Hybrid Ia antigens: Genetic,serologic, and biochemical analyses

Ia specificities 22 and 23 were found to be determinants on hybrid Ia molecules, formed by the noncovalent binding of a 26,000–28,000 dalton beta polypeptide chain (A_e) coded by the I-A subregion and a 32,000–35,000 dalton alpha chain (E_α) coded by the I-E subregion. For expression of Ia. 23 the A_e chain, coded by the I-A subregion, must be derived from the H-2^d haplotype, while A^b, A^s, or A^k can provide the complementing beta chain for the expression of Ia. 22. For expression of Ia. 22 and Ia. 23, most Ia. 7 positive strains can provide the complementing alpha chain (E_α), with the one exception of B 10. PL (E^u), which is Ia. 7 positive but will not complement with A^d to express Ia. 23. Antisera were also produced against hybrid Ia antigens by immunizing with F₁ cells expressing Ia. 22 or Ia. 23 generated by transcomplementation. These antisera detect the same specificities as conventional anti-Ia. 22 and anti-Ia. 23 sera produced against cis-complementing Ia antigens. It is postulated that hybrid Ia determinants are involved in recognition and generation of immune response to antigens under dual Ir gene control. It is also suggested that there are 2 types of Ia specificities: (1) allotypic Ia specificities expressed on the alpha or beta chains (for example, Ia. 7 on the E_α chain) and (2) hybrid Ia specificities, which are unique interaction determinants formed by the association of alpha and beta chains (for example, Ia. 22 and Ia. 23). These interaction gene products may be involved in antigen recognition and presentation.     

Higher numbers of T-bet+ intratumoral lymphoid cells correlate with better survival in gastric cancer

In the present study, we studied the expression of T-bet, a key marker for type 1 immune responses, within the tumor microenvironment of gastric cancer, and analyzed its association with clinicopathological parameters. One hundred and fifty-two archival paraffin-embedded gastric tumor tissues were collected, and the expression of T-bet in these cancer tissue specimens was examined by immunohistochemistry. T-bet⁺ tumor-infiltrating lymphocytes (TILs) in some gastric cancer tissues were further characterized by flow cytometric analysis. The density of T-bet⁺ TILs in gastric cancer tissues in relation to patient’s clinicopathological parameters and postoperative prognosis has been analyzed. Herein, we have found significant increases in T-bet⁺ lymphocytes in tumor tissues as compared with normal stomach tissues, gastritis tissues or gastric polyp specimens. T-bet⁺ cells mainly consisted of CD4⁺, CD8⁺ and CD56⁺ TILs. In addition, lower numbers of T-bet⁺ TILs were associated with poor clinicopathological parameters such as invasion to muscular layer, larger tumor size and advanced cancer stages. Moreover, patients with higher numbers of T-bet⁺ TILs have longer disease-free survival and overall survival. Thus, our study supports the idea that tumor growth elicits spontaneous type 1 cellular immune responses and tumor progression is associated with suppression of antitumor immunity. T-bet expression within tumor can serve as a prognostic indicator for gastric cancer and a potential biomarker for immunotherapy.     

树突状细胞对胃癌前病变的免疫保护分析

摘要 目的：探究树突状细胞(Dendritic cells,DC)对胃癌的免疫保护作用。方法：选择2016年1月至2018年1月于我院接受治疗的145例胃癌、39例慢性萎缩性胃炎、21例不典型增生、27例肠上皮化生以及20例正常对照组患者为研究对象,分别采集其胃粘膜标本进行染色,记录和比较其胃粘膜中S100⁺、CD4⁺和CD8⁺细胞的数量、平均面积以及平均吸光度,并将胃癌患者分为中分化腺癌(49例)、低分化腺癌(53例)和未分化癌(43例)进行对比。结果：(1)胃癌组、慢性萎缩性胃炎组、不典型增生、肠上皮化生组的胃粘膜S100⁺阳性细胞计数明显高于正常对照组（P<0.05）,胃癌组平均吸光度低于对照组,其他3组平均吸光度显著高于对照组,(P<0.05);胃癌组平均面积与正常对照组相比无差异(P>0.05),其他三组平均面积显著高于对照组（P<0.05）;(2)慢性萎缩性胃炎组、肠上皮化生组、不典型增生组患者CD4⁺细胞数均低于对照组(P<0.05);胃癌组、慢性萎缩性胃炎组、肠上皮化生组患者平均面积均低于对照组(P<0.05);胃癌组、慢性萎缩性胃炎组、不典型增生、肠上皮化生组平均吸光度均低于对照组(P<0.05);(3)慢性萎缩性胃炎组、肠上皮化生组、不典型增生组患者CD8⁺细胞数明显高于对照组(P<0.05),胃癌组稍低于对照组(P>0.05);胃癌组患者平均面积低于对照组(P<0.05);胃癌组患者平均吸光值低于对照组,慢性萎缩性胃炎组、肠上皮化生组患者高于对照组(P均<0.05);(4)随着胃癌分化程度的降低,胃癌患者DC细胞数有降低趋势。结论：胃癌前病变患者胃粘膜中DC数量会显著增多,免疫功能加强,DC细胞数量会随胃癌分化程度的降低而减少,分析其原因与DC细胞能够抑制癌前病变有关。     

Intratumor IL-17-Positive Mast Cells Are the Major Source of the IL-17 That Is Predictive of Survival in Gastric Cancer Patients

Interleukin-17 (IL-17) is prevalent in tumor tissue and suppresses effective anti-tumor immune responses. However, the source of the increased tumor-infiltrating IL-17 and its contribution to tumor progression in human gastric cancer remain poorly understood. In this study, we enrolled 112 gastric cancer patients, immunofluorescence was used to evaluate the colocalization of CD3, CD4, CD56, CD20, CD68, and mast cell tryptase (MCT) with IL-17. Immunohistochemistry was used to evaluate the distribution of microvessel density (CD34), CD66b⁺, CD68⁺, and FoxP3⁺ cells in different microanatomical areas. Prognostic value was determined by Kaplan-Meier analysis and a Cox regression model. The results showed that mast cells, but not T cells or macrophages, were the predominant cell type producing IL-17 in gastric cancer. Significant positive correlations were detected between densities of mast cell-derived IL-17 and microvessels, neutrophils, and regulatory T cells (Tregs). Futhermore, we found that the majority of vascular endothelial cells expressing Interleukin-17 receptor (IL-17R). Kaplan-Meier analysis revealed that increasing intratumor infiltrated mast cells and IL-17⁺ cells, as well as MCT⁺ IL-17⁺ cells, were significantly associated with worse overall survival. These findings indicated that mast cells were the major source of IL-17 in gastric cancer, and intratumor IL-17 infiltration may have promoted tumor progression by enhancing angiogenesis in the tumor microenvironment through the axis of IL-17/IL-17R. IL-17-positive mast cells showed a prognostic factor in gastric cancer, indicating that immunotherapy targeting mast cells might be an effective strategy to control intratumor IL-17 infiltration, and consequently reverse immunosuppression in the tumor microenvironment, facilitating cancer immunotherapy.     

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.     

Lymphoma-Like T Cell Infiltration in Liver Is Associated with Increased Copy Number of Dominant Negative Form of TGFβ Receptor II

Hepatosplenic T cell lymphoma (HSTCL) is a distinct and lethal subtype of peripheral T cell lymphoma with an aggressive course and poor outcome despite multiagent chemotherapy. Contradictory literature, an unknown etiology, and poor response to treatment highlight the need to define the malignant process and identify molecular targets with potential for successful therapeutic interventions. Herein, we report that mice homozygously expressing a dominant negative TGFβRII (dnTGFβRII) under the control of the CD4 promoter spontaneously develop lymphoma-like T cell infiltration involving both spleen and liver. Splenomegaly, hepatomegaly and liver dysfunction were observed in homozygous dnTGFβRII mice between 10 weeks and 10 months of age associated with a predominant infiltration of CD4⁻CD8⁻TCRβ⁺NK1.1⁺ or CD8⁺TCRβ⁺NK1.1⁻ T cell subsets. Notch 1 and c-Myc expression at the mRNA levels were significantly increased and positively correlated with the cell number of lymphoid infiltrates in the liver of dnTGFβRII homozygous compared to hemizygous mice. Further, 2×10⁴ isolated lymphoma-like cells transplant disease by adoptive cell transfers. Collectively, our data demonstrate that increased copy number of dnTGFβRII is critical for development of lymphoma-like T cell infiltration.     

p27(kip1) upregulated by hnRNPC1/2 antagonizes CagA (a virulence factor of Helicobacter pylori)-mediated pathogenesis

Background and Aims: Infection by Helicobacter pylori is one of the major contributing factors of chronic active gastritis and peptic ulcer and is closely associated with the occurrence and progression of gastric cancer. CagA protein is a major virulence factor of H. pylori that interacts with SHP‐2, a true oncogene, to interfere with cellular signaling pathways; CagA also plays a crucial role in promoting the carcinogenesis of gastric epithelial cells. However, currently, the molecular mechanisms of gastric epithelial cells that antagonize CagA pathogenesis remain inconclusive. Methods: We showed that AGS gastric cancer cells transfected with CagA exhibited the inhibition of proliferation and increased activity of caspase 3/7 using two‐dimensional gel electrophoresis and secondary mass spectrometry (MS/MS). Results: It was found that the AGS gastric cancer cells stably expressing CagA displayed significantly increased the expression of 16 proteins, including hnRNPC1/2. Further analysis revealed that hnRNPC1/2 significantly boosted the expression of the p27^kip1 protein. Conclusion: Our data suggested that hnRNPC1/2 upregulates p27^kip1 expression and the subsequent suppression of cell proliferation and induction of apoptosis, thereby providing an important mechanism whereby gastric epithelial cells antagonize CagA‐mediated pathogenesis.     

Surface antigen changes occurring in short-term cultures of activated human T lymphocytes: analysis by flow cytometry

Surface antigens of activated and cultured human T cells were studied using peripheral blood lymphocytes activated with conditioned medium from phytohemagglutinin-activated leukocytes and maintained in liquid culture for 2 weeks with conditioned medium containing Interleukin 2. The ensuing cell population was tested for kinetic changes in cell size and for the expression of surface antigens by immunofluorescence staining with a panel of monoclonal antibodies and analysis by flow cytometry. Upon activation, the cell population progressively increased in size to large blasts, with the rapid appearance on all of the large dividing cells of the antigen recognized by OKT9, the transferrin receptor. Cells within the population continued to express the common peripheral T-cell antigens bound by OKT3 and UCHT1, and also the antigen bound by 3A1, but never the antigen bound by OKT6, a thymic cell marker. From the time of activation an increasing proportion of the T cells, up to 80%, expressed the antigen detected with OKIa and FMC4, which recognise nonpolymorphic Ia determinants. This sequence of events was followed by a general decrease in size of the cell population, a process accompanied by further phenotypic changes. The percentage of cells expressing Ia antigens decreased, but most striking was the rapid change in the OKT4:OKT8 ratio of cells within the population, from 60:40 to 40:60. Thereafter the proportions of OKT4⁺ to OKT8⁺ cells within the cultures remained relatively stable and it is suggested that these data provide evidence for a possible change in phenotype of cultured human T lymphoblasts, from OKT4 to OKT8.     

Lymphocyte subpopulations of regional lymph nodes in human colon and gastric adenocarcinomas

 In order to study the host immune response to tumours, previous knowledge of the cellular composition of regional draining lymph nodes is necessary. Enlarged regional lymph nodes are a common finding in colon and gastric adenocarcinomas. We have studied the cellular composition of normal non-reactive and of regional draining lymph nodes of colon and gastric adenocarcinomas. In normal non-reactive lymph nodes, T lymphocytes (CD2⁺, CD7⁺) constituted the largest fraction of the lymphoreticular cells. These lymphocytes were mainly CD4⁺, and there were more cells expressing the CD45RA isoform of the CD45 antigen than CD45RO. Reactive lymph nodes presented a decreased proportion of CD4⁺ CD45RA⁺ cells and an increased number of B cells. Although most of the T cells in the reactive nodes were CD4⁺ CD45RO⁺, their proportion was similar to that found in normal non-reactive nodes. We studied the presence of the molecules CD28 and CD80 involved in the processes of interaction and activation of T and B lymphocytes. The CD28 molecule was found in all the T lymphocytes, while the CD80 molecule was weakly expressed on the B lymphocyte membrane. Received: 4 January 1996 / Accepted: 28 May 1996     

A20 Controls Macrophage to Elicit Potent Cytotoxic CD4+ T Cell Response

Emerging evidence indicates that CD4⁺ T cells possess cytotoxic potential for tumor eradication and perforin/granzyme-mediated cytotoxicity functions as one of the important mechanisms for CD4⁺ T cell-triggered cell killing. However, the critical issue is how the cytotoxic CD4⁺ T cells are developed. During the course of our work that aims at promoting immunostimulation of APCs by inhibition of negative regulators, we found that A20-silenced Mф drastically induced granzyme B expression in CD4⁺ T cells. As a consequence, the granzyme-highly expressing CD4⁺ T cells exhibited a strong cytotoxic activity that restricted tumor development. We found that A20-silenced Mф activated cytotoxic CD4⁺ T cells by MHC class-II restricted mechanism and the activation was largely dependent on enhanced production of IFN-γ.     

Increased prevalence of tumour infiltrating immune cells in oropharyngeal tumours in comparison to other subsites: relationship to peripheral immunity

Background

The nature of the tumour microenvironment immune response in head and neck cancer patients has an important role in tumour development and metastasis, but it is unknown if this differs between cancer subsites or whether it is related to the peripheral immune response.

Methods

Immune cells (CD4, CD8, Foxp3) in head and neck squamous cell carcinoma tissue (HNSCC; n = 66), detected by immunohistochemistry, have been correlated with tumour subsite and immune cells in the peripheral circulation (CD4⁺CD25^HighFoxp3⁺ Treg and CD4⁺ T cells), identified using flow cytometry.

Results

Oropharyngeal tumours had a greater number of infiltrating immune cells in both tumour and stroma compared with other subsites, but no difference was observed in the circulating levels. Immune cells in the stroma were positively related to those in the tumour with consistently higher levels in stroma. A strong relationship was found between the number of CD4⁺ and Foxp3⁺ cells but not between the number of CD8⁺ and Foxp3⁺ cells in the tumour. The number of Foxp3⁺ cells within the tumour was positively correlated with the percentage of circulating CD4⁺CD25^High cells positive for Foxp3. Late stage laryngeal tumours showed a higher number of Foxp3⁺ lymphocytes compared with early stage malignancies, and oropharyngeal tumours had more CD4⁺ cells in node negative tumours compared with node positive ones.

Conclusion

The level of immune cell infiltration in head and neck squamous cell carcinoma appears to be subsite dependent residing primarily in the stroma and is likely to be dependent on the peripheral immune response.     

Cytotoxic activity and phenotypic characteristics of lymphocyte subsets after therapy of cancer patients with interleukin-2

Summary After a 5-day period of continuous intravenous infusion of recombinant interleukin 2 (rIL-2) in seven patients with malignant melanoma or gastric or pancreatic cancer, different lymphocyte subsets were separated from patients' blood and tested ex vivo for cytotoxic activity against various tumour cell lines. Lytic activity was mediated by CD3⁺CD56⁺, CD3^–CD56⁺, CD3^–CD2⁺ and CD8⁺CD56⁺ lymphocytes. No cytotoxic activity could be observed within the CD3⁺CD56^–, CD3⁺CD2⁺ or CD4⁺ T cell subsets. To characterize CD56⁺ cytotoxic cells further, the expression of other antigens on this population was analysed before and after IL-2 therapy. CD3, CD4, CD16 and CD57 antigens were weakly expressed, and the IL-2 receptor (CD25) was not detectable on these cells either before and after treatment with IL-2. In contrast, increased expression of CD2, CD8 and HLA-DR antigens occurred following therapy. The divergence of CD3 and CD8 antigen expression after IL-2 therapy was caused by an increase in CD3^–CD8⁺ cells, detectable as a low-density CD8⁺ subset. This study shows that cytotoxic activity of in vivo IL-2-activated killer cells is predominantly, but not exclusively, mediated by CD3^–CD56⁺ lymphocytes, partially coexpressing the CD8 antigen and lacking the expression of CD 16 antigens.     

Epithelial-mesenchymal transition of ovarian tumor cells induces an angiogenic monocyte cell population

Vasculogenesis, or recruitment of progenitors able to differentiate into endothelial-like cells, may provide an important contribution to neovessel formation in tumors. However, the factors involved in the vasculogenic process and in particular the role of the epithelial-mesenchymal transition of tumor cells have not yet been investigated. We found a CD14⁺/KDR⁺ angiogenic monocyte population in undifferentiated ovarian tumors, significantly increased in the corresponding tumor metastasis. In vitro, monocyte differentiation into CD14⁺/KDR⁺ cells was induced by conditioned media from the primary ovarian tumor cells expressing a mesenchymal phenotype. In contrast, the ovarian tumor cell line SKOV3 expressing an epithelial phenotype was unable to stimulate the differentiation of monocytes into CD14⁺/KDR⁺ cells. When an epithelial-mesenchymal transition was induced in SKOV3, they acquired this differentiative ability. Moreover, after mesenchymal transition pleiotrophin expression by SKOV3 was increased and conversely its blockade significantly reduced monocyte differentiation. The obtained CD14⁺/KDR⁺ cell population showed the expression of endothelial markers, increased the formation of capillary-like structures by endothelial cells and promoted the migration of ovarian tumor cells in vitro. In conclusion, we showed that the epithelial-mesenchymal transition of ovarian tumor cells induced differentiation of monocytes into the pro-angiogenic CD14⁺/KDR⁺ population and thus it may provide a tumor microenvironment that favours vasculogenesis and metastatization of the ovarian cancer.     

Isoform-specific up-regulation of plasma membrane Ca2+ATPase expression during colon and gastric cancer cell differentiation

In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca²⁺ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na⁺/K⁺–ATPase or that of the sarco/endoplasmic reticulum Ca²⁺ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca²⁺ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells.Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.     

Tumour angiogenesis normalized by myo-inositol trispyrophosphate alleviates hypoxia in the microenvironment and promotes antitumor immune response

Pathologic angiogenesis directly responds to tumour hypoxia and controls the molecular/cellular composition of the tumour microenvironment, increasing both immune tolerance and stromal cooperation with tumour growth. Myo-inositol-trispyrophosphate (ITPP) provides a means to achieve stable normalization of angiogenesis. ITPP increases intratumour oxygen tension (pO₂) and stabilizes vessel normalization through activation of endothelial Phosphatase-and-Tensin-homologue (PTEN). Here, we show that the tumour reduction due to the ITPP-induced modification of the tumour microenvironment by elevating pO₂ affects the phenotype and properties of the immune infiltrate. Our main observations are as follows: a relative change in the M1 and M2 macrophage-type proportions, increased proportions of NK and CD8⁺T cells, and a reduction in Tregs and Th2 cells. We also found, in vivo and in vitro, that the impaired access of PD1⁺NK cells to tumour cells is due to their adhesion to PD-L1⁺/PD-L2⁺ endothelial cells in hypoxia. ITPP treatment strongly reduced PD-L1/PD-L2 expression on CD45+/CD31+ cells, and PD1⁺ cells were more numerous in the tumour mass. CTLA-4⁺ cell numbers were stable, but level of expression decreased. Similarly, CD47⁺ cells and expression were reduced. Consequently, angiogenesis normalization induced by ITPP is the mean to revert immunosuppression into an antitumor immune response. This brings a key adjuvant effect to improve the efficacy of chemo/radio/immunotherapeutic strategies for cancer treatment.