Signaling mediated through the IL-2R, in conjunction with signals mediated through the T cell Ag receptor, promotes the proliferation and effector function of T cells (32
). Some of the signals, such as up-regulation of the IL-2R α-chain, result from cooperative signals mediated both through the T cell Ag receptor as well as through the IL-2R itself; however, the relative role of signals delivered through these pathways has not been fully elucidated. Human tumor-reactive effector T cells have been shown to proliferate extensively in vitro in the presence of high-dose IL-2 alone (33
). In addition, between 15 and 20% of melanoma and renal cancer patients treated with high-dose IL-2 alone respond to therapy (6
), which may reflect the ability of IL-2 to maintain the proliferation of T cells that were activated by prior exposure to tumor Ags.
Interactions between the costimulatory receptor CD27 and its ligand, CD70, have also been found to play a key role in T lymphocyte activation, proliferation, survival, and differentiation (10
). The intracytoplasmic domain of CD27 triggered by CD70 mediates these effects through activation of Jun kinase and NF-κB, which is required for optimal effector/memory CD8 T cell generation (34
This study explores the effects of IL-2 on the expression of CD70 and its receptor, CD27, on CD8+
T cells, as well as the role of stimulation mediated through the CD27 pathway in regulating the proliferation and function of effector T cells. Incubation of PBMCs with IL-2 in vitro resulted in the up-regulation of CD70 expression on CD8+
cells and down-regulation of CD27 expression. This outcome did not appear to result from the selective death of CD27+
T cells, as significant levels of apoptosis were not observed in the CD27+
T cells that were present in these cultures (data not shown). Cells that possess a similar phenotype were observed in vivo in patients receiving high-dose IL-2 therapy at the time when IL-2 is present at detectable levels in peripheral blood. Expression of IL-2Rα (CD25) was not detected on CD8+
T cells before the up-regulation of CD70; thus, it appears that relatively high concentrations of IL-2 can up-regulate CD70 expression by signaling through the intermediate affinity IL-2Rβγ receptor, which has previously been shown to activate T cells (35
Previous investigations demonstrated that CD27 expression is down-regulated on the surface of activated T cells following interaction with CD70. In CD70 transgenic mice that constitutively express CD70 on B cells, CD27 was down-regulated because of the continuous interaction of CD70 with CD27+
T cells (10
). In another report, the incubation of CD8+
T cell with anti-CD70 Ab interfered with the down-regulation of CD27 that was normally observed following Ag activation (11
). The results of the present study suggest that down-regulation of CD27 expression on CD8+
T cells from peripheral blood following incubation with IL-2 may have resulted from interactions with CD70 expressed on T cells, which was associated with the acquisition of effector functions on those cells. This hypothesis was supported by the observation that the withdrawal of IL-2 from activated effector CD8+
T cells was associated with the up-regulation of CD27 and the down-regulation of CD70 expression. A similar phenotypic change was observed on CD8+
T cells present in the peripheral blood of IL-2-treated patients soon after the termination of IL-2 administration.
Additional experiments were then conducted to evaluate the nature of the interaction of CD27 with CD70 expressed on CD8+ T cells. The incubation of in vitro cultured effector CD8+ T cells that expressed CD70 with an anti-CD70 blocking Ab significantly inhibited the proliferation of these cells, even in the presence of high-dose IL-2, but did not alter the expression of markers such as CD25 (data not shown). These findings suggest that this Ab interfered with delivery of a signal through the CD27 molecule rather than delivering a direct signal by the cross-linking of CD70 molecules on the surface of T cells. Studies conducted on PBMCs that were cultured in vitro with IL-2 indicated that the subset of CD70+ cells that bound to rhIL-2F proliferated more extensively than cells that failed to bind to the rhIL-2F. The population of CD70+ CD8+ T cells that bound high levels of IL-2 also appeared to selectively down-regulate CD27 expression (data not shown), indicating that the delivery of a strong signal through the IL-2 receptor may play an important role in the down-regulation of CD27 expression by CD70. These observations suggest that engagement of CD27 by CD70 molecules expressed on T cells represents a down-stream mediator of the activation of T cells by IL-2.
The expression of CD27 was then examined on TILs that were adoptively transferred to patients, as this might provide an indication of the size of the effector/memory pool in these polyclonal populations of cells. Previous studies had demonstrated that the differentiation of T cells to end stage effector cells was associated with stable down-regulation of CD27 expression (14
). The fact that IL-2, as well as TCR activation, transiently down-regulated CD27 expression on TILs hampered evaluation of the stage of differentiation of cells present in these polyclonal populations. However, the withdrawal of IL-2 from in vitro TIL cultures that had been grown continuously in the presence of IL-2 resulted in up-regulation of the expression of CD27 on a variable percentage of the T cells present in these cultures. The percentage of T cells that up-regulated CD27 expression varied widely between TILs, and for some cultures little or no up-regulation was observed. The minimal levels of CD27 expression that were observed on some TILs following IL-2 withdrawal may have resulted from the large percentages of end stage effector T cells present in these cultures.
Additional studies were then conducted to evaluate the properties of CD27+
T cell present within TILs. When TILs were cultured for 2 days in vitro in the absence of IL-2, CD27+
T cells proliferated to a greater extent than CD27−
T cells. Previous studies have demonstrated that central memory cells possess an enhanced ability to secrete IL-2 (38
), and results presented in this study demonstrate that CD27−
cells secreted higher levels of IL-2 than CD27−
cells following Ag activation. These factors may help to account for the selective ability of CD27+
T cells to survive in vivo following adoptive transfer under conditions where common γ-chain cytokines such as IL-2 are limiting (17
). In this study, several genes that are selectively expressed in proliferating cells (29
) were up-regulated selectively in CD27+
T cells isolated from TILs. In contrast, the most highly expressed genes that were selectively up-regulated in CD27−
T cells included several genes involved with T cell activation and apoptosis, characteristics that have been associated with late stage effector T cells.
The potential relevance of these findings to clinical observations was then evaluated. Previous studies (31
) suggested that the persistence of adoptively transferred T cells was associated with their proliferative capacity as well as with their ability to mediate tumor regression. Examination of individual clonotypes present within bulk TILs that persisted in patients’ peripheral blood following cell transfer revealed that these cells, when cultured before transfer in the absence of IL-2 for 2 days, expressed higher levels of CD27 than clonotypes that did not persist. An evaluation of the bulk TILs obtained from 33 patients treated with TILs following nonmyeloablative chemotherapy, including 16 responders and 17 nonresponders, demonstrated that the mean of total number of CD27+
T cells present in the TILs that were administered to responders were significantly higher than those administered to nonresponders. The expression of CD70 on cells cultured before transfer in the absence of IL-2 for 2 days also appeared to be significantly higher in TILs that were administered to responders than to nonresponders, implying that the pool of activated memory/effector cells present in TILs that were administered to responders was larger than in TILs that were administered to nonresponders. The expression of CD70 in vivo on other cells, such as B cells and dendritic cells that are present in patients that received adoptive TIL transfer, may also act to costimulate T cells that express CD27. The expression of CD70 on the transferred T cells alone, however, may provide a sufficient signal to promote the in vivo survival and proliferation of transferred CD8+
cells that are also capable of expressing CD27, as suggested by the in vitro studies presented in this report as well as by additional studies (40
). In addition, in mouse model systems the expression of CD27 appeared to be associated with T cell accumulation at tissue effector sites and the survival of activated CD8+
T cells in vivo, whereas expression of CD28 was not associated with T cell migration (41
). It is difficult to determine the relative roles of CD27 and CD70 expression in mediating the function of adoptively transferred T cells, but the expression of both the costimulatory receptor and ligand on T cells may enhance the in vivo function of these cells. These observations provide potential explanations for the association between CD27 expression in TILs and patient response to adoptive immunotherapy.
In summary, IL-2 is a crucial cytokine that modulates CD8+ T cell responses in immunotherapy. Our results suggested that IL-2 may function, at least in part, by promoting CD8+ T cell effector function through the interaction of CD27 with CD70. In addition, evaluation of the pool of T cells present in patients’ TILs that express CD27 and CD70 may facilitate the identification of effective TILs for use in patient treatment.