A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Th1-type cytokine profile and to produce interleukin 12

Leishmania braziliensis causes cutaneous and mucosal leishmaniasis in humans. Most patients with cutaneous leishmaniasis heal spontaneously and may therefore have developed protective immunity. There appears to be a mixed cytokine profile associated with active cutaneous or mucosal disease, and a dominant T helper (Th)1-type response associated with healing. Leishmanial antigens that elicit these potent proliferative and cytokine responses from peripheral blood mononuclear cells (PBMC) are now being identified. Herein, we report on the cloning and expression of a L. braziliensis gene homologous to the eukaryotic ribosomal protein eIF4A (LeIF) and patient PBMC responses to rLeIF. Patients with mucosal and self-healing cutaneous disease had significantly higher proliferative responses than those with cutaneous lesions. Whereas the parasite lysate stimulated patient PBMC to produce a mixed Th1/Th2-type cytokine profile, LeIF stimulated the production of interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor alpha but not IL-4 or IL-10. Recombinant LeIF (rLeIF) downregulated both IL-10 mRNA in the "resting" PBMC of leishmaniasis patients and LPS-induced IL-10 production by patient PBMC. rLeIF also stimulated the production of IL-12 in cultured PBMC from both patients and uninfected individuals. The production of IFN-gamma by patient PBMC stimulated with either rLeIF or parasite lysate was IL-12 dependent, whereas anti-IFN-gamma monoclonal antibody only partially blocked the LeIF-induced production of IL-12. In vitro production of both IFN-gamma and IL-12 was abrogated by exogenous human recombinant IL-10. Therefore, we have identified a recombinant leishmanial antigen that elicits IL-12 production and Th1-type responses in patients as well as IL-12 production in normal human PBMC.     

T cell cytokine responses in persons with tuberculosis and human immunodeficiency virus infection.

Tuberculosis causes more extensive and life-threatening disease in patients with HIV infection than in immunocompetent persons. To investigate the hypothesis that these severe manifestations of tuberculosis may be due to alterations in cytokine production, we evaluated cytokine patterns in HIV-infected tuberculosis patients. Upon stimulation with Mycobacterium tuberculosis in vitro, PBMC from HIV-infected tuberculosis patients had reduced proliferative and type 1 responses, compared with HIV-seronegative tuberculosis patients. The reduction in proliferative responses was independent of the CD4 cell count, but the reduced type 1 response was a direct result of CD4 cell depletion. There was no enhancement of type 2 cytokine production in HIV-infected patients, although production of IL-10 was prominent in all tuberculosis patients. In HIV-infected tuberculosis patients, M. tuberculosis-induced proliferative responses were significantly enhanced by neutralizing antibodies to IL-10 but not by antibodies to IL-4 or by recombinant IL-12. The M. tuberculosis-induced type 1 response was augmented both by antibodies to IL-10 and by recombinant IL-12. Tuberculosis in the context of HIV infection is characterized by diminished type 1 responses, probably induced by immunosuppressive cytokines produced by macrophages/monocytes, rather than by type 2 cells.     

A novel immunogen to modulate cytokine production and promote immune system reconstitution in HIV-AIDS

This 'proof of concept' study was implemented in anticipation of identifying and testing a novel antigen of human origin as a potential immunogen in a paradigm that emphasizes immunomodulation and immune system reconstitution as requisites to the development of an effective human immunodeficiency virus (HIV)-acquired immune deficiency syndrome vaccine. Fifteen HIV-infected, highly active antiretroviral therapy (HAART) naive, otherwise healthy male seropositive patients were stratified by [CD4+] into 3 groups of 5 patients: group 1 >500/mm; group 2 > 250/mm but <500/mm; and group 3 < 250/mm. Five healthy male subjects were used as controls. Replicate peripheral blood mononuclear cell (PBMC) [H]thymidine uptake phytohemaglutinin-stimulated proliferation studies, and serum cytokine assays were carried out in the presence or absence of Kveim antigen at dilutions ranging from 0.001 to 100 μg/mL. Serum cytokines [interleukin-2 (IL-2), IL-4, IL-6, interferon gamma, and tumor necrosis factor alpha] were assayed using standardized methodology. Nonparametric statistical analyses and linear regression analysis were used to test for statistical significance and strength of associations. PBMCs harvested from HIV-infected patients and incubated, ex vivo, demonstrated reproducible, antigen concentration-dependent changes in cytokine production over a range of antigen concentrations (0.001-100 μg/mL) in contrast to antigen-naive PBMCs and controls. Significant correlations were demonstrated between antigen concentration and the amount of cytokines secreted. The magnitude of the cytokine response and the patterns of cytokine secretion were HIV group-specific and could be used to identify and distinguish between the 3 groups of HIV-infected subjects. A shift toward the production of type 1-like (Th1) cytokines characteristically seen in systemic sarcoidosis and associated with effective HAART was seen when patterns of cytokine secretion were compared between antigen exposed and antigen-naive PBMCs. PBMCs harvested from seropositive HIV-infected patients and exposed to the Kveim antigen have the following properties: (1) They demonstrate proliferation and exhibit an antigen concentration-dependent secretion of cytokines. The magnitude of the cytokine response can be used to identify and distinguish between groups of seropositive patients stratified by [CD4+]. (2) These PBMCs secrete cytokines in patterns suggestive of a shift to a type 1-like (Th1) response characteristic of HAART and sarcoidosis as opposed to the type 2-like (Th2) cytokine profile characteristic of HIV-acquired immune deficiency syndrome.     

Priming for high interferon-gamma production induced by interleukin-12 in both CD4+ and CD8+ T cell clones from HIV-infected patients.

HIV-infected patients are defective in their ability to produce interleukin (IL)-12 in vitro in response to pathogenic bacteria and parasites. IL-12 enhances the patient's depressed natural killer cell cytotoxic activity, peripheral blood lymphocyte production of interferon-gamma (IFN-gamma), and proliferative T cell response in vitro to recall antigens, HIV antigens, alloantigens, and mitogens. However, these effects represent short-lived responses and imply the need for chronic IL-12 therapeutic administration in the clinical setting. To identify any long-term effects of IL-12 on T cell differentiation toward Th1 cells, peripheral blood T cells from 10 HIV-infected patients at different stages of disease were cloned by limiting dilution in the presence or absence of IL-12 and tested for cytokine production in response to stimulation with anti-CD3 antibodies and phorbol diesters IL-12 present during the first 2 wk of clonal expansion determined a stable severalfold enhancement on the ability of both CD4+ and CD8+ clones to produce IFN-gamma. Because priming for high IFN-gamma production is probably the most important mechanism by which IL-12 induces generation of efficient T helper type 1 (Th1) cells, these results suggest the possibility that IL-12 treatment in vivo of HIV-infected patients may stimulate a protective Th1 response against opportunistic pathogens and possibly HIV itself.     

IL-1 beta, IL-2, IL-6 and TNF-alpha production by peripheral blood mononuclear cells from patients with Parkinson's disease.

The capacity of peripheral blood mononuclear cells (PBMC) from patients with treated Parkinson's disease (PD) to produce interleukin (IL) IL-1 beta IL-2, IL-6, tumor necrosis factor (TNF)-alpha and the proliferative response to mitogens, was compared with that from cells from healthy subjects. The production of IL-2 and the mitogen response were significantly lower in PD patients, whereas the secretion of IL-1 beta, IL-6 and TNF-alpha were significantly enhanced. To evaluate the role of levodopa in creating immunological alterations, PBMC of patients and controls were incubated with concentrations of the drug extrapolated from those used in clinical practice. Levodopa caused an inhibition of mitogen-induced proliferation, stimulation of IL-6 and TNF-alpha production, whereas the secretion of IL-1 beta and IL-2 was not affected. The results of the study provide a further support for the interrelationship between the central nervous and immune system. In addition, the data indicate that the immunological alterations found in PD may be partially attributed to levodopa administration.     

Activation of antigen-induced lymphocyte proliferation by interleukin-15 without the mitogenic effect of interleukin-2 that may induce human immunodeficiency virus-1 expression.

The newly identified cytokine, IL-15 enhanced antigen-induced proliferation of PBMC obtained from HIV-1-seropositive subjects. When compared to IL-2 which enhanced both spontaneous and antigen-induced lymphocyte proliferative responses, IL-15 rarely increased spontaneous lymphocyte proliferation. Additionally, in cultures of lymphocytes obtained from 15 HIV-1-infected patients with < 300 circulating CD4- lymphocytes/microliter IL-15 induced significant HIV-1 expression (46, 21, and 71 pg/ml) in only 3 of 15 experiments and IL-2 induced significant HIV-1 expression (range 16- > 5000 pg/ml) in 11 of 15 experiments (P < 0.01, Fischer's exact test). Simultaneous assays of cytokine-induced spontaneous lymphocyte proliferation and HIV-1 expression revealed similar dose-response relationships for induction of HIV-1 and lymphocyte proliferation by IL-2. Thus, IL-15 helps to correct the impaired proliferative response of CD4+ lymphocytes from HIV-1-infected persons without the mitogenic effect of IL-2 that also may induce HIV-1 expression.     

Antigen specific suppressor T cells from chronic hepatitis B virus (HBV) carriers inhibit the responsiveness to HBsAg of allogeneic high-responder lymphocytes

Immunoregulatory mechanisms in chronic HBsAg carriers have been investigated through the study of in vitro proliferative responses to HBsAg by allogeneic coculture experiments between T lymphocytes from HBsAg + chronic active hepatitis (CAH) patients (HBsAg no responder) and PBMC from subjects boosted with anti-hepatitis B vaccine (high responder). When high-responder PBMC have been challenged with the hepatitis B surface antigen (HBsAg) in the presence of HBsAg no-responder T lymphocytes, HBsAg no-responder T lymphocytes caused an antigen specific, dose-dependent, suppression of the responsiveness of high-responder PBMC. On the other hand, T cells from patients with autoimmune CAH did not exert any suppressor effect in our system. The suppressor T lymphocytes were mitomycin C resistant and were positive for OKT8, but were negative for OKT4. When T8 + cells were depleted from HBsAg no-responder PBMC, the in vitro immunoproliferative response to HBsAg in chronically HBV infected patients was markedly improved. Out data clearly demonstrate the existence of T8 + suppressor T lymphocytes that can control low responsiveness to HBsAg in chronic HBV patients.     

Chronic exposure to tumor necrosis factor (TNF) in vitro impairs the activation of T cells through the T cell receptor/CD3 complex; reversal in vivo by anti-TNF antibodies in patients with rheumatoid arthritis.

Experiments were designed to test the hypothesis that chronic exposure to tumor necrosis factor alpha (TNF) alters the function of activated T lymphocytes. Pretreatment of tetanus toxoid-specific T cell clones with TNF for up to 16 d impaired rechallenge proliferative responses to antigen in a dose- and time-dependent fashion. IL-2 and PHA responses were preserved. Prolonged treatment with TNF impaired production of IL-2, IL-10, IFN gamma, TNF, and lymphotoxin (LT) following stimulation with immobilized OKT3, and resulted in suboptimal expression of the IL-2R alpha chain (Tac) but not CD3, CD4, or HLA-DR antigens, when compared to untreated control cells. By contrast, pretreatment of T cells for prolonged periods in vitro with neutralizing anti-TNF monoclonal antibodies (mAb) enhanced proliferative responses, increased lymphokine production, and upregulated Tac expression following stimulation with OKT3. To determine whether TNF exerts immunosuppressive effects on T cells in vivo, we studied cell-mediated immunity in patients with active rheumatoid arthritis (RA), before and after treatment with a chimeric anti-TNF mAb. Treatment with anti-TNF restored the diminished proliferative responses of PBMC to mitogens and recall antigens towards normal in all patients tested. These data demonstrate that persistent expression of TNF in vitro and in vivo impairs cell-mediated immune responses.     

In vivo and vitro effects of interferon-alpha 2b on functional activity of T-lymphocytes from patients with rheumatoid arthritis

AIM: To investigate the effect of recombinant alpha 2b-interferon (r alpha 2b-IFN) on functional capacity of peripheral blood (PB) T cells in rheumatoid arthritis (RA) patients and the relationship between functional characteristics of T lymphocytes and the disease activity. MATERIALS AND METHODS: PB mononuclear cells (PBMC) were separated by Ficoll-Verografine++ gradient centrifugation from 24 healthy donors (HD) and 75 RA patients 19 of which were treated with r alpha 2b-IFN (realdiron, Biofa, Lithuania) in the dosage 1 million IU i.m. each other day for 20 days, 10 injections a course. Cell surface markers (CD3, CD4, CD8) and adhesion molecules (CD18, CD54, CD2) were analyzed using specific monoclonal antibodies (MoAbs) and flow cytometry on the PBMC, freshly isolated and treated for 72 hours with medium alone, PHA, r alpha 2b-IFN and their combination. The proliferative response of PBMC to MoAbs for CD3, PHA and r alpha 2b-IFN were assessed by 3H-thymidine incorporation. The percentage of spontaneous and inducing apoptosis was quantified by flow cytometry using propidium iodide staining. RESULTS: The expression of CD18 was lower on RA PB lymphocytes compared to HD PB lymphocytes (p < 0.05). After stimulation of PBMC in both RA patients and HD with PHA, percentages of CD2+, CD3+, CD4+, CD18+ cells significantly diminished (p < 0.05), whereas the percentages of CD54+ and CD18+ (p < 0.05) cells increased. We have found three types of RA PB lymphocytes response to complex factors in vitro: 1) the presence of the proliferative response to T-mitogens but not to r alpha 2b-IFN (56% of the patients); 2) the presence of the increased proliferative response to T-mitogens and r alpha 2b-IFN (17% of the patients); 3) the absence of the proliferative response to T-mitogens and r alpha 2b-IFN (27% of the patients). PBMC of HD demonstrate only the first type of the response. R2 alpha b-IFN demonstrated own mitogenic effect and increased mitogen-induced proliferation in PBMC cultures with a high proliferative response to T-mitogens. The levels of spontaneous and inducing apoptosis were increased in RA PB lymphocytes compared to HD. After stimulation with PHA, RA PB lymphocytes preferentially underwent apoptosis whereas cells of HD proliferated. High disease activity correlated positively with an increase of a proliferative response to mitogens and apoptosis and a decrease in the percentage of lymphocytes, expressed adhesion molecules. The treatment with r alpha 2b-IFN induces changes in T-cell response to mitogens similarly to those after incubation with r alpha 2b-IFN in vitro before treatment. CONCLUSION: Functional capacity of RA PB lymphocytes relates to the disease activity. Inhibitory or stimulatory effects of r alpha 2b-IFN depend on functional activity of RA lymphocytes. Using the test with alpha 2b-IFN incubation, we may predict changes of apoptosis and proliferation levels caused by different agents in RA lymphocytes after treatment with r alpha 2b-IFN.     

Role of interleukin-18 in peripheral blood mononuclear cells infected with human herpes virus type 6

OBJECTIVE: Interleukin 18 (IL-18) production represents a critical step in the polarization of the Th1 immune response. Human herpes virus type 6 (HHV-6) possesses a peculiar tropism for immunocompetent cells. To understand the relationships among immunocompetent cells, HHV-6 and cytokines, the role of IL-18 during infection of peripheral blood mononuclear cells (PBMC) with HHV-6 was evaluated. METHODS: PBMC were obtained from healthy HHV-6-seronegative donors, after centrifugation of heparinized venous blood over a Ficoll-Hypaque gradient. Supernatants from PBMC were analyzed for the presence of cytokines. To study the effects of exogenous recombinant human (rh) IL-18 on HHV-6 replication, the number of cells expressing viral antigens and the amount of extracellular virus were analysed. RESULTS: No basal production of IL-18 was found in supernatants of unstimulated PBMC. Appreciable amounts of the cytokine were produced by lipopolysaccharide (LPS)-stimulated PBMC. HHV-6 infection of LPS-treated PBMC downregulated IL-18 production. It was found that the addition of rhIL-18 to HHV-6-infected PBMC downregulated the percentage of antigen-positive cells and the release of extracellular virus. CONCLUSION: Impairment of IL-18 release, which is involved in the induction of antiviral cytokines, such as interferon-gamma, could represent a strategy of the virus to evade the immune response of the host.     

Limiting dilution analysis of cytotoxic T lymphocytes to human immunodeficiency virus gag antigens in infected persons: in vitro quantitation of effector cell populations with p17 and p24 specificities

The presence of cytotoxic T lymphocytes (CTL) to the gag antigens of human immunodeficiency virus (HIV) has been described in infected populations. We found that the majority of this immune response as measured in bulk CTL assays of unstimulated peripheral blood mononuclear cells (PBMC) is directed against the p24 component of the p55 gag precursor protein. Using limiting dilution analysis of this effector cell population we confirm that the majority of activated gag-specific CTL circulating in the PBMC of infected hemophilic patients are directed at p24 determinants and are present at frequencies of 1/36,000 to 1/86,000 lymphocytes. By performing in vitro stimulation after limiting dilution, the precursor population of gag-specific CTL are characterized and quantitated. HIV gag-specific CTL precursors are identified at frequencies of 1/1700 to 1/17,000 lymphocytes and are made up of cells with both p17 and p24 specificities. No HIV gag-specific CTL precursor cells are identified in the PBMC of HIV-uninfected individuals. These studies demonstrate that CTL directed at both p17 and p24 determinants make up the cellular immune repertoire in HIV-infected individuals but that only the p24-specific CTL are routinely found in an activated state in the circulation.     

Epitope-specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL-15 in vitro.

Bee venom phospholipase A2 (PLA) is the major allergen in bee sting allergy. It displays three peptide and a glycopeptide T cell epitopes, which are recognized by both allergic and non-allergic bee venom sensitized subjects. In this study PLA- and PLA epitope-specific T cell and cytokine responses in PBMC of bee sting allergic patients were investigated before and after 2 mo of rush immunotherapy with whole bee venom. After successful immunotherapy, PLA and T cell epitope peptide-specific T cell proliferation was suppressed. In addition the PLA- and peptide-induced secretion of type 2 (IL-4, IL-5, and IL-13), as well as type 1 (IL-2 and IFN-gamma) cytokines were abolished, whereas tetanus toxoid-induced cytokine production and proliferation remained unchanged. By culturing PBMC with Ag in the presence of IL-2 or IL-15 the specifically tolerized T cell response could be restored with respect to specific proliferation and secretion of the type 1 T cell cytokines, IL-2 and IFN-gamma. In contrast, IL-4, IL-5, and IL-13 remained suppressed. Treatment of tolerized T cells with IL-4 only partially restored proliferation and induced formation of distinct type 2 cytokine pattern. In spite of the allergen-specific tolerance in T cells, in vitro produced anti-PLA IgE and IgG4 Ab and their corresponding serum levels slightly increased during immunotherapy, while the PLA-specific IgE/IgG4 ratio changed in favor of IgG4. These findings indicate that bee venom immunotherapy induces a state of peripheral tolerance in allergen-specific T cells, but not in specific B cells. The state of T cell tolerance and cytokine pattern can be in vitro modulated by the cytokines IL-2, IL-4, and IL-15, suggesting the importance of microenvironmental cytokines leading to success or failure in immunotherapy.     

Enhanced production of neutrophil-activating peptide-1/interleukin-8 in rheumatoid arthritis.

Production of the neutrophil-activating peptide (NAP)-1/IL-8 by mononuclear phagocytes from patients with RA and from control subjects was studied under various conditions. Mononuclear cells from bone marrow (BMMC), PBMC, and synovial fluid (SFMC) were cultured for up to 48 h in the absence or presence of Escherichia coli LPS, different interleukins, interferon-gamma, zymosan, or immune complexes, and the neutrophil-stimulating activity released into the culture medium was determined. As shown by neutralization with an antiserum raised against human recombinant NAP-1/IL-8, over 90% of this activity could be attributed to NAP-1/IL-8. In unstimulated mononuclear cells from control individuals and BMMC from RA patients, the production of NAP-1/IL-8 was very low and was enhanced moderately by stimulation with LPS. By contrast, the spontaneous production of NAP-1/IL-8 was 3- to 10-fold higher in PBMC and even much higher in SFMC from RA patients. In all instances, the yield of NAP-1/IL-8 could be enhanced by stimulation in culture. In addition to LPS, rheumatoid factor-containing immune complexes, zymosan, and IL-1 were highly effective in inducing NAP-1/IL-8 production, while IL-3, GM-CSF, tumor necrosis factor (TNF), and IL-2 were somewhat less potent. An inhibitory effect was obtained with IFN-gamma, which significantly decreased the spontaneous NAP-1/IL-8 release from SFMC and the IL-1- and LPS-induced NAP-1/IL-8 from RA and control PBMC. Inhibition was also observed with glucocorticoids. The production of NAP-1/IL-8 was markedly reduced by dexamethasone in phagocytosis-stimulated PBMC, and almost totally inhibited in SFMC obtained from joints after intraarticular administration of betamethasone. By contrast, the cyclooxygenase inhibitor, indomethacin, tended to increase the NAP-1/IL-8 yield from PBMC in culture.     

Pro-inflammatory cytokine release and mediation of the acute phase protein response in fulminant hepatic failure

Objective: To study the relationship between interleukin-6 (IL-6), tumour necrosis factor (TNF) and the acute phase protein C-reactive protein (CRP) in patients with fulminant hepatic failure (FHF) and to investigate the potential of peripheral blood mononuclear cells (PBMC) isolated from these patients to stimulate CRP production by isolated human hepatocytes in vitro. Setting: Patients with FHF were studied at the time of their admission to the intensive care unit. Study design: Serum TNF and IL-6 were measured in 12 patients with FHF. PBMC from 6 of these patients were then cultured in the presence and absence of lipopolysaccharides (LPS). TNF and IL-6 in serum and supernatants were measured by ELISA. PBMC supernatants were added to isolated human hepatocytes and CRP production was measured. Results: Serum IL-6 (348 ± 172 pg/ml) and TNF (118.5 ± 15.5 pg/ml) were elevated compared with healthy controls (not detected) and these observations were matched by elevated serum CRP in patients with FHF (38.9 ± 7 mg/l). Both the production of IL-6 and TNF by PBMC isolated from patients with FHF and the potential of supernatants from these cells to stimulate CRP production by hepatocytes in vitro was significantly reduced compared with controls. Conclusions: Despite the observation that patients with FHF have an elevated hepatic acute phase response, PBMC from patients with FHF have reduced potential to produce IL-6 and TNF and elicit an acute phase response in vitro by the time of patient admission to the intensive care unit. One explanation for this observation is early activation and exhaustion of PBMC in vivo. Received: 18 August 1997 Accepted: 13 January 1998     

Interleukin 10 production correlates with pathology in human Leishmania donovani infections.

We have found that an important Th2 cytokine, IL-10, is produced by tissues from patients acutely infected with Leishmania donovani. In all individuals tested, IL-10 mRNA production was increased in lymph nodes taken during acute disease over that observed in postacute samples. In contrast, both pre- and posttreatment lymph nodes had readily detected mRNA for IFN-gamma and IL-2. A down-regulating effect of IL-10 on leishmania-induced proliferative responses was demonstrated when Hu rIL-10 was added to cultures of PBMC from clinically cured individuals. PBMC from individuals with acute visceral leishmaniasis responded to stimulation with leishmania lysate by producing IL-10 mRNA. Simultaneously cultured PBMC collected from the same patients after successful chemotherapy produced no detectable IL-10 mRNA after leishmania antigen stimulation. Neutralizing anti-IL-10 mAb added to PBMC from patients with acute visceral leishmaniasis markedly increased the proliferative response to leishmania lysate. Finally, we observed mRNA for IL-10 and IFN-gamma concurrently in a lesion from a patient with post-kala-azar dermal leishmaniasis (PKDL). These results indicate the production of IL-10 during L. donovani infection, and suggest a role for this cytokine in the regulation of immune responsiveness during visceral leishmaniasis.     

Endothelial cells augment T cell interleukin 2 production by a contact-dependent mechanism involving CD2/LFA-3 interaction.

We have demonstrated that endothelial cells (EC) augment IL-2 production by PHA-stimulated PBMC or purified CD4+ T cells and that the increase is apparent both in the amount of soluble IL-2 secreted and in the level of specific mRNA detectable by Northern blot hybridization. The ability of EC to affect levels of IL-2 cannot be reproduced by soluble factors, including the cytokines IL-1, IL-6, IFN-gamma, or TNF, conditioned medium from resting EC or IL-1, IFN-gamma- or TNF-treated EC, or from resting PBMC + EC cultures. Separation of the EC and PBMC by a Transwell membrane demonstrated that cell contact was required for augmentation of IL-2 synthesis and that this effect was unlikely to be mediated by a short-lived soluble signal. The cell-cell interaction required the ligand pair CD2/LFA-3, since augmentation could be inhibited by antibodies to these structures. Antibodies to ICAM-1, LFA-1, CD4, and MHC class II were without effect. A contact-dependent pathway involving CD2/LFA-3 interactions also may be used by EC to augment IL-2 production from T cells stimulated more specifically through the TCR/CD3 complex with antibody OKT3. This pathway provides a proliferative advantage to T cells stimulated with OKT3 in the presence of EC and may also be involved in the proliferative response of resting T cells to allogeneic class II MHC-expressing EC. We propose that EC augmentation of T cell IL-2 synthesis may be critical in the ability of EC to elicit primary T cell antigen responses and may have consequences for the development of localized cell-mediated immune reactions.     

CpG ODN诱导人PBMC增殖的体外实验研究

目的研究CpGODN在体外刺激人PBMC的增殖情况。方法分别以肿瘤抗原（Ag）、白细胞介素-2（IL-2）和CpGODN单独或联合刺激正常人和肿瘤患者PBMC,并对其增殖活性进行测定。结果以CpGODN刺激人PBMC组的细胞增殖活性明显高于对照组（P〈O．05）。结论CpGODN、IL-2与肿瘤抗原共同刺激能有效提高人PBMC的增殖活性。     

Ex vivo expansion of long-term culture initiating marrow cells by IL-10, SCF,and IL-3

BACKGROUND: Ex vivo expansion of progentior cells may shorten hematopoietic regeneration after myeloablative chemoradiotherapy, increase target cells for gene therapy, and improve purging of progenitor cell components. STUDY DESIGN AND METHODS: Marrow cells were incubated for 1 week in suspension culture with and without IL-10, IL-3, and SCF. As long-term culture initiating cells (LTC-ICs) represent early hematopoietic progenitors in vitro, these cells were quantified at initiation and after a 1-week culture period in a limiting dilution assays. Additionally, immunophenotyping of cells before and after culture was performed. RESULTS: In six experiments, marrow cells cultured for 1 week with IL-10, IL-3, and SCF showed a significant increase (almost doubling) in LTC-ICs as compared with marrow cells before expansion. Additionally, an increased proliferative capacity of LTC-ICs was achieved with a sevenfold increase of committed colony-forming cells and a 10-fold proliferation of high proliferative potential colony-forming cells. Immunophenotyping revealed a sevenfold increase of CD34+ CD45 RA- cells in IL-10-, IL-3-, SCF-stimulated suspension cultures. In unstimulated cultures, no LTC-ICs were maintained after 1 week. CONCLUSION: Expansion of LTC-ICs by IL-10, IL-3, and SCF has not been shown so far. This in vitro model allows expansion of LTC-IC if compared with the input of progenitor cells without extensive progenitor cell manipulation. This should be an attractive model for in vitro purging, gene transfer, or expansion of progenitor cells to allow rapid engraftment after myeloablative chemotherapy.     

Role of interleukin 10 in specific immunotherapy.

The induction of allergen-specific anergy in peripheral T cells represents a key step in specific immunotherapy (SIT). Here we demonstrate that the anergic state results from increased IL-10 production. In bee venom (BV)-SIT the specific proliferative and cytokine responses against the main allergen, the phospholipase A2 (PLA), and T cell epitope-containing PLA peptides were significantly suppressed after 7 d of treatment. Simultaneously, the production of IL-10 increased during BV-SIT. After 28 d of BV-SIT the anergic state was established. Intracytoplasmic cytokine staining of PBMC combined with surface marker detection revealed that IL-10 was produced initially by activated CD4(+)CD25(+), allergen-specific T cells, and followed by B cells and monocytes. Neutralization of IL-10 in PBMC fully reconstituted the specific proliferative and cytokine responses. A similar state of IL-10-associated T cell anergy, as induced in BV-SIT, was found in hyperimmune individuals who recently had received multiple bee stings. The addition of IL-10 to soluble CD40 ligand IL-4-stimulated PBMC or purified B cells inhibited the PLA-specific and total IgE and enhanced the IgG4 formation. Accordingly, increased IL-10 production by SIT causes specific anergy in peripheral T cells, and regulates specific IgE and IgG4 production toward normal IgG4-related immunity.