R. Kammerer

Biliary glycoprotein (CD66a), a cell adhesion molecule of the immunoglobulin superfamily, on human lymphocytes: structure, expression and involvement in T cell activation

The biliary glycoproteins (BGP or CD66a), a group of different splice variants of a single gene, are members of the carcinoembryonic antigen family and the immunoglobulin superfamily. Recently, we detected CD66a on IL‐2 activated lymphocytes. In this study we characterized the structure and the expression pattern of BGP on human lymphocytes and investigated its role in T cell activation. Lymphocytes express 2 of the 13 known splice variants, i.e. BGPa and BGPb, which are glycosylated in a lymphocyte‐specific manner. Both BGPa and BGPb have the long cytoplasmic tail, which contains two immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐like motifs, but differ in their extracellular region containing 4 and 3 immunoglobulin‐like domains, respectively. On PBL BGP is expressed in small amounts only on B cells and Th cells. Stimulation with IL‐2 leads to a strong up‐regulation of BGP by these cells, and induces de novo BGP expression on γ δ T cells, CD8+ and CD56+ cells, but not on CD16+ lymphocytes. This up‐regulation of BGP seems to be part of the physiological process of T cell activation, since stimulation with anti‐CD3 mAb is sufficient to induce BGP up‐regulation. Based on the presence of the two ITIM‐like motifs, one may expect that BGP inhibits T cell activation, but surprisingly, engagement of BGP enhances the proliferation of anti‐CD3‐stimulated T cells.

Biliary glycoprotein (BGP) expression on T cells and on a natural-killer-cell sub-population

Human T and natural‐killer (NK) cells, that are thought to be the major cytotoxic effector‐cell populations in the defence against neoplastic cells, were isolated from blood and decidua in order to analyze their expression of carcinoembronic‐antigen‐(CEA)‐family‐member proteins. Biliary glycoprotein (BGP, CD66a) was the only member of the carcinoembryonic antigen family detected. While freshly isolated T‐cells expressed low amounts of BGP, freshly isolated NK cells were negative. After in vitro stimulation for 3 days,T cells up‐regulated their BGP expression and a sub‐group of NK cells (CD16− CD56+), known to predominate in decidua, revealed de novo expression of BGP. In contrast, stimulated CD16+ CD56+ NK cells, which occur exclusively in the blood, remained negative. The expression of BGP could be shown on the protein level by using a panel of 12 well‐defined MAbs and on the transcription level in rt‐PCR and subsequent oligonucleotide hybridization. Interestingly, rIL‐2‐stimulated T cells expressed 3‐fold higher levels of BGP compared with those seen after stimulation with phytohemaglutinine (PHA). PHA, on the other hand, induced a higher expression of HLA‐DR, an activation marker of T cells. The differential regulation implies a distinct function of BGP and HLA‐DR.

A DISTINCT DISTRIBUTION OF NATURAL KILLER CELL SUBGROUPS IN HUMAN TISSUES AND BLOOD

Natural killer (NK) cells have been subdivided according to their CD16/ CD56 expression into at least 2 subgroups. We examined the distribution of these NK subgroups in humans. In the blood of normal individuals, CD16++/ CD56+/ CD3– NK cells predominate, constituting more than 90% of all NK cells. In contrast, decidua is infiltrated almost exclusively by CD16+/–/ CD56+/ CD3– NK cells (>90%), a fact so far seen in context with decidua being an immunoprivileged tissue. However, this NK subgroup can also be detected in the blood, where it comprises about 10% of NK cells. We have found that normal (colon) as well as neoplastic (ovarian and urothelial carcinoma) tissues are also predominantly infiltrated by this CD16+/– NK subgroup. Lymphatic fluid draining solid tissues contains CD16+/– NK cells exclusively, with absolute numbers of NK cells being very low. No predominating NK subgroup was seen in ascites. CD16+/– NK cells, when tested against the target cell lines K562 and JAR, revealed a cytotoxic spectrum different from CD16++ NK cells and from T cells. A change in the CD16/ CD56/ CD3 phenotype was not seen in either subgroup in long‐term cultures containing IL‐2 (1,000 U/ ml). Our data indicate that the decidua is not the only solid tissue infiltrated by CD16+/– NK cells. Other normal and malignant tissues were also infiltrated predominantly by this NK cell subgroup. We suggest that CD16+/– NK cells represent a functionally distinct NK subgroup involved in the surveillance of solid tissues. Int. J. Cancer 78:533–538, 1998.

Comparison of the Effects of Immunosuppressive Factors from Newly Established Colon Carcinoma Cell Cultures on Human Lymphocyte Proliferation and Cytokine Secretion

Tumor cells may influence the host’s immune reactivity by the production of immunosuppressive factors (ISFs). In this study, the effects of ISFs derived from nine polyclonal colorectal carcinoma (CRC) cell lines on PHA-induced lymphocyte proliferation and cytokine secretion was investigated. We found that most of the culture supernatants (8/9) from CRC cell lines contained ISFs, which inhibited T cell proliferation to a variable degree in a dose-dependent manner. Comparison of T cell proliferation in the presence or absence of monocytes showed that monocytes can modulate the effects of tumor-derived ISFs on lymphocyte function. In addition, exposure of activated PBMC to the tumor cell supernatants resulted in an altered secretion of cytokines by these cells, i.e. the secretion of IFN-γ was generally reduced while the secretion of IL-1β, IL-2 and TNF-α was little affected. We further investigated the supernatants’ inhibitory effects on PBMC in respect to the production of prostaglandin E2 (PGE2). It was found that PGE2 was secreted by all tumor cell cultures. Therefore this substance is probably involved in the immunosuppression in vivo. However, the secreted PGE2 was shown not to be solely responsible for the observed suppression of lymphocyte proliferation in vitro. Our results suggest that the secretion of ISF is a common property of CRCs as demonstrated with newly established CRC cell cultures, and therefore this may also be an important immune escape mechanism of colonic carcinomas in vivo.

The carcinoembryonic antigen (CEA) modulates effector-target cell interaction by binding to activated lymphocytes

We and others have shown that the carcinoembryonic antigen (CEA) modulates the susceptibility of human colorectal carcinoma cells to cytotoxic lymphocytes. We now demonstrate that the density of the CEA molecules on the tumor cell surface has a determining influence on its protective function. In contrast, CEA released by tumor cells has no protective effect for CEA‐negative cells. To elucidate the responsible mechanism, we analyzed the binding properties of CEA to lymphokine activated killer (LAK) cells. In agreement with our observation that only membrane‐bound CEA provides protection, we found that intercellular contact between LAK cells and CEA‐expressing tumor cells is required for binding of CEA to LAK cells. Using FACScan analysis, we demonstrate the presence of CEA on lymphocytes co‐cultured with CEA‐transfected cells but not after co‐culture with their parental cells and after incubation with soluble CEA. Interestingly, following overnight co‐culture the amount of bound CEA on LAK cells was identical regardless of adherence on tumor cells or loss of contact with tumor cells. This indicates that CEA is released from the tumor cells after binding to LAK cells. Our results suggest that tumor cells can modulate effector cell adhesion by regulating the turnover rate of CEA on the tumor cell membrane.

Isolated extracellular matrix-based three-dimensional in vitro models to study orthotopically cancer cell infiltration and invasion

An initial event in colon cancer progression is the migration of epithelial cells through the basement membrane (BM) and the invasion of the colon submucosa, where tumour cells enter blood and lymph vessels to spread throughout the body. To interrupt this process would mean the prevention of metastasis. In order to investigate tumour cell invasion orthotopically in the human system, we established novel in vitro models which mimic normal human colon tissue (colon reproductions, CoRes) and primary colon carcinomas (artificial tumours, ArTs). These models are based on the isolated extracellular matrix (iECM) of the respective human tissues. Two isolation methods were established, the Digestion Method and the Lysis Method neither of which destroyed the characteristic architecture of the ECM found in the original tissues. BM components, i.e. laminin, fibronectin and collagen IV, were detectable in the iECM isolated with the Lysis Method but not those isolated with the Digestion Method. Scanning electron microscopic analysis of the normal colon iECM demonstrated that even if the BM was missing, the luminal surface consisted of densely packed ECM filaments which do not allow cell infiltration without degradation of the iECM. Furthermore, we demonstrated that iECM can be separately supplemented with different cell types, i.e. colorectal carcinoma cells, normal fibroblasts and immune cells at any desired concentration, combination and localisation. Therefore, these models could be used to determine the role of the BM and of the tumour cell/normal cell crosstalk in the infiltration process of human colorectal carcinoma cells.

MODULATIONS OF THE EFFECTOR FUNCTION AND CYTOKINE PRODUCTION OF HUMAN LYMPHOCYTES BY SECRETED FACTORS DERIVED FROM COLORECTAL-CARCINOMA CELLS

We investigated the in vitro effects of factors secreted by 3 freshly explanted human colorectal‐carcinoma (CRC) cell lines on lymphocyte proliferation, IL‐2‐receptor expression, LAK‐cell generation and cytokine secretion. We found that the supernatants of all 3 CRC cell lines inhibited T‐cell proliferation in a dose‐dependent manner, due to the secretion of immunosuppressive factors (ISFs). In addition, the supernatants of 2 cell lines were able to inhibit LAK‐cell generation and to depress IL‐2R, but not HLA‐DR expression, on PHA‐activated T cells. Furthermore, the secretion of cytokines, i.e., IFN‐γ, IL‐1β, IL‐2 and TNF‐α, by peripheral‐blood mononuclear cells (PBMC) was differently modulated by the tumor‐cell supernatants, e.g., the production of IFN‐γ was reduced in normal PBMC stimulated with PHA. However, the effects induced by the supernatants were not identical: for example, factors from one CRC cell line (w25) influenced early and late events of T‐cell activation and division, while 2 others (w19 and te6) contributed only to the inhibition of early events. Some biochemical properties of the ISFs were characterized. Our results suggest that colon‐tumor cells can secrete ISFs, which may lead to the in vivo immunosuppression often observed in patients with these tumors. Int. J. Cancer 72:142–148, 1997.

Artificial tumor: a novel heterotypic, polymorphic, three-dimensional in vitro model of individual human solid tumors.

We established a new complex three-dimensional in vitro model called artificial tumor (ArT), which stimulates individual human solid tumors. In contrast to the generally used spheroid models consisting of tumor cells only, the model described in the present report is composed of both tumor cells and tumor stromal cells. We created ArTs as a replica of colon carcinomas, composed of colon carcinoma cells, colon fibroblasts, and as supportive skeleton fibrous tissue from human colon. All cellular components were established from the same surgically obtained colon carcinoma specimen. The ArTs studied came from 4 types of colon carcinomas; mucinous, well, moderately and undifferentiated colon carcinomas. Among all types of colon carcinomas ArTs could be established with a high rate of success, i.e. 81%. Cells growing for 5 days on the fibrous tissue under normal culture conditions formed tumors up to 2,000 microns in diameter. Morphological studies showed that tumor cells grown as ArTs maintained the individual characteristics of the original tumor; for example, a comparable heterogeneity in CEA expression and/or maintenance of the differentiation stage. Polarized tumor cells were linked with the tumor stroma-simulating structure by their basal cell membrane, and formed central lumina lined by the apical membranes of the tumor cells, a structure quite similar to the tissue architecture of colon carcinomas in vivo.

The influence of the local environment on tissue architecture of colorectal carcinoma (CRC) cell aggregates and its consequence for tumour attack by lymphocytes in vitro and in vivo

We analysed colorectal carcinoma (CRC) specimens, tumour cell spheroids and artificial tumours (ArTs) for tissue architecture, carcinoembryonic antigen (CEA) expression and lymphocyte infiltration. Two distinct organisation forms of well-differentiated CRC cells were found in vivo and in vitro. Tumour cells having contact with the tumour stroma in primary tumours, and tumour cells growing within a stroma-like structure in vitro (ArTs) were arranged as pseudoglands. In contrast, tumour cells grown as spheroids or tumour cells having lost contact with the tumour stroma in primary tumours, and most probably in the circulation, showed an inversion of the architecture of these pseudoglands, presenting their apical cell membrane to the environment. These different tumour cell formations affect lymphocytes attacking the tumour, which need contact with specific cellular membranes of polarised tumour cells, depending on the tumour architecture. Recently, we demonstrated that the CEA expression of CRC cells correlated with their resistance against LAK-cell lysis. Since CEA is mainly expressed on the apical membrane of the tumour cells, independent of the tissue architecture, the change from the pseudoglandular to the spheroid-like formation may represent an escape mechanism for malignant cells.

Artificial Tumor (ArT): Rekonstruktion individueller, humaner Primärtumoren in vitro

Zusammenfassung Wir haben ein neues dreidimensionales in vitro-Modell, Artificial Tumor (ArT) genannt, entwickelt, das individuelle solide Humantumore simuliert. Statt ausschlieslich Tumorzellen, wie herkommliche Spheroidmodelle, verwendet dieses System Kolonkarzinom-(CC)-zellen, Fibroblasten und Stroma, also eine CC-Replik. Alle Komponenten stammen aus ein und demselben Operationsmaterial. ArTs wurden aus 4 CC-Arten, namlich muzinosen, hoch-, moderat und entdifferenzierten CCs, gezuchtet. Als ArTs gezuchtete Tumorzellen behielten individuelle Charakteristika des Ausgangsmaterials bei, z.B. vergleichbare Heterogentat bei Antigen CEA-Expression und Beibehaltung des Differen-zierungsgrades. Ein Vergleich der Gewebearchitektur von ArT, Tumorzell-Spheroiden und CC-Zellinien zeigte: in vitro spheroid generierte differenzierte CCs formen Aggregate polarisierter Zellen, deren apikale Membran zum Kulturmedium und deren basale Membran in die Sphero-ide gerichtet sind. Haben CC-Zellen jedoch Kontakt zu Fibroblasten und extrazellularer Matrix (EZM) - wie im Falle von ArTs - formen sie pseudodrusige Strukturen, die fur differenzierte CCs charakteristisch sind. Unsere Hypothese besagt, das in vivo Tumorzellen ohne EZM-Kontakt, z.B. Tumorzellaggregate in Zirkulation, ebenfalls eine inside-out Architektur wie Spheroide aufweisen. Mit diesen beiden Modellen (ArT und Spheroide), einzeln oder in Kombination, konnen wir Tumorzellwachstum in vivo optimal simulieren. Deshalb hoffen wir, das ArT in vielen Studien den Tierversuch ersetzen kann, gab es doch bisher nur den Tierversuch, um Tumorzellwachstum histologisch zu untersuchen.