Med Oncol (2010) 27:29–33

DOI 10.1007/s12032-008-9165-9

ORIGINAL PAPER

CD8+CD282 cells and CD4+CD25+ regulatory T cells

in the peripheral blood of advanced stage lung cancer patients
Bülent Karagöz Æ Oğuz Bilgi Æ Mahmut Gümüs Æ
Alev Akyol Erikçi Æ Özkan Sayan Æ Orhan Türken Æ
Emin Gökhan Kandemir Æ Ahmet Öztürk Æ Mustafa Yaylacı

Received: 18 November 2008 / Accepted: 30 December 2008 / Published online: 16 January 2009
Ó Humana Press Inc. 2009

Abstract Aim To evaluate the CD8?CD28- and CD4? hematological parameters and the distribution of lympho-
CD25? regulatory T (Treg) cells in addition to other some cyte subgroups. In NSCLC patients, the percentage of
lymphocyte subgroups in peripheral blood of advanced CD8? CD28- cells among the lymphocyte population was
stage lung cancer patients. Methods The study group higher in patients with stage IV than those with stage III.
(n = 28) comprised chemotherapy and radiotherapy naı̈ve Conclusion These findings may reflect the possibility of
patients with non-small cell lung cancer (NSCLC) and tumor-induced immunosuppression and they should be
small cell lung cancer (SCLC). The control group (n = 22) complemented with further studies.
consisted of age- and sex-matched healthy volunteers. Flow
cytometry was used to count T cells, natural killer (NK) Keywords Treg
Lung cancer
Immunosuppression

cells and CD4?CD25 Treg cells, and for CD8? T cell CD8?CD28- cells
CD4?CD25? cells
subgroup analysis. Flow cytometry was performed and
annexin V binding was used for apoptotic cell evaluation.
Results In patient group, the percentage of CD8?CD28- Introduction
cells among lymphocytes was elevated, and there was also
an increase in the CD28-/CD28? cell ratio among CD8 Several T lymphocyte subtypes with regulatory functions
lymphocyte population. The distribution of CD8 cells was are described: T cells that secrete interleukin-10, cells that
different in lung cancer patients when compared with the secrete transforming growth factor-b (TGF-b), CD4 and
control group. The absolute count of CD4?CD25bright cells CD8 double negative T cells, natural killer T (NKT) cells,
and the percentages of these cells among total lymphocytes CD8?CD28- cells, and CD4?CD25? regulatory T (Treg)
were higher in the patient group. The Annexin V(?) cell cells [1–3].
percentages among CD8?CD28- and CD8?CD28? CD4?CD25? Treg cells are the most studied cells that
lymphocytes were higher in the patient group than in the have regulatory functions in tumor immunology. They
control group. No differences were found between the constitute 5–10% of peripheral blood CD4? lymphocytes
NSCLC and SCLC patients with respect to the [3–6]. CD4?CD25? Treg cells express CD25 (the alpha
chain of the IL-2 receptor), cytotoxic T lymphocyte-asso-
B. Karagöz (&)
O. Bilgi
O. Türken
E. G. Kandemir ciated antigen-4 (CTLA-4), a glucocorticoid induced TNF-
Department of Medical Oncology, GATA Haydarpaşa Training a receptor (GITR), and a novel member of the forkhead
Hospital, Istanbul, Turkey transcription factors (Foxp3) [3]. Studies presented that
e-mail: bulentkaragoz1968@yahoo.com
these cells played a role in allograft rejection, graft versus
M. Gümüs
M. Yaylacı host disease, autoimmune diseases [7–9], and suppressed
Department of Medical Oncology, Kartal Lütfi Kırdar Hospital, the antitumor immune response [10]. The increase of these
Istanbul, Turkey cells in number and activity has been reported in patients
with cancer [11–15]. Like CD4?CD25? Treg cells, it has
A. A. Erikçi
Ö. Sayan
A. Öztürk
Department of Hematology, GATA Haydarpaşa Training been recently shown that CD8?CD28- cells have also a
Hospital, Istanbul, Turkey suppressive effect [16, 17]. It has been reported that these
30 Med Oncol (2010) 27:29–33

cells suppressed the cytotoxic function of T lymphocytes, centrifugation. Histopaque was added to centrifuge tubes.
and inhibited the T lymphocyte proliferation [16, 17]. On The tube was centrifugated at 400g for exactly 30 min at
the other hand, these cells have not been studied in many room temperature. The mononuclear cell band was care-
cancer patients. Therefore, in this study, we have evaluated fully transferred into a conical centrifuge tube. Phosphate
the absolute number and percentage of CD8?CD28- cells buffer saline (PBS) was added. In total, 100 ll aliquots were
in addition to other T lymphocyte subgroups in peripheral transferred to polypropylene test tubes (12 9 75 mm; BD
blood of advanced stage lung cancer patients. Bioscience Catalog No: 352052) and 20 ll of congugated
monoclonal antibodies or isotype controls was added to
each tube. The PBS with paraformaldehyde was added into
Materials and methods tubes after the appropriate staining protocol. Flow cyto-
metric analysis was performed by BD FACS Calibur.
Patients
Annexin staining
The study group (n = 28) comprised chemotherapy and
radiotherapy naı̈ve patients with non-small cell lung cancer The annexin V binding was used for apoptotic cell evalu-
(NSCLC), classified as stages III to IV, and advanced stage ation in patients and 12 control subjects. Before annexin
small cell lung cancer (SCLC). The control group (n = 22) staining, a surface staining protocol was used for anti-CD28
consisted of age- and sex-matched healthy volunteers. Sub- FITC and anti-CD8 PC5. Next, 1 ml of annexin buffer
jects with autoimmune diseases (e.g. rheumatoid arthritis, solution (BD Pharmingen Catalog No: 556454) and 5 ll PE
systemic lupus erythematosus), chronic infections (e.g. conjugated Annexin V were added to the test tubes. The
human immunodeficiency virus infection, tuberculosis), tubes were stored for 15 min in darkness at room temper-
bone marrow involvement, anticoagulant and antithrombotic ature prior to being analyzed.
drug using, or those who had received immunosuppressive
treatment were excluded. All of the patients and control Analysis
subjects have smoked for least 5 years.
Triple staining with anti-CD28 FITC, anti-CD45RO PE, and
Flow cytometry anti-CD8 PC5 was used for CD8 T cell subtyping. A CD8/
CD28 two-dimensional histogram was used on the lym-
Peripheral blood samples were obtained and studied while phocyte population. CD8bright cells were gated. In CD8bright
still fresh (in lower than 30 min). Flow cytometry was used to lymphocyte gated CD28/CD45RO histogram, CD8bright
count T cells, natural killer (NK) cells and CD4?CD25 Treg CD28- cells, CD8brightCD28?CD45RO? cells, and
cells, and for CD8? T cell subgroup analysis. Flow cytom- CD8brightCD28?CD45RO- cells were counted (Fig. 1).
etry was performed on a Becton Dickinson FACSCalibur. CD8 T cell subtype apoptosis levels were analyzed in a
Data were obtained and analyzed using CellQuest software. CD8-PC5/CD28-FITC/Annexin-PE test tube. Annexin V
positive cell percentages were determined using an annexin
Monoclonal antibodies V (FL-2) histogram on both CD8brightCD28- and
CD8brightCD28? gated cells.
Anti-human monoclonal antibodies conjugated with flu- CD4?CD25? Treg cells were studied with double
orochromes and appropriate isotype controls were used: staining of anti-CD4 FITC/anti-CD25 PC5. CD4?CD25high
Fluorescein isothiocyanate (FITC) conjugated anti-CD56 cells were calculated as CD4?CD25? Treg cells.
(Santa Cruz Catalog No: sc-7326), anti-CD4 (Caltag Lab
Catalog No: MHCD0401), anti-CD28 (BD Pharmingen Statistics
Catalog No: 555728), phycoerythrin-cyanine 5 (PC5)
conjugated anti-CD3 (eBioscience Catalog No: 15-0038), Statistical analysis was performed by using Mann–Whitney
anti-CD25 (BD Pharmingen Catalog No: 555433), anti- U and Fischer exact test in SPSS software.
CD8 (eBioscience Catalog No: 15-0088), phycoerythrin
(PE) anti-CD45RO (BD Pharmingen Catalog No: 555493),
and annexin V (BD Pharmingen Catalog No: 556422). Results

Cell preparation and surface staining Twenty-eight lung cancer patients (19 male, 9 female)
were enrolled in this study (Patients Group). Twenty-three
Human peripheral blood mononuclear cells were isolated were NSCLC patients (15 with stage III and 8 with stage
using Histopaque (Sigma Catalog No: 1077) gradient IV). Five patients had extensive stage SCLC. Twenty-two
Med Oncol (2010) 27:29–33 31

Fig. 1 CD8? lymphocyte

subtypes in patient and control
group. The lymphocytes (in FS/
SS histogram) and CD8bright
cells (in lymphocytes) were
gated. CD8? lymphocyte
subtypes were calculated in
CD8? lymphocyte gated CD28/
CD45RO histogram

healthy volunteers (14 male, 8 female) were included as Flow cytometry indicated significant differences between
Control Group. Patient and control groups were matched the groups with respect to the distribution of CD8? T cell
for age (median 62, range 45–78 vs. median 58.5, range subgroups. CD8?CD28?CD45RO- cells were regarded as
36–68, respectively). CD8? naı̈ve T cells. These cells were reduced in patients. In
Total white blood cell (WBC) counts in patients with the patient group, the percentage of CD8brightCD28- cells
lung cancer were higher than in healthy controls (Table 1). among lymphocytes was elevated, and there was also an
The other hematological parameters were not different increase in the CD28-/CD28? cell ratio among CD8bright
between the groups. lymphocyte population. The CD8? memory T cells
The CD3?, CD4? lymphocytes, and NK cells were not (CD8?CD28?CD45RO? cells) did not differ between the
different, whereas CD8? lymphocytes were higher in the patient and control groups (Table 2, Fig. 1).
patient group compared with the control group (Table 1). The percentage of CD4?CD25bright (Treg) cells among
total lymphocytes and CD4? lymphocytes was higher in
the patient group when compared with the control group
Table 1 NK cells and T lymphocytes (Table 2, Fig. 2).
Patients Healthy Pa
The Annexin V(?) cell percentages among
group controls CD8brightCD28- and CD8brightCD28? lymphocytes were
higher in the patient group than in the control group
WBC (9 103/mm3) 9.71 ± 3.40 7.61 ± 1.57 0.0429 (Table 2).
Total lymphocytes in WBC 26.88 ± 11.23 30.54 ± 6.46 0.2145 No differences were found between the NSCLC and
CD3? in lymphocytes (%) 72.14 ± 9.19 75.26 ± 8.23 0.1400 SCLC patients with respect to the hematological parameters
NK cells in lymphocytes 7.01 ± 6.17 7.53 ± 3.16 0.0819 and the distribution of lymphocyte subgroups (Table 3). In
(%)
NSCLC patients, the percentage of CD8brightCD28- cells
CD4? in lymphocytes (%) 38.59 ± 9.82 34.86 ± 19.24 0.9688
among the lymphocyte population was higher in patients
CD8? in lymphocytes (%) 31.73 ± 6.39 26.42 ± 6.72 0.0014
with stage IV than those with stage III, but other parameters
WBC white blood cells of stage IV patients were similar with stage III patients
a
Mann–Whitney test (Table 4).

Table 2 CD4?CD25? Treg

Patients group Healthy controls Pa
cells and CD8? lymphocyte
subtypes CD4?CD25bright in lymphocytes (%) 4.45 ± 2.39 1.89 ± 1.42 0.0003
CD4?CD25bright in CD4? lymphocytes (%) 11.79 ± 6.3 4.60 ± 2.66 \0.0001
CD8brightCD28- in lymphocytes (%) 23.16 ± 4.93 14.19 ± 7.02 \0.0001
bright bright
CD8 CD28-/CD8 CD28? ratio 4.25 ± 4.07 1.38 ± 0.84 \0.0001
Memory cells in CD8? lymphocytes (%) 25.33 ± 15.9 29.46 ± 10.80 0.2912
Naive cells in CD8? lymphocytes (%) 10.63 ± 7.32 36.82 ± 15.42 \0.0001
a Annexin? cells in CD8brightCD28- lymphocytes (%) 46.81 ± 25.01 23.43 ± 10.49b 0.0026
Mann–Whitney test bright
b Annexin? cells in CD8 CD28? lymphocytes (%) 47.05 ± 28.73 27.11 ± 16.56b 0.0361
n = 12
32 Med Oncol (2010) 27:29–33

like CD4?CD25? Treg cells, inhibit T lymphocyte prolif-

eration and cytotoxicity [18]. CD8?CD28- T lymphocytes
show immunosuppressive effects by secreting IL-10, TGF-
b, or IFN-c25. In our study, elevated Annexin V binding
suggested that CD8 cell turnover was augmented and that the
cellular activation induced apoptosis (activation induced cell
death (AICD)). Therefore, we imply that high Annexin
binding and higher CD8?CD28- cell number (along with
increased CD4?CD25? Treg cells) might cause tumor-
induced immunosuppression.
Fig. 2 CD4 and CD8 regulatory cells in lung cancer patients and Tsukishiro et al. [19] investigated CD8 T cells in cir-
controls
culation of patients with head and neck cancer. They have
shown that among CD8? T cells, the percentage and
Discussion Annexin-binding proportion of CD28- cells were higher.
Our results are similar to their findings; however, while
In this study, we aimed to investigate whether CD8?CD28-, they have found that the percentage of Annexin binding of
CD4?CD25? Treg cells and other some lymphocyte sub- CD8?CD28?CD45RO- cells was decreased, we have
types were different between lung cancer patients and shown that both Annexin?CD8?CD28? cells and
controls. We found that CD8?CD28- and CD4?CD25? Annexin?CD8?CD28- cells were increased.
Treg cells were higher in patients when compared with Meloni et al. [20] studied CD4?CD25? and
healthy subjects. The percentage of Annexin V positivity was CD8?CD28- regulatory cells in peripheral blood of
also higher in both CD8?CD28- and CD8?CD28? cells in patients with pleural mesothelioma and lung cancer. A
patient group. significant increase in CD4?CD25? and CD8?CD28-
There are a few limitations of this study. FoxP3 intra- cells has been observed in patients with respect to healthy
cellular staining was not performed for determination of subjects. Our findings are in accordance with their study and
CD4?CD25? Treg cells. The number of patients enrolled furthermore we have additionally evaluated the Annexin
were small and the suppressive effects of CD4 and CD8 binding of CD8?CD28- cells. Our patient population
regulatory cells were not demonstrated in vitro. contained advanced stage lung cancer patients. The per-
CD8?CD28- T cells, known suppressive CD8 T cells, centages of CD4?CD25? and CD8?CD28- regulatory
have been shown to exist in tumor tissues [18]. These cells, cells were not different either between the patients with

Table 3 Lymphocyte subtypes

NSCLC patients SCLC patients Pa
in lung cancer patients
CD3? in lymphocytes (%) 73.09 ± 9.57 67.78 ± 6.05 0.1732
NK cells in lymphocytes (%) 7.3 ± 6.28 5.68 ± 6.15 0.2636
CD8? in lymphocytes (%) 31.85 ± 6.91 31.15 ± 3.48 0.6838
CD4? in lymphocytes (%) 38.97 ± 9.72 36.85 ± 11.23 0.9075
CD4?CD25bright in lymphocytes (%) 4.23 ± 2.24 5.5 ± 3.07 0.6414
NSCLC non-small cell lung bright
CD8 CD28- in lymphocytes (%) 22.8 ± 5.03 24.78 ± 4.53 0.4128
cancer, SCLC small cell lung Memory cells in CD8? lymphocytes (%) 27.91 ± 15.77 13.46 ± 11.09 0.0615
cancer
a Naive cells in CD8? lymphocytes (%) 10.52 ± 6.93 11.12 ± 9.84 0.9537
Mann–Whitney test

Table 4 Lymphocyte subtypes

Stage III Stage IV Pa
in non-small cell lung cancer
patients CD3? in lymphocytes (%) 72.04 ± 11.15 75.06 ± 5.72 0.591
NK cells in lymphocytes (%) 6.86 ± 7.13 8.11 ± 4.59 0.2132
CD8? in lymphocytes (%) 30.51 ± 7.19 34.36 ± 5.97 0.3246
CD4? in lymphocytes (%) 39.45 ± 9.63 38.07 ± 10.5 0.6813
CD4?CD25bright in lymphocytes (%) 4.05 ± 2.29 4.56 ± 2.24 0.6813
CD8brightCD28- in lymphocytes (%) 21.18 ± 4.47 25.85 ± 4.83 0.0337
Memory cells in CD8? lymphocytes (%) 27.09 ± 16.09 29.45 ± 16.12 0.6813
a Naive cells in CD8? lymphocytes (%) 10.2 ± 6.45 11.12 ± 8.21 0.9748
Mann–Whitney test
Med Oncol (2010) 27:29–33 33

NSCLC and SCLC. In NSCLC patients, the percentage of 10. Terabe M, Berzofsky JA. Immunoregulatory T cells in tumor
CD8?CD28- cells among the lymphocyte population was immunity. Curr Opin Immunol. 2004;16:157–62. doi:10.1016/
j.coi.2004.01.010.
higher in patients with stage IV than those with stage III. 11. Wei WZ, Morris GP, Kong YC. Anti-tumor immunity and auto-
In conclusion, CD8?CD28- cells, as well as CD4? immunity: a balancing act of regulatory T cells. Cancer Immunol
CD25? Treg cells, are higher in lung cancer patients, and the Immunother. 2004;53:73–8. doi:10.1007/s00262-003-0444-1.
distribution of CD8 T cell subgroups is different from heal- 12. Kono K, Kawaida H, Takahashi A, Sugai H, Mimura K, Miya-
gawa N, et al. CD4(?)CD25high regulatory T cells increase with
thy subjects. Ongoing research will lead to our increasing tumor stage in patients with gastric and esophageal cancers.
knowledge about regulatory cells in cancer immunology. Cancer Immunol Immunother. 2006;55:1064–71. doi:10.1007/
s00262-005-0092-8.
13. Chikamatsu K, Sakakura K, Whiteside TL, Furuya N. Relation-
ships between regulatory T cells and CD8? effector populations
in patients with squamous cell carcinoma of the head and neck.
References Head Neck. 2007;29:120–7. doi:10.1002/hed.20490.
14. Lau KM, Cheng SH, Lo KW, Lee SA, Woo JK, van Hasselt CA,
1. Sakaguchi S. Regulatory T cells: key controllers of immunologic et al. Increase in circulating Foxp3?CD4?CD25(high) regula-
self-tolerance. Cell. 2000;101:455–8. doi:10.1016/S0092-8674 tory T cells in nasopharyngeal carcinoma patients. Br J Cancer.
(00)80856-9. 2007;96:617–22. doi:10.1038/sj.bjc.6603580.
2. Yi H, Zhen Y, Jiang L, Zheng J, Zhao Y. The phenotypic char- 15. Peng QQ, Li SP, Xu L, Li JQ. Clinical significance of the pro-
acterization of naturally occurring regulatory CD4?CD25? T portion of CD4?CD25? regulatory T cells in peripheral blood of
cells. Cell Mol Immunol. 2006;3:189–95. hepatocellular carcinoma patients: a report of 117 cases. Ai
3. Zhang L, Zhao Y. The regulation of Foxp3 expression in regu- Zheng. 2007;26:748–51.
latory CD4(?)CD25(?)T cells: multiple pathways on the road. 16. Najafian N, Chitnis T, Salama AD, Zhu B, Benou C, Yuan X,
J Cell Physiol. 2007;211:590–7. doi:10.1002/jcp.21001. et al. Regulatory functions of CD8?CD28- T cells in an auto-
4. Maggi E, Cosmi L, Liotta F, Romagnani P, Romagnani S, An- immune disease model. J Clin Invest. 2003;112:1037–48.
nunziato F. Thymic regulatory T cells. Autoimmun Rev. 2005;4: 17. Filaci G, Fravega M, Negrini S, Procopio F, Fenoglio D, Rizzi M,
579–86. doi:10.1016/j.autrev.2005.04.010. et al. Nonantigen specific CD8? T suppressor lymphocytes
5. Sakaguchi S. The origin of FOXP3-expressing CD4? regulatory originate from CD8?CD28- T cells and inhibit both T-cell
T cells: thymus or periphery. J Clin Invest. 2003;112:1310–2. proliferation and CTL function. Hum Immunol. 2004;65:142–56.
6. Taams LS, Akbar AN. Peripheral generation and function of doi:10.1016/j.humimm.2003.12.001.
CD4?CD25? regulatory T cells. Curr Top Microbiol Immunol. 18. Filaci G, Fenoglio D, Fravega M, Ansaldo G, Borgonovo G,
2005;293:115–31. doi:10.1007/3-540-27702-1_6. Traverso P, et al. CD8? CD28- T regulatory lymphocytes
7. Valencia X, Yarboro C, Illei G, Lipsky PE. Deficient CD4? inhibiting T cell proliferative and cytotoxic functions infiltrate
CD25high T regulatory cell function in patients with active human cancers. J Immunol. 2007;179:4323–34.
systemic lupus erythematosus. J Immunol. 2007;178:2579–88. 19. Tsukishiro T, Donnenberg AD, Whiteside TL. Rapid turnover of
8. Cohen JL, Trenado A, Vasey D, Klatzmann D, Salomon BL. the CD8(?)CD28(-) T-cell subset of effector cells in the cir-
CD4(?)CD25(?) immunoregulatory T cells: new therapeutics culation of patients with head and neck cancer. Cancer Immunol
for graft-versus-host disease. J Exp Med. 2002;196:401–6. doi: Immunother. 2003;52:599–607. doi:10.1007/s00262-003-0395-6.
10.1084/jem.20020090. 20. Meloni F, Morosini M, Solari N, Passadore I, Nascimbene C,
9. Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S. Novo M, et al. Foxp3 expressing CD4? CD25? and CD8?
Donor-type CD4(?)CD25(?) regulatory T cells suppress lethal CD28- T regulatory cells in the peripheral blood of patients with
acute graft-versus-host disease after allogeneic bone marrow lung cancer and pleural mesothelioma. Hum Immunol. 2006;67:
transplantation. J Exp Med. 2002;196:389–99. doi:10.1084/jem. 1–12. doi:10.1016/j.humimm.2005.11.005.
20020399.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.