HLA-G Gene Variability Is Associated with Papillary Thyroid Carcinoma Morbidity and the HLA-G Protein Profile.

Human leukocyte antigen (HLA)-G is an immune checkpoint molecule that is highly expressed in papillary thyroid carcinoma (PTC). The HLA-G gene presents several functional polymorphisms distributed across the coding and regulatory regions (5'URR: 5' upstream regulatory region and 3'UTR: 3' untranslated region) and some of them may impact HLA-G expression and human malignancy. To understand the contribution of the HLA-G genetic background in PTC, we studied the HLA-G gene variability in PTC patients in association with tumor morbidity, HLA-G tissue expression, and plasma soluble (sHLA-G) levels. We evaluated 185 PTC patients and 154 healthy controls. Polymorphic sites defining coding, regulatory and extended haplotypes were characterized by sequencing analyses. HLA-G tissue expression and plasma soluble HLA-G levels were evaluated by immunohistochemistry and ELISA, respectively. Compared to the controls, the G0104a(5'URR)G*01:04:04(coding)UTR-03(3'UTR) extended haplotype was underrepresented in the PTC patients, while G0104a(5'URR)G*01:04:01(coding)UTR-03(3'UTR) was less frequent in patients with metastatic and multifocal tumors. Decreased HLA-G tissue expression and undetectable plasma sHLA-G were associated with the G010102a(5'URR)G*01:01:02:01(coding)UTR-02(3'UTR) extended haplotype. We concluded that the HLA-G variability was associated with PTC development and morbidity, as well as the magnitude of the encoded protein expression at local and systemic levels.

Molecular, metabolic, and functional CD4 T cell paralysis in the lymph node impedes tumor control.

CD4 T cells are central effectors of anti-cancer immunity and immunotherapy, yet the regulation of CD4 tumor-specific T (TTS) cells is unclear. We demonstrate that CD4 TTS cells are quickly primed and begin to divide following tumor initiation. However, unlike CD8 TTS cells or exhaustion programming, CD4 TTS cell proliferation is rapidly frozen in place by a functional interplay of regulatory T cells and CTLA4. Together these mechanisms paralyze CD4 TTS cell differentiation, redirecting metabolic circuits, and reducing their accumulation in the tumor. The paralyzed state is actively maintained throughout cancer progression and CD4 TTS cells rapidly resume proliferation and functional differentiation when the suppressive constraints are alleviated. Overcoming their paralysis established long-term tumor control, demonstrating the importance of rapidly crippling CD4 TTS cells for tumor progression and their potential restoration as therapeutic targets.

BRAF, TERT and HLA-G Status in the Papillary Thyroid Carcinoma: A Clinicopathological Association Study.

As BRAF, TERT, HLA-G, and microRNAs have been individually associated with papillary thyroid carcinoma (PTC), we aimed to evaluate the individual and collaborative role of these markers in PTC in the same patient cohort. HLA-G and BRAF tumor expression was evaluated by immunohistochemistry. Using molecular methods, BRAFV600E and TERT promoter mutations were evaluated in thyroid fine needle aspirates. MicroRNA tumor profiling was investigated using massively parallel sequencing. We observed strong HLA-G (67.96%) while BRAF (62.43%) staining was observed in PTC specimens. BRAF overexpression was associated with poor response to therapy. The BRAFV600E (52.9%) and TERTC228T (13%) mutations were associated with extrathyroidal extension, advanced-age, and advanced-stage cancer. The TERT rs2853669 CC+TC genotypes (38%) were overrepresented in metastatic tumors. Nine modulated microRNAs targeting the BRAF, TERT, and/or HLA-G genes were observed in PTC and involved with cancer-related signaling pathways. The markers were individually associated with PTC features, emphasizing the synergistic effect of BRAFV600E and TERTC228T; however, their collaborative role on PTC outcome was not fully demonstrated. The differentially expressed miRNAs targeting the BRAF and/or HLA-G genes may explain their increased expression in the tumor milieu.

Lenvatinib Plus Anti-PD-1 Combination Therapy for Advanced Cancers: Defining Mechanisms of Resistance in an Inducible Transgenic Model of Thyroid Cancer.

Background: Combination therapy with lenvatinib plus programmed death-1 (PD-1) immune checkpoint blockades (ICBs) is under investigation in many solid tumors, including thyroid cancer. Lenvatinib is known to reduce angiogenesis and may overturn the immunosuppressive effects of vascular endothelial growth factor in the tumor microenvironment. Previous studies investigating the effects of VEGF receptor inhibition on the immune response were performed in rapidly growing tumor models where immune equilibrium is not established before treatment. We hypothesize that physiologically relevant preclinical models are necessary to define mechanisms of resistance to immune-targeted combination therapies. Methods: We utilized the TPO-CreER/BrafV600E/wt/Trp53Δex2-10/Δex2-10 inducible transgenic model of advanced thyroid cancer to investigate lenvatinib treatment in the context of an anti-PD-1 ICB. Following tumor establishment, 3.5 months postinduction, mice were treated with high- (10 mg/kg) or low-dose (2 mg/kg) lenvatinib, anti-PD-1, or combination of lenvatinib with anti-PD-1. Tumor volume and lung metastases were assessed in each group. Immune infiltrate was characterized by flow cytometry and immunohistochemistry, and TCRß sequencing was performed to further investigate the T cell response. Results: Both low- and high-dose lenvatinib reduced tumor volume, while anti-PD-1 had no effect, alone or in combination. Although both low- and high-dose lenvatinib reduced vascular density, low-dose lenvatinib was superior in controlling tumor size. Lung metastases and survival were not improved with therapy despite the effects of lenvatinib on primary tumor size. Low-dose lenvatinib treatment led to a subtle reduction in the dominant Ly6G+CD11b+ myeloid cell population and was associated with increased CD4+ T cell infiltrate and enrichment in 4-1BB+ and granzyme B+ CD4+ T cells and FoxP3+ regulatory T cells. Polyclonal T cell expansion was evident in the majority of mice, suggesting that a tumor-specific T cell response was generated. Conclusions: The effects of lenvatinib on the immune response were most pronounced in mice treated with low-dose lenvatinib, suggesting that dose should be considered in clinical application. While the immune-modulating potential of lenvatinib is encouraging, alterations in the immune milieu and T cell activation status were insufficient to sustain durable tumor regression, even with added anti-PD-1. Additional studies are necessary to develop more effective combination approaches in low-mutation burden tumors, such as thyroid cancer.

Plasma levels of soluble HLA-G and cytokines in papillary thyroid carcinoma before and after thyroidectomy.

BACKGROUND: Increasing evidence shows that chronic inflammation plays an important role in thyroid tumorigenesis. Cytokines as central mediators in inflammatory microenvironment can present both pro-tumour and anti-tumour effects and cytokine release may be influenced by soluble HLA-G (sHLA-G), an immune checkpoint molecule whose expression can also be induced by certain cytokines. AIM: To understand the role of these soluble factors in papillary thyroid cancer (PTC). METHODS: We evaluated plasma levels of sHLA-G and of 13 cytokines using ELISA and flow cytometry, respectively, in PTC patients at two time points: pre- and post-thyroidectomy; and control subjects. RESULTS: Compared with controls, IL-6 levels were increased, while IL-1ß, IFN-α and TGF-ß1 levels were decreased in pre-thyroidectomy PTC patients. IFN-α and TGF-ß1 efficiently discriminated patients from controls and were associated with extrathyroidal extension and lymph node metastasis, respectively. In addition, TNF and IL-13 were associated with male gender, lymph node metastasis and Hashimoto thyroiditis, and sHLA-G with tumour invasion. Compared with pre-thyroidectomy, IL-4, IL-10, TNF, IFN-α and TGF-ß1 levels were increased in post-thyroidectomy. CONCLUSION: There are significant changes in the cytokine profile after surgical removal of the thyroid tumour, and IFN-α e TGF-ß1 showed to be promising cytokines for discriminating PTC patients from controls. We also found that different cytokines are associated with clinicohistopathological characteristics of PTC related to poor prognosis, suggesting that cytokines seem to play an important role in PTC development and management.

Liver HLA-E Expression Is Associated with Severity of Liver Disease in Chronic Hepatitis C.

Hepatitis C virus (HCV) can escape from innate and adaptive immunity, making the immune response ineffective. Human leukocyte antigen E (HLA-E) might regulate the antiviral function of immune response and contribute to the persistence of HCV and the severity of liver disease. This study aimed to evaluate the expression of HLA-E in the liver and its association with the severity of liver disease in HCV patients. We performed a retrospective analysis of liver biopsies from 125 HCV patients and from 20 control subjects without liver disease. Liver biopsies were reviewed and classified according to severity of fibrosis and inflammatory activity. The pathologist assessed the magnitude of HLA-E expression in a semiquantitative way, attributing scores from 0 to 3. Immunohistochemistry showed positive for HLA-E in hepatocyte and Kupffer cells. The rate of HLA-E positivity in hepatocytes and Kupffer cells was significantly higher in HCV patients compared to controls. The liver samples classified as severe fibrosis and necroinflammatory activity presented greater expression of HLA-E on Kupffer cells and hepatocytes, with a significant linear association. It indicates that HLA-E expression may have an immunomodulatory effect and a possible role in the severity of liver disease in chronic hepatitis C.

The genetic diversity within the 1.4 kb HLA-G 5' upstream regulatory region moderately impacts on cellular microenvironment responses.

The HLA-G 5'URR extending 1.4 kb from the ATG presents a unique set of regulatory elements among HLA genes. Several variable sites have been described that coincide with or are close to these elements, thus HLA-G 5'URR polymorphism might influence the HLA-G expression level. We cloned the ten most frequent HLA-G 5'URR haplotypes to evaluate their activity on a luciferase reporter gene in HLA-G+ cell lines (JEG-3/choriocarcinoma and FON+/melanoma). We also investigated associations between the plasma HLA-G (sHLA-G) levels and the HLA-G 5'URR variability in 157 healthy individuals. Cell lines were transfected with pGL3-Basic vector constructions containing HLA-G 5'URR sequences. The G010101a (in JEG-3) and G010101b (in FON+) haplotypes exhibited higher promoter activity, whereas the G010101d (in JEG-3) and G010102a (in FON+) haplotypes exhibited lower promoter activity. In the presence of HLA-G inducers (interferon-ß and progesterone) or repressors (cyclopamine) HLA-G promoter activity was modulated, but certain haplotypes exhibited differential responses. No strict association was observed between plasma sHLA-G levels and the 5'URR haplotypes or genotypes; however, the G010101b haplotype was underrepresented among HLA-G-negative plasmas. Therefore, the HLA-G 5'URR polymorphism may have an impact on the modulation of HLA-G gene expression, but alone provides a limited predictive value for sHLA-G levels in vivo.

IL28B gene polymorphisms in mono- and HIV-coinfected chronic hepatitis C patients.

INTRODUCTION: Single-nucleotide polymorphisms (SNPs) associated with hepatitis C virus (HCV) clearance were identified near the IL28B gene. Coinfection by the human immunodeficiency virus (HIV) influences the course of HCV contributing to liver damage. Nevertheless, little is known about the relationship between these SNPs and HCV/HIV coinfection. Our aim was to estimate the frequencies of the allelic and genotypic variants of the IL28B polymorphisms rs12979860 (C/T) and rs8099917 (T/G) and their possible association with the establishment of HCV infection. METHODOLOGY: A total of 199 non-infected controls and 230 patients with chronic hepatitis C, including 53 coinfected with HIV, participated in the study. Genotyping consisted of polymerase chain reaction and subsequent analysis of the restriction patterns resulting from exposure to endonucleases. RESULTS: Among the controls with established results, 47.4% (90/190) exhibited the rs12979860 CC genotype, 43.7 CT, and 8.9% TT, whereas 29.1% (66/227), 51.5%, and 19.4% of the patients exhibited the CC, CT, and TT genotypes, respectively. With respect to rs8099917, 66.8% (133/199) of the controls exhibited the TT genotype, 31.2% TG, and 2.0% GG, whereas 56.1% (129/230), 40.9%, and 3.0% of the patients exhibited the TT, TG, and GG genotypes, respectively. CONCLUSION: The frequencies of the rs12979860 C allele and CC genotype and of the rs8099917 T allele and TT genotype were significantly higher among controls compared with patients, thus confirming the suggested protective effect against HCV infection. No significant difference was observed in the genotype and allelic distributions between the mono- and coinfected patients.