Influence of cytokines on the expression of fas ligand and CD44 splice variants in colon carcinoma cells.

The expression of Fas ligand (FasL) by malignant cells might be a mechanism for tumor immune escape. We investigated FasL expression by LS 174T colon carcinoma cells. Furthermore, the effects of in vitro stimulation with rIL-2, rIFN-gamma and rTNF-alpha were investigated with regard to a possible regulation of the FasL expression by cytokines. FasL expression was detected by flow cytometry and RT-PCR. We observed a spontaneous expression of FasL by LS 174T cells. Incubation with high-dose rTNF-alpha induced an upregulation of FasL of 23%. rIL-2 and rIFN-gamma did not significantly affect FasL expression. To control whether our cytokine stimulation experiments were suitable to prove an upregulation of membrane proteins by tumor cells, we investigated the expression of ICAM-1, N-CAM, CD44s, CD44v6 and CD44v10. These adhesion molecules were spontaneously expressed by LS 174T cells. Only ICAM-1 and CD44v10 were significantly upregulated by rIFN-gamma and rTNF-alpha, respectively. These results could indicate that cytokines, released by tumor-infiltrating leukocytes, may induce the FasL-dependent apoptotic signal by which tumors downregulate an immunological host response.

[Kidney donation by living donors. Surgical procedure]. / Die Nierenlebendspende. Chirurgische Vorgehen.

The living donation of kidneys is gaining importance as a possible way to give a transplant to patients with terminal renal insufficiency. However we do not yet have experience with all the possibilities arising from this method. In particular, there is caution caused by the risks of the donor operation. In this context, the method is discussed according to the literature and our own experience of 89 living kidney donations. In our own practice with living donations, we have a success rate with 96% after 4 years and 82% after 16 years. We observed complications including wound infections (10.7%), haemorrhage, hernia and neurological complications (each 2.7%). When performed by specialists, the donor operation is safe and is a responsible alternative to the transplantation of cadaver kidneys, which opens up new possibilities in these times of organ shortage.

Analysis of the T cell receptor variability of tumor-infiltrating lymphocytes in colorectal carcinomas.

While there is good clinical and experimental evidence for immunological tumor control in some tumors--malignant melanomas, for instance--the immunogenicity of colorectal carcinoma (CRC) still remains unsettled. We examined surgical specimens from 4 CRC patients for T cell clones among tumor-infiltrating lymphocytes (TIL). The growth of specific lymphocyte clones in a tumor indicates an immunological response in vivo. We used a T cell receptor V beta family-specific semiquantitative PCR with additional sequencing to examine TIL for clonal expansion. In CRC, specific T cell clones could not be demonstrated. However, we observed a predominance of V beta 9 in 3 of 4 tumors.

[Cytokine regulated expression of Fas-Ligand by colon carcinoma cells]. / Zytokinregulierte Expression von Fas-Ligand durch Kolonkarzinomzellen.

We studied the proliferative response of tumor-infiltrating lymphocytes (TIL) from colorectal carcinomas to rIL-2 as well as their cytotoxic activity which was reduced as compared to autologous peripheral blood lymphocytes. We observed TIL to be polyclonal and asked if Fas-ligand-expressing tumor cells are responsible for the elimination of specific T-cells. Therefore we studied the expression of Fas-ligand by LS174T colon carcinoma cells which we showed to be induced by cytokines.

Expression of CD44, ICAM-1 and N-CAM in colorectal cancer. Correlation with the tumor stage and the phenotypical characteristics of tumor-infiltrating lymphocytes.

The cellular adhesion molecules (CAMs) CD44s, CD44v6, CD44v10, ICAM-1 and N-CAM were immunohistologically detected in colorectal cancers using the APAAP method. The expression of CD44s and CD44v6 was associated with the presence of lymph node metastases in the examined tumors. The pattern of ICAM-1 expression was inversely related to that of CD44, i.e. lower numbers of ICAM-1 positive cells were observed in metastasizing tumors. An intense focal staining of N-CAM was observed in the majority of the metastasizing tumors. The expression of CD44v, ICAM-1 or N-CAM on tumor cells did not correlate with the density of the tumor-infiltrating lymphocytes (TIL) within the tumors. The flowcytometric analysis of TIL showed a significant accumulation of CD25+ and HLA-DR+ cells and a reduced number of CD45RA+ cells as compared to autologous peripheral blood lymphocytes (PBL) or intraepithelial lymphocytes of the colon mucosa (IEL). These phenotypic characteristics of TIL did not correlate with the CAMexpression on tumor cells.

Phenotypical and functional differences of tumor-infiltrating lymphocytes from human colorectal cancers induced by costimulation with rIL-2 and rIL-4 in vitro.

The influence of recombinant human interleukin-4 (rIL-4) on the proliferation and cytotoxic activity of tumor-infiltrating lymphocytes (TIL) from human colorectal cancers was investigated TIL and peripheral blood lymphocytes (PBL) were cultured for 3-5 weeks. Under simultaneous stimulation with rIL-2 and rIL-4 the proliferation of TIL was less pronounced compared to stimulation with rIL-2 alone. In rIL-2 expanded TIL an outgrowth of CD56+ cells was observed. Concordantly, the expression of CD3+ cells was low. The number of CD56+ cells could be reduced significantly in TIL and PBL by stimulation with rIL-2 and rIL-4 simultaneously while the number of CD3+ cells increased. In TIL and PBL co-stimulation with rIL2 and rIL-4 resulted in higher CD4/CD8 ratios as compared to expansion with rIL-2 alone. In a 72 hour cytotoxicity assay the rIL-2/rIL-4 expanded TIL and PBL showed a lower lytic activity against autologous tumor targets in comparison to the rIL-2 expanded lymphocytes. In contrast, the cytotoxic activity of rIL-2/rIL-4 cultured TIL against allogeneic tumor cells was higher than in rIL-2 stimulated TIL.

Morphological and functional characteristics of tumor-infiltrating lymphocytes from human colorectal cancers after stimulation with rIL-2.

Tumor-infiltrating lymphocytes (TILs) from colorectal cancers were separated from tumor cells by enzymatic and mechanical tissue disaggregation and discontinuous density gradients. Peripheral blood lymphocytes (PBLs) were isolated using the same procedure. The freshly separated TILs and PBLs were analyzed phenotypically by flow cytometry. The CD4+/CD8+ ratios of the freshly isolated TILs and PBLs were comparable (> 1 in both lymphocyte populations). CD25+, HLA-DR+ and CD56+ cells were significantly more frequent in the TIL than in the PBL population. However, the number of CD45RA+ cells was lower in the TILs as compared to PBLs, while CD29+ accumulated by about 90% in TILs. TILs and autologous PBLs were expanded in vitro with rIL-2. The mean rate of proliferation after 4 weeks was 642-fold in TIL cultures and 335-fold in PBLs. More than 90% of the rIL-2-expanded lymphocytes expressed CD2 and the great majority was CD29+. Stimulation with rIL-2 in vitro induced an outgrowth of CD56+ cells mainly in the TILs. Accordingly the expression of CD3+ and alpha/beta receptor in the TILs was low. Those cells which phenotypically represented lymphokine-activated killer cells displayed a lytic activity against the autologous tumor as well as against allogeneic K562 and Daudi targets. In accordance with the better proliferative response of TILs in long-term cultures with rIL-2, the lytic activity of TILs against autologous and allogeneic tumor targets was significantly higher as compared to PBLs.

Phenotypical and functional characteristics of tumor-infiltrating lymphocytes from colon carcinomas stimulated with rIL-2 and rIL-4 in vitro: comparison with lymphocytes of the normal colon mucosa and the peripheral blood.

The phenotypical composition of tumor-infiltrating lymphocytes (TIL) from colorectal carcinomas was compared with that of intraepithelial lymphocytes of the autologous normal colon mucosa (IEL) and autologous peripheral blood lymphocytes (PBL). The CD4+/CD8+ ratios of the freshly isolated TIL, IEL and PBL were comparable and always > 1. CD25+, HLA-DR+ and CD56+ cells accumulated significantly in the TIL, while CD45RA+ cells were less frequent compared to the autologous IEL and PBL. CD29+ memory-cells were found with the highest frequency in the TIL. Stimulation with rIL-2 in vitro induced an outgrowth of CD56+ cells in the TIL. Concordantly the expression of CD3+ and the alpha/beta T-cell receptor was low. In a standard 51Cr-release assay these phenotypically LAK-cells representing effectors displayed an unspecific lytic activity against the autologous tumor as well as against allogeneic K562 and Daudi targets. The number of CD56+ cells could be reduced in TIL and PBL by simultaneous stimulation with rIL-2 and rIL-4, while the number of CD3+ cells increased.

Phenotyping of human tumor-infiltrating lymphocytes before and after exposure to different in vitro stimulation conditions.

Tumor-infiltrating lymphocytes (TiL) and autologous peripheral blood lymphocytes (PBL), mainly from breast and kidney tumor patients were cultivated with high- and low-dose rIL-2 under addition of autologous tumor cells or nonmalignant epithelial cells. Tumor cells added to TIL-cultures induced an additional proliferative response. Before cultivation, CD8+ and CD25+ lymphocytes were more frequent in TIL when compared to PBL, while CD16+, CD19+ and CD56+ cells were rare. After culture, lymphocytes of both origins showed an increase of CD2+, CD25+, CD56+ and HLA-DR+ cells and a relative decrease of CD3+ cells. The CD4+/CD8+ ratios and a number of CD56+ cells were rIL-2 dose dependent.