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H Kiyono

Publications and source records attributed to H Kiyono.

At least 163 records · Page 9Linked to original sources
PubMed1994

Quantitation of human influenza virus-specific cytotoxic T lymphocytes: correlation of cytotoxicity and increased numbers of IFN-gamma producing CD8+ T cells.

To study virus-specific cytotoxic T lymphocyte (CTL) activity at the single cell level, an IFN-gamma specific ELISPOT assay was adapted to elucidate the frequency of influenza-specific CTLs together with a standard cytotoxic 51Cr-release assay. Peripheral blood mononuclear cells (PBMCs) from human volunteers were cultured with influenza virus-infected autologous cells; following 3 or 7 days of culture, T cell subsets were assessed for IFN-gamma production by IFN-gamma-specific ELISPOT and ELISA, while IFN-gamma mRNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Influenza virus-specific CTL activity was measured in a 4 h 51Cr-release assay. Culture of PBMC with autologous A/Taiwan influenza (H1N1)-infected stimulator cells resulted in IFN-gamma spot forming cells (SFCs) at 3 days that increased after 7 days of incubation. Numbers of IFN-gamma SFCs directly correlated with levels of secreted IFN-gamma and higher levels were seen in supernatants from 7 day cultures. RT-PCR analysis (35 cycles of amplification) showed greater IFN-gamma mRNA in T cells isolated from 7 day cultures. Separate aliquots of T cells from these cultures were also assessed for virus-specific cytotoxicity and T cells from 7 day (but not from 3 day) cultures induced high 51Cr release. Analysis indicated a significant direct correlation between level of cytotoxicity, number of IFN-gamma SFCs, and amount of IFN-gamma in culture supernatants. Studies with purified T cell subsets showed that elevated IFN-gamma SFCs, IFN-gamma synthesis, and cytotoxic activity were associated with CD4-CD8+ T cells but not with the CD4+CD8- T cell subset.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

alpha beta T cell receptor-positive intraepithelial lymphocytes with CD4+, CD8- and CD4+, CD8+ phenotypes from orally immunized mice provide Th2-like function for B cell responses.

Our previous studies have shown that murine alpha beta TCR+ intraepithelial lymphocytes (IEL) contained T cells that can provide B cell help. In this study, we have examined the three subsets of alpha beta TCR+ IEL for Ag-specific helper function and cytokine production because alpha beta TCR+ IEL are divisible into three subsets including CD4+, CD8- T cells, CD4+, CD8+ double positive (DP) T cells, and CD4-, CD8+ T cells. When these three subsets of alpha beta TCR+ IEL from C3H/HeN mice (H-2k) orally immunized with SRBC were cultured with splenic B cells, adherent cells, and SRBC, both CD4+, CD8- and DP T cell fractions supported IgM, IgG1, and IgA anti-SRBC responses, whereas the CD4-, CD8+ T cell subset did not exhibit helper function. Addition of anti-I-Ak mAb resulted in the reduction of SRBC-specific PFC responses, whereas anti-H-2Kk mAb did not affect the CD4+, CD8- and DP T cell-supported B cell responses. Furthermore, when these CD4-bearing T cells from mice orally immunized with SRBC were co-cultured with B cells and adherent cells in the presence of unrelated Ag (e.g., horse RBC), SRBC-specific B cell responses were not induced. When type 1 and type 2 Th cell cytokine production was examined by IFN-gamma, IL-2-, IL-4-, and IL-5-specific enzyme-linked immunospot assays, increased numbers of IL-4- and IL-5-secreting type 2 Th cells, whereas lower numbers of IFN-gamma and IL-2-producing type 1 Th cells were seen in CD4+, CD8- and DP T cell fractions on in vitro stimulation with SRBC. In the case of CD4-, CD8+ T cells, approximately equal and low numbers of type 2 cytokine-producing cells were noted in both SRBC-stimulated and -unstimulated cultures. Furthermore, when different fractions of IEL T cells from Ag-stimulated and -unstimulated cultures were characterized for cytokine-specific mRNA by reverse transcription polymerase chain reaction, stronger bands for IL-4 and IL-5 were detected in both CD4+, CD8- and DP IEL subsets when compared with the unstimulated cells. In contrast, the change in intensity of the band of polymerase chain reaction product for IFN-gamma was slight or unchanged in Ag-stimulated CD4-bearing IEL when compared with unstimulated cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration, Oral↗
PubMed1993

Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues.

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2 and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

Administration, Oral↗
PubMed1993

Application of the ILISPOT-IDIP system for the enumeration of different sizes of IgA spot forming cells in the murine small and large intestine.

The immunofluorescence-linked immunospot (ILISPOT) assay associated with the immunofluorescence digital image processing (IDIP) system was originally developed in our laboratory to allow enumeration of immunoglobulin (Ig) producing, spot forming cells (SFC) in a more objective and quantitative manner. In this study, the ILISPOT-IDIP system was further advanced in order to analyze different sizes of SFC (e.g., IgA producing cells) including large (L), medium (M), and small (S) cells which correspond to high (> 2.4 pg), medium (1.2-2.4 pg) and low (< 1.2 pg) IgA secreting cells by the adaptation of real time image processor and intensified video camera system. When the ILISPOT-IDIP system was used to characterize the frequency of IgA secreting cells among mononuclear cells isolated from different parts of the murine gastrointestinal (GI) tract including the small (upper, middle and lower sections) and large (colon and rectum) intestine, the small intestine contained higher numbers of IgA SFC (approximately 8.4 x 10(5) SFC/10(6) cells) when compared with large intestine (approximately 1.3 x 10(5) SFC/10(6) cells). Among the 3 areas of small intestine, the upper (approximately 3.7 x 10(5) SFC/10(6) cells) and middle (approximately 2.4 x 10(5) SFC/10(6) cells) parts had higher numbers of IgA SFC when compared to the lower small (approximately 2.3 x 10(5) SFC/10(6) cells) intestine. When these IgA producing cells in different parts of the intestine were classified into three groups according to the size of individual spots, the upper and middle intestine contained higher frequencies of large (approximately 20%) and medium (approximately 20%) SFC which corresponded to high and medium IgA secretors in comparison to the lower small (approximately 9%) and large (approximately 6%) intestine. In contrast, the lower small and large intestine were dominated by small SFC since approximately 85% of IgA producing cells were categorized as low secretors. Using the advanced ILISPOT-IDIP system, a unique distribution of different sizes (or secretion rates) of IgA producing SFC was elucidated in the different regions of the mouse small and large intestine.

Animals↗
PubMed1993

Cytokine-specific ELISPOT assay. Single cell analysis of IL-2, IL-4 and IL-6 producing cells.

In order to assess cytokine-producing cells at the single cell level, the cytokine-specific ELISPOT assay has proven to be an important and sensitive method. The purpose of this study was to adapt this method to elucidate individual cells producing murine IL-2, IL-4 or IL-6. In order to establish these cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific cDNA transfected myeloma cell lines, e.g., X63-Ag8-653 X2, X63-Ag8-653 X4 and X63-Ag8-653 X6, respectively, were used as specific cytokine-producing cells. In the IL-2 ELISPOT assay, the coating reagent, monoclonal antibody (mAb) rat IgG2a anti-mouse IL-2 (CR #40014) was used while rabbit IgG polyclonal anti-mouse IL-2 was employed for detection of IL-2 spot forming cells (SFC). The mAbs anti-mouse IL-4, BVD4-1D11 and BVD6-24G2 were selected as capture and detection antibodies for enumeration of IL-4 SFC. For the IL-6 ELISPOT assay, anti-mouse IL-6 (MP5-20F3) mAb was used for coating and MP5-32C11 mAb was used for detection of IL-6 SFC. When IL-2 producing X63-Ag8-653 X2 cells were subjected to these three different ELISPOT assays, IL-2-specific SFC were only noted with the IL-2 ELISPOT system. In the case of IL-4 SFC, only X63-Ag8-653 X4 cells formed specific spots using the tandem of BVD4-1D11 and BVD6-24G2 mAbs. IL-6-specific spots developed in MP5-20F3 mAb pre-coated wells containing X63-Ag8-653 X6 cells, when developed with mAb anti-IL-6 (MP5-32C11). Addition of cycloheximide (50 micrograms/ml) inhibited formation of IL-2, IL-4 and IL-6 SFC by approximately 90%. When an unrelated mAb was used as detection antibody in these three different cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific SFC were not detected. Further, when concanavalin A stimulated T cells from Peyer's patch of normal mice were subjected to the respective cytokine-specific ELISPOT assay, IL-2, IL-4 and IL-6 SFC were enumerated. These results have shown that cytokine-specific IL-2, IL-4 and IL-6 ELISPOT assays have now been established and will allow analysis of the frequency of cytokine-secreting cells at the single cell level.

Animals↗
PubMed1993

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5.

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

Animals↗
PubMed1993

The Mycobacterium tuberculosis 71-kDa heat-shock protein induces proliferation and cytokine secretion by murine gut intraepithelial lymphocytes.

Murine intraepithelial lymphocytes (IEL) respond poorly to T cell mitogens and to monoclonal antibody stimulation of T cell receptor (TCR)- and CD3- associated molecules. In contrast, we found that a soluble extract of Mycobacterium tuberculosis (Mtb), but not purified protein derivative of tuberculin, induced significant proliferative responses in IEL cultures. The active component was apparently a heat shock protein (HSP), since recombinant 71-kDa HSP from Mtb induced IEL to proliferate, while 65-kDa HSP from M. bovis and M. leprae did not. Both alpha/beta and gamma/delta TCR-enriched IEL gave proliferative responses to 71-kDa HSP. Further, culture supernatants from IEL stimulated with 71-kDa HSP contained elevated levels of interleukin-(IL)-3/granulocyte-macrophage colony-stimulating factor, interferon-gamma and IL-6, but not IL-2, IL-4, IL-5 or transforming growth factor-beta. Finally, several IEL T cell clones have been maintained for up to 6 weeks, when stimulated with 71-kDa HSP, IL-2 and feeder cells. Our results show that the 71-kDa HSP of Mtb induces IEL T cells to divide and to secrete cytokines and this may offer a model for cloning and study of IEL T cells in vitro.

Animals↗
PubMed1993

Function of alpha beta TCR+ intestinal intraepithelial lymphocytes: Th1- and Th2-type cytokine production by CD4+CD8- and CD4+CD8+ T cells for helper activity.

The immunobiological function of lymphocytes within the epithelium (IELs) of the small intestine is incompletely understood; however, it has been shown that intestinal IEL T cells exhibit cytotoxicity, produce cytokines, and perform some regulatory roles. In this study, we have focused on CD4+, alpha beta TCR+ IELs, which comprise approximately 15-20% of the total population, for helper functions. We have assessed the profile of type 1 or type 2 Th cell cytokines produced in alpha beta TCR bearing CD4+CD8- and CD4+CD8+ (double positive, DP) intestinal IELs by cytokine-specific ELISPOT and by reverse transcription-polymerase chain reaction. Help for B cells taken from Peyer's patches (PP) allowed us to assess IEL function for mucosal antibody responses. Freshly isolated CD4+CD8- IEL T cells contain Th2-type cells, e.g. high numbers of IL-5 secreting (spot forming) cells (SFC) followed by IL-4 and IL-6, while DP T cells have IL-5 and IL-6 producing cells, but not IL-4. In addition to Th2-like cytokine producing T cells, both CD4+ T cell subsets contain IFN-gamma secreting Th1-type cells but neither subset synthesizes IL-2. Stimulation of CD4+CD8- and DP T cells with solid phase mAb anti-CD3 resulted in production of IL-2 in addition to IFN-gamma, IL-5, and IL-6, and this treatment stimulated DP T cells to produce IL-4. When freshly isolated intestinal IEL T cells were separated into CD4+ and CD4- cells, and co-cultured with PP B cells, the former subset supported Ig synthesis including IgA responses, while the later fraction did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.

Adjuvants, Immunologic↗
PubMed1993

New perspectives in vaccine development: mucosal immunity to infections.

In this review, we focus on six key areas currently receiving attention in the mucosal immune system. These six areas are of considerable importance for development of vaccines. They are (a) the necessity to understand the unique features of the mucosal immune system; (b) the possibility that the common mucosal immune system may contain distinct compartments; (c) differences in antigen uptake and in types of antigen-presenting cells in mucosal inductive and effector sites; (d) more careful consideration of mucosal memory in vaccine development; (e) recent studies, which show that oral vaccines induce T-helper (Th)-cell subsets that regulate mucosal IgA responses; and (f) the mechanisms whereby mucosal S-IgA and T cells provide mucosal immune protection. An example of the above suffices to illustrate why the selected areas are of importance in vaccine development. Oral immunization preferentially induces type 2 Th (Th2) cell responses that directly correlate with antigen-specific IgA responses in mucosal effector sites. It is likely that activated, antigen-specific Th2 cells that are induced in Peyer's patches are continuously supplied to mucosal effector sites for regulation of IgA responses. These Th2 cells are producing cytokines such as interleukin (IL)-5 and IL-6 and these cytokines may direct antigen-specific surface IgA-positive B cells to become IgA-producing plasma cells. Nevertheless, additional studies will be required to establish that IgA responses to T-cell-dependent antigens depend on Th2 cell-derived help. What are the implications of these studies for current oral vaccines, including novel antigen delivery systems? The most obvious would be that vaccines should be optimized for induction of Th2-cell responses in IgA inductive sites such as the gut-associated lymphoreticular tissues. It is now clear that induction of Th2-type responses in both mucosal inductive and mucosal effector sites are essential for oral vaccines to induce S-IgA responses. However, antigens delivered by live vectors such as Salmonella typhimurium in the murine system and S. typhi in humans must consider T-cell responses induced against a live vector in addition to the inserted recombinant antigen. In this regard, it has been shown that these microorganisms induce cell-mediated immunity responses that largely result from Th1-type cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

New perspectives in mucosal immunity with emphasis on vaccine development.

In this review, we have purposely focused on five areas that are currently receiving extensive research attention and will be of major importance for development of mucosal and systemic immunity to oral vaccines. These five areas include the following: (1) helper T-cell (Th) subsets and cytokines for mucosal IgA responses; (2) Th1- and Th2-type subsets in regulation of mucosal IgA responses; (3) antigen uptake and presentation in the mucosal immune system; (4) the importance of memory in mucosal immunity to vaccines; and (5) the determination of whether oral immunization alone induces immunity in all mucosal effector tissues. It is now established that the mucosal immune system can be divided into discrete mucosal inductive sites where vaccines/antigens are encountered and taken up, processed, and presented to B and T cells, and separate areas where immune cells actually function (mucosal effector tissues). Further, through the help provided by Th cells and cytokines, the B cells respond to antigen and undergo expansion including memory cell formation. Following the migration of memory B cells to mucosal effector tissues, the cells rapidly develop into IgA plasma cells, and the prevalence of the latter cell type represents a major characteristic of mucosal effector tissues. It also appears that antigen-specific Th cells and perhaps even CD8+ cytotoxic T lymphocytes can make this circular journey (along with memory/activated B cells) from inductive to mucosal effector sites, and this is termed the common mucosal immune system (CMIS). The major implications of the CMIS for development of vaccines would include each of the five components that are discussed.

Administration, Oral↗
PubMed1993

Cytokines and periodontal disease: immunopathological role of interleukins for B cell responses in chronic inflamed gingival tissues.

Localized and chronically-inflamed gingival tissues of adult periodontitis (AP) are generally characterized as a hyper-responsiveness of B lineage cells where increased numbers of plasma cells occur. It was previously shown that high numbers of IgG subclass antibody-secreting cells (e.g., IgG1 > IgG2 > IgG3 > or = IgG4) with significant numbers of IgA subclass antibody-producing cells were seen in enzymatically dissociated gingival mononuclear cells (GMC) from inflamed periodontal tissues. An interesting finding was that the frequency of IgA2 plasma cells was elevated in the severe stage of AP when compared with the moderate stage. IgM plasma cells were essentially not found in these tissues. To understand the cytokine involvement in these increased B cell responses in inflamed gingiva, GMC isolated from inflamed tissues of AP patients were examined for cytokine production, specifically for IL-2, IL-4, IL-5, and IL-6 at both the protein and mRNA levels, since these cytokines have been shown to be essential interleukins for the regulation of the B cell response. Freshly-isolated GMC and peripheral blood mononuclear cells (PBMC) from AP patients were initially examined for IL-6 production because of its essential role for the terminal differentiation of B cells to become Ig-producing plasma cells. High levels of IL-6 were produced by GMC but not by PBMC unless cells were stimulated with T cell mitogen. A similar findings was also obtained when levels of IL-6 specific mRNA were examined in GMC and PBMC.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗
PubMed1993

Gingival mononuclear cells from chronic inflammatory periodontal tissues produce interleukin (IL)-5 and IL-6 but not IL-2 and IL-4.

A unique characteristic of the localized inflammatory tissue in the periodontium (e.g., adult periodontitis [AP]) is the accumulation of IgG (IgG1 > IgG2 > IgG3 > or = IgG4) followed by IgA plasma cells (IgA1 > IgA2). However, the exact molecular mechanisms contributing to these elevated B-cell responses at the local disease site are still unknown. Thus, this study has examined the production of cytokines of importance in B-cell responses, e.g., interleukin (IL)-2, IL-4, IL-5, and IL-6 by gingival mononuclear cells (GMC) isolated from patients in severe stages of AP. These cytokines were assessed at the protein and messenger (m)RNA levels to understand their importance for the observed increased B-cell responses present in these tissues. Among the four cytokines tested by respective cytokine-specific, polymerase chain reaction and dot-blot hybridization, high levels of IL-5- and IL-6-specific mRNA were noted in GMC freshly isolated from AP patients. On the other hand, specific message for IL-2 and IL-4 were not present. Further, the analysis of culture supernatants of GMC also revealed that cells from AP patients spontaneously produced IL-5 and IL-6 but not IL-2 and IL-4. In contrast, when peripheral blood mononuclear cells isolated from the same patients were examined for these cytokines, no detectable levels of mRNA or secreted cytokines were noted. These results showed that GMC from localized inflammatory tissues in severe stages of AP possess a distinct cytokine profile represented by high levels of IL-5 and IL-6 mRNA expression and protein synthesis, whereas IL-2 and IL-4 were not detected. Further, this study supports the concept that AP is a localized inflammatory disease, because GMC from the inflamed tissue actively produce IL-5 and IL-6, whereas peripheral blood mononuclear cells from the same patients do not.

B-Lymphocytes↗
PubMed1992

Cytokine- and Ig-producing T cells in mucosal effector tissues: analysis of IL-5- and IFN-gamma-producing T cells, T cell receptor expression, and IgA plasma cells from mouse salivary gland-associated tissues.

The present study has focused on the analysis of cytokine- and Ig-producing mononuclear cells (MC) that reside in the salivary glands and their associated tissues (SGAT) in the oral region. The SGAT are located under the mandibular area and consist of submandibular glands, periglandular lymph nodes, and cervical lymph nodes. MC were isolated from individual SGAT and examined for T cell subsets and TCR expression, in comparison with T cells obtained from other mucosa-associated and systemic tissues. Forty to fifty percent of MC in submandibular glands were CD3+ T cells, equally divided into CD4+ CD8- and CD4- CD8+ T cell subsets. On the other hand, the intestinal lamina propria and Peyer's patches possessed a approximately 2 to 3:1 ratio of CD4+ CD8- to CD4- T cells. A high frequency of CD4- CD8- (double negative) (DN) T cells (approximately 6 to 10%) was also isolated from submandibular glands. In contrast, approximately 70 to 90% of MC in periglandular lymph nodes and cervical lymph nodes were CD3+ T cells and like the peripheral lymph nodes consisted of fivefold higher numbers of CD4+ CD8- than CD4- CD8+ T cells, with low numbers of DN cells (less than 5%). When expression of gamma/delta and alpha/beta TCR was examined in individual T cell subsets of submandibular glands, the CD4- CD8+ and DN T cell fractions contained 25% and 100% gamma/delta TCR+ cells, respectively. On the other hand, essentially all CD4+ CD8- T cells in SGAT as well as CD4- CD8+ cells in periglandular lymph nodes and cervical lymph nodes were alpha/beta TCR+ T cells. When cytokine production was examined by using IFN-gamma- and IL-5-specific enzyme-linked immunospot assays, the CD3+ CD4+ CD8- T cells in submandibular glands contained T cells spontaneously producing IFN-gamma and IL-5. Further, IL-5 spot-forming cells (SFC) were two- to threefold greater in number, compared with IFN-gamma SFC. The periglandular lymph node T cells contained cytokine producing cells with a ratio of 2:1 for IL-5 and IFN-gamma SFC cells, whereas cervical lymph node T cells did not produce cytokines unless stimulated with T cell mitogens. When the isotype distribution of Ig-producing cells was examined among SGAT, submandibular glands contained large numbers of IgA-producing cells, with few IgM- and IgG-producing cells, a pattern similar to that of the lamina propria. Further, elevated numbers of IgA-secreting cells were also seen in periglandular lymph nodes but not in cervical lymph nodes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Immunoregulatory functions for murine intraepithelial lymphocytes: gamma/delta T cell receptor-positive (TCR+) T cells abrogate oral tolerance, while alpha/beta TCR+ T cells provide B cell help.

Past work has shown that a subset of effector T cells with unique characteristics could abrogate hapten- or antigen-induced tolerance, and the reconstitution of this immune response has been termed contrasuppression. We have studied contrasuppression in a model of oral tolerance (OT) in which adoptively transferred antigen-specific T contrasuppressor (Tcs) cells reverse OT and result in antibody responses to the eliciting antigen. In the present study, we show that murine intraepithelial lymphocytes (IELs) from mice orally immunized with sheep red blood cells (SRBC) contain T cells that exhibit Tcs cell activity. This effect was mediated by CD3+ gamma/delta T cell receptor-positive (TCR+), but not alpha/beta TCR+ T cells, and gamma/delta TCR+ Tcs cells were associated with both the CD4-,CD8+ and CD4-,CD8- (double-negative) IEL fractions. The CD4-,CD8+ gamma/delta TCR+ IELs were further separated into Vicia villosa-adherent and -nonadherent fractions. Adoptive transfer of V. villosa-adherent gamma/delta TCR+ T cells to mice with OT to SRBC resulted in splenic IgA, IgM, and IgG subclass anti-SRBC responses, while V. villosa-nonadherent gamma/delta TCR+ T cells were without activity. The gamma/delta TCR+ IELs did not support in vitro antibody responses in B cell cultures, while alpha/beta TCR+ IELs were effective T helper cells. Further, cytokine production by the gamma/delta TCR+ IELs was examined, and the gamma/delta TCR+ V. villosa-adherent fraction, which possessed contrasuppressor function, contained low levels of IL-5 mRNA and small numbers of IL-5-producing cells when compared with alpha/beta TCR+ IELs and V. villosa-nonadherent gamma/delta TCR+ IELs. Our results now show that mouse IELs contain two distinct types of T cells that function in the immune response, e.g., alpha/beta TCR+ T cells that produce IL-5 and function as helper cells, and gamma/delta TCR+ T cells that restore antibody responses in mice that had been orally tolerized with antigen.

Animals↗
PubMed1992

Intestinal intraepithelial lymphocyte T cells are resistant to lpr gene-induced T cell abnormalities.

The mucosal immune system of the gastrointestinal (GI) tract consists of Peyer's patches (PP), which are IgA inductive sites, and more diffuse effector regions which include cells in the intraepithelial lymphocyte (IEL) compartment. Since autoimmune MRL lpr/lpr (MRL/lpr) mice develop a proliferating CD3+, CD4-, CD8- (double negative; DN), B220+ T cell subset in systemic lymphoid tissue, we have initiated studies to determine the distribution of CD3+, DN, B220+ T cells (B220+ T cells or lpr/lpr T cells) in the GI immune system. Specifically, we examined T cell subsets separated according to expression of CD4, CD8, Thy-1, B220, alpha/beta T cell receptor (TcR) and gamma/delta TcR in PP and IEL of MRL/lpr mice at 6, 12 and 21 weeks of age. Increased numbers of CD3+ T cells were noted in both PP and spleen of 12- and 21-week-old mice in which the development of autoimmune disorders were also evident. However, normal numbers of CD3+ IEL T cells were seen in MRL/lpr mice in all three age groups tested. When the presence of T cell lymphadenopathy was examined in both IgA inductive and effector tissues, the PP followed the B220+ T cell pattern seen in the spleen, where approximately 30%-50% of CD3+ T cells in the PP of 12- and 21-week-old MRL/lpr mice expressed the phenotype of lpr/lpr T cells and greater than 90% were alpha/beta TcR+. On the other hand, B220+ T cells had not developed in PP or spleen of 6-week-old MRL/lpr mice. Of interest was the finding that IEL from lpr/lpr homozygous mice did not contain B220+ T cells in any age group tested. In this regard, the IEL of MRL/lpr mice comprised an identical pattern and frequency of CD4-/CD8+, CD4+/CD8-, DN and CD4+/CD8+ (double positive, DP) T cell subsets as their normal counterparts (i.e. MRL +/+, BALB/c and C3H/HeN mice) which consisted of approximately 75%, approximately 7.5%, approximately 7.5% and approximately 10%, respectively. Further, Thy-1, gamma/delta TcR and alpha/beta TcR expression in these four subsets of MRL/lpr IEL were very similar to normal mice. These results suggest that the intestinal IEL compartment is minimally affected by the lpr/lpr mutation which induces T cell abnormalities and indicate that B220+ T cells do not preferentially home to IEL. Further, our results support the concept that IEL T cells develop as a separate T cell lineage from thymus-derived cells.

Animals↗
PubMed1992

Induction of T helper cells and cytokines for mucosal IgA responses.

CD4+ Th cells and their derived cytokines play an important role in the regulation of IgA responses in the mucosal immune system. Th1 and Th2 cells induce different Ig isotype and IgG-subclass responses. Further, cytokines produced by Th2-type cells (e.g., IL-5 and IL-6) have been shown to induce PP sIgA+ B cells to secrete IgA. Our studies have now shown that oral immunization with SRBC selectively induces Th2-type cells in PP while systemic (I.P.) immunization with SRBC predominantly induces Th1-type cells. It is tempting to suggest that Th2 cells which produce IL-5 and IL-6 tend to be predominant in mucosal effector regions, such as the salivary glands and LP tissues and account for the predominant IgA responses which characterize these tissues. The PP contain B cell subsets which respond to IL-5 and IL-6, and these are largely restricted to the PNALo non-GC (memory) sIgA+ B cells. The importance of CD4+ Th cells in the regulation of IgA responses has also been shown by the depletion of CD4+ Th cells in anti-L3T4 (CD4)-treated mice. Loss of CD4+ Th cells from mucosal tissues resulted in dramatically decreased numbers of IgA plasma cells in the small intestine and led to a reduction in IgA SFC in isolated LP cells. The overall size of PP was reduced and the GCs were absent; however, the relative frequency of sIgA+ B cells in PP did not change, possibly suggesting that CD4+ Th cells do not influence switches to IgA.

Animals↗