Regulatory T-Cells in the Tumor Microenvironment of B-Cell Non-Hodgkin Lymphoma

Principal Investigator: Stephen Ansell, M.D., Ph.D., Mayo Clinic
Co-Investigators: Richard Bram, M.D., Ph.D. and Keith Knutson, Ph.D., Mayo Clinic; Brian Link, M.D., University of Iowa

B-cell non-Hodgkin lymphomas (NHL) are common lymphoid cancers in which malignant cells arrested at various stages of differentiation proliferate within lymph nodes and occasionally other tissues. However, cells other than tumor cells are commonly present in the tumor microenvironment. These cells include T lymphocytes that seem to be more than simple residual elements from the normal lymph node structure. It is commonly believed that these infiltrating immune cells are targeting the cancer cells, yet they appear unable to eradicate the malignant cells. Despite extensive studies regarding anti-tumor immunity, the significance of infiltrating T cells in B-cell NHL remains poorly understood.

Recent studies in other cancers have suggested that regulatory T (T_reg) cells are involved in the control of anti-tumor immunity by inducing tolerance to the tumor. It has been shown that T_reg cells influence tumor immune responses by suppressing tumor-specific immune cells. However, there are little data regarding the effect of T_reg cells on tumor-specific T cell immunity in B-cell NHL and subsequently on the malignant B-cell growth.

Studies in B-cell lymphoma have described a T-cell or immune signature in the tumor that correlates with patient outcome. The immune infiltrate is usually comprised of CD4+ and CD8+ T-cells as well as monocytes. In previous work supported by a University of Iowa/Mayo Clinic (UI/MC) Lymphoma SPORE development award, UI/MC researchers showed that the presence of activated intratumoral CD4+ T-cells predicts the failure-free and overall survival of patients with B-cell NHL.

Furthermore, they found that a subset of CD4+CD25+ T cells with high Foxp3 expression characteristic of T_reg cells was present in areas of B-cell NHL. Importantly, these intratumoral T_reg cells display the ability to suppress other infiltrating T cells present in B-cell NHL. In preliminary studies, the researchers have shown that these T_reg cells suppress the proliferation and activation of both CD4+ and CD8+ T-cells. They also found that T_reg cells migrate in response to factors produced by the malignant B cells.

The team's hypothesis is that intratumoral T_reg cells in patients with B-cell NHL significantly upregulate their ability to suppress tumor specific T-cell responses as they enter sites infiltrated by malignant B cells. The investigators further postulate that malignant B cells play an active role in this process by direct activation of T_reg cells thereby facilitating immune tolerance to their presence.

To determine whether localization of T_reg cells influences their suppressive capacity and to define the role of malignant B cells in the migration and activation of T_reg cells in patients with B-cell lymphoma, they are pursuing the following aims:

1. To show that T_reg cells are pathologically recruited to areas of B-cell NHL and gain suppressive function when present in the malignant lymph nodes.

   T_reg cells mainly originate from thymic tissue and migrate to and are retained in peripheral target organs or lymph nodes that drain those organs. Whether there are either broad or specific signals to direct these T_reg cells to sites of tumor is poorly understood. Chemokines are the logical candidates to direct the recruitment of T_reg cells. It has been reported that CD4+CD25+ T_reg cells have a unique chemotactic response profile compared to CD4+CD25- T cells. However, limited data from these studies are available and it is hard to draw conclusions regarding the selective expression of chemokine receptors on T_reg cells.

   UI/MC Lymphoma SPORE preliminary data regarding B-cell NHL shows that intratumoral T_reg cells in B-cell NHL express CCR4 and migrate in response to CCL22 produced by malignant B cells in vitro. The investigators found that CCL22 is only partially responsible for the migration of T_reg cells. This raises the question as to what other chemokines and chemokine receptors are responsible for the migration of T_reg cells to these sites and whether the activation status or location of the T_reg cell plays a role. Other researchers have shown that T_reg cells migrate in response to CCL19 or CXCL13 depending on the expression of CD69 on the cells. Furthermore, recent data has suggested that distinct lymphoid compartments may play a critical role in T_reg cell function. These data suggest that certain subsets of T_reg cells inhibit the initiation of the immune response by entering lymphoid tissue, while other subsets, which are able to enter inflamed sites, control established immune reactions. It therefore appears that the migratory behavior and location of T_reg cells crucially influences their suppressive activity.

   In this aim, the investigators will identify the subsets of T_reg cells present in lymphoma patients and determine whether these cells become increasing suppressive as they are recruited to malignant lymph nodes.

2. To determine whether malignant B cells directly interact with T_reg cells to facilitate immune tolerance.

   UI/MC investigators previously showed that T_reg cells migrate in response to chemotactic factors such as CCL22 produced by malignant B cells. Although there are no reports to date that have studied the interaction between T_reg cells and malignant B cells, it is known that lymphoma B cells express HLA class II molecules and are capable of presenting antigens to CD4+ T lymphocytes.

   In lymphoid organs involved by malignant B-cell NHL, it has also been shown that tumor-infiltrating CD4+ T cells are in contact with tumor B cells and that lymphoma B cells are capable of proliferating in response to various signals usually provided by CD4+ T cells, such as IL-4, or CD40L. What is not known, however, is whether malignant B cells directly interact with and activate T_reg cells.

   Due to the fact that cell contact is necessary for T_reg cell-mediated suppression, molecules expressed on the cell surface are more likely play an important role in this process. Possible mechanisms including signaling through TGF-β, NFATc2 and NFATc3, CTLA4, and possibly cytokines, may be involved in the induction of the inhibitory action of intratumoral T_reg cells in B-cell NHL.

   The UI/MC team contends that malignant B cells directly activate T_reg cells and thereby protect themselves from the tumor-directed immune response. They are working to determine whether T_reg cells are activated when co-cultured in the presence of B cells and identify the mechanism for this activation.

3. To test whether therapeutic inhibition of T_reg cell recruitment and function results in significant clinical benefit for patients with B-cell NHL

   Based on preliminary data, the UI/MC researchers hypothesize that tumor T_reg cells regulate the growth of malignant lymphoma B cells by suppressing tumor-infiltrating T cells in the area of B-cell NHL and are recruited by the malignant B cells. They are conducting a clinical trial in newly-diagnosed follicular lymphoma patients using denileukin diftitox in combination with rituximab to test whether T_reg cell inhibition will result in clinically relevant benefit for patients with B-cell follicular NHL. They anticipate that denileukin diftitox will deplete the intratumoral T_reg cells while rituximab will deplete malignant B cells and thereby also prevent further recruitment of T_reg cells to the tumor.

   There is a strong rationale for combining denileukin diftitox with rituximab in patients with newly-diagnosed follicular lymphoma. Rituximab has proven to be an effective biologic therapy in indolent B-cell lymphoma and is now being used as frontline therapy for these patients.

   Thomas Witzig, M.D., a Mayo Clinic investigator, conducted a clinical trial of rituximab as a single agent in newly diagnosed follicular lymphoma. In this North Central Cancer Treatment Group study, 37 patients received rituximab 375 milligrams/square meter (mg/m2) intravenous weekly for four doses and were then followed for response and progression; no maintenance therapy was provided. The overall response rate was 72 percent, with 36 percent complete remissions, and the median time to progression was 2.2 years.

   Others have tested denileukin diftitox in B-cell NHL. Dang et al treated 50 patients with relapsed lymphoma with denileukin diftitox 18 micrograms/kilogram/day (μg/kg/day) for five days and saw a 25 percent response rate in B-cell NHL with a superior result in lymphomas that did not express CD25 on the malignant cells (29 percent versus 22 percent). Although this study did not evaluate the effect of the agent on T_reg cells, the result in CD25 negative lymphomas suggests that the effect of the agent may be through the depletion of other CD25 expressing cells such as T_reg cells. Furthermore, denileukin diftitox has been shown to deplete T_reg cells in cancer patients without significant toxicity to other subset of CD25 expressing cells. Studies in T-cell lymphomas have also utilized denileukin diftitox at 18 μg/kg/day and have shown this dose to be safe and effective either as a single agent or in combination with other agents. Furthermore, denileukin diftitox has been safely combined with rituximab in patients with relapsed B-cell lymphoma.