Sequential production of cytokines by dengue virus-infected human peripheral blood leukocyte cultures.

The study was undertaken to elucidate the sequence of appearance of T helper (Th)1- and Th2-type cytokines in human peripheral blood leucocyte cultures infected in vitro with dengue type 2 virus. Commercial sandwich enzyme-linked immunosorbent assay kits were used to assay the levels of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, and IL-10 in culture supernatants. Culture supernatants were also screened for the cytotoxic factor and the dengue virus titres determined. The cytokines that appeared in the culture supernatants on the first day post-infection (p.i.) were cytotoxic factor, TNF-alpha, IL-2, and IL-6; their levels were highest on the second day p.i. IFN-gamma appeared on the second day with a peak on the third day p.i. The levels of these cytokines declined quickly, except for human cytotoxic factor (hCF) and IL-2. The cytokines that appeared later were IL-10 and IL-5 on the fourth day and IL-4 on the sixth day p.i. Dengue virus replicated in the peripheral blood leucocyte (PBL) cultures and was present throughout the course of the study. The findings of the present study show that dengue virus induced a predominant Th1-type cytokine response during the first 3 days of infection of PBL cultures that was replaced by a Th2-type response later.     

CD40 cross-linking inhibits specific antibody production by human B cells

Ligation of CD40 on B cells is a co-stimulatory signal for proliferation,antibody secretion, heavy chain switching and rescue from apoptosisafter somatic mutation in the germinal centre. The importanceof these manifold responses to CD40 activation for humoral immunityis exemplified by the inability of boys with X-linked hyperIgM syndrome to make IgG, IgE or IgA due to a mutation in inthe gene coding for CD40 ligand (CD40L). In the present study,we have investigated the effect of CD40 ligation on specificantibody production by human B cells to influenza virus. Theantibody. response was T cell dependent and specific for thestrain of influenza virus used as antigen. Addition of eitherCD40 mAb or recombinant trimeric CD40L profoundly inhibitedspecific antibody production. Antibody production by unseparatedtonsillar mononuclear cells and by T-depleted B cells stimulatedwith antigen in the presence of T cell replacing factor wereequally inhibited with CD40 antibody showing that the effectwas due to ligation of CD40 on B cells rather than blockingof T cell help. The specific antibody detected in these experimentswas mostly IgG with little or no IgM and was obtained from surfaceIgM B cells consistent with activation of a secondary (memoryresponse. Co-stimulation of tonsillar B cells with CD40 antibodyand anti-IgG induced proliferation of IgG⁺ B cells. These resultssuggest that CD40 ligation can inhibit specific antibody responsesand stimulate proliferation in the same IgG⁺ (memory) B cellsubpopulation. Addition of CD40 antibody during the first 24–48h of the response was required for inhibition, suggesting thatthe effect was on early B cell activation and/or proliferationrequired for antibody production. There was no correlation,however, between the ability of CD40 mAb to stimulate proliferationand inhibit antibody production. We suggest that early activationof CD40 in the specific antibody response inhibits the formationof plasma cells and promotes instead the generation of memorycells.     

EB病毒体外诱导CD5+B细胞产生IL-6及免疫球蛋白的意义

目的观察EB病毒(Epstein-Bart virus,EBV)及其膜表面糖蛋白350(gp350)对分离的脐带血CD5+B细胞产生IL-6和免疫球蛋白的影响.方法无菌采集新生儿脐带血,RosetteSepTM法分离总B细胞,免疫磁珠分离CD5+B细胞,用EBV及纯化的gp350刺激细胞,ELISA法检测培养上清液中的IL-6和免疫球蛋白IgG、IgM,对照为未加刺激的培养细胞上清液.结果 CD5+B细胞于刺激的第24h开始分泌IL-6,OD值较对照组显著升高(P＜0.01);EBV刺激的CD5+B细胞13 d以后的培养上清液中能检测到免疫球蛋白IgG和抗角蛋白自身抗体(AK auto Ab)IgM;gp350刺激的CD5+B细胞存活15 d,其第13、15天的细胞培养上清液中能检测到免疫球蛋白IgM;未刺激的细胞存活7 d,培养上清液中未检测到IL-6和免疫球蛋白.结论 EBV可能通过其膜表面糖蛋白gp350刺激培养的CD5+B细胞产生IL-6和具有天然自身抗体性质的免疫球蛋白.     

Comparable profiles of the immunoglobulin heavy chain complementarity determining region (CDR)-3 in CD5+ and CD5- human cord blood B lymphocytes.

It has been suggested that CD5+ B cells are closely related to the pathogenesis of autoimmune disease or chronic lymphocytic leukaemia, whereas the origin and physiological role of CD5+ B cells remain controversial. To study the molecular differences between CD5+ and CD5- B cells in terms of immunoglobulin gene structure, we sorted both subsets from new-born cord blood and analysed the complementarity determining region (CDR)-3 profiles of the immunoglobulin heavy chain (IgH) gene. The CDR-3 sequences from both CD5+ and CD5- B cells represented the same incidence and length of N-region, and the same usage of D and JH segments. When translated into amino acids, 24 of 32 clones (75%) and 25 of 37 clones (65.8%) from their respective subset were productive, and the composition of the deduced amino acids were similar between both subsets. These data suggest that CD5+ B cells are not a distinct lineage presenting a biased immunoglobulin repertoire in B cells.     

Effects of boar seminal immunosuppressive fraction on production of cytokines by Concanavalin A-stimulated spleen cells and on proliferation of B lymphoma cell lines

PROBLEM: The immunosuppressive fraction (ISF) of boar seminal vesicle fluid has recently been demonstrated to inhibit mitogen-stimulated proliferation of lymphocytes and antibody response to corpuscular and soluble antigens. The effects of ISF on in vitro and in vivo production of cytokines as well as its possible inhibitory effect on proliferation of B lymphoma cells remain to be elucidated. METHODS: The effect of ISF on proliferation of normal mouse spleen cells stimulated by Concanavalin A (Con A) and on mouse B lymphoma cells was measured by 3H-thymidine incorporation. Cytokines were determined in the supernatants of mouse spleen cells stimulated with Con A in the presence or absence of ISF by enzyme-linked immunosorbent assay (ELISA). In vivo cytokine production in the sera samples of mice treated with ISF and immunized with keyhole limpet hemocyanin (KLH) was followed by ELISA, too. RESULTS: We confirmed the inhibitory effect of ISF on Con A-stimulated lymphocyte proliferation. ISF affected cytokine production in the Con A-stimulated spleen cells: production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was lowered, but production of IL-4, IL-6, and IL-10 was enhanced. Similarly, in the sera samples of mice immunized with keyhole limpet hemocyanin (KLH), IL-2 and IFN-gamma levels were decreased by ISF. ISF inhibited proliferation of Ag 8 and X 63-IL-2 B lymphoma cells as well. CONCLUSIONS: ISF inhibited production of T helper1 (Th1) cytokines (IL-2 and IFN-gamma) and enhanced production of Th2 cytokines (IL-4, IL-6, and IL-10). ISF seems to shift the Th1/Th2 pattern in favor of Th2. ISF exhibited an antiproliferative activity on mouse B lymphoma cells.     

HIV-gp160 modulates differentially the production in vitro of IgG,IgA and cytokines by blood and tonsil B lymphocytes from HIV-negative individuals

HIV1-gp160 holds promises in anti-HIV vaccinal strategies. However, this molecule has been described to exhibit superantigenic activities. The present study aimed at examining the effect(s) of HIV1-gp160 on human B cells and in particular on B cells originating from HIV- donors. We purified human B cells of various origins, i.e. from blood and from tonsils (representing a mucosal-type origin), and we tested these cells (stimulated with a polyclonal B cell activator, interleukin (IL)-2 and IL-10 as cytokines, and recombinant HIV1-gp160) for the production of IgG and IgA in an in vitro model. Gp160 induced significantly less total IgG by blood - but not tonsil-originating - B cells and did not affect total IgA production. Further, HIV1-gp160 up-regulated IL-2-, IL-4- and IL-10-mRNA levels in stimulated blood B cells (these cytokines are known to be active on B cell activation and differentiation). Interestingly, HIV1-gp160 also up-regulated IL-1beta-, transforming growth factor (TGF)-beta-, interferon (IFN)-gamma- and IL-12-mRNA levels in stimulated mucosal-type, tonsil-originating, B cells. As these latter cytokines are involved in proinflammatory activities, HIV-gp160 delivery at the mucosal sites would be compatible with an adjuvant activity.     

IgG1 production by sIgD+ splenic B cells and peritoneal B-1 cells in response to IL-5 and CD38 ligation.

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induces proliferation, IL-5 receptor alpha chain expression and tyrosine phosphorylation of Bruton's tyrosine kinase. Furthermore, stimulation of splenic B cells with IL-5 together with CS/2 induces Blimp-1 expression and differentiation into Ig-producing cells. Here we examined the role of IL-5 in IgG1 and IgA production by B cells isolated from the spleen and peritoneal cavity. CD38 recognized by CS/2 was expressed in the follicular mantle B cells surrounding the germinal center, sIgD+ splenic B cells and peritoneal B cells. IL-5 induced IgG1 production in splenic sIgD+ B cells stimulated with CS/2, while it was ineffective to induce IgA production. Among the various cytokines tested, only IL-5 had a synergistic effect on IgG1 production with CS/2. IL-5 could induce the generation of S micro-Sgamma1 reciprocal recombination DNA products in CS/2-stimulated B cells. IL-4 was ineffective to induce either micro-gamma1 switch recombination or IgG1 secretion with CS/2, demonstrating that IL-5 promotes both micro-gamma1 switch recombination and IgG1 secretion in an IL-4-independent manner. The peritoneal B-2 cells exhibited both IgG1 and IgA production in response to IL-5 plus CS/2, while B-1 cells produced IgG1. These results imply that the pattern of differentiation to Ig-producing cells seen with peritoneal B cells is not identical to the pattern seen with splenic B cells and that peritoneal B-2 cells contain precursors of IgA-producing cells responding to IL-5 plus CS/2.     

Human hybridomas derived from CD5+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies.

Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B-CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT-sensitive heteromyeloma line (CB-F7). A fusion frequency of up to 10(-5) allowed us to analyse hundreds of initial hybridoma lines per fusion. In all culture supernatants in three out of five fusions IgM lambda antibodies were detected, in two experiments only IgM kappa was measured, suggesting monoclonality of the primary hybridoma cell lines. The later fusions resulted in hybridomas producing multi-specific antibodies against both an autoantigen and an infectious agent: (i) dsDNA/influenza virus haemagglutinin; (ii) dsDNA/class V outer membrane protein type C from Neisseria meningitidis. However, no antibodies of the described specificity were detected in blood sera of patients, indicating a 'switch-on' of the immunoglobulin secretion capacity of malignant B cells during fusion to a myeloma partner. We discuss the results as further evidence for the natural multi-reactive antibody repertoire of CD5+ B cells.     

The mycotoxins citrinin and gliotoxin differentially affect production of the pro-inflammatory cytokines tumour necrosis factor-α and interleukin-6, and the anti-inflammatory cytokine interleukin-10

BACKGROUND: Microbial growth is considered one of the major causes of indoor air problems. Moulds have been associated with asthma, allergy and a wide range of diffuse indoor air-related symptoms. However, mechanisms of the adverse health effects are not well understood. OBJECTIVE: We hypothesized that the mycotoxins citrinin and gliotoxin could cause an imbalance between the secretion of the pro-inflammatory cytokines TNF-alpha and IL-6 and the anti-inflammatory cytokine IL-10. METHODS: We investigated the influence of citrinin and gliotoxin on the human monocytic cell line Mono-Mac-6 (MM6) with and without lipopolysaccharide -stimulation. The levels of IL-10, IL-6 and TNF-alpha were analysed in cell culture supernatants by ELISA. Cell viability and cell apoptosis were measured by flow cytometry. RESULTS: The strongest inhibition of cytokine secretion was found for IL-10. IL-6 levels were found to decrease in a dose-dependent manner along with reduced cell viability. TNF-alpha levels increased with low gliotoxin exposure (less than 100 ng/mL), but decreased significantly at 375 ng/mL and higher along with increased cell apoptosis and reduced cell viability. TNF-alpha levels were not reduced by citrinin exposure. CONCLUSION: We observed a cytokine imbalance with a more pronounced reduction of IL-10 concentrations compared with those of TNF-alpha and IL-6. We suggest that low exposure doses of citrinin and gliotoxin (corresponding to less than 100 ng/mL gliotoxin and less than 10 mug/mL citrinin) may inhibit IL-10 and lead to increased risk of an inflammatory response with relative overproduction of TNF-alpha and IL-6. The findings and their clinical implications must be verified by human studies. However, we speculate that the observed biological effects may be of importance as they may partly explain the occurrence of diffuse general indoor air-related symptoms as well as the worsening of asthmatic inflammatory reactions experienced in mouldy environments.     

Polyreactive antigen-binding B (PAB-) cells are widely distributed and the PAB population consists of both B-1+ and B-1- phenotypes

B cells that make polyreactive antibodies (PAB+ cells) express polyreactive Ig receptors on their surface and can bind a variety of different antigens. The present study shows that PAB+ cells are widely distributed, are present in varying numbers in different lymphoid organs and that their phenotype varies depending on the organs from which they are isolated. Up to 10 times more cells in PAB+ enriched populations bind antigens as compared to PAB- populations. Comparison of PAB+ with B-1+ cells showed that a high percentage of PAB+ cells are B-1+, but that many PAB+ cells do not express B-1 cell surface markers and, in fact, are B-1-. It is concluded that the B cell population consists of PAB+/B-1+, PAB+/B-1-, PAB-/B-1+, and PAB-/B-1- cells. The presence of PAB+ cells in the thymus points to the possibility that PAB+ cells may carry endogenous host antigens from peripheral tissues to the thymus where they may contribute to immunological tolerance.     

Impaired production of proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation in elderly humans.

Ageing is associated with decreased resistance to bacterial infections and concomitant increased circulating levels of inflammatory cytokines. The purpose of the present study was to research age-related changes in levels of early mediators of the acute-phase response in whole blood supernatants following LPS stimulation, representing an ex vivo model of sepsis. Levels of tumour necrosis factor-alpha (TNF-alpha), IL-1beta and IL-6 in whole blood supernatants were measured after in vitro LPS stimulation for 24 h in 168 elderly humans aged 81 years from the 1914 cohort in Glostrup, Denmark and in 91 young controls aged 19-31 years. Levels of TNF-alpha and IL-1beta were significantly lower in elderly humans compared with young controls, whereas no difference was detected with regard to IL-6. Elderly humans with low body mass index had the lowest levels of IL-1beta. Young women had lower levels of proinflammatory cytokines compared with young men, but this difference was blurred by ageing. No relation was found between circulating plasma levels of TNF-alpha and levels after in vitro LPS stimulation. In conclusion, decreased production of TNF-alpha and IL-1beta after exposure to LPS may reflect impaired host defence against infections in the elderly and be of importance in elderly humans with underlying health disorders. However, the clinical relevance is questionable in healthy elderly people because decreased levels were found compared with young men but not compared with young women.     

Natural autoantibodies, IgG antibodies to tetanus toxoid and CD5+ B cells in patients with Mediterranean visceral leishmaniasis. The Leishmania Study Group.

Natural autoantibodies (NaAb) and IgG antibodies to tetanus toxoid (TT) were analysed in the sera of 38 children with active visceral leishmaniasis (VL) previously vaccinated with TT and in 30 healthy controls matched for sex and age. Patients exhibited high levels of NaAb to a panel of self antigens (tubulin, myosin, myoglobin, actin) contrasting to a low level of IgG to TT. Analysis of the circulating B cells in 26 untreated patients showed a low percentage of CD5+ per total B cells (3-66%, mean 36.6%) compared with 14 normal controls (17.8-66.6%, mean 52.7%) (P < 0.001). Evaluation of these parameters after antimonial therapy showed a significant decrease of the level of the NaAb (P < 0.0005), and a spontaneous increase of the level of the IgG to TT without any vaccine boosting (P < 0.01). In contrast, there was a significant increase in CD5+ B cells (P < 0.0005). This result suggests that CD5+ B cells may be sequestrated in parasitized lymphoid organs and may be released after remission. These findings show that the polyclonal B cell activation that occurs during active VL involves mainly B cells bearing NaAb and are in favour of a functional dichotomy of B cells.     

Cloning of a human antibody directed against human neuroblastoma cells and specific for human translation elongation factor 1alpha

Human sera have shown antitumor effects mediated by tumor-specific immunoglobulin M (IgM) antibodies. Most people who have cytotoxic serum are in good health and show no evidence of exposure to tumor antigens. We characterized the serum of a healthy female adult that was highly lytic to a neuroblastoma cell line via IgM-activated complement (>60% of malignant cells were killed during the 60-min assay). Complement-dependent lysis was not mediated by other classes of serum antibodies (data not shown) which is consistent with the findings of Ollert et al. To identify the target antigen on neuroblastoma cells, we fractionated neuroblastoma cell lysates by ion-exchange chromatography. In the fraction that showed maximal IgM binding, the dominant protein was identified as the 47-kDa translational elongation factor 1alpha (eEF1alpha). We used the donor's B-cells to create hybridomas producing the antibody (B12.6.22) that bound to neuroblastoma cells and mediated cytotoxicity. This antibody recognized eEF1alpha in a specific manner. Sequence analysis of the heavy chain of B12.6.22 showed usage of VH3-23 and JH6 gene segments, with no somatic mutation. The structural similarity of B12.6.22 to antibodies of the innate immune system supports the assumption that natural antibodies are a potential source of therapeutic antibodies.     

Aberrant T-regulation in rheumatoid arthritis and IgA nephropathy affects CD5+ and CD5- B lymphocytes equally.

T-suppressor function and T-helper function in healthy adults, elderly patients with non-immune diseases, and patients with rheumatoid arthritis (RA) and IgA nephropathy (IgAN) were titrated by adding graded concentrations of CD8+ cells to autologous CD8-depleted peripheral blood mononuclear cells (PBMC), or CD4+ cells to CD8- 4- PBMC, respectively. Following culture with pokeweed mitogen (PWM), numbers of CD5+ and CD5- immunoglobulin-secreting cells were determined using a combination of rosetting with anti-CD5-coated Dynabeads and reverse haemolytic plaque formation (Jones, 1990). Of 11 RA patients studied, eight had slightly reduced suppressor activity for CD5+ and CD5- IgM-secreting cells, and three with active disease and high serum levels of C-reactive protein, could not suppress IgG, IgA or IgM secretion by either B subset. Helper activity for both CD5+ and CD5- B cells was slightly but significantly increased in RA patients. One of eight patients with IgAN could not suppress IgG, IgA or IgM production by CD5+ or CD5- B cells, and all IgAN patients required strikingly fewer CD4+ cells for PWM-induced activation of CD5+ and CD5- B cells than controls. It was concluded that in two immunologically mediated diseases in which some patients have raised numbers of circulating CD5+ B cells, aberrant T-regulation affects CD5+ and conventional CD5- B cells equally.     

Inhibitory receptors CD85j, LAIR-1, and CD152 down-regulate immunoglobulin and cytokine production by human B lymphocytes

Class switching consists in the substitution of the heavy-chain constant region of immunoglobulin M (IgM) with that of IgG, IgA, or IgE. This enables antibodies to acquire new effector functions that are crucial to combat invading pathogens. Class switching usually requires engagement of CD40 on B cells by CD40 ligand (CD40L) on antigen-activated CD4(+) T cells and the production of cytokines. The process must be regulated tightly because abnormal IgG and IgA production favors the onset of autoimmunity, whereas increased switching to IgE leads to atopy. These inflammatory disorders can be triggered or exacerbated by costimulatory signals. Although thoroughly investigated on T cells, the roles of the inhibitory receptors CD85j, LAIR-1, and CD152 on B-cell functions have not been fully elucidated. In this study we show that cross-linking of the B-cell inhibitory receptors by specific monoclonal antibodies inhibits IgG and IgE production, reduces the percentage of IgG- and IgE-expressing B cells, and down-regulates interleukin 8 (IL-8), IL-10, and tumor necrosis factor alpha production. These effects were demonstrated using different B-cell stimulatory pathways (recall antigens, CD40L-transfected cells plus IL-4, and lipopolysaccharide plus IL-4). It thus appears that CD85j, LAIR-1, and CD152 play a central role for the control of IL-4-driven isotype switching.     

Autostimulatory effects of IL-6 on excessive B cell differentiation in patients with systemic lupus erythematosus: analysis of IL-6 production and IL-6R expression.

Introducing avidin-biotin complex ELISA for anti-DNA antibody, the mechanism of in vitro production of anti-ssDNA antibody as well as of polyclonal immunoglobulin mediated by an IL-6-IL-6R loop was studied in patients with systemic lupus erythematosus (SLE). Regardless of the presence or absence of T cells, B cells from SLE patients could produce IgG anti-ssDNA antibody as well as total IgG without any stimulation. Low density B cells obtained by Percoll gradient density centrifugation responded to rIL-6 to produce IgG and IgG anti-ssDNA antibody. rIL-2 and rIL-4 had lesser effects on the differentiation of low density B cells. In fact, IL-6R was preferentially expressed on low density B cells from active SLE patients, as detected by anti-IL-6R MoAb, MT18, which did not inhibit IL-6 binding. SLE B cells, especially high density B cells, produced greater amounts of IL-6 in culture supernatants than did T cells, regardless of whether disease was active or inactive. Normal T cells and B cells did not produce significant amounts of IL-6. Thus, endogenous IL-6 produced by high density B cells bound to the IL-6R preferentially expressed on the low density B cells, and drove them into terminal differentiation, especially in active SLE patients. Further, addition of polyclonal anti-IL-6 or anti-IL-6R MoAb (PM1), which inhibited IL-6 binding, both inhibited IgG anti-ssDNA antibody as well as total IgG production by SLE B cells in a dose-dependent manner. These results suggest that interruption of the autocrine IL-6 loop would be of therapeutic value in SLE.     

Treatment of murine CD5- B cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation pathways.

Anti-Ig stimulated murine B cells express high levels of surface CD5 (ly-1) and increased CD44 while maintaining surface IgD, CD23 and J11d. Sorting of CD5- and CD5+ cells demonstrates that anti-Ig induces CD5 expression rather than the selective expansion of CD5+ cells. Anti Ig plus interleukin-6 (IL-6) induces the CD23, IgD, low ly-5 (B220) (CD45low), J11dhigh phenotype of typical CD5+ peritoneal B cells. In contrast, lipopolysaccharide (LPS)-stimulated B cells have high levels of CD44 but decreased surface IgD, CD23 and J11d and no CD5. Thus LPS and anti-Ig generate activated cells with differing phenotypes. Induced CD5+ cells have increased viability, even in the absence of added exogenous factors, while the viability of CD5- B cells is dependent on factors such as IL-4. We conclude that conventional CD5- B cells can be activated by either of two pathways: one generating CD5+ B cells; the other yielding conventional activated cells. We hypothesize that the first path requires slg cross-linking and corresponds to T-independent (type 2) stimulation, while cognate interaction with helper T cells in the absence of slg cross-linking induces B cells to enter the second path.     

Pyk2 is differentially regulated by β 1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

β₁ integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by β₁ integrin signaling in human T cells. Stimulation of Jurkat T cells with the α₄β₁ integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and α₄β₁ integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4⁺ T cells confirmed the ability of CD3-derived signals to synergize with β₁ integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell costimulation mediated specifically by β₁ integrins.     

Augmentation by transferrin of IL-2-inducible killer activity and perforin production of human CD8+ T cells.

The effects of human transferrin (Tf) on lymphokine (IL-2)-activated killer (LAK) induction from blood lymphocytes of healthy donors was examined. LAK cells were induced by 6-day incubation in medium with recombinant human IL-2 of lymphocytes, and their cytotoxic activity was assessed by measuring 51Cr release from NK-resistant Daudi cells. Tf alone did not induce any LAK activity, but in combination with IL-2, it augmented LAK induction dose- and time-dependently. This augmenting effect was completely abolished by pretreatment with anti-Tf antiserum. Tf augmented the proliferative response of lymphocytes to IL-2 and their expressions of receptors for IL-2 and Tf. CD8+ T cells were isolated from purified blood lymphocytes using antibody-bound magnetic beads. Addition of Tf to cultures of CD8+ cells resulted in significant augmentation of killer cell induction and perforin (PFP) production after 4 days stimulation with IL-2. These results indicate that Tf is important in generation of IL-2-inducible killer properties and PFP activity of human CD8+ T cells.