In the present study, regarding the importance of cytotoxic T (CD8+) and memory (CD45RO+) lymphocytes in eradicating tumor cells and the regulatory role of immune cells expressing FOXP3, we attempted to clarify the role of the immune system based on the frequency of CD3+, CD8+, CD45RO+ and FOXP3+ lymphocytes in both stromal and intratumoral areas of bladder tumors. Our results, similar to previous studies (10–12), revealed a high degree of variation in the prevalence of investigated subsets in different patients. However, in general, CD45RO+ lymphocytes followed by CD3+ cells, were the most frequent subsets in both areas of the bladder tumor tissues, while FOXP3+ cells showed the lowest prevalence.
CD45RO is generally thought to be a marker of memory T cells, which following exposure to antigen provide fast and more severe immune responses. Primary analysis of tumor tissues of BC showed that besides being the most frequent, CD45RO+ lymphocytes were significantly higher in the higher-grade tissues. This increase was also observed in patients with invasive tumors (T2/T3 vs. Ta/T1), higher stages (≥II vs. 0/I), as well as those with muscle invasion. Given the protective role for memory cells in antitumor responses and their association with better prognosis in most cancers (13), the increase in the frequency of CD45RO+ cells can be simply interpreted by the continuous and prolonged contact of immune cells with tumor antigens and their efforts to remove cancer cells. However, Horn et al. have found no significant relationship between infiltrated subsets including CD45RO+ cells and clinicopathological features of the disease (10), but a similar association was obtained in our previous study on breast cancer patients (9). It seems that tumors with fast growth and less differentiation have more antigenic and phenotypic differences in comparison with the normal tissues, lead to more detection and recruitment of immune cells into tumors. In this regard, studies have shown that bladder tumors are highly immunogenic and strongly induce immune responses at least at early stages before tumor-specific suppression (14). It is also important to note that CD45RO might be found on a variety of immune cells other than T cells. Thereby, the evaluation of further markers as well as functional studies seems to be necessary to clarify the precise role of CD45RO+ cells in the context of tumors.
The presence of CD3+ T cells, as the main arm of cellular immunity and their association with desirable clinical outcomes, has been investigated in many cancers (15). Similar results have been obtained in the urological cancers including BC (10). Our primary evaluation of CD3+ lymphocytes in tumor tissue from patients with BC showed that these cells, at the same range of CD45RO+, accounted for the highest level of investigated immune cells in the stromal and intratumoral regions of bladder tumors. The frequency of CD3+ lymphocytes was significantly higher in the intratumoral region of progressed tumors (T2 vs. T1 and stage II vs. I) and tumors having invasion to the muscular layer. Consistently, Karpina et al. have also shown that the number of CD3+ lymphocytes at the time of tumor resection is significantly higher in patients who experience cancer recurrence (16). As previously mentioned, this increase could simply be a sign of stimulating the protective immune system following an inflammatory response induced by tumor cells in the first stages. However, studies on BC and other cancer types have shown that the growth and spread of metastatic tumors were associated with a decrease in the density of T cells (17, 18). Besides tumor type, this difference might be attributed to the stage of tumor progression. It is assumed that in the early stages, tumor growth is followed by immune system attempts to eradicate the tumor cells. Following tumor progression and escape, the frequency of these cells is severely reduced or their function is suppressed. It should be also noted that T lymphocytes are heterogeneous populations with very different effector functions, both protumorigenic [regulatory T (Treg) and type 2 helper T (Th2)] and antitumorigenic (CD8+ cytotoxic T and Th1). Consistently, we observed that the frequency of protumorigenic subtypes, Th2 and FOXP3+ Treg cells significantly increased in the draining lymph nodes of patients with different cancers including BC (19). Hence, in the present study, along with CD3+ T cells two other important functional markers of T cells, CD8 and FOXP3, were also investigated.
CD8+ cells, known as cytotoxic cells, are of the most important subsets of T cells. They contain cytotoxic enzymes and induce death in tumor cells. Although their association with increased survival and recurrence prevention has been reported in many types of cancers, in urological malignancies, there are limited studies with controversial results. We found that, similar to other subtypes, the frequency of CD8+ lymphocytes in both areas of the tumor significantly increased with tumor growth from T1 to T3 or from stage I to II. Masson-Lecomte et al. also have observed similar results in T1 vs. Ta tumors (20). Despite the increase in the recruiting factors, it seems that the tumor inhibitory microenvironment suppressed or changed the functionality of CD8+ T cells in a way to help tumor growth. Consistently, in our previous study on BC, we found that the frequency of protumorgenic subtypes of CD8+ T cells producing IL-4 and IL-17, remarkably increased in the tumor-draining lymph nodes along with tumor progression (21). We also observed similar results in breast and salivary gland tumors (19, 22). In the same way, Karpina et al. have shown that the number of CD8+ lymphocytes was higher in the early-stage patients who underwent transluminal resection and experienced recurrence (16). Furthermore, Zhang et al. have indicated that considering the clinical stage, CD8+ lymphocytes represented a favorable prognostic factor in non-organ-confined disease, but were an independent unfavorable factor in organ-confined ones (23). It shows that the prognostic role of these cells and probably their function change during disease progression.
Along with effector T cells, some subpopulations exert inhibitory functions to regulate the immune response. In most cases, these regulatory cells express the FOXP3 transcription factor (24). Although there are controversial reports the frequency of FOXP3+ Treg cells in most cancers has been associated with a worse prognosis (25). However, the result of our study did not show any difference between the frequency of FOXP3+ cells and the clinicopathological factors, but in our previous studies on bladder and breast tumors, we observed that the frequency of FOXP3+ Treg cells in the draining lymph nodes significantly increased in patients with tumor-affected nodes (19). Controversial observations have been also reported in urothelial carcinoma but there are some reports which consistently showed a poor prognosis in patients with severe infiltration of Treg cells in their tumor tissues (26, 27). In addition, Murai et al. have found that in the non-metastatic tissues, specimens with the high percentage of FOXP3+CD3+ cells had higher recurrence, suggesting a role for the contribution of these cells in tumor progression (26).
We next compared the frequencies of immune cells in tumor tissues between alive and dead patients. Frequency analyses did not represent any significant differences in both groups. None of the investigated markers also showed a direct association with survival time. Meanwhile, Horn et al. have found a correlation between lower OS and higher ratios of FOXP3+/CD3+ and FOXP3+/CD8+, but higher levels of CD3+ and CD8+ showed a greater tendency for better survival (10). However, in our study, such associations were not observed, Sharma et al. have found that patients with higher infiltrated CD8+ cells had better free-disease and overall survival, indicating a predictive role for CD8+ lymphocytes in BC (28). In a study by Winerdal et al., FOXP3 expression in tumor cells was associated with decreased survival, though its expression in tumor-infiltrating lymphocytes correlated with a positive prognosis. They considered the FOXP3 as an activation marker for T cells rather than regulatory molecule, but due to their limited sample size, it should be interpreted with caution (27).
In none of these studies, the tumor areas were separately investigated, while Yu et al. have observed that in patients with advanced BC, those with the higher density of CD3+ and CD8+ in the invasive margins showed better OS and higher DSF in case of more density of CD8+ (11). This observation was consistent with the previous studies on BC which have found CD8+ as a favorable prognostic factor (17, 29, 30). Similar contradictory results have been also obtained in other cancers, which confirmed that the tumor microenvironment even in two patients with the same type of cancer is not uniform and there is a heterogeneity in the different types of cancers. In addition, the tumor microstructure, distribution of immune cells in the margin and center of the tumor, secondary lymphoid structures, as well as the type of inflammatory cells, have a great impact on the prognosis.