清野 宏

The induction and distribution of antigen-specific antibody-secreting cells in various tissues were assessed in BALB/c mice immunized with the purified fimbrial protein of the Porphyromonas gingivalis strain 381. Groups of mice were immunized by gastric intubation of liposomes containing fimbriae and GM-53 on days 0, 1, 27, and 28. Additional groups of mice were immunized with P. gingivalis fimbriae and adjuvant GM-53 in Freund's incomplete adjuvant by subcutaneous injection on days 0 and 28. In the latter group of mice, levels of serum IgM anti-fimbria antibodies were first detected on day 7, while high levels of serum IgG anti-fimbria antibodies were seen after secondary immunization. Fimbria-specific spot-forming cells (SFC) were detected in the spleen, circulating blood mononuclear cells (CBMC), and brachial lymph nodes of immunized mice by ELISPOT. Fimbria-specific IgM SFC appeared by day 5 and antigen-specific IgG SFC were seen later in subcutaneously immunized mice. Mice immunized orally exhibited serum anti-fimbria IgG and IgA antibodies after boosting. Although numerical analysis revealed that the numbers of fimbria-specific SFC were generally lower than in subcuianeously immunized mice, significant numbers of antigen-specific IgA SFC were seen in lamina propria and mesenteric lymph nodes of orally immunized mice. In contrast, antigen-specific IgM and IgG SFC were observed mainly in CBMC. The route of immunization with fimbriae and GM-53 also influenced the total numbers of immunoglobulin-secreting cells. Thus, subcutaneous immunization enhanced the total number of IgM and IgG SFC, including fimbria-specific antibody-secreting cells in CBMC and the spleen. On the other hand, oral immunization augmented the total number of Ig-producing cells in addition to antigen-specific IgA cells in mucosa-associated tissues. Our studies have shown that the route of immunization markedly affects clonal expansion of selective isotypes of B cells, including antigen-specific B cells. © 1992 Oxford University Press.

The induction and distribution of antigen-specific antibody-secreting cells in various tissues were assessed in BALB/c mice immunized with the purified fimbrial protein of the Porphyromonas gingivalis strain 381. Groups of mice were immunized by gastric intubation of liposomes containing fimbriae and GM-53 on days 0, 1, 27, and 28. Additional groups of mice were immunized with P. gingivalis fimbriae and adjuvant GM-53 in Freund's incomplete adjuvant by subcutaneous injection on days 0 and 28. In the latter group of mice, levels of serum IgM anti-fimbria antibodies were first detected on day 7, while high levels of serum IgG anti-fimbria antibodies were seen after secondary immunization. Fimbria-specific spot-forming cells (SFC) were detected in the spleen, circulating blood mononuclear cells (CBMC), and brachial lymph nodes of immunized mice by ELISPOT. Fimbria-specific IgM SFC appeared by day 5 and antigen-specific IgG SFC were seen later in subcutaneously immunized mice. Mice immunized orally exhibited serum anti-fimbria IgG and IgA antibodies after boosting. Although numerical analysis revealed that the numbers of fimbria-specific SFC were generally lower than in subcuianeously immunized mice, significant numbers of antigen-specific IgA SFC were seen in lamina propria and mesenteric lymph nodes of orally immunized mice. In contrast, antigen-specific IgM and IgG SFC were observed mainly in CBMC. The route of immunization with fimbriae and GM-53 also influenced the total numbers of immunoglobulin-secreting cells. Thus, subcutaneous immunization enhanced the total number of IgM and IgG SFC, including fimbria-specific antibody-secreting cells in CBMC and the spleen. On the other hand, oral immunization augmented the total number of Ig-producing cells in addition to antigen-specific IgA cells in mucosa-associated tissues. Our studies have shown that the route of immunization markedly affects clonal expansion of selective isotypes of B cells, including antigen-specific B cells. © 1992 Oxford University Press.

The humoral and genital secretory immune response to chronic SIV infection was compared between female Rhesus macaques inoculated by i.v. or intravaginal routes. Total IgG levels in serum were 10-fold higher in SIV-infected animals when compared with uninfected controls. Vaginal washes from normal macaques contained predominantly IgA and IgG, while those from SIV-infected animals contained high levels of IgG. The SIV-infected animals had high titres of SIV-specific IgG in serum, with lower but detectable IgA and IgM responses. The genital secretory immune response to SIV was similar in intravenously and intravaginally inoculated animals. The anti-SIV response in the vaginal washes consisted mainly of IgG. Within the lamina propria of the reproductive tract of animals chronically infected with SIV there were essentially no IgA or IgG plasma cells and only a small number of IgM plasma cells, while two normal animals had large numbers of IgA plasma cells. These results suggest that the mucosal immune system of the female reproductive tract is impaired in chronic SIV infection.

The humoral and genital secretory immune response to chronic SIV infection was compared between female Rhesus macaques inoculated by i.v. or intravaginal routes. Total IgG levels in serum were 10-fold higher in SIV-infected animals when compared with uninfected controls. Vaginal washes from normal macaques contained predominantly IgA and IgG, while those from SIV-infected animals contained high levels of IgG. The SIV-infected animals had high titres of SIV-specific IgG in serum, with lower but detectable IgA and IgM responses. The genital secretory immune response to SIV was similar in intravenously and intravaginally inoculated animals. The anti-SIV response in the vaginal washes consisted mainly of IgG. Within the lamina propria of the reproductive tract of animals chronically infected with SIV there were essentially no IgA or IgG plasma cells and only a small number of IgM plasma cells, while two normal animals had large numbers of IgA plasma cells. These results suggest that the mucosal immune system of the female reproductive tract is impaired in chronic SIV infection.

Recent studies in experimental animals and humans have shown that the mucosal immune system, which is characterized by secretory IgA (S-IgA) antibodies as the major humoral defence factor, contains specialized lymphoid tissues where antigens are encountered from the environment, are taken up and induce B- and T-cell responses. This event is followed by an exodus of specific lymphocytes, which home to various effector sites such as the lamina propria regions and glands. These responses are regulated by T cells and cytokines and lead to plasma cell differentiation and subsequent production of S-IgA antibodies in external secretions. This knowledge has led to practical approaches for vaccine construction and delivery into mucosal inductive sites in an effort to elicit host protection at mucosal surfaces where the infection actually occurs.

Patients with adult periodontitis (AP) exhibit elevated serum antibody levels to Porphyromonas (Bacteroides) gingivalis; however, it is not known whether these antibodies originate from plasma cells in the local disease site or From peripheral lymphoid tissues. We studied the isotype and subclass levels and origin of antibodies to P. gingivalis fimbriae, since elevated serum anti‐fimbriae responses were seen when compared with sera of healthy controls. IgG unti‐fimbriae litres were dominant and the subclass response was IgG3 ≫ IgG1 > IgG2 ≫ IgG4; however, some IgA anti‐fimbriae antibodies were also seen. The IgA subclass fimbriae‐specific response was mainly IgA1; however, significant IgA2 anti‐fimbrae antibodies were seen. We also assessed numbers of anti‐fimbriae antibody producing cells from peripheral blood mononuclear cells (PMBC) and from either healthy or inflamed gingiva of AP subjects. Gingival mononuclear cells (GMC) of AP patients exhibited high numbers of immunoglobulin‐producing (spot‐forming) cells (SFC) including fimbriae‐specific antibody secreting cells in a pattern of IgG > IgA > > > IgM. However, low numbers of SFC were seen in GMC from healthy gingiva; further, no anti‐timbriac SFC responses were noted in healthy GMC. Although no fimbriae‐specific immunoglobulin‐producing cells were seen in PBMC. low numbers of antigen‐specific SFC were found in pokeweed mitogen‐triggercd PBMC from AP subjects. Treatment of AP patients for plaque and surgical removal of inflamed gingiva resulted in significant reductions in serum anti‐fimbriac responses. These studies show that AP patients exhibit brisk serum IgG and IgA subclass anti‐fimbriac antibodies, whose origin appear to be the plasma cells present in the localized inflamed tissues. Copyright © 1991, Wiley Blackwell. All rights reserved

Our studies have given direct evidence that CD3+, CD4-, CD8+ T cells in the IELs can be separated into at least two subsets that produce IFN-gamma and/or IL-5 and may exhibit Th1- and Th2-type functions in the epithelium and in the underlying lamina propria region. Further, it was also shown that TCR1+ T cells in IELs are capable of producing IFN-gamma and/or IL-5. In addition to production of these cytokines, IELs derived from mice orally primed with TD antigen contain a subset of T cells which posses antigen-specific immunoregulatory function. CD3+; CD4-, CD8+ T cells isolated from IEL of TD antigen-primed mice abrogated systemic unresponsiveness to secondary-type responses including those of the IgA isotype upon adoptive transfer to mice with OT. Our studies further suggested that these CD3+, CD4-, CD8+ IELs use the gamma-delta-form of TCR (TCR1) for their immunoregulatory function.

Antigen-activated peripheral blood B cells were induced by parenteral immunization of healthy individuals with a polyvalent pneumococcal vaccine, or diphtheria toxoid. Seven to nine days after immunization, high frequencies of antigen-specific antibody-secreting cells, representing in vivo activated lymphoblastoid B cells, were detectable in peripheral blood or spleen. The B cellenriched fractions were stimulated for 7 days with different concentrations of rhIL-6. Both the frequency of antibody-secreting cells and the secreted amount of antibody to the immunizing antigen were increased by rhIL-6 in a dose-dependent fashion. Stimulation with rhIL-6 did not alter the isotype distribution of antibody-secreting cells. A polyclonal anti-IL-6 serum completely abrogated the stimulatory effect of rhIL-6 on the in vitro antibody secretion. Fluorescence-activated cell sorter analysis revealed that 25-29% of cells in the large B cell fraction which presumably contained the in vivo activated cells bore the IL-6 receptor. Thus, rhIL-6 enhances the terminal differentiation of in vivo activated B cells into antibody-secreting plasma cells. © 1991.

Our studies have given direct evidence that CD3+, CD4-, CD8+ T cells in the IELs can be separated into at least two subsets that produce IFN-gamma and/or IL-5 and may exhibit Th1- and Th2-type functions in the epithelium and in the underlying lamina propria region. Further, it was also shown that TCR1+ T cells in IELs are capable of producing IFN-gamma and/or IL-5. In addition to production of these cytokines, IELs derived from mice orally primed with TD antigen contain a subset of T cells which posses antigen-specific immunoregulatory function. CD3+; CD4-, CD8+ T cells isolated from IEL of TD antigen-primed mice abrogated systemic unresponsiveness to secondary-type responses including those of the IgA isotype upon adoptive transfer to mice with OT. Our studies further suggested that these CD3+, CD4-, CD8+ IELs use the gamma-delta-form of TCR (TCR1) for their immunoregulatory function.

Antigen-activated peripheral blood B cells were induced by parenteral immunization of healthy individuals with a polyvalent pneumococcal vaccine, or diphtheria toxoid. Seven to nine days after immunization, high frequencies of antigen-specific antibody-secreting cells, representing in vivo activated lymphoblastoid B cells, were detectable in peripheral blood or spleen. The B cellenriched fractions were stimulated for 7 days with different concentrations of rhIL-6. Both the frequency of antibody-secreting cells and the secreted amount of antibody to the immunizing antigen were increased by rhIL-6 in a dose-dependent fashion. Stimulation with rhIL-6 did not alter the isotype distribution of antibody-secreting cells. A polyclonal anti-IL-6 serum completely abrogated the stimulatory effect of rhIL-6 on the in vitro antibody secretion. Fluorescence-activated cell sorter analysis revealed that 25-29% of cells in the large B cell fraction which presumably contained the in vivo activated cells bore the IL-6 receptor. Thus, rhIL-6 enhances the terminal differentiation of in vivo activated B cells into antibody-secreting plasma cells. © 1991.

Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-γ (IFN-γ) or interleukin-5 (IL-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-γ (R4-6A2) and to IL-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-γ (XMG 1.2) or anti-IL-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-γ and IL-5 SFC were seen. Approximately 20-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-γ with recombinant IFN-γ (rIFN-γ) or anti-IL-5 mAbs with rIL-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-γ- and IL-5 specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-γ or IL-5, and should be useful for detection of cytokine secretion at the single cell level. © 1990.

Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-γ (IFN-γ) or interleukin-5 (IL-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-γ (R4-6A2) and to IL-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-γ (XMG 1.2) or anti-IL-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-γ and IL-5 SFC were seen. Approximately 20-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-γ with recombinant IFN-γ (rIFN-γ) or anti-IL-5 mAbs with rIL-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-γ- and IL-5 specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-γ or IL-5, and should be useful for detection of cytokine secretion at the single cell level. © 1990.

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and in Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyconal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb 3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.

Antigens of Streptococcus mutans 6715 (alternatively designated serotype g Streptococcussobrinus), including whole cells (WC g), cell walls (CW g), peptidoglycan (PG g) and serotype carbohydrate (Ml g) were coupled with trinitrophenyl (TNP), and the nature of the immune response to each immunogen was determined in normal and X-linked immunodeficient (xid) murine spleen cell cultures. Responses to TNP-WC g, -CW g and -PG g and to the classical type 1 antigen TNP-Brucella abortus occurred in both xid and normal splenic cultures, while TNP-Ml g only triggered immune responses in normal spleen cell cultures, suggesting that the former three antigens are type 1 and the latter type 2. Further support for the type 2 nature of TNP-Ml g was the finding that Peyer's patch cell cultures from both xid (which contain mature B cells) and normal mice supported responses to TNP-Ml g and TNP-Ficoll, while xid splenic cultures failed to support responses to either type 2 antigen. The three type 1 TNP-S. mutans antigens induced responses in nude spleen cell and in purified splenic B cell cultures, but required T cells for in vitro responses to lower doses of immunogen. On the other hand, TNP-Ml g induced anti-TNP PFC responses at several antigen concentrations in purified B cell cultures, without requirement for added T cells. These studies show that the intact S. mutans cell, as well as CW g and PG g, acts as a T cell-dependent (TD) type 1 antigen, while the serotype carbohydrate (Ml g) induces a T cell-independent (TI) type 2 response. Thus, the intact bacterium is a TD type 1 antigen, whereas its purified components are either type 1 or type 2 antigens and differ significantly in terms of their T cell dependence. © 1987, Gustav Fischer Verlag · Stuttgart · New York. All rights reserved.