- Email: [email protected]
HLA-B44 is associated with progression of premalignant lesions of the cervix uteri, a prospective study
Targeting of immune effector cells in cancer
Tuesday, 24 June 1997 - Oral presentations
0.5.15.7
~ragxllular
cytokines in subsets of CD5* cells in
M.E. North, A.D.B. Webster, J. Farrant. Dept. ofClinical/mmuno/~ Free Hospital School of Medicine,London, UK
Roya/
Introduction:We have previously repotted that some patients with the antibody deficiency disease Common Variable lmmunodefiiiency (CVID) have an aberrant activation of CD4+ and CD8+ T cells with increased surface HLA-DR and intracellular IFNy. We have now analysed the CD8+ T ceils in more detail looking at cytokine content in subsets of CD8+ ceils that are activated or unactivated (assessed by CD89 or HLA-DR positivity) and in subsets defined by the presence or absence of CD28. Materialsand Methods:Mononuclear cells from the patients and normal donors were stimulated with PMA and ionomycin for 18 hours. This longer culture time did allow clear flow cytometric discrimination of CD8+ cells unlike CD4+ cells which lose the CD4+ antfgen on prolonged stimulation though protein kinase C. The mononuclear cells were fixed, permeabilised and stained with FITC-conjugated anti-cytokine monOclonalantibodies or anti-CD89 together with surface markers eg anti-CD8 (PE or PerCP), anti-HLA-DR. or anti-CD28 (PE or PerCP) antibodies using our previous methods with conjugated antibodies. Three colour flow cytometry was done using a Facscan. Patient and normal data of the proportions of cytokine positive cells were compared using Student’s t test. Result% With all three cytokines (IFNy, TNFcr, or IL-2), the mean cytokine positive cells induced in the total CD8+ cells In CVID were in the nolTnalrange. Three markers (CD28, CD89 and HLA-DR) were used to study CD8+ cell subsets. For CVID, the mean sizes of the CD8+ cell subsets were greater than normal for CD28 negative cells, less than normal for CD89 positiie cells and normal for HLA-DR positive or negative cells. There was a significant increase in CVID in the IFNy content within the CD28 positive subset of CD8+ cells. With TNFa and IL-2 there were no differences within the various CD8+ subsets in CVID compared to normal cells. Activation of CVID CD8+ cells measured by CD89 positivii was significantly reduced in both HLA-DR+ and HLA-DRpopulations but for CD28 populations only in the CD28 negative subset. The failure of CD89+ activation in the CD28 negative subset in CVID, was associated with a failure to express IFNy. Conclusion:An aberrant activation in CVID with raised IFNy levels in the CD28 positive subset of CD8+ cells, is accompanied by depressed levels of the early activation marker CD89 and a consequent resbiction of IFNy levels within the pathologically expanded CD28 negative cells. This immunodeficiency disease in which CD4+ cells fail to provide specific memory and fail to help B cell antibody production may thus originate in a defect in CD8+ cells due to an aberrant activation that disturbs the normal cytoklne network. 0.5.15.8
Functional CD4 T cell subsets activity in C57BUlO mice with inherited low IgG responsiveness
B. Rihov& M. &mv& I. hiha. institute of Microbiology AS CR, 142 20 Prague 4, Czech Republic
Introduction: The C57BUlO (BlO) mica exhibit a low IgG production against a variety of T-dependent antigens. Genetic control of the IgG response in BIO mice is multifactorial, with both the H-2 genes and the genes from the background being involved. Low IgG responsiveness is not due to a limitation in potential of B-cell repertoire per se. We have shown earlier that antigen-presenting function of BIO macrophages is impaired. Moreover, we and others (1) have also shown that in vitro production of IL-2 after a mitogenic signal delivered by the CD3-TCR complex is in the 810 strain limited. To further anaiysa the antibody response we studied the the selective activation of Th subsets (T helper type 1 and T helper type 2) during the antigenic stimulation in BlO mice. MaterialandMethods:Mice:Mice of A/J, C57BW OScSn (BIO) and BALB/c strains were obtained from the breeding colony of the Institute of Physiology AS CR. (BALB/c x BiO)Fi hybrids were bred in our own mouse facilities. Mice aged 10-l 5 weeks were used in all experiments. /mmunkation procedure: Mice were immunized with HEL or KLH in CFA S.C.in two sites at the base of the tail (!%mv&et al., 1998). Restimulation of lymph no& cells: Cells isolated from LN were cultivated with different doses of antigens (HEL, KLH) for 3 days. The proliferation was measured using 3H-TdR assay. After 48 h of cultivation, cytokines were estimated. Cyfokine assays: Antigen-driven IL-2 production was measured by a standard bioassay using a subclone of the CTL.L cell line insensitive to murine IL4. IL4 production was monitored by a bioassay based on the ability of IL4 to increase la expression on B cells. IFN-gamma production was evaluated by ELISA test kit. IL-3 actfvity was assayed using IL+dependent cell line DA-I. Reauh Proliferative response in lymph node cells of BlO mice prfmed in vivo and subjected to a restimulation in vitrowas highly restricted and almost no interlet&in-2 and interleukin-4 production was observed, whilst AN cells responded by significant proliferation and cytokine production. The antigen-specific T-cell response of BlO mice could not be increased by lipopolysaccharide treatment in viw or by in vitro cultivation with 11-2.T cells isolated from antigen-stimulated BlO mice have a very limited capacity to produce IL4 in response to stimulation
223
with anti-CD3, even in the presence of added 11-2.However, the T cells of BlO mice produced a high levels of IL-2 and IL4 when stimulated by phorbol myristate acetate (PMA) and Ca2+ ionophore, pmving the existence of a functionally intact signal transduction pathway downstream of protein kinase C (PKC). Conclusion: In viw antigen priming does not effectively activate Th2 cells in BIO mice. The limited activation consequently leads to low IgGl production. [l] M. &mvB, I. Riha, B. RihovB, Stand. J. Immunol. 44,453, 1998
16:30-l
OS.16
10.5.16.1
6:30
Room 0
Targeting of immune effector cells in cancer 1 HLA-B44 is associated with progression of premalignant lesions of the cervix uteri, a prospective study
H.J. Bontkes’, T.D. de Gruijl’, A.J. Remmink*, R.M.M. Verheijen2, T.J.M. Helmerhorst2~3,M.J. St&art’, M.F. Duggan-Keen4, P.L. Stem4, R.J.Scheper’,C.J.L.M. Meijer’, J.M.M. Walboomers’.‘Depf.ofPathd~ Free University Hospital, PO. Box 7057, 1007 MB Amsterdam, The Netherlands, 2Dept of Obstetrics and Gynaeculw Free LJniveMy Hospita/, RO. Box 7057, 1007 MB Amsterdam, The Netherian&, 3Dapt. Gynaecologic Onc&g~ The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands, 4CRC Dept. Immunology; Paterson /nstiMe for Cancer Research, Christie Hospiral NM Test, Manchester M20 96X, UK
Introduction:Cervical carcinomas are closely associated with high risk HPV types and are preceded by cervical intraepithelial neoplasia (CIN). Most CIN lesions regress spontaneously and will not evolve to invasive carcinoma. Cellular immune responses mediated by T-lymphocytes are thought to play an important role in the ultimate decline of CIN lesions. Major Histocompatibility Complex (MHC) class I and II proteins play a central role in the regulation of the T-cell response to foreign antigens. However, MHC class I expression is frequently down-regulated in HPV induced lesions, hereby frustrating tumour cell recognition by infiltrating cytotoxic T-lymphocytes (CTL). Several studies have suggested a role for HlA polymorphisms in CIN and cervical cancer. However these studies are difficult to interpret in relation to premalignant disease because patients with the same pathology will be heterogeneous in terms of disease progression. Patients from a non-intervention prospective follow-up study presenting with cytomorphologically abnonal smears can be classified with respect to HPV status and disease progression, providing an unique population for analysis of HLA polymorphism. Materialsand Methods:HPV 18 positive and HPV negative patients from a prospective cohort study were HLA typed at the class I and II loci using molecular methods. HLA class I expression was studied with immunohistcchemical methods using class I allele specific antibodies. Results: HLA-DRB1’07 was over represented in patients with an HPV18 infection compared to the patients that were negative for all HPV types. HLA-B*44 was over represented in patients that showed clinical progression compared to patients that did not show progression. Furthermore, HIA-B’44 is down-regulated on a substantial number of progressing lesions, while the expression of this allele was normal on non-progressing lesions. Conclusion: HLA-B’44 is associated with disease progression, a significant component of this effect appears to be due to down-regulation of HlA-B’44 on the neoplastic cells. IO.51 6.2 1 Enhanced generation of specific tumor-reactive CTL in vitro by Melan-A/Mart-l immunodominant peptide analogs D. Valmori ‘, C. Marati6n Lizana 2, D. Li6nard 1.3, D. Rimoldi 4, V. Jongeneel 4, J.-C. Cerottini 1.4,P. Romero 1*4.‘Divisionof Clinical Onto-lmmunolog): Ludwig Institute tbr Cancer Research, CHlJV 101 I Lausanne, Swkzerland, 2 lstituto de Patasitologia y Biomedicina, Consejo Superior de Investigaciones Cientikas, 194301Granada, Spain, 3 Centre Pluridicliplinaire d’Oncoimmunologie, CHUY 1011 Lausanne, Switzerland, 4Ludwig Institute for Cancer Research, Lausanne branch, University of Lausanne, 1066 Epalinges, Switzerland
Introduction:The Melan-A/Mart-l gene is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines. It codes for an antigen recognized by cytolytic T lymphocytes (CTL) in the context of
Tuesday, 24 June 1997 - Oral presentations
0.5.15.7
~ragxllular
cytokines in subsets of CD5* cells in
M.E. North, A.D.B. Webster, J. Farrant. Dept. ofClinical/mmuno/~ Free Hospital School of Medicine,London, UK
Roya/
Introduction:We have previously repotted that some patients with the antibody deficiency disease Common Variable lmmunodefiiiency (CVID) have an aberrant activation of CD4+ and CD8+ T cells with increased surface HLA-DR and intracellular IFNy. We have now analysed the CD8+ T ceils in more detail looking at cytokine content in subsets of CD8+ ceils that are activated or unactivated (assessed by CD89 or HLA-DR positivity) and in subsets defined by the presence or absence of CD28. Materialsand Methods:Mononuclear cells from the patients and normal donors were stimulated with PMA and ionomycin for 18 hours. This longer culture time did allow clear flow cytometric discrimination of CD8+ cells unlike CD4+ cells which lose the CD4+ antfgen on prolonged stimulation though protein kinase C. The mononuclear cells were fixed, permeabilised and stained with FITC-conjugated anti-cytokine monOclonalantibodies or anti-CD89 together with surface markers eg anti-CD8 (PE or PerCP), anti-HLA-DR. or anti-CD28 (PE or PerCP) antibodies using our previous methods with conjugated antibodies. Three colour flow cytometry was done using a Facscan. Patient and normal data of the proportions of cytokine positive cells were compared using Student’s t test. Result% With all three cytokines (IFNy, TNFcr, or IL-2), the mean cytokine positive cells induced in the total CD8+ cells In CVID were in the nolTnalrange. Three markers (CD28, CD89 and HLA-DR) were used to study CD8+ cell subsets. For CVID, the mean sizes of the CD8+ cell subsets were greater than normal for CD28 negative cells, less than normal for CD89 positiie cells and normal for HLA-DR positive or negative cells. There was a significant increase in CVID in the IFNy content within the CD28 positive subset of CD8+ cells. With TNFa and IL-2 there were no differences within the various CD8+ subsets in CVID compared to normal cells. Activation of CVID CD8+ cells measured by CD89 positivii was significantly reduced in both HLA-DR+ and HLA-DRpopulations but for CD28 populations only in the CD28 negative subset. The failure of CD89+ activation in the CD28 negative subset in CVID, was associated with a failure to express IFNy. Conclusion:An aberrant activation in CVID with raised IFNy levels in the CD28 positive subset of CD8+ cells, is accompanied by depressed levels of the early activation marker CD89 and a consequent resbiction of IFNy levels within the pathologically expanded CD28 negative cells. This immunodeficiency disease in which CD4+ cells fail to provide specific memory and fail to help B cell antibody production may thus originate in a defect in CD8+ cells due to an aberrant activation that disturbs the normal cytoklne network. 0.5.15.8
Functional CD4 T cell subsets activity in C57BUlO mice with inherited low IgG responsiveness
B. Rihov& M. &mv& I. hiha. institute of Microbiology AS CR, 142 20 Prague 4, Czech Republic
Introduction: The C57BUlO (BlO) mica exhibit a low IgG production against a variety of T-dependent antigens. Genetic control of the IgG response in BIO mice is multifactorial, with both the H-2 genes and the genes from the background being involved. Low IgG responsiveness is not due to a limitation in potential of B-cell repertoire per se. We have shown earlier that antigen-presenting function of BIO macrophages is impaired. Moreover, we and others (1) have also shown that in vitro production of IL-2 after a mitogenic signal delivered by the CD3-TCR complex is in the 810 strain limited. To further anaiysa the antibody response we studied the the selective activation of Th subsets (T helper type 1 and T helper type 2) during the antigenic stimulation in BlO mice. MaterialandMethods:Mice:Mice of A/J, C57BW OScSn (BIO) and BALB/c strains were obtained from the breeding colony of the Institute of Physiology AS CR. (BALB/c x BiO)Fi hybrids were bred in our own mouse facilities. Mice aged 10-l 5 weeks were used in all experiments. /mmunkation procedure: Mice were immunized with HEL or KLH in CFA S.C.in two sites at the base of the tail (!%mv&et al., 1998). Restimulation of lymph no& cells: Cells isolated from LN were cultivated with different doses of antigens (HEL, KLH) for 3 days. The proliferation was measured using 3H-TdR assay. After 48 h of cultivation, cytokines were estimated. Cyfokine assays: Antigen-driven IL-2 production was measured by a standard bioassay using a subclone of the CTL.L cell line insensitive to murine IL4. IL4 production was monitored by a bioassay based on the ability of IL4 to increase la expression on B cells. IFN-gamma production was evaluated by ELISA test kit. IL-3 actfvity was assayed using IL+dependent cell line DA-I. Reauh Proliferative response in lymph node cells of BlO mice prfmed in vivo and subjected to a restimulation in vitrowas highly restricted and almost no interlet&in-2 and interleukin-4 production was observed, whilst AN cells responded by significant proliferation and cytokine production. The antigen-specific T-cell response of BlO mice could not be increased by lipopolysaccharide treatment in viw or by in vitro cultivation with 11-2.T cells isolated from antigen-stimulated BlO mice have a very limited capacity to produce IL4 in response to stimulation
223
with anti-CD3, even in the presence of added 11-2.However, the T cells of BlO mice produced a high levels of IL-2 and IL4 when stimulated by phorbol myristate acetate (PMA) and Ca2+ ionophore, pmving the existence of a functionally intact signal transduction pathway downstream of protein kinase C (PKC). Conclusion: In viw antigen priming does not effectively activate Th2 cells in BIO mice. The limited activation consequently leads to low IgGl production. [l] M. &mvB, I. Riha, B. RihovB, Stand. J. Immunol. 44,453, 1998
16:30-l
OS.16
10.5.16.1
6:30
Room 0
Targeting of immune effector cells in cancer 1 HLA-B44 is associated with progression of premalignant lesions of the cervix uteri, a prospective study
H.J. Bontkes’, T.D. de Gruijl’, A.J. Remmink*, R.M.M. Verheijen2, T.J.M. Helmerhorst2~3,M.J. St&art’, M.F. Duggan-Keen4, P.L. Stem4, R.J.Scheper’,C.J.L.M. Meijer’, J.M.M. Walboomers’.‘Depf.ofPathd~ Free University Hospital, PO. Box 7057, 1007 MB Amsterdam, The Netherlands, 2Dept of Obstetrics and Gynaeculw Free LJniveMy Hospita/, RO. Box 7057, 1007 MB Amsterdam, The Netherian&, 3Dapt. Gynaecologic Onc&g~ The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands, 4CRC Dept. Immunology; Paterson /nstiMe for Cancer Research, Christie Hospiral NM Test, Manchester M20 96X, UK
Introduction:Cervical carcinomas are closely associated with high risk HPV types and are preceded by cervical intraepithelial neoplasia (CIN). Most CIN lesions regress spontaneously and will not evolve to invasive carcinoma. Cellular immune responses mediated by T-lymphocytes are thought to play an important role in the ultimate decline of CIN lesions. Major Histocompatibility Complex (MHC) class I and II proteins play a central role in the regulation of the T-cell response to foreign antigens. However, MHC class I expression is frequently down-regulated in HPV induced lesions, hereby frustrating tumour cell recognition by infiltrating cytotoxic T-lymphocytes (CTL). Several studies have suggested a role for HlA polymorphisms in CIN and cervical cancer. However these studies are difficult to interpret in relation to premalignant disease because patients with the same pathology will be heterogeneous in terms of disease progression. Patients from a non-intervention prospective follow-up study presenting with cytomorphologically abnonal smears can be classified with respect to HPV status and disease progression, providing an unique population for analysis of HLA polymorphism. Materialsand Methods:HPV 18 positive and HPV negative patients from a prospective cohort study were HLA typed at the class I and II loci using molecular methods. HLA class I expression was studied with immunohistcchemical methods using class I allele specific antibodies. Results: HLA-DRB1’07 was over represented in patients with an HPV18 infection compared to the patients that were negative for all HPV types. HLA-B*44 was over represented in patients that showed clinical progression compared to patients that did not show progression. Furthermore, HIA-B’44 is down-regulated on a substantial number of progressing lesions, while the expression of this allele was normal on non-progressing lesions. Conclusion: HLA-B’44 is associated with disease progression, a significant component of this effect appears to be due to down-regulation of HlA-B’44 on the neoplastic cells. IO.51 6.2 1 Enhanced generation of specific tumor-reactive CTL in vitro by Melan-A/Mart-l immunodominant peptide analogs D. Valmori ‘, C. Marati6n Lizana 2, D. Li6nard 1.3, D. Rimoldi 4, V. Jongeneel 4, J.-C. Cerottini 1.4,P. Romero 1*4.‘Divisionof Clinical Onto-lmmunolog): Ludwig Institute tbr Cancer Research, CHlJV 101 I Lausanne, Swkzerland, 2 lstituto de Patasitologia y Biomedicina, Consejo Superior de Investigaciones Cientikas, 194301Granada, Spain, 3 Centre Pluridicliplinaire d’Oncoimmunologie, CHUY 1011 Lausanne, Switzerland, 4Ludwig Institute for Cancer Research, Lausanne branch, University of Lausanne, 1066 Epalinges, Switzerland
Introduction:The Melan-A/Mart-l gene is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines. It codes for an antigen recognized by cytolytic T lymphocytes (CTL) in the context of