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.     

Oral but Not Parenteral Interleukin (IL)-12 Redirects T Helper 2 (Th2)-type Responses to an Oral Vaccine Without Altering Mucosal IgA Responses

Our past studies have shown that the mucosal adjuvant cholera toxin (CT) induces T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (S-IgA) antibodies (Abs). In this study, recombinant murine IL-12 (rmIL-12) was given either parenterally or orally to mice orally immunized with tetanus toxoid (TT) and CT to determine whether this cytokine could redirect the CT-induced Th2-type responses and what effect this shift would have on S-IgA Ab responses. Intraperitoneal administration of rmIL-12 shifted TT-specific responses toward Th1-type and resulted in CD4⁺ T cells producing IFN-γ and IL-2 with markedly reduced levels of Th2-type cytokines. This cytokine profile was accompanied by increased delayed-type hypersensitivity (DTH) and shifts in serum IgG1 to IgG2a and IgG3 anti-TT Ab responses. Further, serum IgE and S-IgA Ab responses were markedly reduced by parenteral IL-12. When IL-12 complexed to liposomes was given orally both shifts to IgG2a and IgG3 and low IgE Abs again occurred concomitant with enhanced serum IFN-γ and DTH responses. Interestingly, oral rmIL-12 did not result in significant levels of serum IL-12 nor altered S-IgA Ab responses and resulted in higher levels of some Th2-type cytokines both in vitro and in vivo when compared with parenteral IL-12. Our results show that the shifts in systemic immune responses with intact S-IgA Abs which occur after oral delivery of IL-12-liposomes are due to cytokine effects in the Peyer's patches and suggest new strategies for the targeted manipulation of Th1- and Th2-type responses to mucosal vaccines.     

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.     

Experimental IgA nephropathy induced by oral immunization

To test the hypothesis that IgA nephropathy can result from a mucosal immune response, mice were orally immunized with one of three protein antigens for 14 wk. Such mice exhibited an essentially pure mucosal antibody response characterized by specific IgA-producing plasma cells in exocrine sites and specific IgA antibodies in serum. Furthermore, 73% of immunized mice had IgA and 88% had immunogen deposited in the glomerular mesangium, and 64% of immunized mice examined ultrastructurally had electron-dense mesangial deposits. All three were present concurrently in 57% of the immunized mice. No differences in regard to IgG or IgM were observed between immunized and control mice for any of these parameters. Mucosal immunization therefore can result in a specific immune response that leads to mesangial deposition of immune complexes containing IgA antibody. In its fundamental features the experimental renal lesion resembles that seen in the human disease IgA nephropathy.     

A novel M cell-specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

Mucosally ingested and inhaled antigens are taken up by membranous or microfold cells (M cells) in the follicle-associated epithelium of Peyer's patches or nasopharynx-associated lymphoid tissue. We established a novel M cell–specific monoclonal antibody (mAb NKM 16–2-4) as a carrier for M cell–targeted mucosal vaccine. mAb NKM 16–2-4 also reacted with the recently discovered villous M cells, but not with epithelial cells or goblet cells. Oral administration of tetanus toxoid (TT)– or botulinum toxoid (BT)–conjugated NKM 16–2-4, together with the mucosal adjuvant cholera toxin, induced high-level, antigen-specific serum immunoglobulin (Ig) G and mucosal IgA responses. In addition, an oral vaccine formulation of BT-conjugated NKM 16–2-4 induced protective immunity against lethal challenge with botulinum toxin. An epitope analysis of NKM 16–2-4 revealed specificity to an α(1,2)-fucose–containing carbohydrate moiety, and reactivity was enhanced under sialic acid–lacking conditions. This suggests that NKM 16–2-4 distinguishes α(1,2)-fucosylated M cells from goblet cells containing abundant sialic acids neighboring the α(1,2) fucose moiety and from non-α(1,2)-fucosylated epithelial cells. The use of NKM 16–2-4 to target vaccine antigens to the M cell–specific carbohydrate moiety is a new strategy for developing highly effective mucosal vaccines.     

Hochuekkito, a Kampo (traditional Japanese herbal) Medicine, Enhances Mucosal IgA Antibody Response in Mice Immunized with Antigen-entrapped Biodegradable Microparticles

The effect of oral administration of Hochuekkito (HET; Bu-Zhong-Yi-Qi-Tang in Chinese), a traditional Japanese herbal medicine, on mucosal IgA immune response was investigated. To induce the antigen-specific antibodies in mucosal site, ovalbumin (OVA)-entrapped biodegradable microparticles (OVA-microparticles) were used as an antigen. Mice were orally immunized with OVA-microparticles for 3 successive days with intragastric gavage. From 7 days after the onset of immunization, the mice were boosted twice a week with the same antigen for 2 weeks. HET or water alone was orally administered to the mice via the intragastric route from 7 days before to 27 days after the onset of immunization. Although no significant change in total secretory IgA antibody level was observed in intestinal and nasal washes, OVA-specific IgA titers in intestinal washes were significantly enhanced by oral administration of HET. When lymphocytes from spleen, peripheral blood and Payer's patches were investigated for cytokines production, it was found that the IFN-γ secretion from the lymphocytes was increased by the administration of HET. Microarray analysis of Peyer's patch cells revealed enhanced expression of L-selectin gene. The increase of L-selectin positive cells in B lymphocytes fraction was observed in Peyer's patch cells and peripheral blood mononuclear cells by flow cytometry. These results suggest that the enhanced IFN-γ secretion and increased population of L-selectin positive B lymphocytes by orally administered HET may partly contribute to enhancement of IgA immune response against intestinal antigens, and orally administered HET may strengthen defensive systems against various pathogens and food antigens in intestine.     

Immunization of rabbits against a bacterial pathogen with an alginate microparticle vaccine.

Pasteurella multocida is an important bacterial pathogen of domestic rabbits. To evaluate the ability of a thiocyanate extract (PTE) of P. multocida to stimulate an immune response and protect against infection with P. multocida, rabbits were immunized subcutaneously or intranasally on Days 7, 21 and 35. Cholera toxin, a potent mucosal adjuvant, was included in one treatment group. Rabbits immunized subcutaneously (SC) or intranasally (IN) had significant increases in serum anti-PTE IgG but not IgA. In contrast, only rabbits immunized IN with PTE developed significant titers of nasal lavage anti-PTE IgA and cholera toxin significantly enhanced this response. In a second study rabbits were immunized via the drinking water with PTE incorporated into alginate microparticles on Days 7, 14 and 21. Mild increases in serum IgG were noted in rabbits immunized with PTE in microparticles, with or without cholera toxin, and this increase was significant (P相似文献   

Dual antigenic recognition by cloned human gamma delta T cells.

The function of gamma delta T cells is still elusive. The nature of the antigens that they recognize and the mode of presentation of these antigens are largely unknown. The majority of human peripheral gamma delta T cells bear a V gamma 9/V delta 2 T cell receptor, and display nonclonal reactivity to mycobacteria, without restriction by MHC. It is unknown whether these cells have clonal antigenic specificity as well. Here we describe rheumatoid arthritis-derived V gamma 9/V delta 2 T cell clones, displaying dual antigenic recognition: a nonclonal, MHC-unrestricted recognition of mycobacteria, and a clonal recognition of a short tetanus toxin peptide presented by HLA-DRw53, a nonpolymorphic class II MHC molecule associated with susceptibility to rheumatoid arthritis. This is the first evidence that V gamma 9/V delta 2 T cells can recognize nominal antigenic peptides presented by class II MHC molecules. These results suggest that much like alpha beta T cells, V gamma 9/V delta 2 cells may contribute to the immune response against foreign antigens in an antigen-specific and MHC-restricted manner. The reactivity of these gamma delta T cells to mycobacteria may represent a superantigen-like phenomenon.     

The role of antigen form and function in the primary and secondary intestinal immune responses to cholera toxin and toxoid in rats

This report describes studies of the mucosal antitoxic response in rats after enteric administration of several forms of cholera toxin or toxoid, proteins which differ primarily in their ability to bind to cell membranes and activate cellular adenyl cyclase. These two characteristics appeared to markedly enhance the local primary response to these antigens. A single dose of toxoid lacking these features was ineffective in local priming even though it was absorbed and induced a systemic immune response. Single dose mucosal priming occurred only with preparations which bind to cell membranes and was enhanced by those which also activate cellular adenyl cyclase. In contrast, single-dose mucosal boosting was best accomplished by materials with these properties but was also seen with a toxoid lacking both of these functions. The property of membrane binding appears to be most advantageous in mucosal priming, perhaps by increasing effective trapping of absorbed antigen in unprimed mucosal lymphoid tissue, whereas the ability to activate adenyl cyclase appears to enhance primary and secondary type responses about equally. Combinations of crude toxoid and toxin were also more effective in mucosal priming than purified materials, a finding which is unexplained. A single dose of this combination induced mucosal priming which was fully developed in 2 wk, undiminished after 4 too, and only modestly diminished after 8 mo, thus demonstrating relatively prolonged memory in the IgA mucosal immune system. Effective two-dose local immunizing regimens were developed, and it was shown that there was no correlation between the mucosal and systemic secondary antitoxin responses provoked by these regimens.     

Mucosal immune responses following oral immunization with rotavirus antigens encapsulated in alginate microspheres.

Availability of effective oral vaccine delivery vehicles should contribute to the success of oral immunization in domestic animals. To achieve this goal, we evaluated alginate microspheres for their capacity to induce mucosal immune responses following oral and enteric immunizations. Mice were immunized with either live porcine rotavirus (PRV) or its recombinant VP6 protein, encapsulated in alginate microspheres or unencapsulated. VP6-specific IgG (but no IgA) antibodies were detected in the sera of mice after a single intraperitoneal (i.p.) immunization with either VP6 in Incomplete Freund's adjuvant (VP6-IFA), VP6 in alginate microspheres (VP6-MS) or with live PRV in incomplete Freund's adjuvant (PRV-IFA). In contrast, VP6-specific IgA (but no IgG) was detected in culture supernatants of mesenteric lymph nodes from mice immunized i.p. with either VP6-IFA or with PRV-IFA. Oral immunization with VP6-MS induced the highest level of VP6-specific fecal IgA antibody, similar to responses induced by oral immunization with live PRV. Furthermore, the VP6-specific fecal IgA could be boosted by a secondary i.p. immunization with VP6. Further experiments were performed in a sheep intestinal 'loop' model to evaluate uptake of microspheres by Peyer's patches. Microspheres containing colloidal carbon were specifically bound and transported by follicle-associated epithelium of Peyer's patches. Additionally, mucosal immune responses were detected following enteric immunization with porcine serum albumin (PSA) encapsulated in alginate microspheres. Our results confirm that alginate microspheres are an effective oral delivery vehicle for protein antigens and intestinal IgA antibody responses are induced by antigens encapsulated in alginate microspheres without any additional mucosal adjuvant. These investigations confirm that alginate microspheres have the potential as an effective delivery vehicle for oral immunization of ruminants.     

The B-cell targeted CTA1-DD vaccine adjuvant is highly effective at enhancing antibody as well as CTL responses

A novel immunomodulating gene-fusion protein, CTA1-DD, has been developed which combines the ADP-ribosylating ability of cholera toxin (CT) with a dimer of an Ig-binding fragment, D, of Staphylococcus aureus protein A. The CTA1-DD adjuvant is non-toxic and greatly augmented T-cell-dependent and T-cell-independent responses to soluble admixed antigens after systemic as well as mucosal immunizations. CTL and antibody responses of all classes were increased by 10- to 100-fold above those observed in control mice immunized without adjuvant. CTA1-DD does not appear to form immune complexes or bind to soluble Ig following injection, but rather it binds directly to B-cells of all isotypes, including na?ve IgD+ cells. No binding was observed to macrophages or dendritic cells, and immunizations in Fc gamma R-deficient mice demonstrated unaltered enhancing effects. As shown by inactive mutants, the CTA1-DD adjuvant is dependent on ADP-ribosyltransferase activity and requires the binding to Ig- via the DD moiety. The enhancing effect is associated with enlarged germinal centers, and binding of CTA1-DD to the B-cells strongly upregulates co-stimulatory molecules and counteracts apoptosis by inducing intracellular Bcl-2.     

Suppression by autogenous complementary idiotypes: the priority of the first response.

Complementary idiotypes or antibodies are considered to have combining site structures which are at least partly directed against each other. Complementary antibodies were induced in A/He mice by immunization with phosphorylcholine (PC)-containing antigens and by immunization with the PC-binding IgA myeloma protein TEPC-15 (T15). Both responses were monitored by enumerating plaque-forming cells (PFC) and assaying serum antibody levels against the corresponding antigens. Mice immunized at least three times with T15 in adjuvants had markedly suppressed responses to subsequent immunization with PC; similarly, mice preimmunized multiple times with PC had suppressed responses to immunizations with T15. In contrast, mice immunized with T15 in the interval between "primary" and "secondary" immunizations with PC had undiminished PFC responses to both antigens but significantly decreased antibody titers to PC. Simultaneous responses were also induced by immunizations with T15 superimposed on weekly immunizations with PC; with this regime, immunization with T15 actually enhanced the PFC response to PC, but serum antibody to PC was significantly lower than for mice immunized with PC only. Levels of serum antibody to PC were probably lower, either because anti-PC antibody was complexed with the complementary antibody directed against T15, or because the antibody directed against T15 prevented synthesis and/or release of anti-PC antibody by cells in vivo. Thus, an established prior autogenous immune response can dramatically suppress a subsequent primary complementary response, but the effects of complementary responses on each other are more complex with different sequences of immunization. Also, the effects of variables such as the amounts and ratios of the classes of antibodies on regulation of complementary responses remain to be defined.     

Amplification of T cell blastogenic responses in healthy individuals and patients with acquired immunodeficiency syndrome.

Human immunodeficiency virus (HIV) infection is associated with a profound impairment of T cell function. Hence, enhancement of T cell reactivity to viral and bacterial antigens is important in the treatment of patients with AIDS. To develop tools for amplifying T cell reactivity, we have immunized mice with human helper T cell clones and selected monoclonal antibodies (MAbs) that enhance in vitro blastogenic responses. MAb NDA5, which recognizes the leukocyte common antigen CD45, amplifies human T cell responses to mitogens and soluble antigens including HIV-1 glycoprotein (gp)-120 and peptides derived from the HIV-1 gp-120 sequence. In the presence of MAb NDA5, peripheral blood mononuclear cells (PBMC) from healthy, HIV-1-seronegative individual displayed augmented blastogenic responses to HIV-1 gp-120 and to HIV-1 gp-120 synthetic peptides. In vitro memory responses to various vaccines and to alloantigens were also enhanced in cultures with MAb. Similarly, the response of PBMC from AIDS patients to pokeweed mitogen, HIV-1 gp-120, and tetanus toxoid was enhanced with MAb NDA5. The finding that the in vitro immune response of patients with AIDS can be amplified with MAb NDA5, suggests that the in vivo immune response of immunodeficient individuals can also be enhanced.     

Special features of the priming process for a secretory IgA response. B cell priming with cholera toxin

Administration of cholera toxin/toxoid by either intraduodenal or parenteral routes increases the frequency of antigen-sensitive B cells in Peyer's patches (PP) and in distant lymphoid tissues greater than 50- fold. The special feature of mucosal priming with toxin is its unique effectiveness at generating secondary B cells, whose progeny express IgA exclusively, and such cells appear in highest frequency in PP and in appreciable numbers in spleen. Thus, this deliberate intraduodenal immunization seems to mimic the natural priming process induced by enteric bacterial colonization, which we have postulated to account for the high frequencies of IgA-committed cells specific for bacterial determinants in the PP of conventionally reared mice. furthermore, as a result of intraduodenal immunization, antigen-specific memory B cells are disseminated to sites distant form that of antigen application, including the lymphoid follicles associated with the respiratory mucosa. Direct antigenic stimulation of cells in the PP therefore results in effective cross-priming among mucosal and systemic sites through division, differentiation, and disemination of antigen- sensitive secondary B cells.     

Isotype-specific immunoregulation. Evidence for a distinct subset of T contrasuppressor cells for IgA responses in murine Peyer''s patches

The ability of murine Peyer's patch (PP) T contrasuppressor cells (Tcs) to reverse oral tolerance to the T cell-dependent (TD) antigen SRBC was studied both in vivo and in vitro. C3H/HeJ mice given SRBC orally for 4 wk are not rendered tolerant to this antigen and were used as a source of PP Tcs cells for adoptive transfer to identically treated, orally tolerized C3H/HeN mice. Transfer of 10(4) or 5 X 10(4) V. villosa-adherent PP T cells resulted in splenic IgM, IgG, and mainly IgA responses in C3H/HeN mice challenged systemically with SRBC. The T cell responsible was Lyt-1+, 2-, L3T4-, I-JK+ and V. villosa lectin-adherent, all characteristics of mature effector Tcs cells. This C3H/HeJ PP Tcs cell subset was also effective when added to in vitro cultures of tolerized spleen cells derived from SRBC-fed, C3H/HeN mice. Interestingly, C3H/HeJ PP Tcs cells restored mainly IgA responses when transferred in vivo or when added to suppressed C3H/HeN splenic cultures. Comparison of the functional activity of Tcs cells derived from spleen or PP of orally immunized C3H/HeJ mice revealed that splenic Tcs cells supported responses of all 3 isotypes; however, PP Tcs cells yielded three-fourfold higher IgA responses, when compared with IgM or IgG anti-SRBC responses. Adherence of C3H/HeJ PP Tcs to an Fc alpha R+ T cell line derived from IgA-specific Th cells resulted in a nonadherent cell fraction that potentiated only IgM and IgG responses, while bound Tcs cells preferentially supported IgA responses. These results suggest that murine PP contain IgA-specific Tcs cells that allow IgA response induction in the presence of Ts cells that mediate oral tolerance.     

Highly restricted expression of the thymus leukemia antigens on intestinal epithelial cells

The TL region of the major histocompatibility complex of the mouse contains dozens of tandemly arranged class I genes, including those encoding the thymus leukemia (TL) antigens. TL antigens have been thought to be expressed only on the surface of some T lineage cells, namely immature thymocytes of some mouse strains (TL+ strains), some leukemia cells, and activated T cells. While the function of TL antigens is unknown, recent studies have implicated the products of at least some TL region class I genes as molecules that present antigens to gamma/delta T cells. Since some gamma/delta T cells are known to be specifically associated with certain epithelial tissues, we have investigated the expression of some TL region class I genes in a variety of epithelium-containing tissues. Our results show that the TL antigen gene of C57BL/6 mice, T3b, and the TL antigen genes of BALB/c mice, T3d (previously T3c) and T18d (previously T13c), are highly expressed in the epithelium of the small intestine. In the case of T3b, we further show, using a T3 product-specific antibody, that its product is expressed on the surface of the columnar epithelial cells. In addition, we demonstrated that two other TL region class I genes of C57BL/6 origin, T9b and T21b, are also expressed nearly exclusively in intestinal epithelial cells. These results are consistent with the hypothesis that the products of these TL region class I genes are recognized by gamma/delta T cell receptors of intestinal intraepithelial lymphocytes, a subset of gamma/delta T cells that is localized in the intestinal epithelium and has a restricted V gamma repertoire. Finally, our study indicates that the relative levels of expression of the two homologous TL antigen genes, T3d and T18d, differ widely between the thymus and the intestine.     

Induction of murine peritoneal gamma/delta T cells and their role in resistance to bacterial infection

Previous studies have reported an association of gamma/delta T cells with microbial infection in both human lesions and murine infectious disease models. In this study we provide a comprehensive analysis of the conditions under which the induction of gamma/delta T cells occurs at a site of infection. We found a site-specific induction of gamma/delta T cells after the injection of Listeria monocytogenes in the peritoneal cavity of C3H mice. No changes were seen in the splenic or lymph node populations after these injections. Both the proportion and the absolute number of gamma/delta T cells increased in the peritoneal cavity. Additionally, when peritoneal T cells from Listeria- immune mice were restimulated in vitro, the induced gamma/delta T cells exhibited a greater expansion potential than the alpha/beta T cells. Neither the induced gamma/delta T cells nor those from normal mice expressed CD4 or CD8 on the cell surface. Thy-1 was expressed on only 29% of normal peritoneal gamma/delta T cells, but after intraperitoneal Listeria injection 65% of induced gamma/delta T cells expressed. Thy-1, Pgp-1 and CD45R expression on both normal and induced gamma/delta T cells was consistent with an activation phenotype. Significant increases in peritoneal gamma/delta T cells were not seen until 5-7 d after Listeria injection. The proportion of the CD3+ population expressing the gamma/delta T cell receptor remained elevated for 6-7 wk, while the absolute numbers of peritoneal gamma/delta T cells declined gradually over this time period, reflecting a decrease in both the number of lymphocytes and the percentage of these that were CD3+. Peak numbers of gamma/delta T cells were seen at day 10 with live microbes such as Listeria. A variety of microbes, toxins, mitogens, antigens, cytokines, and nonspecific inflammatory agents were evaluated for their ability to induce gamma/delta T cells in the peritoneal cavity. Both Gram-positive and Gram-negative bacteria as well as Mycobacteria were able to induce gamma/delta T cells that showed increased in vitro expansion potential. An exotoxin from a Gram- positive organism, listeriolysin-o, and the lipopolysaccharide (LPS) endotoxin from a Gram-negative organism were also effective. gamma/delta T cell responses to LPS were under lps gene control. Peak numbers of gamma/delta T cells were observed at day 3 after injection with exotoxins and endotoxins. Modifications that abrogated the virulence of a bacterial strain also eliminated the inductive effect for gamma/delta T cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogenic development and tissue distribution of V gamma 1-expressing gamma/delta T lymphocytes in normal mice

A hamster monoclonal antibody (mAb) recognizing an epitope in the V gamma 1-J gamma 4-C gamma 4 chain of the gamma/delta T cell receptor has been generated. Using this mAb, we have quantitated the occurrence of V gamma 1-bearing gamma/delta T cells in the developing thymus and in the lymphoid organs and several epithelia of adult mice. The V gamma 1-expressing cells constitute a minor gamma/delta T cell subpopulation during fetal and early postnatal life, but they constitute a major population of gamma/delta T cells in the thymus and in the peripheral lymphoid organs in adult mice. In addition, we found that V gamma 1- bearing cells comprise a large proportion (15-60%) of the gamma/delta T cells present in the intestinal epithelium (i-IEL) in all strains of mice tested. V gamma 1+ i-IEL are present in athymic (nude) mice and in antigen-free mice, demonstrating that they can develop extrathymically and that their presence in the intestinal epithelium is independent of the antigenic load of the gut. Our results show that V gamma 1-bearing lymphocytes account for the largest population of gamma/delta T cells in the mouse. This population includes a thymus-dependent component that homes to the secondary lymphoid organs and a thymus-independent component that constitutes a major fraction of the gamma/delta i-IELs.     

Thymic origin of embryonic intestinal gamma/delta T cells

Current evidence suggests both thymic and extrathymic origins for T cells. Studies in mice favor an in situ origin for a prominent population of intestinal intraepithelial lymphocytes that express gamma/delta T cell receptor (TCR). This developmental issue is explored in an avian model in which the gamma/delta lymphocytes constitute a major T cell subpopulation that is accessible for study during the earliest stages of lymphocyte development. In the chick embryo, cells bearing the gamma/delta TCR appear first in the thymus where they reach peak levels on days 14-15 of embryogenesis, just 2 d before gamma/delta T cells appear in the intestine. Using two congenic chick strains, one of which expresses the ov antigen, we studied the origin and kinetics of intestinal colonization by gamma/delta T cells. The embryonic gamma/delta+ thymocytes homed to the intestine where they survived for months, whereas an embryonic gamma/delta- thymocyte population enriched in thymocyte precursors failed to give rise to intestinal gamma/delta+ T cells. Embryonic hemopoietic tissues, bone marrow, and spleen, were also ineffective sources for intestinal gamma/delta+ T cells. Intestinal colonization by gamma/delta+ thymocytes occurred in two discrete waves in embryos and newly hatched birds. The data indicate that intestinal gamma/delta T cells in the chicken are primarily thymic migrants that are relatively long-lived.     

A protective role of gamma/delta T cells in primary infection with Listeria monocytogenes in mice

We have previously reported that T cells bearing T cell receptors (TCRs) of gamma/delta type appear at a relatively early stage of primary infection with Listeria monocytogenes in mice. To characterize the early-appearing gamma/delta T cells during listeriosis, we analyzed the specificity and cytokine production of the gamma/delta T cells in the peritoneal cavity in mice inoculated intraperitoneally with a sublethal dose of L. monocytogenes. The early-appearing gamma/delta T cells, most of which were of CD4-CD8- phenotype, proliferated and secreted IFN-gamma and macrophage chemotactic factor in response to purified protein derivative from Mycobacterium tuberculosis, or recombinant 65-kD heat-shock protein derived from M. bovis but not to heat-killed Listeria. To further elucidate the potential role of the gamma/delta T cells in the host-defense mechanism against primary infection with Listeria, we examined the effects of in vivo administration of monoclonal antibodies (mAbs) against TCR-gamma/delta or TCR-alpha/beta on the bacterial eradication in mice infected with Listeria. Most of alpha/beta T cells or gamma/delta T cells were depleted in the peripheral lymphoid organs at least for 12 d after an intraperitoneal injection of 200 micrograms TCR-alpha/beta mAb or 200 micrograms TCR-gamma/delta mAb, respectively. An exaggerated bacterial multiplication was evident at the early stage of listerial infection in the gamma/delta T cells-depleted mice, whereas the alpha/beta T cell-depleted mice exhibited much the same resistance level as the control mice at this stage although the resistance was severely impaired at the late stage after listerial infection.(ABSTRACT TRUNCATED AT 250 WORDS)