Heterogeneity in cytokine profiles of Babesia bovis-specific bovine CD4+ T cells clones activated in vitro.

The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T cell-dependent antibody production and as effector cells acting on intracellular parasites through the elaboration of cytokines, has prompted an investigation of the bovine cellular immune response against Babesia bovis antigens. CD4+ T helper (Th) cell clones generated from four B. bovis-immune cattle by in vitro stimulation with a soluble or membrane-associated merozoite antigen were characterized for reactivity against various forms of antigen and against different geographical isolates of B. bovis and B. bigemina and analyzed for cytokine production following mitogenic stimulation with concanavalin A. Biological assays to measure interleukin-2 (IL-2), IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor alpha or tumor necrosis factor beta and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, IFN-gamma, and tumor necrosis factor alpha revealed differential production of cytokines by the Th cell clones. The majority of clones expressed the Th0 pattern of cytokines: IFN-gamma, IL-4, and IL-2. One clone expressed the Th1 profile (IFN-gamma and IL-2 but not IL-4), whereas none of the clones expressed the Th2 profile. All of the Th cell clones examined expressed the low-molecular-weight isoform of the leukocyte common antigen associated with a memory cell phenotype (CD45RO), and all expressed the lymph node homing receptor (L-selectin). These results extend our previous finding of differential cytokine expression by B. bovis-specific Th cell clones and confirm the identity of the specific cytokines produced, showing that a Th0 response is preferentially induced in a panel of 20 CD4+ T cell clones obtained from immune cattle.     

The effect of IL-18 on IL-12-induced CD30 expression and IL-4 and IFN-gamma production by allergen and PPD specific T cells

CD30 is expressed on activated T cells that, as has been suggested, preferentially produce IFN-gamma. Interleukin 12 increases antigen-induced CD30 expression on T cells and IFN-gamma production. Synthesis of IFN-gamma can be augmented further by IL-18. The aim of our study was to investigate whether IL-18 affects the IL-12 induced CD30 expression and cytokine production by allergen or PPD specific T cells. Mononuclear cells of healthy or atopic volunteers were stimulated with PPD or allergen, respectively, to obtain specific T cell lines. T cells were restimulated with appropriate antigen and antigen-presenting cells in the presence of IL-12, IL-18 or a combination of these cytokines. After 3 days, expression of CD30 was investigated on CD4 and CD8 T cells and IFN-gamma and IL-4 cytokine production was estimated in the culture supernatants. Flow cytometric analyses showed no effect of IL-18 on CD30 expression during IL-12 co-stimulation. At the same time after the optimal stimulation for CD30 expression, the levels of IFN-gamma were high in PPD-stimulated cell lines but have not been up-regulated by IL-18. IFN-gamma levels were much lower in allergen-stimulated T cells and although they were up-regulated by IL-12 there was no additional or synergistic effect from IL-18. IL-18, however, increased production of IL-4 in allergen-stimulated cell lines. Our studies provide new information about IL-18 activity on human cells and question its exclusive role in Th1 mediated responses.     

Availability of antigen-presenting cells can determine the extent of CD4 effector expansion and priming for secretion of Th2 cytokines in vivo

Like dendritic cells (DC), activated B cells are effective antigen-presenting cells (APC) for na?ve CD4 cells due to their expression of MHC class II and multiple costimulatory molecules. We showed previously that CD4 cells primed in B cell-deficient micro MT) mice undergo more limited expansion than in normal animals after immunization with keyhole limpet hemocyanin. Here we report that in the absence of B cells, priming of effectors with the capacity to produce the Th2 cytokines, IL-4, IL-5 and IL-13, was profoundly reduced whereas the development of effectors that secrete the Th1 cytokine IFN-gamma was much less affected. A blockade of IL-12 reduced priming of IFN-gamma-secreting effectors but did not reverse the IL-4, IL-5, or IL-13 deficiency of the response. CD4 cell expansion and priming for Th2 cytokines in micro MT mice was reconstituted by adoptive transfer of activated splenic B cells, which were present throughout the primary response. However, transfer of splenic DC from either control or micro MT mice also supported development of Th2 cytokine responses, indicating that an APC deficit rather than a unique contribution of B cells accounted for diminished effector priming. We conclude that CD4 cell expansion must be sustained via APC for the development of Th2 cytokine-secreting effectors in vivo and that in responses to protein antigen, B cells can be a crucial population to serve in this role. The results suggest that the level of APC engagement can not only determine the extent of effector expansion, but also the overall Th1/Th2 cytokine balance.     

Decreased CD40 ligand induction in CD4 T cells and dysregulated IL-12 production during HIV infection.

During HIV infection various cytokines are overproduced in early stages, whereas in advanced disease cytokines of the T helper 1 type (e.g. interferon-gamma (IFN-gamma)) are selectively deficient. During antigenic stimulation, the production of type-1 cytokines is enhanced by IL-12, secreted by antigen-presenting cells (APC) after their interaction with activated CD4 T cells. Two factors are essential in this process: priming APC with IFN-gamma and triggering the CD40 receptor on APC by CD40 ligand (CD40L). In view of the importance of this pathway, we compared its regulation in HIV-infected and control subjects. After cross-linking of the T cell receptor (TCR)/CD3 complex, the proportional expression of CD40L was similar on CD4+ T cells from controls and from patients with high circulating CD4 T counts (> 500/microl), but CD40L up-regulation was significantly reduced in patients with more advanced disease. Simultaneous triggering of the costimulatory receptor CD28 on T cells through its natural ligand CD80 partly corrected the CD40L defect in patients with intermediate CD4 T counts (200-500), but not in AIDS patients. Early production of IFN-gamma was preserved in lymphocytes from HIV+ patients. The expression of CD40 on peripheral monocytes from HIV+ subjects was increased in a disease stage-related fashion. Stimulation of mononuclear cells through cell-bound CD40L and soluble IFN-gamma induced significantly higher IL-12 in cultures from patients with > 200 circulating CD4 T cells, whereas IL-12 production was marginally decreased in cultures from patients with < 200 CD4 T cells, compared with healthy control cultures. In conclusion, our data suggest that impaired CD40L induction on CD4 T cells contributes to deficient type-1 responses through decreased IL-12 production in AIDS infection, whereas enhanced CD40-mediated IL-12 production in less advanced stages might contribute to increased levels of various cytokines in early disease     

Corrective effects of interleukin-12 on age-related deficiencies in IFN-gamma production and IL-12Rbeta2 expression in virus-specific CD8+ T cells.

Interleukin-12 receptor beta2 (IL-12Rbeta2) has been shown to be selectively expressed on Th1 T cell subsets, and we have previously shown that influenza-specific CD8+ cytotoxic T lymphocyte (CTL) deficiency in old mice was associated with deficient Th1 (interferon-gamma [IFN-gamma]) cytokine production. This study tested whether IL-12Rbeta2 expression was also deficient in CD8+ CTL from old mice and the effect of IL-12 treatment on these responses. Splenic lymphocytes from influenza-primed old and young BALB/c mice were stimulated with influenza virus in vitro with and without IL-12 and then enriched for CD8+ T cells. IFN-gamma was significantly reduced, whereas IL-4 and IL-12p40 (an antagonist of IL-12 function) were evaluated in old when compared with young mice. This was true for secreted protein measured by ELISA and for mRNA levels quantitated by RT-PCR. IL-12Rbeta2 mRNA expression in CD8+ CTL was also significantly reduced in old mice. IL-12 treatment in vitro caused significant upregulation of IFN-gamma and IL-12Rbeta2 and downregulation of IL-4 in CD8+ T cells from old mice and young mice. The present demonstration of an age-related downregulation in IL-12Rbeta2 expression and our previous data showing reduced IFN-gamma and elevated IL-4 production provide strong evidence that CD8+ CTL deficiency in aging results from a Th1/Th2 cytokine production switch. Agents that increase IL-12Rbeta2 expression and redirect Th2 to Thl immune responses are likely to enhance CD8+ CTL-mediated control of viral infections in aging.     

Reciprocal effects of IFN-beta and IL-12 on STAT4 activation and cytokine induction in T cells

IL-12 is an immunoregulatory cytokine, which promotes Th1 cell differentiation and is a major inducer of IFN-gamma. IFN-beta, a Type I IFN used in the treatment of multiple sclerosis, has been shown to significantly increase the expression of the anti-inflammatory cytokine IL-10, a major suppressor of Th1 cytokines. The beneficial immunomodulatory effects of IFN-beta may in part be a result of its ability to suppress IL-12. However, IL-12 and IFN-beta signal via the STAT4 pathway. Our aim was to investigate the relationship between IL-12 and IFN-beta by observing the effect of prior exposure to IL-12 or IFN-beta on the ability of T cells to subsequently respond to the other cytokine. We report that IFN-beta increases IL-12-induced STAT4 phosphorylation and up-regulates IL-12 receptor beta1 and beta2 expression. However, despite this up-regulation, IFN-beta suppressed IL-12-induced IFN-gamma expression. Our results suggest that this may be a result of the parallel induction of IL-10 by IFN-beta.     

Normal B cells fail to secrete interleukin-12

Interleukin-12 is a key regulatory cytokine produced by antigen-presenting cells (APC) which drives the development of interferon-γ (IFN-γ)-producing cells and promotes cell-mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL-12 p75 upon antigen-specific interaction with T cells. We have analyzed secretion of IL-12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen-specific CD4⁺ cells. These APC populations do not produce IL-12 constitutively but, upon stimulation with heat-fixed Staphylococcus aureus and IFN-γ, IL-12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL-12. B cells also fail to secrete IL-12 in response to stimulation with LPS and IFN-γ. Co-culture with CD4⁺ T hybridoma cells and antigen induces IL-12 secretion by DC. Up-regulation of IL-12 secretion by interaction with antigen-specific CD4⁺ T cells is abrogated by anti-class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti-CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR-peptide/class II and by CD40-CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4⁺ T cells. Yet, even upon interaction with antigen-specific T cells, B cells fail to secrete IL-12. The capacity of B cells to present antigen but not to secrete IL-12 may explain their propensity to selectively drive T helper type 2 cell development.     

A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4(+) T lymphocyte responses in B. bovis-immune cattle.

Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4(+) T lymphocytes and IFN-gamma production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4(+) T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4(+) lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4(+) T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4(+) T cell clones proliferated in response to several B. bovis strains and produced IFN-gamma. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/alpha-crystallin family.     

Glucocorticoids drive human CD8(+) T cell differentiation towards a phenotype with high IL-10 and reduced IL-4, IL-5 and IL-13 production

Glucocorticoids are highly effective in the treatment of allergy and asthma and inhibit the synthesis of IL-4, IL-5 and IL-13 by disease-promoting CD4(+) Th2 cells. CD8(+) T cells also synthesize these cytokines, and the aim of this study was to investigate how glucocorticoids effect cytokine production by these cells. When CD8(+) T cells are stimulated with anti-CD3 and IL-2 plus IL-4 or dexamethasone, production of the anti-inflammatory cytokine IL-10 is low in both primary and secondary cultures restimulated with anti-CD3 and IL-2 alone. However, when both are present, a synergistic effect on IL-10 synthesis is observed. The additional presence of antigen-presenting cells (APC) in the priming culture maintains IL-10 levels, but inhibits IL-4 and IL-5 production. CD4(+) T cells develop a similar glucocorticoid-induced phenotype. These cells demonstrate regulatory activity and inhibit CD4(+) T cell activation in an IL-10-dependent manner. Earlier reports show glucocorticoids promote a Th2 phenotype by effects on purified naive T cells or pretreatment of APC. This study demonstrates, more critically, that when APC are present, glucocorticoids induce CD4 and CD8 T cell populations synthesizing high levels of IL-10, but greatly reduced amounts of disease-promoting IL-4 and IL-5.     

Control of CD4 T cell fate by antigen re-stimulation with or without CTLA-4 engagement 24 h after priming

After two consecutive inoculations with Staphylococcus enterotoxin B (SEB) at 24 h intervals in vivo, CD4 T cells became anergic to the antigen challenge in vitro. Administration of anti-CTLA-4 mAb in conjunction with the second SEB inoculation 24 h after antigen priming interfered with anergy and CD4 T cells became T(h)2 cells. However, the anergy induction was not ablated when SEB and anti-CTLA-4 mAb were administered 48 or 72 h after antigen priming. Moreover, anti-CTLA-4 mAb without SEB did not interfere with anergy nor promoted the T(h)2 differentiation. T-antigen-presenting cell (APC) interaction in vitro in the presence of high doses of antigen and anti-CTLA-4 mAb induced a T(h)2-polarizing cytokine IL-6 and IL-10. IL-10 then down-modulated a T(h)1-polarizing cytokine IL-12. The results demonstrate that 24 h after the initial antigen stimulation, CD4 T cells enter the critical activation phase where antigen re-stimulation with or without CTLA-4 engagement alters the fate of the cell, anergy or differentiation respectively. Once anergy is interfered with, T(h)2-polarizing cytokines produced upon prolonged T-APC interaction favor the T(h)2 differentiation.     

Resistance to acute babesiosis is associated with interleukin-12- and gamma interferon-mediated responses and requires macrophages and natural killer cells

We examined the role of the cytokines gamma interferon (IFN-gamma) and interleukin-12 (IL-12) in the model of acute babesiosis with the WA1 Babesia. Mice genetically deficient in IFN-gamma-mediated responses (IFNGR2KO mice) and IL-12-mediated responses (Stat4KO mice) were infected with the WA1 Babesia, and observations were made on the course of infection and cytokine responses. Levels of IFN-gamma and IL-12 in serum increased 24 h after parasite inoculation. The augmented susceptibility observed in IFNGR2KO and Stat-4KO mice suggests that the early IL-12- and IFN-gamma-mediated responses are involved in protection against acute babesiosis. Resistance appears to correlate with an increase in nitric oxide (NO) production. In order to assess the contribution of different cell subsets to resistance against the parasite, we also studied mice lacking B cells, CD4+ T cells, NK cells, and macrophages. Mice genetically deficient in B lymphocytes or CD4+ T lymphocytes were able to mount protective responses comparable to those of immunosufficient mice. In contrast, in vivo depletion of macrophages or NK cells resulted in elevated susceptibility to the infection. Our observations suggest that a crucial part of the response that protects from the pathogenic Babesia WA1 is mediated by macrophages and NK cells, probably through early production of IL-12 and IFN-gamma, and induction of macrophage-derived effector molecules like NO.     

IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein,induces proliferation of naive CD4+ T cells

An efficient Th1-driven adaptive immune response requires activation of the T cell receptor and secretion of the T cell stimulatory cytokine IL-12 by activated antigen-presenting cells. IL-12 triggers Th1 polarization of naive CD4(+) T cells and secretion of IFN-gamma. We describe a new heterodimeric cytokine termed IL-27 that consists of EBI3, an IL-12p40-related protein, and p28, a newly discovered IL-12p35-related polypeptide. IL-27 is an early product of activated antigen-presenting cells and drives rapid clonal expansion of naive but not memory CD4(+) T cells. It also strongly synergizes with IL-12 to trigger IFN-gamma production of naive CD4(+) T cells. IL-27 mediates its biologic effects through the orphan cytokine receptor WSX-1/TCCR.     

Survival and cytokine polarization of naive CD4(+) T cells in vitro is largely dependent on exogenous cytokines

Naive CD4(+) T cells differ from memory cells by their heightened expression of the disialoceramide recognized by antibody 3G11. 3G11(bright) cells respond well to immobilized anti-CD3 / anti-CD28 and to their cognate antigens but produce little or no IFN-gamma or IL-4 "acutely" and undergo cell death even in the presence of IL-2. They can be rescued by IL-4, IL-6 or IL-12. IL-6 is particularly notable since it is neutral in regard to Th1 / Th2 priming, allowing an assessment of the role of endogenous IL-4 in priming for IL-4 production. Naive TCR-transgenic BALB / c scid T cells cultured with an ovalbumin peptide and IL-4(- / -) antigen-presenting cells in the presence of IL-6 showed a modest degree of priming for IL-4 production if both IFN-gamma and IL-12 were neutralized. This priming is far less than that observed if IL-4 is added to the priming culture. These results indicate that IL-4 production as a result of TCR engagement is sufficient for only a minor component of the polarization observed when unseparated BALB / c CD4 T cell populations are primed or when IL-4 is intentionally added to the priming culture.     

High-level IL-12 production by human dendritic cells requires two signals

IL-12 is a key cytokine in the development of Th1 responses. IL-12 production by antigen-presenting cells (APC) can be induced by the interaction between CD40 on the APC and CD40 ligand (CD40L) expressed on T cells after activation. Our previous study indicated that in dendritic cells (DC), the only APC that can activate naive T(h) cells efficiently, the mere CD40 engagement is insufficient to induce IL-12 production. The aim of the present study was to dissect the conditions for efficient IL-12 production by DC further. Using populations of naive and memory Th cells, recombinant CD40L, neutralizing and blocking antibodies, and by determining IFN-gamma production and CD40L expression levels, we here show that T cell-induced IL-12 production by DC results from the action of two signals, mediated by CD40L and IFN- gamma, and that the inability of naive T(h) cells to induce IL-12 production resides in their inability to produce IFN-(gamma). Other factors than CD40L and IFN-gamma can provide the required signals for IL-12 production by DC, as either factor could be replaced by lipopolysaccharide (LPS). The two-signal requirement proved unique for the production of IL-12, since either CD40 engagement or LPS was sufficient for the efficient production of tumor necrosis factor-alpha, IL-8 and the p40 subunit of IL-12, and may be considered as a safety mechanism for optimal control of potentially harmful T(h)1 responses.      

High dose allergen stimulation of T cells from house dust mite-allergic subjects induces expansion of IFN-gamma+ T Cells, apoptosis of CD4+IL-4+ T cells and T cell anergy

BACKGROUND: During clinically effective allergen-specific immunotherapy a shift in cytokine dominance from IL-4, IL-5 predominant to IFN-gamma predominant has been observed. As antigen concentration influences Th cell priming, this study aimed to determine the effect of different allergen concentrations on human house dust mite (HDM)-specific T cell production of IL-4 and IFN-gamma, proliferation and apoptosis. METHODS: HDM-allergic donor PBMC were cultured for 14 days with different concentrations of HDM extract (1, 10 and 100 microg/ml). T cell intracellular IL-4 and IFN-gamma, division (CFSE labelling) and apoptosis (active caspase-3 staining) were analysed by flow cytometry. Proliferation was assessed by (3)H-thymidine incorporation. RESULTS: Increased CD4+IFN-gamma+ and CD8+IFN-gamma+ T cell numbers were observed in high allergen concentration cultures compared with low concentration cultures whereas there were no differences in CD4+IL-4+ and CD8+IL-4+ T cell numbers. CFSE cell labelling revealed that high allergen concentration favours the expansion of IFN-gamma-producing CD4+ T cells. The proportion of apoptotic cells increased with allergen concentration and there was preferential apoptosis of CD4+IL-4+ T cells. HDM-induced proliferation was decreased in high allergen concentration cultures; this was reversible by IL-2 consistent with anergy. CONCLUSION: These results show that T cell division and apoptosis contribute to the cytokine skewing to predominant IFN-gamma production by T cells observed at high allergen concentration. Thus the use of hypoallergenic T cell reactive preparations which can be given safely at higher doses than natural extracts may enhance efficacy of allergen-specific immunotherapy.     

Decreased IL-4 producing CD4+ T cells in patients with active systemic lupus erythematosus-relation to IL-12R expression

The balance of interferon-gamma (IFN-gamma) and/or interleukin-4 (IL-4) producing T cells and interleukin-12 receptor (IL-12R) expression on T cells were evaluated in patients with active systemic lupus erythematosus (SLE). Assessment of intracellular IFN-gamma and/or IL-4 were conducted with cytoplasmic staining. IL-12R presenting T cells were also assessed by flowcytometry without in vitro stimulation. In SLE, the number of IFN-gamma producing CD4+ T cells was increased, and the absolute number of IL-4 producing CD4+ T cells was significantly decreased. Although the ratio of IL-12R presenting CD4+ T cells was significantly greater, the absolute number did not increase. The ratio of IFN-gamma/IL-4-producing CD4+ T cells correlated with the SLE disease activity index (SLEDAI) and was significantly higher among patients with lupus nephritis. Therefore, the imbalance of IFN-gamma/IL-4 producing CD4+ T cells was due to the decrease in IL-4 producing CD4+ T cells and may play an important pathogenic role in active SLE.