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Flow Cytometric. Detection of Intracellular Cytokines andChemokines in Acute T Lymphoblastic Leukemia Cells,I

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]ifeng Yul, Liching Zhan~, Hui Sun3, Ling Sun3, Qiutang Zhang3

1 . ED Eiosciences Pharmingen, San Diego, CA 92121, USA

2. General Atomics-Diazyme, San Diego, CA, USA

3 . The First Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, 450052 China

Abstract: Intracellular cytokines/chemokines IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-16,IFN-)', TNF-a, GM-CSF, MCP-1, MCP-3, MIP-1a and RANTES in T lymphocytes from normal donors (n =8)and patients with primary acute T lymphoblastic leukemia (T-ALL) (n = 35) was detected followinga 6-hr stimu-lation with PMA and calcium ionophore A23187. Significantly decreased percentages of cytokine/chemokine posi-tive cells in T-ALL leukemic blasts were observed for IL-2, IL-16, IFN-a, TNF-)' and GM-CSF. In contrast, sig-nificantly increased percentage of cytokines/chemokines positive cells in T-ALL leukemic blasts was observed for IL-4, IL-8, IL-12 and MIP-1a. No significant differences were observed between TALL leukemic blasts and normalT lymphocytes for IL-1a, IL-3, IL-5, IL-6, IL-10, MCP-l, MCP-3 and RANTES. Also, the T-ALL patientscan produce some cytokines/chemokines positive cells that normal donors usually do not produce under the PMAstimulation, such as IL-12 and MIP-1a. In 23 cases of T-ALL patients with the leukemic blast phonotyping of

CD3- CD7+ , a minor populationof CD3+ CD7+ T cells was alsoobserved in 16 patients. Comparedwith T lym-phocytes from normal donors, the CD3 + CD7+ cell populations in these T-ALL patients revealed significant higherpercentages of IL-4, IL-8, IL-12 and MIP-la cytokines/chemokines positive cells as well as significant lower per-centagesof IL-2, IL-16, IFN-a, TNF-)' and GM-CSF cytokines/chemokines positive cells. Compared with theCD3-CD7+ leukemic blasts from these patients, the CD3+ CD7+ cell populations revealed significant higher per-centages of IL-2, lL-4 and IL-12, IL-16 and IFN-a cytokines positive cells. No significant differences of IL-1a,IL-3, IL-5, IL-6, IL-8, IL-10, TNF-)', GM-CSF, MCP-1, MCP-3, MIP-1a and RANTES positive percentages

are found between the CDr CD7+ and CD3+ CD7+ cell populations. Detection of cytokineslchemokines inleukemic blasts may prove useful for predicting and monitoring response to therapies in which cytokines could beused as potential immunomodulators or therapeutic targets. [Life Science Journal. 2006; 3 (1) : 29 - 34] (ISSN:1097 - 8135).

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Keywords: acute T lymphoblastic leu~emia; cytokinel chemokine; intracellular staining; flow cytometry

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~1 Introduction

T-cell acute lymphoblastic leukemia (T-ALL)is a malignant disease resulting from the clonal pro-liferation of T lymphoid precursors. It accounts forabout 15% of all ALL cases in children and 20 %-25% in adults (Rivera, 1995; Uckun, 1998). T-

ALL is thought to originate inside the thymus andleukemic cells express phenotypic features corre-sponding to distinct maturational stages of thymo-cyte development: early (stage I), intermediate(stage II), or late (stage Ill) (Heerema, 1998;Foa, 1986). Because leukemia cells may retain cer-tain features of their normal counterparts, theircharacterization with respect to cytokine respon-siveness can provide valuable information about thedifferentiation state of malignant cells and their de-

pendence on the microenvironment. Cytokinesl

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chemokines and its roles in the T-ALL leukemic

cells have been studied by many researchers(Dibirdik, 1991; Masuda, 1991; Karawajew,2000; Scupoli, 2003). It showed the effects of IL-7, IL-4, and IL-2 regarding the induction of prolif-eration in childhood T-lineage acute lymphoblasticleukemia (T-ALL), and their potential as growthfactors has been pointed out (Dibirdik, 1991; Ma-suda, 1991). Inhibition of in vitro spontaneousapoptosis by IL-7 correlates with Bcl-2 up-regula-tion, cortical/mature immunophenotype, and bet-ter early cytoreduction of childhood T-cell acutelymphoblastic leukemia has been observed( Karawaj ew, 2000; Scupoli, 2003). However,many cytokines/chemokines have not been studiedwith T-ALL leukemic cells. Therefore, in the cur-

rent study we investigated 17 different cytokineslchemokines intracellular expression profiles inleukemic blasts as well as in the non-leukemic cells

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Life Science Journal, 3 (1) ,2006, Yu, et al, Flow Cytornetric Detection of Intracellular Cytokines and Chernokines

from 35 patients with T-ALL. Our data demon-strated that many different cytokines/chemokinescould be detected in leukemic blasts with intracellu-

lar staining by flow cytometry after in vitro stimu-lation. Compared with T lymphocytes from normaldonors, a significant decrease of cytokine/chemokine production in T-ALL was observed forIL-2, IL-16, IFN-a, TNF-y and GM-CSF. Incontrast, a significant increase of cytokines/chemokines production in T-ALL was observed forMIP-1a and IL-8. Furthermore, independent anal-ysis of leukemic blasts and non-leukemic cells of T-ALL patients revealed differences in cytokines/chemokines production.

2 Materials and Methods

.

2. 1 Patients and normal donorsAll of the 35 patients with T-ALL fulfilled the

French-American-British (FAB) CooperativeGroup criteria (Bennett, 1981). Age range of pa-tients was 14 - 52 years with 23 males and 12 fe-males. Eight normal donors with age range of 20-40 years were included in this study as the normalcontrols.2 .2 Cells preparation

Peripheral blood samples were collected, afterinformed consent, from 35 adult patients withnewly diagnosed T-ALL. Immunophenotype analy-sis of T-ALL blasts was performed by direct im-munofluorescence and flow cytometry with a FAC-Scan instrument(Becton-Dickinson, San Jose, CA,USA) . Peripheral blood mononuclear cells(PBMC) were isolated with Ficoll-Hypaque. Be-fore use, the cells' viability consistently exceeded90% in each sample, as assessed by propidium-io-dide (PI) dye exclusion. Then PBMC was stimu-lated with PMA (phorbol 12-myristate 13-acetate,Sigma) 50 ng/ml and Calcium Ionophore A23187(Sigma) 1f-1g/ml in the presence of BD GoI-giStopTMprotein transport inhibitor 2 mM at 37'C,7% CO:?for 6 hours. Stimulated cells were stainedsurface with CD3 or CD3 and CD7, combining in-tracellular with anti-cytokine/chemokine monoclon-al antibodies(See Table 1). Data was acquired on aFACScan instrument (Becton-Dickinson, San Jose,CA, USA) and analyzed with CELLQUEST soft-ware.

Values were expressed as mean i SD. Differ-ences (P values) were evaluated using the 2-tailedStudent's t-test. Differences were considered sta-tistically significant for P < 0.05.

Table 1 Anti-cytokine/chemokine monoclonal antibodies

Table 2. Comparison of cytokine prqducing cells betweennonnal donor T cells and T-ALL leukemic blasts.- ----

Nonnal T lympho T-ALL Blasts P V I(N=8) (N=35) aue

0.HO.1 0.3:t0.252.8:t7.4 15.5:t13.30.5:t0.3 0.HO.23.2:t1.3 5.8:t5.30.4:t0.3 0.4:t0.20.5:t0.1 0.8:t0.79.7:t 2. 6 47. 8 :t 33. 50.3:t0.1 0.4:t0.20.2:t0.1 2.0:t2.4

91.9:t3.8 30.9:t16.019.8 :t 7. 1 8.2:t 6. 133. 2 :t 7. 5 10.4 :t 10. 412. 3 :t 4.0 5. 2 :t 4. 50.HO.1 0.3:t0.20.5:t0.3 0.4:t0.24.3:t2.1 28.2:t23.40.3:t0.1 0.HO.2

IL-1aIL-2IL-3IL-4IL-5IL-6IL-8IL-1OIL-12IL-16IFN-YTNF-a

GM-CSFMCP-1MCP-3MIP-1a

RANTES

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>0.05<0:001>0.05>0.05>0.05>0.05<0.05>0.05<0.05

<0.001< 0.001< 0.001<0.01>0.05>0.05<0.05>0.05

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L,3 Results

t-r,In the T-ALL patients, two different kinds of

immunophenotyping of the leukemic blasts wereobserved in this study. Among these 35 T-ALL pa-tient circulating leukemic blasts, 23 patients' blasts

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used in this studyrnAb Clone name Fonnat

Il-1a 364-3B3-14 PE*

IL-2 MQl-17H12 PE

IL-3 BVD3-1F9 PE

IL-4 MP4-25D2 PE

IL-5 JESl-39D10 PE

IL-6 MQ2-13A5 PE

IL-8 G265-8 PE

IL-1O JES3-9D7 PE

IL-12 Cll.5 PE

IL-16 14.1 PE

IFN-y 4S.B3 PE

TNF-a MAbll PE

GM-CSF BVD2-21Cll PE

MCP-1 5m-F7 PE

MCP-3 9Hll PE

MIP-1a llA3 PE

RANTES 2D5 PE

cm HITIa PE-Cy5CD7 M-T701 FITC* *

* PE: Phycoerythrin. * * FITC: Flourescein isothio-

cyanate. # All monoclonal antibodies are from BD Bio-sciencesPhanningen.

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had the phenotypingof CD3- CD?+ , while 12 pa-tients' blasts had the phenotyping of CD3+ CD?+

(Figure 1). In the 23 patients' blasts with the

CDr CD? + phenotyping, a minor CD3+ CD?+ cell~

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population was observed in 16 patients (Figure 2).After staining with different fluorescence conjugat-ed antibodies, intracellular cytokines/chemokineswere analyzed in these different cell populations us-ing different flow cytometric gating strategies.

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panel showed the leukemic blasts with CD3+Cm+ gated on the lymphocytic ceUs population and analysed with CD3 PE-Cy5 (Y-axis)

and CD7 FITC (X-axis). The lower panel showed leukemic blasts with CD3 - CD7+ phenotyping.

Leukemic blasts produce many different cy-tokines/chemokines that can be detected with in-

tracellular staining by flow cytometry. By gatingon the leukemic blast populations, we analyzed dif-ferent cytokines/chemokines production. We foundthat leukemic blast could produce many differentcytokines/chemokines that could be detected withintracellular staining by flow cytometry, althoughthe variation was quite big between different pa-tients. Compared with normal donor T lympho-cytes, significant decreased percentages of cy-tokine/chemokine positive cells in T-ALL leukemicblasts were observed for IL-2, IL-16, IFN-a,

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TNF-y and GM-CSF. In contrast, significant in-creased percentage of cytokines/chemokines posi-tive cells in T-ALL leukemic blasts was observedfor IL-4, IL-8, IL-12 and MIP-1a. No significantdifferences were observed between T-ALL leukemicblasts and normal T lymphocytes for IL-1a, IL-3,IL-5, IL-6, IL-10, MCP-1, MCP-3andRANTES(Table 2). Also, the cytokines/chemokines pro-files from T-ALL patients were quite different fromnormal donors. Under the PMA stimulation condi-tion, all normal donors produced detectable amountof IL-2, IL-4, IL-16, IFN-y, TNF, GM-CSF andIL-8 positive cells, but no detectable IL-1a, IL-3,

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IL-5, IL-6, IL-lO, IL-12, MCP-1, MCP-3 andRANTES positive cells. However, the T-ALL pa-tients could produce some cytokinesl chemokinespositive cells that normal donors usually do not pro-duce under the PMA stimulation, such as IL-12and MIP-1a.

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served. Independent analysis of the CD3 + CD7 + and CDr em +

cell populations revealed different profiles in cytokines/chemokines

positive cell percentages. These are the typical histogranlS of dif-

ferent cytokines/chemokines from nun-leukemic cells (green) and

the leukemic blasts (pink).

~ In 23 cases of T-ALL patients with theleukemic blast phonotyping of CO3 - CO7+ , a mi-nor population of CO3+ CO7 + T cells was also ob-served in 16 patients. We considered this minorCD3+CD7+ population as the non-leukemic cells or

the normal T cell in these T-ALL patients. Inde-pendent analysis of this CO3+ CD?+ T cell popula-tion revealed differences in cytokines/chemokinespositive cell percentages when compared to eitherthe cor CD7+ leukemic blasts from the same pa-tient or T lymphocytes from normal donors. Com-pared with T lymphocytes from normal donors, theCO3+CO7+ cell populations in these T-ALL pa-tients revealed significantly higher percentages ofIL-4, IL-8, IL-12 and MIP-1a cytokineslchemokines positive cells as well as significantlylower percentages of IL-2, IL-16 , IFN-a, TNF-yand GM-CSF cytokines/chemokines positive cells(Table 3). Compared with the cor CO7+leukemic blasts from these patients, theCO3+ CO7+ cell populations revealed significantlyhigher percentages of IL-2, IL-4 and IL-12, IL-16and IFN-a cytokines positive cells. No significantdifferences of IL-1a, IL-3, IL-S, IL-6, IL-8, IL-10, TNF-y, GM-CSF, MCP-1, MCP-3, MIP-1aand RANTES positive percentages were found be-tween the cor CO7+ and CO3+CO7+ cell popu-lations (Table 4).

Table 3. Comparison of cytokine producing cells betweennormaldonorT cellsand non-leukemiccells(rn3 + CD7+ )from T-ALL patients with leukemic blasts of phenotypingcm-cm+ .

Nonna! T lympho(N=8)

0.3:t0.1

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0.5:1:0.3

3.211.3

0.410.3

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0.210.1

91.9:1:3.8

19.8:1:7.1

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4.3:t2.1

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4 Discussion

Cytokines/chemokines are very important fac-tors in human immuno-regulation. Cytokineslchemokines producing cells detection can provide

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critical infonnation for cell-cell interaction in hu-man body. There are different methods to detectcytokinesl chemokines producing cells, such asELISPOT, flow cytometry, immuno-radioactiveassay, etc. Intracellular cytokines/chemokinesstaining by flow cytometry combining with cell sur-face staining with different monoclonal antibodiesnot only can detect cytoklines/chemokines produc-ing cells, but also can provide further infonnationsuch as which of cell subpopulation can producewhat kind of cytokines/chemokines. It is knownthat different cell populations can produce differentcytokines. Detection of the cytokinesl chemokinesproducing cells would provide further infonnationfor understanding the function of cyteokineslchemokines producing cells in immunoregulation.

Table4. Comparisonof cytokine producing cells betweenleukemic blasts (CDr CD7+) and non-leukemic cells(CD3+ CD7+) from T-ALL patients with leukemic blastphenotyping CD3 - CD7 + .

T-ALLcm+cm+(n=16)

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9.7111.7

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T-ALL is a special type of leukemia involvingthe T-cell clonal expansion of leukemia cells. We u-tilized the well-established cytokines/chemokinesintracellular staining methods as well as thereagents from BD Biosciences to detect cytokineslchemokines producing cells in T-ALL leukemic pa"tients. The different leukemic blasts not only havedifferent morphology and immunophenotyping, butalso have different patterns of cytokine/chemokineproducing cells. Although the role of many differ-ent cytokinesl chemokines in the leukemia still re-

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mains mostly unknown, the detection of cytokinelchemokines producing cells both in the leukemicblasts and non-leukemic cells would provide impor-tant infonnation regarding the cytokineslchemokines production.

Our data demonstrated the abnonnal cy-tokines/chemokines production pattern in the T-ALL patients, compared with nonnal T lympho-cytes. T-ALL leukemic blasts have the significantlower producing ability for IL-2, IL-16, IFN-o:,TNF-y and GM-CSF. In contrast, T-ALLleukemic blasts have the significant lower producingability for IL-4, IL-8, IL-12 and MIP-10:. Inter-estingly, the T-ALL patients can produce some cy-tokines/chemokines positive cells that nonnaldonors usually do not produce under the PMA stim-ulation, such as IL-12 and MIP-10:. These abnor-malities of cytokine/chemokine production coulddue to the clonal expansion of the T-Iymphoid pre-cursors.

Combined with surface staining of CD3 andCD7 monoclonal antibodies, we further classifiedthe patients' lymphocytes in 16 patients, who havethe leukemic blasts with CD3- CD7+ phenotyping,into two different populations: leukemic blast andnon-leukemic cells. The cytokines/chemokines pro-ducing cells are different between these two differ-ent cell populations. Compared witr T lymphocytesfrom nonnal donors, the CD3+ CD7+ cell popula-tions in these T-ALL patients revealed significantlyhigher percentages of IL-4, IL-8, IL-12 and MIP-10: cytokines/chemokines positive cells as wel} assignificant lower percentages of IL-2, IL-16, IFN-0:, TNF-y and GM-CSF cytokines/chemokines pos-itive cells. Compared with the CDrCD7+leukemic blasts from these patients, the CD3 +

CD7+ cell populations revealed significantly higherpercentages of IL- 2 , IL-4 and IL-12, IL" 16 andIFN-o: cytokines positive cells. These cytokines in-volved both the Th1 and Th2 type cytokines thatcan be produced by different kinds of T cells. Theincrease of several different cytokines/chemokinesproducing cells among the non-leukemic cells in T-ALL patients could be the results of stimulation bythe leukemic blasts, or the immuno-regulation ofthe in vivo cell-cell, cell-cytokine interactions. De-tection of cytokinesl chemokines in leukemic blastsmay prove useful for predicting and monitoring re-sponse to therapies in which cytokines could be usedas potential immunomodulators or therapeutic tar-gets.

....

5 Conclusions

In this study, we demonstrated that:

. 33 .

J Haematol1981 ;47: 553 - 8.2. Dibirdik I, Langlie MC, Ledbetter JA, et al. Engage-

ment of interleukin-7 receptor stimulates tyrosine phos-phorylation, phosphoinositide turnover, and clonal prolif-eration of human T-lineage acute lymphoblastic leukemiacells. Blood 1991 ;78 :564 -70.

3. Faa R, Migone N, Gavosto F. Immunological andmolecular classification of acute Iympboblastic leukemia.Haematologica 1986; 71 : 277-85

4. Uckun FM, Sensel MG, Sun L, et al. Biology and treat-ment of childhood T-lineage acute lymphoblastic leukemi-a. Blood 1998;91:735-46.

5. Karawajew L, Ruppert V, Wuchter C, et al. Inhibitionof in vitro spontaneous apoptosis by IL-7 correlates withbcl-2 up-regulation, cortical/mature immunophenotype,and better early cytoreduction of childhood T-cell acutelymphoblasticleukemia. Blood2000 ;96(1) :297 - 306.

6. Masuda M, Takanashi M, Motoji T, et al. Effects of in-terleukins 1 - 7 on the proliferation of T-lineage acutelymphoblasticleukemiacells. Leuk Res 1991; 15: 1091-6.

7. Rivera GK, Crist WM. Acute lymphoblastic leukemia.In: Handin RI, Stossel TP, Lux SE, editors. Blood:Principles and Practice of Hematology. Philadelphia: JBLippincott Company. 1995;743-59.

8. Scupoli MT, Vinante F, Krampera M, et al. Thymic ep-ithelial cells promote survival of human T-cell acute lym-phoblastic leukemia blasts: the role of interleukin-7.Haematologica 2003 ; 88( 11): 1229 - 37.

9. Uckun FM, Sensei MG, Sun L,et al. Biology and treat-ment of childhood T-lineage acute lymphoblastic leukemi-a. Blood 1998;91:735-46.

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( 1) Intracellular cytokinesl chemokines can bedetected in the leukemic cells from T-ALL patientsby multiparameter flow cytometry analysis after 6-hour PMA stimulation.

(2) T-ALL leukemic blasts have abnormal cy-tokine/chemokine producing cell patterns, com-pared with normal T lymphocytes.

(3) Different levels of cytokines/chemokinesproductions were observed between the non-leukemic (CD3 + CD7+) and leukemic

(CD3 - CD7+) cell populations in T-ALL patientswith CDr CD7+ phenotyping of blasts.

AcknowledgmentsThe authors would like to thank Dr. Xifeng

Yang, and Dr. Zhang Chen for important input onthe preliminary study of this paper.

Correspondence to:Jifeng YuBD Biosciences PharmingenSan Diego,CA 92121, USATelephone: 858-812-3773(W)Email: jfy8168@yahoo.com

References1. Bennett JM, Catowsky D, Daniel MT, The French-

American-British (FAB) Cooperative Group. The mor-phologicalclassificationof acute lymphoblasticleukaemia:concordanceamong observers and clinical correlation. Br

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