Tumor infiltration by T lymphocytes is commonly observed in cancers of various histological origin and anatomical localization. Recently, a large body of evidence has uncovered a correlation between the presence of lymphoid infiltration and the survival of patients affected by many types of cancer. In 58 of 60 published studies, the presence of CD8+
memory T cells was associated with a favorable prognosis.14
In colon and breast cancer, the presence of a favorable immunological signature coincides with a good subsequent response to chemotherapy.15-17
Furthermore, although this still requires further verification in Phase III clinical studies, the use of an “immune score” based on the presence of CTLs and CD45RO+
memory cells has proven of an outstanding value in predicting the risk of relapse among patients affected by localized colon cancer without lymph node involvement or distant metastases.18
In fact, the ability of cancer cells to avoid immune recognition and elimination has recently suggested as a common hallmark of progressive cancer.19
Still, the immune system has also been proposed to contribute to tumor growth as it promotes tumor-associated inflammation.20
Therefore, when introducing immunotherapy into treatment protocols, the possible interactions between therapy-induced immunity and naturally occurring inflammatory responses in the tumor milieu should carefully be considered. Current clinical protocols for the management of NB include immunotherapeutic strategies based on the anti-GD2 monoclonal antibody ch14.18 and alternating the administration of GM-CSF and IL-2. This approach combined with high-dose isotretionin resulted in improved event-free survival in children affected by high-risk disease.10
A smaller clinical study has also reported that the infusion of CTLs bearing chimeric antigen receptors (CARs) specific for GD2 and a native TCR specific for the Epstein-Barr virus promoted promising clinical responses in 50% of enrolled patients.21
However, limited data are available describing the immunological signature of primary NBs, which is needed to define the prerequisites for immunotherapy and which will help to identify the patients that may truly benefit from immunotherapy.
The characterization of tumor-infiltrating lymphocytes (TILs) prior to the unspecific and/or antigen-specific triggering ex vivo represents a serious technical challenge. Multiple studies have reported a phenotypic analysis of TILs following rapid T-cell expansion protocols that included the exposure of T cells to a variety of strong unspecific stimuli including fetal calf serum, the exogenous supply of recombinant cytokines and irradiated allogeneic feeders.22-24
Here, we report the ex vivo phenotypic analysis of intratumoral and circulating T cells simultaneously obtained from NB patients. Such a comparison is essential for the rational design of novel immunotherapeutics against NB. To date, the characterization of TILs in pediatric neoplasms in respect to phenotype, clonality, specificity, frequency and response to expansion protocols in vitro is sparse, as compared with other oncological settings such as melanoma. In this latter case, a bulk of detailed studies have contributed to TIL therapy becoming a successful story for the treatment of metastatic melanoma patients, with some clinical trials reporting > 50% response rates.25,26
Interestingly, several features of isolated, non-manipulated TILs from NB patients, such as a skewing toward CD8+
T cells, the presence of a large proportion of effector memory-like T cells and the retention of reactivity against autologous tumor cells have also been observed after the application of rapid expansion protocols to TILs from melanoma patients.27,28
It still needs to be elucidated whether such immunologically favorable features are characteristic of TILs invading neural crest-derived tumors, such as melanoma and NB, or if they extend to TILs of different origin.
The analysis of immune activation and/or suppression in human tumors is hampered by logistical and ethical limitations. Some insights into this process can be gained by comparing the composition, phenotype and functional characteristics of circulating and intratumoral T cells. Here, we present a comparative analysis of T-cell populations from two different compartments, the peripheral blood and the neoplastic lesions of NB patients, performed within a minimal period of time after tumor excision and without exposure to any mitogenic stimulus ex vivo. Importantly, the genetic analysis of tumor specimens demonstrated that patients of all genetic subsets, as defined by Carén et al.,29,30
were included into our study ().
The analysis of the whole cell content of NBs by flow cytometry revealed CD3+
cell populations in all examined specimens, which in some cases represented up to 18% of the total cell number (; ). These findings are in striking contrast with some previous reports, which failed to detect lymphoid infiltration in NBs.31
This surprisingly high degree of tumor-associated lymphocytic infiltration is unlikely to be an artifact due to the contamination of tumor samples with peripheral blood lymphocytes, as our subsequent analysis demonstrated that these two compartments significantly differ in their composition. First, we observed that CD8+
T cells prevail over CD4+
T cells in the tumor lesions derived from most patients, where in some cases the CD4+
T-cell ratio was 4- to 5-fold lower than in the peripheral blood. CD4+
T-cell ratios in the PBLs of NB patients were similar to those previously observed in a large cohort of healthy donors.32
Different molecular events may lead to the skewing of the CD4+
T-cell ratio at the NB site, including a differential homing efficacy, a differential proliferative capacity and/or a differential viability at the tumor site of CD4+
subsets. We found that the physiological CD4+
T-cell ratio in PBLs could be altered by exposure to autologous tumor cells in vitro, notably toward the ratio that we observed in TALs. The actual reason for the altered CD4+
T-cell observed within NB lesions is currently not clear. It has previously been reported that human NB cells induce T-cell apoptosis via FAS-FASL interactions.33
However, we failed to detect FASL expression in the primary NB cells analyzed in this study (data not shown). Although we cannot formally exclude that NB cells possess other factors causing the demise or blocking the proliferation of CD4+
T cells, this appears unlikely as we repeatedly detected the expression of activation markers on CD3+
T cells at levels that were comparable to those observed on CD3+
cells (). Similar results demonstrating a preferential presence of CD8+
T cells in various solid tumors and their correlation with prognosis have been reported in various settings during the last decade.34-36
The presence of T cells with potential regulatory properties (Tregs) was investigated using the intracellular marker FOXP3. Though the ectopic expression of FOXP3 confers regulatory capacities to CD4+
cells, FOXP3 may also be expressed transiently upon T-cell activation.11
Therefore, the regulatory potential of FOXP3+
cells should be measured directly, as their ability to suppress the activation of effector T cells. Due to the limited amount of material that was available for our study, we could not accomplish this. Notably, we detected decreased levels of FOXP3 in TALs as compared with PBLs in two patients (). It was recently shown that patients affected by Stage 4 non-MYCN
amplified NB exhibiting FOXP3
expression levels above the median have a reduced 5-year event-free survival,37
suggesting that the expression of FOXP3 in NB samples is relevant for disease outcome. A previous attempt to define immune responses in high-risk vs. low-risk NBs concluded that adaptive immune responses would play a role in disease progression. However, this study was limited by the fact that the authors did not investigate tumor tissues by flow cytometry, but relied only upon a few peripheral blood samples.38
These data from our cohort suggest that the NB milieu favors the specific activation of T cells at the tumor site rather than the accumulation of immunosuppressive Tregs. This hypothesis is supported by a number of additional lines of experimental evidence. First, a selective enrichment of CD8+
T cells bearing a memory phenotype was detected at the tumor site as compared with the peripheral blood (). Second, a higher proportion of cells expressing the surface IL-2 receptor α chain was observed among TALs as compared with autologous PBLs (). Third, a variable but significant upregulation of CD25 was observed in 5 out of 7 PBL samples exposed to autologous tumor cells in vitro (). Fourth, the exposure of PBLs to tumor cells stimulated the production of various cytokines including IFNγ and TNFα (). Fifth, a decreased production of TGFβ was detected in 6 out of 7 PBL specimens co-cultured with autologous tumors (). Of note, an elevated expression of TGFβ, one of the cytokines believed to mediate the immunosuppressive activity of Tregs,11
has been correlated with a reduced 5-year event-free survival in non-MYCN
Sixth, the memory T-cell pools, central and effector memory T cells in particular, were significantly increased in both the CD8+
cell subsets of CD3+
PBLs exposed to autologous tumor cells (). Finally, we were able to detect a substantial number of proliferating T cells in situ and TCR aggregation at the sites of interaction between T lymphoctyes and tumor cells ().
The exposure of PBLs to autologous tumor resulted in a prominent alteration in cytokine secretion. As mentioned above, a tendency toward the enhanced secretion of TH
1 cytokines such as IFNγ and TNFα was observed, whereas the immunosuppressive factor TGFβ was decreased in most cases. The expression of TH
2 cytokines such as IL-4, IL-5 and IL-10 was low in all primary tumor and PBL specimens, and was not significantly increased upon the exposure of lymphocytes to tumor cells (). IL-8, which is often defined as an angiogenic and metastasis-promoting factor,39
was the dominant cytokine produced by primary tumor cells, which is in agreement with previous data.9
The high levels of IL-8 produced by PBLs indicate that the cellular source of IL-8 in NB patients may reside outside of the tumor microenvironment.
It is important to stress that the interpretation of changes in cytokine production by PBLs exposed to NB in co-culture experiments described herein must consider the presence of TALs that contribute to cytokine secretion. We have noted, however, that in all control tumor samples incubated in parallel with “co-culture” samples, T cells gradually declined in number and were barely detectable at day 8, when the analysis was usually performed. This may possibly be explained by the low dose of IL-2 (10 IU/mL) used in our experiments. Therefore, the contribution of TALs to cytokine production in these experiments is expected to be minor and the observed changes most probably reflect the alterations inflicted by NB cells to PBLs.
The activated phenotype of NB-infiltrating lymphocytes appears paradoxical in the light of obvious tumor progression. Current methodologies are not sufficiently advanced to directly demonstrate the specific recognition of malignant cells by TILs in patients. It is also difficult to assess the impact of T-cell activation on tumor progression in situ. However, several observations discussed above support an ongoing interaction between TALs and tumor cells within NB lesions. Furthermore, our previously published data suggest that even unspecific T-cell activation could result in strong suppressive effects on NB cells.4
One must conclude that the extent of naturally occurring TAL activation in NBs is not sufficient for mediating antitumor effects. This may result from the suppression of CTL activation by factors produced within the tumor microenvironment. Alternatively, an intense proliferation of tumor cells may overcome the antineoplastic effects of local immune responses. In this light, we suggest that the adoptive transfer of activated CTLs capable of resisting an immunosuppressive microenvironment may add up to the TAL pool and limit tumor progression.
In conclusion, our study demonstrates the presence of activated T cells within primary NBs and the possibility for autologous T cells to become activated in the presence of NB cells. Further studies on the crosstalk between TALs and NB cells may help to exploit the immune system for the treatment of children affected by this dreadful disease.