IL-12 reverses anergy to T cell receptor triggering in human lung tumor-associated memory T cells

Abstract

Memory T cells in human non-small cell lung cancer are unresponsive to progressing tumors. T cells were evaluated at the single cell level by imaging the nuclear translocation of NF-κB and NFAT via immunofluorescence confocal microscopy as an early measure of responsiveness to T cell receptor triggering. Little translocation of NF-κB or NFAT occurred in tumor-associated T cells in response to CD3+CD28 cross-linking under conditions which led to maximal translocation in normal donor peripheral blood T cells. TNF-α induced maximal NF-κB translocation in these T cells, indicating that they remain receptive to alternative signaling pathways, and pulsing with IL-12 prior to TCR triggering reversed their apparent anergy. T cells from additional chronic inflammatory microenvironments demonstrated a similar refractoriness to TCR activation, suggesting either that a common regulatory mechanism present within the microenvironment controls these cells or that with continuous antigen exposure, they remain refractory to activation via the TCR.

Introduction

The chronic inflammatory state associated with non-small cell lung cancer and its functional significance and capabilities have remained somewhat of an enigma, as the T lymphocytes, despite their presence within the tumor microenvironment, appear to be quiescent and unresponsive to the progressing tumor [1]. While such T cells, specific for tumor antigens, are present in the immunocompetent tumor-bearing host, they may have defects in T cell receptor signaling pathways [2], [3]. However, the defects may not be uniquely tumor-associated, as changes in signaling molecules have been observed in cells isolated from other chronic inflammatory conditions including HIV [4], chronic viral infections [5], and systemic lupus erythematosus [6].

The T cell signaling pathway culminates with the activation of transcription factors including the nuclear factor of activated T cells (NFAT) and nuclear factor-κB (NF-κB) [7], [8]. NF-κB has been identified in many cell types and its expression used as an indicator of cellular activation, i.e. in response to cytokines, ultraviolet radiation, inhaled particles, and bacterial and viral products [9]. In the resting cell, NF-κB is sequestered in the cytoplasm through binding to the inhibitory protein IκB. Upon cellular stimulation, IκB is phosphorylated and degraded, thereby releasing NF-κB which then translocates to the nucleus [10]. The ubiquitous expression of NF-κB and its activation by a variety of mediators leading to cellular events such as cell–cell adhesion, migration, and amplification of pathogenic signals have generated a profound interest in NF-κB and its role in the initiation and pathogenesis of inflammatory conditions [10]. The majority of these studies, however, have focused upon gel shift analysis of whole tissue lysates [11], [12], thereby including the cell signaling pathways of epithelial cells, stromal cells, and other leukocytes, in addition to the T cells. Therefore, a protocol enabling the direct visualization of T cells within the mixed cell population would be advantageous as the effects of extracellular stimuli specifically on T cells could then be determined.

We recently established that T cells persisting in the tumor microenvironment of human non-small lung cancer were effector memory T cells that could be reactivated in situ by the local and sustained release of IL-12 to proliferate, secrete IFN-γ, and kill tumor cells [13], [14]. Two important questions regarding these findings are (A) is the failure of tumor-associated memory T cells to control tumor progression due to an inability to respond to TCR-induced signals and (B) is IL-12 able to reverse this defect? In the current report, we address these questions by focusing upon T cells at the single cell level, and examining their ability to signal through the T cell receptor prior to and subsequent to pulsing with cytokines. Through immunofluorescence microscopy, these T cells can be probed for their ability to translocate NF-κB and NFAT from the cytosol to the nucleus in response to such extracellular stimulation. We establish that while these T cells are not responsive to T cell receptor cross-linking under conditions which activate peripheral blood T cells from the same tumor-bearing patients and T cells from healthy donors, such T cells are activated via cytokine pathways. We show here that IL-12 is able to reverse the blockade in the T cell receptor signal cascade and that TNF-α activates the quiescent T cells independent of the T cell receptor. Similar anergy (resistance to activation via the TCR) was observed in T cells isolated from other chronic inflammatory microenvironments, namely nasal polyp tissue from patients with chronic hyperplastic rhino-sinusitis with nasal polyposis and synovial fluid from patients with rheumatoid arthritis.