gnosed breast cancers. There is increased resistance of TNBC to conventional chemotherapy and hormonal therapy due to lack of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression. Mutual relationship and interactions between both CD4+ and CD8+ T lymphocytes are very crucial for eliciting adaptive immune system during anti-cancer immune response. This study aimed to determine the ratio of CD4+ and CD8+ T lymphocytes in TNBC by immunohistochemistry assay and to investigate the association of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) with survival outcome. Quantification of both immunostaining TILs subset was done using conventional light microscope. A wide range of CD4+ and CD8+ TILs subset was found within intratumor stroma of TNBC with population mean score of 0.93 and 0.53, respectively. However, the difference of mean population between both TILs subsets was insignificant overall (P-value of CD4+= 0.484; CD8+= 0.835) when compared statistically using independent t-test. The ratio of CD4+/CD8+ in intratumor stroma ranged from 0.50-5.88 among all TNBC subtypes. The high CD4+/CD8+ ratio within intratumor stroma showed favorable association with survival outcome during the 2 years’ follow-up. 

Keywords: CD4+, CD8+, digital pathology,  prognostic biomarkers, triple-negative breast cancer (TNBC), tumor-infiltrating lymphocytes (TILs)  

INTRODUCTION

More than two-thirds of breast cancers express progesterone receptor (PR) and oestrogen receptor (ER) while approximately 20% show overexpression of human epidermal growth factor receptor 2 (HER2) gene (Howlader et al. 2014). Lacking of ER/PR expression and HER2 amplification in triple-negative breast cancer (TNBC) account for 10-15% of all diagnosed breast cancers (Dawson et al. 2009) and this underlie the main reason of their resistance to most conventional chemotherapy (Lin et al. 2012). In cases which are subjected to chemotherapy, more than 50% of TNBC patients do not have complete pathologic response (cPR) in addition to high risk of local recurrence or distant relapse (Soni et al. 2015; Wang et al. 2017). Due to the high rates of non-responders, alternative treatment other than chemotherapy are highly sought following by the need to identify potential novel biomarkers in improving the treatment outcome. 

Recently, T lymphocyte infiltration into primary tumors had been proven as favorable prognostic factor in variety of solid tumors such as colorectal carcinoma, ovarian carcinoma and non-small cell lung carcinoma (Gooden et al. 2011). The tumor-infiltrating lymphocytes (TILs) is an indication that a local immune system is activated in response to tumor progression (Canna et al. 2005). In breast cancer, most of the TILs are mainly composed of CD4+ and CD8+ T lymphocytes, T regulatory cells, mast cells, plasma cells and macrophages (Bense et al. 2017). However, most of the TILs were mainly CD4+ and CD8+ T lymphocytes which mediated pro-inflammatory type 1 immunity. Majority of them expressed effector cytotoxic (CD8+) phenotype according to previous studies that involved patients with various carcinoma and melanoma (Ben-Hur et al. 2002; Leong et al. 2006). The TILs association and their prognostic significance with treatment cPR had also been highlighted in several cohorts studies, both under adjuvant and neoadjuvant settings (Adams et al. 2014; Ali et al. 2014; Loi et al. 2014; Salgado et al. 2015).

The CD4+ T lymphocytes has become increasingly highlighted for their pivotal role in effective anti-tumor immunity and to activate the CD8+ cytotoxic T lymphocytes (CTL) response in cancer immunotherapies. CD4+ T lymphocyte could differentiate into diverse functional subtypes due to their polyfunctional properties, which in turn allowing them to play the central “coordinator” role in eliciting appropriate effector immune response (Tay et al. 2021). CD8+ effector T lymphocytes play essential role in cellular-mediated immune response by killing cancer cells upon interaction with specific antigen presenting cells (APC) whereas CD4+ T lymphocytes facilitate CD8+ anti-tumor function within the tumor microenvironment (Dobrzanski et al. 2006). Mutual relationship and interactions between both CD4+ and CD8+ T lymphocytes are very crucial for eliciting adaptive immune system during anti-cancer immune response.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that suppresses the inflammation arising from immune response. IL-10 deficiency could result in development of inflammatory diseases or autoimmune diseases (Wilson et al. 2005). Originally, IL-10 is synthesised by macrophages, dendritic cell (DC), B lymphocytes, CD4+ and CD8+ T lymphocytes. In previous study, IL-10 was secreted to inhibit the activity of helper T-cells, natural killer (NK) cells and macrophages during infection to prevent tissue damage due to hyperinflammatory responses (Couper et al. 2008). The immunosuppressive effect of IL-10 is mediated by heterodimeric IL-10 receptor (IL-10R1, IL-10R2) which is expressed on effector immune cells through activated JAK/STAT signaling pathway upon receptor binding (Wills-Karp et al. 2010). This in turn, decreases the release of pro-inflammatory cytokines and concomitantly decreases the immune cell activities. Thus, IL-10 is often expressed aberrantly in cancer cells as one of the mechanism to evade immunosurveillance.

The TIL subsets were previously explored in a cohort of 42 TNBC cases at the tumor-host interface within intratumoral stroma and CD4+/CD8+ ratio was found to be significantly associated with TNBC patients overall survival (OS) (Wang et al. 2017).  Intratumoral TILs (iTu-Ly) refer to those lymphocytes which have direct contact  with tumor cell with no intervening stroma (Khoury et al. 2018). Recently, more data has shown that TILs infiltration has been used as a favorable prognostic indicator in a wide range of solid primary tumors such as colorectal, ovarian and non-small cell lung carcinomas (Gooden et al. 2011). Presence of TILs has often been associated with better OS and has response to cancer treatments. The number of TILs could be used as a predictive biomarker for adjuvant treatment outcomes indicating strong association between TILs and better overall survival rates (Adams et al. 2014; Gooden et al. 2011; Liu et al. 2011). 

The aim of this study was to investigate the potential of TILs and IL-10 as prognostic biomarkers by correlating their expression with patients’ OS. The relationship between TILs subsets with the level of IL-10, how IL-10 regulated the level of intratumor TILs in TNBC and possibility of cancer progression caused by impaired IL-10 function were elucidated in this study. We used conventional immunohistochemistry assay to identify TILs and IL-10 within the TNBC intratumor stroma.  

The other objective of this study was to quantify the ratio of CD4+/ CD8+ lymphocytes in TNBC and their correlation with level of IL-10. Semi-quantitative scoring systems were used to convert subjective IHC-marker expression into quantitative data, which was then used for statistical analysis by computer software as adapted from previous study (Akbar et al. 2015; Moratin et al. 2021; Wang et al. 2017). 

MATERIALS AND METHODS

Clinicopathologic Characteristics

Samples were identified and obtained from Universiti Kebangsaan Malaysia Medical Center (UKMMC) database via ILMSV5503_PPUKM software. A total number of 50 samples were obtained from patients’ archived list dated between 2012 and 2018. All samples were scored as negative for ER, PR and HER2 status in immunohistochemical expression. The HER2 status was also considered negative if the ratio of HER2/Chr17 was less than 2.0 (non-amplified) when tested by fluorescent in-situ hybridisation (FISH) or dual-clour dual hapten in-situ hybridisation  (DDISH) test, which was performed when HER2 score was equivocal  (Wolff et al. 2018). All of the cases met the inclusion criteria, which included 28 mastectomy specimens, 15 lumpectomy specimens and 7 wide-local excisional specimens. Biopsies specimen were excluded from this study because they were too scanty to detect adequate amount of TILs for investigation (Wang et al. 2017). All patients had received conventional chemotherapy treatment and radiotherapy.

Immunohistochemistry Assay to Identify IL-10, CD4+ and CD8+ TILs in Paraffin-embedded Tissue Sections 

Primary antibidies included; (i) Rabbit monoclonal CD8+ antibody, Clone SP16 Ready-to-use (Ref. No. RMPD012, Diagnostic BioSystems California USA); (ii) Rabbit monoclonal CD4 antibody, Clone EP204 Ready-to-use (Ref. No. RMPD083, Diagnostic BioSystems California USA); (iii) Mouse monoclonal IL10 antibody, Clone E-10 (Cat. No. sc-8438, Santa Cruz Biotechnology, Inc Texas USA) was used at a dilution of 1:50. Lymph node tissue was used as a positive control.

Immunohistochemical staining was performed on the tissue sections using the protocol from Mouse/Rabbit PolyVue Plus™ HRP/DAB Detection System (Cat. No. PVP250D, Diagnostic BioSystems California USA). Primary antibodies were diluted to an optimal concentration using Antibody Diluent, Dako REAL (Code No. S2022, Dako Agilent Denmark). Washing steps between each reagent were performed using EnVision FLEX Wash Buffer 20X (Code No. K8007, Dako Agilent Denmark) which was diluted to a working solution with deionised water. The DAB-containing Substrate Working Solution was prepared by diluting the 50x concentrated EnVision FLEX DAB+ Chromogen with Envision FLEX Substrate Buffer (Code No. K8023, Dako Agilent Denmark).

Tissue blocks were sectioned approximately 3 µm thickness and mounted on adhesive glass slide, Platinum Pro White (Product No: PRO-01, Matsunami Japan). The slides were left to be air-dried in room temperature overnight. The tissue slides were then incubated on hot plate at 60ºC for 1 hour. Deparaffinisation and pre-treatment step were performed in the Decloaking Chamber™ NxGen (Ref. No: DC2012-220V, Biocare Medical California USA) using the Dako Target Retrieval Solution High pH (Code No. K8023, Dako Agilent Denmark) in the conditions of 110ºC for 30 minutes followed by another 30 minutes of cooling at room temperature and then were rinsed with running tap water for 3 minutes. The slides were subsequently incubated with Tissue Primer (Cat. No. PVP250D, Diagnostic BioSystems California USA) for 10 minutes followed by washing step and then were incubated with Background Blocker (Cat. No. PVP250D, Diagnostic BioSystems California USA) for 10 minutes. No washing step was done after this incubation.

Slides were then directly incubated with primary antibody for 30 minutes at room temperature followed by washing step. Next the slides were incubated with PolyVue™ (Enhancer) (Cat. No. PVP250D, Diagnostic BioSystems California USA) for 30 minutes followed by washing step. The slides were then incubated with PolyVue™ (HRP) (Cat. No. PVP250D, Diagnostic BioSystems California USA) for 30 minutes in room temperature and followed by washing step. Slides were then incubated with DAB-containing Substrate Working Solution for 7 minutes and then rinsed with washing buffer.

The slides were then counterstained with Hematoxylin 2 (Richard-Allan Scientific™ Signature Series™ 16 oz, Ref. No. 7231, Thermo Fisher Scientific USA) for 3-dips after the procedures had been completed followed by dehydration step with increasing alcohol solutions (80%, 90%, and 100%) (Pureview Alcohol 100% Gal. Cat. No. PV1000, Cancer Diagnostics USA) and two times with Xylene (Richard-Allan Scientific™ Xylene, Ref. No. 6615, Thermo Fisher Scientific USA). Finally, the slides were mounted using CoverSeal™-X xylene-based mounting medium (Cat. No. FX2176, Cancer Diagnostics USA).

Evaluation and Quantification of IL-10, CD4+ and CD8+ T Lymphocytes 

Expression of IL-10, CD4+ and CD8+ lymphocytes were identified using immunohistochemistry according to the manufacturer’s protocols. The three strongest intensity of CD4+ and CD8+ T lymphocytes at three regions were selected to access CD4+ and then IL-10. The IL-10 and both TILs subsets at these regions were compared and quantified manually under a 20 high-power-fields (HPFs) scoring method (20x magnification) using a light microscope (OLYMPUS Model BX53) and expressed as mean population, respectively. In this study, semi-quantitative scoring system (modified H-score) which included degree of lymphocytic infiltration and IL-10 immunostaining intensity were used to convert IHC expression into quantitative data (0 to 1) (Table 1). The degree of TILs infiltration was considered positive if brown color  staining was presented at the region and negative if no staining presence. An image processing software ImageJ (NIH, Bethesda, MD, USA) was used to validate the surface area and regional IHC intensity covered by IL-10, CD4+ and CD8+ TILs, thus confirming the degree of immunostaining. The calculated area of IL-10 and lymphocytes were divided by the area of fields to obtain the mean value for each sample. The counting step was performed twice for each slide. The final score was obtained by summing all scores from three regions before being expressed as CD4+ and CD8+ TILs’ mean population, respectively.

Statistical Analysis 

Besides descriptive statistics, population means of TILs among patients were compared statistically using independent t-test to determine any significant difference between population means (high CD4+/CD8+ ratio versus low CD4+/CD8+ ratio). All data and statistical calculation were performed using SPSS version 16.0 (IBM Corp, Armonk, NY, USA). All data were expressed in the form of mean, median and standard deviation (SD). Disease-free survival (DFS) was calculated from the date of diagnosis until the date of death or follow-up cut off point (24 months). The clinicopathological parameters for a total of 50 patients including age, tumor size, histologic grade and histologic subtypes were evaluated with Chi square test which test theirs' significance with population of IL-10, TILs subset as well as CD4+/CD8+ ratio. The association between IL-10 population with both TILs subset and CD4+/CD8+ ratio were analysed using Chi square test as well. Overall survival was  analysed using the log-rank statistical test prior for plotting Kaplan-Meier survival curves. All patients who survived until the study cut off point were censored in the analysis as they did not demonstrated event of interest. P values of <0.05 was considered statistically significant in all analyses.

RESULTS

Quantification IL-10, CD4+ and CD8+ T Lymphocytes

Characteristics of all patients were detailed in Table 2. The TILs were found predominantly in intratumor stroma which consisted of CD4+ and CD8+ TILs subset (population mean score of 0.93 and 0.53 respectively). IL-10 was also found within intratumor stroma which correlated negatively to level of both CD4+ and CD8+ TILs (population mean score of 0.33) surface area. Regional IHC intensity covered by immunohistostaining region for IL-10, CD4+ and CD8+ TILs (Figure 1A, 1B & 1C) were annotate manually by the pathologist in order to confirm the degree of immunostaining under set scale (power of magnification = 20 X = 200 µm scale ; 620 pixel and 3.1 pixel /µm). Then, ImageJ software was used to calculate the region’s area in pixel. The use of ImageJ software improved the data accuracy as it enhanced the quantification process of intratumoral IL-10 and TILs as the intensity of immunostaining was ambiguous (Fedchenko & Reifenrath 2014). This was done through calculating the surface area (pixel/µm) of IHC immunostaining using standardised set scale in this study. 

Immunostaining region of both TILs subset in intratumor (Figure 2A & 2B) and control tissue were visualised and quantified. IHC expression of IL-10, CD4+ and CD8+ TILs for several TNBC samples were compared at the same area of intratumor TNBC tissues where low IL-10 expression correlated with high infiltration of both CD4+ and CD8+ TILs subsets (Figure 3A) or vice versa (Figure 3B). The population means of TILs among patients between those survived and deceased were compared statistically using independent t-test. The result showed that the difference of means population between both subsets were insignificant (P-value of CD4+ = 0.484; CD8+ = 0.835) among patients from survived and deceased groups (Table 3). This was most probably due to the small cohort of samples involved in this study. 

Meanwhile, the ratio of CD4+/CD8+ in intratumor stroma ranged between 0.50-5.88 among all TNBC subtypes (Table 4). CD4+ TILs count was generally significantly higher than CD8+ TILs within intratumor stroma of TNBC. Consequently, the CD4+/CD8+ ratio at intratumor stroma of TNBC was high (mean score of 1.95>1) particularly among invasive carcinoma of non-special type (NST) subtypes which account for 68% of cases (34/50). The median/normal point of   < 2 was selected as low ratio and high ratio represented those with scores of ≥2. On top of that, the data spread between population mean of IL-10 (SD=0.214); CD4+ TILs (SD = 0.167) and CD8+ TILs (SD = 0.158) was minor. In addition, the standard deviation for CD4+/CD8+ ratio in intratumor stroma was 0.932 with less deviation among ratio spread as well. 

Characterisation of Survival Outcomes and Statistical Analysis in relation to CD4+/CD8+ ratio

Log-rank test showed high number of CD4+ TILs to low number of CD8+ TILs (high CD4+/CD8+ ratio) within intratumor stroma was found to be associated with favorable survival outcomes particularly among low-grade patients (Table 5) during the 2 years follow-up (cutoff point). In addition, high CD4+/CD8+ ratio within intratumor stroma also had significant influence on TNBC patients’ survival outcomes or disease-free survival (DFS) as shown in Kaplan-Meier survival curve (Figure 4). Nevertheless, the overall survival between TNBC patients with high CD4+/CD8+ ratio compared with those with low CD4+/CD8+ ratio was not considered statistically significant using log-rank test (P = 0.055), possibly due to small sample size. Overall, the prognosis of TNBC patients was 72% (36/50) with high CD4+/CD8+ ratio (excluding the deceased and low/inversion CD4+/CD8+ ratio) within intratumor stroma survived until the end of study. A total of 7 patients (14%) was found deceased, while remaining 7 patients (14%) were either lost to follow-up or no longer retaining their medical records at the end of study (July 2021).

There was a total of 68% of invasive carcinoma of NST subtypes in this cohort study which demonstrated a relatively high CD4+/CD8+ ratio (>2) within intratumor stroma. Survival outcomes of these patients were longer than follow-up cut off time (24 months) particularly being discharged after mastectomy surgery compared with other TNBC subtypes. Further investigation had found trend of a favorable prognosis among patients with low grade NST subtype TNBC and high CD4+/CD8+ ratio intratumorally. However, there was no prognostic significance found among patients at advanced TNBC stage even though with high CD4+/CD8+ ratio intratumorally. 

In addition, IL-10 had no significant influence (P=0.714) on TNBC patients’ survival outcomes or disease-free survival (DFS) as shown in Kaplan-Meier survival curve (Figure 5). However, patients with low IL-10 level had slightly higher survival period compare to those with high IL-10 level. Finally, no significant link was found between IL-10 with both CD4+ and CD8+ TILs population means with different clinicopathological features (Table 6). However, age parameter was nearly considered significant with CD4+/CD8+ ratio from Chi-square test. No significant association was observed on both TILs subset and CD4+/CD8+ ratio with IL-10 population mean for all samples (Table 7).

DISCUSSION

In this study, both CD4+ and CD8+ TILs subsets were found in the TNBC tissues sections as shown by IHC staining. Generally, the population of CD4+ TILs was higher than the population of CD8+ TILs within all intratumor stroma of TNBC samples, resulting in higher CD4+/CD8+ ratio. However, some cases showed inversion of this ratio, indicating lower CD4+/CD8+ intratumor stroma. The ratio of CD4+/CD8+ TILs was found to have some significance on TNBC patients overall survival and progression rate, which could be potentially applied to indicate the effectiveness of treatment and cPR towards treatments given. 

To quantify the IHC staining, we used scoring system which involved combination of quantitative and qualitative parameters as suggested in previous study (Fedchenko & Reifenrath 2014). The scoring system included measurement of area which was covered by positively stained TILs region in percentage. In addition, we also measured the surface area of the region of interest (ROI), where immunopositive TILs cells were stained to confirm our scoring accuracy based on IHC intensity. The result showed that high CD4+/CD8+ ratio indeed led to longer OS and better prognosis outcome (Table 3). This could be attributed to better response of TNBC tumor to surgery, cancer treatments regimes, recovery and vice versa. On the contrary, majority of patients were found to have worst prognosis, progression-free survival (PFS) and OS in case of inversion ratio of CD4+/CD8+. However, there were some exceptions whereby certain patients with high ratio had short-lived whereas certain patients who had inversion ratio lived longer than expected. This phenomena was largely attributed to other factors beyond control. 

From the result, the ratio of CD4+/CD8+ intratumorally did not have much significant effect on OS or prognosis of patients with advanced stage of TNBC. This was likely due to the rate of proliferation among poorly differentiated cancer cells was faster than the anti-cancer immune response. In other words, the secretion of immune cytokines, activity of phagocytosis, stimulation of antibodies production and lysis caused by CTL could not cope with the rate of tumor growth and regeneration. Consequently, this led to patients’ early demise in less than 2 years follow-up cut-off period. In addition, Chi-square test revealed that there was association between age with CD4+/CD8+ ratio as immune system was getting weaker with age progression. In other words, there was tendency that high ratio of CD4+/CD8+ among younger individuals. This research also highlighted the increased of CD8+ TILs population as a result of acute inflammatory condition, which led to sepsis after tumor removal surgery.  

Previous study used TIL subsets in a cohort of 42 TNBC cases to compare CD4+/CD8­+ ratio in tumor-host interface and within intratumoral stroma. The result showed that the CD4+/CD8­+ ratio at the tumor-host interface was found significantly associated with OS, while no association was observed for the CD4+/CD8­+ ratio within the intratumoral stroma (Wang et al. 2017). However, our findings supported the contention that TILs could be one of the important prognostic biomarker in TNBC progression intratumorally. Our result showed that increased CD4+/CD8+ ratio within intratumor had improved the OS of TNBC patients, as shown in the Kaplan-Meier survival curve (Figure 4) although they were not statistically significant due to small sample size. This study further proved that TILs acted as favorable prognostic biomarkers for TNBC patients as well as good pCR under adjuvant and neoadjuvant chemotherapy setting. Therefore we proposed that identification of TILs in treatment-resistant TNBC was important because they could be potential biomarkers in predicting treatment outcome for standard adjuvant or neoadjuvant therapies and immunotherapy in TNBC patients in the future.  

However, there were some TNBC patients’ survival rate and treatment outcomes that did not conform to the theory due to presence of outlier. These outlier data were probably attributed to artefact during immunostaining protocols, heterogenous nature of breast cancer tissue, location of tumour during lesion tissue being taken for biopsy or other uncontrollable variables, which led to unusually low or almost none of CD8+ TILs infiltration into intratumoral breast tissue. For instance, uneven hematoxylin counterstaining was the common tissue processing artefact where strong staining could impeded the evaluation of both TILs subsets’ immunostaining regions. On the other hand, deep-seated tumor was expected to have lower TILs due to poor perfusion and compromised immune system due to chemotoxicities. This would result in false-favorable survival outcome among TNBC patients. 

Prognostic significance of CD8+ TILs remained debatable. This was due to other modifiable risk factors such as diet, lifestyle, environmental and menopausal status of the TNBC patients, which were beyond control of researchers during follow-up. For instance, some patients passed on even though they had high CD4+/CD8+ ratio. On the other hand, some patients survived despite of the low CD4+/CD8+ ratio. Some patients with high ratio lived shorter than expected. This might due to breast cancer 1 (BRCA1) genetic predisposition carried by these subset of patients (Peshkin et al. 2010). Patients who carried BRCA1 predisposition might experience higher rate of genetic mutation due to altered DNA repairing mechanism via homologous recombination (Gudmundsdottir & Ashworth 2006). Consequently, the TNBC tumors progress faster than normal to advanced stage the more risk factors being carried despite carrying favourable CD4+/CD8+ ratio. On the contrary, TNBC patients who lived longer than expected despite low ratio of CD4+/CD8+. This may be attributed to their normal gene composition that governed the genetic stability and diet with rich of antioxidants might be the factors.

In contrast to the good prognosis associated with higher level of CD8+ T lymphocytes aforementioned, previous study consisted of larger cohort had shown that high CD8+ T lymphocyte density infiltration intratumourally or peritumourally actually had no beneficial effect on OS or PFS among breast cancer patients (Liu et al. 2011). This phenomenon was further investigated in tissue microarray, which latter found out that CD8+ T lymphocytes only demonstrated prognostic significance to breast cancers when high advanced histologic grade and lacked of ER expression (Baker et al. 2011). The information on prognostic significance of CD4+ TILs had not been fully elucidated. Increased CD4+ TIL was associated with better prognosis and survival outcomes, but other subsets had shown no prognostic significance (Chung et al. 2017). Conversely, two earlier studies had demonstrated that higher CD4+ T lymphocyte level correlated with worse prognosis among breast cancer patients (Macchetti et al. 2006; Matkowski et al. 2009). 

Undeniably, decreased CD4+/CD8+ ratio could associate with impaired cellular-mediated immunity. On the other hand, high CD4+/CD8+ ratio was associated with good prognosis in the cases of non-small cell lung cancer and cervical squamous cell carcinoma (Shah et al. 2011).  Increased CD4+ T lymphocytes and declined of CD8+ T lymphocytes population were observed in regional lymph nodes that was invaded by breast cancer cells (Macchetti et al. 2006).  Furthermore, high CD4+/CD8+ ratio was also associated with reduced cancer recurrence in hepatocellular carcinoma after liver transplant (Unitt et al. 2006). Remarkably, a study showed early pre-treatment with CD4+ resulting increased CD4+/CD8+ ratio and inversion of CD4+/CD8+ ratio after treatment would lead to prolonged survival under metastatic melanoma setting (Hernberg et al. 2004). Similarly, inversion of CD4+/CD8+ ratio was also associated with better pCR and prognosis of breast cancer in one study (García-Martínez et al. 2014).

Generally, the population of IL-10 found in TNBCs tissue sections in our study correlated negatively with both CD4+ and CD8+ TILs population. Negative correlation affected both subsets population together whereas influence on either one subset only as single entity was not observed in this study. The inflammatory inhibition of IL-10 appeared to have greater effect on CD4+ TILs compared to CD8+ TILs population. High IL-10 associated with lower CD4+ TILs in some samples, which led to ratio inversion. However, the influence of IL-10 on overall population of both TILs subsets and CD4+/CD8+ ratio was not significantly associated.

IL-10 appeared to have some significant influence on TNBC patients OS. Decreased IL-10 tended to correlate with higher ratio of CD4+/CD8+ which led to favorable prognosis among TNBC patients. To further elaborate this, IL-10's function was to inhibit APC and protect tissue against damage from pro-inflammatory responses from T helper cell type 1 (Th1) and T helper cell type 2 (Th2) responses during pathogen infections (Schopf et al. 2002). This was achieved by reducing expression of MHC class II and co-stimulatory molecules such as B7-1 or B7-2 on monocytes and macrophages. Subsequently, this further reduced the downstream pro-inflammatory cytokines and chemokines production such as interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-18 (IL-18), tumour necrosis factor α (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), CCL12 and Chemokine (C-X-C motif) ligand 2 (CXCL2) (Moore et al. 2001). In previous study that used mice model, it was found that neutralisation of interferon gamma (IFN-γ), signal transducer and activator of transcription 1 (STAT1) gene (one of the main transcription factor that regulated IFN-γ signaling pathway) gene expression will sensitise the mice to methylcholanthrene (MCA) induced carcinogenesis compared to their wild-type counterparts (Kaplan et al. 1998). In addition, cytolytic protein, perforin was synthesised by CD8+ TILs had also been found to inhibit B cell lymphoma development (Bolitho et al. 2009). These key findings suggested that less immuno-competent or immunocompromised individual were more likely to experience high risk of tumorigenicity as well as cancer progression. 

Most cancers depended on 3Es of cancer immuno-editing to refine cancer immunosurveillance i.e. elimination, equilibrium, and escape (Dunn et al. 2004). This was attributed to nature of cancer cell to sculpture their immunogenicity during development, which plays an important role during interaction with host immune cells in later stage. The more immune-selection pressure underwent by cancer cells, the lesser the immunogenicity developed by tumors, resulting in recognition escape by adaptive immune system in later stage. Immuno-editing generated tumors with lesser immunogenicity, which resulted in recognition escape by adaptive immune system. This was also the reason that some cancer cells developed resistance to certain immunotherapy. This situation exacerbated through inhibition of acute pro-inflammatory responses by IL-10 in order to protect tumour tissue against damage from Th1 and Th2 responses. As a result, the cancer cells escaped immunosurveillance as well as anti-tumor effect from TILs, leading to worst prognosis.

Mutual relationships and interactions between both CD4+ and CD8+ T lymphocytes were very crucial for eliciting adaptive immune system during anti-cancer response. In other words, tumor specific antigen (TSA) expressed on cancer cells were required by CD4+ T lymphocyte to activate APC via CD40-CD40L interactions during priming stage prior to generate a more tumour-specific targeting CD8+ T-lymphocyte population (Feau et al. 2012). In the anti-cancer immune cycle, these T lymphocytes' crosstalk generated tumor-directed T cells in the final stage (Chen & Mellman 2013). This process was induced by macrophages that serve as APC after phagocyting TSAs, followed by presenting their antigens to CD4+ T helpers cells in lymph node. Subsequently, tumor-specific CD8+ T lymphocytes was activated by CD4+ T helpers cells were selected and clonally expanded prior migration into tumor site through lymphatic vessels to eliminate tumor cells. However, tumor growth could only be inhibited and eliminated if the lymphocytes had undergone a fully completed immune cycle. This meant that any disruption of cross-talk event during the cancer immune cycle progression could allow cancer cells to escape immunosurveillance, resulting in failure to control tumor growth. 

Meanwhile, chronic inflammation caused by dietary and environmental carcinogens and exposure, was also linked intimately with tumor-promoting microenvironment. It contributed to cancer initiation and progression by inducing cellular proliferation, metastasis through enhanced angiogenesis and invasion and mutagenesis  by generating oxidative genotoxic species (Grivennikov et al. 2010).  Most of the solid tumor depended on intrinsic inflammation to build “pro-tumor” microenvironment (Mantovani et al. 2008). For instance, lung tumor could promote inflammation by secreting proteoglycan versican which acted as macrophage activator through Toll-like receptor (TLR) 2 to enhance metastatic growth (Kim et al. 2009). The inflammatory microenvironment also aided cancer cell renewal and proliferation continuously through secretion of cytokines and chemokines. Furthermore, cancer-related inflammation also led to silencing of mismatch repair which associated with increased genetic instability that caused by microsatellite instability (Colotta et al. 2009). By suppressing inflammation in non-immunocompetent individuals, it will eliminate the “metastatic-niche” for tumour and impede the progression process. However, this paradox role of inflammation which in this case-dampening inflammation would causelow TILs infiltration, resulting in poorer prognosis of TNBC patients. 

CONCLUSION

This study highlight novel emergent aspects of anti-tumor by CD4+ and CD8+ T lymphocytes. Presence of TILs and absence of IL-10 within intratumor stroma strongly correlated with good prognosis and longer PFS outcomes among TNBC patients. Hence, TILs and IL-10 could serve as potent prognostic markers to overcome some of the limitations of current prognostic biomarker for TNBC. In attributing to this finding and translating this knowledge into clinical management, we proposed that pre-treatment with immunotherapy such as autologous T-cell therapy, chimeric antigen receptor T cells (CART) and autologous immune enhancement therapy (AIET) were crucial during pre-surgery neoadjuvent chemotherapy or after adjuvant post-surgery chemotherapy. We would also suggest that prior injection of non-reactive/autologous-cultured T lymphocytes at local tumor area may complement the chemotherapy and radiotherapy for better prognosis and treatment outcomes. Beta-adrenergic drugs family appeares to be contraindicated with the prognosis of TNBC patient as these beta agonists induced an increased in the IL-10 production both in vitro and in vivo in opposed to beta-blockers.

Acknowledgement

The authors thank Ultimed Scientific for the sponsorship of IHC detection systems, CD4+ and CD8+ antibodies (Diagnostic Biosystems) for this study.