Search

Regulatory T (Treg) cells are a suppressive CD4+ T‐cell subset. We generated induced Treg (iTreg) cells and explored their therapeutic potential in a murine model of rapidly progressive glomerulonephritis. Polyclonal naive CD4+ T cells were cultured in vitro with interleukin‐2 (IL‐2), transforming growth factor‐β1, all‐trans‐retinoic acid and monoclonal antibodies against interferon‐γ and IL‐4, generating Foxp3+ iTreg cells. To enhance their suppressive phenotype, iTreg cultures were modified with the addition of a monoclonal antibody against IL‐12p40 or by using ROR γt–/– CD4+ T cells. Induced Treg cells were transferred into models of delayed‐type hypersensitivity and experimental glomerulonephritis. The iTreg cells exhibited comparable surface receptor expression and in vitro suppressive ability to natural Treg cells, but did not regulate antigen‐specific delayed‐type hypersensitivity or systemic inflammatory immune responses, losing Foxp3 expression in vivo. In glomerulonephritis, transferred iTreg cells did not prevent renal injury or modulate systemic T helper type 1 immune responses. Induced Treg cells cultured with anti‐IL‐12p40 had an enhanced suppressive phenotype in vitro and regulated dermal delayed‐type hypersensitivity in vivo, but were not protective against renal injury, losing Foxp3 expression, especially in the transferred cells recruited to the kidney. Use of ROR γt–/– CD4+ T cells or iTreg cells generated from sensitized CD4+ Foxp3– cells did not regulate renal or systemic inflammatory responses in vivo. In conclusion, iTreg cells suppress T‐cell proliferation in vitro, but do not regulate experimental glomerulonephritis, being unstable in this inflammatory milieu in vivo.

Oral tolerance promotes a generalized decrease in specific immune responsiveness to proteins previously encountered via the oral route. In addition, parenteral immunization with a tolerated protein also triggers a significant reduction in the primary responsiveness to a second unrelated antigen. This is generally explained by 'innocent bystander suppression', suggesting that the transient and episodic effects of inhibitory cytokines released by contact with the tolerated antigen would block responses to the second antigen. In disagreement with this view, we have previously shown that: (i) these inhibitory effects do not require concomitance or contiguity of the injections of the two proteins; (ii) that intravenous or intragastric exposures to the tolerated antigen are not inhibitory; and (iii) that the inhibitory effect, once triggered, persists in the absence of further contact with the tolerated protein, possibly by inhibition of secondary responsiveness (immunological memory). The present work confirms that immunological memory of the second unrelated antigen is hindered by exposure to the tolerated antigen and, in addition, shows that this exposure: (i) inhibits the inflammation triggered by an unrelated antigen through the double effect of inhibiting production of leucocytes in the bone marrow and blocking their migration to inflammed sites; and (ii) significantly blocks footpaw swelling triggered by carrageenan. Taken together, these results conclusively demonstrate that inhibitory effects of parenteral injection of tolerated antigens are much more general than suggested by the 'innocent bystander suppression' hypothesis.

Although intraocular tumours reside in an immune-privileged site, they can circumvent immune privilege and undergo rejection. Ocular tumour rejection typically follows one of two pathways. One pathway involves CD4+ T cells, delayed-type hypersensitivity (DTH), and culmination in ischemic necrosis of the tumour and phthisis (atrophy) of the eye. The second pathway is DTH-independent and does not inflict collateral injury to ocular tissues, and the eye is preserved. In this study, we used a well-characterized tumour, Ad5E1, to investigate the role of CD4+ T cells in the non-phthisical form of intraocular tumour rejection. It has been previously documented that CD4+ T cells and interferon (IFN)-gamma are necessary for rejection of these tumours in the eye. In this study, we demonstrate that CD4+ T cells can circumvent immune privilege and infiltrate intraocular Ad5E1 tumours. Following tumour rejection, CD4+ T cells from tumour rejector mice could be adoptively transferred to severe combined immunodeficiency (SCID) mice and protect them from intraocular Ad5E1 tumour growth. Tumour-specific CD4+ T cells produced IFN-gamma in response to Ad5E1 tumour antigens. Macrophages also contributed to rejection, as they were present in intraocular Ad5E1 tumours, and local depletion of macrophages resulted in progressive tumour growth. Ocular macrophages contributed to Ad5E1 tumour rejection, as Ad5E1 tumour rejection did not occur in macrophage-depleted SCID mice reconstituted with rejector CD4+ T cells. This demonstrates that macrophage and CD4+ T-cell co-operation is needed for non-phthisical rejection of intraocular tumours.

Summary The introduction of antigen into the anterior chamber of an eye induces the antigen-specific suppression of cell-mediated immunity and the antigen-induced production of immunoglobulin G2 antibodies. To define further the role of iris monocytic cells in the systemic suppression of cell-mediated immunity that follows the entry of foreign antigen into the anterior chamber, murine iris wholemounts or cell suspensions of iris cells were stained with fluorescent anti-F4/80 and/or anti-CD11c, anti-CD11b antibodies and examined by confocal microscopy or flow cytometry, respectively. Monocytic cells in iris cell suspensions were recovered from mice receiving an injection of trinitrophenylated bovine serum albumin (TNP-BSA) into an anterior chamber and Percoll-enriched iris cells separated into cells expressing F4/80 or CD11c were injected intravenously into TNP-BSA-immunized or naive recipients. The recipients were challenged to induce delayed-type hypersensitivity (DTH) or were provided with splenocytes or thymocytes that transfer the suppression of DTH. The homing of monocytic bone marrow cells to the iris was determined by the intravenous injection of bone marrow cells from green fluorescent protein (GFP)-transgenic donors into C57 mice, and the staining of recipient iris wholemounts with anti-F4/80 antibodies. Iris cells with a dendritic morphology expressing both F4/80 and/or CD11c and CD11b, some cells expressing only F4/80 or CD11c, were detected. The irides of irradiated GFP– mice that received intravenous GFP+ bone marrow cells contained GFP+ F4/80+ cells. F4/80+ and CD11c+ cells from the irides of donors that received intracameral TNP-BSA transferred the suppression of DTH when injected intravenously into TNP-BSA-immunized recipients, activated immunoregulatory thymocytes and activated antigen-specific splenic regulatory effector cells. These results support the hypothesis that iris monocytic cells may participate in the systemic induction of regulatory T cells.

The local immune response is characterized by an increase in the rate of entry of lymphocytes from the blood into regional lymph nodes and changes in the output of cells in lymph. While significant data are available regarding the role of inflammation-induced vascular adhesion processes in regulating lymphocyte entry into inflamed tissues and lymph nodes, relatively little is known about the molecular processes governing lymphocyte exit into efferent lymph. We have defined a novel role for lymphatic endothelial cells in the regulation of lymphocyte exit during a delayed type hypersensitivity (DTH) response to mycobacterial purified protein derivative (PPD). Soluble, pro-adhesive factors were identified in efferent lymph concomitant with reduced lymphocyte output in lymph, which significantly increased lymphocyte binding to lymphatic endothelial cells. While all lymphocyte subsets were retained, CD4+ T cells appeared less susceptible than others. Among a panel of cytokines in inflammatory lymph plasma, interferon (IFN)-gamma alone appeared responsible for this retention. In vitro adhesion assays using physiological levels of IFN-gamma confirmed the interaction between recirculating lymphocytes and lymphatic endothelium. These data demonstrate a new level of immune regulation, whereby the exit of recirculating lymphocytes from lymph nodes is selectively and sequentially regulated by cytokines in a manner equally as complex as lymphocyte recruitment.

Orally induced tolerance is a physiologically relevant form of peripheral tolerance, which is believed to be important for the prevention of pathological immune responses in the gut. Of several mechanisms proposed to mediate oral tolerance, one that has received much attention recently is the concept of regulatory CD4+ T cells. As recent studies have suggested that interleukin (IL)-15 may be important for the differentiation and maintenance of regulatory CD4+ T cells, we have examined the role of IL-15 in oral tolerance, using a soluble form of the IL-15 receptor (sIL-15R) which blocks the biological effects of IL-15 in vivo. Oral tolerance induced by feeding mice ovalbumin (OVA) in a low-dose regimen believed to induce regulatory T cell activity was not affected by the administration of sIL-15R during either the induction or maintenance phase of tolerance. Thus, oral tolerance does not involve an IL-15-dependent mechanism.

We investigated the role of T helper (Th)1- and Th2-type cytokines in delayed-type hypersensitivity to soluble protein antigens elicited early postimmunization. Mice were sensitized by intradermal injection without adjuvants, or subcutaneously with complete Freund's adjuvant, and subsequently ear challenged intradermally. As soon as day 3, antigen-specific eosinophil-rich responses were elicited in wild-type mice, but not in T-cell receptor-alpha-/- mice without adjuvant. Draining lymph node T cells stimulated with antigen secreted interleukin (IL)-4, IL-5 and interferon-gamma (IFN-gamma). IFN-gamma-dependent specific immunoglobulin G (IgG)2a and IL-4-dependent IgG1 were also generated. Delayed-type hypersensitivity ear swelling and local eosinophil recruitment were decreased in IL-5-/-, IL-4-/- and signal transducer and activator of transcription-6 (STAT-6)-/- mice, and with anti-IL-4 treatment of wild-type mice, suggesting Th2 mechanisms. Interestingly, responses were also decreased in IFN-gamma-/- mice, and IFN-gamma protein and the IFN-gamma-inducible CXC chemokine, IP-10, were present in 24-hr ear tissue extracts, suggesting Th1 effects. Finally, ear swelling, total histology and eosinophils were decreased in mice deficient in CXCR3, the chemokine receptor for IP-10. These results suggest that both a Th2-like (IL-5, IL-4 and STAT-6) and a Th1-like (IFN-gamma, IP-10, CXCR3) pathway contribute to eosinophil recruitment in early delayed-type hypersensitivity.

An important factor in the establishment of ocular immune privilege is the dynamic down regulation of T helper 1 (Th1) immune responses that occurs in response to antigens delivered intraocularly; a phenomenon that has been termed anterior chamber-associated immune deviation (ACAID). ACAID is characterized by the generation of splenic regulatory cells that inhibit the expression of delayed-type hypersensitivity. Previous studies have shown that antigens introduced into the anterior chamber of the eye induce the generation of a CD4+ T-cell population that suppress the induction of Th1 immune responses and the appearance of a second population of CD8+ T regulatory cells that suppresses the expression of Th1 inflammatory responses (= efferent suppressor cells). Experiments described here characterized the function of the CD4+ ACAID suppressor cell population and its effect on the generation of CD8+ efferent suppressor cells that inhibit the expression of DTH in situ. Both in vivo and in vitro experiments demonstrated that CD4+ T cells are required for the generation of CD8+ efferent suppressor cells. CD4+ T cells do not require cell contact with CD8+ T cells; instead they produce soluble IL-10 that is sufficient for the generation of ACAID suppressor cells. Finally, the CD4+ afferent T suppressor cells are not natural killer T cells, but do express the CD25 cell surface marker.

Immunization of mice with activated antigen-presenting cells (APC) pulsed ex vivo with cryptococcal capsular polysaccharide, a glucuronoxylomannan (GXM-APC) results in prolongation of survival and delayed-type hypersensitivity (DTH) responsiveness following infection with Cryptococcus neoformans (NU-2). GXM-APC has both non-specific and GXM-specific effects that influence the immune responses that develop in mice after infection with NU-2. Type 1 cytokine responses are augmented after immunization with APC alone, while GXM must be present for the vaccine to influence survival and DTH reactions. This investigation evaluated the role that major histocompatibility complex (MHC) and co-stimulatory molecules play in the non-specific and GXM-specific responses induced by GXM-APC. APC from CD40 knockout mice were as effective as wild-type APC for the induction of non-specific and GXM-specific responses. Blocking activity of B7-1 and B7-2 by treatment of immunized mice with monoclonal antibodies specific for these molecules just before and for 6 days following GXM-APC immunization decreased the splenic interferon-gamma response of mice subsequently infected with NU-2, but only in mice that were treated with both antibodies. These antibody treatments had no effect on DTH reactivity in similarly treated animals. MHC class I molecules were not involved in the antigen non-specific or GXM-specific activities of the vaccine. MHC class II molecules were not required for augmentation of type 1 cytokine responses but were needed for induction of the GXM-specific response that regulates the expression of DTH reactivity. This investigation has shown that an MHC class II-restricted, GXM-specific response is responsible for altering DTH responsiveness which is the correlate of immunity in this model.

SUMMARY Parenteral injection of a lipopeptide containing a human leucocyte antigen (HLA)A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope from the human cytomegalovirus (HCMV) immunodominant matrix protein pp65 efficiently induces systemic CTL responses in HLA-A*0201 transgenic mice. In this study, we demonstrate that intranasal (i.n.) administration of this lipopeptide, covalently linked to a universal T helper (Th) epitope (PADRE), also induces potent systemic CTL responses. Immune responses were substantially reduced when the unlipidated peptide analogue was used (P

In the present study we examine the effects of medroxyprogesterone acetate (MPA) on the specific antibody secretion to T-dependent antigens. Our results show that the in vivo administration of MPA to mice, 7 or 90 days before immunization with sheep red blood cells (SRBC), significantly enhanced both, primary and secondary antibody responses, without affecting delayed-type hypersensitivity (DTH). These effects could be counteracted by the anti-progestin onapristone or ZK 98299 (ZK) suggesting that MPA interacted with progesterone (PRG) receptors to increase B-cell response. To better understand the mechanisms involved in MPA activity we carried out cultures of splenocytes, bone marrow cells or lymph node cells from immunized mice in the presence of MPA, and evaluated the amount of antibody release to supernatants. We found that low doses of MPA (10(-9) M and 10(-10) M) significantly enhanced the in vitro production of specific immunoglobulin G (IgG) antibodies, an effect that appears to involve the interaction of the progestin with PRG receptors, as judged by the inhibition of MPA effects with ZK (10(-8) M) or RU486 (10(-9) M). These receptors were detected by flow cytometry analysis in a proportion of T lymphocytes. Because MPA did not increase the number of immunoglobulin-secreting cells, our findings suggest that MPA enhanced the capacity of individual cells to produce specific immunoglobulin.

BALB/c mice were inoculated with Bartonella henselae by both systemic and mucosal routes. Culture analysis of tissues from mice infected intraperitoneally with a high dose of B. henselae yielded positive results 24 hr after infection. However, culture analysis of blood taken between 6 hr and 7 days after infection from groups receiving live B. henselae were negative. Following intraperitoneal infection, B. henselae was detected by polymerase chain reaction in liver and mesenteric lymph nodes by 6 hr and up to 7 days after infection in liver, kidney and spleen tissue. Enzyme-linked immunosorbent assay (ELISA) of serum samples collected as early as 13 days after infection indicated humoral immune responses to B. henselae. Specific humoral responses remained through week 6. Analysis of faecal samples revealed induction of B. henselae-specific immunoglobulin A by day 28 after infection. In addition, B. henselae-specific cellular responses were indicated by a positive delayed-type hypersensitivity and a T helper 1 (Th1) (CD4+ T cell)-type cytokine response following in vitro stimulation of splenocytes. The significance and implications of these data in relation to B. henselae infections are discussed.

The naturally occurring trans-isomer of urocanic acid (trans-UCA), found in the stratum corneum, absorbs ultraviolet light (UV) and isomerizes to the cis-form. Cis-UCA has been shown to impair some cellular immune responses, and has been proposed as an initiator of the suppression that follows UV irradiation. UVB exposure leads to an increase in cis-UCA in the skin of rats from about 10% to 40% of the total UCA. Previously it has been demonstrated that UVB lowers immune responses to Trichinella spiralis after oral infection of rats with the parasitic worm. In the present study we investigated the role of cis-UCA in the control of this parasitic infection. Rats were infected orally with T. spiralis and injected with different doses of cis- or trans-UCA subcutaneously. Mitogenic responses and the mixed lymphocyte reaction were not affected by either isomer. In contrast, the number of T. spiralis larvae in muscle tissue of infected rats was increased significantly in the cis-UCA-treated animals compared with the trans-UCA-treated animals. In addition, delayed-type hypersensitivity (DTH) to T. antigen in infected rats was significantly impaired by cis-UCA but not by trans-UCA. If rats were injected with a monoclonal antibody with specificity for cis-UCA 2 hr prior to UVB exposure, the UVB-induced suppression in DTH to T. spiralis and the increase in larvae counts were significantly inhibited compared with rats that were similarly injected with a control antibody. Thus cis-UCA can inhibit the specific resistance to parasitic infections and acts as an important mediator of UVB-induced suppression of immunity to T. spiralis in the rat.

Ocular immune privilege is the result of a number of protective mechanisms, including a specialized immune response to antigen encountered in the anterior chamber of the eye. Anterior chamber-associated immune deviation, or ACAID, is characterized by the antigen-specific, selective down-regulation of systemic cell-mediated and humoral immune responses. One current hypothesis of the initiation of ACAID predicts that ocular APC process antigen and then migrate out of the eye and to the spleen where various regulatory T-cell populations are generated. A novel in vitro model of the ACAID spleen was developed to study the cells involved in the generation of suppressed T-cell immunity. ACAID APC co-cultured with whole splenocytes or splenic B and T cells induced efferent suppressors of delayed-type hypersensitivity (DTH). However, ACAID APC co-cultured with splenic T cells did not generate efferent suppressors of DTH. The requirement for B cells was confirmed with B-cell knockout mice. ACAID APC co-cultured with splenocytes from B-cell knockout mice did not induce efferent suppressors of DTH. Moreover, ACAID could not be induced in B-cell knockout mice in vivo. The reconstitution of B-cell knockout mice with wild-type B cells restored ACAID. In summary, these data confirm the role for B cells in the splenic phase of ACAID. A putative mechanism predicts that ACAID APC release antigenic peptides to B cells in the spleen. B cells then present antigen in a tolerogenic manner leading to the generation of regulatory T cells.

Although almost all of the energy contained within the ultraviolet (UV) wavelengths of solar radiation is absorbed within the epidermis and upper layers of the dermis, UV irradiation can suppress the immune response to antigens introduced at distant, non-irradiated body sites. The production of immune modulatory cytokines, such as interleukin-10 (IL-10), by UV-irradiated keratinocytes and its effect on T helper type 1 (Th1)/Th2-cell balance are thought to play a major role in the induction of systemic immune suppression. Because it is suggested that costimulatory molecules, such as CD80 and CD86, differentially stimulate Th1 and Th2 cells we wished to investigate the role of these costimulatory molecules in the activation of immune suppression. We injected UV-irradiated mice with monoclonal antibodies to CD80 and CD86 and asked what effect, if any, this would have on UV-induced immune suppression. Anti-CD86, but not anti-CD80 or control rat IgG, blocked UV-induced immune suppression. Moreover, monoclonal anti-CD86 blocked the induction of suppressor T cells normally found in the spleens of the UV-irradiated mice. Monoclonal anti-CD86 also reversed the UV-induced impairment of systemic antigen-presenting cell function. IL-10 was detectable in the serum of UV-irradiated mice as compared with normal controls, and injecting UV-irradiated mice with anti-CD86, but not anti-CD80 or control rat IgG, blocked the secretion of IL-10 into the serum. We propose that UV exposure favours costimulation by CD86, which enhances the production of serum IL-10, thus suppressing Th1-cell-mediated immune reactions.

This report evaluates the efficacy of eukaryotic expression plasmids encoding cytokines at modulating the induction and expression of cutaneous delayed-type hypersensitivity (DTH) responses to virus infections. Mice given a single intramuscular administration of cytokine DNA were subsequently infected with either herpes simplex virus (HSV) or vaccinia virus, then tested for DTH. Responses in animals given interleukin-10 DNA were markedly suppressed for at least 5 weeks after pretreatment. Animals also expressed diminished T-cell proliferative responses and modest changes in the balance of T helper type 1 and 2 T-cell reactions. Treatment of animals already sensitized to express DTH, also showed inhibited responses, these taking 6-7 days after treatment to become apparent. Our results show the potency and convenience of plasmid DNA encoding cytokines to modulate inflammatory reactions. Advantages and risks of the cytokine DNA approach are briefly discussed.

The adjuvant effect of mannan-coated liposomes on human immunodeficiency virus type-1 (HIV-1) DNA vaccine and the mechanism of this enhancement were studied. Coating of cationic liposomes with mannan significantly enhanced the ability of this vaccine to induce an HIV-specific delayed-type hypersensitivity (DTH) response. HIV-specific cytotoxic T-cell (CTL) activity elicited by DNA vaccination was also significantly enhanced with the mannan-liposome cocktail. This mannan-liposome-mediated activity was greatly inhibited by in vivo injection of anti-interferon (IFN)-gamma antibody, which suggests that IFN-gamma plays an important role in this HIV-specific immune response. The results of both isotype-specific antibody and cytokine analysis revealed that mannan-liposome-mediated DNA vaccination enhances Th1-mediated immunity.

Prolonged exposure to ultraviolet light (UV) is known to lead to premature skin ageing, increased incidence of cataract and a high risk of developing skin cancers. UV-B irradiation, even if given as a single suberythemal dose, suppresses some immune responses, possibly reducing the production of T helper (Th) 1 cytokines [interleukin (IL)-2 and interferon-gamma] and augmenting Th2 cytokines (IL-4, IL-5 and IL-10) in mice. We investigated the role of IL-4 in UV-B induced immunomodulation using IL-4 knockout (IL-4 -/-) mice and the parent strain Bb129. Suberythemal UV-B irradiation (1440 J/m2) led to a reduction in the density and antigen presenting ability of Langerhans' cells in the epidermis of both normal and IL-4 -/- mice. Exposure also induced an accumulation of CD4+ and CD8+ lymphocytes as well as dendritic cells in the lymph nodes draining the irradiated site in both strains. The proliferation of lymph node cells in response to the mitogen concanavalin A was enhanced in the IL-4 -/- mice compared with the parent strain. Following UV-B exposure, this proliferation was increased in lymph node cells of parent mice but was significantly suppressed in the IL-4 -/- mice. The contact hypersensitivity (CH) response to oxazolone was suppressed to the same extent by UV-B irradiation in both strains. In the parent mice, infected with herpes simplex virus (HSV) following UV-B exposure and challenged subsequently with inactivated virus, the delayed hypersensitivity (DH) response was suppressed by about 50% compared with unirradiated mice; no such suppression in DH occurred in irradiated IL-4 -/- mice infected with HSV. Thus, IL-4 may be an important mediator of the UV-B-induced suppression in DH but not in CH, where other cytokines may be involved or may compensate for the lack of IL-4.

Cell-mediated immunity is a primary host resistance mechanism against many infectious organisms and is responsible for leucocyte recruitment to the infection site. Delayed-type hypersensitivity (DTH) reactions are in vivo correlates of cell-mediated immunity and have long been used to assess the level of cell-mediated immune (CMI) responsiveness to specific antigens. It has been difficult to study the kinetics of cellular influx and cytokine composition at the site of an on-going CMI reaction. Consequently, knowledge of the sequential events occurring during the efferent phase of a CMI response is incomplete. Here we report on the use of a gelatin sponge model for evaluating the progression of events during the effector phase of a DTH reaction to antigens of the mycotic organism Cryptococcus neoformans. Previously, we have shown that 24 hr after antigen injection into sponges in infected or immune mice, the leucocyte types infiltrating the sponges are consistent with a classical murine DTH reaction. Through kinetic studies, we show here that neutrophils are the first leucocytes to appear in DTH-reactive sponges, followed by increases in lymphocyte and then monocyte numbers. Tumour necrosis factor (TNF), interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-5 were elevated in DTH-reactive sponges compared with control sponges, and each cytokine had a relatively unique temporal profile. IL-4 was not detectable in the sponges. Together our data indicate that the expression of a CMI response comprises a well-regulated sequential influx of leucocytes that contribute to the lymphokine composition of the reaction.

The adjuvant activity of saponin for T-cell responses was evaluated and compared with that of complete Freund's adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed-type hypersensitivity and T-cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin-10 (IL-10) and low interferon-gamma (IFN-gamma) levels were observed in lymph node (LN) cell cultures from saponin-immunized mice in contrast with the high IFN-gamma and decreased IL-10 production by LN cells from CFA-immunized mice. Production of IL-10 and IFN-gamma in these conditions was CD4-activation dependent. Concanavalin A (Con A)-stimulated interleukin-4 (IL-4) production was higher in saponin-immunized mice than in CFA-immunized mice. IL-10 produced by LN cells from saponin-immunized mice suppressed IFN-gamma production and Con A-induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T-helper (Th)1 and Th2-type cells by immunization with saponin; in vitro a Th2-type cytokine response with high IL-10 production predominates, indicating preferential priming towards a Th2-type response. Immunization with CFA induced a Th1-type cytokine response. To our knowledge this is the first report in which an adjuvant is shown to prime for a dominant IL-10 production.

The immunological privilege of the anterior chamber (AC) of the eye is due, at least in part, to a selective antigen-specific down-regulation of delayed-type hypersensitivity (DTH) and a normal induction of antibody responses: a phenomenon that has been termed anterior chamber-associated immune deviation (ACAID). This dichotomy in the systemic immune responses is suggestive of a T-helper type-2 (Th2)-dominated immune phenotype in which a Th2 cell population is preferentially activated and cross-regulates T-helper type-1 (Th1) effector elements. This hypothesis was tested by comparing the cytokine pattern of antigen-pulsed spleen cells from mice primed in the anterior chamber with antigens that induce ACAID with responses in hosts primed with antigens that do not induce ACAID. The results indicated that CD4+ spleen cells from hosts primed in the AC with antigens that induce ACAID produced significant quantities of interleukin-10 (IL-10) but insignificant levels of IL-2, IL-4 and interferon-gamma (IFN-gamma). In contrast, hosts primed in the AC with antigens that do not induce ACAID, but instead elicit normal DTH, displayed cytokine patterns indicative of a Th1 response significant quantities of IL-2 and IFN-gamma were produced while IL-4 and IL-10 secretion was insignificantly different from normal controls. The immunological phenotype of the AC-primed hosts could be altered by systemic treatment with antibodies against either a Th1 cytokine (IFN-gamma) or a Th2 cytokine (IL-10). Hosts treated with anti-IL-10 antibody and subsequently primed in the AC with ACAID-inducing antigens developed normal DTH responses, while hosts treated with anti-IFN-gamma antibody and primed in the AC with antigens that normally produce positive DTH responses failed to develop positive DTH collectively the results support the proposition that immune privilege in the AC of the eye is due to the selective activation of a Th2 population that cross-regulates Th1 responses.

We have previously reported that the buccal mucosa can support delayed type hypersensitivity (DTH) reactions to contact sensitizers. In the present study, we show that cells isolated from the buccal epithelium are able to present soluble exogenous antigens to specific T cells. Single cell suspensions obtained by enzymatic dispersion of buccal epithelial sheets could present the native protein antigen hen-egg lysozyme (HEL) to the I-Ak-restricted CD4+ T-cell hybridoma specific for a.a 46-61 on HEL. T-cell activation resulted in interleukin-2 (IL-2) production which could be inhibited by anti-major histocompatibility complex (MHC) class-II antibodies of pertinent specificity. Immunohistochemical staining of whole buccal epithelial sheets revealed that all MHC II positive cells had a dendritic morphology and expressed ATPase activity, indicating that these cells represent a major antigen-presenting cell (APC) population in this tissue. Furthermore, single cell suspensions isolated from buccal epithelium (BEC) after local in vivo administration of either a native soluble protein, a synthetic dodecapeptide, or a contact sensitizer were able to activate antigen-specific T cells ex vivo. Kinetic analyses indicated that maximal APC activity in the oral epithelium occurred within 1 hr after local antigen administration, and had essentially vanished after 24 hr. Conversely, APC activity was undetectable in draining cervico-mandibular lymph node cell suspensions recovered 1 hr after local antigen injection but became manifest after 3-24 hr. These observations suggest that dendritic cells can acquire antigens in the buccal epithelium and migrate to draining lymph nodes where they present processed antigen to MHC class II-restricted T cells. This APC population may thus be a critical element in the initiation of Th1-driven DTH responses in the oral mucosa.

CD8(+) regulatory (suppressor) T cells are induced by complex cellular pathways in the spleens of mice that have received an injection of antigen into the anterior chamber (AC) of an eye, an immune-privileged site. Although these CD8(+) regulatory T cells perform an antigen-specific regulatory function for an immune response to self and non-self antigens, the mechanisms of the activation or function of these regulatory cells are not clear. Here, we describe a novel mechanism for the activation of splenic CD8(+) regulatory T cells induced by injection of antigen into the AC. Immunization of mice with trinitrophenyl and bovine serum albumin (TNP-BSA) amplified AC-induced splenic CD8(+) regulatory T cells that suppressed the initiation of contact sensitivity when transferred to immunized, challenged mice. These CD8(+) regulatory T cells were produced independently of perforin, indicating that they are not canonical cytotoxic T cells. Fas ligand (FasL)-deficient CD8(+) regulatory T-cell function was rescued by inclusion of exogenous interferon-gamma (IFN-gamma), demonstrating that the expression of FasL by CD8(+) regulatory T cells was dispensable, but IFN-gamma was not. Ultimately, we demonstrated that the generation of these CD8(+) regulatory T cells occurred independently of IFN-gamma, but their suppressor function required IFN-gamma receptor stimulation.

Epicutaneous immunization is a potential novel technique for topical vaccine delivery. It targets the immunologically rich milieu of the skin while having the advantage of being a non-invasive immunization procedure. By disrupting the stratum corneum of the epidermis a natural adjuvant effect can be achieved through activation of resident Langerhans cells. This negates the normal need for co-application of noxious adjuvants. Epicutaneous immunization on barrier-disrupted skin induces potent antigen-specific systemic immunity with a strong T helper type 2 (Th2) bias. We show here that epicutaneous immunization enhances the vigour of a subsequent T-cell response to the same antigen. The induced systemic Th2 response prevents the development of Th1 responses induced through injection of antigen in complete Freund's adjuvant (CFA). Prior epicutaneous immunization results in reduced production of antigen-specific interferon-gamma and immunoglobulin G2a (IgG2a) and enhanced interleukin-4, IgG1 and IgE responses to immunization with CFA. Moreover, epicutaneous immunization converts an established Th1 response to a Th2 response, as demonstrated by the specific reduction of interferon-gamma and IgG2a and the enhancement of interleukin-4 and IgE. This Th2 dominance of epicutaneous immunization may have direct therapeutic application as an immune-modulating procedure in Th1-dominant diseases such as autoimmune rheumatoid arthritis, type 1 diabetes, Hashimoto's thyroiditis and multiple sclerosis.

The immune privilege that exists in the eye is maintained by various mechanisms. One of the best studied is a form of systemic tolerance termed anterior chamber-associated immune deviation (ACAID). We have investigated the mechanisms by which ocular inflammation associated with the vitreous cavity (VC) is reduced, by injecting either ovalbumin (OVA) or allogeneic splenocytes into the VCs of mice, and assessed the effect of this on delayed type hypersensitivity (DTH) responses. After antigen inoculation into the VC, antigen-specific DTH responses were significantly impaired and we named this phenomenon 'vitreous cavity-associated immune deviation' (VCAID). VCAID could also be induced by inoculating antigen-pulsed macrophages into the VC. However, VCAID did not develop either in mice with inflamed eyes, whether as a result of experimental autoimmune uveitis or coadministration of interleukin (IL)-6 in the VC, or in knockout mice deficient for natural killer T (NKT) cells. Finally, we found that so-called 'hyalocytes' are the only cells present in the VCs of normal mice, uniformly distributed on the retinal surface. Interestingly, they express F4/80, suggesting that hyalocytes are candidate antigen-presenting cells (APCs) responsible for mediating VCAID. As for the anterior chamber model, systemic tolerance can be induced in the VC in non-inflamed eyes and in the presence of invariant NKT cells.

Studies of tuberculosis have suggested a shift in dominance from a T helper type 1 (Th1) towards a Th2 immune response that is associated with suppressed cell-mediated immune (CMI) responses and increased humoral responses as the disease progresses. In this study a natural host disease model was used to investigate the balance of the evolving immune response towards Mycobacterium bovis infection in cattle with respect to pathogenesis. Cytokine analysis of CD4 T-cell clones derived from M. bovis-infected animals gave some indication that there was a possible relationship between enhanced pathogenesis and an increased ratio of Th0 [interleukin-4-positive/interferon-gamma-positive (IL-4(+)/IFN-gamma(+))] clones to Th1 (IFN-gamma(+)) clones. All animals developed strong antimycobacterial CMI responses, but depressed cellular responses were evident as the disease progressed, with the IFN-gamma test failing to give consistently positive results in the latter stages. Furthermore, a stronger Th0 immune bias, depressed in vitro CMI responses, elevated levels of IL-10 expression and enhanced humoral responses were also associated with increased pathology. In minimal disease, however, a strong Th1 immune bias was maintained and an anti-M. bovis humoral response failed to develop. It was also seen that the level of the anti-M. bovis immunoglobulin G1 (IgG1) isotype antibody responses correlated with the pathology scores, whereas CMI responses did not have as strong a relationship with the development of pathology. Therefore, the development and maintenance of a Th1 IFN-gamma response is associated with a greater control of M. bovis infection. Animals progressing from a Th1-biased to a Th0-biased immune response developed more extensive pathology and performed less well in CMI-based diagnostic tests but developed strong IgG1 humoral responses.

The prevalence of food allergic diseases is rising and poses an increasing clinical problem. Peanut allergy affects around 1% of the population and is a common food allergy associated with severe clinical manifestations. The exact route of primary sensitization is unknown although the gastrointestinal immune system is likely to play an important role. Exposure of the gastrointestinal tract to soluble antigens normally leads to a state of antigen-specific systemic hyporesponsiveness (oral tolerance). A deviation from this process is thought to be responsible for food-allergic diseases. In this study, we have developed a murine model to investigate immunoregulatory processes after ingestion of peanut protein and compared this to a model of oral tolerance to chicken egg ovalbumin (OVA). We demonstrate that oral tolerance induction is highly dose dependent and differs for the allergenic proteins peanut and OVA. Tolerance to peanut requires a significantly higher oral dose than tolerance to OVA. Low doses of peanut are more likely to induce oral sensitization and increased production of interleukin-4 and specific immunoglobulin E upon challenge. When tolerance is induced both T helper 1 and 2 responses are suppressed. These results show that oral tolerance to peanut can be induced experimentally but that peanut proteins have a potent sensitizing effect. This model can now be used to define regulatory mechanisms following oral exposure to allergenic proteins on local, mucosal and systemic immunity and to investigate the immunomodulating effects of non-oral routes of allergen exposure on the development of allergic sensitization to peanut and other food allergens.

SUMMARY Initiation of an antitumour immune response is characterized bya complex process of chemokine-mediated cell migration and cell–cell interactions. Overexpression of chemokine CCL19 in tumour cells has been shown to result in accelerated tumour rejection under certain experimental conditions, suggesting a novel approach in the therapyof neoplastic malignancies. To investigate CCL19-mediated modulations of cellular immune responses in vivo, we generated a chimeric CCL19-immunoglobulin G2b (IgG2b) Fc fusion protein, which binds to the chemokine receptor CCR7 comparable to native CCL19. CCL19-IgG2b possesses a long-lasting potent chemotactic activityas a result of the extended half-life of Fc fusion proteins. Stable overexpression of CCL19-IgG2b in BALB ⁄c-derived J558L tumour cells fails to support tumour cell rejection following transplantation in syngeneic mice. Moreover, overexpression of CCL19IgG2b hinders tumour rejection in allogeneic C57BL ⁄6 mice. This phenomenon was accompanied bya six-fold increase of dendritic cells (DCs) isolated from CCL19-IgG2b-secreting tumours when compared to the number of DCs isolated from control parental J558L tumours. While mice bearing the allogeneic parental tumour showed an intense hypercellularity in the draining lymph nodes, no such response could be observed in the draining lymph nodes of mice carrying the CCL19-IgG2b-secreting tumour. We could demonstrate that overexpression of CCL19-IgG2b in tumour cells retains antigen-presenting cells in the tumour mass and prevents DCs from migrating into draining lymph nodes to present antigens and to activate T cells, resulting in an impaired immune response against the tumour.

Recently it has been shown that selective subconjunctival macrophage depletion reduced the incidence and severity of stromal herpes simplex virus (HSV) keratitis in mice. In this study, we examined the effect of conjunctival macrophage depletion on the corneal and systemic T-cell-mediated immune response. BALB/c mice were treated with subconjunctival injections of dichloromethylene diphosphonate (Cl2MDP)-liposomes (Cl2MDP-LIP) or phosphate-buffered saline (PBS) 7 and 2 days before corneal infection with 105 plaque-forming units (PFU) of HSV-1 (KOS strain). Interferon (IFN)-gamma, interleukin (IL)-2, and IL-4 production in the cornea was analysed by enzyme-linked immunosorbent assay (ELISA), and cytokine mRNA levels (IFN-gamma, IL-4) were measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Cell culture supernatants from submandibular lymph nodes were analysed by ELISA for expression of IFN-gamma, IL-2, and IL-4 and by bioassay for IL-6. The HSV-1-specific proliferative response of lymphocytes from regional lymph nodes and the delayed-type hypersensitivity (DTH) response were tested after corneal infection. Virus-neutralizing antibody titres and HSV-1-specific immunoglobulin G (IgG)2a/IgG1-ratios were measured. Cytokine mRNA expression (IFN-gamma, IL-4) and secretion (IFN-gamma, IL-2, IL-4) in the corneas were decreased after HSV-1 corneal infection in the macrophage-depleted mice. The secretion of IFN-gamma and IL-2 was decreased in the regional lymph nodes from Cl2MDP-LIP-treated animals (P0.05). Furthermore, Cl2MDP-LIP-treated mice had decreased HSV-1 specific proliferative responses (P0.05) and DTH response after corneal HSV-1 infection (P0.05). The virus-neutralizing serum-antibody levels (P0.05) increased while the HSV-1 specific IgG2a/IgG1-ratio was unaffected after macrophage depletion. Macrophage depletion did not induce a shift between the T helper 1 (Th1) and Th2 response in this HSK model. The data suggest that conjunctival macrophage functions are enhancing the T-cell-mediated immune response after corneal infection. This effect is at least in part responsible for the impaired course of herpetic keratitis after macrophage depletion.

Summary We demonstrate the induction of antigen-specific interleukin-10 (IL-10)-secreting cells in murine Peyer's patches (PPs) after low-dose β-lactoglobulin (BLG) feeding. In addition, we show that PP cells can inhibit the T-cell proliferative response in vitro as well as T-cell-mediated inflammation in vivo. The active suppression mediated by these regulatory cells was seen only within a narrow range of antigen dosage (feeding), with the most prominent effect at 5 × 1 mg BLG. On either side of this range, T-helper 1-like cytokine responses were observed when PP cells were stimulated with antigen in vitro. This result correlated with reduced production of regulatory cytokines as well as reduced activity of bystander suppression. We found that changes in IL-10 production correlated inversely with changes in interferon-γ production. Inhibitory effects mediated by CD4+ PP cells were partially neutralized by antibodies to IL-10 and transforming growth factor-β. Interestingly, the generation of such regulatory cells after low-dose BLG feeding exhibited organ dependence. Among spleen, lymph node and PP cells derived from orally tolerized mice, PP cells were the most effective in promoting bystander suppression in the presence of BLG, indicating the significance of PPs as an inductive site for antigen-specific regulatory cells upon induction of low-dose oral tolerance. Moreover, PP cells from mice fed 5 × 1 mg BLG were shown to suppress a BLG-specific delayed-type hypersensitivity response induced in footpads, suggesting that IL-10-secreting PP cells regulate systemic inflammation.

Problems of logistics, compliance and drug resistance point to an urgent need for immunotherapeutic strategies capable of shortening the current 6-month chemotherapy regimens used to treat tuberculosis, or of supplementing ineffective therapy. In this study we sought to define the mechanism of action of two immunotherapies, both of which have previously been shown to prolong survival. Secondly, we wished to identify any clinically useful synergy between these therapies. In BALB/c mice infected via the trachea with Mycobacterium tuberculosis H37Rv there is an initial phase of partial resistance dominated by type 1 cytokines plus tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), followed by a phase of progressive disease. This progressive phase is accompanied by increasing expression of IL-4, and diminished expression of IL-1 and TNF-alpha. Animals in this late progressive phase of the disease (day 60) were treated with two injections (day 60 and day 90) of 0.1 or 1.0 mg of heat-killed Mycobacterium vaccae, or with 3beta, 17beta-androstenediol (AED; 25 microg subcutaneously three times/week), or with both therapies. We show here using four techniques in parallel (morphometry, immunohistochemistry with automated cell counting, semiquantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assays of cytokines in lung extracts) that treatment with M. vaccae causes a switch back towards a type 1 cytokine profile, restoration of expression of IL-1alpha and TNF-alpha, and a switch from pneumonia to granuloma. This is very similar to the changes previously seen after treatment with AED. However, there was no evidence for synergy between M. vaccae and AED.

Delayed-type hypersensitivity (DTH) is an important in vivo manifestation of cell-mediated immunity. We examined the DTH response to methylated bovine serum albumin of a novel mutant strain of mice that have soluble CD4 (sCD4) in their circulation without expression of CD4 on the cell surface. The DTH response of the mutant mice was severely impaired, although the response of CD4 knockout (KO) mice, generated by homologous recombination, was comparable to that of wild-type mice. The response of the mutant mice was restored by the neutralization of sCD4 with anti-CD4, and that of CD4KO mice was markedly reduced by the implantation of a diffusion chamber containing sCD4 cDNA transfectant cells. The restored DTH response of the mutant mice treated with anti-CD4 was abolished by treatment with anti-interferon-gamma (IFN-gamma). IFN-gamma production by CD4 mutant and CD4KO mice was consistent with their DTH response and inversely related to the presence of sCD4 in their circulation, indicating that sCD4 impairs the DTH response by blocking the production of IFN-gamma in our mutant mice. These results raise the possibility that sCD4 could impair cell-mediated immunity. Our mutant mice would provide a useful tool with which to analyse the mechanisms of the DTH reaction.

We have investigated whether CD95-CD95 ligand interactions are important in anterior chamber-associated immune deviation (ACAID) induced by soluble protein antigen, and if so, to identify the participating cells on which these molecules are expressed. Peritoneal exudate cells as antigen-presenting cells (APC) obtained from B6.lpr/lpr, B6.gld/gld and C57BL/6 mice were cultured with ovalbumin (OVA) and transforming growth factor-beta2 (TGF-beta2) overnight, then injected intravenously into C57BL/6 or B6.lpr/lpr recipients. Some B6.lpr/lpr mice were reconstituted with naive T cells from wild-type C57BL/6 donors. In other experiments, B6. lpr/lpr and B6.gld/gld mice received an anterior chamber injection of OVA followed 7 days later by subcutaneous immunization with OVA plus adjuvant. Delayed hypersensitivity (DH) was assessed with an ear swelling assay. T cells activated in vitro with OVA-pulsed, TGF-beta-treated APC were tested in vivo for their capacity to suppress DH expression in a local adoptive transfer assay. The results indicate that when ACAID was induced by in-vitro generated ACAID-inducing cells, the APC expressed CD95L, and recipient T cells expressed CD95. The capacity of in vitro generated regulatory T cells to suppress DH expression to OVA in vivo was not governed by CD95-CD95L interactions. When OVA was injected into the anterior chamber of naive mice, CD95 expression was required for ACAID induction, although ACAID was readily induced in CD95L-deficient mice. We conclude that CD95-CD95L interactions are required in ACAID for the initial stage of APC presentation of eye-derived antigens to T cells, and that CD95-CD95L interactions participate at one or more additional step in the process by which ACAID is induced by soluble protein antigens.

Previous studies in this laboratory have shown that mice with a gene disruption to the intracellular adhesion molecule-1 (ICAM-K/O) express normal cell-mediated immunity but cannot mount delayed-type hypersensitivity reactions following Mycobacterium tuberculosis infection. However, even in the absence of any appreciable granuloma formation, these mice control bacterial growth for at least 90 days. While not required to control the infection initially, we hypothesized that granuloma formation was required to control chronic infection, acting by surrounding infected cells to prevent bacterial dissemination. To test this, ICAM-1 knockout mice were infected with a low dose aerosol of M. tuberculosis Erdman and were found to succumb to infection 136+/-30 days later, displaying highly elevated bacterial loads compared to wild-type mice. Lung tissue from ICAM-K/O mice displayed extensive cellular infiltration and widespread tissue necrosis, but no organized granulomatous lesions were evident, whereas the control mice displayed organized compact granulomas. These data demonstrate that while a granulomatous response is not required initially to control M. tuberculosis infection, absence of granulomas during chronic infection leads to increased bacterial growth and host death. Thus these data support the hypothesis that granuloma formation is required to control chronic infection, acting by surrounding and walling off sites of infection to prevent bacterial dissemination and maintain a state of chronic infection.

A recombinant bacillus Calmette-Guérin (BCG) vaccine has been developed, which constitutively secretes interleukin (IL)-2. Groups of deer were immunized with either normal BCG (Pasteur 1173 P2 strain) or recombinant BCG (rBCG/IL-2) and their immune responses were monitored over 3 months. Animals gained weight over this period and showed no signs of adverse reactions to either vaccine. Lymphocyte transformation responses did not differ significantly between the two groups. No antibody that was specific for BCG was detected in any animal. Intradermal skin-test responses to BCG antigens showed that the rBCG/IL-2 induced a smaller delayed-type hypersensitivity response than the normal BCG. Cytokine transcription was determined by reverse transcription-polymerase chain reaction (RT-PCR). While IL-2 and interferon-gamma (IFN-gamma) levels did not differ significantly between the two groups, the level of IL-4 was found to be lower in the group given rBCG/IL-2. This resulted in a strong interferon-gamma:IL-4 ratio, suggesting a skewing of the immune response towards a Type 1 response. The rate at which the vaccine was eliminated from the host was the same regardless of whether BCG or rBCG was used. At autopsy (3 months after vaccination) 99.99% of the organisms had been eliminated. The small number of organisms isolated from the draining lymph node of animals given rBCG/IL-2 were grown in antibiotic-containing media. They were shown to still contain the shuttle plasmid and to secrete biologically active IL-2, indicating that the plasmid was stably maintained despite the host's immune response and in the absence of antibiotic selection.

The delayed-type hypersensitivity (DTH) to purified protein derivative (PPD) test has been used to infer about protective immunity to Mycobacterium tuberculosis and to diagnose tuberculosis. We showed that in memory tuberculosis-immune mice both DTH to PPD and resistance to M. tuberculosis could be effectively elicited in the footpad and both reactions led to the accumulation of reactive T cells in the regional lymph nodes with a CD4+ phenotype and characterized by the secretion of high levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) and no IL-4. By adoptive transfer into nude mice of highly purified CD4+ T cells harvested during the recall of protective immunity it was confirmed that this population mediated both manifestations. However, the specificity of the T cells recruited during these processes were found to differ markedly; T cells involved in protection to a challenge with live tuberculosis bacilli recognized predominantly low-mass culture filtrate antigens below 15 000 MW, while cells recruited during DTH to PPD were directed to molecular mass fractions between 15 000 and 31 000. Using single purified antigens we showed that the latter cells recognized the secreted mycobacterial protein Ag85B and the heat-shock proteins, DnaK and GroEL. Protective T cells, in contrast, were characterized by a very high frequency of T cells directed to the ESAT-6 peptide 1-20.

The contribution of autoimmune responses to the pathogenesis of Theiler's virus-induced demyelinating disease was investigated. Delayed-type hypersensitivity responses to myelin were examined in both symptomatic and asymptomatic mice at different times post-infection, in order to determine whether autoreactivity correlates with the development of demyelination. The results indicate that although autoimmune responses probably do not play a major role in the initiation of demyelination at early times post-infection, autoreactivity to myelin antigens dose eventually develop in symptomatic animals, perhaps through the mechanism of epitope spreading. Autoimmunity to myelin components is therefore an additional factor that may contribute to lesion progression in chronically diseased animals.

A hallmark of infection with Cryptococcus neoformans is depression of the immune system characterized by poor inflammatory responses and loss of delayed-type hypersensitivity (DTH) and antibody responses. T-suppressor cell (Ts) responses, elicited by the capsular polysaccharide (GXM) of the organism, are known to develop during infection. This study was undertaken to develop a method to inhibit the anti-GXM Ts response and thereby study the influence of the Ts response on immune responsiveness and survival in cryptococcosis. Antigen-presenting cells (APC), elicited with complete Freund's adjuvant (CFA), were treated in vitro with GXM (GXM-APC). The GXM-APC were injected intravenously into normal mice. These mice were resistant to induction of anti-GXM Ts cells when soluble GXM was administered in tolerogenic doses or when animals were infected with C. neoformans. Inhibition of the anti-GXM Ts response was specific to GXM as levan-APC did not inhibit induction of anti-GXM Ts cells. Inhibition of the anti-GXM Ts response could not be attributed to increased clearance of GXM due to induction of anti-GXM antibodies or other mechanisms. Anti-cryptococcal DTH responses were lost in mice by the second week of infection. However, treatment with GXM-APC, but not levan-APC, allowed mice to maintain their DTH response. GXM-APC pretreatment enhanced survival of infected mice compared with mice pretreated with levan-APC. These results show that GXM-APC induces immune responses that inhibit the induction of Ts responses and enhances DTH responses in infected mice. These responses correlate with enhanced survival after cryptococcal infection.

We developed a candidate DNA vaccine designated pCMV160IIIB with pcREV (pCMV160IIIB/REV) that encodes gp160 of human immunodeficiency virus (HIV)-1IIIB and Rev driven by the cytomegalovirus (CMV) promotor. This vaccine induced both HIV-1-specific antibodies and cytotoxic T lymphocyte (CTL) activity. In the present study, we inoculated the TCA3 expression plasmid into mouse skeletal muscle with pCMV160IIIB/REV to determine whether this cytokine expression plasmid was able to modify the immune response. Results of a delayed-type hypersensitivity (DTH) assay using footpad swelling as well as those of a CTL assay clearly demonstrated that cell-mediated immunity (CMI) elicited by co-inoculation of pCMV160IIIB/REV with the TCA3 expression plasmid was markedly enhanced compared with that obtained using pCMV160IIIB/REV alone. When TCA3 expression plasmid was inoculated with anti-TCA3 antibody, enhancement of the DTH response was suppressed below the level of that obtained with pCMV160IIIB/REV alone. The titre of HIV-1-specific IgG2a was slightly high when pCMV160IIIB/REV was co-inoculated with this plasmid, suggesting that T-helper 1 (Th1) response was predominant in TCA3-inoculated mice. Infiltration of mononuclear cells was seen in the muscles at sites where TCA3 expression plasmid had been inoculated. Our present data suggest that TCA3 expression plasmid has potent adjuvant activity that results in an augmented CMI response.

Although mice acquire only a slight degree of protection against tuberculosis by immunization with Mycobacterium leprae (M. leprae) hsp65 in incomplete Freund's adjuvant, protection is substantial following immunization by injection with J774 macrophage-like tumour cells that express the protein from the mycobacterial gene via a retroviral vector. We here took the same vector, used it to transfect the gene into normal murine bone marrow cells in vitro, and then used the transfected cells to reconstitute haematopoiesis in lethally irradiated mice. Bone marrow-cell clonal expansion and production of the protein in vivo resulted in specific delayed-type hypersensitivity and protection against challenge with Mycobacterium tuberculosis (M. tuberculosis) in about half of recipients. Counts of live bacteria in liver at 3 weeks were fivefold lower in delayed-type hypersensitivity (DTH)-positive than in DTH-negative mice. Other mice acquired neither DTH nor protection despite the presence of the protein in peripheral blood.

In order to examine the possibility that a local chronic infection can induce organ-specific autoimmune disease, we inoculated unilateral kidneys with viable Listeria monocytogenes (intrarenal infection). The delayed footpad reaction against syngeneic kidney homogenate (KH) became positive from 1 week after initiating the intrarenal infection. A proliferative response of the spleen T cells from the infected mice was also observed against KH from 1 week after initiating the intrarenal infection, but no such response was seen against liver homogenate (LH). In contrast, an intravenous Listeria infection did not induce a delayed footpad reaction or proliferative response against KH, suggesting that these autoimmune responses were not caused by molecular mimicry between renal antigens and Listeria antigens. Furthermore, the ability to transfer the autoimmune response of spleen T cells from intrarenally infected mice was examined. The transferred mice showed a positive delayed footpad reaction against KH and an interstitial infiltration of mononuclear cells in their kidneys. These results demonstrate that the intrarenal Listeria infection induced renal autoantigen-specific T cells, which subsequently induced an autoimmune interstitial nephritis (AIN). The autoreactive T cells were all induced without immunization by autoantigens mixed with complete Freund's adjuvant. Based on these findings, we propose that a local bacterial infection may induce an autoimmune response against autoantigens in the infected organ and subsequently trigger organ-specific autoimmune disease.

Bystander suppression of delayed-type hypersensitivity (DTH) and the antibody response to human serum albumin (HSA) were studied in young normal rats and in young rats made partially tolerant to ovalbumin (OVA) by feeding an OVA-containing diet for 4 weeks from weaning. At 2 months of age, the animals were intracutaneously immunized with a mixture of OVA and HSA in Freund's complete adjuvant (FCA) at one site of the back, or separately at two different sites on the back. All rats made orally tolerant to OVA showed a significantly reduced IgE and IgG anti-OVA antibody production and DTH response to OVA, compared to the controls. OVA-fed rats subsequently immunized with a mixture of OVA + HSA had significantly lower IgE and DTH responses to HSA than the controls. When rats were immunized with OVA and HSA at two different sites, however, there was no difference in the response to HSA between the OVA-fed rats and the control rats, which rules out the possibility of shared epitopes between the antigens. Ear-challenge with the mixture of OVA + HSA gave a significantly lower DTH reaction in the tolerant rats immunized with a mixture of the antigens, compared to the control rats. However, suppression of the DTH reaction was not seen when tolerant and control rats were immunized with HSA alone and challenged with the mixture of OVA + HSA in one ear. These results present evidence that young rats orally tolerant to one antigen show a suppressed T-cell and antibody response to an unrelated antigen, provided that the two antigens are given in a mixture during the inductive phase. There was no evidence for bystander suppression of the T-cell response at the effector site.

The role of the mucosal immune system in the generation of circulating tolerogenic ovalbumin (OVA) moieties has been investigated after a single feed of the protein. Serum collected from SCID mice 1 hr after a 25-mg feed of OVA was unable to transfer tolerance of delayed-type hypersensitivity (DTH) into naive BALB/c recipients. This is in contrast to serum collected from BALB/c mice which was able to transfer DTH tolerance to naive BALB/c recipients. The levels of circulating OVA detected in the serum of SCID mice 60 min after feeding OVA were approximately half those detected in the serum of BALB/c mice at the same time-point. However even dose adjustment of SCID mouse serum to a level of immunoreactive OVA equivalent to that found in BALB/c serum was unable to induce DTH tolerance in BALB/c recipients. This failure of SCID serum to transfer tolerance was shown to be unrelated to the germ-free conditions under which SCID mice are kept. Serum from OVA-fed germ-free BALB/c mice transferred DTH tolerance at equivalent levels to serum from conventionally reared BALB/c mice. When the intestinal morphology and intraepithelial lymphocyte (IEL) numbers in the duodenum of SCID mice were compared to conventionally reared and germ-free BALB/c controls, SCID mice were characterized by a lower number of IEL with a different morphology from the majority of IEL found in BALB/c mice.

T-cell fibronectin (FN) is a unique cellular FN that is rapidly synthesized by memory T cells in response to antigen. Monoclonal anti-T-cell FN antibodies have been used to clarify the role of T-cell FN in the in vivo expression of delayed hypersensitivity. IgGl(kappa) mouse anti-human T-cell FN monoclonal antibodies 231 and 248 recognized epitopes on the FN cell-binding domain, were cross-reactive with plasma FN, and neutralized human and guinea-pig T-cell FN monocyte agglutinating activity. When injected intradermally together with tuberculin or 30 min before topical application of reactive sensitizer, antibody 231 significantly decreased macroscopic expression of guinea-pig delayed hypersensitivity at 24 hr in a dose-dependent manner. Similar doses of antibody 248 caused a slight statistically non-significant enhancement of delayed-type hypersensitivity (DTH) expression. Inhibition of visible skin responses was not associated with qualitative or quantitative changes in cellular infiltrates at the reaction site. Antibody 231 modulated expression of delayed hypersensitivity in a qualitatively and quantitatively similar manner to the FN-binding mycobacterial antigen 85 proteins. This is consistent with anti-T-cell FN and antigen 85 acting on the same molecule in vivo.

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) are cytokines with important functions in regulating immune responses. IFN-gamma may be produced by cells responsible for delayed-type hypersensitivity (DTH), whereas IL-4 is essential for IgE production. Pertussis toxin (PT) from Bordetella pertussis enhances both DTH and IgE responses, and causes enhancement of both IFN-gamma and IL-4 secretion in immunized mice. In the present study, the effects of neutralizing monoclonal antibodies against IFN-gamma or IL-4 on DTH, serum IgE and cytokine production were assessed. Treatment with a monoclonal anti-IL-4 antibody at the time of immunization caused a striking increase in DTH responses, and elicited enhanced IFN-gamma expression, while inhibition of the production of IL-4 and IgE was observed. By contrast, injection of a monoclonal anti-IFN-gamma antibody was followed by significant but not complete suppression of DTH reactions. IFN-gamma secretion was also inhibited, whereas IL-4 production and serum IgE were increased. Thus antibodies to IL-4 and IFN-gamma, given at the time of immunization, can profoundly influence the nature of short-term immune responses elicited by PT in immunized mice.

The role of tumour necrosis factor-alpha (TNF-alpha) in tuberculosis is paradoxical because although there is much evidence for a protective role, there is also evidence that it plays a part in the tissue damage that characterizes human disease. We have shown previously that TNF-alpha frequently induces necrosis when injected into sites undergoing delayed-type hypersensitivity (DTH) responses to mycobacterial antigen. This is dependent on CD4+ T cells. However the presence of this sensitivity to TNF-alpha-induced necrosis depended on the immunization protocol. We have tested the hypothesis that sensitivity to TNF-alpha depends on the cytokine profile of the induced T-cell response. All subcutaneous doses of mycobacterial immunogen used (10(7) to 10(9) organisms) primed spleen cells so that they secreted interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) when cultured in vitro with soluble antigen. However priming for production of IL-4 was dose dependent as in other systems, and was produced at all times from 7 to 30 days after immunization with 10(9) organisms. Time-course studies over 30 days showed that sensitivity to TNF-alpha was found in DTH sites of animals primed for IL-4 and IFN-gamma production, but not in animals primed only for the Th1 cytokines. We suggest therefore that the paradoxical role of TNF-alpha can be resolved. In 'pure' Th1 responses it may act as an additional macrophage-activating factor. In mixed Th1 + Th2 or Th0 responses it may cause tissue damage. This mixed pattern is characteristic of tuberculosis, and of the late stage of many chronic infections where elimination of the infecting organism is failing, and chronic tissue damage is seen.

Mice injected in the footpad with carrageenin developed local inflammation which peaked at 48 hr. This delayed-type footpad swelling was significantly reduced in mice injected intraperitoneally (i.p.) with a specific nitric oxide (NO) synthase inhibitor, L-NGmonomethyl-arginine (L-NMMA). The draining lymph node (DLN) cells from mice injected 48 hr previously with carrageenin produced significantly higher levels of proliferation and interleukin-1 (IL-1), IL-2, IL-6 and interferon-gamma (IFN-gamma), but less IL-10, compared to cells from saline-injected controls, when stimulated with concanavalin A (Con A) in vitro. Treatment of the carrageenin-injected mice with L-NMMA had little effect on the proliferative response of the DLN cells, but significantly reduced the production of IL-1, IL-2, IL-6 and IFN-gamma, and increased the secretion of IL-10. These data demonstrate that NO plays a significant role in local inflammation and the pattern of cytokines induced in this model.

We have examined the range of mucosal and systemic immune responses induced by oral or parenteral immunization with ovalbumin (OVA) entrapped in poly(D,L-lactide-co-glycolide) (PLG) microparticles. A single subcutaneous immunization with OVA-PLG primed significant OVA-specific IgG and delayed-type hypersensitivity (DTH) responses. The DTH responses were of similar magnitude to those obtained using immunostimulating complexes (ISCOMS) as a potent control adjuvant, although ISCOMS stimulated higher serum IgG responses. Both vectors also primed OVA-specific in vitro proliferative responses in draining lymph node cells following a single immunization and strong OVA-specific CTL responses were found after intraperitoneal (i.p.) immunization. ISCOMS were more efficient in inducing cytotoxic T lymphocytes (CTL), requiring much less antigen and only ISCOMS could stimulate primary OVA-specific CTL responses in the draining lymph nodes. Multiple oral immunizations with OVA in PLG microparticles or in ISCOMS resulted in OVA-specific CTL responses and again ISCOMS seemed more potent as fewer feeds were necessary. Lastly, multiple feeds of OVA in PLG microparticles generated significant OVA-specific intestinal IgA responses. This is the first demonstration that PLG microparticles can stimulate CTL responses in vivo and our results highlight their ability to prime a variety of systemic and mucosal immune responses which may be useful in future oral vaccine development.

Orally active synthetic vaccines containing purified antigens would have many benefits for immunizing against systemic and mucosal diseases. However, several factors have limited the development of such vaccines, including the poor immunogenicity of purified proteins and their usual ability to induce tolerance when given orally. Here, we show that incorporation of ovalbumin (OVA) into immune-stimulating complexes (ISCOMS) containing saponin prevents the induction of oral tolerance in mice. In parallel, the spleen and mesenteric lymph node of mice fed OVA ISCOMS are primed for class I major histocompatibility complex (MHC)-restricted cytotoxic T-cell activity which recognizes physiologically processed epitopes on OVA. Oral immunization with OVA ISCOMS also stimulates high secretory IgA antibody responses in the intestine itself, as well as serum IgG antibodies. None of these active immune responses are detectable in mice fed OVA alone. Despite the potent priming of mucosal priming by OVA ISCOMS, re-exposure to antigen does not induce the intestinal immunopathology found in other systems after the breakdown of oral tolerance. Thus, ISCOMS have several unique properties as vectors for oral immunization and could provide a basis for future mucosal vaccines.

Corneal allografts contain few or no Ia+ Langerhans' cells and consistently fail to elicit allospecific delayed-type hypersensitivity (DTH). The present study examined the hypothesis that the inability of corneal allografts to induce allospecific DTH was the result of active suppression. However, the results indicated that an initial exposure to corneal allografts provided a subthreshold stimulus for the induction of DTH because hosts that received a second pair of corneal grafts 7 days later developed DTH. By contrast, hosts that received all four corneal allografts simultaneously, failed to display DTH to donor alloantigens. Hosts that received a single set of corneal allografts did not display DTH to donor alloantigens, yet their spleen cells were capable of transferring DTH to hosts subsequently stimulated with a single set of corneal grafts. The capacity of Langerhans' cell-free corneal grafts to prime the host so that a second set of corneal grafts stimulated DTH to donor alloantigens was termed 'subthreshold stimulation of DTH'. The ability of sequential sets of corneal grafts to induce DTH was abolished by fixation with 0.2% paraformaldehyde. Surprisingly, the single cell-layered corneal endothelium was found to be the crucial corneal component necessary for the induction of allospecific DTH. This unique spectrum of corneal alloimmunogenicity may contribute to the immunological privilege and high success rate of corneal allografts.