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Cystic fibrosis (CF) is caused by mutations to the CF transmembrane conductance regulator (CFTR) gene. CFTR is known to be expressed on multiple immune cell subtypes, dendritic cells, monocytes/macrophages, neutrophils and lymphocytes. We hypothesized that the lack of CFTR expression on peripheral blood innate immune cells would result in an altered cell profile in the periphery and that this profile would reflect lung pathology. We performed a flow cytometric phenotypic investigation of innate immune cell proportions in peripheral blood collected from 17 CF patients and 15 age-matched healthy controls. We observed significant differences between CF patients and controls in the relative proportions of natural killer (NK) cells, monocytes and their subsets, with significant correlations observed between proportions of NK and monocyte cell subsets and lung function (forced expiratory volume in 1 sec, % predicted; FEV1% predicted) in CF patients. This study demonstrates the widespread nature of immune dysregulation in CF and provides a basis for identification of potential therapeutic targets. Modulation of the distinct CF-related immune cell phenotype identified could also be an important biomarker for evaluating CFTR-targeted drug efficacy.

Peroxisome proliferator activated receptor (PPARgamma) has been suggested as a target for anti-inflammatory therapy in chronic lung disease, including infection with Pseudomonas aeruginosa. However, the P. aeruginosa signal molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) has been reported to inhibit function of PPARs in mammalian cells. This suggests that binding of 3-oxo-C12-HSL to PPARs could increase inflammation during P. aeruginosa infection, particularly if it could compete for binding with other PPAR ligands. We investigated the ability of 3-oxo-C12-HSL to bind to a PPARgamma ligand binding domain (LBD) construct, and to compete for binding with the highly active synthetic PPARgamma agonist rosiglitazone. We demonstrate that 3-oxo-C12-HSL binds effectively to the PPARgamma ligand binding domain, and that concentrations of 3-oxo-C12-HSL as low as 1 nM can effectively interfere with the binding of rosiglitazone to the PPARgamma ligand binding domain. Because 3-oxo-C12 HSL has been demonstrated in lungs during P. aeruginosa infection, blockade of PPARgamma-dependent signaling by 3-oxo-C12-HSL produced by the infecting P. aeruginosa could exacerbate infection-associated inflammation, and potentially impair the action of PPAR-activating therapy. Thus the proposed use of PPARgamma agonists as anti-inflammatory therapy in lung P. aeruginosa infection may depend on their ability to counteract the effects of 3-oxo-C12-HSL.

Pseudomonas aeruginosa releases a wide array of toxins and tissue-degrading enzymes. Production of these malicious virulence factors is controlled by interbacterial communication in a process known as quorum sensing. An increasing body of evidence reveals that the bacterial signal molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) exhibits both quorum-sensing signalling and immune-modulating properties. Recently, yet another quorum-sensing signal molecule, the Pseudomonas quinolone signal (PQS), has been shown to affect cytokine release by mitogen-stimulated human T cells. In the present article we demonstrate that both OdDHL and PQS decrease the production of interleukin-12 (IL-12) by Escherichia coli lipopolysaccharide-stimulated bone marrow-derived dendritic cells (BM-DCs) without altering their IL-10 release. Moreover, BM-DCs exposed to PQS and OdDHL during antigen stimulation exhibit a decreased ability to induce T-cell proliferation in vitro. Collectively, this suggests that OdDHL and PQS change the maturation pattern of stimulated DCs away from a proinflammatory T-helper type I directing response, thereby decreasing the antibacterial activity of the adaptive immune defence. OdDHL and PQS thus seem to possess dual activities in the infection process: as inducers of virulence factors as well as immune-modulators facilitating the infective properties of this pathogen.

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.

People with cystic fibrosis (CF) have been reported to make lung T cell responses that are biased towards T helper (Th) 2 or Th17. We hypothesized that CF-related T cell regulatory defects could be detected by analyzing CD4+ lymphocyte subsets in peripheral blood. Peripheral blood mononuclear cells from 42 CF patients (6 months–53 years old) and 78 healthy controls (2–61 years old) were analyzed for Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17+), Treg (FOXP3+), IL-10+ and TGF-β+ CD4+ cells. We observed higher proportions of Treg, IL-10+ and TGF-β+ CD4+ cells in CF adults (≥ 18 years old), but not children/adolescents, compared with controls. Within the CF group, high TGF-β+% was associated with chronic Pseudomonas aeruginosa lung infection (p < 0.006). We observed no significant differences between control and CF groups in the proportions of Th1, Th2 or Th17 cells, and no association within the CF group of any subset with sex, CFTR genotype, or clinical exacerbation. However, high Th17% was strongly associated with poor lung function (FEV1 % predicted) (p = 0.0008), and this association was strongest when both lung function testing and blood sampling were performed within one week. Our results are consistent with reports of CF as a Th17 disease and suggest that peripheral blood Th17 levels may be a surrogate marker of lung function in CF.

Th-cell differentiation is highly influenced by the local cytokine environment. Although cytokines such as IL-12 and IL-4 are known to polarize the Th-cell response towards Th1 or Th2, respectively, it is not known whether these cytokines instruct the developmental fate of uncommitted Th cells or select cells that have already been committed through a stochastic process. We present an individual based model that accommodates both stochastic and deterministic processes to simulate the dynamic behaviour of selective versus instructive Th-cell development. The predictions made by each model show distinct behaviours, which are compared with experimental observations. The simulations show that the instructive model generates an exclusive Th1 or Th2 response in the absence of an external cytokine source, whereas the selective model favours coexistence of the phenotypes. A hybrid model, including both instructive and selective development, shows behaviour similar to either the selective or the instructive model dependent on the strength of activation. The hybrid model shows the closest qualitative agreement with a number of well-established experimental observations. The predictions by each model suggest that neither pure selective nor instructive Th development is likely to be functional as exclusive mechanisms in Th1/Th2 development.

Background Allergic asthma is a T H 2 cell–driven immunological disease, characterized by eosinophilic inflammation. The cytotoxic agent cyclophosphamide paradoxically augments several immune responses. Objective We studied the proposal that cyclophosphamide may aggravate airway inflammation in allergic mice, and these features might result from the loss of regulatory T cells. Methods BALB/c mice were immunized with ovalbumin on days 0 and 14 and challenged with aerosolized ovalbumin from days 21 to 27. Some mice also received cyclophosphamide on days –2 and 12. Results In the lungs of cyclophosphamide-treated animals, pronounced worsening of inflammatory features was noted, including increased eosinophil infiltration, epithelial thickness, mucus occlusion, and eosinophil numbers in bronchoalveolar lavage fluid. There was also increased total and ovalbumin-specific serum IgE, increased IL-4 and IL-5 secretion by peritracheal lymph node cells, and reduced lung mRNA expression of IL-10 and TGF-β in animals treated with cyclophosphamide. The expression of FoxP3, a marker of regulatory T cells, was significantly reduced in lymphoid organs after the second injection of cyclophosphamide, and in the lung tissue after allergen challenge in cyclophosphamide-treated mice. Lung IL-10 + CD4 + T cells and cytotoxic T lymphocyte–associated antigen 4 + CD4 + T cells were reduced after allergen challenge in cyclophosphamide-treated mice. Conclusion Cyclophosphamide worsened features of allergic pulmonary inflammation in this model, in association with increased production of IgE and T H 2 cytokines. The reduced expression of FoxP3 and immunosuppressive cytokines by cyclophosphamide is consistent with the possibility that toxicity to regulatory T cells may contribute to the increased inflammation.

ThePseudomonas aeruginosaquorum-sensing moleculeN-3-(oxododecanoyl)-l-homoserine lactone (OdDHL) has been reported to have immunomodulatory activity in several systems, although the mechanism of that activity remains to be fully characterized. We demonstrate here, using a defined in vitro model of antigen responses by T-cell receptor (TCR)-transgenic mouse splenic CD4 T cells, that the effect of OdDHL on activation and cytokine production is complete within 4 h of antigen or mitogen stimulation and does not depend on the insertion of OdDHL in the cell membrane, despite a previous report that immunosuppression by homoserine lactones required a minimum acyl chain length of 11 carbons (S. R. Chhabra, C. Harty, D. S. W. Hooi, M. Daykin, B. W. Bycroft, P. Williams, and D. Pritchard, J. Med. Chem.46:97-104, 2003). We also demonstrate that while OdDHL can have toxic effects on nonlymphoid leukocytes, it does not induce significant cell death in T cells at the concentrations (≤10 μM) used in these experiments. In addition, we show that primary and secondary antigen-specific cytokine responses are equally susceptible to inhibition by OdDHL and that the compound inhibits the differentiation of both Th1 and Th2 cells. However, the precise balance of cytokine production by CD4 T cells stimulated in the presence of OdDHL varies with both the antigen concentration and its affinity for the transgenic TCR. Thus, conflicting reports of the nature of the immunosuppression by OdDHL may be due in part to the differences in antigen affinity and concentration in different models.

N-3-(oxododecanoyl)-l-homoserine lactone (OdDHL), a quorum-sensing molecule ofPseudomonas aeruginosa, plays an important role in the pathogenesis of the organism through its control of virulence factor expression. Several reports have suggested that OdDHL can also directly modulate host immune responses. However, the nature of the modulation is controversial, with different reports suggesting promotion of either humoral (Th2-mediated) or inflammatory (Th1-mediated) responses. This report describes a series of studies which demonstrate for the first time that in vivo administration of OdDHL can modulate the course of an antibody response, with an increase in ovalbumin (OVA)-specific immunogloblulin G1 (IgG1) but not IgG2a in OdDHL-treated OVA-immunized BALB/c mice compared to levels for controls. In vitro stimulation of lymphocytes from both Th1-biased C57Bl/6 and T-cell receptor transgenic mice and Th2-biased BALB/c mice in the presence of OdDHL demonstrated that OdDHL inhibits in vitro cytokine production in response to both mitogen and antigen, with gamma interferon (IFN-γ) tending to be more inhibited than interleukin-4 (IL-4). In vitro mitogen or antigen restimulation of cells from mice treated with OdDHL in vivo shows effects on cytokine production which depend on the underlying immune bias of the mouse strain used, with a relative increase of IFN-γ in Th1-biased C57Bl/6 mice and a relative increase of IL-4 in Th2-biased BALB/c mice. Thus, the mode of action of OdDHL on T-cell cytokine production is likely to be a relatively nonspecific one which accentuates an underlying immune response bias rather than one which specifically targets either Th1 or Th2 responses.

Members of the Sydney Blood Bank Cohort (SBBC) have been infected with an attenuated strain of HIV-1 with a natural nef/LTR mutation and have maintained relatively stable CD4+ T lymphocyte counts for 14-18 years. Flow cytometric analysis was used to examine the phenotype of CD4+ and CD8+ T lymphocytes in these subjects, including the immunologically important naive (CD45RA+CD62L+), primed (CD45RO+), and activated (CD38+HLA-DR+ and CD28-) subsets. The median values were compared between the SBBC and control groups, comprising age-, sex-, and transfusion-matched HIV-1-uninfected subjects; transfusion-acquired HIV-1-positive LTNPs; and sexually acquired HIV-1-positive LTNPs. Members of the SBBC not only had normal levels of naive CD4+ and CD8+ T lymphocytes, but had primed CD45RO+ CD4+ T lymphocytes at or above normal levels. Furthermore, these primed cells expressed markers suggesting recent exposure to specific antigen. SBBC members exhibited variable activation of CD8+ T lymphocytes. In particular, SBBC members with undetectable plasma HIV-1 RNA had normal levels of activated CD8+ T lymphocytes. Therefore, the result of long-term infection with natural nef/LTR mutant HIV-1 in these subjects suggests a decreased cytopathic effect of attenuated HIV-1 on susceptible activated CD4+ T lymphocyte subsets in vivo, and minimal activation of CD8+ T lymphocytes.

High-dose chemotherapy with autologous stem cell rescue has been proposed as an intensive therapy for severe rheumatoid arthritis (RA). In view of previous observations of abnormal haemopoiesis in RA patients, the composition and function of peripheral blood stem cell harvests (PBSCH) was investigated. Compared with PBSCH from healthy allogeneic donors mobilized with the same dose of G-CSF (filgrastim; 10 microg/kg/d, n = 14), RA PBSCH (n = 9) contained significantly fewer mononuclear cells (375 v 569 x 10(6)/kg, P = 0.03) and CD34+ cells (2.7 v 5.8 x 10(6)/kg, P = 0.003). However, there were increased proportions of CD14+ cells (P = 0.006) and CD14+ CD15+ cells (the phenotype of previously described 'abnormal' myeloid cells, P = 0.002) in the RA PBSCH which translated into 3.5- and 7-fold increases respectively on a per CD34+ cell basis. There were no differences in T-cell activation status as judged by proportions of CD4+ and CD8+ expressing CD45RA, CD45RO, HLA-DR and CD28 (RA PBSCH, n = 7, donor PBSCH, n = 5, P = 0.2-0.7). Phytohaemagglutinin responses determined fluorocytometrically with induction of CD69 expression were reduced in CD4+ and CD8+ cells following filgrastim administration in 3/3 RA patients tested. Compared with bone marrow as a potential source of CD34+ cells, PBSCH contained 11-fold more T cells (P < 0.0005), 8-fold more B cells (P < 0.0005) and 4-fold more monocytes (P = 0.02). In short-term methylcellulose culture there were no differences in colony counts (CFU-GM, CFU-GEMM, BFU-E) per CD34+ cell from PBSCH from RA patients (n = 11) and healthy donors (n = 10). Long-term culture initiator cells were cultured successfully from cryopreserved PBSCH from RA patients (n = 9). In conclusion, PBSCH from RA patients differed significantly in composition from normal individuals, but in vitro studies support normal stem and progenitor cell function. Changes in T-cell function occur during mobilization in RA patients. This work provides reassurance for the use of PBSCH as haematological rescue and baseline data for clinical trials of graft manipulation strategies in patients with RA.

It has been widely reported that quorum-sensing incapable strains of Pseudomonas aeruginosa are less virulent than wild type strains. However, quorum sensing mutants of P. aeruginosa have been shown to develop other spontaneous mutations under prolonged culture conditions, and one of the phenotypes of P. aeruginosa that is frequently affected by this phenomenon is type IV pili-dependent motility, referred to as twitching motility. As twitching motility has been reported to be important for adhesion and colonisation, we aimed to generate a quorum-sensing knockout for which the heritage was recorded and the virulence factor production in areas unrelated to quorum sensing was known to be intact. We created a lasIRrhlIR quadruple knockout in PAO1 using a published technique that allows for the deletion of antibiotic resistance cartridges following mutagenesis, to create an unmarked QS knockout of PAO1, thereby avoiding the need for use of antibiotics in culturing, which can have subtle effects on bacterial phenotype. We phenotyped this mutant demonstrating that it produced reduced levels of protease and elastase, barely detectable levels of pyoverdin and undetectable levels of the quorum sensing signal molecules N-3-oxododecanoly-L-homoserine lactone and N-butyryl homoserine lactone, but retained full twitching motility. We then used a mouse model of acute lung infection with P. aeruginosa to demonstrate that the lasIRrhlIR knockout strain showed equal persistence to wild type parental PAO1, induced equal or greater neutrophil infiltration to the lungs, and induced similar levels of expression of inflammatory cytokines in the lungs and similar antibody responses, both in terms of magnitude and isotype. Our results suggest, in contrast to previous reports, that lack of quorum sensing alone does not significantly affect the immunogenicity, infectiveness and persistence of P. aeruginosa in a mouse model of acute lung infection. © 2013 Lazenby et al.

T lymphocytes spend much of the time as small non-cycling cells. To determine the pattern of cytokine expression in such resting cells, they were purified from human peripheral blood mononuclear cells (PBMC) on the basis of high buoyant density. The cells were stimulated and cytokine mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Expression of interleukin-2 (IL-2), IL-3 and interferon-gamma (IFN-gamma) was similar in high-density lymphocytes and in unfractionated PBMC. In contrast, the high-density lymphocytes expressed less IL-4 than PBMC, and little or no IL-5. Because a substantial minority of the high-density lymphocytes was CD45RO+, the presence of this marker was not an indicator of the ability to express IL-4 and IL-5. In the high-density lymphocytes, IFN-gamma expression was confined to the CD45RO+ fraction, whereas IL-2 was expressed by both CD45RO+ and CD45RO- subsets. To assess whether high-density lymphocytes could give rise to cells with a broader range of inducible cytokine expression, they were activated and then restimulated between 10 and 22 days of culture. Cells derived from both the CD45RO+ and CD45RO- fractions of high-density lymphocytes expressed IL-5 after restimulation. Thus the high-density lymphocyte population has the potential to acquire a broader range of inducible cytokine expression.

The Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxododecanoyl-l-homoserine lactone (3OC(12)HSL) can inhibit function of the mammalian anti-inflammatory transcription factor peroxisome proliferator activated receptor (PPAR)γ, and can be degraded by human paraoxonase (PON)2. Because 3OC(12)HSL is detected in lungs of cystic fibrosis (CF) patients infected with P. aeruginosa, we investigated the relationship between P. aeruginosa infection and gene expression of PPARγ and PON2 in bronchoalveolar lavage fluid (BALF) of children with CF. Total RNA was extracted from cell pellets of BALF from 43 children aged 6 months-5 years and analyzed by reverse transcription-quantitative real time PCR for gene expression of PPARγ, PON2, and P. aeruginosa lasI, the 3OC(12)HSL synthase. Patients with culture-confirmed P. aeruginosa infection had significantly lower gene expression of PPARγ and PON2 than patients without P. aeruginosa infection. All samples that were culture-positive for P. aeruginosa were also positive for lasI expression. There was no significant difference in PPARγ or PON2 expression between patients without culture-detectable infection and those with non-Pseudomonal bacterial infection, so reduced expression was specifically associated with P. aeruginosa infection. Expression of both PPARγ and PON2 was inversely correlated with neutrophil counts in BALF, but showed no correlation with other variables evaluated. Thus, lower PPARγ and PON2 gene expression in the BALF of children with CF is associated specifically with P. aeruginosa infection and neutrophilia. We cannot differentiate whether this is a cause or the effect of P. aeruginosa infection, but propose that the level of expression of these genes may be a marker for susceptibility to early acquisition of P. aeruginosa in children with CF.

Previous studies from this laboratory have shown that PBMC from recipients of an HLA-identical sibling bone marrow transplant produce levels of IL-2 which are 10-100-fold lower than those produced by the same number of PBMC from healthy controls, whereas production of IFN-gamma is normal. The present study examined IL-2 and IFN production over a range of cell numbers for PBMC and for isolated CD4+ and CD8+ cells for controls and marrow transplant recipients. There was a 5-fold lower IL-2 production in marrow transplant recipient CD8+ cells compared with equivalent numbers of control cells, whereas no difference was found in IL-2 production by CD4+ cells. In contrast, IFN production by CD4+ cells from marrow transplant recipients was 4-fold higher than in controls, whereas CD8+ cells from both populations produced similar amounts of IFN. When the observed production of cytokine by PBMC was compared with the expected production based on the CD4+ and CD8+ content of the PBMC, control values were similar, but the expected values for both cytokines were approximately 2-fold higher than the observed values for marrow transplant recipients. The results suggest that the capacity of T cells from marrow transplant recipients to produce IL-2 and IFN is not impaired, but that the frequency of cytokine-producing cells may be reduced, and that a negative interaction present in recipient PBMC, eliminated by isolating T-cell subsets, is responsible for the observed low levels of cytokine production.

BMT from matched unrelated donors or mismatched family members is associated with an increased risk of graft-versus-host disease (GVHD) compared with HLA-identical sibling donors. It has been suggested that the level of patient-specific CTL precursors (CTLp) present in matched unrelated donors correlates with the incidence and severity of GVHD after BMT. This study group consisted of 17 patients who all received unmanipulated bone marrow from an HLA-A,B,DR-matched unrelated donor. Patient-specific CTLp frequencies were estimated in the donor before transplant. The CTLp frequencies were then compared with the incidence and severity of GVHD experienced by the patient after transplantation. Statistical analysis revealed no correlation between donor precursor frequencies and the patient developing clinically significant acute GVHD after transplantation (X2 = 1.16). This study suggests that caution should be used before the inclusion of the CTLp frequency result in the clinical decision of selecting the most suitable matched unrelated donor for BMT. CTLp frequency does not correlate with either the incidence or severity of GVHD after matched unrelated donor BMT.

Our previous finding that heparin-dependent antibodies in heparin- induced thrombocytopenia (HIT) bind to platelets via platelet IgG Fc receptors (FcRs) prompted this study. Platelet FcRs in 16 patients with HIT, 23 control patients, and 42 normal subjects were studied. Patients with HIT had substantially increased platelet FcRs during the acute illness. Those who suffered serious thrombotic complications or died shortly after diagnosis had significantly more FcRs per platelet than those with milder disease. Consistent with their increased FcRs, platelets of patients with HIT showed increased aggregation reactivity to aggregated IgG and heparin-dependent antibodies. Platelet FcRs in patients with HIT remained elevated for 1 to 3 months after the acute illness then stabilized to a mean value not significantly different from either control group. The increased expression of FcRs on HIT platelets and their increased reactivity to heparin-dependent antibodies may contribute to the pathogenesis of thrombocytopenia and thrombosis in HIT.

BALB/c mice (H-2d) given 10 Gy total body irradiation (TBI) followed by 10(7) bone marrow (BM) and 10(6) spleen cells from C57BL/6 (H-2b) donor mice received recombinant cytokines intraperitoneally (IP) twice daily. The effect on neutrophil recovery rate, graft-v-host disease (GVHD), and survival was assessed. Four reagents were used: granulocyte-colony- stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin-1 (IL-1) and IL-4, both alone and in combination. The most effective combination for increasing the circulating absolute neutrophil account (ANC) above the control value at day 7 posttransplant was the combination of G-CSF and IL-1 (mean ANC 2.4 +/- 1.6 x 10(9)/L as compared with control value of 0.07 +/- 0.05, P less than .02), followed by G-CSF alone (mean ANC 1.1 +/- 0.2, P less than .0001), the combination of GM-CSF plus IL-1 (mean ANC 0.8 +/- 0.3, P less than .002), and the combination of G-CSF plus GM-CSF (mean ANC 0.8 +/- 0.3, p less than .005). At day 10 posttransplant, the most effective combination in raising the ANC was the combination of G-CSF plus GM-CSF (mean ANC 7.5 +/- 2.3 as compared with control value of 3.5 +/- 1.1, P less than .01), followed by G-CSF alone (mean ANC 6.9 +/- 2.1, P less than .02). At the doses used, neither G-CSF nor GM-CSF had a deleterious effect on the incidence or severity of GVHD; indeed, GM- CSF was associated with improved survival. In contrast, IL-1 at doses greater than or equal to 100 ng twice daily caused marked early mortality, and there was a suggestion that IL-4 at doses of 500 ng twice daily resulted in increased late mortality, possibly owing to exacerbation of GVHD. This model appears to be of value for exploring the use of hematopoietic growth factors before they are used clinically in marrow allograft recipients.

Communication through quorum sensing (QS) enables bacterial populations to coordinate their behavior. Recent work on N-acylhomoserine lactone-mediated QS has revealed that some soil bacteria exploit host-derived substrates to generate an alternative N-substituted homoserine lactone. New light has also been shed on the mechanism by which N-(3-oxo-dodecanoyl)-l-homoserine lactone modulates host inflammatory signaling pathways to promote bacterial survival.

Th-cell development has been suggested to include selective mechanisms in which certain cytokines select either Th1 or Th2 cells to proliferate and grow. The selective theory is based on the observation that Th2 cells secrete IL-4, a cytokine that promotes Th2 development, whereas Th1 cells secrete interferon-gamma (IFN-gamma) that favours Th1 development, and both positive and negative selective influences have been suggested to operate. In this study, we investigate the role of autocrine secretion and utilization of IL-4 by Th2 cells and address the question of whether an activated Th2 cell can be positively selected by IL-4 secreted from other Th2 cells. We present a spatial three dimensional (3D) modelling approach to simulate the interaction between the IL-4 ligand and its IL-4 receptors expressed on discrete IL-4 secreting cells. The simulations, based on existing experimental data on the IL-4 receptor-ligand system, illustrate how Th-cell development is highly dependent on the distance between cells that are communicating. The model suggests that a single Th2 cell is likely to communicate with possible target cells within a range of approximately 100 microm and that an activated Th2 cell manages to fill most of its own IL-4 receptors, even at a low secretion rate. The predictions made by the model suggest that negative selection against Th1 cells is more effective than positive selection by IL-4 for promoting Th2 dominance.

AIDS patients often contain HIV-1-infected mast cells (MCs)/basophils in their peripheral blood, and in vivo-differentiated MCs/basophils have been isolated from the blood of asthma patients that are HIV-1 susceptible ex vivo due to their surface expression of CD4 and varied chemokine receptors. Because IL-16 is a ligand for CD4 and/or an undefined CD4-associated protein, the ability of this multifunctional cytokine to regulate the development of human MCs/basophils from nongranulated progenitors residing in cord or peripheral blood was evaluated. After 3 wk of culture in the presence of c-kit ligand, IL-16 induced the progenitors residing in the blood of normal individuals to increase their expression of chymase and tryptase about 20-fold. As assessed immunohistochemically, >80% of these tryptase+ and/or chymase+ cells expressed CD4. The resulting cells responded to IL-16 in an in vitro chemotaxis assay, and this biologic response could be blocked by anti-IL-16 and anti-CD4 Abs as well as by a competitive peptide inhibitor corresponding to a sequence in the C-terminal domain of IL-16. The additional finding that IL-16 induces calcium mobilization in the HMC-1 cell line indicates that IL-16 acts directly on MCs and their committed progenitors. IL-16-treated MCs/basophils also are less susceptible to infection by an M/R5-tropic strain of HIV-1. Thus, IL-16 regulates MCs/basophils at a number of levels, including their vulnerability to retroviral infection.

Summary Gastrointestinal hypersensitivity to food allergens is a significant but relatively poorly understood allergic disease. Recent evidence from a rat model of IgE-mediated gastrointestinal hypersensitivity has suggested that hepatic mast cells (HMC) may play an important role in such reactions. The present study was undertaken to better define their phenotype. Livers from Australian albino Wistar (AaW), Brown Norway (BN) and PVG/c rats were examined using traditional histological techniques and reverse transcription‐polymerase chain reaction (RT-PCR). Hepatic mast cells were overwhelmingly Alcian blue positive, sensitive to formalin fixation and predominantly rat mast cell protease (RMCP) 1 + /2 ‐ (AaW 57%; BN 53%). Such a phenotype has previously been associated with an immature mast cell phenotype. A significant number of HMC also stained RMCP 1 ‐ /2 + (AaW 15%; BN 19%) or were RMCP 1 + /2 + (AaW 24%; BN 26%). In contrast to previous reports, RT-PCR showed that the liver expressed mRNA of other mast cell proteases, including the chymase RMCP 5 as well as two tryptases, RMCP 6 and RMCP 7. These results suggest that HMC are a heterogeneous population of mast cells with some characteristics previously associated with immature cells.

Background : Absolute counts of CD4 + T-lymphocytes are used in the management of patients with human immunodeficiency virus infection. Low absolute counts of CD3 + CD4 + cells have also been observed in healthy people-a phenomenon called idiopathic CD4 lymphocytopenia. It is common practice for normal ranges for lymphocyte subsets to be derived from samples taken from blood donors. Study Design and Methods : A sample of EDTA blood was taken through the donation line tubing, after donation from 565 blood donors in Sydney, Australia, who were selected from a range of age groups. An additional 12 donors provided a predonation sample as well as a postdonation sample. Hematologic assays were performed on two analyzers. Samples were stained for CD3, CD4, D8, CD19, and CD56 and analyzed on a flow cytometer. Results : Three donors were found to have absolute CD3 + CD4 + counts

Pertussis toxin (PT), from Bordetella pertussis, causes lymphocytosis and increased IL-4 and IgE secretion. The lymphocytosis is associated with impaired entry of lymphocytes into lymph nodes. The dose response of PT on IL-4 secretion was found to be similar to those for lymphocytosis and IgE production. These findings are consistent with the possibility that increased IL-4 production by PT may be related to its effect on lymphocyte circulation. The possibility that PT may selectively influence the entry into lymph nodes of subsets identified with CD45RB, CD4 or CD8 was tested by assessing the phenotype of lymph node cells. PT had no significant effect on the proportion of cells with these markers, either in unimmunized or in immunized mice. These findings indicate that PT does not selectively impair entry of these lymphocyte subsets into lymph nodes.

The expression of IL5 by CD4+CD45RA+, CD4+CD45R0+ and CD3+CD8+ subsets of human peripheral blood mononuclear cells was assessed. Interleukin 5 expression was detected by RNA extraction, reverse transcription and polymerase chain reaction. Populations of highly purified cells were obtained by a protocol of sequential plastic adherence, magnetic bead separation and flow cytometric cell sorting. IL5 was clearly expressed in the CD4+CD45R0+ subset from 3 to 48 hr after activation. The CD4+CD45RA+ and CD3+CD8+ subsets expressed very much less IL5. By contrast, IL2 expression was readily detected in all sorted populations. Thus, in activated CD4+ cells, IL5 was predominantly expressed in the CD4+CD45R0+ subset, a pattern of expression corresponding to that reported for a number of other cytokines, and differing from that of IL2.

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.

We have demonstrated that both recombinant and purified IL-2 exert a direct effect on quiescent human microvascular endothelial cells in vitro, causing the cells to enter the cell cycle and proliferate (Hicks et al., 1989). In this study we have identified IL-2 receptors (R) on both human umbilical vein (HUVEC) and neonatal foreskin (HCEC) endothelial cells. The techniques used to identify the receptors included proliferation studies, flow cytometry and immunofluorescence. Results indicate that both HUVEC and HCEC possess low numbers of receptors since both cell types proliferate in response to IL-2. The number of receptors on the cell surface vary according to passage number and culture conditions. Immunofluorescent studies show discrete areas of staining on the cell membrane. These combined results suggest that human vascular endothelial cells possess IL-2R.

Spleen cells from B6 mice injected with fetal calf serum (FCS) could be kept proliferating as a continuous cell line in vitro provided they were culture in the presence of irradiated syngeneic spleen cells and FCS. Cells in this cell line showed a strong proliferative response when stimulated with concanavalin A (Con A), and they were able to mediate the following functions: (1)they helped the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) from thymocyte-spleen cell mixed lymphocyte cultures (MLC), (2)they induced the generation of CTL from normal syngeneic spleen cells in the absence of allogeneic stimulator cells, and (3)they induced normal spleen cells to differentiate into anti-sheep erythrocyte (SRBC) plaque-forming cells (PFC), in the absence of SRBC in the cultures. The use of this cell line (called line 12) may thus provide an interesting approach for the study of cellular and molecular requirements for cell-cell interactions and for the differentiation of T and B effector functions.

Thymocytes, in contrast to peripheral T cells, were unable to generate significant numbers of anti-self-TNBS cytotoxic T cells in primary in vitro cultures. The addition to such cultures of syngeneic 1000 rad irradiated lymph node or spleen cells from mice sensitized 6 or 7 days previously with trinitrochlorobenzene (TNCB) permitted the development of an anti-self-TNBS cytotoxic T cell response. This effect was shown to be T-dependent since it was largely abolished by anti-Thy 1 plus complement and was present in nylon-wool column purified T cells. The lymph node cells act as specific helper cells in that a) they must be restimulated in vitro by the priming antigen and b) they do not take part in the lytic phase of the response. The specificity of the helper effect is further indicated a) by the lack of help from nonsensitized lymph node cells and b) by absence of an enhanced helper effect for thymic anti-allogeneic or anti-allogeneic-TNBS responses in the presence of specific T helper cells for the anti-self-TNBS response. Analysis of the thymocyte subpopulation able to provide precursor cells was approached by separating thymocytes into a “medullary-like” minor, peanut-lectin-agglutinable (PNA-) fraction and a “cortical-like” major, peanut-lectin-agglutinable (PNA+) fraction. It was found that the PNA- fraction was enriched in thymocytes able to mount an autonomous anti-self-TNBS cytotoxic response, whereas the PNA+ population was totally unable to generate anti-self-TNBS effector cells in the absence of helper cells. The presence of helper cells from TNCB-painted mice permitted differentiation of anti-self-TNBS cytotoxic precursor cells from the PNA+ thymocyte cultures and enhanced the level of cytotoxic cells generated in PNA- thymocyte cultures.

Normal bone marrow obtained at harvest for bone marrow transplantation was passed over a Ficoll density gradient to obtain the mononuclear cell fraction. These cells were incubated with anti-T cell monoclonal antibodies (MAb) anti-HuLym-1 and anti-HuLym-8 plus rabbit complement, washed, and the degree of T cell depletion was assessed by culture in phytohemagglutinin, by flow cytometry and by limiting dilution analysis. The optimal protocol for T cell depletion was found to be a 2-stage incubation with 2 cycles of complement treatment, using a bone marrow cell concentration as low as possible. The precise conditions of complement dilution and incubation temperature depended on the batch of complement used, and had to be assessed for each batch. Of the 3 methods used for assessing T cell contamination, culture in PHA was found to be inappropriate due to large variation in background proliferation of treated and untreated bone marrow cells. Flow cytometry was accurate for residual T cell content of greater than 1% (corresponding to a 90-95% depletion of T cells) but could not detect lower levels of T cell contamination. The limit dilution assay was found to be by far the most sensitive, being capable of detecting a T cell contamination of 1 in 10,000 cells. When combined with flow cytometry on untreated marrow for calculation of the cloning efficiency, it gave very accurate determinations of residual T cell content of marrow.

Previous studies have shown that interleukin 2 (IL2) production by peripheral blood mononuclear cells (PBMC) is severely impaired post allogeneic bone marrow transplantation, whereas production of interferon-gamma (IFN-gamma) is at most marginally depressed. To investigate the mechanisms behind this apparently differential inhibition of lymphokine production, we stimulated PBMC from recipients of HLA-identical sibling bone marrow transplants with phytohaemagglutinin (PHA), PHA + phorbol ester (PMA) (to bypass accessory cell requirements) or Ca++ ionophore + PMA (to bypass both accessory cell and T cell surface receptor (CD2 and/or CD3/Ti interactions). Increasing the potency of the stimulus increased the amount of IL2 and IFN produced by PBMC from both normal volunteers and from marrow transplant recipients, but for each stimulus the amount of IL2 produced by marrow transplant recipient PBMC remained 10-100-fold lower than that produced by normal PBMC, suggesting an underlying defect in IL2 production by marrow transplant recipient T cells, not due to accessory cell or CD2 defects. Selection experiments showed that CD3+ cells were the primary IL2 producers, and we were unable to demonstrate presence of suppressor cells in marrow transplant PBMC. Statistical analysis of the clinical factors possibly affecting lymphokine synthesis showed that in vivo cyclosporin A did not affect the in vitro capacity of PBMC to produce cytokines, although steroid therapy had a negative effect on IL2 production. The only variable significantly affecting IL2 and IFN production in marrow transplant recipients was increasing time post transplant. It is suggested that the defect in IL2 but not IFN production could be due to either a selective reduction in the frequency of IL2 producing cells as opposed to IFN producing cells, or to a reduction in the amount of IL2 produced per cell in marrow transplant recipients.

The interaction of T helper (Th) cells with syngeneic and allogeneic cytotoxic T lymphocyte precursors (CTL.P) has been investigated. Unprimed and mixed lymphocyte culture-primed peripheral T cells were used as a source of Th. Thymocytes, which depend upon exogenous Th cells for activation, were used as a source of cytotoxic precursors. Data is presented that demonstrates that at least two pathways of T-T interaction can lead to the activation of cytotoxic lymphocytes. The first is an allogeneic effect, in which Th cells recognize and respond to alloantigens expressed on CTL.P. The second is the interaction of Th cells with syngeneic CTL.P, in which both cell types are thought to respond to alloantigens on stimulator cells. The latter interaction can be shown to be restricted by H-2-linked determinants when primed Th cells are used and allogeneic effects against thymocytes are minimized. Restricted interactions between unprimed Th cells and thymocyte CTL.P have never been observed. Mechanisms that may explain the difference between the interaction of unprimed and primed Th cells with CTL.P are discussed.

Sixteen patients with hematological malignancy received cyclophosphamide (120 mg/kg), fractionated total body irradiation (12 Gy), oral cyclosporin, and an HLA-identical sibling marrow transplant depleted of T cells by incubation with the monoclonal antibody anti-HuLy-m1 (CD2) and rabbit complement with (five patients) or without (11 patients) anti-HuLy-m8). These 16 patients were compared historically to 84 patients with hematological malignancy receiving cyclophosphamide (120 mg/kg), fractionated total body irradiation (12 or 14 Gy), oral cyclosporin, and unmanipulated HLA-identical sibling marrow, for parameters of engraftment and graft-versus-host disease (GVHD). Graft failure occurred in one of the 16 T-cell depleted recipients and in one of the 84 nondepleted recipients. Engraftment was slightly but significantly slower in the T-cell depleted group and bacterial infections significantly more frequent and severe than in the unmanipulated group. There was a suggestion that the severity of acute GVHD was reduced in those receiving T depleted marrow. Randomized trials will be necessary to determine if marrow T-cell depletion results in superior long-term leukemia-free survival.

Interferon (IFN) is a component of the immune response and influences immune responses as demonstrated by in vitro experimentation. In this review these complementary aspects of IFN's interaction with the immune response are discussed. Of chief interest is IFN-gamma ('immune' IFN) but IFNs-alpha and -beta are also considered.

The survival time of mouse thyroid, transplanted under the kidney capsule of an H-2 incompatible recipient, is extended by holding the thyroid in organ culture for 12 days prior to transplantation.

The effect of intravenous injection of 10(6) BALB/c spleen cells into C57B1/6J recipients was assayed by both mixed lymphocyte culture (MLC) of recipient lymphocytes, and by grafting donor (BALB/c) thyroids into recipient mice. It was observed that a single intravenous injection produced depression of proliferative and cytotoxic T cell responses in MLC of spleen, lymph node and peripheral blood lymphocytes of the recipients. This effect was specific for the sensitizing genotype (MLC of recipient and third-party CBA/H lymphocytes was unaffected), and persisted for several days after sensitization. The pattern of this diminished response suggested that the effect was due to a combination of recruitment of reactive lymphocytes from peripheral lymphoid populations, and the generation of alloantigen (H-2?)-specific suppressor T cells in the spleen. In contrast to these findings in vitro, a similar sensitization led only to accelerated rejection of thyroid allografts in vivo.

An immune response induced by the presentation of the hapten trinitrophenyl (TNP) on syngeneic cells can result from in vitro stimulation of T cells with syngeneic cells after trinitrobenzenesulfonate (TNBS) treatment (1) or after TNP-protein adsorption (2), or in vivo by trinitrochlorobenzene (TNCB) skin sensitization (3,4). The heterogeneity of T cell functions (5–9) is evident from the description of effector cells, including cytotoxic T cells (CTL) (1,2,4), helper T cells (TH) for CTL precursors (pCTL) (5) or B cells (6,7), as well as suppressor cells (Ts) (8–10) and delayed-type hypersensitivity (DTH) effector cells (3). In terms of the fine specificity of the response to TNBS-treated cells, it was not clear whether the apparently non-major histo-compatibility complex (MHC) -restricted components of CTL responses in mice (11,12) and humans (13) could originate from T cell recognition of non-MHC and/or nonpolymorphic cell surface antigens (13) in addition to the recognition of the known class I MHC antigens. In order to analyse such a heterogenous response in terms of functional T cell subsets (14,15) and fine specificity of T cells (16,17) we established T cells lines proliferating in response to TNBS-treated syngeneic cells (14) in which we analysed interacting T cell populations (15) and from which we derived individual T cell clones (15–17). The study of the mechanism of action of TH for the development of CTL from thymic precursor cells (5,18) led us to hope that T cell hybridomas might help in the analysis of products secreted by such TH (18). Here we will briefly summarize results obtained with such procedures.

The role of T helper cells (Th) and their soluble products in the generation of a cytotoxic T-cell (CTL) response of thymocytes to trinitrophenyl (TNP)-modified syngeneic cells was investigated. The Th have a Thy 1 + Lyt 1 + 2 − surface phenotype, and produce at least two soluble helper factors. Production of factors requires stimulation of primed Th by specific antigen (self-TNP), and depends on a Thy 1 + Lyt 1 + 2 − cell. Factors present in supernatants after 5 hr of stimulation act preferentially on antiallogeneic precursor CTL (pCTL); factors present at 24 hr act preferentially on self-TNP-specific pCTL with a variable activity for alloantigen-specific pCTL. These results are interpreted as suggesting a possible role for helper factors having selective action in generation of CTL responses.

Mouse T lymphocytes sensitized to alloantigens were cloned by limiting dilution in the presence of interleukin 2. Clones were tested for surface markers Thy-1, Lyt-1 and Lyt-2, and for cytotoxic function. Production of interferon (IFN) by clones either (a) stimulated with allogeneic cells; (b) activated with concanavalin A (Con A); or (c) infected with Semliki Forest virus or Newcastle disease virus were assayed. All clones produced IFN upon Con A stimulation and most after virus infection. Analysis of the IFN produced by a single clone, using anti-IFN antisera, showed that while Con A stimulation induced production of type II IFN (IFN-gamma), the IFN produced after virus infection was type I IFN (IFN-alpha/beta).

THE theory of allogeneic stimulation1 postulates that transplantation antigens, such as H-2 in the mouse or H-LA in man, are not strong immunogens for allogeneic animals, but are rendered highly immunogenic when presented to the immune system in conjunction with an allogeneic stimulus. The capacity to provide an allogeneic stimulus is the property of metabolically active2–4 immunocompetent cells1, and it has been suggested that allogeneic stimulation requires the transfer of some cytoplasmic component from the stimulating to the responsive cell1. According to this hypothesis, any treatment that inactivates the metabolic activity of lymphoid cells will reduce their immunogenicity for other members of the same species.