Solid tumors are not just composed of malignant cells, but are complex microcosms of many cell types, including a wide range of hematopoietic cells . Anti-tumor immune responses may be one of the most important weapons in the arsenal against cancer . Galon et al. [38, 39] suggested that once human colorectal tumors become clinically detectable, the adaptive immune response plays a role in preventing tumor recurrence and metastasis. Intratumoral T cells could modify tumor stroma or tumor cells in ways that attenuate the metastatic potential of tumor cells [40, 41]. However, it is still unclear if the presence of tumor infiltrating lymphocytes represents the result of an inflammatory response that facilitates either tumor progression or a protective host response against cancer .
The present study confirms that tumor infiltrating lymphocytes are present in colorectal cancer and, as expected, a strong correlation was found between CD3+ and CD8+ T cells. Additionally, a strong association was also seen between the presence of CD8+ T lymphocytes and the expression of their associated cytotoxic molecule granzyme B in all examined areas of the tumor. Granzyme B expression was much sparser which can be explained by the fact that only those CD8+ T lymphocytes which are cytolytic (about 50% in this study) will express their associated cytotoxic molecule granzyme B. When exposed to dysfunctional cells, cytotoxic T lymphocytes will release granzyme B and will eventually induce apoptosis of the proximal tumor cells bearing the appropriate antigen determinants [7, 17]. This might hold promise in the field of immunotherapy [16, 42]. Unfortunately, at this time we were unable to investigate T cell surface activation markers or the apoptosis status of the infiltrated tumor cells in order to confirm the lytic function of these infiltrating T cells.
As found in several other studies [7, 39, 43–48], the presence of a pronounced lymphocytic infiltration within the tumor is associated with improved survival in the current study. Specifically, CD3+ and CD8+ T lymphocytes within cancer cell nests of colorectal cancer and of CD3+ in the colorectal cancer stroma had a major impact on the patients' overall survival by univariate analysis in the overall population. When colon and rectal tumors were investigated separately, the results of the overall population were confirmed in the group of colon cancer patients but not in the group of the rectal cancer patients, even when the rectal cancer patients receiving pre-operative radiotherapy were excluded from the analysis.
The improved survival associated with infiltration of lymphocytes may be the result of an effective suppression of micrometastases in distant organs or near the primary site. In other words, the number of CD8+ T cells within the primary tumor might be a good indicator of the presence of a systemic immunosurveillance mechanism [40, 41]. In addition, tumor cells can secrete substances in the stroma, which might be recognized by the immune system to destroy the tumor.
In accordance to Pages et al. , Ropponen et al  and Koch et al , but in contrast to Ling et al. , stage was a major determinant of infiltration of activated cytotoxic intraepithelial lymphocytes (as shown by multivariate analysis) with a higher infiltration in earlier stages (HR: 0.582, p = 0.006). This might indicate that immune reactions may be more prominent in early stages of disease and might have a stage specific influence on survival [31, 50], possibly irrespective of the MSI-status. There might be a protective local immune response in these earlier tumor stages, preventing further tumor growth and spread. However, this hypothesis has to be substantiated by further analysis of activation status of tumor infiltrating lymphocytes in patients with early tumor stages. Alternatively, host immune response against cancer cells may decrease with increasing tumor growth .
Several groups found stage specific beneficial effects of infiltrating lymphocytes on clinical outcome [30–33]. Guidoboni et al. described a correlation between the presence of cytotoxic T lymphocytes and improved survival in stage II and III colorectal tumors irrespective of the MSI-status . These results were confirmed by Prall et al.  for stage III disease. However, when MSI-status was taken into account, the correlation with survival was most pronounced when both MSI-H and intratumoral-activated cytotoxic T lymphocytes were present in stage III tumors .
In this study, stage specific survival analysis did not show a major influence of tumor infiltration on overall survival. However, given the relative small number of cases per stage group, such an influence might be difficult to detect. Nevertheless, this could be of interest since we previously showed a significant beneficial effect of active specific immunotherapy in adjuvant treatment of patients with stage II colon cancer only .
Accumulating evidence indicates that most MSI-positive colorectal tumors are characterized by the presence of a pronounced intratumoral inflammatory reaction, the nature of which, however, is still poorly understood [30, 51]. Within these tumors, tumor infiltrating lymphocytes have been identified as predominantly activated CD8+ T cells. The presence of these cytotoxic T lymphocytes has been attributed to the inherently greater production of abnormal peptides as a result of unreliable DNA repair in MSI-positive tumors . In the current study, among the different clinicopathological parameters analyzed, MSI was strongly correlated with a high presence of activated cytotoxic intraepithelial lymphocytes. Nevertheless, survival analysis by MSI-status revealed that, despite the high infiltration of lymphocytes in MSI-positive colorectal tumors, no association was found with a favorable prognosis within this subset. These data are in agreement with some studies [31, 37, 50, 52–57], but they are in conflict with a number of other reports [13, 24–27, 30, 34, 58] in which a predominant presence of intra-tumor cell infiltrating lymphocytes in MSI-H cancers was significantly correlated with a better prognosis. In accordance to Baker et al. , MSS colorectal tumors with infiltration of CD8+ tumor infiltrating lymphocytes showed a better overall survival. One explanation might be that the functions of tumor infiltrating lymphocytes differ between these subgroups. It is possible that the high prevalence of recruitment and retention of tumor infiltrating lymphocytes in MSI-positive colorectal tumors is inherent to the unique aspect of the tumor biology, supporting the hypothesis that the genetic instability of MSI-positive colorectal tumors may lead to the production of a greater number of tumor specific antigens that trigger the immune system [7, 11–16, 27, 30]. As described previously , direct contact between tumor cells, presenting these aberrant peptides, and leucocytes plays a crucial role in the immune reaction. However, the concept of cross-priming in which antigen presenting cells pick up antigens released by dead tumor cells and subsequently present them to T cells may also be important in vivo .
On the other hand, MSI-positive tumors can undergo immuno-editing, which dictates that tumors reaching the stage of clinical detection have been shaped antigenically by the initial immune responses mounted against them to a point where they are no longer recognized as foreign to the body. In MSS tumors, which arise in a much less immunologically stimulating environment, the tumors are less likely to have adapted antigenically and may thus be more sensitive to late stage immune attack. Thus, even though MSI-positive tumors produce more tumor specific antigens that might trigger the immune system, the elevated levels of these antigens in combination with the lack of appropriate costimulatory molecules on the tumor cells may generate a microenvironment which leads to a state of tumor infiltrating lymphocyte anergy, thereby preempting any beneficial effect on survival [12, 37]. In addition, the expression of HLA class I proteins, presenting tumor associated antigens on the tumor cell surface, is considered a prerequisite for an effective T cell immune response . It has been described that genetically unstable tumors are often HLA class I negative and might escape T-cell mediated immune killing. However, in the absence of any surface HLA class I these tumors would be susceptible to a natural killer cell attack. In contrast, those tumors that downregulate specific HLA class I alleles may avoid both T cell and natural killer cell activation . Therefore, Speetjens et al.  hypothesized that both oncogenic pathway and HLA class I expression might dictate clinical tumor progression. Furthermore, insufficient evidence exists to recommend routine use of biological factors as MSI for either determining prognosis or predicting response to therapy in colorectal cancer patients .
When entered into a multiple Cox regression analysis adjusting for other possible important confounding factors, only early stage and young age (borderline significant for overall population only) were associated with a better overall survival (early stage also with disease-free survival). The beneficial effect of tumor infiltrating lymphocytes in colorectal cancer could not be confirmed in the Cox regression survival analysis.
In summary, this study confirms that tumor infiltrating lymphocytes are indeed important clinical and prognostic indicators in colorectal cancer, in univariate analysis, irrespective of the MSI-status. Therefore, it was considered that tumor infiltration could reflect a general principle of antitumor immunity as mentioned by Prall et al. . In addition, in accordance with Baker et al., we also demonstrated an important disease-specific survival advantage for patients with MSS tumors containing intraepithelial CD8+ tumor infiltrating lymphocytes . Although no stage specific survival difference of tumor infiltrating lymphocytes was noted in colorectal cancer, it was demonstrated that stage was also a major determinant of the presence of tumor infiltrating lymphocytes in colorectal cancer, with a higher infiltration in earlier stages.
We would like to note that the tumor immune response does not only depend on infiltration and activation of T lymphocytes. Therefore, the minute balanced association of all infiltrating cells, their capability to synthesize and release tumor-modifying substances, and their content of presynthesized, granule-associated bioactive substances deserves intensive study. By doing so, new biological tumor-host responses may be disclosed, leading to the development of new approaches to the treatment of malignant disease .