Enhancement of B7-1 (CD80) expression on B-lymphoma cells by irradiation

Irradiation of A20.2J mouse B-lymphoma cells enhanced their antigen-presenting ability to induce interleukin-2 (IL-2) production by 42-6A T cells specific for ovalbumin (OVA)323-339/I-Ad. Irradiated and fixed A20.2J cells were more efficient antigen-presenting cells (APC) to present OVA323-339 peptide than the unirradiated and fixed cells. Irradiation selectively increased the expression of B7-1 molecules, but not of the major histocompatibility complex class II molecules, B7-2, lymphocyte function-associated antigen-1, or intracellular adhesion molecule-1. Irradiation of A20.2J cells with 100 Gy followed by overnight incubation was optimal for the enhancement of B7-1 expression. Anti-B7-1 monoclonal antibody inhibited the irradiation-induced enhancement of APC function. Irradiation of A20.2J cells induced the accumulation of B7-1 mRNA. Thus, it was concluded that the enhancement of APC function by irradiation was due to the up-regulation of B7-1 molecules through the accumulation of its mRNA. Although partial inhibition of protein synthesis has been shown to enhance the accumulation of B7-1 mRNA and its expression, irradiation did not decrease the protein synthesis in A20.2J cells. The incubation with irradiated A20.2J cells stimulated unirradiated A20.2J cells to increase B7-1 expression, suggesting that irradiation of A20.2J cells induced expression or secretion of some molecule(s) to enhance B7-1 expression.     

Cholera toxin increases intracellular pH in B lymphoma cells and decreases their antigen-presenting ability.

Cholera toxin (CT) can function as a potent adjuvant in the mucosal immune response. However, we have found that treatment of A20-HL murine B lymphoma cells with CT severely inhibits the presentation of ovalbumin (OVA) to cells of the T cell clone 42-6A specific for OVA(323-339)/I-Ad, whereas it does not affect the presentation of OVA(323-339) peptide. CT treatment did not affect the expression of B7-1, B7-2, ICAM-1, LFA-1 or MHC class II on, or the internalization of OVA into A20-HL cells. In CT-treated A20-HL cells, degradation of OVA was decreased, and intracellular pH was raised to a level approximately equivalent to that in CH3NH2-treated cells. Treatment with CH3NH2 is known to raise the pH in endocytic structures and thus inhibits antigen processing. Treatment of A20-HL cells with dibutyryl-cAMP similarly increased intracellular pH. The increase in intracellular pH following CT treatment was inhibited by a cAMP inhibitor, 2',3'-dideoxyadenosine. These results strongly suggest that CT treatment of A20-HL cells inhibits their antigen-presenting cell function by triggering the cAMP cascade, increasing intracellular pH, and reducing the degradation of OVA.     

Irradiation up-regulates CD80 expression through two different mechanisms in spleen B cells, B lymphoma cells, and dendritic cells

We have previously demonstrated irradiation-induced up-regulation of CD80 expression in A20-HL B lymphoma cells by inducing expression of tumour necrosis factor-alpha (TNF-alpha) and CD154. In the present study, we investigated whether irradiation also up-regulates CD80 expression in mouse spleen B cells. Because freshly prepared spleen B cells are highly sensitive to irradiation, we employed spleen B cells stimulated with lipopolysaccharide (LPS-B cells). X-irradiation (8 Gy) followed by incubation (9-12 hr) highly and selectively up-regulated CD80 expression in LPS-B cells, whereas the same treatment slightly increased expression of CD54 and did not affect expression of CD86, major histocompatibility complex class II, CD11a or surface immunoglobulin M. The irradiation-induced up-regulation of CD80 expression resulted in enhanced APC function of LPS-B cells. Up-regulation of CD80 expression on LPS-B cells was accompanied by an increase in CD80 mRNA accumulation and nuclear factor (NF)-kappaB activation. Activation of NF-kappaB was shown to be critical for up-regulation of CD80 expression as pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, severely decreased the observed up-regulation. X-irradiation of LPS-B cells induced expression of TNF-alpha but not CD154. However, anti-TNF-alpha monoclonal antibody (mAb) with anti-CD154 mAb did not inhibit X-irradiation-induced up-regulation of CD80 expression in LPS-B cells, whereas these mAbs almost completely inhibited this up-regulation in A20-HL cells and bone marrow-derived dendritic cells (DCs). In contrast, a thiol antioxidant, N-acetyl-l-cysteine, completely blocked X-irradiation-induced up-regulation of CD80 expression in LPS-B cells, but not in A20-HL cells or in DCs. Based on these findings, we concluded that X-irradiation up-regulates CD80 expression not only in A20-HL cells and DCs but also in LPS-B cells, and that this up-regulation in LPS-B cells via NF-kappaB activation is dependent on the generation of reactive oxygen species, while that in A20-HL cells and DCs is not.     

Differences in antigen processing with haplotype-mismatched MHC class II heterodimers: Aalpha(d)Abeta(b) heterodimers participate in early endosomal processing

MHC class II heterozygotes form haplotype-mismatched heterodimers (combining alpha and beta chains of different alleles). Transfected L-cells expressing Aalpha(d)Abeta(d) or Aalpha(d)Abeta(b) presented exogenous OVA(323-339) peptide to T cells with similar high efficiency, while Aalpha(b)Abeta(b) was less efficient and Aalpha(b)Abeta(d) was ineffective. In contrast, Aalpha(d)Abeta(b) greatly exceeded Aalpha(d)Abeta(d) in processing of intact OVA for presentation of OVA(323-339); Aalpha(b)Abeta(b) was even less efficient and Aalpha(b)Abeta(d) was ineffective. Of macrophages from C57BL/6 (H-2(b)), DBA/2 (H-2(d)) and B6D2F1 (H-2(bxd))mice, B6D2F1 macrophages had highest I-A expression and efficiency for OVA processing or presentation of exogenous OVA(323-339) peptide. Blocking antibodies specific for I-A chains showed that OVA processing by B6D2F1 macrophages primarily involved haplotype-mismatched Aalpha(d)Abeta(b) heterodimers, whereas Aalpha(d)Abeta(d) and Aalpha(b)Abeta(b) contributed more to presentation of exogenous OVA(323-339) peptide. OVA(323-339):I-A complexes were formed from OVA within 10 min with B6D2F1 macrophages but not until 20 min with C57BL/6 or DBA/2 macrophages, and OVA processing was more resistant to inhibition of late endocytic function by hypothermia (18 degrees C) in B6D2F1 than C57BL/6 or DBA/2 macrophages. These results indicate that Aalpha(d)Abeta(b) haplotype-mismatched heterodimers may contribute to antigen processing in early endocytic compartments.     

CD4+ T cells specific for a model latency-associated antigen fail to control a gammaherpesvirus in vivo

CD4(+) T cells play a major role in containing herpesvirus infections. However, their cellular targets remain poorly defined. In vitro CD4(+) T cells have been reported to kill B cells that harbor a latent gammaherpesvirus. We used the B cell-tropic murine gammaherpesvirus-68 (MHV-68) to test whether this also occurred in vivo. MHV-68 that expressed cytoplasmic ovalbumin (OVA) in tandem with its episome maintenance protein, ORF73, stimulated CD8(+) T cells specific for the H2-K(b)-restricted OVA epitope SIINFEKL and was rapidly eliminated from C57BL/6 (H2(b)) mice. However, the same virus failed to stimulate CD4(+) T cells specific for the I-A(d)/I-A(b)-restricted OVA(323-339) epitope. We overcame any barrier to the MHC class II-restricted presentation of an endogenous epitope by substituting OVA(323-339) for the CLIP peptide of the invariant chain (ORF73-IRES-Ii-OVA), again expressed in tandem with ORF73. This virus presented OVA(323-339) but showed little or no latency deficit in either BALB/c (H2(d)) or C57BL/6 mice. Latent antigen-specific CD4(+) T cells therefore either failed to recognize key virus-infected cell populations in vivo or lacked the effector functions required to control them.     

Selective enhancement of B cell antigen receptor-mediated antigen presentation by treatment with transforming growth factor-beta

TGF-beta1 was examined for the ability to regulate Ag-presentation by B cells, using A20-HL B lymphoma cells bearing TNP-specific IgM receptors. Treatment of A20-HL cells with TGF-beta1 at 1 ng/ml, a concentration that inhibited proliferation, enhanced presentation of Ag internalized via surface IgM (sIgM), but not via fluid-phase pinocytosis. TGF-beta1-treatment slightly enhanced surface expression of sIgM, but not of MHC class II molecules. The treatment accelerated recovery of sIgM expression after its removal by ligation with TNP-OVA, and induced prolonged intracellular residence of TNP-OVA internalized via sIgM, which co-localized with intracellular MHC class II molecules. TGF-beta1-treatment increased accumulation of newly synthesized intracellular MHC class II molecules that were localized in compartments positive for lysosome-associated membrane protein 1, although cellular protein synthesis was decreased by the treatment. The accumulated intracellular MHC class II molecules were triggered to the cell surface by ligation of sIgM. Finally, TGF-beta1-treatment induced Igalpha-phosphorylation in response to lower concentrations of TNP-OVA. On the basis of these findings, we conclude that TGF-beta1-treatment of A20-HL cells selectively enhances the ability to present Ag internalized via sIgM, not via fluid-phase pinocytosis, through accelerating sIgM recovery, increasing accumulation of intracellular MHC class II molecules and enhancing the ability of sIgM ligation to induce Igalpha-phosphorylation.     

抗原特异性T细胞活化后Foxp3、CTLA-4及细胞因子mRNA表达水平的动态分析

目的研究抗原特异性T细胞活化后,其所表达的Foxp3、CTLA-4和细胞因子的动态变化,为深入了解特异性免疫应答调控奠定基础。方法体外采用树突状细胞(DC)系D2SC/1作为抗原提呈细胞(APC)将OVA323-339直接提呈给OVA特异性TCR转基因小鼠DO11.10来源的CD4~+T细胞。采用CCK-8法测定细胞活化增殖情况,并应用Real time-PCR动态分析细胞因子IL-2、IFN-γ、IL-10、TGF-β,CTLA-4和Foxp3的mRNA表达水平。结果OVA特异性T细胞活化所需的最佳OVA剂量为2μmol/L,T细胞活化后IL-2、IFN-γ、IL-10和TGF-βmRNA表达水平最高峰值分别在8、4、2 h和8 h,Foxp3 mRNA在2 h时有一过性的表达峰,随后在24 h又有所升高,而CTLA-4 mRNA在静止时有较高水平的表达,但活化后迅速下调,其在24 h内无明显变化。结论初步了解了适应性免疫应答中Foxp3、CTLA-4和主要免疫调节相关的细胞因子表达趋势,为深入研究适应性免疫应答调控机制奠定初步基础。     

Comparison between Ovalbumin and Ovalbumin Peptide 323‐339 Responses in Allergic Mice: Humoral and Cellular Aspects

Ovalbumin (OVA) is widely used in allergy research. OVA peptide 323‐339 has been reported to be responsible for 25–35% of isolated BALB/c mouse T‐cell response to intact OVA. An investigation of whether OVA and OVA 323‐339 molecules can induce equivalent in vivo and in vitro immune responses was conducted. Eight‐week‐old BALB/c mice were randomly divided into three groups: OVA, OVA 323‐339 and saline. On days 0, 7, 14, mice were intraperitoneally injected with 25 μg OVA or OVA 323‐339 absorbed on 300 μg Alum, or saline; on days 21–23, all groups were challenged intranasally with either 20 μl of 1% OVA, 1% OVA 323‐339 or saline. On day 28, after killing, splenocytes were isolated and cultured under the stimulus of each allergen or medium. Evaluated by hematoxylin/eosin and major basic protein immunohistochemical stainings, OVA and OVA 323‐339 induced similar lung inflammation. Interestingly, significant serum total IgE and OVA‐specific IgE were observed in OVA mice when compared to saline control. OVA 323‐339 mice showed higher serum OVA‐specific IgE, OVA 323‐339‐specific IgE, IL‐4 and lower IFN‐γ similar to OVA mice. The proliferative response to OVA was found in cultured splenocytes of both OVA and OVA 323‐339 mice, while the similar proliferative response to OVA 323‐339 was only observed in the splenocytes of OVA 323‐339‐sensitized and challenged mice. Although OVA 323‐339 induced a Th2‐like response in the mouse model as did OVA, OVA 323‐339 has clearly limited immunogenic potency to activate OVA‐sensitized and challenged mice splenocytes, unlike OVA.     

1142280946Effect of estradiol and PAMPs on class II mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

Problem:  Antigen presenting cells (APC) in the female reproductive tract play important roles in innate immune defense and activation of the adaptive immune responses. The objective of this study was to examine the effects of estradiol and PAMP on antigen presentation in the female reproductive tract.
Method of Study:  DO11.10 T cell antigen receptor transgenic mice specific for the MHC class II-restricted OVA323–339peptide were used to study the effects of estradiol and PAMP on antigen presentation of OVA by uterine epithelial (EC) and stromal cells as well as vaginal cells to OVA specific memory-T cells.
Results:  Estradiol inhibited antigen presentation of OVA by uterine EC, uterine stromal cells and vaginal cells to OVA specific memory-T cells. When ovariectomized animals were treated with estradiol for 1 or 3 days, antigen presentation decreased by 20–80%. In contrast, incubation with TLR agonists increased antigen presentation by EC (Poly (I:C), Pam3Cys), stromal cells (PGN, Pam3Cys) and vaginal cells (LPS, Pam3Cys). Analysis of mRNA expression by real time RT-PCR indicated that estradiol inhibited CD40, CD80/86 and class II in the uterus and vagina. In contrast, stimulation of antigen presentation by PAMP did not correlate with changes in costimulatory molecule mRNA expression.
Conclusions:  These results indicate that APC in the uterus and vagina are responsive to estradiol, which inhibits antigen presentation and costimulatory molecule expression. These findings suggest that whereas APC in the uterus and vagina respond to TLR agonists with increased antigen presentation, which initiates an adaptive immune response, their effects appear to be at levels other than the expression of costimulatory molecules.
Acknowledgement:  Supported by AI-13541 from NIH.     

Synthesized OVA323-339MAP octamers mitigate OVA-induced airway inflammation by regulating Foxp3 T regulatory cells

ABSTRACT: BACKGROUND: Antigen-specific immunotherapy (SIT) has been widely practiced in treating allergic diseases such as asthma. However, this therapy may induce a series of allergic adverse events during treatment. Peptide immunotherapy (PIT) was explored to overcome these disadvantages. We confirmed that multiple antigen peptides (MAPs) do not cause autoimmune responses, which led to the presumption that MAPs intervention could alleviate allergic airway inflammation without inducing adverse effects. RESULTS: In this study, synthesized OVA323-339MAP octamers were subcutaneously injected into ovalbumin (OVA)-sensitized and -challenged Balb/c mice to observe its effect on allergic airway inflammation, Th2 immune response, and immune regulating function. It was confirmed that OVA sensitization and challenge led to significant peritracheal inflammatory, cell infiltration, and intensive Th2 response. Treatment of OVA323-339MAP octomers in the airway inflammation mice model increased CD4+CD25+Foxp3+ T regulatory (Treg) cells and their regulatory function in peripheral blood, mediastinal draining lymph nodes, and the spleen. Furthermore, OVA323-339MAP increased IL-10 levels in bronchial alveolar lavage fluid (BALF); up-regulated the expression of IL-10,membrane-bound TGF-beta1, as well as Foxp3 in lung tissues; and up-regulated programmed death-1 (PD-1) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4) on the surface of Treg cells. These results were further correlated with the decreased OVA specific immunoglobulin E (sIgE) level and the infiltration of inflammatory cells such as eosinophils and lymphocytes in BALF. However, OVA323-339 peptide monomers did not show any of the mentioned effects in the same animal model. CONCLUSIONS: Our study indicates that OVA323-339MAP had significant therapeutic effects on mice allergic airway inflammation by regulating the balance of Th1/Th2 response through Treg cells in vivo. Key words Allergic airway inflammation; Specific immunotherapy; Multiple antigen peptide.     

Murine neutrophils present Class II restricted antigen

Neutrophils were originally described as short lived, terminally differentiated phagocytes that contribute only to the innate immune response. Recent evidence of neutrophil cytokine production and expression of numerous cell surface proteins has suggested that neutrophils are likely to influence adaptive responses and may satisfy the criteria of antigen presenting cells. Under certain inflammatory conditions human neutrophils express major histocompatibilty complex (MHC) Class II and the costimulatory molecules CD80 and CD86. We have employed a murine T cell hybridoma with a transgenic T cell receptor specific for ovalbumin peptide 323-339 (OVA(323-339)), and a green fluorescent reporter of T cell receptor ligation, to directly investigate neutrophil-T cell interactions. These cells provide an ideal model system, allowing precise identification of antigen specificity and a clear readout of T cell activation. Additionally, whilst murine neutrophils have previously been shown to stimulate MHC Class I-dependent CD8(+) T cell activation, CD4(+) T cells stimulation via MHC Class II-expressing neutrophils has not been investigated. We addressed this by isolating murine neutrophils, loading with OVA(323-339) and co-culturing with T cells specific for the OVA(323-339)/MHC Class II complex, and this resulted in T cell activation, as determined by expression of the green-fluorescent protein reporter. Antigen-pulsed neutrophils were also able to prime na?ve OVA-specific CD4(+) T cells in a contact-dependent manner, as shown by proliferation and cytokine production. Activation of lymphocytes was not due to contaminating macrophages. These studies demonstrate that murine neutrophils present MHC Class II-restricted peptides and induce T cell proliferation, confirming findings in human neutrophils, and demonstrate a novel pro inflammatory effect of murine neutrophils.     

Requirement of certain epitope specificities of glycosylation inhibiting factor for the suppression of in vivo IgE and IgG antibody responses.

The ovalbumin (OVA)-specific T cell hybridoma 71B1, which constitutively secretes glycosylation inhibiting factor (GIF) and is specific for the immunogenic epitope represented by amino acids 323-339 in the OVA molecules, failed to form GIF having affinity for nominal antigen upon stimulation with OVA-pulsed antigen-presenting cells (APC). However, the GIF produced by the antigen-stimulated 71B1 cells bound to the mAb 14-12, which is specific for the antigen-binding chain of effector type suppressor T cell factor (TseF), and to mAb specific for TCR. The GIF constitutively released from unstimulated 71B1 cells failed to bind to any of these antibodies. Gel filtration of GIF preparations showed that the 14-12+ GIF from the antigen-stimulated 71B1 cells are composed of 80-100 and 25-35 kDa species, while the GIF from unstimulated cells was 12-15 kDa. Reduction and alkylation treatment of the GIF from the antigen-stimulated cells resulted in the disappearance of the 80-100 and 25-35 kDa GIF, which was accompanied by the formation of the 12-15 kDa GIF. Thus, the GIF from the antigen-stimulated 71B1 cells was similar to the previously described OVA-binding GIF from the 231F1 cells with respect to their antigenic structures and molecular size, and both factors appear to be composed of the 14-12+ polypeptide chain and 12-15 kDa non-specific GIF. However, the GIF from the antigen-stimulated 71B1 cells lacked affinity for the native OVA or synthetic peptide 323-339, and failed to suppress the in vivo antibody response to dinitrophenyl (DNP)-OVA. In contrast, the OVA-binding GIF has affinity for native OVA and the peptide 307-317, to which the cell source of the factor is specific, and suppressed the in vivo anti-hapten antibody response to DNP-OVA. The results suggest that formation of antigen-specific TsF is confined to T cells with certain epitope specificities. It was also found that the OVA-binding GIF failed to suppress the in vivo anti-hapten antibody response to DNP-conjugates of urea-denatured OVA (UD-OVA), which does not bind OVA-binding GIF. However, APC pulsed with UD-OVA appear to express the epitope 307-317 for which the OVA-binding GIF has affinity. The results collectively suggest that the affinity of GIF for an immunizing antigen, rather than processed antigen, is required for immunosuppression.     

Sel1L 对小鼠骨髓源树突状细胞的影响

目的:研究Sel1L(Suppressor/ enhancer of Lin-12-like)基因对小鼠骨髓源树突状细胞(BMDCs)分化及其功能的影响。方法:利用Cre-Loxp 重组系统构建Sel1L 基因敲除小鼠模型,用ELISA 和实时荧光定量法检测BMDCs 分泌细胞因子IL-6、IL-12 的表达;用免疫印迹法(Western bolt)检测BMDCs 细胞中Sel1L蛋白的表达;用流式细胞术检测BMDCs 细胞CFSE、细胞表面分子CD80、CD86、MHC-Ⅰ、MHC-Ⅱ及其对特异性CD4⁺ T 细胞抗原提呈能力的影响。结果:Sel1L的缺失抑制BMDC诱导分化过程中的增殖效率,上调DCs 分泌因子的能力和MHC-Ⅰ的表达,减少MHC-Ⅱ的表达,并抑制BMDCs 细胞对OVA323-339 抗原特异性T 细胞的增殖。结论:Sel1L 缺失可以抑制小鼠骨髓源树突状细胞的分化,下调DC 对OVA 特异性的CD4⁺ T细胞的抗原提呈功能。     

Tumour necrosis factor-alpha but not lipopolysaccharide enhances preference of murine dendritic cells for Th2 differentiation

Using murine spleen-derived dendritic cells (DC) and DO11.10 T cells specific for ovalbumin (OVA), the influences of maturational condition and antigen dose on the capability of DC to induce helper T-cell (Th) differentiation were analysed. Immature DC (iDC) with high- or low-dose OVA(323-339) predominantly induced Th1 or Th2 responses in DO11.10 T cells, respectively. DC matured by tumour necrosis factor-alpha (TNF/DC) induced a significantly higher Th2 response in the presence of low-dose OVA(323-339) than iDC and DC matured by lipopolysaccharide (LPS) (LPS/DC). In the presence of high-dose OVA(323-339), LPS/DC induced significantly lower levels of Th1 response than iDC. Under these conditions no difference in the Th1 response was noted between TNF/DC and iDC. The enhanced capability of TNF/DC with a low-dose antigen for Th2 polarization and the decreased preference of LPS/DC with a high-dose antigen to Th1 polarization were not related to the amount of IL-12 produced in these cultures. These results demonstrate for the first time that TNF/DC with a low-dose antigen are potent inducers of Th2 differentiation.     

Opposite effects of immunotherapy with ovalbumin and the immunodominant T-cell epitope on airway eosinophilia and hyperresponsiveness in a murine model of allergic asthma.

In the present study, we investigated immunotherapy using an entire protein or an immunodominant epitope in a murine model of allergic asthma. Immunotherapy was performed in ovalbumin (OVA)-sensitized mice before OVA challenge. Mice were treated subcutaneously with OVA, the immunodominant epitope OVA323-339, or vehicle. In vehicle-treated animals, repeated OVA challenge induced increased serum levels of OVA-specific immunoglobulin (Ig)G1, IgE, airway eosinophilia, and hyperresponsiveness, compared with saline-challenged animals. In addition, interleukin (IL)-4 and IL-5 production upon OVA restimulation of lung-draining lymph node cells in vitro were significantly increased in OVA-challenged animals. Immunotherapy using OVA significantly reduced airway eosinophilia and hyperresponsiveness. This finding was accompanied by significantly reduced OVA-specific IL-4 and IL-5 production. Further, OVA immunotherapy induced increased serum levels of OVA-specific IgG1, whereas OVA-specific IgG2a and IgE levels were not affected. In contrast to OVA immunotherapy, immunotherapy with OVA323-339 aggravated airway eosinophilia and hyperresponsiveness. OVA-specific IgG1, IgG2a, and IgE serum levels, and in vitro IL-4 and IL-5 production, were not affected. Thus, immunotherapy with protein resulted in beneficial effects on airway eosinophilia and hyperresponsiveness, which coincided with a local reduced T-helper 2 (Th2) response. In contrast, peptide immunotherapy aggravated airway hyperresponsiveness and eosinophilia, indicating a local enhanced Th2 response.     

Relative efficiency of microglia, astrocytes, dendritic cells and B cells in naive CD4+ T cell priming and Th1/Th2 cell restimulation.

We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323 - 339-dependent activation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells, followed by IFN-gamma-preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN-gamma-pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN-gamma-pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4(+) T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.     

Immunomodulatory effects of antigen-pulsed macrophages in a murine model of allergic asthma

Macrophages (Mphi) play an unique role in the activation and regulation of T cells through their ability to modulate specific costimulatory and cytokine signals. Here we investigated the immunomodulatory effects of allergen presentation by Mphi in a murine model of allergic asthma. Purified peritoneal Mphi were pulsed with ovalbumin (OVA) (OVA-Mphi), or the immunodominant epitope OVA(323-339) (OVA(323-339)-Mphi), and characterized for cell surface markers, cytokine production, and antigen-presenting capacity toward OVA(323-339)-specific DO11.10 T cells. Antigen-pulsed Mphi were injected (intravenously) in OVA-sensitized Balb/c mice that were repeatedly challenged with OVA or saline aerosol. Administration of OVA-Mphi inhibited airway eosinophilia and hyperresponsiveness to methacholine concomitant with a reduced interleukin (IL)-4 and IL-5 production by T cells upon OVA stimulation in vitro. Interestingly, OVA-induced IL-10 levels remained unchanged, whereas interferon-gamma could not be detected. In contrast to OVA-Mphi, OVA(323-339)-Mphi administration had no effects on these asthma manifestations. Additional in vitro studies demonstrated that OVA-Mphi, but not OVA(323-339)-Mphi, produced high levels of IL-10 upon interaction with the DO11.10 T cells. This IL-10 production by the OVA-Mphi was dependent on MHC-TCR and CD86-CD28, but not CD80-CD28 or CD40-CD154 interactions. Our data suggest that IL-10 production by allergen presenting Mphi plays a crucial role in successful immunotherapy.     

CD4+ CD25+调节性T细胞AICD机制的研究

目的探讨CD4^＋CD25^＋调节性T细胞活化诱导的细胞死亡（AICD）发生的机制。方法CD4^＋CD25^＋T细胞以磁性细胞分离器（MACS）从BALB／c小鼠或DO11．10小鼠的静息T细胞分离纯化。体外细胞增殖抑制实验证实其免疫调节作用。CD4^＋CD25^＋T细胞的AICD以CD3／CD28单克隆抗体活化或以特异性OVA323-339肽、抗原提呈细胞活化等两种方法获得。CD4^＋CD25^＋T细胞凋亡相关基因的表达通过实时定量PCR检测。流式细胞仪检测细胞的凋亡率。进一步观察FasL中和抗体、TRAIL中和抗体及caspase抑制剂zVAD-fmk对CD4^＋CD25^＋T细胞凋亡的影响。结果MACS成功分离CD4^＋CD25^＋T细胞，纯度可达98％，该细胞可特异性表达Foxp3基因，能明显抑制效应性T细胞的体外增殖。CD3／CD28抗体以及OVA特异性抗原活化8d的CD4^＋CD25^＋调节性T细胞AICD达39％～45％。活化前后的CD4^＋CD25^＋调节性T细胞死亡受体家族表达发生明显变化；FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4^＋CD25^＋调节性T细胞的凋亡。结论FasL／Fas及其他凋亡相关分子可能参与了CD4^＋CD25^＋调节性T细胞的凋亡。