There has been increasing evidence that regulatory T cells play an important role in fostering an immune privileged tumor microenvironment thereby promoting tumor progression 32
. In support of this, studies have shown that the numeration and distribution of Treg in the tumor microenvironment are correlated with clinical outcome 4
. Treg can be recruited, expanded or converted in the tumor microenvironment 4,6–9
. However, it is still not clear whether Treg conversion in the tumor microenvironment is a property of the tumor cells or that of the tumor-infiltrating T cells.
In this study, we have shown that FL B cells can convert conventional T cells to Treg. Our results confirmed the observation of other investigators 27
. In addition, we demonstrated that Treg conversion in follicular lymphoma is clearly dictated by tumor cells. We observed, for the first time, that in contrast to malignant B cells, normal B cells did not have the ability to convert Treg in our experimental setting. Furthermore, tumor-infiltrating T cells were as susceptible to Treg conversion as normal T cells from PBMC (). Follicular lymphoma has distinct clinical and pathological properties, such that immunological factors of both the host and the tumor microenvironment have been linked to clinical outcome 33–37
. Our results demonstrated how tumor B cells can impact the immunological factors in the tumor microenvironment,
Previous studies have established that artificial TCR stimulation by antiCD3 along with costimulation by CD28 can convert conventional T cells to Treg. Under these experimental conditions, the presence of FL-derived malignant B cells is not necessary but helpful to Treg conversion 23, 26
. In contrast to these studies, Treg conversion in our experiments was achieved without artificial TCR stimulation. Instead, the stimulation of conventional T cells was solely provided by normal or malignant B cells. This observation is important for two reasons: First, this observation suggested that only malignant B cells are uniquely equipped with the ability to modify the tumor microenvironment, which may play a role in tumor progression. Secondly, it is possible that TCR stimulation by natural antigens, such as tumor-associated antigens, presented by malignant B cells may be the key factor leading to Treg conversion. The mechanism of the B and T cell interaction in Treg conversion is the focus of our future studies.
Treg conversion observed in our experiment described a phenomenon, where the percentage of CD4+
cells increased during the co-culture of malignant B cells with tumor-infiltrating conventional T cells (CD4+
). The tumor-infiltrating conventional T cells were sorted to at least 90% purity before co-cultured with B cells. We and others have observed that 5–10% tumor-infiltrating conventional T cells from FL do express FoxP3 (unpublished data and ref 26
). During the 96 to 120-hour co-culture, we observed 2 fold increase of the total cell number (data not shown). The question arises whether Treg conversion we described was due to preferential proliferation of the CD4+
cells over the conventional T cells (the expansion scenario); or FoxP3 expression was induced in true conventional, CD4+
, T cells (the conversion scenario), or the combination of both. Our data showed that both the total cell number and the frequency of FoxP3 expressing CD4 T cells increased during the co-culture period, which is in concordance with the observation from other investigators 27
. Since we could not label the 5–10% of the CD4+
cells separately from the true conventional T cells (CD4+
), and observe their behavior independently in the co-culture, we were not able to distinguish the conversion from the expansion scenario. However, we believe that the pure expansion scenario is unlikely, given that the total cell number doubled during the co-culture period, but the percentage of CD4+
cells increased from 5–10% to 50–90% (Supplemental Table 1
). In support of this reasoning, it should be mentioned that true human Treg (CD4+
) are difficult to expand in vitro
, because even a minute amount of conventional T cells mixed in the sorted Treg can outgrow the Treg in expansion cultures. As such, Treg expansion in vitro
usually requires robust stimulation of TCR and special conditions such as addition of Rapamycin to preferentially suppress proliferation of conventional T cells (ref 38
and unpublished results by Hou et al.). Taken together, we believe that the increased frequency of CD4+
cells during the co-culture of conventional T cells with malignant B cells likely represents Treg conversion and perhaps expansion of these Treg as well.
It has been shown that FoxP3 can be transiently expressed in T cells activated by artificial TCR along with co-stimulation of CD28 30, 31
. Although it is unknown whether the disconnection between FoxP3 expression and acquisition of regulatory function is also present in Treg induced by antigen presenting cells, we felt that it was important to determine whether converted Treg indeed had regulatory activity. Gavin and colleagues have shown that Th-1 production can distinguish FoxP3-expressing effector T cells from FoxP3+ regulatory T cells. Thus, single-cell cytokine profiles can serve as an ideal surrogate for Treg activity 31
. This FACS-based single cell analysis is easy to do on a small number of converted Treg available from our conversion experiment. Based on this, we analyzed the production of IFN-γ and IL-2 in converted Treg by flow cytometry and showed they indeed did not produce Th-1 cytokines, suggesting that they have acquired Treg activity in addition to expression of FoxP3. Using a T cell suppression assay, Mittal et al., have shown that Treg converted by FL B cells have suppressive function 27
. Thus, our assays were complementary to these investigators’ in demonstrating the regulatory function of converted Treg.
Preferential trafficking is another potential mechanism by which Treg are accumulated in the tumor microenvironment. Chemokines and chemokine receptors that are involved in Treg trafficking are CCL17, CCL22 and CCR4, CCR5, respectively. CCL17 and CCL22 are secreted by macrophages; and CCR4 and CCR5 are expressed on CD4 T cells, including Treg. Normal and malignant B cells can secrete CCL17 and CCL22 when stimulated by soluable CD40 ligand or viral antigens 39–41
; however, it is not clear whether malignant B cells can secrete more such cytokines that can account for the high frequency of Treg in tumors. For this reason, we compared the production of CCL17 and CCL22 by purified B cells and expression of CCR4 and CCR5 on Treg from FL specimens and benign tonsils (). To our surprise, with soluble CD40 ligand stimulation, there was no difference in CCL17 and CCL22 production between B cells from tonsils and B cells FL. Furthermore, without soluable CD40 stimulation, tonsils B cells appeared to produce more CCL22, which would be unlikely to account for less frequency of Treg in tonsils. Our results indicated that preferential trafficking of Treg would be unlikely the mechanism for increased frequency in FL as compared with that in tonsils.
It is important to recognize that malignant B cells do produce CCL17 and CCL22 ( and ref 7
), as the production of these cytokines may play an important role in architectural localization of Treg within the tumor microenvironment. The architectural localization of Treg appeared to be clinically important, as a recent study demonstrated that the number and the location of Treg relative to follicles in a lymph node were correlated with the risk of histological transformation in FL 42
. It should be mentioned that we did not measure the secretion of CCL17 and CCL22 by tumor infiltrating macrophages, because they generally do not survive the freeze-and-thaw process (data not shown).
In summary, our results provided evidence that malignant B cells possess intrinsic properties to modify the tumor microenvironment through the induction of Treg. Studies are underway to identify molecules that are essential in Treg conversion. To this end, we used the anti-TGF-β blocking antibody in the conversion experiment, but it failed to inhibit Treg conversion. Similar results were reported by other investigators 27
. However, some studies suggested that CD70 and PD-1 on B cells may play a role in Treg induction 26, 27
. Identification of the critical molecules involved in Treg induction by lymphoma tumor cells may lead to specific interventions to prevent the negative immune regulatory effects of the lymphoma cells.