A Subpopulation of Follicular Lymphoma Tumor Infiltrating T Cells Shows Suppressed Common Gamma Chain Cytokine Signaling.

Tumor infiltrating T cells present within biopsy specimens of human B cell non-Hodgkin's lymphomas (NHL) provide a valuable opportunity to examine immune system function in the presence of cancer. We recently used flow cytometry to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a novel lymphoma cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome. Here, we turned our attention to signaling differences in subsets of the tumor-infiltrating T cells from FL and two other NHLs, diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Signaling differences that distinguish the tumor infiltrating T cells from each malignancy might be expected to be a reflection of the specific disease microenvironment, whereas T cell signaling differences distinguishing cases of the same malignancy might be related to the biology of each patient's tumor.

Single cell flow cytometry measurements of signaling were acquired for samples of DLBCL (N=13), MCL (N=20), and FL (N=14). Phosphorylation of 14 signaling proteins was measured under 12 stimulation conditions in every cell, including lymphoma B cells and tumor-infiltrating T cells within the same specimen. Stimulation conditions included those that were B cell specific (BCR crosslinking, CD40 ligand), T cell specific (IL-7), and those that stimulated both B and T cells (IL-4, IL-10, IL-21, PMA + ionomycin, and IFN-γ).

Striking differences were observed in the signaling responses of tumor infiltrating T cells. T cells infiltrating FL patient samples showed significantly lower responses to cytokines where signal transduction is mediated by the common γ chain receptor. Specifically, we observed significant lower induction of p-STAT6 after IL-4 stimulation, p-STAT5 after IL-7 stimulation, and p-STAT3 after IL-21 stimulation (p < 0.001 for FL vs. MCL in all cases). In contrast, receptor-independent signaling was not significantly different as FL tumor infiltrating T cells responded at a level comparable to MCL and DLBCL tumor infiltrating T cells when stimulated with PMA and ionomycin. The lower response to common γ chain family cytokines could be the result of a partial suppression of all tumor infiltrating T cells or a complete suppression of a distinct subset. To distinguish between these possibilities, we analyzed signaling in tumor infiltrating T cell subsets. This single cell approach showed that tumor infiltrating T cells were a heterogeneous mixture of non-responsive cells and highly responsive T cells in response to cytokines. Specifically, the mean percentage of T cells that did not induce p-STAT3 after IL-21 stimulation was 50.3% in FL samples in contrast to only 26.2% in MCL samples. Phenotypic analysis showed that the vast majority of T cells infiltrating FL patient samples were CD4+CD45RO memory cells, and the single cell signaling approach revealed that the FL nonresponsive T cell subset had this phenotype. Furthermore, FL T cells were composed of a significantly larger fraction of T regulatory cells than MCL T cells, on average 17% FoxP3+CD25+ cells compared to only 9% in MCL (p<0.0002). Experiments are ongoing to test whether the prevalence of T regulatory cells influence the signaling capacity of the remaining CD4 conventional T cells.

A subpopulation of tumor infiltrating T cells within FL patient samples has reduced responsiveness to the common gamma chain family members IL-4, IL-7 and IL-21, and distinguishes FL from DLBCL and MCL. These results may reflect a more suppressive microenvironment in FL.

No relevant conflicts of interest to declare.