Specific Delayed-type Hypersensitivity Responses Research Articles

The Combination of 2′-Fucosyllactose with Short-Chain Galacto-Oligosaccharides and Long-Chain Fructo-Oligosaccharides that Enhance Influenza Vaccine Responses Is Associated with Mucosal Immune Regulation in Mice

ABSTRACTBackgroundA critical role for host-microbe interactions and establishment of vaccine responses has been postulated. Human milk oligosaccharides, of which 2′-fucosyllactose (2′FL) is the most prevalent, are known to alter host-associated microbial communities and play a critical role in the immunologic development of breastfed infants.ObjectivesDietary supplementation with a combination of 2′FL and prebiotic short-chain (sc) galacto-oligosaccharides (GOS) and long-chain (lc) fructo-oligosaccharides (FOS) was employed to examine human milk oligosaccharide effects on immune responsiveness, within a murine influenza vaccination model.MethodsFemale mice (6 wk old, C57Bl/6JOlaHsd) were fed either control diet (CON) or scGOS/lcFOS/2′FL-containing diet (GF2F) for 45 d. After starting dietary intervention (day 14), mice received a primary influenza vaccination (day 0) followed by a booster vaccination (day 21), after which ear challenges were conducted to measure vaccine-specific delayed type hypersensitivity (DTH). Serum immunoglobulin (Ig) levels, fecal and cecal microbial community structure, short-chain fatty acids, host intestinal gene expression and cellular responses in the mesenteric lymph nodes (MLNs) were also measured.ResultsRelative to CON, mice fed the GF2F diet had increased influenza vaccine–specific DTH responses (79.3%; P < 0.01), higher levels of both IgG1 (3.2-fold; P < 0.05) and IgG2a (1.2-fold; P < 0.05) in serum, and greater percentages of activated B cells (0.3%; P < 0.05), regulatory T cells (1.64%; P < 0.05), and T-helper 1 cells (2.2%; P < 0.05) in their MLNs. GF2F-fed mice had elevated cecal butyric (P < 0.05) and propionic (P < 0.05) acid levels relative to CON, which correlated to DTH responses (R2 = 0.22; P = 0.05 and R2 = 0.39; P < 0.01, respectively). Specific fecal microbial taxa altered in GF2F diet fed mice relative to CON were significantly correlated with the DTH response and IgG2a level increases.ConclusionsDietary GF2F improved influenza vaccine–specific T-helper 1 responses and B cell activation in MLNs and enhanced systemic IgG1 and IgG2a concentrations in mice. These immunologic changes are correlated with microbial community structure and metabolites.

Human Milk Oligosaccharide 2'-Fucosyllactose Improves Innate and Adaptive Immunity in an Influenza-Specific Murine Vaccination Model.

Human milk is uniquely suited to provide optimal nutrition and immune protection to infants. Human milk oligosaccharides are structural complex and diverse consisting of short chain and long chain oligosaccharides typically present in a 9:1 ratio. 2'-Fucosyllactose (2'FL) is one of the most prominent short chain oligosaccharides and is associated with anti-infective capacity of human milk. To determine the effect of 2'FL on vaccination responsiveness (both innate and adaptive) in a murine influenza vaccination model and elucidate mechanisms involved. A dose range of 0.25-5% (w/w) dietary 2'FL was provided to 6-week-old female C57Bl/6JOlaHsd mice 2 weeks prior primary and booster vaccination until the end of the experiment. Intradermal (i.d.) challenge was performed to measure the vaccine-specific delayed-type hypersensitivity (DTH). Antigen-specific antibody levels in serum as well as immune cell populations within several organs were evaluated using ELISA and flow cytometry, respectively. In an ex vivo restimulation assay, spleen cells were cocultured with influenza-loaded bone marrow-derived dendritic cells (BMDCs) to study the effects of 2'FL on vaccine-specific CD4+ and CD8+ T-cell proliferation and cytokine secretions. Furthermore, the direct immune regulatory effects of 2'FL were confirmed using in vitro BMDCs T-cell cocultures. Dietary 2'FL significantly (p < 0.05) enhanced vaccine specific DTH responses accompanied by increased serum levels of vaccine-specific immunoglobulin (Ig) G1 and IgG2a in a dose-dependent manner. Consistently, increased activation marker (CD27) expression on splenic B-cells was detected in mice receiving 2'FL as compared to control mice. Moreover, proliferation of vaccine-specific CD4+ and CD8+ T-cells, as well as interferon-γ production after ex vivo restimulation were significantly increased in spleen cells of mice receiving 2'FL as compared to control mice, which were in line with changes detected within dendritic cell populations. Finally, we confirmed a direct effect of 2'FL on the maturation status and antigen presenting capacity of BMDCs. Dietary intervention with 2'FL improves both humoral and cellular immune responses to vaccination in mice, which might be attributed in part to the direct effects of 2'FL on immune cell differentiation.

Abstract 5307: Indomethacin reverts tumor mediated immune suppression in a murine lung adenocarcinoma

Abstract Tumor-induced immune suppression is widespread in patients and experimental animals with malignant tumors and is likely to be a significant impediment to immunotherapy. Multiple mechanisms are thought to facilitate this suppression state, being Myeloid-Derived Suppressor Cells (MDSC) and T regulatory cells (Tregs) major contributors. Our aim was to analyze immune parameters indicative of immune suppression in BALB/C mice bearing LP07 (COX+) lung tumor (TBM) during tumor growth and the effect of a Non Steroidal Anti-inflammatory drug (NSAID), Indomethacin, which is a non-specific COX inhibitor, on these parameters. We determined 1) the effect of Indomethacin on LP07 tumor growth; 2) the content of MDSC and Treg cells in lymphoid organs (spleen, tumor-draining lymph nodes-TDLN-) and in the tumors at different times by FACS; 3) arginase activity in lung, spleen, liver and tumor measured as µg Urea/mg protein; 4) specific Delayed Type Hypersensitivity (DTH) response with formalinized LP07 cells, by the foot pad swelling assay. Results: Indomethacin treatment significantly inhibited tumor growth. The percentage of MDSC (CD11b+ Gr1+) augmented significantly in the spleen of TBM in advanced stages of tumor progression (33 days): Normal: 2,6%; TBM 10 days: 2,4%; TBM 20 days: 5,6%; TBM 33 days: 8,3%, p&amp;lt;0,05 vs control. This rise was prevented by Indomethacin treatment (4,6%). Intratumoral and TDLN CD4+CD25+Foxp3+ Tregs frequency augmented during tumor growth, peaking at day 20. Interestingly, Indomethacin treatment decreased Treg cell and MDSC induction (p&amp;lt;0.05) in TDLN. Arginase activity increased in all analyzed tissues of TBM compared to those of control mice. Furthermore arginase activity increase in the spleen was in line with MDSC induction. Specific DTH response decreased in late stages of tumor progression, while Indomethacin treatment reversed this inhibition (TBM: 0,06 ± 0,045 mm; Indo:0,14 ± 0,038 mm). Conclusion: In our model, the increment in MDSC and Tregs frequency during tumor progression correlates with specific immunesuppression demonstrated by DTH responses and increased arginase activity. We suggest that Indomethacin treatment prevented this immunesuppression by modulating COX activity in the tumor microenvironment, which is known to activate MDSC and differentiate Treg cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5307.

Suppression of the inflammatory response in experimental arthritis is mediated via estrogen receptor α but not estrogen receptor β

IntroductionThe immune modulatory role of estrogens in inflammation is complex. Both pro- and anti-inflammatory effects of estrogens have been described. Estrogens bind both estrogen receptor (ER)α and β. The contribution of ERα and ERβ to ER-mediated immune modulation was studied in delayed type hypersensitivity (DTH) and in experimental arthritisMethodsER-mediated suppression of rat adjuvant arthritis (AA) was studied using ethinyl-estradiol (EE) and a selective ERβ agonist (ERB-79). Arthritis was followed for 2 weeks. Next, effects of ER agonists (ethinyl-estradiol, an ERα selective agonist (ERA-63) and a selective ERβ agonist (ERB-79) on the development of a tetanus toxoid (TT)-specific delayed type hypersensitivity response in wild type (WT) and in ERα - or ERβ-deficient mice were investigated. Finally, EE and ERA-63 were tested for their immune modulating potential in established collagen induced arthritis in DBA/1J mice. Arthritis was followed for three weeks. Joint pathology was examined by histology and radiology. Local synovial cytokine production was analyzed using Luminex technology. Sera were assessed for COMP as a biomarker of cartilage destruction.ResultsEE was found to suppress clinical signs and symptoms in rat AA. The selective ERβ agonist ERB-79 had no effect on arthritis symptoms in this model. In the TT-specific DTH model, EE and the selective ERα agonist ERA-63 suppressed the TT-specific swelling response in WT and ERβKO mice but not in ERαKO mice. As seen in the AA model, the selective ERβ agonist ERB-79 did not suppress inflammation. Treatment with EE or ERA-63 suppressed clinical signs in collagen induced arthritis (CIA) in WT mice. This was associated with reduced inflammatory infiltrates and decreased levels of proinflammatory cytokines in CIA joints.ConclusionsERα, but not ERβ, is key in ER-mediated suppression of experimental arthritis. It remains to be investigated how these findings translate to human autoimmune disease.

Sulfur mustard induces immune sensitization in hairless guinea pigs

Sulfur mustard (SM, bis-(2-chloroethyl) sulfide) is a well known chemical warfare agent that may cause long-term debilitating injury. Because of the ease of production and storage, it has a strong potential for chemical terrorism; however, the mechanism by which SM causes chronic tissue damage is essentially unknown. SM is a potent protein alkylating agent, and we tested the possibility that SM modifies cellular antigens, leading to an immunological response to “altered self” and a potential long-term injury. To that end, in this communication, we show that dermal exposure of euthymic hairless guinea pigs induced infiltration of both CD4 + and CD8 + T cells into the SM-exposed skin and strong upregulated expression of proinflammatory cytokines and chemokines (TNF-α, IFN-γ, and IL-8) in distal tissues such as the lung and the lymph nodes. Moreover, we present evidence for the first time that SM induces a specific delayed-type hypersensitivity response that is associated with splenomegaly, lymphadenopathy, and proliferation of cells in these tissues. These results clearly suggest that dermal exposure to SM leads to immune activation, infiltration of T cells into the SM-exposed skin, delayed-type hypersensitivity response, and molecular imprints of inflammation in tissues distal from the site of SM exposure. These immunological responses may contribute to the long-term sequelae of SM toxicity.

Role of T-cell receptor V beta 8.3 peptide vaccine in the prevention of experimental autoimmune uveoretinitis

T-cell receptor (TCR) plays an important role in the development of autoimmune diseases. Recently, it was reported that immunization of animals with TCR peptide derived from the pathogenic cells could prevent autoimmune diseases. The aim of this study was to investigate whether vaccination with a synthetic peptide from the hypervariable region of TCR V(beta) 8.3, an experimental autoimmune uveoretinitis (EAU)-associated gene, was able to prevent the disease. EAU was induced in Lewis rats by immunization with IRBP R16 peptide emulsified in complete Freund's adjuvant (CFA). The clinical and histological appearances were scored. Delayed type hypersensitivity (DTH) and lymphocyte proliferation were detected. Cytokine levels of aqueous humour, supernatants of cells from spleen and draining lymph nodes were measured by enzyme linked immunosorbent assay (ELISA). Gene expression of TCR V(beta) 8.3 on CD(4)(+) T cells was examined by real time quantitative polymerase chain reaction (PCR). After vaccination, the intraocular inflammation was significantly mitigated, antigen specific DTH and lymphocyte proliferation responses were suppressed, interleukin (IL)-2 in aqueous humour, interferon (IFN)-gamma and IL-2 produced by the spleen and draining lymph node cells were significantly decreased, whereas the production of IL-4 and IL-10 were increased. The response of draining lymph node cells to TCR V(beta) 8.3 peptide was enhanced after vaccination. Inoculation with CFA alone did not affect the severity of EAU and the above parameters. The suppression of EAU was much stronger in the group of four fold inoculations than the group of two fold inoculations. The expression of TCR V(beta) 8.3 gene was significantly reduced in the group of fourfold inoculations. Vaccination with the synthetic TCR V(beta) 8.3 peptide could remarkably inhibit the development of EAU.

From transcriptome to immunome: Identification of DTH inducing proteins from a Phlebotomus ariasi salivary gland cDNA library

Delayed-type hypersensitivity (DTH) response to arthropod vector salivary proteins is associated with protection against pathogen transmission. Massive cDNA sequencing, high-throughput DNA plasmid construction and DNA immunisation were used to identify twelve DTH inducing proteins isolated from a Phlebotomus ariasi salivary gland cDNA library. Additionally, nine P. ariasi DNA plasmids produced specific anti-saliva antibodies, four of these showed a Th1 immune response while the other two exhibited a Th2 profile as determined by IgG2a and IgG1 isotype switching, respectively. In order to validate the specificity of sand fly DNA plasmids, mice previously exposed to sand fly saliva were intradermally injected once with selected P. ariasi plasmids and a specific DTH response consisting of infiltration of mononuclear cells in varying proportions was observed at 24 and 48 h. This approach can help to identify DTH inducing proteins that may be related to host protection against vector-borne diseases or other disease agents where cellular immune response is protective.

A randomized trial of PSA-peptide based, specific active immunotherapy in HLA-A2+ patients with prostate cancer: Comparison of two different vaccination strategies

2519 Background: Standard treatments for prostate cancer (Pca) cause undesirable side-effects and often fail. Specific T cell immunotherapy against defined Pca-associated antigens offers the potential for less toxic, more effective outcomes. Methods: A pilot trial was conducted to determine whether PSA 146–154 peptide vaccination can induce specific T cell immunity without associated toxicity. The trial compared two different vaccination methods. Arm1-PSA peptide and GM-CSF injected i.d. versus Arm 2-PSA peptide-pulsed, autologous dendritic cells infused i.v. Two groups of HLA-A2+ patients [Group A (n=14): high-risk, local disease; Group B (n=14): metastatic, hormone-sensitive disease in remission] were randomized 1:1 to the two treatment arms. Patients were vaccinated on weeks 1, 4, and 10. Immune responses were monitored by delayed-type hypersensitivity (DTH) skin testing and in vitro T cell assays on weeks 4, 14, 26, and 52. Tumor status was monitored by serial PSA levels and clinical tests. Results: Vaccinations were completed in April of 2004. The majority of patients have completed 52 weeks of observation. Both vaccination methods have been well tolerated with no grade 3/4 toxicities. Fourteen patients (9 on Arm 1 and 5 on Arm 2) have developed specific DTH responses to PSA 146–154 peptide. Eleven DTH responders (78%) have had stable or declining serum PSA levels. Nine DTH non-responders (64%) have had rising PSA levels and/or clinical progression. Both vaccination methods elicited specific T cell responses per cytokine release and tetramer assays. Conclusion: Vaccination of HLA-A2+ patients with PSA146–154 peptide is well tolerated and elicits specific T cell immunity. Intradermal administration of PSA 146–154 peptide with GM-CSF and intravenous administration of PSA 146–154 peptide-pulsed dendritic cells are both effective immunization strategies. Induction of specific T cell immunity to PSA 146–154 peptide in patients with Pca correlates with less disease progression. No significant financial relationships to disclose.

Conjunctival macrophage-mediated influence of the local and systemic immune response after corneal herpes simplex virus-1 infection.

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 (P<0.05). Furthermore, Cl2MDP-LIP-treated mice had decreased HSV-1 specific proliferative responses (P<0.05) and DTH response after corneal HSV-1 infection (P<0.05). The virus-neutralizing serum-antibody levels (P<0.05) 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.

Cancer immunotherapy in clinical oncology.

The identification of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer therapy. Different groups of cancer-associated antigens have been described as targets for cytotoxic T lymphocytes (CTLs) in vitro and in vivo: 1) cancer-testis (CT) antigens, which are expressed in different tumors and normal testis; 2) melanocyte differentiation antigens; 3) point mutations of normal genes; 4) antigens that are overexpressed in malignant tissues; and 5) viral antigens. Clinical studies with peptides derived from these antigens have been initiated to induce specific CTL responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined, i.e., delayed-type hypersensitivity (DTH), CTL, autoimmmune, and tumor regression responses. Preliminary results demonstrate that tumor-associated peptides alone elicit specific DTH and CTL responses leading to tumor regression after intradermal injection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was proven effective in enhancing peptide-specific immune reactions by amplification of dermal peptide-presenting dendritic cells. Long-lasting complete tumor regressions have been observed after induction of peptide-specific CTLs. However, in single cases with disease progression after an initial tumor response, either a loss of the respective tumor antigen targeted by CTLs or of the presenting major histocompatibility complex (MHC) class I allele was detected as a mechanism of immune escape under immunization. Based on these observations, cytokines to enhance antigen and MHC class I expression in vivo are being evaluated to prevent immunoselection. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1, which is regarded as one of the most immunogenic antigens known today inducing spontaneous immune responses in 50% of patients with NY-ESO-1-expressing cancers. Clinical studies involving antigenic constructs that induce both antibody and CTL responses will show whether these are more effective for immunotherapy of cancer.

Expression in cattle of epitopes of a heterologous virus using a recombinant rinderpest virus.

We have investigated the bovine immune response to heterologous proteins expressed using a recombinant rinderpest virus (RPV). A new gene unit was created in a cDNA copy of the genome of the vaccine strain of RPV, and an open reading frame inserted that encodes the polymerase (3Dpol) and parts of the capsid protein VP1 from foot-and-mouth disease virus (FMDV). Infectious recombinant RPV was rescued and shown to express the FMDV-derived protein at good levels in infected cells. The rescued virus was only slightly more attenuated in tissue culture than the original virus. Cattle infected with this recombinant generated a normal immune response to RPV, and were protected from lethal challenge by that virus. Experimental animals showed a specific delayed-type hypersensitivity response to FMDV 3Dpol, similar to that seen in FMDV infection; however, no antibodies were detected recognizing either of the components of the FMDV-derived protein, nor was any proliferative response to these epitopes found in isolated peripheral blood lymphocytes from infected animals. No protection was seen against FMDV infection.

MECHANISMS OF TOLERANCE TO A TRANSGENIC MINOR TRANSPLANTATION ANTIGEN

937 β-galactosidase can behave as a minor transplantion antigen in C57BL/6 (B6, H-2b) mice as they reject congenic B6 β-galactosidase transgenic (β-gal tg) skin grafts by day 25. Recall immune responses after rejection reveal β-gal specific proliferation and type 1 cytokine production, cytotoxicity of β-gal tg cells and β-gal specific delayed type hypersensitivity (DTH) responses. We induced immunologic tolerance to β-gal tg skin grafts by pretreatment of the B6 recipients with i.v. injection of soluble β-gal. B6 β-gal tg skin graft survival was prolonged for > 5 months, while third party grafts were rejected within 14 days. Recall responses in tolerized mice revealed an absence of β-gal-specific IFNγ, IL-2, IL-4 or IL-5 production (no type 2 immune deviation). Spleen cells obtained from the tolerant animals were unable to mediate CTL activity or DTH responses. In an effort to discern whether clonal deletion, anergy and/or suppression were active mechanisms of the tolerance, we next tested whether we could recover β-gal specific immunity from tolerant animals by in vitro incubation of tolerized immune cells in the presence of exogenous IL-2. Although the cells were unable to respond to β-gal in primary recall responses, incubation with IL-2 resulted in recovery of β-gal specific, IFNγ - producing T cells, thus excluding clonal deletion as an operative mechanism. Furthermore, spleen cells obtained from tolerized mice acted as suppressor cells, in that co-injection of these spleen cells inhibited ovalbumin (OVA) specific DTH responses normally produced by OVA-primed T cells. Finally, β-gal stimulation of immune cells from the tolerant animals induced significant upregulation of TGFβ message. These results suggest that the i.v. immunization resulted in expansion of a population of β-gal specific, TGFβ producing T cells capable of suppressing the pathogenic, anti-skin graft immune response. Taken together these data demonstrate the feasibility of inducing graft tolerance to a transgenic minor antigen and implicate TGFβ mediated suppression and/or anergy as mechanisms of such tolerance. Lessons obtained from this experimental model may translate into approaches for inducing graft tolerance in human organ transplant recipients.

Strategies for the development of vaccines to treat breast cancer.

The characterization of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer therapy. Several categories of cancer-associated antigens have been described as targets for cytotoxic T lymphocytes (CTLs) in vitro and in vivo: "cancer-testis" (CT) antigens expressed in different tumors and normal testis, melanocyte differentiation antigens, point mutations of normal genes, antigens that are overexpressed in malignant tissues, and viral antigens. Clinical studies using peptides derived from these antigens have been initiated to induce specific CTL responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined, i.e., induction of delayed-type hypersensitivity (DTH), CTL, autoimmune, and tumor regression responses. Preliminary results demonstrate that tumor-associated peptides alone elicit specific DTH and CTL responses leading to tumor regression after intradermal injection. GM-CSF was proved to be effective in enhancing peptide-specific immune reactions by amplification of dermal peptide-presenting dendritic cells. Long-lasting complete tumor regressions have been observed after induction of CTLs by peptide immunization. However, in a few cases where there was disease progression after initial tumor response, loss of either the tumor antigen targeted by CTLs or of the presenting MHC class I molecule was detected as the mechanism of immune escape under immunization in vivo. Based on these observations, cytokines to enhance antigen and MHC class I expression in vivo are being evaluated to prevent immunoselection. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1. In a melanoma patient with high titer antibody against NY-ESO-1, strong HLA-A2-restricted CTL reactivity against the same antigen was also found. Clinical studies involving tumor antigens that induce both antibody and CTL responses will show whether these are better candidates for immunotherapy of cancer.

Role of indirect allorecognition in experimental late acute rejection.

Late acute rejection affects up to 28% of renal allograft recipients and remains a major risk factor for late graft loss. As donor-origin antigen-presenting cells are depleted with time, T-cell recognition of donor-derived alloantigenic peptides presented by self antigen-presenting cells (the "indirect pathway" of allorecognition) may play a key role in the initiation of late acute rejection episodes. To test this hypothesis, we developed a clinically relevant experimental model in the rat (Wistar-Furth/Lewis) in which allograft recipients received cyclosporine for 1 month after transplantation and were then allowed to reject the graft upon discontinuation of immunosuppression. Lymphocyte proliferation assays to synthetic class II MHC allopeptides of donor origin and also to intact donor (Wistar-Furth) cells were performed at this time. The effector mechanisms studied included delayed-type hypersensitivity (DTH) responses, lymphocyte-mediated cytotoxicity, and alloantibody production. Lymphocytes from recipients undergoing late acute rejection had marked suppression of mixed lymphocyte reaction proliferation to intact donor cells. Significant proliferation to donor-derived 25-mer polymorphic class II MHC allopeptides was elicited, however. In vivo, significant DTH responses were observed to both MHC allopeptides and intact Wistar-Furth cells. Recipient lymphocytes also exhibited significant killing of donor cells, although not third-party cells, and anti-donor alloantibodies were detected by flow cytometry. Our results indicate that T cells primed via the indirect pathway are present during acute rejection that occurs after discontinuation of cyclosporine. Mixed lymphocyte reactivity is markedly reduced at this time. Furthermore, there is an association between such allopeptide-primed T cells and the elicitation of specific DTH responses and provision of help to B cells to produce alloantibodies and activation of CD8+ T cells to become effector cytotoxic cells.

Alloreactive delayed-type hypersensitivity in graft recipients: complexity of responses and divergence from acute rejection.

Immunocompetent allograft recipients typically exhibit evidence of sensitization to graft antigens through alloantibody production and allograft rejection, as well as delayed-type hypersensitivity (DTH) reactivity to donor antigens. Most previous studies have relied on whole donor splenocytes, which primarily elicit allorestricted allogeneic responses, to test specific DTH responses and overlook the independent element of self-restricted responses in host-allograft interactions. We tested expression of self-MHC-restricted versus allo-MHC-restricted allogeneic DTH responses in both nonimmunosuppressed and tolerized C57BL/6 mice. Mice were sensitized for allogeneic DTH either by rejection of skin or cardiac allografts, or by subcutaneous injection of intact allogeneic splenocytes. Patterns of alloreactive DTH were compared in allosensitized, tolerant, and naive hosts. All three methods of allosensitization resulted in equivalent self-restricted and allorestricted allogeneic DTH responses in nonimmunosuppressed mice. Gallium nitrate blocked acute rejection of cardiac allografts, and also blocked allosensitization of both self-restricted and allorestricted DTH responses, but did not influence the expression of DTH responses in presensitized mice. Gallium nitrate treatment could not block acute rejection of skin allografts, but interfered with sensitization for self-restricted, but not allorestricted, DTH responses in these recipients. This divergence of self- versus allo-MHC-restricted allosensitization for DTH was observed in two additional situations: the rates of allosensitization for self-restricted versus allorestricted DTH, and the acquisition of allorestricted, but not self-restricted, alloreactive DTH responses in cardiac allograft tolerant mice subsequently challenged with a skin allograft. These studies demonstrate that acute rejection correlates generally with allogeneic DTH, whereas tolerance is associated with a lack of alloreactive DTH. However, self-restricted and allorestricted allosensitization can operate independently in allograft recipients. Thus, the relationships between alloreactive DTH and graft-induced allosensitization, acute rejection, or tolerance are more complicated than previously appreciated.

Chemical denaturation of ovalbumin abrogates the induction of oral tolerance of specific IgG antibody and DTH responses in mice.

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA, for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265) Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses against P. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.

Adjuvant effect of liposomes in the autoimmune response to rat male accessory glands

Wistar rats submitted to 3 intravenous (i.v.) or intraperitoneal (i.p.) immunizations with saline extract of rat male accessory glands (RAG) associated to liposomes developed a delayed type hypersensitivity (DTH) response to RAG after the first immunization, a remission state after the second immunization and a specific DTH response after the third injection. In a further study we transferred spleen mononuclear (SpM) cells from i.p. immunized rats taken 10 days after the second immunization (DTH negative) to normal or immunized recipients 24 h before or 10 days after the first immunization with RAG liposomes, respectively. The DTH response was reduced only in recipients previously immunized. Besides, it was possible to show that the transfer of SpM cells present when the response increased after the third injection in i.p. immunized donors reduced the suppression observed after the second injection. Rat accessory gland biopsies taken 10 days after the last immunization showed in nearly all cases mast cells, plasma cells and eosinophils with scarce lymphoid elements and increased acinar desquamation. This kind of infiltration had characteristics of cutaneous basophil hypersensitivity.

Characterization of a Listeria monocytogenes‐specific protein capable of inducing delayed hypersensitivity in Listeria‐immune mice

Recovery of the host after infection by the intracellular pathogen Listeria monocytogenes is dependent on cell-mediated immunity. Little is known of the nature of listerial antigens that induce cell-mediated responses in the infected host. In this study we report on the identification and cloning of an Escherichia coli recombinant encoding a listerial antigen, designated ImaA, capable of eliciting a specific delayed-type hypersensitivity response in Listeria-immune mice. Nucleotide sequencing of the Listeria DNA insert in plasmid pLM10 showed that the ImaA gene product consisted of 170 amino acids with a molecular weight of 17,994. The predicted amino acid sequence suggests that the protein is localized to the bacterial plasma membrane or cell wall. The ImaA gene was unique to the pathogenic species L. monocytogenes and Listeria ivanovii; it was not present in any other species of the genus Listeria.

Evidence of T-cell recognition in mice of a purified lipophosphoglycan from Leishmania major.

We have previously reported that a Leishmania major lipophosphoglycan (LPG), given with killed Corynebacterium parvum as an adjuvant, can vaccinate mice against cutaneous leishmaniasis. In order to analyze whether T cells are able to recognize this important parasite antigen, we have studied both humoral and cellular immune responses to L. major LPG that had been isolated from promastigotes by sequential solvent extraction and hydrophobic chromatography. The data show that immunization of mice with highly purified LPG induced an increase in frequency of L. major-reactive T cells and the production of immunoglobulin G antibodies to LPG. Furthermore, genetically resistant mice infected with L. major were able to develop a specific delayed-type hypersensitivity response in the ear to L. major LPG. These findings strongly suggest that T cells can recognize and respond to glycolipid antigens, in this case a host-protective Leishmania LPG, even though such antigens appear not to be potent T-cell stimulators in mice.