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Expression of core and E1 proteins from hepatitis C virus in dendritic cells impairs T cell induction in vivo
15
Monday, March 31,2003
Parallel Session 4: Immunology
I37
EXPRESSION
OF CORE AND El PROTEINS
C VIRUS IN DENDRITIC
FROM HEPATITIS
CELLS IMPAIRS T CELL INDUCTION
IN
VIVO F! &robe, J.J. Lasarte, A. Zabaleta, L. Arribillaga, A. Arina, I. Melero, F. Bon-as-Cuesta, .I. Prieto. ‘Division Of Hepatology And Gene Therapy, FIMA, University Of Navarra,
Pamplona,
Spain
HCV infection is characterized by low or undetectable cellular immune responses against viral antigens. Some reports suggest that viral proteins may have an immunomodulatory role, suppressing these responses. Thus, dendritic cells (DC) expressing viral antigens have shown low in vitro stimulatory ability. The aim of this work was to study the in viva effect of HCV core and El proteins (CEl) on the induction of cellular immune responses. Immunization of mice with an adenovirus encoding HCV CEl proteins (AdCEl), induced CD4 T-cell responses against core protein as efficiently as an adenovirus encoding HCV NS3 (AdNS3) did against NS3 protein. Moreover, immune responses against adenoviral antigens, were similar in AdCEl and in AdNS3 immunized mice. However, immunization with immature DC transduced with AdCEl (DC-CEl) induced a lower CD4 T-cell response than immunization with DC transduced with AdNS3 (DCNS3). This low CD4 T-cell response was accompanied by a weaker CD8 T-cell response in DC-CEl immunized mice. Experiments of CD8 T-cell response induction carried out in a CD4-independent system showed that DC-CEl induced similar CD8 responses than DC-NS3, suggesting that the defect in CD8 responses is concomitant to the low CD4 responses. Finally, immunization with DC matured previously to infection with AdCEl or AdNS3, revealed that there were not differences in the induction ability between mature DC-CEl and mature DC-NS3. These results suggest that HCV CEl proteins have an immunomodulatory effect on T-cell responses, by decreasing the stimulatory ability of DC in viva, possibly by interfering with the maturation of these cells.
I38
DENDRITIC
CELLS
ENVELOPE
PROTEINS
RESPONSE
AND DECREASE
PREVENTING
PULSED WITH RECOMBINANT
HBV
ELICIT A TH2 TYPE IMMUNE
THE INDUCTION
NKT LYMPHOCYTES
THUS
OF VIRAL SPECIFIC
B. Thalenfeld2, 0. Shibolet’, A. Roslana’, L. Zolotarov’, D. Engelhardt2, E. Rabbani2, Y. Ilan’. ‘Liver Unit, Department Medicine,
Hadassah
Israel; 2ENZ0.
Hebrew
Biochem,
University Medical
IMMUNITY
Of
Centec Jerusalem,
New York, NI: USA
Background: Recovery from HBV infection requires an antigen specific Thl immune response. Dendritic cells (DC) can activate helper and suppressor T-cells and may elicit either Thl or Th2 responses. Aim: To assess the role of HBV antigen pulsed DC in anti-HBV immunity. Methods: DCs were separated using magnetic beads, pulsed overnight with HBV envelope proteins (HBsAg+Pre Sl+Pre S2), and injected intraperitoneally (1.P). Control mice were injected IP with HBV proteins. Booster HBV injections were administered at 3 weeks. Mice were tested for anti-HBs antibody levels, FACS analysis for CD4+, CD8+, and NKl.l+, HBV specific T cell IFNy ELISPOT assay, and serum cytokines. Results: Adoptive transfer of HBV pulsed DCs prevented the induction of
anti-HBV immunity. Anti-HBs antibody levels were undetectable in DCs treated vs 7.5 IU in immunized controls. Similarly, HBV antigen pulsed DCs prevented the induction of HBV specific T cell response. A marked decrease in IFNy producing T-cells clones was noted (0 vs. 6.6 spot forming colonies, in DCs treated vs. controls, respectively). A significant reduction in the CD4+/CD8+ T cell ratio, and in peripheral NKT lymphocytes, was observed (1.5 vs. 1.78, and 5.5% vs. 8%, in DCs treated and control group, respectively). The IFNy/ILlO cytokine ratio decreased significantly in DC treated mice (0.49 vs. 236, respectively). Conclusions: Adoptive transfer of HBV pulsed DCs induced antigen specific Th2 type of immune response, and prevented the induction of effective anti viral immunity. These findings suggest that the HBV immune escape mechanism may involve a Th2 immune shift.
I39
DYNAMICS
OF ADHESION
OF LYMPHOCYTES
WITH THE NOVEL CHEMOKINE
M. Hevdtmann’, ‘Birmingham
l? Lalor’,
CXCLl6
S.G. H’ubscher’,
University, Birmingham,
ACTIVATED
TO VCAM
M. Briskin2, D.H. Adams’.
UK; 2Millenium
Inc., Boston MA,
USA
Background: CXCL16 exists in a transmembrane and soluble form and is present in liver. Its receptor, CXCR6 is expressed on intrahepatic lymphocytes and we have recently shown a role for CXCL16/CXCR6 interactions in recruitment of lymphocytes to the liver. Here we show that CXCL16 mediated signaling leads to activation of VLA-4 with adhesion to VCAM-1 under static and flow conditions. Methods: PBLCs and CXCR6 transfected L1.2 cells were activated and resultant VLA-4 activation was analyzed by the conformational antibodies 9EG7 (mouse) and 12GlO (human) using flow cytometry. In static and dynamic adhesion assays the dynamics of CXCL16 mediated adhesion on VCAM + CXCL16 versus VCAM alone were analyzed. Results: Human recombinant CXCL16 led to activation of VLA-4 on CXCR6 transfected L1.2 cells. The activation was blocked by pertussis toxin, confirming a G-protein coupled signaling process. Cross-linking of CXCR6 on human PBLCs similarly led to VLA-4 activation. Static and dynamic adhesion assays showed that CXCR6 interactions with immobilized CXCL16 were unable to support rolling or static adhesion in the absence of VCAM In static adhesion assays 2 kg/ml CXCL16 triggered VLA-4 dependent adhesion. Flow-based adhesion assays revealed rapid triggering of firm adhesion on co-immobilized CXCL16 and VCAM with absence of a rolling step. Conclusions: CXCL16 is unable to support adhesion alone but when coimmobilized with VCAM-1 it is a potent trigger of VLA-4 dependent adhesion in lymphocytes via a CXCR6 G-protein dependent mechanism Co-expression of CXCL16 and VCAM-1 in the liver suggests this may be an important mechanism of liver damage.
I40
ANALYSIS
AND FUNCTION
VIRUS-SPECIFIC
OF DELTA-HEPATITIS
CELLULAR
IMMUNE RESPONSES
N. Aslan’,
C. Yurdaydin2, H. Bozkaya2, l? Baglan2, A.M. Bozdayi2, H.L. Tillmann’, M.P. Manns’, H. Wedemeyer’. ‘Dept. Of Gastroenterology School, Hannoves
Ankara,
And Hepatology
And Endocrinology,
Get-many; 2Ankara
Hannover
Medical
University, School Of Medicine,
Turkey
Infection with the hepatitis D virus (HDV) is a major health problem in Turkey and the Middle East. Since HDV genotype 1 is non-cytopathic, hep-
Monday, March 31,2003
Parallel Session 4: Immunology
I37
EXPRESSION
OF CORE AND El PROTEINS
C VIRUS IN DENDRITIC
FROM HEPATITIS
CELLS IMPAIRS T CELL INDUCTION
IN
VIVO F! &robe, J.J. Lasarte, A. Zabaleta, L. Arribillaga, A. Arina, I. Melero, F. Bon-as-Cuesta, .I. Prieto. ‘Division Of Hepatology And Gene Therapy, FIMA, University Of Navarra,
Pamplona,
Spain
HCV infection is characterized by low or undetectable cellular immune responses against viral antigens. Some reports suggest that viral proteins may have an immunomodulatory role, suppressing these responses. Thus, dendritic cells (DC) expressing viral antigens have shown low in vitro stimulatory ability. The aim of this work was to study the in viva effect of HCV core and El proteins (CEl) on the induction of cellular immune responses. Immunization of mice with an adenovirus encoding HCV CEl proteins (AdCEl), induced CD4 T-cell responses against core protein as efficiently as an adenovirus encoding HCV NS3 (AdNS3) did against NS3 protein. Moreover, immune responses against adenoviral antigens, were similar in AdCEl and in AdNS3 immunized mice. However, immunization with immature DC transduced with AdCEl (DC-CEl) induced a lower CD4 T-cell response than immunization with DC transduced with AdNS3 (DCNS3). This low CD4 T-cell response was accompanied by a weaker CD8 T-cell response in DC-CEl immunized mice. Experiments of CD8 T-cell response induction carried out in a CD4-independent system showed that DC-CEl induced similar CD8 responses than DC-NS3, suggesting that the defect in CD8 responses is concomitant to the low CD4 responses. Finally, immunization with DC matured previously to infection with AdCEl or AdNS3, revealed that there were not differences in the induction ability between mature DC-CEl and mature DC-NS3. These results suggest that HCV CEl proteins have an immunomodulatory effect on T-cell responses, by decreasing the stimulatory ability of DC in viva, possibly by interfering with the maturation of these cells.
I38
DENDRITIC
CELLS
ENVELOPE
PROTEINS
RESPONSE
AND DECREASE
PREVENTING
PULSED WITH RECOMBINANT
HBV
ELICIT A TH2 TYPE IMMUNE
THE INDUCTION
NKT LYMPHOCYTES
THUS
OF VIRAL SPECIFIC
B. Thalenfeld2, 0. Shibolet’, A. Roslana’, L. Zolotarov’, D. Engelhardt2, E. Rabbani2, Y. Ilan’. ‘Liver Unit, Department Medicine,
Hadassah
Israel; 2ENZ0.
Hebrew
Biochem,
University Medical
IMMUNITY
Of
Centec Jerusalem,
New York, NI: USA
Background: Recovery from HBV infection requires an antigen specific Thl immune response. Dendritic cells (DC) can activate helper and suppressor T-cells and may elicit either Thl or Th2 responses. Aim: To assess the role of HBV antigen pulsed DC in anti-HBV immunity. Methods: DCs were separated using magnetic beads, pulsed overnight with HBV envelope proteins (HBsAg+Pre Sl+Pre S2), and injected intraperitoneally (1.P). Control mice were injected IP with HBV proteins. Booster HBV injections were administered at 3 weeks. Mice were tested for anti-HBs antibody levels, FACS analysis for CD4+, CD8+, and NKl.l+, HBV specific T cell IFNy ELISPOT assay, and serum cytokines. Results: Adoptive transfer of HBV pulsed DCs prevented the induction of
anti-HBV immunity. Anti-HBs antibody levels were undetectable in DCs treated vs 7.5 IU in immunized controls. Similarly, HBV antigen pulsed DCs prevented the induction of HBV specific T cell response. A marked decrease in IFNy producing T-cells clones was noted (0 vs. 6.6 spot forming colonies, in DCs treated vs. controls, respectively). A significant reduction in the CD4+/CD8+ T cell ratio, and in peripheral NKT lymphocytes, was observed (1.5 vs. 1.78, and 5.5% vs. 8%, in DCs treated and control group, respectively). The IFNy/ILlO cytokine ratio decreased significantly in DC treated mice (0.49 vs. 236, respectively). Conclusions: Adoptive transfer of HBV pulsed DCs induced antigen specific Th2 type of immune response, and prevented the induction of effective anti viral immunity. These findings suggest that the HBV immune escape mechanism may involve a Th2 immune shift.
I39
DYNAMICS
OF ADHESION
OF LYMPHOCYTES
WITH THE NOVEL CHEMOKINE
M. Hevdtmann’, ‘Birmingham
l? Lalor’,
CXCLl6
S.G. H’ubscher’,
University, Birmingham,
ACTIVATED
TO VCAM
M. Briskin2, D.H. Adams’.
UK; 2Millenium
Inc., Boston MA,
USA
Background: CXCL16 exists in a transmembrane and soluble form and is present in liver. Its receptor, CXCR6 is expressed on intrahepatic lymphocytes and we have recently shown a role for CXCL16/CXCR6 interactions in recruitment of lymphocytes to the liver. Here we show that CXCL16 mediated signaling leads to activation of VLA-4 with adhesion to VCAM-1 under static and flow conditions. Methods: PBLCs and CXCR6 transfected L1.2 cells were activated and resultant VLA-4 activation was analyzed by the conformational antibodies 9EG7 (mouse) and 12GlO (human) using flow cytometry. In static and dynamic adhesion assays the dynamics of CXCL16 mediated adhesion on VCAM + CXCL16 versus VCAM alone were analyzed. Results: Human recombinant CXCL16 led to activation of VLA-4 on CXCR6 transfected L1.2 cells. The activation was blocked by pertussis toxin, confirming a G-protein coupled signaling process. Cross-linking of CXCR6 on human PBLCs similarly led to VLA-4 activation. Static and dynamic adhesion assays showed that CXCR6 interactions with immobilized CXCL16 were unable to support rolling or static adhesion in the absence of VCAM In static adhesion assays 2 kg/ml CXCL16 triggered VLA-4 dependent adhesion. Flow-based adhesion assays revealed rapid triggering of firm adhesion on co-immobilized CXCL16 and VCAM with absence of a rolling step. Conclusions: CXCL16 is unable to support adhesion alone but when coimmobilized with VCAM-1 it is a potent trigger of VLA-4 dependent adhesion in lymphocytes via a CXCR6 G-protein dependent mechanism Co-expression of CXCL16 and VCAM-1 in the liver suggests this may be an important mechanism of liver damage.
I40
ANALYSIS
AND FUNCTION
VIRUS-SPECIFIC
OF DELTA-HEPATITIS
CELLULAR
IMMUNE RESPONSES
N. Aslan’,
C. Yurdaydin2, H. Bozkaya2, l? Baglan2, A.M. Bozdayi2, H.L. Tillmann’, M.P. Manns’, H. Wedemeyer’. ‘Dept. Of Gastroenterology School, Hannoves
Ankara,
And Hepatology
And Endocrinology,
Get-many; 2Ankara
Hannover
Medical
University, School Of Medicine,
Turkey
Infection with the hepatitis D virus (HDV) is a major health problem in Turkey and the Middle East. Since HDV genotype 1 is non-cytopathic, hep-