Troublesome T Cells: The Role of Regulatory T Cells in Dogs with Cancer
ACVIM 2008
Barb Biller, DVM, PhD, DACVIM
Fort Collins, CO, USA

Introduction

Identification and Function of Regulatory T Cells (Treg)

Treg are a distinct subset of T cells that comprise between 5-8% of the total population of T cells in the normal mouse or human lymph node, or 10-12% of the total CD4+ population (1,2). Treg directly suppress the proliferation of harmful self-reactive T cells and play a critical role in prevention of autoimmune disease and maintenance of peripheral tolerance (2,3). In mice, humans and cats, Treg can be identified based on surface expression of high levels of the IL-2-receptor-α chain (CD25) (4). Although other cell surface molecules such as glucocorticoid-induced TNF-receptor (GITR), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and CD103 aid in Treg identification, definitive delineation from activated CD4+ non-regulatory T cells using cell surface marker expression alone is not possible (5-7). Recently, however, intracellular detection of the transcription factor FoxP3 has been shown to uniquely identify the murine and human Treg populations (8-10). Identification of CD4+/FoxP3+ T cells permit distinction of Treg from activated non-regulatory CD4+ T cells and has greatly facilitated the study of Treg in various diseases such as cancer and autoimmune disorders. We have recently reported the detection of canine Treg using a flow cytometry-based assay that employs a cross-reactive anti-mouse-Foxp3 antibody and an anti-dog-CD4 antibody to identify FoxP3+/CD4+ T cells (11). Figure 1 gives representative dot plots of FoxP3 expression by CD4+ T cells (the cell population in the upper right quadrant of each plot) from the blood (A), an uninvolved lymph node (B), and a tumor-draining lymph node (C) in a dog with an oral melanoma. In (D), staining of blood from the same dog with an irrelevant isotype-matched antibody (control for FoxP3 staining) is shown. The percentages of lymphocytes contained within each quadrant are also given for comparison. Using this technique we are investigating the role of Treg in dogs with different types of cancer.

Figure 1.
Figure 1.

Flow cytometric identification of canine Treg using a FoxP3 antibody.
 

Treg are Increased in Human Cancer PATIENTS and Negatively Correlated with Survival

Multiple studies have shown that Treg are increased in people with malignancies such as melanoma, ovarian carcinoma, gastric carcinoma, and squamous cell carcinoma of the head and neck (12-15). Direct interference with antitumor immune responses by Treg has been clearly documented. CD4+/Foxp3+ Treg have been shown, for example, to inhibit the proliferation and cytokine production of tumor-specific cytotoxic T cells in people with metastatic melanoma (15). In mouse tumor models, Treg depletion leads to effective CTL-mediated immune responses that destroy tumor cells and protect against metastatic disease (5,16). Evaluation of Treg numbers can also provide valuable prognostic information; in women with advanced ovarian carcinoma increased numbers of tumor-infiltrating Treg were associated with a 25-fold higher risk of death and 4-fold reduction in survival time (12). In this study, the number of tumor-infiltrating Treg was a significant predictor of death even after controlling for stage of disease and type of therapy. Treg levels, particularly in tumor tissue and tumor-draining lymph nodes, may more accurately predict survival, duration of the disease-free interval and response to treatment than other factors such as stage and grade of disease (5,12,17-18).

Results of Current Studies Evaluating Treg in Dogs with Cancer

Treg are Increased in the Blood and Tumor Draining Lymph Nodes of Dogs with Cancer Compared to Healthy Dogs

Using the flow cytometry assay described above, we evaluated Treg within the blood and lymph nodes of 10 dogs with different types of cancer and 10 healthy, age-matched control dogs. We found that both the percentage of Treg and the absolute number of Treg were significantly higher in the peripheral blood of dogs with cancer than in healthy dogs (Table 1). For example, the mean percentage of Treg in peripheral blood in all 10 dogs with cancer was 7.5%, compared to 4.3% in the 10 normal dogs. The absolute numbers of Treg in peripheral blood of dogs with cancer were also significantly increased (mean of 173 cells per ml blood in dogs with cancer versus 98 cells per ml blood in control dogs, p < 0.04, not shown). Next, we assessed whether the lymph nodes of dogs with cancer contained more Treg than the lymph nodes of healthy dogs and whether the tumor draining lymph node had more Treg than non-tumor draining lymph nodes. The percentage of Treg in the tumor draining lymph nodes of seven dogs with cancer (excluding three dogs with osteosarcoma (OSA) where a tumor draining lymph node was no longer present due to limb amputation) was significantly higher ( p = 0.04) than the percentage of Treg in the non-tumor draining lymph nodes of the same dogs. However, we found that the percentage of Treg in the non-tumor draining lymph nodes of dogs with cancer (11.1%) was not significantly different from the percentage of Treg in lymph nodes of healthy control dogs (9.8%) (Figure 2 and Table 1). These findings suggest that Treg are higher in canine cancer patients than healthy dogs and may preferentially accumulate within tumor draining lymph nodes.

Table 1. Treg levels in healthy dogs and dogs with cancer.

Tumor type

Number of dogs

% Treg in
peripheral BLOOD
(+/- SD)

% Treg in
non- draining node
(+/- SD)

% Treg in
draining node
(+/- SD)

Healthy dogs

10

4.3 (+/- 0.7)

9.8 (+/- 2.4)

ND

Oral melanoma

4

11.1 (+/- 2.1)

12.0 (+/- 0.5)

19.5 (+/- 8.0)

Osteosarcoma

3

7.2 (+/- 1.9)

10.8 (+/- 0.5)

ND

Mast cell tumor

2

6.3

10.0

15.5

Soft tissue sarcoma

1

5.5

11.6

16.4

Figure 2.
Figure 2.

%Treg in lymph nodes of healthy dogs and dogs with cancer.
 

Evaluation of the Prognostic Potential of Treg Numbers in Dogs with OSA and Oral Melanoma

Studies to determine whether particular tumor types might be associated with higher numbers of Treg and to assess the potential prognostic value of Treg are currently ongoing in our laboratory. As an initial step, we compared Treg numbers in the blood and lymph nodes of dogs with melanoma (n = 4) and those with OSA (n = 3) to healthy control dogs (n = 10). We found that dogs with melanoma had significantly (p < 0.05) more Treg in their blood than either dogs with OSA or control dogs (Table 1). When Treg obtained from aspirates of the same lymph node (submandibular) in all three groups of dogs were compared, we also found that dogs with melanoma had significantly (p < 0.05) more Treg (19.5%) than both control dogs (9.8%) and dogs with osteosarcoma (10.8%). However, it should be noted that the submandibular lymph node was the tumor draining lymph node for dogs with oral melanoma; interestingly, metastatic disease to the submandibular lymph node was not observed on cytology in any of these patients. Although preliminary, these results suggest that certain canine cancers may be associated with higher numbers of Treg in blood and lymph nodes and that Treg elevation may precede the development of regional metastases.

Conclusion and Future Directions

The development of a diagnostic method to reliably identify Treg in dogs is facilitating the study of the role of Treg in important diseases of dogs such as autoimmune diseases, chronic infections and cancer. Our observation that Treg levels are higher in the peripheral blood and tumor-draining lymph nodes of dogs with cancer than in healthy dogs is remarkably similar to findings for human cancer patients. In addition, our preliminary data suggests that highly metastatic tumors may be associated with greater numbers of Treg (Table 1) and supports our hypothesis that Treg numbers will provide important prognostic information in canine cancer patients as they do in people. We anticipate that further investigations into the role of Treg in various malignancies will offer important insights into the immunological control of cancer and illuminate new pathways to harness the power of the immune system in controlling cancer.

References

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Speaker Information
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Barbara Biller, DVM, PhD, DACVIM
Colorado State University
Fort Collins, CO


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