There is a need to improve treatment of CRC patients to reduce the high rate of disease recurrence following potentially curative surgery. In many cases malignant cells may seed other organs and expand following surgery to cause patient death. Removing these relatively small numbers of cells that remain after surgery is difficult and is currently only partially aided by postoperative chemotherapy when indicated by histological staging of resected malignant bowel. There is strong indirect evidence that T cells can be effective at controlling CRC metastases.1
How the activity of these cells can be harnessed for the purpose of immunotherapy is not yet understood. Indeed, several early phase trials of vaccination in CRC have been disappointing,31
possibly reflecting the persistence of regulatory mechanisms which serve to down-modulate otherwise effective anti-tumour immune responses.
The current study was undertaken to gain insight into the impact of tumours on the immune response and in particular, on Tregs. For this purpose we compared anti-tumour immune responses and Tregs in patients pre- and postoperatively. This longitudinal analysis revealed that Foxp3 expression levels are higher in CRC patients compared to healthy controls and strikingly, that in most subjects expression levels realign with those observed in healthy controls, following tumour excision. This finding may point to an effect of tumours in driving the suppressive capacity of Tregs. In support of this premise, detailed functional characterisation of Treg subsets and our own findings (data not shown) indicate that Tregs expressing high levels of Foxp3 have higher suppressive capacity than those expressing lower levels.23
Tumour recurrence was however observed in some of the patients studied despite a decrease in Foxp3 expression levels. Prior to surgery, the tumour represents a large bulk of tissue that may have developed over several years, leading to an increase in Tregs. On the other hand, relatively small deposits of metastatic tissue may not lead to detectable differences in Treg phenotype when assessed in whole PBMC. Thus, in the case of disease recurrence, it may not be possible to measure alterations in Tregs, particularly when detected at an early stage such as 12 months postoperatively.
Our study also demonstrated that Treg proportions within the CD4+
T cell population differ markedly in PBMCs compared to tumour stroma. Data indicating that a significantly higher proportion of Foxp3+
cells express CD49d in cancer patients compared to healthy controls support this finding as CD49d expression implies that the cells are actively migrating to a site of immune activity. Deng et al
, demonstrating a similar highly significant increase of Foxp3+
cells in the tumour draining lymph nodes and particularly the tumours of CRC patients compared to PBMCs, corroborate these findings.32
Overall, the enrichment of Tregs within tumour-infiltrating leucocytes strongly implies that this is an important site of tumour immunosuppression.
Previous studies have documented 5T4- and CEA-specific responses in CRC patients.17
However, these studies did not take the effect of surgery into account. Thus, the second part of this study examined longitudinal effector T cell responses to CEA and 5T4 pre- and postoperatively and measured the impact of Tregs on these responses at the same time-points. The data demonstrate that at least two thirds of patients studied responded to one or both of the antigens under study but that Tregs suppressed these responses in the majority of cases. When the preoperative 5T4-specific responses of individual patients were compared to responses 6 months after surgery, a significant elevation of 5T4 responses was observed at the latter time-point, suggesting that removal of the tumour burden facilitates the unmasking of a previously suppressed CD4+
T cell response. Our data imply that Tregs are at least partly responsible for this suppression as a lower proportion of 5T4-specific responses were suppressed postoperatively. Overall these data support the earlier findings of the study, which indicate that tumours expand the Treg population, driving suppression of CD4+
T cell responses to tumour-associated antigens. Furthermore, the results offer some insight into Treg specificity. Since suppression was assessed in assays using 5T4 (and CEA) pulsed PBMCs as stimulators, it suggests that both the effector cells and the Tregs recognise the same protein antigen. The pattern of responses to CEA was similar to 5T4. However, the extent to which Tregs suppressed CEA responses did not alter after surgery. It is possible that CEA-specific Tregs are maintained by influences other than the tumour for the purpose of limiting self-reactivity in the healthy host, as CEA, unlike 5T4, is expressed at low levels in normal tissue. While some degree of non-specific immunosuppression was detected, overall the data indicate that the greatest extent of suppression is observed when measuring responses to tumour antigens.
An extremely important aspect of this study is the absolute concordance of tumour recurrence at a 12-month postoperative time-point and Treg-mediated suppression of CEA- and 5T4-specific responses, measured preoperatively. No such concordance was observed within the group of patients remaining tumour-free or with respect to Treg mediated suppression of responses to unrelated control antigens. Recent studies have however, reported that high numbers of intratumoural Foxp3+
T cells are associated with better survival of CRC patients.18
More than one explanation can account for these seemingly contradictory findings. The role of Tregs may depend on the type of immune response present in the tumour microenvironment. When inflammatory cells that promote tumour progression dominate the immune response, Tregs may be beneficial in suppressing this process.36
However, in the case where the immune response is dominated by T cells, Tregs may promote disease progression by suppressing their anti-tumour effects. More simplistically, Treg numbers may accumulate in parallel with the conventional T cell response, thus their presence could just reflect robust anti-tumour T cell activity known to correlate with an improved prognosis.1
The data presented here underpin the importance of conducting further studies of CRC patients to include more antigens and larger patient groups in order to identify T cell specificities involved in controlling tumour growth and those that might be involved in promoting tumour growth. A better understanding of the relationship between tumours and Tregs may prove instrumental to the success of anticancer immunotherapy.