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Keywords:

  • dog;
  • metastatic tumor;
  • Tc1;
  • Th1;
  • Treg

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

It is well known that lymphocytes from patients with advanced-stage cancer have impaired immune responsiveness and that type1 T lymphocyte subsets in tumor bearing hosts are suppressed. Treg have been reported to comprise a subgroup which inhibits T cell mediated immune responses. In the present study, the percentage of Treg, Th1 and Tc1 in the peripheral blood of tumor bearing dogs with or without metastases was evaluated. The percentages of Th1 and Tc1 in dogs with metastatic tumor were significantly less, and that of Treg was significantly greater, than those of dogs without metastatic tumor. The percentage of Treg showed an inverse correlation with that of Th1 and Tc1 in tumor bearing dogs. It was concluded that an increase in Treg in the peripheral blood of dogs with metastatic tumor may induce suppression of tumor surveillance by the Type1 immune response and lead to metastasis of tumor[0][0].[0]

List of Abbreviations: 
ECOG

Eastern Cooperative Oncology Group

FITC

fluorescein isothiocyanate

FSC

forward scatter

IFN-γ

ιnterferon-gamma

mAb

monoclonal antibody

PBL

peripheral blood lymphocyte(s)

PBMC

peripheral blood mononuclear cell(s)

PE

phycoerythrin

PMA

phorbol 12-myristate 13-acetate

SSC

side scatter

Tc1

Type1 cytotoxic T lymphocyte(s)

Th1

Type1 helper T lymphocyte(s)

Treg

regulatory T cell(s)

It is well known that lymphocytes from patients with advanced-stage cancer have impaired immune responsiveness (1) and that cell-mediated immunity in cancer-bearing hosts is suppressed by many factors (2). IFN-γ acts to enhance recognition of transformed cells by the immune system. Thus IFN-γ plays a central role in providing an immunocompetent host with a mechanism for tumor surveillance (3). IFN-γ is produced mainly by Th1 and Tc1. Recent studies in humans have demonstrated that a low percentage of Th1 and Tc1 may contribute to the escape of tumor cells from immunosurveillance (4–7). In veterinary medical science, we have reported that the percentage of Th1 in PBMC from tumor bearing dogs is significantly less than that of healthy dogs. In addition, the percentage of Th1 in dogs with metastatic tumor is significantly less than that in dogs without metastatic tumor (8).

Treg, which is one of the subtypes of the CD4+ T cell lineage, is characterized by coexpression of CD25. Recently, intracellular detection of the transcription factor Foxp3 has been shown to uniquely identify a highly enriched Treg population in rodents, and this is considered to be the most specific Treg marker (9–11). Treg have been identified in dogs using antibodies specific for dog CD4 and murine Foxp3 (12). Treg are thought to comprise a functionally unique subset of T lymphocytes which play an important function in maintaining immune homeostasis (13, 14). Of note, Treg can inhibit the immune response mediated by CD4+CD25- and CD8+ T cells. Accordingly it has been reported that Treg play an important role in preventing allograft rejection, graft-versus-host disease, and autoimmune disease (15, 16). In addition, it has been shown in an experimental model with cancer that Treg down-regulates the activity of effector function against tumors, resulting in T cell dysfunction in cancer-bearing hosts (17–19). Combined with other indirect evidence, these studies lead us to formulate the hypothesis that tumor bearing hosts with advanced cancer have an increased population of Treg, which might inhibit the T cell mediated anti-tumor immune response. In fact, an increased population of Treg has been reported in patients with various cancers (20–25). Moreover, Chikamatsu et al. have reported that the percentage of Treg is inversely correlated with that of Tc1 in PBMC of patients with squamous cell carcinoma of the head and neck (26). In veterinary medical science, Biller et al. have reported an increased population of Treg in dogs with various cancers (12). However, there are no previous reports describing relationships between Treg and process of metastasis or type1 T cell populations in dogs with tumors.

In the present study, we evaluated the percentage of Treg, Th1, and Tc1 in the peripheral blood of tumor bearing dogs with or without metastases, and evaluated the correlation between the percentage of Treg and that of Th1 or Tc1 in these dogs.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Research animals

Blood samples from a total of fourteen dogs with advanced cancer were examined in this study. All animals were at the Japan Animal Referral Medical Center and had histologically confirmed malignant tumors. None of the animals had received any immunosuppressive treatment, including anticancer drugs, radiotherapy or corticosteroids.

The histologically confirmed clinical diagnoses were mammary adenocarcinoma in one dog, pheochromocytoma in one, hepatocellular carcinoma in one, cerumino carcinoma in one, thyroid gland carcinoma in one, transitional cell carcinoma of the urinary bladder in one, seminoma in one, soft tissue sarcoma in three and oral melanoma in four.

All dogs were clinically staged according to the WHO staging system by palpation and imaging techniques (Table 1). The cases that were diagnosed as metastatic on the basis of these examinations were one of seminoma, one of cerumino carcinoma, one of hepatocellular carcinoma, two of oral melanoma and three of soft tissue sarcoma. There was no significant difference between dogs with and without metastatic tumor in regard to performance status according to classification by the ECOG scoring system (data not shown) (27).

Table 1.  Characteristics of research animals
Clinical diagnosesWHO Stage
Mammary adenocarcinomaIII
PheochromocytomaIII
Hepatocellular carcinomaIV
Cerumino carcinomaIV
Thyroid gland carcinomaIII
Transitional cell carcinoma of the urinary bladderII
SeminomaIV
Soft tissue sarcomaIV
IV
IV
Oral melanomaI
I
IV
IV

All experiments in this study were carried out in accordance with the guidelines for animal experiments issued by the College of Bioresource Sciences, Nihon University.

Blood sample preparation

Blood samples were collected by heparinized syringe before biopsy. PBMC were isolated as we have previously described (28).

Intracellular cytokine analysis

Th1 and Tc1 were measured by flow cytometric analysis using intracellular cytokine staining. Intracellular cytokine staining was performed as we have previously described (28). In brief, PBMC were stimulated with a combination of PMA; (Sigma-Aldrich Milwaukee, WI, USA), and ionomycin (Sigma-Aldrich) for 4 hr at 37oC in the presence of Brefeldin A (Sigma-Aldrich) for the last 2 hr. After incubation, non-adherent cells were collected as PBL. Then the PBL were stained with FITC –conjugated monoclonal antibody for each cell surface marker: CD4 as a Th marker, CD8 as a Tc marker.

For staining of intracellular cytokine, PBL were fixed and permeabilized using Intraprep (Immunotech, Marseilles, France). PBL were incubated with the respective PE-labeled anti-bovine IFN-γ mAb (Serotec, Oxford, UK).

Th1 and Tc1 subpopulations were analyzed by flow cytometry (FACS Cant, Becton Dickenson, San Jose, CA, USA). Lymphocytes were gated according to the region determined by FSC and SSC, then CD4+ or CD8+, as Th or Tc respectively, were gated according to FITC intensity. Th1 cell and Tc1 cell subpopulations were determined by PE intensity. Th1 or Tc1 percentage was calculated as the percentage of IFN-γ positive cells of the total CD4 or CD8 positive cell population (Fig. 1a,b).

image

Figure 1. Characterization of Th1, Tc1 and Treg in peripheral blood from tumor bearing dogs shown as dot plot patterns of intracellular staining. (a, b) PBMC were cultured with PMA and ionomycin for 4 hr in the presence of Brefeldin A for the last 2 hr and stained with FITC-conjugated mAb to CD4 or CD8 and PE-conjugated mAb to IFN-γ. (c) PBMC were stained with FITC-conjugated mAb to CD4 and PE-conjugated mAb to FoxP3. X-axis, FITC intensity of CD4 (a, c) or CD8 (b); Y-axis, PE intensity of IFN-γ (a, b) or FoxP3 (c).

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Intracellular Foxp3 analysis

Treg was measured by flow cytometric analysis using intracellular FoxP3 staining. PBMC were stained with FITC conjugated anti-canine CD4 mAb. For staining of intracellular FoxP3, PBMC were fixed and permeabilized using FoxP3 Staining Set (eBioscience, San Diego, CA, USA) following a set protocol. PBMC were incubated with the respective phycoerythrin PE-labeled mAb to FoxP3. Anti-FoxP3 mAbs used in this study were anti-murine FoxP3 (clone FJK-16s, eBioscience). Cross-reactivity of this mAb has previously been clarified by B. J. Biller et al. (12).

The Treg subpopulation was analyzed by flow cytometry (FACS Canto, Becton Dickenson, San Jose, CA, USA). Lymphocytes were gated according to the region determined by FSC and SSC, then CD4 positive cells were gated according to FITC intensity. The Treg cell subpopulation was determined by PE intensity. The Treg percentage was the percentage of FoxP3 positive cells of the total CD4 positive cell population (Fig. 1c).

Statistical analysis

Results are presented as mean values with associated standard deviations. Statistical analyses were done with MS-Excel (Microsoft). The differences between the two groups were assessed by the Mann–Whitney U test. Spearman's correlation coefficient by rank test was used to analyze correlations. P values less than 0.05 were accepted as statistically significant.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Comparison of the percentage of Th1, Tc1 and Treg in tumor bearing dogs with or without metastases

PBMC obtained from dogs with and without metastatic tumors were stained with surface and intracellular markers for T cell subpopulations (CD4+ IFN-γ+, Th1; CD8+ IFN-γ+, Tc1; CD4+ FoxP3+, Treg; Fig. 1). The percentage of Th1 in dogs with metastatic tumor (13.66%± 5.45) was significantly less than that in dogs without metastatic tumor (22.95%± 9.53; P < 0.05, Fig. 2a). Moreover the percentage of Tc1 in dogs with metastatic tumor (22.75%± 13.65) was also significantly less than that in dogs without metastatic tumor (48.48%± 8.80; P < 0.05, Fig. 2b).

image

Figure 2. (a) The percentage of Th1 in CD4+ cells, (b) of Tc1 in CD8+ cells and (c) of Treg in CD4+ cells. (a,b) Th1 and Tc1were significantly less in dogs with metastases than in dogs without metastases (means ± S.D., P < 0.05) while (c) Treg was significantly greater in dogs with metastases than that in dogs without metastases (means ± S.D., P < 0.01).

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On the other hand, the percentage of Treg in dogs with metastatic tumor (13.03%± 3.74) was significantly greater than that in dogs without metastatic tumor (7.20%± 1.67; P < 0.01, Fig. 2c).

Relation between Treg, Th1 and Tc1 in tumor bearing dogs

There was significant correlation between Th1 and Tc1 (rs = 0.53, P < 0.05; Fig. 3a). The percentage of Treg inversely correlated with that of both Th1 (rs =−0.55, P < 0.05; Fig. 3b) and Tc1 (rs =−0.91, P < 0.001; Fig. 3c).

image

Figure 3. Correlation between percentage of Th1, Tc1 and Treg. (a) The correlation coefficient of both the percentage of Th1 and Tc1 was rs = 0.53. (b) The correlation coefficient of both the percentage of Treg and Th1 rs =−0.55. (c) The correlation coefficient of both the percentage of Treg and Tc1 rs =−0.91.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

In this study, we showed a smaller population of Th1 and Tc1 and a larger population of Treg in the peripheral blood of dogs with metastatic tumor in comparison with dogs without metastatic tumor. Moreover, there was a highly significant correlation between the percentage of Tc1 and the percentage of Treg in dogs with tumors.

The percentage of Th1 and Tc1 in dogs with metastatic tumor was significantly less than that in dogs without metastatic tumor. A similar result has been reported for human breast cancer (29). It may be a trend that patients with metastatic tumor show strongly suppressed Type1 immune responses. IFN-γ, which is one of the Type1 cytokines, acts to enhance recognition by the immune system of transformed cells (3). Thus IFN-γ plays a central role in providing an immunocompetent host with a mechanism for tumor surveillance. Therefore, suppression of Type1 immune responses may cause escape of tumor cells from immunosurveillance and the development of metastatic tumor.

The percentage of Treg in dogs with metastatic tumor was significantly greater than that in dogs without metastatic tumor. In human medical science, there are several reports which are similar to our findings. Ikemoto et al. have reported that the percentage of Treg in pancreatic cancer patients with metastases is significantly greater than that in pancreatic cancer patients without metastases. (30). Beyer and Schultze described in their review that Treg are increased in most human solid tumors, and that there seems to be a stage-dependent increase in Treg (31). Furthermore, we found a highly significant inverse correlation between the percentage of Treg and that of Tc1 (rs =−0.91, P < 0.001). In human patients with squamous cell carcinoma of the head and neck, the percentage of Treg inversely correlates with that of Tc1 (26). We consider that the trend of Treg in dogs with tumors may be similar to the trend of Treg in humans with tumors.

Liyanage et al. have assessed the suppressor function of human Treg by coculturing CD8+ or CD4+CD25- cells with CD4+CD25+ cells. In their report, as the ratio of Treg increases, IFN-γ secretion by CD4+CD25- and CD8+ cells is suppressed. Moreover the proliferation of CD4+CD25- and CD8+ cells, as measured by the 3H thymidine incorporation test, is also suppressed (18). The cause for the significant inverse correlation between the percentage of Treg and that of Tc1 or Th1 which we found is similar to the mechanisms described by Liyanage et al. There is increasing evidence that Treg play a key role in suppressing T cell mediated immunity in cancer-bearing hosts. In several animal models, it has been suggested that the efficacy of therapeutic cancer vaccination could be enhanced by depleting Treg (32), and that adoptive transfer of Treg impaired tumor specific immunity results in tumor progression (33). In light of the present study and previous reports, the fact that an increased population of Treg is observed in the peripheral blood in advanced stage tumor suggests that Treg provide immune tolerance for tumor cells by suppressing Type1 immunity and may be connected with tumor metastasis.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We thank Miss Rie Shirai, Mr. Ryudo Gouhara, Mr. Sho Kimijima, Miss Yoko Okumura and Miss Madoka Nakamura for veterinary technical support.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  • 1
    Miescher S., Whiteside T.L., Carrel S., Von Fliender V. (1986) Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors: effects of tumor cells and their supernatants on proliferative responses of lymphocytes. J immunol 136: 1899907.
  • 2
    Kiessling R., Wasserman K., Horiguchi S., Kono K., Sjoberg J., Pisa P., Petersson M. (1999) Tumor induced immune dysfunction. Cancer Immunol Immunother 48: 35362.
  • 3
    Kaplan D.H., Shankaran V., Dighe A.S., Stochert E., Aguet M., Old L.T., Schreiber R.D. (1998) Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice. Proc Natl Acad Sci U S A 95: 755661.
  • 4
    Agarwal A., Verma S., Burra U., Murthy N.S., Mohanty N.K., Saxena S. (2006) Flow cytometric analysis of Th1 and Th2 cytokines in PBMC as a parameter of immunological dysfunction in patients with Superficial Transitional cell carcinoma of bladder. Cancer Immunol Immunother 55: 73443.
  • 5
    Botella-Estrada R., Escudero M., O’Connor J.E., Nagore E., Fenollosa B., Sanmartin O., Requena C., Guillen C. (2005) Cytokine production by peripheral lymphocytes in melanoma. Eur Cytokine Netw 16: 4755.
  • 6
    Ito N., Nakamura H., Tanaka Y., Ohgi S. (1999) Lung carcinoma: analysis of T helper type 1 and 2 cells and T cytotoxic type 1 and 2 cells by intracellular cytokine detection with flow cytometry. Cancer 85: 235967.
  • 7
    Nakayama H., Kitayama J., Muto T., Nagawa H. (2000) Characterization of intracellular cytokine profile of CD4(+) T cells in peripheral blood and tumor-draining lymph nodes of patients with Gastrointestinal cancer. Jpn J Clin Oncol 30(7): 3015.
  • 8
    Horiuchi Y., Hanazawa A., Nakajima Y., Nariai Y., Asanuma H., Kuwabara M., Yukawa M., Ito H. (2007) T-helper (Th) 1/Th2 imbalance in the peripheral blood of dogs with malignant tumor. Microbiol Immunol 51(11): 11358.
  • 9
    Fontenot J.D., Gavin M.A., Rudensky A.Y. (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4: 3306.
  • 10
    Fontenot J.D., Rudensky A.Y. (2005) A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat Immunol 6: 3317.
  • 11
    Ramsdell F. (2003) Foxp3 and natural regulatory T cell: key to a cell lineage? Immunity 19: 1658.
  • 12
    Biller B.J., Elmslie R.E., Burnett R.C., Avery A.C., Dow S.W. (2007) Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer. Vet Immunol Immunopathol 116(1–2): 6978.
  • 13
    Dieckmann D., Plottner H., Berchtold S., Berger T., Schuler G. (2001) Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood. J Exp Med 193: 130310.
  • 14
    Sakaguchi S., Sakaguchi N., Asano M., Itoh M., Toda M. (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155: 115164.
  • 15
    Asano M., Toda M., Sakaguchi N., Sakaguchi S. (1996) Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med 184: 38796.
  • 16
    Sakaguchi S., Sakaguchi N., Shimizu J., Yamazaki S., Sakihama T., Itoh M., Kuniyasu Y., Nomura T., Toda M., Takahashi T. (2001) Immunologic tolerance maintained by CD25+ CD4+ regulatory cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 182: 1832.
  • 17
    Awwad M., North R.J. (1989) Cyclophosphamide-induced immunologically mediated regression of a cyclophosphamide-resistant murine tumor: a consequence of eliminating precursor L3T4+ suppressor T-cells. Cancer Res 49: 164954.
  • 18
    Liyanage U.K., Moore T.T., Joo H.G., Tanaka Y., Herrmann V., Doherty G., Drebin J.A., Strasberg S.M., Eberlein T.J., Goedegebuure P.S., Linehan D.C. (2002) Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 169: 275661.
  • 19
    Somasundaram R., Jacob L., Swoboda R., Caputo L., Song H., Basak S., Monos D., Peritt D., Marincola F., Cai D., Birebent B., Bloome E., Kim J., Berencsi K., Mastorabgelo M., Herlyn D. (2002) Inhibition of cytolytic T lymphocyte proliferation by Autologous CD4+/CD25+ regulatory T cells in a colorectal carcinoma patients is mediated by transforming growth factor-β. Cancer Res 62: 526772.
  • 20
    Gray C.P., Arosio P., Hersey P. (2003) Association of increased levels of heavy-chain ferritin with increased CD4+CD25+ regulatory T-cell levels in patients with melanoma. Clin Cancer Res 9(7): 25519.
  • 21
    Hiraoka N., Onozato K., Kosuge T., Hirohashi S. (2006) Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions. Clin Cancer Res 12(18): 542334.
  • 22
    Ichihara F., Kono K., Takahashi A., Kawaida H., Sugai H., Fujii H. (2003) Increased populations of regulatory T cells in peripheral blood and tumor-infiltrating lymphocytes in patients with gastric and esophageal cancers. Clin Cancer Res 9(12): 44048.
  • 23
    Ling K.N., Pratap S.E., Bates G.J., Singh B., Mortensen N.J., George B.D., Warren B.F., Piris J., Roncador G., Fox S.B., Banham A.H., Cerundolo V. (2007) Increased frequency of regulatory T cells in peripheral blood and tumor infiltrating lymphocytes in colorectal cancer patients. Cancer Immun 7: 7.
  • 24
    Miller A.M., Lundberg K., Ozenci V., Banham A.H. Hellstrom M., Egevad L., Pisa P. (2006) CD4+CD25high T cells are enriched in the tumor and peripheral blood of prostate cancer patients. J Immunol 177(10): 7398405.
  • 25
    Sasada T., Kimura M., Yoshida Y., Kanai M., Takabayashi A. (2003) CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies. CANCER 98(5): 108999.
  • 26
    Chikamatsu K., Sakakura K., Whiteside T.L., Furuva N. (2007) Relationships between regulatory T cells and CD8+ effector populations in patients with squamous cell carcinoma of the head and neck. Head Neck 29(2): 1207.
  • 27
    Oken M.M., Creech R.H., Tormey D.C., Horton J., Davis T.E., McFadden E.T., Carbone P.P. (1982) Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 5(6): 64955.
  • 28
    Horiuchi Y., Nakajima Y., Nariai Y., Asanuma H., Kuwabara K., Yukawa M. (2007) Th1/Th2 balance in canine peripheral blood lymphocytes - a flow cytometric study. Vet Immunol Immunopathol 118(3–4): 17985.
  • 29
    Campbell M.J., Scott J., Maecker H.T., Park J.W., Esserman L.J. (2005) Immune dysfunction and micrometastases in women with breast cancer. Breast Cancer Res Treat 91: 163371.
  • 30
    Ikemoto T., Yamaguchi T., Morine Y., Imura S., Soejima Y., Fujii M., Maekawa Y., Yasutomo K., Shimada M. (2006) Clinical roles of increased populations of Foxp3+CD4+ T Cells in peripheral blood from advanced pancreatic cancer patients. Pancreas 33(4): 38690.
  • 31
    Beyer M., Schultze J.L. (2006) Regulatory T cells in cancer. Blood 108(3): 80411.
  • 32
    Onizuka S., Tawara I., Shimizu J., Sakaguchi S., Fujita T., Nakayama E. (1999) Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor α) monoclonal antibody. Cancer Res. 59: 312833.
  • 33
    Fujimoto S., Greene M., Sehon A.H. (1975) Immunosuppressor T cells in tumor bearing host. Immunol Commun 4: 20117.