Activation of the receptor NKG2D leads to production of Th17 cytokines in CD4+ T cells of patients with Crohn's disease.

BACKGROUND & AIMS: The natural killer group 2 member D (NKG2D) is a stimulatory receptor expressed on a subset of mucosal and peripheral CD4+ T cells in patients with Crohn's disease (CD) and other inflammatory diseases. Ligand activation of NKG2D in patients induces CD4+ T cells to release T-helper (Th) 1 cytokines and become cytotoxic. We investigated the Th17 cytokines produced by T cells that express NKG2D in blood and intestinal mucosa samples from patients with CD. METHODS: We isolated CD4+ T cells from peripheral blood and lamina propria samples of patients with CD or ulcerative colitis (UC) and healthy individuals (controls). We analyzed the phenotype and functions of the CD4+NKG2D+ T cells and the cytokines they produce in response to NKG2D stimulation. RESULTS: In patients with CD, CD4+ T cells that express NKG2D produced high levels of interleukin (IL)-17 and IL-22 and expressed high levels of CCR6, the IL-23 receptor, CD161, and RORC (a transcription factor that regulates expression of Th17 cytokines). CD4+ T cells that produced IL-17 expressed high levels of NKG2D and CD161. Costimulation of NKG2D and the T-cell receptor (TCR) significantly increased production of IL-17 and tumor necrosis factor α by CD4+ T cells, compared with activation of only the TCR. CD4+NKG2D+ T cells also responded to Th17 polarization. CONCLUSIONS: NKG2D is a functional marker of CD4+ T cells that produce IL-17 in patients with CD, via costimulation of the TCR and NKG2D. Reagents developed to block NKG2D might reduce gastrointestinal inflammation in patients with CD.

Biomolecular network reconstruction identifies T-cell homing factors associated with survival in colorectal cancer.

BACKGROUND & AIMS: Colorectal cancer is a complex disease involving immune defense mechanisms within the tumor. Herein, we used data integration and biomolecular network reconstruction to generate hypotheses about the mechanisms underlying immune responses in colorectal cancer that are relevant to tumor recurrence. METHODS: Mechanistic hypotheses were formulated on the basis of data from 108 patients and tested using different assays (gene expression, phenome mapping, tissue-microarrays, T-cell receptor [TCR] repertoire). RESULTS: This integrative approach revealed that chemoattraction and adhesion play important roles in determining the density of intratumoral immune cells. The presence of specific chemokines (CX3CL1, CXCL10, CXCL9) and adhesion molecules (ICAM1, VCAM1, MADCAM1) correlated with different subsets of immune cells and with high densities of T-cell subpopulations within specific tumor regions. High expression of these molecules correlated with prolonged disease-free survival. Moreover, the expression of certain chemokines associated with particular TCR repertoire and specific TCR use predicted patient survival. CONCLUSIONS: Data integration and biomolecular network reconstruction is a powerful approach to uncover molecular mechanisms. This study shows the utility of this approach for the investigation of malignant tumors and other diseases. In colorectal cancer, the expression of specific chemokines and adhesion molecules were found as being critical for high densities of T-cell subsets within the tumor and associated with particular TCR repertoire. Intratumoral-specific TCR use correlated with the prognosis of the patients.

Memory T-cell responses and survival in human cancer: remember to stay alive.

Cancer is a major public health problem worldwide. Accumulating evidence suggests that Gtumor-host interactions may in part impact on tumor progression. However, the role of inflammation and adaptive immune reaction in cancer emergence, local and metastatic invasion and recurrence are still not dearly defined. Pro-inflammatory mediators are suspected to favor tumor growth and angiogenesis and naturally generated T cells with antigenic specificity to tumor associated antigens were usually in a state of anergy. Nevertheless, experiments in mouse and human showed a significant association between high density of tumor infiltrating T cells and improved cancer prognosis. Recently, the global analysis of colorectal cancer microenvironment demonstrated that a strong and coordinated Th1 adaptive immune response within primary tumors dramatically reduced the risks of relapse events. Interestingly the absence of early signs of metastatic invasion (lymphovascular emboli) correlated with a significant increase of the density of memory T cells in situ. This chapter presents the arguments supporting the existence of immunosurveillance mechanisms in human cancer. We will discuss the potent role of memory T cells in cancer immunity as well as the opportunities of therapeutic strategies uncovered by this new area of investigation.

Effector memory T cells, early metastasis, and survival in colorectal cancer.

BACKGROUND: The role of tumor-infiltrating immune cells in the early metastatic invasion of colorectal cancer is unknown. METHODS: We studied pathological signs of early metastatic invasion (venous emboli and lymphatic and perineural invasion) in 959 specimens of resected colorectal cancer. The local immune response within the tumor was studied by flow cytometry (39 tumors), low-density-array real-time polymerase-chain-reaction assay (75 tumors), and tissue microarrays (415 tumors). RESULTS: Univariate analysis showed significant differences in disease-free and overall survival according to the presence or absence of histologic signs of early metastatic invasion (P<0.001). Multivariate Cox analysis showed that an early conventional pathological tumor-node-metastasis stage (P<0.001) and the absence of early metastatic invasion (P=0.04) were independently associated with increased survival. As compared with tumors with signs of early metastatic invasion, tumors without such signs had increased infiltrates of immune cells and increased levels of messenger RNA (mRNA) for products of type 1 helper effector T cells (CD8, T-BET [T-box transcription factor 21], interferon regulatory factor 1, interferon-gamma, granulysin, and granzyme B) but not increased levels of inflammatory mediators or immunosuppressive molecules. The two types of tumors had significant differences in the levels of expression of 65 combinations of T-cell markers, and hierarchical clustering showed that markers of T-cell migration, activation, and differentiation were increased in tumors without signs of early metastatic invasion. The latter type of tumors also had increased numbers of CD8+ T cells, ranging from early memory (CD45RO+CCR7-CD28+CD27+) to effector memory (CD45RO+CCR7-CD28-CD27-) T cells. The presence of high levels of infiltrating memory CD45RO+ cells, evaluated immunohistochemically, correlated with the absence of signs of early metastatic invasion, a less advanced pathological stage, and increased survival. CONCLUSIONS: Signs of an immune response within colorectal cancers are associated with the absence of pathological evidence of early metastatic invasion and with prolonged survival.

Coordination of intratumoral immune reaction and human colorectal cancer recurrence.

A role for the immune system in controlling the progression of solid tumors has been established in several mouse models. However, the effect of immune responses and tumor escape on patient prognosis in the context of human cancer is poorly understood. Here, we investigate the cellular and molecular parameters that could describe in situ immune responses in human colorectal cancer according to clinical parameters of metastatic lymph node or distant organ invasion (META- or META+ patients). Primary tumor samples of colorectal carcinoma were analyzed by integrating large-scale phenotypic (flow cytometry, 39 patients) and gene expression (real time reverse transcription-PCR, 103 patients) data sets related to immune and protumoral processes. In META- colorectal cancer primary tumors with high densities of T cells, we observed significant positive correlations between markers of innate immune cells [tumor-associated macrophages, dendritic cells, natural killer (NK) cells, and NKT cells] and markers of early-activated T cells. Significant correlations were also observed between markers of cytotoxic and effector memory T-cell subpopulations. These correlation profiles were absent in tumors with low T-cell infiltrates and were altered in META+ tumors with high T-cell infiltrates. We show that the coexpression of genes mediating cytotoxicity (GNLY) and Th1 adaptive immune responses (IRF1) accurately predicted patient survival independently of the metastatic status. High intratumoral mRNA expression of the proangiogenic mediator vascular endothelial growth factor was associated with significantly reduced survival rates in patients expressing high mRNA levels of GNLY. Investigation of the colorectal cancer primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response.

Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.

The role of the adaptive immune response in controlling the growth and recurrence of human tumors has been controversial. We characterized the tumor-infiltrating immune cells in large cohorts of human colorectal cancers by gene expression profiling and in situ immunohistochemical staining. Collectively, the immunological data (the type, density, and location of immune cells within the tumor samples) were found to be a better predictor of patient survival than the histopathological methods currently used to stage colorectal cancer. The results were validated in two additional patient populations. These data support the hypothesis that the adaptive immune response influences the behavior of human tumors. In situ analysis of tumor-infiltrating immune cells may therefore be a valuable prognostic tool in the treatment of colorectal cancer and possibly other malignancies.