CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11

The chemokine receptor CXCR3 is predominantly expressed on T lymphocytes, and its agonists CXCL9, CXCL10 and CXCL11 are IFN-gamma-inducible chemokines that promote Th1 responses. In contrast, the CCR3 agonists CCL11, CCL24 and CCL26 are involved in the recruitment of cells such as eosinophils and basophils during Th2 responses. Here, we report that although CCL11, CCL24 and CCL26 are neither agonists nor antagonists of CXCR3, CCL11 binds with high affinity to CXCR3. This suggests that, in vivo, CXCR3 may act as a decoy receptor, sequestering locally produced CCL11. We also demonstrate that the CXCR3 ligands inhibit CCR3-mediated functional responses of both human eosinophils and CCR3 transfectants induced by all three eotaxins, with CXCL11 being the most efficacious antagonist. The examination of CCR3-CCR1 chimeric constructs revealed that CCL11 and CXCL11 share overlapping binding sites contained within the CCR3 extracellular loops, a region that was previously shown to be essential for effective receptor-activation. Hence, eosinophil responses mediated by chemokines acting at CCR3 may be regulated by two distinct mechanisms: the antagonistic effects of CXCR3 ligands and the sequestration of CCL11 by CXCR3-expressing cells. Such interplay may serve to finely tune inflammatory responses in vivo.     

FasL expression in hepatic antigen-presenting cells and phagocytosis of apoptotic T cells by FasL+ Kupffer cells are indicators of rejection activity in human liver allografts

Fas-Fas ligand (FasL) interaction and apoptosis are important in the mechanism of allograft rejection. However, the interaction between donor and recipient cells, specifically focusing on antigen-presenting cells (APCs), under various conditions is poorly understood in human liver allografts. FasL expression on APCs, its association with apoptosis, and the origin of apoptotic lymphocytes in human liver allografts were assessed by immunohistochemistry and in situ hybridization. We found increased expression of FasL on Kupffer cells (KCs) and endothelium in acute cellular rejection (n = 20) and to lesser extent in chronic rejection (n = 6) and septic cholangitis (n = 5) compared with stable grafts and normal controls. In addition, the graft specificity of infiltrating T cells was confirmed by polymerase chain reaction examination of T-cell receptor-gamma loci. T-cell apoptosis occurred at a higher rate in acute cellular rejection than in chronic rejection or septic cholangitis. The number of apoptotic bodies derived from recipient lymphocytes correlated with the severity of rejection and was reversed by treatment. FasL(+) KCs phagocytosed CD4(+) interferon-gamma(+) T cells, rather than CD4(+) interleukin-4(+) T cells, suggesting a role of KCs in regulating CD4(+) T-cell subset differentiation. In conclusion, our data suggest that FasL expression on APCs and phagocytosis of apoptotic T cells by FasL(+) KCs are indicators of rejection activity in human liver allografts.     

Ultra-localization of Foxp3+ T cells within renal allografts shows infiltration of tubules mimicking rejection

Kidney transplant recipients are monitored for rejection by measurement of serum creatinine and graft biopsies. Biopsy samples are evaluated according to the Banff classification, which states that infiltration of tubules by mononuclear cells is an indicator of acute rejection. However, regulatory T cells play a crucial role in the overall immune response and are also present within transplanted tissue. We hypothesize that infiltration of mononuclear cells within kidney grafts is not always associated with rejection, especially if a high proportion of this infiltrate is regulatory T cells. Using a life-sustaining mouse kidney transplant model, we found that mononuclear cell tubular infiltration can occur in both rejecting and tolerant grafts. However, tolerant kidney grafts demonstrated a higher and sustained level of Foxp3+ regulatory cells. Importantly, a significant proportion of these cells were found within tubules. In cases in which graft function was normal, these cells were not harmful to the kidney and could be said to be mimicking, rather than causing, rejection.     

The roles of host and donor cells in the rejection of skin allografts by T cell-deprived rats injected with syngeneic T cells

Monoclonal antibodies that define rat T helper cells and cytotoxic T cell precursors were used to deplete thoracic duct lymphocytes of one or other of these subsets and the residual cells injected into syngeneic T cell-deprived rats bearing skin allografts. The subsequent fate of the grafts was compared with that of grafts on rats injected with unfractionated donor cells. Removal of cytotoxic T cell precursors from the donor inoculum did not affect the ability of the injected cells to mediate destruction of the grafts whereas removal of T helper cells led to prolonged graft survival. Using monoclonal antibodies to label cryostat sections, the phenotypes of the cells infiltrating rejecting grafts and healthy grafts were also established. Seven days after grafting syngeneic or allogeneic skin on T cell-deprived rats, all grafts were heavily infiltrated with Ia⁺ macrophages, but by 4 weeks this infiltrate had subsided and the grafts, whether syngeneic or allogeneic, were in perfect condition. At this time animals bearing grafts were injected with thoracic duct lymphocytes depleted of cytotoxic T cell precursors. Within 6 days of these cell transfers the allogeneic grafts, but not the syngeneic ones, were infiltrated with large numbers of mononuclear cells. Many of these cells were T cells, as judged by their expression of a pan T cell antigen, defined by the monoclonal antibody MRC OX-19, and many were Ia' macrophages. In addition there was a very heavy infiltrate of cells labeled by the MRC OX-8 monoclonal antibody which detects both rat cytotoxic T cells and natural killer cells. T cell-deprived rats contained elevated numbers of nonspecific cytotoxic cells. Evidence was obtained that these cells were MRC OX-8⁺. The possible role of these cells and of Ia⁺ macrophages in allograft rejection is discussed. As part of these studies the phenotypes of the cytotoxic T cell precursor and the cytotoxic effector cell were determined. It was shown that both cell types were indistinguishable, in terms of their reactivities with monoclonal anti-rat T cell antibodies, from rat suppressor T cells.     

Expression of CXCR6 and its ligand CXCL16 in the lung in health and disease

BACKGROUND: Chemokine receptors (CR) play an important role in T cell migration, but their contribution to lung trafficking is unclear. OBJECTIVE: We hypothesized that if a particular CR was involved in T cell homing its expression would be enriched on lung T cells compared with peripheral blood T cells (PBT). METHODS: We have measured the CR expression on BAL T cells from patients with sarcoid, other interstitial lung diseases (ILD), asthma and healthy volunteers. RESULTS: Of 14 CR studied in sarcoid, CXCR6 expression was the most markedly increased in the lung compared with the blood, a finding that was also seen in ILD patients. A striking although lesser increase was also seen in asthmatics and healthy controls. Analysis of expression of the CXCR6 ligand, CXCL16, by immunohistochemistry suggested that alveolar macrophages (AM) were the major source of CXCL16 in the lung. AM expressed mRNA for CXCL16 and released nanogram quantities after adhesion to plastic as shown by RT-PCR, Western blotting and ELISA. Bronchoalveolar lavage (BAL) fluid from all subjects contained large amounts of CXCL16. The full-length CXCL16 was the predominant isoform in AM lysates, supernatants and BAL. CONCLUSION: This data suggests that CXCR6 and CXCL16 may play a role in T cell recruitment to the lung.     

The expression of the chemokine receptor CXCR3 and its ligand, CXCL10, in human breast adenocarcinoma cell lines

The acquisition of a metastatic phenotype in breast epithelial cells is a progressive process, influenced by a large variety of cellular and soluble factors. Of these, members of the chemokine superfamily, such as CCL2, CCL5, CXCL8 and CXCL12 have been recently suggested to promote breast cancer progression. A pre-requisite for elucidation of the role of other chemokines in breast cancer progression is the characterization of chemokine and chemokine receptor expression by breast tumor cells. The present study focuses on CXCL10, a CXC chemokine that was recently suggested to have anti-malignant properties, and its corresponding receptor CXCR3. CXCR3 expression was detected in three human breast adenocarcinoma cell lines, MDA-MB-231, MCF-7 and T47D. CXCR3 expression was potently up-regulated by growing the cells under stress conditions, imposed by serum starvation. Unlike many other chemokine receptors, CXCR3 expression was not down-regulated by exposure to high concentrations (500ng/ml) of its ligand, CXCL10, but rather was promoted. CXCL10-induced up-regulation of CXCR3 expression in the three cell lines was inhibited by cycloheximide, indicating that de novo protein synthesis is required for this process. In addition to CXCR3, the secretion of CXCL10 was noted in the MDA-MB-231, MCF-7 and T47D cells. CXCL10 secretion was found to be down-regulated by IL-6, a potentially pro-malignant cytokine in breast cancer. The concomitant expression of CXCR3 and CXCL10 in breast tumor cells suggests that a CXCR3-CXCL10 axis may function in these cells, and paves the way for an in depth analysis of CXCL10-CXCR3 interactions in breast tumor cells.     

Antigens shared by thymic stromal cells and T lymphocytes are abnormally expressed in AKR thymuses

Accumulating evidence has shown that thymic stromal-lymphoid interactions play a major role in the development of AKR thymic leukemia. A normal thymic stromal cell (TSC) line B6TE-A, which has been shown to support the in vitro growth of AKR leukemic T cells by forming multicellular complexes with them, was used to raise monoclonal antibodies. Three of these mAb, MTS 31, 32 and 38, in addition to 2 other MTS mAb, are abnormally expressed in the preleukemic and/or leukemic stages in AKR mice. These 5 MTS mAb, which detect antigens on both subpopulations of TSC and T cells, show some reduced cortical reactivity from the pre-leukemic period (MTS 32 and 35) to markedly depressed reactivity in the leukemic period (MTS 31, 32, 33, 35 and 38). While it appears that the major reduction is due to the loss of antigens from the cortical thymocytes, there is some indication that the stromal elements may be affected also. In addition, MTS 29, which was also produced in this study, while only reacting with rare thymic medullary cells in situ was densely distributed on cultured stromal cells from both normal and leukemic thymuses. In this report, the value of these MTS mAb for documenting various stages of AKR leukemogenesis has been clearly demonstrated: their possible modulatory effects on in vitro T cell leukemia growth is currently being investigated.     

Claudins 10 and 18 are predominantly expressed in lung adenocarcinomas and in tumors of nonsmokers

Aims

We investigated the expression of claudins 18 and 10 in a large set of primary lung carcinomas.

Methods and results

Immunohistochemical expression of claudin 18 was seen in 12.7 % and claudin 10 in 12.5 % of lung carcinomas. Their expression significantly associated with each other (p<0.001). The expression of claudin 18 and 10 was most prominent in lung adenocarcinomas which displayed positivity in 21.2% and 23.4 % of cases. Female patients had more often claudin 18 and 10 positive tumors, also separately in adenocarcinomas. Interestingly, claudin 10 (p=0.036) and claudin 18 (p=0.001) were more common in tumours of nonsmokers. In adenocarcinomas claudin 18 predicted a better survival (p=0.032). In Cox multivariate analysis, claudin 18 had an independent prognostic value (p=0.027).

Conclusion

The results show that both claudins are most commonly expressed in lung adenocarcinomas and they are more occasionally detected in other histological tumour types. Curiously, female patients and non-smokers express these claudins more commonly suggesting that they may play a part in the carcinogenesis of tobacco unrelated carcinoma. Claudin 18 associated with a better survival in lung adenocarcinoma and had an independent prognostic value and may thus be used in the evaluation of patient prognosis.     

Binding sites for Lewis antigens are expressed by human colon cancer cells and negatively affect their migration

In colon cancer, endothelial cell selectins can promote tumor cell attachment via interactions with sialylated Lewis antigens present at the surface of tumor cells, thereby facilitating tumor cell arrest and transmigration into the extravascular space. However, it is not known whether Lewis antigens interact with colon tumor cells and modify their migration. Our aim was to detect the presence of binding sites on human tumor cells for Lewis(a/x) antigens and their sialylated derivatives in vitro and in vivo and to analyze their influence on migration of colon cancer cells. The immunocytochemical and histochemical levels of expression of the four Lewis antigens were quantitatively determined in four human colon cancer cell lines and in in vivo nude mice xenografts. The levels of expression of specific binding sites for these sugar epitopes were determined by synthetic neoglycoconjugates. The influence of binding of these carbohydrate ligands on cancer cell migration was quantitatively evaluated by computer-assisted phase-contrast videomicroscopy performed on Matrigel culture supports either left uncoated or coated with neoglycoconjugate presenting synthetic Lewis(a), sialyl Lewis(a), Lewis(x), or sialyl Lewis(x) antigens. The influence of the calcium concentration in the culture medium on the Lewis antigen-mediated effects was checked. Human colon cancer cells expressed significant amounts of specific binding sites detected by the synthetic probes in addition to the oligosaccharide epitopes. The expression levels differed considerably between the four cell lines and between in vitro and in vivo specimens. Cell migration analysis revealed that the four Lewis antigens markedly decreased the levels of migration of the HCT-15 and LoVo cancer cells. This effect depends on the calcium concentration in the culture medium. Binding sites for Lewis epitopes are present on colon cancer cells. The functional relevance of these sites is indicated by the negative influence on cell migration of a matrix containing the oligosaccharides as ligand parts.     

Flt-3 and its ligand are expressed in neural crest-derived tumors and promote survival and proliferation of their cell lines.

Flt-3 ligand (FL) is a cytokine that promotes the survival, proliferation, and differentiation of hematopoietic progenitors in synergy with other growth factors, such as stem cell factor. Previously we have demonstrated that stem cell factor and its receptor c-kit are expressed in neural crest-derived tumor cells and that a c-kit block induces their apoptosis. Here we have evaluated the expression of flt-3 and its ligand in 12 neuroectodermal tumor cell lines from neuroblastoma (NB), neuroepithelioma (NE), Ewing sarcoma (ES), and peripheral neuroectodermal tumor (PNET) and in 38 biopsies: 19 from NB and 19 from ES and PNET. RT-PCR demonstrated the expression of flt-3 and FL in all lines. Coexpression was observed in 42% of NB and in 74% of ES and PNET biopsies. Flow cytometry confirmed the presence of membrane and cytoplasmic flt-3 and membrane FL in all lines, whereas soluble FL protein was not measurable in their supernatants. Microphysiometric demonstration of acidification of the medium provided evidence of the specific response of cell lines to FL stimulation. Specific flt-3 phosphorylation after FL treatment was also demonstrated by Western blotting analysis. In cells growing in RPMI plus 1% fetal calf serum, FL revealed a significant proliferating activity, more evident in NB and NE lines (mean increase of viable cells, 73 +/- 26% after 1 day). Treatment with flt-3 antisense oligonucleotides significantly inhibited cell growth. FL also displayed an antiapoptotic activity: after a 12-hour culture in the presence of 0.1% fetal calf serum, FL caused a 50% reduction of apoptotic cells. These results provide further evidence that neuroectodermal and hematopoietic cells share common regulatory pathways, and could be of interest in the clinical management of neuroectodermal tumors.     

穿孔素和Fas配体在柯萨奇B3病毒性心肌炎小鼠心肌浸润细胞中表达

目的探讨穿孔素（ＰＦＰ）和Ｆａｓ配体（Ｌ）介导的细胞毒作用在病毒性心肌炎发病中的作用。方法将４０只ＢＡＬＢ／ｃ小鼠随机等分为实验组和对照组，分别用柯萨奇Ｂ３病毒（ＣＶＢ３）及不含病毒的病毒稀释液经腹腔接种，于接种后７天处死，取其心脏。应用免疫组化、逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）和原位杂交等方法，检测细胞介导的细胞毒作用的主要效应分子ＰＦＰ和ＦａｓＬ在心肌浸润细胞中的表达。结果（１）实验组小鼠的心肌组织中均有ＰＦＰ和ＦａｓＬ抗原阳性细胞浸润，对照组则无；（２）ＲＴ－ＰＣＲ检测发现实验组鼠的心肌组织中ＰＦＰ和ＦａｓＬｍＲＮＡ的阳性率均为１００％，明显高于对照组的２０％和３０％（Ｐ均＜０．０１）；（３）原位杂交检查显示，实验组小鼠心肌组织中均可见ＰＦＰ和ＦａｓＬｍＲＮＡ阳性的浸润细胞，而对照组则均未发现。结论ＣＶＢ３小鼠心肌炎急性期，其心肌浸润细胞中有ＰＦＰ和ＦａｓＬ表达，提示它们在病毒性心肌炎发病机制中可能有重要作用     

IL-5 and eosinophils mediate the rejection of fully histoincompatible vascularized cardiac allografts: regulatory role of alloreactive CD8(+) T lymphocytes and IFN-gamma

CD8(+) T lymphocytes are known to inhibit the development of eosinophilia and IL-5 synthesis in models of experimental lung disease. In transplantation, the rejection of fully mismatched cardiac allografts by recipients depleted of CD8(+) T cells is characterized by the recruitment of eosinophils in the rejected organs. We show here that this intragraft eosinophilia is dependent on the production of IL-5 since hearts transplanted into IL-5-deficient recipients depleted of CD8(+) cells did not contain eosinophils. More importantly, allograft survival was significantly extended in these animals. In mixed lymphocyte cultures (MLC), the presence of CD8(+) T cells in the responding cell population inhibited the secretion of IL-5. This inhibition was IFN-gamma dependent since adding neutralizing anti-IFN-gamma antibodies induced the production of IL-5. Furthermore, spleen cells isolated from IFN-gamma receptor (IFN-gammaR)-deficient mice secreted IL-5 upon allogeneic stimulation in primary MLC. In vivo, eosinophilia was observed in allografts rejected by IFN-gammaR-deficient recipients. On the contrary, grafts rejected by IFN-gammaR-deficient mice treated with neutralizing anti-IL-5 antibodies did not exhibit eosinophilic infiltration. Our study reveals the capacity of IL-5-secreting CD4(+) T cells and eosinophils to promote the rejection of heart allograft and demonstrates the importance of CD8(+) T cells and IFN-gamma in regulating this pathway of rejection.     

CXCL10-producing mucosal CD4+ T cells, NK cells, and NKT cells are associated with chronic colitis in IL-10(-/-) mice, which can be abrogated by anti-CXCL10 antibody inhibition

We have shown previously that there is a temporal increase in the levels of CXCL10 and CXCR3 mRNA during spontaneous murine colitis. We now show that CXCL10 is significantly expressed by mucosal CD4+ T cells, natural killer (NK) cells, and NKT cells, but not by dendritic cells (DCs), during chronic murine colitis. CXCL10 blockade alleviated chronic colitis and attenuated the associated increase in serum amyloid A (SAA), interleukin-12 (IL-12)p40, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-1 alpha, and IL-1 beta levels as well as in the number of CD4+ T, NKT, and NK cells that express CXCL10 and CXCR3, compared with groups treated with control antibody (Ab). After CXCL10 blockade, the number of CXCR3+ DCs in the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) were increased to levels found before the onset of colitis. In contrast, the numbers of splenic and intestinal lamina propria (LP) CXCR3+ DCs were reduced after anti-CXCL10 Ab treatment, compared with controls. Ex vivo antigen and CXCL10 stimulation of mucosal cells caused an increase in MHC class II, CD40, and CD86 as well as a decrease in CD30 ligand (CD30L) expression by DCs. This study provides insights into CXCL10 expression during inflammatory bowel disease (IBD) and the cellular and molecular mechanisms of CXCL10-mediated colitis. Our data also support the premise that CXCL10 blockade can attenuate chronic colitis by preventing the activation and recruitment of CXCR3+ leukocytes during IBD.     

IL-10 downregulates CXCR3 expression on Th1 cells and interferes with their migration to intestinal inflammatory sites

Inflammatory bowel disease (IBD) is characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the etiology is poorly understood, it is widely accepted that loss of tolerance is involved in the development of IBD. Therefore, re-establishing tolerance or gut homeostasis is one of the key features in the development of new therapeutic strategies. Here we show that antigen targeting to DEC-205 on dendritic cells leads to an interleukin (IL)-10-dependent downregulation of C-X-C chemokine receptor 3 (CXCR3) expression on differentiated antigen-specific T helper type 1 (Th1) cells in vivo. This downregulation interferes with the migration of Th1 cells into the gut and protects mice against severe acute and relapsing intestinal inflammation. Moreover, CD4⁺CXCR3⁺ T cells are highly enriched in the inflamed mucosa of IBD patients. Interference with this pathway may therefore be a promising approach for the treatment of IBD. In conclusion, we propose a hitherto undescribed mechanism by which IL-10 can act on effector T cells and orchestrate intestinal immune responses.     

融合蛋白CXCL10-loop3- EGF的可溶性表达及其抗肿瘤效应研究

目的： 构建重组趋化因子CXCL10及表皮生长因子受体干扰序列融合蛋白（CXCL10-loop3-EGF）,验证其靶向性抗肿瘤效应及抑制血管形成活性。方法: 运用RT-PCR从经IFN-γ刺激活化的PBMC中扩增人CXCL10基因,运用重叠延伸PCR法扩增CXCL10-loop3-EGF融合基因,并将其与载体pTG19-T连接、转化大肠杆菌DH5α、筛选阳性克隆,CXCL10-loop3-EGF融合基因与载体pET32a（+）连接、转化大肠杆菌Origami B(DE3),诱导可溶性表达重组his-CXCL10- loop3-EGF融合蛋白,并经镍柱亲和层析、酶切、超滤及透析等方法获得纯化的重组CXCL10- loop3-EGF融合蛋白。通过Transwell细胞趋化实验及HUVEC血管形成抑制实验验证此融合蛋白的抗肿瘤效应。结果: SDS-PAGE和Western blotting证实CXCL10- loop3-EGF融合蛋白构建成功,纯化后的融合蛋白具有显著的趋化活化PBMC活性及抑制HUVEC血管形成活性。结论: 成功构建融合蛋白CXCL10- loop3-EGF,该蛋白具有较好的靶向性抗肿瘤活性。     

Interleukin-10 expressed at early tumour sites induces subsequent generation of CD4(+) T-regulatory cells and systemic collapse of antitumour immunity

We investigated the relationship between transforming growth factor-beta (TGF-beta)-secreting T-regulatory (Tr) cells and anti-B16 melanoma immunity, and studied the association of early cytokines expressed at tumour sites with the generation of Tr cells. A large number of CD4(+) Tr cells producing interleukin (IL)-4, IL-10 and TGF-beta accumulated with functionally depressed CD8(+) cytotoxic T lymphocytes (CTLs) at tumour sites on day 20 after subcutaneous (s.c.) inoculation of B16 tumour cells. Tr cells consisted of two populations, which were termed T helper 3 (Th3) and Tr1 cells. B16-infiltrating Tr cells strongly inhibited the generation of B16-specific T helper 1 (Th1) cells in a TGF-beta-dependent manner and were assumed to suppress effective generation of CTLs. In addition, B16 cells markedly progressed in mice transferred adoptively by the cultured B16-infiltrating Tr cells compared with untreated mice. The capacity of these Tr cells to produce TGF-beta was hampered by neutralizing anti-IL-10 and partly anti-IL-4 monoclonal antibodies (mAbs) injected intralesionally during the early development of B16 tumours, and this treatment markedly attenuated B16 growth. Furthermore, a lesional injection of recombinant mouse IL-10 at an early tumour site resulted in the vigorous progression of B16 tumours. These results provide evidence that Tr cells, belonging to the T helper 3/T-regulatory 1 (Th3/Tr1) type, are activated in B16-bearing hosts under the influence of T helper 2 (Th2) cytokines, mainly IL-10 (produced at early tumour lesions), and that this regulatory T-cell population functions as a suppressor of anti-B16 immunity.     

Indoleamine 2,3-dioxygenase and metabolites protect murine lung allografts and impair the calcium mobilization of T cells

The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan into kynurenine metabolites that suppress effector T-cell function. In this study, we investigated IDO and its metabolite, 3-hydroxyanthranilic acid (3HAA), in regulating lung allograft rejection, using a murine orthotopic lung transplant model with a major mismatch (BALB/c donor and C57BL6 recipient). IDO was overexpressed in murine donor lungs, using an established nonviral (polyethylenimine carrier)-based gene transfer approach, whereas 3HAA was delivered daily via intraperitoneal injection. Increased IDO expression or its metabolite, 3HAA, resulted in a remarkable therapeutic effect with near normal lung function and little acute rejection, approximately A1, compared with A3 in untreated allografts (grading based on International Society for Heart and Lung Transplantation guidelines). We found that a high IDO environment for 7 days in lung allografts resulted in impaired T-cell activation, the production of multiple effector cytokines (IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, IL-12, and IL-13), and the generation of effector memory T cells (CD62L(lo)CD44(hi) phenotype). In isolated murine splenocytes, we observed that IDO/3HAA impaired T-cell receptor (TCR)-mediated T-cell activation, and more importantly, a decrease of intracellular calcium, phospholipase C-γ1 phosphorylation, and mitochondrial mass was evident. This work further illustrates the potential role of a high IDO environment in lung transplantation, and that the high IDO environment directly impairs TCR activation via the disruption of calcium signaling.     

Antigen processing by endosomal proteases determines which sites of sperm-whale myoglobin are eventually recognized by T cells.

This study reports an identification of the major processing products of an exogenous protein antigen, viz, sperm-whale myoglobin, as obtained after cell-free processing with partially purified macrophage endosomes. It is demonstrated that such a system yields fragments that are indistinguishable by high performance liquid chromatography analysis from those generated after uptake of myoglobin inside live macrophages. The concerted action of the endosomal proteases cathepsin D and cathepsin B can account for nearly all cleavages observed. Cathepsin D appears to be mainly responsible for the initial cleavage of myoglobin, while cathepsin B catalyzes the C-terminal trimming of initially released fragments. The fragments released by cathepsin D contain most, if not all, major epitopes for murine myoglobin-specific helper T cells. Interestingly, each known T cell epitope of myoglobin is located at the very N terminus of a different myoglobin fragment released upon processing. In order to explain this correspondence, noted also in several other protein antigens, a structural relationship is proposed between antigen processing by cathepsin D and antigen recognition by major histocompatibility complex (MHC) class II products. As is demonstrated here, this relationship may be used as a predictive tool for the identification of MHC-binding sequences as well as of T cell epitopes in their naturally occurring form.