Primary Stimulation of CD4+ Cells in the Presence of IL-4 or IFN-γ Alters the Frequencies of Cytokine-Producing Cells at Restimulation

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8⁺ cells and of exogenousiy added cytokines (interleukin (IL)-2, IL-4 and interferon (IFN)-γ) on the cylokine production in splenic CD4⁺ T cells. IL-2, IL-4, IL-5 and IFN-γ production in CD4⁺ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8⁺ cells. On day 5 the cells were restimulated with mitogen alone and analysed to evaluate the short-term development of cytokine-producing cells in such cultures. Preactivation in the presence of either exogenous IL-4 or IFN-γ led to an increased production of IL-4 and IFN-γ respectively at restimtilation, and the effects of both IL-4 and IFN-γ were augmented by IL-2. After preactivation in the presence of IL-2 and IL-4, every third CD4⁺ cell could be induced to produce IL-4. Exogenous IL-4 or IFN-γ further decreased each other's production. Depletion of CD8⁺ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8⁺ cells will influence lymphokine production in CD4⁺ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4⁺ and CD8⁺ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines.     

Rheumatoid Inflammatory T-Cell Clones express mostly Th1 but also Th2 and Mixed (Th0-Like) Cytokine Patterns

This study was performed in order to characterize whether T cells from rheumatoid synovial inflammation belong to the Th1- or Th2-like functional subsets. Cytokine production was studied in 26 CD4⁺αβ⁺ and 2 CD8⁺αβ T-cell clones from the synovial fluid, the synovial membrane and peripheral blood of 5 patients. Fifteen of the CD4⁺ clones were raised against various mycobacterial antigens and 11 CD4⁺ clones and 2 CD8⁺ clones were raised unspecifically using PHA and/or IL-2. The specificities of these clones are not known. In the mycobacterial antigen-specific group, all CD4⁺'αβ T-cell clones produced IFN-γ at high levels, while the production of IL-4 was generally absent or low (< 1 ng/ml), consistent with a Thl-like profile. Some of these clones, however, also produced various amounts of IL-10 which has been regarded as a Th2 product but can be produced also in lower amounts by Thi cells. One HSP-65-specific clone produced levels of IL-4 and IL-10 in the same order as that of IFN-γ, thus appearing to be Th0-like. Among the 11 unspecific CD4⁺ clones, 7 showed a Thl-like pattern but with lower levels of IFN-γ than the antigen-specific clones. However, three clones did not produce any IFN-γ activity but produced IL-4 and one of them also produced distinct amounts of IL-10, compatible with a Th2-like pattern. In addition, one of the clones also showed an almost equally strong IFN-γ and IL-4 production, thus most likely representing a Th0-like clone.     

Schistosoma mansoni Egg-Primed ThO and Th2 Cells: Failure to Down-regulate IFN-γ Production Following In Vitro Culture

Schistosoma mansoni eggs induce a rapid and pronounced T_h response which, based on cytokine secretion patterns, at day 3 post priming is T_h0)-like and at day 10 is T_h2-like. To establish whether or not the day-3 cells have been programmed in vivo to develop into T_h2 cells, they were cultured for 7 days to become in vitro equivalents of day-10 in vivo cells. Following this culture period, the population was approximately 75% CD4⁺, 22% CD8⁺, 6% B220⁺ and capable of producing IL-2, IFN-γ, IL-4, -5 and -10 upon stimulation. This T_h0-like status was confirmed by the observations that in response to mitogen IL-4 and IFN-γ production are both CD4⁺ -cell dependent and that IFN-γ and IL-4 are produced concomitantly by single cells. These data suggest that T^hO cells persist in vivo , but are incapable of secreting IFN-γ at day 10 due loan inhibitory factor which does not develop or is labile in vitro . This concept is supported by ihc surprising observation that day-10 LN cells, which are T_h2-like immediately ex-vivo , rapidly gain the ability to secrete IFN-γ following a short period of culture.     

Lack of Polarized Type 1 or Type 2 Cytokine Profile in Asymptomatic HIV-1-Infected Patients During a Two-Year Bimonthly Follow-Up

The production of type 1 (interferon or IFN-γ) and type 2 (interleukin or IL-4 and IL-10) cytokines by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic human immunodeficiency virus-seropositive (HIV⁺) patients untreated with any antiviral, antibacterial or antimycotic drugs, and from healthy individuals, was evaluated by quantitative ELISA. Patients who were HIV⁺ were characterized by the absence of abnormal cytokine production. The level of each cytokine differed among individuals in the same group with intersubject variations greater for HIV⁺ patients than for healthy individuals. The longitudinal evaluation of IFN-γ, IL-4 and IL-10 production showed intrasubject variations which were particularly marked in HIV⁺ patients. Accordingly, HIV⁺ patients and, to a lesser extent, healthy individuals were characterized by a wide spectrum of possible profiles, which were confined to type 0 phenotype. In HIV⁺ patients no correlation was found between each cytokine level and the number of CD4⁺ T cells, not even in those with a falling CD4⁺ T-cell count and clinical symptoms.     

Immunophenotypic and Functional Characteristics of Haemopoietic Cells from Human Cord Blood

Cord blood (CB) as a new source for bone marrow transplantation represents advantageous features concerning stem cell and leucocyte compartments and function. We attempted to get more information about the phenotypes and function of CB cells by investigating their cell surface markers and also the production of IL-2, IFN-γ and IL-6 by mitogen and alloantigen stimulation. The CB cells were characterized by a low proportion of CD3⁺ T cells, CD4⁺ T subpopulation, activated T cells and CD3⁺ CD16/CD56⁺ cytotoxic cells, suggesting reduced graft versus host potential. The significant increase of CD19/CD3 double positive cells and decrease of CD19/HLA-DR double positive mature B cells reflect that immature B cells exist in CB. In the functional studies, a 27- and 5-fold reduction was observed in the production of IFN-γ by CB cells stimulated with PHA and allogeneic cells, respectively. The production of IL-2 in PHA-stimulated CB cells also showed a 50% determination. Decrease in the production of these cytokines by CB cells is supported by the decline of the proportion of CD3⁺ T cells. However, an increase was observed in the production of IL-6 by CB cells stimulated with allogeneic cells as compared with the controls. These results suggest a difference in the functional activity of the T helper cell subsets between the CB and peripheral blood and/or differences in the functional maturity of T helper cell subsets and B cells in these compartments.     

Interleukin-6 is essential for Staphylococcal exotoxin B-induced T regulatory cell insufficiency in nasal polyps

Background The pathogenesis of nasal polyps is still unclear. There is increasing evidence indicating that Staphylococcal aureus (S. aureus) is associated with the formation of nasal polyps, but the mechanism has not been well documented to date.
Methods We stimulated cultured nasal polyps and turbinate tissues with Staphylococcal exotoxin B (SEB), detected the expression of pro-inflammatory cytokines (IL-2, IL-6, and IL-8) and T cell cytokines (IFN-γ, IL-4, IL-5, IL-10, and IL-17) in the supernatants, and evaluated mRNA expression (T-bet, GATA-3, Foxp3, and RORγt) and frequencies of CD4⁺CD25⁺ T regulatory cells (Tregs) in nasal tissues. We also evaluated the effects of blocking IL-6 with monoclonal antibodies to T cell profiles in cultured nasal tissues stimulated by SEB.
Results Levels of IL-6, IFN-γ and IL-4 increased significantly in SEB-stimulated nasal polyps. Meanwhile, mRNA expressions of T-bet and GATA-3 were significantly up-regulated, while Foxp3 was inhibited and the frequencies of CD4⁺CD25⁺ Tregs were decreased after SEB stimulation. After blocking IL-6, the levels of IL-10 and Foxp3 mRNA, as well as the frequencies of CD4⁺CD25⁺ Tregs, were significantly increased, while IFN-γ and IL-4 production and the mRNA expression of T-bet and GATA-3 were significantly inhibited.
Conclusions SEB is able to modulate pro-inflammatory factors, T-helper type 1/Th2 profiles and suppress Treg activity in cultured nasal polyps, which were rescued by blocking IL-6 activity. Therefore, IL-6 is essential for SEB-induced Treg insufficiency in nasal polyps.     

Suppression of allergic inflammation by allergen-DNA-modified dendritic cells depends on the induction of Foxp3+ Regulatory T cells

CD4⁺CD25⁺Foxp3⁺Regulatory T cells (Tregs) play important roles in regulating allergic inflammation. To analyse if allergen-DNA-modified dendritic cells (DC) can suppress allergic responses and what roles Treg cells play in DC-based allergen-specific immunotherapy. Immature DC were transfected with retrovirus encoding Der p2 DNA, and administered to mice that sensitized and challenged with Der p2 protein. After Treg cells were depleted with anti-CD25 mAb, mice were re-challenged to observe the airway inflammation, and Treg cells in spleen CD4⁺ T cells. And responses of spleen CD4⁺ T cells to Der p2 were determined. Co-culture of naïve CD4⁺ T cells with allergen-modified DC induced Foxp3+ Tregs. Sensitized and challenged mice developed allergic airway inflammation and Th2 responses, and decreased Foxp3⁺ Tregs. Treatment with allergen-modified-DC suppressed airway inflammation and Th2 responses, and increased IL-10 and IFN-γ production and Foxp3⁺ Tregs significantly; and eliminated the responses of CD4⁺ T cells to allergen. Administration of anit-CD25 mAb eliminated all the effects of modified-DC except for the increasing of IFN-γ. Allergen-modified DC can induce immune tolerance to allergens and reverse the established Th2 responses induced by allergen, with dependence on the induction of Foxp3⁺ Tregs.     

Suppression of Experimental Autoimmune Myasthenia Gravis After CD8 Depletion is Associated with Decreased IFN-γ and IL-4

CDS⁺ T cells can perform both Th1 - and Th2-like functions by producing cytokines such as interferonγ (IFN-γ) and interleukin-4 (IL-4), as well as the immune response down-regulating transforming growth factor-β (TGF-β), which are all involved in the development of experimental autoimmune myasthenia gravis (EAMG), a model for human MG. We have reported that depletion of CD8⁺ T cells results in the suppression of EAMG accompanied by the down-regulation of AChR-specific B cell responses and AChR-reactive IFN-γ secreting Th1-like cells. To identify the involvement of IFN-γ, IL-4 and TGF-β in the development of EAMG after CD8⁺ T cell depletion, the expression of mRNA for these cytokines was studied in mononuclear cells from popliteal, inguinal and mesenteric lymph nodes, spleen and thymus by adopting in situ hybridization with complementary DNA oligonucleotide probes. Depletion of CD8⁺ T cells resulted in decreased levels of IFN-7 and IL-4 mRNA expressing cells in different lymphoid organs except thymus, but no change in the numbers of TGF-β mRNA expressing cells. The results imply that the suppression of EAMG after depletion of CD8⁺ T cells is caused by decreasing the effector factors but not by increasing the suppressor factor(s).     

Peripheral Immune Response in Pulmonary Tuberculosis

Cellular immune response and delayed-type hypersensitivity reactions are considered to play a major role in the immunopathogenesis of pulmonary tuberculosis (PTB). But the exact mechanism is still to be clarified. Th1 cells are mainly involved in cellular immune responses in PTB and provide a normal healing process with minimal or no sequela whereas Th2 cell and CD8⁺ T lymphocyte responses may lead to more severe type of disease. In this study, we investigated the peripheral blood immune responses in PTB. The study group consisted of acid fast positive young male soldiers with PTB and a negative HIV serology. The control group included healthy young volunteer male soldiers without a history of PTB. Intracytoplasmic cytokine content of CD8⁺ T cells and lymphocytes, including IL-2, IL-4, IL-5, IL-10 and IFN-γ were determined by flow cytometry, and IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-α serum levels were measured by cytometric bead array (CBA). No difference was observed between the percentages of T, B, NK cells and HLA-DR expression in both groups, however, the number of CD3⁺HLA-DR⁺ activated T cell percentages was higher in PTB group as compared to healthy subjects. IL-2, IL-4, IL-5, IL-10 contents of lymphocytes and IFN-γ⁺CD8⁺ T cells were found to be significantly lower in PTB patients when compared with healthy subjects, and in parallel, serum IL-2, IL-4, IL-5 and TNF-α levels were also significantly lower in PTB patients. In conclusion we suggest that, CD8⁺ T cells producing both Th1 and Th2 type cytokines, may play important role in the peripheral immune response to mycobacteria.     

Augmented Lung Adenocarcinoma Cytotoxicity by the Combination of a Genetically Modified Anti-Lewis Y Antibody and Antibodies to Complement Regulatory Proteins

Nine vervet monkeys ( Cercopithecus aethiops ) were infected intradermally with 8 × 10⁷ virulent L. donovani promastigotes. Four animals developed clinical visceral leishmaniasis and died over a period of 18 months. The remaining five animals have remained asymptomatic for a period of 3 years now. Attempts to isolate parasites from spleen and liver through biopsies were fruitless. Immunological respotises of these subclinically infected animals were examined. Enzyme-linked itnmunosorbent assay ( ELISA ) and western blot analyses demonstrated Leishmania specific antibodies in these animals, but the antibody titres were low. When proliferation of peripheral blood monocytes ( PBMC ) to Con-cunavalin A (Con A) of these animals was compared with control 'disease free animals' there were no significant differences iti response. However L. donovani antigen (fixed promastigotes) specific proliferation was demonstrated in the five subclinically infected animals. High and varying levels of interferon gamma (IFN-γ) were secreted in PBMC cultures from the five vervet monkeys when stimulated with either Con A or L. donovani antigens. In control animals, IFN-γ was only detected wheti PBMC were stimulated with Con A. Marked delayed-type hypersensitivity ( DTH ) responses were demonstrated in the five subclinically infected animals 48 h after injection with formalin fixed promastgotes.
It was concluded that the visceral Leishmania disease spectrum due to L. donovani observed in humans could be induced in vervet monkeys and that L. donovani asymptomatic/cryptic infected animals have competent humoral and cellular responses to homologous parasites.     

Suppression of Tumour-Specific Cytotoxic T-Cell Responses Against the Syngeneic BALB/c Plasmacytoma ADJ-PC-5 by Tumour-Induced CD8+ Regulatory T Cells Via IFN-γ

The mechanisms of tolerance induction by tumour cells during early stages of tumourigenesis were analysed in a murine model system using the highly immunogenic BALB/c plasmacytoma ADJ-PC-5. Early stages of tumourigenesis were simulated in syngeneic BALB/c mice by repeated intraperitoneal injections with subimmunogenic doses of X-irradiated ADJ-PC-5 tumour cells. This treatment causes a state of tumour-specific tolerance in a high percentage of mice, involving a population of CD8⁺ peritoneal T cells which are able to suppress a protective tumour-specific Tc response against this tumour. Using a primary mixed lymphocyte tumour cell culture (MLTC) as an in vitro system to study suppressive mechanisms of such regulatory T cells, the role of production or consumption of a number of cytokines was analysed. The data presented here demonstrate that inhibition of a protective Tc response against ADJ-PC-5 tumour cells is due to IFN-γ production by suppressive T cells from tolerized mice, but not to IL-2 consumption. In contrast to typical CD8⁺ Tc cells, ADJ-PC-5-specific CD8⁺ Tc cells do not produce IFN-γ and are furthermore suppressed by IFN-γ. Thus, tumour-induced suppressive T cells and tumour-specific Tc cells seem to represent functionally and phenotypically different subsets of CD8⁺ T cells, possibly pointing towards a differential activation of type-1 and type-2 CD8⁺ T cells depending on the dose of tumour cells.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus , bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4⁺ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25⁺ and CD69⁺ lymphocytes were higher. Seventy-four percent of the CD25⁺ PBMC but only 55% of the CD69⁺ cells were CD3⁺ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69⁺ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

Phenotypic and Functional Analysis of Activated B Cells of Autoimmune NZB × NZW F1 Mice

Polyclonal B-cell activation is the central theme in the production of autoantibodies and possible activation of autoreactive T cells in both human and murine lupus. The abnormal expansion of CD5⁺ B cells in murine lupus has been suggested, in particular, to be one of the most characteristic findings in these mice. Activated B cells can be separated from the B cells of resting stage by the difference in cell density. The aim of this study was to investigate the characteristics of different densities of the spleen cells separated by gradient density. Furthermore, the ability of anti-DNA antibody secretion in each percoll gradient fraction of B cells was also analysed. The results showed: a higher percentage of CD5⁺ B cells, which corresponded to the activated B-cell population, in percoll gradient 1 and 2 fractions; that splenic B cells of NZB/W F₁ mice had proliferative response to interleukin (IL)-4 or IL-5 but not to IL-10 or interferon-γ (IFN-γ); and that B cells isolated by percoll gradient produced anti-DNA antibody after stimulation with lipopolysaccharide (LPS) plus IL-5 and IFN-γ, but not IL-4 and IL-10. These data suggest that B cells at different stages of activation express differential characteristics and functions.     

Anti-GD 3 Antibodies are Potent Activators of Human γ/δ and α/β Positive T Cells

The ganglioside GD3 has a variety of biological functions. These include stimulatory effects is on proliferation, natural killer activity and cytokine production by freshly isolated peripheral T cells. In this study we have characterized anti-GD3 antibody (MoAb Z21) mediated effects on T cell clones. Our data indicate that α/β TCR CD4⁺ and CD8⁺ as well as γ/δ TCR positive T cells can be stimulated resulting in proliferation and cytokine production. This effect could be blocked by cyclosporin A and did not involve the LFA-3 or CD4 molecule. Apart from IFN-γ and IL-2 production by T helper I and T helper 0 cells we have observed production of IL-4 and IL-10 by T helper 2 cells indicating that the GD3 molecule is not a marker for a certain functional T cell subset. In contrast to anti-CD3 mediated activation, the responsiveness of T cells to stimulation via GD3 was dependent on the cell surface expression of the molecule and could be enhanced by costimulation via CD2, CD3, CD26 or CD28. In addition, anti-GD3 antibodies delivered a potent costimulatory signal for antigen-induced proliferation of CD4⁺ T lymphocytes. In summary, our experiments illuminate the mechanisms of anti-GD3 antibody induced T cell activation.     

Local T-Cell Proliferation and Differentiation in Inflammatory Myopathies

Our objective was to investigate the patterns of proliferation and differentiation of infiltrating cells in inflammatory myopathies. Immunohistochemical staining was performed on muscle biopsy specimens from 18 patients with inclusion body myositis, polymyositis and dermatomyositis using monoclonal and polyclonal antibodies.
An abundance of cells were TNF-α⁺ (4–8%), ICAM-1⁺ (7–65%). IFN-γ⁺ (3–6%), and Ki-67⁺ (4–8%). It was shown that 70% of the Ki-67⁺ cells were Ki-67⁺CD3⁺ cells. Very few mononuclear cells were IL-2R⁺. MHC-I expression was found on nearly all muscle fibres in all cases, while MHC-II expression was found on occasional muscle fibres in 1/3 of cases. Analysis of repeated biopsies from four IBM patients after prednisolone treatment showed no change in the proportions of TNF-α, ICAM-1, IFN-γ or Ki-67 positive cells. In inflammatory myopathies there is an intense proliferation and differentiation of inflammatory cells in situ , indicating a local stimulation of the inflammatory process.     

Contrary Roles of IL-4 and IL-12 on IL-10 Production and Proliferation of Human Tumour Reactive T Cells

The cytokine profile of tumour reactive T cells is likely to play a central role in their function. However, little is known about how cytokine patterns of tumour reactive T cells can be regulated. Here, the authors investigated the influence of exogenous regulatory cytokines in addition to interleukin-2 (IL-2) on cytokine patterns and the proliferation of T cells recognizing an autologous sarcoma cell line. In this system, IL-4 and IL-12 showed the most polarizing influences on tumour reactive T cells. Exogenous IL-4 induced a predominant production of IL-4 while decreasing the interferon-γ (IFN-γ) and IL-10 production by tumour reactive T cells. It also stimulated the growth of tumour reactive CD4⁺ T cell clones. In contrast, IL-12 substantially increased the production of IL-10 and IFN-γ. This was accompanied by a growth inhibition of tumour reactive T cells. The growth of CD4⁺ tumour reactive T cells was also suppressed by exogenous IL-10. This study shows that cytokine patterns and proliferation tumour reactive T cells can be significantly influenced by exogenous cytokines and confirms the hypothesis of a negative feedback loop of IL-12 by the induction of IL-10 in the context of human tumour reactive T cells.     

Ig-Isotype Patterns of Primary and Secondary B Cell Responses to Plasmodium Chabaudi Chabaudi Correlate with IFN-γ and IL-4 Cytokine Production and with CD45RB Expression by CD4+ Spleen Cells

In this work, the authors analysed T and B lymphocyte subsets and cytokine production in the spleen of BALB/c mice during polyclonal lymphocyte activation (primary infection) and parasite-specific response to Plasmodium chabaudi chabaudi (secondary infection). The secondary response was evaluated in fully immunoprotected animals, 60 days after a chloroquine-cured infection. The authors observed that in polyclonal lymphocyte activation antibody-secreting cells of all isotypes increased, with predominance of IgG2a and IgG3 classes. At that time, IFN-γ was largely produced, but IL-4/IL-5 were just slightly enhanced. In mice re-infected after 60 days, the Ig-isotype pattern was restricted to IgG1 and only IL–4/IL-5 were produced. In both responses, however, the levels of IL-2 were greatly reduced, while those of IL-10 were enhanced to similar levels. The different involvement of Th1 and Th2 cells in both responses was confirmed through analysis of CD45RB expression by CD4⁺ cells. The authors observed that CD45RB^high cells were the major CD4⁺ subpopulation in primary infected mice, while CD45RB^low cells predominated in 60 days re-infected animals. Moreover, the great majority of activated (large) CD4⁺ cells in the primary infection belonged to the CD45RB^high subset, while after re-infection most of the CD4⁺ large had a CD45RB^low phenotype.     

Delayed Elimination of the LCM Virus from Acid Sphingomyelinase-Deficient Mice due to Reduced Expansion of Virus-Specific CD8+ T Lymphocytes

The phagolysosomally localized acid sphingomyelinase (ASMase) activated by proinflammatory cytokines such as TNF and IFN-γ generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin D. These characteristics of ASMase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. We show here that ASMase^–/– mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (LCM) virus as rapidly as littermate wildtype mice. Investigation of the immune response revealed a reduced expansion of CD8⁺ T cells. The secretion of IFN-γ in response to contact with target cells as well as the cytolytic activity of virus-specific CD8⁺ T cells was severely impaired. Additionally, both phases of the LCM virus-specific DTH response, mediated by CD8⁺ and CD4⁺ T cells consecutively, were diminished in ASMase^–/– mice. However, the secondary memory response of virus-specific CTL was not altered, and the virus was effectively controlled for at least 3 months by ASMase^–/– mice. In conclusion, the results of this study suggest an involvement of the ASMase in the activation, expansion or maturation of virus-specific CD8⁺ T cells during the acute infection of mice with the LCM virus.     

CD8+ Cells are the Main Producers of IL10 and IFNγ after Superantigen Stimulation

Purified CD8⁺ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8⁺ cells would contribute to cytokine production in the presence of cytokine secreting CD4⁺ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4⁺ and CD8⁺ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8⁺ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4⁺ subset. When priming with SEB in vivo before subsequent restimulation in vitro , a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8⁺ cells and indicate that CD4⁺ and CD8⁺ T cells may, in a given situation, produce distinct cytokines.     

Probiotic Bacteria Induce Regulatory Cytokine Production via Dendritic Cells

Probiotic bacteria, e.g. Lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. We examined cytokine production and phenotypic change after in vitro stimulation of T cells from healthy volunteers using different probiotic strains.
Methods:  T cells were cultured from colonic biopsies from eight healthy volunteers (Agnholt and Kaltoft, Exp Clin Immunogenet 2001;18:213–25), and dendritic cells were matured from their peripheral blood mononuclear cells. T-cell cultures were stimulated with autologous bacterial sonicate or strains of Lactobacillus spp., with and without the addition of dendritic cells. Cytokine levels (TNF-α, IFN-γ, IL-10 and GM-CSF) and phenotype (CD3, CD4, CD25 and CD69) were measured on day 4.
Results:  Lactobacillus spp. induced higher productions of TNF-α and IL-10 than did autologous bacteria. In presence of dendritic cells, the production of all cytokines increased. However, the increases of IFN-γ and TNF-α were more pronounced in wells with autologous bacteria than in wells with Lactobacillus spp. The addition of dendritic cells upregulated CD25 expression without simultaneous upregulation of CD69. The upregulation was pronounced after stimulation with Lactobacillus rhamnosus GG compared with autologous bacteria and other lactobacilli.
Discussion:  In presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. Lactobacillus rhamnosus GG induced a regulatory phenotype (cd25⁺), in part mediated by dendritic cells. Future studies will address whether this shift to a CD25⁺ phenotype represents a differentiation into competent regulatory T cells. In a clinical context, such cells might be used for treatment of inflammatory diseases.