Abstract
Abstract 3645
Introduction:
The bulk of the tumour infiltrate in classical Hodgkin lymphoma (CHL) is composed of immune cells, predominantly CD4+ T cells, with the malignant Hodgkin Reed Sternberg cell (HRS) representing <1% of cells. The lymphoid microenvironment has been described as anergic and hypoproliferative with suppressive properties (Marshall et al. Blood 2004 103:1755–62) but the functional significance of this is unclear. This study set out to examine the proliferative capacity and phenotype of T cells derived from CHL-diagnostic lymph node tissue taken at diagnosis.
Method:
Frozen single cell suspensions (SCS) from 6 patients were selected from the tissue bank of our Institute. T cell growth-augmenting and/or Th2 polarising cytokines were added in various combinations (IL2, IL4 only, IL2+4 or no added cytokine) to SCS-derived cells in 96 well plates at 0.3 × 106 cells per well in 200mcl of optimized lymphocyte culture media. No CD4+ enrichment step was carried out: all recovered cells were plated at baseline to maintain potential interactions between CD4+ cells and other cells, and no mitogen or T-cell receptor-stimulating or costimulating agents were added at any point. As controls, SCS derived from normal tonsil, and ÔreactiveÕ lymph nodes (n=4) (confirmed by histological report at the time of diagnosis) were also plated. Plates were examined daily for cell/colony morphology to estimate growth and split with fresh media and cytokines once every 7 days, with estimated proliferation (by haemocytometry) plotted. Cultures were assessed at baseline, 10 days, 28 days, 50 days and 100 days.
Results:
Proliferation, based on formation of discrete colonies and blastoid cell morphology, occurred in the majority of wells by day 7 in all CHL-derived cultures, and in a minority of wells, and to a lesser extent in all control cultures. CHL-derived T cells from one patient continue to expand after 200 days, doubling every 3–5 days, while the other 5 continue after 50–100 days. In contrast, no tonsil or reactive node-derived T cells survived beyond 50 days and none showed a net expansion in cell numbers. Growth was superior in the IL2+4 and IL2-only conditions, with no growth in the media-only or IL4-only conditions. The most favorable condition was with the addition of IL2+4. By day 21 a net increase in CHL-derived T cells was apparent, but not in any control T cell populations (Figure). At baseline, composition of the CHL-derived cells revealed a majority of CD3+ cells as expected, of which 60–80% were CD4+ and the remainder CD8+. By D21 the CD4+ component had outgrown all other cells in the CHL-derived cultures, being CD3+CD4+CD45RO+ consistent with antigen-experienced T helper cells, while all tonsil and reactive node-derived cells were CD8+CDRO+. Markers of central memory (CCR7 & CD62-L), Th2 (CCR4 and IL4), Treg (FOXP3 and CD25) and anergy (CD57) were absent after expansion, while markers of activation were upregulated (CD28, CD27, CD69, CD40L, CD30 & CD95). This phenotype persisted in the ongoing T cell lines.
Conclusions:
The CD4+ compartment of the CHL microenvironment contains a primed subset of cells capable of massive expansion without further mitogenic stimulation and of generating cytokine-dependent continuous cell lines with an antigen-experienced, activated phenotype. This challenges the assumption of T cell anergy and hypoproliferation in the tissue microenvironment of CHL. We are currently assessing the function and anti-tumor specific or tumor supportive nature of these T cells.
Disclosures:
Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.
Improving the quality cell yield of T-cell immunotherapies through selective pressures imparted by culture media supplements
