CONTROL-T RESEARCH PROJECT 7

The team

From left:
Prof. Sylvia Hartmann
Olga Goncharova

Second Funding Period

RP 7 joined the Control-T consortium in the second funding period.

Abstract

Anaplastic large cell lymphoma (ALCL) is a CD30-expressing T-cell lymphoma, showing morphologic overlap with Hodgkin lymphoma (HL). However, important differences in clinical behavior have been observed: ALCL usually presents with advanced stage and has a worse prognosis. Whereas in HL the cellular microenvironment as well as the chemokines and their corresponding receptors, which together contribute to the composition of the microenvironment, have been extensively studied, very little is known about the microenvironment and the expression of chemokines and their receptors in ALCL. Chemokines and their receptors crucially impact the distribution and trafficking of leukocytes. Therefore, the aim of the present study is to characterize chemokine ligand and receptor expression on tumor and bystander cells in ALCL and to characterize the composition of the microenvironment in primary ALCL samples in order to better understand the microenvironmental niche which provides survival advantage for ALCL tumor cells. The findings in primary ALCL specimens can then be applied on in vitro experiments and experiments in native human lymphoid tissue with ALCL cell lines. Chemokine receptor expression will be modulated in ALCL cell lines and behavior of the ALCL tumor cells will be monitored in coculture with reactive bystander cells as well as in migration assay chamber experiments. Furthermore, GFP-labeled ALCL cell lines with and without expression of certain chemokine receptors will be applied onto primary thick slides of human lymphoid tissue. With this assay, additionally reactive bystander cells can be highlighted and the reactions, movements and interactions of all labeled cells will be visualized using live cell imaging. These experiments shall lead to a better understanding of the migration behavior and the distribution of ALCL tumor cells in human lymphoid tissue as well as their interaction with the cellular microenvironment. Differences in the microenvironmental interactions and chemokine expression may give an explanation of the differing clinical behavior when compared with cHL. Furthermore, the effects of novel therapeutic approaches in ALCL can be tested in primary human tissue under visual monitoring.

Aims

Objectives

Aim 1: Determination of the composition of the microenvironment and expression of chemokines and corresponding receptors in primary ALCL

To better understand the role of the microenvironment in ALCL, primary cases of ALK + and ALK - ALCL will be analyzed for the composition of their microenvironment with respect to T cells, B cells and histiocytes. The most frequent and most important cell types will be determined. Since we expect that either the reactive bystander cells secrete chemokines which attract ALCL tumor cells or vice versa that ALCL cells attract certain cell types of the microenvironment by cytokine secretion, immunohistochemical stainings for chemokines and the corresponding receptors will be performed with focus on their expression in tumor cells and specialized cell types in the microenvironment.

Aim 2A: Clarification which chemokine receptors and ligands are important for the migration behavior of ALCL

Since ALCL tumor cells are usually found in the sinuses of the lymph node or the T zone, we assume that the chemokine milieu in these areas must provide a particular niche to attract ALCL cells to these places. We therefore aim to clarify if the chemokine receptor expression observed in primary ALCL tumor cells in Aim 1 contributes to their typical distribution in the lymph node. Consequently, we first aim to mimic the chemokine situation observed in lymph nodes in cell culture experiments. For this purpose, the ALK + ALCL cell lines Karpas-299, SR786, JB6, DEL, SUDHL-1, SUP-M2, KI-JK and the ALK - ALCL cell line Mac-1 will be tested for expression of chemokine receptors by qRT-PCR, Western blot and flow cytometry (FACS). Corresponding ligands in the supernatants will be analyzed via ELISA or bead arrays. Thereafter, the appropriate cell lines will be studied in a cell migration assay using a chemokine gradient and performing time-lapse microscopy. For the chemokine receptors that are expressed in none of the ALCL cell lines, the respective chemokine receptors will be reexpressed using lentiviral transduction (in collaboration with RP1). In contrast, chemokine receptors, which are expressed in certain cell lines, like CCR4 and CCR7 in the Mac-1, will be downregulated using shRNAs or the CRISPR/Cas9 technology (in collaboration with RP1). Hence, the most important chemokine receptors for ALCL migration behavior will be determined.

Aim 2B: Determination in how far the interaction with primary human non-malignant cells influences the expression of CCRs, the migration behavior and the cell survival of ALCL cell lines

Here, we want to elucidate the functional role of the reactive bystander cells in the ALCL microenvironment on growth behavior and chemokine receptor expression in vitro. The most important cell types in the microenvironment of ALCL identified in Aim 1 will be purified from peripheral blood and will be studied in coculture with ALCL cell lines. The growth behavior of ALCL cell lines will be determined in the presence and absence of the reactive bystander cells. Furthermore, it will be tested if the expression of chemokine receptors, which are expressed in primary ALCL but are lost in the cell lines, can be reintroduced after coculture with reactive bystander cells. Since immune checkpoint inhibitors have recently entered the therapeutic spectrum and expression of PD-L1 was shown in primary ALCL and the ALCL cell lines SUDHL-1 and Karpas-299, growth of these cell lines will be studied after checkpoint inhibition in coculture with T cells.

Aim 3A: Determination of the migration behavior and microenvironmental interaction of ALCL cell lines in primary human tissue with respect to the expression of specific CCRs

In this aim we would like to clarify if the most important chemokine receptors for the migration of ALCL tumor cells identified in Aim 2A are really responsible for the distribution of ALCL tumor cells in human lymphoid tissue as observed in primary ALCL cases. For this purpose, the most appropriate ALCL cell lines identified in Aim 2A and ALCL cell lines over expressing certain chemokine receptors after viral gene transfer will be studied by in situ imaging to analyze migratory behavior of ALCL cells in lymphoid tissue. Therefore, fluorescently labeled ALCL cells will be plated on thick slices of human tonsils and lymph nodes. In preliminary studies, we already observed infiltration of labeled tumor cells into primary human tissue (in collaboration with the group of E. Donnadieu, Paris). Moreover, thick slices of lymphoid tissue will also be stained with fluorescently labeled antibodies against different cell types (microenvironment) and movements of these cells will be analyzed by spinning disk microscopy. Polyclonal T cells will be used in control experiments. Using this technique, we will focus on the ALCL migration behavior with respect to the expression of certain chemokine receptors and we will be able to visualize the interaction between ALCL cells and the cells of the microenvironment.

Aim 3B: Clarification whether treatment of ALCL cell lines with Brentuximab Vedotin influences cell survival and behavior in primary human tissue coculture assays

Applying the approach from Aim 3A, we would like to elucidate the effects of novel therapies employed in ALCL by visualization of the reaction of tumor and bystander cells, their interaction and movements. Brentuximab Vedotin, a specific anti-CD30 antibody-drug conjugate, has been implemented in the therapy of cHL as well as ALCL in recent years and has shown a direct toxic effect on the CD30-positive tumor cells. However, its therapeutic reaction has not been visualized in situ so far, as an experimental setup enabling real-time observation was missing. Therefore, ALCL cell lines and the microenvironment will be monitored in primary human tissue by spinning disk microscopy after application of Brentuximab Vedotin.