Human CD4+ T lymphocytes with increased intracellular cAMP levels exert regulatory functions by releasing extracellular cAMP

We have previously shown that cholera toxin (CT) and other cAMP-elevating agents induce up-regulation of the inhibitory molecule CTLA-4 on human resting T lymphocytes. In this study, we evaluated the function of these cells. We found that purified human CD4(+) T lymphocytes pretreated with CT were able to inhibit proliferation of autologous PBMC in a dose-dependent manner. It is interesting that this phenomenon was not mediated by inhibitory cytokines such as IL-10, IL-4, or TGF-beta but was in part caused by the release of extracellular cAMP by the CD4(+) T lymphocytes. Purified CD4(+) T cells pretreated with forskolin, a transient cAMP inducer, or with dibutyryl cAMP, an analog of cAMP, did not exert suppressive functions, suggesting that a sustained production of cAMP, such as that induced by CT, was required to identify a novel regulatory function mediated by CD4(+) T cells. Our results show that CD4(+) T lymphocytes can exert regulatory functions through the release of extracellular cAMP and that the cyclic nucleotide acts as a primary messenger, which could play a biological role in the modulation of immune responses.     

Expression of the costimulator molecules, CD40 and CD154, on lymphocytes from neonates and young children

Differential expression of the costimulator molecules CD40 and CD154 on neonatal lymphocytes may be one explanation for limited T-dependent antibody responses in human neonates. CD40 was expressed at similar levels on resting B cells from adults, young children (2-20 months of age) or cord blood. CD40 expression was higher on cord blood B cells compared to adult B cells after stimulation with PMA and ionomycin, but similar on adult and cord blood B cells activated by CD3-stimulated T cells. In contrast to previous reports, cord blood T cells stimulated with PMA and ionomycin expressed adult levels of CD154 initially, but this expression was more transient on cord blood T cells. When adult and cord blood mononuclear cells were stimulated with CD3 mAb, T cells from some cord blood specimens showed different kinetics of CD154 expression compared with adult T cells. However, some cord blood specimens showed adult patterns of T cell CD154 expression. When mononuclear cells were depleted of B cells and monocytes prior to stimulation with CD3 mAb, the MFI and percentage of T cells expressing CD154 increased, with adult and cord T cells showing similar patterns of expression. These results show some differences in expression of CD40 and CD154 between neonatal and adult lymphocytes, but do not directly account for the relative deficiencies of humoral immunity in neonates.     

Expression of the costimulator molecules, CD80, CD86, CD28, and CD152 on lymphocytes from neonates and young children

The expression of CD80, CD86, CD28, and CD152 were examined on peripheral blood lymphocytes from adults, neonates (cord blood lymphocytes) and young children (2-20 months of age). There was no difference in the expression of CD80 or CD86 between adult and neonatal B cells, either resting or activated. A higher percentage of resting T cells expressed CD28 in neonates and young children compared to adults. CD28 expression was similar on adult and neonatal T cells activated with PMA and ionomycin. However, CD28 was expressed at greater intensity on a higher percentage of neonatal T cells than adult T cells stimulated with CD3. CD152 expression was lower on neonatal T cells than adult T cells stimulated with PMA and ionomycin and undetectable on neonatal T cells stimulated with CD3. In contrast, intracellular CD152 was equivalent in adult and neonatal T cells stimulated with PMA and ionomycin, suggesting trafficking of CD152 to the cell surface may be differentially regulated in neonatal T cells. Since the T cell response is determined by the balance of signals received from CD28 and CD152, high levels of CD28 expression and lower surface expression of CD152 on neonatal T cells may represent specialisation to promote activation of neonatal T cells.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Decreased expression of the ligand for CD40 in newborn lymphocytes

Immune responses in newborn lymphocytes show a defect in isotype switching from IgM to IgG and IgA. Immunoglobulin isotype switching in B lymphocytes requires a contact-dependent signal from T lymphocytes which is delivered by the ligand for the B cell surface antigen CD40. We investigated the capacity of newborn lymphocytes to express the CD40 ligand and to undergo CD40 ligand-dependent immunoglobulin isotype switching. After stimulation by phorbol ester and ionomycin, newborn lymphocytes expressed markedly decreased amounts of CD40 ligand on their surface compared to normal adult lymphocytes. Northern blot analysis of mRNA derived from activated cord blood lymphocytes also revealed markedly decreased amounts of CD40 ligand mRNA. Decreased expression of CD40 ligand in newborn lymphocytes was associated with a severely decreased ability to undergo T cell-dependent immunoglobulin isotype switching. Newborn lymphocytes synthesized little or no detectable IgE in response to T cell-dependent stimulation by interleukin-4 but synthesized IgE in response to T cell-independent stimulation by CD40 monoclonal antibody and interleukin-4. These results indicate that decreased expression of CD40 ligand in newborn lymphocytes may be the underlying cause of deficient immunoglobulin isotype switching in newborns.     

Lack of Correlation Between Membrane CD30 Expression and Cytokine Secretion Pattern in Allergen-Primed Naive Cord Blood T-Cell Lines and Clones

Various surface molecules are expressed by activated T cells. Among them, the CD30 antigen has been proposed as a reproducible marker that identifies a subset of differentiated and/or activated T lymphocytes that produce T helper (Th)-2-type cytokines, i.e. interleukin (IL)-4 and IL-5. However, because CD30 has mainly been detected on established T-cell clones, it is still unclear whether a priming allergen and/or cytokine can induce its membrane expression on naive T cells, perhaps in parallel with the up-regulation of other relevant activation markers, such as CD25, HLA-DR and L-selectin. It is also unknown whether proper allergen stimulation affects the cytokine secretion pattern by CD30⁺ T-cell clones derived from antigen-unprimed (naive) T lymphocytes. More information on these questions was sought by adopting a model that used cord blood as a source of virgin T cells and exposing them to native cypress allergen or cytokine (IL-2 or IL-4) stimulation, as well as to conventional polyclonal activators such as PHA or anti-CD3. Peripheral blood MC from four adult cypress-sensitive patients was also assayed and used as controls for all culture experiments. Freshly isolated cord and adult T cells did not express the CD30 antigen on their membrane. Many of the stimulating agents tested were able to up-regulate the expression of CD30. However, despite high expression of this molecule, cloned allergen-specific cord CD4⁺ T lymphocytes were unable to produce IFN-γ and/or IL-4. In contrast, they retained the capability to produce IL-2. Thus, expression of the CD30 antigen on virgin T cells does not correlate with a polarized model of T helper (Th)-1 or Th-2 cytokine-producing cells, suggesting that these types of lymphokine-secreting lymphocytes are not a paradigmatic example of T-cell subpopulations that display stable phenotypical features.     

Impact of cAMP on the T-cell response to type II collagen

There is considerable interest in the possible use of cAMP-elevating agents in the treatment of autoimmune diseases such as rheumatoid arthritis. The objective of this study was to evaluate the impact of different cAMP-elevating agents on the T-cell response to type II collagen within the context of collagen-induced arthritis, a murine model of rheumatoid arthritis. Spleen cells or lymph node cells from type-II-collagen-immunized DBA/1 mice were cultured in the presence of type II collagen plus one of five different cAMP-elevating agents: rolipram, forskolin, prostaglandin E2, 8-bromo-cAMP, or cholera toxin. Levels of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and IL-5 were measured in culture supernatants by enzyme-linked immunosorbent assay. All of the cAMP-elevating agents tested were found to profoundly suppress IFN-gamma production in a dose-dependent manner. IL-4 and IL-5 production was slightly up-regulated at low concentrations of the cAMP-elevating agents and was modestly suppressed at the highest concentrations of cAMP-elevating agents. Experiments were then carried out to determine whether T cells were directly affected by cAMP-elevating agents or whether the immunomodulatory effects were mediated via antigen-presenting cells. Pulsing T cells alone for a brief period with cholera toxin produced an almost identical effect to pulsing antigen-presenting cells alone, i.e. down-regulation of proliferation, down-regulation of IFN-gamma production with little effect on IL-5 production. It was concluded that cAMP-elevating agents suppressed T helper type 1 responses to type II collagen to a greater extent than T helper type 2 responses. The cAMP-elevating agents could directly influence the activity of T cells but, in addition, influenced the ability of antigen-presenting cells to support T helper type 1 responses.     

佛波醇酯加离子霉素诱导脐血和成人外周血CD4+CD25+ T细胞分泌IL-2的相关机制研究

目的：以佛波醇酯加离子霉素作为刺激剂,验证CD4+CD25+ 调节性T细胞本身并不存在分泌IL-2障碍；同时通过对脐血和成人外周 血的比较性研究，了解脐血CD4+CD25+ T细胞的成熟度。方法：以au toMACS从足月婴儿脐血（CB）和成人外周血（PB）分选CD4+CD25+和CD4+CD25-T细 胞，以PDB+ionomycin作为刺激剂，培养45 h后流式细胞术检测各组细胞表达CD69和CD25水 平，并以Luminex多重细胞因子检测技术检测培养上清中7种细胞因子的浓度。结果:经PDB+ionomycin刺激后，CB、PB的CD4+CD25+ 和CD4+CD25- T细胞均 发生增殖，但在培养 45 h 后CD4+CD25+ T细胞均出现细胞状态变差或死亡倾向。C B、PB 的CD4+CD25+ T细胞活化后CD25分子表达进一步上调，高于CD25-细胞活化后的CD25分 子密度。经PDB+ionomycin刺激后，PB CD4+CD25+和CD4+CD25- T细胞均分泌高水平 的IFN-γ、IL-2和TNF-α，但CD25+ 细胞分泌IL-5、IL-4和IL-10水平远远高于CD25-细 胞；CB CD4+CD25+和CD4+CD25- T细胞亦分泌高水平的IL-2和TNF-α，但IFN-γ水 平远远低于PB，基本不分泌IL-5、IL-4和IL-10。结论：CD4+CD25+ T细胞本身并不存在合成和分泌IL-2障碍，其可能具有与传统T细胞不同的T细胞受体信息转 导模式；脐血CD4+CD25+ T细胞功能尚未完全成熟。     

Expansion of regulatory CD8+ CD25+ T cells after neonatal alloimmunization

Transplantation tolerance induced by neonatal injection of semi-allogeneic spleen cells is associated with a pathological syndrome caused by T helper type 2 (Th2) differentiation of donor-specific CD4(+) T lymphocytes. We have shown previously that this Th2-biased response is inhibited by host CD8(+) T cells. Herein, we demonstrate that upon neonatal immunization with (A/J × BALB/c)F(1) spleen cells, BALB/c mice expand a population of CD8(+) T cells expressing both CD25 and forkhead box P3 (FoxP3) markers. In this setting, CD8(+) CD25(+) T cells predominantly produce interferon (IFN)-γ and interleukin (IL)-10 and are efficient in controlling IL-4, IL-5 and IL-13 production by donor-specific CD4(+) T cells in vitro. CD8(+) FoxP3(-) T cells are single producers of IFN-γ or IL-10, whereas CD8(+) FoxP3(+) T cells are double producers of IFN-γ and IL-10. We further demonstrate that IFN-γ and IL-10 are two major cytokines produced by CD8(+) T cells involved in the in vivo regulation of Th2-type pathology. In this setting, we conclude that neonatal alloimmunization induces the expansion of several regulatory CD8(+) T cells which may control Th2 activities via IFN-γ and IL-10.     

Interleukin-15 up-regulates the expression of CD154 on synovial fluid T cells

To investigate the role of the CD40-CD154 interaction in rheumatoid arthritis (RA), we analysed the expression of CD154 on CD3+ and CD4+ T cells in synovial fluid (SF) from patients with RA and in peripheral blood (PB) from patients and normal controls. As interleukin (IL)-15 is a potent activator of synovial T cells we wanted to study whether IL-15 also regulated the expression of CD154 on these T cells. Freshly isolated synovial T cells did not express significant levels of CD154, as evaluated using flow cytometry, whereas the expression of CD86 and human leucocyte antigen (HLA)-DR was significantly elevated on SF T cells when compared with PB T cells from patients or controls. Synovial T cells could up-regulate their CD154 expression following activation with phorbol 12-myristate 13-acetate (PMA) + ionomycin or anti-CD3 + anti-CD28 monoclonal antibodies (mAbs), but the maximal level of expression remained lower than in control T cells. IL-15 significantly increased the expression of CD154 on SF and PB T cells from patients, whereas IL-2 had minimal effects. Furthermore, IL-15 induced extensive proliferation in SF T cells. Our results show that SF T cells up-regulate the expression of CD154 in the presence of IL-15, a cytokine present in the synovium of patients with RA. These results further emphasize the role of IL-15 in the pathogenesis of RA.     

新生儿脐血淋巴细胞IL-2R的表达

白细胞介素2(IL-2)及其受体(IL-2R)系统在免疫调节中发挥着重要的作用。本研究采用间接免疫荧光技术检测了新生儿脐血淋巴细胞IL-2R表达情况及其T细胞亚类水平。结果表明健康足月新生儿脐血Tac阳性细胞百分率明显低于正常成人,且异常新生儿的减低更为明显;而健康足月新生儿脐血CD_4阳性细胞和CD_4 CD_8比值却明显高于正常成人。说明尽管新生儿具有足够数量的T细胞(CD_3阳性细胞)和辅助性T细胞(CD_4阳性细胞),且CD_4/CD_3比值还高于成人,但其活化淋巴细胞上IL-2R的表达不足,故影响IL-2作用的发挥;加之新生儿细胞表面CD抗原标志与自身功能发育的非同步性,导致其细胞免疫功能低下。     

IL-7 promotes the survival and maturation but not differentiation of human post-thymic CD4+ T cells

This study examines the influence of IL-7 on post-thymic CD4⁺ T cells using cord blood as a model system. Survival of naive cord blood T cells in the presence of IL-7 alone was significantly prolonged by up-regulating bcl-2, thereby preventing apoptosis while maintaining maximal cell viability. Cultures without IL-7 showed high rates of apoptosis resulting in 50 % cell death by day 5 of culture. Upon phorbol 12-myristate 13-acetate + ionomycin stimulation, accumulation of cytoplasmic IL-2 was similar to that observed in freshly isolated cells, but no IL-4- or IFN-γ-positive cells were detected. IL-7 maintained the naive T cells in a quiescent state expressing the CD45RA antigen. A significant finding was the loss of CD38 antigen expression on the naive cord blood T cells to levels similar to that observed on adult naive T cells. In contrast to the reduced proliferative response of fresh cord blood T cells to anti-CD2 + CD28 stimulation, the proliferative response of IL-7-treated cells was similar to that of adult naive T cells. This study shows that as well as maintaining the naive T cell pool by enhancing cell survival and up-regulating bcl-2 expression, IL-7 also functions as a maturation factor for post-thymic naive T cells.     

Signalling via CD28 of human naive neonatal T lymphocytes.

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the 'naive' neonatal T cell response to anti-CD2-mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti-CD2 antibodies in the presence or absence of anti-CD28 MoAb. With anti-CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL-2, whereas adult T cells proliferated vigorously, with significant IL-2 production. Costimulation with anti-CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL-2 secretion was increased in the presence of anti-CD28 MoAb, neonatal T cell IL-2 production remained lower than in adults. In contrast, enhancement of IL-2 mRNA expression in neonates was similar to adult levels. Anti-CD28 MoAb costimulation increased NF kappa B levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti-CD2 may therefore be due to decreased NF kappa B induction, reduced IL-2 mRNA expression and deficient IL-2 production. Although anti-CD28 MoAb costimulation enhances all of the above signals, NF kappa B and IL-2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate.     

Changes in intracellular cytokine levels in newborn and adult lymphocytes induced by HSV-1

Changes in the expression of intracellular interleukin-2 (IL-2), interleukin-4 (IL-4), interferon (IFN)-γ, and tumor necrosis factor (TNF)-α in newborn and adult lymphocytes induced by herpes simplex virus (HSV)-1 were examined. Cord blood mononuclear cells (CBMC) or adult peripheral blood mononuclear cells (PBMC) were infected with HSV-1 and cultured with phorbol 2-myristate 13-acetate (PMA) plus ionomycin in the presence of monensin for 4 hr. Surface antigen and intracellular cytokines were stained simultaneously and analyzed by flow cytometry. The percentage of cells that expressed IL-2, IFN-γ, and TNF-α was significantly increased in HSV-1-infected CD3⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45R0⁺ lymphocytes compared with uninfected lymphocytes from adult PBMC. The percentage of cells that expressed IL-2 and TNF-α was increased significantly in HSV-1-infected CD3⁺, CD4⁺, CD8⁺, and CD45RA⁺ lymphocytes compared with uninfected lymphocytes from CBMC. IFN-γ was under the detectable level in HSV-1-infected and uninfected lymphocytes from CBMC. Intracellular IL-4 was not detected in HSV-1 or in uninfected lymphocytes from PBMC and CBMC. These results demonstrate that HSV-1 enhances intracellular levels of IL-2, IFN-γ, and TNF-α in adult lymphocytes and defective IFN-γ production in cord blood. J. Med. Virol. 56:145–150, 1998. © 1998 Wiley-Liss, Inc.     

Rapid conversion of naive to effector T cell function counteracts diminished primary human newborn T cell responses.

The reduced incidence of graft versus host disease following the use of human cord blood as a source of stem cells for bone marrow reconstitution challenges our understanding of the immunocompetence of newborn T cells. Newborn CD4+ T cells express mainly the CD45RA phenotype and have been considered to respond comparably to adult CD4+ T cells exhibiting the CD45RA phenotype. We compared the in vitro kinetics of phenotypic conversion of newborn and adult CD4+CD45RA+ T cells to CD4+CD45RO+ T cells. The cytokine profile and B cell helper activity of the converted CD4+CD45RO+ T cell population were also determined. Newborn CD4+CD45RA+ T cells were converted to CD4+CD45RO+ with significantly faster time kinetics than adult CD4+CD45RA+ T cells, following either phytohaemagglutinin (PHA) or anti-CD2 activation. Freshly purified newborn naive T cells did not produce IL-2, IL-4 or interferon-gamma (IFN-gamma) following stimulation, whereas adult naive T cells secreted IL-2 and adult-derived CD4+CD45RO+ T cells secreted all three cytokines under the same stimulatory conditions. However, newborn and adult CD4+CD45RA+ T cells, following primary stimulation and maturation in vitro, acquired the ability to secrete a Th1-type cytokine profile of IL-2 and IFN-gamma after secondary stimulation. Newborn CD4+ naive T cells that acquired the CD45RO phenotype in vitro also gained B cell helper activity equivalent to that of adult in vitro matured CD4+ naive T cells. These findings suggest that newborn and adult CD4+CD45RA+ T cell subsets are differentially responsive to various stimuli. They show that newborn CD4+CD45RA+ naive T cells can transform more quickly than their adult counterparts into functionally equivalent CD4+CD45RO+ T cells, a process that may be important to counteract the immature immune environment which exists in the newborn.     

Interleukin-1 Augments the Diminished Interleukin-2 mRNA Expression and Proliferative Response of Neonatal T Lymphocytes to Anti-CD2 Antibodies

Expression of IL-2 mRNA by unstimulated and stimulated purified T cells and mononuclear cells from adult and cord blood was investigated in an attempt to better understand the underlying defective neonatal host immune defense system. Using RNA dot-blot analysis, IL-2 mRNA expression in anti CD2-stimulaled neonatal T cells revealed significantly reduced levels when compared to adult T cells (P<0.01). Purified neonatal T cells also showed a significantly reduced proliferative response to antiCD2 antibodies (P<0.01). Addition of IL-1ß enhanced the hyporesponsiveness of neonatal T cells at both the level of proliferation and IL-2 mRNA expression. Unseparated mononuclear cells from adult and cord blood revealed similar IL-2 mRNA levels and proliferation when activated by anti-CD2 stimulation. The reduced IL-2 mRNA expression observed in neonatal T cells may explain, in part, the difference in host defense between the newborn and adult during states of increased demand such as infection.