Nature of hapten-modified determinants involved in induction of T cell tolerance and suppressor T cells to NDFB contact sensitivity

Unresponsiveness to DNFB contact sensitivity induced by DNP-modified lymphoid cells (DNP-LC) is mediated by two separable pathways: a rapidly induced, long lasting inhibition of reactive T cell clones (donor tolerance), and a transient period of suppressor T cell (Ts) activity. The present report has examined the nature of the hapten-modified determinants responsible for the induction of these pathways by utilizing soluble DNP-LC cell lysate preparations as tolerogens. The results indicate that both DNP-modified MHC and non-MHC encoded determinants can mediate donor tolerance 7 days after tolerization. On the other hand, the induction of Ts requires DNP-modified MHC determinants, since DNP-LC lysates passed over lentil lectin or specific anti-H-2 immunoabsorbent columns lost their ability to induce Ts. Additional experiments showed that the injection of DNP-LC lysate compatible with the recipient strain at the H-2K and H-2D region of the MHC was sufficient for the induction of Ts. We propose that Ts induction involves the direct presentation of DNP-H-2 determinants to Ts precursors, whereas the induction of donor tolerance may involve host processing and presentation of DNP-modified membrane determinants in conjunction with host MHC structures.     

Infection of mice with Newcastle disease virus inhibits the T suppressor afferent cell circuit which regulates contact sensitivity to picryl chloride

The interaction between Newcastle disease virus (NDV) and the suppressor cell circuit which regulates the induction phase of contact sensitivity reaction to picryl chloride (Pcl) was investigated. NDV infection impairs the activity of the T suppressor afferent cells (Ts-aff) which inhibit DNA synthesis in the draining lymph nodes of mice specifically sensitized with Pcl and the development of contact sensitivity. The inhibitory effect of NDV was evident when the virus was administered up to 2 days before or at the same time as the injection of picrylsulfonic acid; this effect required infectious virus, as NDV inactivated by ultraviolet irradiation failed to inhibit Ts-aff activity. Taken together with the previous finding that the T suppressor efferent cell is unaffected by NDV, the present results support the view that contact sensitivity reaction to picryl chloride is regulated by two distinct T-suppressor-cell circuits.     

Control of suppressor cell activity: autoanti-idiotype B cells produced by painting with picryl chloride inhibit the T-suppressor cell which blocks the efferent stage of contact sensitivity

This paper describes a B “suppressor of suppressor” cell which blocks the production or action of the T-suppressor cell, Ts-eff (cs), which acts at the efferent stage of the contact sensitivity reaction. Ts-eff (cs) occur in mice 7 days after injecting picrylsulfonic acid (PSA) and are assayed by their ability to block the passive transfer of contact sensitivity in a 24-hr experiment. These Ts-eff (cs) cannot be demonstrated in mice painted with picryl chloride and injected with PSA 8 days later. In fact, 8 days after painting mice contain B cells which prevent the appearance of Ts-eff (cs) following the injection of PSA. Moreover, the serum of mice 12 days after painting contains antibody which inactivates Ts-eff (cs). This antibody is anti-idiotypic as shown by its absorption to and elution from insolubilized mouse anti-picryl antibody and the lack of effect of absorption with insolubilized picryl groups. The antibody belongs to the IgG2a class and requires an intact Fc moiety for its action.     

Suppression of delayed-type hypersensitivity to syngeneic testicular cells in mice: involvement of suppressor T cells which act at the induction stage

Suppressor cells for delayed footpad reaction (DFR) against syngeneic testicular cells (TC) were detected in the spleen cells of donor mice immunized intravenously (iv) with viable syngeneic TC. Cyclophosphamide (CY)-pretreated recipients were given spleen cells from donors iv, immunized subcutaneously (sc) with syngeneic TC, and the footpad reaction at 24 hr was elicited with syngeneic TC 6 days after immunization. DFR in the recipients was suppressed by the transfer of spleen suppressor cells. The suppressor cells induced were Thy-1+, CY-sensitive, adult thymectomy (ATx)-resistant and act only at the induction stage. They directly suppress the generation of effector T cells for delayed-type hypersensitivity (DTH). When mice pretreated with CY were actively immunized with syngeneic TC, DFR could be provoked to a measurable level only when they were immunized sc. However, peritoneal exudate cells of those tolerant mice immunized sc without CY pretreatment or immunized iv with CY pretreatment also passively transferred DFR locally, suggesting the existence of effector T cells for DTH even in tolerant mice.     

Regulatory responses in contact sensitivity: afferent suppressor T cells inhibit the activation of efferent suppressor T cells.

Two types of suppressor cells regulate the contact sensitivity (CS) response to picryl chloride (PCL). Afferent suppressor T cells (Ts-aff) inhibit the generation of CS responses to PCL, while efferent suppressor T cells (Ts-eff) inhibit the activity of Th 1 cells that mediate CS reaction. Intravenous injection of mice with TNP-substituted peritoneal exudate cells (TNP-PEC) induces Ts-eff cells that block the adoptive transfer of contact sensitivity. The induction of Ts-eff cells is prevented by the presence of Ts-aff cells, which in turn are induced by the injection of TNP-PEC coupled with antibodies of the IgG2a and IgG2b isotype (TNP-PEC-Ab). If an animal is injected with TNP-PEC prior to or simultaneously with TNP-PEC-Ab, it generates only Ts-aff cells, while if it is injected with TNP-PEC alone or TNP-PEC prior to TNP-PEC-Ab, it generates Ts-eff cells. Ts-aff cells effect only the generation of Ts-eff cells, as the addition of Ts-eff cells to assays for Ts-eff cells has no inhibitory effect on the suppressive effects of Ts-eff cells in adoptive transfer. Our experiments show that Ts-aff cells induced by TNP-PEC-Ab are phenotypically either Lyt 1+2- or Lyt 1-2+, but only the latter inhibit the generation of Ts-eff cells in vivo. The Ts-aff cells that inhibit Ts-eff activity adhere to the lectin Vicia villosa (VV), while Ts-eff cells are VV nonadherent. In addition, Ts-aff cells can prevent the generation of Ts-eff to linked haptens presented on the same PEC. It appears that a cascade of Ts cell interactions are involved in the regulation of CS responses.     

T cells recognizing polysaccharide-specific B cells function as contrasuppressor cells in the generation of T cell immunity to Pseudomonas aeruginosa

The cellular interactions involved in the development of T cell-mediated immunity to Pseudomonas aeruginosa have been examined. T cell immunity can be generated by immunizing mice with 10 micrograms of P. aeruginosa polysaccharide (PS) plus the antimitotic agent vinblastine sulfate. Vinblastine is required to inactivate a population of Ts cells generated by immunization with 10 micrograms of PS alone. Immunization with either live bacteria or with higher dose (50 micrograms) of PS without vinblastine also generates T cell immunity; these protocols activate a population of Lyt-1+, 2-, I-J+ T cells which, like vinblastine, counteract the effect of Ts cells. Immunization with 10 micrograms PS alone fails to activate this T cell subpopulation. When administered at the time of immunization, this subpopulation can render the tolerogenic 10-micrograms immunization protocols immunogenic. Like previously described contrasuppressor T cells, this T cell subpopulation exhibits an affinity for the lectin Vicia villosa. We have determined, however, that the T cells that act as contrasuppressor cells in this system are directly activated by PS-immune B cells and not by PS Ag. Furthermore, their activity can be removed by adsorption to PS-specific B cell hybridomas. Our studies indicate an important role for B cells in the development of T cell immunity to P. aeruginosa and suggest that a complex idiotype network controls the development of this response.     

Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization

Background

Porcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).

Results

Five genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.

Conclusions

These results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.     

Increased susceptibility of purinergic receptor-deficient mice to lung infection with Pseudomonas aeruginosa

Purinergic receptors are expressed throughout the respiratory system in diverse cell types. The efficiency of mucus clearance in the airways, the cascade leading to tissue injury, and inflammation are modulated by autocrine/paracrine release of nucleotides and signaling by purinergic receptors. We assessed the role of purinergic receptors in innate host defense of the lung in vivo by infecting mice deficient in P2Y1, P2Y2, or both receptors with intratracheal instillation of Pseudomonas aeruginosa. After P. aeruginosa challenge, all double knockout (P2Y1/P2Y2-/-) mice succumbed within 30 h of challenge, whereas 85% of the wild-type mice survived. Thirty-three percent of wild-type mice survived beyond 96 h. Single knockout mice, P2Y1-/-, or P2Y2-/-, exhibited intermediate survivals. Twenty-four hours following intratracheal instillation of a sublethal dose of P. aeruginosa, the level of total protein in bronchoalveolar lavage fluid was 1.8-fold higher in double knockout than in wild-type mice (P < 0.04). Total cell count in bronchoalveolar lavage fluids at 4 h and levels of IL-6 and macrophage inflammatory protein-2 in lung homogenates at 24 h postchallenge were significantly reduced in P2Y1/P2Y2-/- mice relative to wild-type mice. These findings suggest that purinergic receptors exert a protective role against infection of the lungs by P. aeruginosa by decreasing protein leak and enhancing proinflammatory cytokine response.     

NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice

CD4(+) T cells produce IFN-gamma contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-gamma and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (-/-; no T/NKT cells), B6.TCR Jalpha281(-/-) (NKT cell deficient), alpha-galactosylceramide (alphaGalCer) (anergized NKT cells) injected and IL-12p40(-/-) vs B6 controls were tested. IFN-gamma mRNA was undetectable in RAG-1(-/-)- and alphaGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jalpha281(-/-), alphaGalCer-treated, and IL-12p40(-/-) (activated CD4(+) T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mphi; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-kappaB, and CD1d, and addition of rIFN-gamma potentiated Mphi CD1d levels. Together, these data suggest that Langerhans cell/Mphi recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-gamma production by NKT cells, that IFN-gamma is required to optimally activate NK cells to produce IFN-gamma, and that depletion of both NKT/NK cells results in earlier corneal perforation.     

Tolerance and contact sensitivity to DNFB in mice. VI. Inhibition of afferent sensitivity by suppressor T cells in adoptive tolerance.

Tolerance in contact sensitivity to DNFB can be adoptively transferred to normal mice with lymph node cells from tolerant donors. This tolerance is antigen specific and is mediated by T cells, i.e., "suppressor" T cells. Experiments were carried out to investigate the mechanism(s) by which the suppressor T cells induce tolerance to DNFB contact sensitivity. The suppressor cells were effective only if they were present during the early stages of the afferent limb of sensitization. As measured by DNA synthesis, cell proliferation in the draining lymph nodes of recipients of suppressor cells was found to be significantly less than in control animals indicating that the suppressor cells acted, at least in part, by limiting or inhibiting DNFB-induced cell proliferation. This inhibition was shown to be antigen specific since the DNFB suppressor cells did not inhibit cell proliferation induced by oxazolone, an unrelated contact sensitizer. The ability to DNFB tolerant cells to block afferent sensitization pathways differs from the mechanism of tolerance to picryl chloride, reported by others, where efferent pathways are blocked.     

Role of Cftr genotype in the response to chronic Pseudomonas aeruginosa lung infection in mice

Patients with cystic fibrosis have a lesion in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which is associated with abnormal regulation of other ion channels, abnormal glycosylation of secreted and cell surface molecules, and vulnerability to bacterial infection and inflammation in the lung usually leading to the death of these patients. The exact mechanism(s) by which mutation in CFTR leads to lung infection and inflammation is not clear. Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and DeltaF508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads. Body weights of mice bearing mutations in Cftr were significantly smaller than wild-type mice at most ages. The inflammatory responses to P. aeruginosa-laden agarose beads were comparable in mice of all four Cftr mutant genotypes with respect to absolute and relative cell counts in bronchoalveolar lavage fluid, and cytokine levels (TNF-alpha, IL-1beta, IL-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant) and eicosanoid levels (PGE2 and LTB4) in epithelial lining fluid: the few small differences observed occurred only between cystic fibrosis mice bearing the S489X mutation and those bearing the knockout mutation Y122X. Thus we cannot implicate either misprocessing of CFTR or failure of CFTR to reach the plasma membrane in the genesis of the excess inflammatory response of CF mice. Therefore, it appears that any functional defect in CFTR produces comparable inflammatory responses to lung infections with P. aeruginosa.     

IL-23 mediates inflammatory responses to mucoid Pseudomonas aeruginosa lung infection in mice

Patients with cystic fibrosis (CF) develop chronic Pseudomonas aeruginosa lung infection with mucoid strains of P. aeruginosa; these infections cause significant morbidity. The immunological response in these infections is characterized by an influx of neutrophils to the lung and subsequent lung damage over time; however, the underlying mediators to this response are not well understood. We recently reported that IL-23 and IL-17 were elevated in the sputum of patients with CF who were actively infected with P. aeruginosa; however, the importance of IL-23 and IL-17 in mediating this inflammation was unclear. To understand the role that IL-23 plays in initiating airway inflammation in response to P. aeruginosa, IL-23p19(-/-) (IL-23 deficient) and wild-type (WT) mice were challenged with agarose beads containing a clinical, mucoid isolate of P. aeruginosa. Levels of proinflammatory cytokines, chemokines, bacterial dissemination, and inflammatory infiltrates were measured. IL-23-deficient mice had significantly lower induction of IL-17, keratinocyte-derived chemokine (KC), and IL-6, decreased bronchoalveolar lavage (BAL) neutrophils, metalloproteinase-9 (MMP-9), and reduced airway inflammation than WT mice. Despite the reduced level of inflammation in IL-23p19(-/-) mice, there were no differences in the induction of TNF and interferon-gamma or in bacterial dissemination between the two groups. This study demonstrates that IL-23 plays a critical role in generating airway inflammation observed in mucoid P. aeruginosa infection and suggests that IL-23 could be a potential target for immunotherapy to treat airway inflammation in CF.     

Demonstration of antigen-specific suppressor cells in unresponsive nude mice: a model for the induction of antigen-specific suppressor cells

Nude spleen cells were rendered incapable of giving an in vitro response in the presence of TRF by pretreatment of nude mice with antigen, followed 1 week later by T-cell injection. It is shown that this unresponsiveness is caused by antigen-specific suppressor cells which affect not only the stimulation of nude spleen cells but also the activation of memory cells and the production of a T-cell-replacing factor. The appearance in nude mice of suppressing activity rather than helper activity, after administration of T cells, is dependent on the sequence of treatment. These results suggest a model for the induction of antigen-specific suppressor cells by activated B cells.     

Contact sensitivity to picryl chloride: the occurrence of B suppressor cells in the lymph nodes and spleen of immunized mice.

Mice were immunized for contact sensitivity and antibody production by painting the skin with picryl chloride. Lymph node and spleen cells taken 4 days later transferred contact sensitivity. However, cells taken at 7–8 days failed to transfer but were able to block the transfer by 4 day immune cells. These suppressor cells occurred in the regional lymph nodes, spleen and thymus. The suppressor activity of lymph node and spleen cells was due to B cells as shown by the effect of anti-θ serum and complement, nylon wool filtration and separation of EAC positive and negative cells by centrifugation on a discontinuous gradient. The transfer of fractions rich or poor in macrophages showed that the suppressor cell in the transferred population was not a macrophage. Separation using EAC rosettes suggested that B cells were responsible for the suppressor activity in the thymus.T cells isolated from the lymph nodes and spleen 7–8 days after immunization transferred contact sensitivity although the initial population was inactive. This indicates that passive transfer cells are present in the regional lymph nodes and spleen at later times after immunization but cannot be demonstrated because of the presence of suppressor B cells. However, no passive transfer cells were found in the thymus. The production of B suppressor cells required little or no T cell help and following immunization the spleens of reconstituted (B) mice were at least as active as control cells in causing suppression. There are several different suppressor cells which act in the picryl system and the B suppressor cells in immunized mice described here are distinct from the T suppressor cells in mice injected with picryl sulphonic acid.     

Soluble factors in tolerance and contact sensitivity to DNFB in mice. VIII. Regulation of T suppressor cell function by autoreactive T helper cells

When cultured with DNP-labeled I-A+ cells, Lyt 2+ T suppressor cells (Ts) from 2,4,-dinitrobenzene sulfonate (DNBS)-tolerized mice are activated to synthesize and release a suppressor factor (SSF) which suppresses the transfer of contact sensitivity to DNFB. The signals required to activate the DNBS-primed Ts to produce SSF were studied in greater detail. As previously observed with fixed DNP-labeled spleen cell stimulators, the supernatants from cultures of DNBS-primed spleen cells and glutaraldehyde-fixed DNP-labeled P388D1 cell monolayers did not contain SSF. When the tolerant cells were harvested from these monolayers and were treated with IL-1, the Ts released the synthesized SSF. Synthesis and release of SSF required Ts recognition of DNP/class I MHC on the hapten-presenting cells followed by interaction with the costimulator IL-1. When the tolerant cells were cultured with fixed DNP-labeled I-A+ or I-A- stimulators to induce SSF synthesis, release was induced by adding either unlabeled or TNP-labeled unprimed spleen cells to the cultures. The release of SSF was blocked when the second stimulators were pretreated with anti-I-A antibody but not with anti-DNP or anti-class I MHC antibodies. These results indicate that the release of SSF by DNBS-primed Lyt 2+ Ts is regulated by the activity of a self-I-A-reactive (i.e., autoreactive) T cell in the tolerant spleen cell population.