Thyroid Cancer Screening Using Tumor-Associated DN T Cells as Immunogenomic Markers.

Background: Thyroid nodules are an extremely common entity, and surgery is considered the ultimate diagnostic strategy in those with unclear malignant potential. Unfortunately, strategies aiming to predict the risk of malignancy have inadequate specificity. Our group recently found that the microenvironment of thyroid cancer is characterized by an enhanced immune invasion and activated immune response mediated by double-negative T lymphocytes (DN T) (CD3+CD4-CD8-), which are believed to enable or promote tumorigenesis. In the present work, we try to use the DN T cells' proportion in thyroid fine-needle aspiration (FNA) material as a predictor of the risk of malignancy. Methods: We recruited 127 patients and obtained ultrasound-guided FNA samples from subjects with cytology-positive or suspicious for malignancy and from those with benign nodular goiter associated with compressive symptoms (such as dysphagia, shortness of breath, or hoarseness), Hashimoto thyroiditis, and Graves' disease. Out of 127, we investigated 46 FNA samples of patients who underwent total thyroidectomy and for which postoperative histological diagnosis by the academic pathologists was available. We specifically measured the number of cells expressing CD3+CD4-CD8- (DN T) as a function of total CD3+ cells in FNA samples using flow cytometry. We correlated their FNA DN T-cell proportions with the pathological findings. Results: The DN T cells were significantly more abundant in lymphocytic infiltrates of thyroid cancer cases compared to benign nodule controls (p < 0.0001). When the DN T-cell population exceeded a threshold of 9.14%, of total CD3+ cells, the negative likelihood ratio of being cancer-free was 0.034 (96.6% sensitivity, 95% CI, 0.915-1.000, p < 0.0001). DN T cells at <9.14% were not found in any subject with benign disease (specificity 100%). The high specificity of the test is promising, since it abolishes a false-positive diagnosis and in turn unnecessary surgical procedures. Conclusion: The present study proposes DN T cells' proportion as a preoperative diagnostic signature for thyroid cancer that with integration of RNA transcriptomics can provide a simplified technology based on the PCR assay for the ease of operation.

Immune Cell Plasticity Allows for Resetting of Phenotype From Effector to Regulator With Combined Inhibition of Notch/eIF5A Pathways.

Type 1 diabetes (T1D) results from the destruction of pancreatic ß-cells caused by an altered immune balance in the pancreatic microenvironment. In humans as well as in mouse models, T cells are well recognized as key orchestrators of T1D, which is characterized by T helper (Th) 1 and Th17 cell bias and/or low/defective T-regulatory cells (Treg), and culminates in cytotoxic T-cell (CTL)-mediated destruction of ß-cells. Refitting of immune cells toward the non-inflammatory phenotype in the pancreas may represent a way to prevent/treat T1D. Recently we developed a unique spontaneous humanized mouse model of type 1 diabetes, wherein mouse MHC-II molecules were replaced by human DQ8, and ß-cells were made to express human glutamic acid decarboxylase (GAD) 65 auto-antigen. The mice spontaneously developed T1D resembling the human disease. Humanized T1D mice showed hyperglycemic (250-300 mg/dl) symptoms by the 4th week of life. The diabetogenic T cells (CD4, CD8) present in our model are GAD65 antigen-specific in nature. Intermolecular antigen spreading recorded during 3rd-6th week of age is like that observed in the human preclinical period of T1D. In this paper, we tested our hypothesis in our spontaneous humanized T1D mouse model. We targeted two cell-signaling pathways and their inhibitions: eIF5A pathway inhibition influences T helper cell dynamics toward the non-inflammatory phenotype and Notch signaling inhibition enrich Tregs and targets auto-reactive CTLs, rescues the pancreatic islet structure, and increases the functionality of ß-cells in terms of insulin production. We report that inhibition of (eIF5A + Notch) signaling mediates suppression of diabetogenic T cells by inducing plasticity in CD4 + T cells co-expressing IL-17 and IFNγ (IL-17 + IFNγ +) toward the Treg cells phenotype.

eIF5A inhibition influences T cell dynamics in the pancreatic microenvironment of the humanized mouse model of Type 1 Diabetes.

We have developed a transgenic mouse model of Type 1 Diabetes (T1D) in which human GAD65 is expressed in pancreatic ß-cells, and human MHC-II is expressed on antigen presenting cells. Induced GAD65 antigen presentation activates T-cells, which initiates the downstream events leading to diabetes. In our humanized mice, we have shown downregulation of eukaryotic translation initiation factor 5 A (elF5A), expressed only in actively dividing mammalian cells. In-vivo inhibition of elF5A hypusination by deoxyhypusine synthase (DHS) inhibitor "GC7" was studied; DHS inhibitor alters the pathophysiology in our mouse model by catalyzing the crucial hypusination and the rate-limiting step of elF5A activation. In our mouse model, we have shown that inhibition of eIF5A resets the pro-inflammatory bias in the pancreatic microenvironment. There was: (a) reduction of Th1/Th17 response, (b) an increase in Treg numbers, (c) debase in IL17 and IL21 cytokines levels in serum, (d) lowering of anti-GAD65 antibodies, and (e) ablation of the ER stress that improved functionality of the ß-cells, but minimal effect on the cytotoxic CD8 T-cell (CTL) mediated response. Conclusively, immune modulation, in the case of T1D, may help to manipulate inflammatory responses, decreasing disease severity, and may help manage T1D in early stages of disease. Our study also demonstrates that without manipulating the CTLs mediated response extensively, it is difficult to treat T1D.

Nature of coexisting thyroid autoimmune disease determines success or failure of tumor immunity in thyroid cancer.

BACKGROUND: Thyroid cancer and thyroid autoimmunity are considered opposite extremes of immune-responses. However, several studies have suggested that thyroid cancer coexists with autoimmune thyroid diseases like Hashimoto Thyroiditis (HT) and Graves disease (GD). We have shown that the risk of developing thyroid cancer is higher in patients with a silent form of autoimmune thyroid disease -Euthyroid Hashimoto Thyroiditis-(EHT). METHODS: We analyzed data from 2633 consecutive patients with GD, HT, EHT and non-Autoimmune Thyroid Disease (Non-AITD) for the presence of Differentiated Thyroid Cancer (DTC). We further investigated the microenvironment, and cellular mechanism of protection from DTC in GD/EHT by ex-vivo aspirating infiltrates from thyroid samples. We also re-constituted in vitro the in-vivo microenvironment to mimic an in-vivo context. We isolated NK cells and differentiated macrophages into M1 and M2 phenotype from healthy human peripheral blood monocytes. RESULTS: DTC was less frequent/aggressive in GD as compared to EHT or Non-AITD. Intra-thyroidal immune-cell profiling revealed differential Natural Killer (NK) cell activity and macrophage polarization in the settings of GD versus EHT. In GD, NK-cells were activated, and macrophages showed M1-like phenotype whereas, in EHT, NK-cells were less active and macrophages displayed M2-like phenotype. Furthermore, in vitro co-cultures of NK-cells with differentiated macrophage subsets revealed that the presence of activated NK (NA) cells favors M1 macrophages, boosts macrophage action and amplifies the innate defense mechanisms. Moreover, co-culture of M2 macrophages with NA, increases the cytotoxicity of NK-cells and favors a pro-inflammatory microenvironment that reverts the anti-inflammatory M2 towards pro-inflammatory M1. CONCLUSION: Surveillance innate immune-cells like Natural Killer (NK) cells and macrophages are complementary to each other in their actions. We discovered here that activated NK-cells in the background of the thyroid autoimmune disease, GD, drive macrophage differentiation to the M1/killer phenotype which in turn is cytotoxic to cancer cells and down regulates the M2/repair phenotype. Understanding the molecular basis of macrophage-NK cell interface in Thyroid Cancer, ETH and GD will open new vistas for immunopathology and therapeutic intervention. Macrophages/innate immunity can be modulated from M2 to M1 phenotype to help treat thyroid cancer as naturally done by GD.