role of ccr4 in regulang cell surface glycan expression on ......of the final poster. all text and...

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Glycans (oligosaccharides) are one of the four fundamental structures that make up all living systems. Cell surface glycans have been shown to play an important role in bacteria and viral recognition, cell signaling, and cancer development. In previous studies, Chemokine Receptor 4 (CCR4) has been shown to be key components to T cell recruitment and it has been demonstrated that changes in glycan structure can act as a regulator of migration (Faustino et al., 2013; Gu & Taniguchi, 2008). For this reason, we characterized T-lymphocyte cell surface glycan structures from two different cell lines with a flow-based lectin array after the engagement of chemokine receptor CCR4. By further characterizing the role of chemokine receptors on glycans in T cells, we believe we can better understand the pathogenesis of immune-related diseases.

ABSTRACT

OBJECTIVES

Cell Lines: -HuT 78 (TIB-161) from ATCC -MJ CRL-8294 from ATCC C-type plant lectins: binding for GalNac, Galactose, a-methylglucoside, and L-Fucose Flow Cytometer: BD LSR Fortessa

Data Analysis software: FlowJo 1)  Cell lines are cultured and treated with or

without TARC 2)  Stained with fluorescently labeled

antibodies CD4 (PE), CCR4 (PE-Cy7), and four different lectins (FITC)

3)  Stained cells are analyzed by flow cytometry to determine the lectin-binding profile of each T-cell population expressing CD4 and CCR4

4)  Fold change is calculated according to the below formula. MFI: Mean Flourescence Intensitiy, MFIR: Mean Fluorescence Intensity Ratio.

MATERIALSandMETHODS

RESULTS CONCLUSIONSCCR4 stimulated by TARC did not cause significant changes (1.06 – 1.1 fold change) in the CTL cell line whereas a slight decrease in L-Fucose (0.7 fold change) was found in the TIB cell line. The different response between two cell lines may be related to the expression levels of CCR4. Future directions include testing the assay with longer treatment of TARC, expanding the lectin panels to detect other types of glycan changes, and characterizing glycosylation gene expressions with targeted glycomic gene qPCR analysis.

Faustino, L., da Fonseca, D. M., Takenaka, M. C., Mirotti, L., Florsheim, E. B., Guereschi, M. G., … Russo, M. (2013). Regulatory T cells migrate to airways via CCR4 and attenuate the severity of airway allergic inflammation. Journal of Immunology (Baltimore, Md.  : 1950), 190(6), 2614–21. doi:10.4049/jimmunol.1202354 Gu, J., & Taniguchi, N. (2008). Potential of N-glycan in cell adhesion and migration as either a positive or negative regulator. Cell Adhesion & Migration, 2(4), 243–245. doi:10.4161/cam.2.4.6748

ACKNOWLEDGEMENTSThanks to Dr. Michiko Shimoda and Dr. Emanual Maverakis for their mentorship, advice, and support. Also, thanks to Dr. Guillaume Luxardi and Dr. Khiem Tran for their input on this project. Lastly, thanks to UC Davis School of Medicine for funding.

The overall hypothesis is that chemokine receptor-ligand interactions change the T cell surface glycans through regulating the expression of glycosylation enzymes, which ultimately impact the T cell migratory functions. Specifically, our aim is to characterize T-lymphocyte cell surface glycan structures with a flow-based lectin array before and after the engagement of chemokine receptor CCR4 using Thymus and Activation Regulated Chemokine (TARC).

CTL-FITC-LECTINBINDING

Lec/nforGalNac

MeanFluorescenceIntensityRa/o

Lec/nforGalactose


Lec/nforα-methylglucoside


Lec/nforL-Fucose


MFIofIsotypeControl

CTL - PE-Cy7-CCR4

Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 7880.84549356

2 8060.86480686

7 8170.87660944

2 8920.95708154

5 932CCL17(-)MFI 1399

17.98200514 1196

15.37275064 1227

15.77120823 1268

16.29820051 77.8

CCL17(+) MFI 6970.99856733

5 6700.95988538

7 6630.94985673

4 7091.01575931

2 698CCL17(+) MFI 1049

17.74957699 866

14.65313029 926

15.66835871 857

14.50084602 59.1

%orfoldchange:

-11.54822335

1.181046645

-16.87344913

1.109941911-18.8494492

1.083557498

-20.51569507

1.061309057

%orfoldchange:

-25.01786991

0.987074403

-27.59197324

0.953188576

-24.53137734

0.993478654

-32.41324921

0.889720679

TIB-FITC-LECTINBINDING

Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

TIB - PE-Cy7-CCR4

Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 838 1.309375 786 1.228125 781 1.2203125 871 1.3609375 640CCL17(-)MFI 1733

31.85661765 1438

26.43382353 1404

25.80882353 1293

23.76838235 54.4

CCL17(+) MFI 9341.21614583

3 7691.00130208

3 7550.98307291

7 7220.94010416

7 768CCL17(+) MFI 504

10.14084507 1364

27.44466801 1447

29.11468813 1408

28.32997988 49.7

%orfoldchange:

11.455847260.92879872

7-2.1628498

730.81530958

4-3.32906

53010.80559112

2-17.1067

73820.69077688

5%orfoldchange:

-70.91748413

0.318327739

-5.1460361611.03824057

3.0626780631.12809048

8.8940448571.19191872


Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CTL - PE-Cy7-CCR4

Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 7880.84549356

2 8060.86480686

7 8170.87660944

2 8920.95708154

5 932CCL17(-)MFI 1399

17.98200514 1196

15.37275064 1227

15.77120823 1268

16.29820051 77.8

CCL17(+) MFI 6970.99856733

5 6700.95988538

7 6630.94985673

4 7091.01575931

2 698CCL17(+) MFI 1049

17.74957699 866

14.65313029 926

15.66835871 857

14.50084602 59.1

%orfoldchange:

-11.54822335

1.181046645

-16.87344913

1.109941911-18.8494492

1.083557498

-20.51569507

1.061309057

%orfoldchange:

-25.01786991

0.987074403

-27.59197324

0.953188576

-24.53137734

0.993478654

-32.41324921

0.889720679


Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

TIB - PE-Cy7-CCR4

Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 838 1.309375 786 1.228125 781 1.2203125 871 1.3609375 640CCL17(-)MFI 1733

31.85661765 1438

26.43382353 1404

25.80882353 1293

23.76838235 54.4

CCL17(+) MFI 9341.21614583

3 7691.00130208

3 7550.98307291

7 7220.94010416

7 768CCL17(+) MFI 504

10.14084507 1364

27.44466801 1447

29.11468813 1408

28.32997988 49.7

%orfoldchange:

11.455847260.92879872

7-2.1628498

730.81530958

4-3.32906

53010.80559112

2-17.1067

73820.69077688

5%orfoldchange:

-70.91748413

0.318327739

-5.1460361611.03824057

3.0626780631.12809048

8.8940448571.19191872


Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CTL - PE-Cy7-CCR4

Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 7880.84549356

2 8060.86480686

7 8170.87660944

2 8920.95708154

5 932CCL17(-)MFI 1399

17.98200514 1196

15.37275064 1227

15.77120823 1268

16.29820051 77.8

CCL17(+) MFI 6970.99856733

5 6700.95988538

7 6630.94985673

4 7091.01575931

2 698CCL17(+) MFI 1049

17.74957699 866

14.65313029 926

15.66835871 857

14.50084602 59.1

%orfoldchange:

-11.54822335

1.181046645

-16.87344913

1.109941911-18.8494492

1.083557498

-20.51569507

1.061309057

%orfoldchange:

-25.01786991

0.987074403

-27.59197324

0.953188576

-24.53137734

0.993478654

-32.41324921

0.889720679


Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

TIB - PE-Cy7-CCR4

Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 838 1.309375 786 1.228125 781 1.2203125 871 1.3609375 640CCL17(-)MFI 1733

31.85661765 1438

26.43382353 1404

25.80882353 1293

23.76838235 54.4

CCL17(+) MFI 9341.21614583

3 7691.00130208

3 7550.98307291

7 7220.94010416

7 768CCL17(+) MFI 504

10.14084507 1364

27.44466801 1447

29.11468813 1408

28.32997988 49.7

%orfoldchange:

11.455847260.92879872

7-2.1628498

730.81530958

4-3.32906

53010.80559112

2-17.1067

73820.69077688

5%orfoldchange:

-70.91748413

0.318327739

-5.1460361611.03824057

3.0626780631.12809048

8.8940448571.19191872


Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 788 0.845493562 806 0.864806867 817 0.876609442 892 0.957081545 932

CCL17(+) MFI 697 0.998567335 670 0.959885387 663 0.949856734 709 1.015759312 698

%orfoldchange: -11.54822335 1.181046645-16.8734491

3 1.109941911 -18.8494492 1.083557498-20.5156950

7 1.061309057

CTL - PE-Cy7-CCR4 Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 1399 17.98200514 1196 15.37275064 1227 15.77120823 1268 16.29820051 77.8

CCL17(+) MFI 1049 17.74957699 866 14.65313029 926 15.66835871 857 14.50084602 59.1

%orfoldchange:

-25.01786991 0.987074403

-27.59197324 0.953188576

-24.53137734 0.993478654

-32.41324921 0.889720679

TIB - PE-Cy7-CCR4 Lec/nforGalNac


Lec/nforGalactose




Lec/nforL-Fucose


MFIofIsotypeControl

CCL17(-)MFI 1733 31.85661765 1438 26.43382353 1404 25.80882353 1293 23.76838235 54.4

CCL17(+) MFI 504 10.14084507 1364 27.44466801 1447 29.11468813 1408 28.32997988 49.7

%orfoldchange:

-70.91748413 0.318327739

-5.146036161 1.03824057 3.062678063 1.12809048 8.894044857 1.19191872

ChristopherY.Itoh1,MichikoShimoda1,EmanualMaverakis11DepartmentofDermatology,SchoolofMedicine,UniversityofCaliforniaDavis,Sacramento,CA,USA

RoleofCCR4inRegula/ngCellSurfaceGlycanExpressiononT-Lymphocytes

GalNacα-methylglucoside Galactose L-Fucose

CTL

TIB

!"#$ !ℎ!"#$ = !"#$ !"#ℎ !"#$!"#$ !"#ℎ!"# !"#$ =

!"# !" !"#$ + !"#ℎ !"#$%&!"# !" !"#$%&' !"#$%"&

!!" !" !"#$ – !"#ℎ !"#$%&!"# !" !"#$%&' !"#$%"&

CTLTIB

REFERENCES

CD4 CD4CCR4 CCR4

SSC

SSC

Lec/nbinding

Figure2.CD4andCCR4expressiononCTLandTIBcelllines.Representa/veFACSprofilesofSSCvsCD4orCCR4(red)comparedtoisotypecontrols(blue)areshown.

+TARC

coun

t

-TARC

+TARC-TARC

Figure3.Lec/nbindingprofilesofCTLandTIBcelllines.Representa/vehistogramsoflec/nbindingonCTLorTIBcellswith(red)orwithout(bue)TARCtreatmentareshown.

Table1.SummaryoffoldchangeoftheMFIforeachlec/nbindingonCTLandTIBcellswithandwithoutTARCtreatment

Figure1.FluorescentspectraviewerofemissionwavelengthforPE,PE-Cy7,andFITC

role of ccr4 in regulang cell surface glycan expression on ......of the final poster. all text and...

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