travels from the blood to the bone marrow. We predict, and our data support the view, that B lineage cells localized in the bone marrow are predominantly end stage non-proliferating cells which have little or no propensity or capability to migrate, whose future options consist mainly of entry into apoptotic pathways and eventual death. If this view is correct, it predicts that the malignant stem cell in myeloma may be located outside the bone marrow. If the circulating monoclonal B lineage cells in blood are in fact malignant cells, then they either represent the stem cells themselves or are early descendents in the process of trafficking to the marrow where they will terminally differentiate. Spleen might be considered the most likely place of origin for myeloma as plasma cells of normal bone marrow originate in the spleen, migrate as antigen-activated B cells to the bone marrow via the blood and undergo terminal differentiation to antigen-specific plasma cells (1, 2), a pattern remarkably similar to that of the monoclonal B lineage in myeloma (3). Also consistent with this view is work indicating that the accumulation of plasma cells in normal bone marrow is the major site of antibody synthesis in the body, and increases with age (1). In myeloma, analysis of autopsy material indicated the least mature cells of the malignant lineage were in the spleen whereas considerably more terminally differentiated B lineage cells were found in blood (Jensen et al, submitted). This highly speculative hypothesis is a difficult one to test as events in the spleen would be predicted to occur in the earliest stages of the disease and cells might become extra-splenic even before diagnosis. Thus the properties and potential of the circulating blood B lineage cells have become an important target of investigation, and an even more crucial consideration in the design of new modes of therapy for myeloma patients.

Effects of hemorrhage and resuscitation on bacterial antigen-specific pulmonary plasma cell function

Nosocomial pneumonia is frequent after hemorrhage and trauma, and often contributes to multiple organ system failure, morbidity, and mortality in this setting. Although the percentages and numbers of resting pulmonary B cells (clonal precursors) able to be stimulated to produce antibodies to bacterial antigens are markedly decreased after hemorrhage, the effects of hemorrhage on the pulmonary plasma cells actually producing antibody to bacterial antigens have not been examined. To investigate this question, mice were bled 30% blood volume, then resuscitated with the shed blood 1 hr later. At predetermined times after hemorrhage, the mice were intranasally immunized with liposomes containing the bacterial polysaccharide antigen levan (from Aerobacter levanicum). One week later, lung lavages were performed to measure bacterial antigen-specific secretory immunoglobulin A (sIgA) titers and the numbers of intraparenchymal pulmonary plasma cells producing antibody against the bacterial antigen were determined. Reduced numbers of pulmonary plasma cells producing antibody against the immunizing bacterial polysaccharide antigen were found between 1 and 14 days after blood loss, and titers of bacterial antigen-specific secretory IgA were decreased for greater than 2 wks after hemorrhage. The importance of these abnormalities in pulmonary B-cell function was demonstrated by an increased susceptibility to Pseudomonas aeruginosa pneumonia in mice infected 4 days after hemorrhage, when bacterial antigen-specific pulmonary plasma cell numbers were at their lowest point. Resuscitated mice showed the same increased susceptibility to P. aeruginosa pneumonia as did hemorrhaged but unresuscitated animals. Hemorrhage, even if resuscitated, results in alterations in bacterial antigen-specific pulmonary B-cell function and secretory IgA production that are profound, long lasting, and associated with increased susceptibility to infection at this mucosal surface. Because these effects on pulmonary B-cell function do not occur immediately after hemorrhage, immunization techniques able to enhance bacterial antigen-specific secretory IgA titers at pulmonary surfaces may be able to increase resistance to nosocomial pneumonia if administered shortly after injury and blood loss.

Transfer of T or CD8 + Cells from Hemorrhaged Mice Produce Alterations in Bacterial Antigen Specific Plasma Cell Repertoires in Normal Syngeneic Recipients

Hemorrhage has multiple effects on immunologic response, including alteration of B cell repertoires and T cell function.This study examined possible relationships between these two phenomena by determining the effects of T cells and T cell subsets transferred from hemorrhaged donors into normal, unhemorrhaged syngeneic recipients on B cell repertoires.Mice given total T or CD8 + cells from hemorrhaged animals then immunized with the bacterial polysaccharide antigen levan had a decreased percentage of plasma cells producing antibody to levan compared to that in mice given T or CD8 + cells from unhemorrhaged animals.These effects of post hemorrhage CD8 + cells were not seen after transfer into nu/nu mice, indicating that these cells did not directly affect B cell function, but rather required other T cell populations in order to alter the B cell repertoire.These results demonstrate that hemorrhage-induced alterations in bacterial antigen specific B cell repertoires may result from T and CD8 + cell mediated changes in T-B interactions.

Effects of intravenous immunoglobulin on hemorrhage-induced alterations in plasma cell repertoires

Hemorrhage produces decreases in serum immunoglobulin (Ig) levels and alterations in the number and frequency of B cells producing antibodies against bacterial antigens. These abnormalities in immune response may contribute to the increased susceptibility to infection after injury and hemorrhage. To examine the relationship between serum Ig levels and bacterial antigen-specific plasma cell numbers and frequencies after blood loss, we treated hemorrhaged mice with intravenous Ig (IVIG). Hemorrhaged mice given IVIG had increased total numbers of splenic plasma cells compared with normal or hemorrhaged, untreated mice. Immunization with the bacterial polysaccharide antigen levan immediately after hemorrhage resulted in approximately 60% fewer levan-specific splenic plasma cells than those seen in normal unhemorrhaged mice. Treatment of hemorrhaged mice with IVIG did not correct the decrease in levan-specific plasma cells. These results demonstrate that hemorrhage-induced alterations in the numbers and frequencies of bacterial antigen-specific B cells are not related to changes in serum Ig levels and cannot be corrected through administration of IVIG.

Antibody synthesis specific for nonoral antigens in inflamed gingiva.

In vitro experimentation indicates that periodontitis-associated bacteria contain potent polyclonal B-cell activators (PBA). We reasoned that if PBA were operative in vivo, plasma cells specific for nonoral antigens should be present in the inflamed gingival tissues, which are characterized by a plasma cell infiltrate. To test this, rabbits with experimental periodontitis were immunized in the hind legs with the histochemically detectable antigen horseradish peroxidase (HRP) or glucose oxidase (GO). At various times after secondary immunization, inflamed gingival tissue was removed, sectioned, and treated histochemically to reveal plasma cells that specifically bound HRP or GO. Remarkably, by 9 days after secondary immunization, hundreds of HRP- or GO-binding plasma cells were found in the inflamed gingival tissue of immunized rabbits. The presence of these plasma cells, observed 7 to 10 days after booster immunization, was further substantiated by the presence of large amounts of locally produced HRP- or GO-specific antibody in gingival crevicular fluid. By 1 month after secondary immunization, the number of antigen-binding plasma cells had decreased dramatically, but a small number of antigen-specific plasma cells were detected for as long as 9 months after secondary immunization. The large number of HRP- or GO-specific plasma cells observed 9 days after immunization led us to see whether recently stimulated cells were more susceptible to PBA. Peripheral blood lymphocytes (PBL) were obtained at different times after booster immunization and cultured in the presence or absence of a PBA from Fusobacterium nucleatum. At 7 days after immunization, PBL spontaneously differentiated into antibody-forming cells in culture, and this process was enhanced by PBA. In contrast, PBL taken months after immunization produced little antibody in culture, and enhancement by PBA was difficult to detect. Compared with resting B cells, the recently stimulated B cells clearly differentiated more readily into antibody-forming cells. In conclusion, antibody synthesis specific for nonoral antigens did occur in inflamed gingival tissue, and a number of mechanisms, including PBA, probably contributed to this phenomenon.

The regulation of lymphocyte traffic.

The distribution of lymphocytes among the tissues of the body is not random. Both the number and the representation of particular functional subsets of lymphocytes are carefully controlled, differing in each lymphoid organ or tissue in a manner that presumably reflects local immune requirements. The tissue distribution of lymphocytes is a function of lymphocyte class, of their previous history or stage of differentiation, and of their antigenic specificity. For example, those lymphocyte populations primarily responsible for humoral immunity (B cells) predominate in the spleen and in the gut-associated Peyer’s patches, whereas T cells, which are primarily regulatory and cytotoxic cells, are the major lymphocyte type in the peripheral lymph nodes and skin (Stevens et al. 1982; Streilein 1978). Functionally and antigenically defined T-cell subsets are also unequally distributed between mucosal and nonmucosal lymphoid tissues (Elson et al. 1979; Kraal et al. 1983). The distribution of certain effector and effector-precursor populations can be even more restricted: especially dramatic is the segregation of IgA- vs. IgG-expressing B cells. Surface IgA-bearing lymphocytes are highly represented in the mucosa-associated lymphoid organs, and the mucosal surfaces attract predominantly IgA-secreting plasma cells (Guy-Grand et al. 1974; reviewed by Lamm 1976). In nonmucosal sites, such as peripheral lymph nodes or the skin, IgA-bearing cells are rare, and most plasma cells secrete IgM or IgG. Lymphocytes can also segregate in vivo on the basis of antigen specificity: antigen-specific B and T cells are disproportionately represented in lymph nodes or spleen challenged with antigen (Kraal et al. 1982; Sprent 1980) and antigen-specific plasma cells accumulate in tissue sites of specific antigen deposition (Husband and Gowans 1978; Husband 1982).

Stimulation of immunoglobulin-containing cells and isotype-specific antibody response in experimental Mycoplasma hyopneumoniae infection in specific-pathogen-free pigs.

The development of immunoglobulin-containing cells and antigen-specific plasma cells in retropharyngeal and bronchial lymph nodes, in lung tissue, and in nasal mucous membrane was followed during an experimental infection of pigs with Mycoplasma hyopneumoniae. An enzyme-linked immunosorbent assay was used to follow stimulation of the immunoglobulin isotypes (classes) M, G, and A directed against the infectious agent in tracheobronchial secretions and systemically. A significant increase of antigen-specific plasma cells in lymph nodes and lung tissue as well as antibodies in tracheobronchial secretions was demonstrated 2 weeks after infection. This specific reaction was followed by a marked increase of immunoglobulin-containing cells in lymph nodes and lung tissue. The data are discussed in terms of autoimmune reactions in the pathogenesis of enzootic pneumonia.

Specific antibody-containing cells in the mammary gland of non-lactating sheep after intraperitoneal and intramammary immunisation

Ewes were immunised intraperitoneally with ovalbumin and Brucella abortus in Freund's complete adjuvant, followed seven days later by intramammary immunisation in which ovalbumin was presented to one mammary gland and Brucella abortus to the other. Mammary tissue taken after a further seven days contained more antigen-specific plasma cells than ewes given intraperitoneal or intramammary immunisation alone. These cells were found predominantly in the specifically immunised gland and only a few were found in the contralateral gland. Most of these cells were of the IgG1 isotype. There was also an increase in the total number of IgG1- and IgG2-containing cells in mammary gland tissues of these ewes, indicating a non-specific response to immunisation. Following either intraperitoneal or intramammary immunisation there was also a significant increase in the number of antigen-specific IgA cells in the lamina propria of the jejunum. The gut response following intramammary immunisation alone was abrogated by chronic drainage of intestinal lymph but not mammary lymph. This suggests that antigen may relocate from the mammary gland to the intestine where an IgA response is generated from gut associated lymphoid tissue. These data provide evidence for interaction between the gut and mammary gland of sheep in response to antigen.