Regulatory T cell frequency in peripheral blood of women with advanced cervical Cancer including women living with HIV

Abstract

Background: Persistent high-risk Human papillomavirus (HR-HPV) infections are the main cause of cervical cancer. Cumulative evidence implicates regulatory T cells (Tregs) as a critical factor in the failure to eliminate HPV-induced cancers leading to their persistence and progression to cancer. Also, the WHO recognised cervical cancer as 100% attributable to persistent HR-HPV infection. The province of KwaZulu-Natal (KZN) in South Africa has a high prevalence of cervical cancer and HIV infection.

Materials and methods: We evaluated Treg frequency in dual infection of HR HPV and HIV coinfection using phenotypic markers, CD4, CD25 and intracellular Foxp3, in the peripheral blood of 51 cervical cancer and 46 non-cervical cancer participants and evaluated the effect of HIV on regulatory T cell proportion. Peripheral blood mononuclear cells were surface stained with a cocktail fluorescent labelled CD4 and CD25 and subsequently with APC anti-human FoxP3 (eBioscience). Flow cytometry was performed with FACS analysis. Statistical analysis of results was done using Instat 3 program (GraphpadR). Tregs results were expressed as median ± interquartile range (IQR). Associations of cervical cancer with demographic, clinical and laboratory variables were evaluated by univariate and multivariate logistic regression analysis using SPSS version 27 (IBM).

Results: Tregs frequency was significantly higher in individuals with cervical cancer (11.00 ± 19.79%) compared to controls (1.71 ± 8.91%) (p < 0.0001). HIV infection was associated with an increase in Tregs frequency. In controls a significant difference in Tregs frequency was noted between women living with HIV (6.00 ± 10.57%, n = 9) and those without HIV (1.30 ± 6.10%, n = 37), p = 0.0023. In multivariate logistic regression, Tregs frequency was significantly associated with cervical cancer after controlling for age, smoking, weight loss, presence of STI, HIV and HPV genotype.

Discussion/conclusion: Higher Tregs frequency was significantly associated with cervical cancer highlighting the immunosuppressive role of Tregs in cervical cancer. Treg frequency was more strongly associated with cervical cancer than HIV infection. We provide baseline data for monitoring Treg frequencies in response to new preventive and therapeutic strategies in the management of cervical cancer.

Keywords: Cervical cancer; HIV; Human papillomavirus; Immunosuppression; Regulatory T cells.

Fig. 1

Dot Plot demonstrating 3.02% of non-specific binding (NSB) cells as a proportion of CD4 + CD25 + cells (Upper Right quadrant, Panel C) in a cervical cancer participant. Panel A: Dot plot representation of PBMC according to size (x-axis = forward scatter) and granularity (y-axis = side scatter); the gated region corresponds to lymphocytes. Panel B: Dot plot showing gated lymphocytes from panel A, stained with CD4 (x-axis) and CD25 (y-axis). Panel C: Dot plot showing gated double CD4 and CD25 positive cells (upper right quadrant in panel B) further stained with Isotype control (NSB/dead cells). The upper right quadrant represents cells (positive for CD4, CD25 and NSB)

Fig. 2

Dot Plot demonstrating FoxP3 in the same cervical cancer participant shown in Fig. 1. 47.33% positivity of FoxP3 as a proportion of CD4 + CD25 + cells (upper right quadrant, Panel C). Panel A: Dot plot representation of PBMC according to size (x-axis = forward scatter) and granularity (y-axis = side scatter); the gated region corresponds to lymphocytes. Panel B: Dot plot showing gated lymphocytes from panel A, stained with CD4 (x-axis) and CD25 (y-axis). Panel C: Dot plot showing gated double CD4 and CD25 positive cells (upper right quadrant in panel B) further stained with FoxP3. The upper right quadrant represents Treg cells (positive for CD4, CD25 and FoxP3).

Fig. 3

The bar graph demonstrates the frequency of T regulatory cells as a frequency of CD4 + CD25 + FoxP3 T cells according to HPV types in cervical cancer patients and controls women. No significant difference in Tregs frequency in controls for HPV infections (Kruskal-Wallis (p = 0.7788). Tregs frequency was significantly high in HR HPV cervical cancer compared to controls using the Mann-Whitney test, p < 0.0001). Bars represent the median, and error bars represent the interquartile range (IQR).

Fig. 4

T regulatory cell frequency in cervical cancer women according to HIV status. The bar graph demonstrates a significant difference in Treg frequency between HIV (+) controls and HIV (+) cervical cancer, p = 0.0006. In controls, Treg values between HIV (+) and HIV (-) were significant p = 0.0023. However, Treg frequency was significant between HIV (-) cervical cancer and HIV (-) controls, p < 0.0001. No significant difference between HIV (+) cervical cancer and HIV (-) cervical cancer, p = 0.547and between HIV (+) cervical cancer and HIV (-) controls, p = 0.395. Non-parametric Mann-Whitney test. Bars represent the median, and error bars represent the interquartile range (IQR)

Fig. 5

A graphic representation of Treg frequency in cervical cancer (CC) and controls according to age. In the cervical cancer group, Treg frequency was significantly different between the 3 age groups, p = 0.0044. (ANOVA) (not shown). In controls, no difference was recorded between any of the age groups (p = 0.4136 (not shown). No difference in Treg frequency was found in controls. In comparing Treg frequency between cervical cancer and controls in 36–50 and > 50 age groups, Treg frequency was significantly higher in the cervical cancer women than the controls (p = 0.0003 and (p = 0.0059), respectively. Bars represent the median, and error bars represent the interquartile range (IQR)