[Stimulatory and inhibitory signaling pathways of the T cell-APC interaction and the effect of TLR agonists on APCs]. / Stimulierende und inhibierende Signalwege der APZ- und T-Zell-Interaktion sowie Einfluss von TLR-Agonisten auf APZ.

BACKGROUND: CD8+ cells are key players in the identification and elimination of cancer cells. Cancers can escape an effective T cell response by inducing an exhausted cell state, which limits the cytotoxic capacity of the effector cells. Among other mechanisms, new checkpoint inhibitors reactivate exhausted, dysfunctional T cells. CD8+ T cells can eliminate tumor cells after presentation of tumor-specific antigens via antigen-presenting cells (APCs). APC-mediated tumor recognition is mainly stimulated by Toll-like receptors (TLRs). OBJECTIVE: This study investigates the effect of TLR agonists on APCs as well as stimulatory and inhibitory signaling pathways of the T cell-APC interaction. MATERIALS AND METHODS: Gene expression of interleukin (IL)12 and programmed death ligand 1 (PD-L1) was analyzed by quantitative polymerase chain reaction (qPCR) after 0, 8, 24, and 48 h of CD14+ cell stimulation with CpG. Protein expression of inhibitor of nuclear factor kappa B (IκBα) after CpG stimulation was investigated by western blot. CD8+ T cells were stimulated for 72 h with or without programmed cell death protein 1 (PD-1) checkpoint blockade and analyzed for expression of PD­1, Tim­3, CTLA4, and Lag3 by flow cytometry. RESULTS: TLR stimulation (by unmethylated CpG DNA) of APCs upregulates immunostimulatory signals such as IL12 expression but also activates immunoinhibitory signaling pathways such as PD-L1 expression. This signaling is NF-κB dependent. After blockade of the PD-1/PD-L1 signaling pathway, overexpression of other immune checkpoint inhibitory receptors was observed-a potential explanation for lacking therapeutic responses after TLR stimulation with PD­1 checkpoint blockade. CONCLUSION: TLR stimulation causes APCs in the tumor microenvironment to upregulate PD-L1 in an NF-κB-mediated fashion, thereby contributing to CD8+ T cell exhaustion. The effect of PD­1 blockade after TLR stimulation might be impaired due to upregulation of other checkpoint inhibitors.

[Establishing three dimensional tumors in vitro and the reaction of the lymphokine-activated killer cells (LAK) to the three dimensional tumors]. / Etablierung dreidimensionaler Tumoren in vitro und Wirkung von lymphokin-aktivierten Killer (LAK)-Zellen auf die dreidimensionalen Tumoren.

In this study, we attempted to develop a technique, by which three-dimensional tumors were produced from two cultured head and neck tumor cell lines (Hep2, KB) and a colon adenocarcinoma cell line (HT29) using fibrinogen, thrombin, and double layered agar system. The three-dimensional tumor was large enough to perform the histologic study, which showed no significant histologic difference in comparison with the histologic findings of the xenografted tumor on nude mice. Furthermore, we applied this assay model to evaluate the antitumor effect of lymphokine activated killer (LAK) cells on the three-dimensional tumor produced by the technique. When tumor cells were cocultured with LAK cells, the damage of the three-dimensional structure due to the degeneration of tumor cells was observed. These findings suggest that the three-dimensional tumor may be useful to evaluate the antitumor effect of LAK cells in term of head and neck solid tumors.

[Cellular immunity in 3 siblings with cancers of the upper aerodigestive tract]. / Die zelluläre Immunität bei drei Geschwistern mit Karzinomen des oberen Aerodigestivtraktes.

Although the number of carcinomas of the upper aerodigestive tract is increasing, there are not many reports in the literature about familial occurrence. Epidemiological studies have shown that the major causative factors are tobacco and alcohol abuse. But the possibility of an endogenous susceptibility is also discussed. A possible explanation is failure of the immune defense system. The author was able to examine immunologic patterns in three siblings with cancer of the upper aerodigestive tract. The results obtained so far indicate normal numbers of lymphocyte subpopulations in the peripheral blood, but with impaired function.

Effect of lymphokine-activated killer cells on head and neck tumours in nude mouse model.

The present study was designed to investigate the in vivo effect of local application of lymphokine-activated killer (LAK) cells on the growth of tumours implanted under the renal capsule in nude mice, and especially to test whether large granular lymphocytes (LGL), regarded as natural killer (NK) cells, are the main precursor of LAK cells in vivo. Our results showed that the local application of LAK cells inhibited the growth of tumours in the head and neck region. The growth of tumours implanted under the renal capsule was inhibited by local application of 1 x 10(7) recombinant interleukin-2 (rIL-2) activated non-adherent lymphocytes, but the inhibitory effect was almost the same as produced by 3 x 10(6) rIL-2-activated LGL application. The findings indicate that the rIL-2-activated LGL are the main effectors in inhibiting tumour growth. In addition, rIL-2-activated non-adherent lymphocytes as well as LGL significantly prolonged the number of days of 50% survival and mean survival time of nude mice, in which HLaC78 cells, from a laryngeal tumour cell line, were injected into the subrenal capsule space with effector cells at various effector: target (E:T) ratios. The results indicate that the application of LAK cells may be useful in the treatment of patients with head and neck tumours.

[Initial results of immunotherapy approaches to tumor treatment in the animal model]. / Erste Ergebnisse immuntherapeutischer Ansätze zur Tumorbehandlung im Tiermodell.

The possibilities of using immunocytes in cancer therapy have been increasing during the last few years. Contrary to the promising results gained by in vitro experiments, several clinical trials have shown that, on the one hand, it is difficult to preserve a quantity of cells big enough to inhibit tumours, and they have also shown that, on the other hand, antitumour lymphocytes do not get into the tumour. That is why we concentrated on improving the tumour selectivity of the antitumour lymphocytes. We carried this out practising two sets of experiments. First we incubated patient lymphocytes with tumour extract and vaccinated them in this manner. Secondly, by binding antitumour antibodies to lymphocytes, we could improve the ability of lymphocytes to bind with tumour cells. We tested these therapy models on human tumours and on tumour cell lines. Both were implanted in the renal capsule space of nude mice.

The anti-tumor effect of in vitro tumor-stimulated autologous peripheral blood lymphocytes measured by the subrenal capsule assay.

Lymphokine-activated killer cells (LAK) are able to kill natural killer (NK)-resistant fresh bioptic tumor cells. We have tried to increase the antitumor activity of peripheral blood lymphocytes by the simultaneous stimulation with interleukin-2 and autologous tumor extract (TE). The influence of LAK cells and LAK cells stimulated with TE was compared in the subrenal capsule assay in nude mice. Experiments were performed with eight head and neck tumors following their surgical extirpation. The tumors were first grown in the renal capsule space while lymphocytes were being stimulated in vitro. Following this, the lymphocytes were injected into the growing tumors. The autologous TE-stimulated LAK cells were more effective in treating tumors than were the LAK cells. Tumors regressed in some cases so treated, a finding which was never observed with LAK cells alone.

Use of the subrenal capsule assay to measure antibody-dependent, cell-mediated cytotoxicity against head and neck tumors.

Experience with antibody-dependent, cell-mediated cytotoxicity (ADCC) has shown that antibody can increase the localization and killing capacity of lymphocytes. We tested the possibility of improving the activity of lymphokine-activated killer cells (LAK) on human tumor using the subrenal capsule assay in nude mice. The tumors were first grown in the renal capsule space and the effector cells injected later. In the model experiment we used M21 melanoma and monoclonal antibody against melanoma-associated antigen GD3. This antibody increases the tumor inhibitory activity of LAK cells from healthy donors in comparison to LAK alone. We have been able to prove the clinical relevance of such an approach. Tumor bioptic material from five tumor patients was tested with various monoclonal antibodies, following which the highly reactive antibodies were selected and incubated with the patient's LAK cells. Such pretreated LAK cells have a high growth-inhibitory effect on autologous tumor growing in the renal capsule space of the test mice.