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Women with cervical cancer have high levels of citrulline that are inversely correlated with percentages of circulating IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells

Astrid M. Bedoya1, 2, David J. Tate3, John R. Patterson3, Carlos M. Córdoba4, 5, Mauricio Borrero4, 6, René Pareja6, Fredy Rojas6, Beatriz Finkel-Jimenez7, Constance Porretta8, Cristiam M. Alvarez9, Armando Baena1, Rolando Herrero10, Arnold H. Zea3 and Gloria I. Sánchez1*
  • 1 Universidad de Antioquia, School of Medicine and Corporación Académica para el Estudio de Patologías Tropicales, Colombia
  • 2 Universidad de Antioquia, School of Microbiology, Colombia
  • 3 Louisiana State University Health Sciences Center, Stanley S. Scott Cancer Center, United States
  • 4 Universidad de Antioquia, Departament of Gynecology and Obstetrics, Colombia
  • 5 Hospital San Vicente Fundación, Departament of Gynecology and Obstetrics, Colombia
  • 6 Clínica Las Américas, Instituto de Cancerología Las Américas, Colombia
  • 7 American University of the Caribbean, School of Medicine, United States
  • 8 Louisiana State University Health Sciences Center, Alcohol Research Center, United States
  • 9 Universidad de Antioquia, Instituto de Investigaciones Medicas, Colombia
  • 10 International Agency for Research on Cancer, France

INTRODUCTION: Cervical cancer remains a serious public health problem. Globally, 528.000 new cases and 265.000 deaths were reported in 2012. About 90% of these cases and deaths occurred in developing countries. High-Risk Human Papillomavirus (HR-HPV) infection is the necessary but not sufficient cause of cervical cancer. This common sexually transmitted virus infects the genital epithelium of almost 50% of the sexually active population in the world. The majority of women who acquire HPV, develop cervical cytological abnormalities or do not present any clinical evidence and clear the infection within one to two years. However, the small percentage of women that do not clear the infection spontaneously, develop persistent infection, which severely increases the risk of high-grade cervical lesions and cancer. The immune response plays an important role in the elimination of HPV infection and possibly in the regression of pre-neoplastic lesions. We have previously observed that sera of women with cervical cancer have high levels of arginase and low levels of L-arginine. Reduction of L-arginine by the enzyme arginase is an immune mechanism associated with T-cell dysfunction by inhibiting the expression of the CD3 zeta chain of the T-cell receptor. We also observed that in spite of low levels of L-arginine, in women with cervical cancer the majority of circulating E6 HPV 16-specific CD8+ T-cells express IFN-γ and perforin suggesting that other mechanisms could be overcoming L-arginine deficiency. The nitric oxide synthase (NOS) enzyme converts L-arginine into Nitric Oxide (NO) and citrulline. Citrulline is a naturally occurring non-essential amino acid that in turn can be converted to L-arginine by the sequential action of the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). In vitro observations have shown that citrulline, can substitute for L-arginine deficiency, prevent the loss of CD3 zeta chain expression in CD8+ T-cells and restore T-cell function. Since women with cervical cancer have low levels of L-arginine and the majority of their circulating E6 HPV 16-specific CD8+ T-cells express IFN-γ and perforin; our hypothesis is that women with cervical cancer have high levels of citrulline which correlate with the high percentage of circulating IFN-γ+/perforin+ E6 HPV 16-specific CD8+ T-cells. OBJECTIVES: 1. To evaluate the association between high levels of citrulline and cervical intraepithelial neoplasia 3 (CIN3) or invasive cervical cancer (ICC) 2. To describe in women with cervical cancer the relationship between citrulline and L-arginine levels with the percentage of IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells. METHODS: The levels of L-arginine and citrulline were evaluated by High Performance Liquid Chromatography (HPLC) in the serum of 84, 41 and 79 women with ICC, CIN3 and normal cytology respectively. Percentages of IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells were estimated in 50 ICC cases by flow cytometry using PE-labeled HLA-A*0201 E6(18-26), and HLA-A*2401 E6(49-57), (87-95) tetramers and Anti-CD3-Alexa fluor 700 (clone 17A2), anti-CD8-PerCPCy5.5 (clone HIT8a), anti-IFN-γ-APC (clone XMG1.2) and anti-perforin-FITC (clone δG9) (Beckton Dickinson). Kruskal-Wallis test was used to compare the median among groups. Logistic regression models, adjusted by age and HPV were used to evaluate the association of CIN3 and invasive cervical cancer with high levels of citrulline (upper tertile). Pearson Correlation was used to evaluate the relationship between levels of arginine, citrulline and percentages of IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells. RESULTS: Circulating citrulline levels were significantly higher in women with CIN3 and invasive cervical cancer compared to controls (Kruskal-Wallis test, p <0.0001, Dunn's test p <0.001, CIN3 and invasive cervical cancer vs. controls). In the logistic regression analysis an association between higher levels (upper tertile) of citrulline and CIN3 (OR: 270.4; 95%CI: 15.6-4686) or invasive cervical cancer (OR: 156; 95%CI: 9.4-2600) was observed. In the analysis of correlation between levels of citrulline and L-arginine, we noted that in both CIN3 (Pearson test, r= 0.8749, p <0.001) and invasive cervical cancer (Pearson test, r= 0.4636, p= 0.001) there is a positive correlation between the levels of L-arginine and citrulline. However in the correlation analysis between the levels of citrulline and the percentages of IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells in peripheral blood of women with invasive cervical cancer, a significant inverse correlation (Spearman r= -0.4323, p-value = 0.0011) was observed. That is, individuals with high levels of citrulline have lower percentages of IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells. CONCLUSIONS: In spite of the association between high levels of citrulline and cervical cancer and the positive correlation between levels of citrulline and L-arginine in women with this cancer, we observed a negative correlation between levels of citrulline and percentages of IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells. Our results suggest that contrary to what has been observed in vitro, citrulline does not seem to abrogate the ability of arginase-induced L-arginine depletion and therefore may have not a role in restoring the dysfunction of T-cells mediated by the action of arginase. Subsequent studies testing the expression of ASS in cervical cancer, will be important to elucidate the role of citrulline as a compensation mechanism due to the effects of the depletion of L-arginine by arginase. On the other hand, our results also suggest that it may be more relevant to target the arginase producing cells, rather than supplementing patients with L-arginine or citrulline, in order to overcome the immunosuppression observed in this disease.

Keywords: Citrulline, cervical cancer, HPV-16-specific CD8+ T-cells, L-arginine, Human papillomavirus 16

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Oral Presentation

Topic: Tumor immunology

Citation: Bedoya AM, Tate DJ, Patterson JR, Córdoba CM, Borrero M, Pareja R, Rojas F, Finkel-Jimenez B, Porretta C, Alvarez CM, Baena A, Herrero R, Zea AH and Sánchez GI (2015). Women with cervical cancer have high levels of citrulline that are inversely correlated with percentages of circulating IFN-γ+/perforin+ E6 HPV-16-specific CD8+ T-cells
. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00290

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Received: 31 May 2015; Published Online: 15 Sep 2015.

* Correspondence: Prof. Gloria I Sánchez, Universidad de Antioquia, School of Medicine and Corporación Académica para el Estudio de Patologías Tropicales, Medellin, Antioquia, Colombia, sanchezg@une.net.co