GM-CSF modulates the migratory phenotype of vaccine-induced T cells by enhancing CXCR3 expression

Il-Kang Na Carmen Scheibenbogen Ulrich Keilholz

Department of Hematology, Oncology and Transfusion Medicine, Charité Berlin, Campus Benjamin Franklin, Germany. Background

Selective receptor

Inflammatory Local tissue microenvironment CXCR3 Tumor tissue

CCR4 Skin

CCR9 Tissue specific dendritic cells Small intestine Aim of the study

Analysis of • Expression of chemokine receptors CXCR3 and CCR4 on vaccine-induced T cells.

• Influence of GM-CSF as vaccine adjuvant on expression. Materials & Methods

• Patient samples: stage III/ IV melanoma

1) Tyrosinase peptide + KLH + GM-CSF 2 cohorts 2) Tyrosinase peptide + KLH

• T cell response assessment by IFNγ flow cytometry analysis IFNg-secreting T cells / 106 PBMC Phase Itrialoftyrosinase peptidewith 250 100 150 200 50 10 20 30 40 50 0 0

P2 P31 1

P P32 2 2 P33 P2

3 P34 Tyr +KLH P 2

4 P35

P2 P35 5 P36 P 2 6 P36 P2

7 P37 P

2 P38 8

P2 P38 9

Patient P39 P 3 0 P40 ident 50 250 20 30 40 10 100 150 200 0 50 0

P3 P11 1 i f Tyr +KLHGM-CSF P11 i cation P

3 P12 2 P12 P3 3 P13 adjuvants P

3 P13 4 P14 P3

5 P15

P P15 3 6 P16

P3 P17 7

P17 G P 3 8 P18 M-CSF andKLH

P3 P18 9 P19 Schei b KLH specific Tyr specific enbogen et al. 2003 # 6 # 4 # 2 befo r e CXCR3 expression on KLH-specific T cells

no antigen KLH

25.2 0.02 24.7 0.06 74.7 0.04 75.1 0.2 Tyr + KLH cohort CXCR3 46.6 0.02 46.0 0.09 52.1 0.01 51.9 0.06 Tyr + KLH + GM-CSF cohort

IFNγ CXCR3, CCR4 and CCR9 expression on KLH-specific T cells

with GM-CSF without GM-CSF with GM-CSF without GM-CSF

s n=5 n=6

l

l

n=8 n=7 e 60

c

T

40 s 80

l

+ l p=0.001

4

e

R c 60 20

C

T

C

+ 0 40

3

%

R

C 20 total KLH- total KLH- X CD4+ specific CD4+ specific

C 0 T cells T cells T cells T cells

%

s

total KLH- total KLH- l l n=4 n=7

CD4+ specific CD4+ specific e 60

c T cells T cells T cells T cells

T

40

+

9

R 20

C

C

0

% Follow up of CXCR3 profile of KLH-specific T cells

total CD4+ T cells KLH-specific T cells 60 With Without 50 GM-CSF GM-CSF 40

30

20 % CXCR3 + T cells 10

0 1 6 17 1 4 18 time [months] Summary

• These results show for the first time in humans that it is possible to modulate chemokine receptor expression on T cells generated by vaccination by modifying the local vaccine milieu.

• Immunization protocols that induce T cells expressing CXCR3 may be of considerable value for improvement of immune therapy strategies. Ulrich Keilholz Sandra Bauer Anne Letsch Eckhard Thiel Anne Marie Asemissen Carmen Scheibenbogen Dirk Nagorsen